Helicobacter pylori genotype I is strongly associated with different gastric pathologies and with the development of gastric cancer. There are several virulence factors that act in synergy to damage the gastric epithelium. Recently, the participation of the vapD gene in the persistence of Helicobacter pylori inside the gastric cell has been described. The objective of present study was to determine the VapD protein expression and location of Helicobacter pylori, when the microorganism is inside the eukaryote cell. Our research group had to development the recombinant VapD protein, polyclonal antibodies against VapD, cocultures of Helicobacter pylori and AGS cells and an immunofluorescence assay. Our results show that the VapD protein is expressed when the bacteria are inside the eukaryote cell. Probably maintaining homeostasis in the bacteria to resist the stress exerted by the intracellular environment of the gastric cell.

Biomaterials such as exopolysaccharides have been of great interest for their diverse biological activities in controlling or preventing chronic degenerative diseases, such as cancer. Previously, we isolated four dextrans produced by four strains isolated from Agave salmiana, which were named SF3, SF2, SD1, and SD23. The objective was to evaluate the antitumor activity of these dextrans on prostate (PC3) and colon (SW480) cancer cells. Growth inhibition, morphological changes, mitochondrial metabolism, and cell apoptosis were evaluated by sulforhodamine B, transmission electron microscopy, Seahorse XF and TUNEL assays, respectively. To gene expression was used qPCR and to protein ELISA and immunofluorescence. The cells treated with the dextrans to a dose of 8 mg/mL presented an inhibition of cell growth. Studies of the metabolism cell indicated a disruption in mitochondrial function and a diminished ability of the cells to respond to energy demands through glycolysis. These changes indicate mitochondrial damage resulting in oxidative stress or metabolic alterations. Survivin gen decreased and caspase-3 and -8 increased, key regulators of the apoptotic response with treatment. Moreover, the TUNEL assays indicated cell apoptosis. In conclusion, our findings suggest that dextrans could be considered as potential compounds for cancer treatment.

Due to the distinctive characteristics of probiotics, it is essential to pinpoint strains originating from diverse sources that prove efficacious in addressing a range of pathologies linked to dysfunction of the intestinal barrier. Nine strains of lactic acid bacteria were isolated from two different sources of tepache kefir grains (KAS2, KAS3, KAS4, KAS7, KAL4, KBS2, KBS3, KBL1 and KBL3), and were categorized to the genus Lacticaseibacillus, Liquorilactobacillus, and Lentilactobacillus by 16S rRNA gene. Kinetic behaviors of these strains were evaluated in MRS medium, and their probiotic potential was performed: resistance to low pH, tolerance to pepsin, pancreatin, bile salts, antibiotic resistance, hemolytic activity, and adhesion ability. KAS7 strain presented a higher growth rate (0.50 h-1) compared with KAS2 strain, who presented a lower growth rate (0.29 h-1). KBS2 strain was the only strain that survived the in vitro stomach simulation conditions (29.3%). Strain KBL1 demonstrated significantly higher viability (90.6%) in the in vitro intestine simulation conditions. Strain KAS2 demonstrated strong hydrophilic character with chloroform (85.6%) and xylol (57.6%) and a higher percentage of mucin adhesion (87.1%). However, strains KBS2 (84.8%) and KBL3 (89.5%) showed the highest autoaggregation values. In terms of adhesion to the intestinal epithelium in rats, strains KAS2, KAS3 and KAS4 showed values above 80%. The growth of the strains KAS2, KAS3, KAS4, KBS2, and KBL3 was inhibited by cefuroxime, cefotaxime, tetracycline, ampicillin, erythromycin, and cephalothin. Strains KBS2 (41.9% and 33.5%) and KBL3 (42.5% and 32.8%) had the highest co-aggregation values with S. aureus and E. coli. The results obtained in this study indicate that lactic acid bacteria isolated from tepache can be considered as candidates for potentially probiotic bacteria, laying the foundations to evaluate their probiotic functionality in vivo and thus to be used in the formulation of functional foods.

Male infertility presents a substantial challenge in reproductive medicine, often attributed to impaired sperm motility. The present study investigates the role of CCDC28A, a protein expressed specifically in male germ cells, whose paralog CCDC28B has been implicated in ciliogenesis. We identify unique expression patterns for CCDC28A and CCDC28B within the mouse testes, where CCDC28A is expressed in germ cells, whereas CCDC28B is expressed in supporting somatic cells. Through knockout mouse models and histological analyses, we reveal that CCDC28A deficiency results in diminished sperm motility and structural aberrations in sperm tails, notably affecting the head-tail coupling apparatus (HTCA), thereby causing male infertility. Fine structural analyses by transmission electron microscopy reveal disruptions at the capitulum-basal plate junction of the HTCA in the CCDC28A mutants. This results in the bending of the head within the neck region, often accompanied by thickening of the tail midpiece. Our discovery demonstrates that CCDC28A plays an essential role in male fertility and sperm tail morphogenesis through the formation of HTCA.

The extremotolerant red yeast Rhodotorula mucilaginosa displays resilience to diverse environmental stressors, including cold, osmolarity, salinity, and oligotrophic conditions. Particularly, this yeast exhibits a remarkable ability to accumulate lipids and carotenoids in response to stress conditions. However, research into lipid biosynthesis has been hampered by limited genetic tools and a scarcity of studies on adaptive responses to nutrient stressors stimulating lipogenesis. This study investigated the impact of nitrogen stress on the adaptive response in Antarctic yeast R. mucilaginosa M94C9. Varied nitrogen availability reveals a nitrogen-dependent modulation of biomass and lipid droplet production, accompanied by significant ultrastructural changes to withstand nitrogen starvation. In silico analysis identifies open reading frames of genes encoding key lipogenesis enzymes, including acetyl-CoA carboxylase (Acc1), fatty acid synthases 1 and 2 (Fas1/Fas2), and acyl-CoA diacylglycerol O-acyltransferase 1 (Dga1). Further investigation into the expression profiles of RmACC1, RmFAS1, RmFAS2, and RmDGA1 genes under nitrogen stress revealed that the prolonged up-regulation of the RmDGA1 gene is a molecular indicator of lipogenesis. Subsequent fatty acid profiling unveiled an accumulation of oleic and palmitic acids under nitrogen limitation during the stationary phase. This investigation enhances our understanding of nitrogen stress adaptation and lipid biosynthesis, offering valuable insights into R. mucilaginosa M94C9 for potential industrial applications in the future.

In Saccharomyces cerevisiae, the transcriptional repressor Nrg1 (Negative Regulator of Glucose-repressed genes) and the β-Zip transcription factor Rtg3 (ReTroGrade regulation) mediate glucose repression and signalling from the mitochondria to the nucleus, respectively. Here, we show a novel function of these two proteins, in which alanine promotes the formation of a chimeric Nrg1/Rtg3 regulator that represses the ALT2 gene (encoding an alanine transaminase paralog of unknown function). An NRG1/NRG2 paralogous pair, resulting from a post-wide genome small-scale duplication event, is present in the Saccharomyces genus. Neo-functionalization of only one paralog resulted in the ability of Nrg1 to interact with Rtg3. Both nrg1Δ and rtg3Δ single mutant strains were unable to use ethanol and showed a typical petite (small) phenotype on glucose. Neither of the wild-type genes complemented the petite phenotype, suggesting irreversible mitochondrial DNA damage in these mutants. Neither nrg1Δ nor rtg3Δ mutant strains expressed genes encoded by any of the five polycistronic units transcribed from mitochondrial DNA in S. cerevisiae. This, and the direct measurement of the mitochondrial DNA gene complement, confirmed that irreversible damage of the mitochondrial DNA occurred in both mutant strains, which is consistent with the essential role of the chimeric Nrg1/Rtg3 regulator in mitochondrial DNA maintenance.

Calcitriol and transforming growth factor beta 1 (TGF-β1) are unrelated molecules that regulate biological processes according to the genetic target, cell type, and context. Several studies have shown independent effects of calcitriol and TGF-βs on the placenta, but there is no information regarding the impact of their combination on these cells. Therefore, this study analyzed the effects of calcitriol, TGF-β1, and their combination in primary cultures of human trophoblast cells using a whole genome expression microarray. Data analysis revealed a set of differentially expressed genes induced by each treatment. Enrichment pathway analysis identified modulatory effects of calcitriol on genes related to metabolic processes such as vitamin D, steroid, and fat-soluble vitamins as well as antimicrobial and immune responses. In relation to TGF-β1, the analysis showed a few differentially expressed genes that were mainly associated with the neutrophil immune response. Lastly, the analysis revealed that the combination of calcitriol and TGF-β1 up-regulated genes involving both immunologic processes and the biosynthesis of unsaturated fatty acids, eicosanoids, and lipoxins, among others. In contrast, pathways down-regulated by the combination were mostly associated with the catabolic process of acylglycerols and peptides, PPAR signaling pathway, cellular response to low-density lipoprotein stimulus, renin angiotensin system and digestion, mobilization and transport of lipids. Consistent with these results, the combined treatment on human trophoblast cells induced the accumulation of intracellular neutral lipid droplets and stimulated both gene and protein expression of 15-hydroxyprostaglandin dehydrogenase. In conclusion, the results revealed that differentially expressed genes induced by the combination modified the transcriptional landscape compared to each treatment alone, mainly altering the storage, activity and metabolism of lipids, which might have an impact on placental development.

BACKGROUND INFORMATION:Autophagy is a conserved process that functions as a cytoprotective mechanism; it may function as a cell death process called programmed cell death type II. There is considerable evidence for the presence of autophagic cell death during oocyte elimination in prepubertal rats. However, the mechanisms involved in this process have not been deciphered.RESULTS:Our observations revealed autophagic cell death in oocytes with increased labeling of the autophagic proteins Beclin 1, light chain 3 A (LC3 A), and lysosomal-associated membrane protein 1 (Lamp1). Furthermore, mTOR and phosphorylated (p)-mTOR (S2448) proteins were significantly decreased in oocytes with increased levels of autophagic proteins, indicating autophagic activation. Moreover, phosphorylated protein kinase B (p-AKT) was not expressed by oocytes, but mitogen-activated protein kinase/extracellular signalregulated kinase (MAPK/ERK) signaling was observed. Additionally, selective and elevated mitochondrial degradation was identified in altered oocytes.CONCLUSIONS:All these results suggest that mTOR downregulation, which promotes autophagy, could be mediated by low energy levels and sustained starvation involving the phosphoinositide 3-kinase (PI3K)/AKT/mTOR and MAPK/ERK pathways.SIGNIFICANCE:In this work, we analyzed the manner in which autophagy is carried out in oocytes undergoing autophagic cell death by studying the behavior of proteins involved in different steps of the autophagic pathway.

Polycystic ovary syndrome (PCOS) is a disease whose diagnosis is based on the detection of hyperandrogenism (HA) and ovulatory dysfunction. Women with PCOS frequently develop insulin resistance (IR), which generates a metabolic condition that involves a decrease in the action of insulin at the cellular level and is linked to compensatory hyperinsulinemia (HI). In PCOS, the ovary remains sensitive to the action of insulin. Additionally, it has been observed that the main effect of insulin in the ovary is the stimulation of androgen synthesis, resulting in HA, one of the fundamental characteristics of the PCOS. In this sense, the excess of androgens favors the development of IR, thus perpetuating the cycle of IR-HI-HA, and therefore PCOS. Moreover, mitochondrial dysfunction is present in PCOS patients and is a common feature in both IR and HA. This review places electron transfer as a key element in HA and IR development, with emphasis on the relationship between androgen biosynthesis and mitochondrial function. Indeed, metformin has been involved in repair mitochondrial dysfunction, decrease of oxidative stress, reduction of androgens levels and the enhancing of insulin sensitivity. Therefore, we propose that treatment with metformin could decrease HI and consequently HA, restoring, at least in part, the metabolic and hormonal disorders of PCOS.

Polycystic ovary syndrome (PCOS) is an endocrine–metabolic disorder of unknown etiology. Hyperandrogenism (HA) is the main diagnostic criteria for PCOS, in addition to being a risk factor for developing several disorders throughout the patient’s life, including pregnancy. However, the impact on offspring is little known. Therefore, the aim of this work was to evaluate the effect of maternal HA on glucose metabolism and hepatic lipid accumulation in adult offspring. We used Balb/c mice treated with dehydroepiandrosterone (DHEA) for 20 consecutive days. The ovary of DHEA-treated mice showed hemorrhagic bodies, an increased number of atretic follicles, and greater expression of genes related to meiotic cell cycle and DNA repair. The DHEA offspring (O-DHEA) had low birth weight, and some pups showed malformations. However, O-DHEA individuals gained weight rapidly, and the differences between them and the control group became significantly greater in adulthood. Moreover, O-DHEA presented higher serum glucose after a 6 h fast and a larger area under glucose, insulin, and pyruvate tolerance test curves. Oil Red O staining showed a more significant accumulation of fat in the liver but no changes in serum cholesterol and triacylglycerol levels. In summary, our results show that HA, induced by DHEA, affects gene expression in oocyte, which in turn generates defects in embryonic development, insulin resistance, and alteration in hepatic gluconeogenesis and lipid metabolism in O-DHEA, thereby increasing the risk of developing metabolic diseases.

Intraventricular and extraventricular choroid plexuses are neuroepithelial folds which arise from the roof of the diencephalon. We describe the circumventricular structure of the diencephalon roof (paraphysis cerebri) during the various development stages of Ambystoma mexicanum. The parasagittal sections of the larvae epithalamus exhibit the presence, in addition to the epiphysis, of two dorsal primordia in nearby areas, which appear to be extraventricular saccular evaginations of different origin that give rise to two structures we define as the anterior extraventricular choroid plexus (AEP) and posterior extraventricular choroid plexus (PEP). During larvae development, the primordia arise perpendicular to each other, grow and show luminal folds and invaginations. Later, the two extraventricular evaginations, which are separate units, become interrelated. As the PEP grows, it covers the AEP dorsally, but it is difficult to define the borders of these organs. AEP is formed by alveolar-acinar epithelial aggregates with evidence of secretion-like content. PEP structure is like a choroid plexus, but its position is extraventricular and dorsal to the AEP. The PEP is always between the AEP and the meninges and can be small or large in size. This means that in A. mexicanum, the paraphysis cerebri is made up of two adjacent organs, which arise almost simultaneously from two different primordia (the AEP and the PEP) and as the posterior one grows, it overlaps the anterior one and masks itself. In conclusion, we suggest that AEP and PEP are homologous to paraphysis cerebri and the dorsal sac, respectively.

Apoptosis is the best-known programmed cell death that maintains tissue homeostasis in eukaryotic cells. The morphological characteristics include nuclear and cytoplasmic contraction and cytoplasmic blebbing, its biochemical hallmarks include caspase protease activity and DNA fragmentation. In rat ovaries, cell death is a normal process that occurs throughout the organism's life. Granulosa cells, the more abundant cell type forming the ovarian follicles, are eliminated via different routes of cell death. Most granulosa cells are eliminated through apoptotic cell death. In this work, we analyzed the behavior of nuclear components throughout the apoptotic process and determined how they are regionalized and conserved during follicular atresia in rat ovaries. Apoptosis was detected based on caspase-3 activity and DNA fragmentation using the TUNEL technique. We identified the transcription markers H3ac and RNA Pol II, and splicing factor SC35 by immunodetection. The nucleolar components were analyzed via light microscopy and transmission electron microscopy through immunodetection of the proteins nucleolin and nucleophosmin-1. The nuclear ultrastructure was analyzed using standard contrast and preferential ribonucleoprotein contrast. Our results demonstrate that during the pro-gression of apoptosis, chromatin is remodeled to constitute apoptotic bodies; transcription and spliceosome el-ements are reorganized along with the nucleolar components. Additionally, the splicing and transcription factors are segregated into specific territories inside the apoptotic bodies, suggesting that transcriptional elements are reorganized during the apoptotic process. Our results indicate that apoptotic bodies not only are compacted, and chromatin degraded but all the nuclear components are progressively reorganized during cell elimination; moreover, the transcriptional components are preserved.

Yeasts are microscopic fungi inhabiting all Earth environments, including those inhospitable for most life forms, considered extreme environments. According to their habitats, yeasts could be extremotolerant or extremophiles. Some are polyextremophiles, depending on their growth capacity, tolerance, and survival in the face of their habitat's physical and chemical constitution. The extreme yeasts are relevant for the industrial production of value-added compounds, such as biofuels, lipids, carotenoids, recombinant proteins, enzymes, among others. This review calls attention to the importance of yeasts inhabiting extreme environments, including metabolic and adaptive aspects to tolerate conditions of cold, heat, water availability, pH, salinity, osmolarity, UV radiation, and metal toxicity, which are relevant for biotechnological applications. We explore the habitats of extreme yeasts, highlighting key species, physiology, adaptations, and molecular identification. Finally, we summarize several findings related to the industrially-important extremophilic yeasts and describe current trends in biotechnological applications that will impact the bioeconomy.

It was almost 150 years ago that Golgi revolutionised histology with silver-based stains. Major advances in knowledge of the nervous system became possible because of silver impregnations. Silver staining combined with classical histological staining, cytochemistry methods, and electron microscopy is useful for studying mechanisms and components at subcellular, cellular, and tissue levels. Despite the advantages of silver staining, its use has decreased over time. The aim of this work was to use argentic staining to study the cerebellar effects of controversial prenatal glucocorticoid (GC) therapy. At postnatal day 12 (P12), the cerebellum of corticosterone (CC)-treated rats impregnated with AgNOR staining exhibited diminished thickness of the external granule layer (EGL) and irregular Purkinje cell arrangement. There was a greater number of nucleoli and nucleolar organiser regions (NORs) in 24% of Purkinje cells. Cerebellar granule neuron progenitor (CGNP) cells of the EGL showed a decrease in cellular density (confirmed by proliferating cell nuclear antigen [PCNA] immunolocalization) and NORs. At postnatal day 6 (P6), the Golgi-Kopsch technique allowed us to observe disturbances in the distribution pattern of CGNP cells (during proliferation, migration, and differentiation) and premature growth of the Bergmann glia. Our findings reveal disturbances in the cerebellar development program with early cellular and tissue changes.

A typical nucleolus structure is shaped by three components. A meshwork of fine fibers forming the fibrillar center (FC) is surrounded by densely packed fibers forming the dense fibrillar component (DFC). Meanwhile, wrapping the FC and DFC is the granular component (GC). During the mitotic prophase, the nucleolus undergoes disassembling of its components. On the contrary, throughout the first meiotic prophase that occurs in the cells of the germ line, small nucleoli are assembled into one nucleolus by the end of the prophase. These nucleoli are transcriptionally active, suggesting that they are fully functional. Electron microscopy analysis has suggested that these nucleoli display their three main components but a typical organization has not been observed. Here, by immunolabeling and electron microscopy, we show that the nucleolus has its three main components. The GC is interlaced with the DFC and is not as well defined as previously thought during leptotene and zygotene stage.

Follicles are ovarian structures that contain a single germ cell. During the mammalian reproductive lifetime, ovarian follicles mature through the process of follicular development, with the aim of selecting oocytes for ovulation. As part of this process, several follicles are eliminated by means of follicular atresia, a mechanism that mainly involves apoptosis. Nevertheless, it has been shown that there are other routes of programmed cell death in the ovary including autophagy, paraptosis, and necroptosis. Surprisingly, the endoplasmic reticulum is involved in these different programmed cell death pathways. Moreover, there are several evidences for the pathways triggered by intra- and extracellular signals in endoplasmic reticulum-induced cell death. Thus, it is important to analyze the participation of endoplasmic reticulum in follicular atresia.

ABSTRACTApoptosis is a type of cell death responsible for maintaining tissue homeostasis that can occur in male gonads. The morphological and biochemical characteristics of apoptosis include cellular contraction, caspase activation, and DNA fragmentation. Dynamic processes of cell renewal and differentiation occur inside the seminiferous tubules, which are regulated by mitosis and meiosis, respectively. During meiosis, recombination is caused by assembly of the synaptonemal complex, which involves the participation of constitutive proteins, such as synaptonemal complex protein‐3 (SYCP3). The present study evaluated germinal cell death in immature male rats and the distribution of the SYCP3 protein. Our results indicate that as germinal cells progress to the second meiotic stage, significant numbers of them are eliminated by apoptosis. We determined that the SYCP3 protein is not always incorporated into the structure of the synaptonemal complex but rather forms a nuclear cumulus near the inner nuclear membrane, causing many of these cells to undergo apoptosis. We propose that both the excess of the SYCP3 protein and its accumulation during the first meiotic division could contribute to the cell death of primary spermatocytes during the first spermatogenic wave in prepubertal Wistar rats. Anat Rec, 302:2082–2092, 2019. © 2019 American Association for Anatomy

Follicular atresia, a common process present in all mammals, involves apoptotic and autophagic cell death. However, the participation of paraptosis, a type of caspase-independent cell death, during follicular atresia is unknown. This study found swollen endoplasmic reticulum in the granulosa cells of adult Wistar rats. Calnexin was used as a marker of the endoplasmic reticulum at the ultrastructural and optical levels. The cells with swelling of the endoplasmic reticulum were negative to the TUNEL assay and active caspase-3 immunodetection, indicating that this swelling is not part of any apoptotic or autophagic process. Additionally, immunodetection of the CHOP protein was used as a marker of endoplasmic reticulum stress, and this confirmed the presence of the paraptosis process. These data suggest that paraptosis-like cell death is associated with the death of granulosa cells during follicular atresia in adult Wistar rats.

Aims: Excessive energy uptake of dietary carbohydrates results in their storage as fat and requires glucose-6-phosphate dehydrogenase (G6PD)-mediated NADPH production. We sought to assess whether the nicotinamide-induced reduction of G6PD activity might modulate redox balance and lipid accumulation in 3T3-L1 cells.Main methods: 3T3-L1 preadipocytes (days 4 and 6 of differentiation) and adipocytes were cultured in the presence of 5 or 25 mM glucose. The cells cultured in 25 mM glucose were supplemented with nicotinamide (5-15 mM). Next, we evaluated the following parameters: cell viability, apoptosis, lipid accumulation, lipolysis, reducing power, reactive oxygen species (ROS), NAD(P)H and NAD(P), isocitrate dehydrogenase (IDP), malic enzyme and G6PD, as well as the protein and mRNA levels of G6PD. We also analysed the kinetics of the nicotinamide-induced inhibition of G6PD.Key findings: G6PD mRNA levels increased at day 4 of adipocyte differentiation, whereas G6PD activity progressively increased at days 4 and 6 of differentiation and was reduced in adipocytes. Concomitantly, ROS, reducing power and lipid accumulation increased gradually as the preadipocytes matured into adipocytes. High glucose increased the activity of G6PD, which coincided with an increase in ROS, reducing power and lipid accumulation. All of these changes are prevented by nicotinamide, with the exception of lipid accumulation in adipocytes. Nicotinamide increased IDP activity without affecting NADPH levels. Lastly, nicotinamide inhibited G6PD in a non-competitive mixed way.Significance: Nicotinamide modulates G6PD via a non-competitive mixed inhibition and decreases high glucose-dependent oxidative stress and lipid accumulation. Nicotinamide maintains NADPH levels by increasing the activity of IDP. (C) 2013 Elsevier Inc. All rights reserved.

Reactive oxygen species derived from abdominal fat and uncontrolled glucose metabolism are contributing factors to both oxidative stress and the development of metabolic syndrome (MetS). This study was designed to evaluate the effects of daily administration of an oral glycine supplement on antioxidant enzymes and lipid peroxidation in MetS patients. The study included 60 volunteers: 30 individuals that were supplemented with glycine (15 g/day) and 30 that were given a placebo for 3 months. We analysed thiobarbituric acid reactive substances (TBARS) and S-nitrosohemoglobin (SNO-Hb) in plasma; the enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) in erythrocytes; and the expression of CAT, GPX, and SOD2 in leukocytes. Individuals treated with glycine showed a 25% decrease in TBARS compared with the placebo-treated group. Furthermore, there was a 20% reduction in SOD-specific activity in the glycine-treated group, which correlated with SOD2 expression. G6PD activity and SNO-Hb levels increased in the glycine-treated male group. Systolic blood pressure (SBP) also showed a significant decrease in the glycine-treated men (p = 0.043). Glycine plays an important role in balancing the redox reactions in the human body, thus protecting against oxidative damage in MetS patients.