Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation

Genome editing with site-specific endonucleases has implications for basic biomedical research as well as for gene therapy. We generated helper-dependent, capsid-modified adenovirus (HD-Ad5/35) vectors for zinc-finger nuclease (ZFN)- or transcription activator-like effector nuclease (TALEN)-mediated genome editing in human CD34+ hematopoietic stem cells (HSCs) from mobilized adult donors. The production of these vectors required that ZFN and TALEN expression in HD-Ad5/35 producer 293-Cre cells was suppressed. To do this, we developed a microRNA (miRNA)-based system for regulation of gene expression based on miRNA expression profiling of 293-Cre and CD34+ cells. Using miR-183-5p and miR-218-5p based regulation of transgene gene -expression, we first produced an HD-Ad5/35 vector expressing a ZFN specific to the HIV coreceptor gene ccr5. We demonstrated that HD-Ad5/35. ZFNmiR vector conferred ccr5 knock out in primitive HSC (i.e., long-term culture initiating cells and NOD/SCID repopulating cells). The ccr5 gene disruption frequency achieved in engrafted HSCs found in the bone marrow of transplanted mice is clinically relevant for HIV therapy considering that these cells can give rise to multiple lineages, including all the lineages that represent targets and reservoirs for HIV. We produced a second HD-Ad5/35 vector expressing a TALEN targeting the DNase hypersensitivity region 2 (HS2) within the globin locus control region. This vector has potential for targeted gene correction in hemoglobinopathies. The miRNA regulated HD-Ad5/35 vector platform for expression of site-specific endonucleases has numerous advantages over currently used vectors as a tool for genome engineering of HSCs for therapeutic purposes.

Systematic comparison of plasma EBV DNA, anti-EBV antibodies and miRNA levels for early detection and prognosis of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is originated from the epithelial cells of nasopharynx, Epstein-Barr virus (EBV)-associated and has the highest incidence and mortality rates in Southeast Asia. Late presentation is a common issue and early detection could be the key to reduce the disease burden. Sensitivity of plasma EBV DNA, an established NPC biomarker, for Stage I NPC is controversial. Most newly reported NPC biomarkers have neither been externally validated nor compared to the established ones. This causes difficulty in planning for cost-effective early detection strategies. Our study systematically evaluated six established and four new biomarkers in NPC cases, population controls and hospital controls. We showed that BamHI-W 76 bp remains the most sensitive plasma biomarker, with 96.7% (29/30), 96.7% (58/60) and 97.4% (226/232) sensitivity to detect Stage I, early stage and all NPC, respectively. Its specificity was 94.2% (113/120) against population controls and 90.4% (113/125) against hospital controls. Diagnostic accuracy of BamHI-W 121 bp and ebv-miR-BART7-3p were validated. Hsa-miR-29a-3p and hsa-miR-103a-3p were not, possibly due to lower number of advanced stage NPC cases included in this subset. Decision tree modeling suggested that combination of BamHI-W 76 bp and VCA IgA or EA IgG may increase the specificity or sensitivity to detect NPC. EBNA1 99 bp could identify NPC patients with poor prognosis in early and advanced stage NPC. Our findings provided evidence for improvement in NPC screening strategies, covering considerations of opportunistic screening, combining biomarkers to increase sensitivity or specificity and testing biomarkers from single sampled specimen to avoid logistic problems of resampling

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Worldwide trends in underweight and obesity from 1990 to 2022: a pooled analysis of 3663 population-representative studies with 222 million children, adolescents, and adults

Background Underweight and obesity are associated with adverse health outcomes throughout the life course. We estimated the individual and combined prevalence of underweight or thinness and obesity, and their changes, from 1990 to 2022 for adults and school-aged children and adolescents in 200 countries and territories. Methods We used data from 3663 population-based studies with 222 million participants that measured height and weight in representative samples of the general population. We used a Bayesian hierarchical model to estimate trends in the prevalence of different BMI categories, separately for adults (age ≥20 years) and school-aged children and adolescents (age 5–19 years), from 1990 to 2022 for 200 countries and territories. For adults, we report the individual and combined prevalence of underweight (BMI <18·5 kg/m2) and obesity (BMI ≥30 kg/m2). For schoolaged children and adolescents, we report thinness (BMI <2 SD below the median of the WHO growth reference) and obesity (BMI >2 SD above the median). Findings From 1990 to 2022, the combined prevalence of underweight and obesity in adults decreased in 11 countries (6%) for women and 17 (9%) for men with a posterior probability of at least 0·80 that the observed changes were true decreases. The combined prevalence increased in 162 countries (81%) for women and 140 countries (70%) for men with a posterior probability of at least 0·80. In 2022, the combined prevalence of underweight and obesity was highest in island nations in the Caribbean and Polynesia and Micronesia, and countries in the Middle East and north Africa. Obesity prevalence was higher than underweight with posterior probability of at least 0·80 in 177 countries (89%) for women and 145 (73%) for men in 2022, whereas the converse was true in 16 countries (8%) for women, and 39 (20%) for men. From 1990 to 2022, the combined prevalence of thinness and obesity decreased among girls in five countries (3%) and among boys in 15 countries (8%) with a posterior probability of at least 0·80, and increased among girls in 140 countries (70%) and boys in 137 countries (69%) with a posterior probability of at least 0·80. The countries with highest combined prevalence of thinness and obesity in school-aged children and adolescents in 2022 were in Polynesia and Micronesia and the Caribbean for both sexes, and Chile and Qatar for boys. Combined prevalence was also high in some countries in south Asia, such as India and Pakistan, where thinness remained prevalent despite having declined. In 2022, obesity in school-aged children and adolescents was more prevalent than thinness with a posterior probability of at least 0·80 among girls in 133 countries (67%) and boys in 125 countries (63%), whereas the converse was true in 35 countries (18%) and 42 countries (21%), respectively. In almost all countries for both adults and school-aged children and adolescents, the increases in double burden were driven by increases in obesity, and decreases in double burden by declining underweight or thinness. Interpretation The combined burden of underweight and obesity has increased in most countries, driven by an increase in obesity, while underweight and thinness remain prevalent in south Asia and parts of Africa. A healthy nutrition transition that enhances access to nutritious foods is needed to address the remaining burden of underweight while curbing and reversing the increase in obesit

The Development of an Automated Multi-Spit Lamb Rotisserie Machine for Improved Productivity

Innovations in food manufacturing support the agenda for sustainable development goal 9 (SDG9) on industry, innovation and infrastructure. Pursuant to this goal, this study aims to develop an automated multi-spit lamb rotisserie machine that potentially improves the lamb-roasting productivity for small and medium enterprises (SMEs). The conceptualisation involved patents, scholarly literature and product reviews of lamb-roasting devices. The design and analysis are performed using Autodesk Inventor 2019. A scaled-down prototype is developed and tested with (1) roasting output, (2) roasting time and (3) temperature stability tests. The data for test (1) are analysed by comparing the means between control and experimental groups. The data for tests (2) and (3) are analysed using the t-test and Mann–Whitney U test, respectively. Significant differences are observed in tests (1) and (2), with outcomes being in favour of the proposed invention. The prototype cooks 92.27% faster with 700% more meat than a regular lamb roaster. It also cooks at a stable temperature. The cost analysis indicated that this invention could be sold at USD 278 if mass-produced. The design is structurally simple, inexpensive and easy to manufacture, allowing SMEs that rely on traditional spit-based machines to enhance their ability in producing roast lamb

Clinical Characteristics and In-Hospital Outcomes in Dialysis Patients with Septic Arthritis

Background and Objectives: Septic arthritis is a medical emergency associated with high morbidity and mortality. The incidence rate of septic arthritis among dialysis patients is higher than the general population, and dialysis patients with bacteremia frequently experience adverse outcomes. The aim of this study was to identify the clinical features and risk factors for longer hospital length of stay (LOS), positive blood culture, and in-hospital mortality in dialysis patients with septic arthritis. Materials and Methods: The medical records of 52 septic arthritis dialysis patients admitted to our hospital from 1 January 2009 to 31 December 2020 were analyzed. The primary outcomes were bacteremia and in-hospital mortality. Variables were compared, and risk factors were evaluated using linear and logistic regression models. Results: Twelve (23.1%) patients had positive blood cultures. A tunneled cuffed catheter for dialysis access was used in eight (15.4%) patients, and its usage rate was significantly higher in patients with positive blood culture than in those with negative blood culture (41.7 vs. 7.5%, p = 0.011). Fever was present in 15 (28.8%) patients, and was significantly more frequent in patients with positive blood culture (58.3 vs. 20%, p = 0.025). The most frequently involved site was the hip (n = 21, 40.4%). The most common causative pathogen was Gram-positive cocci, with MRSA (n = 7, 58.3%) being dominant. The mean LOS was 29.9 ± 25.1 days. The tunneled cuffed catheter was a significant predictor of longer LOS (Coef = 0.49; Cl 0.25–0.74; p p = 0.037) and tunneled cuffed catheter (OR = 7.60; Cl 1.31–44.02; p = 0.024). The predictor of mortality was tunneled cuffed catheter (OR = 14.33; Cl 1.12–183.18; p = 0.041). Conclusions: In the dialysis population, patients with tunneled cuffed catheter for dialysis access had a significantly longer hospital LOS. Tunneled cuffed catheter and fever were independent predictors of positive blood culture, and tunneled cuffed catheter was the predictor of in-hospital mortality. The recognition of the associated factors allows for risk stratification and determination of the optimal treatment plan in dialysis patients with septic arthritis

Systematic comparison of plasma EBV DNA, anti‐EBV antibodies and miRNA levels for early detection and prognosis of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is originated from the epithelial cells of nasopharynx, Epstein–Barr virus (EBV)‐associated and has the highest incidence and mortality rates in Southeast Asia. Late presentation is a common issue and early detection could be the key to reduce the disease burden. Sensitivity of plasma EBV DNA, an established NPC biomarker, for Stage I NPC is controversial. Most newly reported NPC biomarkers have neither been externally validated nor compared to the established ones. This causes difficulty in planning for cost‐effective early detection strategies. Our study systematically evaluated six established and four new biomarkers in NPC cases, population controls and hospital controls. We showed that BamHI‐W 76 bp remains the most sensitive plasma biomarker, with 96.7% (29/30), 96.7% (58/60) and 97.4% (226/232) sensitivity to detect Stage I, early stage and all NPC, respectively. Its specificity was 94.2% (113/120) against population controls and 90.4% (113/125) against hospital controls. Diagnostic accuracy of BamHI‐W 121 bp and ebv‐miR‐BART7‐3p were validated. Hsa‐miR‐29a‐3p and hsa‐miR‐103a‐3p were not, possibly due to lower number of advanced stage NPC cases included in this subset. Decision tree modeling suggested that combination of BamHI‐W 76 bp and VCA IgA or EA IgG may increase the specificity or sensitivity to detect NPC. EBNA1 99 bp could identify NPC patients with poor prognosis in early and advanced stage NPC. Our findings provided evidence for improvement in NPC screening strategies, covering considerations of opportunistic screening, combining biomarkers to increase sensitivity or specificity and testing biomarkers from single sampled specimen to avoid logistic problems of resampling