Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannan-binding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP. Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays. Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05). Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

The regulation of spermatogonial proliferation and apoptosis is of great significance for maintaining spermatogenesis. The single-cell RNA sequencing (scRNA-seq) analysis of the testis was performed to identify genes upregulated in spermatogonia. Using scRNA-seq analysis, we identified the spermatogonia upregulated gene origin recognition complex subunit 6 (Orc6), which is involved in DNA replication and cell cycle regulation; its protein expression in the human and mouse testis was detected by western blot and immunofluorescence. To explore the potential function of Orc6 in spermatogonia, the C18-4 cell line was transfected with control or Orc6 siRNA. Subsequently, 5-ethynyl-2-deoxyuridine (EdU) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, flow cytometry, and western blot were used to evaluate its effects on proliferation and apoptosis. It was revealed that ORC6 could promote proliferation and inhibit apoptosis of C18-4 cells. Bulk RNA sequencing and bioinformatics analysis indicated that Orc6 was involved in the activation of wingless/integrated (Wnt)/ β-catenin signaling. Western blot revealed that the expression of β-catenin protein and its phosphorylation (Ser675) were significantly decreased when silencing the expression of ORC6. Our findings indicated that Orc6 was upregulated in spermatogonia, whereby it regulated proliferation and apoptosis by activating Wnt/β-catenin signaling.

Objective: To investigate the effects and clinical significance of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activated by interleukin (IL)-17A in chronic rhinosinusitis with nasal polyps (CRSwNP). Methods: Patients underwent nasal endoscopic surgery in the Third Affiliated Hospital of Sun Yat-sen University from January 2020 to December 2021 were collected, including 28 CRSwNP (including 19 males and 9 females, aged 19 to 67 years), 22 chronic rhinosinusitis without nasal polyps (CRSsNP) and 22 controls. qRT-PCR was used to detect the expressions of IL-17A, NLRP3, IL-1β and IL-18 in the three groups, and their correlations were analyzed. The positions of IL-17A, NLRP3 and IL-18 in nasal polys were analyzed by immunofluorescence. Western Blotting and ELISA were employed to detect the expression of NLRP3, IL-1β and IL-18 in the human nasal epithelial cells after using IL-17A stimulation or IL-17A receptor inhibitor. Immunofluorescence was used to observe the NLRP3, IL-1β, and IL-18 protein expression after IL-17A stimulating human nasal epithelial cells, and after the use of IL-17A receptor inhibitor and NLRP3 inhibitor MCC950. The correlations between NLRP3, IL-1β, IL-18 and CT scores, nasal endoscopic scores, visual analogue scale (VAS) scores, and sino-nasal outcome test (SNOT) 22 scores of CRSwNP patients were analyzed. SPSS 20.0 software was used for statistical analysis. Results: The expressions of IL-17A, NLRP3, IL-1β and IL-18 in the tissues of CRSwNP patients were significantly higher than those in CRSsNP group(P=0.018,P<0.001,P=0.005, P=0.016) and the control group(all P<0.001). IL-17A was positively correlated with the expression of NLRP3, IL-1β, and IL-18(r ralue was 0.643,0.650,0.629,respectively, all P<0.05). IL-17A, NLRP3, and IL-18 were co-localized in the epithelial propria of polyp tissue. IL-17A stimulated the expressions of NLRP3, IL-1β, and IL-18 in human nasal epithelial cells. After the use of IL-17A receptor inhibitor, the expressions of NLRP3, IL-1β, and IL-18 were significantly down-regulated. After the use of NLRP3 inhibitor MCC950, IL-17A was significantly down-regulated to promote the expression of NLRP3, IL-1β, and IL-18. The expressions of NLRP3, IL-1β and IL-18 were positively correlated with CT, nasal endoscopy, VAS, and SNOT22 scores in patients with CRSwNP. Conclusions: IL-17A promotes the release of IL-1β and IL-18 by activating the NLRP3 inflammasome and aggravates the severity of the disease in CRSwNP.
Female ; Humans ; Male ; Chronic Disease ; Clinical Relevance ; Inflammasomes ; Interleukin-17/metabolism* ; Interleukin-18 ; Nasal Polyps/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rhinitis/metabolism* ; Sinusitis/metabolism* ; Young Adult ; Adult ; Middle Aged ; Aged

Objective: To observe the efficacy and adverse reactions of a combination therapy regimen based on bortezomib and glucocorticoids in recurrent/refractory immune thrombocytopenic purpura (iTTP) . Methods: Six patients with recurrent/refractory TTP were included and treated with a glucocorticoid and two courses of bortezomib-based regimen. The clinical remission status of patients, changes in ADAMTS13 activity/ADAMTS13 inhibitor, and the occurrence of treatment-related adverse reactions were observed. Results: Of the 6 patients, 2 were males and 4 were females, with a median age of 21.5 (18-68) years. Refractory TTP was found in 1 case and recurrent TTP in 5 cases. Glucocorticoids were administered with reference to prednisone at 1 mg·kg(-1)·d(-1), and gradually reduced in dosage after achieving clinical remission. Bortezomib is subcutaneously administered at 1.3 mg/m(2) on days 1, 4, 8, and 11 with a 28-day treatment course consisting of 2 courses. Six patients achieved clinical remission after receiving bortezomib as the main treatment. ADMATS13 activity returned to normal in all patients with TTP after treatment, and the ADAMTS13 inhibitor turned negative. Thrombocytopenia is the most common adverse reaction after treatment, with other adverse reactions, including peripheral neuritis and abdominal pain, but ultimately all patients returned to normal. In a median follow-up of 26 (9-41) months, 5 patients maintained sustained remission, and 1 patient relapsed after 16 months of bortezomib treatment. Conclusion: Combination therapy of bortezomib and glucocorticoids has a satisfactory therapeutic effect and controllable adverse reactions for recurrent/refractory iTTP.
Male ; Female ; Humans ; Young Adult ; Adult ; Middle Aged ; Aged ; Bortezomib/therapeutic use* ; Glucocorticoids/therapeutic use* ; Rituximab/therapeutic use* ; Purpura, Thrombotic Thrombocytopenic/drug therapy* ; Purpura, Thrombocytopenic, Idiopathic/drug therapy* ; ADAMTS13 Protein/therapeutic use*

Objective: To report the clinical manifestations and laboratory features of five patients with congenital thrombotic thrombocytopenic purpura (cTTP) and explore its standardized clinical diagnosis and treatment along with a review of literature. Methods: Clinical data of patients, such as age of onset, disease manifestation, personal history, family history, and misdiagnosed disease, were collected. Treatment outcomes, therapeutic effects of plasma infusion, and organ function evaluation were observed. The relationship among the clinical manifestations, treatment outcomes, and ADAMTS13 gene mutation of patients with cTTP was analyzed. Additionally, detection of ADAMTS13 activity and analysis of ADAMTS13 gene mutation were explored. Results: The age of onset of cTTP was either in childhood or adulthood except in one case, which was at the age of 1. The primary manifestations were obvious thrombocytopenia, anemia, and different degrees of nervous system involvement. Most of the patients were initially suspected of having immune thrombocytopenia. Acute cTTP was induced by pregnancy and infection in two and one case, respectively. ADAMTS13 gene mutation was detected in all cases, and there was an inherent relationship between the mutation site, clinical manifestations, and degree of organ injury. Therapeutic or prophylactic plasma transfusion was effective for treating cTTP. Conclusions: The clinical manifestations of cTTP vary among individuals, resulting in frequent misdiagnosis that delays treatment. ADAMTS13 activity detection in plasma and ADAMTS13 gene mutation analysis are important bases to diagnose cTTP. Prophylactic plasma transfusion is vital to prevent the onset of the disease.
Female ; Pregnancy ; Humans ; Adult ; Blood Component Transfusion ; Plasma ; Purpura, Thrombotic Thrombocytopenic/therapy* ; Mutation ; Purpura, Thrombocytopenic, Idiopathic ; ADAMTS13 Protein/therapeutic use*

Objective:To summarize and analyze the practice of the local practice on covering drugstore bills by pooled funds of basic medical insurance,and provide a reference for improving relevant policies.Methods:The medical insurance policies from various provinces,municipalities,autonomous regions,and coordination areas were systematically retrieved.ROST CM6 software was applied to analyze the high-frequency words and semantic network of the policy text,and combined with the interview and field investigation resultss,the key dimensions of the policy practice were identified and summarized.Then the regional differences,existing problems,and their causes were analyzed to put forward policy recommendations.Result:The selection of pharmacies covered in the payment system with outpatient expenses reimbursed by the pooled fund,the formulation of drug reimbursement list,the design of benefit plans,the management of drug prices and payments,and the supervision of hospital outflow prescriptions were five key dimensions of policy practice.There were significant differences in practice among different regions,and the problems mainly included the overall arrangement of covering pharmacies in the payment system,the mechanism of drug prices in pharmacies,and the coordination with other medical insurance policies.Conclusion:To improve the convenience of buying drugs for the insured,it is necessary to make full use of the advantages of pharmacies to meet the demand for outpatient medicine,promote the transparency of drug prices in pharmacies,coordinate the relevant medical insurance policies,strengthen the collaborative management between the healthcare security administration and relevant departments such as the health commission and the medical products administration,analyze and evaluate the potential effects of policy measures,and adjust policy measures promptly according to local conditions.

The vagina contains at least a billion microbial cells,dominated by lactobacilli.Here we perform metagenomic shotgun sequencing on cervical and fecal samples from a cohort of 516 Chinese women of reproductive age,as well as cervical,fecal,and salivary samples from a second cohort of 632 women.Factors such as pregnancy history,delivery history,cesarean section,and breastfeeding were all more important than menstrual cycle in shaping the microbiome,and such information would be necessary before trying to interpret differences between vagino-cervical micro-biome data.Greater proportion of Bifidobacterium breve was seen with older age at sexual debut.The relative abundance of lactobacilli especially Lactobacillus crispatus was negatively associated with pregnancy history.Potential markers for lack of menstrual regularity,heavy flow,dysmenor-rhea,and contraceptives were also identified.Lactobacilli were rare during breastfeeding or post-menopause.Other features such as mood fluctuations and facial speckles could potentially be predicted from the vagino-cervical microbiome.Gut and salivary microbiomes,plasma vitamins,metals,amino acids,and hormones showed associations with the vagino-cervical microbiome.Our results offer an unprecedented glimpse into the microbiota of the female reproductive tract and call for international collaborations to better understand its long-term health impact other than in the settings of infection or pre-term birth.

Drug resistance resulting from bacterial biofilms can invalidate antibacterial agents. Therefore,eradicating bacterial biofilms to reverse drug resistance is a hotspot in the pharmaceutical research. In recent years,numerous studies have revealed the complicated mechanism of bacterial biofilm formation and strong drug resistance with multiple influential factors involved. This paper gives a comprehensive review on the process of biofilm formation and intervention by natural drugs,which can provide some reference and evidence for the following studies.
Anti-Bacterial Agents/pharmacology* ; Bacteria/genetics* ; Biofilms ; Drug Resistance ; Pharmaceutical Preparations

Methyl-CpG binding protein 2 (MeCP2) is a basic nuclear protein involved in the regulation of gene expression and microRNA processing. Duplication of MECP2-containing genomic segments causes MECP2 duplication syndrome, a severe neurodevelopmental disorder characterized by intellectual disability, motor dysfunction, heightened anxiety, epilepsy, autistic phenotypes, and early death. Reversal of the abnormal phenotypes in adult mice with MECP2 duplication (MECP2-TG) by normalizing the MeCP2 levels across the whole brain has been demonstrated. However, whether different brain areas or neural circuits contribute to different aspects of the behavioral deficits is still unknown. Here, we found that MECP2-TG mice showed a significant social recognition deficit, and were prone to display aversive-like behaviors, including heightened anxiety-like behaviors and a fear generalization phenotype. In addition, reduced locomotor activity was observed in MECP2-TG mice. However, appetitive behaviors and learning and memory were comparable in MECP2-TG and wild-type mice. Functional magnetic resonance imaging illustrated that the differences between MECP2-TG and wild-type mice were mainly concentrated in brain areas regulating emotion and social behaviors. We used the CRISPR-Cas9 method to restore normal MeCP2 levels in the medial prefrontal cortex (mPFC) and bed nuclei of the stria terminalis (BST) of adult MECP2-TG mice, and found that normalization of MeCP2 levels in the mPFC but not in the BST reversed the social recognition deficit. These data indicate that the mPFC is responsible for the social recognition deficit in the transgenic mice, and provide new insight into potential therapies for MECP2 duplication syndrome.

OBJECTIVES: To develop a new Chinese medicine (CM)-based drug and to evaluate its safety and effect for suppressing acute respiratory distress syndrome (ARDS) in COVID-19 patients. METHODS: A putative ARDS-suppressing drug Keguan-1 was first developed and then evaluated by a randomized, controlled two-arm trial. The two arms of the trial consist of a control therapy (alpha interferon inhalation, 50 µg twice daily; and lopinavir/ritonavir, 400 and 100 mg twice daily, respectively) and a testing therapy (control therapy plus Keguan-1 19.4 g twice daily) by random number table at 1:1 ratio with 24 cases each group. After 2-week treatment, adverse events, time to fever resolution, ARDS development, and lung injury on newly diagnosed COVID-19 patients were assessed. RESULTS: An analysis of the data from the first 30 participants showed that the control arm and the testing arm did not exhibit any significant differences in terms of adverse events. Based on this result, the study was expanded to include a total of 48 participants (24 cases each arm). The results show that compared with the control arm, the testing arm exhibited a significant improvement in time to fever resolution (P=0.035), and a significant reduction in the development of ARDS (P=0.048). CONCLUSIONS Keguan-1-based integrative therapy was safe and superior to the standard therapy in suppressing the development of ARDS in COVID-19 patients. (Trial registration No. NCT04251871 at www.clinicaltrials.gov ).
Administration, Inhalation ; Adult ; China ; Coronavirus Infections ; diagnosis ; drug therapy ; mortality ; Dose-Response Relationship, Drug ; Drug Administration Schedule ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Follow-Up Studies ; Humans ; Integrative Medicine ; Interferon-alpha ; administration & dosage ; Lopinavir ; administration & dosage ; Male ; Middle Aged ; Pandemics ; Pneumonia, Viral ; diagnosis ; drug therapy ; mortality ; Risk Assessment ; Severe Acute Respiratory Syndrome ; diagnosis ; drug therapy ; mortality ; Severity of Illness Index ; Survival Rate