Genome-Wide Identification of the Hypericum perforatum WRKY Gene Family Implicates HpWRKY85 in Drought Resistance.

WRKY, named for its special heptapeptide conserved sequence WRKYGOK, is one of the largest transcription factor families in plants and is widely involved in plant responses to biotic, abiotic, and hormonal stresses, especially the important regulatory function in response to drought stress. However, there is no complete comprehensive analysis of this family in H. perforatum, which is one of the most extensively studied plants and is probably the best-known herbal medicine on the market today, serving as an antidepressant, neuroprotective, an antineuralgic, and an antiviral. Here, we identified 86 HpWRKY genes according to the whole genome database of H. perforatum, and classified them into three groups through phylogenetic analysis. Gene structure, conserved domain, motif, cis-elements, gene ontology, and expression profiling were performed. Furthermore, it was found that HpWRKY85, a homologous gene of AtWRKY75, showed obvious responses to drought treatment. Subcellular localization analysis indicated that this protein was localized in the nucleus by the Arabidopsis protoplasts transient transfection. Meanwhile, HpWRKY85-overexpressing Arabidopsis plants showed a stronger ability of root growth and scavenging endogenous reactive oxygen species. The results provide a reference for further understanding the role of HpWRKY85 in the molecular mechanism of drought resistance of H. perforatum.

Impacts of manganese bio-based nanocomposites on phytochemical classification, growth and physiological responses of Hypericum perforatum L. shoot cultures.

We report a new green route for preparing MnO2/perlite nanocomposites (NCs) by leaf extract of Hypericum perforatum. Characterization of the physicochemical properties of the MnO2/perlite-NCs was performed using XRD, FESEM, EDX, FT-IR, and DLS techniques. Furthermore, their effects on the phytochemical classification and growth parameters of H. perforatum shoot cultures were assessed. According to the FESEM image, the synthesized spherical MnO2 nanoparticles on the sheet-like structure of nano-perlite were formed, ranging about 20-50 nm. In addition, based on the EDX spectra, the elemental analysis showed the presence of Carbon, Oxygen, Silicon, Aluminum, and Manganese elements in the as-synthesized MnO2/perlite-NCs. Biological studies confirmed that nano-perlite and MnO2/perlite-NCs were non-toxic to H. perforatum shoot cultures and showed positive effects on plant growth in specific concentrations. Overall, phytochemical classification demonstrated that the terpenoids decreased in the evaluated treatments, while hypericin and pseudohypericin were increased in some treatments (25, 50 and 150 mg/L of nano-perlite) relative to control. Metabolomics results suggested that both nano-perlite and MnO2/perlite-NCs can be used as elicitors and new nanofertilizers for generating some secondary metabolites.

Beyond rest and quiescence (endodormancy and ecodormancy): A novel model for quantifying plant-environment interaction in bud dormancy release.

Bud dormancy of plants has traditionally been explained either by physiological growth arresting conditions in the bud or by unfavourable environmental conditions, such as non-growth-promoting low air temperatures. This conceptual dichotomy has provided the framework also for developing process-based plant phenology models. Here, we propose a novel model that in addition to covering the classical dichotomy as a special case also allows the quantification of an interaction of physiological and environmental factors. According to this plant-environment interaction suggested conceptually decades ago, rather than being unambiguous, the concept of "non-growth-promoting low air temperature" depends on the dormancy status of the plant. We parameterized the model with experimental results of growth onset for seven boreal plant species and found that based on the strength of the interaction, the species can be classified into three dormancy types, only one of which represents the traditional dichotomy. We also tested the model with four species in an independent experiment. Our study suggests that interaction of environmental and physiological factors may be involved in many such phenomena that have until now been considered simply as plant traits without any considerations of effects of the environmental factors.

Soil Microbiomes Underlie Population Persistence of an Endangered Plant Species.

Microbiomes can dramatically alter individual plant performance, yet how these effects influence higher-order processes is not well resolved. In particular, little is known about how microbiome effects on individual plants alter plant population dynamics, a question critical to imperiled species conservation. Here we integrate bioassays, multidecadal demographic data, and integral projection modeling to determine how the presence of the natural soil microbiome underlies plant population dynamics. Simulations indicated that the presence of soil microbiomes boosted population growth rates (λ) of the endangered Hypericum cumulicola by 13% on average, the difference between population growth versus decline in 76% of patches. The greatest benefit (47% increase in λ) occurred in low-nutrient, high-elevation habitats, suggesting that the soil microbiome may help expand H. cumulicola's distribution to include these stressful habitats. Our results demonstrate that soil microbiomes can significantly affect plant population growth and persistence and support the incorporation of soil microbiomes into conservation planning.

Temperature-dependent growth and hypericin biosynthesis in Hypericum perforatum.

Hypericum perforatum is a perennial herb that produces the anti-depression metabolite hypericin (Hyp). While several efforts to increase Hyp production have been made, the effects of temperatures on growth and Hyp biosynthesis are still limited. In this study, the growth morphophysiological traits, Hyp biosynthesis and their related genes expression, as well as major bioactive compounds accumulation and antioxidant capacity were assessed by exposing H. perforatum seedlings to three different temperatures (15, 22 and 30 °C). The results showed that aerial parts biomass was greater at 15 °C with 1.3 and 1.6-fold increase compared to at 22 and 30 °C, in large part because of greater increase in chlorophyll content, stem number and leaf area on a per plant basis. Hyp content in the aerial parts was greater 1.9 and 5.6-fold on a per plant basis compared to 22 and 30 °C treatments, and the contents of other bioactive compounds (flavonoids and phenolics) as well as antioxidant capacity in the aerial parts, on dry weight and per plant basis, also exhibited significant increases with the temperatures decrease. The mRNA expressions of eight genes (psbA, psbB, psbC, psbD, ycf3, ycf4, ycf5 and matK) related to photosynthesis and two genes (Polyketide synthase, PKS; Phenolic oxidative coupling protein, Hyp-1) involved in Hyp biosynthesis were also up-regulated at 15 °C. The findings are useful in guiding cultivation and regulating Hyp biosynthesis in H. perforatum.

Targeted metabolomic profiling reveals interspecific variation in the genus Hypericum in response to biotic elicitors.

Shoot cultures of eight Hypericum species belonging to the sections Hypericum, Oligostema, Ascyreia and Webbia were evaluated for their phytochemical profiles by high-performance liquid chromatography. In total, 17 secondary metabolites assigned to the groups of anthraquinones, phloroglucinols, hydroxycinnamic acids and flavonoids were detected. Furthermore, the elicitation potential of 18 biotic factors derived from saccharides, endophytic fungi and Agrobacterium rhizogenes was examined and statistically analysed with the paired two-sample t-test and principal component analysis. The production of naphthodianthrones and emodin was predominantly stimulated by elicitors derived from Fusarium oxysporum and Trichoderma crassum, while Piriformospora indica promoted the phloroglucinols production. Among flavonoids, the aglycone amentoflavone was readily increased by several elicitors up to 15.7-fold in H. humifusum treated by potato-dextrose broth. However, the chlorogenic acid proved to be the most susceptible metabolite to elicitation, when 31.7-times increase was detected in H. maculatum shoots upon D-glucose treatment. In spite of several biotic factors have been tested, no metabolite was commonly induced in all Hypericum spp. as a response to elicitor treatments.

Renoprotective effect of Hypericum perforatum against diabetic nephropathy in rats: Insights in the underlying mechanisms.

Oxidative stress and inflammation play a key role in the initiation and progression of diabetic nephropathy (DN). The present study aimed to investigate the possible protective effect of hypericum perforatum (HP) against DN. Rats were allocated into six groups: control, received normal saline; diabetic untreated (DM), received single dose of streptozotocin (STZ) after injection of nicotinamide (NA); gliclazide, received STZ,NA + gliclazide (10 mg/kg); DM + HP50, DM + HP100, DM + HP200, received STZ,NA and HP 50, 100, 200 mg/kg, respectively. Gliclazide and HP were administered daily via gavage for 8 weeks. Serum glucose, insulin, kidney function and histopathological picture were assessed. Furthermore, oxidative/nitrosative stress, inflammatory cytokines, apoptotic and fibrotic markers were measured. Diabetic untreated group showed increase in serum glucose, urea, creatinine with albuminurea. Renal expression of protein for nuclear factor kappa-B (NF-кB), renal expression of inducible nitric oxide synthase (iNOS), cyclooxygenase II (COXII), collagen IV, fibronectin were elevated. Malondialdehyde (MDA), nitric oxide (NO), tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), intracellular adhesion molecule (ICAM-1), monocellular chemoattractant protein-1 (MCP-1), tumour growth factor- ß (TGF-ß), caspase-3 and cytochrome c contents were also increased consequently with decline of serum insulin, expression of peroxisome proliferator-activated receptor (PPARγ), renal reduced glutathione (GSH) content and superoxide dismutase (SOD) activity. Treatment with either gliclazide or HP mitigated the deleterious effects of STZ on the tested parameters. These findings indicate for the first time that HP may have a renoprotective effect against DN through reduction of oxidative/nitrosative stress, enhancement of antioxidant defense mechanisms, decline of inflammatory cytokines, antifibrotic, antiapoptotic and blood glucose lowering properties.

Shoot Tip Meristem Cryopreservation of Hypericum Species.

Based on our long-standing experience with in vitro culture of Hypericum perforatum, a clonal multiplication system and vitrification-based cryopreservation protocols have been applied to several Hypericum species: H. humifusum L., H. annulatum Moris, H. tomentosum L., H. tetrapterum Fries, H. pulchrum L., and H. rumeliacum Boiss. The shoot tips were cryopreserved using a uniform procedure that includes pretreatment with abscisic acid (ABA), PVS3 cryoprotection, and direct immersion into the liquid nitrogen (LN). The freezing-tolerant Hypericum species were pre-exposed to the cold acclimation conditions performed by a 7-day exposure to 4 °C. The content of naphtodianthrones (hypericins) including hypericin, pseudohypericin, and their protoforms was quantified by HPLC. Ploidy of plants was determined by both flow cytometry of leaf tissue and chromosome counts of root tip meristematic cells. We have shown that the post-thaw recovery rate of the shoot tips, pretreated with 0.076 µM ABA for 7 days at room temperature, led to the post-cryogenic survival from 5 % in H. tomentosum to 21 % in H. annulatum. As compared to the untreated (control) plants, the content of hypericins in plants regenerated after cryopreservation remained unchanged or decreased in H. perforatum, H. humifusum, H. annulatum, H. tomentosum, H. tetrapterum, and H. rumeliacum. However, the pre-exposition of the freezing-tolerant H. perforatum to cold acclimation prior to excision of the shoot tips has improved the post-thaw recovery to 45 % and resulted in threefold increase of the total hypericin content.

Local climate explains degree of seed dormancy in Hypericum elodes L. (Hypericaceae).

Seed dormancy and germination characteristics may vary within species in response to several factors. Knowledge of such variation is crucial to understand plant evolution and adaptation to environmental changes. We examined the correlation of climate and population genetic differentiation (ISSR) with primary seed dormancy and germination behaviour in populations of the Atlantic-European soft-water pool specialist Hypericum elodes. Primary dormancy was measured by analysing seed germination response of fresh seeds and after various periods of cold stratification. Laboratory germination experiments revealed that the single most important factor for promoting germination was cold stratification prior to placing at the germination temperature. However, in agreement with their weaker primary dormancy, the seeds germinated well when fresh, and the benefit of cold stratification was more relaxed for the southern populations. Seeds of all populations demonstrated a near absolute requirement for a light and alternating temperature regime in order to germinate. The promoting effect of alternating temperatures was particularly effective at warm temperatures (mean 20 °C) but not at cool temperatures. Whilst seed germination requirements were similar among populations, the degree of primary dormancy varied considerably and was not associated with population genetic differentiation. Primary dormancy degree was instead associated with local climate: higher temperature in summer and rainfall in winter predicted weak and rapid loss of dormancy. These results suggest that seed maturation environment may play a substantial role in explaining the degree of dormancy in H. elodes, highlighting that physiological dormancy can be modulated by local climate.

Oligocene niche shift, Miocene diversification - cold tolerance and accelerated speciation rates in the St. John's Worts (Hypericum, Hypericaceae).

BACKGROUND: Our aim is to understand the evolution of species-rich plant groups that shifted from tropical into cold/temperate biomes. It is well known that climate affects evolutionary processes, such as how fast species diversify, species range shifts, and species distributions. Many plant lineages may have gone extinct in the Northern Hemisphere due to Late Eocene climate cooling, while some tropical lineages may have adapted to temperate conditions and radiated; the hyper-diverse and geographically widespread genus Hypericum is one of these. RESULTS: To investigate the effect of macroecological niche shifts on evolutionary success we combine historical biogeography with analyses of diversification dynamics and climatic niche shifts in a phylogenetic framework. Hypericum evolved cold tolerance c. 30 million years ago, and successfully colonized all ice-free continents, where today ~500 species exist. The other members of Hypericaceae stayed in their tropical habitats and evolved into ~120 species. We identified a 15-20 million year lag between the initial change in temperature preference in Hypericum and subsequent diversification rate shifts in the Miocene. CONCLUSIONS: Contrary to the dramatic niche shift early in the evolution of Hypericum most extant species occur in temperate climates including high elevations in the tropics. These cold/temperate niches are a distinctive characteristic of Hypericum. We conclude that the initial release from an evolutionary constraint (from tropical to temperate climates) is an important novelty in Hypericum. However, the initial shift in the adaptive landscape into colder climates appears to be a precondition, and may not be directly related to increased diversification rates. Instead, subsequent events of mountain formation and further climate cooling may better explain distribution patterns and species-richness in Hypericum. These findings exemplify important macroevolutionary patterns of plant diversification during large-scale global climate change.

Relation between frost tolerance and post-cryogenic recovery in Hypericum spp.

BACKGROUND: The species of the Hypericum genus are markedly variable in morphological, physiological and biochemical traits. They significantly differ in their area of distribution, which may determine their natural tolerance to environmental conditions, such as temperature extremes. OBJECTIVE: To test the hypothesis that the species growing worldwide in different regions and altitudes would be better able to withstand cryopreservation than the endemics. METHODS: The frost tolerance of 10 selected Hypericum species was evaluated. A possible stimulatory effect of cold-acclimation and vitrification-associated stressors on the content of hypericins was also investigated RESULTS: We found that frost tolerance of 10 selected Hypericum species expressed by LT50 ranged between -11 degree C for the species occurring worldwide and -4 degree C for sub/tropical frost sensitive taxons which corresponded with their natural habitats. CONCLUSIONS: Although the mean recoveries for all species cryopreserved with the same vitrification procedure did not exceed 30%, the effect of genetic predisposition to cold tolerance should be considered for optimisation of cryopreservation protocol. Our data neither proved an elicitation effect of cold on hypericin biosynthesis, nor correlation between hypericin content and quantitative characteristics of the hypericin-accumulating black nodules.

Biogeographic variation in genetic variability, apomixis expression and ploidy of St. John's wort (Hypericum perforatum) across its native and introduced range.

BACKGROUND AND AIMS: St. John's wort (Hypericum perforatum) is becoming an important model plant system for investigations into ecology, reproductive biology and pharmacology. This study investigates biogeographic variation for population genetic structure and reproduction in its ancestral (European) and introduced (North America) ranges. METHODS: Over 2000 individuals from 43 localities were analysed for ploidy, microsatellite variation (19 loci) and reproduction (flow cytometric seed screen). Most individuals were tetraploid (93%), while lower frequencies of hexaploid (6%), diploid (<1%) and triploid (<1%) individuals were also identified. KEY RESULTS: A flow cytometric analysis of 24 single seeds per individual, and five individuals per population demonstrated opposite patterns between ploidy types, with tetraploids producing more apomictic (73%) than sexual (24%) seed, while hexaploids produced more sexual (73%) than apomictic (23%) seed. As hexaploids are derived from tetraploids, these data imply that gene dosage, in addition to the effects of hybridization, influences the switch from apomictic to sexual reproduction. No significant differences in seed production were found between Europe and North America. An analysis of population structure based upon microsatellite profiling demonstrated three major genetic clusters in Europe, whose distribution was reflective of Pleistocene glaciation (e.g. refugia) and post-glacial recolonization of Europe. CONCLUSIONS: The presence of pure and mixed populations representing all three genetic clusters in North America demonstrates that H. perforatum was introduced multiple times onto the continent, followed by gene flow between the different gene pools. Taken together, the data presented here suggest that plasticity in reproduction has no influence on the invasive potential of H. perforatum.

Oxidative stress and antioxidant response in Hypericum perforatum L. plants subjected to low temperature treatment.

Extreme low temperatures cause plants multiple stresses, among which oxidative stress is presumed to be the major component affecting the resultant recovery rate. Plants of Hypericum perforatum L., which are known especially for the photodynamic activities of hypericins capable of producing reactive oxygen species under exposure to visible light, were observed to display a substantial increase and persistence in active oxygen production at least two months after recovery from cryogenic treatment. In an effort to uncover the causative mechanism, the individual contributions of wounding during explant isolation, dehydration and cold were examined by means of antioxidant profiling. The investigation revealed activation of genes coding for enzymatic antioxidant catalase and superoxide dismutase at both the transcript and protein levels. Interestingly, plants responded more to wounding than to either low-temperature associated stressor, presumably due to tissue damage. Furthermore, superoxide dismutase zymograms showed the Cu/Zn isoforms as the most responsive, directing the ROS production particularly to chloroplasts. Transmission electron microscopy revealed chloroplasts as damaged structures with substantial thylakoid ruptures.

[Study on the growth, development and artificial propagation of Hypericum ascyron].

OBJECTIVE: To explore the morphological changes, growth conditions and artificial propagation of Hypericum ascyron. METHODS: The morphological changes were observed and recorded in the scene, the height and diameter of the plants were measured; the growth Verhaulst model was set up with the SPSS 17.0 software; the sexual reproduction and asexual reproduction were carried out in artificial cultivation. RESULTS: Hypericum ascyron started germinating in late April each year, branching in late May, flowering in late June, the period of full bearing was in early August, seeds were mature in early October. The Verhaulst models of the increase in the height (H), the quantity of leaf pairs (L) and the branching (B) were, H = 127.109/(1 + 23.744 x e(-0.062t)), L = 23.343/(1 + 11.303 x e(-0.062t)), B = 22.037/(1 + 73.068 x e(-0.068t)). The survival rate of whole graft and segmentation plant were 100% and 67.2% respectively on asexual reproduction; on the sexual reproduction, the seed germination rate was 15.2%, the survival rate of transplant seedlings was 36%. CONCLUSIONS: The period of growth and development of Hypericum ascyron is from April to October and it can be carried out artificial propagation.

Differences among Epilobium and Hypericum species revealed by four IR spectroscopy modes: transmission, KBr tablet, diffuse reflectance and ATR.

INTRODUCTION: Quality control of dried plant material in assessments of suitability of herbal medicinal products is of extreme importance. Commonly used procedures for identification of species are time consuming and expensive. The development of multivariate statistical methods has enabled application of vibrational spectroscopy for establishing plant species membership. OBJECTIVE: To determine which infrared spectroscopy mode gives most informative spectra for plant species identification. METHODOLOGY: Different modes of infrared spectroscopy were applied for investigation of differences among Epilobium and Hypericum species: diffuse reflectance (DR), attenuated total reflectance (ATR), direct transmission of whole leaves and KBr tablet transmission with comminuted leaves. The same chemometrical methods were applied to all spectra. The informative wave numbers were chosen by one-way analysis of variance. Afterwards the colinearity was reduced with principal component analysis. In the final step the species identification was determined with discriminant analysis. RESULTS: Transmission and diffuse reflectance mode did not give satisfactory results. Best results for discrimination among Epilobium species were obtained with ATR mode (98%), while best results for Hypericum species were obtained with KBr transmission mode (97%). This might be explained by the morphological properties of the two genera. Epilobium species differ in distribution and morphology of trichomes on the surface of the leaves. Hypericum species differ in structures and secondary metabolites in the interior of the leaves. CONCLUSION: For best results in using infrared spectroscopy for plant species identification in quality control, the morphological properties of plant material should be taken into consideration.

The cytohistological basis of apospory in Hypericum perforatum L.

St. John's wort (Hypericum perforatum L., 2n = 4x = 32) is a medicinal plant that produces pharmaceutically important metabolites with antidepressive, anticancer and antiviral activities. It is also regarded as a serious weed in many countries. H. perforatum is furthermore an attractive model system for the study of apomixis. Natural populations of H. perforatum are predominantly composed of tetraploid individuals, although diploids and hexaploids are known to occur. It has been demonstrated that while diploids are sexual, polyploids are facultative apomictic whereby a single individual can produce both sexual and apomictic seeds. Despite our increasing understanding of gamete formation in sexually reproducing species, relatively little is known regarding the cytological basis of reproduction in H. perforatum. Here, we have studied embryo sac formation and the genetic constitution of seeds by means of staining-clearing of ovules/ovaries, DIC microscopy and flow cytometric seed screening (FCSS) of embryo and endosperm DNA contents. Comparisons of female sporogenesis and gametogenesis between sexual and apomictic accessions have enabled the identification of major phenotypic differences in embryo sac formation, in addition to complex fertilization scenarios entailing reduced and unreduced male and female gametes. These data provide new insights into the production of aposporous seeds in H. perforatum, and complement ongoing population genetic, genomic and transcriptomic studies.

Identification and genetic analysis of the APOSPORY locus in Hypericum perforatum L.

The introduction of apomixis - seed formation without fertilization - into crop plants is a long-held goal of breeding research, since it would allow for the ready fixation of heterozygosity. The genetic basis of apomixis, whether of the aposporous or the diplosporous type, is still only poorly understood. Hypericum perforatum (St John's wort), a plant with a small genome and a short generation time, can be aposporous and/or parthenogenetic, and so represents an interesting model dicot for apomixis research. Here we describe a genetic analysis which first defined and then isolated a locus (designated HAPPY for Hypericum APOSPORY) associated with apospory. Amplified fragment length polymorphism (AFLP) profiling was used to generate a cleaved amplified polymorphic sequence (CAPS) marker for HAPPY which co-segregated with apospory but not with parthenogenesis, showing that these two components of apomixis are independently controlled. Apospory was inherited as a dominant simplex gene at the tetraploid level. Part of the HAPPY sequence is homologous to the Arabidopsis thaliana gene ARI7 encoding the ring finger protein ARIADNE7. This protein is predicted to be involved in various regulatory processes, including ubiquitin-mediated protein degradation. While the aposporous and sexual alleles of the HAPPY component HpARI were co-expressed in many parts of the plant, the gene product of the apomict's allele is truncated. Cloning HpARI represents the first step towards the full characterization of HAPPY and the elucidation of the molecular mechanisms underlying apomixis in H. perforatum.

Factors affecting polyphenol biosynthesis in wild and field grown St. John's Wort (Hypericum perforatum L. Hypericaceae/Guttiferae).

The increasing diffusion of herbal products is posing new questions: why are products so often different in their composition and efficacy? Which approach is more suitable to increase the biochemical productivity of medicinal plants with large-scale, low-cost solutions? Can the phytochemical profile of a medicinal plant be modulated in order to increase the accumulation of its most valuable constituents? Will polyphenol-rich medicinal crops ever be traded as commodities? Providing a proactive answer to such questions is an extremely hard task, due to the large number of variables involved: intraspecific chemodiversity, plant breeding, ontogenetic stage, post-harvest handling, biotic and abiotic factors, to name but a few. An ideal path in this direction should include the definition of optimum pre-harvesting and post-harvesting conditions and the availability of specific Good Agricultural Practices centered on secondary metabolism enhancement. The first steps to be taken are undoubtedly the evaluation and the organization of scattered data regarding the diverse factors involved in the optimization of medicinal plant cultivation, in order to provide an interdisciplinary overview of main possibilities, weaknesses and drawbacks. This review is intended to be a synopsis of the knowledge on this regard focused on Hypericum perforatum L. (Hypericaceae/Guttiferae) secondary metabolites of phenolic origin, with the aim to provide a reference and suggest an evolution towards the maximization of St. John's Wort bioactive constituents. Factors considered emerged not only from in-field agronomic results, but also from physiological, genetical, biotic, abiotic and phytochemical data that could be scaled up to the application level. To increase quality for final beneficiaries, growers' profits and ultimately transform phenolic-rich medicinal crops into commodities, the emerging trend suggests an integrated and synergic approach. Agronomy and genetics will need to develop their breeding strategies taking account of the suggestions of phytochemistry, biochemistry, pharmacognosy and pharmacology, without losing sight of the economic balance of the production.

An Iranian herbal-marine medicine, MS14, ameliorates experimental allergic encephalomyelitis.

Multiple sclerosis is an inflammatory and demyelinating disease of the central nervous system which mainly affects young adults. To overcome wide spectrum troublesome symptoms of multiple sclerosis which affects the quality of life both in patients and their families, new drugs and remedies have been examined and offered. The preclinical beneficial effects of different medicines have mostly been examined in an animal model of multiple sclerosis called experimental allergic encephalomyelitis (EAE). In this study we have tested a traditionally used natural (herbal-marine) product called MS(14) in EAE mice. EAE mice were fed with MS(14) containing diet (30%) on the immunization day and monitored for 20 days. The results show that while clinical scores and therefore severity of the disease was progressive in normal-fed EAE mice, the disease was slowed down in MS(14)-fed EAE mice. Moreover, while there were moderate to severe neuropathological changes in normal fed mice, milder changes were seen in MS(14) fed mice.

Global warming and flowering times in Thoreau's Concord: a community perspective.

As a result of climate change, many plants are now flowering measurably earlier than they did in the past. However, some species' flowering times have changed much more than others. Data at the community level can clarify the variation in flowering responses to climate change. In order to determine how North American species' flowering times respond to climate, we analyzed a series of previously unstudied records of the dates of first flowering for over 500 plant taxa in Concord, Massachusetts, USA. These records began with six years of observations by the famous naturalist Henry David Thoreau from 1852 to 1858, continued with 16 years of observations by the botanist Alfred Hosmer in 1878 and 1888-1902, and concluded with our own observations in 2004, 2005, and 2006. From 1852 through 2006, Concord warmed by 2.4 degrees C due to global climate change and urbanization. Using a subset of 43 common species, we determined that plants are now flowering seven days earlier on average than they did in Thoreau's times. Plant flowering times were most correlated with mean temperatures in the one or two months just before flowering and were also correlated with January temperatures. Summer-flowering species showed more interannual variation in flowering time than did spring-flowering species, but the flowering times of spring-flowering species correlated more strongly to mean monthly temperatures. In many cases, such as within the genera Betula and Solidago, closely related, co-occurring species responded to climate very differently from one another. The differences in flowering responses to warming could affect relationships in plant communities as warming continues. Common St. John's wort (Hypericum perforatum) and highbush blueberry (Vaccinium corymbosum) are particularly responsive to changes in climate, are common across much of the United States, and could serve as indicators of biological responses to climate change. We discuss the need for researchers to be aware, when using data sets involving multiple observers, of how varying methodologies, sample sizes, and sampling intensities affect the results. Finally, we emphasize the importance of using historical observations, like those of Thoreau and Hosmer, as sources of long-term data and to increase public awareness of biological responses to climate change.