Engineering of vitamin prototrophy in Clostridium ljungdahlii and Clostridium autoethanogenum

Clostridium autoethanogenum and Clostridium ljungdahlii are physiologically and genetically very similar strict anaerobic acetogens capable of growth on carbon monoxide as sole carbon source. While exact nutritional requirements have not been reported, we observed that for growth, the addition of vitamins to media already containing yeast extract was required, an indication that these are fastidious microorganisms. Elimination of complex components and individual vitamins from the medium revealed that the only organic compounds required for growth were pantothenate, biotin and thiamine. Analysis of the genome sequences revealed that three genes were missing from pantothenate and thiamine biosynthetic pathways, and five genes were absent from the pathway for biotin biosynthesis. Prototrophy in C. autoethanogenum and C. ljungdahlii for pantothenate was obtained by the introduction of plasmids carrying the heterologous gene clusters panBCD from Clostridium acetobutylicum, and for thiamine by the introduction of the thiC-purF operon from Clostridium ragsdalei. Integration of panBCD into the chromosome through allele-coupled exchange also conveyed prototrophy. C. autoethanogenum was converted to biotin prototrophy with gene sets bioBDF and bioHCA from Desulfotomaculum nigrificans strain CO-1-SRB, on plasmid and integrated in the chromosome. The genes could be used as auxotrophic selection markers in recombinant DNA technology. Additionally, transformation with a subset of the genes for pantothenate biosynthesis extended selection options with the pantothenate precursors pantolactone and/or beta-alanine. Similarly, growth was obtained with the biotin precursor pimelate combined with genes bioYDA from C. acetobutylicum. The work raises questions whether alternative steps exist in biotin and thiamine biosynthesis pathways in these acetogens

A genome-scale model of Clostridium autoethanogenum reveals optimal bioprocess conditions for high-value chemical production from carbon monoxide

Clostridium autoethanogenum is an industrial microbe used for the commercial-scale production of ethanol from carbon monoxide. While significant progress has been made in the attempted diversification of this bioprocess, further improvements are desirable, particularly in the formation of the high-value platform chemicals, such as 2,3-butanediol. A new, experimentally parameterised genome scale model of C. autoethanogenum predicts dramatically increased 2,3-butanediol production under non-carbon-limited conditions when thermodynamic constraints on hydrogen production are considered

Whole genome sequence and manual annotation of Clostridium autoethanogenum, an industrially relevant bacterium

Clostridium autoethanogenum is an acetogenic bacterium capable of producing high value commodity chemicals and biofuels from the C1 gases present in synthesis gas. This common industrial waste gas can act as the sole energy and carbon source for the bacterium that converts the low value gaseous components into cellular building blocks and industrially relevant products via the action of the reductive acetyl-CoA (Wood-Ljungdahl) pathway. Current research efforts are focused on the enhancement and extension of product formation in this organism via synthetic biology approaches. However, crucial to metabolic modelling and directed pathway engineering is a reliable and comprehensively annotated genome sequence

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Engineering of a Fully Human Anti-MUC-16 Antibody and Evaluation as a PET Imaging Agent

Antibodies that recognize cancer biomarkers, such as MUC16, can be used as vehicles to deliver contrast agents (imaging) or cytotoxic payloads (therapy) to the site of tumors. MUC16 is overexpressed in 80% of epithelial ovarian cancer (EOC) and 65% of pancreatic ductal adenocarcinomas (PDAC), where effective &lsquo;theranostic&rsquo; probes are much needed. This work aims to develop fully human antibodies against MUC16 and evaluate them as potential immuno-PET imaging probes for detecting ovarian and pancreatic cancers. We developed a fully human monoclonal antibody, M16Ab, against MUC16 using phage display. M16Ab was conjugated with p-SCN-Bn-DFO and radiolabeled with 89Zr. 89Zr-DFO-M16Ab was then evaluated for binding specificity and affinity using flow cytometry. In vivo evaluation of 89Zr-DFO-M16Ab was performed by microPET/CT imaging at different time points at 24&ndash;120 h post injection (p.i.) and ex vivo biodistribution studies in mice bearing MUC16-expressing OVCAR3, SKOV3 (ovarian) and SW1990 (pancreatic) xenografts. 89Zr-DFO-M16Ab bound specifically to MUC16-expressing cancer cells with an EC50 of 10nM. 89Zr-DFO-M16Ab was stable in serum and showed specific uptake and retention in tumor xenografts even after 120 h p.i. (microPET/CT) with tumor-to-blood ratios &gt; 43 for the SW1990 xenograft. Specific tumor uptake was observed for SW1990/OVCAR3 xenografts but not in MUC16-negative SKOV3 xenografts. Pharmacokinetic study shows a relatively short distribution (t1/2&alpha;) and elimination half-life (t1/2&szlig;) of 4.4 h and 99 h, respectively. In summary, 89Zr-DFO-M16Ab is an effective non-invasive imaging probe for ovarian and pancreatic cancers and shows promise for further development of theranostic radiopharmaceuticals

Blood pressure—lowering medication prescribing, its adherence to guidelines and relationship with blood pressure control at a family medicine department

Abstract Background In many resource‐constrained countries, control of blood pressure (BP) is low. Antihypertensive drug prescribing practices may influence BP control. However, adherence of prescribing to treatment guidelines may not be optimal in resource‐constrained settings. The aim of this study was to evaluate the pattern of blood pressure‐lowering medication prescribing, and how it adheres to treatment guidelines, and to identify the relationship between medication prescriptions and BP control. Methods It was a cross‐sectional study of hypertensive outpatients at the Korle Bu Teaching Hospital (KBTH) Family Medicine department (FMD)/Polyclinic. Data was collected with a validated structured form. Adherence of “prescribing” to recommendations of the 2017 Standard Treatment Guidelines of Ghana and 2018 European Society of Cardiology guidelines was assessed using a composite measure. We analyzed data with SPSS. Results About 81% (247/304) of patients received two or more antihypertensive drugs. Most patients (41%; 267/651) received calcium channel blockers (CCB), and 21.8% (142/651), 15.7% (102/651) and 12.7% (83/651) were on diuretics, angiotensin‐receptor blockers (ARBs) and angiotensin converting enzyme (ACE) inhibitors respectively. CCB plus RAS inhibitor (50%) was the most prescribed two‐drug combination. Number of BP drugs per patient had a statistically significant inverse relationship with BP control (beta Coefficient = –0.402; 95% Cl: 1.252–2.470; p = 0.015). The composite adherence score was 0.73 (moderate adherence) but Single‐pill combination (SPC) was poor (3.2%; n = 8). Conclusion Most patients received multiple‐pill combination treatment, and overall adherence to guidelines was suboptimal, largely owing to complex drug therapy. Number of drugs predicted BP control. Our findings suggest a need to prioritize simplified treatment, and implement other strategies to improve hypertension guideline adherence. Further research on the influence of SPC on BP control may inform future hypertension guidelines in Ghana and elsewhere in Africa

Quality of Life after Mastectomy with or without Breast Reconstruction and Breast-Conserving Surgery in Breast Cancer Survivors: A Cross-Sectional Study at a Tertiary Hospital in Ghana

(1) Background: Breast cancer is the leading malignancy worldwide, and in Ghana, it has a poor overall survival rate. However, approximately 50% of cases are cases of early-stage disease, and with advances in breast cancer treatment and improvements in survival, quality of life (QOL) is becoming as important as the treatment of the disease. (2) Methodology: This was a cross-sectional study of survivors who had breast-conserving surgery (BCS), mastectomy only (M) and mastectomy with breast reconstruction (BRS) from 2016 to 2020 at a tertiary hospital in Ghana, comparatively assessing their QOL using EORTC QLQ C-30 and EORTC QLQ BR-23. (3) Results: The study participants had an overall global health status (GHS) median score of 83.3 [IQR: 66.7–91.7] with no significant differences between the surgery types. The BRS group had statistically significant lower median scores for the functional scale (82.8 and 51.0) and the highest scores for the symptomatic scale (15.7 and 16.5). Body image was significantly lowest for the BRS group (83.3) [68.8–91.7] and highest (100) [91.7–100] for the BCS group (p < 0.001). (4) Conclusion: There is a need to develop support systems tailored at improving the QOL of breast cancer survivors taking into consideration the type of surgery performed

Mental health and psychosocial support in humanitarian settings: research priorities for 2021-30.

We describe an effort to develop a consensus-based research agenda for mental health and psychosocial support (MHPSS) interventions in humanitarian settings for 2021-30. By engaging a broad group of stakeholders, we generated research questions through a qualitative study (in Indonesia, Lebanon, and Uganda; n=101), consultations led by humanitarian agencies (n=259), and an expert panel (n=227; 51% female participants and 49% male participants; 84% of participants based in low-income and middle-income countries). The expert panel selected and rated a final list of 20 research questions. After rating, the MHPSS research agenda favoured applied research questions (eg, regarding workforce strengthening and monitoring and evaluation practices). Compared with research priorities for the previous decade, there is a shift towards systems-oriented implementation research (eg, multisectoral integration and ensuring sustainability) rather than efficacy research. Answering these research questions selected and rated by the expert panel will require improved partnerships between researchers, practitioners, policy makers, and communities affected by humanitarian crises, and improved equity in funding for MHPSS research in low-income and middle-income countries

Additional file 1: of Whole genome sequence and manual annotation of Clostridium autoethanogenum, an industrially relevant bacterium

Discrepancies occurring between the current and Brown et al. finished genome sequence of C. autoethanogenum. This table shows all of the discrepancies that occur when our finished genome sequence (CLAU) is mapped against the Brown et al. finished genome sequence (BRO). Mutation column describes the mutation occurring in the CLAU genome compared to the BRO genome. Gene / region gives the gene name where the discrepancy occurs, ← / ← or similar denotes that the discrepancy occurred in a non-coding region between the named genes. Homopolymer length indicates the number of the same base occurring consecutively at the site of the discrepancy. Amino acid length gives the annotated protein length of the gene in which the discrepancy occurs, *indicates protein codes for multiple stop codons and ^indicates that no stop codon was found in the annotation. The sequence identity is relative to the CLAU C. autoethanogenum genome sequence when protein BLAST searched on the NCBI database. CLAU, C. autoethanogenum finished genome sequence in present study; CLJU, C. ljungdahlii DSM 13528 finished genome sequence (GCA_000143685.1); BRO, Brown et al. C. autoethanogenum finished genome sequence (GCA_000484505.1); CAUT, Bruno-Barcena et al. C. autoethanogenum draft genome sequence (GCA_000427255.1); NF, not found. (DOCX 73 kb