Thermally resistant nuclease in Serratia marcescens hinders PCR reactions and degrades PCR products.

Polymerase chain reaction (PCR) is an important tool for exogenous gene acquisition and recombinants identification. There exist two problems when using Serratia marcescens as a template for PCR amplification: amplified PCR products are rapidly degraded, and the results of PCR amplification are unstable. The aim of the present work was to elucidate the reasons for this. By mixing PCR products amplified from Escherichia coli DH5α with S. marcescens supernatant or pellet, we found that the DNA-degrading substance in S. marcescens is thermally resistant and present both intracellularly and extracellularly. We then determined that it is protein, and most likely S. marcescens nuclease, that degrades PCR products since the addition of SDS and EDTA can effectively inhibit or block the degradation of PCR products. By knocking out the S. marcescens nuclease encoding gene, nucA, we confirmed that the nuclease is responsible for the degradation of PCR products and the instability of PCR amplification. This work is the first to show that the S. marcescens nuclease is temporarily and partially inhibited by high temperatures during PCR and recovers rapidly at room temperature after PCR.

Endothelial cyclin I reduces vulnerability to angiotensin II-induced vascular remodeling and abdominal aortic aneurysm risk.

BACKGROUND: Retinoblastoma protein (Rb) supports vasoprotective E2F Transcription Factor 1 (E2f1)/Dihydrofolate Reductase (Dhfr) pathway activity in endothelial cells. Cyclin I (Ccni) promotes Cyclin-Dependent Kinase-5 (Cdk5)-mediated Rb phosphorylation. Therefore, we hypothesized that endothelial Ccni may regulate cardiovascular homeostasis, vessel remodeling, and abdominal aortic aneurysm (AAA) formation. METHODS: Aortic CCNI mRNA expression was analyzed in the Gene Expression Omnibus (GEO) GSE57691 cohort consisting of AAA patients (n = 39) and healthy controls (n = 10). We employed wild-type (WT) mice and endothelial Ccni knockout (Ccnifl/flTie2-Cre) mice to conduct in vivo and ex vivo experimentation using an Angiotensin (Ang) II hypertension model and a CaCl2 AAA model. Mice were assessed for Rb/E2f1/Dhfr signaling, biopterin (i.e., biopterin [B], dihydrobiopterin [BH2], and tetrahydrobiopterin [BH4]) production, cardiovascular homeostasis, vessel remodeling, and AAA formation. RESULTS: Aortic CCNI mRNA expression was downregulated in AAA patients. Both Ang II- and CaCl2-induced WT mice showed aortic Ccni upregulation coupled with vasculoprotective upregulation of Rb/E2f1/Dhfr signaling and biopterins. Endothelial Ccni knockout downregulated medial Rb/E2f1/Dhfr signaling and biopterins in Ang II-induced hypertensive mice, which exacerbated eNos uncoupling and H2O2 production. Endothelial Ccni knockout impaired in vivo hemodynamic responses and endothelium-dependent vasodilatation in ex vivo mesenteric arteries in response to Ang II. Endothelial Ccni knockout exacerbated mesenteric artery remodeling and AAA risk in response to Ang II and CaCl2. CONCLUSIONS: Endothelial Ccni acts as a critical negative regulator of eNos uncoupling-mediated ROS generation and thereby reduces vulnerability to hypertension-induced vascular remodeling and AAA development in mice.

Epidemiological and Whole-Genome Sequencing Analysis of a Gastroenteritis Outbreak Caused by a New Emerging Serotype of Vibrio parahaemolyticus in China.

Vibrio parahaemolyticus is an important foodborne pathogen with diverse serotypes. In May 2021, we investigated a gastroenteritis outbreak that occurred in China, caused by V. parahaemolyticus O10:K4 infection. Based on the epidemiological curve, this outbreak was identified as a homologous exposure event. A case-control study demonstrated that emperor crab with mashed garlic (odds ratio [OR] = 4.60, p = 0.030; 95% confidence interval [95% CI]: 1.11-19.14), goose liver geoduck (OR = 4.50, p = 0.029; 95% CI: 1.12-18.13), shrimp (OR = 4.89, p = 0.021; 95% CI: 1.22-19.65), and sea cucumber (OR = 7.36, p = 0.005; 95% CI: 1.68-32.26) were the potential sources of the food poisoning. V. parahaemolyticus isolates from 18 laboratory-confirmed cases were all serotyped O10:K4, and determined to be sequence type ST3 via multilocus sequence typing. Pulsed field gel electrophoresis and whole-genome sequencing analysis revealed the identical pattern and 0-2 single nucleotide variation among these isolates. tdh was positive in all isolates, while trh and Orf8 were absent. Seven essential base positions in toxRS for pandemic clone identification were identical between the O10:K4 and O3:K6 pandemic clones. Phylogenetic analysis with 45 additional genomes of 13 different serotypes showed the closest genetic relationship between O10:K4 and O1: KUT. O10:K4 was thought to evolve from the O3:K6 pandemic clone. The new serovariant of O3:K6 poses a challenge for the prevention and control of V. parahaemolyticus disease outbreaks, or even epidemics, in the future.

Rapid fluorescent color analysis of copper ions on a smart phone via ratiometric fluorescence sensor.

A smartphone-assisted fluorescence color sensing system for rapid, convenient, and on-site detection of copper ions was developed. The ratiometric fluorescence sensor was fabricated by using silica-coated blue-light-emitting carbon dots and surface-grafted red-light-emitting cadmium-telluride quantum dots. After exposure to Cu2+ in 20 s, the red fluorescence was quenched obviously, while the blue fluorescence remained unchanged, and the sensor color changes continuously from red to blue under the ultraviolet lamp. The concentration (50-1200 nM) of copper ions could be measured by the fluorescence spectrum (excitation at 360 nm, dual-emission at 441 and 640 nm) with a detection limit of 7.7 nM. The fluorescence colors were converted to digital RGB values to calculate the concentration of copper ions by a smartphone with a detection limit of 9.6 nM. The method was applied to detecting copper ions spiked in real samples with recovery from 97.9 to 108.0% and RSD from 3.8 to 8.9%. Thus, this convenient and practical fluorescence color sensing system presents a new strategy for rapid, sensitive, and on-site determination of copper ions in environmental or biological samples.

Prevalence and Population Analysis of Vibrio parahaemolyticus Isolated from Freshwater Fish in Zhejiang Province, China.

Objectives: The previous researches revealed that Vibrio parahaemolyticus has been detected in freshwater fish samples. However, the molecular characteristics of V. parahaemolyticus isolated from freshwater fish, including pathogenic and pandemic strains, are still unknown. This study aims to characterize and identify molecular properties of the bacterium. In addition, it identifies the source of V. parahaemolyticus from freshwater fish samples in Zhejiang Province, China. Methods: Four hundred and twenty-one freshwater fish samples (from fishing farms, retail markets, and restaurants) and 212 seafood samples (from retail markets) were collected in 10 cities of Zhejiang Province. V. parahaemolyticus strains were isolated from these samples and comparatively analyzed by multilocus sequence typing, serotyping, antimicrobial susceptibility test, and polymerase chain reaction, targeting common toxin genes (tdh, trh) and markers for pandemic strains (orf8, toxRS/new). Results: Sixty-eight V. parahaemolyticus strains were isolated from the 421 freshwater fish samples, and 89 V. parahaemolyticus isolates were identified out of 212 seafood samples. The detection rate of V. parahaemolyticus was significantly different (p < 0.05) between the fishing farms, the retail markets, and the restaurants. The isolates from freshwater fish samples were divided into eight O serotypes with three O3:K6 isolates, which contain three pandemic complexes (tdh+, orf8+, toxRS/new+). A total of 53 different sequence types (STs) were identified among the 68 isolates, including 28 novel STs. Antimicrobial susceptibility results indicated that 76.5% of the strains were resistant to ampicillin. A third (3/9) of the isolates from fishing farm sources shared the same STs with their counterparts from retail markets. Compared with the isolates from the seafood samples collected in the same sampling sites, 13.2% (9/68) freshwater fish isolates overlapped with seafood isolates. Conclusions: Our study showed that V. parahaemolyticus population in freshwater fish is genetically diverse. The V. parahaemolyticus contaminates might have come from both fishing farm sources and cross-contamination from seafood in the closed area at the markets. Freshwater fish may work as a reservoir of pathogenic and pandemic V. parahaemolyticus isolates, indicating potential public health and food safety risks associated with the consumption of freshwater fish.

A Framework for the Automatic Integration and Diagnosis of Building Energy Consumption Data.

Buildings account for a majority of the primary energy consumption of the human society, therefore, analyses of building energy consumption monitoring data are of significance to the discovery of anomalous energy usage patterns, saving of building utility expenditures, and contribution to the greater environmental protection effort. This paper presents a unified framework for the automatic extraction and integration of building energy consumption data from heterogeneous building management systems, along with building static data from building information models to serve analysis applications. This paper also proposes a diagnosis framework based on density-based clustering and artificial neural network regression using the integrated data to identify anomalous energy usages. The framework and the methods have been implemented and validated from data collected from a multitude of large-scale public buildings across China.

Production, Characterization and Immunomodulatory Activity of an Extracellular Polysaccharide from Rhodotorula mucilaginosa YL-1 Isolated from Sea Salt Field.

A novel exopolysaccharide from marine-derived red yeast Rhodotorula mucilaginosa strain YL-1 was produced and characterized. The highest yield of polysaccharide reached 15.1 g/L after medium and culture parameter optimization. This exopolysaccharide, composed of four neural monosaccharides including glucose, mannose, galactose and fucose, had an average molecular weight of 1200 KDa. It had good immunomodulatory activity on RAW256.7 cell lines. ELISA (enzyme linked immunosorbent assay) and Q-PCR (quantitative real-time PCR) results showed that the cell was stimulated to express more IL-6, IL-18, IL-1ß and TNFα cytokines than the control group. This is the first report of an exopolysaccharide with immunomodulatory activity from marine-derived Rhodotorula mucilaginosa.

Epidemiological and Whole Genomic Sequencing Analysis of a Campylobacter jejuni Outbreak in Zhejiang Province, China, May 2019.

Campylobacter is well recognized as the leading cause of bacterial foodborne diarrheal disease worldwide with a very low outbreak reported in China. In May 2019, we investigated an outbreak of Campylobacter jejuni infections among students in a junior high school in Eastern China. Cases were interviewed to identify a common source of contamination. As cases were identified in the same school during a period of time, menus were reviewed and food items included in the questionnaire. Rectal swabs from school kitchen staff and suspected food items (raw chicken) from a local market from where the school food came were examined for C. jejuni. Pulsed-field gel electrophoresis and whole genome sequencing were performed to determine the relatedness of the isolates. To identify the source of the contamination, a case-control study was conducted. Forty-five cases were reported with diarrhea among 1696 students and staff. Stool samples for 10 of the 45 and 5 tested positive for C. jejuni. WGS analysis revealed a 0-4 single nucleotide variation in case-patient isolates. Although we were unable to identify the specific food item, a specific menu was identified as the potential source of the contamination (odds ratios = 20.82; 95% confidence interval = 6.472-66.957). In this menu, chicken was served. A food isolate collected from chicken in Zhejiang province in 2018 was positive for the same identical strain (5-7 single nucleotide polymorphisms). This is one of the few reports in China about outbreak caused by C. jejuni. This investigation illustrates the potential risk of outbreaks caused by Chinese cold dishes of chicken.

Dietary nano cerium oxide promotes growth, relieves ammonia nitrogen stress, and improves immunity in crab (Eriocheir sinensis).

Oxidative stress plays a crucial role in ammonia nitrogen toxicity. In this study, the beneficial effects of dietary nano cerium oxide (nano CeO2) as a potent antioxidant were examined in the Chinese mitten crab (Eriocheir sinensis). Crabs were fed a diet supplemented with 0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, or 12.8 mg/kg nano CeO2 for 60 d. The optimum supplementation level of nano CeO2 that significantly increased weight gain rate and decreased feed coefficient was 0.8 mg/kg. This level also offered immune protection when crabs were kept under ammonia nitrogen stress and/or exposed to pathogen infection (Aeromonas hydrophila). Supplementation with 0.8 mg/kg of CeO2 (i) relieved pathological damage to the hepatopancreas; (ii) increased hemocyte counts, including total number of hemocytes, granulocytes, and hyalinocytes; (iii) decreased malondialdehyde content and increased antioxidant enzyme activities of superoxide dismutase and catalase in the hemolymph; (iv) increased the activities of lysozyme, acid phosphatase, and alkaline phosphatase in the hemolymph; and (v) increased gene and protein expression of cathepsin L in the hepatopancreas. Mortality increased when crabs were injected with bacteria under ammonia nitrogen stress, but dietary supplementation with 0.8 mg/kg nano CeO2 decreased the mortality rate. Thus, the results of this study suggested that dietary supplementation with nano CeO2 in crabs promoted growth and up-regulated immunity to bacterial infection under ammonia nitrogen stress.

[RAM study on general standard of maximum residue limits for pollution-free traditional Chinese medicine based on Chinese Pharmacopoeia formula].

The excessive pesticide residues and heavy metals in traditional Chinese medicine seriously endanger human health and the sustainable development of Chinese medicine industry. In order to improve the quality of traditional Chinese medicine and establish a general standard for maximum residue limits(MRL) of pesticides in pollution-free traditional Chinese medicine and decoction pieces, and to ensure the safety of clinical medication from its origin, MRLs were calculated based on the formula(MRL=A×W/100M) from Chinese Pharmacopeia, comparing it with the current Chinese and international standards as well as literature review, the RAND/UCLA appropriateness method(RAM) was applied to determine the categories and MRLs of pesticides in pollution-free traditional Chinese medicine and decoction pieces. Two questionnaires were drafted for expert panel and appropriateness analysis was carried out with the 9-point Likert scale to determine the general standard for MRLs of pollution-free traditional Chinese medicine and decoction pieces. The results showed that a total of nine experts from different fields scored the necessity of standard-setting and 206 pesticide residue limits respectively. The appropriateness scores of 206 pesticides were greater than 7, and appropriateness rate was 100%, which signifies that the expert panel has reached consensus. In summary, based on the RAM, the general standard for maximum residue limits of pesticides in pollution-free Chinese medicines and decoction pieces has reached an expert consensus. Comparing with the MRLs of medicinal plants and plant-sourced food from CAC, Europe Union, the United States, South Korea, Japan, Australia, New Zealand and Canada, 206 MRLs from this general standard share 88.8% in common, 4.4% of which is higher and 6.8% lower than those international standards. This has provided a basis for standardizing the use of pesticides in pollution-free traditional Chinese medicine.

High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide.

An alginate lyase encoding gene sagl from Flavobacterium sp. H63 was codon optimized and recombinantly expressed at high level in P.pastoris through high cell-density fermentation. The highest yield of recombinant enzyme of sagl (rSAGL) in yeast culture supernatant reached 226.4 µg/mL (915.5 U/mL). This was the highest yield record of recombinant expression of alginate lyase so far. The rSAGL was confirmed as a partially glycosylated protein through EndoH digestion. The optimal reaction temperature and pH of this enzyme were 45 °C and 7.5; 80 mM K⁺ ions could improve the catalytic activity of the enzyme by 244% at most. rSAGL was a thermal stable enzyme with T5015 of 57⁻58 °C and T5030 of 53⁻54 °C. Its thermal stability was better than any known alginate lyase. In 100 mM phosphate buffer of pH 6.0, rSAGL could retain 98.8% of the initial activity after incubation at 50 °C for 2 h. Furthermore, it could retain 61.6% of the initial activity after 48 h. The specific activity of the purified rSAGL produced by P. pastoris attained 4044 U/mg protein, which was the second highest record of alginate lyase so far. When the crude enzyme of the rSAGL was directly used in transformation of sodium alginate with 40 g/L, 97.2% of the substrate was transformed to di, tri, tetra brown alginate oligosaccharide after 32 h of incubation at 50 °C, and the final concentration of reducing sugar in mixture reached 9.51 g/L. This is the first report of high-level expression of thermally stable alginate lyase using P. pastoris system.

Controlled synthesis of polydopamine: A new strategy for highly sensitive fluorescence turn-on detection of acetylcholinesterase activity.

A new water soluble fluorescent coronene probe (CTCA) was synthesized and is shown to display strong fluorescence (with excitation/emission maxima at 313/450 nm) in aqueous solution. Dopamine was oxidized under air to form polydopamine (PDA) which quenches the fluorescence of CTCA. The enzyme acetylcholinesterase (AChE) is known catalyze the hydrolysis of acetylthiocholine to produce thiocholine. Thiocholine inhibits the polymerization of DA, and this leads to recovery in CTCA fluorescence. These findings form the basis for a new method for detection of AChE activity. The assay has a detection limit as low as 0.05 mU·mL-1 of AChE. It is highly selective, and other enzymes do no noticeably interfere. It was applied to the determination of AChE activity in (spiked) human serum, and of AChE inhibitors in (spiked) lake water samples. Graphical abstract Controlled synthesis of polydopamine for the highly sensitive and selective sensing of AChE activity is reported for the first time.

Fluorescence turn-on detection of alkaline phosphatase activity based on controlled release of PEI-capped Cu nanoclusters from MnO2 nanosheets.

A fluorescence turn-on assay for alkaline phosphatase (ALP) activity is developed through the controlled release of polyethyleneimine-capped copper nanoclusters (PEI-capped CuNCs) from the MnO2 nanosheets. In an aqueous solution, the positively charged PEI-capped CuNCs could be adsorbed onto the surface of the negatively charged MnO2 nanosheets. Such adsorption through favorable electrostatic interactions could efficiently quench the nanocluster fluorescence emission via resonance energy transfer from the PEI-capped CuNCs to the MnO2 nanosheets. 2-Phospho-L-ascorbic acid (AAP) could be hydrolyzed to L-ascorbic acid (AA) in the presence of ALP. AA could reduce MnO2 into Mn2+ and trigger the disintegration of the MnO2 nanosheets. As a result, the CuNCs were released and the quenched fluorescence was recovered efficiently. The detection strategy is simple, inexpensive, sensitive, selective, with low toxicity, and has better biocompatibility. The newly fabricated biosensor for ALP activity will potentially make it a robust candidate for numerous biological and biomedical applications.

Polyphosphoric acid-induced perylene probe self-assembly and label-free fluorescence turn-on detection of alkaline phosphatase.

A label-free fluorescence turn-on strategy for alkaline phosphatase (ALP) detection was established based on its enzymatic catalyzed hydrolysis of polyphosphoric acid (PPA, an anionic polymer) that had been utilized for aggregation with our homemade positively charged perylene derivative (Probe-1) via noncovalent interactions. The disaggregation caused turn-on fluorescence signal which was recovered by the released Proble-1 molecules whose original strong fluorescence in an aqueous buffer solution had been quenched due to their previous aggregation induced by PPA. Such method presents its great advantages of free labeling, convenience and simplicity, cost effectiveness, high selectivity, and high sensitivity, with a detection limit of 0.5 mU/mL of ALP. Graphical Abstract A label-free fluorescence turn-on strategy for alkaline phosphatase based on its enzymatic catalyzed hydrolysis of polyphosphoric acid that had been utilized for aggregation with our homemade positively charged perylene derivative via noncovalent interactions.

Multilocus sequence type profiles of Bacillus cereus isolates from infant formula in China.

Bacillus cereus sensu stricto is an opportunistic foodborne pathogen. The multilocus sequence type (MLST) of 74 B. cereus isolated from 513 non-random infant formula in China was analyzed. Of 64 sequence types (STs) detected, 50 STs and 6 alleles were newly found in PubMLST database. All isolates except for one singleton (ST-1049), were classified into 7 clonal complexes (CC) by BURST (n-4), in which CC1 with core ancestral clone ST-26 was the largest group including 86% isolates, and CC2, 3, 9, 10 and 13 were first reported in China. MLST profiles of the isolates from 8 infant formula brands were compared. It was found the brands might be potentially tracked by the variety of STs, such as ST-1049 of singleton and ST-1062 of isolate from goat milk source, though they could not be easily tracked just by clonal complex types of the isolates.

Quantitative Prevalence, Phenotypic and Genotypic Characteristics of Bacillus cereus Isolated from Retail Infant Foods in China.

Bacillus cereus is an important foodborne pathogen, which can cause severe food poisoning. The aim of this study was (i) to evaluate the quantitative prevalence of B. cereus in retail prepackaged infant formula and ready-to-eat rice flour in China and (ii) to gain the basic information on pheno- and genotypic characteristics of B. cereus isolates. We found that 40 out of the 587 samples were positive for B. cereus. B. cereus in 3.5% of infant formula samples and 1.0% of rice flour samples outnumbered 100 Colony-Forming Units (CFU)/g. B. cereus level even attained 103-104 CFU/g in four infant formula samples and one rice flour sample. Furthermore, we identified the distribution patterns of toxin genes in B. cereus isolates. The results showed that 97.5% of B. cereus isolates harbored at least one enterotoxin gene. Antibiotic susceptibility tests revealed that all isolated B. cereus strains were resistant to penicillin and 50% of them were multidrug resistant. Thirteen new sequence types (STs) and four new alleles were identified via multilocus sequence typing. Clonal Complex (CC) ST-205 and CC ST-142 were predominant clonal complexes. Interestingly, we revealed the special relationship between STs of B. cereus isolates and the geographical distributions of infant food manufacturers for the first time. The data implied that B. cereus of different STs might have a distinct ecological niche in China. In view of relatively high contamination level of enterotoxin- producing B. cereus in a proportion of infant foods, especially in those suitable for the ≤6-month-old infant group, appropriate safety criteria and hygienic control measures for infant foods should be drafted in China to prevent B. cereus infection.

[Effect of Bupleurum polysaccharides on splenocytes from lupus like mouse re-challenged with Campylobacter jejuni-S(131) in vitro].

Mice were immunized with Campylobacter jejuni-S(131) (CJ-S(131)) to establish the lupus-like model. Splenocytes from lupus like mice were challenged with CJ-S(131) to induce inflammatory response in vitro. Bupleurum smithii var. parvifolium polysaccharides (BPs) was added in the inflammatory model to observe its underlying mechanisms of action on lupus. BALB/c mice were randomly divided into three groups including normal control group, adjuvant control group and lupus-like model. Mice were immunized on Day 0 and 14 with CJ-S(131) to establish lupus-like syndrome, and sacrificed on Day 19. Splenocytes from each group were collected and divided into blank control group, BPs added group (BPs 5, 10, 20, 40 µg·m L(-1)), CJ-S(131) stimulated group, and CJ-S(131) plus BPs group. The levels of total IgG, anti-ds DNA antibody, interferon-γ, interleukin-10 (IL-10) and IL-17 were quantified by ELISA. The proliferation of splenocytes was determined in the MTT assay. BPs significantly suppressed the high levels of total IgG, anti-ds DNA antibody, IFN-γ and IL-10 stimulated by CJ-S131 and had no significant effects on increased IL-17 secretion and splenocytes proliferation. The results suggest that re-stimulation of splenocytes with CJ-S(131) could establish an inflammatory model in vitro. The effect of BPs on lupus might is related to its inhibition of the production of autoantibodies and associated cytokines.

Identification of three extra-chromosomal replicons in Leptospira pathogenic strain and development of new shuttle vectors.

BACKGROUND: The genome of pathogenic Leptospira interrogans contains two chromosomes. Plasmids and prophages are known to play specific roles in gene transfer in bacteria and can potentially serve as efficient genetic tools in these organisms. Although plasmids and prophage remnants have recently been reported in Leptospira species, their characteristics and potential applications in leptospiral genetic transformation systems have not been fully evaluated. RESULTS: Three extrachromosomal replicons designated lcp1 (65,732 bp), lcp2 (56,757 bp), and lcp3 (54,986 bp) in the L. interrogans serovar Linhai strain 56609 were identified through whole genome sequencing. All three replicons were stable outside of the bacterial chromosomes. Phage particles were observed in the culture supernatant of 56609 after mitomycin C induction, and lcp3, which contained phage-related genes, was considered to be an inducible prophage. L. interrogans-Escherichia coli shuttle vectors, constructed with the predicted replication elements of single rep or rep combined with parAB loci from the three plasmids were shown to successfully transform into both saprophytic and pathogenic Leptospira species, suggesting an essential function for rep genes in supporting auto-replication of the plasmids. Additionally, a wide distribution of homologs of the three rep genes was identified in L. interrogans isolates, and correlation tests showed that the transformability of the shuttle vectors in L. interrogans isolates depended, to certain extent, on genetic compatibility between the rep sequences of both plasmid and host. CONCLUSIONS: Three extrachromosomal replicons co-exist in L. interrogans, one of which we consider to be an inducible prophage. The vectors constructed with the rep genes of the three replicons successfully transformed into saprophytic and pathogenic Leptospira species alike, but this was partly dependent on genetic compatibility between the rep sequences of both plasmid and host.

Postoperative intra-abdominal hypertension predicts worse hospital outcomes in children after cardiac surgery: a pilot study†.

OBJECTIVES: Our goal was to determine the incidence and characteristics of postoperative intra-abdominal hypertension (IAH) in paediatric patients undergoing open-heart surgery. METHODS: This single-centre study included consecutive children (aged <16 years) who underwent open-heart surgery between July 2020 and February 2021. Patients who entered the study were followed until in-hospital death or hospital discharge. The study consisted of 2 parts. Part I was a prospective observational cohort study that was designed to discover the association between exposures and IAH. Postoperative intra-abdominal pressure was measured immediately after admission to the intensive care unit and every 6 h thereafter. Part II was a cross-sectional study to compare the hospital-related adverse outcomes between the IAH and the no-IAH cohorts. RESULTS: Postoperatively, 24.7% (38/154) of the patients exhibited IAH, whereas 3.9% (6/154) developed abdominal compartment syndrome. The majority (29/38, 76.3%) of IAH cases occurred within the first 24 h in the intensive care unit. Multivariable analysis showed that the Society of Thoracic Surgeons-European Association for Cardio-Thoracic Surgery score [odds ratio (OR) = 1.86, 95% confidence interval (CI) 1.23-2.83, P = 0.004], right-sided heart lesion (OR = 5.60, 95% CI 2.34-13.43, P < 0.001), redo sternotomy (OR = 4.35, 95% CI 1.64-11.57, P = 0.003), high baseline intra-abdominal pressure (OR = 1.43, 95% CI 1.11-1.83, P = 0.005), prolonged cardiopulmonary bypass duration (OR = 1.01, 95% CI 1.00-1.01, P = 0.005) and deep hypothermic circulatory arrest (OR = 5.14, 95% CI 1.15-22.98, P = 0.032) were independent predictors of IAH occurrence. IAH was associated with greater inotropic support (P < 0.001), more gastrointestinal complications (P = 0.001), sepsis (P = 0.003), multiple organ dysfunction syndrome (P < 0.001) and prolonged intensive care unit stay (z = -4.916, P < 0.001) and hospitalization (z = -4.710, P < 0.001). The occurrence of a composite outcome (P = 0.009) was significantly increased in patients with IAH. CONCLUSIONS: IAH is common in children undergoing cardiac surgery and is associated with worse hospital outcomes. Several factors may be associated with the development of IAH, including basic cardiac physiology and perioperative factors. TRIAL INFORMATION: This study was registered in the Chinese Clinical Trial Registry (Trial number: ChiCTR2000034322)URL site: https://www.chictr.org.cn/hvshowproject.html?id=41363&v=1.4.

Genetic and molecular biological characterization of two homologous cheR genes from Leptospira interrogans.

The Leptospira interrogans genome encodes two copies of cheR genes and each of them is able to complement for the swarming defective phenotype of Escherichia coli cheR null mutant RP1254 to certain extent, while over-expression of either of them inhibits the swarming of the chemotactic wild-type E. coli strain, RP437. Therefore, both CheR1 and CheR2 ought to bear the methyltransferase activities, although CheR1 has only one instead of two conserved basic amino acid residues located on the positively charged face of α2-helix. When this residue as well as the Lys48 and Arg55 of CheR2 was mutated, none of the CheRs was able to maintain aforementioned complementation functions, suggesting their critical roles in recognition of methyl-accepting chemotaxis proteins similar to that of E. coli. Demonstrated by microarray assay, the expression of cheR1 in L. interrogans cultured at 28°C in Ellinghausen-McCullough-Johnson-Harris medium was significantly lower than the average transcription level of all other genes, while the transcription of cheR2 was significantly higher than that of cheR1 in accordance with real-time reverse transcriptase-polymerase chain reaction assay. Tandem MS-MS data mining for the proteome of the same culture detected 16 peptides derived from CheR2 but none from CheR1. Therefore, although both genes were shown to be functional in E. coli, the structurally more conserved CheR2 rather than CheR1 might be the major functional component of L. interrogans chemotaxis adaptation system under our laboratory culture conditions.