Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection

We have systematically made a set of precisely defined, single-gene deletions of all nonessential genes in Escherichia coli K-12. Open-reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one-step method for inactivation of chromosomal genes and primers designed to create in-frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high-throughput studies, two independent mutants were saved for every deleted gene. These mutants—the ‘Keio collection'—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome-wide testing of mutational effects in a common strain background, E. coli K-12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist-nara.ac.jp/)

Generation of Functional CLL-Specific Cord Blood CTL Using CD40-Ligated CLL APC

PMCID: PMC3526610This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

IL-2 Regulates SEB Induced Toxic Shock Syndrome in BALB/c Mice

BACKGROUND:Toxic Shock Syndrome (TSS) is characterized by fever, rash, hypotension, constitutional symptoms, and multi-organ involvement and is caused by Staphylococcus aureus enterotoxins such as Staphylococcal Enterotoxin B (SEB). SEB binds to the MHC-IIalpha chain and is recognized by the TCRbeta chain of the Vbeta8 TCR(+) T cells. The binding of SEB to Vbeta chain results in rapid activation of T cells and production of inflammatory cytokines, such as Interleukin-2 (IL-2), Interferon-gamma and Tumor Necrosis Factor-alpha which mediate TSS. Although IL2 was originally identified as the T cell growth factor and was proposed to contribute to T cell differentiation, its role in TSS remains unexplored. METHODOLOGY/PRINCIPAL FINDINGS:Mice were injected with D-Gal (25 mg/mouse). One hour after D-Galactosamine (D-Gal) injection each mouse was injected with SEB (20 microg/mouse. Mice were then observed for 72 hrs and death was recorded at different times. We tested Interleukin-12, IFNgamma, and IL-2 deficient mice (IL-2(-/-)), but only the IL-2 deficient mice were resistant to SEB induced toxic shock syndrome. More importantly reconstitution of IL-2 in IL-2 deficient mice restored the shock. Interestingly, SEB induced IL-2 production from T cells was dependent on p38MAPK activation in macrophages as inhibition of it in macrophages significantly inhibited IL-2 production from T cells. CONCLUSION:This study shows the importance of IL -2 in TSS which has not been previously explored and it also shows that regulating macrophages function can regulate T cells and TSS

Association of Variants at UMOD with Chronic Kidney Disease and Kidney Stones—Role of Age and Comorbid Diseases

Chronic kidney disease (CKD) is a worldwide public health problem that is associated with substantial morbidity and mortality. To search for sequence variants that associate with CKD, we conducted a genome-wide association study (GWAS) that included a total of 3,203 Icelandic cases and 38,782 controls. We observed an association between CKD and a variant with 80% population frequency, rs4293393-T, positioned next to the UMOD gene (GeneID: 7369) on chromosome 16p12 (OR = 1.25, P = 4.1×10−10). This gene encodes uromodulin (Tamm-Horsfall protein), the most abundant protein in mammalian urine. The variant also associates significantly with serum creatinine concentration (SCr) in Icelandic subjects (N = 24,635, P = 1.3×10−23) but not in a smaller set of healthy Dutch controls (N = 1,819, P = 0.39). Our findings validate the association between the UMOD variant and both CKD and SCr recently discovered in a large GWAS. In the Icelandic dataset, we demonstrate that the effect on SCr increases substantially with both age (P = 3.0×10−17) and number of comorbid diseases (P = 0.008). The association with CKD is also stronger in the older age groups. These results suggest that the UMOD variant may influence the adaptation of the kidney to age-related risk factors of kidney disease such as hypertension and diabetes. The variant also associates with serum urea (P = 1.0×10−6), uric acid (P = 0.0064), and suggestively with gout. In contrast to CKD, the UMOD variant confers protection against kidney stones when studied in 3,617 Icelandic and Dutch kidney stone cases and 43,201 controls (OR = 0.88, P = 5.7×10−5)

Educational outreach in an integrated clinical management tool for nurse-led non-communicable chronic disease management in primary care in South Africa: pragmatic cluster randomised controlled trial

Background: In many low-income countries, care for patients with non-communicable diseases (NCDs) and mental health conditions is provided by nurses. The benefits of nurse substitution and supplementation in NCD care in high income settings are well recognised, but evidence from low- and middle-income countries is limited. Primary Care 101 (PC101) is a programme designed to support and expand nurses’ role in NCD care, comprising a clinical management tool with enhanced prescribing provisions for nurses, and educational outreach. We evaluated the effectiveness of the programme on primary care nurses’ capacity to manage NCDs (ISRCTN20283604). Methods and findings: In a cluster randomised controlled trial design, 38 public sector primary care clinics in the Western Cape province, South Africa, were randomised. Nurses in the intervention clinics were trained to use the PC101 management tool during educational outreach sessions delivered by health department trainers and authorised to prescribe an expanded range of drugs for several NCDs. Control clinics continued use of the Practical Approach to Lung Health and HIV /AIDS in South Africa (PALSA PLUS) management tool and usual training. Patients attending these clinics with one or more of hypertension (3227), diabetes (1842), chronic respiratory disease (1157) or screened positive for depression (2466), totalling 4393 patients, were enrolled between March 2011 and October 2011. Primary outcomes were treatment intensification for hypertension, diabetes, and chronic respiratory disease cohorts, defined as the proportion of patients in whom treatment was escalated during follow-up over 14 months, and case detection in the depression cohort. Primary outcome data were analysed for 2110 (97%) intervention and 2170 (97%) control group patients. Treatment intensification rates in intervention clinics were not superior to those in the control group clinics [hypertension: 44% in the intervention group versus 40% in the controls, risk ratio (RR) 1.08 (95% CI: 0.94 to 1.24; p=0.252); diabetes: 57% v 50%, RR 1.10 (0.97 to 1.24;p=0.126); chronic respiratory disease: 14% v 12%, RR 1.08 (0.75 to 1.55; p=0.674); and case detection of depression: 18% v 24%, RR 0.76 (0.53 to 1.10; p=0.142)]. No adverse effects of the nurses’ expanded scope of practice were observed. Limitations of the study include dependence on self-reported diagnoses for inclusion in the patient cohorts, limited data on uptake of PC101 by users, reliance on process outcomes, and insufficient resources to measure important health outcomes, such as HbA1c, at follow-up. Conclusions: Educational outreach to primary care nurses through use of a management tool involving an expanded role in managing NCDs, is feasible and safe but was not associated with treatment intensification or case detection for index diseases. This notwithstanding, the intervention, with adjustments to improve its effectiveness, has been adopted for implementation in primary care clinics throughout South Africa

Suppression of p75 Neurotrophin Receptor Surface Expression with Intrabodies Influences Bcl-xL mRNA Expression and Neurite Outgrowth in PC12 Cells

Background: Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies. Results: Three monoclonal recombinant antibody fragments (scFv) with affinities in the low nanomolar range to murine p75NTR were isolated by antibody phage display. To suppress p75NTR cell surface expression, the encoding genes of these scFvs extended by the ER retention peptide KDEL were transiently transfected into the neuron-like rat pheochromocytoma cell line PC12 and the mouse neuroblastoma x mouse spinal cord hybrid cell line NSC19. The ER retained intrabody construct, SH325-G7-KDEL, mediated a downregulation of p75NTR cell surface expression as shown by flow cytometry. This effect was maintained over a period of at least eight days without activating an unfolded protein response (UPR). Moreover, the ER retention of p75NTR resulted in downregulation of mRNA levels of the anti-apoptotic protein Bcl-xL as well as in strong inhibition of NGF-induced neurite outgrowth in PC12 cells. Conclusion: The ER retained intrabody SH325-G7-KDEL not only induces phenotypic knockdown of this p75NTR but als

The nuclear receptor LXR modulates interleukin-18 levels through multiple mechanisms

IL-18 is a member of the IL-1 family involved in innate immunity and inflammation. Deregulated levels of IL-18 are involved in the pathogenesis of multiple disorders including inflammatory and metabolic diseases, yet relatively little is known regarding its regulation. Liver X receptors or LXRs are key modulators of macrophage cholesterol homeostasis and immune responses. Here we show that LXR ligands negatively regulate LPS-induced mRNA and protein expression of IL-18 in bone marrow-derived macrophages. Consistent with this being an LXR-mediated process, inhibition is abolished in the presence of a specific LXR antagonist and in LXR-deficient macrophages. Additionally, IL-18 processing of its precursor inactive form to its bioactive state is inhibited by LXR through negative regulation of both pro-caspase 1 expression and activation. Finally, LXR ligands further modulate IL-18 levels by inducing the expression of IL-18BP, a potent endogenous inhibitor of IL-18. This regulation occurs via the transcription factor IRF8, thus identifying IL-18BP as a novel LXR and IRF8 target gene. In conclusion, LXR activation inhibits IL-18 production through regulation of its transcription and maturation into an active pro-inflammatory cytokine. This novel regulation of IL-18 by LXR could be applied to modulate the severity of IL-18 driven metabolic and inflammatory disorders

Lives saved by Global Fund-supported HIV/AIDS, tuberculosis and malaria programs: estimation approach and results between 2003 and end-2007

<p>Abstract</p> <p>Background</p> <p>Since 2003, the Global Fund has supported the scale-up of HIV/AIDS, tuberculosis and malaria control in low- and middle-income countries. This paper presents and discusses a methodology for estimating the lives saved through selected service deliveries reported to the Global Fund.</p> <p>Methods</p> <p>Global Fund-supported programs reported, by end-2007, 1.4 million HIV-infected persons on antiretroviral treatment (ARV), 3.3 million new smear-positive tuberculosis cases detected in DOTS (directly observed TB treatment, short course) programs, and 46 million insecticide-treated mosquito nets (ITNs) delivered. We estimated the corresponding lives saved using adaptations of existing epidemiological estimation models.</p> <p>Results</p> <p>By end-2007, an estimated 681,000 lives (95% uncertainty range 619,000-774,000) were saved and 1,097,000 (993,000-1,249,000) life-years gained by ARV. DOTS treatment would have saved 1.63 million lives (1.09 - 2.17 million) when compared against no treatment, or 408,000 lives (265,000-551,000) when compared against non-DOTS treatment. ITN distributions in countries with stable endemic <it>falciparum </it>malaria were estimated to have achieved protection from malaria for 26 million of child-years at risk cumulatively, resulting in 130,000 (27,000-232,000) under-5 deaths prevented.</p> <p>Conclusions</p> <p>These results illustrate the scale of mortality effects that supported programs may have achieved in recent years, despite margins of uncertainty and covering only selected intervention components. Evidence-based evaluation of disease impact of the programs supported by the Global Fund with international and in-country partners must be strengthened using population-level data on intervention coverage and demographic outcomes, information on quality of services, and trends in disease burdens recorded in national health information systems.</p