New Antibiotic Binding Resin Bottles Collected from Children Enhances Rapid Microbial Identification.

BACKGROUND: Bloodstream infections (BSI) represent a common cause of sepsis and mortality in children. Blood culture (BC) is the gold standard for diagnosis of BSI. The low sensitivity of BC in the pediatric population is usually due to the small volume of blood used for inoculation and to the antibiotics used before sampling. Here, we explore the ways to effectively reduce antibiotic activity to maximize the chances of pathogen recovery, and to enhance the growth of microorganisms in lower blood volume and bacterial counts. METHODS: The recovery of common pathogens causing blood stream infections was analyzed after exposure to cefo-perazone/sulbactam, vancomycin, and caspofung by using resin-containing or not BacT/Alert PF Plus and BD FX400 peds plus pediatric bottles. The microbial growth in the resin-containing bottles was assessed using 0.5 colony-forming units (CFU) bacterial inoculum to mimic the bacteremia/fungemia condition. The usefulness of a diagnosis to confirm or exclude BSI was evaluated by lower than recommended blood culture sampling (102 CFU/mL, 0.3 mL). RESULTS: Staphylococcus aureus (S. aureus), and Candida glabrata (C. glabrata) were recovered from 100% of two types of resin-containing bottles in the presence of a sufficient antibiotic dose, while Escherichia coli (E. coli) was not restored to 100% in BD FX400 peds plus pediatric bottles. The shorter TTD for S. aureus, C. glabrata, and E. coli were observed in antibiotic-containing BacT/Alert PF Plus bottles. Both the PF Plus and BD resin test bottles showed consistently good TTD performances to Gram-negative, Gram-positive, and yeast species in low inoculum levels, with the exception of S. aureus. The lower volume of blood inoculated into culture bottles hardly affected the growth of most bacteria, but optimized PF Plus resin-bottles accelerated the detection of infectious agents, especially S. aureus, Streptococcus pneumoniae, and C. glabrata. CONCLUSIONS: It is possible to enhance recovery from antibiotic-containing pediatric bottles and shorten TTD for the identification of pathogens by using the BacT/Alert blood culture system combination with new resin-containing media.

Bacillus Co-culture Inhibits Quorum Sensing in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a widespread source of hospital-acquired infections and a top priority antibiotic-resistant pathogen as it has developed robust immunity to most traditional antibiotics. Quorum sensing (QS) enables P. aeruginosa to modulate virulence functions and is important for pathogenesis. QS relies on the production and perception of autoinducing chemical signal molecules. Acyl-homoserine lactones are the key autoinducer molecules that mediate P. aeruginosa-associated QS, and N-(3-oxododecanoyl)-L-homoserine lactone (3-O-C12-HSL) and N-butyryl-L-homoserine lactone (C4-HSL) are the two types. This study aimed to identify potential quenching targets of QS pathways that may reduce the chances of resistance developing in P. aeruginosa using co-culture approaches. In co-cultures, Bacillus reduced the production of 3-O-C12-HSL/C4-HSL signal molecules by inactivating acyl- homoserine lactone-based QS to inhibit important virulence factor expression. Moreover, Bacillus is subject to complex crosstalk with other regulatory systems, such as the integrated QS system and the Iqs system. The results showed that blocking one or more QS pathways was insufficient to reduce infection with multidrug resistant P. aeruginosa.

Genomic variations in polymyxin-resistant Pseudomonas aeruginosa clinical isolates and their effects on polymyxin resistance.

Infection with P. aeruginosa, one of the most relevant opportunistic pathogens in hospital-acquired infections, can lead to high mortality due to its low antibiotic susceptibility to limited choices of antibiotics. Polymyxin as last-resort antibiotics is used in the treatment of systemic infections caused by multidrug-resistant P. aeruginosa strains, so studying the emergence of polymyxin-resistant was a must. The present study was designed to define genomic differences between paired polymyxin-susceptible and polymyxin-resistant P. aeruginosa strains and established polymyxin resistance mechanisms, and common chromosomal mutations that may confer polymyxin resistance were characterized. A total of 116 CRPA clinical isolates from patients were collected from three tertiary care hospitals in China during 2017-2021. Our study found that polymyxin B resistance represented 3.45% of the isolated carbapenem-resistant P. aeruginosa (CRPA). No polymyxin-resistant isolates were positive for mcr (1-8 and 10) gene and efflux mechanisms. Key genetic variations identified in polymyxin-resistant isolates involved missense mutations in parR, parS, pmrB, pmrA, and phoP. The waaL and PA5005 substitutions related to LPS synthesis were detected in the highest levels of resistant strain (R1). The missense mutations H398R in ParS (4/4), Y345H in PmrB (4/4), and L71R in PmrA (3/4) were the predominant. Results of the PCR further confirmed that mutation of pmrA, pmrB, and phoP individually or simultaneously did affect the expression level of resistant populations and can directly increase the expression of arnBCADTEF operon to contribute to polymyxin resistance. In addition, we reported 3 novel mutations in PA1945 (2129872_A < G, 2130270_A < C, 2130272_T < G) that may confer polymyxin resistance in P. aeruginosa. Our findings enriched the spectrum of chromosomal mutations, highlighted the complexity at the molecular level, and multifaceted interplay mechanisms underlying polymyxin resistance in P. aeruginosa.

Deletion of Pr72 causes cardiac developmental defects in Zebrafish.

The alpha regulator subunit B'' of protein phosphatase 2 (PPP2R3A), a regulatory subunit of protein phosphatase 2A (PP2A), was reported to present a special subcellular localization in cardiomyocytes and elevate in non-ischemia failing hearts. PPP2R3A has two transcriptions PR72 and PR130. PR72 acts as a negative regulator of the Wnt signaling cascade, while the Wnt signaling cascade plays a pivotal role in cardiac development. And PR130 was found to be involved in cardiac development of zebrafish in our previous study. Thus, to investigate the function of PR72 in heart, two stable pr72 knockout (KO) zebrafish lines were generated using Transcription Activator-Like Effector Nuclease (TALEN) technology. Homozygous pr72 KO fish struggled to survive to adulthood and exhibited cardiac developmental defects, including enlarged ventricular chambers, reduced cardiomyocytes and decreased cardiac function. And the defective sarcomere ultrastructure that affected mitochondria, I bands, Z lines, and intercalated disks was also observed. Furthermore, the abnormal heart looping was detected in mutants which could be rescued by injection with wild type pr72 mRNA. Additionally, it was found that Wnt effectors were elevated in mutants. Those indicated that deletion of pr72 in zebrafish interrupted cardiac development, probably through activation of the Wnt pathway.

Genome-Wide Analysis of mRNA Expression Profiling Identified Lung Cancer-Related Gene in Xuanwei, China.

BACKGROUND: The morbidity and mortality of lung cancer in Xuanwei (LCXW) is among the highest in China for both males and females and increases constantly. The underlying molecular changes associated with LCXW are still unclear. The purpose of this study was to screen out potential cancer-related genes which may become promising biomarkers of LCXW. METHODS: Differentially expressed genes (DEGs) were detected by the expression microarrays in 29 paired LCXW tissues (tumor tissue and matched adjacent non-cancerous lung tissues). Integrated with bioinformatic analyses, a literature review was applied for screening out important cancer-related genes. An additional 44 paired LCXW samples were collected to verify the transcription and expression levels of the candidate gene by qRT-PCR and Western blotting. The relationship between the candidate genes and clinical pathological features were evaluated using Fisher's test. RESULTS: mRNA expression microarrays revealed that the LCXW patients harbored 2,424 differentially expressed genes (fold change ≥ 2.0). Of these genes, 1,636 were up-regulated while the other 788 genes were down-regulated. Forty previously reported DNA repair genes associated with PAHs exposure were identified by microarrays. Bioinformatic analyses indicated down-regulated ANXA3 in LCXW patients which was closely related to pathological stage and involved in the regulation of a variety of biological responses. Review of the literature on ANXA3 found its down-regulation was a distinct expression pattern compared with lung cancer occurring in other geographic areas. qRT-PCR and Western blotting confirmed the down-regulation of ANXA3 was associated with LCXW. The correlation analysis of clinical features showed down-regulated ANXA3 was correlated to the progression of pathological stage. CONCLUSIONS: The LCXW patients harbored more DEGs, and these DEGs were involved in a wide range of pathways and biological function damage. We preliminarily suggested ANXA3 may be a potential LCXW related gene. ANXA3 may serve as a promising biomarker for diagnosis of LCXW.

Expression and Function Analysis of MicroRNA-29b in Xuanwei Lung Cancer.

BACKGROUND: In lung cancer, miR-29b can act as a tumor suppressor, influencing epigenetic regulation, cell proliferation, differentiation, apoptosis, metastasis, and chemosensitivity. However, its expression and biological function in Xuanwei lung cancer (XWLC) remains unknown. METHODS: Fifty pairs of XWLC and matched normal tissues were collected and analyzed for the expression of miR29b by quantitative RT-PCR. MiR-29b precursor and inhibitor were transfected into XWLC-05 cells to investigate its role on regulating cell proliferation by MTT assay and cell apoptosis by caspase 3/7/9 assay in vitro. The target genes of miR-29b were predicted and determined by bioinformatics analysis and luciferase reporter assay. RESULTS: qRT-PCR results showed that the expression level of miR-29b in XWLC tissues was significantly lower than that in the matched normal tissues, with a proportion of 86%. The low expression of miR-29b was significantly associated with lymphatic metastasis. MiR-29b could suppress cell proliferation and promote apoptosis in XWLC-05 cells. According to the bioinformatics analysis and luciferase reporter assay results, PUMA was a target gene of miR-29b in XWLC. CONCLUSIONS: Our studies provided evidence for the involvement of miR-29b in XWLC lymphatic metastasis and suggested that miR-29b may serve as a new biomarker for diagnosis of XWLC or therapeutic targets.

The effect of plasma exchange on entecavir-treated chronic hepatitis B patients with hepatic de-compensation and acute-on-chronic liver failure.

BACKGROUND AND AIMS: Various studies showed that entecavir (ETV) failed to improve the short-term survival in chronic hepatitis B (CHB) patients with severe acute exacerbation (SAE) and hepatic de-compensation or acute-on-chronic liver failure (ACLF). One study concluded that plasma exchange (PE) significantly decreased the short-term mortality of CHB patients with ACLF who were treated with lamivudine (LAM). Our study was designed to examine the effect of PE on CHB patients with ACLF who were treated with ETV. METHODS: From August 2010 to January 2015, 38 (PE group) and 120 (control group) consecutive CHB-naïve patients with hepatic de-compensation and ACLF treated with PE plus ETV and ETV, respectively, were recruited. The primary endpoint was liver-related mortality at week 12. Biochemical and virological responses were also studied. RESULTS: At baseline, the PE group had higher serum alanine aminotransferase (ALT) levels and model for end-stage liver disease (MELD) scores, and had lower albumin levels than the control group. The cumulative survival rate at week 4 and week 12 in the PE group and control group were, respectively, 37 and 18 %, and 29 and 14 % (p < 0.001, by log rank test). While the bilirubin levels in the PE group were more quickly lowered by PE therapy (p < 0.001), the decrease of ALT levels and virological response were similar in the two groups (p > 0.05). Univariate analysis showed that the control group had a higher liver-related mortality (p = 0.038) at week 12 than the PE group. Multivariate analysis showed that hepatic encephalopathy, ascites, PE treatment, and MELD scores were independent factors for liver-related mortality at week 12. CONCLUSIONS: PE significantly improved the short-term survival of CHB patients with hepatic de-compensation and ACLF who were treated with ETV. Hepatic encephalopathy, ascites, PE treatment, and MELD scores were independent factors for liver-related mortality at week 12.

[Analysis of genomic copy number variations in two sisters with primary amenorrhea and hyperandrogenism].

OBJECTIVE: To analyze genomic copy number variations (CNVs) in two sisters with primary amenorrhea and hyperandrogenism. METHODS: G-banding was performed for karyotype analysis. The whole genome of the two sisters were scanned and analyzed by array-based comparative genomic hybridization (array-CGH). The results were confirmed with real-time quantitative PCR (RT-qPCR). RESULTS: No abnormality was found by conventional G-banded chromosome analysis. Array-CGH has identified 11 identical CNVs from the sisters which, however, overlapped with CNVs reported by the Database of Genomic Variants (http://projects.tcag.ca/variation/). Therefore, they are likely to be benign. In addition, a -8.44 Mb 9p11.1-p13.1 duplication (38,561,587-47,002,387 bp, hg18) and a -80.9 kb 4q13.2 deletion (70,183,990-70,264,889 bp, hg18) were also detected in the elder and younger sister, respectively. The relationship between such CNVs and primary amenorrhea and hyperandrogenism was however uncertain. RT-qPCR results were in accordance with array-CGH. CONCLUSION: Two CNVs were detected in two sisters by array-CGH, for which further studies are needed to clarify their correlation with primary amenorrhea and hyperandrogenism.

Genome-Wide Analysis of Long Noncoding RNA Expression Profiles in Human Xuanwei Lung Cancer.

BACKGROUND: Long noncoding RNAs (lncRNAs) play an important role in various biological processes involved the development and progression of lung cancer. However, their expression signature in Xuanwei lung cancer (XWLC) remains unknown. METHODS: High throughput microarray assay was performed to detect lncRNA and mRNA expression profiles in five paired human XWLC and adjacent normal tissues. Bioinformatic analyses (gene ontology, pathway, and net-work analysis) were applied for further study of these differentially expressed mRNAs. An additional 33 paired XWLC samples were collected to verify the expression levels of 6 candidate lncRNAs using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: Using abundant and varied probes, about 37,000 lncRNAs and 34,000 mRNAs were assessed in our micro-array. 1,484 lncRNAs and 1,997 mRNAs were differentially expressed in XWLC and adjacent normal samples (fold change ≥ 2.0). Using qRT-PCR validation, six candidate lncRNAs were differentially expressed in XWLC and adjacent normal tissues. The qRT-PCR results were consistent with the microarray data. Among these, FENDRR and SGOL1-AS1 were the most aberrantly expressed lncRNAs. CONCLUSIONS: This study firstly ascertained the expression profile of lncRNAs in XWLC by microarray. The results revealed that many lncRNAs were differentially expressed in XWLC and adjacent normal tissues. Further investigation of the differentially expressed lncRNAs may serve as new biomarkers for diagnosis of XWLC or novel therapeutic targets.

Novel insertion mutation p.Asp610GlyfsX23 in APC gene causes familial adenomatous polyposis in Chinese families.

OBJECTIVE: The aim of the study was to identify the causative gene defects associated with familial adenomatous polyposis (FAP) in two Chinese pedigrees. METHODS: The diagnosis of FAP patients was confirmed by clinical manifestations, family histories, colonoscopy and pathology examinations. Blood samples were collected and genomic DNA was extracted. The mutation analysis of the adenomatous polyposis coli (APC) and human mutY homolog (MUTYH) genes was conducted by direct polymerase chain reaction (PCR) sequencing and multiplex ligation-dependent probe amplification (MLPA). RESULTS: In pedigree A, the results of direct PCR sequencing revealed a heterozygous insertion mutation at codon 610 in exon 15 of APC gene (c.1828_1829insG), which resulted in frameshift change (p.Asp610GlyfsX23) in all 4 patients, but was absent in the unaffected familial members and controls. In pedigree B, we didn't identify that causative mutations cosegregated with the clinical phenotype in the APC and MUTYH genes. CONCLUSIONS: We identified a novel insertion mutation as the pathogenic gene of FAP in Chinese population, which could enrich the germline mutation spectrum of APC gene, and the prophylactic proctocolectomy for the mutation carrier in family should be considered.

Molecular diagnosis of two families with classic congenital adrenal hyperplasia.

We investigated the genetic defects in two families with classic congenital adrenal hyperplasia (CAH). Clinical data and vein blood of the family members were collected, hormonal evaluation, karyotype analysis, ultrasound and CT scans were performed, and a direct-sequencing of PCR products of the candidate genes was used to identify the mutations. In family A, Patients A-II:1 and A-II:2 were found to be in a compound heterozygous state for mutations of p.I172N (g.1004T>A) in exon 4 and IVS2-13A/C>G (g.659A/C>G) in intron 2 in CYP21A2 gene, their father A-I:1 and mother A-I:2 were found to carry a heterozygous mutation of IVS2-13A/C>G (g.659A/C>G) and p.I172N (g.1004T>A) respectively. In family B, Patient B-II:1 was detected to carry only one heterozygous mutation of p.I172N; no other mutations in CYP11B1, CYP17A1 or HSD3B2 genes were detected. Her father B-I:1 carried a heterozygous mutation of p.I172N (g.1004T>A) and her mother B-I:2 was found to be a wild type in all the candidate genes. Obviously, patients A-II:1 and A-II:2 inherited the p.I172N (g.1004T>A)-bearing maternal allele and the IVS2-13A/C>G (g.659A/C>G)-bearing paternal allele. And this kind of compound heterozygous mutations caused simple virilising (SV) CAH. While patient B-II:1, whose phenotype was SV CAH too, was found to carry only one heterozygous mutation of the p.I172N (g.1004T>A)-bearing paternal allele, and needed further studies.