Identification of 3-(9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acids as promising DNMT1 inhibitors.

DNA methyltransferase 1 (DNMT1) is the primary enzyme responsible for maintaining DNA methylation patterns during cellular division, crucial for cancer development by suppressing tumor suppressor genes. In this study, we retained the phthalimide structure of N-phthaloyl-l-tryptophan (RG108) and substituted its indole ring with nitrogen-containing aromatic rings of varying sizes. We synthesized 3-(9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acids and confirmed them as DNMT1 inhibitors through protein affinity testing, radiometric method using tritium labeled SAM, and MTT assay. Preliminary structure-activity relationship analysis revealed that introducing substituents on the carbazole ring could enhance inhibitory activity, with S-configuration isomers showing greater activity than R-configuration ones. Notably, S-3-(3,6-di-tert-butyl-9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (7r-S) and S-3-(1,3,6-trichloro-9H-carbazol-9-yl)-2-(1,3-dioxoisoindolin-2-yl)propanoic acid (7t-S) exhibited significant DNMT1 enzyme inhibition activity, with IC50 values of 8.147 µM and 0.777 µM, respectively (compared to RG108 with an IC50 above 250 µM). Moreover, they demonstrated potential anti-proliferative activity on various tumor cell lines including A2780, HeLa, K562, and SiHa. Transcriptome analysis and KEGG pathway enrichment of K562 cells treated with 7r-S and 7t-S identified differentially expressed genes (DEGs) related to apoptosis and cell cycle pathways. Flow cytometry assays further indicated that 7r-S and 7t-S induce apoptosis in K562 cells and arrest them in the G0/G1 phase in a concentration-dependent manner. Molecular docking revealed that 7t-S may bind to the methyl donor S-adenosyl-l-methionine (SAM) site in DNMT1 with an orientation opposite to RG108, suggesting potential for deeper penetration into the DNMT1 pocket and laying the groundwork for further modifications.

Microglia-derived exosomes selective sorted by YB-1 alleviate nerve damage and cognitive outcome in Alzheimer's disease.

BACKGROUND: Neuroinflammation is a characteristic pathological change of Alzheimer's Diseases (AD). Microglia have been reported to participate in inflammatory responses within the central nervous system. However, the mechanism of microglia released exosome (EXO) contribute to communication within AD microenvironment remains obscure. METHODS: The interaction between microglia and AD was investigated in vitro and in vivo. RNA-binding protein immunoprecipitation (RIP) was used to investigate the mechanisms of miR-223 and YB-1. The association between microglia derived exosomal YB-1/miR-223 axis and nerve cell damage were assessed using Western blot, immunofluorescence, RT-PCR, ELISA and wound healing assay. RESULTS: Here, we reported AD model was responsible for the M1-like (pro-inflammatory) polarization of microglia which in turn induced nerve cell damage. While M2-like (anti-inflammatory) microglia could release miR-223-enriched EXO which reduced neuroinflammation and ameliorated nerve damage in AD model in vivo and in vitro. Moreover, YB-1 directly interacted with miR-223 both in cell and EXO, and participated in microglia exosomal miR-223 loading. CONCLUSION: These results indicate that anti-inflammatory microglia-mediated neuroprotection form inflammatory damage involves exporting miR-223 via EXO sorted by YB-1. Consequently, YB-1-mediated microglia exosomal sorting of miR-223 improved the nerve cell damage repair, representing a promising therapeutic target for AD.

Exosomal YB-1 facilitates ovarian restoration by MALAT1/miR-211-5p/FOXO3 axis.

Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. Mesenchymal stem cells-derived small extracellular vesicles (MSCs-sEVs) are attractive candidates for ovarian function restoration and folliculogenesis for POF due to their safety and efficacy, however, the key mediator in MSCs-sEVs that modulates this response and underlying mechanisms remains elusive. Herein, we reported that YB-1 protein was markedly downregulated in vitro and in vivo models of POF induced with H2O2 and CTX respectively, accompanied by granulosa cells (GCs) senescence phenotype. Notably, BMSCs-sEVs transplantation upregulated YB-1, attenuated oxidative damage-induced cellular senescence in GCs, and significantly improved the ovarian function of POF rats, but that was reversed by YB-1 depletion. Moreover, YB-1 showed an obvious decline in serum and GCs in POF patients. Mechanistically, YB-1 as an RNA-binding protein (RBP) physically interacted with a long non-coding RNA, MALAT1, and increased its stability, further, MALAT1 acted as a competing endogenous RNA (ceRNA) to elevate FOXO3 levels by sequestering miR-211-5p to prevent its degradation, leading to repair of ovarian function. In summary, we demonstrated that BMSCs-sEVs improve ovarian function by releasing YB-1, which mediates MALAT1/miR-211-5p/FOXO3 axis regulation, providing a possible therapeutic target for patients with POF.

Facile hydrothermal assembly of three-dimensional GO-MTZE composite and its adsorption properties toward Cu2.

Three-dimensional (3D) graphene oxide (GO)-based aerogels, GO and 4-methyl-5-thiazoleethanol (MTZE) composites, were prepared by a facile hydrothermal method. Due to the hydrogen bonding and π-π stacking interactions, the produced 3D GO-MTZE composites possessed large cylindrical structures. The morphologies, composition, and chemical states of 3D GO-MTZE3:1 composite were characterized by Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), and N2 adsorption-desorption isotherms based on the Brunauer-Emmett-Teller (BET) method. The existence of nitrogen (N)-containing heterocyclic system and oxygen (O)-containing branched chain of MTZE contributed to the formation of 3D structures, while the complexation effect of heterocyclic sulfur (S)- and N-containing functional groups of MTZE for metal cations dominated the adsorption performance of 3D GO-MTZE3:1 composite, which could selectively adsorb copper ions (Cu2+). In addition, the better hydrophobic property of 3D GO-MTZE3:1 composite facilitates its facile recycling from aqueous solution after adsorption. The adsorption data of 3D GO-MTZE3:1 composite toward Cu2+ fitted well (R2 = 0.9996) with the linear pseudo-second-order kinetic model, giving an equilibrium rate constant (k2) of 0.0187 g mg-1 min-1. The linear Langmuir isothermal model could more accurately describe the experimental data, indicating the adsorption process is mainly dominated by the complexation interactions between MTZE and Cu2+. The thermodynamic parameters of ΔG° (< 0), ΔH° (> 0), and ΔS° (> 0) further indicate that the adsorption is a spontaneous and endothermic, confirming that the complexation between Cu2+ and 3D GO-MTZE3:1 composite occurs. Due to its high selectivity for Cu2+, good hydrophobicity, and excellent stability, the developed 3D GO-MTZE3:1 composite possesses might be promisingly used in the aqueous selective enrichment/removal of Cu2+.

Cardiac resident macrophages: Spatiotemporal distribution, development, physiological functions, and their translational potential on cardiac diseases.

Cardiac resident macrophages (CRMs) are the main population of cardiac immune cells. The role of these cells in regeneration, functional remodeling, and repair after cardiac injury is always the focus of research. However, in recent years, their dynamic changes and contributions in physiological states have a significant attention. CRMs have specific phenotypes and functions in different cardiac chambers or locations of the heart and at different stages. They further show specific differentiation and development processes. The present review will summarize the new progress about the spatiotemporal distribution, potential developmental regulation, and their roles in cardiac development and aging as well as the translational potential of CRMs on cardiac diseases. Of course, the research tools for CRMs, their respective advantages and disadvantages, and key issues on CRMs will further be discussed.

Effects of prolactin on the proliferation and hormone secretion of ovine granulosa cells in vitro.

Objective: The objective of this study was to investigate the effects of prolactin (PRL) on the proliferation and apoptosis of ovine ovarian granulosa cells (GCs) and the secretion of estrogen (E2) and progesterone (P4), as well as to explore the effects of PRL on related genes and proteins. Methods: We isolated ovarian GCs from 1-year-old small-tail Han sheep and identified PRL receptor (PRLR) on ovaries and follicle stimulating hormone receptor (FSHR) on ovarian GCs, respectively, using immunohistochemistry. PRL (0, 0.05, 0.50, 5.00 µg/mL) were added to GCs in vitro along with FSH, cell proliferation was measured by Cell Counting Kit-8 (CCK-8) and apoptosis by flow cytometry. The measurement of E2 and P4 content by ELISA after 24h and 48h. The expression of functional genes and proteins was identified by RT-qPCR and Western-blot after 24h. Results: PRLR was expressed in both follicular GCs and corpus luteum, whereas FSHR was expressed specifically. The proliferative activity was lower on day 1 while higher on day 4 and day 5. The apoptosis rate of GCs in the 0.05 µg/mL group was significantly higher than that in the control group after treatment with PRL for 24 h (p<0.05). Compared with the control group, the secretion of E2 in GCs was reduced significantly (p<0.05) in PRL treatment for 24h and 48h, while the secretion of P4 was significantly increased (p<0.05). The mRNA expression levels of PRLR, FSHR, LHR, CYP11A1, HSD3B7 and STAR were significantly higher than those in the control group (p<0.01), and the relative abundance of BCL2 in all PRL group were increased after PRL treatment. Conclusion: PRL promoted the proliferation of GCs and supraphysiological concentrations inhibited apoptosis caused by down-regulation of BAX and up-regulation of BCL2. PRL inhibited E2 by down-regulating CYP19A1 and promoted P4 by up-regulating CYP11A1, STAR and HSD3B7.

CD40 Expression by B cells is Required for Optimal Immunity to Murine Pneumocystis Infection.

CD40-CD40L interactions are critical for controlling Pneumocystis infection. However, which CD40-expressing cell populations are important for this interaction have not been well-defined. We used a cohousing mouse model of Pneumocystis infection, combined with flow cytometry and qPCR, to examine the ability of different populations of cells from C57BL/6 mice to reconstitute immunity in CD40 knockout (KO) mice. Unfractionated splenocytes, as well as purified B cells, were able to control Pneumocystis infection, while B cell depleted splenocytes and unstimulated bone-marrow derived dendritic cells (BMDCs) were unable to control infection in CD40 KO mice. Pneumocystis antigen-pulsed BMDCs showed early, but limited, control of infection. Consistent with recent studies that have suggested a role for antigen presentation by B cells, using cells from immunized animals, B cells were able to present Pneumocystis antigens to induce proliferation of T cells. Thus, CD40 expression by B cells appears necessary for robust immunity to Pneumocystis.

The Effects of Electrolytic Multivitamins and Neomycin on Antioxidant Capacity and Intestinal Damage in Transported Lambs.

Transport stress can cause damage to animals. In this experiment, 60 four-month-old lambs were randomly divided into three groups: CG (basal diet), EG (basal diet + 375 mg/d/lamb electrolytic multivitamin), and NG (basal diet + 200 mg/d/lamb neomycin). The results were as follows: during road transport, in all groups, the levels of SOD, T-AOC, and GSP-Px, and mRNA expressions of CAT, SOD, Nrf2, HO-1, and Bcl-2 in the jejunum and colon decreased (p < 0.01). However, mRNA expressions of Keap1, IL-1ß, IL-2, IL-12, Bax, and Caspase3 in the jejunum and colon and the level of MDA increased (p < 0.01). The concentrations of IgA, IgG, and sIgA in the jejunum and colon also decreased (p < 0.01). In the EG and NG, the levels of SOD (p < 0.05) and T-AOC (p < 0.01) increased, and the level of MDA decreased (p < 0.01). However, in the jejunum, the levels of SOD and T-AOC, the concentrations of IgA and IgG, and mRNA expression of Bcl-2 increased (p < 0.05). mRNA expressions of IL-1, IL-2, and Caspase 3 (p < 0.05), and mRNA expression of IL-12 (p < 0.01) decreased. In the colon, SOD activity and the concentration of sIgA increased (p < 0.01). The level of MDA and mRNA expressions of IL-2 and Caspase 3 also decreased (p < 0.05). In the jejunum and colon, mRNA expression of SOD (p < 0.05) and mRNA expression of Nrf2 increased (p < 0.01). mRNA expression of Keap1 (p < 0.05) and Bax (p < 0.01) decreased. In summary, road transport can cause a decrease in antioxidant activity and immunity of lambs and an increase in oxidative damage. Electrolytic multivitamins and neomycin can improve immune function and potentially reduce oxidative damage to the jejunum and colon.

Extracellular Proximity Labeling Reveals an Expanded Interactome for the Matrisome Protein TIMP2.

Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within the cell or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein tissue inhibitor of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM-targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.

CD40 Expression by B cells is Required for Optimal Immunity to Murine Pneumocystis Infection.

CD40-CD40L interactions are critical for controlling Pneumocystis infection. However, which CD40-expressing cell populations are important for this interaction have not been well-defined. We used a cohousing mouse model of Pneumocystis infection, combined with flow cytometry and qPCR, to examine the ability of different populations of cells from C57BL/6 mice to reconstitute immunity in CD40 knockout (KO) mice. Unfractionated splenocytes, as well as purified B cells, were able to control Pneumocystis infection, while B cell depleted splenocytes and unstimulated bone-marrow derived dendritic cells (BMDCs) were unable to control infection in CD40 KO mice. Pneumocystis antigen-pulsed BMDCs showed early, but limited, control of infection. Consistent with recent studies that have suggested a role for antigen presentation by B cells, using cells from immunized animals, B cells were able to present Pneumocystis antigens to induce proliferation of T cells. Thus, CD40 expression by B cells appears necessary for robust immunity to Pneumocystis.

Application Effect of Patient-centered Health Education in Patients with Type 2 Diabetes Accompanied by Hyperlipidemia.

Objective: This study aims to investigate the impact of patient-centered health education on individuals with type 2 diabetes coexisting with hyperlipidemia. Methods: A cohort of 80 patients with type 2 diabetes and hyperlipidemia attending our hospital from February 2022 to August 2022 were randomly assigned to either the health education group or the control group. While the control group received routine health education, the health education group received additional patient-centered health education. Subsequently, we compared blood glucose and lipid levels, negative emotions, quality of life, and the incidence of unhealthy eating or overweight between the two groups post-education. Results: Following the health education intervention, the health education group exhibited superior improvements in blood glucose and lipid levels compared to the control group. Moreover, there was a significant decrease in SAS and SDS scores and a notable increase in quality of life compared to the control group. The health education group also demonstrated a lower incidence of unhealthy eating or overweight. Conclusions: Patient-centered health education for individuals with type 2 diabetes and hyperlipidemia proves effective in enhancing glucose and lipid metabolism, mitigating negative emotions, improving quality of life, and reducing unhealthy habits.

Core microbiota for nutrient digestion remained and ammonia utilization increased after continuous batch culture of rumen microbiota in vitro.

Introduction: This study aimed to investigate the digestive function, urea utilization ability, and bacterial composition changes in rumen microbiota under high urea (5% urea in diet) over 23 days of continuous batch culture in vitro. Methods: The gas production, dry matter digestibility, and bacterial counts were determined for the continuously batch-cultured rumen fluid (CRF). The changes in fermentation parameters, NH3-N utilization efficiency, and microbial taxa were analyzed in CRF and were compared with that of fresh rumen fluid (RF), frozen rumen fluid (FRF, frozen rumen fluid at -80°C for 1 month), and the mixed rumen fluid (MRF, 3/4 RF mixed with 1/4 CRF) with in vitro rumen fermentation. Results: The results showed that the dry matter digestibility remained stable while both the microbial counts and diversity significantly decreased over the 23 days of continuous batch culture. However, the NH3-N utilization efficiency of the CRF group was significantly higher than that of RF, FRF, and MRF groups (p < 0.05), while five core genera including Succinivibrio, Prevotella, Streptococcus, F082, and Megasphaera were retained after 23 days of continuous batch culture. The NH3-N utilization efficiency was effectively improved after continuous batch culture in vitro, and Streptococcus, Succinivibrio, Clostridium_sensu_stricto_1, p.251.o5, Oxalobacter, Bacteroidales_UCG.001, and p.1088.a5_gut_group were identified to explain 75.72% of the variation in NH3-N utilization efficiency with the RandomForest model. Conclusion: Thus, core bacterial composition and function retained under high urea (5% urea in diet) over 23 days of continuous batch culture in vitro, and bacterial biomarkers for ammonia utilization were illustrated in this study. These findings might provide potential applications in improving the efficiency and safety of non-protein nitrogen utilization in ruminants.

Regional centromere configuration in the fungal pathogens of the Pneumocystis genus.

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.

Effects of Electrolyte Multivitamins and Neomycin on Immunity and Intestinal Barrier Function in Transported Lambs.

Animals experience stress when they are transported. In this experiment, sixty 4-month-old lambs were randomly divided into three groups: CG (basal diet), EG (basal diet + 375 mg/d/lamb electrolytic multivitamin) and NG (basal diet + 200 mg/d/lamb neomycin). The transportation day was recorded as the 0th day. Blood, liver, spleen, jejunum and colon were collected on the 0th, 7th and 14th day. The results were as follows: In EG and NG groups, the lamb weights (p < 0.01), IgA and IgG (p < 0.05) increased significantly. The concentrations of ACTH, E, COR, IL-1ß, IL-6 and IFN-γ decreased significantly (p < 0.01). The content of colonic propionate increased significantly (p < 0.05). The villus height and V/C increased, and crypt depth decreased significantly (p < 0.01). The mRNA expressions of Occludin and MUC1, and the protein expression of Occludin in the jejunal mucosa, the mRNA expressions of ZO-1 and Occludin, and the protein expression in the colonic mucosa increased significantly (p < 0.01). The mRNA expression of TRAF6 and the protein expression of TLR4 in the jejunum decreased significantly (p < 0.05), as well as the mRNA expressions of TLR4, MyD88 and NF-kB, and the protein expression of NF-kB p65 and the mRNA expressions of TRAF6, TLR4 and NF-kB in the colon (p < 0.01). In conclusion, an electrolytic multivitamin could potentially improve the immunity and intestinal barrier function, and when it was added with 375 mg/d in the basal diet for each lamb from 2 d before transportation to 7 d after transportation, it had a better effect than neomycin.

The effect of early colonized gut microbiota on the growth performance of suckling lambs.

The early colonized gut microbiota during the newborn period has been reported to play important roles in the health and immunity of animals; however, whether they can affect the growth performance of suckling lambs is still unclear. In this study, a total of 84 newborn lambs were assigned into LF-1 (top 15%), LF-2 (medium 70%), and LF-3 (bottom 15%) groups according to their average body weight gain at 30 days of age. Fecal samples of lambs (LF) as well as feces (MF), vagina (VAG), colostrum (COL), teat skin (TEAT) samples of ewes, and the air sediment (AIR) in the delivery room were collected 72 h after birth, and then the 16S rRNA gene was sequenced on the Illumina MiSeq platform. The results showed that the early colonized gut microbiota had a significant effect on the growth performance of suckling lambs with alpha and beta diversity (p < 0.05), and we observed that the contribution of early colonized bacteria on the growth performance of lambs increased with age (from BW30 at 25.35% to BW45 at 31.10%; from ADG30 at 33.02% to ADG45 at 39.79% by measuring the relative effects of factors that influence growth performance). The early colonized gut microbiota of suckling lambs with high growth performance was similar to that in VAG, MF, and AIR (p < 0.05). With the RandomForest machine learning algorithm, we detected 11, 11, 6, and 4 bacterial taxa at the genus level that were associated with BW30, BW45, ADG30, and ADG45 of suckling lambs, respectively, and the correlation analysis showed that Butyricicoccus, Ruminococcus_gnavus_group, Ruminococcaceae_Other, and Fusobacterium could significantly affect the growth performance (BW30, BW45, ADG30, and ADG45) of suckling lambs (p < 0.05). In conclusion, the early colonized gut microbiota could significantly affect the growth performance of suckling lambs, and targeting the early colonized gut microbiota might be an alternative strategy to improve the growth performance of suckling lambs.

Prolactin inhibitor changes testosterone production, testicular morphology, and related genes expression in cashmere goats.

Prolactin has multifaceted roles in lactation, growth, metabolism, osmoregulation, behavior, and the reproduction of animals. This study aimed to investigate the involvement of prolactin in testicular function in cashmere goats. Twenty cashmere goats were randomly assigned to either the control group (CON) or the bromocriptine treatment group (BCR, bromocriptine, prolactin inhibitor). Blood and testis samples collected for analysis after 30 days of treatment. The results indicated that, compared with the CON group, BCR significantly decreased (p < 0.05) the serum concentrations of prolactin, and significantly increased (p < 0.05) the levels of testosterone and luteinizing hormone (LH) on day 30. The serum level of the follicle-stimulating hormone (FSH) was not affected (p > 0.05) by the treatment. The mean seminiferous tubule diameter and spermatogenic epithelium thickness were increased (p < 0.05) in the BCR group. Subsequently, we performed RNA sequencing and bioinformatics analysis to identify the key genes and pathways associated with the regulation of spermatogenesis or testosterone secretion function. A total of 142 differentially expressed genes (DEGs) were identified (91 were upregulated, 51 were downregulated). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the DEGs were mainly involved in the extracellular matrix (ECM), hippo, and steroid hormone biosynthesis, which are related to testicular function. The expression of the genes SULT2B1, CYP3A24, and CYP3A74 in the steroid hormone biosynthesis pathway significantly increased (p < 0.05) in the BCR group, which was validated by qRT-PCR. These results provide a basis for understanding the mechanisms underlying the regulation of testicular function by prolactin in cashmere goats.

[Preparation of Mycobacterium tuberculosis EsxV lipid nanoparticles subunit vaccine and its immunological characteristics].

To prepare a lipid nanoparticle (LNP)-based subunit vaccine of Mycobacterium tuberculosis (Mtb) antigen EsxV and study its immunological characteristics, the LNP containing EsxV and c-di-AMP (EsxV: C: L) was prepared by thin film dispersion method, and its encapsulation rate, LNP morphology, particle size, surface charge and polyphase dispersion index were measured. BALB/c mice were immunized with EsxV: C: L by nasal drops. The levels of serum and mucosal antibodies, transcription and secretion of cytokines in lung and spleen, and the proportion of T cell subsets were detected after immunization. EsxV: C: L LNPs were obtained with uniform size and they were spherical and negatively charged. Compared with EsxV: C immunization, EsxV: C: L mucosal inoculation induced increased sIgA level in respiratory tract mucosa. Levels of IL-2 secreted from spleen and ratios of memory T cells and tissue-resident T cells in mice were also elevated. In conclusion, EsxV: C: L could induce stronger mucosal immunity and memory T cell immune responses, which may provide better protection against Mtb infection.

High Prolactin Concentration Induces Ovarian Granulosa Cell Oxidative Stress, Leading to Apoptosis Mediated by L-PRLR and S-PRLR.

High prolactin (PRL) concentration has been shown to induce the apoptosis of ovine ovarian granulosa cells (GCs), but the underlying mechanisms are unclear. This study aimed to investigate the mechanism of apoptosis induced by high PRL concentration in GCs. Trial 1: The optimal concentration of glutathion was determined according to the detected cell proliferation. The results showed that the optimal glutathione concentration was 5 µmol/mL. Trial 2: 500 ng/mL PRL was chosen as the high PRL concentration. The GCs were treated with 0 ng/mL PRL (C group), 500 ng/mL PRL (P group) or 500 ng/mL PRL, and 5 µmol/mL glutathione (P-GSH group). The results indicated that the mitochondrial respiratory chain complex (MRCC) I-V, ATP production, total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and thioredoxin peroxidase (TPx) in the C group were higher than those in the P group (p < 0.05), while they were lower than those in the P-GSH group (p < 0.05). Compared to the C group, the P group exhibited elevated levels of reactive oxygen species (ROS) and apoptosis (p < 0.05) and increased expression of ATG7 and ATG5 (p < 0.05). However, MRCC I-V, ATP, SOD, A-TOC, TPx, ROS, and apoptosis were decreased after the addition of glutathione (p < 0.05). The knockdown of either L-PRLR or S-PRLR in P group GCs resulted in a significant reduction (p < 0.05) in MRCC I-V, ATP, T-AOC, SOD and TPx, while the overexpression of either receptor showed an opposite trend (p < 0.05). Our findings suggest that high PRL concentrations induce apoptotic cell death in ovine ovarian GCs by downregulating L-PRLR and S-PRLR, activating oxidative stress and autophagic pathways.

The Host Adapted Fungal Pathogens of Pneumocystis Genus Utilize Genic Regional Centromeres.

Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. How centromeres form in strongly host-adapted fungal pathogens has yet to be investigated. Here, we characterized the centromere structures in closely related species of mammalian-specific pathogens of the fungal phylum of Ascomycota. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of Schizosaccharomyces pombe. Using organisms from a short-term in vitro culture or infected animal models and ChIP-seq, we identified centromeres in three Pneumocystis species that diverged ~100 million years ago. Each species has a unique short regional centromere (< 10kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. CENP-C, a scaffold protein that links the inner centromere to the kinetochore appears dispensable in one species, suggesting a kinetochore rewiring. Despite the loss of DNA methyltransferases, 5-methylcytosine DNA methylation occurs in these species, though not related to centromere function. These features suggest an epigenetic specification of centromere function.