Integration of a non-homologous end-joining pathway into prokaryotic cells to enable repair of double-stranded breaks induced by Cpf1

The CRISPR-Cpf1 induces DNA double stranded breaks (DSBs) that need to be repaired by either homology-directed repair (HDR) or non-homologous end joining (NHEJ) repair mechanisms. Most prokaryotes lack the NHEJ repair system and repair DSBs by HDR when template DNA is provided. The human bacterial commensal Escherichia coli can repair DNA DSB damage by HDR or by the alternative method of microhomology mediated end joining (MMEJ). MMEJ can enable end resection after RecBCD exonuclease activity deletes DNA between microhomologous regions. This can result in the formation of small to undesirable large deletions between these microhomologies. Up until this point, a specific and precise gene knockout tool that does not induce extensive deletions has not been developed for use in E. coli or other NHEJ deficient bacteria. Hence, a novel strategy was developed in this study to induce DSBs by CRISPR-Cpf1, inhibit RecBCD exonuclease activity and enable repair by the expression of compatible plasmid-borne genes supplied in trans. This strategy required the development of a compatible plasmid system that could encode an arabinose inducible FnCpf1 effector from a chloramphenicol resistant p15A oriV replicon and a T7 RNA polymerase expressed crRNA (to guide FnCpf1 to its DNA target site) from a Cpf1 guide module residing on an ampicillin resistant promoterless ColE1 plasmid vector to induce DSB formation within E. coli BL21 DE3 lacZω. Surprisingly, the results revealed that the Cpf1 induction did not compromise cell viability in the induced compared to the uninduced state. In addition, a LacI de-repressible plasmid-borne multi-component NHEJ repair system containing the gam gene from phage λ (to repress the RecBCD complex), the Ku DNA end-binding protein from Mycobacterium smegmatis (to protect DSB ends from exonuclease attack) and the bacteriophage T4 DNA ligase gene (to promote ligation of DSB termini) was developed to enhance DSB end resection. The repair system was incorporated into the broad host range (BHR) plasmid pBBR1 which could be conjugally transferred into a wide variety of bacterial backgrounds. Further work needs to be conducted now to investigate if the constructed system can be used to effectively edit the genomes of not only E. coli but also other Gram-negative bacteria

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

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 middle-income 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 low-income 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 low-income, 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

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

Preparation and Characterization of Poly(ethylene-co-vinyl alcohol)/poly(ε-caprolactone) Blend for Bioscaffolding Applications

In order to improve the cell adhesion on poly(&epsilon;-caprolactone) (PCL) scaffolds, poly(ethylene-co-vinyl alcohol) (E-VAL) which has hydroxyl groups capable of developing hydrogen bonds with celling was blended with this polymer. To reach this goal, a series of E-VAL/PCL blends with different compositions were prepared by the solvent casting method. The miscibility of the polymer blend was proved by differential scanning calorimetry and Fourier-transform infrared spectroscopy spectrometry. Furthermore, the mechanical properties of the polymer blends were assessed in their wet state by dynamic mechanical analysis. The surfaces wettability of blends and their components were examined through static contact angle measurements. The pore interconnections in the resulted scaffolds were achieved by the incorporation of naphthalene microparticles which were used as porogen and then removed in its gas state by sublimation under reduced pressure. The presence of pores interconnected inside the polymeric materials and their surface morphologies was examined by scanning electron microscopy. The in-vitro cytotoxicity and cell adhesion on the prepared materials were examined by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay

Enhanced CO2 Adsorption by Nitrogen-Doped Graphene Oxide Sheets (N-GOs) Prepared by Employing Polymeric Precursors

Nitrogen-doped graphene oxide sheets (N-GOs) are prepared by employing N-containing polymers such as polypyrrole, polyaniline, and copolymer (polypyrrole-polyaniline) doped with acids such as HCl, H2SO4, and C6H5-SO3-K, which are activated using different concentrations of KOH and carbonized at 650 °C; characterized using SEM, TEM, BET, TGA-DSC, XRD, and XPS; and employed for the removal of environmental pollutant CO2. The porosity of the N-GOs obtained were found to be in the range 1–3.5 nm when the KOH employed was in the ratio of 1:4, and the XRD confirmed the formation of the layered like structure. However, when the KOH employed was in the ratio of 1:2, the pore diameter was found to be in the range of 50–200 nm. The SEM and TEM analysis reveal the porosity and sheet-like structure of the products obtained. The nitrogen-doped graphene oxide sheets (N-GOs) prepared by employing polypyrrole doped with C6H5-SO3-K were found to possess a high surface area of 2870 m2/g. The N-GOs displayed excellent CO2 capture property with the N-GOs; PPy/Ar-1 displayed ~1.36 mmol/g. The precursor employed, the dopant used, and the activation process were found to affect the adsorption property of the N-GOs obtained. The preparation procedure is simple and favourable for the synthesis of N-GOs for their application as adsorbents in greenhouse gas removal and capture

Evaluation of prognostic risk models for postoperative pulmonary complications in adult patients undergoing major abdominal surgery: a systematic review and international external validation cohort study

Background Stratifying risk of postoperative pulmonary complications after major abdominal surgery allows clinicians to modify risk through targeted interventions and enhanced monitoring. In this study, we aimed to identify and validate prognostic models against a new consensus definition of postoperative pulmonary complications. Methods We did a systematic review and international external validation cohort study. The systematic review was done in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We searched MEDLINE and Embase on March 1, 2020, for articles published in English that reported on risk prediction models for postoperative pulmonary complications following abdominal surgery. External validation of existing models was done within a prospective international cohort study of adult patients (≥18 years) undergoing major abdominal surgery. Data were collected between Jan 1, 2019, and April 30, 2019, in the UK, Ireland, and Australia. Discriminative ability and prognostic accuracy summary statistics were compared between models for the 30-day postoperative pulmonary complication rate as defined by the Standardised Endpoints in Perioperative Medicine Core Outcome Measures in Perioperative and Anaesthetic Care (StEP-COMPAC). Model performance was compared using the area under the receiver operating characteristic curve (AUROCC). Findings In total, we identified 2903 records from our literature search; of which, 2514 (86·6%) unique records were screened, 121 (4·8%) of 2514 full texts were assessed for eligibility, and 29 unique prognostic models were identified. Nine (31·0%) of 29 models had score development reported only, 19 (65·5%) had undergone internal validation, and only four (13·8%) had been externally validated. Data to validate six eligible models were collected in the international external validation cohort study. Data from 11 591 patients were available, with an overall postoperative pulmonary complication rate of 7·8% (n=903). None of the six models showed good discrimination (defined as AUROCC ≥0·70) for identifying postoperative pulmonary complications, with the Assess Respiratory Risk in Surgical Patients in Catalonia score showing the best discrimination (AUROCC 0·700 [95% CI 0·683–0·717]). Interpretation In the pre-COVID-19 pandemic data, variability in the risk of pulmonary complications (StEP-COMPAC definition) following major abdominal surgery was poorly described by existing prognostication tools. To improve surgical safety during the COVID-19 pandemic recovery and beyond, novel risk stratification tools are required. Funding British Journal of Surgery Society