Antiproliferative and immunomodulative potential of Citrullus colocynthis and its bioactive compounds in human lymphocytes and lung cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Citrullus colocynthis (L.) Schrad is a member of the Cucurbitaceae plant family which has been used in traditional medicine for the treatment of lung diseases such as asthma and bronchitis. AIM OF THE STUDY: The study was conducted to investigate antiproliferative and immunomodulating effects of C. colocynthis and isolated cucurbitacins on human T lymphocytes and lung epithelial cells in order to evaluate their potential in the treatment of airway diseases. MATERIALS AND METHODS: Different concentrations of an ethanolic extract of C. colocynthis fruits and cucurbitacins B (CuB), E (CuE) and E-glucopyranoside (CuE-Glu) were analysed for their cytotoxicity and immunomodulatory potential on Peripheral Blood Mononuclear Cells (PBMCs) of healthy donors and on the epithelial lung cancer cell line A549. Viability and proliferation were tested using WST1 and CFSE assays. Flow cytometric analysis of AnnexinV/PI staining was used to investigate cell death through apoptosis/necrosis. Effects on regulatory mechanisms of T lymphocytes, such as CD69 and CD25 marker activation, cytokine production of the cytokines interleukin 2 (IL2), tumor necrosis factor α (TNFα) and interferon γ (IFNy) were also analysed via flow cytometry. Influences on the activator protein 1 (AP1), nuclear factor of activated T-cells (NFAT) or nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NFκB) pathways were analysed in the Jurkat reporter cell line. Cytokine secretion in A549 cells stimulated with virus-like particles was analysed using the bead-based Legendplex™ assay. RESULTS: Non-toxic concentrations of C. colocynthis and CuE-Glu showed dose-dependent effects on viability and proliferation in both T lymphocytes and A549 cells. The extracts inhibited lymphocyte activation and suppressed T cell effector functions, which was also shown by lower production of cytokines IL2, TNFα and IFNy. A dose dependent inhibition of the pathways NFκB, NFAT and AP1 in Jurkat cells could be observed. In A549 cells, especially CuE and CuE-Glu showed inhibitory effects on cytokine production following a simulated viral infection. Unglycosylated cucurbitacins were more effective in suppressing the immune function in lymphocytes than glycosylated cucurbitacins, however this activity is limited to cytotoxic concentrations. CONCLUSION: In our study we could confirm the immunmodulating effect of C. colocynthis and cucurbitacins B, E and E-glucopyranoside in vitro by suppression of different pathways of inflammation and T cell proliferation. Activity in a lung cell model using a virus-like stimulation shows promise for further research regarding cucurbitacins in airway diseases.

Bowel health, defecation patterns and nutrient intake following adoption of a vegan diet: a randomized-controlled trial.

BACKGROUND: The beneficial effects of a plant-based diet on gut microbiota diversity are well documented, however, its impact on clinical bowel health and defecation patterns are less well understood. Vegetarian diets have been associated with a higher bowel movement (BM) frequency as well as softer stools in cross-sectional studies. The effects of the de-novo adoption of a vegan diet on bowel health, however, have never been investigated in a randomized-controlled trial. MATERIALS AND METHODS: The present study examined bowel health and defecation patterns in relation to diet and nutrient intake in a young and healthy sample of n = 65 physically-active German university students who were randomly assigned to either a vegan or a meat-rich diet for eight weeks. Bowel health assessment included the Bristol Stool Form Scale (BSFS), the Gastrointestinal Quality of Life Index (GIQLI) and the Cleveland Clinic Fecal Incontinence Score (CCFIS). Nutrient intake was assessed using weighed food diaries. The study was prospectively registered at the German Clinical Trial Register (DRKS00031541). RESULTS: Weekly BM frequency slightly increased in vegans, whereas it remained unaltered in participants assigned to a meat-rich diet. Fiber intake increased significantly in vegans (34.89 (18.46) g/d) whereas it decreased in those assigned to the meat-rich group (22.79 (12.5) g/d). No significant intergroup differences in BSFS and CCFIS patterns were observed. Adoption of a vegan diet neither resulted in a transient increase in abdominal discomfort nor in a decreased gastrointestinal quality of life, which was comparable across the diet groups. CONCLUSIONS: The short-term de-novo adoption of a vegan diet did not negatively affect markers of bowel health in this study.

A Physcomitrella PIN protein acts in spermatogenesis and sporophyte retention.

The auxin efflux PIN-FORMED (PIN) proteins are conserved in all land plants and important players in plant development. In the moss Physcomitrella (Physcomitrium patens), three canonical PINs (PpPINA-C) are expressed in the leafy shoot (gametophore). PpPINA and PpPINB show functional activity in vegetative growth and sporophyte development. Here, we examined the role of PpPINC in the life cycle of Physcomitrella. We established reporter and knockout lines for PpPINC and analysed vegetative and reproductive tissues using microscopy and transcriptomic sequencing of moss gametangia. PpPINC is expressed in immature leaves, mature gametangia and during sporophyte development. The sperm cells (spermatozoids) of pinC knockout mutants exhibit increased motility and an altered flagella phenotype. Furthermore, the pinC mutants have a higher portion of differentially expressed genes related to spermatogenesis, increased fertility and an increased abortion rate of premeiotic sporophytes. Here, we show that PpPINC is important for spermatogenesis and sporophyte retention. We propose an evolutionary conserved way of polar growth during early moss embryo development and sporophyte attachment to the gametophore while suggesting the mechanical function in sporophyte retention of a ring structure, the Lorch ring.

Axenic in vitro cultivation of 19 peat moss (Sphagnum L.) species as a resource for basic biology, biotechnology, and paludiculture.

Sphagnum farming can substitute peat with renewable biomass and thus help mitigate climate change. Large volumes of the required founder material can only be supplied sustainably by axenic cultivation in bioreactors. We established axenic in vitro cultures from sporophytes of 19 Sphagnum species collected in Austria, Germany, Latvia, the Netherlands, Russia, and Sweden: S. angustifolium, S. balticum, S. capillifolium, S. centrale, S. compactum, S. cuspidatum, S. fallax, S. fimbriatum, S. fuscum, S. lindbergii, S. medium/divinum, S. palustre, S. papillosum, S. rubellum, S. russowii, S. squarrosum, S. subnitens, S. subfulvum and S. warnstorfii. These species cover five of the six European Sphagnum subgenera; namely, Acutifolia, Cuspidata, Rigida, Sphagnum and Squarrosa. Their growth was measured in suspension cultures, whereas their ploidy was determined by flow cytometry and compared with the genome size of Physcomitrella patens. We identified haploid and diploid Sphagnum species, found that their cells are predominantly arrested in the G1 phase of the cell cycle, and did not find a correlation between plant productivity and ploidy. DNA barcoding was achieved by sequencing introns of the BRK1 genes. With this collection, high-quality founder material for diverse large-scale applications, but also for basic Sphagnum research, is available from the International Moss Stock Center.

Chloroplasts require glutathione reductase to balance reactive oxygen species and maintain efficient photosynthesis.

Thiol-based redox-regulation is vital for coordinating chloroplast functions depending on illumination and has been throroughly investigated for thioredoxin-dependent processes. In parallel, glutathione reductase (GR) maintains a highly reduced glutathione pool, enabling glutathione-mediated redox buffering. Yet, how the redox cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and detoxification of reactive oxygen species remains largely unresolved because null mutants of plastid/mitochondrial GR are embryo-lethal in Arabidopsis thaliana. To investigate whether maintaining a highly reducing stromal glutathione redox potential (EGSH ) via GR is necessary for functional photosynthesis and plant growth, we created knockout lines of the homologous enzyme in the model moss Physcomitrella patens. In these viable mutant lines, we found decreasing photosynthetic performance and plant growth with increasing light intensities, whereas ascorbate and zeaxanthin/antheraxanthin levels were elevated. By in vivo monitoring stromal EGSH dynamics, we show that stromal EGSH is highly reducing in wild-type and clearly responsive to light, whereas an absence of GR leads to a partial glutathione oxidation, which is not rescued by light. By metabolic labelling, we reveal changing protein abundances in the GR knockout plants, pinpointing the adjustment of chloroplast proteostasis and the induction of plastid protein repair and degradation machineries. Our results indicate that the plastid thioredoxin system is not a functional backup for the plastid glutathione redox systems, whereas GR plays a critical role in maintaining efficient photosynthesis.

Clonal in vitro propagation of peat mosses (Sphagnum L.) as novel green resources for basic and applied research.

As builders and major components of peatlands, Sphagnopsida (peat mosses) are very important organisms for ecosystems and world's climate. Nowadays many Sphagnum species as well as their habitats are largely protected, while their scientific and economic relevance remains considerable. Advanced methods of in vitro cultivation provide the potential to work in a sustainable way with peat mosses and address aspects of basic research as well as biotechnological and economical topics like biomonitoring or the production of renewable substrates for horticulture (Sphagnum farming). Here, we describe the establishment of axenic in vitro cultures of the five peat moss species Sphagnum fimbriatum Wils. and Hook., Sphagnum magellanicum Brid., Sphagnum palustre L., Sphagnum rubellum Wils. and Sphagnum subnitens Russ. and Warnst. with specific focus on large-scale cultivation of S. palustre in bioreactors. Axenic, clonal cultures were established to produce high quantities of biomass under standardized laboratory conditions. For advanced production of S.palustre we tested different cultivation techniques, growth media and inocula, and analyzed the effects of tissue disruption. While cultivation on solid medium is suitable for long term storage, submerse cultivation in liquid medium yielded highest amounts of biomass. By addition of sucrose and ammonium nitrate we were able to increase the biomass by around 10- to 30-fold within 4 weeks. The morphology of in vitro-cultivated gametophores showed similar phenotypic characteristics compared to material from the field. Thus the tested culture techniques are suitable to produce S. palustre material for basic and applied research.