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1.  Structure and Specificity of the Bacterial Cysteine Methyltransferase Effector NleE Suggests a Novel Substrate in Human DNA Repair Pathway 
PLoS Pathogens  2014;10(11):e1004522.
Enteropathogenic E. coli (EPEC) and related enterobacteria rely on a type III secretion system (T3SS) effector NleE to block host NF-κB signaling. NleE is a first in class, novel S-adenosyl-L-methionine (SAM)-dependent methyltransferase that methylates a zinc-coordinating cysteine in the Npl4-like Zinc Finger (NZF) domains in TAB2/3 adaptors in the NF-κB pathway, but its mechanism of action and other human substrates are unknown. Here we solve crystal structure of NleE-SAM complex, which reveals a methyltransferase fold different from those of known ones. The SAM, cradled snugly at the bottom of a deep and narrow cavity, adopts a unique conformation ready for nucleophilic attack by the methyl acceptor. The substrate NZF domain can be well docked into the cavity, and molecular dynamic simulation indicates that Cys673 in TAB2-NZF is spatially and energetically favorable for attacking the SAM. We further identify a new NleE substrate, ZRANB3, that functions in PCNA binding and remodeling of stalled replication forks at the DNA damage sites. Specific inactivation of the NZF domain in ZRANB3 by NleE offers a unique opportunity to suggest that ZRANB3-NZF domain functions in DNA repair processes other than ZRANB3 recruitment to DNA damage sites. Our analyses suggest a novel and unexpected link between EPEC infection, virulence proteins and genome integrity.
Author Summary
Pathogens often manipulate host functions by posttranslational modifications such as ubiquitination and methylation. The NF-κB pathway is most critical for immune defense against infection, thereby frequently targeted by bacterial virulence factors. NleE, a virulence effector from EPEC, is a SAM-dependent methyltransferase that modifies a zinc-finger cysteine in TAB2/3 in the NF-κB pathway. NleE is not homologous to any known methyltransferases. We present the crystal structure of SAM-bound NleE that shows a novel methyltransferase fold with a unique SAM-binding mode. Computational docking and molecular dynamics simulation illustrate a structural and chemical mechanism underlying NleE recognition of the NZF and catalyzing site-specific cysteine methylation. Subsequent substrate specificity analyses identify an N-terminal region in TAB3 required for efficient NleE recognition as well as another NZF protein ZRANB3 being a new substrate of NleE. NleE-catalyzed cysteine methylation also disrupts the ubiquitin chain-binding of ZRANB3-NZF domain, providing new insights into ZRANB3-NZF functioning in DNA damage repair. These results reinforce the idea of harnessing bacterial effectors as a tool for dissecting eukaryotic functions.
doi:10.1371/journal.ppat.1004522
PMCID: PMC4239114  PMID: 25412445
2.  Approach to Analyze the Diversity of Myxobacteria in Soil by Semi-Nested PCR-Denaturing Gradient Gel Electrophoresis (DGGE) Based on Taxon-Specific Gene 
PLoS ONE  2014;9(10):e108877.
The genotypic diversity of insoluble macromolecules degraded myxobacteria, provided an opportunity to discover new bacterial resources and find new ecological functions. In this study, we developed a semi-nested-PCR-denaturing gradient gel electrophoresis (DGGE) strategy to determine the presence and genotypic diversity of myxobacteria in soil. After two rounds of PCR with myxobacteria-specific primers, an 194 bp fragment of mglA, a key gene involved in gliding motility, suitable for DGGE was obtained. A large number of bands were observed in DGGE patterns, indicating diverse myxobacteria inhabiting in soils. Furthermore, sequencing and BLAST revealed that most of the bands belonged to the myxobacteria-group, and only three of the twenty-eight bands belonged to other group, i.e., Deinococcus maricopensis. The results verified that myxobacterial strains with discrepant sequence compositions of gene mglA could be discriminated by DGGE with myxobacteria-specific primers. Collectively, the developed semi-nested-PCR-DGGE strategy is a useful tool for studying the diversity of myxobacteria.
doi:10.1371/journal.pone.0108877
PMCID: PMC4184826  PMID: 25280065
3.  Metadherin contribute to BCR signaling in chronic lymphocytic leukemia 
We have reported Metadherin (MTDH) was proven to be overexpression and involved in malignance of chronic lymphocytic leukemia (CLL) via Wnt signaling pathway. In this study, we further investigate the role of MTDH in regulation of BCR signaling pathway in CLL. Six CLL samples whose cells were proliferation after BCR activation were chosen from patients with unmutated IgVH. CCK-8 method used to evaluate the proliferation rate. MTDH expression was measured by quantitative PCR and Western blot. After BCR activation, there exist upregulation of MTDH expression in mRNA and protein level in all six CLL patients (P<0.05). In cell line MEC-1, we observed the same pro-proliferation effect accompanying with elevated MTDH expression. The proliferation effects of BCR activation to MEC-1 can be inhibited by MTDH interference. The results of this study indicate that MTDH involved in the pro-proliferation effect of BCR activation in CLL. And the results imply that MTDH can be a potential therapy target of CLL.
PMCID: PMC4014239  PMID: 24817955
Chronic lymphocytic leukemia; B cell receptor; Metadherin
4.  Detection and Analysis of Human Papillomavirus (HPV) DNA in Breast Cancer Patients by an Effective Method of HPV Capture 
PLoS ONE  2014;9(3):e90343.
Despite an increase in the number of molecular epidemiological studies conducted in recent years to evaluate the association between human papillomavirus (HPV) and the risk of breast carcinoma, these studies remain inconclusive. Here we aim to detect HPV DNA in various tissues from patients with breast carcinoma using the method of HPV capture combined with massive paralleled sequencing (MPS). To validate the confidence of our methods, 15 cervical cancer samples were tested by PCR and the new method. Results showed that there was 100% consistence between the two methods.DNA from peripheral blood, tumor tissue, adjacent lymph nodes and adjacent normal tissue were collected from seven malignant breast cancer patients, and HPV type 16(HPV16) was detected in 1/7, 1/7, 1/7and 1/7 of patients respectively. Peripheral blood, tumor tissue and adjacent normal tissue were also collected from two patients with benign breast tumor, and 1/2, 2/2 and 2/2 was detected to have HPV16 DNA respectively. MPS metrics including mapping ratio, coverage, depth and SNVs were provided to characterize HPV in samples. The average coverage was 69% and 61.2% for malignant and benign samples respectively. 126 SNVs were identified in all 9 samples. The maximum number of SNVs was located in the gene of E2 and E4 among all samples. Our study not only provided an efficient method to capture HPV DNA, but detected the SNVS, coverage, SNV type and depth. The finding has provided further clue of association between HPV16 and breast cancer.
doi:10.1371/journal.pone.0090343
PMCID: PMC3948675  PMID: 24614680
7.  A Subanesthetic Dose of Isoflurane during Postconditioning Ameliorates Zymosan-Induced Neutrophil Inflammation Lung Injury and Mortality in Mice 
Mediators of Inflammation  2013;2013:479628.
Anesthetic isoflurane (ISO) has immunomodulatory effects. In the present study, we investigated whether a subanesthetic dose of ISO (0.7%) protected against zymosan (ZY) induced inflammatory responses in the murine lung and isolated neutrophils. At 1 and 6 hrs after ZY administration intraperitoneally, ISO was inhaled for 1 hr, and 24 hrs later, lung inflammation and injury were assessed. We found that ISO improved the survival rate of mice and mitigated lung injury as characterized by the histopathology, wet-to-dry weight ratio, protein leakage, and lung function index. ISO significantly attenuated ZY-induced lung neutrophil recruitment and inflammation. This was suggested by the downregulation of (a) endothelial adhesion molecule expression and myeloperoxidase (MPO) activity in lung tissue and polymorphonuclear neutrophils (b) chemokines, and (c) proinflammatory cytokines in BALF. Furthermore, ZY-induced nuclear translocation and DNA-binding activity of NF-κB p65 were also reduced by ISO. ISO treatment inhibited iNOS expression and activity, as well as subsequent nitric oxide generation. Consistent with these in vivo observations, in vitro studies confirmed that ISO blocked NF-κB and iNOS activation in primary mouse neutrophils challenged by ZY. These results provide evidence that 0.7% ISO ameliorates inflammatory responses in ZY-treated mouse lung and primary neutrophils.
doi:10.1155/2013/479628
PMCID: PMC3863458  PMID: 24369446
8.  Biomechanical effects of semi-constrained integrated artificial discs on zygapophysial joints of implanted lumbar segments 
This study aimed to optimize the design and application of semi-constrained integrated artificial discs (SIADs) using a finite element (FE) analysis following implantation, wherein the zygapophysial joints of the segment were biomechanically reconstructed. An FE model of the L4–L5 segment was constructed. Variations in the stresses on the discs and zygapophysial joints were observed during 5° anteflexion, 5° extension and 5° rotation under the 400-N applied axial load. Stresses and load translation analyses of the discs and zygapophysial joints were conducted during anteflexion, extension and rotation under the 400-N applied axial load. Following implantation of the lumbar segments, the stresses on the SIAD zygapophysial joints were not significantly different from those of physiological discs during anteflexion, and these were both marginally greater compared with those of non-constrained artificial discs (NADs). During extension, the increase in the stress on the SIAD zygapophysial joints was less than that on NAD zygapophysial joints. Stresses on the NAD zygapophysial joints were higher than those on SIAD and physiological discs during rotation. The stress on the SIAD zygapophysial joints was not significantly different from that on physiological discs during rotation. For SIADs and NADs, the stresses on the zygapophysial joints and the displacements of the discs were greater compared with those of the physiological discs during extension. The SIADs affected the variations in the stresses on the implanted segment more than the NADs, and the SIADs protected the zygapophysial joints of the implanted segment to a higher degree than the NADs.
doi:10.3892/etm.2013.1313
PMCID: PMC3829729  PMID: 24255672
lumbar vertebrae; zygapophysial joints; artificial disc; semi-constrained disc; biomechanics
9.  Palliative surgery for primary sarcoma in the abdominal aorta: A case report and review of the literature 
Oncology Letters  2013;6(6):1738-1740.
Primary sarcoma of the aorta is extremely rare and accounts for <1% of all sarcomas. The present study describes the case of a 45-year-old male who presented with lower limb and abdominal pain. Abdominal computed tomography (CT) and magnetic resonance (MR) arteriography revealed a tumor that extended from the infrarenal aorta to the aortic bifurcation. The external and internal iliac arteries were occluded by the tumor incursion. Palliative surgery was performed for the sarcoma since the patient refused a radical resection. To improve the blood supply to the lower limbs, an axillary bifemoral bypass was established. Following the surgery, the pain was significantly reduced. However, the patient succumbed due to extensive metastasis 6 months after this surgery. Aortic sarcoma is an extremely rare disease with a poor prognosis. A diagnosis at a relatively early stage is necessary for a longer survival time. Radical surgery is the most significant treatment. Patients at advanced stages should consider palliative surgery in order to improve their quality of life.
doi:10.3892/ol.2013.1594
PMCID: PMC3834868  PMID: 24273607
abdominal aortic aneurysm; palliative surgery
11.  Isolation and Identification of Myxobacteria from Saline-Alkaline Soils in Xinjiang, China 
PLoS ONE  2013;8(8):e70466.
Fifty-eight terrestrial and salt-tolerant myxobacteria were isolated from the saline-alkaline soils collected from Xinjiang, China. Based on the morphologies and the 16S rRNA gene sequences, these isolates were assigned into 6 genera, Myxococcus, Cystobacter, Corallococcus, Sorangium, Nannocystis and Polyangium. All the strains grew better with 1% NaCl than without NaCl. Some Myxococcus strains were able to grow at 2% NaCl concentration, suggesting that these strains may be particular type of terrestrial myxobacteria.
doi:10.1371/journal.pone.0070466
PMCID: PMC3735578  PMID: 23936436
12.  Type-Specific Detection of 30 Oncogenic Human Papillomaviruses by Genotyping both E6 and L1 Genes 
Journal of Clinical Microbiology  2013;51(2):402-408.
Human papillomavirus (HPV) is the principal cause of invasive cervical cancer and benign genital lesions. There are currently 30 HPV types linked to cervical cancer. HPV infection also leads to other types of cancer. We developed a 61-plex analysis of these 30 HPV types by examining two genes, E6 and L1, using MassARRAY matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (PCR-MS). Two hundred samples from homosexual males (HM) were screened by PCR-MS and MY09/MY11 primer set-mediated PCR (MY-PCR) followed by sequencing. One hundred thirty-five formalin-fixed, paraffin-embedded (FFPE) cervical cancer samples were also analyzed by PCR-MS, and results were compared to those of the commercially available GenoArray (GA) assay. One or more HPV types were identified in 64.5% (129/200) of the samples from HM. Comprising all 30 HPV types, PCR-MS detected 51.9% (67/129) of samples with multiple HPV types, whereas MY-PCR detected only one single HPV type in these samples. All PCR-MS results were confirmed by MY-PCR. In the cervical cancer samples, PCR-MS and GA detected 97% (131/135) and 90.4% (122/135) of HPV-positive samples, respectively. PCR-MS and GA results were fully concordant for 122 positive and 4 negative samples. The sequencing results for the 9 samples that tested negative by GA were completely concordant with the positive PCR-MS results. Multiple HPV types were identified in 25.2% (34/135) and 55.6% (75/135) of the cervical cancer samples by GA and PCR-MS, respectively, and results were confirmed by sequencing. The new assay allows the genotyping of >1,000 samples per day. It provides a good alternative to current methods, especially for large-scale investigations of multiple HPV infections and degraded FFPE samples.
doi:10.1128/JCM.01170-12
PMCID: PMC3553860  PMID: 23152557
13.  Hepatitis B Virus Surface Antigen (HBsAg)-Positive and HBsAg-Negative Hepatitis B Virus Infection among Mother-Teenager Pairs 13 Years after Neonatal Hepatitis B Virus Vaccination 
It is unclear whether a mother who is negative for hepatitis B virus surface antigen (HBsAg) but positive for hepatitis B virus (HBV) is at potential risk for mother-to-child transmission of HBV. This study, using a paired mother-teenager population, aimed to assess whether maternal HBsAg-negative HBV infection (hnHBI) is a significant source of child HBV infection (HBI). A follow-up study with blood collection has been conducted on the 93 mother-teenager pairs from the initial 135 pregnant woman-newborn pairs 13 years after neonatal HBV vaccination. Serological and viral markers of HBV have been tested, and phylogenetic analysis of HBV isolates has been done. The HBI prevalence was 1.9% (1 hnHBI/53) for teenage children of non-HBI mothers, compared with 16.7% (1 hnHBI/6) for those of hnHBI mothers and 2.9% (1 HBsAg-positive HBV infection [hpHBI]/34) for those of hpHBI mothers. Similar viral sequences have been found in one pair of whom both the mother and teenager have had hnHBI. In comparison with the hpHBI cases, those with hnHBI had a lower level of HBV load and a higher proportion of genotype-C strains, which were accompanied by differentiated mutations (Q129R, K141E, and Y161N) of the “a” determinant of the HBV surface gene. Our findings suggest that mother-to-teenager transmission of hnHBI can occur among those in the neonatal HBV vaccination program.
doi:10.1128/CVI.00539-12
PMCID: PMC3571284  PMID: 23254298
14.  Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP 
Cell Research  2013;23(6):775-787.
Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.
doi:10.1038/cr.2013.54
PMCID: PMC3674391  PMID: 23588383
Legionella; Rab GTPases; GTPase-activating protein (GAP); type IV secretion system; TBC GAP; membrane trafficking
15.  Neoadjuvant rh-endostatin, docetaxel and epirubicin for breast cancer: efficacy and safety in a prospective, randomized, phase II study 
BMC Cancer  2013;13:248.
Background
Recombinant human endostatin (rh-endostatin) is a novel antiangiogenesis drug developed in China. Previous experiments have shown that rh-endostatin can inhibit the proliferation and migration of endothelial cells and some types of tumor cells. In this study, we evaluated the efficacy and safety profiles of combination therapy of rh-endostatin and neoadjuvant chemotherapy for breast cancer patients in a prospective, randomized, controlled, phase II trial.
Methods
Sixty-eight patients with core-biopsy confirmed breast cancer were allocated randomly to two groups to receive 3 cycles of intravenous administration of either neoadjuvant DE (docetaxel: 75 mg/m2, d1, epirubicin: 75 mg/m2, d1, every 3 weeks), or neoadjuvant DE combined with rh-endostatin (7.5 mg/m2, d1-d14, every 3 weeks). The primary end point was clinical response based upon Response Evaluation Criteria in Solid Tumors, and the secondary end point was safety and quality of life.
Results
All patients were assessable for toxicity and 64 (94.2%) were assessable for efficacy evaluation. The objective response rate was 67.7% for chemotherapy (n = 31) and 90.9% for rh-endostatin plus chemotherapy (n = 33) (P = 0.021). A retrospective subset analysis revealed that rh-endostatin was more effective in premenopausal patients and patients with ECOG score of zero (P = 0.002 and P = 0.049, respectively). Five patients in the rh-endostatin plus chemotherapy arm achieved pathologic complete response compared with 2 in the chemotherapy arm (P = 0.428). No significant difference was identified in quality of life score and side effects (P > 0.05).
Conclusion
The combination of rh-endostatin with chemotherapy produced a higher tumor response rate without increasing toxicity in breast cancer patients.
Trial registration
ClinicalTrials.gov Identifier, NCT00604435
doi:10.1186/1471-2407-13-248
PMCID: PMC3664086  PMID: 23693018
Breast cancer; Recombinant human endostatin; Neoadjuvant chemotherapy; Clinical trial
16.  Distribution and Properties of the Genes Encoding the Biosynthesis of the Bacterial Cofactor, Pyrroloquinoline Quinone† 
Biochemistry  2012;51(11):2265-2275.
Pyrroloquinoline quinone (PQQ) is a small, redox-active molecule that serves as a cofactor for several bacterial dehydrogenases, introducing pathways for carbon utilization that confer a growth advantage. Early studies had implicated a ribosomally translated peptide as the substrate for PQQ production. This study presents a sequence and structure based analysis of the components of the pqq operon. We find the necessary components for PQQ production are present in 126 prokaryotes, most of which are Gram- negative and a number of which are pathogens. A total of five gene products, PqqA, PqqB, PqqC, PqqD and PqqE, are concluded to be obligatory for PQQ production. Three of the gene products in the pqq operon, PqqB, PqqC and PqqE, are members of large protein superfamilies. By combining evolutionary conservation patterns with information from three-dimensional structures, we are able to differentiate the gene products involved in PQQ biosynthesis from those with divergent functions. The observed persistence of a conserved gene order within analyzed operons strongly suggests a role for protein/protein interactions in the course of cofactor biosynthesis. These studies propose previously unidentified roles for several of the gene products as well as possible new targets for antibiotic design and application.
doi:10.1021/bi201763d
PMCID: PMC3334298  PMID: 22324760
pyrroloquinoline; quinone; pathogenicity; phylogenomic analysis; metallo-beta-lactamase; radical SAM domain; cofactorless oxidase
17.  Induction of Thoracic Aortic Remodeling by Endothelial-Specific Deletion of MicroRNA-21 in Mice 
PLoS ONE  2013;8(3):e59002.
MicroRNAs (miRs) are known to have an important role in modulating vascular biology. MiR21 was found to be involved in the pathogenesis of proliferative vascular disease. The role of miR21 in endothelial cells (ECs) has well studied in vitro, but the study in vivo remains to be elucidated. In this study, miR21 endothelial-specific knockout mice were generated by Cre/LoxP system. Compared with wild-type mice, the miR21 deletion in ECs resulted in structural and functional remodeling of aorta significantly, such as diastolic pressure dropping, maximal tension depression, endothelium-dependent relaxation impairment, an increase of opening angles and wall-thickness/inner diameter ratio, and compliance decrease, in the miR21 endothelial-specific knockout mice. Furthermore, the miR21 deletion in ECs induced down-regulation of collagen I, collagen III and elastin mRNA and proteins, as well as up-regulation of Smad7 and down-regulation of Smad2/5 in the aorta of miR21 endothelial-specific knockout mice. CTGF and downstream MMP/TIMP changes were also identified to mediate vascular remodeling. The results showed that miR21 is identified as a critical molecule to modulate vascular remodeling, which will help to understand the role of miR21 in vascular biology and the pathogenesis of vascular diseases.
doi:10.1371/journal.pone.0059002
PMCID: PMC3601125  PMID: 23527070
18.  MicroRNA-195 plays a tumor-suppressor role in human glioblastoma cells by targeting signaling pathways involved in cellular proliferation and invasion 
Neuro-Oncology  2012;14(3):278-287.
Accumulating evidence has implicated the deregluation of miRNAs in tumorigenesis. Previous studies have reported that microRNA-195 (miR-195) is markedly down-regulated in human glioblastoma cells, compared with normal brain tissue, but the biological role of miR-195 in glioblastoma development is currently unknown. In this study, we define a tumor-suppressor role for miR-195 in human glioblastoma cells. Over-expression of miR-195 in glioblastoma cell lines robustly arrested cell cycle progression and significantly repressed cellular invasion. We identified E2F3 and CCND3 as functional downstream targets of miR-195 in glioblastoma cells. Through knockdown studies, we demonstrated that E2F3 was the dominant effector of miR-195-mediated cell cycle arrest and that CCND3 was a key mediator of miR-195–induced inhibition of glioblastoma cell invasion. Furthermore, we showed that p27Kip1 was an important regulator downstream of CCND3 and that the accumulation of p27Kip1 in the cytoplasm might be responsible for the miR-195–mediated cell invasion inhibition in glioblastoma cells. This work provides evidence for the initial mechanism by which miR-195 negatively regulates both the proliferation and invasion of glioblastoma cells, suggesting that the down-regulation of miR-195 might contribute to the malignant transformation of glioblastoma cells and could be a molecular signature associated with glioblastoma progression.
doi:10.1093/neuonc/nor216
PMCID: PMC3280801  PMID: 22217655
glioblastoma; invasion; microRNA-195; proliferation; tumor-suppressor
19.  Involvement of Rab28 in NF-κB Nuclear Transport in Endothelial Cells 
PLoS ONE  2013;8(2):e56076.
Our previous proteomic analysis revealed the expression of Rab28 in arteries of rats. However, the function of Rab28 in mammalian cells, and its role in vessels are still unknown. Coarctation of abdominal aorta above left kidney artery in rat was used as hypertensive animal model. FX-4000 cyclic strain loading system was used to mimic the mechanical condition on vascular cells during hypertension in vitro. Immunofluorescence and co-immunoprecipitation (Co-IP) were used to identify distribution and interaction of Rab28 and nuclear factor kappa B (NF-κB). Rab28 expression was significantly increased in carotid arteries of hypertensive rats. High cyclic strain induced Rab28 expression of endothelial cells (ECs) through a paracrine control of vascular smooth muscles cells (VSMCs), which at least partly via angiotensin II (Ang II). Rab28 knockdown decreased proliferation of ECs, while increased apoptosis and migration. Immunofluorescence revealed that Ang II stimulated the co-translocation of Rab28 and NF-κB from cytoplasm into nucleus. Knockdown of Rab28 attenuated NF-κB activation. Co-IP of NF-κB p65 and Rab28 indicated their interaction. Our results revealed that Rab28, as a novel regulator of NF-κB nuclear transport, might participate in the disturbance of EC homeostasis.
doi:10.1371/journal.pone.0056076
PMCID: PMC3573041  PMID: 23457503
20.  Burkholderia dabaoshanensis sp. nov., a Heavy-Metal-Tolerant Bacteria Isolated from Dabaoshan Mining Area Soil in China 
PLoS ONE  2012;7(12):e50225.
Heavy-metal-tolerant bacteria, GIMN1.004T, was isolated from mine soils of Dabaoshan in South China, which were acidic (pH 2–4) and polluted with heavy metals. The isolation was Gram-negative, aerobic, non-spore-forming, and rod-shaped bacteria having a cellular width of 0.5−0.6 µm and a length of 1.3−1.8 µm. They showed a normal growth pattern at pH 4.0–9.0 in a temperature ranging from 5°C to 40°C.The organism contained ubiquinone Q-8 as the predominant isoprenoid quinine, and C16∶0, summed feature 8 (C18∶1ω7c and C18∶1ω6c), C18∶0, summed feature 3 (C16∶1ω7c or iso-C15∶0 2-OH), C17∶0 cyclo, C18∶1ω9c, C19∶0 cyclo ω8c, C14∶0 as major fatty acid. These profiles were similar to those reported for Burkholderia species. The DNA G+C % of this strain was 61.6%. Based on the similarity to 16S rRNA gene sequence, GIMN1.004T was considered to be in the genus Burkholderia. The similarities of 16S rRNA gene sequence between strain GIMN1.004T and members of the genus Burkholderia were 96−99.4%, indicating that this novel strain was phylogenetically related to members of that genus. The novel strain showed the highest sequence similarities to Burkholderia soli DSM 18235T (99.4%); Levels of DNA-DNA hybridization with DSM 18235T was 25%. Physiological and biochemical tests including cell wall composition analysis, differentiated phenotype of this strain from that closely related Burkholderia species. The isolation had great tolerance to cadmium with MIC of 22 mmol/L, and adsorbability of 144.94 mg/g cadmium,and it was found to exhibit antibiotic resistance characteristics. The adsorptive mechanism of GIMN1.004T for cadmium depended on the action of the amide,carboxy and phosphate of cell surface and producing low-molecular-weight (LMW ) organic acids to complex or chelated Cd2+.Therefore, the strain GIMN1.004T represented a new cadmium resistance species, which was tentatively named as Burkholderia dabaoshanensis sp. nov. The strain type is GIMN1.004T ( = CCTCC M 209109T =  NRRL B-59553T ).
doi:10.1371/journal.pone.0050225
PMCID: PMC3514224  PMID: 23226514
21.  Is phototherapy exposure associated with better or worse outcomes in 501–1000 gram birth weight infants? 
Aim
To compare risk-adjusted outcomes at 18–22 months corrected age for extremely low birth weight (ELBW) infants who never received phototherapy (NoPTx) to those who received any phototherapy (PTx) in the NICHD Neonatal Research Network randomized trial of Aggressive vs. Conservative Phototherapy.
Methods
Outcomes at 18–22 months corrected age included death, neurodevelopmental impairment (NDI), and Bayley Scales Mental Developmental Index (MDI). Regression models evaluated the independent association of PTx with adverse outcomes controlling for center and other potentially confounding variables.
Results
Of 1972 infants, 216 were NoPTx and 1756 were PTx. For the entire 501–1000 g BW cohort, PTx was not independently associated with death or NDI (OR 0.85, 95% CI 0.60 –1.20), death, or adverse neurodevelopmental endpoints. However, among infants 501–750 g BW, the rate of significant developmental impairment with MDI<50 was significantly higher for NoPTx (29%) than PTx (12%) (p=0.004).
Conclusions
Phototherapy did not appear to be independently associated with death or NDI for the overall ELBW group. Whether PTx increases mortality could not be excluded due to bias from deaths before reaching conservative treatment threshold. The higher rate of MDI<50 in the 501–750g BW NoPTx group is concerning, and consistent with NRN Trial results.
doi:10.1111/j.1651-2227.2011.02175.x
PMCID: PMC3505994  PMID: 21272067
22.  Identification of Extremely Premature Infants at High Risk of Rehospitalization 
Pediatrics  2011;128(5):e1216-e1225.
OBJECTIVE:
Extremely low birth weight infants often require rehospitalization during infancy. Our objective was to identify at the time of discharge which extremely low birth weight infants are at higher risk for rehospitalization.
METHODS:
Data from extremely low birth weight infants in Eunice Kennedy Shriver National Institute of Child Health and Human Development Neonatal Research Network centers from 2002–2005 were analyzed. The primary outcome was rehospitalization by the 18- to 22-month follow-up, and secondary outcome was rehospitalization for respiratory causes in the first year. Using variables and odds ratios identified by stepwise logistic regression, scoring systems were developed with scores proportional to odds ratios. Classification and regression-tree analysis was performed by recursive partitioning and automatic selection of optimal cutoff points of variables.
RESULTS:
A total of 3787 infants were evaluated (mean ± SD birth weight: 787 ± 136 g; gestational age: 26 ± 2 weeks; 48% male, 42% black). Forty-five percent of the infants were rehospitalized by 18 to 22 months; 14.7% were rehospitalized for respiratory causes in the first year. Both regression models (area under the curve: 0.63) and classification and regression-tree models (mean misclassification rate: 40%–42%) were moderately accurate. Predictors for the primary outcome by regression were shunt surgery for hydrocephalus, hospital stay of >120 days for pulmonary reasons, necrotizing enterocolitis stage II or higher or spontaneous gastrointestinal perforation, higher fraction of inspired oxygen at 36 weeks, and male gender. By classification and regression-tree analysis, infants with hospital stays of >120 days for pulmonary reasons had a 66% rehospitalization rate compared with 42% without such a stay.
CONCLUSIONS:
The scoring systems and classification and regression-tree analysis models identified infants at higher risk of rehospitalization and might assist planning for care after discharge.
doi:10.1542/peds.2011-1142
PMCID: PMC3208965  PMID: 22007016
logistic models; infant; premature; predictive value of tests
23.  t(4;11) leukemias display addiction to MLL-AF4 but not to AF4-MLL 
Leukemia research  2010;35(3):305-309.
The most frequent MLL-gene rearrangement found in leukemia is a reciprocal translocation with AF4 on chromosome 4 resulting in the formation of the MLL-AF4 and the AF4-MLL fusion genes. The oncogenic role of MLL-AF4 is documented but the significance of the reciprocal product - AF4-MLL in leukemia is less clear. In the human leukemia cell lines – RS4;11 and SEMK2-M1, both of which express MLL-AF4 and AF4-MLL, we knocked down the expression of AF4-MLL using siRNA. Loss of AF4-MLL had no effect on the growth of either RS4;11 or SEMK2-M1 cells. Furthermore, in SEMK2-M1 cells there were no changes in cell cycle or apoptosis with loss of AF4-MLL. In contrast, knockdown of MLL-AF4 significantly inhibited growth of both RS4;11 and SEMK2-M1. Additionally, in SEMK2-M1 cells, loss of MLL-AF4 led to G2/M cell cycle arrest and increased apoptosis. Overall, these results demonstrate that in t(4;11) leukemia, the MLL-AF4 fusion protein is critical for leukemia cell proliferation and survival while the AF4-MLL fusion product is dispensable.
doi:10.1016/j.leukres.2010.08.011
PMCID: PMC3011030  PMID: 20869771
MLL; AF4; t(4;11); leukemia; 11q23
24.  Evaluation of a randomized intervention to delay sexual initiation among fifth-graders followed through the sixth grade 
Sex education  2011;11(1):27-46.
US adolescents initiate sex at increasingly younger ages, yet few pregnancy prevention interventions for children as young as 10–12 years old have been evaluated. Sixteen Washington, DC schools were randomly assigned to intervention versus control conditions. Beginning in 2001/02 with fifth-grade students and continuing during the sixth grade, students completed pre-intervention and post-intervention surveys each school year. Each year, the intervention included 10–13 classroom sessions related to delaying sexual initiation. Linear hierarchical models compared outcome changes between intervention and control groups by gender over time. Results show the intervention significantly decreased a rise over time in the anticipation of having sex in the next 12 months among intervention boys versus control boys, but it had no other outcome effects. Among girls, the intervention had no significant outcome effects. One exception is that for both genders, compared with control students, intervention students increased their pubertal knowledge. In conclusion, a school-based curriculum to delay sexual involvement among fifth-grade and sixth-grade high-risk youths had limited impact. Additional research is necessary to outline effective interventions, and more intensive, comprehensive interventions may be required to counteract adverse circumstances in students’ lives and pervasive influences toward early sex.
ClinicalTrials. gov identifier: NCT00341471
doi:10.1080/14681811.2011.538146
PMCID: PMC3158595  PMID: 21857793
25.  Management of severe sepsis in patients admitted to Asian intensive care units: prospective cohort study 
Objectives To assess the compliance of Asian intensive care units and hospitals to the Surviving Sepsis Campaign’s resuscitation and management bundles. Secondary objectives were to evaluate the impact of compliance on mortality and the organisational characteristics of hospitals that were associated with higher compliance.
Design Prospective cohort study.
Setting 150 intensive care units in 16 Asian countries.
Participants 1285 adult patients with severe sepsis admitted to these intensive care units in July 2009. The organisational characteristics of participating centres, the patients’ baseline characteristics, the achievement of targets within the resuscitation and management bundles, and outcome data were recorded.
Main outcome measure Compliance with the Surviving Sepsis Campaign’s resuscitation (six hours) and management (24 hours) bundles.
Results Hospital mortality was 44.5% (572/1285). Compliance rates for the resuscitation and management bundles were 7.6% (98/1285) and 3.5% (45/1285), respectively. On logistic regression analysis, compliance with the following bundle targets independently predicted decreased mortality: blood cultures (achieved in 803/1285; 62.5%, 95% confidence interval 59.8% to 65.1%), broad spectrum antibiotics (achieved in 821/1285; 63.9%, 61.3% to 66.5%), and central venous pressure (achieved in 345/870; 39.7%, 36.4% to 42.9%). High income countries, university hospitals, intensive care units with an accredited fellowship programme, and surgical intensive care units were more likely to be compliant with the resuscitation bundle.
Conclusions While mortality from severe sepsis is high, compliance with resuscitation and management bundles is generally poor in much of Asia. As the centres included in this study might not be fully representative, achievement rates reported might overestimate the true degree of compliance with recommended care and should be interpreted with caution. Achievement of targets for blood cultures, antibiotics, and central venous pressure was independently associated with improved survival.
doi:10.1136/bmj.d3245
PMCID: PMC3113333  PMID: 21669950

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