Decentralizing Cell-Free RNA Sensing With the Use of Low-Cost Cell Extracts.

Cell-free gene expression systems have emerged as a promising platform for field-deployed biosensing and diagnostics. When combined with programmable toehold switch-based RNA sensors, these systems can be used to detect arbitrary RNAs and freeze-dried for room temperature transport to the point-of-need. These sensors, however, have been mainly implemented using reconstituted PURE cell-free protein expression systems that are difficult to source in the Global South due to their high commercial cost and cold-chain shipping requirements. Based on preliminary demonstrations of toehold sensors working on lysates, we describe the fast prototyping of RNA toehold switch-based sensors that can be produced locally and reduce the cost of sensors by two orders of magnitude. We demonstrate that these in-house cell lysates provide sensor performance comparable to commercial PURE cell-free systems. We further optimize these lysates with a CRISPRi strategy to enhance the stability of linear DNAs by knocking-down genes responsible for linear DNA degradation. This enables the direct use of PCR products for fast screening of new designs. As a proof-of-concept, we develop novel toehold sensors for the plant pathogen Potato Virus Y (PVY), which dramatically reduces the yield of this important staple crop. The local implementation of low-cost cell-free toehold sensors could enable biosensing capacity at the regional level and lead to more decentralized models for global surveillance of infectious disease

A collaboratively derived environmental research agenda for Galapagos

Galápagos is one of the most pristine archipelagos in the world and its conservation relies upon research and sensible management. In recent decades both the interest in, and the needs of, the islands have increased, yet the funds and capacity for necessary research have remained limited. It has become, therefore, increasingly important to identify areas of priority research to assist decision-making in Galápagos conservation. This study identified 50 questions considered priorities for future research and management. The exercise involved the collaboration of policy makers, practitioners and researchers from more than 30 different organisations. Initially, 360 people were consulted to generate 781 questions. An established process of preworkshop voting and three rounds to reduce and reword the questions, followed by a two-day workshop, was used to produce the final 50 questions. The most common issues raised by this list of questions were human population growth, climate change and the impact of invasive alien species. These results have already been used by a range of organisations and politicians and are expected to provide the basis for future research on the islands so that its sustainability may be enhanced. </jats:p

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Effect of SGLT2 inhibitors on stroke and atrial fibrillation in diabetic kidney disease: Results from the CREDENCE trial and meta-analysis

BACKGROUND AND PURPOSE: Chronic kidney disease with reduced estimated glomerular filtration rate or elevated albuminuria increases risk for ischemic and hemorrhagic stroke. This study assessed the effects of sodium glucose cotransporter 2 inhibitors (SGLT2i) on stroke and atrial fibrillation/flutter (AF/AFL) from CREDENCE (Canagliflozin and Renal Events in Diabetes With Established Nephropathy Clinical Evaluation) and a meta-Analysis of large cardiovascular outcome trials (CVOTs) of SGLT2i in type 2 diabetes mellitus. METHODS: CREDENCE randomized 4401 participants with type 2 diabetes mellitus and chronic kidney disease to canagliflozin or placebo. Post hoc, we estimated effects on fatal or nonfatal stroke, stroke subtypes, and intermediate markers of stroke risk including AF/AFL. Stroke and AF/AFL data from 3 other completed large CVOTs and CREDENCE were pooled using random-effects meta-Analysis. RESULTS: In CREDENCE, 142 participants experienced a stroke during follow-up (10.9/1000 patient-years with canagliflozin, 14.2/1000 patient-years with placebo; hazard ratio [HR], 0.77 [95% CI, 0.55-1.08]). Effects by stroke subtypes were: ischemic (HR, 0.88 [95% CI, 0.61-1.28]; n=111), hemorrhagic (HR, 0.50 [95% CI, 0.19-1.32]; n=18), and undetermined (HR, 0.54 [95% CI, 0.20-1.46]; n=17). There was no clear effect on AF/AFL (HR, 0.76 [95% CI, 0.53-1.10]; n=115). The overall effects in the 4 CVOTs combined were: Total stroke (HRpooled, 0.96 [95% CI, 0.82-1.12]), ischemic stroke (HRpooled, 1.01 [95% CI, 0.89-1.14]), hemorrhagic stroke (HRpooled, 0.50 [95% CI, 0.30-0.83]), undetermined stroke (HRpooled, 0.86 [95% CI, 0.49-1.51]), and AF/AFL (HRpooled, 0.81 [95% CI, 0.71-0.93]). There was evidence that SGLT2i effects on total stroke varied by baseline estimated glomerular filtration rate (P=0.01), with protection in the lowest estimated glomerular filtration rate (45 mL/min/1.73 m2]) subgroup (HRpooled, 0.50 [95% CI, 0.31-0.79]). CONCLUSIONS: Although we found no clear effect of SGLT2i on total stroke in CREDENCE or across trials combined, there was some evidence of benefit in preventing hemorrhagic stroke and AF/AFL, as well as total stroke for those with lowest estimated glomerular filtration rate. Future research should focus on confirming these data and exploring potential mechanisms

Effects of alirocumab on types of myocardial infarction: insights from the ODYSSEY OUTCOMES trial

Aims  The third Universal Definition of Myocardial Infarction (MI) Task Force classified MIs into five types: Type 1, spontaneous; Type 2, related to oxygen supply/demand imbalance; Type 3, fatal without ascertainment of cardiac biomarkers; Type 4, related to percutaneous coronary intervention; and Type 5, related to coronary artery bypass surgery. Low-density lipoprotein cholesterol (LDL-C) reduction with statins and proprotein convertase subtilisin–kexin Type 9 (PCSK9) inhibitors reduces risk of MI, but less is known about effects on types of MI. ODYSSEY OUTCOMES compared the PCSK9 inhibitor alirocumab with placebo in 18 924 patients with recent acute coronary syndrome (ACS) and elevated LDL-C (≥1.8 mmol/L) despite intensive statin therapy. In a pre-specified analysis, we assessed the effects of alirocumab on types of MI. Methods and results  Median follow-up was 2.8 years. Myocardial infarction types were prospectively adjudicated and classified. Of 1860 total MIs, 1223 (65.8%) were adjudicated as Type 1, 386 (20.8%) as Type 2, and 244 (13.1%) as Type 4. Few events were Type 3 (n = 2) or Type 5 (n = 5). Alirocumab reduced first MIs [hazard ratio (HR) 0.85, 95% confidence interval (CI) 0.77–0.95; P = 0.003], with reductions in both Type 1 (HR 0.87, 95% CI 0.77–0.99; P = 0.032) and Type 2 (0.77, 0.61–0.97; P = 0.025), but not Type 4 MI. Conclusion  After ACS, alirocumab added to intensive statin therapy favourably impacted on Type 1 and 2 MIs. The data indicate for the first time that a lipid-lowering therapy can attenuate the risk of Type 2 MI. Low-density lipoprotein cholesterol reduction below levels achievable with statins is an effective preventive strategy for both MI types.For complete list of authors see http://dx.doi.org/10.1093/eurheartj/ehz299</p

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

Effect of alirocumab on mortality after acute coronary syndromes. An analysis of the ODYSSEY OUTCOMES randomized clinical trial

Background: Previous trials of PCSK9 (proprotein convertase subtilisin-kexin type 9) inhibitors demonstrated reductions in major adverse cardiovascular events, but not death. We assessed the effects of alirocumab on death after index acute coronary syndrome. Methods: ODYSSEY OUTCOMES (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab) was a double-blind, randomized comparison of alirocumab or placebo in 18 924 patients who had an ACS 1 to 12 months previously and elevated atherogenic lipoproteins despite intensive statin therapy. Alirocumab dose was blindly titrated to target achieved low-density lipoprotein cholesterol (LDL-C) between 25 and 50 mg/dL. We examined the effects of treatment on all-cause death and its components, cardiovascular and noncardiovascular death, with log-rank testing. Joint semiparametric models tested associations between nonfatal cardiovascular events and cardiovascular or noncardiovascular death. Results: Median follow-up was 2.8 years. Death occurred in 334 (3.5%) and 392 (4.1%) patients, respectively, in the alirocumab and placebo groups (hazard ratio [HR], 0.85; 95% CI, 0.73 to 0.98; P=0.03, nominal P value). This resulted from nonsignificantly fewer cardiovascular (240 [2.5%] vs 271 [2.9%]; HR, 0.88; 95% CI, 0.74 to 1.05; P=0.15) and noncardiovascular (94 [1.0%] vs 121 [1.3%]; HR, 0.77; 95% CI, 0.59 to 1.01; P=0.06) deaths with alirocumab. In a prespecified analysis of 8242 patients eligible for ≥3 years follow-up, alirocumab reduced death (HR, 0.78; 95% CI, 0.65 to 0.94; P=0.01). Patients with nonfatal cardiovascular events were at increased risk for cardiovascular and noncardiovascular deaths (P<0.0001 for the associations). Alirocumab reduced total nonfatal cardiovascular events (P<0.001) and thereby may have attenuated the number of cardiovascular and noncardiovascular deaths. A post hoc analysis found that, compared to patients with lower LDL-C, patients with baseline LDL-C ≥100 mg/dL (2.59 mmol/L) had a greater absolute risk of death and a larger mortality benefit from alirocumab (HR, 0.71; 95% CI, 0.56 to 0.90; Pinteraction=0.007). In the alirocumab group, all-cause death declined wit h achieved LDL-C at 4 months of treatment, to a level of approximately 30 mg/dL (adjusted P=0.017 for linear trend). Conclusions: Alirocumab added to intensive statin therapy has the potential to reduce death after acute coronary syndrome, particularly if treatment is maintained for ≥3 years, if baseline LDL-C is ≥100 mg/dL, or if achieved LDL-C is low. Clinical Trial Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01663402

Engineering of the Filamentous Fungus Penicillium chrysogenum as Cell Factory for Natural Products

Penicillium chrysogenum (renamed P. rubens) is the most studied member of a family of more than 350 Penicillium species that constitute the genus. Since the discovery of penicillin by Alexander Fleming, this filamentous fungus is used as a commercial beta-lactam antibiotic producer. For several decades, P. chrysogenum was subjected to a classical strain improvement (CSI) program to increase penicillin titers. This resulted in a massive increase in the penicillin production capacity, paralleled by the silencing of several other biosynthetic gene clusters (BGCs), causing a reduction in the production of a broad range of BGC encoded natural products (NPs). Several approaches have been used to restore the ability of the penicillin production strains to synthetize the NPs lost during the CSI. Here, we summarize various re-activation mechanisms of BGCs, and how interference with regulation can be used as a strategy to activate or silence BGCs in filamentous fungi. To further emphasize the versatility of P. chrysogenum as a fungal production platform for NPs with potential commercial value, protein engineering of biosynthetic enzymes is discussed as a tool to develop de novo BGC pathways for new NPs

Optimising promoters and subcellular localisation for constitutive transgene expression in Marchantia polymorpha.

Marchantia polymorpha has become an important model system for comparative studies and synthetic biology. The systematic characterisation of genetic elements would make heterologous gene expression more predictable in this testbed for gene circuit assembly and bioproduction. Yet, the toolbox of genetic parts for Marchantia includes only a few constitutive promoters that need benchmarking to assess their utility. We compared the expression patterns of previously characterized and new constitutive promoters. We found that driving expression with the double enhancer version of the cauliflower mosaic virus 35S promoter (pro35S×2) provided the highest yield of proteins although it also inhibits the growth of transformants. In contrast, promoters derived from the Marchantia ETHYLENE RESPONSE FACTOR 1 (MpERF1) and the CLASS II HOMEODOMAIN-LEUCINE ZIPPER (MpC2HDZ) genes drove expression to higher levels across all tissues without growth penalty and can provide intermediate levels of gene expression. In addition, we showed that the cytosol is the best subcellular compartment to target heterologous proteins for higher levels of expression without a significant growth burden. To demonstrate the potential of these promoters in Marchantia, we expressed the polycistronic RUBY betalain synthesis cassette to demonstrate coordinated expression of metabolic enzymes. A heat-shock inducible promoter was used to further mitigate growth burdens associated with high amounts of betalain accumulation. We have expanded the existing toolkit for gene expression in Marchantia and provide new resources for the Marchantia research community.This work was funded as part of the BBSRC/EPSRC OpenPlant Synthetic Biology Research Centre Grant BB/L014130/1 to J.H., BBSRC BB/F011458/1 for confocal microscopy, BBSRC BB/T007117/1 to J.H. SWT is funded by the Doris Zimmern HKU-Cambridge Hughes Hall Scholarshi

Construction of DNA Tools for Hyperexpression in Marchantia Chloroplasts.

Chloroplasts are attractive platforms for synthetic biology applications since they are capable of driving very high levels of transgene expression, if mRNA production and stability are properly regulated. However, plastid transformation is a slow process and currently limited to a few plant species. The liverwort Marchantia polymorpha is a simple model plant that allows rapid transformation studies; however, its potential for protein hyperexpression has not been fully exploited. This is partially due to the fact that chloroplast post-transcriptional regulation is poorly characterized in this plant. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 bryophyte species and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilization. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplastic transformed plants. We have produced novel DNA tools for protein hyperexpression in this facile plant system that is a test-bed for chloroplast engineering