Coexistence of a nonresistance-conferring IncI1 plasmid favors persistence of the blaCTX-M-bearing IncFII plasmid in Escherichia coli.

The interaction between coexisting plasmids can affect plasmid-carried resistance gene persistence and spread. However, whether the persistence of the blaCTX-M gene in clinical Enterobacteriaceae is related to the interaction of coresident nonresistance-conferring plasmids has not been reported. This study was initiated to elucidate how a nonresistance-conferring IncI1 plasmid affected the blaCTX-M-bearing IncFII plasmid colocated on the same cell. Herein, we constructed three isogenic derivatives of E. coli C600, designated as C600FII, C600I1, and C600FII+I1, which harbored the blaCTX-M-IncFII plasmid and/or the nonresistance-IncI1 one. We discovered that strain C600FII+I1 conferred higher fitness advantages than strain C600FII; also, the stability of the blaCTX-M-IncFII plasmid was noticeably improved in an antibiotic-free environment when it coexisted with the IncI1 plasmid. To further explore why the IncI1 plasmid enhanced the persistence of the blaCTX-M-IncFII plasmid, we assessed the blaCTX-M-IncFII plasmid's copy numbers, conjugation frequencies, and rep gene expressions in strains C600FII and C600FII+I1. The results demonstrated that the rep expressions of the blaCTX-M-IncFII plasmid in strain C600FII+I1 was greatly decreased, along with the plasmid's copy numbers and mating efficiencies, compared to those in strain C600FII. Moreover, further study revealed that the intracellular ATP levels of strain C600FII+I1 were far lower than those of strain C600FII. Our findings confirmed that coexistence of the nonresistance-IncI1 plasmid can keep the blaCTX-M-IncFII plasmid more stable by increasing the fitness advantages of the host bacteria, which will pose a threat to preventing the long-term presence of the plasmid-carried blaCTX-M gene in clinical Enterobacteriaceae. IMPORTANCE: So far, plasmid-carried blaCTX-M is still the most common extended-spectrum beta-lactamase (ESBL) genotype in clinical settings worldwide. Except for the widespread use of third-generation cephalosporins, the interaction between coexisting plasmids can also affect the long-term stable existence of the blaCTX-M gene; however, the study on that is still sparse. In the present study, we assess the interaction of coinhabitant plasmids blaCTX-M-IncFII and nonresistance-IncI1. Our results confirmed that the increased fitness advantages of strain C600FII+I1 were attributable to the cohabitant nonresistance-IncI1 plasmid, which largely reduced the intracellular ATP levels of host bacteria, thus decreasing the rep gene expression of the blaCTX-M-IncFII plasmid, its copy numbers, and mating efficiencies, while the higher fitness advantages of strain C600FII+I1 enhanced the persistence of the blaCTX-M-IncFII plasmid. The results indicate that the nonresistance-IncI1 plasmid contributes to the long-term existence of the blaCTX-M-IncFII plasmid, implying a potentially new strategy for controlling the spread of resistance plasmids in clinical settings by targeting nonresistance plasmids.

Horizontal transfer characterization of ColV plasmids in blaCTX-M-bearing avian Escherichia coli.

Extended-spectrum-ß-lactamases (ESBLs)-producing Escherichia coli conferred resistance to most ß-lactams, except for carbapenems. To date, the transmission mechanism of blaCTX-M, as the most common ESBLs subtype, in E. coli has received sustained attention around the worldwide, but the research on the pathogenicity of blaCTX-M-bearing E. coli is still scarce. The aims of this study were to discern the spread characteristics of ColV (encoding colicin V) plasmids in blaCTX-M-positive E. coli. The multi-drug resistance traits, phylogroups, and ColV plasmid profilings were screened in 76 blaCTX-M-positive E. coli. Thereafter, the genetic profiles of E. coli G12 and GZM7 were determined by whole genome sequencing, conjugation and S1-pulsed-field gel electrophoresis. The median lethal dose was analyzed in E. coli G12 and TG12A, the ColV-plasmid transconjugant of G12. Of all 76 blaCTX-M-bearing E. coli, 67.11% exhibited resistance to at least 2 drugs in addition to ceftiofur, 14.47% carried ColV-positive plasmids, and 53.95% were phylogroup C. Further studies demonstrated that the blaCTX-M-bearing E. coli G12 was assigned to the predominant lineage O78:H4-ST117 of phylogroup G. In addition, its ColV-positive plasmid simultaneously carried multiple resistance genes, and could be independently transferred to confer partial pathogenicity on its host by plasmid mating. E. coli GZM7 was O53:H9-ST23 of phylogroup C, which belonged to another representative lineage of APEC (avian pathogenic E. coli). Its ColV-positive plasmid could complete conjugation with the help of the other coexisting-resistance conjugative plasmid, although it failed to transfer alone. Our findings highlight the flexibly horizontal transfer of ColV plasmids along with multidrug-resistant genes among blaCTX-M-bearing E. coli poses a threat to poultry health and food safety, which contributes to elucidate the concept of "One Health" and deserves particular concern.

Curcumin sensitizes response to cytarabine in acute myeloid leukemia by regulating intestinal microbiota.

PURPOSE: To address whether Curcumin has synergistic effect with cytarabine (Ara-C) in treating acute myeloid leukemia (AML). METHODS: A xenograft AML mouse model was established by injecting HL-60 cells into tail vein of mice to assess the function of Curcumin. Mononuclear cells (MNCs) isolated from AML mice and AML cell lines were used to examine the effect of Curcumin. Metagenomics and metabolomics were used to evaluate the alteration of intestinal microbiota and the change of metabolites in MNCs. RESULTS: Curcumin treatment sensitized response to Ara-C in MNCs of AML mice, but had no direct effect on AML cell lines. Metagenomics revealed an alteration of intestinal microbiota with Curcumin treatment, which contributes to sensitized response to Ara-C. Curcumin treatment led to enhanced intestinal intact to sensitize response to Ara-C in AML mice, through reducing mucus degrading bacteria. Metabolomics demonstrated that Curcumin treatment led to decreased cholesterol in MNCs of AML mice. Further study proved that Curcumin treatment resulted in inhibition of SQLE, a key enzyme of cholesterol biosynthesis, to increase sensitivity to Ara-C. CONCLUSION: Curcumin sensitizes response to Ara-C through regulating microbiota, highlighting the importance of intestinal intact strengthening in chemoresistant therapy. Moreover, aiming at cholesterol synthesis is promising in AML treatment.

Structural basis for ligand recognition of the neuropeptide Y Y2 receptor.

The human neuropeptide Y (NPY) Y2 receptor (Y2R) plays essential roles in food intake, bone formation and mood regulation, and has been considered an important drug target for obesity and anxiety. However, development of drugs targeting Y2R remains challenging with no success in clinical application yet. Here, we report the crystal structure of Y2R bound to a selective antagonist JNJ-31020028 at 2.8 Å resolution. The structure reveals molecular details of the ligand-binding mode of Y2R. Combined with mutagenesis studies, the Y2R structure provides insights into key factors that define antagonistic activity of diverse antagonists. Comparison with the previously determined antagonist-bound Y1R structures identified receptor-ligand interactions that play different roles in modulating receptor activation and mediating ligand selectivity. These findings deepen our understanding about molecular mechanisms of ligand recognition and subtype specificity of NPY receptors, and would enable structure-based drug design.

Iron overload as a risk factor for poor graft function following allogeneic hematopoietic stem cell transplantation.

Hematological malignancies are increasingly treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). Unfortunately, iron overload is a frequent adverse effect of allo-HSCT and is associated with poor prognosis. In the present study, we investigated hematopoiesis in iron-overloaded mice and elucidated the effects of iron overload on the bone marrow (BM) microenvironment. Iron-overloaded BALB/C mice were generated by injecting 20 mg/mL saccharated iron oxide intraperitoneally. Hematoxylin-eosin staining was performed to evaluate the effects of an iron overload in mice. BM cells obtained from C57BL/6 mice were transplanted into irradiated BALB/C mice (whole-body irradiation of 4 Gy, twice with a 4-hours interval) by tail vein injection. Two weeks after allo-HSCT, the hematopoietic reconstitution capacity was evaluated in recipients by colony-forming assays. Histopathological examinations showed brown-stained granular deposits, irregularly arranged lymphocytes in the liver tissues, and blue-stained blocks in the BM collected from mice received injections of high-dose saccharated iron oxide (20 mg/mL). Iron-overloaded mice showed more platelets, higher-hemoglobin (HGB) concentration, fewer granulocyte-macrophage colony-forming units (CFU-GM), erythrocyte colony-forming units (CFU-E), and mixed granulocyte/erythrocyte/monocyte/megakaryocyte colony-forming units (CFU-mix) than healthy mice. Iron-overloaded recipients presented with reduced erythrocytes and HGB concentration in peripheral blood, along with decreased marrow stroma cells, CFU-GM, CFU-E, and CFU-mix relative to healthy recipients. Taken together, our findings demonstrate that iron overload might alter the number of red blood cells after transplantation in mice by destroying the BM microenvironment, thereby affecting the recovery of BM hematopoietic function.

Epidermal growth factor stimulates exosomal microRNA-21 derived from mesenchymal stem cells to ameliorate aGVHD by modulating regulatory T cells.

Regulatory T cells (Tregs), a subset of CD4+ T cells, may exert inhibitory effects on alloimmune responses including acute graft-versus-host disease (aGVHD), and several microRNAs are implicated in the pathophysiological process of GVHD. Therefore, we aimed in the present study to characterize the functional relevance of epidermal growth factor (EGF)-stimulated microRNA-21 (miR-21) in regulating bone marrow-derived mesenchymal stem cells (BMSCs) in a mouse model of aGVHD. We first isolated and cultured BMSCs and Tregs. Then, we examined effects of miR-21 knockdown or overexpression and EGF on cell activities of BMSCs and the expression of PTEN, Foxp3, AKT phosphorylation, and extent of c-jun phosphorylation by gain- and loss-of-function approaches. The results showed that miR-21 promoted the proliferation, invasion, and migration of BMSCs. Furthermore, miR-21 in BMSCs-derived exosomes inhibited PTEN, but enhanced AKT phosphorylation and Foxp3 expression in Tregs. In addition, EGF enhanced c-jun phosphorylation to elevate the miR-21 expression. Furthermore, EGF significantly increased the efficacy of BMSCs in a mouse model of aGVHD, manifesting in reduced IFN-γ expression and lesser organ damage. Moreover, EGF treatment promoted the Foxp3 expression of Tregs in BMSCs-treated aGVHD mice. Taken together, EGF induced the BMSCs-derived exosomal miR-21 expression, which enhanced Foxp3 expression in Tregs, thereby improving the therapeutic effect of BMSCs on aGVHD.

Bone marrow mesenchymal stem cells alleviate the daunorubicin-induced subacute myocardial injury in rats through inhibiting infiltration of T lymphocytes and antigen-presenting cells.

INTRODUCTION: Bone marrow mesenchymal stem cells (BMSCs) have been extensively investigated from a perspective on cardiac regeneration therapy. The current study aimed to investigate the protective effect conferred by BMSCs in subacute myocardial injury, and to identify an appropriate BMSC reinfusion time. METHODS: BMSCs were isolated from human bone marrow blood. Daunorubicin (DNR)-induced subacute myocardial models were subsequently established. The rats with DNR-induced subacute myocardial injury were injected with dexrazoxane (DZR) and/or BMSCs at varying time points, after which cardiac function was evaluated by assessing left ventricular ejection fraction (LVEF) and fraction shortening (FS). The myocardial structural changes were analyzed, after which the levels of CD3 and human leukocyte antigen DR (HLA-DR) were examined to further validate the mechanism by which BMSCs could influence subacute myocardial injury. RESULTS: BMSCs combined with DZR treatment enhanced the cardiac function of rats with DNR-induced myocardial injury, as reflected by increased LVEF and FS. DNR-induced myocardial injuries were mitigated via the application of BMSCs combined with treatment of DZR, accompanied by diminished infiltration or vacuolization. Moreover, BMSCs were observed to alleviate infiltration of T lymphocyte and antigen-presenting cells, as evidenced by reduced expression of CD3 and HLA-DR. CONCLUSION: Taken together, this study demonstrates that BMSCs could protect against DNR-induced myocardial injury, especially in the first three days of DNR administration. BMSCs combined with DZR exert a better therapeutic effect, but there are individual differences.

G-CSF-primed haplo-identical HSCT with intensive immunosuppressive and myelosuppressive treatments does not increase the risk of pre-engraftment bloodstream infection: a multicenter case-control study.

A multicenter retrospective study in 131 patients (44 females/87 males) with hematological disorders who underwent G-CSF-primed/haplo-identical (Haplo-ID) (n = 76) or HLA-identical (HLA-ID) HSCT (n = 55) from February 2013 to February 2016 was conducted to compare the incidence and risk factors for pre-engraftment bloodstream infection (PE-BSI). In the Haplo-ID group, 71/76 patients with high-risk (n = 28) or relapsed/refractory hematological malignancies (n = 43) received FA5-BUCY conditioning (NCT02328950). All received trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis. Blood cultures and catheter tip cultures were obtained to confirm the BSI. PE-BSI was detected in 24/131 HSCT patients (18/76 in Haplo-ID and 6/55 in HLA-ID) after 28 febrile neutropenic episodes. Among 28 isolates for the 24 patients, 21 (75%) were Gneg bacteria, 6 (21.4%) Gpos and 1 (3.6%) fungi. Bacteria sources were central venous line infection (7/29.2%), gastroenteritis (6/25%), lower respiratory tract infection (LRTI; 5/20.8%), perianal skin infection (4/16.7%), and unknown (2/8.3%). The duration of neutropenia (P = 0.046) and previous Gneg bacteremia (P = 0.037) were important risk factors by univariate analysis, while the type of HSCT was not. A trend of TMP-SMX-resistant BSI in both groups may be due to routine antibacterial prophylaxis strategies. Our data show that G-CSF-primed Haplo-ID HSCT did not increase the risk of PE-BSI, even with intensive immunosuppressive treatments.

Bone marrow-derived mesenchymal stem cells overexpressing MiR-21 efficiently repair myocardial damage in rats.

OBJECTIVE: We investigated the ability of bone marrow derived mesenchymal stem cells (BMSCs) overexpressing microRNA-21 (miR-21) to repair cardiac damage induced by anthracyclines in rats. METHODS: Sprague-Dawley (SD) rats of 2~3 weeks old were selected to isolate and culture BMSCs. A lentivirus harboring pLVX-miR-21 was generated and transfected into rat BMSCs. The rats were assigned into an untreated negative control group, and groups injected with adriamycin alone or with adriamycin followed by BMSCs, pLVX-BMSCs or pLVX-miR-21-BMSCs (n = 10 each). Proliferation and migration of cells were detected by cholecystokinin-8 (CCK- 8) and transwell. MiR-21 expression, mRNA expressions of B cell lymphoma 2 (Bcl2), BAX (BCL-2-associated X protein) and vascular endothelial growth factor (VEGF) were tested by qRT-PCR. Western blotting was applied to detect protein expressions of Bcl-2, Bax and VEGF. RESULTS: Using CCK- 8 and transwell assays, we found that pLVX-miR-21-BMSCs, which overexpressed miR-21, exhibited greater proliferation and migration than untransfected BMSCs or pLVX-BMSCs. Ultrasonic cardiograms and immunohistochemical analysis demonstrated that among the five groups, the pLVX-miR-21-BMSC group exhibited the most improved heart function and enhanced angiogenesis. Moreover, the pLVX-miR-21-BMSC group showed enhanced expression of Bcl-2, VEGF and Cx43 and reduced expression of Bax, BNP and troponin T. CONCLUSION: These findings suggest miR-21 overexpression enhanced the proliferation, invasiveness and differentiation of BMSCs as well as expression of key factors (Bcl-2, VEGF and Bax) essential for repairing the cardiac damage induced by anthracyclines and restoring heart function.

The Construction and Identification of Induced Pluripotent Stem Cells Derived from Acute Myelogenous Leukemia Cells.

OBJECTIVE: The present study aimed to establish an induced pluripotent stem cell (iPSC) line from acute myelogenous leukemia (AML) cells in vitro and identify their biological characteristics. METHODS: Cells from the AML-infiltrated skin from an M6 patient were infected with a lentivirus carrying OCT4, SOX2, KLF4 and C-MYC to induce iPSCs. The characteristics of the iPSCs were confirmed by alkaline phosphatase (ALP) staining. The proliferation ability of iPSCs was detected with a CCK-8 assay. The expression of pluripotency markers was measured by immunostaining, and the expression of stem cell-related genes was detected by qRT-PCR; distortion during the induction process was detected by karyotype analysis; the differentiation potential of iPSCs was determined by embryoid body-formation and teratoma-formation assays. ALP staining confirmed that these cells exhibited positive staining and had the characteristics of iPSCs. RESULTS: The CCK-8 assay showed that the iPSCs had the ability to proliferate. Immunostaining demonstrated that iPSC clones showed positive expression of NANOG, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. qRT-PCR results revealed that the mRNA expression of Nanog, Lin28, Cripto, FOX3, DNMT3b, DPPA2, and DPPA4 significantly increased in iPSCs. Karyotype analysis found no chromosome aberration in the iPSCs. The results of the embryoid body-formation and teratoma-formation assays indicated that the iPSCs had the potential to differentiate into all three germ layers. CONCLUSION: Our study provided evidence that an iPSC line derived from AML cells was successfully established.

Investigating the validation of the Chinese Mandarin version of the Social Responsiveness Scale in a Mainland China child population.

BACKGROUND: Researchers from several different countries have found the Social Responsiveness Scale (SRS) to have good psychometric properties. However, to our knowledge, no studies on this subject have been reported in Mainland China. In this study, we investigated the psychometric properties of the Chinese Mandarin version of the SRS when used in Mainland China. METHODS: The reliability and validity of the parent-report SRS in a sample of 749 children of 4- to 14-year-olds: 411 typically developing and 338 clinical participants (202 with autism spectrum disorder (ASD)) were examined. RESULTS: Internal consistency for total scale (0.871-0.922), test-retest reliability (0.81-0.94), and convergent validity with the Autism Behavior Checklist (ABC) (0.302-0.647) were satisfactory. The SRS total score discriminated between the ASD and other developmental disorders. Receiver operating characteristic (ROC) analyses revealed that the SRS was predicted to accurately classify 69.2-97.2% of youth ASD. Exploratory factor analysis (EFA) supported a single-factor solution for the ASD subsample. Confirmatory factor analysis (CFA) did not confirm the theoretical construct of five factors model with inadequate fit in the ASD subsample. CONCLUSIONS: Overall, our findings supported the reliability and validity of the parent-report SRS as one ASD screening instrument. In addition, we also suggest that the use of separate cut-offs for screening purposes (optimizing sensitivity) vs. clinical confirmation (optimizing specificity) should be considered.