Efficient detection of Streptococcus pyogenes based on recombinase polymerase amplification and lateral flow strip.

PURPOSE: This article aims to establish a rapid visual method for the detection of Streptococcus pyogenes (GAS) based on recombinase polymerase amplification (RPA) and lateral flow strip (LFS). METHODS: Utilizing speB of GAS as a template, RPA primers were designed, and basic RPA reactions were performed. To reduce the formation of primer dimers, base mismatch was introduced into primers. The probe was designed according to the forward primer, and the RPA-LFS system was established. According to the color results of the reaction system, the optimum reaction temperature and time were determined. Thirteen common clinical standard strains and 14 clinical samples of GAS were used to detect the selectivity of this method. The detection limit of this method was detected by using tenfold gradient dilution of GAS genome as template. One hundred fifty-six clinical samples were collected and compared with qPCR method and culture method. Kappa index and clinical application evaluation of the RPA-LFS were carried out. RESULTS: The enhanced RPA-LFS method demonstrates the ability to complete the amplification process within 6 min at 33 °C. This method exhibits a high analytic sensitivity, with the lowest detection limit of 0.908 ng, and does not exhibit cross-reaction with other pathogenic bacteria. CONCLUSIONS: The utilization of RPA and LFS allows for efficient and rapid testing of GAS, thereby serving as a valuable method for point-of-care testing.

Bio-nanoparticles loaded with synovial-derived exosomes ameliorate osteoarthritis progression by modifying the oxidative microenvironment.

BACKGROUND AND AIMS: Osteoarthritis (OA) is a prevalent degenerative joint disorder, marked by the progressive degeneration of joint cartilage, synovial inflammation, and subchondral bone hyperplasia. The synovial tissue plays a pivotal role in cartilage regulation. Exosomes (EXOs), small membrane-bound vesicles released by cells into the extracellular space, are crucial in mediating intercellular communication and facilitating the exchange of information between tissues. Our study aimed to devise a hydrogel microsphere infused with SOD3-enriched exosomes (S-EXOs) to protect cartilage and introduce a novel, effective approach for OA treatment. MATERIALS AND METHODS: We analyzed single-cell sequencing data from 4247 cells obtained from the GEO database. Techniques such as PCR, Western Blot, immunofluorescence (IF), and assays to measure oxidative stress levels were employed to validate the cartilage-protective properties of the identified key protein, SOD3. In vivo, OA mice received intra-articular injections of S-EXOs bearing hydrogel microspheres, and the effectiveness was assessed using safranine O (S.O) staining and IF. RESULTS: Single-cell sequencing data analysis suggested that the synovium influences cartilage via the exocrine release of SOD3. Our findings revealed that purified S-EXOs enhanced antioxidant capacity of chondrocytes, and maintained extracellular matrix metabolism stability. The S-EXO group showed a significant reduction in mitoROS and ROS levels by 164.2% (P < 0.0001) and 142.7% (P < 0.0001), respectively, compared to the IL-1ß group. Furthermore, the S-EXO group exhibited increased COL II and ACAN levels, with increments of 2.1-fold (P < 0.0001) and 3.1-fold (P < 0.0001), respectively, over the IL-1ß group. Additionally, the S-EXO group showed a decrease in MMP13 and ADAMTS5 protein expression by 42.3% (P < 0.0001) and 44.4% (P < 0.0001), respectively. It was found that S-EXO-containing hydrogel microspheres could effectively deliver SOD3 to cartilage and significantly mitigate OA progression. The OARSI score in the S-EXO microsphere group markedly decreased (P < 0.0001) compared to the OA group. CONCLUSION: The study demonstrated that the S-EXOs secreted by synovial fibroblasts exert a protective effect on chondrocytes, and microspheres laden with S-EXOs offer a promising therapeutic alternative for OA treatment.

The specific viral composition in triple-negative breast cancer tissue shapes the specific tumor microenvironment characterized on pathological images.

Numerous studies have shown that different subtypes of breast cancer (BC) have significant differences in terms of the tumor microbiome, host gene expression, and histopathological image, whereas the biological links between these cancer-associated indicators are still unknown. Here, we performed a comprehensive analysis with 610 patients of the four subtypes of BC with matched tissue microbiota, host transcriptome, and histopathological image samples. Correlation analysis showed that the composition of intratumoral viruses shaped the tumor microenvironment (TME) of patients with BC, and the TME was further reflected in the histopathological images. Of the four subtypes, patients with triple-negative breast cancer (TNBC) had unique intratumoral viral community composition, non-cancer cell infiltration in the TME, and histopathological image characteristics. Furthermore, we detected multiple virus-cell-image association axes in TNBC, in which tumor-associated macrophages (TAMs) have clinical prognostic implication. This study provides a comprehensive map of the associations between the intratumoral virome, TME, and histopathological image of TNBC, as well as insights into disease prognosis that can be crucial for precise therapeutic intervention strategies.

Lipid-coated albumin-paclitaxel nanoparticles loaded with sorcin-siRNA reverse cancer chemoresistance via restoring intracellular calcium ion homeostasis.

Chemoresistance is often a cause of the failure of chemotherapy in cancer treatment. Sorcin (SRI) is a soluble resistance-related calcium-binding protein involved in chemoresistant processes and is overexpressed in many chemoresistant cancer cells, including paclitaxel (PTX)-resistant ovarian cancer. Increased SRI can reduce the concentration of calcium ions in the cytosol and mitochondria and the decrease of calcium ion concentration prevents the occurrence of apoptosis. Here we examined the SRI expression in multiple cancers using a human TissueArray and found that SRI expression was significantly higher in malignant tumor tissues. Furthermore, SRI was overexpressed, while intracellular calcium concentration was decreased, in chemoresistant cancer cells. To restore intracellular calcium homeostasis and overcome chemoresistance, we developed lipid-coated albumin-PTX nanoparticles loaded with SRI-siRNA (LANP-PTX-siSRI) for PTX and SRI-siRNA co-delivery. LANP-PTX-siSRI had dual-target roles in the regulation of SRI and the delivery of PTX into chemoresistant cells. The LANP-PTX-siSRI inhibited the expression of SRI and enhanced intracellular calcium, leading to the induction of apoptosis and the inhibition of the growth of PTX-resistant cancer cells in vitro and in vivo. In addition, the mechanism study revealed that the overexpression of SRI was associated with an impaired TGF-ß signaling pathway. The administration of TGF-ß1 inhibited two calcium-binding proteins SRI and S100A14. In conclusion, our data unveil that restoring intracellular calcium ion homeostasis via reducing SRI expression can reverse chemoresistance. Thus, the fabricated LANP-PTX-siSRI has a potentially therapeutical application.

Protective Effects of Scutellarin on Acute Alcohol Intestinal Injury.

The present study aims to investigate the roles of scutellarin (SCU) on acute alcohol intestinal injury. Mice were divided into six groups: alcohol, three administration, negative control and positive drug bifendate control. The administration group mice were intraperitoneally injected with SCU for 3 consecutive days followed by alcohol gavage at an interval of 1 h. After the mice were sacrificed, colon tissue damage was evaluated by histopathological examination; the activities of inducible nitric oxide synthase (iNOS) and catalase (CAT), as well as the content of malondialdehyde (MDA) were detected using biochemical kits; the levels of inflammatory cytokines mRNA were determined by real-time fluorescence quantitative PCR; the protein expression levels of hemeoxygenase-1 (HO-1) and phosphorylated nuclear factor-ĸB p65 were measured via western blotting. The results showed that alcohol induced severe colon morphological degradation, epithelia atrophy, and more inflammatory cells infiltration in the submucosa. SCU treatment prevented this process, especially in the middle and high dose groups. Alcohol treatment caused excessive lipid peroxidation product accumulation of MDA, restrained the activity of antioxidant enzyme CAT, induced HO-1 expression in the colon, whereas low dose SCU treatment significantly down-regulated the MDA level, enhanced the CAT level, and accelerated HO-1 signals. SCU prevented alcohol stimulation triggered inflammatory response in colon tissues through significantly downregulating the iNOS activity, transcript levels of Tnf-α, Il-1ß and Il-6, and phosphorylation levels of NF-κB p65. These findings suggest that SCU protects the colon via antioxidant and anti-inflammatory mechanisms, making it a promising drug against alcohol-induced colon damage.

A three-dimensional model of RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

Ribonuclease P (RNase P) is an endoribonuclease involved in maturation of the 5'-end of tRNA. We found previously that RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNase P RNA (PhopRNA) and five protein cofactors designated PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38. The crystal structures of the five proteins have been determined, a three-dimensional (3-D) model of PhopRNA has been constructed, and biochemical data, including protein-RNA interaction sites, have become available. Here, this information was combined to orient the crystallographic structures of the proteins relative to their RNA binding sites in the PhopRNA model. Some alterations were made to the PhopRNA model to improve the fit. In the resulting structure, a heterotetramer composed of PhoPop5 and PhoRpp30 bridges helices P3 and P16 in the PhopRNA C-domain, thereby probably stabilizing a double-stranded RNA structure (helix P4) containing catalytic Mg2+ ions, while a heterodimer of PhoRpp21 and PhoRpp29 locates on a single-stranded loop connecting helices P11 and P12 in the specificity domain (S-domain) in PhopRNA, probably forming an appropriate conformation of the precursor tRNA (pre-tRNA) binding site. The fifth protein PhoRpp38 binds each kink-turn (K-turn) motif in helices P12.1, P12.2, and P16 in PhopRNA. Comparison of the structure of the resulting 3-D model with that of bacterial RNase P suggests transition from RNA-RNA interactions in bacterial RNase P to protein-RNA interactions in archaeal RNase P. The proposed 3-D model of P. horikoshii RNase P will serve as a framework for further structural and functional studies on archaeal, as well as eukaryotic, RNase Ps.

Pentatricopeptide repeat motifs in the processing enzyme PRORP1 in Arabidopsis thaliana play a crucial role in recognition of nucleotide bases at TψC loop in precursor tRNAs.

Proteinaceous RNase P (PRORP1) in Arabidopsis thaliana is an endoribonuclease that catalyzes hydrolysis to remove the 5'-leader sequence of precursor tRNAs (pre-tRNAs). PRORP1 is composed of pentatricopeptide repeat (PPR) motifs, a central linker region, and a metal nuclease domain, the NYN domain. The PPR motifs are single-stranded RNA-binding motifs that recognize bases in a modular fashion. To obtain insight into the mechanism by which the PPR motifs in PRORP1 recognize a target sequence in catalysis, N-terminal successive deletion mutants were overproduced in Escherichia coli, and the resulting proteins were characterized in terms of enzymatic activity using chloroplast pre-tRNA(Phe) as a substrate. Although Δ89, in which all PPR motifs are present, retained the pre-tRNA cleavage activity, Δ129 devoid of the first PPR motif (PPR1) had significantly reduced cleavage activity. Likewise, deletions of the second (PPR2) or third PPR (PPR3) motif abolished the cleavage activity, suggesting that PPR motifs play a crucial role in catalysis. A proposed recognition code for PPR motifs predicted that PPR2-PPR5 in PRORP1 recognize C, A/U, A, and U, respectively, whose sequence is in good agreement with C56-A57-A58-A59 in the TψC loop in pre-tRNA(Phe). Mutational analyses of nucleotide residues in the TψC loop as well as nucleotide-specifying residues (NSRs) in PPR motifs further suggested that PPR2 and PPR3 in PRORP1 favorably recognize nucleotide bases C56 and A57 at the TψC loop in pre-tRNA(Phe), respectively. This prediction and previous biochemical data were combined to construct a fitting model of tRNA onto PRORP1, showing that the mechanism by which PRORP1 recognizes pre-tRNAs appears to be distinct from that by bacterial RNase P.

[Primary premature ejaculation of kidney deficiency and liver stagnation treated with warm acupuncture of large-quantity moxibustion: a randomized controlled trial].

OBJECTIVE: To explore the clinical effect of warm acupuncture with large-quantity moxibustion on primary premature ejaculation (kidney deficiency and liver stagnation). METHODS: A total of 240 patients with primary premature ejaculation (kidney deficiency and liver stagnation) were randomly divided into a warm acupuncture group (80 cases, 5 cases dropped out), an acupuncture group (80 cases, 4 cases dropped out) and a western medication group (80 cases, 6 cases dropped out). In the warm acupuncture group, a large quantity of moxibustion was delivered after acupuncture at Baihui (GV 20), Qihai (CV 6), Guanyuan (CV 4) and Zhongji (CV 3), as well as bilateral Fengchi (GB 20), lateral line 3 of forehead (MS 4), neishengzhiqi (TF2), Ganshu (BL 18), Shenshu (BL 23), and etc. One treatment with warm acupuncture took 40 min, once daily; five treatments were given per week and 4 weeks of treatment was required. In the acupuncture group, moxibustion was not delivered, and the rest operation of acupuncture was same as the warm acupuncture group. In the western medication group, dapoxetine hydrochloride tablets were administered orally, 30 mg each time, taken with warm water 1 h to 3 h before sexual intercourse. Medication was administered at most once within 24 h, twice per week, and 6 times within 4 weeks. Before and after treatment, the score of TCM symptoms, the score of premature ejaculation diagnostic tool (PEDT), intravaginal ejaculation latency time (IELT) and the serum sex hormone content (testosterone [T], luteinizing hormone [LH] and follicule stimulating hormone [FSH]) were observed and the clinical effect was evaluated in the three groups. RESULTS: After treatment, the scores for less duration of intercourse (<1 min), post-ejaculation fatigue, low spirit and decreased libido, and the total scores of TCM symptoms, as well as PEDT scores were reduced when compared with those before treatment in each group (P<0.01, P<0.05), and IELT was prolonged (P<0.01) in the three groups. The serum T content was increased when compared with that before treatment in the warm acupuncture group (P<0.05). After treatment, in comparison with the acupuncture group and the western medication group, the scores for post-ejaculation fatigue, soreness and weakness in the lumbar region and knee joints, decreased libido, insomnia, dream-disturbed sleep and frequent nocturnal enuresis, as well as the total score of TCM symptoms were lower (P<0.05, P<0.01) and the serum T content was increased (P<0.05) in the warm acupuncture group. When compared with the acupuncture group, PEDT scores were lower and IELT prolonged in the warm acupuncture group and the western medication group (P<0.05, P<0.01). The total effective rate was 82.7% (62/75) in the warm acupuncture group, higher than that of the acupuncture group (68.4%, 52/76) and the western medication group (64.9%, 48/74, P<0.05) respectively. CONCLUSION: Warm acupuncture with large-quantity moxibustion ameliorates the clinical symptoms and increases intravaginal ejaculation latency time and the levels of sex hormone in the patients with primary premature ejaculation (kidney deficiency and liver stagnation).

Establishment and application of a rapid visual diagnostic method for Streptococcus agalactiae based on recombinase polymerase amplification and lateral flow strips.

This study aims to establish a rapid diagnostic method for Streptococcus agalactiae (GBS) based on recombinase polymerase amplification (RPA) and lateral flow strips (LFS). The best primer pairs designed by SIP gene were screened according to the basic RPA reaction, then the probe was designed. The reaction condition was optimized based on the color development of the LFS detection line. To ascertain the reaction specificity, 10 common clinical pathogens and 10 clinical specimens of GBS were tested. Furthermore, the reaction sensitivity was assessed by utilizing a tenfold gradient dilution of GBS genomic DNA as templates. RPA-LFS method was compared to the qPCR assay and biochemical culture method for the Kappa consistency test. The RPA-LFS technique was able to complete the amplification process within 30 min and the results were observed on lateral flow strips. The method is highly sensitive, with a minimum detection limit of 1.31 ng for GBS. The RPA-LFS method showed consistent accuracy of results compared to qPCR and the culture-biochemical method. The establishment of this method is conducive to the development of on-site immediate detection, which can provide information for the timely development of a reasonable antimicrobial treatment plan, and has a greater potential for clinical application.

Anti-papillary thyroid carcinoma effects of dioscorea bulbifera L. through ferroptosis and the PI3K/AKT pathway based on network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Papillary thyroid carcinoma (PTC) is the predominant form of thyroid cancer with a rising global incidence. Despite favorable prognoses, a significant recurrence rate persists. Dioscorea bulbifera L. (DBL), a traditional Chinese medicine, has been historically used for thyroid-related disorders. However, its therapeutic effects and mechanisms of action on PTC remain unclear. AIM OF THE STUDY: To explore the potential therapeutic effects, principal active components, and molecular mechanisms of DBL in the treatment of PTC through network pharmacology and molecular docking, with experimental validation conducted to corroborate these findings. MATERIALS AND METHODS: The Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) was utilized as a systematic tool for collecting and screening the phytochemical components of DBL, and for establishing associations between these components and molecular targets. Based on this, network data was visually processed using Cytoscape software (version 3.8.0). Concurrently, precise molecular docking studies of the principal active components of DBL and their corresponding targets were conducted using Autodock software. Additionally, PTC-related genes were selected through the GeneCards and GEO databases. We further employed the DAVID bioinformatics resources to conduct comprehensive Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses on the intersecting genes between DBL and PTC. These analyses aid in predicting the potential therapeutic actions of DBL on PTC and its mechanisms of action. To validate these findings, corresponding in vitro experimental studies were also conducted. RESULTS: In this investigation, 14 bioactive compounds of DBL and 195 corresponding molecular targets were identified, with 127 common targets shared between DBL and PTC. Molecular docking revealed strong binding affinities between major bioactive compounds and target proteins. GO enrichment analysis unveiled key processes involved in DBL's action. KEGG analysis highlighted DBL's modulation of the PI3K/AKT signaling pathway. Experimental outcomes demonstrated DBL's potential in inhibiting PTC cell proliferation and migration, suppressing PI3K/AKT pathway activation, and promoting ferroptosis. CONCLUSION: In conclusion, DBL offers a multifaceted therapeutic approach for PTC, targeting multiple molecular entities and influencing diverse biological pathways. Network pharmacology and molecular docking shed light on DBL's potential utility in PTC treatment, substantiated by experimental validation. This study contributes valuable insights into using DBL as a promising therapeutic agent for PTC management.

The emerging roles of miRNA-mediated autophagy in ovarian cancer.

Ovarian cancer is one of the common tumors of the female reproductive organs. It has a high mortality rate, is highly heterogeneous, and early detection and primary prevention are very complex. Autophagy is a cellular process in which cytoplasmic substrates are targeted for degradation in lysosomes through membrane structures called autophagosomes. The periodic elimination of damaged, aged, and redundant cellular molecules or organelles through the sequential translation between amino acids and proteins by two biological processes, protein synthesis, and autophagic protein degradation, helps maintain cellular homeostasis. A growing number of studies have found that autophagy plays a key regulatory role in ovarian cancer. Interestingly, microRNAs regulate gene expression at the posttranscriptional level and thus can regulate the development and progression of ovarian cancer through the regulation of autophagy in ovarian cancer. Certain miRNAs have recently emerged as important regulators of autophagy-related gene expression in cancer cells. Moreover, miRNA analysis studies have now identified a sea of aberrantly expressed miRNAs in ovarian cancer tissues that can affect autophagy in ovarian cancer cells. In addition, miRNAs in plasma and stromal cells in tumor patients can affect the expression of autophagy-related genes and can be used as biomarkers of ovarian cancer progression. This review focuses on the potential significance of miRNA-regulated autophagy in the diagnosis and treatment of ovarian cancer.

Establishment of portable Pseudomonas aeruginosa detection platform based on one-tube CRISPR/Cas12a combined with recombinase polymerase amplification technology.

Pseudomonas aeruginosa, a common Gram-negative bacterium, is associated with diverse diseases. Its increasing resistance to antibiotics presents challenges in clinical treatment. The predominant diagnostic approach involves conventional biochemical cultures, known for their time and labor intensiveness. Despite progress in isothermal amplification studies, limitations persist, including reliance on specialized equipment, intricate primer design, and aerosol contamination. Therefore, there is a demand for enhanced clinical assays. This study successfully combined RPA and CRISPR/Cas12a techniques. Through a series of experiments involving the design and screening of lasB crRNA, the creation of lasB RPA primers, and the establishment of a streamlined RPA-CRISPR/Cas12a assay, the study developed a one-tube detection method targeting P. aeruginosa's lasB gene. The assay demonstrated inclusive behavior across standard and 21 isolates, while specifically discerning P. aeruginosa from diverse strains. Sensitivity reached 15.9 CFU/reaction. Clinical validation revealed a 97.62% concordance with traditional methods. The one-tube assay's protocol mitigated aerosol contamination. Offering precision, specificity, and sensitivity, this method shows promise for field applications in resource-scarce regions, enabling early detection and improved management of P. aeruginosa infections.

Cascaded controlled delivering growth factors to build vascularized and osteogenic microenvironment for bone regeneration.

The process of bone regeneration is intricately regulated by various cytokines at distinct stages. The establishment of early and efficient vascularization, along with the maintenance of a sustained osteoinductive microenvironment, plays a crucial role in the successful utilization of bone repair materials. This study aimed to develop a composite hydrogel that would facilitate the creation of an osteogenic microenvironment for bone repair. This was achieved by incorporating an early rapid release of VEGF and a sustained slow release of BMP-2. Herein, the Schiff base was formed between VEGF and the composite hydrogel, and VEGF could be rapidly released to promote vascularization in response to the early acidic bone injury microenvironment. Furthermore, the encapsulation of BMP-2 within mesoporous silica nanoparticles enabled a controlled and sustained release, thereby facilitating the process of bone repair. Our developed composite hydrogel released more than 80% of VEGF and BMP-2 in the acidic medium, which was significantly higher than that in the neutral medium (about 60%). Moreover, the composite hydrogel demonstrated a significant improvement in the migratory capacity and tube formation ability of human umbilical vein endothelial cells (HUVECs). Furthermore, the composite hydrogel exhibited an augmented ability for osteogenesis, as confirmed by the utilization of ALP staining, alizarin red staining, and the upregulation of osteogenesis-related genes. Notably, the composite hydrogel displayed substantial osteoinductive properties, compared with other groups, the skull defect in the composite hydrogels combined with BMP-2 and VEGF was full of new bone, basically completely repaired, and the BV/TV value was greater than 80%. The outcomes of animal experiments demonstrated that the composite hydrogel effectively promoted bone regeneration in cranial defects of rats by leveraging the synergistic effect of an early rapid release of VEGF and a sustained slow release of BMP-2, thereby facilitating vascularized bone regeneration. In conclusion, our composite hydrogel has demonstrated promising potential for vascularized bone repair through the enhancement of angiogenesis and osteogenic microenvironment.

A rapid and visual detection of Staphylococcus haemolyticus in clinical specimens with RPA-LFS.

Staphylococcus haemolyticus (S. haemolyticus), which is highly prevent in the hospital environment, is an etiological factor for nosocomial infections. Point-of-care rapid testing (POCT) of S. haemolyticus is not possible with the currently used detection methods. Recombinase polymerase amplification (RPA) is a novel isothermal amplification technology with high sensitivity and specificity. The combination of RPA and lateral flow strips (LFS) can achieve rapid pathogen detection, enabling POCT. This study developed an RPA-LFS methodology using a specific probe/primer pair to identify S. haemolyticus. A basic RPA reaction was performed to screen the specific primer from 6 primer pairs targeting mvaA gene. The optimal primer pair was selected based on agarose gel electrophoresis, and the probe was designed. To eliminate false-positive results caused by the byproducts, base mismatches were introduced in the primer/probe pair. The improved primer/probe pair could specifically identify the target sequence. To explore the optimal reaction conditions, the effects of reaction temperature and duration of the RPA-LFS method were systematically investigated. The improved system enabled optimal amplification at 37 °C for 8 min, and the results were visualized within 1 min. The S. haemolyticus detection sensitivity of the RPA-LFS method, whose performance was unaffected by contamination with other genomes, was 0.147 CFU/reaction. Furthermore, we analyzed 95 random clinical samples with RPA-LFS, quantitative polymerase chain reaction (qPCR), and traditional bacterial-culture assays and found that the RPA-LFS had 100% and 98.73% compliance rates with the qPCR and traditional culture method, respectively, which confirms its clinical applicability. In this study, we designed an improved RPA-LFS assay based on the specific probe/primer pair for the detection of S. haemolyticus via rapid POCT, free from the limitations of the precision instruments, helping to make diagnoses and treatment decisions as soon as possible.

Functional regulation of syntaxin-1: An underlying mechanism mediating exocytosis in neuroendocrine cells.

The fusion of the secretory vesicle with the plasma membrane requires the assembly of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes formed by synaptobrevin, syntaxin-1, and SNAP-25. Within the pathway leading to exocytosis, the transitions between the "open" and "closed" conformations of syntaxin-1 function as a switch for the fusion of vesicles with the plasma membranes; rapid assembly and disassembly of syntaxin-1 clusters on the plasma membrane provide docking and fusion sites for secretory vesicles in neuroendocrine cells; and the fully zippered trans-SNARE complex, which requires the orderly, rapid and accurate binding of syntaxin-1 to other SNARE proteins, play key roles in triggering fusion. All of these reactions that affect exocytosis under physiological conditions are tightly regulated by multiple factors. Here, we review the current evidence for the involvement of syntaxin-1 in the mechanism of neuroendocrine cell exocytosis, discuss the roles of multiple factors such as proteins, lipids, protein kinases, drugs, and toxins in SNARE complex-mediated membrane fusion, and present an overview of syntaxin-1 mutation-associated diseases with a view to developing novel mechanistic therapeutic targets for the treatment of neuroendocrine disorders.

Downregulation of miR-221-3p promotes the ferroptosis in gastric cancer cells via upregulation of ATF3 to mediate the transcription inhibition of GPX4 and HRD1.

BACKGROUND: Gastric cancer (GC) is an aggressive gastrointestinal tumor. MiRNAs participate in the tumorigenesis of GC. Nevertheless, the function of miR-221-3p in GC remains largely unknown. METHODS: RNA levels were assessed by RT-qPCR. Western blot was performed to test the protein levels. The relation between miR-221-3p and ATF3 was investigated by dual-luciferase reporter assay. ChIP and dual-luciferase reporter assay were applied to assess the interaction between ATF3 and HRD1 or GPX4. Meanwhile, cell proliferation was investigated by CCK8 and colony formation assay. The content of erastin-induced Fe2+ was investigated by iron assay kit. Erastin-induced lipid ROS level was assessed by C11-BODIPY 581/591. Co-immunoprecipitation was used to detect the interaction between HRD1 and ACSL4. In addition, xenograft mice model was established to detect the effect of miR-221-3p in GC. RESULTS: Depletion of miR-221-3p greatly attenuated GC cell proliferation through promoting ferroptosis. Meanwhile, ATF3 was downregulated in GC, and it was identified to be the downstream mRNA of miR-221-3p. MiR-221-3p downregulation could promoted the ferroptosis in GC cells through upregulation of ATF3. HRD1 mediates ubiquitination and degradation of ACSL4 to inhibit ferroptosis. ATF3 upregulation could reduce GC cell proliferation via downregulating the transcription of GPX4 and HRD1. Furthermore, downregulation of miR-221-3p markedly attenuated the growth of GC in mice. CONCLUSION: HRD1 mediates ubiquitination and degradation of ACSL4 to inhibit ferroptosis. MiR-221-3p depletion upregulates the ferroptosis in GC cells via upregulation of ATF3 to mediate the transcription inhibition of GPX4 and HRD1. Our study might provide a novel target for GC treatment.

Current status of clinical trial research and application of immune checkpoint inhibitors for non-small cell lung cancer.

Immunotherapy has emerged as a hot topic in the treatment of non-small cell lung cancer (NSCLC) with remarkable success. Compared to chemotherapy patients, the 5-year survival rate for immunotherapy patients is 3-fold higher, approximately 4%-5% versus 15%-16%, respectively. Immunotherapies include chimeric antigen receptor T-cell (CAR-T) therapy, tumor vaccines, immune checkpoint inhibitors, and so forth. Among them, immune checkpoint inhibitors are in the spotlight. Common immune checkpoint inhibitors (ICIs) currently in clinical use include programmed death receptor-1(PD-1)/programmed death ligand-1(PD-L1) and cytotoxic T lymphocyte-associated antigen 4(CTLA-4). This article focuses on monotherapy and combination therapy of CTLA-4 and PD-1/PD-L1 immune checkpoint inhibitors. In particular, the combination therapy of ICIs includes the combination of ICIs and chemotherapy, the combination therapy of dual ICIs, the combination of ICIs and anti-angiogenic drugs, the combination of ICIs and radiotherapy, and the combination of ICIs inhibitors and tumor vaccines and so forth. This article focuses on the combination therapy of ICIs with chemotherapy, the combination therapy of dual ICIs, and the combination therapy of ICIs with anti-angiogenic drugs. The efficacy and safety of ICIs as single agents in NSCLC have been demonstrated in many trials. However, ICIs plus chemotherapy regimens offer significant advantages in the treatment of NSCLC with little to no dramatic increase in toxicity, while combined dual ICIs significantly reduce the adverse effects (AEs) of chemotherapy. ICIs plus anti-angiogenic agents regimen improves anti-tumor activity and safety and is expected to be the new paradigm for the treatment of advanced NSCLC. Despite some limitations, these agents have achieved better overall survival rates. In this article, we review the current status and progress of research on ICIs in NSCLC in recent years, aiming to better guide the individualized treatment of NSCLC patients.

An improved recombinase polymerase amplification assay for the visual detection of Staphylococcus epidermidis with lateral flow strips.

Staphylococcus epidermidis is an opportunistic pathogenic microorganism that is an important cause of cross-infection in hospitals. The development of rapid and effective detection techniques is important for its control. The application of traditional identification and PCR-based methods is limited by their requirements for both laboratory instrumentation and trained personnel. To overcome this issue, we developed a fast detection approach for S. epidermidis that was based on recombinase polymerase amplification (RPA) and lateral flow strips (LFS). First, five pairs of primers were designed for molecular diagnosis using the sesB gene as the target, and were screened for their amplification performance and the formation of primer dimers. Specific probes were then designed based on the best primer pairs screened, which were susceptible to primer-dependent artifacts and generated false-positive signals when used for LFS detection. This weakness of the LFS assay was overcome by modifying the sequences of the primers and probes. The efficacy of these measures was rigorously tested, and improved the RPA-LFS system. Standardized systems completed the amplification process within 25 min at a constant temperature of 37 °C, followed by visualization of the LFS within 3 min. The approach was very sensitive (with a detection limit of 8.91 CFU/µL), with very good interspecies specificity. In the analysis of clinical samples, the approach produced results consistent with PCR and 97.78% consistent with the culture-biochemical method, with a kappa index of 0.938. Our method was rapid, accurate, and less dependent on equipment and trained personnel than traditional methods, and provided information for the timely development of rational antimicrobial treatment plans. It has high potential utility in clinical settings, particularly in resource-constrained locations.

Cyclin dependent kinase 14 as a paclitaxel-resistant marker regulated by the TGF-ß signaling pathway in human ovarian cancer.

Cyclin dependent kinase 14 (CDK14) plays a central role in the control of cell proliferation and cell cycle progression. However, the specific function and regulatory mechanism of CDK14 on paclitaxel (PTX) resistance in ovarian cancer (OC) remain unclear. The present study demonstrated that CDK14 was overexpressed in OC tissues and cells at mRNA and protein levels detected by qRT-PCR, Western blot, and immunohistochemistry. Survival analysis showed that elevated CDK14 was related to the poor prognosis of OC patients. Overexpression of CDK14 was correlated with chemoresistance in OC. The expression level of CDK14 was higher in PTX-resistant OC cells (SK3R-PTX and OV3R-PTX) than in their counterpart-sensitive cells (SK-OV-3 and OVCAR-3). Knockdown of CDK14 decreased multidrug resistance 1 (MDR1) and ß-catenin expression in SK3R-PTX and OV3R-PTX cells and resensitized OC cells to PTX by decreasing cell proliferation and inducing cell apoptosis. Administration of transforming growth factor (TGF)-ß1 decreased CDK14 protein in PTX-resistant OC cells. The inhibitory effect of TGF-ß1 on CDK14 expression was abolished in the presence of a TGF-ß type I receptor kinase inhibitor (SB-431542). Furthermore, TGF-ß signal transducer Smad2 protein directly bound to the region -437 to -446 upstream of the CDK14 transcription start site (TSS), resulting in downregulating the expression of CDK14. These data indicate that CDK14 is a PTX-resistant marker and is regulated by the TGF-ß signaling pathway. Targeting CDK14 to enhance the sensitivity of PTX may provide a new therapeutic strategy for reversing the PTX resistance in OC.

Target Finder of Transcription Factor (TFoTF): a novel tool to predict transcription factor-targeted genes in cancer.

Transcription factors (TFs) are key players in the regulation of gene transcription in mammalian cells. Although high-throughput screening can be used to identify differentially expressed genes between comparable groups, the precision of the corresponding datasets is far from optimal. Here, we establish Target Finder of Transcription Factor (TFoTF), a method for the prediction of TF-targeted genes from genomic and cancer-related transcriptomic data. TFoTF can identify potential TF-targeted genes in large cancer datasets and efficiently estimate correlations between TFs and their targeted genes with a significant level of specificity, sensitivity, and precision. Overall, TFoTF is an easy-to-use tool that can be utilized to generate testable hypotheses in the context of cancer research projects.