Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

BACKGROUND: Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes. RESULTS: C. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands. CONCLUSIONS: C. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide modified antigens for immunological studies and vaccination, including live subunit vaccines, and might be useful to co-express MOMP with other chlamydial membrane proteins

Multiple Palmitoyltransferases Are Required for Palmitoylation-dependent Regulation of Large Conductance Calcium- and Voltage-activated Potassium Channels

Palmitoylation is emerging as an important and dynamic regulator of ion channel function; however, the specificity with which the large family of acyl palmitoyltransferases (zinc finger Asp-His-His-Cys type-containing acyl palmitoyltransferase (DHHCs)) control channel palmitoylation is poorly understood. We have previously demonstrated that the alternatively spliced stress-regulated exon (STREX) variant of the intracellular C-terminal domain of the large conductance calcium- and voltage-activated potassium (BK) channels is palmitoylated and targets the STREX domain to the plasma membrane. Using a combined imaging, biochemical, and functional approach coupled with loss-of-function (small interfering RNA knockdown of endogenous DHHCs) and gain-of-function (overexpression of recombinant DHHCs) assays, we demonstrate that multiple DHHCs control palmitoylation of the C terminus of STREX channels, the association of the STREX domain with the plasma membrane, and functional channel regulation. Cysteine residues 12 and 13 within the STREX insert were the only endogenously palmitoylated residues in the entire C terminus of the STREX channel. Palmitoylation of this dicysteine motif was controlled by DHHCs 3, 5, 7, 9, and 17, although DHHC17 showed the greatest specificity for this site upon overexpression of the cognate DHHC. DHHCs that palmitoylated the channel also co-assembled with the channel in co-immunoprecipitation experiments, and knockdown of any of these DHHCs blocked regulation of the channel by protein kinase A-dependent phosphorylation. Taken together our data reveal that a subset of DHHCs controls STREX palmitoylation and function and suggest that DHHC17 may preferentially target cysteine-rich domains. Finally, our approach may prove useful in elucidating the specificity of DHHC palmitoylation of intracellular domains of other ion channels and transmembrane proteins

Obesogenic and diabetogenic effects of high-calorie nutrition require adipocyte BK channels:Adipocyte BK protects from overwhelming BW gain

Elevated adipose tissue expression of the Ca2+- and voltage-activated K+ (BK) channel was identified in morbidly obese men carrying a BK gene variant supporting the hypothesis that K+ channels affect metabolic responses of fat cells to nutrients. To establish the role of endogenous BKs for fat cell maturation, storage of excess dietary fat and body-weight (BW) gain we studied a gene-targeted mouse model with a global ablation of the BK channel (BKL1/L1) and adipocyte-specific BK-deficient (adipoqBKL1/L2) mice. Global BK deficiency afforded protection from high-fat-diet (HFD) induced BW gain and excessive fat accumulation. Expansion of white adipose tissue-derived epididymal BKL1/L1 pre-adipocytes and their differentiation to lipid-filled mature adipocytes in vitro, however, were improved. Moreover, BW gain and total fat masses of usually super-obese ob/ob mice were significantly attenuated in the absence of BK together supporting a central or peripheral role for BKs in the regulatory system that controls adipose tissue and weight. Accordingly, HFD-fed adipoqBKL1/L2 mutants presented with a reduced total BW and overall body fat mass, smaller adipocytes and reduced leptin levels. Protection from pathologic weight gain in the absence of adipocyte BKs was beneficial for glucose handling and related to an increase in body core temperature due to higher levels of uncoupling protein 1 as well as low abundance of the proinflammatory interleukin-6 as a common risk factor for diabetes and metabolic abnormalities. This suggests that adipocyte BK activity is at least partially responsible for excessive BW gain under high-caloric conditions suggesting BK channels as promising drug targets for pharmacotherapy of metabolic disorders and obesity

Twelve-month observational study of children with cancer in 41 countries during the COVID-19 pandemic

Introduction Childhood cancer is a leading cause of death. It is unclear whether the COVID-19 pandemic has impacted childhood cancer mortality. In this study, we aimed to establish all-cause mortality rates for childhood cancers during the COVID-19 pandemic and determine the factors associated with mortality. Methods Prospective cohort study in 109 institutions in 41 countries. Inclusion criteria: children <18 years who were newly diagnosed with or undergoing active treatment for acute lymphoblastic leukaemia, non-Hodgkin's lymphoma, Hodgkin lymphoma, retinoblastoma, Wilms tumour, glioma, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, medulloblastoma and neuroblastoma. Of 2327 cases, 2118 patients were included in the study. The primary outcome measure was all-cause mortality at 30 days, 90 days and 12 months. Results All-cause mortality was 3.4% (n=71/2084) at 30-day follow-up, 5.7% (n=113/1969) at 90-day follow-up and 13.0% (n=206/1581) at 12-month follow-up. The median time from diagnosis to multidisciplinary team (MDT) plan was longest in low-income countries (7 days, IQR 3-11). Multivariable analysis revealed several factors associated with 12-month mortality, including low-income (OR 6.99 (95% CI 2.49 to 19.68); p<0.001), lower middle income (OR 3.32 (95% CI 1.96 to 5.61); p<0.001) and upper middle income (OR 3.49 (95% CI 2.02 to 6.03); p<0.001) country status and chemotherapy (OR 0.55 (95% CI 0.36 to 0.86); p=0.008) and immunotherapy (OR 0.27 (95% CI 0.08 to 0.91); p=0.035) within 30 days from MDT plan. Multivariable analysis revealed laboratory-confirmed SARS-CoV-2 infection (OR 5.33 (95% CI 1.19 to 23.84); p=0.029) was associated with 30-day mortality. Conclusions Children with cancer are more likely to die within 30 days if infected with SARS-CoV-2. However, timely treatment reduced odds of death. This report provides crucial information to balance the benefits of providing anticancer therapy against the risks of SARS-CoV-2 infection in children with cancer

Harvestplus : Breeding crops for better nutrition

Micronutrient malnutrition, the so-called hidden hunger, affects more than one-half of the world's population, especially women and preschool children in developing countries. Despite past progress in controlling micronutrient decencies through supplementation and food fortification, new approaches are needed to expand the reach of food-based interventions. Biofortification, a new approach that relies on conventional plant breeding and modern biotechnology to increase the micronutrient density of staple crops, holds great promise for improving the nutritional status and health of poor populations in both rural and urban areas of the developing world. HarvestPlus, a research program implemented with the international research institutes of the CGIAR, targets a multitude of crops that are a regular part of the staple-based diets of the poor and breeds them to be rich in iron, zinc, and provitamin A. This paper emphasizes the need for interdisciplinary research and addresses the key research issues and methodological considerations for success. The major activities to be undertaken are broadly grouped into research related to nutrition research and impact analysis, and research considerations for delivering biofortified crops to end-users effectively. The paper places particular emphasis on the activities of the plant breeding and genetics component of this multidisciplinary program. The authors argue that for biofortification to succeed, product profiles developed by plant breeders must be driven by nutrition research and impact objectives and that nutrition research must understand that the probability of success for biofortified crops increases substantially when product concepts consider farmer adoption and, hence, agronomic superiority

Biofortification of durum wheat with zinc and iron

Ismail Cakmak,1,2 Wolfgang H. Pfeiffer,3 and Bonnie McClafferty4 ABSTRACT Cereal Chem. 86(0):000–000 Micronutrient malnutrition affects over 2 billion people in the developing world. Iron (Fe) deficiency alone affects >47% of all preschool aged children globally, often leading to impaired physical growth, mental development, and learning capacity. Zinc (Zn) deficiency, like iron, is thought to affect billions of people, hampering growth and development, and destroying immune systems. In many micronutrient-deficient regions, wheat is the dominant staple food making up >50% of the diet. Biofortification, or harnessing the powers of plant breeding to improve the nutritional quality of foods, is a new approach being used to improve the nutrient content of a variety of staple crops. Durum wheat in particular has been quite responsive to breeding for nutritional quality by making full use of the genetic diversity of Fe and Zn concentrations in wild and synthetic parents. Micronutrient concentration and genetic diversity has been well explored under the HarvestPlus biofortification research program, and very positive associations have been confirmed between grain concentrations of protein, Zn, and Fe. Yet some work remains to adequately explain genetic control and molecular mechanisms affecting the accumulation of Zn and Fe in grain. Further, evidence suggests that nitrogen (N) nutritional status of plants can have a positive impact on root uptake and the deposition of micronutrients in seed. Extensive research has been completed on the role of Zn fertilizers in increasing the Zn density of grain, suggesting that where fertilizers are available, making full use of Zn fertilizers can provide an immediate and effective option to increase grain Zn concentration, and productivity in particular, under soil conditions with severe Zn deficienc

Surface expression, single-channel analysis and membrane topology of recombinant Major Outer Membrane Protein-9

<p><b>Copyright information:</b></p><p>Taken from "Surface expression, single-channel analysis and membrane topology of recombinant Major Outer Membrane Protein"</p><p>BMC Microbiology 2005;5():5-5.</p><p>Published online 26 Jan 2005</p><p>PMCID:PMC549562.</p><p>Copyright © 2005 Findlay et al; licensee BioMed Central Ltd.</p>o 3 channels are open during ramp (a), and 2 of the channels close in succession at about +80 mV. In ramp (b), 1 channel is open at 0 mV, and 2 channels are open at +100 mV. B. Channel currents in the presence of 500 mM KCl and during two voltage ramps (as in A). Note the reduction in single-current amplitudes, and current reversal at 0 mV as in A. C. Currents during two voltage ramps in asymmetric KCl, 500 mM 50 mM . Note the shift in equilibrium potential (E) from 0 mV to -30 mV (arrowed). Positive (upgoing) currents at 0 mV indicate a net flux of Kto in the absence of an electrical driving force. Equilibrium currents in symmetric 500 mM KCl at a constant holding potential of +100 mV, showing brief single-channel closures and voltage-dependent inactivation of all 3 channels within 5 s