The impact of nucleic acid testing to detect human immunodeficiency virus, hepatitis C virus, and hepatitis B virus yields from a single blood center in China with 10-years review.

BACKGROUND: Since 2010, the Blood Center of Zhejiang province, China, has conducted a pilot nucleic acid amplification testing (NAT) screening of blood donors for Hepatitis B virus (HBV), Hepatitis C virus (HCV), and Human immunodeficiency virus (HIV). This study aims to assess the results of NAT testing over 10 years to establish the effects and factors influencing NAT yields of HBV, HCV, and HIV. METHODS: Blood donations from seven different blood services were screened for HBV DNA, HCV RNA, and HIV RNA using 6 mini pools (6MP) or individual donation (ID)-NAT method between August 1, 2010, and December 31, 2019, at the NAT centralized screening center. We compared 3 transcription-mediated amplification (TMA) assays and 2 polymerase chain reaction (PCR) assays. Further, HBV, HCV, and HIV NAT yields were calculated and donor characteristics and prevalence of HBV NAT yields analyzed. Donors with HCV and HIV NAT yield were also followed up. RESULTS: 1916.31 per million donations were NAT screening positive overall. The NAT yields for HBV, HCV, HIV and non-discriminating reactive were 1062.90 per million, 0.97 per million, 1.45 per million, and 850.99 per million, respectively, which varied in the seven blood services and different years. HBV NAT yields were higher than those of HCV and HIV and varied across demographic groups. Risk factors included being male, old age, low education level, and first-time donors. We found no differences in NAT yields of HBV, HCV, and HIV between the 3 TMA and 2 PCR assays; nonetheless, statistically, significant differences were noted between the five assays. CONCLUSION: In summary, NAT screening in blood donations reduces the risk of transfusion-transmitted infections and shortens the window period for serological marker screening. Therefore, a sensitive NAT screening method, ID-NAT workflow, and recruitment of regular low-risk donors are critical for blood safety.

Molecular evolution of chloroplast genomes in subfamily Zingiberoideae (Zingiberaceae).

BACKGROUND: Zingiberoideae is a large and diverse subfamily of the family Zingiberaceae. Four genera in subfamily Zingiberoideae each possess 50 or more species, including Globba (100), Hedychium (> 80), Kaempferia (50) and Zingiber (150). Despite the agricultural, medicinal and horticultural importance of these species, genomic resources and suitable molecular markers for them are currently sparse. RESULTS: Here, we have sequenced, assembled and analyzed ten complete chloroplast genomes from nine species of subfamily Zingiberoideae: Globba lancangensis, Globba marantina, Globba multiflora, Globba schomburgkii, Globba schomburgkii var. angustata, Hedychium coccineum, Hedychium neocarneum, Kaempferia rotunda 'Red Leaf', Kaempferia rotunda 'Silver Diamonds' and Zingiber recurvatum. These ten chloroplast genomes (size range 162,630-163,968 bp) possess typical quadripartite structures that consist of a large single copy (LSC, 87,172-88,632 bp), a small single copy (SSC, 15,393-15,917 bp) and a pair of inverted repeats (IRs, 29,673-29,833 bp). The genomes contain 111-113 different genes, including 79 protein coding genes, 28-30 tRNAs and 4 rRNA genes. The dynamics of the genome structures, gene contents, amino acid frequencies, codon usage patterns, RNA editing sites, simple sequence repeats and long repeats exhibit similarities, with slight differences observed among the ten genomes. Further comparative analysis of seventeen related Zingiberoideae species, 12 divergent hotspots are identified. Positive selection is observed in 14 protein coding genes, including accD, ccsA, ndhA, ndhB, psbJ, rbcL, rpl20, rpoC1, rpoC2, rps12, rps18, ycf1, ycf2 and ycf4. Phylogenetic analyses, based on the complete chloroplast-derived single-nucleotide polymorphism data, strongly support that Globba, Hedychium, and Curcuma I + "the Kaempferia clade" consisting of Curcuma II, Kaempferia and Zingiber, form a nested evolutionary relationship in subfamily Zingiberoideae. CONCLUSIONS: Our study provides detailed information on ten complete Zingiberoideae chloroplast genomes, representing a valuable resource for future studies that seek to understand the molecular evolutionary dynamics in family Zingiberaceae. The identified divergent hotspots can be used for development of molecular markers for phylogenetic inference and species identification among closely related species within four genera of Globba, Hedychium, Kaempferia and Zingiber in subfamily Zingiberoideae.

Characteristic of HBV nucleic acid amplification testing yields from blood donors in China.

BACKGROUND: Nucleic acid amplification testing (NAT) for blood screening has been previously performed in some countries to determine NAT yields. The current study sought to explore the non-discriminating reactive NAT yields using individual-NAT (ID-NAT) and characteristics of HBV NAT yields through a 10-year retrospective analysis in Zhejiang, China. METHODS: Blood donations were analyzed using individual-NAT mode by the transcription-mediated amplification (TMA) method. Supplementary HBV serological tests were performed using chemiluminescent immunoassay, and HBV viral load assay was performed by real-time polymerase chain reaction. Follow-up studies were performed in partial donors with low HBV viral loads. RESULTS: Non-discriminating reactive NAT yields and HBV NAT yields varied in different years. The yields ranged from 853.73 per million to 2018.68 per million and 624.60 per million to 1669.50 per million, respectively. In the 476 NAT yields, 19 were probable window periods (WP), 33 probable occult hepatitis B virus infections (OBIs), 409 were confirmed OBIs and 15 were chronic HBV infections. ID-NAT results were categorized in four groups, and the findings showed that the levels of HBV DNA viral loads were different in the four different groups (χ2 = 275.02, p < 0.01). HBV viral load distribution was significantly different between anti-HBs positive and anti-HBc positive samples (χ2 = 49.429, p < 0.01). Notably, only 42.03% donors were NAT repeated positive in the 138 repeat donors' follow up tests. CONCLUSION: NAT screening of blood donations can reduce the risk of transfusion-transmitted HBV infections. Positive proportions of anti-HBs and anti-HBc are correlated with the HBV viral load level. However, low level of viral load donors pose risks in HBV NAT assays, and show fluctuating state for HBV viral load and leads to non-repeated NAT results during follow up studies.

Ligustilide suppresses RANKL-induced osteoclastogenesis and bone resorption via inhibition of RANK expression.

Osteoclast (OC) is the only cell involved in bone resorption. Dysfunction of OCs leads to a variety of bone diseases. Ligustilide (LIG) is the main component of the volatile oil isolated and purified from Angelica sinensis. LIG exerts many pharmacological activities, but its effects on osteoclastogenesis and bone resorption are still unclear. Our study showed that LIG inhibited receptor activator of nuclear factor-κB (NF-κB) ligand-induced OC formation and activation in a dose-dependent manner. Additionally, LIG downregulated the messenger RNA (mRNA) expression of OC-specific genes, such as V-ATPase d2, tartrate-resistant acid phosphatase, a dendritic cell-specific transmembrane protein, cathepsin K, and nuclear factor of activated T cells cl. Furthermore, LIG blocked the activation of NF-κB/extracellular signal-regulated kinase/p38/immunoreceptor tyrosine-based activation motif signaling pathways. Crucially, the expression of tumor necrosis factor receptor-associated factor 6 proteins and the expression of receptor activator of NF-κB mRNA were inhibited by LIG. However, LIG did not affect the formation and mineralization of osteoblasts. Collectively, this observation suggests that LIG may serve as a promising agent for the prevention and treatment of diseases caused by abnormal bone resorption.

Annular ligament reconstruction by suture anchor for treatment of radial head dislocation in children.

BACKGROUND: We investigated the efficacy of annular ligament reconstruction by suture anchor in the treatment of radial head dislocation (RHD) in children. METHOD: A total of 20 RHD children nderwent annular ligament reconstruction surgery using suture anchor. Preoperative and postoperative elbow functions were evaluated according to Broberg and Morrey 100-point scale. Recovery of radial nerve function was assessed using the Chinese Medical Association of Hand Surgery Branch of Upper Limb Functional Assessment Standard. All statistical analyses were performed using SPSS version 17.0 software. RESULTS: All 20 RHD children who underwent the procedure were followed up for a median duration of 24 months. At the last follow-up, the average Broberg-Morrey score was 94.3, with 12 children (60.0%) showing excellent outcomes (score range, 95 to 100), 7 children (35.0%) showing good outcomes (score range, 80 to 94), 1 child (5.0%) displayed a fair outcome (score range, 60 to 79), and 0 (0%) poor outcome. A significant difference in the excellent-good rate was observed when the elbow function before surgery was compared to after surgery (χ(2) = 5.559, P = 0.018). The radial nerve function of the 13 RHD children with radial nerve injury also recovered to normal. Among these 13 RHD children, nine exhibited excellent outcomes, 3 showed good outcomes, 1 displayed a fair outcome, and no patient showed a poor outcome. A significant difference in the excellent-good rate of radial nerve function was also observed when before surgery was compared to after surgery in these RHD children (χ(2) = 4.887, P = 0.027). CONCLUSION: Our results strongly indicated that suture anchor is highly effective for reconstruction of the annular ligament and to promote full functional recovery in RHD children, demonstrating that the procedure is an excellent treatment choice in RHD children.

Genetic model of selective COX2 inhibition reveals novel heterodimer signaling.

Selective inhibitors of cyclooxygenase-2 (COX2) have attracted widespread media attention because of evidence of an elevated risk of cardiovascular complications in placebo-controlled trials, resulting in the market withdrawal of some members of this class. These drugs block the cyclooxygenase activity of prostaglandin H synthase-2 (PGHS2), but do not affect the associated peroxidase function. They were developed with the rationale of conserving the anti-inflammatory and analgesic actions of traditional nonsteroidal anti-inflammatory drugs (tNSAIDs) while sparing the ability of PGHS1-derived prostaglandins to afford gastric cytoprotection. PGHS1 and PGHS2 coexist in the vasculature and in macrophages, and are upregulated together in inflammatory tissues such as rheumatoid synovia and atherosclerotic plaque. They are each believed to function as homodimers. Here, we developed a new genetic mouse model of selective COX2 inhibition using a gene-targeted point mutation, resulting in a Y385F substitution. Structural modeling and biochemical assays showed the ability of PGHS1 and PGHS2 to heterodimerize and form prostaglandins. The heterodimerization of PGHS1-PGHS2 may explain how the ductus arteriosus closes normally at birth in mice expressing PGHS2 Y385F, but not in PGHS2-null mice.

Composition of Sphagnum palustre L. extracts using different extraction methods.

Sphagnum palustre L. is a Chinese herbal medicine with a long history, however, few studies have been performed on its chemical composition and active effects. In this study, we investigated the composition and antibacterial and antioxidant capacities of extracts obtained from Sphagnum palustre L. phytosomes extracted with conventional solvents (water, methanol, and ethanol) and two different hydrogen bond donors (citric acid and 1,2-propanediol) modified with choline chloride-type deep eutectic solvents (DESs). The results show that Sphagnum palustre extracts contained 253 compounds, including citric acid, ethyl maltol, and thymol. The highest total phenolic content (TPC) was obtained with a DES extraction method combining 1,2-propanediol and choline chloride (39.02 ± 7.08 mg gallic acid equivalent/g dried weight (DW). This shows the composition of Sphagnum palustre as a natural product and the application of DESs in the extraction of active ingredients, demonstrating the potential of peat moss extracts in cosmetics and health products.

COX2 in CNS neural cells mediates mechanical inflammatory pain hypersensitivity in mice.

A cardinal feature of peripheral inflammation is pain. The most common way of managing inflammatory pain is to use nonsteroidal antiinflammatory agents (NSAIDs) that reduce prostanoid production, for example, selective inhibitors of COX2. Prostaglandins produced after induction of COX2 in immune cells in inflamed tissue contribute both to the inflammation itself and to pain hypersensitivity, acting on peripheral terminals of nociceptors. COX2 is also induced after peripheral inflammation in neurons in the CNS, where it aids in developing a central component of inflammatory pain hypersensitivity by increasing neuronal excitation and reducing inhibition. We engineered mice with conditional deletion of Cox2 in neurons and glial cells to determine the relative contribution of peripheral and central COX2 to inflammatory pain hypersensitivity. In these mice, basal nociceptive pain was unchanged, as was the extent of peripheral inflammation, inflammatory thermal pain hypersensitivity, and fever induced by lipopolysaccharide. By contrast, peripheral inflammation-induced COX2 expression in the spinal cord was reduced, and mechanical hypersensitivity after both peripheral soft tissue and periarticular inflammation was abolished. Mechanical pain is a major symptom of most inflammatory conditions, such as postoperative pain and arthritis, and induction of COX2 in neural cells in the CNS seems to contribute to this.

DNA microarray SNP associations with clinical efficacy and side effects of domperidone treatment for gastroparesis.

BACKGROUND: Domperidone treatment for gastroparesis is associated with variable efficacy as well as the potential for side effects. DNA microarray single nucleotide polymorphism (SNP) analysis may help to elucidate the role of genetic variability on the therapeutic effectiveness and toxicity of domperidone. AIM: The aim of this study was to identify SNPs that are associated with clinical efficacy and side effects of domperidone treatment for gastroparesis from DNA microarray experiments. This will help develop a strategy for rational selection of patients for domperidone therapy. METHODS: DNA samples extracted from the saliva of 46 patients treated with domperidone were analyzed using Affymetrix 6.0 SNP microarrays. Then least angle regression (LARS) was used to select SNPs that are related to domperidone efficacy and side effects. Decision tree based prediction models were constructed with the most correlated features selected by LARS. RESULTS: Using the most stable SNP selected by LARS a prediction model for side effects of domperidone achieved (95 ± 0)% true negative rate (TN) and (78 ± 11)% true positive rate (TP) in nested leave-one-out tests. For domperidone efficacy, the prediction based on five most stable SNPs achieved (85 ± 7)% TP and (61±4)% TN. Five identified SNPs are related to ubiquitin mediated proteolysis, epithelial cell signaling, leukocyte, cell adhesion, and tight junction signaling pathways. Genetic polymorphisms in three genes that are related to cancer and hedgehog signaling were found to significantly correlate with efficacy of domperidone. CONCLUSION: LARS was found to be a useful tool for statistical analysis of domperidone-related DNA microarray data generated from a small number of patients.

Cardiomyocyte cyclooxygenase-2 influences cardiac rhythm and function.

Nonsteroidal anti-inflammatory drugs selective for inhibition of COX-2 increase heart failure and elevate blood pressure. The COX-2 gene was floxed and crossed into merCremer mice under the alpha-myosin heavy-chain promoter. Tamoxifen induced selective deletion of COX-2 in cardiomyocytes depressed cardiac output, and resulted in weight loss, diminished exercise tolerance, and enhanced susceptibility to induced arrhythmogenesis. The cardiac dysfunction subsequent to pressure overload recovered progressively in the knockouts coincident with increasing cardiomyocyte hypertrophy and interstitial and perivascular fibrosis. Inhibition of COX-2 in cardiomyocytes may contribute to heart failure in patients receiving nonsteroidal anti-inflammatory drugs specific for inhibition of COX-2.

Emergence of a KPC-90 Variant that Confers Resistance to Ceftazidime-Avibactam in an ST463 Carbapenem-Resistant Pseudomonas aeruginosa Strain.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) has become a serious challenge in the clinic. Recently, the prevalence of CRPA isolates carrying the blaKPC-2 gene has been increasing in China. Ceftazidime-avibactam (CZA) has shown good efficacy against large portions of KPC-2-producing CRPA strains. However, with the increasing usage of this drug, CZA resistance in CRPA strains has been reported. Here, we reported for the first time that resistance of the ST463 CRPA strain to CZA was caused by a novel variant in the KPC gene that arose after CZA exposure. The CRPA strain PA2207 is a carbapenem- and CZA-resistant strain that harbors a mutated blaKPC gene, named blaKPC-90. Cloning and expression of blaKPC-90 in Escherichia coli DH5α revealed that KPC-90 led to a 64-fold increase in the MIC value of CZA. Conjugation experiments further confirmed that blaKPC-90 was located on a conjugative plasmid. Whole-genome sequencing analysis showed that this plasmid had high sequence similarity to a previously reported novel blaKPC-2-harboring plasmid in a clinical P. aeruginosa strain isolated in China. In addition, overexpression of an efflux pump (MexXY-OprM) might be associated with the CZA resistance phenotype, as determined by reverse transcription-quantitative PCR and efflux pump inhibition experiments. For the first time, we reported a KPC variant, KPC-90, in a clinical ST463 CRPA strain with CZA resistance that was mediated by a 2 amino acid insertion outside the KPC omega-loop region. Our study further highlights that diverse KPC variants that mediate CZA resistance have emerged in the CRPA strain. Furthermore, KPC-90 mutation combined with efflux pump overexpression resulted in a high level of resistance to CZA in the PA2207 isolate. Effective surveillance should be conducted to prevent CZA resistance from spreading in the CRPA strain. IMPORTANCE For the first time, we reported a KPC variant, KPC-90, in a clinical ST463 CRPA strain with CZA resistance. CZA resistance was mediated by a 2 amino acid insertion outside the KPC omega-loop region in CRPA. Our study further emphasized that CZA resistance caused by blaKPC gene mutation could be selected in CRPA after CZA therapy. Considering the widespread presence of the ST463 CRPA strain in China, clinicians should pay attention to the risk of the development of CZA resistance in CRPA strains under treatment pressure.

Chrysin Protects Against Titanium Particle-Induced Osteolysis by Attenuating Osteoclast Formation and Function by Inhibiting NF-κB and MAPK Signaling.

Bone homeostasis only exists when the physical function of osteoblast and osteoclast stays in the balance between bone formation and resorption. Bone resorption occurs when the two processes are uncoupled, shifting the balance in favour of bone resorption. Excessive activation of osteoclasts leads to a range of osteolytic bone diseases including osteoporosis, aseptic prosthesis loosening, rheumatoid arthritis, and osteoarthritis. Receptor activator of nuclear factor kappa-B ligand (RANKL) and its downstream signaling pathways are recognized as key mediators that drive the formation and activation of osteoclastic function. Hence, osteoclast formation and/or its function remain as dominant targets for research and development of agents reaching the treatment towards osteolytic diseases. Chrysin (CHR) is a flavonoid with a wide range of anti-inflammatory and anti-tumor effects. However, its effect on osteoclasts remains unknown. In this study, we found the effects of CHR on inhibiting osteoclast differentiation which were assessed in terms of the number and size of TRAcP positive multinucleated osteoclasts (OCs). Further, the inhibitory effects of CHR on bone resorption and osteoclast fusion of pre-OC were assessed by hydroxyapatite resorption pit assay and F-actin belts staining; respectively. Western blotting analysis of RANKL-induced signaling pathways and immunofluorescence analysis for p65 nuclear translocation in response to RANKL-induced osteoclasts were used to analyze the mechanism of action of CHR affecting osteoclasts. Lastly, the murine calvarial osteolysis model revealed that CHR could protect against particle-induced bone destruction in vivo. Collectively, our data strongly suggested that CHR with its promising anti-tumor effects would also be a potential therapeutic agent for osteolytic diseases.

Targeted deletions of cyclooxygenase-2 and atherogenesis in mice.

BACKGROUND: Although the dominant product of vascular Cyclooxygenase-2 (COX-2), prostacyclin (PGI(2)), restrains atherogenesis, inhibition and deletion of COX-2 have yielded conflicting results in mouse models of atherosclerosis. Floxed mice were used to parse distinct cellular contributions of COX-2 in macrophages and T cells (TCs) to atherogenesis. METHODS AND RESULTS: Deletion of macrophage-COX-2 (Mac-COX-2KOs) was attained with LysMCre mice and completely suppressed lipopolysaccharide-stimulated macrophage prostaglandin (PG) formation and lipopolysaccharide-evoked systemic PG biosynthesis by approximately 30%. Lipopolysaccharide-stimulated COX-2 expression was suppressed in polymorphonuclear leukocytes isolated from MacKOs, but PG formation was not even detected in polymorphonuclear leukocyte supernatants from control mice. Atherogenesis was attenuated when MacKOs were crossed into hyperlipidemic low-density lipoprotein receptor knockouts. Deletion of Mac-COX-2 appeared to remove a restraint on COX-2 expression in lesional nonleukocyte (CD45- and CD11b-negative) vascular cells that express vascular cell adhesion molecule and variably alpha-smooth muscle actin and vimentin, portending a shift in PG profile and consequent atheroprotection. Basal expression of COX-2 was minimal in TCs, but use of CD4Cre to generate TC knockouts depressed its modest upregulation by anti-CD3epsilon. However, biosynthesis of PGs, TC composition in lymphatic organs, and atherogenesis in low-density lipoprotein receptor knockouts were unaltered in TC knockouts. CONCLUSIONS: Macrophage-COX-2, primarily a source of thromboxane A(2) and prostaglandin (PG)E(2), promotes atherogenesis and exerts a restraint on enzyme expression by lesional cells suggestive of vascular smooth muscle cells, a prominent source of atheroprotective prostacyclin. TC COX-2 does not detectably influence TC development or function or atherogenesis in mice.

Whole genome sequence of an Escherichia coli ST131 strain isolated from a patient with bloodstream infection in China co-harbouring blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6')-Ib-cr genes.

OBJECTIVES: Escherichia coli sequence type 131 (ST131) is an international multiresistant high-risk clone associated with a large number of clinical infections. Here we report the draft genome sequence of a ST131 clinical isolate (EC538) obtained from a patient with bloodstream infection (BSI) in China co-harbouring the blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6')-Ib-cr genes. METHODS: Antimicrobial susceptibility testing of E. coli EC538 was performed. DNA of E. coli EC538 was extracted and was sequenced using an Illumina HiSeqTM X Ten platform. Generated sequence reads were assembled using CLC Genomics Workbench. Contigs were annotated using Rapid Annotation using Subsystem Technology (RAST), and further bioinformatics analyses were performed. RESULTS: The total number of assembled bases was 5 420 040bp, with 5611 protein-coding sequences. Escherichia coli EC538 belongs to ST131 by multilocus sequence typing (MLST). The presence of blaKPC-2, blaCTX-M-3 and blaCTX-M-14 genes was detected in addition to other antimicrobial resistance genes conferring resistance to fluoroquinolones, aminoglycosides, trimethoprim, sulfonamides, tetracyclines, macrolides and rifampicin. CONCLUSION: To our knowledge, this is the first report of an E. coli ST131 strain from a BSI co-harbouring blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6')-Ib-cr genes in China. The presented genome sequence of a carbapenemase-producing E. coli ST131 strain could provide further insight into the genomic diversity of this highly virulent, multiresistant and successfully pandemic bacterial pathogen.

Emergence of a Clinical Escherichia coli Sequence Type 131 Strain Carrying a Chromosomal bla KPC-2 Gene.

Objectives: Bacteria carrying the Klebsiella pneumoniae carbapenemase genes have rapidly spread worldwide and have become a great threat to public health. The bla KPC-2 gene has been primarily located on plasmids cocirculating in various strains. However, chromosomal integration of the bla KPC -2 gene in Escherichia coli has not been reported. In the present study, we report the detection of the first clinical strain of E. coli ST131 with a bla KPC -2 gene, which integrated in the chromosome. E. coli strain EC3385 was identified and subjected to susceptibility testing and genotyping. The complete genome sequences of this strain and four Proteus mirabilis strains were obtained. Chromosomal integration of the bla KPC-2 gene was confirmed using a combination of short- and long-read sequencing. Comparative genetic analyses were performed and the origin of the chromosomal location of the bla KPC-2 gene was further analyzed. Whole-genome sequencing revealed that strain EC3385 belonged to the ST131 type and possessed various resistance and virulence genes. Sequence analysis showed that the bla KPC-2 gene was carried in a 24-kb insertion sequence on the chromosome. This insertion sequence possessed high sequence similarity to previously reported bla KPC-2 -habouring plasmids of P. mirabilis in China. To the best of our knowledge, this is the first report of a clinical ST131 E. coli strain carrying bla KPC-2 on the chromosome. The bla KPC-2 gene was probably horizontally transferred from the P. mirabilis plasmid to the E. coli chromosome by the IS26 element, indicating that P. mirabilis might be an important reservoir of bla KPC-2 gene for E. coli. Furthermore, the E. coli ST131 strain carrying the chromosomal bla KPC -2 gene could be further spread due to its carbapenem resistance and high virulence. It is imperative to perform active surveillance to prevent further dissemination of KPC-2 type carbapenemase-producing isolates.

Hederagenin protects mice against ovariectomy-induced bone loss by inhibiting RANKL-induced osteoclastogenesis and bone resorption.

AIMS: Postmenopausal osteoporosis and other osteolytic bone diseases are often caused by the elevation in osteoclastogenesis and/or increased osteoclastic bone resorption, leading to excessive bone loss. Hederagenin (Hed) is a pentacyclic triterpenoid saponin extracted from various natural medicinal plants and exhibits numerous biological activities and may offer benefits against bone-related conditions. We evaluated the effects of Hed on osteoclast formation and bone resorption in vitro and the in vivo therapeutic benefits in the mouse model of ovariectomy (OVX)-induced bone loss. MAIN METHODS: In vitro, osteoclast formation were determined by TRAcp staining; bone resorption were examined using Hydroxyapatite resorption assay and Podosomal actin belt formation assay; Related molecular mechanisms were determined by western blot assay. Construction of OVX mice by bilateral oophorectomy to simulate bone loss in vivo. KEY FINDINGS: In vitro cellular assays showed that Hed inhibited RANKL-induced osteoclast formation and osteoclast bone (hydroxyapatite) resorption as well as marker gene expression from BMM culture. Mechanistically, Hed attenuated RANKL-induced intracellular reactive oxygen species (ROS) production, and MAPK signaling pathway (ERK and p38) activation which curbed the downstream induction of c-Fos and NFATc1. Consistent with the in vitro findings, Hed administration effectively protected OVX mice from bone loss by reducing osteoclast number and activity on bone surface. SIGNIFICANCE: Our data provided promising evidence for the potential use of Hederagenin in the treatment of osteoclast-mediated osteolytic bone diseases such as postmenopausal osteoporosis.

Molecular Epidemiology of Plasmid-Mediated Fosfomycin Resistance Gene Determinants in Klebsiella pneumoniae Carbapenemase-Producing Klebsiella pneumoniae Isolates in China.

The Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae has become a serious problem because the species is wide ranging and there are few treatment options. Fosfomycin has attracted renewed interest in combination therapy for infections caused by KPC-producing K. pneumoniae isolates. Because of the increasing use of fosfomycin, resistant isolates have been continually reported in carbapenem-resistant K. pneumoniae (CRKP). At present, multiple mechanisms can result in fosfomycin resistance. However, there is limited knowledge with respect to plasmid-mediated fosfomycin resistance gene (fosA3) determinants in KPC-producing K. pneumoniae isolates. In this study, a total of 101 CRKP strains were collected from four hospitals in Zhejiang province from January 2013 to August 2014; 28.7% (29/101) of CRKP isolates were resistant to fosfomycin. Gene fosA3 was detected in 29 fosfomycin-resistant KPC-producing K. pneumoniae isolates, whereas genes fosA, fosB, fosB2, fosC, fosC2, and fosX were all negative among the resistant isolates. In addition, among 29 fosfomycin-resistant KPC-producing K. pneumoniae isolates, pulsed-field gel electrophoresis (PFGE) analysis revealed five pulsotypes. S1-PFGE and Southern blot showed that the fosA3 gene was located on an approximately 140-kb plasmid in all isolates. Eight of the 29 isolates (27.6%) tested could successfully transfer their fosfomycin-resistant phenotype to Escherichia coli strain J53. All fosA3-positive isolates were determined to have an identical genetic background, IS26-tetR-cadC-orf1-fosA3-IS26, which is the same as that of the fosA3-positive plasmid pFOS18 in China. The primary resistance mechanism to fosfomycin was caused by a plasmid-mediated fosA3. Furthermore, it is noteworthy that the plasmid genetically carrying a combination of the fosA3 and blaKPC-2 genes could accelerate the spread of antibiotic resistance. Effective and persistent monitoring and surveillance will be vital to prevent further dissemination of these resistance genes.

Draft genome sequence of Enterobacter cloacae HBY, a ST128 clinical strain co-producing KPC-2 and NDM-1 carbapenemases.

OBJECTIVES: Enterobacter cloacae is one of the major pathogens responsible for a variety of human infections. Here we report the draft genome sequence of multidrug-resistant E. cloacae strain HBY isolated from a female patient in China. METHODS: Whole genomic DNA of E. cloacae strain HBY was extracted and was sequenced using an Illumina HiSeq™ 2000 platform. The generated sequence reads were assembled using CLC Genomics Workbench. The draft genome was annotated using Rapid Annotations using Subsystems Technology (RAST), and the presence of antimicrobial resistance genes was identified. RESULTS: The 5799439-bp genome contains various antimicrobial resistance genes conferring resistance to aminoglycosides, ß-lactams, fosfomycin, macrolides, sulphonamides and fluoroquinolones. Notably, the strain was identified to carry two main carbapenemase genes (blaKPC-2 and blaNDM-1). CONCLUSIONS: The genome sequence reported in this study will provide valuable information to understand antibiotic resistance mechanisms in this strain. It is important to monitor the spread strains of Enterobacter sp. encoding both of these carbapenemase genes.

Dissemination of blaNDM-5 gene via an IncX3-type plasmid among non-clonal Escherichia coli in China.

Background: The emergence and spread of New Delhi metallo-ß-lactamase-producing Enterobacteriaceae has been a serious challenge to manage in the clinic due to its rapid dissemination of multi-drug resistance worldwide. As one main type of carbapenemases, New Delhi metallo-ß-lactamase (NDM)is able to confer resistance to almost all ß-lactams, including carbapenems, in Enterobacteriaceae. Recently, New Delhi metallo-ß-lactamase-5 attracted extensive attention because of increased resistance to carbapenems and widespread dissemination. However, the dissemination mechanism of blaNDM-5 gene remains unclear. Methods: A total of 224 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected from different hospitals in Zhejiang province. NDM-5-positive isolates were identified and subjected to genotyping, susceptibility testing, and clinical data analysis. We established the genetic location of blaNDM-5 with southern blot hybridisation, and analysed plasmids containing blaNDM-5 with filter mating and DNA sequencing. Results: Eleven New Delhi metallo-ß-lactamase-5 (NDM-5)-producing strains were identified, including 9 Escherichia coli strains, 1 Klebsiella pneumoniae strain, and 1 Citrobacter freundii strain. No epidemiological links for E. coli isolates were identified by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). S1-PFGE and southern blot suggested that the blaNDM-5 gene was located on a 46-kb IncX3-type plasmid in all isolates. Nine of the 11 isolates (81.8%) tested could successfully transfer their carbapenem-resistant phenotype to E. coli strain C600. Moreover, sequence analysis further showed that this plasmid possessed high sequence similarity to most of previously reported blaNDM-5-habouring plasmids in China. Conclusion: The present data in this study showed the IncX3 type plasmid played an important role in the dissemination of blaNDM-5 in Enterobacteriaceae. In addition, to the best of our knowledge, this report is the first to isolate both E. coli and C. freundii strains carrying blaNDM-5 from one single patient, which further indicated the possibility of blaNDM-5 transmission among diverse species. Close surveillance is urgently needed to monitor the further dissemination of NDM-5-producing isolates.

Cardiovascular hazard and non-steroidal anti-inflammatory drugs.

Selective inhibitors of cyclooxygenase (COX)-2 depress prostacyclin (PGI(2)) without a concomitant inhibition of platelet COX-1-derived thromboxane (Tx)A(2). Experiments in gene-deleted mice have shown that ablation of the PGI(2) receptor (the IP) predisposes to an exaggerated response to agonists which elevate blood pressure, accelerate atherogenesis and induce thrombosis. Such a class-based effect would be expected to be modulated by the underlying risk of cardiovascular disease in patients, elements of drug exposure, such as dose, duration of action and duration of dosing, and inter-individual variability of drug response. Five placebo-controlled trials of three structurally distinct selective inhibitors of COX-2 have revealed an increased hazard of myocardial infarction and stroke consistent with a mechanism-based class-specific cardiovascular hazard. Sustained inhibition of platelet TxA(2) by aspirin affords cardiovascular benefit, despite concomitant inhibition of PGI(2). Although there is no information from randomized placebo-controlled trials, traditional non-steroidal anti-inflammatory drugs, such as naproxen, dicofenac and ibuprofen, might differ in their effects of cardiovascular biology.