Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background: Homologous recombination mediated by the lambda-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the lambda-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these lambda-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. \ud \ud Results: Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the lambda-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6xHis, 3xFLAG, 4xProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the lambda-Red system, which can lead to unwanted secondary alterations to the chromosome. \ud \ud Conclusion: We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in E. coli. We have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic O157:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains

Design considerations and analysis planning of a phase 2a proof of concept study in rheumatoid arthritis in the presence of possible non-monotonicity

BACKGROUND: It is important to quantify the dose response for a drug in phase 2a clinical trials so the optimal doses can then be selected for subsequent late phase trials. In a phase 2a clinical trial of new lead drug being developed for the treatment of rheumatoid arthritis (RA), a U-shaped dose response curve was observed. In the light of this result further research was undertaken to design an efficient phase 2a proof of concept (PoC) trial for a follow-on compound using the lessons learnt from the lead compound. METHODS: The planned analysis for the Phase 2a trial for GSK123456 was a Bayesian Emax model which assumes the dose-response relationship follows a monotonic sigmoid "S" shaped curve. This model was found to be suboptimal to model the U-shaped dose response observed in the data from this trial and alternatives approaches were needed to be considered for the next compound for which a Normal dynamic linear model (NDLM) is proposed. This paper compares the statistical properties of the Bayesian Emax model and NDLM model and both models are evaluated using simulation in the context of adaptive Phase 2a PoC design under a variety of assumed dose response curves: linear, Emax model, U-shaped model, and flat response. RESULTS: It is shown that the NDLM method is flexible and can handle a wide variety of dose-responses, including monotonic and non-monotonic relationships. In comparison to the NDLM model the Emax model excelled with higher probability of selecting ED90 and smaller average sample size, when the true dose response followed Emax like curve. In addition, the type I error, probability of incorrectly concluding a drug may work when it does not, is inflated with the Bayesian NDLM model in all scenarios which would represent a development risk to pharmaceutical company. The bias, which is the difference between the estimated effect from the Emax and NDLM models and the simulated value, is comparable if the true dose response follows a placebo like curve, an Emax like curve, or log linear shape curve under fixed dose allocation, no adaptive allocation, half adaptive and adaptive scenarios. The bias though is significantly increased for the Emax model if the true dose response follows a U-shaped curve. CONCLUSIONS: In most cases the Bayesian Emax model works effectively and efficiently, with low bias and good probability of success in case of monotonic dose response. However, if there is a belief that the dose response could be non-monotonic then the NDLM is the superior model to assess the dose response

Antibody to P. falciparum in Pregnancy Varies with Intermittent Preventive Treatment Regime and Bed Net Use

Antibodies towards placental-binding P. falciparum are thought to protect against pregnancy malaria; however, environmental factors may affect antibody development.Using plasma from pregnant Malawian women, we measured IgG against placental-binding P. falciparum parasites by flow cytometry, and related results to intermittent preventive treatment (IPTp) regime, and bed net use. Bed net use was associated with decreased antibody levels at mid-pregnancy but not at 1 month post partum (1 mpp). At 1 mpp a more intensive IPTp regime was associated with decreased antibody levels in primigravidae, but not multigravidae.Results suggest bed nets and IPTp regime influence acquisition of pregnancy-specific P. falciparum immunity

Attrition among Human Immunodeficiency Virus (HIV)- Infected Patients Initiating Antiretroviral Therapy in China, 2003–2010

BACKGROUND: Mortality and morbidity from HIV have dramatically decreased in both high- and low-income countries. However, some patients may not benefit from combination antiretroviral therapy (cART) because of inadequate access to HIV care, including attrition after care initiation. METHODOLOGY/PRINCIPAL FINDINGS: The study population included all HIV-infected patients receiving cART through the Chinese National Free Antiretroviral Treatment Program from 1 January 2003 to 31 December 2010 (n = 106,542). We evaluated retention in HIV care and used multivariable Cox proportional hazard models to identify independent factors predictive of attrition. The cumulative probability of attrition from cART initiation was 9% at 12 months, 13% at 18 months, 16% at 24 months and 24% at 60 months. A number of factors were associated with attrition, including younger age, male gender, and being single or divorced. Patients with higher CD4 cell counts at cART initiation were more likely to drop out of HIV care. The proportion of patients remaining in HIV care increased in more recent calendar years and among patients who initiated modern cART regimens. CONCLUSIONS/SIGNIFICANCE: Retention in HIV care is essential for optimizing individual and public health outcomes. Attrition, even the degree observed in our study, can lead to premature morbidity and mortality, and possibly affect further transmission of HIV and HIV resistant drug variants. Effective strategies to promote retention in HIV care programs are needed. In China these strategies may include focusing particularly on younger male patients and those with higher CD4 cell counts at therapy initiation

The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders

The health disparities cancer collaborative: a case study of practice registry measurement in a quality improvement collaborative

<p>Abstract</p> <p>Background</p> <p>Practice registry measurement provides a foundation for quality improvement, but experiences in practice are not widely reported. One setting where practice registry measurement has been implemented is the Health Resources and Services Administration's Health Disparities Cancer Collaborative (HDCC).</p> <p>Methods</p> <p>Using practice registry data from 16 community health centers participating in the HDCC, we determined the completeness of data for screening, follow-up, and treatment measures. We determined the size of the change in cancer care processes that an aggregation of practices has adequate power to detect. We modeled different ways of presenting before/after changes in cancer screening, including count and proportion data at both the individual health center and aggregate collaborative level.</p> <p>Results</p> <p>All participating health centers reported data for cancer screening, but less than a third reported data regarding timely follow-up. For individual cancers, the aggregate HDCC had adequate power to detect a 2 to 3% change in cancer screening, but only had the power to detect a change of 40% or more in the initiation of treatment. Almost every health center (98%) improved cancer screening based upon count data, while fewer (77%) improved cancer screening based upon proportion data. The aggregate collaborative appeared to increase breast, cervical, and colorectal cancer screening rates by 12%, 15%, and 4%, respectively (p < 0.001 for all before/after comparisons). In subgroup analyses, significant changes were detectable among individual health centers less than one-half of the time because of small numbers of events.</p> <p>Conclusions</p> <p>The aggregate HDCC registries had both adequate reporting rates and power to detect significant changes in cancer screening, but not follow-up care. Different measures provided different answers about improvements in cancer screening; more definitive evaluation would require validation of the registries. Limits to the implementation and interpretation of practice registry measurement in the HDCC highlight challenges and opportunities for local and aggregate quality improvement activities.</p

Assessing risk of breast cancer in an ethnically South-East Asia population (results of a multiple ethnic groups study)

10.1186/1471-2407-12-529BMC Cancer12-BCMA

An apoptosis targeted stimulus with nanosecond pulsed electric fields (nsPEFs) in E4 squamous cell carcinoma

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was >95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. Bid cleavage was caspase-dependent (55–60%) and calcium-dependent (40–45%). Intracellular calcium as an intrinsic mechanism and extracellular calcium as an extrinsic mechanism were responsible for about 30 and 70% of calcium dependence for Bid cleavage, respectively. The results reveal electric field-mediated cell death induction and progression, activating pro-apoptotic-like mechanisms and affecting plasma membrane and intracellular functions, primarily through extrinsic-like pathways with smaller contributions from intrinsic-like pathways. Nanosecond second pulsed electric fields trigger heterogeneous cell death mechanisms in E4 SCC populations to delete them, with caspase-associated cell death as a predominant, but not an unaccompanied event

Co-producing across organizational boundaries: promoting asylum seeker integration in Scotland

This paper questions whether asylum seeker integration is promoted through inter-organisational relationships between non-profit and voluntary organisations (NPVOs) and government agencies. It focuses particularly on the role of NPVOs in service delivery (co-management) and in the delivery and planning of public services (co-governance). It presents a research study on the public services provided to asylum seekers in Glasgow and asks the following questions: What role do NPVOs play in the planning and delivery of public services? When planning and delivering public services, to what extent do NPVOs work across organisational boundaries and what kind of relationships exist? And in practice, what makes inter-organisational relationships work? This paper offers new empirical evidence and also contributes to the theoretical debate around the integration of asylum seekers

Antibodies That Induce Phagocytosis of Malaria Infected Erythrocytes: Effect of HIV Infection and Correlation with Clinical Outcomes

HIV infection increases the burden of disease of malaria in pregnancy, in part by impairing the development of immunity. We measured total IgG and phagocytic antibodies against variant surface antigens of placental-type CS2 parasites in 187 secundigravidae (65% HIV infected). In women with placental malaria infection, phagocytic antibodies to CS2VSA were decreased in the presence of HIV (p = 0.011) and correlated positively with infant birth weight (coef = 3.57, p = 0.025), whereas total IgG to CS2VSA did not. Phagocytic antibodies to CS2VSA are valuable tools to study acquired immunity to malaria in the context of HIV co-infection. Secundigravidae may be an informative group for identification of correlates of immunity