Britannin inhibits cell proliferation, migration and glycolysis by downregulating KLF5 in lung cancer.

Lung cancer is a harmful type of malignancy and the leading cause of cancer-associated mortality. It is therefore imperative to develop novel drugs effective for treating this cancer. The Traditional Chinese Medicine compound Britannin has been previously reported to inhibit the development of certain cancers, such as pancreatic, breast and liver cancer. Moreover, Kruppel-like factor 5 (KLF5) has been identified an on oncogene in lung cancer. In the present study, the possible regulatory effects and underlying mechanism of Britannin in lung cancer were investigated. A549 and 16HBE cells were treated with different concentrations of Britannin. Subsequently, Cell counting kit-8, EdU staining and colony formation assays were used to detect the proliferative ability of these cells. Cell migration was detected by wound healing and Transwell assays, respectively. XF96 extracellular flux analyzer was used to analyze the extent of extracellular acidification and oxygen consumption rate in cells, whereas assay kits were used to detect glucose and lactic acid levels in the cell supernatant. The targeting effect between Britannin and the KLF5 protein was investigated using molecular docking technology. The protein expression levels of KLF5 in cells challenged with Britannin was detected by western blotting. Finally, overexpression of KLF5 in A549 cells was performed before cell proliferation, migration and the glycolysis rate were measured to explore the regulatory effects of Britannin. Britannin was found to inhibit the proliferation, migration and glycolysis of lung cancer cells, during which the protein expression levels of KLF5 were decreased. This suggests that Britannin regulated the expression of KLF5 in A549 cells. Overexpression of KLF5 reversed the inhibitory effects of Britannin on the proliferation, migration and glycolysis in lung cancer cells. In conclusion, these results suggest that Britannin can inhibit cell proliferation, migration and glycolysis by downregulating KLF5 expression in lung cancer cells.

Palmitoylation of vacuole membrane protein 1 promotes small extracellular vesicle secretion via interaction with ALIX and influences intercellular communication.

BACKGROUND: Small extracellular vesicles (EVs), exemplified by exosomes, mediate intercellular communication by transporting proteins, mRNAs, and miRNAs. Post-translational modifications are involved in controlling small EV secretion process. However, whether palmitoylation regulates small EV secretion, remains largely unexplored. METHODS: Vacuole Membrane Protein 1 (VMP1) was testified to be S-palmitoylated by Palmitoylation assays. VMP1 mutant plasmids were constructed to screen out the exact palmitoylation sites. Small EVs were isolated, identified and compared between wild-type VMP1 or mutant VMP1 transfected cells. Electron microscope and immunofluorescence were used to detect multivesicular body (MVB) number and morphology change when VMP1 was mutated. Immunoprecipitation and Mass spectrum were adopted to identify the protein that interacted with palmitoylated VMP1, while knock down experiment was used to explore the function of targeted protein ALIX. Taking human Sertoli cells (SCs) and human spermatogonial stem cell like cells (SSCLCs) as a model of intercellular communication, SSCLC maintenance was detected by flow cytometry and qPCR at 12 days of differentiation. In vivo, mouse model was established by intraperitoneal injection with palmitoylation inhibitor, 2-bromopalmitate (2BP) for 3 months. RESULTS: VMP1 was identified to be palmitoylated at cysteine 263,278 by ZDHHC3. Specifically, palmitoylation of VMP1 regulated its subcellular location and enhanced the amount of small EV secretion. Mutation of VMP1 palmitoylation sites interfered with the morphology and biogenesis of MVBs through suppressing intraluminal vesicle formation. Furthermore, inhibition of VMP1 palmitoylation impeded small EV secretion by affecting the interaction of VMP1 with ALIX, an accessory protein of the ESCRT machinery. Taking SCs and SSCLCs as a model of intercellular communication, we discovered VMP1 palmitoylation in SCs was vital to the growth status of SSCLCs in a co-culture system. Inhibition of VMP1 palmitoylation caused low self-maintenance, increased apoptosis, and decreased proliferation rate of SSCLCs. In vivo, intraperitoneal injection of 2BP inhibited VMP1 palmitoylation and exosomal marker expression in mouse testes, which were closely associated with the level of spermatogenic cell apoptosis and proliferation. CONCLUSIONS: Our study revealed a novel mechanism for small EV secretion regulated by VMP1 palmitoylation in Sertoli cells, and demonstrated its pivotal role in intercellular communication and SSC niche.

miRNA profiling of granulosa cell-derived exosomes reveals their role in promoting follicle development.

To explore whether granulosa cell (GC)-derived exosomes (GC-Exos) and follicular fluid-derived exosomes (FF-Exos) have functional similarities in follicle development and to establish relevant experiments to validate whether GC-Exos could serve as a potential substitute for follicular fluid-derived exosomes to improve folliculogenesis. GC-Exos were characterized. MicroRNA (miRNA) profiles of exosomes from human GCs and follicular fluid were analyzed in depth. The signature was associated with folliculogenesis, such as phosphatidylinositol 3 kinases-protein kinase B signal pathway, mammalian target of rapamycin signal pathway, mitogen-activated protein kinase signal pathway, Wnt signal pathway, and cyclic adenosine monophosphate signal pathway. A total of five prominent miRNAs were found to regulate the above five signaling pathways. These miRNAs include miRNA-486-5p, miRNA-10b-5p, miRNA-100-5p, miRNA-99a-5p, and miRNA-21-5p. The exosomes from GCs and follicular fluid were investigated to explore the effect on folliculogenesis by injecting exosomes into older mice. The proportion of follicles at each stage is counted to help us understand folliculogenesis. Exosomes derived from GCs were isolated successfully. miRNA profiles demonstrated a remarkable overlap between the miRNA profiles of FF-Exos and GC-Exos. The shared miRNA signature exhibited a positive influence on follicle development and activation. Furthermore, exosomes derived from GCs and follicular fluid promoted folliculogenesis in older female mice. Exosomes derived from GCs had similar miRNA profiles and follicle-promoting functions as follicular fluid exosomes. Consequently, GC-Exos are promising for replacing FF-Exos and developing new commercial reagents to improve female fertility.

A case report of encephalitis induced by SARS-CoV-2 confirmed by etiology: first case in Qingdao, China.

The neurological manifestations of SARS-CoV-2-infected patients are receiving increasing attention with the global spread of SARS-CoV-2. Here, we report the first case of SARS-CoV-2-induced encephalitis in Qingdao, China. We detected SARS-CoV-2 in nasopharyngeal swabs and cerebrospinal fluid from this 68-year-old female patient.

Sertoli cell-only syndrome: advances, challenges, and perspectives in genetics and mechanisms.

Male infertility can be caused by quantitative and/or qualitative abnormalities in spermatogenesis, which affects men's physical and mental health. Sertoli cell-only syndrome (SCOS) is the most severe histological phenotype of male infertility characterized by the depletion of germ cells with only Sertoli cells remaining in the seminiferous tubules. Most SCOS cases cannot be explained by the already known genetic causes including karyotype abnormalities and microdeletions of the Y chromosome. With the development of sequencing technology, studies on screening new genetic causes for SCOS are growing in recent years. Directly sequencing of target genes in sporadic cases and whole-exome sequencing applied in familial cases have identified several genes associated with SCOS. Analyses of the testicular transcriptome, proteome, and epigenetics in SCOS patients provide explanations regarding the molecular mechanisms of SCOS. In this review, we discuss the possible relationship between defective germline development and SCOS based on mouse models with SCO phenotype. We also summarize the advances and challenges in the exploration of genetic causes and mechanisms of SCOS. Knowing the genetic factors of SCOS offers a better understanding of SCO and human spermatogenesis, and it also has practical significance for improving diagnosis, making appropriate medical decisions, and genetic counseling. For therapeutic implications, SCOS research, along with the achievements in stem cell technologies and gene therapy, build the foundation to develop novel therapies for SCOS patients to produce functional spermatozoa, giving them hope to father children.

Rapid identification and relative quantification of disaccharide isomers by three fragment ion pairs using ESI-MS/MS and its application in yellow rice wine.

Small structural differences bring great difficulties on carbohydrates identification, especially in terms of their quantification. Herein, a novel ESI-MS/MS based strategy was established to discriminate and relatively quantified protonated PMP-disaccharides with different composition and glycosidic bond. Interestingly, protonated PMP labeled-disaccharides provided abundant fragment ions arising from cross-ring cleavage and glycosidic bond cleavage, which could afford diagnostic fragment patterns for isomers differentiation in combination of statistical analysis. It was worth to note that the relative intensity ratios (RIR) of three ion pairs could completely discriminate 16 disaccharides, and subsequently used to relatively quantified isomers in a binary mixture. Ultimately, this method was applied for the discrimination of yellow rice wine, and then the relative content of maltose and isomaltose were confirmed as well. In general, this method was easy to operation and effective for rapid differentiation and quantification of isomeric disaccharides in complex matrices.

A homozygous PIWIL2 frameshift variant affects the formation and maintenance of human-induced pluripotent stem cell-derived spermatogonial stem cells and causes Sertoli cell-only syndrome.

BACKGROUND: The most serious condition of male infertility is complete Sertoli cell-only syndrome (SCOS), which refers to the lack of all spermatogenic cells in the testes. The genetic cause of SCOS remains to be explored. We aimed to investigate the genetic cause of SCOS and assess the effects of the identified causative variant on human male germ cells. METHODS: Whole-exome sequencing was performed to identify potentially pathogenic variants in a man with complete SCOS, and Sanger sequencing was performed to verify the causative variant in this man and his father and brother. The pathogenic mechanisms of the causative variant were investigated by in vitro differentiation of human-induced pluripotent stem cells (hiPSCs) into germ cell-like cells. RESULTS: The homozygous loss-of-function (LoF) variant p.His244ArgfsTer31 (c.731_732delAT) in PIWIL2 was identified as the causative variant in the man with complete SCOS, and the same variant in heterozygosis was confirmed in his father and brother. This variant resulted in a truncated PIWIL2 protein lacking all functional domains, and no PIWIL2 expression was detected in the patient's testes. The patient and PIWIL2-/- hiPSCs could be differentiated into primordial germ cell-like cells and spermatogonial stem cell-like cells (SSCLCs) in vitro, but the formation and maintenance of SSCLCs were severely impaired. RNA-seq analyses suggested the inactivation of the Wnt signaling pathway in the process of SSCLC induction in the PIWIL2-/- group, which was validated in the patient group by RT-qPCR. The Wnt signaling pathway inhibitor hindered the formation and maintenance of SSCLCs during the differentiation of normal hiPSCs. CONCLUSIONS: Our study revealed the pivotal role of PIWIL2 in the formation and maintenance of human spermatogonial stem cells. We provided clinical and functional evidence that the LoF variant in PIWIL2 is a genetic cause of SCOS, which supported the potential role of PIWIL2 in genetic diagnosis. Furthermore, our results highlighted the applicability of in vitro differentiation models to function validation experiments.

Enhancing User Experience of Eye-Controlled Systems: Design Recommendations on the Optimal Size, Distance and Shape of Interactive Components from the Perspective of Peripheral Vision.

For special populations with motor impairments, eye-controlled interaction may be the only way for them to communicate with the outside world. Because of the dominance of vision in the motor mechanism, eye-controlled interaction has high usability and important research value. During eye-controlled interaction, the visual channel needs to receive information from the graphical user interface (GUI) and transmit the user's eye-controlled instructions, which overburdens the visual channel and reduces the efficiency of eye-controlled interaction. This study presents an ergonomic experiment to study how to design interactive GUI components in an eye-controlled user interface. The experiments were conducted based on the shape, size, and distance (from the object to the center of the screen) of the visual interactive components. The experiment comprised three parts: (1) the pre-experiment determined the evaluation index and selected the icon material; (2) the formal experiment was a three-factor within-subjects experiment, which included a search task using participants' peripheral vision; and (3) after the experiment, subjective evaluations were conducted using a questionnaire. The results showed that the shape, size, and distance of the interactive object significantly affected the reaction time, and the size factor significantly affected the movement time of the eye-controlled interaction. Finally, combined with the results of the subjective evaluation, we concluded that the recommended sizes of the interactive components were 2.889°, 3.389°, and 3.889°, and the recommended distances were 5.966° and 8.609°. Additionally, designers should utilize components with simple concrete shapes as much as possible to improve user recognition efficiency. Our study provides enlightening recommendations on how to design components in eye-controlled interactive interfaces, and has great guiding significance for building design standards of the eye-controlled systems.

Lipid-induced S-palmitoylation as a Vital Regulator of Cell Signaling and Disease Development.

Lipid metabolites are emerging as pivotal regulators of protein function and cell signaling. The availability of intracellular fatty acid is tightly regulated by glycolipid metabolism and may affect human body through many biological mechanisms. Recent studies have demonstrated palmitate, either from exogenous fatty acid uptake or de novo fatty acid synthesis, may serve as the substrate for protein palmitoylation and regulate protein function via palmitoylation. Palmitoylation, the most-studied protein lipidation, encompasses the reversible covalent attachment of palmitate moieties to protein cysteine residues. It controls various cellular physiological processes and alters protein stability, conformation, localization, membrane association and interaction with other effectors. Dysregulation of palmitoylation has been implicated in a plethora of diseases, such as metabolic syndrome, cancers, neurological disorders and infections. Accordingly, it could be one of the molecular mechanisms underlying the impact of palmitate metabolite on cellular homeostasis and human diseases. Herein, we explore the relationship between lipid metabolites and the regulation of protein function through palmitoylation. We review the current progress made on the putative role of palmitate in altering the palmitoylation of key proteins and thus contributing to the pathogenesis of various diseases, among which we focus on metabolic disorders, cancers, inflammation and infections, neurodegenerative diseases. We also highlight the opportunities and new therapeutics to target palmitoylation in disease development.

Valproic acid promotes the in vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells.

BACKGROUND: Studying human germ cell development and male infertility is heavily relied on mouse models. In vitro differentiation of human pluripotent stem cells into spermatogonial stem cell-like cells (SSCLCs) can be used as a model to study human germ cells and infertility. The current study aimed to develop the SSCLC induction protocol and assess the effects of the developed protocol on SSCLC induction. METHODS: We examined the effects of valproic acid (VPA), vitamin C (VC) and the combination of VPA and VC on the SSCLC induction efficiency and determined the expression of spermatogonial genes of differentiated cells. Haploid cells and cells expressed meiotic genes were also detected. RNA-seq analysis was performed to compare the transcriptome between cells at 0 and 12 days of differentiation and differently expressed genes were confirmed by RT-qPCR. We further evaluated the alteration in histone marks (H3K9ac and H3K27me3) at 12 days of differentiation. Moreover, the SSCLC induction efficiency of two hiPSC lines of non-obstructive azoospermia (NOA) patients was assessed using different induction protocols. RESULTS: The combination of low concentrations of VPA and VC in the induction medium was most effective to induce SSCLCs expressing several spermatogonial genes from human pluripotent stem cells at 12 days of differentiation. The high concentration of VPA was more effective to induce cells expressing meiotic genes and haploid cells. RNA-seq analysis revealed that the induction of SSCLC involved the upregulated genes in Wnt signaling pathway, and cells at 12 days of differentiation showed increased H3K9ac and decreased H3K27me3. Additionally, two hiPSC lines of NOA patients showed low SSCLC induction efficiency and decreased expression of genes in Wnt signaling pathway. CONCLUSIONS: VPA robustly promoted the differentiation of human pluripotent stem cells into SSCLCs, which involved the upregulated genes in Wnt signaling pathway and epigenetic changes. hiPSCs from NOA patients showed decreased SSCLC induction efficiency and Wnt signaling pathway gene expression, suggesting that SSC depletion in azoospermia testes might be associated with inactivation of Wnt signaling pathway. Our developed SSCLC induction protocol provides a reliable tool and model to study human germ cell development and male infertility.

LC-MS/MS-based non-isotopically paired labeling (NIPL) strategy for the qualification and quantification of monosaccharides.

Investigation into monosaccharides is critical for studies of oligosaccharides structure and function in biological processes. However, monosaccharides quantification is still challenge due to their isomeric structure and high hydrophilic properties. Besides, it was difficult to obtain isotopic internal standards (IS) of each monosaccharide in complex matrixes. Herein, we developed a novel strategy for the qualification and quantification of monosaccharides in urine using two structure analogs 1-(4-methylphenyl)-3-methyl-5-pyrazolone (MPMP) and1-phenyl-3-methyl-5-pyrazolone (PMP) as non-isotopically paired labeling (NIPL) reagents by liquid chromatograph-tandem mass spectrometry (LC-MS/MS). The derivatized monosaccharides by NIPL method not only had sufficient retention time differences on reversed-phase column, but also exhibited predominant product ion pairs (m/z 189 & m/z 175) in the multiple reaction monitoring (MRM) mode. In this method, PMP labeled standards were adopted as one-to-one internal standards (ISs). 12 urinary monosaccharides were successfully determined and the linear ranges expanded five orders of magnitude with limit of quantification (LOQ) varied from 0.09 ng mL-1 to 0.36 ng mL-1 as well as the accuracy higher than 98.15% and the relative standard derivation (RSD) lower than 7.92%. With assistance of multivariate analysis, the targeted monosaccharide biomarkers were firstly obtained for the diagnosis of bladder cancer. By the inexpensive NIPL reagents-MPMP/PMP, the developed strategy possessed the specific advantages of low cost, simple operation, high sensitivity and high accuracy for the qualification and quantitation of monosaccharides. As expected, this method will provide an alternative application potential for targeted metabolomics analysis.

Association of Serum Testosterone and Luteinizing Hormone With Blood Pressure and Risk of Cardiovascular Disease in Middle-Aged and Elderly Men.

Background The age-related decline in testosterone levels is thought to be of great importance for male aging and cardiovascular diseases. However, data are controversial on whether abnormal sex hormones are linked to the presence of cardiovascular diseases and it is also uncertain how blood pressure modifies the association between testosterone levels and major cardiovascular diseases. Methods and Results This is a multicenter, population-based, cross-sectional study of 6296 men conducted between 2013 and 2016. Basic information and clinical symptoms were obtained by questionnaires. Blood pressure and plasma levels of total testosterone, sex hormone-binding globulin, luteinizing hormone, and free testosterone were determined in men in a multistage random, cluster sampling in 6 provinces of China. There were 5786 Chinese men (mean [SD] age 55.0 [10.1] years) included after exclusion criteria were applied; 37.2% (2150) of them were diagnosed with hypertension. Total testosterone, free testosterone, and sex hormone-binding globulin were inversely associated with the prevalence of hypertension. Age >65 years or body mass index ≥24 negatively impacted the inverse correlation between testosterone levels and hypertension, whereas smoking and family history of hypertension strengthened the correlation. In participants with grade 2 hypertension, total testosterone was positively associated with the presence of stroke, and luteinizing hormone was also positively correlated with cardiovascular and cerebrovascular diseases. Conclusions Lower total testosterone could be a promising risk marker for prevalent hypertension. Both low and high levels of testosterone are associated with greater cardiovascular risk. Primary hypogonadism may be a risk marker for major cardiovascular diseases in men with severe hypertension.

Androgen-dependent miR-125a-5p targets LYPLA1 and regulates global protein palmitoylation level in late-onset hypogonadism males.

Late-onset hypogonadism (LOH) is defined as a clinical and biochemical syndrome with multiple symptoms caused by testosterone deficiency in aging males. An in-depth exploration of the molecular mechanism underlying LOH development is insufficient. We previously identified miR-125a-5p as a dysregulated microRNA in LOH patients and potential diagnostic biomarker for LOH. The present study demonstrated that plasma miR-125a-5p was upregulated after testosterone supplementation in both LOH patients and castrated mice, and positively associated with the testosterone concentrations, suggesting direct regulation of miR-125a-5p expression by testosterone. Androgen response element in the promoter of miR-125a-5p was subsequently identified. Target gene screening and confirmation verified that LYPLA1, encoding acyl-protein thioesterase 1 which catalyzed protein depalmitoylation process, was a target gene of miR-125a-5p. Furthermore, in cells cultured with testosterone deprivation and organs from castrated mice, testosterone deficiency led to decreased global protein palmitoylation level. In aging males, global protein palmitoylation in peripheral blood showed a notable decline in LOH patients contrast to the normal elderly males. And the palmitoylation level was positively correlative with serum testosterone concentrations. Our results suggested that testosterone could regulate global palmitoylation level through miR-125a-5p/LYPLA1 signaling pathway. Given that protein palmitoylation is pivotal for protein function and constitutes the pathogenesis of various diseases, testosterone/miR-125a-5p/LYPLA1 may contribute to the molecular mechanism underlying multiple symptoms caused by testosterone deficiency in LOH patients, and aberrant global palmitoylation could be a potential biomarker for LOH.

The timeline and risk factors of clinical progression of COVID-19 in Shenzhen, China.

BACKGROUND: The novel coronavirus disease 2019 (COVID-19) broke out globally. Early prediction of the clinical progression was essential but still unclear. We aimed to evaluate the timeline of COVID-19 development and analyze risk factors of disease progression. METHODS: In this retrospective study, we included 333 patients with laboratory-confirmed COVID-19 infection hospitalized in the Third People's Hospital of Shenzhen from 10 January to 10 February 2020. Epidemiological feature, clinical records, laboratory and radiology manifestations were collected and analyzed. 323 patients with mild-moderate symptoms on admission were observed to determine whether they exacerbated to severe-critically ill conditions (progressive group) or not (stable group). We used logistic regression to identify the risk factors associated with clinical progression. RESULTS: Of all the 333 patients, 70 (21.0%) patients progressed into severe-critically ill conditions during hospitalization and assigned to the progressive group, 253 (76.0%) patients belonged to the stable group, another 10 patients were severe before admission. we found that the clinical features of aged over 40 (3.80 [1.72, 8.52]), males (2.21 [1.20, 4.07]), with comorbidities (1.78 [1.13, 2.81]) certain exposure history (0.38 [0.20, 0.71]), abnormal radiology manifestations (3.56 [1.13, 11.40]), low level of T lymphocytes (0.99 [0.997, 0.999]), high level of NLR (0.99 [0.97, 1.01]), IL-6 (1.05 [1.03, 1.07]) and CRP (1.67 [1.12, 2.47]) were the risk factors of disease progression by logistic regression. CONCLUSIONS: The potential risk factors of males, older age, with comorbidities, low T lymphocyte level and high level of NLR, CRP, IL-6 can help to predict clinical progression of COVID-19 at an early stage.