Involvement of Tn3 transposon in formation and transmission of hypervirulent and carbapenem-resistant Klebsiella pneumoniae.

IMPORTANCE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is resistant to most common antibiotics, becoming the most important and prevalent nosocomial opportunity pathogen. Besides, K. pneumoniae can also cause severe community-acquired infections, such as primary liver abscess and endophthalmitis. These pathogens are commonly referred to as hvKp. CRKP and hvKp have evolved separately, each occupying its own clonal lineage and exhibiting a variety of properties. Our study provides important insights into the evolutionary events related to the arousal of virulence and drug resistance in K. pneumoniae through plasmid transmission, mediated by Tn3 transposon. Our study also provides evidence that multiple mechanisms contribute to the successful transfer of non-conjugative virulence plasmid, and the involvement of transposons enhances the efficiency. A good knowledge of its transmission mechanisms is fundamental to finding effective strategies to combat these threatening pathogens. Transposons are widely present in bacteria, spreading resistance and virulence genes between the environment and humans. Therefore, emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens should be highly valued.

Influence of roasted flaxseed marc flour on rheological, structural, fermentation, water distribution, and migration properties of wheat dough.

Roasted flaxseed (RF) marc, which is a by-product obtained from RF oil extraction, has high nutritional value. This study evaluated the impact of RF marc flour on rheological, structural, fermentation, water distribution, and migration properties of the wheat dough. Results showed that adding RF into wheat flour (WF) could effectively increase the water absorption in the dough and retard the retrogradation of starch. The fermentation results revealed that adding RF could improve the gas retention coefficient of dough. Compared to the dough prepared with WF, the doughs enriched with RF had higher tan Î´ values (ratio of loss modulus G″ to storage modulus G'), indicating a more liquid-like property. The mobility of tightly bound and free water in dough was decreased by adding RF, whereas the distribution of free water was increased. On the one hand, adding RF would dilute the gluten content in dough, resulting in a weaker protein network. On the other hand, the dietary fiber and proteins in RF could offset the gluten dilution effect to some extent. Overall, the results suggested that the substitution level of RF in WF should be below 25% to avoid serious dough quality deterioration, and the RF-WF blended flour could be a potential ingredient to produce wheat products with moist taste. These findings could be useful for guiding the future usage of RF marc in wheat-based products.

Association of adiposity indices with cardiometabolic multimorbidity among 101,973 chinese adults: a cross-sectional study.

BACKGROUND: Cardiometabolic multimorbidity (CMM) and obesity represent two major health problems. The relationship between adiposity indices and CMM, however, remains understudied. This study aimed to investigate the associations of body mass index (BMI), waist circumference (WC), waist-to-height ratio (WHtR), a body shape index (ABSI), body roundness index (BRI), and conicity index (CI) with CMM among Chinese adults. METHODS: Data of 101,973 participants were collected from a population-based screening project in Southern China. CMM was defined as having two or more of the following diseases: coronary heart disease, stroke, hypertension, and diabetes. The relationship between the six adiposity indices and CMM was investigated by multivariate logistic regression and restricted cubic splines. Receiver operator characteristic curve, C-statistic and net reclassification index were used to estimate the discriminative and incremental values of adiposity indices on CMM. RESULTS: Logistic regression models showed the six adiposity indices were all significantly associated with the odds of CMM with non-linear relationships. For per SD increment, WC (Odds ratio [OR]: 1.66; 95% confidence interval (CI): 1.62-1.70) and WHtR (OR, 1.61; 95% CI, 1.58-1.65) were more significantly associated with a higher prevalence of CMM than BMI (OR, 1.55; 95% CI, 1.52-1.58) (all P < 0.05). In addition, WC, WHtR, and BRI displayed significantly better performance in detecting CMM compared with BMI (all P < 0.05). Their respective area under the curve (AUC) values were 0.675 (95% CI: 0.670-0.680), 0.679 (95% CI: 0.675-0.684), and 0.679 (95% CI: 0.675-0.684), while BMI yielded an AUC of 0.637 (95% CI: 0.632-0.643). These findings hold true across all subgroups based on sex and age. When Adding WC, WHtR, or BRI to a base model, they all provided larger incremental values for the discrimination of CMM compared with BMI (all P < 0.05). CONCLUSIONS: Adiposity indices were closely associated with the odds of CMM, with WC and WHtR demonstrating stronger associations than BMI. WC, WHtR, and BRI were superior to BMI in discriminative ability for CMM. Avoidance of obesity (especially abdominal obesity) may be the preferred primary prevention strategy for CMM while controlling for other major CMM risk factors.

Stage 1 hypertension defined by the 2017 ACC/AHA blood pressure guideline and cardiometabolic multimorbidity in Chinese adults.

The association of blood pressure (BP) classification defined by the 2017 American College of Cardiology/American Heart Association (ACC/AHA) guideline with cardiometabolic multimorbidity (CMM) remains unclear. The present study aimed to investigate this research gap in the Chinese adults. Cross-sectional data were collected from a population-based cohort conducted in Southern China. Participants were categorized as having normal BP, elevated BP, stage 1 hypertension, and stage 2 hypertension according to the 2017 ACC/AHA guideline. CMM was defined as having two or more of the following diseases: coronary heart disease, stroke, and diabetes. The relationship between the BP classifications and CMM was examined by multivariate logistic regression. A total of 95 649 participants (mean age: 54.3 ± 10.2 years, 60.7% were women) were enrolled in this study. Multivariable-adjusted logistic regression models revealed that stage 1 hypertension (odds ratio [OR], 1.35; 95% confidence interval [CI], 1.03-1.78) and stage 2 hypertension (OR, 3.53; 95% CI, 2.82-4.47) were significantly associated with a higher prevalence of CMM compared with normal BP. The association between stage 1 hypertension and CMM were profound in women (OR, 1.76; 95% CI, 1.17-2.67) and in the middle-aged group (OR, 1.53; 95% CI, 1.02-2.35) compared with men and older individuals, respectively. Our study showed that among Chinese adults, stage 1 hypertension defined by the 2017 ACC/AHA guideline was already associated with higher odds of CMM compared with normal BP, particularly in women and middle-aged participants. Managing stage 1 hypertension may be an important measure to prevent CMM in Chinese adults.

microRNA-1-3p and T-synthase mRNA have high diagnostic efficacy on intestinal mucosal barrier dysfunction in patients with severe acute pancreatitis.

Acute pancreatitis (AP) is an inflammatory disorder of the pancreas that can be complicated by intestinal mucosal barrier dysfunction (SAP&IBD). The current study sought to examine the diagnostic efficacy of miR-1-3p and T-synthase mRNA in SAP&IBD patients. First, SAP patients were assigned to SAP&IBD and SAP groups. Serum miR-1-3p expression and T-synthase mRNA expression patterns in peripheral blood B lymphocytes were measured using RT-qPCR. Pearson tests, ROC curve analysis, and multivariate logistic regression were used to analyze the correlation between miR-1-3p/T-synthase mRNA and clinical data, their diagnostic efficiency, and independent risk factors for SAP&IBD patients, respectively. The results showed that serum miR-1-3p in the SAP&IBD group was elevated, and T-synthase mRNA expression in peripheral blood B lymphocytes was diminished. Additionally, serum miR-1-3p expression in SAP&IBD patients was negatively correlated with T-synthase mRNA expression, and positively correlated with their Ranson score, CRP, IL-6, DAO, and D-Lactate levels. Meanwhile, T-synthase mRNA level was negatively correlated with IL-6, DAO, and D-Lactate levels. Both, serum miR-1-3p, T-synthase mRNA, and their combination were found to exhibit diagnostic efficiency for SAP&IBD patients, and were independently associated with IBD in SAP patients. Collectively, our findings suggest that miR-1-3p and T-synthase serve as independent risk factors for SAP&IBD patients and can aid the diagnosis of IBD in SAP patients.

Different regulatory mechanisms of the capsule in hypervirulent Klebsiella pneumonia: "direct" wcaJ variation vs. "indirect" rmpA regulation.

Introduction: Hypervirulent Klebsiella pneumoniae produce an increased amount of capsular substance and are associated with a hypermucoviscous phenotype. Capsule production is regulated by capsular regulatory genes and capsular gene cluster variations. In the present study, we focus on the effect of rmpA and wcaJon capsule biosynthesis. Methods: Phylogenetic trees were constructed to analyze wcaJ and rmpA sequence diversity in different serotypes hypervirulent strains. Then mutant strains (K2044ΔwcaJ, K2044K1wcaJ, K2044K2wcaJand K2044K64wcaJ) were used to verify the effects of wcaJ and its diversity on capsule synthesis and strain virulence. Furthmore, the role of rmpA in capsular synthesis and its mechanisms were detected in K2044ΔrmpA strain. Results: RmpA sequences are conversed in different serotypes. And rmpA promoted the production of hypercapsules by simultaneously acting on three promoters in cps cluster. Whereas wcaJ, its sequences are different in different serotypes, and its loss result in the termination of capsular synthesis. Moreover, the results verified that K2 wcaJ could form hypercapsule in K2044 strains (K1 serotype), but K64 wcaJ could not. Discussion: The interaction of multiple factors is involved in capsule synthesis, including wcaJ and rmpA. RmpA, an known conserved capsular regulator gene, acts on cps cluster promoters to promote the production of the hypercapsule. WcaJ as initiating enzyme of CPS biosynthesis, its presence determines the synthesis of capsule. Besides, different from rmpA, wcaJ sequence consistency is limited to the same serotype, which cause wcaJ functioning in different serotype strains with sequence recognition specificity.

Hypercapsule is the cornerstone of Klebsiella pneumoniae in inducing pyogenic liver abscess.

Purpose: To investigate the mechanisms of Klebsiella pneumoniae-induced pyogenic liver abscess (PLA). Methods: Forty-three K. pneumoniae strains from PLAs and 436 from non-PLAs were collected. Their differences were compared for virulence genes and factors, sequence types, and serotypes. Virulence genes wzi, wzy-K1, and wzi+wzy-K1 were deleted in K. pneumoniae NTUH-K2044. Various analyses, such as transmission electron microscopy, neutrophil killing tests, and mouse lethality tests, were used to confirm the consequent changes. Results: Differences were found between K. pneumoniae strains from PLA and non-PLA samples for virulence genes and factors, including metabolism genes (allS and peg-344), capsular polysaccharide (CPS)-synthesis channel gene (wzy-K1), CPS-regulating genes (p-rmpA, p-rmpA2, and c-rmpA), and siderophore genes (iucA and iroN). When wzy-K1 was positive, the difference between PLA and non-PLA samples was only observed with c-rmpA. Δwzi, Δwzy-K1, and ΔwziΔwzy-K1 strains reverted to hypovirulence. In the Kupffer cell stimulation assay, interleukin (IL)-6, IL-12, IL-10, and transforming growth factor-ß secretions were found to be equivalent in NTUH-K2044, Δwzi, Δwzy-K1, and ΔwziΔwzy-K1 groups. Lower IL-1ß and higher tumor necrosis factor-α secretions were observed for Δwzi, Δwzy-K1, and ΔwziΔwzy-K1. Conclusions: Hypercapsule production is the cornerstone of hypervirulence, regardless of exopolysaccharides. K1 K. pneumoniae-induced PLA may decrease core inflammatory cytokines rather than increase anti-inflammatory cytokines. Exopolysaccharides could also attenuate the inflammatory response to aid in the immune escape of K. pneumoniae.

Exploiting a conjugative endogenous CRISPR-Cas3 system to tackle multidrug-resistant Klebsiella pneumoniae.

BACKGROUND: Mobile plasmids play a key role in spurring the global dissemination of multidrug-resistant (MDR) K. pneumoniae, while plasmid curing has been recognized as a promising strategy to combat antimicrobial resistance. Here we exploited a K. pneumoniae native CRISPR system to cure the high-risk IncFII plasmids. METHODS: We examined matched protospacers in 725 completely sequenced IncFII plasmids from K. pneumoniae genomes. Then, we re-engineered a native CRISPR-Cas3 system and deliver the CRISPR-Cas3 system via conjugation. Plasmid killing efficiency and G. mellonella infection model were applied to evaluate the CRISPR-Cas3 immunity in vitro and in vivo. FINDINGS: Genomic analysis revealed that most IncFII plasmids could be targeted by the native CRISPR-Cas3 system with multiple matched protospacers, and the targeting regions were highly conserved across different IncFII plasmids. This conjugative endogenous CRISPR-Cas3 system demonstrated high plasmid curing efficiency in vitro (8-log decrease) and in vivo (∼100% curing) in a Galleria mellonella infection model, as well as provided immunization against the invasion of IncFII plasmids once the system entering a susceptible bacterial host. INTERPRETATION: Overall, our work demonstrated the applicability of using native CRISPR-mediated plasmid curing to re-sensitize drug-resistant K. pneumoniae to multiple antibiotics. This work provided strong support for the idea of utilizing native CRISPR-Cas systems to tackle AMR in K. pneumoniae. FUNDING: This work was supported by research grants National Natural Science Foundation of China [grant numbers 81871692, 82172315, 82102439, and 82202564], the Shanghai Science and Technology Commission [grant number 19JC1413002], and Shanghai Sailing Program [grant number 22YF1437500].

A mutant of Monascus purpureus obtained by carbon ion beam irradiation yielded yellow pigments using various nitrogen sources.

The industrial production of monascus yellow pigments (MYPs) has not yet been done due to the lack of high-quality industrial Monascus strains. In this work, we employed carbon ion beam (12C6+) irradiation to screen Monascus strains that produce high-quality extracellular MYPs (extr-MYPs). One genetically stable M. purpureus mutant of BWY-5 with extr-MYPs accumulation was obtained under 12C6+ irradiation (80 MeV/u, 200 Gy). M. purpureus BWY-5 could use various nitrogen sources to produce single pH stable extr-MYPs (around 80 AU at 370 nm). Moreover, citrinin was not detected by HPLC method. Transcriptomics of the MYP production strain suggested that Carbon ion beam irradiation led to deletion (MpigF, MpigG and MpigH), downregulation (CtnE, CtnH and CtnI) and upregulation (MpigM) of genes related with biosynthesis of MOPs and MRPs, citrinin, and extr-MYPs, respectively. The results showed that M. purpureus BWY-5 has the potential to be used as an industrial Monascus strain and platform for extr-MYPs production and monascus polyketide synthetic pathway studies, respectively.

Correlation between vaginal flora and cervical immune function of human papilloma virus-infected patients with cervical cancer.

Background: To analyse the correlation between vaginal flora and cervical immune function of HPV-infected patients with cervical cancer. Methods: Six hundred females with genital tract infections treated in Xuzhou Hospital of Traditional Chinese Medicine from January 2014 to December 2016 were selected and divided into a high-risk HPV group (n=246) and a control group (n=354). The vaginal flora and human T lymphocyte subsets (CD3+, CD4+, CD8+) were detected. Multivariate logistic regression analysis was performed to explore the risk factors for HPV infection. Results: The numbers of CD4+ and CD4+/CD8+ T cells of the high-risk HPV group were significantly lower than those of the control group (P<0.05). The two groups had similar numbers of CD3+ and CD8+ T cells. In the high-risk HPV group, the positive rates of Lactobacillus, Chlamydia trachomatis, Mycoplasma hominis, mycetes, Ureaplasma urealyticum and bacterial vaginosis were significantly higher than those of the control group (P<0.05). There was no significant difference in the positive rates of trichomonads between the two groups. Multivariate logistic regression analysis revealed that C. trachomatis and U. urealyticum were independent risk factors for high-risk HPV infection (P<0.05). Conclusion: High-risk HPV infection in patients with cervical cancer was associated with vaginal flora and immune function. C. trachomatis and U. urealyticum were independent risk factors for high-risk HPV infection.

Prevalence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae under divergent evolutionary patterns.

K1/K2 hvKP strains acquire carbapenem-resistance plasmids, known as CR-hvKp, and carbapenem-resistant Klebsiella pneumoniae (CRKP) strains obtain virulence plasmids, recognized as hv-CRKP. The two different evolution patterns of hypervirulent combined carbapenem-resistant Klebsiella pneumoniae may lead to their different prevalence in hospitals. Our study aimed to investigate the prevalence of hv-CRKP and CR-hvKp strains and to analyze factors influencing their evolution and prevalence. We collected 890 K. pneumoniae genomes from GenBank and 530 clinical K. pneumoniae isolates from nine hospitals. Our study found that hv-CRKP strains were more prevalent than CR-hvKp strains and both were dominated by blaKPC-2 gene. The blaKPC-2-carrying plasmids could mobilize non-conjugative virulence plasmids from hvKp strains to CRKP strains. The conserved oriT of virulence plasmids and the widespread of conjugative helper plasmids were potential factors for the mobilization of non-conjugative virulence plasmids. HvKp strains with KPC plasmid could hardly simultaneously exhibit hypervirulence and carbapenem resistance as CRKP strains with virulence plasmid, and we found that rfaH mutation reduced capsular synthesis and increased carbapenem resistance of the CR-hvKp strain. In summary, this study revealed that hv-CRKP strains were more suitable for survival in hospital settings than CR-hvKp strains and the widespread conjugative KPC-producing plasmids contributed to the emergence and prevalence of hv-CRKP strains.

Protocol for Home-Based Solution for Remote Atrial Fibrillation Screening to Prevent Recurrence Stroke (HUA-TUO AF Trial): a randomised controlled trial.

INTRODUCTION: Current international guidelines recommend ECG monitoring after an ischaemic stroke to detect atrial fibrillation (AF) in order to prevent stroke recurrence. However, optimal strategies to detect AF and the downstream management to prevent stroke recurrence remain to be established. The objective of the study was to explore the use of long-term home-based ECG monitoring for AF detection and stroke prevention in patients with a history of stroke. METHODS AND ANALYSIS: This prospective, randomised, open-label trial with blinded endpoint adjudication aimed to evaluate the efficacy of long-term home-based ECG monitoring for AF detection and stroke prevention in a 24-month period. Patients aged >18 years with a history of ischaemic stroke will be stratified according to the time from the index ischaemic stroke: <1, 1-3 and >3 years and then randomised in 1:1 to (1) home-based AF screening and (2) control. The home-based AF screening system comprises (1) a handheld single-lead ECG recorder (Comfit Healthcare Devices, Hong Kong SAR, China) and (2) a patient-facing smartphone application specially designed for the study. Patients randomised to the home-based AF group will record a 30 s single-lead ECG using a specially designed handheld ECG device every morning or when symptomatic. All remotely obtained data will be automatically transmitted in real-time through the study smartphone application to a secured cloud hosting and analysed using an artificial intelligence-based diagnostic system. When a diagnosis of AF is made with the system, the patients will be called back for a formal cardiology consultation within 1 week. The primary endpoint is the time to first detection of AF at 24 months of follow-up. Secondary endpoints include recurrent stroke or transient ischaemic attack, initiation of long-term anticoagulation therapy, hospitalisation for heart failure, cardiovascular death and all-cause death. ETHICS AND DISSEMINATION: The study protocol has been approved by the institutional review board of The University of Hong Kong, and Hong Kong West Cluster, Hospital Authority, Hong Kong SAR, China. Results will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04523649.

Prevalence of Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae and Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae in China Determined via Mouse Lethality Tests.

Objective: To investigate the epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) and hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP). Methods: Totally 436 K. pneumoniae strains were collected from 7 hospitals in mainland China between 2017.01 and 2018.02. Sequence types, serotypes, antimicrobial-resistance and virulence genes were analyzed. Additionally, string test, capsule stain, Periodic Acid Schiff stain, fitness analysis, quantitative real-time PCR and mouse lethality test were also performed. Molecular combinations were used to screen putative blaKPC(+)-HvKP and Hv-blaKPC(+)-KP, followed by the confirmation of mouse lethality test. Results: Diverse detection rates were found for the virulence genes, ranging from c-rmpA (0.0%) to entB (100.0%). According to the molecular criteria, 127, 186, 9 and 26 strains were putatively denoted as HvKP, blaKPC(+)-KP, blaKPC(+)-HvKP and Hv-blaKPC(+)-KP. Mouse lethality test confirmed 2 blaKPC(+)-HvKP strains (JS184 and TZ20) and no Hv-blaKPC(+)-KP. JS184 showed K2 serotype, thin capsule, positive exopolysaccharid and string test. TZ20 presented K20 serotype, thin capsule, negative exopolysaccharide and string test. Compared with the positive control NTUH-K2044, equal galF expression and growth curves were confirmed for JS184 and TZ20. Conclusions: Molecular determination of CR-HvKP and Hv-CRKP brings remarkable bias compared with mouse lethality test. The exact prevalence of CR-HvKP is less than 1.0%, which of Hv-CRKP is much lower.

DBFU-Net: Double branch fusion U-Net with hard example weighting train strategy to segment retinal vessel.

Background: Many fundus imaging modalities measure ocular changes. Automatic retinal vessel segmentation (RVS) is a significant fundus image-based method for the diagnosis of ophthalmologic diseases. However, precise vessel segmentation is a challenging task when detecting micro-changes in fundus images, e.g., tiny vessels, vessel edges, vessel lesions and optic disc edges. Methods: In this paper, we will introduce a novel double branch fusion U-Net model that allows one of the branches to be trained by a weighting scheme that emphasizes harder examples to improve the overall segmentation performance. A new mask, we call a hard example mask, is needed for those examples that include a weighting strategy that is different from other methods. The method we propose extracts the hard example mask by morphology, meaning that the hard example mask does not need any rough segmentation model. To alleviate overfitting, we propose a random channel attention mechanism that is better than the drop-out method or the L2-regularization method in RVS. Results: We have verified the proposed approach on the DRIVE, STARE and CHASE datasets to quantify the performance metrics. Compared to other existing approaches, using those dataset platforms, the proposed approach has competitive performance metrics. (DRIVE: F1-Score = 0.8289, G-Mean = 0.8995, AUC = 0.9811; STARE: F1-Score = 0.8501, G-Mean = 0.9198, AUC = 0.9892; CHASE: F1-Score = 0.8375, G-Mean = 0.9138, AUC = 0.9879). Discussion: The segmentation results showed that DBFU-Net with RCA achieves competitive performance in three RVS datasets. Additionally, the proposed morphological-based extraction method for hard examples can reduce the computational cost. Finally, the random channel attention mechanism proposed in this paper has proven to be more effective than other regularization methods in the RVS task.

Optimal ablation index parameters for radiofrequency ablation therapy of atrial fibrillation.

Objectives: To explore the optimal ablation index (AI) parameters for radiofrequency catheter ablation (RA) for treating atrial fibrillation (AF). Methods: Patients with AF (186) who underwent bilateral PVAI in the Department of Cardiology, Zhuhai People's Hospital, Guangdong Province, from March 2018 to October 2019 and received catheter ablation as first-round treatment, were grouped according to the received AI. Control group included patients (95) who received the recommended AI ablation (350-400 for posterior wall, 400-450 for non-posterior wall). Patients in optimal AI group were ablated with optimal AI (300-330 for posterior wall, 350-380 for non-posterior wall). Recurrence was defined as any AF, atrial tachycardia, or atrial flutter lasting more than 30 seconds without anti-arrhythmic drugs after the 3-month blank period. Results: Of 186 patients, 66 patients had paroxysmal atrial fibrillation and a mean CHA2DS2-VASc score of 2.83±1.64. Isolation rates of bilateral PVI in both groups were 91.4% and 93.6%, for patients with paroxysmal atrial fibrillation, and 81.7% and 80% for patients with persistent atrial fibrillation (P > 0.05). Left atrial function index (LAFI) decreased under the condition of sinus rhythm at the 3rd and 6th months (P < 0.05). LAFI improvement was significantly better in the optimal AI group than in the control group (P < 0.05). Rates of pain and cough during the ablation, and postoperative gastrointestinal discomfort and use of PPIs were higher in the control group (P < 0.05). The recurrence rate was 14.7% and 14.3% after 12 months of follow-up, respectively, and the difference was not statistically significant (P > 0.05). Conclusion: Radiofrequency ablation of AF, guided by optimal AI combined with impedance, can minimize atrial injury, prevent atrial failure, promote the recovery of atrial function, reduces intraoperative cough, pain, and postoperative gastrointestinal discomfort and use of PPIs.

Whole Genome Sequencing of Single-Circulating Tumor Cell Ameliorates Unraveling Breast Cancer Heterogeneity.

Objective: Because tumor tissues are most frequently heterogeneous and hard to characterize, the resulting therapeutic strategy could be misled. The most active and invasive tumor cells are circulating tumor cells (CTCs). In this study, we investigated the feasibility of individualized treatment of breast cancer patients based on whole genome sequencing (WGS) of single cell CTC. Methods: Twenty-four CTCs were identified in three breast cancer patients. For each patient, one polyploid CTC was captured, and on which the WGS was performed. WGS was considered due to its sequencing robustness compared to conventional sequencing approaches. Based on the histopathological Her-2 status in tumor tissue and the HER2 gene status in WGS results of CTC, we adjusted treatment strategies and monitored disease progression. Results: Patients ID1 and ID2 are found to be Her-2 positive in primary tumors and HER2 gene amplification in the DNA of CTCs. In-patient ID3, histopathological examination of the primary tumor and liver metastases revealed Her-2 negative, but the WGS analysis of CTC showed HER2 gene amplification. After adjusting treatment by adding Her-2 inhibitors according to the results of CTC sequencing, liver metastases and pleural effusion were significantly reduced two months later, CTC number and ctDNA burden were decreased, and 18-months progression-free survival (PFS) was recorded. In addition, some potential therapeutic targets and mutations in drug-resistant genes were detected. Conclusion: The results of CTC sequencing effectively guided the treatment of a patient with HER2 gene amplification in CTC but with Her-2 negative on tumor tissue. Therefore, CTC sequencing could help resolve the heterogeneity of tumors and provide precision medicine for patients.

Bu-shen-zhu-yun decoction inhibits granulosa cell apoptosis in rat polycystic ovary syndrome through estrogen receptor α-mediated PI3K/AKT/mTOR pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu-shen-zhu-yun decoction (BSZYD) is a traditional chinese herbal prescription is widely used in the treatment of infertility. AIM OF THE STUDY: We aimed to elucidate the impact of a traditional herbal prescription BSZYD on polycystic ovary syndrome (PCOS). MATERIALS AND METHODS: The candidate active compounds in BSZYD and their putative targets were investigated by bioinformatics analysis. A deydroepiandrosterone (DHEA)-induced PCOS rat model was then constructed using female Sprague-Dawley (SD) rats. Serum hormone levels were measured by enzyme-linked immunosorbent assay (ELISA). Pathological changes in ovarian tissues were analyzed with hematoxylin and eosin (H&E) staining. The expressions of estrogen receptor α (ER α)-mediated PI3K/AKT/mTOR pathway were measured by immunofluorescence and western blotting. RESULTS: Bioinformatics analysis showed that the putative targets of active compound candidates in BSZYD were enriched in PI3K/AKT and estrogen signaling pathways related to regulating ovarian ovulation. Animal experiments showed that BSZYD significantly alleviated pathological changes in the ovary, altered hormone levels of serum and reduced apoptosis rate of granulosa cells. In addition, BSZYD treatment notably upregulated the expressions of proteins in ER α-mediated PI3K/AKT/mTOR pathway and downregulated apoptosis-related proteins in PCOS rats. CONCLUSION: BSZYD can restore ovary lesions and ameliorate apoptosis through ER α-mediated PI3K/AKT/mTOR pathway, which might partly contribute to the treatment of PCOS.

Replicative transposition contributes to the evolution and dissemination of KPC-2-producing plasmid in Enterobacterales.

ABSTRACTKlebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales are prevalent worldwide and pose an alarming threat to public health. The incidence and transmission of blaKPC-2 gene via horizontal gene transfer (e.g. transposition) have been well documented. However, the dynamics of transposon structure bearing blaKPC-2 and their exact effects on the evolution and dissemination of blaKPC-2 gene are not well characterized. Here, we collected all 161 carbapenem-resistant Enterobacterales (CRE) isolates during the early stage of CRE pandemic. We observed that the prevalence of KPC-2-producing Enterobacterales was mediated by multiple species and sequence types (STs), and that blaKPC-2 gene was located on three diverse variants of Tn1721 in multi-drug resistance (MDR) region of plasmid. Notably, the outbreak of KPC-2-producing plasmid is correlated with the dynamics of transposon structure. Furthermore, we experimentally demonstrated that replicative transposition of Tn1721 and IS26 promotes horizontal transfer of blaKPC-2 and the evolution of KPC-2-producing plasmid. The Tn1721 variants appearing concurrently with the peak of an epidemic (A2- and B-type) showed higher transposition frequencies and a certain superior ability to propagation. Overall, our work suggests replicative transposition contributes to the evolution and transmission of KPC-2-producing plasmid and highlights its important role in the inter- and intra-species dissemination of blaKPC-2 gene in Enterobacterales.

Crosstalk between Circulatory Microenvironment and Vascular Endothelial Cells in Acute Myocardial Infarction.

BACKGROUND: The reason of high mortality of acute myocardial infarction (AMI) was the lack of exploring the cellular and molecular mechanism of AMI. Therefore, we explored the crosstalk among cells, as well as its potential molecular mechanism of mediating AMI. METHODS: The gene expression profile of peripheral blood, endothelial, platelets and mononuclear cells were applied to differentially expressed genes (DEGs) analysis. ClusterProfiler and the package of gene set enrichment analysis (GSEA) were applied to explore the potential functional pathways of DEGs in 3 types of intravascular cells (endothelial, platelets and mononuclear cells) and peripheral blood. Subsequently, we extracted the surface receptors, secreted proteins and extracellular matrix from the up-regulated DEGs to explore their potential interactions mechanism of AMI by crosstalk and pivot analysis. FINDINGS: A total 11 common regulated DEGs (CDEGs) were identified, which might be potential biomarkers for AMI diagnosis. The abnormal pathways involved in DEGs of 3 types of intravascular cells and peripheral blood were shown, which also verified by GSEA. Afterwards, it was found that there was crosstalk in 3 types of intravascular cells and peripheral blood. Furthermore, we constructed a cell-cell interaction map among cells in AMI regulated by exosome lncRNA, which was involved in the development of AMI. Finally, we identified 8 hub genes, which might be potential biomarkers of AMI. INTERPRETATION: The result of this study can not only be used as a reference for subsequent experiments and further exploration, but also contribute to the development of novel cell and molecular therapies.

Loss of exosomal LncRNA HCG15 prevents acute myocardial ischemic injury through the NF-κB/p65 and p38 pathways.

Exosomes are nanosized bilayer membrane vesicles that may mediate intercellular communication by transporting bioactive molecules, including noncoding RNAs, mRNAs, and proteins. Research has shown that exosomes play an important role in acute myocardial infarction (AMI), but the function and regulation of exosomal long noncoding RNAs (lncRNAs) in AMI are unclear. Thus, RNA sequencing (RNA-Seq) was conducted to investigate the exosomal lncRNA transcriptome from MI patients and identified 65 differentially expressed lncRNAs between the MI and control groups. HCG15, one of the differentially expressed lncRNAs, was verified to have the highest correlation with cTnT by qRT-PCR, and it also contributed to the diagnosis of AMI by receiver operating characteristic (ROC) analysis. Upregulation of HCG15 expression facilitated cardiomyocyte apoptosis and inflammatory cytokine production and inhibited cell proliferation. We also confirmed that HCG15 was mainly wrapped in exosomes from AC16 cardiomyocytes under hypoxia, which contributed to cardiomyocyte apoptosis, the release of inflammatory factors, and inhibition of cell proliferation via the activation of the NF-κB/p65 and p38 pathways, whereas suppressing the expression of HCG15exerted opposite effects, In addition, Overexpression of HCG15 aggravated cardiac IR injury in C57BL/6J mice. This study not only helps elucidate exosomal lncRNA function in AMI pathogenesis but also contributes to the development of novel therapeutic strategies.