Impact of pre-culture on short- and long-term in vitro recovery of the biosynthetic potential and enzymatic and non-enzymatic antioxidant defense of Hypericum rumeliacum Boiss. after cryostorage

Due to its high hypericin and pseudohypericin in vitro biosynthetic capacity, the Balkan endemic Hypericum rumeliacum was selected as a prospective candidate for long-term preservation of valuable medicinal plant germplasm. Initial cryopreservation experiments were previously conducted based on the successful protocol established and reported for the widely studied H. perforatum. This is the first report on the impact of pre-culture duration on the short- and long-term in vitro recovery of the biosynthetic potential and antioxidant defense system of H. rumeliacum cryopreserved by vitrification. Cryopreservation did not impair the phenolics and flavonoids production of the regenerated plants. Moreover, hypericin and pseudohypericin levels even increased substantially in one of the regenerated lines, reaching yields from 0.107 and 0.752?mg?g^?1?DW in the control up to 0.277 and 1.112?mg?g^?1?DW for hypericin and pseudohypericin, respectively. However, the physical injury stress of the pre-culture treatment manipulations affected the physiological status of regenerants in a time dependent manner. Within 6?months after thawing, regenerants with the highest oxidative stress after pre-culture, were characterized with an augmentation of antioxidant metabolites such as phenolics, flavonoids, glutathione and ascorbic acid as well as increased antioxidant enzymatic activities in comparison with both the non-frozen control and the regenerants with the lowest pre-culture oxidative stress. Then, after 18?months of recovery, the same first H. rumeliacum group displayed a marked drop of enzymatic antioxidant activity as compared with the other groups of plants. Further research is needed to target oxidative stress alleviation to optimize H. rumeliacum cryopreservation protocol.     

Sympatric population divergence within a highly pelagic seabird species complex (Hydrobates spp.)

Both physical and non‐physical barriers can restrict gene flow among seabird populations. Understanding the relative importance of non‐physical barriers, such as breeding phenology, is key to understanding seabird biodiversity. We investigated drivers of diversification in the Leach's storm‐petrel species complex (Hydrobates spp.) by examining population genetic structure across its range. Variation in the mitochondrial control region and six microsatellite loci was assayed in birds sampled from breeding colonies throughout the North Atlantic and North Pacific (H. leucorhoa leucorhoa), as well as from San Benito Islands (H. l. chapmani), and two seasonal populations in Guadalupe (summer breeding H. socorroensis and winter breeding H. cheimomnestes), Mexico. Weak but significant differentiation was found between populations of H. l. leucorhoa breeding in the Atlantic versus North Pacific, as well as between H. l. chapmani and H. l. leucorhoa, and between H. socorroensis and H. cheimomnestes within Guadalupe. In contrast, strong differentiation in both mitochondrial DNA and microsatellites was found between H. leucorhoa and both H. socorroensis and H. cheimomnestes. Phylogenetic reconstruction suggested the Guadalupe seasonal breeding populations are sister taxa, at least in their mitochondrial DNA. Non‐physical barriers to gene flow appear to be more important than physical barriers in driving divergence within the Leach's storm‐petrel species complex. In particular, allochronic speciation may have occurred between the seasonal populations within Guadalupe. Further work should include higher resolution sequencing to confirm results, and an increased sampling effort, particularly within the California area, to fully resolve the relationship between H. l. leucorhoa and H. l. chapmani.     

Preferential Secretion of Thymic Stromal Lymphopoietin (TSLP) by Terminally Differentiated Esophageal Epithelial Cells: Relevance to Eosinophilic Esophagitis (EoE)

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (<15 eos/hpf) and non-EoE control subjects, and found that TSLP expression was restricted to the differentiated suprabasal layer of the epithelium in actively inflamed EoE biopsies. Consistent with these results in vivo, inducible TSLP protein secretion was higher in CaCl₂ differentiated telomerase-immortalized esophageal epithelial cells (EPC2-hTERT) compared to undifferentiated cells of the basal phenotype, following stimulation with the TLR3 ligand poly(I:C). To determine whether food antigens could directly induce epithelial TSLP secretion, differentiated and undifferentiated primary esophageal epithelial cells from EoE and non-EoE subjects were challenged with food antigens clinically relevant to EoE: Chicken egg ovalbumin (OVA), wheat, and milk proteins beta-lactoglobulin (blg) and beta-casein. Food antigens failed to induce TSLP secretion by undifferentiated cells; in contrast, only OVA induced TSLP secretion in differentiated epithelial cells from both EoE and control cell lines, an effect abolished by budesonide and NF-κb inhibition. Together, our study shows that specific food antigens can trigger innate immune mediated esophageal TSLP secretion, suggesting that esophageal epithelial cells at the barrier surface may play a significant role in the pathogenesis of EoE by regulating TSLP expression.     

The interaction of human neuroglobin with hydrogen sulphide

Neuroglobin has been identified to protect brain neurons from apoptotic stress. Hydrogen sulphide has a role in the brain as a neuromodulator, involving NMDA receptor activation. Here we report on studies of the in vitro interaction of ferric neuroglobin with hydrogen sulphide. Hydrogen sulphide binds very tightly to the heme group of neuroglobin in a biphasic reaction. The faster of the two reaction processes is concentration dependent whilst the slower process is not. The rate of hydrogen sulphide binding is pH sensitive and as the pH is reduced over the physiological range the rate of reaction increases by a factor of approximately 10. This change in reactivity appears to reflect the ionisation of the heme distal His ligand rather than a preference for the binding of H(2)S. We discuss the potential role of neuroglobin in the modulation of hydrogen sulphide sensitivity of neurons in the brain.     

Early seed fall and seedling emergence: precursors to tropical restoration

We explore processes of seed immigration and seedling recruitment before an experimental rainforest restoration matures enough to affect either. Twenty-four 30 × 30-m plots were fenced in 12 ha of pasture in 2006. Seeds were collected in ninety-six 1-m⁻² seed traps; recruits were censused in ~12,000 m² in which establishment was allowed. We tested effects of distance from forest, living trees, and stumps of trees cut during site preparation on seed rain in 2007 and effects of these and soil depth on recruits through June 2008. Seed fall and recruitment were not correlated with distance to forest 90–400 m away, nor to living shade trees outside the 160 × 485-m experimental grid. Recruitment differed for animal- and wind-dispersed species in a topographically complex landscape. Recruitment of wind-dispersed species was random with respect to soil depth or distance to recent stumps. Recruitment of animal-dispersed species was multimodal; partial correlations with number of stumps within 30 m of plots were significant with soil depth held constant (P < 0.025), as were correlations of recruitment with soil depth with number of stumps held constant (P < 0.01). Animal-dispersed recruits were often not conspecifics of adults that had been cut, indicating a legacy of attraction by fruiting trees of animals bearing seeds from distant sources. Ecological implications are that recruitment in pastures released from grazing reflects a mix of widely scattered wind-dispersed pioneers and, where animal-dispersed trees exist, multi-modal and decidedly non-random recruitment of pioneer and later successional animal-dispersed trees from seed banks.     

Phenoloxidase activity and thermostability of Cancer pagurus and Limulus polyphemus hemocyanin

The intrinsic and inducible o-diphenoloxidase (o-diPO) activity of Cancer pagurus hemocyanin (CpH) and Limulus polyphemus hemocyanin (LpH) were studied using catechol, l-Dopa and dopamine as substrates. The kinetic analysis shows that dopamine is a more specific substrate for CpH than catechol and l-Dopa (K_m value of 0.01 mM for dopamine versus 0.67 mM for catechol, and 2.14 mM for l-Dopa), while k_cat is highest for catechol (2.44 min^? 1 versus 0.67 min^? 1 for l-Dopa and 0.71 min^? 1 for dopamine). On treatment with 4 mM sodium dodecyl sulfate (SDS) or by proteolysis the o-diPO activity of CpH increases about twofold. In contrast, native LpH shows no o-diPO activity, and exhibits only a slight activity after incubation with SDS. Neither CpH nor LpH show intrinsic mono-PO activity with l-tyrosine and tyramine as substrates. To explore the possible correlation between the degree of PO activity and protein stability of arthropod hemocyanins, the thermal stability of CpH and LpH was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy. CpH is found to be less thermostable (T_m ~ 80 °C), suggesting that the dicopper active sites are more accessible, thereby allowing the hemocyanin to show PO activity in the native state. The LpH, on the other hand, is more thermostable (T_m ~ 92 °C), suggesting the existence of a correlation between the thermal stability and the intrinsic PO activity of arthropod hemocyanins.     

Rhinovirus and dsRNA Induce RIG-I-Like Receptors and Expression of Interferon β and λ1 in Human Bronchial Smooth Muscle Cells

Rhinovirus (RV) infections cause exacerbations and development of severe asthma highlighting the importance of antiviral interferon (IFN) defence by airway cells. Little is known about bronchial smooth muscle cell (BSMC) production of IFNs and whether BSMCs have dsRNA-sensing receptors besides TLR3. dsRNA is a rhinoviral replication intermediate and necrotic cell effect mimic that mediates innate immune responses in bronchial epithelial cells. We have explored dsRNA-evoked IFN-β and IFN-λ₁ production in human BSMCs and potential involvement of TLR3 and RIG-I-like receptors (RLRs). Primary BSMCs were stimulated with 0.1–10 µg/ml dsRNA, 0.1–1 µg/ml dsRNA in complex with the transfection agent LyoVec (dsRNA/LyoVec; selectively activating cytosolic RLRs) or infected with 0.05–0.5 MOI RV1B. Both dsRNA stimuli evoked early (3 h), concentration-dependent IFN-β and IFN-λ₁ mRNA expression, which with dsRNA/LyoVec was much greater, and with dsRNA was much less, after 24 h. The effects were inhibited by dexamethasone. Further, dsRNA and dsRNA/LyoVec concentration-dependently upregulated RIG-I and MDA5 mRNA and protein. dsRNA and particularly dsRNA/LyoVec caused IFN-β and IFN-λ₁ protein production (24 h). dsRNA- but not dsRNA/LyoVec-induced IFN expression was partly inhibited by chloroquine that suppresses endosomal TLR3 activation. RV1B dose-dependently increased BSMC expression of RIG-I, MDA5, IFN-β, and IFN-λ₁ mRNA. We suggest that BSMCs express functional RLRs and that both RLRs and TLR3 are involved in viral stimulus-induced BSMC expression of IFN-β and IFN-λ₁.     

Planarian MBD2/3 is required for adult stem cell pluripotency independently of DNA methylation

Planarian adult stem cells (pASCs) or neoblasts represent an ideal system to study the evolution of stem cells and pluripotency as they underpin an unrivaled capacity for regeneration. We wish to understand the control of differentiation and pluripotency in pASCs and to understand how conserved, convergent or divergent these mechanisms are across the Bilateria. Here we show the planarian methyl-CpG Binding Domain 2/3 (mbd2/3) gene is required for pASC differentiation during regeneration and tissue homeostasis. The genome does not have detectable levels of 5-methylcytosine (5^mC) and we find no role for a potential DNA methylase. We conclude that MBD proteins may have had an ancient role in broadly controlling animal stem cell pluripotency, but that DNA methylation is not involved in planarian stem cell differentiation.     

Effects of light intensity and dilution rate on the semicontinuous cultivation of Arthrospira (Spirulina) platensis. A kinetic Monod-type approach

Semicontinuous cultures were carried out at different dilution rates (D) and light intensities (I) to determine the maximum productivity of Arthrospira platensis cultivated in helicoidal photobioreactor up to the achievement of pseudo-steady-state conditions. At I=108 μmol photons m(-2) s(-1), the semicontinuous regime ensured the highest values of maximum cell concentration (X(m)=5772±113 mg L(-1)) and productivity (P(XS)=1319±25 mg L(-1) d(-1)) at the lowest (D=0.1 day(-1)) and the highest (D=0.3 day(-1)) dilution rates, respectively. A kinetic model derived from that of Monod was proposed to determine the relationship between the product of light intensity to dilution rate (ID) and the cell productivity, which were shown to exert a combined influence on this parameter. This result put into evidence that pseudo-steady-state conditions could be modified according to circumstances, conveniently varying one or other of the two independent variables.