Protozoa populations are ecosystem engineers that shape prokaryotic community structure and function of the rumen microbial ecosystem

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community—either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.

Insights into in vivo follicle formation: a review of in vitro systems

In vitro systems capable of reconstituting the process of mouse oogenesis are now being established to help develop further understanding of the mechanisms underlying oocyte/follicle development and differentiation. These systems could also help increase the production of useful livestock or genetically modified animals, and aid in identifying the causes of infertility in humans. Recently, we revealed, using an in vitro system for recapitulating oogenesis, that the activation of the estrogen signaling pathway induces abnormal follicle formation, that blocking estrogen-induced expression of anti-Müllerian hormone is crucial for normal follicle formation, and that the production of α-fetoprotein in fetal liver tissue is involved in normal in vivo follicle formation. In mouse fetuses, follicle formation is not carried out by factors within the ovaries but is instead orchestrated by distal endocrine factors. This review outlines findings from genetics, endocrinology, and in vitro studies regarding the factors that can affect the formation of primordial follicles in mammals.

Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

The benzene impact on programmed death of sperm cell

Introduction. Benzene and its derivatives enter organism from the natural environment are the sources of constant health danger, including reproductive system disorders. Purpose of the study. In vitro study of the modifying effect of benzene on the indices controlling the apoptosis of sperm cells. Materials and methods. The study includes data on 27 men from 41 to 51 years of age, apparently healthy, not in contact with harmful factors, and living in relatively favourable conditions. Using flow cytometry in the ejaculated semen, we determined CD25+ and CD95+ markers, p53, bax, caspase-3 activity, percentage of AnnexinV-FITC+PI-, and AnnexinV-FITC+PI+-spermatozoa. The seminal fluid samples were incubated with benzene at a concentration of 0.001 µg/ml (experimental samples) for 72 hours at 370C to evaluate the effect of aromatic hydrocarbons on the indices controlling the apoptosis. Sperm samples incubated under identical conditions without benzene addition were used as control samples. Results. p53 content, caspase-3 activity, and the number of dead male germ cells (AnnexinV-FITC+PI+-spermatozoa) were found to decrease statistically significantly (p=0.023-0.026) in experimental semen samples after the addition of benzene (by 50% of the initial levelon average). At the same time, the addition of benzene to the ejaculated semen samples did not change the early activation profile of gametes (according to CD25+ criteria), cells readiness to start FAS-dependent apoptosis (CD95+), and the number of spermatozoa marked for death by apoptosis (AnnexinV-FITC+PI- - spermatozoa). Conclusion. The results of immunological testing demonstrated that in vitro system benzene inhibits apoptosis and interferes with gamete life cycle . On the example of benzene, it was been demonstrated that haptenic contamination could alter sperm fertility associated with an imbalance of proapoptotic factors and impair ed male reproductive system function. The indices of programmed cell death bax, p53, caspase-3, CD25-positive, FAS-positive, AnnexinV-FITC+PI--sperm, and AnnexinV-FITC+PI+-sperm in the ejaculate are recommended for diagnosis and monitoring of sperm fertility disorders.

An in vitro co-culture system for rapid differential response to Fusarium oxysporum f. sp. vasinfectum race 4 in three cotton cultivars

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a devastating fungus pathogen that causes Fusarium wilt in both domesticated cotton species, Gossypium hirsutum (Upland) and G. barbadense (Pima). Greenhouse and field-based pathogenicity assays can be a challenge due to non-uniform inoculum levels, the presence of endophytes, and varying environmental factors. Therefore, an in vitro co-culture system was designed to support the growth of both domesticated cotton species and FOV4 using an inert polyphenolic foam substrate with a liquid medium. A Fusarium wilt-susceptible Pima cotton cultivar, G. barbadense ‘GB1031’, a highly resistant Pima cotton cultivar, G. barbadense ‘DP348RF’, and a susceptible Upland cotton cultivar, G. hirsutum ‘TM-1’, were evaluated for 30 days during co-culture with FOV4 in this foam-based system. Thirty days after inoculation, disease symptoms were more severe in both the susceptible cultivars, which displayed higher percentages of foliar damage, and greater plant mortality, than observed in ‘DP348RF’, the resistant Pima cotton cultivar. This foam-based in vitro system may be useful for screening cotton germplasm for resistance to a variety of fungus pathogens and to facilitate the study of biotic interactions in domesticated cotton species under controlled environmental conditions.

Tension-driven multi-scale self-organisation in human iPSC-derived muscle fibers

Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. By directly probing tension we found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse followed by a disassembly of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.

IN VITRO EXPERIMENTAL REWIRING OF 4 NEUTROPHILIC GRANULOCYTE SUBSETS FROM THE PRO-INFLAMMATORY TO THE ANTI-INFLAMMATORY PHENOTYPE IN CHILDREN WITH SURGICAL PURULENT INFECTION OF SOFT TISSUE

Treatment of young children with atypical or recurrent purulent soft tissue infections (PSTD) that do not respond well to surgery and antibiotics is most challenging. PSTD occurs against the background of impaired functioning of the immune system and, first of all, the system of neutrophilic granulocytes (NG). The vector effect of immunotropic therapy on a specific NG subsets may allow the correction of NG dysfunctions without compromising host protection, including strategies to enhance, inhibit or restore their functions.The aim of study: to evaluate in vitro the modulating effects of arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine (HP) on the transformed phenotype of 4 NG subsets, as well as on the functional activity of NG in children with purulent-inflammatory soft tissue diseases.We studied samples of peripheral blood (PB) from young children 2-4 years old: 17 children with atypical acute PSTD and 10 apparently healthy children. At stage I, a comparative assessment of the content and phenotype of 4 NG subsets CD16+CD62L+СD63- , CD16+CD62L+СD63+, СD64- CD16+CD32+CD11b+, СD64+CD16+CD32+CD11b+, phagocytic and microbicidal functions of NG was carried out. At stage II, the in vitro system determined the effects of HP on NG in children with PSTD according to the studied parameters. By the method of flow cytometry (FC500 “Beckman Coulter” (USA), conjugates of MkAT “Beckman Coulter International S.A.” (France)), the relative number of NGs of the studied subsets and the density of receptor expression (MFI) were determined. To assess the phagocytic function of NG a microbiological method was used to assess the completeness of phagocytosis with S. aureus (strain 209). The activity of NG NADPH oxidase was investigated in the NBT-spontaneous test (NBTsp.) and in the in vitro NBT-induced test (NBTind.). A comparative study of PB samples from conventionally healthy children and children with PSTD made it possible to identify various variants of transformation of the phenotype of the studied NG subsets, associated with defects in their functional activity. In the in vitro system the effects of HP were demonstrated, manifested by a decrease in the amount of CD16+CD62L+CD63+NG and an increase in CD16+CD62L+CD63- NG, modulation of the negatively altered phenotype of subsets CD64- CD32+CD16+CD11b+NG and CD64+CD32+CD16+CD11b+NG, aimed at restoring phagocytic function and maintaining the tension of NADPH oxidases.As a result of the study it was found the immunomodulatory effects of HP, which is manifested in the reorientation of NG from the pro-inflammatory phenotype to the anti-inflammatory one, which can be used in the future when creating personalized targeted immunotherapy aimed at correcting defective functioning NG in early children, suffering from PSTD.

Is Endogenous Ethylene Production Associated with Increased Shoot Number in Gentian Cultured In Vitro? Developing a Method for Ethylene Measurement at Nano Level within a Gas-exchangeable In Vitro System

We investigated the possibility of either exogenous ethylene or endogenous ethylene production having an association with the increase in shoot number when nodal explants of Gentiana spp. ‘Little Pinkie’ were cultured in an in vitro medium supplemented with ethephon (10 mg⋅L–1). For the first time within an in vitro system, we report the application of laser ethylene detector technology, and optimization of the methodology to quantify concentrations of ethylene (in the part-per-billion range) released from ethephon decomposition within the atmosphere of gas-exchangeable culture vessels including nodal explants. Compared with continuous (continuous measurements on the same replicate of vessels) and repeated (sampling same replicate of vessels every 48 hours) sampling methodologies, the nonrepeated (sampling fresh replicate of vessels every 48 hours) method of measurement of ethylene concentration was more representative of the actual condition within vessels. Although no prior published data exist showing the positive or negative effect of gaseous ethylene in the headspace of culture vessels on bud outgrowth in gentian, our study shows gaseous ethylene in the headspace of culture vessels was not effective in increasing shoot formation in gentian explants cultured in vitro, whereas ethephon supplementation in agar was effective. Plant material in culture vessels did not have a significant effect on ethylene production regardless of the presence or absence of ethephon. Therefore, although ethephon supplementation in the medium produced gaseous ethylene in the headspace, it was unlikely to cause endogenous ethylene production in explants, but it did trigger shoot formation in ‘Little Pinkie’, perhaps through decomposition to ethylene within the explant tissue, enhancing the internal ethylene level possibly at a locally high concentration.

Effects of Different Herbal Teas on Reducing the Bioaccessibility of Methylglyoxal in Crackers under Stimulated Gastrointestinal Digestive System

The present study aims to determine the bioaccessibility of methylglyoxal (MGO) in crackers and investigate the effects of herbal teas on reducing the formation of MGO under in vitro digestion system. Different herbal teas were added in crackers to reduce MGO formation under in vitro system. MGO levels were determined by High-Performance Liquid Chromatography. The amounts of MGO in crackers samples at initial and after in vitro digestion were ranged from 51 to 104 µg/100 g and 274 to 408 µg / 100 g, respectively. After in vitro digestion, the bioaccessibility of MGO values was increased up to 628%. Also, it was found that polyphenol-rich herbal teas such as black, green, turmeric, and rosehip significantly reduced MGO bioaccessibility in crackers. The addition of these herbal teas as antioxidants in cracker formulations or consumption of herbal teas along with snack foods may be recommended.