Epigenetic regulation during cancer transitions across 11 tumour types.

Chromatin accessibility is essential in regulating gene expression and cellular identity, and alterations in accessibility have been implicated in driving cancer initiation, progression and metastasis1-4. Although the genetic contributions to oncogenic transitions have been investigated, epigenetic drivers remain less understood. Here we constructed a pan-cancer epigenetic and transcriptomic atlas using single-nucleus chromatin accessibility data (using single-nucleus assay for transposase-accessible chromatin) from 225 samples and matched single-cell or single-nucleus RNA-sequencing expression data from 206 samples. With over 1 million cells from each platform analysed through the enrichment of accessible chromatin regions, transcription factor motifs and regulons, we identified epigenetic drivers associated with cancer transitions. Some epigenetic drivers appeared in multiple cancers (for example, regulatory regions of ABCC1 and VEGFA; GATA6 and FOX-family motifs), whereas others were cancer specific (for example, regulatory regions of FGF19, ASAP2 and EN1, and the PBX3 motif). Among epigenetically altered pathways, TP53, hypoxia and TNF signalling were linked to cancer initiation, whereas oestrogen response, epithelial-mesenchymal transition and apical junction were tied to metastatic transition. Furthermore, we revealed a marked correlation between enhancer accessibility and gene expression and uncovered cooperation between epigenetic and genetic drivers. This atlas provides a foundation for further investigation of epigenetic dynamics in cancer transitions.

Predictive models for sepsis in children with Staphylococcus aureus bloodstream infections: a retrospective cohort study.

BACKGROUND: The presence of Staphylococcus aureus in the bloodstream can lead to the development of sepsis; however, the severity and risk factors of the systemic inflammatory response to Staphylococcus aureus bloodstream infections were unclear. This study is aimed to build a model to predict the risk of sepsis in children with Staphylococcus aureus bloodstream infections. METHODS: A retrospective analysis of hospitalized pediatric patients diagnosed with Staphylococcus aureus bloodstream infections was performed between January 2013 and December 2019. Each patient was assessed using the pediatric version of the Sequential Organ Failure Assessment score (pSOFA) within 24 h of blood culture collection. A nomogram based on logistic regression models was constructed to predict the risk factors for sepsis in children with Staphylococcus aureus bloodstream infections. It was validated using the area under the receiver-operating characteristic curve (AUC). RESULTS: Of the 94 patients included in the study, 35 cases (37.2%) developed sepsis. The pSOFA scores ranged from 0 to 8, with 35 patients having a pSOFA score of ≥ 2. Six children (6.4%) died within 30 days, who were all from the sepsis group and had different pSOFA scores. The most common organs involved in sepsis in children with staphylococcal bloodstream infections were the neurologic system (68.6%), respiratory system (48.6%), and coagulation system (45.7%). Hospital-acquired infections (adjusted odds ratio [aOR], 3.0; 95% confidence interval [CI], 1.3-7.2), implanted catheters (aOR, 10.4; 95% CI, 3.8-28.4), procalcitonin level ≥ 1.7 ng/mL (aOR, 15.4; 95% CI, 2.7-87.1), and underlying diseases, especially gastrointestinal malformations (aOR, 14.0; 95% CI, 2.9-66.7) were associated with Staphylococcus aureus sepsis. However, methicillin-resistant Staphylococcus aureus infection was not a risk factor for sepsis. The nomogram had high predictive accuracy for the estimation of sepsis risk, with an AUC of 0.85. CONCLUSIONS: We developed a predictive model for sepsis in children with Staphylococcus aureus infection.

A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population.

Engineered gene drives are being explored as a new strategy in the fight against vector-borne diseases due to their potential for rapidly spreading genetic modifications through a population. However, CRISPR-based homing gene drives proposed for this purpose have faced a major obstacle in the formation of resistance alleles that prevent Cas9 cleavage. Here, we present a homing drive in Drosophila melanogaster that reduces the prevalence of resistance alleles below detectable levels by targeting a haplolethal gene with two guide RNAs (gRNAs) while also providing a rescue allele. Resistance alleles that form by end-joining repair typically disrupt the haplolethal target gene and are thus removed from the population because individuals that carry them are nonviable. We demonstrate that our drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no functional resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals within a few generations. These results show that a haplolethal homing drive can provide an effective tool for targeted genetic modification of entire populations.

Safety and immunogenicity of heterologous boost immunization with an adenovirus type-5-vectored and protein-subunit-based COVID-19 vaccine (Convidecia/ZF2001): A randomized, observer-blinded, placebo-controlled trial.

BACKGROUND: Heterologous boost vaccination has been proposed as an option to elicit stronger and broader, or longer-lasting immunity. We assessed the safety and immunogenicity of heterologous immunization with a recombinant adenovirus type-5-vectored Coronavirus Disease 2019 (COVID-19) vaccine (Convidecia, hereafter referred to as CV) and a protein-subunit-based COVID-19 vaccine (ZF2001, hereafter referred to as ZF). METHODS AND FINDINGS: We conducted a randomized, observer-blinded, placebo-controlled trial, in which healthy adults aged 18 years or older, who have received 1 dose of Convidecia, with no history of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, were recruited in Jiangsu, China. Sixty participants were randomly assigned (2:1) to receive either 1 dose of ZF2001 or placebo control (trivalent inactivated influenza vaccine (TIV)) administered at 28 days after priming, and received the third injection with ZF2001 at 5 months, referred to as CV/ZF/ZF (D0-D28-M5) and CV/ZF (D0-M5) regimen, respectively. Sixty participants were randomly assigned (2:1) to receive either 1 dose of ZF2001 or TIV administered at 56 days after priming, and received the third injection with ZF2001 at 6 months, referred to as CV/ZF/ZF (D0-D56-M6) and CV/ZF (D0-M6) regimen, respectively. Participants and investigators were masked to the vaccine received but not to the boosting interval. Primary endpoints were the geometric mean titer (GMT) of neutralizing antibodies against wild-type SARS-CoV-2 and 7-day solicited adverse reactions. The primary analysis was done in the intention-to-treat population. Between April 7, 2021 and May 6, 2021, 120 eligible participants were randomly assigned to receive ZF2001/ZF2001 (n = 40) or TIV/ZF2001 (n = 20) 28 days and 5 months post priming, and receive ZF2001/ZF2001 (n = 40) or TIV/ZF2001 (n = 20) 56 days and 6 months post priming. Of them, 7 participants did not receive the third injection with ZF2001. A total of 26 participants (21.7%) reported solicited adverse reactions within 7 days post boost vaccinations, and all the reported adverse reactions were mild, with 13 (32.5%) in CV/ZF/ZF (D0-D28-M5) regimen, 7 (35.0%) in CV/ZF (D0- M5) regimen, 4 (10.0%) in CV/ZF/ZF (D0-D56-M6) regimen, and 2 (10.0%) in CV/ZF (D0-M6) regimen, respectively. At 14 days post first boost, GMTs of neutralizing antibodies in recipients receiving ZF2001 at 28 days and 56 days post priming were 18.7 (95% CI 13.7 to 25.5) and 25.9 (17.0 to 39.3), respectively, with geometric mean ratios of 2.0 (1.2 to 3.5) and 3.4 (1.8 to 6.4) compared to TIV. GMTs at 14 days after second boost of neutralizing antibodies increased to 107.2 (73.7 to 155.8) in CV/ZF/ZF (D0-D28-M5) regimen and 141.2 (83.4 to 238.8) in CV/ZF/ZF (D0-D56-M6) regimen. Two-dose schedules of CV/ZF (D0-M5) and CV/ZF (D0-M6) induced antibody levels comparable with that elicited by 3-dose schedules, with GMTs of 90.5 (45.6, 179.8) and 94.1 (44.0, 200.9), respectively. Study limitations include the absence of vaccine effectiveness in a real-world setting and current lack of immune persistence data. CONCLUSIONS: Heterologous boosting with ZF2001 following primary vaccination with Convidecia is more immunogenic than a single dose of Convidecia and is not associated with safety concerns. These results support flexibility in cooperating viral vectored and recombinant protein vaccines. TRIAL REGISTRATION: Study on Heterologous Prime-boost of Recombinant COVID-19 Vaccine (Ad5 Vector) and RBD-based Protein Subunit Vaccine; ClinicalTrial.gov NCT04833101.

A Facile machine learning multi-classification model for Streptococcus agalactiae clonal complexes.

BACKGROUND: The clinical significance of group B streptococcus (GBS) was different among different clonal complexes (CCs), accurate strain typing of GBS would facilitate clinical prognostic evaluation, epidemiological investigation and infection control. The aim of this study was to construct a practical and facile CCs prediction model for S. agalactiae. METHODS: A total of 325 non-duplicated GBS strains were collected from clinical samples in Xinhua Hospital, Shanghai, China. Multilocus sequence typing (MLST) method was used for molecular classification, the results were analyzed to derive CCs by Bionumeric 8.0 software. Antibiotic susceptibility test was performed using Vitek-2 Compact system combined with K-B method. Multiplex PCR method was used for serotype identification. A total of 45 virulence genes associated with adhesion, invasion, immune evasion were detected by PCR method and electrophoresis. Three types of features, including antibiotic susceptibility (A), serotypes (S) and virulence genes (V) tests, and XGBoost algorithm was established to develop multi-class CCs identification models. The performance of proposed models was evaluated by the receiver operating characteristic curve (ROC). RESULTS: The 325 GBS were divided into 47 STs, and then calculated into 7 major CCs, including CC1, CC10, CC12, CC17, CC19, CC23, CC24. A total of 18 features in three kinds of tests (A, S, V) were significantly different from each CC. The model based on all the features (S&A&V) performed best with AUC 0.9536. The model based on serotype and antibiotic resistance (S&A) only enrolled 5 weighed features, performed well in predicting CCs with mean AUC 0.9212, and had no statistical difference in predicting CC10, CC12, CC17, CC19, CC23 and CC24 when compared with S&A&V model (all p > 0.05). CONCLUSIONS: The S&A model requires least parameters while maintaining a high accuracy and predictive power of CCs prediction. The established model could be used as a promising tool to classify the GBS molecular types, and suggests a substantive improvement in clinical application and epidemiology surveillance in GBS phenotyping.

Hepatotoxicity of cantharidin is associated with the altered bile acid metabolism.

Cantharidin (CTD) is an effective antitumor agent. However, it exhibits significant hepatotoxicity, the mechanism of which remains unclear. In this study, biochemical and histopathological analyses complemented with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)-based targeted metabolomic analysis of bile acids (BAs) were employed to investigate CTD-induced hepatotoxicity in rats. Sixteen male and female Sprague-Dawley rats were randomly divided into two groups: control and CTD (1.0 mg/kg) groups. Serum and liver samples were collected after 28 days of intervention. Biochemical, histopathological, and BA metabolomic analyses were performed for all samples. Further, the key biomarkers of CTD-induced hepatotoxicity were identified via multivariate and metabolic pathway analyses. In addition, metabolite-gene-enzyme network and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to identify the signaling pathways related to CTD-induced hepatotoxicity. The results revealed significantly increased levels of biochemical indices (alanine aminotransferase, aspartate aminotransferase, and total bile acid). Histopathological analysis revealed that the hepatocytes were damaged. Further, 20 endogenous BAs were quantitated via UHPLC-MS/MS, and multivariate and metabolic pathway analyses of BAs revealed that hyocholic acid, cholic acid, and chenodeoxycholic acid were the key biomarkers of CTD-induced hepatotoxicity. Meanwhile, primary and secondary BA biosynthesis and taurine and hypotaurine metabolism were found to be associated with the mechanism by which CTD induced hepatotoxicity in rats. This study provides useful insights for research on the mechanism of CTD-induced hepatotoxicity.

Mapping QTL for Adult-Plant Resistance to Stripe Rust in a Chinese Wheat Landrace.

Wheat stripe (yellow) rust is a worldwide disease that seriously reduces wheat grain yield and quality. Adult-plant resistance (APR) to stripe rust is generally more durable but usually controlled by multiple genes with partial resistance. In this study, a recombinant inbred line population was developed from a cross between a Chinese wheat landrace, Tutoumai, with APR to stripe rust, and a highly susceptible wheat cultivar, Siyang 936. The population was genotyped by genotyping-by-sequencing and phenotyped for APR to stripe rust in four consecutive field experiments. Three QTLs, QYr.sdau-1BL, QYr.sdau-5BL, and QYr.sdau-6BL, were identified for APR to stripe rust, and explained 8.0-21.2%, 10.1-22.7%, and 11.6-18.0% of the phenotypic variation, respectively. QYr.sdau-1BL was further mapped to a 21.6 Mb region using KASP markers derived from SNPs identified by RNA-seq of the two parents. In the QYr.sdau-1BL region, 13 disease-resistance-related genes were differently expressed between the two parents, and therefore were considered as the putative candidates of QYr.sdau-1BL. This study provides favorable gene/QTL and high-throughput markers to breeding programs for marker-assisted selection of the wheat stripe rust APR genes.

Comparative mitogenomics and phylogenetics of the stinging wasps (Hymenoptera: Aculeata).

The stinging wasps (Hymenoptera: Aculeata) include diverse groups such as vespid wasps, ants and bees. Phylogenetic relationships among major lineages of stinging wasps have been inferred from molecular and morphological data. However, the genomic features of the mitochondrial genomes and their phylogenetic utility remain to be explored. In this study, we determined 23 mitochondrial genomes from the Aculeata. Four Mutillidae species showed relatively low A + T content compared to other species of the Aculeata (69.7%-77.4%). Eleven out of 44 species, mainly from the Chrysididae and the Pompilidae, showed reversals of GC skews. Gene rearrangements occurred across the species. Patterns of tRNA rearrangement were conserved in some groups, including the Chrysididae, Bethylidae, Pompilidae, Scolioidea and Vespoidea. Rearrangement of protein-coding genes were found in 12 out of 44 species of the Aculeata, including all four species from the Chrysididae, both species from the Bethylidae, one species from the Dryinidae, all three Scolioidea species and two Apoidea species. Phylogenetic inference showed a long branch in species with unusual genomic features, such as in the Mutillidae and Bethylidae. By excluding these species, we found paraphyly of the Chrysidoidea and a sister group relationship between the Formicoidea and Vespoidea. These results improve our understanding of the evolution of mitochondrial genomes in the Aculeata and, in general, the evolution across this subclade.

Alternative Splicing Plays a Critical Role in Maintaining Mineral Nutrient Homeostasis in Rice (Oryza sativa).

Alternative splicing (AS) of pre-mRNAs promotes transcriptome and proteome diversity and plays important roles in a wide range of biological processes. However, the role of AS in maintaining mineral nutrient homeostasis in plants is largely unknown. To clarify this role, we obtained whole transcriptome RNA sequencing data from rice (Oryza sativa) roots grown in the presence or absence of several mineral nutrients (Fe, Zn, Cu, Mn, and P). Our systematic analysis revealed 13,291 alternatively spliced genes, representing ∼53.3% of the multiexon genes in the rice genome. As the overlap between differentially expressed genes and differentially alternatively spliced genes is small, a molecular understanding of the plant's response to mineral deficiency is limited by analyzing differentially expressed genes alone. We found that the targets of AS are highly nutrient-specific. To verify the role of AS in mineral nutrition, we characterized mutants in genes encoding Ser/Arg (SR) proteins that function in AS. We identified several SR proteins as critical regulators of Zn, Mn, and P nutrition and showed that three SR protein-encoding genes regulate P uptake and remobilization between leaves and shoots of rice, demonstrating that AS has a key role in regulating mineral nutrient homeostasis in rice.

Reducing resistance allele formation in CRISPR gene drive.

CRISPR homing gene drives can convert heterozygous cells with one copy of the drive allele into homozygotes, thereby enabling super-Mendelian inheritance. Such a mechanism could be used, for example, to rapidly disseminate a genetic payload in a population, promising effective strategies for the control of vector-borne diseases. However, all CRISPR homing gene drives studied in insects thus far have produced significant quantities of resistance alleles that would limit their spread. In this study, we provide an experimental demonstration that multiplexing of guide RNAs can both significantly increase the drive conversion efficiency and reduce germline resistance rates of a CRISPR homing gene drive in Drosophila melanogaster We further show that an autosomal drive can achieve drive conversion in the male germline, with no subsequent formation of resistance alleles in embryos through paternal carryover of Cas9. Finally, we find that the nanos promoter significantly lowers somatic Cas9 expression compared with the vasa promoter, suggesting that nanos provides a superior choice in drive strategies where gene disruption in somatic cells could have fitness costs. Comparison of drive parameters among the different constructs developed in this study and a previous study suggests that, while drive conversion and germline resistance rates are similar between different genomic targets, embryo resistance rates can vary significantly. Taken together, our results mark an important step toward developing effective gene drives capable of functioning in natural populations and provide several possible avenues for further control of resistance rates.

Serum Cytokine and Chemokine Profile in Relation to the Severity of Coronavirus Disease 2019 in China.

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated the serum cytokine and chemokine levels in asymptomatic, mild, moderate, severe, and convalescent SARS-CoV-2-infected cases. Proinflammatory cytokine and chemokine production induced by SARS-CoV-2 were observed not only in symptomatic patients but also in asymptomatic cases, and returned to normal after recovery. IL-6, IL-7, IL-10, IL-18, G-CSF, M-CSF, MCP-1, MCP-3, IP-10, MIG, and MIP-1α were found to be associated with the severity of COVID-19. Moreover, a set of cytokine and chemokine profiles were significantly higher in SARS-CoV-2-infected male than female patients. The serum levels of MCP-1, G-CSF, and VEGF were weakly and positively correlated with viral titers. We suggest that combinatorial analysis of serum cytokines and chemokines with clinical classification may contribute to evaluation of the severity of COVID-19 and optimize the therapeutic strategies.

A risk score based on pediatric sequential organ failure assessment predicts 90-day mortality in children with Klebsiella pneumoniae bloodstream infection.

BACKGROUND: Klebsiella pneumoniae bloodstream infection (Kp-BSI) is a serious threat to pediatric patients. The objective of this study was to explore the risk factors, validate the prediction efficiency of pediatric Sequential Organ Failure Assessment (SOFA) and establish better early predictors of mortality in pediatric patients with Kp-BSI. METHODS: All children diagnosed with Kp-BSI were included in this retrospective cohort study from January 2009 to June 2019. Basic characteristics, symptoms and physical examinations, treatments, laboratory statistics, and SOFA at the onset of Kp-BSI were recorded. The Cox proportional hazard model and receiver operating characteristic curves were used to assess the association between the variables and the 90-day mortality and their predictive value. DeLong's test of receiver operating characteristic curves and integrated discrimination improvement index were used to determine the improvement in predictive capacity of the modified SOFA models. A predictive score was developed using multivariate logistic regression. RESULTS: Of the 146 children enrolled, 33 (22.6%) patients died within 90 days. Hospitalization in the last 6 months, intra-abdominal source of infection, presence of organ failure, and altered levels of blood biomarkers, including C-reactive protein, albumin, and lactate were significant risk factors for 90-day mortality. The area under the curve (AUC) of SOFA for predicting 90-day mortality was 0.80 (95% CI 0.71-0.89). Moreover, we found that a prediction model combining SOFA with two other parameters, namely hospitalization in the last 6 months and intra-abdominal source of infection, was better at predicting mortality (AUC = 0.89, 95% CI 0.82-0.96; sensitivity = 0.86; specificity = 0.84). According to this novel risk model, we defined three statistically different groups: low-risk, medium-risk and high-risk groups, with an observed 90-day mortality of 5.4, 35.7, and 72.0%, respectively. With reference to the low-risk patients, the medium-risk and high-risk groups had a higher mortality, with hazard ratios of 8.36 (95% CI 3.60-27.83) and 20.27 (95% CI 7.47-54.95), respectively. CONCLUSIONS: The modified SOFA may be better than the original score to predict 90-day mortality in pediatric patients with Kp-BSI. Future prospective studies are required to validate this novel scoring system in external cohorts.

Nanoparticle reinforced bacterial outer-membrane vesicles effectively prevent fatal infection of carbapenem-resistant Klebsiella pneumoniae.

Infection resulting from carbapenem-resistant Klebsiella pneumoniae (CRKP) is an intractable clinical problem. Outer membrane vesicles (OMVs) from CRKP are believed to be potential vaccine candidates. However, their immune response remains elusive due to low structural stability and poor size homogeneity. In this study, hollow OMVs were reinforced internally by size-controlled BSA nanoparticles to obtain uniform and stable vaccines through hydrophobic interaction. The result showed that the BSA-OMV nanoparticles (BN-OMVs) were homogenous with a size around 100 nm and exhibited a core-shell structure. Remarkably, subcutaneous BN-OMVs vaccination mediated significantly higher CRKP specific antibody titers. The survival rate of the mice infected with a lethal dose of CRKP was increased significantly after BN-OMV immunization. The adoptive transfer experiment demonstrated that the protective effect of BN-OMVs was dependent on humoral and cellular immunity. This study demonstrated that the structure optimization improved the immune efficacy of OMVs for vaccine development against CRKP.

Novel CRISPR/Cas9 gene drive constructs reveal insights into mechanisms of resistance allele formation and drive efficiency in genetically diverse populations.

A functioning gene drive system could fundamentally change our strategies for the control of vector-borne diseases by facilitating rapid dissemination of transgenes that prevent pathogen transmission or reduce vector capacity. CRISPR/Cas9 gene drive promises such a mechanism, which works by converting cells that are heterozygous for the drive construct into homozygotes, thereby enabling super-Mendelian inheritance. Although CRISPR gene drive activity has already been demonstrated, a key obstacle for current systems is their propensity to generate resistance alleles, which cannot be converted to drive alleles. In this study, we developed two CRISPR gene drive constructs based on the nanos and vasa promoters that allowed us to illuminate the different mechanisms by which resistance alleles are formed in the model organism Drosophila melanogaster. We observed resistance allele formation at high rates both prior to fertilization in the germline and post-fertilization in the embryo due to maternally deposited Cas9. Assessment of drive activity in genetically diverse backgrounds further revealed substantial differences in conversion efficiency and resistance rates. Our results demonstrate that the evolution of resistance will likely impose a severe limitation to the effectiveness of current CRISPR gene drive approaches, especially when applied to diverse natural populations.

Casticin inhibits nasopharyngeal carcinoma growth by targeting phosphoinositide 3-kinase.

BACKGROUND: Casticin, an isoflavone compound extracted from the herb Fructus Viticis, has demonstrated anti-inflammatory and anticancer activities and properties. The aim of this study was to investigate the effects and mechanisms of casticin in nasopharyngeal carcinoma (NPC) cells and to determine its potential for targeted use as a medicine. METHODS: NPC cells were used to perform the experiments. The CCK­8 assay and colony formation assays were used to assess cell viability. Flow cytometry was used to measure the cell cycle and apoptosis analysis (annexin V/PI assay). A three-dimensional (3D) tumour sphere culture system was used to characterize the effect of casticin on NPC stem cells. In silico molecular docking prediction and high-throughput KINOME scan assays were used to evaluate the binding of casticin to phosphoinositide 3-kinase (PI3K), including wild-type and most of mutants variants. We also used the SelectScreen assay to detect the IC50 of ATP activity in the active site of the target kinase. Western blotting was used to evaluate the changes in key proteins involved cell cycle, apoptosis, stemness, and PI3K/protein kinase B (AKT) signalling. The effect of casticin treatment in vivo was determined by using a xenograft mouse model. RESULTS: Our results indicate that casticin is a new and novel selective PI3K inhibitor that can significantly inhibit NPC proliferation and that it induces G2/GM arrest and apoptosis by upregulating Bax/BCL2 expression. Moreover, casticin was observed to affect the self-renewal ability of the nasopharyngeal carcinoma cell lines, and a combination of casticin with BYL719 was observed to induce a decrease in the level of the phosphorylation of mTORC1 downstream targets in BYL719-insensitive NPC cell lines. CONCLUSION: Casticin is a newly emerging selective PI3K inhibitor with potential for use as a targeted therapeutic treatment for nasopharyngeal carcinoma. Accordingly, casticin might represent a novel and effective agent against NPC and likely has high potential for combined use with pharmacological agents targeting PI3K/AKT.

Emergence and establishment of KPC-2-producing ST11 Klebsiella pneumoniae in a general hospital in Shanghai, China.

The aim of this study was to investigate the characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) collected during an outbreak in a Chinese teaching hospital and to provide insights into the prevention and control of nosocomial infection. We collected unique CRKP clinical isolates from 2009 to 2013. Antibiotic-resistant genes were identified by polymerase chain reaction (PCR) and sequencing. The isolates were typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Plasmids were classified using a PCR-based incompatibility/replicon typing method and a replicon sequence typing method. Conjugation experiments were performed to evaluate the transferability of carbapenem-resistant genes. Whole genome sequencing (WGS) was conducted to further investigate the genetic background of the isolates. Infection control practices were reviewed throughout the study period. Klebsiella pneumoniae sequence type (ST) 11 emerged in 2010 and acquired the bla KPC-2 gene by 2011. From 2011 to 2013, ST11 KPC-2-producing CRKP (G type) prevailed as the most common CRKP in our hospital, causing a prolonged outbreak. The majority of these CRKP strains possess an IncFII plasmid, with Tn1721-bla KPC-2-ΔTn3-IS26 bearing the genetic structure for bla KPC-2. Infection prevention control measures available at the time contained the initial outbreak, but had no effect on the spread of CRKP later. This study demonstrated the seriousness concerning the spread of KPC-2-producing ST11 CRKP in a Chinese hospital, indicating that current prevention and control strategies for carbapenem-resistant Enterobacteriaceae (CRE) nosocomial infection need to be investigated and adjusted.

Rapid detection of carbapenemase activity of Enterobacteriaceae isolated from positive blood cultures by MALDI-TOF MS.

BACKGROUND: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been proved to be a useful tool for identification of pathogens directly isolated from blood cultures in clinical microbiology laboratories. ß-Lactam antibiotics are commonly used for treatment of bloodstream infections caused by Enterobacteriaceae strains, and carbapenem is the superlative class of ß-lactam antibiotics. Since the carbapenem resistance rate of Enterobacteriaceae strains raised year by year, efficient detection of carbapenemase activity and timely delivery of carbapenem susceptibility reports of Enterobacteriaceae strains isolated from blood cultures is important for clinicians. METHODS: We used 64 simulated blood cultures to establish the method of MALDI-TOF MS based ertapenem hydrolysis assay. The cutoff value of logRQ calculated from the peaks intensity of ertapenem and its hydrolysate was first set to identify the strains with carbapenemase activity. Then, we detected and calculated the logRQ values of 385 Enterobacteriaceae strains from positive clinical blood cultures to distinguish the carbapenemase producers and noncarbapenemase producers. RESULTS: The mean logRQ value of 32 noncarbapenemase producers was - 0.85 ± 0.14 in simulated blood cultures, while the logRQ value of 32 carbapenemase producers was 0.87 ± 0.55. Thus, the cutoff value of logRQ was set at - 0.45 with sensitivity of 100% and specificity of 100%. In 385 clinical positive blood cultures, the logRQ values of all carbapenem-susceptible Enterobacteriaceae strains (81.3%, 313/385) were < - 0.45. Comparing with the detection of carbapenemase genes, carbapenem-resistant Enterobacteriaceae strains (18.7%, 72/385) were well distinguished by MALDI-TOF MS based ertapenem hydrolysis assay with a sensitivity of 92.5% and specificity of 100%. CONCLUSIONS: Our data show that MALDI-TOF MS based ertapenem hydrolysis assay is a rapid and accurate method to detect carbapenemase activity of Enterobacteriaceae strains from positive blood cultures, and can be routinely performed in clinical microbiology laboratories.

Activating killer cell immunoglobulin-like receptor 2DS2 binds to HLA-A*11.

Inhibitory killer cell Ig-like receptors (KIRs) are known to recognize HLA ligands mainly of the HLA-C and Bw4 groups, but the ligands for KIRs are poorly understood. We report here the identification of the cognate ligand for the activating KIR 2DS2 as HLA-A*11:01. The crystal structure of the KIR2DS2-HLA-A*11:01 complex was solved at 2.5-Å resolution and revealed residue-binding characteristics distinct from those of inhibitory KIRs with HLA-C and the critical role of residues Tyr45 and Asp72 in shaping binding specificity to HLA-A*11:01. Using KIR2DS2 tetramers, binding to surface HLA-A*11:01 on live cells was demonstrated and, furthermore, that binding can be altered by residue changes at p8 of the peptide, indicating the influence of peptide sequence on KIR-HLA association. In addition, heteronuclear single quantum coherence NMR was used to map the involvement of critical residues in HLA binding at the interface of KIR and HLA, and validates the data observed in the crystal structure. Our data provide structural evidence of the recognition of A*11:01 by the activating KIR2DS2 and extend our understanding of the KIR-HLA binding spectrum.

A Dimensionality Reduction-Based Multi-Step Clustering Method for Robust Vessel Trajectory Analysis.

The Shipboard Automatic Identification System (AIS) is crucial for navigation safety and maritime surveillance, data mining and pattern analysis of AIS information have attracted considerable attention in terms of both basic research and practical applications. Clustering of spatio-temporal AIS trajectories can be used to identify abnormal patterns and mine customary route data for transportation safety. Thus, the capacities of navigation safety and maritime traffic monitoring could be enhanced correspondingly. However, trajectory clustering is often sensitive to undesirable outliers and is essentially more complex compared with traditional point clustering. To overcome this limitation, a multi-step trajectory clustering method is proposed in this paper for robust AIS trajectory clustering. In particular, the Dynamic Time Warping (DTW), a similarity measurement method, is introduced in the first step to measure the distances between different trajectories. The calculated distances, inversely proportional to the similarities, constitute a distance matrix in the second step. Furthermore, as a widely-used dimensional reduction method, Principal Component Analysis (PCA) is exploited to decompose the obtained distance matrix. In particular, the top k principal components with above 95% accumulative contribution rate are extracted by PCA, and the number of the centers k is chosen. The k centers are found by the improved center automatically selection algorithm. In the last step, the improved center clustering algorithm with k clusters is implemented on the distance matrix to achieve the final AIS trajectory clustering results. In order to improve the accuracy of the proposed multi-step clustering algorithm, an automatic algorithm for choosing the k clusters is developed according to the similarity distance. Numerous experiments on realistic AIS trajectory datasets in the bridge area waterway and Mississippi River have been implemented to compare our proposed method with traditional spectral clustering and fast affinity propagation clustering. Experimental results have illustrated its superior performance in terms of quantitative and qualitative evaluations.