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Curcumin is among the most well-studied natural substances, known for its biological actions within the central nervous system, its antioxidant and anti-inflammatory properties, and human health benefits. However, challenges persist in effectively utilising curcumin, addressing its metabolism and passage through the blood-brain barrier (BBB) in therapies targeting cerebrovascular diseases. Current challenges in curcumin's applications revolve around its effects within neoplastic tissues alongside the development of intelligent formulations to enhance its bioavailability. Formulations have been discovered including curcumin's complexes with brain-derived phospholipids and proteins, or its liposomal encapsulation. These novel strategies aim to improve curcumin's bioavailability and stability, and its capability to cross the BBB, thereby potentially enhancing its efficacy in treating cerebrovascular diseases. In summary, this review provides a comprehensive overview of molecular pathways involved in interactions of curcumin and its metabolites, and brain vascular homeostasis. This review explores cellular and molecular current aspects, of curcumin-based effects with an emphasis on curcumin's metabolism and its impact on pathological conditions, such as neurodegenerative diseases, schizophrenia, and cerebral angiopathy. It also highlights the limitations posed by curcumin's poor bioavailability and discusses ongoing efforts to surpass these impediments to harness the full therapeutic potential of curcumin in neurological disorders.

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Soil organic carbon (SOC) is crucial for ecosystem function and carbon storage, especially in mountain regions where cooler temperatures limit microbial activity, leading to higher SOC stocks compared to lowlands. However, the available data are insufficient to fully understand the distribution of SOC properties along elevation and snow cover duration gradients. Given that climate change models predict a reduction in snow cover duration, it is essential to better characterize these properties at a finer, mesotopographic scale (e.g., ridges and slopes), corresponding to the distribution of mountain plant communities. This study investigates the impact of microclimate on SOC content and stability in European mountain grasslands. We focused on two types of grasslands on acidic soils to maintain homogeneity in key parameters such as soil properties and plant communities. These grasslands, located across temperate European mountain ranges (Alps, Pyrenees, Vosges, Balkans, Carpathians, Black Forest, Bohemian Forest, and Sudetes), span a gradient of snow cover duration, ranging from frost-exposed ridges dominated by Carex curvula, to intermediate grasslands, without frost, dominated by Nardus stricta. SOC content and stability were assessed using Rock-Eval (R) thermal analysis across all sites. The results indicate that microclimate significantly influences SOC properties. Cooler temperatures, driven by elevation and reduced snow cover duration, were associated with increased SOC content but decreased stability. On windy ridges, extended growing seasons combined with intense winter freezing led to higher SOC lability, as freezing slows down mineralization processes. In contrast, intermediate grasslands, with longer growing seasons, showed enhanced SOC stability due to higher decomposition activity. These findings provide valuable insights into how SOC properties may evolve under climate change, particularly in relation to rising temperatures and shifting snow cover dynamics.

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To ensure food and feed security, modern maize hybrids must not only perform well under changing climate conditions but also consistently achieve higher and stable yields, exhibit maximum tolerance to stress factors, and produce high quality grains. In a study conducted in 2022 and 2023, 50 maize hybrids were developed from crosses of five elite (highly productive) inbred lines and ten lines possessing favorable genes for carotenoid content. These hybrids were tested under particularly unfavorable conditions for maize cultivation. The aim was to identify which lines effectively transmit the desired traits to the offspring (general combining ability-GCA), and to identify superior hybrids in terms of productivity, adaptability, and quality (specific combining ability-SCA). The study revealed that total carotenoids ranged from 2.30 to 40.20 mu g/g for the inbred lines and from 7.45 to 25.08 mu g/g for hybrids. A wider distribution of values was observed in the inbred lines compared to the hybrids for key carotenoids such as lutein, zeaxanthin, beta-cryptoxanthin, and beta-carotene. Among the hybrids, notable performers in yield, adaptability, and carotenoid content included E390xD302, A452xD302, and A447xD302. The paternal inbred line D302 exhibited a high general combining ability for yield (1446 kg ha-1) and, when crossed with several inbred lines, produced hybrids with enhanced yields and higher levels of zeaxanthin, lutein, and beta-carotene, as well as improved unbroken plants percent.

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This study aimed to investigate the biotransformation capabilities of a hydroquinone-tolerant Digitalis purpurea cell line (DpHQ) for bioconverting hydroquinone (HQ) into arbutin, a compound with significant therapeutic and cosmetic applications. The research evaluated the influence of various HQ concentrations, feeding protocols, and carbon sources on arbutin bioconversion yield. By using HPLC-MS for the quantification of arbutin in biomass and medium, the study revealed that higher precursor (HQ) concentration led to a more pronounced growth inhibition under single dosing than sequential dosing. At lower sugar (3%) and precursor (4 mM HQ) levels, arbutin predominantly remained within the cells, whereas higher sugar (6%) and HQ (5-6 mM) levels promoted its release into the medium. Arbutin production ranged from 591 mg/L under single dosing to 3049 mg/L with sequential dosing, with the highest yield being achieved with 5 mM HQ in divided doses and 6% glucose. This study holds novelty for being the first to demonstrate the DpHQ's tolerance to high concentrations of HQ and its efficient capabilities to bioconvert HQ to arbutin, indicating that D. purpurea is equipped with the enzymes required for this process. These aspects highlight its potential as a biotechnological source for arbutin synthesis.

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Background and aims: Hepatocellular carcinoma (HCC) is a growing global concern with an increasing incidence rate. The intestinal microbiota has been identified as a potential culprit in modulating the effects of antitumoral drugs. We aimed to assess the impact of adding Lactobacillus rhamnosus probiotic to regorafenib in mice with HCC. Methods: Cirrhosis and HCCs were induced in 56 male Swiss mice via diethylnitrosamine injection and carbon tetrachloride administration. Mice were divided into four groups: treated with vehicle (VC), regorafenib (Rego), L. rhamnosus probiotic, and a combination of regorafenib and probiotic (Rego-Pro). After 3 weeks of treatment, liver and intestinal fragments were collected for analysis. Results: Regorafenib elevated gut permeability, an effect mitigated by probiotic intervention, which exhibited a notable correlation with reduced inflammation (p < 0.01). iNOS levels were also reduced by adding the probiotic with respect to the mice treated with regorafenib only (p < 0.001). Notably, regorafenib substantially increased IL-6, TNF-a and TLR4 in intestinal fragments (p < 0.01). The administration of the probiotic effectively restored IL-6 to its initial levels (p < 0.001). Conclusion: Reducing systemic and intestinal inflammation by administering L. rhamnosus probiotic may alleviate tumoral resistance and systemic adverse effects.

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Epilobium hirsutum L., commonly known as hairy willowherb, is a perennial herbaceous plant native to Europe and Asia. In Romania, the Epilobium genus includes 17 species that are used in folk medicine for various purposes. This study aimed to investigate the anti-inflammatory and antitumor potential of the optimized extract of Epilobium hirsutum (EH) in animal models. The first study investigated the anti-inflammatory properties of EH optimized extract and the model used was carrageenan-induced paw inflammation. Wistar rats were divided into three groups: negative control, positive control treated with indomethacin, and a group treated with the extract. Oxidative stress markers, cytokine levels, and protein expressions were assessed. The extract demonstrated anti-inflammatory properties comparable to those of the control group. In the second study, the antitumor effects of the extract were assessed using the tumor model of Ehrlich ascites carcinoma. Swiss albino mice with Ehrlich ascites were divided into four groups: negative, positive treated with cyclophosphamide (Cph), Group 3 treated with Cph and EH optimized extract, and Group 4 treated with extract alone. Samples from the ascites fluid, liver, and heart were analyzed to evaluate oxidative stress, inflammation, and cancer markers. The extract showed a reduction in tumor-associated inflammation and oxidative stress. Overall, the EH optimized extract exhibited promising anti-inflammatory and antitumor effects in the animal models studied. These findings suggest its potential as a natural adjuvant therapeutic agent for addressing inflammation and oxidative stress induced by different pathologies.

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TMAO, a gut microbiota derived byproduct, has been associated with various cardiometabolic diseases by promoting oxidative stress and inflammation. The liver is the main organ for TMAO production and chronic exposure to high doses of TMAO could alter its function. In this study, we evaluated the effect of chronic exposure of high TMAO doses on liver oxidative stress, inflammation, and fibrosis. TMAO was administered daily via gastric gavage to laboratory rats for 3 months. Blood was drawn for the quantification of TMAO, and liver tissues were harvested for the assessment of oxidative stress (MDA, GSH, GSSG, GPx, CAT, and 8-oxo-dG) and inflammation by quantification of IL-1 alpha, TNF-alpha, IL-10, TGF-(3, NOS and COX- 2 expression. The evaluation of fibrosis was made by Western blot analysis of alpha-SMA and Collagen-3 protein expression. Histological investigation and immunohistochemical staining of iNOS were performed in order to assess the liver damage. After 3 months of TMAO exposure, TMAO serum levels enhanced in parallel with increases in MDA and GSSG levels in liver tissue and lower values of GSH and GSH/GSSG ratio as well as a decrease in GPx and CAT activities. Inflammation was also highlighted, with enhanced iNOS, COX-2, and IL-10 expression, without structural changes and without induction of liver fibrosis.

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Microsatellites are codominant markers that, due to their high polymorphism, are a common choice for detecting genetic variability in various organisms, including fungi, plants, and animals. However, the process of developing these markers is both costly and time-consuming. As a result, the cross-species amplification has become a more rapid and more affordable alternative in biological studies. The objective of this study was to assess the applicability of 13 SSR markers, originally designed for Carex curvula, in other 14 species belonging to different sections of the genus. All the markers were successfully transferred with a mean of 90.76%, and 100% transferability was reached in two species (C. baldensis and C. rupestris). The lowest transferability was registered in the G165 marker, which did not produce amplification in six species. Together, the microsatellites amplified a total of 183 alleles, ranging from 10 to 19 alleles per locus, with an average of 14.07. The mean number of different alleles ranged from 0.846 to a maximum of 2.077 per locus. No significant departures from the Hardy-Weinberg equilibrium were detected in polymorphic loci. The transferability of the 13 SSR markers proved highly successful in various Carex species, across different clades and sections of the genus.

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The intricate cooperation between cancer cells and nontumor stromal cells within melanoma microenvironment (MME) enables tumor progression and metastasis. We previously demonstrated that the interplay between tumor-associated macrophages (TAMs) and melanoma cells can be disrupted by using long-circulating liposomes (LCLs) encapsulating prednisolone phosphate (PLP) (LCL-PLP) that inhibited tumor angiogenesis coordinated by TAMs. In this study, our goal was to improve LCL specificity for protumor macrophages (M2-like (i.e., TAMs) macrophages) and to induce a more precise accumulation at tumor site by loading PLP into IL-13-conjugated liposomes (IL-13-LCL-PLP), since IL-13 receptor is overexpressed in this type of macrophages. The IL-13-LCL-PLP liposomal formulation was obtained by covalent attachment of thiolated IL-13 to maleimide-functionalized LCL-PLP. C57BL/6 mice bearing B16.F10 s.c melanoma tumors were used to investigate the antitumor action of LCL-PLP and IL-13-LCL-PLP. Our results showed that IL-13-LCL-PLP formulation remained stable in biological fluids after 24h and it was preferentially taken up by M2 polarized macrophages. IL-13-LCL-PLP induced strong tumor growth inhibition compared to nonfunctionalized LCL-PLP at the same dose, by altering TAMs-mediated angiogenesis and oxidative stress, limiting resistance to apoptosis and invasive features in MME. These findings suggest IL-13-LCL-PLP might become a promising delivery platform for chemotherapeutic agents in melanoma.

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(1) Background: Peripheral nerve injuries (PNI) can generate important medium-- and long-term disability, as patients mostly complain about associated pain, sensibility and, or motor deficit, and even psychological manifestations. Chitosan and different nanoparticles types were previously used in several studies as treatment of peripheral nerve injuries. The present study aimed to assess the hepatic responses at oral administration of simple chitosan solution and of magnetic iron oxide nanoparticles functionalized with chitosan (CMNPs) solution in an experimentally induced peripheral nerve injury. (2) Methods: Chitosan or CMNPs were oral administrated, for 21 days, to animals with peripheral nerve injury. The treated groups were compared with a control group (peripheric nerve injury without any treatment). The hepatic toxicity of administered solutions was analyzed histologically, through transmission electron microscopy (TEM) and through the oxidative stress parameters, in comparison with the control group. (3) Results: Liver tissue histological evaluation showed non-significant degeneration of hepatocytes in Chitosan group and, in CMNPs group, slight periportal inflammation. TEM investigation revealed nuclear and mitochondrial polymorphism and lipid accumulation in hepatocytes in Chitosan group, and in CMNPs group, irregular nucleus profiles and increased glycogen storage in cytosol. Oxidative stress analysis showed antioxidant hepatic effect of both treatments. (4) Conclusions: Compared to control group, both treatments produced significant increases of hepatic antioxidant protection, probably induced by chitosan properties. Histological modifications of the liver were minimal for both treatment groups. TEM investigation showed unspecific alterations of the hepatocytes structure.

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The label-free detection of biological molecules was demonstrated for different types of DNA. Surface-enhanced Raman spectroscopy (SERS) investigation on nucleic acids extracted from leaves of different Allium cepa cultivars (cvs.) De Buz & abreve;u, Aurie de Buz & abreve;u, Rubiniu, Ro & scedil;ie de Arie & scedil; as well as taxa A. ursinum, A. senescens subsp. montanum, A. schoenoprasum, A. obliquum, and A. fistulosum was performed using 532-nm laser excitation. Main SERS vibrations of these nucleic acids have been characterized, highlighting those at 658, 704, 1089, and 1125 cm-1 attributed to dG and dA phosphodiester groups as well as deoxyribose, specific constituents of DNA. The mild basic pH conditions of the samples can induce a partial breaking of the hydrogen bonds from the dsDNA, which affects the A = T/T = A as well as C equivalent to G/G equivalent to C base pairs and causes the aggregation of AgNPs mixed with DNA of the Allium cultivars and species. Also, SERS profiles of genomic DNA from E. coli 1559 were analyzed at decreasing pHs from 7 to 4. Protonation of the nitrogen N3 of the aromatic six-membered ring of both deoxyadenosine and deoxyguanosine, respectively, induces the decrease of the peak's intensity at 1588 cm-1. Furthermore, SERS characteristics of plasmidic DNA from E. coli 7832 and of chromosomal DNA from E. coli MG 1655 are provided, with specific SERS modes indicated and new SERS bands found upon nucleic acids in vitro aging. Principal component analysis (PCA) in the full SERS spectrum range of bacterial DNA was performed.

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Medicinal plants are a valuable reservoir of novel pharmacologically active compounds. ROS and free radicals are primary contributors to oxidative stress, a condition associated with the onset of degenerative diseases such as cancer, coronary heart disease, and vascular disease. In this study, we used different spectrophotometry methods to demonstrate the antioxidant properties of 6 Allium extracts: Allium fistulosum; Allium ursinum; Allium cepa: Aries red cultivar of A. cepa, and white variety of A. cepa; Allium sativum; and Allium senescens subsp. montanum. HPLC-MS determined the chemical composition of the extracts. Among the tested extracts, the Aries red cultivar of A. cepa stands out as having the best antioxidant activity, probably due to the high content of polyphenols and alliin (12.67 mu g/mL and 3565 ng/mL, respectively). The results obtained in this study show that Allium extracts have antioxidant activity, but also free radical scavenging capabilities. Also, their interactions with cytochrome c and hemoglobin can be the basis of future studies to create treatments for oxidative stress-related diseases.

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Telekia speciosa (Schreb.) Baumg. was selected to investigate the phylogeography of tall-herb species, an ecological group in which the Quaternary history has been rarely studied. This species is a characteristic component of mountain forest communities in the Romanian Carpathian Mountains. The analysis employing Amplified Fragment Length Polymorphism (AFLP) fingerprinting has revealed a notable absence of a well-defined phylogeographical structure among the populations assessed. This finding implies that the various regions of the current distribution range of Telekia speciosa in the SE Carpathians have not undergone extended periods of isolation. Such a pattern suggests that this mountain tall-herb species might have experienced the possibility of long-term persistence along a wide altitudinal gradient in isolated mountain ranges, also encompassing the lowlands between them in glacial periods. Beside the unique vegetation history experienced during the most recent glaciation in the SE Carpathians, the observed weak phylogeographical structure of Telekia speciosa can be reasonably attributed to its inherent biological traits, particularly its proficiency in long-distance dispersal. Additionally, the species' ecological adaptations, which facilitated its survival in riparian zones of relatively low, forested mountain ranges, might contributed further to maintaining genetic connectivity among populations, thereby mitigating substantial genetic differentiation across its distribution. The study concludes that the genetic variation observed in T. speciosa is relatively evenly distributed across the species' distribution area. © 2024, Babes-Bolyai University, "Alexandru Borza" Botanic Garden. All rights reserved.

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BackgroundMicrosatellite primers were developed and tested to genotype several populations of Carex curvula s. l. (Cyperaceae), in order to infer the phylogeographic relationships of the populations within species and the boundaries between the two described subspecies: C. curvula subsp. curvula and C. curvula subsp. rosae.Methods and resultsCandidate microsatellite loci were isolated based on next-generation sequencing. We tested 18 markers for polymorphism and replicability in seven C. curvula s. l. populations and identified 13 polymorphic loci with dinucleotide repeats. Genotyping results showed the total number of alleles per locus varied from four to 23 (including both infrataxa), and the observed and expected heterozygosity ranged between 0.1 to 0.82 and 0.219 to 0.711, respectively. Furthermore, the NJ tree showed a clear separation between C. curvula subsp. curvula and C. curvula subsp. rosae.ConclusionThe development of these highly polymorphic markers proved to be very efficient not only in delineating between the two subspecies, but also in genetic discriminating at population level within each infrataxon. They are promising tools for evolutionary studies in Cariceae section, as well as in providing knowledge on patterns of the species phylogeography.

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Background and Aims Studying trait variability and restricted gene flow between populations of species can reveal species dynamics. Peripheral populations commonly exhibit lower genetic diversity and trait variability due to isolation and ecological marginality, unlike central populations experiencing gene flow and optimal conditions. This study focused on Carex curvula, the dominant species in alpine acidic meadows of European mountain regions. The species is sparser in dry areas such as the Pyrenees and Balkans, compared to the Central-Eastern Alps and Carpathians. We hypothesized that distinct population groups could be identified based on their mean functional trait values and their correlation with the environment; we predicted that ecologically marginal populations would have stronger trait correlations, lower within-population trait variability (intraspecific trait variability, ITV) and lower genetic diversity than populations of optimal habitats. Methods Sampling was conducted in 34 populations that spanned the entire distribution range of C. curvula. We used hierarchical clustering to identify emergent functional groups of populations, defined by combinations of multiple traits associated with nutrient economy and drought tolerance (e.g. specific leaf area, anatomy). We contrasted the geographical distribution of these groups in relation to environment and genetic structure. We compared pairwise trait relationships, within-population trait variation (ITV) and neutral genetic diversity between groups. Key Results Our study identified emergent functional groups of populations. Those in the southernmost ranges, specifically the Pyrenees and Balkan region, showed drought-tolerant trait syndromes and correlated with indicators of limited water availability. While we noted a decline in population genetic diversity, we did not observe any significant changes in ITV in ecologically marginal (peripheral) populations. Conclusions Our research exemplifies the relationship between ecological marginality and geographical peripherality, which in this case study is linked to genetic depauperation but not to reduced ITV. Understanding these relationships is crucial for understanding the biogeographical factors shaping trait variation.

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Alzheimer's disease (AD) is known as the primary and most common cause of dementia in the middle-aged and elderly population worldwide. Chemical analyses of B. pendula leaf extract (BPE), performed using spectrophotometric and chromatographic methods (LC/MS), revealed high amounts of polyphenol carboxylic acids (gallic, chlorogenic, caffeic, trans-p-coumaric, ferulic, and salicylic acids), as well as flavonoids (apigenin, luteolin, luteolin-7-O-glucoside, naringenin, hyperoside, quercetin, and quercitrin). Four groups of Wistar rats were used in this experiment (n = 7/group): control (untreated), A beta(1-42) (2 mu g/rat intracerebroventricular (i.c.v.), A beta(1-42) + BPE (200 mg/Kg b.w.), and DMSO (10 mu L/rat). On the first day, one dose of A beta(1-42) was intracerebroventricularly administered to animals in groups 2 and 3. Subsequently, BPE was orally administered for the next 15 days to group 3. On the 16th day, behavioral tests were performed. Biomarkers of brain oxidative stress Malondialdehyde (MDA), (Peroxidase (PRx), Catalase (CAT), and Superoxid dismutase (SOD) and inflammation (cytokines: tumor necrosis factor -alpha (TNF-alpha), Interleukin 1 beta (IL-1 beta), and cyclooxygenase-2 (COX 2)) in plasma and hippocampus homogenates were assessed. Various protein expressions (Phospho-Tau (Ser404) (pTau Ser 404), Phospho-Tau (Ser396) (pTau Ser 396), synaptophysin, and the Nuclear factor kappa B (NFkB) signaling pathway) were analyzed using Western blot and immunohistochemistry in the hippocampus. The results show that BPE diminished lipid peroxidation and neuroinflammation, modulated specific protein expression, enhanced the antioxidant capacity, and improved spontaneous alternation behavior, suggesting that it has beneficial effects in AD.

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Odd ploidy-level cytotypes in sexually reproducing species are considered a dead end due to absent or reduced fertility. If sterility is only partial, however, their contribution to the population gene pool can be augmented by longevity and clonal growth. To test this, we investigated the cytotype origin and spatial pattern, and pollen viability in three relict shrub species of the genus Daphne (Thymelaeaceae Juss.) in central Europe. Daphne cneorum subsp. cneorum is a widespread European species that has a broad ecological amplitude, whereas D. cneorum subsp. arbusculoides and D. arbuscula are narrow endemics of the western Pannonian Plain and the Western Carpathians, respectively. Our study confirmed that all three taxa are diploid. However, of more than a thousand analysed individuals of D. cneorum subsp. cneorum, five in four different populations were triploid. Our data indicate that these triploids most likely originate from recurrent autopolyploidization events caused by the fusion of reduced and unreduced gametes. High pollen viability was observed in all three taxa and in both diploid and triploid cytotypes, ranging from 65 to 100 %. Our study highlights the significant role of odd ploidy-level cytotypes in interploidy gene flow, calling for more research into their reproduction, genetic variability, and overall fitness. Interestingly, while the endemic D. arbuscula differs from D. cneorum based on genetic and genome size data, D. cneorum subsp. arbusculoides was indistinguishable from D. cneorum subsp. cneorum. However, our study reveals that the subspecies differ in the number of flowers per inflorescence. This is the first comprehensive cytogeographic study of this intriguing genus at a regional scale, and in spite of its karyological stability, it contributes to our understanding of genomic evolution in plant species with a wide ecological amplitude. The presented study sheds light on the emergence of odd-ploidy cytotypes in diploid species, specifically in long-lived relic shrubs from the genus Daphne (Thymeleaceae). Rare odd-ploidy cytotypes are generally considered maladaptive in sexual diploid species due to reduced fitness and fertility. However, our research revealed the independent and recurring evolution of triploid cytotypes, most likely arising via the fusion of reduced and unreduced gametes in exclusively diploid systems and exhibiting significant pollen fertility. Their even partial pollen fertility, especially, in the context of their longevity, can contribute to a more diverse gene pool in their parental diploid populations or even to the spontaneous emergence of new cytotypes. Additionally, we demonstrated that although Daphne cneorum exhibits a large ecological amplitude, spanning various bedrock substrates and altitudinal ranges, its diploid genomes remain highly stable with no ecologically driven genome size expansions or reductions, as previously observed in many species with large ecological amplitudes.

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Surface-enhanced Raman spectroscopy (SERS) applications in clinical diagnosis and spectral pathology are increasing due to the potential of the technique to bio-barcode incipient and differential diseases via real-time monitoring of biomarkers in fluids and in real-time via biomolecular fingerprinting. Additionally, the rapid advancements in micro/nanotechnology have a visible influence in all aspects of science and life. The miniaturization and enhanced properties of materials at the micro/nanoscale transcended the confines of the laboratory and are revolutionizing domains such as electronics, optics, medicine, and environmental science. The societal and technological impact of SERS biosensing by using semiconductor-based nanostructured smart substrates will be huge once minor technical pitfalls are solved. Herein, challenges in clinical routine testing are addressed in order to understand the context of how SERS can perform in real, in vivo sampling and bioassays for early neurodegenerative disease (ND) diagnosis. The main interest in translating SERS into clinical practice is reinforced by the practical advantages: portability of the designed setups, versatility in using nanomaterials of various matter and costs, readiness, and reliability. As we will present in this review, in the frame of technology readiness levels (TRL), the current maturity reached by semiconductor-based SERS biosensors, in particular that of zinc oxide (ZnO)-based hybrid SERS substrates, is situated at the development level TRL 6 (out of 9 levels). Three-dimensional, multilayered SERS substrates that provide additional plasmonic hot spots in the z-axis are of key importance in designing highly performant SERS biosensors for the detection of ND biomarkers.

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Alkylating agents such as cyclophosphamide (CPA) are commonly used in cytotoxic or immunosuppressive therapies for different types of diseases. One of the main secondary effects of such therapy is the reduction in follicular reserve, targeting the primordial follicles. The aim of this study was to investigate the antioxidant and protective effects of Plantago sempervirens extract on the follicular pool. The experiment was performed on Wistar female rats, for 21 days. They were divided into five groups according to the treatment they received (Control, CPA (200 mg/kg BW), P. sempervirens extract in three different doses-25/50/100 dw/mL ethanol extract (5 mL/kg BW)). The investigations regarded enzymatic and non-enzymatic nitro-oxidative stress, hormone levels (FSH and estrogen), TEM sections of the ovaries, and oestrus cycle monitorization. CPA strongly increased the oxidative stress (TOS-32.8 mmol H2O2/L; NO-68.60 mu mol/L; OSI-6.82), alongside a depletion of FSH and a blockage of the oestrus cycle in the metestrus phase. The follicular pool was strongly depleted during CPA exposure, but FSH concentration and the TEM images of the cells revealed a significant improvement after treatment, as the dose increases (P3). Therefore, these findings reveal that P. sempervirens therapy could have a potential protective effect against CPA-induced acute damages in the ovaries, as well as maintaining a functional oestrus cycle.

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Iron dysmetabolism affects a great proportion of heart failure patients, while chronic hypertension is one of the most common risk factors for heart failure and death in industrialized countries. Serum data from reduced ejection fraction heart failure patients show a relative or absolute iron deficiency, whereas cellular myocardial analyses field equivocal data. An observed increase in organellar iron deposits was incriminated to cause reactive oxygen species formation, lipid peroxidation, and cell death. Therefore, we studied the effects of iron chelation on a rat model of cardiac hypertrophy. Suprarenal abdominal aortic constriction was achieved surgically, with a period of nine weeks to accommodate the development of chronic pressure overload. Next, deferiprone (100 mg/kg/day), a lipid-permeable iron chelator, was administered for two weeks. Pressure overload resulted in increased inflammation, fibrotic remodeling, lipid peroxidation, left ventricular hypertrophy and mitochondrial iron derangements. Deferiprone reduced cardiac inflammation, lipid peroxidation, mitochondrial iron levels, and hypertrophy, without affecting circulating iron levels or ejection fraction. In conclusion, metallic molecules may pose ambivalent effects within the cardiovascular system, with beneficial effects of iron redistribution, chiefly in the mitochondria.

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Oxidative stress is linked to a series of diseases; therefore, the development of efficient antioxidants might be beneficial in preventing or ameliorating these conditions. Based on the structure of a previously reported compound with good antioxidant properties and on computational studies, we designed several catechol derivatives with enhanced antioxidant potential. The compounds were synthesized and physicochemically characterized, and their antioxidant activity was assessed through different antiradical, electron transfer and metal ions chelation assays, their electrochemical behavior and cytotoxicity were studied. The results obtained in the in vitro experiments correlated very well with the in silico studies; all final compounds presented very good antioxidant properties, generally superior to those of the reference compounds used. Similarly, the results obtained from studying the compounds' electrochemical behavior were in good agreement with the results of the antioxidant activity evaluation assays. Regarding the compounds' cytotoxicity, compound 7b had a dose-dependent inhibitory effect against all cell lines. In conclusion, through computer-aided design, we developed several catechol thiazolyl-hydrazones with excellent antioxidant properties, of which compound 7b, with two catechol moieties in its structure, exhibited the best antioxidant activity.

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The Carpathians represent not only a European hotspot of plant diversity for both species richness and endemism, but also an important stepping-stone area in historical migrations between the flora of the Asian and European mountain systems and a starting point of postglacial recolonizations for many species. Yet, until recent years, phylogeographical studies for alpine or arctic-alpine plants were focused on the Alps, whereas peripheral mountain ranges, including the Carpathians, were either neglected or insufficiently sampled. In this study, we aimed to complement the Alpine phylogeographic structure of an emblematic high-alpine European endemic taxon, Eritrichium nanum, by focusing on the Carpathian range of the species. We sampled nine populations from the South-Eastern Carpathians and performed ITS1 sequencing and AFLP fingerprinting. In case of ITS1 region, all the populations, no matter of their geographic origin, presented the same ribotype. The AFLP analysis indicated that, within the Carpathians, the extant populations of E. nanum comprised two major allopatric lineages. One important result of the research was the discovery that the species' sole important genetic break was located in the Southern Carpathians, separating populations of the Retezat Mountains from all the others in the Carpathians.

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Tomato (Lycopersicon esculentum Mill.) as the most economically important vegetable crop worldwide has been investigated intensively for the development of new and improved varieties. Most of these technologies require efficient protocols for in vitro regeneration and propagation of plant material. In the present study, an efficient and reproducible in vitro regeneration system for five Romanian tomato genotypes (cvs. 'Capriciu', 'Darsirius', 'Kristin', 'Pontica' and 'Siriana') has been established. The tomato genotypes were selected based on their horticultural and economically valuable traits. To study the in vitro morphogenic response, various explants, such as cotyledons, cotyledonary nodes, hypocotyls, leaf explants, internodes, stem nodes and apical buds have been selected. The highest efficiency in terms of direct shoot organogenesis was obtained in cv. 'Capriciu' (98% for apical buds and 94% for stem nodes) on culture media with zeatin and indole-3-butyric acid. One advantage of this regeneration procedure is beside its feasibility in handling, the high percentage of regenerated shoots and their rooting. The present protocol contributes to the existing information regarding the response of tomato cultivars to in vitro culture conditions.

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The Arieş River (Western Romania) represents one of the most important affluents of the Mureş River, with great significance in the Mureş Tisza basin. The environmental quality of the Arieş basin is significantly affected by both historic mining activities and contemporary impacts. Thus, an evaluation of the effects of the main contaminants found in water (organochlorine pesticides—OCPs, monocyclic aromatic hydrocarbons—MAHs, polycyclic aromatic hydrocarbons—PAHs, and metals) on cyanobacteria and plants was performed. Among OCPs, hexachlorocyclohexane isomers, dichlorodiphenyltrichloroethane, and derivatives were detected in plants while admissible concentrations were detected in water. Among MAHs, high levels of benzene were detected both in water and in plants. The levels of PAHs exceeded the allowable values in all samples. Increased concentrations of metals in water were found only at Baia de Arieş, but in plants, all metal concentrations were high. The pH, nitrates, nitrites, and phosphates, as well as metals, pesticides, and aromatic hydrocarbons, influenced the physiological characteristics of algae, test plants, and aquatic plants exposed to various compounds dissolved in water. Considering that the Arieş River basin is the site of intense past mining activities, these data provide information about the impact on water quality as a consequence of pollution events. © 2023 by the authors.

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Enterococcus faecium is emerging as an important cause of multidrug resistance and hospital acquired infections, special attention being paid to the vancomycin resistant species. Therefore, the characterization of pathogenic strains/isolates plays an important role in the epidemiology of infectious diseases. The enterococcal rate was determined from wastewaters in Cluj-Napoca area. As presence of E. faecium was detected, a number of isolates from wastewater, birds and humans were epidemiologically analyzed according to the MLST website. Comparisons were performed against a collection of available isolates, with multiple origins, contained in the MLST database. Out of the Enterococcus isolates collected from wastewater, 11 were identified as E. faecalis (40.74%); 8 as E. casseliflavus (29.62%); 5 as E. faecium (18.50%); 2 as E. gallinarum (7.40%) and one isolate as E. durans. Based on the MLST data and using the eBURST algorithm, the isolates of E. faecium sampled from Romania were split in three groups: one group comprised isolates from human hosts and wastewater (Cj316, 106/6, Cj197, Cj22, 129/6, Cj117, Cj24, 284/7, and 43/7), while the second (G9, G10-2, G7, G3-2, and G9-1) and the third group (G8, G6, and 40/7) originated from bird hosts. The rest of the isolates were not joined in a particular group, assuming the lack of a phylogenetic bond between these isolates. The obtained data suggested the existence of at least two phylogenetic lines of E. faecium in Romania: a line that had mainly human host prevalence, while in the other line the animal hosts dominated. © Articles by the authors; Licensee SMTCT, Cluj-Napoca, Romania.

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Microsatellite markers previously developed for other leguminous species were tested for cross-genus transferability and evaluated for their potential usefulness in providing an improved assessment of the genetic relationships between two closely related taxa belonging to Onobrychis genus (Fabaceae). Candidate microsatellite markers were tested for polymorphism and replicability in sixteen populations of O. montana DC. subsp. transsilvanica (Simonk.) Jáv. and O. montana. Out of the 23 SSRs, there were identified seven polymorphic loci. In total 32 alleles were detected and the number of alleles per locus varied from two to six. PIC values ranged from 0.375 to 0.6454, and four SSRs displayed a PIC > 0.5. Relative uniform rates of genetic diversity were obtained. In case of O. montana DC. subsp. transsilvanica (Simonk.) Jáv. the observed and expected heterozygosity ranged from 0.100 to 0.952 and from 0.219 to 0.525, respectively, while for O. montana ranged from 0.166 to 0.750 and from 0.083 to 0.375, respectively. Seven polymorphic SSRs with clear and reproducible amplification were identified. These markers proved to be very efficient for unambiguous population discrimination based on both geographic and taxonomic criteria. Hereafter, these SSR markers can be used as tools for evolutionary studies in Onobrychis genus, as well in providing knowledge on patterns of the species phylogeography. © Articles by the authors.

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Onobrychis montana DC. Subsp. transsilvanica (Simonk.) Jáv. (Fabaceae) is a plant taxon, endemic to the South-Eastern Carpathians, whose taxonomic status is treated inconsistently by the classic taxonomy based on morphological traits. In a previous study, by sequencing several chloroplast DNA regions and AFLP genotyping, we tried to elucidate the controversy concerning its taxonomic status in relation to the more widespread O. montana DC., and determined its phylogeographic structure within the Carpathians. The present study, based on DNA fingerprinting by seven SSR markers, brings more insights on this problematic taxon by conferring higher resolution and sub-clustering previously identified phylogeographic groups. However, the newly SSR genetic data are in agreement with molecular evidence revealed by AFLP and chloroplastic SNPs and do not support O. montana DC. Subsp. transsilvanica (Simonk.) Jáv. As a distinct species from O. montana DC. © 2023, Horticulture and Forestry Society from Transylvania. All rights reserved.

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Grapevine (Vitis vinifera subsp. vinifera) is one of the most widespread and economically important perennial fruit crops in the world. Viticulture has changed over the years in response to changing environmental conditions and market demands, triggering the development of new and improved varieties to ensure the crop’s sustainability. The aim of this review is to provide a perspective on the recent developments in biotechnology and molecular biology and to establish the potential of these technologies for the genetic improvement of grapevine. The following aspects are discussed: (i) the importance of molecular marker-based methods for proper cultivar identification and how NGS-based high-throughput technologies have greatly benefited the development of genotyping techniques, trait mapping, and genomic selection; (ii) the recent advances in grapevine regeneration, genetic transformation, and genome editing, such as new breeding technology approaches for enhanced grapevine yield, quality improvement, and the selection of valuable varieties and cultivars. The specific problems and challenges linked to grapevine biotechnology, along with the importance of integrating classical and new technologies, are highlighted. © 2023 by the authors.

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Curcumin (CCM) is one of the most frequently explored plant compounds with various biological actions such as antibacterial, antiviral, antifungal, antineoplastic, and antioxidant/anti-inflammatory properties. The laboratory data and clinical trials have demonstrated that the bioavailability and bioactivity of curcumin are influenced by the feature of the curcumin molecular complex types. Curcumin has a high capacity to form molecular complexes with proteins (such as whey proteins, bovine serum albumin, beta-lactoglobulin), carbohydrates, lipids, and natural compounds (e.g., resveratrol, piperine, quercetin). These complexes increase the bioactivity and bioavailability of curcumin. The current review provides these derivatization strategies for curcumin in terms of biological and physico-chemical aspects with a strong focus on different type of proteins, characterization methods, and thermodynamic features of protein-curcumin complexes, and with the aim of evaluating the best performances. The current literature review offers, taking into consideration various biological effects of the CCM, a whole approach for CCM-biomolecules interactions such as CCM-proteins, CCM-nanomaterials, and CCM-natural compounds regarding molecular strategies to improve the bioactivity as well as the bioavailability of curcumin in biological systems.

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(1) Background: The study aimed to assess neurobehavioral, ultrastructural, and biochemical changes induced by silver nanoparticles synthesized with Cornus mas L. extract (AgNPs-CM) in rat brains. (2) Methods: The study included 36 male adult rats divided into three groups. Over a period of 45 days, AgNPs-CM (0.8 and 1.5 mg/kg b.w.) were administered daily by gavage to two of the groups, while the control group received the vehicle used for AgNP. After treatment, OFT and EPM tests were conducted in order to assess neurobehavioral changes. Six of the animals from each group were sacrificed immediately after completion of treatment, while the remaining six were allowed to recuperate for an additional 15 days. Transmission electron microscopy (TEM), GFAP immunohistochemistry, and evaluation of TNF alpha, IL-6, MDA, and CAT activity were performed on the frontal cortex and hippocampus. (3) Results: Treated animals displayed a dose- and time-dependent increase in anxiety-like behavior and severe ultrastructural changes in neurons, astrocytes, and capillaries in both brain regions. Immunohistochemistry displayed astrogliosis with altered cell morphology. TNF alpha, IL-6, MDA, and CAT activity were significantly altered, depending on brain region and time post exposure. (4) Conclusions: AgNPs-CM induced neurobehavioral changes and severe cell lesions that continued to escalate after cessation of exposure.

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The present work shows the biochemical and structural fundamentals for the stress induced anxiety and stress adjustment response of the CA3 hippocampus area. Adult male Wistar rats were repeatedly exposed to a 3 h day restraint stress, for either 3 or 6 days. The concentration of corticosterone and testosterone in the CA3 hippocampus area was divergent, while oxidative stress was progressively increased during the stress exposure. The mitochondrial lysis in the CA3 neurons confirmed the oxidative stress events. Immunohistochemical findings showed that oligodendrocytes (OCs) proliferation and neuroglobin (Ngb) expression were stimulated, whereas MeCP2 expression was decreased as a balance reaction in stress exposure under corticosterone signaling. Remarkably, ultrastructural changes such as mitochondrial lysis, endoplasmic reticulum swelling, and perivascular lysis with platelets adherence to endothelium in the CA3 area were seen in the 6th day of restraining. The anxiety-like behavior was noticed 6 days later after stress exposure. These results suggest that the duration of the exposure, but not the intensity of the stress, is the key factor in the stress-buffering function by the CA3 hippocampus area via up-regulation of the Ngb-OCs bionome. The imbalance of the Ngb-OCs communication may be involved in the development of CA3-dependent anxious behavior.

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Primary melanoma aggressiveness is determined by rapid selection and growth of cellular clones resistant to conventional treatments, resulting in metastasis and recurrence. In addition, a reprogrammed tumor-immune microenvironment supports melanoma progression and response to therapy. There is an urgent need to develop selective and specific drug delivery strategies for modulating the interaction between cancer cells and immune cells within the tumor microenvironment. This study proposes a novel combination therapy consisting of sequential administration of simvastatin incorporated in IL-13-functionalized long-circulating liposomes (IL-13-LCL-SIM) and doxorubicin encapsulated into PEG-coated extracellular vesicles (PEG-EV-DOX) to selectively target both tumor-associated macrophages and melanoma cells. To this end, IL-13 was conjugated to LCL-SIM which was obtained via the lipid film hydration method. EVs enriched from melanoma cells were passively loaded with doxorubicin. The cellular uptake of rhodamine-tagged nano-particles and the antiproliferative potential of the treatments by using the ELISA BrdU-colorimetric immunoassay were investigated in vitro. Subsequently, the therapeutic agents were administered i.v in B16.F10 melanoma-bearing mice, and tumor size was monitored during treatment. The molecular mechanisms of antitumor activity were investigated using angiogenic and inflammatory protein arrays and western blot analysis of invasion (HIF-1) and apoptosis markers (Bcl-xL and Bax). Quantification of oxidative stress marker malondialdehyde (MDA) was determined by HPLC. Immunohistochemical staining of angiogenic markers CD31 and VEGF and of pan-macrophage marker F4/80 was performed to validate our findings. The in vitro data showed that IL-13-functionalized LCL were preferentially taken up by tumor-associated macrophages and indicated that sequential administration of IL-13-LCL-SIM and PEG-EV-DOX had the strongest antiproliferative effect on tumor cells co-cultured with tumor-associated macrophages (TAMs). Accordingly, strong inhibition of tumor growth in the group treated with the sequential combination therapy was reported in vivo. Our data suggested that the antitumor action of the combined treatment was exerted through strong inhibition of several pro-angiogenic factors (VEGF, bFGF, and CD31) and oxidative stress-induced upregulation of pro-apoptotic protein Bax. This novel drug delivery strategy based on combined active targeting of both cancer cells and immune cells was able to induce a potent antitumor effect by disruption of the reciprocal interactions between TAMs and melanoma cells.

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Atherosclerosis is a chronic inflammatory disease of the arterial wall involving inflammation, redox imbalance, and impaired cholesterol transport. A high level of trimethylamine-N-oxide (TMAO) produced by meat and fat metabolism are involved in atherosclerosis development, but the exact relationship with inflammation is not completely clear. The study aimed to identify a possible association between TMAO; atherosclerotic changes in the aortic root; oxidative stress; and inflammation quantified by highly sensitive C-reactive protein (hsCRP), tumor necrosis factor alpha (TNFa), and interleukin-1beta (IL-1b) levels. TMAO dihydrate was administered via gastric gavage to 20 male Wistar rats for 90 days; one separate group received vehicle. The TMAO-treated animals were divided into two groups: one group received a low dose of TMAO (20 mg/day) and the other group received a high dose of TMAO (40 mg/day). Malondialdehyde (MDA), proinflammatory markers - IL-1b, TNF-a, and hsCRP, total cholesterol, high-density lipoprotein (HDL)-cholesterol, triglycerides, and glucose were assessed 30 and 90 days after TMAO administration. Additionally, conventional histopathology and immunohistochemistry for collagen I distribution were performed. MDA, hsCRP, TNF-a, and IL-1b levels increased after 90 days of TMAO administration in conjunction with significant changes suggestive of incipient atherosclerosis and inflammation of the aortic root. The increase was higher in the group treated with 40 mg/day TMAO compared with the group treated with 20 mg/day TMAO. Additionally, blood levels of TMAO were significantly correlated with hsCRP, TNF-a, IL-1b levels, but also with MDA, low HDL-cholesterol levels, and high triglyceride levels. The increase in MDA and inflammatory cytokines and modification of lipid metabolism markers may explain the pro-atherogenic effect of TMAO.

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Hemoglobin-based oxygen carriers (HBOCs) have been proposed and tested for several decades for the treatment of hemorrhage. We have previously proposed replacing hemoglobin (Hb) in HBOC with the oxygen-carrying protein hemerythrin (Hr), from marine worms, showing that Hr-based derivatives can perform at least as well or even better than Hb-based HBOC in a range of in vitro assays involving oxidative and nitrosative stress as well as in top-up animal models, where small amounts of Hr- or Hb-HBOC were injected into rats. Here, these experiments are extended to a hemorrhage experiment, in which Hr polymerized with glutaraldehyde, alone or conjugated with human serum albumin, is administered after a loss of 20-30% blood volume. The performance of these preparations is compared with that of Hb-based HBOC measured under the same conditions. Polymerized Hr is found to decrease the survival rate and can hence cannot be used as an oxygen carrier in transfusions. On the other hand, an Hr-albumin copolymer restores survival rates to 100% and generally yields biochemical and histological parameters similar to those of glutaraldehyde-polymerized bovine hemoglobin, with the exception of an acid-base imbalance. The latter may be solved by employing an allogeneic albumin as opposed to the human albumin employed in the present study.

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Acute cardiac pathologies represent one of the leading causes of death, while iron metabolism is recognized to be implicated in reactive oxygen species production, lipid peroxidation, and inflammation. The aim of the present study was to assess iron chelation effects in isoproterenol (ISO) induced acute cardiac stress. We divided male Wistar rats into preventive and secondary treatment groups, with the active arm consisting in deferiprone (DFP), a lipid permeable chelator. Mortality of ISO was 10-18.18% in both preventive and secondary groups. We analyzed serum and myocardial tissue parameters of inflammation, iron dynamics, and lipid peroxidation, accompanied by ultramicroscopy, histological, and ultrasound-derived parameters of left ventricular function. Results reveal that ISO-mediated lipid peroxidation and inflammation are alleviated by administration of DFP, with negligible effect on systemic ferroregulation dynamics and global ventricular function (as assessed by ultrasound). DFP administration after cardiovascular stress is associated with a decrease in lipid peroxidation and inflammation, without an improvement in gross left ventricular parameters.

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Maize occupies a significant place in the world agriculture. Yellow kernel maize contains mainly non-provitamin A carotenoids: lutein and zeaxanthin. The accumulation of provitamin A carotenoids is regulated by favourable alleles of lcyE and crtRB1 genes and could be used for the enhancement of these carotenoids in the maize grain through breeding. In this study, molecular screening of the Romanian germplasm was performed, looking for favourable alleles of the crtRB1 and lcyE genes, and the level of carotenoids was determined in a few selected lines. A number of 2746 inbred lines from seven research stations were subjected to a PCR amplification of crtRB1 and lcyE genes in order to identify the favourable alleles. It was selected 27 lines carrying the favourable alleles and nine lines with unfavourable alleles (four groups in total), from which total carotenoids, lutein, zeaxanthin, beta-cryptoxanthin, beta-carotene and retinol equivalents were determined by HPLC. Out of 2746 inbred lines analysed, 23.53% contained one or both genes with favourable alleles. The favourable allele of the crtRB1 gene was the most widespread (584 lines), followed by the lcyE gene (55 lines), while alleles favourable for both genes were detected in only 7 lines. Inbred lines with the favourable allele of the crtRB1 gene showed the highest levels of beta-carotene and beta-cryptoxanthin, while those with favourable allele of lcyE gene showed a high level of beta-cryptoxanthin; the lines with favourable alleles for both genes had a level of beta-carotene 60% higher than the lines with two unfavourable alleles.

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In Romania, Prunus species have great economic and social importance. With the introduction of new cultivars arises the need to preserve and characterize the local Prunus germplasm. Thus, a set of 24 polymorphic SSRs were selected for the overall characterization, including 10 peach, 11 apricot and 5 nectarine cultivars. The average number of alleles per locus (Na = 1.958), in addition to overall observed (Ho = 0.299) and expected heterozygosity (He = 0.286) were lower or comparable to those reported in similar studies, probably explained by the smaller number of analyzed cultivars restricted to a smaller geographic area. Among 26 genotypes a total of 101 alleles were identified, of which 46 alleles were in peach, 55 in apricot and 40 in nectarine, respectively. Six alleles from six loci (CPPCT-030, Pchgms-003, Pchgms-004, Pchgms-010, UDP97-401, UDP98-405) were common to all taxonomic groups. The most informative loci were BPPCT-025, Pchgms-021 and UDP96-001 in peach; BPPCT-025, BPPCT-001 and UDP96-001 in nectarine; and BPPCT-002, BPPCT-025, Pchgms-004, Pchgms-020 and Pchgms-021 in apricot. Clustering and genetic similarity analysis indicated that the degree of interspecific divergence in peach and nectarine cultivars was less than that in peach and apricot. These results will be useful to prevent confusion between cultivars, to improve breeding strategies and to benefit the management of Prunus cultivars bred in Romania.

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In this work surface-enhanced Raman spectra of nucleic acids from in vitro grown Solanum tuberosum L. cultivars and populations (Buzau population, Lazarea population, Patraque d'Auvergne, RFA Roclas Clone 2.6 Ferma, Vitelotte Negresse, Roclas Clone C, Blue Congo) were measured with 532 nm laser line. Main surface-enhanced Raman modes of these DNAs have been analyzed. Also, DNA from two grapevine (Vitis vinifera L.) varieties were studied at acidic pHs by surface-enhanced Raman spectroscopy. Modified SERS intensities and wavenumber shifts of nucleic acids bands were observed upon lowering the pH, being a proof of binding affinity changes of DNA with silver nanoparticles (AgNPs) and of structural modifications induced at acidic pHs in DNA molecular groups. Furthermore, the (sub)picosecond surface dynamics of DNA extracted from leaf tissues of grapevine (Vitis vinifera L.) varieties was investigated. In this work, the bands full widths at half-maximum (FWHMs) have values in the wavenumber range from 8 to 34 cm(-1). (Sub)picosecond molecular dynamics of DNA groups with global relaxation times between 0.31 ps 1.33 ps has been found.

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Background Previous investigations carried out in ultramafic habitats emphasized the greater importance of site conditions over soil toxic metal content for vegetation composition. Very little is known about the floristic structure of the Southern Carpathian ultramafic grasslands and there is no information on the local environmental drivers of their composition and coenotic features. Here, we aim to fill these knowledge gaps by referring to similar phytocoenoses described in the Balkan Peninsula and central Europe. In particular, we searched for: (i) floristic and ecological patterns supporting the classification and taxonomic assignment of these grasslands, and (ii) simple relationships between serpentine vegetation characteristics and its physiographic environment. A total of 120 phytosociological releves, of which 52 performed in the Southern Carpathians, were analysed through cluster, ordination and regression procedures. Results Despite some floristic similarities with their Balkan counterparts, the Southern Carpathian ultramafic grasslands were clustered into four distinct groups, which were assigned to as many new syntaxa: Plantago serpentinae-Armerietum halleri, Asplenio serpentini-Achnatheretum calamagrostis, Minuartio frutescentis-Plantaginetum holostei and Sileno saxifragae-Plantaginetum holostei. The latter was best individualised through the occurrence of several Carpathian endemic taxa. The first two ordination axes were significantly related with the terrain slope/presence of xerophilous species and respectively, with site elevation/presence of calcifugous species. The total plant cover showed a unimodal relationship with respect to site elevation. While controlling for the effect of the sampled area, species richness showed a unimodal response to both elevation and slope of the terrain, although their effects were not singular. Conclusions The syntaxonomic distinctiveness of the Southern Carpathian ultramafic grasslands is mainly supported by their overall species composition rather than regional differential species. The main limiting factors driving the composition, cover and species richness of all studied ultramafic grasslands are the water deficit at low elevation and on steep slopes, and the low soil fertility at higher elevations. Our results confirm the previous findings according to which physiographic conditions and, to a lesser extent, soil base nutrients are more important than heavy metal concentrations in structuring the ultramafic vegetation.

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In this study, we compare two rapid cryopreservation (-196 degrees C) procedures, droplet-vitrification and encapsulation-dehydration for rose (Rosa x hybrida L., cultivars 'loana', 'Mariana', 'Vulcan'). Significant factors for cryopreservation, such as sucrose concentration during osmoprotection, treatment duration with plant vitrification solution 2 (PVS2) in droplet-vitrification, duration of air desiccation and moisture content of alginate beads in encapsulation-dehydration, were investigated. In addition, the morphogenetic response to in vitro culture and to liquid nitrogen storage and the content in photosynthetic pigments have been assessed. The in vitro cultures were initiated from plant material originating from field collection. The highest regeneration frequencies were obtained for cv. 'Vulcan' in both of the cryopreservation procedures tested, 72% in droplet-vitrification and 65% following encapsulation-dehydration. The morphogenetic response (multiplication index and height of shoots) to liquid nitrogen storage was direct multiple shoot formation per initial shoot tip for all genotypes. The content in chlorophyll a and b was statistically comparable in plant material resulting from cryopreserved and non-cryopreserved shoot tips in all cultivars. The findings expand the information on Rosa's response to in vitro culture conditions and cryopreservation, providing protocols with a high regeneration capacity for the storage of genotypes with high ornamental value.

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Romania hosts a relatively high species diversity, including 3,829 vascular and 979 non-vascular spontaneous plant taxa. Multiple national red lists exist, with the number of taxa assessed as threatened varying greatly between them, from 548 to 1,438, and with number of taxa assigned to a given threat category also varying between the different sources. A composite list including all taxa mentioned in at least one of the selected red listings from Romania is required in order to compensate for this lack of consensus and to assess their ex situ conservation status. In this study, we synthesized data from the national red lists and counted 1,220 spontaneous vascular plant species and 201 subspecies, of which 77 are endemic and 76 subendemic for Romania. In addition, 18 non-red-listed endemics and 14 subendemics have been added, bringing the total to 1,453 threatened and (sub)endemic plant taxa, representing almost 38% of the total native vascular flora of Romania. Despite the large network of protected areas in Romania, many taxa are still being threatened with extinction in the region mainly due to anthropogenic pressure. Several ex situ conservation measures have been employed to assure a more substantial buffer against plant extinction in the wild, supported by thorough and adequate conservation strategies and multiple means to reintroduce taxa back to their natural habitats. Consequently, our second aim was to evaluate the ex situ conservation status of these threatened and (sub)endemic plants from Romania, focussing on both conventional methods (cultivation in botanic gardens, seed banking) and biotechnological approaches (in vitro tissue culture, medium-term storage and cryostorage). Of the 1,453 taxa included in our list, 642 (44.2%) are conserved by ex situ approaches. Of these, 524 are harboured in the most important botanic gardens throughout Romania, while 156 are currently held in long-term seed banks locally or in the Millennium Seed Bank of the Royal Botanic Garden, Kew (UK). Conversely, only 64 taxa from the list are preserved at the national level through in vitro cultures, and cryopreservation protocols have been developed for only 8 taxa. Overall, more than half of the threatened and (sub)endemic vascular flora from Romania remains unprotected outside the classical in situ conservation measures. For red-listed bryophytes, only 0.6% are preserved in national ex situ collections. Moreover, some aspects related to population genetic studies and the genetic stability of ex situ conserved plants are also briefly discussed, as essential prerequisites for applied biodiversity conservation programs. Finally, considering the distribution range of targeted taxa, we included a synthesis of biotechnological approaches at both national and international level. Our study presents not only a first assessment of the ex situ conservation status of national red listed flora, but also, to our knowledge, the most comprehensive and updated overview of the rare, threatened and (sub)endemic taxa from Romania. This evaluation will provide a supporting tool for national decision-and policy-making actions for biodiversity conservation, using both in situ and ex situ approaches. We also highlight the need for an updated red list for the Romanian flora that accurately follows the IUCN assessment criteria and protocols.

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Oligotrophic peat bogs reach the southern boundary of their European distribution area in the Romanian Carpathians. They shelter boreal species considered glacial relicts, surviving from the Würmian III period. Such areas (habitats) conserving relict species have major phytohistorical importance. They provide insights into the history of forest vegetation in the region and past climatic conditions. Based on floristic, phytocoenological and phytogeographical data, we propose to include two active raised bogs, namely “Calul de Piatră” and “Izvorul văii Șoimului” in the ROSCI0116 Molhașurile Căpățânei site of community interest. The inclusion of the two bogs in the ROSCI0116 Molhașurile Căpățânei protected area will increase the peat bog surface in the Apuseni Mountains and will be beneficial for the conservation of the wetland habitats and their typical flora, both very vulnerable to natural and anthropogenic disturbances. © 2022, Babes-Bolyai University, "Alexandru Borza" Botanic Garden. All rights reserved.

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The present vegetation study was elaborated on the basis of geo-referenced phytocoenological relevés carried out in 2007–2010 on the northern slope of the Vlădeasa Mountain. 150 representative relevés for the woody and herbaceous vegetation were selected from a total of 387. These were grouped in 16 plant associations, based on their coenotic affinity and ecological requirements, and represented in a large-scale vegetation map. Of these, six scrub and forest associations were grouped in the class Vaccinio-Picetea and four forest associations in the class Carpino-Fagetea. Among the six herbaceous associations included in the classes Nardetea strictae and Molinio-Arrhenatheretea, only the association Potentillo aureae-Festucetum supinae has a primary character. © 2022, Babes-Bolyai University, "Alexandru Borza" Botanic Garden. All rights reserved.

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The European Alps are highly rich in species, but their future may be threatened by ongoing changes in human land use and climate. Here, we reconstructed vegetation, temperature, human impact and livestock over the past ~12,000 years from Lake Sulsseewli, based on sedimentary ancient plant and mammal DNA, pollen, spores, chironomids, and microcharcoal. We assembled a highly-complete local DNA reference library (PhyloAlps, 3923 plant taxa), and used this to obtain an exceptionally rich sedaDNA record of 366 plant taxa. Vegetation mainly responded to climate during the early Holocene, while human activity had an additional influence on vegetation from 6 ka onwards. Land-use shifted from episodic grazing during the Neolithic and Bronze Age to agropastoralism in the Middle Ages. Associated human deforestation allowed the coexistence of plant species typically found at different elevational belts, leading to levels of plant richness that characterise the current high diversity of this region. Our findings indicate a positive association between low intensity agropastoral activities and precipitation with the maintenance of the unique subalpine and alpine plant diversity of the European Alps. © 2022, The Author(s).

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Hypericum perforatum, H. umbellatum, H. maculatum, and H. hircinum accessions originating from botanical gardens across Europe were examined by flow cytometry and molecular markers. 2C DNA content of 17 Hypericum perforatum accessions (Hp) and the H. perforatum cultivar Topaz amounted to between 1.56 pg and 1.62 pg. In four Hp accessions some individual plants were found with a DNA content corresponding to 6Cx (2.34 - 2.39 pg). All plants of accession Hp8 showed a DNA content of 6Cx (2.41 pg). In root tips of Hp plants with an average DNA amount of 1.58 pg, 32 chromosomes were detected, corresponding to 2n = 4x. This is the first ploidy and/or DNA content report for H. umbellatum, H. maculatum and H. hircinum. H. umbellatum and H. maculatum, each contained 0.76 pg DNA and 16 chromosomes were counted. The 2C DNA content of H. hircinum was 1.00 pg with the best metaphase plate revealing 32 chromosomes. Additionally, a combined marker analysis, based on inter-simple sequence repeats (ISSR) and sequence related amplified polymorphism (SRAP), was conducted to gain a better understanding of diversity especially within the accessions of H. perforatum. A total of 27 (11 ISSR and 16 SRAP) primer combinations were screened, showing 699 bands, of which 661 were polymorphic. UPGMA clustering revealed that accessions from the same geographic area tended to be more closely related, while H. maculatum was grouped separately from all H. perforatum accessions. Both methods have shown similar sensitivities in detecting the genetic diversity of the analyzed genotypes. Our results may be useful for Hypericum breeding programs and the development of effective conservation strategies. © 2022 Sciendo. All rights reserved.

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In recent years, escitalopram (ESC) has been suggested to have different mechanisms of action beyond its well known selective serotonin reuptake inhibition. The aim of this study is to investigate the effects of escitalopram on oxidative stress, apoptosis, brain-derived neurotrophic factor (BDNF), Methyl-CpG-binding protein 2 (MeCP2), and oligodendrocytes number in the brain of chronic unpredictable mild stress-induced depressed rats. The animals were randomised in four groups (8 in each group): control, stress, stress + ESC 5 and stress + ESC 5/10. ESC was administered for 42 days in a fixed dose (5 mg/kg b.w.) or in an up-titration regimen (21 days ESC 5 mg/kg b.w. then 21 days ESC 10 mg/kg b.w.). Sucrose preference test (SPT) and elevated plus maze (EPM) were also performed. ESC improved the percentage of sucrose preference, locomotion and anxiety. ESC5/10 reduced the oxidative damage in the hippocampus and improved the antioxidant defence in the hippocampus and frontal lobe. ESC5/10 lowered caspase 3 activity in the hippocampus. Escitalopram had a modulatory effect on BDNF and the number of oligodendrocytes in the hippocampus and frontal lobe and also improved the MeCP2 expressions. The results confirm the multiple pathways implicated in the pathogenesis of depression and suggest that escitalopram exerts an antidepressant effect via different intricate mechanisms.

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The concern for implementing bioactive nutraceuticals in antioxidant-related therapies is of great importance for skin homeostasis in benign or malignant diseases. In order to elucidate some novel insights of Lycium barbarum (Goji berry) activity on skin cells, the present study focused on its active compound zeaxanthin. By targeting the stemness markers CD44 and CD105, with deep implications in skin oxidative stress mechanisms, we revealed, for the first time, selectivity in zeaxanthin activity. When applied in vitro on BJ human fibroblast cell line versus the A375 malignant melanoma cells, despite the moderate cytotoxicity, the zeaxanthin-rich extracts 1 and 2 were able to downregulate significantly the CD44 and CD105 membrane expression and extracellular secretion in A375, and to upregulate them in BJ cells. At mechanistic level, the present study is the first to demonstrate that the zeaxanthin-rich Goji extracts are able to influence selectively the mitogen-activated protein kinases (MAPK): ERK, JNK and p38 in normal BJ versus tumor-derived A375 skin cells. These results point out towards the applications of zeaxanthin from L. barbarum as a cytoprotective agent in normal skin and raises questions about its use as an antitumor prodrug alone or in combination with standard therapy.

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Allium sativum L. (garlic bulbs) and Allium fistulosum L. (Welsh onion leaves) showed quantitative differences of identified compounds: allicin and alliin (380 mu g/mL and 1410 mu g/mL in garlic; 20 mu g/mL and 145 mu g/mL in Welsh onion), and the phenolic compounds (chlorogenic acid, p-coumaric acid, ferulic acid, gentisic acid, 4-hydroxybenzoic acid, kaempferol, isoquercitrin, quercitrin, quercetin, and rutin). The chemical composition determined the inhibitory activity of Allium extracts in a dose-dependent manner, on human normal cells (BJ-IC50 0.8841% garlic/0.2433% Welsh onion and HaCaT-IC50 1.086% garlic/0.6197% Welsh onion) and tumor cells (DLD-1-IC50 5.482%/2.124%; MDA-MB-231-IC50 6.375%/2.464%; MCF-7-IC50 6.131%/3.353%; and SK-MES-1-IC50 4.651%/5.819%). At high concentrations, the cytotoxic activity of each extract, on normal cells, was confirmed by: the 50% of the growth inhibition concentration (IC50) value, the cell death induced by necrosis, and biochemical determination of LDH, catalase, and Caspase-3. The four tumor cell lines treated with high concentrations (10%, 5%, 2.5%, and 1.25%) of garlic extract showed different sensibility, appreciated on the base of IC50 value for the most sensitive cell line (SK-MES-1), and the less sensitive (MDA-MB-231) cell line. The high concentrations of Welsh onion extract (5%, 2.5%, and 1.25%) induced pH changes in the culture medium and SK-MES-1 being the less sensitive cell line.

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Anti-angiogenic therapies for melanoma have not yet been translated into meaningful clinical benefit for patients, due to the development of drug-induced resistance in cancer cells, mainly caused by hypoxia-inducible factor 1 alpha (HIF-1 alpha) overexpression and enhanced oxidative stress mediated by tumor-associated macrophages (TAMs). Our previous study demonstrated synergistic antitumor actions of simvastatin (SIM) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on an in vitro melanoma model via suppression of the aggressive phenotype of melanoma cells and inhibition of TAMs-mediated angiogenesis. Therefore, we took the advantage of long circulating liposomes (LCL) superior tumor targeting capacity to efficiently deliver SIM and DMXAA to B16.F10 melanoma in vivo, with the final aim of improving the outcome of the anti-angiogenic therapy. Thus, we assessed the effects of this novel combined tumor-targeted treatment on s.c. B16.F10 murine melanoma growth and on the production of critical markers involved in tumor development and progression. Our results showed that the combined liposomal therapy almost totally inhibited (> 90%) the growth of melanoma tumors, due to the enhancement of anti-angiogenic effects of LCL-DMXAA by LCL-SIM and simultaneous induction of a pro-apoptotic state of tumor cells in the tumor microenvironment (TME). These effects were accompanied by the partial re-education of TAMs towards an M1 phenotype and augmented by combined therapy-induced suppression of major invasion and metastasis promoters (HIF-1 alpha, pAP-1 c-Jun, and MMPs). Thus, this novel therapy holds the potential to remodel the TME, by suppressing its most important malignant biological capabilities.

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Oxidative stress represents the underlying cause of many chronic diseases in human; therefore, the development of potent antioxidant compounds for preventing or treating such conditions is useful. Starting from the good antioxidant and antiradical properties identified for the previously reported Dihydroxy-Phenyl-Thiazol-Hydrazinium chloride (DPTH), we synthesized a congeneric series of phenolic thiazoles. The radical scavenging activity, and the antioxidant and chelation potential were assessed in vitro, a series of quantum descriptors were calculated, and the electrochemical behavior of the synthesized compounds was studied to evaluate the impact on the antioxidant and antiradical activities. In addition, their antibacterial and antifungal properties were evaluated against seven aerobic bacterial strains and a strain of C. albicans, and their cytotoxicity was assessed in vitro. Compounds 5a-b, 7a-b and 8a-b presented remarkable antioxidant and antiradical properties, and compounds 5a-b, 7a and 8a displayed good Cu+2 chelating activity. Compounds 7a and 8a were very active against P. aeruginosa ATCC 27853 compared to norfloxacin, and proved less cytotoxic than ascorbic acid against the human keratinocyte cell line (HaCaT cells, CLS-300493). Several phenolic compounds from the synthesized series presented excellent antioxidant activity and notable anti-Pseudomonas potential.

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(1) Background: Peripheral nerve injuries have a great impact on a patient's quality of life and a generally poor outcome regarding functional recovery. Lately, studies have focused on different types of nanoparticles and various natural substances for the treatment of peripheral nerve injuries. This is the case of chitosan, a natural compound from the crustaceans' exoskeleton. The present study proposes to combine chitosan benefic properties to the nanoparticles' ability to transport different substances to specific locations and evaluate the effects of magnetic nanoparticles functionalized with chitosan (CMNPs) on peripheral nerve injuries' rehabilitation by using an in vivo experimental model. (2) Methods: CMNPs treatment was administrated daily, orally, for 21 days to rats subjected to right sciatic nerve lesion and compared to the control group (no treatment) by analyzing the sciatic functional index, pain level, body weight, serum nerve growth factor levels and histology, TEM and EDX analysis at different times during the study. (3) Results: Animals treated with CMNPs had a statistically significant functional outcome compared to the control group regarding: sciatic functional index, pain-like behavior, total body weight, which were confirmed by the histological and TEM images. (4) Conclusions: The results of the study suggest that CMNPs appear to be a promising treatment method for peripheral nerve injuries.

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Raman microspectroscopy was tested as an alternative/complementary method for biochemical evaluation of the synaptosomes obtained from neonatal rat brain prenatally exposed to sodium valproate and treated with allicin. Spectrophotometric assays of several oxidative stress markers (catalase, superoxide dismutase, total thiols) and acetylcholine esterase activity revealed the redox balancing function and pro-cholinergic effect of the allicin as compared to the valproate effect. Raman evaluation showed no significant changes in our experimental conditions. Different concentrations and volumes of the synaptosomes vesicles must be tested for the optimal Raman examination of these purified synaptosomes.

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In this work structural and (sub)picosecond surface dynamical changes of genomic DNA isolated from different medicinal plants (Hyssopus officinalis, Majorana hortensis, Melissa officinalis, Mentha piperita, Mentha piperita cv Cristal, Monarda didyma and Matricaria chamomilla), as probed with surface-enhanced Raman spectroscopy (SERS), are discussed upon modifying the acidic pH of mixtures consisting of silver colloidal suspension and DNA samples, respectively. Binding affinity changes of DNA with silver NPs and nucleic acids protonation are supposed to take place upon lowering the pH. A small percentage of Hoogsteen GC basepairs was found in Mentha piperita cv Cristal DNA, at low acidic pH. As a general observation, the global relaxation times corresponding to different functional groups of the investigated genomic DNAs, respectively, show a decrease of their values upon lowering the pH. (c) 2021 Elsevier B.V. All rights reserved.

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Potential medical application of UV laser pulses for cancer treatment is one of the main motivations behind this study. Surface-enhanced Raman signatures-based structural and (sub)picosecond dynamical characterization of genomic DNAs from different medicinal plants are presented. Moreover, structural and surface dynamical changes induced in genomic DNAs from selected medicinal plants species by UV femtosecond laser pulses (lambda = 280 nm, 170 fs) were investigated between 300 and 1800 cm(-1). As a general observation, after nucleic acids irradiation some modes became higher in intensity especially in the wavenumber range between 1050 cm(-1)-1650 cm(-1) or new profiles appeared in this interval in the surface-enhanced Raman spectroscopy (SERS) characteristics. Changes in the structure of dA, dG, dC dT residues, nucleic acid backbone and deoxyribose or unstacking of DNA bases were found. Large wavenumber shifts of the marker bands, which appear in the SERS spectra upon nucleic acids irradiation might be associated with loss of essential structural organization of DNA. As far as surface dynamics of DNA is concerned, for control nucleic acids functional groups, the global relaxation times varied between 0.39-1.77 ps and for UV femtosecond laser pulses-irradiated DNAs, this parameter was slower than 0.35 ps and faster than 1.63 ps. A decrease of the limit values of global relaxation times variation interval is to be observed upon UV treatment of nucleic acids. This faster surface dynamics might be explained by the presence of broken bonds (lesions) in DNA systems after laser pulses irradiation. (C) 2021 Elsevier B.V. All rights reserved.

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Graphene/AgNPs-based surface dynamics of native DNA functional groups at different acidic pH values was discussed using surface-enhanced Raman spectroscopy (SERS). Also,ab initiodynamics of Verlet type was investigated for nucleic acid nitrogenous bases adsorbed on a graphene surface, respectively. The experimental dynamical parameters were given in terms of full widths at half-maximum (FWHMs) and (sub)picosecond global relaxation times, associated with SERS bands. Furthermore, using density functional theory (DFT) as implemented in SIESTA and the velocity autocorrelation function (VACF), we have obtained the vibrational density of states (VDOS) for each of the four DNA bases placed on a pristine graphene layer.

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Effective conservation of biodiversity should build on a strong basis of taxonomic and spatial distribution knowledge. The Carpathian Mountains, an iconic centre of biodiversity in temperate Europe, harbour a remarkable number of endemic vascular plants. Current knowledge on their taxonomic status, spatial distribution and genetic diversity is, however, incomplete. Research and conservation efforts have mostly been country specific, resulting in contrasting chorological knowledge and taxonomic acceptance between neighbouring countries, and differing conservation policies. Urgent synchronisation of cross-border conservation measures is required. Here, we present an overview of a new international project that will address these issues, outlining the current state of knowledge and theoretical background concerning Carpathian subendemics and endemics in order to improve their conservation status. The project involves partners from eight countries, working collaboratively in conservation, research and sharing of standardised data for the Carpathian flora. Long-term seed conservation of 500 species, with a focus on endemics, regionally endangered species and range-margin populations from the Carpathians, will be used as an effective measure for the conservation of Carpathian endemics and endangered species. Research focused on the biosystematics of selected taxa will increase our knowledge of the evolutionary processes involved in the origin of the Carpathian flora. Finally, the establishment of the Carpathian Research Network (CRN) provides an official framework for pooling, sharing and standardising scientific data on Carpathian endemics from different countries. The CRN is developing an online database of the distribution of selected species (The Carpathian Endemics Distribution Database, CEDD) including their intrinsic and extrinsic traits and current opinion on their taxonomic status. This will be used to produce a monograph on the Carpathians' endemic flora.

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Genome skimming has the potential for generating large data sets for DNA barcoding and wider biodiversity genomic studies, particularly via the assembly and annotation of full chloroplast (cpDNA) and nuclear ribosomal DNA (nrDNA) sequences. We compare the success of genome skims of 2051 herbarium specimens from Norway/Polar regions with 4604 freshly collected, silica gel dried specimens mainly from the European Alps and the Carpathians. Overall, we were able to assemble the full chloroplast genome for 67% of the samples and the full nrDNA cluster for 86%. Average insert length, cover and full cpDNA and rDNA assembly were considerably higher for silica gel dried than herbarium-preserved material. However, complete plastid genomes were still assembled for 54% of herbarium samples compared to 70% of silica dried samples. Moreover, there was comparable recovery of coding genes from both tissue sources (121 for silica gel dried and 118 for herbarium material) and only minor differences in assembly success of standard barcodes between silica dried (89% ITS2, 96% matK and rbcL) and herbarium material (87% ITS2, 98% matK and rbcL). The success rate was > 90% for all three markers in 1034 of 1036 genera in 160 families, and only Boraginaceae worked poorly, with 7 genera failing. Our study shows that large-scale genome skims are feasible and work well across most of the land plant families and genera we tested, independently of material type. It is therefore an efficient method for increasing the availability of plant biodiversity genomic data to support a multitude of downstream applications.

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Intraspecific trait variability (ITV) is an important component of functional ecology studies. While functional biogeography requires us to understand broad-scale patterns of trait distribution, ITV remains inadequately studied. Due to isolation of ranges, habitat fragmentation and sharp topoclimatic gradients over short distances, temperate mountains offer opportunities to study the ITV in widespread species. In this study, we explored ITV in geographically separated populations of matgrass (Nardus stricta), a clonal dominant plant species of mountain pastures. We measured plant height and leaf traits in 55 populations distributed across six European mountain ranges (from the Pyrenees to the Carpathians) to examine the structure of ITV. We hypothesised that the leaf economics spectrum (LES) and plant size exhibit a strong convergence across a prevailing gradient of growing season length (GSL). We then partitioned the ITV into between ranges, between populations nested in ranges and between individuals nested in populations, and examined the role of climate and local variables on the spatial variation and covariation of traits. Traits showed large ITV and higher between- than within-population variability. Leaf traits displayed a high dependency on height reflecting the growing phase climate and other local factors, while range identity alone (differentiation between ranges) explained a low to moderate amount of the variability in the traits. The strength of coordination between several of the LES traits was higher under shorter GSL due to late snowmelt, suggesting higher phenotypic integration. In the context of functional biogeography of mountain ecosystems, allometric effects and growing phase climate are both key to understanding ITV. We discuss the implications of these findings as they relate to future comparative studies of ITV in seasonal environments.

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Background: Photodynamic therapy (PDT) is a treatment of cancer due to its ability to induce cell death, oxidative stress and acute inflammatory reaction in targeted sites. To optimize the effect of PDT the addition of some compounds with supplementary cytotoxic effect on tumor cells was tried. Methods: The study was performed on 35 Wistar male albino rats with Walker 256 carcinosarcoma. The animals were randomly assigned in seven groups (n = 5) and treated as follows: group 1 - control; group 2 - Cornus mas (CM) extract 15 mg/kg b.w., administered for 7 days; group 3 - CM extract administered for 7 days followed by irradiation (CM + IR); group 4 - one dose of tetra-p-sulfonato-phenyl-porphyrin (TSPP) 10 mg/kg b.w.; group 5 - TSPP + IR; group 6 - CM extract administered daily for 7 days before TSPP and IR (CM + TSPP + IR); group 7 - TSPP + IR followed by CM administered for 7 days (TSPP + IR + CM). Results: The results showed that MDA and GSSG levels increased after PDT in parallel with the increasing of COX-2 expression and DNA damage. Apoptotic and necrotic index enhanced in TSPP + IR, effect improved by CM association before PDT. CM + TSPP + IR regimen also induced more intense inflammatory reactions, increased COX-2 expression, determined DNA damage, apoptosis and necrosis, compared to the TSPP + IR + CM group. Both combined therapeutic regimens reduced MDA levels in tumor tissue, especially CM + TSPP + IR and increased the antioxidant defense and iNOS expression. Conclusions: Our results demonstrated that CM associated before PDT had beneficial effects in PDT and may represent a promising option in PDT strategies.

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Regardless of recent progress, melanoma is very difficult to treat, mainly due to the drug resistance modulated by tumor cells as well as by the tumor microenvironment (TME). Among the immune cells recruited at the tumor site, tumor associated macrophages (TAMs) are the most abundant, promoting important tumorigenic processes: angiogenesis, inflammation and invasiveness. Furthermore, it has been shown that TAMs are involved in mediating the drug resistance of melanoma cells. Thus, in the present study, we used liposomal formulation of prednisolone disodium phosphate (LCL-PLP) to inhibit the protumor function of TAMs with the aim to sensitize the melanoma cells to the cytotoxic drug doxorubicin (DOX) to which human melanoma has intrinsic resistance. Consequently, we evaluated the in vivo effects of the concomitant administration of LCL-PLP and liposomal formulation of DOX (LCL-DOX) on B16.F10 melanoma growth and on the production of key molecular markers for tumor development. Our results demonstrated that the concomitant administration of LCL-PLP and LCL-DOX induced a strong inhibition of tumor growth, primarily by inhibiting TAMs-mediated angiogenesis as well as the tumor production of MMP-2 and AP-1. Moreover, our data suggested that the combined therapy also affected TME as the number of infiltrated macrophages in melanoma microenvironment was reduced significantly.

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5-Fluorouracil-based therapy remains the main approach in colorectal cancer, even though there are still some drawbacks, such as chemoresistance. In this study we combined 5-fluorouracil encapsulated in long-circulating liposomes with simvastatin, also encapsulated in long-circulating liposomes, that was previously proved to exert antitumor actions on the same tumor model. The production of angiogenic/inflammatory proteins was assessed by protein array and the production of markers for tumor aggressiveness (Bcl-2, Bax, and nuclear factor [NF]-kappa B) were determined by western blot analysis. Intratumor oxidative stress was evaluated through measurement of malondialdehyde level by HPLC, and through spectrophotometric analysis of catalytic activity of catalase and of total antioxidant capacity. Immunohistochemical analysis of tumors for CD31 expression was assessed. Intratumor activity of MMP-2 by gelatin zymography was also carried out. Our results revealed that combined therapies based on liposomal formulations exerted enhanced antitumor activities compared with combined treatment with free drugs. Sequential treatment with liposomal simvastatin and liposomal 5-fluorouracil showed the strongest antitumor activity in C26 colon carcinoma in vivo, mainly through inhibition of tumor angiogenesis. Important markers for cancer progression (Bcl-2, Bax, NF-kappa B, and intratumor antioxidants) showed that liposomal simvastatin might sensitize C26 cells to liposomal 5-fluorouracil treatment in both regimens tested. The outcome of simultaneous treatment with liposomal formulations was superior to sequential treatment with both liposomal types as the invasive capacity of C26 tumors was strongly increased after the latest treatment. The antitumor efficacy of combined therapy in C26 colon carcinoma might be linked to the restorative effects on proteins balance involved in tumor angiogenesis.

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The study investigated the potential neuroprotective effects of metformin (MET) on alcohol-induced neurotoxicity in adult Wistar rats. The animals were randomized in four groups (n = 10): control, alcohol (ALC), ALC + MET, and MET. ALC (2 g/kg b.w.) and MET (200 mg/kg b.w.) were orally administered for 21 days, once daily. For the ALC + MET group, MET was administered 2 h after ALC treatment. On day 22, the open field test (OFT) and elevated plus maze (EPM) were performed. MET improved global activity and increased the time spent in unprotected open arms, decreased oxidative stress, both in the frontal lobe and in the hippocampus, and increased neuroglobin expression in the frontal cortex. Histopathologically, an increased neurosecretory activity in the frontal cortex in the ALC + MET group was noticed. Thus, our findings suggest that metformin has antioxidant and anxiolytic effects and may partially reverse the neurotoxic effects induced by ethanol.

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Hemoglobin- (Hb-) based oxygen carriers (HBOC) have for several decades been explored for treatment of hemorrhage. In our previous top-up tests, HBOC with lowerin vitroprooxidant reactivity (incorporating a peroxidase or serum albumin to this end) showed a measurable but small improvement of oxidative stress-related parameters. Here, such HBOCs are tested in a hemorrhage set-up; ovine hemoglobin is also tested for the first time in such a setting, based onin vitrodata showing its improved performance versus bovine Hb against oxidative and nitrosative stress agents. Indeed, ovine Hb performs better than bovine Hb in terms of survival rates, arterial tension, immunology, and histology. On the other hand, unlike in the top-up models, where the nonheme peroxidase rubrerythrin as well as bovine serum albumin copolymerized with Hb were shown to improve the performance of HBOC, in the present hemorrhage models rubrerythrin fails dramatically as HBOC ingredient (with a distinct immunological reaction), whereas serum albumin appears not feasible if its source is a different species (i.e., bovine serum albumin fares distinctly worse than rat serum albumin, in HBOC transfusions in rats). An effect of the matrix in which the HBOCs are dissolved (PBS versus gelofusine versus plasma) is noted.

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Site-directed spin labeling and EPR characterization (continuous wave as well as DEER) of two blood substitute candidates is described for the first time: glutaraldehyde-polymerized bovine hemoglobin and the related hemoglobin-albumin copolymer. In vitro, with two different types of cell cultures, these spin-labeled candidates are relatively stable; however, they appear to be rapidly reduced in vivo, most likely via removal or reduction of the spin label (as opposed to removal of the blood substitute candidate from the circulatory system altogether).

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Aim: To assess ultrastructural changes, alterations in matrix metalloproteinase activity and apoptosis induced by silver nanoparticles (AgNPs) in the rat testicle. Materials & methods: For 45 days, two groups of animals received different doses of AgNPs (0.8 and 1.5 mg/kg b.w.), and a control group was given the buffer used as vehicle for AgNPs. At 7 and 15 days post-treatment, transmission electron microscopy, TUNEL assay, evaluation of NFkB, pNFkB, p53, Bcl-2 and Nrf2 expressions were performed on the removed testes. Results: Transmission electron microscopy revealed severe ultrastructural changes of interstitial tissue and seminiferous epithelium sustained by positive signal for apoptosis. The promatrix metalloproteinase-2 activity and NFkB, Bcl-2 expressions were increased, mainly at 7 days. Conclusion: AgNPs induced severe cell lesions identified even a long time after the exposure.

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Rare and endemic plant species represent important components of plant biodiversity which require protection to ensure their sustainable conservation. Cerastium banaticum (Rochel) Heuff. is such an endemic and rare species from Romania, for which the genetic variability of two natural populations was studied by SSR markers. Shannon's information index revealed low levels of genetic diversity in both populations (I = 0.296). As the first attempt in a conservation program a reproducible micropropagation protocol was established starting from seeds. followed by multiplication, rooting. and ex vitro acclimatization. Among the various plant growth regulators tested the highest multiplication coefficient was achieved on a culture medium with 0.5 mg L-1 6-furfurylaminopurine (K) and 1 mg L-1 alpha-naphthaleneacetic acid (NAA). On this PGRs concentration a number of 26.6 shoots/individual explant with a mean length of 7.9 cm for new generated shoots was registered. The highest number of roots/individual initiated shoot was 2.6 and it was recorded on a culture medium with 0.5 mg L-1 2-isopentyl-adenine (2iP) and 0.1 mg L-1 NAA. The outdoor acclimatization was successfully performed in a specially designed rocky area in the 'Alexandru Borza' Botanical Garden, Cluj-Napoca (Romania).

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Premise Disjunct distributions have been commonly observed in mountain plant species and have stimulated phylogeographic and phylogenetic research. Here we studied Alyssum repens, a member of the polyploid species complex A. montanum-A. repens, which exhibits SE Alpine-Carpathian disjunctions with a large elevational span and consists of diploid and tetraploid populations. We aimed to investigate the species' genetic and cytotype structure in the context of its distribution patterns, to elucidate the polyploid origins and to propose an appropriate taxonomic treatment. Methods We combined AFLP fingerprinting markers, sequence variation of the highly repetitive ITS region of rDNA and the low-copy DET1 nuclear gene, genome size, and morphometric data. Results We identified four geographically structured genetic lineages. One consisted of diploid populations from the foothills of the Southeastern Alps and neighboring regions, and the three others were allopatric montane to alpine groups comprising diploids and tetraploids growing in the Southeastern Carpathians and the Apuseni Mts. in Romania. Conclusions We inferred a vicariance scenario associated with Quaternary climatic oscillations, accompanied by one auto- and two allopolyploidization events most likely involving a northern Balkan relative. Whereas genetic differentiation and allopatric distribution would favor the taxonomic splitting of this species, the genetic lineages largely lack morphological distinguishability, and their ecological, cytotype and genome size divergence is only partial. Even though we probably face here a case of incipient speciation, we propose to maintain the current taxonomic treatment of Alyssum repens as a single, albeit variable, species.

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Apple (Malus x domestica Borkh.) genotypes originating from different plant collections (field collection, in vitro plant collections undergoing or not undergoing cryopreservation) were screened and characterized by SSR markers. Shoot tips excised from plants grown in vitro were successfully cryopreserved by encapsulation-dehydration. 'the highest regrowth frequency (69%, cultivar Goldrush) of cryopreserved apices was achieved after 24 h of osmoprotection in 0.5 M sucrose, 3 h of desiccation, and 24% water content of alginate beads. No differences in morphological characteristics including shoot length and number and length of roots were observed between controls and plants recovered after cryopreservation. SSR markers were used for calculation of genetic similarities between plants from the field collection, in vitro-micropropagated plants, or plants regenerated after liquid nitrogen storage. The set of microsatellite markers showed a low level of polymorphism among the studied genotypes, which could be distinguished by a specific combination of alleles generated by CH03g07, CH05c02, CH05d11, and CH05e03 primers. The CH03g07, CH05c02, CH05d11, CH05e03, GD96, GD147, and GD162 SSR markers exhibited low levels of polymorphism, while CH04AE07, CH04g10, GD100, and GD142 were nonpolymorphic. The Dice coefficient confirmed the effectiveness of SSRs for distinguishing between plants from ex situ collections and preserved plants. No major differences between ex situ plants, micropropagated plants, and plants recovered after cryopreservation were observed.

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Apple (Malus × domestica Borkh.) genotypes originating from different plant collections (field collection, in vitro plant collections undergoing or not undergoing cryopreservation) were screened and characterized by SSR markers. Shoot tips excised from plants grown in vitro were successfully cryopreserved by encapsulation-dehydration. The highest regrowth frequency (69%, cultivar Goldrush) of cryopreserved apices was achieved after 24 h of osmoprotection in 0.5 M sucrose, 3 h of desiccation, and 24% water content of alginate beads. No differences in morphological characteristics including shoot length and number and length of roots were observed between controls and plants recovered after cryopreservation. SSR markers were used for calculation of genetic similarities between plants from the field collection, in vitro-micropropagated plants, or plants regenerated after liquid nitrogen storage. The set of microsatellite markers showed a low level of polymorphism among the studied genotypes, which could be distinguished by a specific combination of alleles generated by CH03g07, CH05c02, CH05d11, and CH05e03 primers. The CH03g07, CH05c02, CH05d11, CH05e03, GD96, GD147, and GD162 SSR markers exhibited low levels of polymorphism, while CH04AE07, CH04g10, GD100, and GD142 were nonpolymorphic. The Dice coefficient confirmed the effectiveness of SSRs for distinguishing between plants from ex situ collections and preserved plants. No major differences between ex situ plants, micropropagated plants, and plants recovered after cryopreservation were observed. © TÜBİTAK.

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Anxiety disorders can associate with oxidative stress and immune system alterations. Our study aimed to chemically analyze Hypericum maculatum (HM) and Hypericum perforatum (HP) dry extracts and to evaluate their effects along with quercetin (Q), on brain oxidative stress biomarkers: malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD), inflammatory cytokines: interleukin-1 alpha, (IL-1 alpha), IL-1 beta, regulated on activation normal T cell expressed and secreted (RANTES), interferon (IFN), monocyte chemoattractant protein-1 (MCP1), macrophage inflammatory protein (MIP) and serum corticosterone levels. Nuclear transcription factor kB (NFkB) signaling pathway in the hippocampus and frontal lobe in rats with N-methyl-9H-pyrido[5,4-b]indole-3-carboxamide (FG-7142) experimental-induced anxiety were also investigated. The chemical analyses of total hypericins were performed by spectrophotometric analysis and hypericin, hyperforin and polyphenols derivatives were quantified by chromatographic methods. The animals were divided in 6 groups: carboxymethylcellulose 2% (CMC); CMC + FG; alprazolam (APZ) + FG; Q + FG; HM + FG; HP + FG. APZ (0.08 mg/kg b.w.), Q (30 mg/kg b.w.), HM and HP (350 mg/kg b.w.) were orally administered for 21 days. FG (7.5 mg/kg b.w.) was intraperitoneally (i.p.) injected in a single dose. Q and hypericum extracts (HpE) exerted anti-inflammatory (decreased IL-l alpha, IL-1 beta, MCP1, IFN and MIP mainly in hippocampus) and antioxidant effects (decreased MDA levels, increased CAT and SOD activity), enhanced NFkB and pNFkB expressions in the brain and reduced serum corticosterone levels. Our findings suggest that HpE may improve anxiety-like behavior, offer brain protection by modulation of oxidative stress and inflammation, and can contribute to overall biological activity of natural compounds-rich diet.

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We have previously proposed the annelid-derived protein, hemerythrin, as a viable replacement for hemoglobin in the synthesis of semi-synthetic oxygen carriers (blood substitutes). Here, we report the first in vivo tests for potential hemerythrin-based oxygen carriers (HrBOC), using a battery of experiments involving Wistar rats and previously tested on a series of hemoglobin-based oxygen carrier candidates (HBOC). At the concentrations tested, hemerythrin appears to behave similarly to hemoglobin including, importantly, immunological effects. The antioxidant strategies based on albumin as well as based on rubrerythrin appear to offer observable physiological advantages. (C) 2017 Elsevier B.V. All rights reserved.

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Galium verum is a well-known medicinal plant which is used in various pathologies. G. verum extracts are characterized here using chromatography, where among the rich pool of phenolic acids of flavonoids two known anti-stress modulators, chlorogenic acid and rutin are identified in high quantities. Additionally, the extracts are characterized using a series of in vitro assays (EPR, DPPH, TPC and TEAC). Considering the chemical findings, the potential beneficial effects of the G. verum extract are explored here in a living organism exposed to stress induced oxidative damages. Thus, the biochemical-modulatory and antioxidant roles of two doses of G. verum extract are examined in animals exposed to acute restraint and dark stress (S). The animals were divided in groups [control, S, SG1 (exposed to 25 mg G. verum extract), SG2 (50 mg extract)]. Increased levels of lipid peroxidation (TBARS from 4.43 to 8.06 nmol/mL), corticosterone from 0.43 to 1.96 mu g/dL and epinephrine from 44.43 to 126.7 mu g/mL, as well as decreased antioxidant enzymes activities (SOD/ CAT) were observed in the S group. The G. verum extract afforded a near-normal equilibrium within the biochemical parameters of animals exposed to RS, by reducing oxidative damage (TBARS at a 3.73 nmol/mL; CS at 0.90 mu g/dL; EP at 63.72 mu g/mL) and by restoring the antioxidant balance.

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Rats exposed to repeated restraint stress exhibit structural and functional deficits in hippocampus that are similar to those observed in patients with depressive illnesses. Blood corticosterone concentrations are proportionally increased with catalase and glutathione-peroxidase activity and are inversely proportional with 3-nitrotyrosine concentrations. Cytochrome c oxidase, adenosin tryphosphatase and monoamine oxidase activities of CA3 hippocampal field mark a stress-time dependent decrease. Acridine-orange labeling of the CA3 field reveals an enhancing green fluorescence of glyocites in stress conditions. After three days of restraint stress, the secretory activity of CA3 neurons did not show significant decrease, and neurons appeared with normal shapes and distribution. CA3 neurons after seven days of restraint stress have marked a slight decrease of secretory activity. In contrast to a well-preserved histological appearance of the CA3 neurons, local and blood stress-related reactions are observed. CA3-glial activation and disturbance of blood oxidative homeostasis are tandem processes during three and seven days of RS. This study depicts the balancing role of CA3 area in time-varying stress conditions. (C) 2016 Elsevier Inc. All rights reserved.

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The ultrastructural changes in tomato shoot apices and the severity of cumulative effects related to the successive stages of a cryopreservation protocol and ex vitro acclimatization of recovered plants have been assessed using imaging methods. Even though after osmoprotection, features like a dense cytoplasm, numerous plastids with starch grains in tunica (L1, L2) cells or the presence of Golgi complex in corpus (L3) cells suggest an intact cell structure, dehydration in the vitrification solution left traces especially in some basal corpus (L3) cells. Shortly after thawing following liquid nitrogen exposure tunica (L1, L2) cells displayed a regular structure with dense cytoplasm, while in basal corpus (L3) cells high vacuolation and signs of plasmolysis were observed. The findings presented in this study have shown that the injuries occurred at cellular and subcellular level cannot be attributed with certainty to a single stress element but rather they may act cumulatively and become evident some time later. As a consequence after 3 weeks of post-thawing on recovery culture medium tunica (L1, L2) and corpus (L3) cells exhibited cellular heterogeneity. To our knowledge this is the first report on ultrastructural observations on shoot apices of acclimatized plants regenerated from cryostored plant material.

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Two distinct microwave power levels and techniques have been studied in two cases: low-power microwave (LPM) irradiation on in vitro Sequoia plants and high-power microwave (HPM) exposure on recovery rates of cryostored (-196A degrees C) Sequoia shoot apices. Experimental variants for LPM exposure included: (a) in vitro plants grown in regular conditions (at 24 +/- 1A degrees C during a 16-h light photoperiod with a light intensity of 39.06 mu Em(-2) s(-1) photosynthetically active radiation), (b) in vitro plants grown in the anechoic chamber with controlled environment without microwave irradiation, and (c) in vitro plants grown in the anechoic chamber with LPM irradiation for various times (5, 15, 30, 40 days). In comparison to control plants, significant differences in shoot multiplication and growth parameters (length of shoots and roots) were observed after 40 days of LPM exposure. An opposite effect was achieved regarding the content of total soluble proteins, which decreased with increasing exposure time to LPM. HPM irradiation was tested as a novel rewarming method following storage in liquid nitrogen. To our knowledge, this is the first report using this type of rewarming method. Although, shoot tips subjected to HPM exposure showed 28% recovery following cryostorage compared to 44% for shoot tips rewarmed in liquid medium at 22 +/- 1 A degrees C, we consider that the method represent a basis and can be further improved. The results lead to the overall conclusion that LPM had a stimulating effect on growth and multiplication of in vitro Sequoia plants, while the HPM used for rewarming of cryopreserved apices was not effective to achieve high rates of regrowth after liquid nitrogen exposure.

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The paper presents the results of the studies performed in the Natura 2000 site Pădurea Bârnova-Repedea (ROSCI0135), in order to identify and map habitat types and plant species of community interest. Palynological and archaeo-botanical studies in the region were reviewed, in order to assess the presence of the ancient forests. This protected area was designed to preserve forests from two habitats types of community interest, namely Asperulo-Fagetum beech forests and Dacian oak-hornbeam forests, and to shelter a species of wild and endangered orchid, Cypripedium calceolus L. During the field work other Natura 2000 habitats of community interest have been identified: mountain hay meadows, Ponto-Sarmatic steppes and Ponto-Sarmatic deciduous thickets. The information obtained provided new data on the habitats and plant species distribution. Field data were correlated with existing topographic maps and aerial photography. The use of the Geographic Information System allowed translating the field distribution of the species and habitat types into accurate maps, which can be used in the future by the managers of this site for implementing adaptive conservation measures. The human activities with potential impact on this Natura 2000 site have been recorded. The map of current pressures on the site was generated based on these impact categories. © 2017, Babes-Bolyai University, "Alexandru Borza" Botanic Garden. All rights reserved.

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In the European Alpine System, the Carpathian Mountains are recognized as one of the major centres of diversity and endemism. In the present study, we aimed to explain the spatial structure of plant endemism in its South-Eastern subunit by the complementary use of diversity indices, parsimony analysis of endemicity (PAE), biotic element analysis (BEA), and barrier analysis. We analyzed the available information on 111 plant taxa confined to the South-Eastern Carpathians, mapped using two different sets of operational geographical units (OGUs): 71 geomorphological units and 64 quadrats. Our results showed that centres of endemics diversity largely corresponded to the areas of endemism and biotic elements. PAE consensus cladogram outlined four major areas of endemism (with three nested ones): (1) Danubian; (2) western part of the Southern Carpathians; (3) eastern part of the Southern Carpathians; and (4) Pocutico-Marmarossian. Out of the seven identified biotic elements, five were spatially clustered and overlapped the major areas of endemism, with one notable exception: the calcareous massifs from the Eastern Carpathians, not identified through PAE. Conversely, the latter outlined a nested area of endemism (Cozia - Buila-Vanturari?a), omitted by BEA. Barrier analysis identified three major breaks in the distribution of endemics: (1) south of the Retezat - ?arcu - Godeanu mountain group; (2) north of the Piatra Craiului - Bucegi - Ciucas mountain group; and (3) north of the Rodna massif. The results obtained in here using different methods are generally spatially convergent, indicating highly structured patterns of endemism in the South-Eastern Carpathians. These patterns mostly follow the present-day distribution of alpine habitats and calcareous bedrock, which might have acted as isolating factors through insularity. Interestingly, three of the spatial clusters of OGUs obtained from the endemics distribution analyses (the Eastern Carpathians, as well as eastern and western parts of the Southern Carpathians) largely also correspond to the mid-Miocene archipelago configuration of landmasses in this part of the Carpathians. This might suggest the existence of older migration barriers that emerged throughout the Neogene Period. Differences in the spatial patterns outlined by PAE and BEA could stem from partial sympatry of endemics caused by post-speciation processes such as dispersal or extinction. Additionally, sympatric distribution of taxa with disjunct populations may be caused by the absence of divergence among segregated populations, such as the patterns of relict distributions seen in alpine plants. Finally, the complementary use of these methods may prove to be an efficient approach for better understanding the geographical structure of endemism and provide a starting point for further testing of hypotheses on evolutionary processes.

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The genomes of the two plant organelles encode for a relatively small number of proteins. Thus, nuclear genes encode the vast majority of their proteome. Organelle-to-nucleus communication takes place through retrograde signaling (RS) pathways. Signals relayed through RS pathways have an impact on nuclear gene expression but their target-genes remain elusive in a normal state of the cell (considering that only mutants and stress have been used so far). Here, we use maize cytolines as an alternative. The nucleus of a donor line was transferred into two other cytoplasmic environments through at least nine back-crosses, in a time-span of > 10 years. The transcriptomes of the resulting cytolines were sequenced and compared. There are 96 differentially regulated nuclear genes in two cytoplasm-donor lines when compared with their nucleus-donor. They are expressed throughout plant development, in various tissues and organs. One-third of the 96 proteins have a human homolog, stressing their potential role in mitochondrial RS. We also identified syntenic orthologous genes in four other grasses and homologous genes in Arabidopsis thaliana. These findings contribute to the paradigm we use to describe the RS in plants. The 96 nuclear genes identified here are not differentially regulated as a result of mutation, or any kind of stress. They are rather key players of the organelle-to-nucleus communication in a normal state of the cell.

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In this work, surface-enhanced Raman spectra of ten genomic DNAs extracted from leaf tissues of different grapevine (Vitis vinifera L.) varieties, respectively, are analyzed in the wavenumber range 300-1800 cm(-1). Furthermore, structural changes induced in grapevine genomic nucleic acids upon femtosecond (170 fs) infrared (IR) laser pulse irradiation (lambda = 1100 nm) are discussed in detail for seven genomic DNAs, respectively. Surface-enhanced Raman spectroscopy (SERS) signatures, vibrational band assignments and structural characterization of genomic DNAs are reported for each case. As a general observation, the wavenumber range between 1500 and 1660 cm(-1) of the spectra seems to be modified upon laser treatment. This finding could reflect changes in the base-stacking interactions in DNA. Spectral shifts are mainly attributed to purines (dA, dG) and deoxyribose. Pyrimidine residues seem to be less affected by IR femtosecond laser pulse irradiation. Furthermore, changes in the conformational properties of nucleic acid segments are observed after laser treatment. We have found that DNA isolated from Feteasca Neagra grapevine leaf tissues is the most structurally-responsive system to the femtosecond IR laser irradiation process. In addition, using unbiased computational resources by means of principal component analysis (PCA), eight different grapevine varieties were discriminated.

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In this work, Fourier transform infrared (FT-IR) spectra of untreated and microwaves irradiated genomic DNAs extracted from leaves of different grapevine varieties, respectively, have been analyzed between 1800-800 cm(-1), in order to investigate their screening characteristic features and their structural response to microwaves treatment at 869-894 MHz frequency band. FT-IR vibrational modes for each of these cases, spectroscopic band assignments and structural interpretations of genomic DNAs are reported. No significant microwaves damage, concerning DNA bases structure, base pairing and base unstacking was found. Conformational changes of specific DNA segments and effects on deoxyribose seem to appear after microwaves irradiation. Grapevine varieties dependent microwaves influence on the structure of DNA has been found. Best nucleic acids structural tolerance upon microwave irradiation, as compared with the non-exposed DNA has been found in the case of genomic DNA isolated from 'Chasselas Dore' grapevine variety.

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DNA barcoding represents an accurate, fast and automatable tool that enhanced species identification and species delineation. It relies on the use of standardized gene regions as internal species tags. The barcode markers should meet the followings requirements: they must have short length, conserved flanking sites and display significant genetic variability. Due to the complexity of plant genomes, all attempts to find a universal marker failed. For different plant species, different barcode markers or combinations of markers have been reported. In this study, we aimed to discover barcode markers for an efficient differentiation of two close related species from the genus Pulmonaria (Boraginaceae). Pulmonaria rubra Schott. and the Carpathian endemic P. filarszkyana Jáv. share a strong morphological resemblance and controversial taxonomic relationships, the latter being considered either a subspecies or a variety of the former, or a full ranked independent species. We tested fourteen target regions, of which thirteen belonged to the chloroplast and one was nuclear. Most regions lacked polymorphism or did not have a reliable, reproductible amplification. Only three regions displayed genetic variability: ITS1, rpL16 and psbD-trnT. Along the full length of the three sequences, six SNPs were detected, but they proved to be sufficient for a significant delimitation of the two taxa within subsequent phylogenetic analysis. © 2016, Babes-Bolyai University, "Alexandru Borza" Botanic Garden. All rights reserved.

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Pollen datasets from central-eastern Europe, a region that for long time has been lacking quality palaeoecological records, are becoming increasingly available and these allow an evaluation of past vegetation sensitivity to climate change. Here, we use a new pollen and non-pollen palynomorph (NPP) record supported by eight AMS radiocarbon measurements from the Ic Ponor peat sequence, NW Romania, covering the end of the Lateglacial to mid-Holocene to examine the vegetation dynamics and associated climatic conditions. The vegetation at the Lateglacial to Holocene transition was composed of mixed forest (Pinus dominated) - tundra-steppe vegetation. Following the decline of Pinus from 11,200 cal BP, Ulmus and Picea abies expanded and formed dense forests, then by Corylus avellana and Quercus from 10,200 cal BP. The forests were mainly composed of C. avellana and P. abies along with Ulmus, Quercus and Fraxinus between 9800 and 7440 cal BP. Starting with 7200 cal BP, P. abies dominated the landscape in the area probably favored by the increasingly cooler and moist conditions. After a considerable hiatus between 5830 and 40 cal BP, the most recent changes in the forest composition in the area were primarily related to forest clearings and grazing. The absence of relevant occurrences of Fagus sylvatica and Carpinus betulus in our sequence prior to 8800 cal BP, fail to give support to the glacial survival of these two taxa in the Apuseni Mountains, as hypothesized in the previous pollen and genetic studies. However, their continuous occurrence in small percentages after 8800 cal BP most probably reflects locally present, small populations of these taxa. Short-term phases of changes in the vegetation composition that appeared to be the response to cooler and/or wetter climate were recorded between 11,800 and 11,700, 11,500-11,200, 10,300-10,200, 9350-9250, 8250-8140, 7700-7400, 6800-6600 and 6100-5900 cal BP. These intervals correlate well to other centennial-scale excursions recorded in Romania and Europe. (C) 2015 Elsevier Ltd and INQUA. All rights reserved.

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In this work the surface-enhanced Raman scattering (SERS) spectra of five genomic DNAs from non-cryopreserved control tomato plants (Lycopersicon esculentum Mill. cultivars Siriana, Darsirius, Kristin, Pontica and Capriciu) respectively, have been analyzed in the wavenumber range 400-1800 cm(-1). Structural changes induced in genomic DNAs upon cryopreservation were discussed in detail for four of the above mentioned tomato cultivars. The surface-enhanced Raman vibrational modes for each of these cases, spectroscopic band assignments and structural interpretations of genomic DNAs are reported. We have found, that DNA isolated from Siriana cultivar leaf tissues suffers the weakest structural changes upon cryogenic storage of tomato shoot apices. On the contrary, genomic DNA extracted from Pontica cultivar is the most responsive system to cryopreservation process. Particularly, both C2'-endo-anti and C3'-endo-anti conformations have been detected. As a general observation, the wavenumber range 1511-1652 cm(-1), being due to dA, dG and dT residues seems to be influenced by cryopreservation process. These changes could reflect unstacking of DNA bases. However, not significant structural changes of genomic DNAs from Siriana, Darsirius and Kristin have been found upon cryopreservation process of tomato cultivars. Based on this work, specific plant DNA-ligand interactions or accurate local structure of DNA in the proximity of a metallic surface, might be further investigated using surface-enhanced Raman spectroscopy. (C) 2015 Elsevier B.V. All rights reserved.

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Shoot tips of five tomato (Lycopersicon esculentum Mill.) genotypes were successfully cryopreserved by droplet vitrification. Recovered plants were studied for genetic stability by two different assays: amplified fragment length polymorphism (AFLP) using 4 primer combinations and sequencing of lycopene cryopreserved beta-cyclase gene (LCY-B) from leaves. The highest shoot regeneration after cryopreservation was 70% (Pontica) following 24 h osmoprotection in 0.5 M sucrose and 30 min dehydration in plant vitrification solution 2 (PVS2). The data inferred from AFLP showed no genetic dissimilarities between in vitro regenerants derived from cryopreserved tissues compared with the noncryopreserved controls. Although a single nucleotide polymorphism, a G -> T transversion, was identified in position 1139 in Capriciu and Darsirius, this was not due to the cryopreservation process itself, since it was detected in both cryopreserved and control samples. Thus, sequencing of LCY-B gene from leaves revealed no DNA damages after cryopreservation and subsequent in vitro regeneration. Our results indicate that cryostorage by droplet vitrification is an efficient and reliable technique to preserve the selected tomato genotypes and to regenerate true-to-type plants.

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Although the Carpathians represent one of the main elements of the European Alpine System and an important area of endemism, only a few phylogeographic studies concerning this region have focused on the detailed intraspecific variation in alpine plant species. Using two molecular marker systems, we (1) aimed to elucidate the controversy concerning the status of the endemic Carpathian taxon Onobrychis transsilvanica Simonk. in relation to the more widespread Onobrychis montana DC. and (2) determined the phylogeographic structure of O. transsilvanica within the Carpathians. For O. transsilvanica, our data suggest either a recent postglacial speciation with incomplete lineage sorting or genetic divergence followed by subsequent continuous gene flow during the glacial period. The genetic structure of the complex does not support O. transsilvanica as a distinct species from O. montana. Within the Carpathians, the extant populations of O. transsilvanica comprise two major allopatric lineages, which have been isolated from each other for a long period of time. Unexpectedly, the major genetic break was not in line with a classical biogeographical boundary in the Carpathians but rather separated a group from the southwestern edge of the mountains. We also discovered an additional divergent haplotype lineage, with weaker genetic support, within the O. transsilvanica populations.

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24 female Wistar rats (120 ± 10 g) were divided into 4 groups: - control group (C); - Galium verum treated group (E), (25 mg extract/100 g bw); - anakinetic stressed group (S); - stress + Galium verum extract (25 mg extract/100 g bw) treated group (SE). Results revealed different reactivity of hypothalamo-hypophyseal-adrenal axis in the three experimental groups. The extract administration, in conditions of exposure to stress, resulted in an enhancement of NPV neurosecretory activity – associated with a possible stimulation of CRH release, a possible activation of adenohipophyseal hormones, as well as stimulation of adrenal steroid hormones. In S group, the effects of stress were opposite to those of E and SE groups. Histological results of the study proved that the administration of Galium verum vegetal extract in condition of anakinetic stress exposure induced important morphological changes at the all constitutive assembly of hypothalamo-hypophyseal-adrenal axis. These results justify the stimulation of secretory activity of the axis. © 2015 Vasile Goldis University Press.

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In this work, Fourier transform infrared (FT-IR) spectra of seven untreated and UV irradiated genomic DNAs extracted from leaves of different in vitro grown plant species, respectively, have been analyzed in the 800-1800 cm(-1) spectral range, in order to investigate their screening characteristic features and their structural response to UV treatment at 253.7 nm. As far as the untreated genomic plant DNAs are concerned, both A and B backbone vibrational modes were detected. A low amount of Z-DNA was also found in the untreated nucleic acids, especially for DNA from Rosa. Besides, in vitro grown plant species dependent UV degradation of DNA has been observed. The major UV influence on nucleic acids from leaf tissues was observed in the case of DNA isolated from Drosera, Hypericum, Leontopodium and Rosa, respectively. It seems, that in a lesser extent is affected Sequoia DNA by UV irradiation at 253.7 nm. Particularly, alterations in nucleic acid bases, base pairing and base stacking have been found. Also, changes in DNA conformation and sugar groups were detected. The DNA conformation seems to be in our cases, a mixture of conformational states. However, A-form DNA is the predominant conformation in both non-irradiated and irradiated samples. (C) 2014 Elsevier Ltd. All rights reserved.

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Ex vitro and in vitro germination in relation to seed quality, cryopreservation and seedling regeneration were examined following liquid nitrogen storage of Jatropha curcas seeds and zygotic embryonic axes. Six seed weight categories were used: (1) <= 0.399 g; (2) 0.400 - 0.499 g; (3) 0.500 - 0.599 g; (4) 0.600 - 0.699 g; (5) 0.700 - 0.799 g; (6) >= 0.800 g. Germination was related to the weight and quality of seeds, the presence / absence of the seed coat and type of explant. The moisture content and water uptake during five hours imbibition were related to the initial fresh weight of seeds. The highest germination (90%) was similar under ex vitro and in vitro conditions for seeds with seed coat in the weight category 0.600 - 0.699 g. Shoot regeneration following liquid nitrogen (-196 degrees C) storage was influenced by the type of explant used. Zygotic embryonic axes excised from cryopreserved seeds displayed the highest regeneration percentages with 95% compared with 64% obtained for isolated embryonic axes cryopreserved in plant vitrification solution droplets. Plants developed from cryopreserved seeds and isolated zygotic embryo axes were morphologically similar to the non-cryopreserved controls.

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Romanian tomato (Lycopersicon esculentum Mill.) cultivars have been cryopreserved by encapsulation-dehydration and successfully acclimatized to ex vitro growth conditions. Shoot rips were excised from in vitro grown plants then precultured for 24 h in various sucrose concentrations, dehydrated up to 6 h in laminar air flow prior to direct immersion in liquid nitrogen (-196 degrees C) for 24 h. Different parameters have been studied: the effects of osmoprotection and desiccation duration on the regrowth of cryopreserved shoot rips, the effects of various IBA concentrations on rooting and the ex vitro acclimatization of plants recovered from liquid nitrogen. The highest frequency of regrowth (72% cv. 'Pontica') was obtained when encapsulated explants were precultured in 0.5 M sucrose and the moisture content (fresh weight basis) of alginate beads was 23%. The highest rooting rates (58% to 77%) for all cultivars were observed for shoots grown on MS medium supplemented with 1.0 mg/l IBA. The rooted plants could be easily acclimatized ex vitro with up to 100% survival.

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In this work, Fourier transform infrared (FT-IR) spectra of seven untreated and UV irradiated genomic DNAs extracted from leaves of different in vitro grown plant species, respectively, have been analyzed in the 800-1800 cm -1 spectral range, in order to investigate their screening characteristic features and their structural response to UV treatment at 253.7 nm. As far as the untreated genomic plant DNAs are concerned, both A and B backbone vibrational modes were detected. A low amount of Z-DNA was also found in the untreated nucleic acids, especially for DNA from Rosa. Besides, in vitro grown plant species dependent UV degradation of DNA has been observed. The major UV influence on nucleic acids from leaf tissues was observed in the case of DNA isolated from Drosera, Hypericum, Leontopodium and Rosa, respectively. It seems, that in a lesser extent is affected Sequoia DNA by UV irradiation at 253.7 nm. Particularly, alterations in nucleic acid bases, base pairing and base stacking have been found. Also, changes in DNA conformation and sugar groups were detected. The DNA conformation seems to be in our cases, a mixture of conformational states. However, A-form DNA is the predominant conformation in both non-irradiated and irradiated samples. © 2014 Elsevier Ltd. All rights reserved.

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Aim Species distribution models (SDMs) based on current species ranges underestimate the potential distribution when projected in time and/or space. A multi-temporal model calibration approach has been suggested as an alternative, and we evaluate this using 13,000 years of data. Location Europe. Methods We used fossil-based records of presence for Picea abies, Abies alba and Fagus sylvatica and six climatic variables for the period 13,000 to 1000yr bp. To measure the contribution of each 1000-year time step to the total niche of each species (the niche measured by pooling all the data), we employed a principal components analysis (PCA) calibrated with data over the entire range of possible climates. Then we projected both the total niche and the partial niches from single time frames into the PCA space, and tested if the partial niches were more similar to the total niche than random. Using an ensemble forecasting approach, we calibrated SDMs for each time frame and for the pooled database. We projected each model to current climate and evaluated the results against current pollen data. We also projected all models into the future. Results Niche similarity between the partial and the total-SDMs was almost always statistically significant and increased through time. SDMs calibrated from single time frames gave different results when projected to current climate, providing evidence of a change in the species realized niches through time. Moreover, they predicted limited climate suitability when compared with the total-SDMs. The same results were obtained when projected to future climates. Main conclusions The realized climatic niche of species differed for current and future climates when SDMs were calibrated considering different past climates. Building the niche as an ensemble through time represents a way forward to a better understanding of a species' range and its ecology in a changing climate.

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In this work, the surface-enhanced Raman scattering (SERS) spectra of seven genomic DNAs from leaves of chrysanthemum (Dendranthema grandiflora Ramat.), common sundew (Drosera rotundifolia L.), edelweiss (Leontopodium alpinum Cass), Epilobium hirsutum L., Hypericum richeri ssp. transsilvanicum (elak) Ciocarlan, rose (Rosa x hybrida L.) and redwood (Sequoia sempervirens D. Don. Endl.), respectively, have been analyzed in the wavenumber range 200-1800cm-1. The surface-enhanced Raman vibrational modes for each of these cases, spectroscopic band assignments and structural interpretations of genomic DNAs are reported. A high molecular structural information content can be found in the SERS spectra of these DNAs from leaf tissues. Based on this work, specific plant DNA-ligand interactions or accurate local structure of DNA might be further investigated using surface-enhanced Raman spectroscopy. Besides, this study will generate information which is valuable in the development of label-free DNA detection for chemical probing in living cell. Copyright (c) 2013 John Wiley & Sons, Ltd.

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Maize has always been under constant human selection ever since it had been domesticated. Intensive breeding programs that resulted in the massive use of hybrids nowadays have started in the 60s. That brought significant yield increases but reduced the genetic diversity at the same time. Consequently, breeders and researchers alike turned their attention to national germplasm collections established decades ago in many countries, as they may hold allelic variations that could prove useful for future improvements. These collections are mainly composed of inbred lines originating from well-adapted local open pollinated varieties. However, there is an overall lack of data in the literature about the genetic diversity of maize in SE Europe, and its potential for future breeding efforts. There are no data, whatsoever, on the nutritional quality of the grain, primarily dictated by the zein proteins. We therefore sought to use the Romanian maize germplasm as an entry point in understanding the molecular make-up of maize in this part of Europe. By using 80 SSR markers, evenly spread throughout the genome, on 82 inbred lines from various parts of the country, we were able to decipher population structure and the existing relationships between those and the eight international standards used, including the reference sequenced genome B73. Corroborating molecular data with a standardized morphological, physiological, and biochemical characterization of all 90 inbred lines, this is the first comprehensive such study on the existing SE European maize germplasm. The inbred lines we present here are an important addition to the ever-shrinking gene pool that the breeding programs are faced-with, because of the allelic richness they hold. They may serve as parental lines in crosses that will lead to new hybrids, characterized by a high level of heterosis, nationwide and beyond, due to their existing relationship with the international germplasm.

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Efficient micropropagation and cryopreservation of Hypericum richeri ssp. transsilvanicum, an endemic species in Romania, and Hypericum umbellatum, a rare and endangered Daco-Balkan species, was achieved. The effects of type of explant and cytokinin on in vitro plant regeneration were investigated. Shoot organogenesis was achieved in all explants, but stem nodes regenerated best. Organogenesis from nodal segments was promoted by incubating these explants on Murashige and Skoog (MS) medium in the presence of cytokinins (6-benzyladenine, thidiazuron, kinetin or 6-gamma,gamma-dimethylallylaminopurine), each tested at four concentrations. The best morphogenic response for both Hypericum species (number of shoots per explant, shoot length, axillary branching of shoot, and frequency of shoot organogenesis) was observed when explants were incubated on MS medium containing 0.44 or 1.11 mu M 6-benzyladenine. Root induction was achieved only when regenerated shoots were transferred to fresh medium with or without auxin. Maximum rooting was recorded on MS medium supplemented with 2.45 mu M indole-3-butyric acid. Plantlets grown in vitro were successfully acclimatized in the greenhouse and showed normal development. Shoot tips and axillary buds excised from the in vitro regenerated plants were successfully cryopreserved in liquid nitrogen by the droplet-vitrification method. Following preculture in 0.25 M sucrose, dehydration and cryopreservation, the highest regeneration rates were obtained in both species by using axillary buds (68 % for H. richeri ssp. transsilvanicum and 71 % for H. umbellatum).

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Primula intricate: Gren. et Godr. has an unclear taxonomic status: it was originally described as a distinct species but subsequently was considered a subspecies (Primula elatior subsp. intricata) or even a variety (P. elatior var.intricata) of Primula elatior (L.) Hill. No prior genetic studies were performed on this group of Primulaceae, therefore we considered useful to investigate taxonomies boundaries within the P elatior-intricata group. We explored genetic differences between Primula intricate: and Primula elatior group by applying three different types of molecular markers: nuclear ribosomal DNA (ITSI), chloroplast DNA (spacer trnH-psbA and intron trnL) and Amplified Fragment Length Polymorphisms (AFLP). We found a solid differentiation between P intricata and P elatior group, differentiation that was confirmed by all the employed molecular markers. This finding enabled us to propose a valid species rank for Primula intricata, as a separate taxon from the P. elatior group.

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Ultrasensitive detection of nucleic acids is demonstrated through exploiting the effect of surface-enhanced Raman scattering (SERS). In this work the SERS spectra of eight genomic DNAs from leaves of apple trees grown in the field (Malus domestica Borkh., Fam. Rosaceae, cultivars. Florina, Idared, Rebra, Goldrush, Romus 3, Romus 4 and the rootstocks M9 and M26) have been analyzed in the wavenumber range 200-1800 cm(-1). SERS signatures, spectroscopic band assignments and structural interpretations of these plant genomic DNAs are reported. SERS spectra of nucleic acids are compared here with caution, because these signals are time-dependent and are strongly influenced by DNA amount in the measured sample volume. Similarities of the SERS spectra of genomic DNAs extracted from apple leaves of Goldrush, Rebra and Florina cultivars, respectively, have been detected. Besides, the SERS spectra, corresponding to DNA from M9, Romus 4 and M26, leaves, respectively, show similar features and well resolved bands. Based on this work, specific plant DNA-ligand interactions or DNA structural changes induced by plant stress conditions associated with their natural environment, might be further investigated using surface-enhanced Raman spectroscopy.

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Ultrasensitive Raman measurements of nucleic acids are possible by exploiting the effect of surface-enhanced Raman scattering (SERS). In this work, the vibrational spectra of eight genomic DNAs from in vitro grown apple leaf tissues (Malus domestica Borkh., Fam Rosaceae, cvs. Florina, Idared, Rebra, Goldrush, Romus 3, Romus 4 and the rootstocks M9 and M26) were analyzed using surface-enhanced Raman spectroscopy, in the wavenumber range 200-1800 cm(-1). SERS signatures, spectroscopic band assignments and structural interpretations of these plant genomic DNAs are reported. Strong dependences of the SERS spectra on genomic DNA amount in the measured sample volume and on time were observed. Similarities of the SERS signals of DNAs from Rebra and Romus 3 leaves were detected. To our knowledge, this is the first SERS study on genomic DNA from leaf tissues. The present work provides a basis for future use of surface-enhanced Raman spectroscopy to analyze specific plant DNA-ligand interactions or DNA structural changes induced by plants' stress conditions associated with their natural environment. Besides, this study will generate information that is valuable in the development of low-level plant DNA-based analytical sensors. Copyright (C) 2010 John Wiley & Sons, Ltd.

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Surface-enhanced Raman scattering (SERS) effect was used to demonstrate ultrasensitive optical detection of nucleic acids. In this work, the SERS spectra of seven genomic DNAs from leaves of Arnica montana (L.), Fam. Compositae, Astragalus peterfii (Jav.), Fam. Fabaceae, Kalanchoe x hybrida, Fam. Crassulaceae, strawberry (Fragaria x ananassa Duch.), Fam. Rosaceae, carnation (Dianthus caryophyllus L.), Fam. Caryophyllaceae, apple (Malus domestica Borkh.), Fam. Rosaceae and Persian violet (Exacum affine Balf.), Fam. Gentianaceae were analyzed in the wavenumber range 200-1800 cm(-1). SERS signatures, spectroscopic band assignments and structural interpretations of these plant genomic DNAs are reported. SERS spectra of nucleic acids are compared here with caution, because these signals are time-dependent. The SERS spectra corresponding to DNA from Arnica, Dianthus, Fragaria and Kalanchoe leaves show well-resolved, accurate bands, providing thus a high molecular structural information content. Based on this work, specific plant DNA-ligand interactions or DNA structural changes induced by plant stress conditions associated with their natural environment might be further investigated using SERS spectroscopy. Besides, this study will generate information that is valuable in the development of label-free DNA-based nanosensors for chemical probing in the living cell. Copyright (C) 2011 John Wiley & Sons, Ltd.

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Dianthus giganteus D'Urv ssp. banaticus (Heuff.) Tutin (fam. Caryophylaceae) is an endemic species of the SW Carpathians, considered vulnerable/rare in Romania. Besides the importance of these species (it is an endemic species, rare and vulnerable) its ornamental value gives it a plus for population conservation both ex situ and in situ. Ex situ collections constitute insurance for the conservation of certain species belonging to several genotypes, from different areas. The ex situ conservation method used in our study is cryopreservation. The plant material was collected from natural sites. The initiation of the in vitro cultures was done from stolons, using three sterilizing methods based on sodium hypochlorite (Domestos) and mercuric chloride. Sterilization with sodium hypochlorite was less efficient (18-63% sterile explants), compared to the one with mercuric chloride (37-84% sterile explants), and the combined method gave the best results (60-100% sterile explants). Two combinations of plant growth regulators were used in culture media: (i) for initiation (NAA 1 mg/L; BAP 1 mg/L); (ii) for multiplication (NAA 0.1 mg/L; BAP 1 mg/L). For the cryopreservation experiments (in liquid nitrogen, at -196 degrees C) apical meristems from in vitro-plants were used. These meristems had 2-4 pairs of foliar primordia and a length of approximately 3-4 mm. We also studied the influence of the treatment with different concentrations of sucrose solutions on the regeneration ability of the apices before and after cryopreservation. The highest regeneration rate after cryopreservation was obtained with 0.5 M sucrose solution (43% regenerated apices).

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Alliinase (Alliin lyase EC 4.4.1.4), a pyridoxal phosphate-dependent lyase, represents one of the major protein components of Allium species. The enzyme is a homodimeric glycoprotein and catalyzes the synthesis of allicin (diallyl thiosulfinate, a biologically active compound), pyruvate, and ammonia starting from the specific non-protein sulfur-containing amino acid alliin ((+S)-allyl-L-cysteine sulfoxide). Using newly developed specific primers two new alliinase genes from Allium obliquum and Allium senescens sop. montanum were amplified and sequenced, as well as their homologs, from Allium fistulosum and Allium schoeonoprasum. The G+C content of the alliinase region ranges between that of other dicot plants and that reported in monocot cereal plants, in all four species. Investigations of gene expression revealed a significantly higher enzyme expression level in bulbs than in leaves in all four taxa. The deduced alliinase sequences displayed a, high variability among different species, since the lowest sequence similarity was found to be 55.5% between senescent var. montanum and Allium cepa, while the highest similarity is 77.5%, between ilium senescent var. montanum and Allium fistulosum. Leucine is the most common amino acid in all four alliinases, while cysteine is also more frequent that in other enzymes, suggesting a high stability of the molecules due to the possible disulfide bonds.

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We investigated the effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 6-gamma,gamma-dimethylallylaminopurine (2iP), thidiazuron (TDZ) and alpha-naphthaleneacetic acid (NAA)], modified Murashige and Skoog (MS) medium containing 10 mM NH4 (+) and 5 mM NO3 (-) and supplemented with 2iP, BA, Kin and NAA (MSM medium), and two elicitors [jasmonic acid (JA), and salicylic acid (SA)], on plant growth and accumulation of hypericins (hypericin and pseudohypericin) and hyperforin in shoot cultures of Hypericum hirsutum and H. maculatum. Our data suggested that culture of shoots on MS medium supplemented with BA (0.4 mg l(-1)) or Kin (0.4 mg l(-1)) enhanced production of hypericins in H. maculatum and hyperforin in H. hirsutum. Hypericins and hyperforin concentrations decreased in both species when TDZ (0.4 mg l(-1)) was added to the MS medium. Also, TDZ induced hyperhydric malformations and necrosis of regenerated shoots. Cultivation of H. maculatum on MSM medium resulted in approximately twofold increased production of hypericins compared to controls, and the growth of H. hirsutum shoots on the same medium led to a 6.16-fold increase in hyperforin production. Of the two elicitors, SA was more effective in stimulating the accumulation of hypericins. At 50 mu M, SA enhanced the production of hypericin (7.98-fold) and pseudohypericin (13.58-fold) in H. hirsutum, and, at 200 mu M, enhanced the production of hypericin (2.2-fold) and pseudohypericin (3.94-fold) in H. maculatum.

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Experiments were performed to determine the influence of medium composition and explant characteristics on somatic embryogenesis in Peucedanum oreoselinum (L.) Moench. Somatic embryogenesis occurred directly without an intervening callus proliferation phase from leaf petiole and root explants cultured on Murashige and Skoog (1962) (MS) medium supplemented with 4.5 mu M 2,4-dichlorophenoxyacetic acid (2,4-D) in darkness. Somatic embryos were directly formed at cut edges or on the surface of leaf explains, around cut ends or along side surfaces of petiole and root explants. Embryo formation was significantly affected by explant position. Adaxial-side-up orientation of leaf explants significantly promoted embryogenesis in comparison with abaxial-side-up orientation. The development of proembryos to globular, heart-shaped, torpedo and cotyledonary stages was achieved on the MS medium without plant growth regulators (PGRs) under light conditions. Embryo formation and maturation was further enhanced by addition of proline (5 mM) in MS medium. The frequency of embryo formation was significantly higher for root explains (85%) in comparison with leaf explants (57%) or petiole explants (11%). This protocol facilitated the recovery of 38 plantlets per root explant within 80 days.

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