The lysine residue in the membrane-spanning domain of the beta chain is necessary for cell surface expression of the T cell antigen receptor.

The TCR is a complex receptor composed of seven polypeptide chains consisting of a ligand-binding subunit, Ti, and a putative signal-transducing subunit, CD3. Phylogenetically conserved charged amino acid residues within the membrane-spanning domains present in all seven chains of the TCR have been proposed to be important in the association between Ti and CD3. Using a Ti beta chain-deficient mutant of the cell line Jurkat, site-directed mutagenesis and transfection of Ti beta chain cDNA was performed to assess the importance of the lysine residue at position 290 within the membrane-spanning domain of the Ti beta chain to expression of the TCR complex. These studies demonstrated that the lysine residue, and not simply conservation of either basic charge or secondary structure, is important at this position

Whole-Genome Sequence Analysis of an Extensively Drug-Resistant Salmonella enterica Serovar Agona Isolate from an Australian Silver Gull (Chroicocephalus novaehollandiae) Reveals the Acquisition of Multidrug Resistance Plasmids.

Although most of the approximately 94 million annual human cases of gastroenteritis due to Salmonella enterica resolve without medical intervention, antimicrobial therapy is recommended for patients with severe disease. Wild birds can be natural hosts of Salmonella that pose a threat to human health; however, multiple-drug-resistant serovars of S. enterica have rarely been described. In 2012, silver gull (Chroicocephalus novaehollandiae) chicks at a major breeding colony were shown to host Salmonella, most isolates of which were susceptible to antibiotics. However, multiple-drug-resistant (MDR) Escherichia coli with resistance to carbapenems, ceftazidime, and fluoroquinolones was reported from this breeding colony. In this paper, we describe a novel MDR Salmonella strain subsequently isolated from the same breeding colony. SG17-135, an isolate of S. enterica with phenotypic resistance to 12 individual antibiotics but only nine antibiotic classes including penicillins, cephalosporins, monobactams, macrolides, fluoroquinolones, aminoglycosides, dihydrofolate reductase inhibitors (trimethoprim), sulfonamides, and glycylcyclines was recovered from a gull chick in 2017. Whole-genome sequence (WGS) analysis of SG17-135 identified it as Salmonella enterica serovar Agona (S Agona) with a chromosome comprising 4,813,284 bp, an IncHI2 ST2 plasmid (pSG17-135-HI2) of 311,615 bp, and an IncX1 plasmid (pSG17-135-X) of 27,511 bp. pSG17-135-HI2 housed a complex resistance region comprising 16 antimicrobial resistance genes including blaCTX-M-55 The acquisition of MDR plasmids by S. enterica described here poses a serious threat to human health. Our study highlights the importance of taking a One Health approach to identify environmental reservoirs of drug-resistant pathogens and MDR plasmids.IMPORTANCE Defining environmental reservoirs hosting mobile genetic elements that shuttle critically important antibiotic resistance genes is key to understanding antimicrobial resistance (AMR) from a One Health perspective. Gulls frequent public amenities, parklands, and sewage and other waste disposal sites and carry drug-resistant Escherichia coli Here, we report on SG17-135, a strain of Salmonella enterica serovar Agona isolated from the cloaca of a silver gull chick nesting on an island in geographic proximity to the greater metropolitan area of Sydney, Australia. SG17-135 is closely related to pathogenic strains of S Agona, displays resistance to nine antimicrobial classes, and carries important virulence gene cargo. Most of the antibiotic resistance genes hosted by SG17-135 are clustered on a large IncHI2 plasmid and are flanked by copies of IS26 Wild birds represent an important link in the evolution and transmission of resistance plasmids, and an understanding of their behavior is needed to expose the interplay between clinical and environmental microbial communities

QuantiFERON®-TB gold in-tube performance for diagnosing active tuberculosis in children and adults in a high burden setting.

To determine whether QuantiFERON®-TB Gold In-Tube (QFT) can contribute to the diagnosis of active tuberculosis (TB) in children in a high-burden setting and to assess the performance of QFT and tuberculin skin test (TST) in a prospective cohort of TB suspect children compared to adults with confirmed TB in Tanzania. Sensitivity and specificity of QFT and TST for diagnosing active TB as well as indeterminate QFT rates and IFN-γ levels were assessed in 211 TB suspect children in a Tanzanian district hospital and contrasted in 90 adults with confirmed pulmonary TB. Sensitivity of QFT and TST in children with confirmed TB was 19% (5/27) and 6% (2/31) respectively. In adults sensitivity of QFT and TST was 84% (73/87) and 85% (63/74). The QFT indeterminate rate in children and adults was 27% and 3%. Median levels of IFN-γ were lower in children than adults, particularly children <2 years and HIV infected. An indeterminate result was associated with age <2 years but not malnutrition or HIV status. Overall childhood mortality was 19% and associated with an indeterminate QFT result at baseline. QFT and TST showed poor performance and a surprisingly low sensitivity in children. In contrast the performance in Tanzanian adults was good and comparable to performance in high-income countries. Indeterminate results in children were associated with young age and increased mortality. Neither test can be recommended for diagnosing active TB in children with immature or impaired immunity in a high-burden setting

Negative DNA supercoiling induces genome-wide Cas9 off-target activity

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites

Immunization coverage and risk factors for failure to immunize within the Expanded Programme on Immunization in Kenya after introduction of new Haemophilus influenzae type b and hepatitis b virus antigens

Background: Kenya introduced a pentavalent vaccine including the DTP, Haemophilus influenzae type b and hepatitis b virus antigens in Nov 2001 and strengthened immunization services. We estimated immunization coverage before and after introduction, timeliness of vaccination and risk factors for failure to immunize in Kilifi district, Kenya. Methods: In Nov 2002 we performed WHO cluster-sample surveys of > 200 children scheduled for vaccination before or after introduction of pentavalent vaccine. In Mar 2004 we conducted a simple random sample (SRS) survey of 204 children aged 9 - 23 months. Coverage was estimated by inverse Kaplan-Meier survival analysis of vaccine- card and mothers' recall data and corroborated by reviewing administrative records from national and provincial vaccine stores. The contribution to timely immunization of distance from clinic, seasonal rainfall, mother's age, and family size was estimated by a proportional hazards model. Results: Immunization coverage for three DTP and pentavalent doses was 100% before and 91% after pentavalent vaccine introduction, respectively. By SRS survey, coverage was 88% for three pentavalent doses. The median age at first, second and third vaccine dose was 8, 13 and 18 weeks. Vials dispatched to Kilifi District during 2001 - 2003 would provide three immunizations for 92% of the birth cohort. Immunization rate ratios were reduced with every kilometre of distance from home to vaccine clinic (HR 0.95, CI 0.91 - 1.00), rainy seasons ( HR 0.73, 95% CI 0.61 - 0.89) and family size, increasing progressively up to 4 children ( HR 0.55, 95% CI 0.41 - 0.73). Conclusion: Vaccine coverage was high before and after introduction of pentavalent vaccine, but most doses were given late. Coverage is limited by seasonal factors and family siz

T-cell subpopulations αβ and γδ in cord blood of very preterm infants : The influence of intrauterine infection

Open Access: This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are creditedPreterm infants are very susceptible to infections. Immune response mechanisms in this group of patients and factors that influence cord blood mononuclear cell populations remain poorly understood and are considered insufficient. However, competent immune functions of the cord blood mononuclear cells are also described. The aim of this work was to evaluate the T-cell population (CD3+) with its subpopulations bearing T-cell receptor (TCR) αβ or TCR γδ in the cord blood of preterm infants born before 32 weeks of gestation by mothers with or without an intrauterine infection. Being a pilot study, it also aimed at feasibility check and assessment of an expected effect size. The cord blood samples of 46 infants age were subjected to direct immunofluorescent staining with monoclonal antibodies and then analyzed by flow cytometry. The percentage of CD3+ cells in neonates born by mothers with diagnosis of intrauterine infection was significantly lower than in neonates born by mothers without infection (p = 0.005; Mann-Whitney U test). The number of cells did not differ between groups. Infection present in the mother did not have an influence on the TCR αβ or TCR γδ subpopulations. Our study contributes to a better understanding of preterm infants' immune mechanisms, and sets the stage for further investigations.Peer reviewedFinal Published versio

Birthweight and risk markers for type 2 diabetes and cardiovascular disease in childhood: the Child Heart and Health Study in England (CHASE).

AIMS/HYPOTHESIS: Lower birthweight (a marker of fetal undernutrition) is associated with higher risks of type 2 diabetes and cardiovascular disease (CVD) and could explain ethnic differences in these diseases. We examined associations between birthweight and risk markers for diabetes and CVD in UK-resident white European, South Asian and black African-Caribbean children. METHODS: In a cross-sectional study of risk markers for diabetes and CVD in 9- to 10-year-old children of different ethnic origins, birthweight was obtained from health records and/or parental recall. Associations between birthweight and risk markers were estimated using multilevel linear regression to account for clustering in children from the same school. RESULTS: Key data were available for 3,744 (66%) singleton study participants. In analyses adjusted for age, sex and ethnicity, birthweight was inversely associated with serum urate and positively associated with systolic BP. After additional height adjustment, lower birthweight (per 100 g) was associated with higher serum urate (0.52%; 95% CI 0.38, 0.66), fasting serum insulin (0.41%; 95% CI 0.08, 0.74), HbA1c (0.04%; 95% CI 0.00, 0.08), plasma glucose (0.06%; 95% CI 0.02, 0.10) and serum triacylglycerol (0.30%; 95% CI 0.09, 0.51) but not with BP or blood cholesterol. Birthweight was lower among children of South Asian (231 g lower; 95% CI 183, 280) and black African-Caribbean origin (81 g lower; 95% CI 30, 132). However, adjustment for birthweight had no effect on ethnic differences in risk markers. CONCLUSIONS/INTERPRETATION: Birthweight was inversely associated with urate and with insulin and glycaemia after adjustment for current height. Lower birthweight does not appear to explain emerging ethnic difference in risk markers for diabetes

Do anti-malarials in Africa meet quality standards? The market penetration of non quality-assured artemisinin combination therapy in eight African countries

BACKGROUND: Quality of artemisinin-based combination therapy (ACT) is important for ensuring malaria parasite clearance and protecting the efficacy of artemisinin-based therapies. The extent to which non quality-assured ACT (non-QAACT), or those not granted global regulatory approval, are available and used to treat malaria in endemic countries is poorly documented. This paper uses national and sub-national medicine outlet surveys conducted in eight study countries (Benin, Kinshasa and Kantanga [Democratic Republic of the Congo, DRC], Kenya, Madagascar, Nigeria, Tanzania, Uganda and Zambia) between 2009 and 2015 to describe the non-QAACT market and to document trends in availability and distribution of non-QAACT in the public and private sector. RESULTS: In 2014/15, non-QAACT were most commonly available in Kinshasa (83%), followed by Katanga (53%), Nigeria (48%), Kenya (42%), and Uganda (33%). Non-QAACT accounted for 20% of the market share in the private sector in Kenya, followed by Benin and Uganda (19%), Nigeria (12%) and Zambia (8%); this figure was 27% in Katanga and 40% in Kinshasa. Public sector non-QAACT availability and distribution was much lower, with the exception of Zambia (availability, 85%; market share, 32%). Diverse generics and formulations were available, but non-QAACT were most commonly artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DHA PPQ), in tablet formulation, imported, and distributed in urban areas at either pharmacies or drug stores. The number of unique manufacturers supplying non-QAACT to each country ranged from 9 in Uganda to 92 in Nigeria. CONCLUSIONS: Addressing the availability and distribution of non-QAACT will require effective private sector engagement and evidence-based strategies to address provider and consumer demand for these products. Given the variation in non-QAACT markets observed across the eight study countries, active efforts to limit registration, importation and distribution of non-QAACT must be tailored to the country context, and will involve addressing complex and challenging aspects of medicine registration, private sector pharmaceutical regulation, local manufacturing and drug importation. These efforts may be critical not only to patient health and safety, but also to effective malaria control and protection of artemisinin drug efficacy in the face of spreading resistance

The action of a multidisciplinary team in the nutritional care of critically ill patients

Hospitalized patients may have special nutrient requirements imposed by a combination of malnutrition and enhanced utilization of nutrients resulting from the disease process. Nutritional support, mainly during critical stages of disease, should be provided safely and effectively. Several studies have evaluated the paper of a multidisciplinary team in the administration of a nutritional therapy. Individually, the majority of these studies are underpowered to evaluate an effect on the quality of nutritional care. With the objective to identify problems inherent to the supply of nutritional support to hospitalized patients and verify the impact of the actions of a multidisciplinary team on the quality of these procedures, we analysed articles that have been published between 1980 and 2004 about the role of the action of multidisciplinary teams in the care and nutritional outcome of hospitalized patients, especially those undergoing intensive care. The terms used for the search were: multidisciplinary team, nutritional support, parenteral nutrition, enteral feeding, critically ill, intensive care unit, critically ill child. Of 130 studies, intially identified, just 24 were selected, of which 14 compared the standard of nutritional therapy with and without the presence of a multidisciplinary team. The inadequate supply of nutrients, infection and metabolic complications and the excessive use of parenteral nutrition were the main problems detected in the supply of nutritional support to hospitalized patients. In the comparative studies, the presence of the multidisciplinary team improved the pattern of nutritional support, and reduced the incidence of complications and the costs.Pacientes hospitalizados podem ter necessidades nutricionais especiais em função da desnutrição e dos desequilíbrios metabólicos impostos pelas doenças. A terapia nutricional, principalmente nos estágios críticos das enfermidades, deve ser administrada de modo seguro e eficaz. Vários estudos têm avaliado o papel da equipe multidisciplinar na administração da terapia nutricional. Com o objetivo de identificar os problemas inerentes à administração da terapia nutricional em pacientes hospitalizados e verificar o impacto da atuação de uma equipe multidisciplinar na qualidade dos procedimentos, foi realizada uma revisão que analisou artigos publicados entre 1980 e 2004 sobre o papel da atuação de equipes multidisciplinares no cuidado e na evolução nutricional de pacientes hospitalizados, principalmente os que se encontravam sob cuidados intensivos. Os termos utilizados na pesquisa foram: multidisciplinary team, nutritional support, parente-ral nutrition, enteral feeding, critically ill, intensive care unit,critically ill child. Dos 130 estudos inicialmente identificados, foram selecionados 24, dos quais 14 compararam o padrão de terapia nutricional com e sem a presença da equipe multidisciplinar. Os principais problemas detectados na administração de terapia nutricional em pacientes hospitalizados foram a oferta inadequada de nutrientes, as complicações infecciosas e metabólicas e o uso excessivo de nutrição parenteral. Nos estudos comparativos, a presença da equipe multidisciplinar melhorou o padrão de oferta nutricional, reduziu a incidência de complicações e os custos.Universidade Federal de São Paulo (UNIFESP) Departamento de PediatriaHospital São Paulo Unidade de Cuidados Intensivos PediátricosHospital São Paulo Equipe Multidisciplinar de Terapia NutricionalUNIFESP, Depto. de PediatriaHospital São Paulo Unidade de Cuidados Intensivos PediátricosHospital São Paulo Equipe Multidisciplinar de Terapia NutricionalSciEL

Assessing a sustainable manufacturing route to lapatinib

A synthetic route to an anti-cancer drug, lapatinib, was devised to support the development of a sustainable manufacturing process in South Africa. Quantitative metrics were employed to evaluate the sustainability of the key steps of the reaction