Development of an integrated and project-based laboratory course in upper-level biochemistry and molecular biology.

An integrated and projected-based laboratory course was described, integrating interconnected knowledge points and biochemistry and molecular biology techniques on a research project-based system. The program, which served as an essential extension of theoretical courses to practice, was conducted with a sophomore of basic medical science who had completed the course in medical biochemistry and molecular biology. This course engaged students in learning "genetic manipulation" and "recombinant DNA technology" to understand the target gene's role in disease mechanics, thus altering evaluation and treatment for clinical disease. Students could master applied and advanced techniques, such as cell culture, transfection, inducing exogenous fusion protein expression, purifying protein and its concentration assay, quantitative polymerase chain reaction, and western bot analysis. This laboratory exercise links laboratory practices with the methods of current basic research. Students need to complete the experimental design report and laboratory report, which could be advantageous for improving their ability to write lab summaries and scientific papers in the future. The reliability and validity analyses were conducted on the questionnaire, and we examined students' satisfaction with the course and their gains from the course. The student feedback was generally positive, indicating that the exercise helped consolidate theoretical knowledge, increase scientific research enthusiasm, and provide a powerful tool to be a better person and make informed decisions.

Metformin protects ovarian granulosa cells in chemotherapy-induced premature ovarian failure mice through AMPK/PPAR-γ/SIRT1 pathway.

Premature ovarian failure (POF) caused by chemotherapy is a growing concern for female reproductive health. The use of metformin (MET), which has anti-oxidative and anti-inflammatory effects, in the treatment of POF damaged by chemotherapy drugs remains unclear. In this study, we investigated the impact of MET on POF caused by cyclophosphamide (CTX) combined with busulfan (BUS) and M1 macrophages using POF model mice and primary granule cells (GCs). Our findings demonstrate that intragastric administration of MET ameliorates ovarian damage and alleviates hormonal disruption in chemotherapy-induced POF mice. This effect is achieved through the reduction of inflammatory and oxidative stress-related harm. Additionally, MET significantly relieves abnormal inflammatory response, ROS accumulation, and senescence in primary GCs co-cultured with M1 macrophages. We also observed that this protective role of MET is closely associated with the AMPK/PPAR-γ/SIRT1 pathway in cell models. In conclusion, our results suggest that MET can protect against chemotherapy-induced ovarian injury by inducing the expression of the AMPK pathway while reducing oxidative damage and inflammation.

Inhibition of PRKAA/AMPK (Ser485/491) phosphorylation by crizotinib induces cardiotoxicity via perturbing autophagosome-lysosome fusion.

Crizotinib, a small-molecule tyrosine kinase inhibitor targeting ALK, MET and ROS1, is the first-line drug for ALK-positive metastatic non-small cell lung cancer and is associated with severe, sometimes fatal, cases of cardiac failure, which increases the risk of mortality. However, the underlying mechanism remains unclear, which causes the lack of therapeutic strategy. We established in vitro and in vivo models for crizotinib-induced cardiotoxicity and found that crizotinib caused left ventricular dysfunction, myocardial injury and pathological remodeling in mice and induced cardiomyocyte apoptosis and mitochondrial injury. In addition, we found that crizotinib prevented the degradation of MET protein by interrupting autophagosome-lysosome fusion and silence of MET or re-activating macroautophagy/autophagy flux rescued the cardiomyocytes death and mitochondrial injury caused by crizotinib, suggesting that impaired autophagy activity is the key reason for crizotinib-induced cardiotoxicity. We further confirmed that recovering the phosphorylation of PRKAA/AMPK (Ser485/491) by metformin re-activated autophagy flux in cardiomyocytes and metformin rescued crizotinib-induced cardiomyocyte injury and cardiac complications. In summary, we revealed a novel mechanism for crizotinib-induced cardiotoxicity, wherein the crizotinib-impaired autophagy process causes cardiomyocyte death and cardiac injury by inhibiting the degradation of MET protein, demonstrated a new function of impeded autophagosome-lysosome fusion in drugs-induced cardiotoxicity, pointed out the essential role of the phosphorylation of PRKAA (Ser485/491) in autophagosome-lysosome fusion and confirmed metformin as a potential therapeutic strategy for crizotinib-induced cardiotoxicity.Abbreviations and Acronyms: AAV: adeno-associated virus; ACAC/ACC: acetyl-Co A carboxylase; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATG7: autophagy related 7; CHX: cycloheximide; CKMB: creatine kinase myocardial band; CQ: chloroquine; c-PARP: cleaved poly (ADP-ribose) polymerase; DAPI: 4'6-diamidino-2-phenylindole; EF: ejection fraction; FOXO: forkhead box O; FS: fractional shortening; GSEA: gene set enrichment analysis; H&E: hematoxylin and eosin; HF: heart failure; HW: TL: ratio of heart weight to tibia length; IR: ischemia-reperfusion; KEGG: Kyoto encyclopedia of genes and genomes; LAMP2: lysosomal-associated membrane protein 2; LDH: lactate dehydrogenase; MCMs: mouse cardiomyocytes; MMP: mitochondrial membrane potential; mtDNA: mitochondrial DNA; MYH6: myosin, heavy peptide 6, cardiac muscle, alpha; MYH7: myosin, heavy peptide 7, cardiac muscle, beta; NPPA: natriuretic peptide type A; NPPB: natriuretic peptide type B; PI: propidium iodide; PI3K: phosphoinositide 3-kinase; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; qPCR: quantitative real-time PCR; SD: standard deviation; SRB: sulforhodamine B; TKI: tyrosine kinase inhibitor; WGA: wheat germ agglutinin.

ceRNA networks in gynecological cancers progression and resistance.

Gynecological cancers are the second most common types of cancer in women. Clinical diagnosis of these cancers is often delayed or misdiagnosed due to lack of insight into their tumorigenesis mechanism and specific diagnostic biomarkers. Many studies have demonstrated that competing endogenous RNAs (ceRNAs) modulate the progression and resistance of gynecological cancer through microRNA (miRNA)-mediated mechanisms, which affect gene expression in multiple cancer-related pathways. Here we review studies on the involvement of the ceRNA hypothesis in the progression and resistance of gynaecological cancers to validate some ceRNAs as therapeutic targets and predictive biomarkers.

Mechanisms of and Potential Medications for Oxidative Stress in Ovarian Granulosa Cells: A Review.

Granulosa cells are essential for follicle initiation and development, and their abnormal function or apoptosis is a crucial factor leading to follicular atresia. A state of oxidative stress occurs when the balance between the production of reactive oxygen species and the regulation of the antioxidant system is disturbed. Oxidative stress is one of the most important causes of the abnormal function and apoptosis of granulosa cells. Oxidative stress in granulosa cells causes female reproductive system diseases, such as polycystic ovary syndrome and premature ovarian failure. In recent years, studies have confirmed that the mechanism of oxidative stress in granulosa cells is closely linked to the PI3K-AKT signaling pathway, MAPK signaling pathway, FOXO axis, Nrf2 pathway, NF-κB signaling pathway, and mitophagy. It has been found that drugs such as sulforaphane, Periplaneta americana peptide, and resveratrol can mitigate the functional damage caused by oxidative stress on granulosa cells. This paper reviews some of the mechanisms involved in oxidative stress in granulosa cells and describes the mechanisms underlying the pharmacological treatment of oxidative stress in granulosa cells.

Crizotinib induces pulmonary toxicity by blocking autophagy flux in alveolar epithelial cells.

Crizotinib is the first-line drug for advanced non-small cell lung cancer with the abnormal expression of anaplastic lymphoma kinase gene. Severe, life-threatening, or fatal interstitial lung disease/pneumonia has been reported in patients treated with crizotinib. The clinical benefit of crizotinib is limited by its pulmonary toxicity, but the underlying mechanisms have not been adequately studied, and protective strategies are relatively scarce. Here, we established an in vivo mouse model in which crizotinib was continuously administered to C57BL/6 at 100 mg/kg/day for 6 weeks and verified that crizotinib induced interstitial lung disease in vivo, which was consistent with the clinical observations. We further treated BEAS-2B and TC-1 cells, the alveolar epithelial cell lines, with crizotinib and found the increased apoptosis rate. We proved that crizotinib-blocked autophagic flux caused apoptosis of the alveolar epithelial cells and then promoted the recruitment of immune cells, suggesting that limited autophagy activity was the key reason for pulmonary injury and inflammation caused by crizotinib. Subsequently, we found that metformin could reduce the macrophage recruitment and pulmonary fibrosis by recovering the autophagy flux, thereby ameliorating impaired lung function caused by crizotinib. In conclusion, our study revealed the mechanism of crizotinib-induced apoptosis of alveolar epithelial cells and activation of inflammation during the onset of pulmonary toxicity and provided a promising therapeutic strategy for the treatment of crizotinib-induced pulmonary toxicity.

Evaluating the impact of radiofrequency spectroscopy on reducing reoperations after breast conserving surgery: A meta-analysis.

BACKGROUND: The benefits of breast conserving surgery for breast cancer patients are well established. To achieve adequate margins of excision, intraoperative management of breast margins is a critical factor through reducing reoperation for inadequate positive margin excision and associated morbidity and cost. Radiofrequency spectroscopy is a technology that could significantly reduce positive margins when used intraoperatively as an adjunct to other margin management methods. METHODS: A meta-analysis was completed with 10 publications comparing use of radiofrequency spectroscopy technology (MarginProbe) with standard margin assessment procedures. Three randomized controlled studies and seven retrospective studies comparing MarginProbe to historical controls were included. The primary endpoint was reduction of re-excision rates. Statistical significance level was set at the two-sided 5% level corresponding to two-sided 95% confidence intervals (CIs) of the pooled relative risk estimates. RESULTS: A total of 2335 patients from 10 publications were included in this meta-analysis. The overall relative reduction in re-excision rate was 0.49 (95% CI: 0.38-0.64, p < 0.001). Statistical methods were used to examine publication bias. CONCLUSION: Despite the limited randomized controlled trials available comparing radiofrequency spectroscopy to standard operation procedures, the data from the 10 studies demonstrate a statistically significant reduction in re-excision rate of 49% for MarginProbe usage, currently the only technology indicated for intraoperative identification of breast cancer tissue at the lumpectomy specimen margin.

PTX3 from vascular endothelial cells contributes to trastuzumab-induced cardiac complications.

AIMS: Trastuzumab, the first humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (ERBB2/HER2), is currently used as a first-line treatment for HER2 (+) tumours. However, trastuzumab increases the risk of cardiac complications without affecting myocardial structure, suggesting a distinct mechanism of cardiotoxicity. METHODS AND RESULTS: We used medium from trastuzumab-treated human umbilical vein endothelial cells (HUVECs) to treat CCC-HEH-2 cells, the human embryonic cardiac tissue-derived cell lines, and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to assess the crosstalk between vascular endothelial cells (VECs) and cardiomyocytes. Protein mass spectrometry analysis was used to identify the key factors from VECs that regulate the function of cardiomyocytes. We applied RNA-sequencing to clarify the mechanism, by which PTX3 causes cardiac dysfunction. We used an anti-human/rat HER2 (neu) monoclonal antibody to generate a rat model that was used to evaluate the effects of trastuzumab on cardiac structure and function and the rescue effects of lapatinib on trastuzumab-induced cardiac side effects. Medium from trastuzumab-treated HUVECs apparently impaired the contractility of CCC-HEH-2 cells and iPSC-CMs. PTX3 from VECs caused defective cardiomyocyte contractility and cardiac dysfunction in mice, phenocopying trastuzumab treatment. PTX3 affected calcium homoeostasis in cardiomyocytes, which led to defective contractile properties. EGFR/STAT3 signalling in VECs contributed to the increased expression and release of PTX3. Notably, lapatinib, a dual inhibitor of EGFR/HER2, could rescue the cardiac complications caused by trastuzumab by blocking the release of PTX3. CONCLUSION: We identified a distinct mode of cardiotoxicity, wherein the activation of EGFR/STAT3 signalling by trastuzumab in VECs promotes PTX3 excretion, which contributes to the impaired contractility of cardiomyocytes by inhibiting cellular calcium signalling. We confirmed that lapatinib could be a feasible preventive agent against trastuzumab-induced cardiac complications and provided the rationale for the combined application of lapatinib and trastuzumab in cancer therapy.

Role of ceRNAs in non-tumor female reproductive diseases†.

The molecular mechanism of non-tumor female reproductive diseases is complicated and needs to be further elucidated. Recently, increasing evidence indicates that non-coding RNAs(ncRNAs) which are extremely rich in the female reproductive system are crucial factors in the pathogenesis of some female reproductive disorders. In fact, these ncRNAs such as lncRNAs, circRNAs, snoRNAs, and pseudogenes that share the same miRNA response elements (MREs) with mRNAs could compete for miRNA binding site to regulate gene expression, this phenomenon is known as the competing endogenous RNAs(ceRNAs) mechanism. This review aims to summarize the role of ceRNAs in cell proliferation, apoptosis, migration, and invasion of non-tumor female reproductive diseases such as polycystic ovary syndrome (PCOS), premature ovarian failure (POF), pre-eclampsia (PE), recurrent implantation failure (RIF), recurrent spontaneous abortion (RSA), endometriosis (EM), and endometritis, and list ceRNAs regulatory axes as well as downstream related signaling pathway. Additionally, based on certain ncRNAs that have already been proven to exist at differential levels in patient tissue samples, we also generalize some ncRNAs that can be used as potential biomarkers and therapeutic targets for these diseases in the future.

UFL1 alleviates ER stress and apoptosis stimulated by LPS via blocking the ferroptosis pathway in human granulosa-like cells.

Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) is a unique E3 ligase of the UFMylation system. Recent studies have shown that this enzyme plays a crucial role in the processes of endoplasmic reticulum stress (ER stress) and apoptosis. Lipopolysaccharide (LPS) can cause injury to ovarian granule cells and hinder follicular development by triggering ER stress and apoptosis. Our study aimed to investigate the mechanism by which UFL1 alleviates ER stress and apoptosis caused by LPS in human granulosa-like cells (KGNs). In this study, we found that the protein levels of UFL1 were increased obviously under LPS stimulation in KGNs and that ER stress and apoptosis were further aggravated when UFL1 was knocked down; in contrast, these events were rescued when UFL1 was overexpressed. Next, we showed that the levels of ferroptosis-related proteins were relatively altered, accompanied by the accumulation of reactive oxygen species (ROS) and Fe2+, following the inhibition of UFL1 expression. In contrast, the overexpression of UFL1 reversed the ferroptosis process by regulating the P53/SLC7A11 (solute carrier family 7, member 11, SLC7A11) system and autophagy in response to LPS stimulation. Furthermore, apoptosis and ER stress in KGNs are rescued by the administration of the ferroptosis inhibitor ferrostatin-1 (Fer-1). Collectively, our research demonstrated a new mechanism for UFL1 that can alleviate ER stress and apoptosis stimulated by LPS; this occurred via the regulation of the ferroptosis pathway in KGNs and may provide a new strategy for research in the field of reproduction.

Application of artificial intelligence in the diagnosis and prognostic prediction of ovarian cancer.

In recent years, the wide application of artificial intelligence (AI) has dramatically improved the work efficiency of clinicians and reduced their workload. This review provides a glance at the latest advances in AI-assisted diagnosis and prognostic prediction of ovarian cancer (OC). We performed an advanced search in PubMed and IEEE/IET Electronic Library, and included 39 articles in this review. A comprehensive and objective criterion was built to assess the reliability and quality of all studies from four aspects: the size of datasets for model development, research design, the division of training sets and test sets, and the type of quantitative performance indicators. This review analyzed the construction of AI models, including data pre-processing methods, feature selection techniques, AI classifiers, or algorithms. Additionally, we compared the performance of these models built on different datasets, which may support researchers for further iteration and development of AI. Finally, we discussed the challenges and future directions for AI application in medicine.

Ubiquitin-like modifier 1 ligating enzyme 1 relieves cisplatin-induced premature ovarian failure by reducing endoplasmic reticulum stress in granulosa cells.

BACKGROUND: Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1), the ligase of the UFMylation system, has recently been reported to be involved in apoptosis and endoplasmic reticulum stress (ER stress) in a variety of diseases. Premature ovarian failure (POF) is a gynecological disease that severely reduces the fertility of women, especially in female cancer patients receiving chemotherapy drugs. Whether UFL1 is involved in protection against chemotherapy-induced POF and its mechanism remain unclear. METHODS: In this study, we examined the function of UFL1 in ovarian dysfunction and granulosa cell (GC) apoptosis induced by cisplatin through histological examination and cell viability analysis. We used western blotting, quantitative real-time PCR (qPCR) and immunofluorescence (IF) to detect the expression of UFL1 and the levels of ER stress specific markers. Enzyme linked immunosorbent assays were used to detect the levels of follicle-stimulating hormone (FSH) and estrogen (E2) in ovaries and GCs. In addition, we used infection with lentiviral particle suspensions to knock down and overexpress UFL1 in ovaries and GCs, respectively. RESULTS: Our data showed that the expression of UFL1 was reduced in POF model ovaries, accompanied by ER stress. In vitro, cisplatin induced a stress-related increase in UFL1 expression in GCs and enhanced ER stress, which was aggravated by UFL1 knockdown and alleviated by UFL1 overexpression. Furthermore, UFL1 knockdown resulted in a decrease in ovarian follicle number, an increase in atretic follicles, and decreased expression of AMH and FSHR. Conversely, the overexpression of UFL1 reduced cisplatin-induced damage to the ovary in vitro. CONCLUSIONS: Our research indicated that UFL1 regulates cisplatin-induced ER stress and apoptosis in GCs, and participates in protection against cisplatin-induced POF, providing a potential therapeutic target for the clinical prevention of chemotherapeutic drug-induced POF.

Bisdemethoxycurcumin alleviates vandetanib-induced cutaneous toxicity in vivo and in vitro through autophagy activation.

High incidence of cutaneous toxicity ranging from 29.2% to 71.2% has been reported during clinical use of vandetanib, which is a multi-target kinase inhibitor indicated for the treatment of unresectable medullary thyroid carcinoma. The cutaneous toxicity of vandetanib has limited its clinical benefits, but the underlying mechanisms and protective strategies are not well studied. Hence, we firstly established an in vivo model by continuously administrating vandetanib at 55 mg/kg/day to C57BL/6 for 21 days and verified that vandetanib could induce skin rash in vivo, which was consistent with the clinical study. We further cultured HaCaT and NHEK cells, the immortalized or primary human keratinocyte line, and investigated vandetanib (0-10 µM, 0-24 h)-caused alteration in cellular survival and death processes. The western blot showed that the expression level of apoptotic-related protein, c-PARP, c-Caspase 3 and Bax were increased, while the anti-apoptotic protein Bcl2 and MCL1 level were decreased. Meanwhile, vandetanib downregulated mitochondrial membrane potential which in turn caused the release of Cytochrome C, excessive production of reactive oxygen species and DNA damage. Furthermore, we found that 5 µM bisdemethoxycurcumin partially rescued vandetanib-induced mitochondria pathway-dependent keratinocyte apoptosis via activation of autophagy in vivo and in vitro, thereby ameliorated cutaneous toxicity. Conclusively, our study revealed the mechanisms of vandetanib-induced apoptosis in keratinocytes during the occurrence of cutaneous toxicity, and suggested bisdemethoxycurcumin as a potential protective drug. This work provided a potentially promising therapeutic strategy for the treatment of vandetanib-induced cutaneous toxicity.

Hydrogen-rich saline mitigates pressure overload-induced cardiac hypertrophy and atrial fibrillation in rats via the JAK-STAT signalling pathway.

OBJECTIVE: To investigate if hydrogen-rich saline (HRS), which has been shown to have antioxidant and anti-inflammatory properties, could mitigate cardiac remodelling and reduce the incidence of atrial fibrillation (AF) in the rat model of cardiac hypertrophy. METHODS: Pressure overload was induced in rats by abdominal aortic constriction (AAC). The animals were separated into four groups: sham; AAC group; AAC plus low dose HRS (LHRS); AAC plus high dose HRS (HHRS). The sham and AAC groups received normal saline intraperitoneally and the LHRS and HHRS groups received 3 or 6 ml/kg HRS daily for six weeks, respectively. In vitro research was also performed using cardiotrophin-1 (CT-1)-induced hypertrophy of cultured neonatal rat cardiomyocytes. RESULTS: Cardiac hypertrophy was successfully induced by AAC and low and high dose HRS mitigated the pressure overload as shown by lower heart and atrial weights in these treatment groups. AF incidence and duration of the HRS groups were also significantly lower in the HRS groups compared with the AAC group. Atrial fibrosis was also reduced in the HRS groups and the JAK-STAT signalling pathway was down-regulated. In vitro experiments showed that hydrogen-rich medium mitigated the CT-1-induced cardiomyocyte hypertrophy with a similar effect as the JAK specific antagonists AG490. CONCLUSIONS: HRS was found to mitigate cardiac hypertrophy induced by pressure overload in rats and reduce atrial fibrosis and AF which was possibly achieved via inhibition of the JAK-STAT signalling pathway.

Essential Role of Ubiquitin-Fold Modifier 1 Conjugation in DNA Damage Response.

Both endogenous and exogenous factors can cause DNA damage that compromises genomic integrity and cell viability. A proper DNA damage response (DDR) plays a role in maintaining genome stability and preventing tumorigenesis. DNA double-strand breaks (DSBs) are the most toxic DNA lesion, whose response is dominated by the ataxia-telangiectasia mutated (ATM) protein kinase. After being activated by the sensor Mre11-Rad50-Nbs1 (MRN) complex or acetyltransferase Tip60, ATM rapidly phosphorylates downstream targets to launch DDR signaling when DNA is damaged. However, the exact mechanism of DDR is complex and ambiguous. Ufmylation, one type of ubiquitin-like modification, proceeds mainly through a three-step enzymatic reaction to help ubiquitin-fold modifier 1 (Ufm1), attach to substrates with ubiquitin-like modifier-activating enzyme 5 (Uba5), Ufm1-conjugating enzyme 1 (Ufc1) and Ufm1-specific ligase 1 (Ufl1). Although ubiquitination is essential to the DSBs response, the potential function of ufmylation in DDR is largely unknown. Herein, we review the relationship between ufmylation and DDR to elucidate the function and mechanism of ufmylation in DDR, which would reveal the pathogenesis of some diseases and provide new guidance to create a therapeutic method.

Hedgehog pathway inhibition causes primary follicle atresia and decreases female germline stem cell proliferation capacity or stemness.

BACKGROUND: Follicle depletion is one of the causes of premature ovarian failure (POF) and primary ovarian insufficiency (POI). Hence, maintenance of a certain number of female germline stem cells (FGSCs) is optimal to produce oocytes and replenish the primordial follicle pool. The mechanism that regulates proliferation or stemness of FGSCs could contribute to restoring ovarian function, but it remains uncharacterized in postnatal mammalian ovaries. This study aims to investigate the mechanism by which inhibiting the activity of the hedgehog (Hh) signaling pathway regulates follicle development and FGSC proliferation. METHODS AND RESULTS: To understand the role of the Hh pathway in ovarian aging, we measured Hh signaling activity at different reproductive ages and the correlation between them in physiological and pathological mice. Furthermore, we evaluated the follicle number and development and the changes in FGSC proliferation or stemness after blocking the Hh pathway in vitro and in vivo. In addition, we aimed to explain one of the mechanisms for the FGSC phenotype changes induced by treatment with the Hh pathway-specific inhibitor GANT61 via oxidative stress and apoptosis. The results show that the activity of Hh signaling is decreased in the ovaries in physiological aging and POF models, which is consistent with the trend of expression levels of the germline stem cell markers Mvh and Oct4. In vitro, blocking the Hh pathway causes follicular developmental disorders and depletes ovarian germ cells and FGSCs after treating ovaries with GANT61. The proliferation or stemness of cultured primary FGSCs is reduced when Hh activity is blocked. Our results show that the antioxidative enzyme level and the ratio of Bcl-2/Bax decrease, the expression level of caspase 3 increases, the mitochondrial membrane potential is abnormal, and ROS accumulate in this system. CONCLUSIONS: We observed that the inhibition of the Hh signaling pathway with GANT61 could reduce primordial follicle number and decrease FGSC reproductive capacity or stemness through oxidative damage and apoptosis.

Resveratrol Plays a Protective Role against Premature Ovarian Failure and Prompts Female Germline Stem Cell Survival.

This study was designed to investigate the protective effect of resveratrol (RES) on premature ovarian failure (POF) and the proliferation of female germline stem cells (FGSCs) at the tissue and cell levels. POF mice were lavaged with RES, and POF ovaries were co-cultured with RES and/or GANT61 in vitro. FGSCs were pretreated with Busulfan and RES and/or GANT61 and co-cultured with M1 macrophages, which were pretreated with RES. The weights of mice and their ovaries, as well as their follicle number, were measured. Ovarian function, antioxidative stress, inflammation, and FGSCs survival were evaluated. RES significantly increased the weights of POF mice and their ovaries as well as the number of follicles, while it decreased the atresia rate of follicles. Higher levels of Mvh, Oct4, SOD2, GPx, and CAT were detected after treatment with RES in vivo and in vitro. RES treatment resulted in significantly lower TNF-α and IL-6 concentrations and an obviously higher IL-10 concentration in the ovaries. In FGSCs, higher Mvh, Oct4, and SOD2 concentrations and lower TNF-α, IL-6, and MDA concentrations were measured in the RES group. Blockage of the Hh signaling pathway reversed the protective effect of RES on FGSCs. In conclusion, RES effectively improved the ovarian function of the POF model and the productive capacity of FGSCs via relieving oxidative stress and inflammation and a mechanism involving the Hh signaling pathway, suggesting that RES is a potential agent against POF and can aid in the survival of FGSCs.

Ufl1/RCAD, a Ufm1 E3 ligase, has an intricate connection with ER stress.

Ufmylation is a type of post-translational modification that deals with complex and fine-tuned cellular activities. This modification proceeds mainly through a three-step enzymatic reaction with ubiquitin-fold modifier 1 (Ufm1), ubiquitin-like modifier-activating enzyme 5 (Uba5), Ufm1-conjugating enzyme 1 (Ufc1) and Ufm1-specific ligase 1 (Ufl1). Ufl1 has previously been reported to function as a Ufm1 E3 ligase in the ufmylation system, but knowledge of its physiological functions remains poor. At the subcellular level, Ufl1 is enriched in the endoplasmic reticulum (ER), implying that it may regulate events closely associated with the ER and ER functions, such as protein synthesis, folding, and secretion, compounding lipids or sterols, and maintaining calcium homeostasis. Different physiological or pathological stress circumstances can, however, disrupt ER homeostasis, giving rise to an incongruous condition that is harmful to cellular activity and ultimately causes ER stress. Understanding the relationship between Ufl1 and ER stress in physiology and pathology may reveal the pathogenesis of some diseases and provide new guidance to create a therapeutic method. Herein, we review the current literature and discuss the relationship between Ufl1 and ER stress (in hematopoietic disease, heart disease, etc.), thus providing insight into additional diseases.

Indispensable role of the Ubiquitin-fold modifier 1-specific E3 ligase in maintaining intestinal homeostasis and controlling gut inflammation.

Intestinal exocrine secretory cells, including Paneth and goblet cells, have a pivotal role in intestinal barrier function and mucosal immunity. Dysfunction of these cells may lead to the pathogenesis of human diseases such as inflammatory bowel disease (IBD). Therefore, identification and elucidation of key molecular mechanisms that regulate the development and function of these exocrine cells would be crucial for understanding of disease pathogenesis and discovery of new therapeutic targets. The Ufm1 conjugation system is a novel ubiquitin-like modification system that consists of Ufm1 (Ubiquitin modifier 1), Uba5 (Ufm1-activating enzyme, E1), Ufc1 (Ufm1-conjugating enzyme, E2) and poorly characterized Ufm1 E3 ligase(s). Recent mouse genetic studies have demonstrated its indispensable role in embryonic development and hematopoiesis. Yet its role in other tissues and organs remains poorly defined. In this study, we found that both Ufl1 and Ufbp1, two key components of the Ufm1 E3 ligase, were highly expressed in the intestinal exocrine cells. Ablation of either Ufl1 and Ufbp1 led to significant loss of both Paneth and goblet cells, which in turn resulted in dysbiotic microbiota and increased susceptibility to experimentally induced colitis. At the cellular and molecular levels, Ufbp1 deficiency caused elevation of endoplasmic reticulum stress and activation of the Unfolded Protein Response (UPR) and cell death program. Administration of small molecular chaperone partially prevented loss of Paneth cells caused by acute Ufbp1 deletion. Taken together, our results have provided unambiguous evidence for the crucial role of the Ufm1 E3 ligase in maintenance of intestinal homeostasis and protection from inflammatory diseases.

Role of the Hedgehog Signaling Pathway in Regulating the Behavior of Germline Stem Cells.

Germline stem cells (GSCs) are adult stem cells that are responsible for the production of gametes and include spermatogonial stem cells (SSCs) and ovarian germline stem cells (OGSCs). GSCs are located in a specialized microenvironment in the gonads called the niche. Many recent studies have demonstrated that multiple signals in the niche jointly regulate the proliferation and differentiation of GSCs, which is of significance for reproductive function. Previous studies have demonstrated that the hedgehog (Hh) signaling pathway participates in the proliferation and differentiation of various stem cells, including GSCs in Drosophila and male mammals. Furthermore, the discovery of mammalian OGSCs challenged the traditional opinion that the number of primary follicles is fixed in postnatal mammals, which is of significance for the reproductive ability of female mammals and the treatment of diseases related to germ cells. Meanwhile, it still remains to be determined whether the Hh signaling pathway participates in the regulation of the behavior of OGSCs. Herein, we review the current research on the role of the Hh signaling pathway in mediating the behavior of GSCs. In addition, some suggestions for future research are proposed.