Metabolomics revealed mechanism for the synergistic effect of sulbactam, polymyxin-B and amikacin combination against Acinetobacter baumannii.

Introduction: The emergence of multidrug-resistant (MDR) Acinetobacter baumannii prompts clinicians to consider treating these infections with polymyxin combination. Methods: Metabolomic analysis was applied to investigate the synergistic effects of polymyxin-B, amikacin and sulbactam combination therapy against MDR A. baumannii harboring OXA-23 and other drug resistant genes. The drug concentrations tested were based on their clinical breakpoints: polymyxin-B (2 mg/L), amikacin (16 mg/L), polymyxin-B/amikacin (2/16 mg/L), and polymyxin-B/amikacin/sulbactam (2/16/4 mg/L). Results: The triple antibiotic combination significantly disrupted levels of metabolites involved in cell outer membrane structure including fatty acids, glycerophospholipids, nucleotides, amino acids and peptides as early as 15 min after administration. Amikacin and polymyxin-B alone perturbed a large number of metabolites at 15 min and 1 h, respectively, but the changes in metabolites were short-lived lasting for less than 4 h. In contrast, the combination treatment disrupted a large amount of metabolites beyond 4 h. Compared to the double-combination, the addition of sulbactam to polymyxin-B/amikacin combination produce a greater disorder in A. baumannii metabolome that further confer susceptibility of bacteria to the antibiotics. Conclusion: The metabolomic analysis identified mechanisms responsible for the synergistic activities of polymyxin-B/amikacin/sulbactam against MDR A. baumannii.

Physiologically-based pharmacokinetic modeling to inform dosing regimens and routes of administration of rifampicin and colistin combination against Acinetobacter baumannii.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) is resistant to major antibiotics such as penicillin, cephalosporin, fluoroquinolone and aminoglycoside, and has become a significant nosocomial pathogen. The efficacy of rifampicin and colistin combination against CRAB could be dependent on the administration routes and drug concentrations at the site of infection. OBJECTIVE: The objective is to predict drug disposition in biological tissues. Treatment efficacy is extrapolated by assessing respective pharmacodynamic (PD) indices, as well as parameters associated with the emergence of resistance. METHODS: Physiologically-based pharmacokinetic models of rifampicin and colistin were utilized to predict tissue exposures. Dosing regimens and administration routes for combination therapy were evaluated in terms of in vitro antimicrobial susceptibility of A. baumannii associated with targeted PD indices and resistance parameters. RESULTS: Simulated exposures in blood, heart, lung, skin and brain were consistent with reported penetration rates. The results demonstrated that a combination of colistin and rifampicin using conventional intravenous (i.v.) doses could achieve effective exposures in the blood and skin. However, for lung infections, colistin by inhalation would be required due to low lung penetration from intravenous route. Inhaled colistin alone provided good PD coverage but this practice could encourage the emergence of additional resistance which may be overcome by a combination regimen that includes inhaled rifampicin. CONCLUSION: This in silico extrapolation provides valuable information on dosing regimens and routes of administration against CRAB infections in specific tissues. The PBPK modeling approach could be a non-invasive way to inform therapeutic benefits of combination antimicrobial therapy.

Pharmacokinetic/Pharmacodynamic Evaluation of Aztreonam/Amoxicillin/Clavulanate Combination against New Delhi Metallo-ß-Lactamase and Serine-ß-Lactamase Co-Producing Escherichia coli and Klebsiella pneumoniae.

This study aimed to examine specific niches and usage for the aztreonam/amoxicillin/clavulanate combination and to use population pharmacokinetic simulations of clinical dosing regimens to predict the impact of this combination on restricting mutant selection. The in vitro susceptibility of 19 New-Delhi metallo-ß-lactamase (NDM)-producing clinical isolates to amoxicillin/clavulanate and aztreonam alone and in co-administration was determined based on the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC). The fractions of a 24-h duration that the free drug concentration was within the mutant selection window (fTMSW) and above the MPC (fT>MPC) in both plasma and epithelial lining fluid were determined from simulations of 10,000 subject profiles based on regimens by renal function categories. This combination reduced the MIC of aztreonam and amoxicillin/clavulanate to values below their clinical breakpoint in 7/9 K. pneumoniae and 8/9 E. coli, depending on the ß-lactamase genes detected in the isolate. In the majority of the tested isolates, the combination resulted in fT>MPC > 90% and fTMSW < 10% for both aztreonam and amoxicillin/clavulanate. Clinical dosing regimens of aztreonam and amoxicillin/clavulanate were sufficient to provide mutant restriction coverage for MPC and MIC ≤ 4 mg/L. This combination has limited coverage against NDM- and extended-spectrum ß-lactamase co-producing E. coli and K. pneumoniae and is not effective against isolates carrying plasmid-mediated AmpC and KPC-2. This study offers a potential scope and limitations as to where the aztreonam/amoxicillin/clavulanate combination may succeed or fail.

The combination effect of meropenem/sulbactam/polymyxin-B on the pharmacodynamic parameters for mutant selection windows against carbapenem-resistant Acinetobacter baumannii.

The objective of this study was to evaluate whether combinations of sulbactam, meropenem, and polymyxin-B could reduce or close the gap of mutant selection window (MSW) of individual antibiotics against Acinetobacter baumannii harboring OXA-23. MICs of three antimicrobials used alone and in combination (meropenem/polymyxin-B or meropenem/polymyxin-B/sulbactam) were obtained in 11 clinical isolates and mutant prevention concentrations were determined in 4 of the 11 isolates. All isolates were resistant to meropenem or polymyxin-B. Combining meropenem and polymyxin-B with or without sulbactam resulted in synergistic bactericidal activities. Pharmacokinetic (PK) simulations of drug concentrations in the blood and epithelial lining fluid coupled with pharmacodynamic (PD) evaluations revealed that the fractions of time over the 24-h in terms of free drug concentration within the MSW (fTMSW) and above the MPC (fT>MPC) were optimized by combination therapy. The resultant clinical regimens of meropenem, polymyxin-B, and sulbactam evaluated in the PK-PD analysis were 2 g q8h, 2.5 mg/kg loading dose followed by 1.5 mg/kg q12h, and 3 g q8h, respectively, in patients with normal renal function. Subsequent corresponding equivalent exposure regimens would depend on the extent of renal failure. The overall results indicate that combination antibiotics consisting of sulbactam/meropenem/polymyxin-B can confer potential efficacy against A. baumannii harboring OXA-23, and reduce the opportunity for bacteria to develop further resistance. This study provides a framework for pharmacodynamic evaluation of drug-resistant mutant suppression in an antimicrobial co-administration setting. The results thereby lay the groundwork for additional studies and future clinical confirmation is warranted.

Expression and purification of soluble recombinant ß-lactamases using Escherichia coli as expression host and pET-28a as cloning vector.

BACKGROUND: Due to its high expression capability, recombination of Escherichia coli and pET vector has become the bioengineering preferred expression system. Because ß-lactamases mediate bacterial antimicrobial resistance, these enzymes have a substantial clinical impact. Using the E. coli expression system, several kinds of ß-lactamases have been produced. However, previous studies have been focused on characterizing target ß-lactamases, and the effects of cultivation and induction conditions on the expression efficiency of target enzymes were not addressed. RESULTS: Using pET-28a as the cloning vector and E. coli BL21(DE3) as the expression host, this study originally elucidated the effects of IPTG concentration, culture temperature, induction time, and restriction sites on recombinant ß-lactamase expression. Moreover, the effects of the target protein length and the 6 × His-tag fusion position on enzyme purification were also explored, and consequently, this study yielded several important findings. (i) Only the signal peptide-detached recombinant ß-lactamase could exist in a soluble form. (ii) Low-temperature induction was beneficial for soluble ß-lactamase expression. (iii) The closer to the rbs the selected restriction site was, the more difficult it was to express soluble ß-lactamase. (iv) The short-chain recombinant protein and the protein with His-tag fused at its C-terminus showed high affinity to the Ni2+ column. CONCLUSIONS: Based on our findings, researchers can easily design an effective program for the high production of soluble recombinant ß-lactamases to facilitate other related studies.

Effects of amikacin, polymyxin-B, and sulbactam combination on the pharmacodynamic indices of mutant selection against multi-drug resistant Acinetobacter baumannii.

Amikacin and polymyxins as monotherapies are ineffective against multidrug-resistant Acinetobacter baumannii at the clinical dose. When polymyxins, aminoglycosides, and sulbactam are co-administered, the combinations exhibit in vitro synergistic activities. The minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined in 11 and 5 clinical resistant isolates of A. baumannii harboring OXA-23, respectively, in order to derive the fraction of time over the 24-h wherein the free drug concentration was within the mutant selection window (fTMSW) and the fraction of time that the free drug concentration was above the MPC (fT>MPC) from simulated pharmacokinetic profiles. The combination of these three antibiotics can confer susceptibility in multi-drug resistant A. baumannii and reduce the opportunity for bacteria to develop further resistance. Clinical intravenous dosing regimens of amikacin, polymyxin-B, and sulbactam were predicted to optimize fTMSW and fT>MPC from drug exposures in the blood. Mean fT>MPC were ≥ 60% and ≥ 80% for amikacin and polymyxin-B, whereas mean fTMSW was reduced to <30% and <15%, respectively, in the triple antibiotic combination. Due to the low free drug concentration of amikacin and polymyxin-B simulated in the epithelial lining fluid, the two predicted pharmacodynamic parameters in the lung after intravenous administration were not optimal even in the combination therapy setting.

Metabolomic profiling of polymyxin-B in combination with meropenem and sulbactam against multi-drug resistant Acinetobacter baumannii.

Empirical therapies using polymyxins combined with other antibiotics are recommended in the treatment of Acinetobacter baumannii infections. In the present study, the synergistic activities of polymyxin-B, meropenem, and sulbactam as combination therapy were investigated using metabolomic analysis. The metabolome of A. baumannii was investigated after treatment with polymyxin-B alone (2 mg/l), meropenem (2 mg/l) alone, combination of polymyxin-B/meropenem at their clinical breakpoints, and triple-antibiotic combination of polymyxin-B/meropenem and 4 mg/l sulbactam. The triple-antibiotic combination significantly changed the metabolite levels involved in cell outer membrane and cell wall biosynthesis, including fatty acid, glycerophospholipid, lipopolysaccharide, peptidoglycan, and nucleotide within 15 min of administration. In contrast, significant changes in metabolome were observed after 1 h in sample treated with either meropenem or polymyxin-B alone. After 1 h of administration, the double and triple combination therapies significantly disrupted nucleotide and amino acid biosynthesis pathways as well as the central carbon metabolism, including pentose phosphate and glycolysis/gluconeogenesis pathways, and tricarboxylic acid cycle. The addition of sulbactam to polymyxin-B and meropenem combination appeared to be an early disruptor of A. baumannii metabolome, which paves the way for further antibiotic penetration into bacteria cells. Combination antibiotics consisting of sulbactam/meropenem/polymyxin-B can effectively confer susceptibility to A. baumannii harboring OXA-23 and other drug resistant genes. Metabolomic profiling reveals underlying mechanisms of synergistic effects of polymyxin-B combined with meropenem and sulbactam against multi-drug resistant A. baumannii.

Aztreonam/avibactam effect on pharmacodynamic indices for mutant selection of Escherichia coli and Klebsiella pneumoniae harbouring serine- and New Delhi metallo-ß-lactamases.

OBJECTIVES: Ceftazidime/avibactam is not active against MBL-producing bacteria. Combining ceftazidime/avibactam or avibactam with aztreonam can counter the resistance of MBL-producing Enterobacterales. The aim of this study was to evaluate whether the addition of avibactam could reduce or close the mutant selection window (MSW) of aztreonam in Escherichia coli and Klebsiella pneumoniae harbouring MBLs; MSW is a pharmacodynamic (PD) parameter for the selection of emergent resistant mutants. METHODS: In vitro susceptibility of 19 clinical isolates to ceftazidime/avibactam, aztreonam alone, and in co-administration (aztreonam/ceftazidime/avibactam and aztreonam/avibactam) was determined, as well as the mutant prevention concentration (MPC). The fraction of time within 24 h that the free drug concentration was within the MSW (fTMSW) and the fraction of time that the free drug concentration was above the MPC (fT>MPC) in both plasma and epithelial lining fluid (ELF) were determined from simulations of 10 000 profiles. The joint PTA was used to derive a joint cumulative fraction of response (CFR). RESULTS: All isolates were resistant to ceftazidime/avibactam or aztreonam. Combining aztreonam and avibactam or ceftazidime/avibactam resulted in synergistic bactericidal activities against all isolates. Synergism was primarily due to the aztreonam/avibactam combination. For aztreonam/avibactam dosing regimens evaluated in clinical trials, fT>MPC values were >90% and >80%, whereas fTMSW measures were <10% and <20% in plasma and ELF, respectively. The CFR was 100% for aztreonam/avibactam against the collection of clinical isolates. CONCLUSIONS: Effective antimicrobial combination optimized the PD parameters measuring selection for emergent mutants by increasing fT>MPC and reducing fTMSW.

Development of a standardized Gram stain procedure for bacteria and inflammatory cells using an automated staining instrument.

Gram stain is a subjective and poorly controlled test, and the resultant errors often perplex laboratory scientists. To reduce errors and make Gram stain a precisely controllable and meritorious test, a standardized Gram stain procedure for bacteria and inflammatory cells was developed using an automated staining instrument in this study. Freshly expectorated sputum specimens, used as the optimized targets, were smeared on slides by laboratory technicians, defining each slide loaded with uniform matrix and monolayer cell. And then, the staining and decolorizing time, as well as the stain and decolorant volume, were optimized as 15, 105, 1, and 25 s and 1.1, 1.4, 0.3, and 0.7 ml, respectively. Culture-positive blood specimens and original purulent fluids were used for confirming the developed standardized Gram stain procedure. Distinct tinctures of bacteria and inflammatory cells adhered to slide uniformly in a monolayer were observed, and the obtained staining results of these samples were highly consistent with their cultured results. Furthermore, according to the staining results under different staining conditions, an updated molecular mechanism of Gram stain for bacteria and the probable staining mechanism for inflammatory cells were also proposed in this study.

Novel IncR/IncP6 Hybrid Plasmid pCRE3-KPC Recovered from a Clinical KPC-2-Producing Citrobacter braakii Isolate.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae have become widespread in hospitals and the environment. Here, we describe a blaKPC-2-carrying plasmid called pCRE3-KPC, which was recovered from a clinical multidrug-resistant Citrobacter braakii CRE3 strain in China. The complete nucleotide sequence of pCRE3-KPC was determined by combining MiSeq and MinION sequencing and then compared with those of three related plasmids. Plasmid conjugal transfer and electroporation tests, modified carbapenem inactivation method, and bacterial antimicrobial susceptibility test were carried out. We compared this plasmid with three related plasmids to verify that the backbone of pCRE3-KPC was composed of the backbones of the IncR plasmid and IncP6 plasmid. Further bioinformatics analysis showed that pCRE3-KPC carried two resistance-related regions (the blaKPC-2 gene cluster and the aacC2-tmrB-related region). The aacC2-tmrB-related region included two novel insertion sequences (ISCfr28 and ISCfr16).IMPORTANCE Reports of human-pathogenic C. braakii strains, especially of strains showing resistance to carbapenems, are rare. To the best of our knowledge, our results represent the first detection of carbapenemase gene blaKPC-2 in C. braakii strains. In addition, we have studied detailed genetic characteristics of the novel IncR/IncP6 hybrid plasmid pCRE3-KPC, which was isolated from a clinical multidrug-resistant Citrobacter braakii CRE3 strain. Our results may provide further insight into the horizontal transfer of multidrug resistance genes in bacteria and into the genomic diversity and molecular evolution of plasmids.

Characterization of a NDM-1- Encoding Plasmid pHFK418-NDM From a Clinical Proteus mirabilis Isolate Harboring Two Novel Transposons, Tn6624 and Tn6625.

Acquisition of the bla NDM- 1 gene by Proteus mirabilis is a concern because it already has intrinsic resistance to polymyxin E and tigecycline antibiotics. Here, we describe a P. mirabilis isolate that carries a pPrY2001-like plasmid (pHFK418-NDM) containing a bla NDM- 1 gene. The pPrY2001-like plasmid, pHFK418-NDM, was first reported in China. The pHFK418-NDM plasmid was sequenced using a hybrid approach based on Illumina and MinION platforms. The sequence of pHFK418-NDM was compared with those of the six other pPrY2001-like plasmids deposited in GenBank. We found that the multidrug-resistance encoding region of pHFK418-NDM contains ΔTn10 and a novel transposon Tn6625. Tn6625 consists of ΔTn1696, Tn6260, In251, ΔTn125 (carrying bla NDM- 1), ΔTn2670, and a novel mph(E)-harboring transposon Tn6624. In251 was first identified in a clinical isolate, suggesting that it has been transferred efficiently from environmental organisms to clinical isolates. Genomic comparisons of all these pPrY2001-like plasmids showed that their relatively conserved backbones could integrate the numerous and various accessory modules carrying multifarious antibiotic resistance genes. Our results provide a greater depth of insight into the horizontal transfer of resistance genes and add interpretive value to the genomic diversity and evolution of pPrY2001-like plasmids.

T Cell Receptor Immune Repertoires Are Promptly Reconstituted After Methicillin-Resistant Staphylococcus aureus Infection.

T cells represent a subset of lymphocytes characterized by immunosurveillance and immunoregulation function. Peripheral blood mononuclear cells (PBMCs) are enriched in T cells, which exert critical antimicrobial roles in infectious diseases. High-throughput sequencing of the T cell receptor (TCR) provides deep insight into monitoring the immune microenvironment. Flow cytometry was used to analyse the distribution of αß/γδ T cells and their CD69, IFN-γ/IL-17 expression from PBMCs. Here, we utilized next-generation sequencing (NGS) to detect the complementarity determining region 3 (CDR3) of TCRß (TRB) and TCRδ (TRD) chain after methicillin-resistant Staphylococcus aureus (MRSA) infection. Our data demonstrated a significant increase in the activation of αß and γδ T cells after MRSA infection. Simultaneously, significantly high CDR3 amino acid (AA) diversity and markedly reconstituted TCR immune repertoires were observed after MRSA infection. Finally, we identified several MRSA-specific initial CDR3 AA motifs after MRSA infection. Our work reveals the profiles of TRB and TRD immune repertoires in response to MRSA and demonstrates a reconstitution of the TCR immune repertoire after MRSA infection.

Analysis of the structures of confirmed and questionable CRISPR loci in 325 Staphylococcus genomes.

The CRISPR-Cas (clustered regular interspaced short palindromic repeats and CRISPR-associated proteins) system is a newly discovered immune defense system in the genome of prokaryotes, which can resist the invasion of foreign genetic elements, such as plasmids or phage. In this study, 154 strains of Staphylococcus published in the CRISPRDatabase and 171 strains included in NCBI were downloaded, the confirmed and questionable CRISPR loci of which were analyzed by bioinformatics methods, including their distribution, characteristics of the structure (including the direct repeats, spacers and cas genes), and the relationship between the presence of CRISPR and the mecA gene. Meanwhile, a comprehensive analysis of orphan CRISPR arrays was performed on this basis. A total of 196 confirmed and 1757 questionable CRISPR loci were found in 325 Staphylococcus genomes. Only 25 strains contained cas genes, which were classified into III-A (48.1%) and II-C (51.9%). The difference between the presence of the cas gene and the carrying rate of mecA was statistically significant, and they were negatively correlated. A total of 137 confirmed and 1755 questionable CRISPR loci were assumed to be false-CRISPR. The present study also analyzed the questionable CRISPR array for the first time while analyzing the confirmed CRISPR array in the Staphylococcal genome and screened the false-CRISPR elements in the orphan CRISPR array.

Identification of an NDM-5-producing Escherichia coli Sequence Type 167 in a Neonatal Patient in China.

Emergence of New Delhi metallo-ß-lactamase-producing Enterobacteriaceae has become a challenging threat to public health. Two carbapenem-resistant Escherichia coli, strain QD28 and QD29, were recovered from the aspirating sputum of a neonate and the urine of an adult in a Chinese hospital in 2013. Molecular typing revealed that both isolates belonged to the sequence type 167, but they were clonally diverse. Both isolates exhibited resistance to carbapenems, cephalosporins, ciprofloxacin, gentamicin, piperacillin-tazobactam and trimethoprim-sulfamethoxazole. In addition, strain QD28 was also resistant to aztreonam, and strain QD29 was resistant to amikacin, fosfomycin and minocycline. Antimicrobial resistance gene screening revealed that strain QD28 harbored aac(6')-Ib, blaCTX-M-14, blaNDM-5, blaTEM-1 and sul1 genes, and strain QD29 harbored aac(6')-Ib, blaCTX-M-3, blaNDM-5, blaTEM-1, rmtB, sul1 and sul2 genes. The blaNDM-5 gene was found to be located on a 46-kb plasmid in two isolates, and further sequence analysis showed that this plasmid was highly similar to the previously reported IncX3 plasmid pNDM-MGR194 in India. This is the first identification of blaNDM-5-carrying E. coli in the neonatal infection.

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak.

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre-gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.

Identification of an integron containing the quinolone resistance gene qnrA1 in Shewanella xiamenensis.

This study investigated multidrug resistance in Shewanella xiamenensis isolated from an estuarine water sample in China during 2014. This strain displayed resistance or decreased susceptibility to ampicillin, aztreonam, cefepime, cefotaxime, chloramphenicol, ciprofloxacin, erythromycin, kanamycin and trimethoprim-sulfamethoxazole. The antimicrobial resistance genes aacA3, blaOXA-199, qnrA1 and sul1 were identified by PCR amplification and by sequencing. Pulsed-field gel electrophoresis and DNA hybridization experiments showed that the quinolone resistance gene qnrA1 was chromosomally located. qnrA1 was located in a complex class 1 integron, downstream from an ISCR1, and bracketed by two copies of qacEΔ1-sul1 genes. This integron is similar to In825 with four gene cassettes aacA3, catB11c, dfrA1z and aadA2az. An IS26-mel-mph2-IS26 structure was also detected in the flanking sequences, conferring resistance to macrolides. This is the first identification of the class 1 integron in S. xiamenensis. This is also the first identification of the qnrA1 gene and IS26-mediated macrolide resistance genes in S. xiamenensis. Presence of a variety of resistance genetic determinants in environmental S. xiamenensis suggests the possibility that this species may serve as a potential vehicle of antimicrobial resistance genes in aquatic environments.

Distinct role of Tim-3 in systemic lupus erythematosus and clear cell renal cell carcinoma.

Tim-3 is considered as one of the T-cell immunoglobulin mucin (TIM) gene family members, which contributes to the activating or silencing genes, but the mechanism of Tim-3 function in mediating SLE or tumor metastasis has not been well explored. Here, we reported Tim-3 was high expressed in the peripheral blood mononuclear cells (PBMCs) of patients with SLE, detected by RT-PCR, significantly, GATA-3 mRNA expression also increased in patients with SLE, compared with the healthy control groups. The bioinformatics used to detect the TCGA database indicated the abnormal expression of Tim-3 was involved in several different cancer types. Further, the higher expression of Tim-3 in kidney renal clear cell carcinoma TCGA database indicated it was a marker for worse 5-year survival. The high expression of Tim-3 in different ccRCC cell lines was detected in both RNA level and protein level. Further, two kinds of relative Tim-3 siRNAs in ccRCC cell lines inhibit cell migration and invasion in vitro, However, the inhibition could be partially rescued by the additional GATA3 knockdown. Further, the down regulation in the RNA and protein levels of GATA3, and the negative correlation between Tim-3 and GATA3 implied that suppression of downstream GATA3 was an important mechanism by which Tim-3 triggered metastasis in ccRCC cell lines. Together, our experiments reveal the role for Tim-3 in facilitating SLE or invasive potential of ccRCC cells by either activating GATA3 or inhibiting GATA3, suggesting that Tim-3 might be a potential therapeutic target for treating SLE or clear cell renal cell carcinoma.

Coexistence of SFO-1 and NDM-1 ß-lactamase genes and fosfomycin resistance gene fosA3 in an Escherichia coli clinical isolate.

This study aims to characterize antimicrobial resistance and antimicrobial resistance genetic determinants of an Escherichia coli clinical isolate HD0149 from China in 2012. This strain displayed high-level resistance to cephalosporins, carbapenems, fluoroquinolones, aminoglycosides and fosfomycin. A range of antimicrobial resistance genes was detected responsible for its multiple antimicrobial resistances, involving the blaCMY-2, blaCTX-M-65, blaNDM-1, blaSFO-1, blaTEM-1, fosA3, rmtB, sul1 and sul2 genes. Four amino acid substitutions were detected in the quinolone resistance-determining regions (QRDRs) of GyrA (S83L and D87N), ParC (S80I) and ParE (S458A). Conjugation experiments revealed two multiresistance plasmids present in E. coli HD0149. The blaSFO-1 gene associated with blaNDM-1 gene was located in a 190 kb IncA/C plasmid and the blaCTX-M-65, fosA3 and rmtB genes were located in a 110 kb IncF plasmid. This is the first identification of the blaSFO-1 gene in an E. coli isolate and on a conjugative IncA/C plasmid. This may dramatically enhance the international prevalence and dissemination of blaSFO-1 among Enterobacteriaceae.

The carbohydrate-binding domain of overexpressed STBD1 is important for its stability and protein-protein interactions.

STBD1 (starch-binding domain-containing protein 1) belongs to the CBM20 (family 20 carbohydrate binding module) group of proteins, and is implicated in glycogen metabolism and autophagy. However, very little is known about its regulation or interacting partners. Here, we show that the CBM20 of STBD1 is crucial for its stability and ability to interact with glycogen-associated proteins. Mutation of a conserved tryptophan residue (W293) in this domain abolished the ability of STBD1 to bind to the carbohydrate amylose. Compared with the WT (wild-type) protein, this mutant exhibited rapid degradation that was rescued upon inhibition of the proteasome. Furthermore, STBD1 undergoes ubiquitination when expressed in COS cells, and requires the N-terminus for this process. In contrast, inhibition of autophagy did not significantly affect protein stability. In overexpression experiments, we discovered that STBD1 interacts with several glycogen-associated proteins, such as GS (glycogen synthase), GDE (glycogen debranching enzyme) and Laforin. Importantly, the W293 mutant of STBD1 was unable to do so, suggesting an additional role for the CBM20 domain in protein-protein interactions. In HepG2 hepatoma cells, overexpressed STBD1 could associate with endogenous GS. This binding increased during glycogenolysis, suggesting that glycogen is not required to bridge this interaction. Taken together, our results have uncovered new insights into the regulation and binding partners of STBD1.

Functional effects of genetic polymorphisms in the N-acetyltransferase 1 coding and 3' untranslated regions.

BACKGROUND: The functional effects of N-acetyltransferase 1 (NAT1) polymorphisms and haplotypes are poorly understood, compromising the validity of associations reported with diseases, including birth defects and numerous cancers. METHODS: We investigated the effects of genetic polymorphisms within the NAT1 coding region and the 3'-untranslated region (3'-UTR) and their associated haplotypes on N- and O-acetyltransferase catalytic activities, and NAT1 mRNA and protein levels following recombinant expression in COS-1 cells. RESULTS: 1088T>A (rs1057126; 3'-UTR) and 1095C>A (rs15561; 3'-UTR) each slightly reduced NAT1 catalytic activity and NAT1 mRNA and protein levels. A 9-bp (TAATAATAA) deletion between nucleotides 1065 and 1090 (3'-UTR) reduced NAT1 catalytic activity and NAT1 mRNA and protein levels. In contrast, a 445G>A (rs4987076; V149I), 459G>A (rs4986990; T153T), and 640T>G (rs4986783; S214A) coding region haplotype present in NAT1*11 increased NAT1 catalytic activity and NAT1 protein, but not NAT1 mRNA levels. A combination of the 9-bp (TAATAATAA) deletion and the 445G>A, 459G>A, and 640T>G coding region haplotypes, both present in NAT1*11, appeared to neutralize the opposing effects on NAT1 protein and catalytic activity, resulting in levels of NAT1 protein and catalytic activity that did not differ significantly from the NAT1*4 reference. CONCLUSIONS: Because 1095C>A (3'-UTR) is the sole polymorphism present in NAT1*3, our data suggest that NAT1*3 is not functionally equivalent to the NAT1*4 reference. Furthermore, our findings provide biologic support for reported associations of 1088T>A and 1095C>A polymorphisms with birth defects.