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Ocean acidification (OA) driven by anthropogenic CO 2 emissions affects marine ecosystems, fisheries and aquaculture. Assessing the impacts of OA using projection models facilitates the development of future scenarios and potential solutions. Here, we explored various ways to incorporate OA impacts into a multi-stressor dynamic bioclimatic envelope model to project biogeographic changes of ten commercially exploited invertebrate species. We examine three dimensions of uncertainties in modelling biophysical OA effects: model structure, parameterization, and scenario uncertainty. Our results show that projected OA impacts are most sensitive to the choice of structural relationship between OA and biological responses, followed by the choice of climate change emission scenarios and parameterizations of the size of OA effects. Species generally showed negative effects to OA but sensitivity to the various sources of uncertainty were not consistent across or within species. For example, some species showed higher sensitivity to structural uncertainty and very low sensitivity to parameter uncertainty, while others showed greatest sensitivity to parameter uncertainty. This variability is largely due to geographic variability and difference in life history traits used to parameterize model simulations. Our model highlights the variability across the sources of uncertainty and contributes to the development of integrating OA impacts in species distribution models. We further stress the importance of defining the limitations and assumptions, as well as exploring the range of uncertainties associated with modelling OA impacts. [ABSTRACT FROM AUTHOR]

Between March and May 2012, three neonates born at a regional maternity hospital developed Elizabethkingia meningoseptica (previously Chryseobacterium meningosepticum) sepsis with meningitis aged <10 days, and were treated successfully with intravenous ciprofloxacin plus vancomycin or piperacillin-tazobactam for three to six weeks. Four (16.6%) of 24 environmental specimens obtained from the nursery and delivery room were positive for this organism. All of the clinical isolates and two isolates from storage boxes for pacifiers and pacifier covers were genetically identical. After changing the storage boxes to stainless steel boxes that underwent regular autoclave sterilization, there were no further cases of infection with E. meningoseptica. [ABSTRACT FROM AUTHOR]

Abstract: Macrophages are centrally involved during atherosclerosis development and are the predominant cell type that accumulates cholesterol in the plaque. Macrophages however, are heterogeneous in nature reflecting a variety of microenvironments and different phenotypes may be more prone to contribute towards atherosclerosis progression. Using primary human monocyte-derived macrophages, we sought to evaluate one aspect of atherogenic potential of different macrophage phenotypes by determining their propensity to associate with and accumulate oxidized low density lipoprotein (oxLDL). Classically-activated macrophages treated simultaneously with interferon γ (IFNγ) and tumor necrosis factor α (TNFα) associated with less oxLDL and accumulated less cholesterol compared to untreated controls. The combined treatment of IFNγ and TNFα reduced the mRNA expression of CD36 and the expression of both cell surface CD36 and macrophage scavenger receptor 1 (MSR1) protein. Under oxLDL loaded conditions, IFNγ and TNFα did not reduce macrophage protein expression of the transcription factor peroxisome proliferator-actived receptor γ (PPARγ) which is known to positively regulate CD36 expression. However, macrophages treated with IFNγ attenuated the ability of the PPARγ-specific agonist rosiglitazone from upregulating cell surface CD36 protein expression. Our results demonstrate that the observed reduction of cholesterol accumulation in macrophages treated with IFNγ and TNFα following oxLDL treatment was due at least in part to reduced cell surface CD36 and MSR1 protein expression. [Copyright &y& Elsevier]

Abstract: To mitigate hyperacute rejection, pigs have been generated with α-Gal transferase gene knockout and transgenic expression of human decay accelerating factor (hDAF), MCP, and CD59. Additionally, heme-oxygenase-1 (HO-1) has been suggested to defend endothelial cells. Sera (MS) (0%, 1%, 5%, 10%, and 15%) from Formosan macaques (Macaca cyclopis, MC), an Old World monkey wildly populated in Taiwan, was used to test the protective in vitro, effects of hDAF or hDAF/hHO-1 on porcine aortic endothelial cells (pAEC) derived from hDAF+, hDAF+/hHO-1+, and hDAF+/hHO-1− and 1 nontransgenic pAEC. Ten percent human serum (HS) served as a positive control. When MS addition increased to 10% or 15%, all transgenic pAEC exhibited a greater survival than nontransgenic pAEC. Noticeably, 15% MS reduced survived to <10% versus >40% in nontransgenic and transgenic pAEC, respectively. These results revealed that hDAF exerted protective effects against MC complement activation. However, comparing with 10% MS and HS in pAEC of nontransgenic pigs, the survivability was higher in HS, suggesting that complement activation by MS was more toxic than that by HS. Furthermore, hDAF+/hHO-1+ showed no further protection against effects of MS on transgenic pAEC. [ABSTRACT FROM AUTHOR]

Abstract: Obesity is a growing problem that threatens the health and welfare of a large proportion of the human population. The n-3 polyunsaturated fatty acids (PUFA) are dietary factors that have potential to facilitate reduction in body fat deposition and improve obesity-induced metabolic syndromes. The n-3 PUFA up-regulate several inflammation molecules including serum amyloid A (SAA), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in hepatocytes and adipocytes. Actions of these inflammation mediators resemble those of n-3 PUFA in the modulation of many lipid metabolism-related genes. For instance, they both suppress expressions of perilipin, sterol regulatory element binding protein-1 (SREBP-1) and lipoprotein lipase (LPL) to induce lipolysis and reduce lipogenesis. This review will connect these direct or indirect regulating pathways between n-3 PUFA, inflammation mediators, lipid metabolism-related genes and body fat reduction. A thorough knowledge of these regulatory mechanisms will lead us to better utilization of n-3 PUFA to reduce lipid deposition in the liver and other tissues, therefore presenting an opportunity for developing new strategies to treat obesity. [Copyright &y& Elsevier]

Serum amyloid A (SAA) reduces fat deposition in adipocytes and hepatoma cells. Human SAA1 mRNA is increased by docosahexaenoic acid (DHA) treatment in human cells. These studies asked whether DHA decreases fat deposition through SAAJ and explored the mechanisms involved. We demonstrated that DHA increased human SAA1 and C/EBPβ mRNA expression in human hepatoma cells, SK-HEP-1. Utilizing a promoter deletion assay, we found that a CCAAT/enhancer-binding protein 13 (C/EBPβ)-binding site in the SAA1 promoter region between -242 and -102 bp was critical for DHA-mediated SAA1 expression. Mutation of the putative C/EBPβ-binding site suppressed the DHA-induced SAA1 promoter activity. The addition of the protein kinase A inhibitor H89 negated the DHA-induced increase in C/EBPβ protein expression. The up-regulation of SAA1 mRNA and protein by DHA was also inhibited by H89. We also demonstrated that DHA increased protein kinase A (PKA) activities. These data suggest that C/EBPβ is involved in the DHA-regulated increase in SAA1 expression via PKA-dependent mechanisms. Furthermore, the suppressive effect of DHA on triacylglycerol accumulation was abolished by H89 in SKHEP-1 cells and adipocytes, indicating that DHA also reduces lipid accumulation via PKA. The observation of increased SAA1 expression coupled with reduced fat accumulation mediated by DHA via PKA suggests that SAA1 is involved in DHA-induced triacyiglycerol breakdown. These findings provide new insights into the complicated regulatory network in DHA-mediated lipid metabolism and are useful in developing new approaches to reduce body fat deposition and fatty liver. [ABSTRACT FROM AUTHOR]

Abstract: Background: Posttransplant lymphoproliferative disease (PTLD) is a serious complication in transplant recipients. Abdominal PTLD has been reported, but the prognosis remains undefined. The purpose of this study was to identify the incidence, predisposing factors, and outcome of abdominal PTLD in pediatric cardiothoracic transplant patients. Methods: Retrospective chart review of 134 transplant patients (50 heart, 77 lung, 7 heart/lung) at our institution (1995-2005). Results: Posttransplant lymphoproliferative disease was diagnosed in 14 patients. Most were Epstein-Barr virus naive initially, but all had seroconverted when diagnosed with PTLD. Eight had abdominal involvement; 4 required surgical interventions—1 for intussusception and for bowel perforation, 2 for bowel perforation, and 1 for tumor debulking. All had lifelong follow-up, with an average follow-up of 3 years. Of 8 patients with abdominal PTLD, 4 died of complications related to PTLD, whereas 1 of 6 patients with extraabdominal PTLD died of PTLD. Conclusions: Epstein-Barr virus infection after transplantation is a major risk factor for PTLD. Pediatric patients with PTLD who present with abdominal involvement are more likely to die of PTLD than those without abdominal disease. Delay in diagnosis may contribute to the high mortality. Therefore, prompt evaluation and surveillance for possible abdominal PTLD may decrease mortality associated with this devastating problem. [Copyright &y& Elsevier]

Abstract: In allotransplantation, donor–recipient human leukocyte antigen (HLA) matches improve graft survival. For studies of the role of donor–recipient HLA II matching on xenotransplantation, we successfully generated HLA-DR15+ transgenic pigs the the skins of which were transplanted to SCID mice, which were thereafter reconstituted with HLA-DR15+ or -DR15− hPBMC. Cyclosporine was given intraperitoneally to SCID mice for 12 days. Human T cells were observed in SCID mice after reconstitution. Mixed lymphocytes responses showed greater responses by HLA-DR15− human peripheral blood mononuclear cells (hPBMC) against HLA-DR15+ porcine PBMC. HLA-DR15+ porcine skins survived more than 100 days in all SCID mice. HLA-DR15+ porcine skins were rejected in all non-SCID (Balb/c) mice. The histologic pictures of transplanted HLA-DR15+ porcine skins showed surviving porcine epithelium in remodeling murine dermis and little lymphocyte infiltration into the murine dermis. The long-term survival of HLA-DR15+ pig skin in all hPBMC-SCID mice might be due to poor engraftment or function of reconstituted T cells. Further studies are needed to clarify the role of donor-recipient matching of HLA-DR15. [Copyright &y& Elsevier]

Characterizes Cys[sup 32] and His[sup 105] which are the critical residues for the calcium-dependent cysteine proteolytic activity of CvaB, an ATP-binding cassette transporter. Central membrane transporter of the colicin V secretion system; Cystein protease activity; Escherichia coli; Antipain; Protease inhibitors.

We examined the effect of transient covert attention on the psychometric function for contrast sensitivity in an orientation discrimination task when the target was presented alone in the absence of distracters and visual masks. Transient covert attention decreased both the threshold (consistent with a contrast gain mechanism) and, less consistently, the slope of the psychometric function. We assessed performance at 8 equidistant locations (4.5° eccentricity) and found that threshold and slope depended on target location—both were higher on the vertical than the horizontal meridian, particularly directly above fixation. All effects were robust across a range of spatial frequencies, and the visual field asymmetries increased with spatial frequency. Notwithstanding the dependence of the psychometric function on target location, attention improved performance to a similar extent across the visual field.Given that, in this study, we excluded all sources of external noise, and that we showed experimentally that spatial uncertainty cannot explain the present results, we conclude that the observed attentional benefit is consistent with signal enhancement. [Copyright &y& Elsevier]

Abstract: A 53-year-old man who underwent successful kidney transplantation for stage 5 chronic kidney disease presented to our clinic with intermittent painless gross hematuria. Urachal adenocarcinoma, stage III A by Sheldon system, was diagnosed after serial histopathologic and radiological studies. The patient was treated with extended partial cystectomy, en bloc resection of urachus and umbilicus, pelvic lymphadenectomy, and ileocystoplasty. There were no complications seen in this patient. Neither urachal adenocarcinoma recurrence, metastasis, nor de novo uroileal cancer developed during 48-month follow-up. His reconstructed bladder functioned efficiently, without compromising the transplanted kidney function. Our case demonstrated that conservative surgery and augmentation ileocystoplasty could be offered to kidney transplant recipients with localized urachal carcinoma. [Copyright &y& Elsevier]

Abstract: The shortage of human organs has encouraged scientists to develop genetically modified pigs for xenotransplantation, such as CD55 or CD46, and CD59 transgenesis as well as α-galactosyl transferase gene knockouts. In allotransplantation, the match of human leukocyte antigen class II (HLA-II) may improve graft survival although the role of HLA-II in xenotransplantation is unknown. HLA-II transgenic pigs, including DP, DQ, and DR, have been successfully generated and HLA-DR15+ transgenic pig skin pieces grafted onto severe congenital immunodeficiency (SCID) mice reconstituted intraperitoneally with HLA-DR15+ or HLA-DR15− human peripheral blood mononuclear cells (hPBMCs). This study sought to develop an animal model to evaluate the effects of HLA-DR matching on xenograft survival. Human CD4+ and CD8+ were detected from days 7 to 29 after hPBMC reconstitution in SCID mice. Both CD4+ and CD8+ cells of HLA-DR15− reconstituted SCID mice were significantly higher at day 29 postgrafting compared with HLA-DR15+ reconstituted SCID mice. An HLA-DR15+ transgenic pig dermal graft survived and integrated into SCID mice reconstituted with hPBMCs/HLA-DR15+ as proven by the histopathological finding that the collagen layer remained intact with little lymphocytic response. In contrast, the transgenic pig dermal graft showed more collagen disruption as well as mild to moderate lymphocytic infiltration when reconstituted in an hPBMC/HLA-DR15− SCID mouse. The results suggested that HLA-DR matching eased xenograft rejection; however, it was not yet clear that the response was mediated by T cells. [Copyright &y& Elsevier]

The Arctic remains one of the most pristine marine regions in the world, however climate change and increasing favourable conditions is triggering increasing exploration and development of commercial fisheries. Canada's Arctic marine capture fisheries are currently small relative to fisheries in other regions in Canada but small scale, predominantly Inuit fisheries are more wide spread. In this study, catch data was first used to estimate the current state of Arctic marine fisheries. Next, an integrated modelling approach was used to estimate the current and future fisheries potentials under high and low climate change scenarios. Comparisons of the current (2004–2015) annual reported tonnage and modelled estimates (±standard deviation) suggest that annual sustainable fisheries catch potential could be much greater at 4.07 (±2.86) million tonnes than the current catch of 189 (±6.26) thousand tonnes. Under a high climate change scenario, future (2091–2100) fisheries potential was projected to increase to 6.95 (±5.07) million tonnes of catch, while under low climate change scenario catch potential was similar to estimates of current catch potential. However, the greatest source of variance in catch potential estimates came from parameter uncertainty, followed by scenario and model uncertainty. These results contribute to understanding Canada's Arctic marine ecosystems in the face of a rapidly changing environment, yet proper steps must be taken to ensure cultural preservation for Inuit communities as well as ecological, economic, and social sustainability. • Climate change will increase access to Arctic marine fish stocks in Canada. • Projections show positive increases in fisheries catch and value potential with climate change. • Range shifts driven by ocean warming will lead to increased catch potential. • Ocean acidification may reduce projected increase in catch potential. • Ecological, economic, social and cultural impacts of exploitation must be considered. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Sirolimus (SRL), an immunosuppressant shown to possess anti-proliferative properties, was hypothesized to mitigate the occurrence of posttransplantation malignancy. We examined its effect on posttransplantation urothelial carcinoma (UC). Methods: This retrospective case analysis included renal allograft recipients with UC treated with SRL in a single institute. Results: Among 90 renal recipients treated with SRL, 6 had previous/new-onset UC in the native kidney/ureter or bladder: at a mean period of 28 months (range, 7–49) of administering SRL for these recipients, UC occurred/recurred in 4 of the 6 patients. Individual cases are presented in detail. Conclusion: SRL does not absolutely abolish the occurrence/recurrence of UC among renal transplant recipients. Its potency as an anti-cancerous immunosuppressant for transplant recipients with UC deserves to be further defined in larger studies, probably by controlling SRL blood levels at lower or much higher ranges than used herein. [Copyright &y& Elsevier]

The article presents a case study of an 80-year-old Taiwanese man with poor appetite, vomiting, leg edema, dry cough and progressive dyspnea. He subsequently received hemodialysis on an emergent basis. Abdominal ultrasonography showed marked bilateral hydronephrosis. A plain X-ray of his abdomen disclosed two radiopaque lesions, in unusual lateral and horizontal positions.

Abstract: Traditional techniques for investigating cultured neural networks, such as the patch clamp and multi-electrode array, are limited by: (1) the number of identified cells which can be simultaneously electrically contacted, (2) the length of time for which cells can be studied, and (3) the lack of one-to-one neuron-to-electrode specificity. Here, we present a new device – the caged neuron multi-electrode array – which overcomes these limitations. This micro-machined device consists of an array of neurocages which mechanically trap a neuron near an extracellular electrode. While the cell body is trapped, the axon and dendrites can freely grow into the surrounding area to form a network. The electrode is bi-directional, capable of both stimulating and recording action potentials. This system is non-invasive, so that all constituent neurons of a network can be studied over its lifetime with stable one-to-one neuron-to-electrode correspondence. Proof-of-concept experiments are described to illustrate that functional networks form in a neurochip system of 16 cages in a array, and that suprathreshold connectivity can be fully mapped over several weeks. The neurochip opens a new domain in neurobiology for studying small cultured neural networks. [Copyright &y& Elsevier]

Endothelial nitric-oxide synthase (eNOS) mRNA levels are abnormal in diseases of the cardiovascular system, but changes in gene expression cannot be accounted for by transcription alone. We found evidence for the existence of an antisense mRNA (sONE) that is derived from a transcription unit (NOS3AS) on the opposite DNA strand from which the human eNOS (NOS3) mRNA is transcribed at human chromosome 7q36. The genes are oriented in a tail-to-tail configuration, and the mRNAs encoding sONE and eNOS are complementary for 662 nucleotides. The mRNA for sONE could be detected in a variety of cell types, both in vivo and in vitro, but not vascular endothelial cells. In contrast, expression of eNOS is highly restricted to vascular endothelium. Most surprisingly, interrogation of transcriptional events across NOS3/NOS3AS genomic regions, using single- and double-stranded probes for nuclear run-off analyses and chromatin immunoprecipitation-based assessments of RNA polymerase II distribution, indicated that NOS3 and NOS3AS gene transcription did not correlate with steady-state mRNA levels. We found strong evidence supporting a role for NOS3AS in the post-transcriptional regulation of NOS3 expression. RNA interference-mediated inhibition of sONE expression in vascular smooth muscle cells increased eNOS expression. Overexpression of sONE in endothelial cells blunted eNOS expression. Finally, the histone deacetylase inhibitor trichostatin A is known to regulate the expression of eNOS via a post-transcriptional mechanism. We found that trichostatin A treatment of vascular endothelial cells increased expression of sONE mRNA levels prior to the observed decrease in eNOS mRNA expression. Taken together, these results indicate that an antisense mRNA (sONE) participates in the post-transcriptional regulation of eNOS and provide a newer model for endothelial cell-specific gene expression. [ABSTRACT FROM AUTHOR]

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide expressed in the vascular endothelium. Stringent control over ET-1 expression is achieved through a highly regulated promoter and rapid mRNA turnover. Since little is known about mechanisms governing ET-1 post.transcriptional regulation, and changes in ET-1 mRNA stability are implicated in disease processes, we characterized these pathways using a variety of functional approaches. We expressed human ET-I and luciferase transcripts with or without a wild type ET-1 3′-untranslated region (3′-UTR) and found that the 3′-UTR had potent mRNA destabilizing activity. Deletion analysis localized this activity to two domains of the 3′-UTR we have termed destabilizing elements 1 and 2 (DE1 and DE2). Mutational studies revealed that DE1 functions as an AU-rich element (ARE) dependent on a 100-nucleotide region. This activity was further localized to a 10nucleotide region at position 978–987 of the 3′-UTR. Depletion of AUF1 by RNA interference up-regulated ET-I in endothelial cells suggesting AUFl-dependent regulation. Since AUF1 functions through the ubiquitin-proteasome pathway, we disrupted this pathway with heat shock and proteasome inhibitor in endothelial cells and observed stabilization of endogenous ET-1 mRNA. Chimeric transcripts bearing wild type ET-1 3′-UTRs were also stabilized in response to proteasome inhibition whereas DE 1 mutants failed to respond. Taken together, these findings suggest a complex model of ARE-mediated mRNA turnover dependent on two 3′-UTR domains, DE1 and DE2. Furthermore, DE1 functions as an ARE directing mRNA half-life through the proteasome. Finally, this data provides evidence for a novel pathway of ET-1 mRNA stabilization by heat shock. [ABSTRACT FROM AUTHOR]

Two-layer and multi-layer depth-averaged models have become popular for simulating exchange flows, seawater currents and geophysical flows. The partial differential equation systems associated with these models are similar to the single-layer shallow-water model. Yet, their eigenstructures are more complex owing to the pressure coupling between the layers. Such models occasionally lose their hyperbolic character, which may lead to numerical issues. A physical explanation is that Kelvin-Helmholtz type instabilities arise at the layers' interface, if the velocity difference between the layers becomes sufficiently large. A way to avoid the hyperbolicity loss is to locally introduce an extra momentum exchange between the layers, assessable from the system eigenstructure and aimed at roughly mimicking the dynamical effects of such instabilities. To better understand the hyperbolicity conditions, the eigenstructure of the two-layer model is methodically studied by an asymptotic analysis. The analysis for the limiting cases, where the layers' thicknesses are either comparable or very different from each other, reveals new stability criteria. These analytical criteria are, then, exploited to design a new family of approximate criteria, valid for any flow condition. Numerical investigations demonstrate the reliability of this approach, which can be easily implemented in numerical schemes for preserving the hyperbolicity. • The two-layer depth-averaged model with potential loss of hyperbolicity is focused • The model eigenstructure is methodically studied by asymptotic analysis • From the limiting exact conditions a new family of stability criteria is proposed • Such criteria are used to preserve the model hyperbolicity in numerical simulations [ABSTRACT FROM AUTHOR]

Abstract: Xenoreactive antibody-induced complement activation and cytotoxicity poses a major obstracle to xenograft survival in humans. Previously, we have generated transgenic pigs carrying the hDAF exogene to help overcome this problem. In this study, we examined whether the hDAF exogene in various swine cells shows an equally protective effect for the complement-mediated cytotoxicity induced by human xenoreactive antibodies. Pig peripheral blood mononuclear cells (PBMCs) and aortic endothelial cells (PAECs) were used as targets. Fresh human serum was harvested from a single healthy human donor as the source of human xenoreactive antibodies and complement. The target cells cocultured with medium containing various concentrations of human serum for 24 hours were evaluated using the 3-[4,5-dimethylthiaolyl]-2,5-diphenyl-tetrazolium bromide assay for cellular viability. We observed that xenoreactive antibody plus human complement-mediated cellular cytoxicity dose-dependently correlated with the concentration of human serum in the culture medium. As compared with the PBMCs from the normal pigs, PBMCs from hDAF transgenic pigs showed significantly better survival after treatment with human serum (P < .05). Similarly, the survival of PAECs from the hDAF transgenic pig were also significantly higher than that from normal pigs (P < .05). Our data demonstrated a protective effect from human xenoreactive antibodies and complement-mediated cytoxicity of hDAF exogenes in pig PBMCs and PAECs. These observations support the clinical value of the hDAF transgenic pig as an organ donor in xenotransplantation. [Copyright &y& Elsevier]

Multi-layer depth-averaged models are widely employed in various hydraulic engineering applications. Yet, such models are not strictly hyperbolic. Their equation systems typically lose hyperbolicity when the relative velocities between layers become too large, which is associated with Kelvin–Helmholtz instabilities involving turbulent momentum exchanges between the layers. Focusing on the two-layer case, we present a numerical improvement that locally avoids the loss of hyperbolicity. The proposed modification introduces an additional momentum exchange between layers, whose value is iteratively calculated to be strictly sufficient to keep the system hyperbolic. The approach can be easily implemented in any finite volume scheme and there is no limitation concerning the density ratio between layers. Numerical examples, employing both HLL-type and Roe-type approximate Riemann solvers, are reported to validate the method and its key features. [ABSTRACT FROM AUTHOR]

Due to the emergence and rapid spread of drug resistance in bacteria, there is an urgent need for the development of novel antimicrobials. SecA, a key component of the general bacterial secretion system required for viability and virulence, is an attractive antimicrobial target. Earlier we reported that systematical dissection of a SecA inhibitor, Rose Bengal (RB), led to the development of novel small molecule SecA inhibitors active against Escherichia coli and Bacillus subtilis . In this study, two potent RB analogs were further evaluated for activities against methicillin-resistant Staphylococcus aureus (MRSA) strains and for their mechanism of actions. These analogs showed inhibition on the ATPase activities of S. aureus SecA1 (SaSecA1) and SecA2 (SaSecA2), and inhibition of SaSecA1-dependent protein-conducting channel. Moreover, these inhibitors reduce the secretion of three toxins from S. aureus and exert potent bacteriostatic effects against three MRSA strains. Our best inhibitor SCA-50 showed potent concentration-dependent bactericidal activity against MRSA Mu50 strain and very importantly, 2–60 fold more potent inhibitory effect on MRSA Mu50 than all the commonly used antibiotics including vancomycin, which is considered the last resort option in treating MRSA-related infections. Protein pull down experiments further confirmed SaSecA1 as a target. Deletion or overexpression of NorA and MepA efflux pumps had minimal effect on the antimicrobial activities against S. aureus , indicating that the effects of SecA inhibitors were not affected by the presence of these efflux pumps. Our studies show that these small molecule analogs target SecA functions, have potent antimicrobial activities, reduce the secretion of toxins, and have the ability to overcome the effect efflux pumps, which are responsible for multi-drug resistance. Thus, targeting SecA is an attractive antimicrobial strategy against MRSA. [ABSTRACT FROM AUTHOR]

Protein translocation is essential for bacterial survival and the most important translocation mechanism is the secretion (Sec) pathway in which SecA is a central core driving force. Thus targeting SecA is a promising strategy for developing novel antibacterial therapeutics. Herein, we report the syntheses and evaluation of a series of nearly 60 4-oxo-5-cyano thiouracil derivatives based upon our previously reported core pyrimidine structure. Introduction of polar group such as –N 3 and linker groups such as –CH 2 –O– enhanced the potency several fold. Apart from being potential antibacterial agents, these inhibitors can be indispensable tools for biologists to probe the mechanism of protein translocation via the SecA machinery in bacteria. [ABSTRACT FROM AUTHOR]

SecA is an essential component of the Sec-dependent protein translocation pathway across cytoplasmic membranes in bacteria. Escherichia coli SecA binds to cytoplasmic membranes at SecYEG high affinity sites and at phospholipid low affinity sites. It has been widely viewed that SecYEG functions as the essential protein-conducting channel through which precursors cross the membranes in bacterial Sec-dependent pathways, and that SecA functions as a motor to hydrolyze ATP in translocating precursors through SecYEG channels. We have now found that SecA alone can promote precursor translocation into phospholiposomes. Moreover, SecA-liposomes elicit ionic currents in Xenopus oocytes. Patch-clamp recordings further show that SecA alone promotes signal peptide- or precursor-dependent single channel activity. These activities were observed with the functional SecA at about 1-2 μm. The results show that SecA alone is sufficient to promote protein translocation into liposomes and to elicit ionic channel activity at the phospholipids low affinity binding sites, thus indicating that SecA is able to form the protein-conducting channels. Even so, such SecA-liposomes are less efficient than those with a full complement of Sec proteins, and lose the signal-peptide proofreading function, resembling the effects of PrlA mutations. Addition of purified SecYEG restores the signal peptide specificity and increases protein translocation and ion channel activities. These data show that SecA can promote protein translocation and ion channel activities both when it is bound to lipids at low affinity sites and when it is bound to SecYEG with high affinity. The latter of the two interactions confers high efficiency and specificity. [ABSTRACT FROM AUTHOR]

Abstract: SecA ATPase is a critical member of the Sec family, which is important in the translocation of membrane and secreted polypeptides/proteins in bacteria. Small molecule inhibitors can be very useful research tools as well as leads for future antimicrobial agent development. Based on previous virtual screening work, we optimized the structures of two hit compounds and obtained SecA ATPase inhibitors with IC50 in the single digit micromolar range. These represent the first low micromolar synthetic inhibitors of bacterial SecA and will be very useful for mechanistic studies. [Copyright &y& Elsevier]

SecA is an obligatory component of the Escherichia coli general secretion pathway. However, the oligomeric structure of SecA and SecA conformational changes during translocation processes are still unclear. Here we obtained the three-dimensional structure of E. coli wild-type full-length SecA in solution by single particle cryo-electron microscopy and determined its oligomeric organization. In this structure, SecA occurs as a dimer in which the two protomers are arranged in an antiparallel mode, with a novel electrostatic interface, and both protomers are in closed conformation. The system developed here may provide a promising technique for studying dynamic structural changes in SecA. [ABSTRACT FROM AUTHOR]

DLC (a-C:H) films were deposited by the plasma enhanced chemical vapor deposition (PECVD) on silicon substrate and then post-implanted by plasma immersion ion implantation (PIII) at different voltages and ion species (Ar, N2 and C2H2). Microstructure, dielectric constant and nano-hardness of the modified DLC films were studied. It is found that implanted C2H2 ions can effectively increase the nano-hardness of DLC films from 13.5 to 25.3 GPa and reduce the dielectric constant to 2.5 in the bias voltage range of −35 to −40 kV. The improved properties are mainly associated with the increase in the ratio of sp3 C—C/sp2 C=C bonds and the reaction mechanisms in the implantation zone are discussed. [Copyright &y& Elsevier]

To facilitate the therapeutic application of antigen-presenting cells (APCs), we have developed a cell-based artificial APC (aAPC) system by engineering K562 cells with lentiviruses to direct the stable expression and secretion of a variety of co-stimulatory molecules and cytokines. Here we report the use of a combinatorial lentiviral gene transfer approach to achieve long-term stable expression of at least seven genes in the K562 parental cell line. Expression of various combinations of genes on the aAPC enables the precise determination of human T-cell activation requirements, such that aAPCs can be tailored for the optimal propagation of T-cell subsets with specific growth requirements and distinct functions. The aAPCs support ex vivo growth and long-term expansion of functional human CD8 T cells without requiring the addition of exogenous cytokines, in contrast to the use of natural APCs. Distinct populations of T cells can be expanded with aAPCs expressing CD137L (4-1BBL) and/or CD80. Finally, the aAPCs provide an efficient platform to expand genetically modified T cells and to maintain CD28 expression on CD8 T cells. Therefore, K562-based aAPCs have therapeutic potential for adoptive immunotherapies and vaccinations.Molecular Therapy (2007) 15 5, 981–988. doi:10.1038/mt.sj.6300134 [ABSTRACT FROM AUTHOR]

Abstract: Understanding the sequence-to-structure relationship is a central task in bioinformatics research. Adequate knowledge about this relationship can potentially improve accuracy for local protein structure prediction. One of approaches for protein local structure prediction uses the conventional clustering algorithms to capture the sequence-to-structure relationship. The cluster membership function defined by conventional clustering algorithms may not reveal the complex nonlinear relationship adequately. Compared with the conventional clustering algorithms, Support Vector Machine (SVM) can capture the nonlinear sequence-to-structure relationship by mapping the input space into another higher dimensional feature space. However, SVM is not favorable for huge datasets including millions of samples. Therefore, we propose a novel computational model called Clustering Support Vector Machines (CSVMs). Taking advantage of both theory of granular computing and advanced statistical learning methodology, CSVMs are built specifically for each information granule partitioned intelligently by the clustering algorithm. This feature makes learning tasks for each CSVM more specific and simpler. CSVMs modeled for each granule can be easily parallelized so that CSVMs can be used to handle complex classification problems for huge datasets. Average accuracy for CSVMs is over 80%, which indicates that the generalization power for CSVMs is strong enough to recognize the complicated pattern of sequence-to-structure relationships. Compared with the conventional clustering algorithm, our experimental results show that accuracy for local structure prediction has been improved noticeably when CSVMs are applied. [Copyright &y& Elsevier]

Abstract: Maintaining a proper balance of metal concentrations is critical to the survival of bacteria. We have designed and synthesized a series of conjugates of metal chelators and efflux transporter substrates aimed at disrupting bacterial metal homeostasis to achieve bacterial killing. Biological studies showed that two of the compounds had very significant antimicrobial effect with an MIC value of 7.8μg/mL against Gram-positive Bacillus subtilis. [Copyright &y& Elsevier]