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Whole-exome sequencing (WES) data are frequently used for cancer diagnosis and genome-wide association studies (GWAS), based on high-coverage read mapping, informative variant calling, and high-quality reference genomes. The center position of the currently used genome assembly, GRCh38, is now challenged by two newly published telomere-to-telomere (T2T) genomes, T2T-CHM13 and T2T-YAO, and it becomes urgent to have a comparative study to test population specificity using the three reference genomes based on real case WES data. Here we report our analysis along this line for 19 tumor samples collected from Chinese patients. The primary comparison of the exon regions among the three references reveals that the sequences in up to ∼ 1% target regions in T2T-YAO are widely diversified from GRCh38 and may lead to off-target in sequence capture. However, T2T-YAO still outperforms GRCh38 genomes by obtaining 7.41% more mapped reads. Due to more reliable read-mapping and closer phylogenetic relationship with the samples than GRCh38, T2T-YAO reduces half of variant calls of clinical significance which are mostly benign, while maintaining sensitivity in identifying pathogenic variants. T2T-YAO also outperforms T2T-CHM13 in reducing calls of Chinese-specific variants. Our findings highlight the critical need for employing population-specific reference genomes in genomic analysis to ensure accurate variant analysis and the significant benefits of tailoring these approaches to the unique genetic backgrounds of each ethnic group., (© The Author(s) 2024. Published by Oxford University Press and Science Press on behalf of the Beijing Institute of Genomics, Chinese Academy of Sciences / China National Center for Bioinformation and Genetics Society of China.)

Introduction: Thromboembolism is a recognized complication of nephrotic syndrome (NS). Evidence supporting the use of rivaroxaban to prevent NS-related thrombosis is limited and controversial. This study aimed to explore the impact of NS on rivaroxaban pharmacokinetics and to collect observational data on the efficacy and safety of rivaroxaban as primary thromboprophylaxis in patients with NS., Methods: This prospective study analyzed 141 patients with NS who received rivaroxaban (10 mg/day) for thromboprophylaxis. High-performance liquid chromatography-tandem mass spectrometry was used to measure the trough and peak plasma concentrations (C trough and C max ) of rivaroxaban. The influence of clinical and genetic factors on these concentrations was examined using multivariate logistic regression., Results: The median C max and C trough were 68.5 ng/mL (interquartile range [IQR], 31.7-105.5 ng/mL) and 4.4 ng/mL (IQR, 1.2-11.9 ng/mL), respectively. The incidence of thromboembolic events (TEs) was 12.8%, while that of bleeding events was 14.2%, although all were classified as minor. Albumin level was the most significant factor affecting C max (ρ = 0.55; p < 0.001) and was also significantly associated with TEs (0.81; 0.71-0.91 per 1.0 g/dL increase; p = 0.001) and bleeding risks (1.11; 1.03-1.19 per 1.0 g/dL increase; p = 0.008). Single nucleotide polymorphisms in the ABCB1 gene significantly influenced C trough but were not associated with clinical outcomes., Conclusion: Hypoalbuminemia significantly affects the pharmacokinetics of rivaroxaban in NS patients. A dose-adjustment strategy based on rivaroxaban concentrations, accounting for variable albumin levels, may improve the safety and efficacy of thromboprophylaxis in this population., Competing Interests: The authors have no competing interests to declare that are relevant to the content of this article., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Purpose: This study aimed to assess the efficacy of surgery as a treatment option for patients with signet-ring cell carcinoma (SRCC) in the gastrointestinal tract (GI-SRCC)., Methods: Using the Surveillance, Epidemiology, and End Results database, patients with GI-SRCC who underwent surgery or received nonsurgical treatment were included. Propensity score matching (PSM) analysis was used to balance baseline characteristics and reduce bias. Overall survival (OS) was calculated in matching cohorts to estimate prognosis for patients with GI-SRCC. Nomogram was established to predict metastasis for patients with GI-SRCC., Results: The study enrolled a total of 9428 patients with GI-SRCC, with 1689 patients in the nonsurgery group and 7739 patients in the surgery group. After 1:1 PSM, we analyzed 743 patients from each group. Our survival analyses revealed that surgery independently correlated with improved OS for patients with GI-SRCC (hazard ratio, 0.37; 95% CI, 0.33-0.42; P < .001). Subgroup analysis further confirmed the positive impact of surgery on the prognosis of patients with nonmetastatic GI-SRCC. Notably, distinct subsets of patients with metastasis, particularly those originating from the upper GI (esophagus, proximal stomach, and distal stomach) and left colon, demonstrated a significant improvement in OS after surgery. However, no significant survival difference was observed for patients with metastatic right colon and rectum SRCC. Using nomogram, we quantitatively assessed the risk of metastasis in patients with right colon and rectum SRCC, which exhibited robust predictive accuracy, with area under the curve values of 0.829., Conclusion: Our study highlighted surgery's positive impact on prognosis for both patients with nonmetastatic and metastatic upper GI-SRCC and left colon SRCC. Hence, we recommend surgery as a treatment option for these groups. In addition, for patients with metastatic right colon and rectum SRCC ineligible for surgery, our predictive nomogram can offer a convenient tool to aid early intervention and improve prognosis., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Society for Surgery of the Alimentary Tract. Published by Elsevier Inc. All rights reserved.)

Liver fibrosis occurs in response to chronic damage and inflammation to the liver. Leaving untreated, it can lead to decreased liver function and can eventually progress to cirrhosis, a more advanced and irreversible state of liver damage. Clinical investigations showed that chronic liver disease associated with neurological symptoms including anxiety, depression, and cognitive decline. However, few therapeutic options are available for treating liver and related brain pathologies simultaneously. In this study, we aim to find therapeutic candidates that target the liver-brain axis. Gossypetin, a flavonoid from sedum, shows promising capability in treating liver and brain pathologies in CCl 4 -induced mouse model. Short term of gossypetin administration is sufficient to ameliorate impaired liver function and pre-existing liver fibrosis, suppress MKK3/6-p38 MAPK and p53 activation, and abolish the activation of hepatic stellate cells and Kupffer cells. Although we observe no neuronal loss in the brain of mice with liver fibrosis, we do observe astrogliosis and microglial activation in certain brain regions, especially the hippocampus. Brief gossypetin administration also shows potential in alleviating neuroinflammation in these regions. These results suggest that gossypetin can target the liver-brain axis and be a promising candidate for treating chronic liver fibrosis patients with neurological symptoms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xu, Tai, Chu, Liu, He, Wang, Su and Li.)

This work developed DNA amplifier logic gates (AND-OR, OR-AND, FAN-IN, FAN-OUT, and 4-bit square-root circuits) using a flap endonuclease 1 (FEN1)-catalyzed signal amplification reaction, for the fastest and compact DNA computing. Moreover, the logic circuit can use input strands with concentrations of less than 1 nM, which is more than 100 times lower than the input concentration of other DNA logic circuits, providing a promising methodology for constructing fast and compact DNA computations.

The chromosome structure of different bacteria has its unique organization pattern, which plays an important role in maintaining the spatial location relationship between genes and regulating gene expression. Conversely, transcription also plays a global role in regulating the three-dimensional structure of bacterial chromosomes. Therefore, we combine RNA-Seq and Hi-C technology to explore the relationship between chromosome structure changes and transcriptional regulation in E. coli at different growth stages. Transcriptome analysis indicates that E. coli synthesizes many ribosomes and peptidoglycan in the exponential phase. In contrast, E. coli undergoes more transcriptional regulation and catabolism during the stationary phase, reflecting its adaptability to changes in environmental conditions during growth. Analyzing the Hi-C data shows that E. coli has a higher frequency of global chromosomal interaction in the exponential phase and more defined chromosomal interaction domains (CIDs). Still, the long-distance interactions at the replication termination region are lower than in the stationary phase. Combining transcriptome and Hi-C data analysis, we conclude that highly expressed genes are more likely to be distributed in CID boundary regions during the exponential phase. At the same time, most high-expression genes distributed in the CID boundary regions are ribosomal gene clusters, forming clearer CID boundaries during the exponential phase. The three-dimensional structure of chromosome and expression pattern is altered during the growth of E. coli from the exponential phase to the stationary phase, clarifying the synergy between the two regulatory aspects., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Liquid biopsy as well as genotyping plays important roles in guiding the use of tumor-targeted drugs and monitoring the generation of drug resistance. However, current methods, such as next-generation sequencing (NGS) and pyrosequencing, require long analysis time and complicated steps. To achieve ultrafast and highly specific detection of cell-free DNA (cfDNA) from blood, we improved our recently developed FEN1-aided RPA (FARPA), which combined flap endonuclease 1 (FEN1)-catalyzed invasive reactions with recombinase polymerase amplification (RPA) by inactivating the RPA enzymes before invasive reactions, designing short RPA primers, and changing invasive reaction conditions. Using the L858R and T790M mutations as examples, FARPA was sensitive to detect 5 copies of targeted mutants, specific to sense the mutants with an abundance as low as 0.01% from blood, and ultrafast to get results within 40 min. The method was readily expended to genotyping, and 15 min was enough to report the allele species directly from oral swab samples by coupling quick DNA extraction reagents. Validation was carried out by detecting clinical samples, including 20 cfDNA from patients with non-small cell lung cancer (NSCLC) for liquid biopsy and 43 human genomic DNA (gDNA) purified from blood (33) or lysed from oral swabs (10) for genotyping, giving 100% agreement with NGS and pyrosequencing, respectively. Furthermore, a portable battery-driven device with dual-channel fluorescence detection was successfully constructed to facilitate point-of-care testing (POCT) of liquid biopsy and genotyping, providing doctors with a potential tool to achieve genotyping- or mutant-guided personalized medicine at emergency or source-limited regions.

Aim: Differences are existed in the bioactivity among various vitamin K (VK) forms. To investigate the correlation between clinical parameters of initial anticoagulation and plasma levels of VK1 and VK2 (MK-4 and MK-7), it was necessary to establish a quantitative method for simultaneous determination. Materials & methods: Plasma samples in cardiovascular patients were extracted by cyclohexane and analyzed using a C18 column. Baseline concentrations of VK1, MK-4 and MK-7 were 0.98 ± 0.52 ng/ml, 0.45 ± 0.13 ng/ml and 0.65 ± 0.31 ng/ml, respectively. The concentrations of MK-7 and total VKs were significantly relevant to INR0, respectively (p = 0.010 and p = 0.048, respectively). Conclusion: Thus, when adjusting anticoagulation dosage, concentrations of various VK homologues might be considered.

Atrial fibrillation (AF) is common in pulmonary hypertension (PH), whereas the mechanisms and treatments remain to be explored. TRPV2 regulates the structure and function of the cardiovascular system; however, little attention has been given to its role in AF. This study was to determine whether TRPV2 was involved in PH-induced AF and the effects of TRPV2 inhibitor tranilast on AF in rat models of PH. Monocrotaline (MCT) and SU5416/hypoxia (SuHx)-induced PH models were performed to detect atrial electrophysiological parameters. Daily tranilast (a TRPV2 inhibitor) or saline was given starting 1 day before PH establishment. PH increased the susceptibility to AF, with TRPV2 up-regulated in the right atria. Compared to PH rats, tranilast reduced AF inducibility and the prolongations of ERP and APD; mitigated cardiopulmonary remodeling and the increases in P-wave duration and P-R interval; partially reversed the down-regulation of ion channels such as Cav1.2, Nav1.5, Kv4.3, Kv4.2, Kv1.5, Kir2.1, Kir3.1, Kir3.4 as well as connexin (Cx) 40 and Cx43; improved right atrial (RA) fibrosis, enlargement, and myocardial hypertrophy; decreased the accumulation of inflammatory cells; down-regulated inflammatory indicators such as TNF-α, IL-1β, CXCL1, and CXCL2; and inhibited the activation of the PI3K-AKT-NF-κB signaling pathway. Our results reveal that TRPV2 participates in PH-induced AF, and TRPV2 inhibitor tranilast prevents PH-induced RA remodeling. TRPV2 might be a promising target for PH-induced AF., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Since its initial release in 2001, the human reference genome has undergone continuous improvement in quality, and the recently released telomere-to-telomere (T2T) version - T2T-CHM13 - reaches its highest level of continuity and accuracy after 20 years of effort by working on a simplified, nearly homozygous genome of a hydatidiform mole cell line. Here, to provide an authentic complete diploid human genome reference for the Han Chinese, the largest population in the world, we assembled the genome of a male Han Chinese individual, T2T-YAO, which includes T2T assemblies of all the 22 + X + M and 22 + Y chromosomes in both haploids. The quality of T2T-YAO is much better than those of all currently available diploid assemblies, and its haploid version, T2T-YAO-hp, generated by selecting the better assembly for each autosome, reaches the top quality of fewer than one error per 29.5 Mb, even higher than that of T2T-CHM13. Derived from an individual living in the aboriginal region of the Han population, T2T-YAO shows clear ancestry and potential genetic continuity from the ancient ancestors. Each haplotype of T2T-YAO possesses ∼ 330-Mb exclusive sequences, ∼ 3100 unique genes, and tens of thousands of nucleotide and structural variations as compared with CHM13, highlighting the necessity of a population-stratified reference genome. The construction of T2T-YAO, an accurate and authentic representative of the Chinese population, would enable precise delineation of genomic variations and advance our understandings in the hereditability of diseases and phenotypes, especially within the context of the unique variations of the Chinese population., (Copyright © 2023 Beijing Institute of Genomics, Chinese Academy of Sciences and Genetics Society of China. Published by Elsevier B.V. All rights reserved.)

Recombinase polymerase amplification (RPA) running at 37-42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Single nucleotide polymorphism (SNP) typing is crucial for drug dosage and disease progression. Therefore, a simple and convenient genotyping assay is essential for personalised medicine. Herein, we developed a non-invasive, closed-tube, and visualised method for genotyping. In this method, oral swabs were lysed to directly perform PCR coupled with nested invasive reaction and visualisation based on gold nanoparticle probes in a closed tube. The strategy for genotyping assay depends on the single base recognition property of invasive reaction. This assay allowed quick and simple sample preparation and the detection of 25 copies/μL of CYP2C19*2 and 100 copies/μL of CYP2C19*3 within 90 min. Further, 20 oral swab samples for CYP2C19*2 and CYP2C19*3 were correctly typed, which agreed with pyrosequencing, indicating that this method has great potential for SNP typing in source-limited regions to guide personalised medicine., (© 2023 The Authors. IET Nanobiotechnology published by John Wiley & Sons Ltd on behalf of The Institution of Engineering and Technology.)

Currently, organic solvents are necessary for the preparation of anionic liposomes for siRNA delivery. The removal of organic solvent is time-consuming and the residual organic solvent is not only a hidden danger, but also affects the stability of anionic liposomes. Glycerol, which is physiologically compatible and does not need to be removed, is used to promote the dispersion of lipids and the formation of anionic liposomes. Additionally, the preparation process is simple and not time-consuming. The results showed that anionic liposomes, which were typically spherical with a particle size of 188.9 nm were successfully prepared with glycerol. And with the help of Ca 2+ , siRNA was encapsulated in anionic liposomes. The highest encapsulation efficiency at 2.4 mM Ca 2+ reached 91%. And the formation of calcium phosphate could promote the endosomal escape of siRNA effectively. The results from cell viability showed that the anionic liposomes had no obvious cytotoxicity. It was also verified that anionic liposomes could improve the resistance of siRNA against degradation. Additionally, siRNA delivered by anionic liposomes could play an effective role in knockout. Therefore, anionic liposomes prepared with glycerol will be a safe and effective delivery platform for siRNA and even other nucleic acid drugs., (© 2023 The Authors. IET Nanobiotechnology published by John Wiley & Sons Ltd on behalf of The Institution of Engineering and Technology.)

The vast population of bacterial phages or viruses (virome) plays pivotal roles in the ecology of human microbial flora and health conditions. Obstacles, including poor viral sequence inference, strain-sensitive virus-host relationship, and the high diversity among individuals, hinder the in-depth understanding of the human virome. We conducted longitudinal studies of the virome based on constructing a high-quality personal reference metagenome (PRM). By applying long-read sequencing for representative samples, we could build a PRM of high continuity that allows accurate annotation and abundance estimation of viruses and bacterial species in all samples of the same individual by aligning short sequencing reads to the PRM. We applied this approach to a series of fecal samples collected for 6 months from a 2-year-old boy who had experienced a 2-month flare-up of atopic eczema (dermatitis) in this period. We identified 31 viral strains in the patient's gut microbiota and deciphered their strain-level relationship to their bacterial hosts. Among them, a lytic crAssphage developed into a dozen substrains and coordinated downregulation in the catabolism of aromatic amino acids (AAAs) in their host bacteria which govern the production of immune-active AAA derivates. The metabolic alterations confirmed based on metabolomic assays cooccurred with symptom remission. Our PRM-based analysis provides an easy approach for deciphering the dynamics of the strain-level human gut virome in the context of entire microbiota. Close temporal correlations among virome alteration, microbial metabolism, and disease remission suggest a potential mechanism for how bacterial phages in microbiota are intimately related to human health. IMPORTANCE The vast populations of viruses or bacteriophages in human gut flora remain mysterious. However, poor annotation and abundance estimation remain obstacles to strain-level analysis and clarification of their roles in microbiome ecology and metabolism associated with human health and diseases. We demonstrate that a personal reference metagenome (PRM)-based approach provides strain-level resolution for analyzing the gut microbiota-associated virome. When applying such an approach to longitudinal samples collected from a 2-year-old boy who has experienced a 2-month flare-up of atopic eczema, we observed thriving substrains of a lytic crAssphage, showing temporal correlation with downregulated catabolism of aromatic amino acids, lower production of immune-active metabolites, and remission of the disease. The PRM-based approach is practical and powerful for strain-centric analysis of the human gut virome, and the underlying mechanism of how strain-level virome dynamics affect disease deserves further investigation.

Precise nanostructure geometry that enables the optical biomolecular delivery of nanosensors to the living intracellular environment is highly desirable for precision biological and clinical therapies. However, the optical delivery through membrane barriers utilizing nanosensors remains difficult due to a lack of design guidelines to avoid inherent conflict between optical force and photothermal heat generation in metallic nanosensors during the process. Here, we present a numerical study reporting significantly enhanced optical penetration of nanosensors by engineering nanostructure geometry with minimized photothermal heating generation for penetrating across membrane barriers. We show that by varying the nanosensor geometry, penetration depths can be maximized while heat generated during the penetration process can be minimized. We demonstrate the effect of lateral stress induced by an angularly rotating nanosensor on a membrane barrier by theoretical analysis. Furthermore, we show that by varying the nanosensor geometry, maximized local stress fields at the nanoparticle-membrane interface enhanced the optical penetration process by four-fold. Owing to the high efficiency and stability, we anticipate that precise optical penetration of nanosensors to specific intracellular locations will be beneficial for biological and therapeutic applications.

Background: Reports show that the left ventricular myocardial work index (LVMWI) is a novel parameter for evaluating cardiac function. Decompensated heart failure leads to a high rate of early mortality in advanced patients with light-chain cardiac amyloidosis (AL-CA) and prevents them from a relatively delayed response to chemotherapy. This study aimed to assess the association of the LVMWI with short-term outcomes and to construct a simple model for risk stratification., Methods: A total of 79 patients with an initial diagnosis of AL-CA were included in this retrospective cohort study. LVMWI was calculated by integrating brachial artery cuff blood pressure and left ventricular longitudinal strain (LVLS). The short-term outcome was defined as 6-month all-cause mortality. Receiver operating characteristic (ROC), logistic regression, and Kaplan-Meier analysis were used in this study., Results: The median follow-up time was 21 months (3-36 months), and 23 (29%) patients died in the first 6 months. The time-dependent ROC and the area under the curve (AUC) showed that the LVMWI had the best predictive potential at the 6-month time point [AUC =0.805; 95% confidence interval (CI): 0.690-0.920]. A bivariate prognostic model based on the LVMWI was constructed, and D-dimer showed a synergistic effect with optimum predicted potential (AUC =0.877; 95% CI: 0.791-0.964). Kaplan-Meier analysis demonstrated that patients with two, one, and none of the variates beyond the cut-off value bore a different risk of 6-month all-cause mortality (accumulated mortality was 86%, 30%, 3%, respectively; log-rank, P<0.001). Multivariate nested logistic regression showed that the level of D-dimer provided an incremental prognostic value (Δχ 2 =10.3; P=0.001) to the value determined from New York Heart Association (NYHA) classification and the LVMWI., Conclusions: The LVMWI is associated with the short-term outcome of patients with AL-CA. The D-dimer test provides additional prognostic information for the LVMWI., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-22-386/coif). The authors have no conflicts of interest to declare., (2023 Quantitative Imaging in Medicine and Surgery. All rights reserved.)

Background: Characterization of early breast cancer circulating tumor cells (CTCs) may provide valuable information on tumor metastasis., Methods: We used immunomagnetic nanospheres to capture CTCs from the peripheral blood of eight early breast cancer patients and then performed single-cell RNA sequencing using our proposed bead-dd-seq method., Results: CTCs displayed obvious tumor cell characteristics, such as the activation of oxidative stress, proliferation, and promotion of metastasis. CTCs were clustered into two subtypes significantly correlated with the lymph node metastasis status of patients. CTCs in subtype 1 showed a strong metastatic ability because these CTCs have the phenotype of partial epithelial-mesenchymal transition and enriched transcripts, indicating breast cancer responsiveness and proliferation. Furthermore, DNA damage repair pathways were significantly upregulated in subtype 1. We performed in vitro and in vivo investigations, and found that cellular oxidative stress and further DNA damage existed in CTCs. The activated DNA damage repair pathway in CTCs favors resistance to cisplatin. A checkpoint kinase 1 inhibitor sensitized CTCs to cisplatin in mouse models of breast cancer metastasis., Conclusion: The present study dissects the molecular characteristics of CTCs from early-stage breast cancer, providing novel insight into the understanding of CTC behavior in breast cancer metastasis., (© 2022 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.)

Objective: Reciprocal cellular crosstalk within the tumour microenvironment (TME) actively participates in tumour progression. The anterior gradient-2 (AGR2) can be secreted to extracellular compartments and contribute to colorectal cancer (CRC) metastasis. We investigated the cellular source for secreted AGR2 in the TME and underlying mechanisms mediating secreted AGR2's effects., Design: Tissue microarray, tumour tissues, blood samples and tumour-associated neutrophils (TANs) from patients with CRC were isolated for phenotypical and functional analyses. The role of TAN-secreted AGR2 was determined in neutrophil-specific Agr2 knockout ( Agr2 f/f ;Mrp-Cre ) mice. The biological roles and mechanisms of secreted AGR2 in CRC metastasis were determined in vitro and in vivo., Results: TANs were a predominant cell type for secreting AGR2 in the TME of CRC. TANs-secreted AGR2 promoted CRC cells' migration. Neutrophils-specific ablation of Agr2 in mice ameliorated CRC liver metastases. The heavy chain of CD98 (CD98hc) served as the functional receptor for secreted AGR2. Mechanistically, secreted AGR2 increased xCT activity in a CD98hc-dependent manner, subsequently activating Ras homologue family member A/Rho-associated protein kinase 2 cascade. CRC cells actively recruited TANs through the C-X-C motif chemokine 2. Moreover, CRC-derived transforming growth factor beta 1 (TGF-β1) educated peripheral blood neutrophils to become AGR2 + TANs that secrete AGR2. Abundant infiltration of AGR2 + TANs and high expression of TGF-β1 and CD98hc-xCT were correlated with poor prognosis of patients with CRC., Conclusions: Our study unveils a novel crosstalk between TANs and CRC cells involving the secreted AGR2-CD98hc-xCT axis that promotes metastasis and impacts the outcomes of patients with CRC., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Growing evidence suggests altered oral and gut microbiota in autism spectrum disorder (ASD), but little is known about the alterations and roles of phages, especially within the oral microbiota in ASD subjects. We enrolled ASD (n = 26) and neurotypical subjects (n = 26) with their oral hygiene controlled, and the metagenomes of both oral and fecal samples (n = 104) are shotgun-sequenced and compared. We observe extensive and diverse oral phageome comparable to that of the gut, and clear signals of mouth-to-gut phage strain transfer within individuals. However, the overall phageomes of the two sites are widely different and show even less similarity in the oral communities between ASD and control subjects. The ASD oral phageome exhibits significantly reduced abundance and alpha diversity, but the Streptococcal phages there are atypically enriched, often dominating the community. The over-representation of Streptococcal phages is accompanied by enriched oral Streptococcal virulence factors and Streptococcus bacteria, all exhibiting a positive correlation with the severity of ASD clinical manifestations. These changes are not observed in the parallel sampling of the gut flora, suggesting a previously unknown oral-specific association between the excessive Streptococcal phage enrichment and ASD pathogenesis. The findings provide new evidence for the independent microbiome-mouth-brain connection, deepen our understanding of how the growth dynamics of bacteriophages and oral microbiota contribute to ASD, and point to novel effective therapeutics., (© 2022. The Author(s).)

Over 80% proteins consumed by the local people in Nujiang Prefecture of Southwest China, a remote and mountainous area in the Eastern Himalayas, are from pork, or Gaoligongshan pig (a landrace of Sus scrofa domestica Brisson). Previous research on the Gaoligongshan pig has focused on nutritional composition, production performance, and genetic resource characteristics, but neglected the reasons behind the local people's practice. From 2019 to 2022, we have used ethnobiological research methods to comprehensively document the traditional rearing and management patterns and the traditional culture associated with Gaoligongshan pigs. The results show that Gaoligongshan pigs graze in mixed herds with cattle and sheep during the day and prefer to eat 23 wild plant species, in which 17 species have medicinal values. At night, the pigs are artificially fed and rest in the pigsty. The local Bai and Lisu people have developed a creative food culture, rituals, and festivals culture associated with Gaoligongshan pigs. Overall, the biocultural diversity of Gaoligongshan pig contributes to the in situ conservation of genetic diversity of this important hog landrace, and supports rural development in this remote area.

A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)-induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the single-stranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and β within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.

Purpose: The emergence of ceftazidime-avibactam (CZA) resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) has been increasingly reported in recent years. We aimed to identify the risk factors of CZA-resistant CRKP infection and assess clinical outcomes of the patients., Methods: The study retrospectively analyzed the clinical and microbiological data of patients with CRKP infection to identify risk factors, clinical features, and outcomes using multivariate logistic regression analysis., Results: A total of 103 patients with CRKP infection were enrolled in this study. Multivariate analysis showed previous renal replacement therapy (OR 3.966, 95% CI 1.301-12.090, P = 0.015) was an independent risk factor for CZA-resistant CRKP infection. The 28-day mortality was higher in patients infected with CZA-resistant CRKP (27.9%) than those with CZA-susceptible CRKP (7.1%) (P = 0.009). CZA-resistant CRKP infection (OR 20.308, 95% CI 1.461-282.293, P = 0.025), and mechanical ventilation (OR 14.950, 95% CI 1.034-216.212, P = 0.047) were independent predictors for 28-day mortality in patients with CRKP infection. Lower level of platelet count (OR 0.987, 95% CI 0.975-0.999, P = 0.032) on the day of CRKP infection onset was related to 28-day mortality. Kaplan-Meier curves showed that the CZA-resistant CRKP group had a shorter survival time than the CZA-susceptible CRKP group., Conclusion: The prevalence and mortality of CZA-resistant CRKP are still increasing. Strengthening the hospital infection control of renal replacement therapy and mechanical ventilation may help to prevent CZA-resistant CRKP., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Background: Atrial fibrillation (AF) is one of the most prevalent sustained cardiac arrhythmias. The latest studies have revealed a tight correlation between nonalcoholic fatty liver disease (NAFLD) and AF. However, the exact molecular mechanisms underlying the association between NAFLD and AF remain unclear. The current research aimed to expound the genes and signaling pathways that are related to the mechanisms underlying the association between these two diseases., Materials and Methods: NAFLD- and AF- related differentially expressed genes (DEGs) were identified via bioinformatic analysis of the Gene Expression Omnibus (GEO) datasets GSE63067 and GSE79768, respectively. Further enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), the construction of a protein-protein interaction (PPI) network, the identification of significant hub genes, and receiver operator characteristic curve analysis were conducted. The gene-disease interactions were analyzed using the Comparative Toxicogenomics Database. In addition, the hub genes were validated by quantitative Real-Time PCR (qRT-PCR) in NAFLD cell model., Results: A total of 45 co-expressed differentially expressed genes (co-DEGs) were identified between the NAFLD/AF and healthy control individuals. GO and KEGG pathway analyses revealed that the co-DEGs were mostly enriched in neutrophil activation involved in the immune response and cytokine-cytokine receptor interactions. Moreover, eight hub genes were selected owing to their high degree of connectivity and upregulation in both the NAFLD and AF datasets. These genes included CCR2, PTPRC, CXCR2, MNDA, S100A9, NCF2, S100A12, and S100A8., Conclusions: In summary, we conducted the gene differential expression analysis, functional enrichment analysis, and PPI analysis of DEGs in AF and NAFLD, which provides novel insights into the identification of potential biomarkers and valuable therapeutic leads for AF and NAFLD., (© 2022. The Author(s).)

Antimicrobial susceptibility testing (AST) is an effective way to guide antibiotic selection. However, conventional culture-based phenotypic AST is time-consuming. The key point to shorten the test is to quantify the small change in the bacterial number after the antibiotic exposure. To achieve rapid AST, we proposed a combination of multiplexed PCR with barcoded pyrosequencing to significantly shorten the time for antibiotic exposure. First, bacteria exposed to each antibiotic were labeled with a unique barcode. Then, the pool of the barcoded products was amplified by PCR with a universal primer pair. Finally, barcodes in the amplicons were individually and quantitatively decoded by pyrosequencing. As pyrosequencing is able to discriminate as low as 5% variation in target concentrations, as short as 7.5 min was enough for cultivation to detect the susceptibility of Escherichia coli to an antibiotic. The barcodes enable more than six kinds of drugs or six kinds of concentrations of a drug to be tested at a time. The susceptibility of 6 antibiotics to 43 E. coli -positive samples from 482 clinical urine samples showed a consistency of 99.3% for drug-resistant samples and of 95.7% for drug-sensitive samples in comparison with the conventional method. In addition, the minimum inhibitory concentration (MIC) of 29 E. coli samples was successfully measured. The proposed AST is dye free (pyrosequencing), multiplexed (six antibiotics), fast (a half-working day for reporting the results), and able to detect the MIC, thus having a great potential for clinical use in quick antibiotic selection.

Cancer-derived exosomes carry a variety of important biomarkers specific to the formation, invasion and metastasis of tumor tissue. Dynamic monitoring of exosomes originated from cancer cells has clinical significance. Here we proposed a novel method to employ zirconium-metal-organic frameworks (Zr-MOFs) for extracting and identifying exosomes from blood. At first UiO-66 was magnetically modified as the adsorbent to anchor exosomes by forming Zr-O-P bonds. Then UiO-66-NH 2 modified with anti-EpCAM was used to construct the fluorescent probe to recognize the extracted EpCAM-positive exosomes by forming a "MOF-exosome-MOF" structure. The proposed fluorescence detection method was evaluated by quantifying MCF-7 cell-derived exosomes at the concentration as low as 16.72 particles/μl. This method was successfully applied to analyze exosomes in the plasma samples from healthy donors and breast cancer patients, demonstrating that our method might have a great potential in assisting the early diagnosis and in dynamically monitoring the efficacy of cancer treatment. We believe that the method could be extended to the detection of other biomarkers in exosomes derived from cancer cell., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Digital nucleic acid analysis technology has shown great application potential due to its excellent performance. However, most current digital nucleic acid detection methods are based on PCR or other template amplification strategies. Here, we present an alternative analysis platform based on digital nucleic acid signal amplification in droplets termed dNASA. Using a bead-based controllable extension bridged cascade signal amplification reaction, we achieved an ultralow background, high efficiency, and highly specific nucleic acid signal amplification analysis. As a "proof of concept", we demonstrated the feasibility of the proposed dNASA platform in single-base DNA mutation analysis using artificially synthesized samples. This platform provides innovative ideas for the field of digital nucleic acid analysis.

The imbalance between pathogenic and beneficial species of the intestinal microbiome and metabolism in rheumatoid arthritis (RA) remains unclarified. Here, using shotgun-based metagenome sequencing for a treatment-naïve patient cohort and a "quasi-paired cohort" method, we observed a deficiency of butyrate-producing species and an overwhelming number of butyrate consumers in RA patients. These outcomes mainly occurred in patients with positive ACPA, with a mean AUC of 0.94. This panel was also validated in established RA with an AUC of 0.986 in those with joint deformity. In addition, we showed that butyrate promoted T regs , while suppressing T convs and osteoclasts, due to potentiation of the reduction in HDAC expression and down-regulation of proinflammatory cytokine genes. Dietary butyrate supplementation conferred anti-inflammatory benefits in a mouse model by rebalancing T FH cells and T regs , as well as reducing antibody production. These findings reveal the critical role of butyrate-metabolizing species and suggest the potential of butyrate-based therapies for RA patients.

Background: Emerging findings highlighted the associations of mental illness to nutrition and dysbiosis in the intestinal microbiota, but the underlying mechanisms, especially in schizophrenia (SZ), remain unclarified., Methods: We conducted a case-control study of SZ patients (case to control=100:52) by performing sequencing of the gut metagenome; measurement of fecal and plasma non-targeted metabolome; including short-, medium-, and long-chain fatty acids; and targeted metabolites, along with recorded details of daily intakes of food., Results: The metagenome analysis uncovered enrichment of asaccharolytic species and reduced abundance of carbohydrate catabolism pathways and enzymes in the gut of SZ patients, but increased abundance of peptidases in contrast to their significantly reduced protein intake. Fecal metabolome analysis identified increased concentrations of many protein catabolism products, including amino acids (AAs), urea, branched short-chain fatty acids, and various nitrogenous derivates of aromatic AAs in SZ patients. Protein synthesis, represented by the abundance of AA-biosynthesis pathways and aminoacyl-tRNA transferases in metagenome, was significantly decreased. The AUCs (area under the curve) of the diagnostic random forest models based on their abundance achieved 85% and 91%, respectively. The fecal levels of AA-fermentative enzymes and products uniformly showed positive correlations with the severity of psychiatric symptoms., Conclusions: Our findings revealed apparent dysbiosis in the intestinal microbiome of SZ patients, where microbial metabolism is dominated by protein fermentation and shift from carbohydrate fermentation and protein synthesis in healthy conditions. The aberrant macronutrient metabolism by gut microbes highlights the importance of nutrition care and the potential for developing microbiota-targeted therapeutics in SZ., (© 2022. The Author(s).)

A simple and rapid genotyping method with less-instrumentation is essential for realizing point-of-care detection of personalized medicine-related gene biomarkers. Herein, we developed a rapid and visualized genotyping method by coupling recombinase polymerase amplification (RPA) with allele-specific invader reaction assisted gold nanoparticle probes assembling. In the method, the DNA targets were firstly amplified by using RPA, which is a rapid isothermal amplification technology. Then an allele-specific invasion reaction was performed to recognize the single nucleotide polymorphisms (SNPs) site in the amplicons, to produce signal molecules that caused discoloration of gold nanoparticle probes. As a result, genotyping was achieved by observing the color change of the reaction by using naked eye without the requirement for any expensive instrument. In order to achieve rapid genotyping detection, the genomic DNA from oral swab lysate samples were used for the RPA templates amplification. In this way, a visualized genotyping from "samples to results" within 25 min was realized. Two clopidogrel related SNPs CYP2C19*2 and CYP2C19*3 of 56 clinical samples were correctly genotyped by using this rapid visualized genotyping assay. In addition, the feasibility for this pathogen genotyping method was also verified by detecting plasmid DNA containing three SARS-COV-2 gene mutation sites, indicating that this method has the potential for clinical sample detection.

Anticoagulation response to warfarin during the initial stage of therapy varies among individuals. In this study, we aimed to combine pharmacometabolomic and pharmacogenetic data to predict interindividual variation in warfarin response, and, on this basis, suggest an initial daily dose range. The baseline metabolic profiles, genotypes, and clinical information of 160 patients with heart valve disease served as the variables of the function of the last international normalized ratio measured before a patient's discharge (INR day7 ) to screen for potential biomarkers. The partial least-squares model showed that two baseline metabolites (uridine and guanosine), one single-nucleotide variation (VKORC1), and four clinical parameters (weight, creatinine level, amiodarone usage, and initial daily dose) had good predictive power for INR day7 (R 2  = 0.753 for the training set, 0.643 for the test set). With these biomarkers, a machine learning algorithm (two-dimensional linear discriminant analysis-multinomial logit model) was used to predict the subgroups with extremely warfarin-sensitive or less warfarin-sensitive patients with a prediction accuracy of 91% for the training set and 90% for the test set, indicating that individual responses to warfarin could be effectively predicted. Based on this model, we have successfully designed an algorithm,"IniWarD," for predicting an effective dose range in the initial 7-day warfarin therapy. The results indicate that the daily dose range suggested by the IniWarD system is more appropriate than that of the conventional genotype-based method, and the risk of bleeding or thrombus due to warfarin could thus be avoided., (© 2021 The Authors. Clinical Pharmacology & Therapeutics © 2021 American Society for Clinical Pharmacology and Therapeutics.)

Recombination is one of the most important molecular mechanisms of prokaryotic genome evolution, but its exact roles are still in debate. Here we try to infer genome-wide recombination within a species, utilizing a dataset of 149 complete genomes of Escherichia coli from diverse animal hosts and geographic origins, including 45 in-house sequenced with the single-molecular real-time platform. Two major clades identified based on physiological, clinical and ecological characteristics form distinct genetic lineages based on scarcity of interclade gene exchanges. By defining gene-based syntenies for genomic segments within and between the two clades, we build a fine-scale recombination map for this representative global E. coli population. The map suggests extensive within-clade recombination that often breaks physical linkages among individual genes but seldom interrupts the structure of genome organizational frameworks as well as primary metabolic portfolios supported by the framework integrity, possibly due to strong natural selection for both physiological compatibility and ecological fitness. In contrast, the between-clade recombination declines drastically when phylogenetic distance increases to the extent where a 10-fold reduction can be observed, establishing a firm genetic barrier between clades. Our empirical data suggest a critical role for such recombination events in the early stage of speciation where recombination rate is associated with phylogenetic distance in addition to sequence and gene variations. The extensive intraclade recombination binds sister strains into a quasisexual group and optimizes genes or alleles to streamline physiological activities, whereas the sharply declined interclade recombination split the population into clades adaptive to divergent ecological niches., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

DNA walkers have shown superior performance in biosensing due to their programmability to design molecular walking behaviors with specific responses to different biological targets. However, it is still challenging to make DNA walkers capable of distinguishing DNA targets with single-base differences, so that DNA walkers that can be used for circulating tumor DNA sensing are rarely reported. Herein, a flap endonuclease 1 (FEN 1)-assisted DNA walker has been proposed to achieve mutant biosensing. The target DNA is captured on a gold nanoparticle (AuNP) as a walking strand to walk by hybridizing to the track strands on the surface of the AuNP. FEN 1 is employed to report the walking events by cleaving the track strands that must form a three-base overlapping structure recognized by FEN 1 after hybridizing with the captured target DNA. Owing to the high specificity of FEN 1 for structure recognition, the one-base mutant DNA target can be discriminated from wild-type DNA. By constructing a sensitivity-enhanced DNA walker system, as low as 1 fM DNA targets and 0.1% mutation abundance can be sensed, and the theoretical detection limits for detecting the DNA target and mutation abundance achieve 0.22 fM and 0.01%, respectively. The results of epidermal growth factor receptor (EGFR) L858R mutation detection on cell-free DNA samples from 15 patients with nonsmall cell lung cancer were completely consistent with that of next-generation sequencing, indicating that our DNA walker has potential for liquid biopsy.

The proteolysis targeting chimeras (PROTACs), which are composed of a target protein binding moiety, a linker, and an E3 ubiquitin ligase binder, have been a promising strategy for drug design and discovery. Given the advantages of potency, selectivity, and drug resistance over inhibitors, several PROTACs have been reported in literature, which mostly focus on noncovalent or irreversible covalent binding to the target proteins. However, it must be noted that noncovalent or irreversible PROTACs have several drawbacks such as weak binding affinity and unpredictable off-target effects. Reversible covalent PROTACs, with properties of enhanced potency, selectivity, and long duration of action, have attracted an increasing amount of attention. Here, we propose a comparison between these three patterns and highlight that reversible covalent PROTACs could pave the way for a wide variety of challenging target degradations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yuan, Chu and Duan.)

To determine the prognostic value of the timing of circulating breast tumour cell measurement during treatment, peripheral blood from 164 patients with breast disease was collected. Circulating tumour cells (CTCs) were enriched by using immunomagnetic nanospheres (IMNs) and were identified by using immunofluorescent staining. The CTC shows nuclear-positive, EpCAM-positive, CK19-positive, and CD45-negative. Patients with CTC positivity (> 19/7.5 mL blood) had shorter progression-free survival (PFS) and overall survival (OS) than those with negative results (≤ 19/7.5 mL blood) at baseline. Surgery caused an increase in the number and prevalence of CTCs, and the effect disappeared on day 14 after surgery. During adjuvant chemotherapy, CTCs detected before therapy was only correlated with PFS; however, CTCs at the end of adjuvant chemotherapy were correlated with both PFS and OS. The PFS and OS of the CTC-positive group were significantly shorter than those of the CTC-negative group at the end-point follow-up visit. The prognostic value of CTCs at different measurement time points was demonstrated during breast cancer treatment. Surgery and chemotherapy affected the prevalence of CTCs, leading to different prognostic relevance of CTCs at different treatment stages. CTCs detected at baseline or in the late phase of treatment are preferable for prognosis.

Anti-retroviral therapy (ART) effectively suppresses viral replication in HIV-infected patients, however CD4 + cell restoration to normal value is not achieved by 15-20% of patients who are called immune non-responders. Gut microbiota composition has been shown to influence host immunity. Herein, to identify intestinal microbial agents that may influence the CD4 recovery in HIV-infected patients, we utilized a "Quasi-paired cohort" method to analyze intestinal metagenome data from immunological responders (IRs) and immunological non-responders (INRs). This method identified significant enrichment for Streptococcus sp. and related lactate-producing bacteria (LAB) in IRs. In a validation cohort, positive correlations between the abundance of these LAB and the post-ART CD4 + recovery was observed, and a prediction model based on these LAB performed well in predicting immune recovery. Finally, experiments using a germ-free mouse model of antibody-induced CD4 + cell depletion showed that supplementation with a lactate-producing commensal Streptococcus thermophilus strongly promoted CD4 recovery. In conclusion, our study identified a group of LAB that was associated with enhanced immune recovery in post-ART HIV-infected patients and promotes CD4 + cell restoration in a mouse model. These findings favour supplementation of LAB commensal as a therapeutic strategy for CD4 + cell count improvement in HIV-infected patients., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

The biological function of TRIM39, a member of TRIM family, remains largely unexplored in cancer, especially in colorectal cancer (CRC). In this study, we show that TRIM39 is upregulated in tumor tissues compared to adjacent normal tissues and associated with poor prognosis in CRC. Functional studies demonstrate that TRIM39 deficiency restrains CRC progression in vitro and in vivo. Our results further find that TRIM39 is a positive regulator of autophagosome-lysosome fusion. Mechanistically, TRIM39 interacts with Rab7 and promotes its activity via inhibiting its ubiquitination at lysine 191 residue. Depletion of TRIM39 inhibits CRC progression and autophagic flux in a Rab7 activity-dependent manner. Moreover, TRIM39 deficiency suppresses CRC progression through inhibiting autophagic degradation of p53. Thus, our findings uncover the roles as well as the relevant mechanisms of TRIM39 in CRC and establish a functional relationship between autophagy and CRC progression, which may provide promising approaches for the treatment of CRC.

Human coronaviruses (CoVs) can cause respiratory infection epidemics that sometimes expand into globally relevant pandemics. All human CoVs have sister strains isolated from animal hosts and seem to have an animal origin, yet the process of host jumping is largely unknown. RNA interference (RNAi) is an ancient mechanism in many eukaryotes to defend against viral infections through the hybridization of host endogenous small RNAs (miRNAs) with target sites in invading RNAs. Here, we developed a method to identify potential RNAi-sensitive sites in the viral genome and discovered that human-adapted coronavirus strains had deleted some of their sites targeted by miRNAs in human lungs when compared to their close zoonic relatives. We further confirmed using a phylogenetic analysis that the loss of RNAi-sensitive target sites could be a major driver of the host-jumping process, and adaptive mutations that lead to the loss-of-target might be as simple as point mutation. Up-to-date genomic data of severe acute respiratory syndrome coronavirus 2 and Middle-East respiratory syndromes-CoV strains demonstrate that the stress from host miRNA milieus sustained even after their epidemics in humans. Thus, this study illustrates a new mechanism about coronavirus to explain its host-jumping process and provides a novel avenue for pathogenesis research, epidemiological modeling, and development of drugs and vaccines against coronavirus, taking into consideration these findings., (© The Author(s) 2021. Published by Oxford University Press.)

Nucleic acid detection is important for clinical diagnostics; however, it is challenging to perform genetic testing at the point-of-care due to the tedious steps involved in DNA extraction and the risk of cross-contamination from amplicons. To achieve a fully-automated and contamination-free nucleic acid detection, we propose a closed-type cassette system which enables the following steps to be operated automatically and sequentially: sample preparation based on magnetic beads, target amplification using multiplex polymerase chain reaction, and colorimetric detection of amplicons using a serial invasive reaction coupled with the aggregation of gold nanoparticle probes. The cassette was designed to be round and closed, and 10 targets in a sample could be simultaneously detected by the naked eye or using a spectrophotometer in the system. In addition, a cassette-driven device was fabricated to transfer reagents between wells, to control the temperature of each reaction, and to sense the colour in the detection wells. The cassette system was sensitive enough to detect 10 genotypes at 5 single nucleotide polymorphism sites related to the anticoagulant's usage, by using a 0.5 μL blood sample. The accuracy of the system was evaluated by detecting 12 whole blood samples, and the results obtained were consistent with those obtained using pyrosequencing. The cassette is airtight and the whole system is fully automatic; the only manual operation is the addition of the sample to the cassette, performing point-of-care genetic testing in a sample-in/answer-out way., Competing Interests: The authors report no declarations of interest., (© 2020 Published by Elsevier B.V.)

Background: Obesity has been widely reported to be associated with the disease progression of coronavirus disease 2019 (COVID-19); however, some studies have reported different findings. We conducted a systematic review and meta-analysis to investigate the association between obesity and poor outcomes in patients with COVID-19 pneumonia., Methods: A systematic review and meta-analysis of studies from the PubMed, Embase, and Web of Science databases from 1 November 2019 to 24 May 2020 was performed. Study quality was assessed, and data extraction was conducted. The meta-analysis was carried out using fixed-effects and random-effects models to calculate odds ratios (ORs) of several poor outcomes in obese and non-obese COVID-19 patients., Results: Twenty-two studies (n = 12,591 patients) were included. Pooled analysis demonstrated that body mass index (BMI) was higher in severe/critical COVID-19 patients than in mild COVID-19 patients (MD 2.48 kg/m 2 , 95% CI [2.00 to 2.96 kg/m 2 ]). Additionally, obesity in COVID-19 patients was associated with poor outcomes (OR = 1.683, 95% CI [1.408-2.011]), which comprised severe COVID-19, ICU care, invasive mechanical ventilation use, and disease progression (OR = 4.17, 95% CI [2.32-7.48]; OR = 1.57, 95% CI [1.18-2.09]; OR = 2.13, 95% CI [1.10-4.14]; OR = 1.41, 95% CI [1.26-1.58], respectively). Obesity as a risk factor was greater in younger patients (OR 3.30 vs. 1.72). However, obesity did not increase the risk of hospital mortality (OR = 0.89, 95% CI [0.32-2.51])., Conclusions: As a result of a potentially critical role of obesity in determining the severity of COVID-19, it is important to collect anthropometric information for COVID-19 patients, especially the younger group. However, obesity may not be associated with hospital mortality, and efforts to understand the impact of obesity on the mortality of COVID-19 patients should be a research priority in the future.

Background: The CHADS 2 and CHA 2 DS 2 -VASc scores are well-established clinical scales to estimate the risk of stroke in patients with atrial fibrillation (AF). However, the predictive power of the two scales concerning left atrial thrombus (LAT) or spontaneous echo contrast (SEC) has not been well investigated. Therefore, we investigated the predict power of CHADS 2 and CHA 2 DS 2 -VASc scores concerning LAT/SEC; identified clinical, echocardiographic and laboratory predictors of LAT/SEC in addition to the CHADS 2 and CHA 2 DS 2 -VASc scores; and derived a new scale to predict LAT/SEC accurately, it might improve thromboembolic risk stratification in patients with nonvalvular atrial fibrillation., Methods: We identified 1102 consecutive AF patients who underwent transesophageal echocardiography (TEE) for the purpose of the exclusion of LAT before catheter ablation, cardioversion or left atrial appendage occlusion. The clinical, echocardiographic and laboratory characteristics of patients were collected from the electronic medical record system., Results: In the study, the prevalence of LAT/SEC was only 4.36%. In the multivariate logistic analysis, hypertension, left atrial enlargement, prior stroke/TIA, left ventricular dysfunction, and renal dysfunction were predictors of LAT/SEC. Receiver operating characteristic curve analysis showed that c-statistics of the CHADS 2 and CHA 2 DS 2 -VASc scores concerning LAT/SEC were 0.673 and 0.643, respectively. We derived a new scale composed of variables from the multivariate logistic analysis that showed a higher c-statistic value (0.761) than the CHADS 2 and CHA 2 DS 2 -VASc scores for the prediction of LAT/SEC., Conclusion: In our cohort, we found two variables not included in the CHA 2 DS 2 -VASc score (renal dysfunction, left atrial enlargement) were independent predictors of LAT/SEC. A new scale combining clinical, echocardiographic and laboratory predictors might improve thromboembolic risk stratification. And there is a great need to carry out a new prospective and multicenter study, with a population more homogenous and including all the determinants for LAT/SEC to establish the independent degree of each variable and the applicability in clinical practice, facilitating the emergence of a new score of thromboembolic risk in patients with nonvalvular atrial fibrillation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Growing evidence suggests that autism spectrum disorder (ASD) is strongly associated with dysbiosis in the gut microbiome, with the exact mechanisms still unclear. We have proposed a novel analytic strategy-quasi-paired cohort-and applied it to a metagenomic study of the ASD microbiome. By comparing paired samples of ASD and neurotypical subjects, we have identified significant deficiencies in ASD children in detoxifying enzymes and pathways, which show a strong correlation with biomarkers of mitochondrial dysfunction. Diagnostic models based on these detoxifying enzymes accurately distinguished ASD individuals from controls, and the dysfunction score inferred from the model increased with the clinical rating scores of ASD. In summary, our results suggest a previously undiscovered potential role of impaired intestinal microbial detoxification in toxin accumulation and mitochondrial dysfunction, a core component of ASD pathogenesis. These findings pave the way for designing future therapeutic strategies to restore microbial detoxification capabilities for patients with ASD., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The development of new bioelectronic platforms for direct interactions with oral fluid could open up significant opportunities for healthcare monitoring. A tongue depressor is a widely used medical tool that is inserted into the mouth, where it comes into close contact with saliva. Glucose is a typical salivary biomarker. Herein, we report-for the first time-a tongue depressor-based biosensor for the detection of glucose in both phosphate buffer and real human saliva. Carbon nanotubes (CNTs) are attractive electronic materials, with excellent electrochemical properties. The sensor is constructed by printing CNTs and silver/silver chloride (Ag/AgCl) to form three electrodes in an electrochemical cell: Working, reference, and counter electrodes. The enzyme glucose oxidase (GOD) is immobilized on the working electrode. The glucose detection performance of the sensor is excellent, with a detection range of 7.3 μM to 6 mM. The glucose detection time is about 3 min. The discretion between healthy people's and simulated diabetic patients' salivary samples is clear and easy to tell. We anticipate that the biosensor could open up new opportunities for the monitoring of salivary biomarkers and advance healthcare applications.

Dysbiosis of airway microbiomes has been found in various respiratory diseases, but its molecular details in terms of taxonomic profile, metabolic characteristics, defensive function, and inhabit adaption are far from clear. Shotgun metagenome sequencing provides detailed information for microbes, whereas its application is rather limited in airways due to host DNA contaminants that overwhelm a minute amount of microbial content. Here, we describe a m icrofluidics-based e nrichment device and an e mulsion-based whole-genome a mplification procedure (MEEA) for the preparation of DNA from sputa for shotgun sequencing in a metagenomics study. The two protocols coupled in MEEA are first separately assayed with mock samples and are both promising in efficiency and bias. The efficiency and consistency of MEEA are further evaluated in six clinical sputum samples against direct sequencing without enrichment, and MEEA enables 2 to 14 times enrichment for microbial reads, which take 14.68% to 33.52% of total reads. The dominant pathogens detected in MEEA are in excellent agreement with those from clinical etiological tests. Meanwhile, MEEA presents much more microbiome complexity and genome information at a strain level than direct sequencing, exhibiting high sensitivity for identifying prophages and DNA viruses. MEEA provides better microbiome profiling than direct sequencing without a preference for specific microorganisms. The more detailed functional and taxonomic characterization of their species constituents, including both bacterium and virus, facilitates metagenomics studies on the pathogenesis of respiratory microbiomes. IMPORTANCE The airway microbial community, which takes important pathogenic roles for respiratory diseases, is far from clear in terms of taxonomy and gene functions. One of the critical reasons is the heavy contamination of host cell/DNA in airway samples, which hinders the subsequent sequencing of the whole genomic contents of the microbial community-the metagenome. Here, we describe a protocol for airway sample preparation which couples a microbe enrichment microfluidic device and a DNA amplification method performed in numerous droplets. When evaluated with mock and clinical sputum samples, the microfluidics-based enrichment device and emulsion-based whole-genome amplification (MEEA) procedure efficiently removes host cells, amplifies the microbial genome, and shows no obvious bias among microbes. The efficiency of MEEA makes it a promising method in research of respiratory microbial communities and their roles in diseases., (Copyright © 2019 Shi et al.)

A topic of confusion over the interactions between arbuscular mycorrhizal (AM) fungi and plants is the mycorrhizal status of some plant families such as Cyperaceae, which is generally considered to be non-mycorrhizal. Here, we conducted experiments to explore how the abiotic environmental conditions and AM network influence the interactions between AM fungi and Carex capillacea. We grew Carex capillacea alone or together with a mycorrhizal host species Medicago sativa in the presence or absence of AM fungi (soil inoculum from Mount Segrila and Rhizophagus intraradices from the Chinese Bank of the Glomeromycota, BGC). Plants were grown in a growth chamber and at two elevational sites of Mount Segrila, respectively. The results indicate that mycorrhizal host plants ensured the presence of an active AM fungal network whether under growth chamber or alpine conditions. The AM fungal network significantly depressed the growth of C. capillacea, especially when native inocula were used and the plants grew under alpine site conditions, although root colonization of C. capillacea increased in most cases. Moreover, the colonization level of C. capillacea was much higher (≤ 30%) when growing under alpine conditions compared with growth chamber conditions (< 8.5%). Up to 20% root colonization by Rhizophagus intraradices was observed in monocultures under alpine conditions. A significant negative relationship was found between shoot phosphorus concentrations in M. sativa and shoot dry mass of C. capillacea. These results indicate that growing conditions, AM network, and inoculum source are all important factors affecting the susceptibility of C. capillacea to AM fungi, and growing conditions might be a key driver of the interactions between AM fungi and C. capillacea.

Human anterior gradient-2 (AGR2), a member of protein disulfide isomerase (PDI) family, is present in both intracellular and extracellular compartments. Although AGR2 is overexpressed in various human cancers and reported to promote aggressive tumor features, little is known regarding AGR2's extracellular functions during tumorigenesis. Here, we demonstrate that secreted AGR2 promotes cell migration and metastasis of colorectal cancer (CRC) in vitro and in vivo. Mechanistically, secreted AGR2 elevated Wnt11 expression, triggering non-canonical Wnt signaling: the Ca 2+ /Calmodulin-dependent protein kinase II (CaMKII) and c-jun amino-terminal kinase (JNK) pathways. Knockdown of Wnt11 or pretreatment with CaMKII and JNK inhibitors reversed the secreted AGR2's migration-promoting effect. Further studies revealed that AGR2 antagonized canonical Wnt/β-catenin signaling via activating CaMKII. Collectively, our study uncovers a critical role of Wnt11-mediated non-canonical Wnt signaling (CaMKII and JNK pathways) in secreted AGR2's promoted migration of CRC cells. These results raise the possibility that secreted AGR2 may be a potential therapeutic target towards inhibiting CRC metastasis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background/aims: Mesenchymal stem cells (MSCs) do not readily migrate to appropriate sites, and this creates a major obstacle for their use in the treatment of graft-versus-host disease (GVHD). Intercellular adhesion molecule-1 (ICAM-1) can guide the homing of various immune cells to the proper anatomical location within secondary lymphoid organs (SLOs), which are the major niches for generating immune responses or tolerance. MSCs rarely migrate to SLOs after intravenous infusion, and are constitutively low expression of ICAM-1. So in our previous work, ICAM-1 was engineered into a murine MSC line C3H10T1/2 by retrovirus transfection system (ICAM-1MSCs). Here, we hypothesized that ICAM-1highMSCs may significantly improve their immunomodulatory effect., Methods: We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate ICAM-1highMSCs immunomodulatory effect on dendritic cells (DCs) and T cells in vitro and in vivo. MSCs were labeled with carboxyfluorescein diacetate succinimidylester (CFSE) to detect its distribution in mouse model., Results: Our in vitro analyses revealed ICAM-1 MSCs could suppress DCs maturation according to co-culture methods and suppress the T cell immune response according to the mixed lymphocyte response (MLR) and lymphoblast transformation test (LTT) tests. We found that infusion of ICAM-1highMSCs potently prolonged the survival of GVHD mouse model. The infused ICAM-1highMSCs migrate to SLOs in vivo, and suppressed DCs maturation, suppressed CD4+ T cell differentiation to Th1 cells, and increased the ratios of Treg cells., Conclusions: Taken together, these data demonstrate that ICAM-1highMSCs had an enhanced immunosuppressive effect on DCs and T cells, which may help explain the protective effect in a GVHD model. This exciting therapeutic strategy may improve the clinical efficacy of MSC-based therapy for GVHD., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The aim of the present study was to determine the repairing effects of intercellular adhesion molecule (ICAM)-1-expressing mesenchymal stem cells (MSCs) in mice with autoimmune thyroiditis. Following induction of an experimental autoimmune thyroiditis (EAT) model, mice were randomly divided into the following groups (n=10 each): i) Normal control; and experimental groups that were subject to EAT induction, including ii) EAT model; and iii) primary MSC; iv) C3H10T1/2/MSC; v) C3H10T1/2-MIGR1/MSC; and vi) C3H10T1/2-MIGR1-ICAM-1/MSC, which were all administered the relevant cells. MSCs were administered via the caudal vein. A blood sample was harvested from the angular vein of each animal 28 days post-treatment and ELISA was used to determine the serum total triiodothyronine, total thyroxine (T4), thyroid-stimulating hormone (TSH), anti-thyroid peroxidase (TPOAb), anti-thyroid microsomal (TMAb) and anti-thyroglobulin (TGAb) antibodies. Hematoxylin and eosin staining was performed to evaluate injury of the thyroid gland by determining the size of the follicle, inflammatory infiltration, colloidal substance retention and epithelial injury. Reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression of interleukin (IL)-4, IL-10, IL-17 and interferon (INF)-γ. Western blot analysis was performed to determine the expression of p38 mitogen-activated protein kinase (p38) and extracellular signal-regulated kinase (ERK). To observe cellular migration in vivo , mice were divided into the following groups, (n=10 each), which were subject to EAT induction: i) CM-DiI-labeled primary MSC; ii) CM-DiI-labeled C3H10T1/2/MSC; iii) CM-DiI-labeled C3H10T1/2-MIGR1/MSC; and iv) CM-DiI-labeled C3H10T1/2-ICAM-1/MSC, which were all administered the relevant cells via the caudal vein. C3H10T1/2-ICAM-1/MSCs were able to ameliorate the expression of T4, TSH, TPOAb, TMAb and TGAb in vivo , attenuate thyroid follicle injury and decrease the splenic index in mice. They were also able to ameliorate the mRNA expression of IL-4, IL-10, IL-17 and INF-γ, and the modulation of the P38 and ERK-signaling pathways in the mouse spleen. Furthermore, ICAM-1 overexpression was able to modulate the nesting of MSCs in the thyroid gland and lung. These findings suggest that C3H10T1/2-ICAM-1/MSC may affect the differentiation, proliferation and migration of immunocytes through modulating the p38 and ERK signaling pathways, and that ICAM-1 may modulate the immunoregulatory effects of MSCs by affecting the migration of MSCs in vivo .

Objectives: To analyze the microbial diversity and gene content of a thermophilic cellulose-degrading consortium from hot springs in Xiamen, China using 454 pyrosequencing for discovering cellulolytic enzyme resources., Results: A thermophilic cellulose-degrading consortium, XM70 that was isolated from a hot spring, used sugarcane bagasse as sole carbon and energy source. DNA sequencing of the XM70 sample resulted in 349,978 reads with an average read length of 380 bases, accounting for 133,896,867 bases of sequence information. The characterization of sequencing reads and assembled contigs revealed that most microbes were derived from four phyla: Geobacillus (Firmicutes), Thermus, Bacillus, and Anoxybacillus. Twenty-eight homologous genes belonging to 15 glycoside hydrolase families were detected, including several cellulase genes. A novel hot spring metagenome-derived thermophilic cellulase was expressed and characterized., Conclusions: The application value of thermostable sugarcane bagasse-degrading enzymes is shown for production of cellulosic biofuel. The practical power of using a short-read-based metagenomic approach for harvesting novel microbial genes is also demonstrated.

The γ-secretase complex catalyzes the final cleavage step of amyloid β-protein precursor (APP) to generate amyloid β (Aβ) peptide, a pathogenic component of senile plaques in the brain of Alzheimer's disease (AD) patients. Recent studies have shown that presenilin enhancer-2 (Pen-2), presenilin (PS, including PS1 and PS2), nicastrin, and anterior pharynx-defective 1 are essential components of the γ-secretase. The structure and function of Pen-2 in vitro have been well defined. However, little is known about the neuroanatomical distribution and expression of Pen-2 in the central nervous system (CNS) of AD model mice. We report here, using various methods such as immunohistochemical staining and immunoblotting, that Pen-2 is widely expressed at specific neuronal cells of major areas in AD model mice, including the olfactory bulb, basal forebrain, striatum, cortex, hippocampus, amygdala, thalamus, hypothalamus, cerebellum, brainstem, and spinal cord. It is co-expressed with PS1 in specific neuronal cells in mouse brain. Pen-2 is distributed much more extensively than extracellular amyloid deposits, suggesting the importance of other factors in localized amyloid deposition. Pen-2 is localized predominantly in cell membrane and cytoplasma in adult AD mice, but only distributed at cell membrane in controls. At the early stages of postnatal development, the expression level of Pen-2 is relatively high in CNS, but declines, gradually in adult mice. The present study provides an anatomical basis for Pen-2 as a key component of γ-secretase complex in the brain of developing and adult mice, and Pen-2 might be closely related to Aβ burden in aging nervous system., (© The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.)