Characterization of an aspartate aminotransferase encoded by YPO0623 with frequent nonsense mutations in Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a genetically monomorphic bacterial pathogen that evolved from Yersinia pseudotuberculosis approximately 7,400 years ago. We observed unusually frequent mutations in Y. pestis YPO0623, mostly resulting in protein translation termination, which implies a strong natural selection. These mutations were found in all phylogenetic lineages of Y. pestis, and there was no apparent pattern in the spatial distribution of the mutant strains. Based on these findings, we aimed to investigate the biological function of YPO0623 and the reasons for its frequent mutation in Y. pestis. Our in vitro and in vivo assays revealed that the deletion of YPO0623 enhanced the growth of Y. pestis in nutrient-rich environments and led to increased tolerance to heat and cold shocks. With RNA-seq analysis, we also discovered that the deletion of YPO0623 resulted in the upregulation of genes associated with the type VI secretion system (T6SS) at 26°C, which probably plays a crucial role in the response of Y. pestis to environment fluctuations. Furthermore, bioinformatic analysis showed that YPO0623 has high homology with a PLP-dependent aspartate aminotransferase in Salmonella enterica, and the enzyme activity assays confirmed its aspartate aminotransferase activity. However, the enzyme activity of YPO0623 was significantly lower than that in other bacteria. These observations provide some insights into the underlying reasons for the high-frequency nonsense mutations in YPO0623, and further investigations are needed to determine the exact mechanism.

Characterization of the follicular fluid microbiota based on culturomics and sequencing analysis.

Introduction. The human oocyte microenvironment is follicular fluid, which is important for follicle growth, ovulation and maturation of the oocyte. The micro-organisms present in follicular fluid could be a predictor of in vitro fertilization outcomes.Hypothesis/Gap Statement. Women with follicular fluid colonized with micro-organisms can be asymptomatic, but the presence of some genera in the follicular fluid correlates with in vitro fertilization.Aim. To confirm the existence of micro-organisms in follicular fluid, and to profile the micro-organisms present in follicular fluid sampled from women undergoing in vitro fertilization with different outcomes.Methodology. Women undergoing in vitro fertilization (n=163) were divided into different subgroups according to their in vitro fertilization outcomes. Their follicular fluid samples were collected, and among them, 157 samples were analysed by 16S rDNA sequencing, and 19 samples were analysed using culturomics.Results. The culturomics results suggested that the 19 follicular fluid samples were not sterile. The isolation rates for Streptococcus, Finegoldia and Peptoniphilus were >50 % in the 19 samples. Linear discriminant analysis effect size analysis showed differential bacteria abundance according to the pregnancy rate, the rate of normal fertilization, the rate of high-quality embryos and the rate of available oocytes. The sequencing results showed that micro-organisms could be detected in all 157 samples. Pseudomonas, Lactobacillus, Comamonas, Streptococcus and Acinetobacter were detected in all of the samples, but with a wide range of relative abundance. Pseudomonas, Lactobacillus, Ralstonia and Vibrio constituted a notable fraction of the microbiota.Conclusions. Follicular fluid is not sterile. Micro-organisms in follicular fluid could be a predictor of in vitro fertilization outcomes.

Comparative Lysine Acetylome Analysis of Y. pestis YfiQ/CobB Mutants Reveals that Acetylation of SlyA Lys73 Significantly Promotes Biofilm Formation of Y. pestis.

Increasing evidence shows that protein lysine acetylation is involved in almost every aspect of cellular physiology in bacteria. Yersinia pestis is a flea-borne pathogen responsible for millions of human deaths in three global pandemics. However, the functional role of lysine acetylation in this pathogen remains unclear. Here, we found more acetylated proteins and a higher degree of acetylation in Y. pestis grown under mammalian host (Mh) conditions than under flea vector (Fv) conditions, suggesting that protein acetylation could significantly change during fleabite transmission. Comparative acetylome analysis of mutants of YfiQ and CobB, the major acetyltransferase and deacetylase of Y. pestis, respectively, identified 23 YfiQ-dependent and 315 CobB-dependent acetylated proteins. Further results demonstrated that acetylation of Lys73 of the SlyA protein, a MarR-family transcriptional regulator, inhibits its binding to the promoter of target genes, including hmsT that encodes diguanylate cyclase responsible for the synthesis of c-di-GMP, and significantly enhances biofilm formation of Y. pestis. Our study presents the first extensive acetylome data of Y. pestis and a critical resource for the functional study of lysine acetylation in this pathogen. IMPORTANCE Yersinia pestis is the etiological agent of plague, historically responsible for three global pandemics. The 2017 plague epidemic in Madagascar was a reminder that Y. pestis remains a real threat in many parts of the world. Plague is a zoonotic disease that primarily infects rodents via fleabite, and transmission of Y. pestis from infected fleas to mammals requires rapid adaptive responses to adverse host environments to establish infection. Our study provides the first global profiling of lysine acetylation derived from mass spectrometry analysis in Y. pestis. Our data set can serve as a critical resource for the functional study of lysine acetylation in Y. pestis and provides new molecular insight into the physiological role of lysine acetylation in proteins. More importantly, we found that acetylation of Lys73 of SlyA significantly promotes biofilm formation of Y. pestis, indicating that bacteria can use lysine acetylation to fine-tune the expression of genes to improve adaptation.

Interplays of mutations in waaA, cmk, and ail contribute to phage resistance in Yersinia pestis.

Plague caused by Yersinia pestis remains a public health threat worldwide. Because multidrug-resistant Y. pestis strains have been found in both humans and animals, phage therapy has attracted increasing attention as an alternative strategy against plague. However, phage resistance is a potential drawback of phage therapies, and the mechanism of phage resistance in Y. pestis is yet to be investigated. In this study, we obtained a bacteriophage-resistant strain of Y. pestis (S56) by continuously challenging Y. pestis 614F with the bacteriophage Yep-phi. Genome analysis identified three mutations in strain S56: waaA* (9-bp in-frame deletion 249GTCATCGTG257), cmk* (10-bp frameshift deletion 15CCGGTGATAA24), and ail* (1-bp frameshift deletion A538). WaaA (3-deoxy-D-manno-octulosonic acid transferase) is a key enzyme in lipopolysaccharide biosynthesis. The waaA* mutation leads to decreased phage adsorption because of the failure to synthesize the lipopolysaccharide core. The mutation in cmk (encoding cytidine monophosphate kinase) increased phage resistance, independent of phage adsorption, and caused in vitro growth defects in Y. pestis. The mutation in ail inhibited phage adsorption while restoring the growth of the waaA null mutant and accelerating the growth of the cmk null mutant. Our results confirmed that mutations in the WaaA-Cmk-Ail cascade in Y. pestis contribute to resistance against bacteriophage. Our findings help in understanding the interactions between Y. pestis and its phages.

Curtanaerobium respiraculi gen. nov., sp. nov., a novel anaerobic bacterium isolated from human bronchoalveolar lavage fluid.

Two related anaerobic strains, designated as SWB101512T and SWB19611, were isolated from the bronchoalveolar lavage fluid of two lung cancer patients. Cells were Gram-stain-positive, non-motile and non-spore-forming. Growth could be observed at 26-45 °C (optimum, 37 °C), pH 5.0-8.5 (optimum, pH 7.0) and with 0.5-2.0 % (v/w) NaCl (optimum, 1.0%). The 16S rRNA gene sequences of SWB101512T and SWB19611 showed the highest similarities to Denitrobacterium detoxificans DSM 21843T (91.1 and 91.3 %, respectively). The phylogenetic tree based on the 16S rRNA gene sequences and the core genome sequences demonstrated that the two strains clustered together and formed a distinct lineage within the family Eggerthellaceae. The DNA G+C contents of strains SWB101512T and SWB19611 were 62.0 and 61.9 mol%, respectively. The predominant cellular fatty acids of strains SWB101512T and SWB19611 were C16 : 0 DMA (27.8 and 28.8 %, respectively). The respiratory menaquinone in both strains was menaquinone 6 and the polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, two phospholipids, three glycolipids and three unidentified lipids. Based on evidence from phenotypic, chemotaxonomic and genomic analyses, a new genus and species belonging to the family Eggerthellaceae, named Curtanaerobium respiraculi gen. nov., sp. nov. is proposed. The type strain is SWB101512T (=GDMCC 1.2991T=JCM 35330T).

Characterization of Lung and Oral Microbiomes in Lung Cancer Patients Using Culturomics and 16S rRNA Gene Sequencing.

Recently, microbiota dysbiosis in lung cancer has attracted immense attention. Studies on lung microbes are mostly based on sequencing, which has left the potentially functional bacteria with extremely low abundance uncovered. In this study, we characterized and compared the lung and oral cavity microbiotas using culturomics and 16S rRNA gene sequencing. Of the 198 bacteria identified at the species level from bronchoalveolar lavage fluid (BALF) samples, Firmicutes was predominant (39.90%). Twenty bacterial species isolated from BALF samples were present in at least half of the patients and were also highly abundant in oral samples. Of all isolated strains, Streptococcus and Veillonella were highly dominant. The abundance of Prevotella and Veillonella decreased from the oral cavity to the lung, whereas that of Pseudomonas increased. Linear discriminant analysis effect size demonstrated that Prevotella was more abundant in the healthy samples than in the cancerous ones, which is in accordance with the isolation of Prevotella oralis only from the healthy group using culturomics. Moreover, Gemella sanguinis and Streptococcus intermedius were isolated only from the non-small-cell lung cancer (NSCLC) group, and 16S rRNA gene sequencing showed that they were higher in the NSCLC than in the small-cell lung cancer group. Furthermore, while Bacillus and Castellaniella were enriched in lung adenocarcinoma, Brucella was enriched in lung squamous cell carcinoma. Overall, alterations were observed in the microbial community of patients with lung cancer, whose diversity might be site and pathology dependent. Using culturomics and 16S rRNA gene amplicon sequencing, this study has provided insights into pulmonary and oral microbiota alterations in patients with lung cancer. IMPORTANCE The relationship between lung microbiota and cancer has been explored based on DNA sequencing; however, culture-dependent approaches are indispensable for further studies on the lung microbiota. In this study, we applied a comprehensive approach combining culturomics and 16S rRNA gene amplicon sequencing to detect members of the microbiotas in saliva and BALF samples from patients with unilateral lobar masses. We found alterations in the microbial community of patients with lung cancer, whose diversity might be site and pathology dependent. These features may be potential bacterial biomarkers and new targets for lung cancer diagnosis and treatment. In addition, a lung and oral microbial biobank from lung cancer patients was established, which represents a useful resource for studies of host-microbe interactions.

Shexiang Tongxin Dropping Pill Allieviates Heart Failure via Extracellula Matrix-Receptor Interaction Pathways Based on RNA-Seq Transcriptomics and Experimental Studies.

OBJECTIVE: To investigate the protective mechanisms of Chinese medicine Shexiang Tongxin Dropping Pills (STDP) on heart failure (HF). METHODS: Isoproterenol (ISO)-induced HF rat model and angiotensin II (Ang II)-induced neonatal rat cardiac fibroblast (CFs) model were used in the present study. HF rats were treated with and without STDP (3 g/kg). RNA-seq was performed to identify differentially expressed genes (DEGs). Cardiac function was evaluated by echocardiography. Hematoxylin and eosin and Masson's stainings were taken to assess cardiac fibrosis. The levels of collagen I (Col I) and collagen III (Col III) were detected by immunohistochemical staining. CCK8 kit and transwell assay were implemented to test the CFs' proliferative and migratory activity, respectively. The protein expressions of α-smooth muscle actin (α-SMA), matrix metalloproteinase-2 (MMP-2), MMP-9, Col I, and Col III were detected by Western blotting. RESULTS: The results of RNA-seq analysis showed that STDP exerted its pharmacological effects on HF via multiple signaling pathways, such as the extracellular matrix (ECM)-receptor interaction, cell cycle, and B cell receptor interaction. Results from in vivo experiments demonstrated that STDP treatment reversed declines in cardiac function, inhibiting myocardial fibrosis, and reversing increases in Col I and Col III expression levels in the hearts of HF rats. Moreover, STDP (6, 9 mg/mL) inhibited the proliferation and migration of CFs exposed to Ang II in vitro (P<0.05). The activation of collagen synthesis and myofibroblast generation were markedly suppressed by STDP, also the synthesis of MMP-2 and MMP-9, as well as ECM components Col I, Col III, and α-SMA were decreased in Ang II-induced neonatal rats' CFs. CONCLUSIONS: STDP had anti-fibrotic effects in HF, which might be caused by the modulation of ECM-receptor interaction pathways. Through the management of cardiac fibrosis, STDP may be a compelling candidate for improving prognosis of HF.

Safety Evaluation and Probiotic Potency Screening of Akkermansia muciniphila Strains Isolated from Human Feces and Breast Milk.

Akkermansia muciniphila is considered a next-generation probiotic because of its immense potential to regulate disorders. We isolated 31 strains of A. muciniphila from feces or breast milk of healthy people. After genome sequencing, assembly, and analysis, we selected six strains (AM01 to AM06) for further exploration. We first analyzed their general characteristics, including morphological description, growth characteristics, and physiological and biochemical characteristics, and then confirmed their genetic characteristics, including GC content, putative virulence factors, and antibiotic resistance genes. We next investigated the tolerance of these strains to artificial gastric and intestinal fluids and bile salts to evaluate their survival potential in the digestive tract. Drug sensitivity tests were also conducted based on the analysis of the antibiotic resistance genes of these strains. Furthermore, we examined the genetic stability and acute toxicity of two strains (AM02 and AM06) in mice. Finally, the safety of AM06 was evaluated in normal mice and nude mice. AM06 exhibited adaptability to pH changes. Since AM02 and AM03 showed more resistance to antibiotics than AM01 and AM04 to AM06, their potential clinical application may be limited. Both AM02 and AM06 were genetically and phenotypically stable and safe in normal mice, and AM06 was safe in nude mice. Considering all this together, AM06 is a safe A. muciniphila strain and exhibits a great potential for use as a probiotic strain among the isolated strains. IMPORTANCE In this study, we isolated 30 strains of Akkermansia muciniphila from different samples of human feces, and for the first time we isolated an A. muciniphila strain from human breast milk. This isolation verified the existence of microbes in human breast milk, which suggests that A. muciniphila can be vertically propagated from mother to infant and participates in the formation of the early gut microbiome. We then systematically evaluated the potential for use as a probiotic of this A. muciniphila strains according to the FAO/WHO recommendation. We confirmed that the AM06 strain isolated from breast milk has no virulence factors and is genetically stable and nonpathogenic for both normal mice and nude mice. Moreover, its tolerance to pH changes and bile salts indicates its desirable probiotic properties. Thus, we propose that the AM06 strain of A. muciniphila is safe for use as a probiotic candidate.

Parvimonas micra activates the Ras/ERK/c-Fos pathway by upregulating miR-218-5p to promote colorectal cancer progression.

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer in the world, and a strong relationship exists between CRC and gut microbiota, which affects the occurrence, development, and metastasis of cancer. Bioinformatics-based analyses revealed that the abundance of Parvimonas micra (P. micra) in the feces of patients with cancer is significantly higher than that in healthy people. Therefore, an important relationship may exist between P. micra and CRC. METHODS: We first confirmed that P. micra can promote the proliferation of cell lines through cell experiments and mouse models. Then we selected the signaling pathways and content of exosomes to promote the development of CRC by transcriptomics and microRNA sequencing. Finally, we confirmed that P. micra promoted CRC development through miR-218-5p/Ras/ERK/c-Fos pathway through the in vivo and in vitro experiments. RESULTS: First, it was confirmed by in vitro and in vivo experiments that P. micra can promote the development of CRC. Transcriptome analysis after the coincubation of bacteria and cells revealed that P. micra promoted cell proliferation by activating the Ras/ERK/c-Fos pathway. Furthermore, microRNA sequencing analysis of the cells and exosomes showed that miR-218-5p and protein tyrosine phosphatase receptor R (PTPRR) were the key factors involved in activating the Ras/ERK/c-Fos pathway, and the miR-218-5p inhibitor was used to confirm the role of microRNA in xenograft mice. CONCLUSION: This experiment confirmed that P. micra promoted the development of CRC by upregulating miR-218-5p expression in cells and exosomes, inhibiting PTPRR expression, and ultimately activating the Ras/ERK/c-Fos signaling pathway.

Development and evaluation of a multiplex droplet digital polymerase chain reaction method for simultaneous detection of five biothreat pathogens.

Biothreat agents pose a huge threat to human and public health, necessitating the development of rapid and highly sensitive detection approaches. This study establishes a multiplex droplet digital polymerase chain reaction (ddPCR) method for simultaneously detecting five high-risk bacterial biothreats: Yersinia pestis, Bacillus anthracis, Brucella spp., Burkholderia pseudomallei, and Francisella tularensis. Unlike conventional multiplex real-time PCR (qPCR) methods, the multiplex ddPCR assay was developed using two types of probe fluorophores, allowing the assay to perform with a common two-color ddPCR system. After optimization, the assay performance was evaluated, showing a lower limit of detection (LOD) (0.1-1.0 pg/µL) and good selectivity for the five bacteria targets. The multiplex assay's ability to simultaneously detect two or more kinds of targets in a sample was also demonstrated. The assay showed strong sample tolerance when testing simulated soil samples; the LOD for bacteria in soil was 2 × 102-2 × 103 colony-forming unit (CFU)/100 mg soil (around 5-50 CFU/reaction), which was 10-fold lower than that of the single-target qPCR method. When testing simulated soil samples at bacterial concentrations of 2 × 103-2 × 104 CFU/100 mg soil, the assay presented a higher sensitivity (100%, 35/35) than that of the qPCR method (65.71%, 23/35) and a good specificity (100%, 15/15). These results suggest that the developed 5-plex ddPCR method is more sensitive than conventional qPCR methods and is potentially suitable for rapidly detecting or screening the five selected bacterial biothreats in suspicious samples.

Subversion of GBP-mediated host defense by E3 ligases acquired during Yersinia pestis evolution.

Plague has caused three worldwide pandemics in history, including the Black Death in medieval ages. Yersinia pestis, the etiological agent of plague, has evolved a powerful arsenal to disrupt host immune defenses during evolution from enteropathogenic Y. pseudotuberculosis. Here, we find that two functionally redundant E3 ligase of Y. pestis, YspE1 and YspE2, can be delivered via type III secretion injectisome into host cytosol where they ubiquitinate multiple guanylate-binding proteins (GBPs) for proteasomal degradation. However, Y. pseudotuberculosis has no such capability due to lacking functional YspE1/2 homologs. YspE1/2-mediated GBP degradations significantly promote the survival of Y. pestis in macrophages and strongly inhibit inflammasome activation. By contrast, Gbpchr3-/-, chr5-/- macrophages exhibit much lowered inflammasome activation independent of YspE1/2, accompanied with an enhanced replication of Y. pestis. Accordingly, Gbpchr3-/-, chr5-/- mice are more susceptible to Y. pestis. We demonstrate that Y. pestis utilizes E3 ligases to subvert GBP-mediated host defense, which appears to be newly acquired by Y. pestis during evolution.

The role of AMPK-Sirt1-autophagy pathway in the intestinal protection process by propofol against regional ischemia/reperfusion injury in rats.

Intestinal ischemia/reperfusion (II/R) is a clinical event associated with high morbidity and mortality. AMP-activated protein kinase (AMPK), a central cellular energy sensor, is associated with oxidative stress and inflammation. However, whether the AMPK is involved in the II/R-induced intestinal injury and the underlying mechanism is yet to be elucidated. Propofol has a protective effect on organs; yet, its specific mechanism of action remains unclear. This study explored the role of the AMPK-Sirt1-autophagy pathway in intestinal injury, and whether propofol could reduce intestinal injury and investigated the mechanisms in a rat model of II/R injury as well as a cell model (IEC-6 cells) of hypoxia/reoxygenation (H/R). Propofol, AMPK agonist (AICAR) and AMPK inhibitor (Compound C) were then administered, respectively. The histopathological changes, cell viability and apoptosis were detected. Furthermore, the levels of proinflammatory factors, the activities of oxidative stress, diamine oxidase, and signaling pathway were also analyzed. The results demonstrated that the AMPK-Sirt1-autophagy pathway of intestine was activated after II/R or H/R. Propofol could further activate the pathway, which reduced intestinal injury, inhibited apoptosis, reversed inflammation and oxidative stress, and improved the 24-hour survival rate in II/R rats in vivo, and attenuated H/R-induced IEC-6 cell injury, oxidative stress, and apoptosis in vitro, as fine as changes in AICAR treatment. Compound C abrogated the protective effect of propofol on II/R and H/R-induced injury. These results suggested a crucial effect of AMPK on the mechanism of intestinal injury and might provide a new insight into the mechanism of propofol reducing II/R injury.

Yersinia pestis-Induced Mitophagy That Balances Mitochondrial Homeostasis and mROS-Mediated Bactericidal Activity.

Manipulating mitochondrial homeostasis is essential for host defense against infection and pathogen survival in cells. This study reports for the first time that Y. pestis infection caused mitochondria damage that subsequently leads to the activation of Pink1/Parkin-independent mitophagy in macrophage, and the effector YopH from the type III secretion system was required for these effects. The generation of mitochondrial reactive oxygen species (mROS) by damaged mitochondria enhances the antibacterial activity of macrophages against Y. pestis and promotes apoptosis of the infected cells. Therefore, Y. pestis-induced mitophagy was employed to eliminate dysfunctional mitochondria and relieve the mROS accumulation. This study reveals a novel role for YopH of Y. pestis in damaging host macrophage mitochondria during plague infection and underlines the vital role of mitophagy in maintaining mitochondrial homeostasis by clearing bacteria-damaged mitochondria. The results show that mitophagy or mitochondrial fission manipulation could be used as a new strategy to treat plague. IMPORTANCE Y. pestis, the pathogen of plague, also known as the "Black Death," has caused millions of deaths throughout history. This study reports that Y. pestis infection induces mitochondrial fragmentation and abnormal mROS accumulation, and releases mitochondrial contents into the cytoplasm in macrophages. mROS promotes the antibacterial activity of macrophages against Y. pestis and increases apoptosis of the infected cells. PINK-Parkin-independent mitophagy is activated to balance mitochondrial homeostasis and mROS-induced bactericidal activity in Y. pestis-infected macrophages. These findings deepen the understanding of Y. pestis pathogenesis on mitochondria damage to disturb the host cellular immune elimination. Manipulating mitophagic activity or mitochondrial fission may be a novel therapeutic approach to treat plague.

Attenuation of Yersinia pestis fyuA Mutants Caused by Iron Uptake Inhibition and Decreased Survivability in Macrophages.

Yersinia pestis is the etiological agent of plague, a deadly infectious disease that has caused millions of deaths throughout history. Obtaining iron from the host is very important for bacterial pathogenicity. Y. pestis possesses many iron uptake systems. Yersiniabactin (Ybt) plays a major role in iron uptake in vivo and in vitro, and in virulence toward mice as well. FyuA, a ß-barrel TonB-dependent outer membrane protein, serves as the receptor for Ybt. In this study, we examined the role of the fyuA gene in Y. pestis virulence using different challenging ways and explored the underlying mechanisms. The BALB/c mouse infection assay showed that the virulence of the mutant strains (ΔfyuA and ΔfyuAGCAdel) was lower when compared with that of the wild-type (WT) strain 201. Furthermore, the attenuation of virulence of the mutant strains via subcutaneous and intraperitoneal challenges was far greater than that via intravenous injection. Iron supplementation restored lethality during subcutaneous challenge with the two mutants. Thus, we speculated that the attenuated virulence of the mutant strains toward the mice may be caused by dysfunctional iron uptake. Moreover, ΔfyuA and ΔfyuAGCAdel strains exhibited lower survival rates in murine RAW264.7 macrophages, which might be another reason for the attenuation. We further explored the transcriptomic differences between the WT and mutant strains at different temperatures and found that the expressions of genes related to Ybt synthesis and its regulation were significantly downregulated in the mutant strains. This finding indicates that fyuA might exert a regulatory effect on Ybt. Additionally, the expressions of the components of the type III secretion system were unexpectedly upregulated in the mutants, which is inconsistent with the conventional view that the upregulation of the virulence genes enhances the virulence of the pathogens.

Genetic diversity and transmission patterns of Burkholderia pseudomallei on Hainan island, China, revealed by a population genomics analysis.

Burkholderia pseudomallei is a Gram-negative soil-dwelling bacillus that causes melioidosis, a frequently fatal infectious disease, in tropical and subtropical regions. Previous studies have identified the overall genetic and evolutionary characteristics of B. pseudomallei on a global scale, including its origin and transmission routes. However, beyond its known hyperendemicity foci in northern Australia and Southeast Asia, the distribution and genetic characteristics of B. pseudomallei in most tropical regions remain poorly understood, including in southern China. Here, we sequenced the genomes of 122 B. pseudomallei strains collected from Hainan, an island in southern China, in 2002-2018, to investigate the population structure, relationships with global strains, local epidemiology, and virulence and antimicrobial-resistance factors. A phylogenetic analysis and hierarchical clustering divided the Hainan strains into nine phylogenic groups (PGs), 80 % of which were concentrated within five major groups (group 1: corresponding to minor sequence types [STs], 12.3 %; group 3: ST46 and ST50, 31.1 %; group 9: ST58, 13.1 %; group 11: ST55, 8.2 %; group 15: mainly ST658, 15.6%). A phylogenetic analysis that included global strains suggested that B. pseudomallei in Hainan originated from Southeast Asian countries, transmitted in multiple historical importation events. We also identified several mutual transmission events between Hainan and Southeast Asian countries in recent years, including three importation events from Thailand and Singapore to Hainan and three exportation events from Hainan to Singapore, Malaysia, and Taiwan island. A statistical analysis of the temporal distribution showed that the Hainan strains of groups 3, 9, and 15 have dominated the disease epidemic locally in the last 5 years. The spatial distribution of the Hainan strains demonstrated that some PGs are distributed in different cities on Hainan island, and by combining phylogenic and geographic distribution information, we detected 21 between-city transmission events, indicating its frequent local transmission. The detection of virulence factor genes showed that 56 % of the Hainan strains in group 1 encode a B. pseudomallei-specific adherence factor, boaB, confirming the specific pathogenic characteristics of the Hainan strains in group 1. An analysis of the antimicrobial-resistance potential of B. pseudomallei showed that various kinds of alterations were identified in clinically relevant antibiotic resistance factors, such as AmrR, PenA and PBP3, etc. Our results clarify the population structure, local epidemiology, and pathogenic characteristics of B. pseudomallei in Hainan, providing further insight into its regional and global transmission networks and improving our knowledge of its global phylogeography.

Highly Specific and Sensitive Detection of Yersinia pestis by Portable Cas12a-UPTLFA Platform.

The recent discovery of collateral cleavage activity of class-II clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas) makes CRISPR-based diagnosis a potential high-accuracy nucleic acid detection method. Colloidal gold-based lateral flow immunochromatographic assay (LFA), which has been combined with CRISPR/Cas-based nucleic detection, usually associates with drawbacks of relative high background and the subjectivity in naked-eye read-out of the results. Here, we developed a novel system composed of Cas12a-based nucleic acid detection and up-converting phosphor technology (UPT)-based LFA (UPT-LFA), termed Cas12a-UPTLFA. We further demonstrated the utility of this platform in highly sensitive and specific detection of Yersinia pestis, the causative agent of the deadly plague. Due to high infectivity and mortality, as well as the potential to be misused as bioterrorism agent, a culture-free, ultrasensitive, specific, and rapid detection method for Y. pestis has long been desired. By incorporating isothermal recombinase polymerase amplification, the Cas12a-UPTLFA we established can successfully detect genomic DNA of Y. pestis as low as 3 attomolar (aM) and exhibited high sensitivity (93.75%) and specificity (90.63%) for detection of spiked blood samples with a detection limit of 102 colony-forming unit per 100 µl of mouse blood. With a portable biosensor, Cas12a-UPTLFA assay can be operated easily by non-professional personnel. Taken together, we have developed a novel Cas12a-UPTLFA platform for rapid detection of Y. pestis with high sensitivity and specificity, which is portable, not expensive, and easy to operate as a point-of-care method. This detection system can easily be extended to detect other pathogens and holds great promise for on-site detection of emerging infectious pathogens.

Secretome and Comparative Proteomics of Yersinia pestis Identify Two Novel E3 Ubiquitin Ligases That Contribute to Plague Virulence.

Plague is a zoonotic disease that primarily infects rodents via fleabite. Transmission from flea to host niches requires rapid adaption of Yersinia pestis to the outer environments to establish infection. Here, quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking the two typical niches, i.e., the mammalian host (Mh) and the flea vector (Fv), were performed to understand the adaption strategies of this deadly pathogen. A secretome of Y. pestis containing 308 proteins has been identified using TMT-labeling mass spectrometry analysis. Although some proteins are known to be secreted, such as the type III secretion substrates, PsaA and F1 antigen, most of them were found to be secretory proteins for the first time. Comparative proteomic analysis showed that membrane proteins, chaperonins and stress response proteins are significantly upregulated under the Mh condition, among which the previously uncharacterized proteins YP_3416∼YP_3418 are remarkable because they cannot only be secreted but also translocated into HeLa cells by Y. pestis. We further demonstrated that the purified YP_3416 and YP_3418 exhibited E3 ubiquitin ligase activity in in vitro ubiquitination assay and yp_3416∼3418 deletion mutant of Y. pestis showed significant virulence attenuation in mice. Taken together, our results represent the first Y. pestis secretome, which will promote the better understanding of Y. pestis pathogenesis, as well as the development of new strategies for treatment and prevention of plague.

Protective Mechanisms of Suxiao Jiuxin Pills () on Myocardial Ischemia-Reperfusion Injury in vivo and in vitro.

OBJECTIVE: To study the protective mechanism of Chinese medicine Suxiao Jiuxin Pills (, SXJ) on myocardial ischemia and reperfusion (I/R) injury. METHODS: Mouse myocardial I/R injury model was created by 30-min coronary artery occlusion followed by 24-h reperfusion, the mice were then divided into the sham group (n=7), the I/R group (n=13), the tirofiban group (TIR, positive drug treatment, n=9), and the SXJ group (n=11). Infarct size (IS), risk region (RR), and left ventricle (LV) were analyzed with double staining methods. In addition, H9C2 rat cardiomyocytes were cultured with Na2S2O4 to simulate I/R in vitro. The phosphorylation of extracellular regulated protein kinases1/2 (ERK1/2), protein kinase B (AKT), glycogen synthase kinase-3ß (GSK3ß), and protein expression of GATA4 in nucleus were detected with Western blot assay. RESULTS: The ratio of IS/RR in SXJ and TIR groups were lower than that in I/R group (SXJ, 22.4% ±6.6%; TIR, 20.8%±3.3%; vs. I/R, 35.4%±3.7%, P<0.05, respectively). In vitro experiments showed that SXJ increased the Na2S2O4-enhanced phosphorylation of AKT/GSK3ß and nuclear expression of GATA4. CONCLUSION: SXJ prevents myocardial I/R injury in mice by activating AKT/GSK3ß and GATA4 signaling pathways.

Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients' B Cells.

The COVID-19 pandemic urgently needs therapeutic and prophylactic interventions. Here, we report the rapid identification of SARS-CoV-2-neutralizing antibodies by high-throughput single-cell RNA and VDJ sequencing of antigen-enriched B cells from 60 convalescent patients. From 8,558 antigen-binding IgG1+ clonotypes, 14 potent neutralizing antibodies were identified, with the most potent one, BD-368-2, exhibiting an IC50 of 1.2 and 15 ng/mL against pseudotyped and authentic SARS-CoV-2, respectively. BD-368-2 also displayed strong therapeutic and prophylactic efficacy in SARS-CoV-2-infected hACE2-transgenic mice. Additionally, the 3.8 Å cryo-EM structure of a neutralizing antibody in complex with the spike-ectodomain trimer revealed the antibody's epitope overlaps with the ACE2 binding site. Moreover, we demonstrated that SARS-CoV-2-neutralizing antibodies could be directly selected based on similarities of their predicted CDR3H structures to those of SARS-CoV-neutralizing antibodies. Altogether, we showed that human neutralizing antibodies could be efficiently discovered by high-throughput single B cell sequencing in response to pandemic infectious diseases.

Songling Xuemaikang Capsule inhibits isoproterenol-induced cardiac hypertrophy via CaMKIIδ and ERK1/2 pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiac hypertrophy is a key pathologic process in heart failure. Songling Xuemaikang Capsule (SXC), is a formulae of Chinese Medicine commonly used in China to treat hypertension and heart failure. However, its mechanism of effects on cardiac hypertrophy is still unclear. AIM OF THE STUDY: The aims of the present study were to investigate the cardio-protection roles and detailed mechanisms of SXC on cardiac hypertrophy in vivo and in vitro. MATERIALS AND METHODS: A rat model of cardiac hypertrophy was constructed by isoproterenol (ISO) intraperitoneal injection (i.p), 10 mg/kg/day for 3 days, and 4 groups were compared: CON (n = 8), ISO (n = 8), MET (metoprolol, positive drug treatment, n = 7), and SXC (SXC treatment, n = 6). Cardiac structure and function were evaluated with echocardiography in vivo. Dose-dependent curve was obtained with SXC different concentrations. In addition, H9C2 rat cardiomyocytes were cultured in vitro and the phosphorylation of ERK1/2, p38, JNK, AKT, and protein expression of CaN, CaMKIIδ, GATA4 were detected with Western blot test. RESULTS: The results showed that SXC reduced diastolic thickness of left ventricular posterior wall, while did not change ejection fraction and fraction shortening significantly (P > 0.05). SXC inhibit ISO-induced cardiac hypertrophy dose-dependently with 50% inhibiting concentration (IC50) is 0.504 g/kg/day. Moreover, SXC inhibited the protein expression of CaMKIIδ, and the phosphorylation of ERK1/2, so inhibiting protein expression of GATA4 in nucleus, and brain natriuretic peptide in serum (P < 0.001). CONCLUSION: The mechanism of SXC in the treatment of heart diseases involves SXC dose-dependently inhibited the ISO-induced cardiac hypertrophy via inhibiting CaMKIIδ and ERK1/2/GATA4 signaling pathway.