Cellular Activity of \u3ci\u3eSalmonella\u3c/i\u3e Typhimurium ArtAB Toxin and Its Receptor-Binding Subunit

Salmonellosis is among the most reported foodborne illnesses in the United States. The Salmonella enterica Typhimurium DT104 phage type, which is associated with multidrug-resistant disease in humans and animals, possesses an ADP-ribosylating toxin called ArtAB. Full-length artAB has been found on a number of broad-host-range non-typhoidal Salmonella species and serovars. ArtAB is also homologous to many AB5 toxins from diverse Gram-negative pathogens, including cholera toxin (CT) and pertussis toxin (PT), and may be involved in Salmonella pathogenesis, however, in vitro cellular toxicity of ArtAB has not been characterized. artAB was cloned into E. coli and initially isolated using a histidine tag (ArtABHIS) and nickel chromatography. ArtABHIS was found to bind to African green monkey kidney epithelial (Vero) cells using confocal microscopy and to interact with glycans present on fetuin and monosialotetrahexosylganglioside (GM1) using ELISA. Untagged, or native, holotoxin (ArtAB), and the pentameric receptor-binding subunit (ArtB) were purified from E. coli using fetuin and D-galactose affinity chromatography. ArtAB and ArtB metabolic and cytotoxic activities were determined using Vero and Chinese hamster ovary (CHO) epithelial cells. Vero cells were more sensitive to ArtAB, however, incubation with both cell types revealed only partial cytotoxicity over 72 h, similar to that induced by CT. ArtAB induced a distinctive clustering phenotype on CHO cells over 72 h, similar to PT, and an elongated phenotype on Vero cells, similar to CT. The ArtB binding subunit alone also had a cytotoxic effect on CHO cells and induced morphological rounding. Results indicate that this toxin induces distinctive cellular outcomes. Continued biological characterization of ArtAB will advance efforts to prevent disease caused by non-typhoidal Salmonella

Mapping local patterns of childhood overweight and wasting in low- and middle-income countries between 2000 and 2017

A double burden of malnutrition occurs when individuals, household members or communities experience both undernutrition and overweight. Here, we show geospatial estimates of overweight and wasting prevalence among children under 5 years of age in 105 low- and middle-income countries (LMICs) from 2000 to 2017 and aggregate these to policy-relevant administrative units. Wasting decreased overall across LMICs between 2000 and 2017, from 8.4% (62.3 (55.1–70.8) million) to 6.4% (58.3 (47.6–70.7) million), but is predicted to remain above the World Health Organization’s Global Nutrition Target of <5% in over half of LMICs by 2025. Prevalence of overweight increased from 5.2% (30 (22.8–38.5) million) in 2000 to 6.0% (55.5 (44.8–67.9) million) children aged under 5 years in 2017. Areas most affected by double burden of malnutrition were located in Indonesia, Thailand, southeastern China, Botswana, Cameroon and central Nigeria. Our estimates provide a new perspective to researchers, policy makers and public health agencies in their efforts to address this global childhood syndemic

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

Probing Stability of Lyophilized Vaccine Against Bovine Mastitis

Staphylococcus aureus is a leading cause of mastitis, or infections in the udder, in dairy cows. Mastitis causes significant financial losses and with the rapid increase of antibiotic resistant bacteria, such as Methicillin-resistant Staphylococcus aureus (MRSA), it is vital to create alternative ways to fight these pathogens. Our lab is developing and testing a mucosal chimeric vaccine against bovine mastitis containing two surface antigens from S. aureus. The genes for the adhesins isdA and clfA were cloned with those for Vibrio cholerae cholera toxin A2/B (CTA2/B) to create the intranasally administered vaccine. The purification of this vaccine was scaled up using 1L culture volumes and D-galactose agarose affinity purification. Purified proteins were analyzed by SDS-PAGE and bicinchoninic (BCA) assay. Currently we have produced over 15 mg of vaccine for use in future bovine vaccine challenge studies. Lyophilization is a well-recognized method in the pharmaceutical industry used to store biologically active drugs that are not stable in solution, or to prolong the shelf-lives of drugs. Excipients can have a great influence on performance and stability of lyophilized drugs. IsdA chimera was lyophilized using a variety of excipients and stored at different temperatures. The stability was analyzed using SDS-PAGE and BCA assay

Cloning and Purification of the ArtB Binding Subunit From a Novel Toxin in Salmonella enterica Typhimurium

According to the CDC, Salmonella is responsible for about 1.35 million infections, 26,500 hospitalizations, and 420 deaths in the United States every year. Food is the source for most of these illnesses. Salmonella enterica is common in cattle and has a number of serovars that are pathogenic in humans. Many Salmonella serovars encode for the AB5 toxin, or ArtAB, which is similar in structure to pertussis toxin. The pentameric B subunit is non-covalently linked to the A subunit, thus allowing separation of the receptor-binding B subunit from the holotoxin. We hypothesize that being able to extract and use ArtAB or the non-toxic ArtB subunit for vaccines could decrease prevalence of Salmonella infections in cows and potentially prevent transmission from cattle and agriculture to humans. The B subunit can be transformed into E. coli using a vector plasmid and then purified and extracted using D-galactose and fetuin affinity chromatography methods. Our results show that we have successfully cloned the B subunit into a plasmid, transformed the plasmid into E. coli, and had ArtB protein expression. Further research will examine the toxicity of ArtAB and ArtB to determine whether or not they are suitable for use in vaccines

Role of Autophagy in Osteogenic Differentiation of Mesenchymal Stem Cells Under Simulated Microgravity

Microgravity-induced bone loss is a significant problem for astronauts during spaceflight, and will limit the potential for future long duration spaceflight. The increased bone resorption by osteoclasts and the reduced bone formation by osteoblasts are largely responsible for this problem. Mesenchymal stem cells (MSCs) are multipotent cells that are capable of osteogenic, chondrogenic, adipogenic, and myogenic differentiation. MSCs play important roles in the regeneration of connective tissues, including bone. Microgravity has been shown to inhibit osteogenic differentiation of MSCs while increasing adipogenic differentiation. Autophagy is a key catabolic process that targets cellular contents for degradation in cellular stress responses and survival. Autophagy plays important roles in the maintenance of pluripotency and the commitment of MSCs to different lineages, especially in the osteoblastic lineage. Therefore, we hypothesized that modulation of autophagy regulates microgravity-induced inhibition of osteogenic differentiation of MSCs and manipulation of autophagy levels can promote MSC differentiation into osteoblasts. We examined autophagy levels MSCs and the effect of autophagy modulation on MSC differentiation into osteoblasts under simulated microgravity using custom-made Clinostat. Osteoblast formation was assessed by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR), Next Generation RNA Sequencing (NGS) and Quantitative Shotgun Proteomics

Effects of Doxorubicin on Cardiac Fibroblasts and the Extracellular Matrix

Cardiotoxicity has been associated with various types of chemotherapeutic drugs contributing to a plethora of cardiac insults and is a significant side effect when treating cancer. Many highly effective anticancer drugs are severely dose dependent, and at higher doses can lead to: cardiac arrhythmias, hypertension, and lethal cardiomyopathy. A well known example of this cardiotoxic side effect is Doxorubicin, a common chemotherapeutic used to treat cancers of the breast, ovary, bladder, and thyroid. Extensive research has shown that high doses of doxorubicin detrimentally alters the normal function of cardiac fibroblasts and cardiomyocytes. In contrast to the extensive research on the toxic effects of chemotherapeutics like doxorubicin in cardiomyocytes, little is known on the effects in cardiac fibroblasts and mechanisms of these drugs on the cardiac extracellular matrix (cECM). We show that doxorubicin has a direct impact on cardiac fibroblasts and in turn the function of the cECM, indicating that the cECM plays an important role in cardiac toxicity induced by doxorubicin

Wound Healing Potential of Human Umbilical Cord Blood and Placental Membrane Derived Products

Wound Healing is a complex process involving hemostasis, inflammation, proliferation and maturation stages. Treatment of chronic wound is a significant challenge for clinicians and warrant urgent need for alternate therapies. Stem cell-based therapy is one of the techniques employed for wound healing but, a successful transplantation is depended upon homing, engraftment and repopulation of stem cells. Due to involvement of multiple and complex pathways the success of transplantation can be hindered. Another approach is to use the cell signaling molecules and growth factors of stem cells to regenerate the damaged site by recruiting host’s stem cells. Cellular allograft derived from umbilical cord blood and dehydrated amniotic membranes are an enriched source of cytokines and growth factors needed for activation of mesenchymal stromal cells to participate in wound healing process. Using bone marrow derived mesenchymal stromal cells as model system, we assessed the regenerative potential of BioBurst Fluid (an umbilical cord blood derived product) and Burst Binate Patch (an amniotic tissue derived product) using cellular proliferation, migration, osteogenesis and angiogenesis assays. Our data shows that the cytokines and growth factors associated with these products support biological processes involved in bone healing

Cellular Activity of Salmonella Typhimurium ArtAB Toxin and Its Receptor-Binding Subunit

Salmonellosis is among the most reported foodborne illnesses in the United States. The Salmonellaenterica Typhimurium DT104 phage type, which is associated with multidrug-resistant disease in humans and animals, possesses an ADP-ribosylating toxin called ArtAB. Full-length artAB has been found on a number of broad-host-range non-typhoidal Salmonella species and serovars. ArtAB is also homologous to many AB5 toxins from diverse Gram-negative pathogens, including cholera toxin (CT) and pertussis toxin (PT), and may be involved in Salmonella pathogenesis, however, in vitro cellular toxicity of ArtAB has not been characterized. artAB was cloned into E. coli and initially isolated using a histidine tag (ArtABHIS) and nickel chromatography. ArtABHIS was found to bind to African green monkey kidney epithelial (Vero) cells using confocal microscopy and to interact with glycans present on fetuin and monosialotetrahexosylganglioside (GM1) using ELISA. Untagged, or native, holotoxin (ArtAB), and the pentameric receptor-binding subunit (ArtB) were purified from E. coli using fetuin and d-galactose affinity chromatography. ArtAB and ArtB metabolic and cytotoxic activities were determined using Vero and Chinese hamster ovary (CHO) epithelial cells. Vero cells were more sensitive to ArtAB, however, incubation with both cell types revealed only partial cytotoxicity over 72 h, similar to that induced by CT. ArtAB induced a distinctive clustering phenotype on CHO cells over 72 h, similar to PT, and an elongated phenotype on Vero cells, similar to CT. The ArtB binding subunit alone also had a cytotoxic effect on CHO cells and induced morphological rounding. Results indicate that this toxin induces distinctive cellular outcomes. Continued biological characterization of ArtAB will advance efforts to prevent disease caused by non-typhoidal Salmonella

Temporal distribution and zoning of nitrate and fluoride concentrations in Behbahan drinking water distribution network and health risk assessment by using sensitivity analysis and Monte Carlo simulation

International audienceThe present study aimed to investigate the spatial distribution and zoning of nitrate and fluoride concentrations in the drinking water distribution network of Behbahan city and the health risk caused by their concentration using sensitivity analysis and the Monte Carlo simulation during 2018 to 2019.In this cross-sectional study, 90 samples were collected from 45 points of the city's drinking water distribution network in the two high-rainy and low-rainy seasons.Nitrate and fluoride concentration was measured byDR 5000 spectrophotometer and its non-carcinogenic impacts were calculated using EDI and HQ.The results showed that the average nitrate concentrations in high and low rainy seasons in the study area were 15.05 and 13.35 mg l(-1), respectively.Also, the average concentrations of fluoride in high and low-rainy seasons were 0.67 and 0.76 mg L-1, respectively.The fluoride concentration in the high-rainy season showed a decreasing trend from north to south.The nitrate concentration decreased from north to south and southwest.The amount of nitrate HQ in the age groups corresponding to children, adolescents, adults and infants were 0.5, 0.4, 0.34, and 0.07, respectively.The fluoride HQ levels in the age groups corresponding to children, adolescents, adults and infants were 0.7, 0.5, 0.05, and 0.1, respectively.These results showed that children were the significant at-risk group due to these ions.According to the Monte Carlo simulator, 95th percentile caused by exposure to nitrate and fluoride ions in all age groups was more than one.Based on the sensitivity analysis, the most efficient factor in the health risk caused by nitrate and fluoride ions was the concentration of these ions in the drinking water distribution network