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OBJECTIVE: To obtain prescription eyewear in Ontario, eye exams must be performed by optometrists or ophthalmologists (eye care providers [ECPs]). In 2004, government-insured routine eye exams were delisted for Ontarians aged 20-64 leaving eye exam coverage only for those aged ≤19 and 65+. We assessed whether having eyewear insurance impacts Ontarians' utilization of ECPs. DESIGN: Cross-sectional survey. PARTICIPANTS: Ontarians aged 12+ without diabetes responding to the Canadian Community Health Survey in 2003, 2005, and 2013/2014. METHODS: We compared the utilization of ECPs by eyewear insurance status and eligibility for government-funded eye exams. Individuals with eyewear insurance funded by employers, government or privately were considered to have insurance. RESULTS: ECP utilization was significantly higher in Ontarians with eyewear insurance versus those without, in all survey years and all age groups, including those eligible for government-funded eye exams (e.g., 66.4% vs 59.1% [p < 0.05] for the 65+). This higher level of utilization was particularly evident among Ontarians aged 20-64 in 2013/2014, when this group no longer had government-funded eye exams (34.9% vs 19.9% among 20-39-year-olds, 43.4% vs 32.9% among 40-64-year-olds, p < 0.05 for both). Adjusting for confounding effects, the likelihood of visiting an ECP was greater among Ontarians with eyewear insurance than those without (adjusted prevalence ratio 1.26 for Ontarians aged 12+ and 1.41 for those aged 20-64; p < 0.05 for both). CONCLUSIONS: Lack of eyewear insurance negatively impacts the utilization of ECPs, even among Ontarians eligible for government-funded eye exams, where the cost barrier for eye exams has been removed by the Ontario government.
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(1) Background: One method of eradicating brucellosis is to cull cattle that test positive for antibodies 12 months after being vaccinated with the 19-strain vaccine. Variations in immunization regimens and feeding practices may contribute to differences in the rate of persistent antibodies. We conducted this study to investigate the real positive rate of Brucella antibody in field strains of Brucella spp. after immunization over 12 months in dairy cows. This research aims to provide data to support the development of strategies for preventing, controlling, and eradicating brucellosis. (2) Method: We employed the baseline sampling method to collect samples from cows immunized with the A19 vaccine for over 12 months in Lingwu City from 2021 to 2023. Serological detection was conducted using the RBPT method. An established PCR method that could distinguish between 19 and non-19 strains of Brucella was utilized to investigate the field strains of Brucella on 10 dairy farms based on six samples mixed into one using the Mathematical Expectation strategy. (3) Results: We analyzed the rates of individual seropositivity and herd seropositive rates in dairy cattle in Lingwu City from 2021 to 2023 and revealed that antibodies induced by the Brucella abortus strain A19 vaccine persist in dairy herds for more than 12 months. We established a PCR method for identifying both Brucella A19 and non-A19 strains, resulting in the detection of 10 field strains of Brucella abortus from 1537 dairy cows. By employing a Mathematical Expectation strategy, we completed testing of 1537 samples after conducting only 306 tests, thereby reducing the workload by 80.1%. (4) Conclusions: There was a certain proportion of cows with a persistent antibody titer, but there was no evidence that all of these cattle were naturally infected with Brucella. The established PCR method for distinguishing between Brucella abortus strain 19 and non-19 strains can be specifically utilized for detecting natural Brucella infection in immunized cattle. We propose that relying solely on the detection of antibodies in cattle immunized with the A19 vaccine more than 12 months previously should not be solely relied upon as a diagnostic basis for brucellosis, and it is essential to complement this approach with PCR analysis to specifically identify field Brucella spp. Brucella abortus was the predominant strain identified in the field during this study. Detection based on the Mathematical Expectation strategy can significantly enhance detection efficiency.
ABSTRACT
Persister cells are transiently tolerant to antibiotics and are associated with recalcitrant chronic infections due to recolonization of host cells after antibiotic removal. Brucella spp. are facultative pathogens that establish intracellular infection cycles in host cells which results in chronic persistent infections. Brucella abortus forms multi-drug persister cells which are promoted by the (p)ppGpp synthetase Rsh during rifampicin exposure. Here, we confirmed that Rsh promoted persister cells formation in B. abortus stationary phase treated with rifampicin and enrofloxacin. Deletion of the gene for Rsh decreased persister cells level in the presence of these drugs in different growth phases. However, persister cells formation by deletion strain varied in different growth phases in the presence of other antibiotics. Rsh also was involved in persister cells formation during rifampicin treatment under certain stress conditions, including acidic conditions, exposure to PBS, and heat stress. Moreover, Rsh impacted persister cell levels during rifampicin or enrofloxacin treatment in RAW264.7 macrophages. Certain typeIItoxin-antitoxin modules were upregulated under various stress conditions in B. abortus. We established that Rsh positively regulated the type II toxin-antitoxin mbcTA. Moreover, rifampicin-tolerant persister cells formation was elevated and ATP levels were decreased when mbcTA promoter was overexpressed in Rsh deletion background in stationary phase. Our results establish that (p)ppGpp synthetase Rsh plays a key role in B. abortus persistence and may serve as a potent novel target in combination with rifampicin in the development of new therapeutic approaches and prevention strategies to treat chronic infections of Brucella.
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Brucella is an intracellular parasitic bacterium lacking typical virulence factors, and its pathogenicity primarily relies on replication within host cells. In this study, we observed a significant increase in spleen weight in mice immunized with a Brucella strain deleted of the gene for alanine racemase (Alr), the enzyme responsible for alanine racemization (Δalr). However, the bacterial load in the spleen markedly decreased in the mutant strain. Concurrently, the ratio of white pulp to red pulp in the spleen was increased, serum IgG levels were elevated, but no significant damage to other organs was observed. In addition, the inflammatory response was potentiated and the NF-κB-NLRP3 signaling pathway was activated in macrophages (RAW264.7 Cells and Bone Marrow-Derived Cells) infect ed with the Δalr mutant. Further investigation revealed that the Δalr mutant released substantial amounts of protein in a simulated intracellular environment which resulted in heightened inflammation and activation of the TLR4-NF-κB-NLRP3 pathway in macrophages. The consequent cytoplasmic exocytosis reduced intracellular Brucella survival. In summary, cytoplasmic exocytosis products resulting from infection with a Brucella strain deleted of the alr gene effectively activated the TLR4-NFκB-NLRP3 pathway, triggered a robust inflammatory response, and reduced bacterial survival within host cells. Moreover, the Δalr strain exhibits lower toxicity and stronger immunogenicity in mice.
Subject(s)
Brucella suis , Brucellosis , Macrophages , NF-kappa B , NLR Family, Pyrin Domain-Containing 3 Protein , Toll-Like Receptor 4 , Animals , Mice , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/immunology , Toll-Like Receptor 4/genetics , Toll-Like Receptor 4/metabolism , NF-kappa B/metabolism , Brucellosis/immunology , Brucellosis/microbiology , Brucellosis/genetics , RAW 264.7 Cells , Brucella suis/immunology , Brucella suis/genetics , Brucella suis/pathogenicity , Virulence/genetics , Macrophages/immunology , Gene Deletion , Signal Transduction/immunology , Female , Mice, Inbred BALB C , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Spleen/immunology , Inflammation/immunologyABSTRACT
Mesenchymal Stem Cells are ideal seed cells for tissue repair and cell therapy and have promising applications in regenerative medicine and tissue engineering. Using Platelet-Rich Plasma as an adjuvant to create and improve the microenvironment for Mesenchymal Stem Cells growth can enhance the biological properties of Mesenchymal Stem Cells and improve the efficacy of cell therapy. However, the mechanism by which Platelet-Rich Plasma improves the biological performance of Mesenchymal Stem Cells is still unknown. In this study, by examining the effects of Platelet-Rich Plasma on the biological performance of Mesenchymal Stem Cells, combined with multiomics analysis (Transcriptomics, Proteomics and Metabolomics) and related tests, we analyzed the specific pathways, related mechanisms and metabolic pathways of Platelet-Rich Plasma to improve the biological performance of Mesenchymal Stem Cells. In an in vitro cell culture system, the biological performance of Mesenchymal Stem Cells was significantly improved after replacing Foetal Bovine Serum with Platelet-Rich Plasma, and the genes (ESM1, PDGFB, CLEC7A, CCR1 and ITGA6 et al.) related to cell proliferation, adhesion, growth, migration and signal transduction were significantly upregulated. Platelet-Rich Plasma can enhance the secretion function of MSC exosomes, significantly upregulate many proteins related to tissue repair, immune regulation and anti-infection, and enhance the repair effect of exosomes on skin injury. After replacing Foetal Bovine Serum with Platelet-Rich Plasma, Mesenchymal Stem Cells underwent metabolic reprogramming, the metabolism of amino acids and fatty acids and various signaling pathways were changed, the anabolic pathways of various proteins were enhanced. These results provide a theoretical and technical reference for optimizing the Mesenchymal Stem Cells culture system, improving the biological characteristics and clinical application effects of Mesenchymal Stem Cells.
Subject(s)
Cell Proliferation , Mesenchymal Stem Cells , Platelet-Rich Plasma , Proteomics , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Platelet-Rich Plasma/metabolism , Humans , Metabolomics , Animals , Cells, Cultured , Gene Expression Profiling , Exosomes/metabolism , MultiomicsABSTRACT
The global challenge of male infertility is escalating, notably due to the decreased testosterone (T) synthesis in testicular Leydig cells under stress, underscoring the critical need for a more profound understanding of its regulatory mechanisms. CREBZF, a novel basic region-leucine zipper transcription factor, regulates testosterone synthesis in mouse Leydig cells in vitro; however, further validation through in vivo experiments is essential. Our study utilized Cyp17a1-Cre to knock out CREBZF in androgen-synthesis cells and explored the physiological roles of CREBZF in fertility, steroid hormone synthesis, and behaviors in adult male mice. Conditional knockout (cKO) CREBZF did not affect fertility and serum testosterone level in male mice. Primary Leydig cells isolated from CREBZF-cKO mice showed impaired testosterone secretion and decreased mRNA levels of Star, Cyp17a1, and Hsd3b1. Loss of CREBZF resulted in thickening of the adrenal cortex, especially X-zone, with elevated serum corticosterone and dehydroepiandrosterone levels and decreased serum dehydroepiandrosterone sulfate levels. Immunohistochemical staining revealed increased expression of StAR, Cyp11a1, and 17ß-Hsd3 in the adrenal cortex of CREBZF-cKO mice, while the expression of AR was significantly reduced. Along with the histological changes and abnormal steroid levels in the adrenal gland, CREBZF-cKO mice showed higher anxiety-like behavior and impaired memory in the elevated plus maze and Barnes maze, respectively. In summary, CREBZF is dispensable for fertility, and CREBZF deficiency in Leydig cells promotes adrenal function in adult male mice. These results shed light on the requirement of CREBZF for fertility, adrenal steroid synthesis, and stress response in adult male mice, and contribute to understanding the crosstalk between testes and adrenal glands.
Subject(s)
Adrenal Cortex , Leydig Cells , Mice, Knockout , Animals , Male , Mice , Leydig Cells/metabolism , Adrenal Cortex/metabolism , Androgens/metabolism , Testosterone/blood , Testosterone/metabolism , Behavior, Animal , Mice, Inbred C57BLABSTRACT
Background: Multiple studies have shown that skeletal muscle index (SMI) measured on abdominal computed tomography (CT) is strongly associated with bone mineral density (BMD) and fracture risk as estimated by the fracture risk assessment tool (FRAX). Although some studies have reported that SMI at the level of the 12th thoracic vertebra (T12) measured on chest CT images can be used to diagnose sarcopenia, it is regrettable that no studies have investigated the relationship between SMI at T12 level and BMD or fracture risk. Therefore, we further investigated the relationship between SMI at T12 level and FRAX-estimated BMD and fracture risk in this study. Methods: A total of 349 subjects were included in this study. After 1â¶1 propensity score matching (PSM) on height, weight, hypertension, diabetes, hyperlipidemia, hyperuricemia, body mass index (BMI), age, and gender, 162 subjects were finally included. The SMI, BMD, and FRAX score of the 162 participants were obtained. The correlation between SMI and BMD, as well as SMI and FRAX, was assessed using Spearman rank correlation. Additionally, the effectiveness of each index in predicting osteoporosis was evaluated through the receiver operating characteristic (ROC) curve analysis. Results: The BMD of the lumbar spine (L1-4) demonstrated a strong correlation with SMI (r = 0.416, p < 0.001), while the BMD of the femoral neck (FN) also exhibited a correlation with SMI (r = 0.307, p < 0.001). SMI was significantly correlated with FRAX, both without and with BMD at the FN, for major osteoporotic fractures (r = -0.416, p < 0.001, and r = -0.431, p < 0.001, respectively) and hip fractures (r = -0.357, p < 0.001, and r = -0.311, p < 0.001, respectively). Moreover, the SMI of the non-osteoporosis group was significantly higher than that of the osteoporosis group (p < 0.001). SMI effectively predicts osteoporosis, with an area under the curve of 0.834 (95% confidence interval 0.771-0.897, p < 0.001). Conclusion: SMI based on CT images of the 12th thoracic vertebrae can effectively diagnose osteoporosis and predict fracture risk. Therefore, SMI can make secondary use of chest CT to screen people who are prone to osteoporosis and fracture, and carry out timely medical intervention.
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Ulcerative colitis (UC) is a relapsing and reoccurring inflammatory bowel disease. The treatment effect of Alhagi maurorum and stem cell extracts on UC remains unclear. The aim of the present study was to investigate the protective role of Alhagi maurorum combined with stem cell extract on the intestinal mucosal barrier in an intestinal inflammation mouse model. Sixty mice were randomly divided into a control group, model group, Alhagi group, MSC group, and MSC/Alhagi group. MSC and Alhagi extract were found to reduce the disease activity index (DAI) scores in mice with colitis, alleviate weight loss, improve intestinal inflammation in mice (p < 0.05), preserve the integrity of the ileal wall and increase the number of goblet cells and mucin in colon tissues. Little inflammatory cell infiltration was observed in the Alhagi, MSC, or MSC/Alhagi groups, and the degree of inflammation was significantly alleviated compared with that in the model group. The distribution of PCNA and TNF-alpha in the colonic tissues of the model group was more disperse than that in the normal group (p < 0.05), and the fluorescence intensity was lower. After MSC/Alhagi intervention, PCNA and TNF-alpha were distributed along the cellular membrane in the MSC/Alhagi group (p < 0.05). Compared with that in the normal control group, the intensity was slightly reduced, but it was still stronger than that in the model group. In conclusion, MSC/Alhagi can alleviate inflammatory reactions in mouse colonic tissue, possibly by strengthening the protective effect of the intestinal mucosal barrier.
Subject(s)
Colitis, Ulcerative , Fabaceae , Mesenchymal Stem Cells , Animals , Mice , Colitis, Ulcerative/drug therapy , Stem Cell Factor , Proliferating Cell Nuclear Antigen , Tumor Necrosis Factor-alpha , Inflammation , Plant Extracts/pharmacology , Plant Extracts/therapeutic useABSTRACT
The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.
Subject(s)
Brucellosis , Flagellin , Macrophages , Virulence Factors , Animals , Mice , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Brucella suis/pathogenicity , Brucella suis/immunology , Brucellosis/immunology , Brucellosis/microbiology , Caspase 1/metabolism , Flagellin/metabolism , Inflammasomes/metabolism , Inflammasomes/immunology , Inflammation/immunology , Lipopolysaccharides/immunology , Macrophages/immunology , Macrophages/microbiology , Myeloid Differentiation Factor 88/metabolism , Myeloid Differentiation Factor 88/genetics , NF-kappa B/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , RAW 264.7 Cells , Virulence , Virulence Factors/metabolism , Virulence Factors/geneticsABSTRACT
Introduction: Optimizing the management of dairy cattle reproduction can reduce postpartum ovarian disease in high-yielding dairy cows and thus enhance ranch economic benefit. The hypothesis of this study was that the Double-Ovsynch (DO) protocol in high-producing dairy cows would result in a lower incidence of follicular cysts but a higher incidence of luteal cysts compared to those undergoing the Presynch-Ovsynch (PS) protocol. Methods: In this experiment, 384 cows (204 primiparous and 180 multiparous) were allocated to the DO group, which followed the protocol: GnRH-7d-PGF2α-3d-GnRH-7d-Ovsynch-56 h (GnRH-7d-PGF2α-56 h-GnRH-16hTAI), starting on 39 ± 3 days in milk (DIM). Additionally, 359 cows (176 primiparous and 183 multiparous) were assigned to the PS group, which followed the protocol: PGF2α-14d-PGF2α-12d-Ovsynch-56 h, starting on 31 ± 3 DIM. In DO, B-mode ultrasound examinations were conducted 1 day after the GnRH-7d-PGF2α-3d-GnRH protocol to diagnose the presence of ovarian diseases followed by reexamination after 7 days of suspected cases. In PS, B-mode ultrasound examinations were conducted 1 day after the PGF2α-14d-PGF2α protocol to diagnose the presence of ovarian diseases followed by reexamination after 7 days. For all cows confirmed to having ovarian diseases, a second B-mode ultrasound examination was conducted at the time of the second GnRH and timed artificial insemination (TAI). If the ovary showed a normal developing follicle in combination with normal ovulation, the ovarian disease was considered to be cured. Results: The current study revealed no significant difference in the overall incidence and cure rate of postpartum ovarian diseases between DO and PS (incidence rate: 3.9% vs. 6.7%, cure rate: 50% vs. 41.7%, DO vs. PS). Also, there was no significant difference in the incidence and cure rate of luteal cysts between DO and PS (incidence rate: 2.9% vs. 2.2%, cure rate: 50.0% vs. 50.0%). The incidence of follicular cysts was significantly lower in the DO group than in the PS group (0.8% vs. 2.8%, DO vs. PS, p = 0.037), but there was no significant difference in the cure rates (66.7% vs. 50%). The occurrence of inactive ovary was lower in DO compared to PS (0.2% vs. 1.7%, p = 0.047). There was no significant difference in the pregnancy rate between the DO and PS groups (48.2% vs. 41.8%), although the DO group had a higher rate. What is different from our assumption is that PS did not effectively reduce the incidence of postpartum luteal cysts.
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Endometrial receptivity is critical for the successful establishment of pregnancy in ruminants. Interferon tau (IFNT) plays a key role in promoting embryo attachment by activating the Janus kinase/signal transducer and activator of transcription pathway, which induces the expression of a series of interferon-stimulated genes (ISGs). In our previous study, sequencing analysis of goat endometrial epithelial cells (gEECs) treated with 20 ng/mL IFNT revealed a differentially expressed long non-coding RNA located on the STAT3 antisense chain, which we designated STAT3-AS. The aim of this study was to investigate the role and mechanism of STAT3-AS in establishing endometrial receptivity in goats. The results showed that STAT3-AS was expressed in both the nucleus and cytoplasm of gEECs, and its expression increased significantly in the uterus on day 15 of pregnancy. STAT3-AS expression was upregulated in gEECs treated with IFNT alone or in combination with progesterone and estradiol. Knockdown of STAT3-AS using specific short interfering RNA significantly inhibited the expression of classical ISGs such as interferon-stimulated gene 15 and 2',5'-oligodenylate synthetase 2, as well as uterine endometrial receptivity-related genes including homeobox gene A11, integrin beta 3, and vascular endothelial growth factor. Moreover, gEEC proliferation and the STAT3 pathway were suppressed in the absence of STAT3-AS. However, pretreatment with the STAT3 activator RO8191 restored the effect of silencing STAT3-AS on endometrial receptivity. Overall, these results suggest that STAT3-AS is an important regulator of endometrial receptivity in goats and that it regulates endometrial receptivity through the STAT3 pathway.
Subject(s)
RNA, Long Noncoding , Pregnancy , Female , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Vascular Endothelial Growth Factor A/metabolism , Endometrium/metabolism , Signal Transduction , Ruminants , Goats , Embryo Implantation/physiologyABSTRACT
Identification and functional analysis of key genes regulated by the circadian clock system will provide a comprehensive understanding of the underlying mechanisms through which circadian clock disruption impairs the health of living organisms. The initial phase involved bioinformatics analysis, drawing insights from three RNA-seq datasets (GSE184303, GSE114400, and GSE199061) derived from wild-type mouse liver tissues, which encompassed six distinct time points across a day. As expected, 536 overlapping genes exhibiting rhythmic expression patterns were identified. By intersecting these genes with differentially expressed genes (DEGs) originating from liver RNA-seq data at two representative time points (circadian time, CT: CT2 and CT14) in global Bmal1 knockout mice (Bmal1-/-), hepatocyte-specific Bmal1 knockout mice (L-Bmal1-/-), and their corresponding control groups, 80 genes potentially regulated by BMAL1 (referred to as BMAL1-regulated genes, BRGs) were identified. These genes were significantly enriched in glycolipid metabolism, immune response, and tumorigenesis pathways. Eight BRGs (Nr1d1, Cry1, Gys2, Homer2, Serpina6, Slc2a2, Nmrk1, and Upp2) were selected to validate their expression patterns in both control and L-Bmal1-/- mice livers over 24 h. Real-time quantitative polymerase chain reaction results demonstrated a comprehensive loss of rhythmic expression patterns in the eight selected BRGs in L-Bmal1-/- mice, in contrast to the discernible rhythmic patterns observed in the livers of control mice. Additionally, significant reductions in the expression levels of these selected BRGs, excluding Cry1, were also observed in L-Bmal1-/- mice livers. Chromatin immunoprecipitation (ChIP)-seq (GSE13505 and GSE39860) and JASPAR analyses validated the rhythmic binding of BMAL1 to the promoter and intron regions of these genes. Moreover, the progression of conditions, from basic steatosis to non-alcoholic fatty liver disease, and eventual malignancy, demonstrated a continuous gradual decline in Bmal1 transcripts in the human liver. Combining the aforementioned BRGs with DEGs derived from human liver cancer datasets identified Gys2 and Upp2 as potential node genes bridging the circadian clock system and hepatocellular carcinoma (HCC). In addition, CCK8 and wound healing assays demonstrated that the overexpression of human GYS2 and UPP2 proteins inhibited the proliferation and migration of HepG2 cells, accompanied by elevated expression of p53, a tumor suppressor protein. In summary, this study systematically identified rhythmic genes in the mouse liver, and a subset of circadian genes potentially regulated by BMAL1. Two circadian genes, Gys2 and Upp2, have been proposed and validated as potential candidates for advancing the prevention and treatment of HCC.
Subject(s)
Carcinoma, Hepatocellular , Circadian Clocks , Liver Neoplasms , Animals , Humans , Mice , ARNTL Transcription Factors/genetics , ARNTL Transcription Factors/metabolism , Carcinoma, Hepatocellular/pathology , Circadian Clocks/genetics , Circadian Rhythm/genetics , CLOCK Proteins/genetics , Gene Expression Regulation , Homer Scaffolding Proteins/metabolism , Liver/metabolism , Liver Neoplasms/pathology , Mice, Knockout , Uridine Phosphorylase/metabolism , Glycogen Synthase/metabolismABSTRACT
Sertoli cells play a key role in testicular development and spermatogenesis. It has been suggested that Sertoli cells differentiate after their proliferation ceases. Our previous study showed that miR-34b inhibits proliferation by targeting MAP2K1 mediated MEK/ERK signaling pathway in bovine immature Sertoli cells. Subsequent studies have revealed that the differentiation marker androgen receptor is upregulated during this process. However, the effect of the miR-34b/MEK/ERK pathway on immature bovine Sertoli cell differentiation and the underlying molecular mechanisms are yet to be explored. In this study, we determined that the miR-34b/MEK/ERK pathway was involved in the differentiation of primary Sertoli cells (PSCs) in response to retinoic acid. Transfection of an miR-34b mimic into PSCs promoted cell differentiation, whereas transfection of an miR-34b inhibitor into PSCs delayed it. Pharmacological inhibition of MEK/ERK signaling by AZD6244 promoted PSCs differentiation. Mechanistically, miR-34b promoted PSCs differentiation by inhibiting the MEK/ERK signaling pathway. Through a combination of bioinformatics analysis, dual-luciferase reporter assay, quantitative real-time PCR, and western blotting, nuclear receptor subfamily 5 group A member 1 (NR5A1) was identified as an upstream negative transcription factor of miR-34b. Furthermore, NR5A1 knockdown promoted Sertoli cell differentiation, whereas NR5A1 overexpression had the opposite effect. Together, this study revealed a new NR5A1/miR-34b/MEK/ERK axis that plays a significant role in Sertoli cell differentiation and provides a theoretical and experimental framework for further clarifying the regulation of cell differentiation in bovine PSCs.
Subject(s)
MAP Kinase Signaling System , MicroRNAs , Male , Animals , Cattle , MicroRNAs/genetics , MicroRNAs/metabolism , Sertoli Cells/metabolism , Cell Proliferation , Cell Differentiation , Mitogen-Activated Protein Kinase Kinases/metabolismABSTRACT
BACKGROUND: Diabetes, a leading cause of visual impairment, is on the rise in Canada. We assessed trends in the prevalence of visual impairment among people in Canada with and without diabetes to inform the development of strategies and policies for the management of visual impairment. METHODS: We analyzed self-reported data from respondents aged 45 years and older in 7 cycles of nationwide surveys (National Population Health Survey and Canadian Community Health Survey) from 1994/95 to 2013/14. The age- and sex-standardized prevalence of visual impairment was calculated. We assessed comparisons by levels of education and income, using sex-standardized prevalence owing to sparse data. RESULTS: Among people in Canada with diabetes, the age- and sex-standardized prevalence of visual impairment was 7.37% (95% confidence interval [CI] 5.31%-9.43%) in 1994/95 and 1996/97 combined, decreasing to 3.03% (95% CI 2.48%-3.57%) in 2013/14, giving a standardized prevalence ratio of 0.41 (95% CI 0.30-0.56) comparing 2013/14 with 1994/95 and 1996/97 combined. Among people in Canada without diabetes, visual impairment prevalence decreased from 3.72% (95% CI 3.31%-4.14%) in 1994/95 and 1996/97 combined to 1.69% (95% CI 1.52%-1.87%) in 2013/14, with a standardized prevalence ratio of 0.45 (95% CI 0.40-0.52). Decreased sex-standardized prevalence of visual impairment was observed among people with high and low education levels and incomes among those with and without diabetes. INTERPRETATION: Visual impairment prevalence was roughly 2 times higher among those with versus without diabetes in all survey years; from 1994 to 2014, visual impairment prevalence decreased among those with and without diabetes irrespective of education and income levels. These results suggest effective collective efforts by clinicians, researchers, the public and government.
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After parturition, bovine endometrial epithelial cells (BEECs) undergo serious inflammation and imbalance between oxidation and antioxidation, which is widely acknowledged as a primary contributor to the development of endometritis in dairy cows. Nevertheless, the mechanism of oxidative stress-mediated inflammation and damage in bovine endometrial epithelial cells remains inadequately defined, particularly the molecular pathways associated with mitochondria-dependent apoptosis. Hence, the present study was designed to explore the mechanism responsible for mitochondrial dysfunction-induced BEEC damage. In vivo, the expressions of proapoptotic protein caspase 3 and cytochrome C were increased significantly in dairy uteri with endometritis. Similarly, the levels of proapoptotic protein caspase 3, BAX, and cytochrome C were markedly increased in H2O2-treated BEECs. Our findings revealed pronounced BEEC damage in dairy cows with endometritis, accompanied by heightened expression of cyto-C and caspase-3 both in vivo and in vitro. The reduction in apoptosis-related protein of BEECs due to oxidant injury was notably mitigated following N-acetyl-L-cysteine (NAC) treatment. Furthermore, mitochondrial vacuolation was significantly alleviated, and mitochondrial membrane potential returned to normal levels after the removal of ROS. Excessive ROS may be the main cause of mitochondrial dysfunction. Mitochondrial permeability transition pore (mPTP) blockade by cyclophilin D (CypD) knockdown with CSA significantly blocked the flow of cytochrome C (cyto-C) and Ca2+ to the cytoplasm from the mitochondria. Our results indicate that elevated ROS and persistent opening of the mPTP are the main causes of oxidative damage in BEECs. Collectively our results reveal a new mechanism involving ROS-mPTP signaling in oxidative damage to BEECs, which may be a potential avenue for the clinical treatment of bovine endometritis.
ABSTRACT
Brucella, a zoonotic facultative intracellular pathogenic bacterium, poses a significant threat both to human health and to the development of the livestock industry. Alanine racemase (Alr), the enzyme responsible for alanine racemization, plays a pivotal role in regulating virulence in this bacterium. Moreover, Brucella mutants with alr gene deletions (Δalr) exhibit potential as vaccine candidates. However, the mechanisms that underlie the detrimental effects of alr knockouts on Brucella pathogenicity remain elusive. Here, initially, we conducted a bioinformatics analysis of Alr, which demonstrated a high degree of conservation of the protein within Brucella spp. Subsequent metabolomics studies unveiled alterations in amino acid pathways following deletion of the alr gene. Furthermore, alr deletion in Brucella suis S2 induced decreased resistance to stress, antibiotics, and other factors. Transmission electron microscopy of simulated macrophage intracellular infection revealed damage to the cell wall in the Δalr strain, whereas propidium iodide staining and alkaline phosphatase and lactate dehydrogenase assays demonstrated alterations in cell membrane permeability. Changes in cell wall properties were revealed by measurements of cell surface hydrophobicity and zeta potential. Finally, the diminished adhesion capacity of the Δalr strain was shown by immunofluorescence and bacterial enumeration assays. In summary, our findings indicate that the alr gene that regulates amino acid metabolism in Brucella influences the properties of the cell wall, which modulates bacterial adherence capability. This study is the first demonstration that Alr impacts virulence by modulating bacterial metabolism, thereby providing novel insights into the pathogenic mechanisms of Brucella spp.
Subject(s)
Alanine Racemase , Brucella , Brucellosis , Humans , Alanine Racemase/genetics , Alanine Racemase/chemistry , Alanine Racemase/metabolism , Brucella/metabolism , Anti-Bacterial Agents , Cell Wall/metabolism , Amino AcidsABSTRACT
Brucellosis is a notorious zoonotic disease caused by Brucella, which can lead to reproductive diseases in humans and animals, such as infertility and abortion. Lipopolysaccharides (LPS) are the main virulence factor of Brucella. LPS derived from Brucella are different and non-classical and are less toxic and less active than LPS isolated from E. coli. However, the effects and possible mechanisms of Brucella LPS-caused pregnancy loss remain to be revealed. In the present study, we investigated the effects of Brucella suis S2 LPS on early pregnancy loss in mice. The results indicated that embryo implantation failure was induced by Brucella LPS treatment in a dose-dependent manner. The injection of Brucella LPS mainly resulted in fibrinolysis in the decidual area of the uterus on the 6th day post coition (dpc), infiltration of large granular cells among the decidual cells near the embryo on the 8th dpc, a large number of gaps in the decidual area, and cell necrosis around the embryo. In addition, the expression of Cyclin D3 mRNA in the uterus on the 7th and 8th dpc and IGFBP-1 mRNA and the progesterone receptor in the uterus on the 6th and 7th dpc were also inhibited. Moreover, the expression of decidualization marker Cyclin D3 and decidualization prolactin-associated protein (dPRP) in endometrial stromal cells were also inhibited by Brucella LPS treatment in vitro. In summary, Brucella LPS affect the process of endometrial decidualization in mice by affecting the structure of the decidua and the expression of decidual marker factors in endometrial stromal cells.
Subject(s)
Brucella suis , Decidua , Pregnancy , Humans , Female , Mice , Animals , Decidua/metabolism , Lipopolysaccharides/pharmacology , Brucella suis/metabolism , Cyclin D3/metabolism , Escherichia coli/metabolism , Uterus , RNA, Messenger/metabolismABSTRACT
Trophoblast plays a crucial role in gestation maintenance and embryo implantation, partly due to the synthesis of progesterone. It has been demonstrated that hypoxia regulates invasion, proliferation, and differentiation of trophoblast cells. Additionally, human trophoblasts display rhythmic expression of circadian clock genes. However, it remains unclear if the circadian clock system is present in goat trophoblast cells (GTCs), and its involvement in hypoxia regulation of steroid hormone synthesis remains elusive. In this study, immunofluorescence staining revealed that both BMAL1 and NR1D1 (two circadian clock components) were highly expressed in GTCs. Quantitative real-time PCR analysis showed that several circadian clock genes were rhythmically expressed in forskolin-synchronized GTCs. To mimic hypoxia, GTCs were treated with hypoxia-inducing reagents (CoCl2 or DMOG). Quantitative real-time PCR results demonstrated that hypoxia perturbed the mRNA expression of circadian clock genes and StAR. Notably, the increased expression of NR1D1 and the reduction of StAR expression in hypoxic GTCs were also detected by western blotting. In addition, progesterone secretion exhibited a notable decline in hypoxic GTCs. SR9009, an NR1D1 agonist, significantly decreased StAR expression at both the mRNA and protein levels and markedly inhibited progesterone secretion in GTCs. Moreover, SR8278, an NR1D1 antagonist, partially reversed the inhibitory effect of CoCl2 on mRNA and protein expression levels of StAR and progesterone synthesis in GTCs. Our results demonstrate that hypoxia reduces StAR expression via the activation of NR1D1 signaling in GTCs, thus inhibiting progesterone synthesis. These findings provide new insights into the NR1D1 regulation of progesterone synthesis in GTCs under hypoxic conditions.
Subject(s)
Progesterone , Trophoblasts , Animals , Humans , Trophoblasts/metabolism , Goats/genetics , Hypoxia , RNA, Messenger , Cobalt , Nuclear Receptor Subfamily 1, Group D, Member 1/genetics , Nuclear Receptor Subfamily 1, Group D, Member 1/metabolismABSTRACT
Brucella abortus is facultative intracellular pathogen that causes chronic persistent infections and results in abortion and infertility in food animals. Recurrent infections can be one of the results of persister cells formation that transiently displays phenotypic tolerance to high dose of antibiotics treatment. We examined persister cells formation of B. abortus strain A19 in stationary phase and investigated a potential role for the (p)ppGpp synthetase Rsh in this process. We found that B. abortus stationary phase cells can produce higher levels of multi-drugs tolerant persister cells in vitro under high dose of antibiotics (20 × MIC) exposure than do exponential phase cells. Persister cell formation was also induced with environmental stressors pH 4.5, 0.01 M PBS (pH7.0), 2% NaCl and 25 °C, upon exposure to ampicillin, enrofloxacin and rifampicin. Persister cells were not formed following exposure to 1 mM H2O2. The numbers of persister cells were significantly increased following uptake of B. abortus stationary phase cells by RAW264.7 macrophages in contrast with cultures in TSB liquid medium. Environmental stressors to B. abortus significantly increased expression of rsh mRNA level. The rsh null mutant (Δrsh) formed significantly fewer persister cells than the complemented (CΔrsh) and wildtype (WT) strains under high dose of rifampicin in vitro. These data for the first time demonstrate that B. abortus can produce multi-drug tolerant persister cells in stationary phase. The (p)ppGpp synthetase Rsh is necessary for persister cell formation in B. abortus in the presence of rifampicin. On this basis, a new understanding of the recurrent infections of Brucella was advanced, thus provided a new basis for revelation of pathogenic mechanism of the chronic persistent infection in Brucella.
Subject(s)
Brucella abortus , Rifampin , Female , Pregnancy , Animals , Brucella abortus/genetics , Rifampin/pharmacology , Hydrogen Peroxide , Reinfection , Anti-Bacterial Agents/pharmacologyABSTRACT
Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain's rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella's survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.