Sustained cancer-relevant alternative RNA splicing events driven by PRMT5 in high-risk neuroblastoma.

Protein arginine methyltransferase 5 (PRMT5) is over-expressed in a wide variety of cancers and is implicated as having a key oncogenic role, achieved in part through its control of the master transcription regulator E2F1. We investigated the relevance of PRMT5 and E2F1 in neuroblastoma (NB) and found that elevated expression of PRMT5 and E2F1 occurs in poor prognosis high-risk disease and correlates with an amplified Myelocytomatosis viral-related oncogene, neuroblastoma-derived (MYCN) gene. Our results show that MYCN drives the expression of splicing factor genes that, together with PRMT5 and E2F1, lead to a deregulated alternative RNA splicing programme that impedes apoptosis. Pharmacological inhibition of PRMT5 or inactivation of E2F1 restores normal splicing and renders NB cells sensitive to apoptosis. Our findings suggest that a sustained cancer-relevant alternative RNA splicing programme desensitises NB cells to apoptosis, and identify PRMT5 as a potential therapeutic target for high-risk disease.

Protein-based oral rehydration solutions for patients with an ileostomy: A randomised, double-blinded crossover study.

BACKGROUND & AIM: Patients with an ileostomy are at increased risk of dehydration and sodium depletion. Treatments recommended may include oral rehydration solutions (ORS). We aimed to investigate if protein type or protein hydrolysation affects absorption from iso-osmolar ORS in patients with an ileostomy. METHODS: This was a randomised, double-blinded, active comparator-controlled 3 × 3 crossover intervention study. We developed three protein-based ORS with whey protein isolate, caseinate or whey protein hydrolysate. The solutions contained 40-48 g protein/L, 34-45 mmol sodium/L and had an osmolality of 248-270 mOsm/kg. The patients ingested 500 mL/d. The study consisted of three 4-week periods with a >2-week washout between each intervention. The primary outcome was wet-weight ileostomy output. Ileostomy output and urine were collected for a 24-h period before and after each intervention. Additionally, blood sampling, dietary records, muscle-strength tests, bioimpedance analyses, questionnaires and psychometric tests were conducted. RESULTS: We included 14 patients, of whom 13 completed at least one intervention. Ten patients completed all three interventions. Wet-weight ileostomy output did not change following either of the three interventions and did not differ between interventions (p = 0.38). A cluster of statistically significant improvements related to absorption was observed following the intake of whey protein isolate ORS, including decreased faecal losses of energy (-365 kJ/d, 95% confidence interval (CI), -643 to -87, p = 0.012), potassium (-7.8 mmol/L, 95%CI, -12.0 to -3.6, p = 0.001), magnesium (-4.0 mmol/L, 95%CI, -7.4 to -0.7, p = 0.020), improved plasma aldosterone (-4674 pmol/L 95%CI, -8536 to -812, p = 0.019), estimated glomerular filtration rate (eGFR) (2.8 mL/min/1.73 m2, 95%CI, 0.3 to 5.4, p = 0.03) and CO2 (1.7 mmol/L 95%CI, 0.1 to 3.3, p = 0.04). CONCLUSION: Ingestion of 500 mL/d of iso-osmolar solutions containing either whey protein isolate, caseinate or whey protein hydrolysate for four weeks resulted in unchanged and comparable ileostomy outputs in patients with an ileostomy. Following whey protein isolate ORS, we observed discrete improvements in a series of absorption proxies in both faeces and blood, indicating increased absorption. The protein-based ORS were safe and well-tolerated. Treatments should be tailored to each patient, and future studies are warranted to explore treatment-effect heterogeneity and whether different compositions or doses of ORS can improve absorption and nutritional status in patients with an ileostomy. GOV STUDY IDENTIFIER: NCT04141826.

Osmolality in oral supplements drives ileostomy output: Defining the Goldilocks zone.

BACKGROUND: Patients with an ileostomy often have impaired quality of life, sodium depletion, secondary hyperaldosteronism, and other organ-specific pathologies. The osmolality of oral supplements influences ileostomy output and increases sodium loss. We hypothesized the existence of an osmolality range in which fluid absorption and secondary natriuresis are optimal. METHODS: This was a single-center, quasi-randomized crossover intervention study, including patients with an ileostomy and no home parenteral support. After an 8-h fasting period, each patient ingested 500 mL of 3-18 different oral supplements and a standardized meal during the various intervention periods, followed by a 6-h collection of ileostomy and urine outputs. The primary outcome was 6-h ileostomy output. RESULTS: A total of 14 ileostomy patients with a median age of 65 years (interquartile range 38-70 years) were included. The association between osmolalities (range 5-1352 mOsm/kg) and ileostomy output forecasted an S-curve. A linear association between osmolality of oral supplements (range 290-600 mOsm/kg) and ileostomy output was identified and assessed with a mixed-effects model. Ileostomy output increased by 57 g/6 h (95% confidence interval (CI) 21-94) when the oral supplement osmolality increased by 100 mOsm/kg (p = 0.005). CONCLUSION: Osmolality in oral supplements correlated with ileostomy output. Our results indicate that patients with an ileostomy may benefit from increased ingestion of oral supplements with osmolalities between 100 and 290 mOsm/kg. We define this range as the Goldilocks zone, equivalent to optimal fluid and electrolyte absorption.

The live biotherapeutic SYNB1353 decreases plasma methionine via directed degradation in animal models and healthy volunteers.

Methionine is an essential proteinogenic amino acid, but its excess can lead to deleterious effects. Inborn errors of methionine metabolism resulting from loss of function in cystathionine ß-synthase (CBS) cause classic homocystinuria (HCU), which is managed by a methionine-restricted diet. Synthetic biotics are gastrointestinal tract-targeted live biotherapeutics that can be engineered to replicate the benefits of dietary restriction. In this study, we assess whether SYNB1353, an E. coli Nissle 1917 derivative, impacts circulating methionine and homocysteine levels in animals and healthy volunteers. In both mice and nonhuman primates (NHPs), SYNB1353 blunts the appearance of plasma methionine and plasma homocysteine in response to an oral methionine load. A phase 1 clinical study conducted in healthy volunteers subjected to an oral methionine challenge demonstrates that SYNB1353 is well tolerated and blunts plasma methionine by 26%. Overall, SYNB1353 represents a promising approach for methionine reduction with potential utility for the treatment of HCU.

Fecal microbiota transplantation for recurrent C. difficile infection in patients with inflammatory bowel disease: A systematic review and meta-analysis.

Fecal microbiota transplantation (FMT) is known to be highly effective in patients with recurrent Clostridioides difficile infection (rCDI), but its role in patients who also suffer from inflammatory bowel disease (IBD) is unclear. Therefore, we performed a systematic review and meta-analysis to evaluate the efficacy and safety of FMT for the treatment of rCDI in patients with IBD. We searched the available literature until November 22, 2022 to identify studies that included patients with IBD treated with FMT for rCDI, reporting efficacy outcomes after at least 8 weeks of follow-up. The proportional effect of FMT was summarized with a generalized linear mixed-effect model fitting a logistic regression accounting for different intercepts among studies. We identified 15 eligible studies, containing 777 patients. Overall, FMT achieved high cure rates of rCDI, 81% for single FMT, based on all included studies and patients, and 92% for overall FMT, based on nine studies with 354 patients, respectively. We found a significant advantage of overall FMT over single FMT in improving cure rates of rCDI (from 80% to 92%, p = 0.0015). Serious adverse events were observed in 91 patients (12% of the overall population), with the most common being hospitalisation, IBD-related surgery, or IBD flare. In conclusion, in our meta-analysis FMT achieved high cure rates of rCDI in patients with IBD, with a significant advantage of overall FMT over single FMT, similar to data observed in patients without IBD. Our findings support the use of FMT as a treatment for rCDI in patients with IBD.

Left Atrioventricular Transvalvular Pressure Gradients Derived from Intraoperative and Postoperative Echocardiograms following Atrioventricular Septal Defect Repair.

BACKGROUND: Left atrioventricular valve (LAVV) stenosis following an atrioventricular septal defect (AVSD) repair is a rare but potentially life-threatening complication. While echocardiographic quantification of diastolic transvalvular pressure gradients is paramount in the evaluation of a newly corrected valve function, it is hypothesized that these measured gradients are overestimated immediately following a cardiopulmonary bypass (CPB) due to the altered hemodynamics when compared to postoperative valve assessments using awake transthoracic echocardiography (TTE) upon recovery after surgery. METHODS: Out of the 72 patients screened for inclusion at a tertiary center, 39 patients undergoing an AVSD repair with both intraoperative transesophageal echocardiograms (TEE, performed immediately after a CPB) and an awake TTE (performed prior to hospital discharge) were retrospectively selected. The mean (MPGs) and peak pressure gradients (PPGs) were quantified using a Doppler echocardiography and other measures of interest were recorded (e.g., a non-invasive surrogate of the cardiac output and index (CI), left ventricular ejection fraction, blood pressures and airway pressures). The variables were analyzed using the paired Student's t-tests and Spearman's correlation coefficients. RESULTS: The MPGs were significantly higher in the intraoperative measurements when compared to the awake TTE (3.0 ± 1.2 vs. 2.3 ± 1.1 mmHg; p < 0.01); however, the PPGs did not significantly differ (6.6 ± 2.7 vs. 5.7 ± 2.8 mmHg; p = 0.06). Although the assessed intraoperative heart rates (HRs) were also higher (132 ± 17 vs. 114 ± 21 bpm; p < 0.001), there was no correlation found between the MPG and the HR, or any other parameter of interest, at either time-point. In a further analysis, a moderate to strong correlation was observed in the linear relationship between the CI and the MPG (r = 0.60; p < 0.001). During the in-hospital follow-up period, no patients died or required an intervention due to LAVV stenosis. CONCLUSIONS: The Doppler-based quantification of diastolic transvalvular LAVV mean pressure gradients using intraoperative transesophageal echocardiography seems to be prone to overestimation due to altered hemodynamics immediately after an AVSD repair. Thus, the current hemodynamic state should be taken into consideration during the intraoperative interpretation of these gradients.

Cross-generational bacterial strain transfer to an infant after fecal microbiota transplantation to a pregnant patient: a case report.

BACKGROUND: Fecal microbiota transplantation (FMT) effectively prevents the recurrence of Clostridioides difficile infection (CDI). Long-term engraftment of donor-specific microbial consortia may occur in the recipient, but potential further transfer to other sites, including the vertical transmission of donor-specific strains to future generations, has not been investigated. Here, we report, for the first time, the cross-generational transmission of specific bacterial strains from an FMT donor to a pregnant patient with CDI and further to her child, born at term, 26 weeks after the FMT treatment. METHODS: A pregnant woman (gestation week 12 + 5) with CDI was treated with FMT via colonoscopy. She gave vaginal birth at term to a healthy baby. Fecal samples were collected from the feces donor, the mother (before FMT, and 1, 8, 15, 22, 26, and 50 weeks after FMT), and the infant (meconium at birth and 3 and 6 months after birth). Fecal samples were profiled by deep metagenomic sequencing for strain-level analysis. The microbial transfer was monitored using single nucleotide variants in metagenomes and further compared to a collection of metagenomic samples from 651 healthy infants and 58 healthy adults. RESULTS: The single FMT procedure led to an uneventful and sustained clinical resolution in the patient, who experienced no further CDI-related symptoms up to 50 weeks after treatment. The gut microbiota of the patient with CDI differed considerably from the healthy donor and was characterized as low in alpha diversity and enriched for several potential pathogens. The FMT successfully normalized the patient's gut microbiota, likely by donor microbiota transfer and engraftment. Importantly, our analysis revealed that some specific strains were transferred from the donor to the patient and then further to the infant, thus demonstrating cross-generational microbial transfer. CONCLUSIONS: The evidence for cross-generational strain transfer following FMT provides novel insights into the dynamics and engraftment of bacterial strains from healthy donors. The data suggests FMT treatment of pregnant women as a potential strategy to introduce beneficial strains or even bacterial consortia to infants, i.e., neonatal seeding. Video Abstract.

Prognosis of Patients with Bronchopulmonary Neuroendocrine Neoplasms in a Tertiary Neuroendocrine Tumor Centre of Excellence.

INTRODUCTION: The European Neuroendocrine Tumor Society (ENETS) reports variables of prognostic significance in bronchopulmonary neuroendocrine neoplasms (BP-NENs). The aim of this study was to investigate prognostic factors, recurrence-free survival (RFS), and overall survival (OS) for patients with typical carcinoid (TC), atypical carcinoid (AC), and large-cell neuroendocrine carcinoma (LCNEC). Current follow-up practices vary as the evidence is sparse, and we aimed to explore the relevance of routine bronchoscopy in follow-up. METHODS: This was a cohort study of 208 patients with BP-NENs followed at Aarhus University Hospital in 2008-2019. RFS and OS were determined using the Kaplan-Meier method for the variables such as primary tumor, primary treatment, smoking status, gender, and histological subtypes. RESULTS: The study included 153 patients with TC, 29 with AC, and 26 with LCNEC. Median follow-up was 48 months. The majority of patients (n = 191) received surgical resection, of which 22 (11%) recurred over time. Seventeen patients had nonsurgical treatment, of which 10 (59%) progressed. The 5-year OS rate was 86% for operated and 9% for nonoperated patients (p < 0.05). Patients with TC had a 5-year OS of 90% compared with 63% and 39% for AC and LCNEC, respectively. As for prognostic factors, nonsmokers did not secure a significant difference in OS compared with current/previous smokers (p = 0.51). In the follow-up period, only 2 (9%) of the 22 recurrences were found on a routine bronchoscopy. Both of these recurrences were also found by diagnostic imaging. CONCLUSIONS: Surgical treatment, especially, and diagnosis of TC were associated with a good prognosis. Furthermore, our data did not support routine bronchoscopy as part of a follow-up program for bronchial carcinoids.

Methionine Restriction: Ready for Prime Time in the Cancer Clinic?

Attempts to selectively starve cancers in the clinic have been made at least since the time of Warburg beginning 100 years ago. Calorie-restriction or low-carbohydrate diets have had limited success with cancer patients. Methionine restriction is another strategy to selectively starve cancer cells, since cancers are addicted to methionine, unlike normal cells. Methionine addiction of cancer is termed the Hoffman effect. Numerous preclinical studies over the past half century have shown methionine restriction to be highly effective against all major cancer types and synergistic with chemotherapy. Low-methionine medical diets can be effective in lowering methionine and have shown some clinical promise, but they are not palatable and thereby not sustainable. However, selectively choosing among plant-based foods allows a variety of low-methionine diets that are sustainable. Our laboratory has developed a methioninase that can be administered orally as a supplement and has resulted in anecdotal positive results in patients with advanced cancer, including hormone-independent prostate cancer, and other recalcitrant cancers. The question is whether methionine restriction through a low-methionine diet, or even greater methionine restriction with methioninase in combination with a low-methionine diet, is ready for prime time in the clinic, especially in combination with other synergistic therapy. The question will hopefully be answered in the near future, especially for advanced cancer patients who have failed all standard therapy.

PRMT5 promotes cancer cell migration and invasion through the E2F pathway.

The pRb-E2F pathway is a critical point of regulation in the cell cycle and loss of control of the pathway is a hallmark of cancer. E2F1 is the major target through which pRb exerts its effects and arginine methylation by PRMT5 plays a key role in dictating E2F1 activity. Here we have explored the functional role of the PRMT5-E2F1 axis and highlight its influence on different aspects of cancer cell biology including viability, migration, invasion and adherence. Through a genome-wide expression analysis, we identified a distinct set of genes under the control of PRMT5 and E2F1, including some highly regulated genes, which influence cell migration, invasio and adherence through a PRMT5-dependent mechanism. Most significantly, a coincidence was apparent between the expression of PRMT5 and E2F1 in human tumours, and elevated levels of PRMT5 and E2F1 correlated with poor prognosis disease. Our results suggest a causal relationship between PRMT5 and E2F1 in driving the malignant phenotype and thereby highlight an important pathway for therapeutic intervention.

Fecal Microbiota Transplantation Is Superior to Fidaxomicin for Treatment of Recurrent Clostridium difficile Infection.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is recommended for treatment of recurrent Clostridium difficile infection (rCDI). We performed a single-center randomized trial to compare the effects of FMT with those of fidaxomicin and vancomycin. METHODS: We studied consecutive adults with rCDI seen at a gastroenterology clinic in Denmark from April 5, 2016 through June 10, 2018. Patients were randomly assigned to a group that received FMT, applied by colonoscopy or nasojejunal tube, after 4-10 days of vancomycin (125 mg 4 times daily; FMTv; n = 24), 10 days of fidaxomicin (200 mg twice daily; n = 24), or 10 days of vancomycin (125 mg 4 times daily; n = 16). Patients who had rCDI after this course of treatment and patients who could not be randomly assigned to groups were offered rescue FMTv. The primary outcome was combined clinical resolution and a negative result from a polymerase chain reaction test for Clostridium difficile (CD) toxin 8 weeks after the allocated treatment. Secondary end points included clinical resolution at week 8. RESULTS: All 64 patients received their assigned treatment. The combination of clinical resolution and negative results from the test for CD were observed in 17 patients given FMTv (71%), 8 patients given fidaxomicin (33%), and 3 patients given vancomycin (19%; P = .009 for FMTv vs fidaxomicin; P = .001 for FMTv vs vancomycin; P = .31 for fidaxomicin vs vancomycin). Clinical resolution was observed in 22 patients given FMTv (92%), 10 patients given fidaxomicin (42%), and 3 patients given vancomycin (19%; P = .0002; P < .0001; P = .13). Results did not differ significantly between patients who received FMTv as their initial therapy and patients who received rescue FMTv. There was 1 serious adverse event that might have been related to FMTv. CONCLUSIONS: In a randomized trial of patients with rCDI, we found the FMTv combination superior to fidaxomicin or vancomycin based on end points of clinical and microbiological resolution or clinical resolution alone. ClinicalTrials.gov, number NCT02743234; EudraCT, j.no 2015-003004-24.

The role of macrophages in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) and its inflammatory and often progressive subtype nonalcoholic steatohepatitis (NASH) are becoming the leading cause of liver-related morbidity and mortality worldwide, and a primary indication for liver transplantation. The pathophysiology of NASH is multifactorial and not yet completely understood; however, innate immunity is a major contributing factor in which liver-resident macrophages (Kupffer cells) and recruited macrophages play a central part in disease progression. In this Review, we assess the evidence for macrophage involvement in the development of steatosis, inflammation and fibrosis in NASH. In this process, not only the polarization of liver macrophages towards a pro-inflammatory phenotype is important, but adipose tissue macrophages, especially in the visceral compartment, also contribute to disease severity and insulin resistance. Macrophage activation is mediated by factors such as endotoxins and translocated bacteria owing to increased intestinal permeability, factors released from damaged or lipoapoptotic hepatocytes, as well as alterations in gut microbiota and defined nutritional components, including certain free fatty acids, cholesterol and their metabolites. Reflecting the important role of macrophages in NASH, we also review studies investigating drugs that target macrophage recruitment to the liver, macrophage polarization and their inflammatory effects as potential treatment options for patients with NASH.

The global prevalence and genetic spectrum of lysosomal acid lipase deficiency: A rare condition that mimics NAFLD.

BACKGROUND & AIMS: Lysosomal acid lipase deficiency (LAL-D) is an autosomal recessive condition that may present in a mild form (cholesteryl ester storage disease [CESD]), which mimics non-alcoholic fatty liver disease (NAFLD). It has been suggested that CESD may affect 1 in 40,000 and is under-diagnosed in NAFLD clinics. Therefore, we aimed to estimate the prevalence of LAL-D using analysis of genetic variation in LIPA. METHODS: MEDLINE and EMBASE were systematically searched for previously reported disease variants and prevalence estimates. Previous prevalence estimates were meta-analysed. Disease variants in LIPA were annotated with allele frequencies from gnomAD and combined with unreported major functional variants found in humans. Pooled ethnicity-specific prevalences for LAL-D and CESD were calculated using the Hardy-Weinberg equation. RESULTS: Meta-analysis of existing genetic studies estimated the prevalence of LAL-D as 1 per 160,000 (95% CI 1 per 65,025-761,652) using the allele frequency of c.894G>A in LIPA. A total of 98 previously reported disease variants in LIPA were identified, of which 32/98 were present in gnomAD, giving a prevalence of 1 per 307,482 (95% CI 257,672-366,865). Wolman disease was associated with more loss-of-function variants than CESD. When this was combined with 22 previously unreported major functional variants in LIPA identified in humans, the pooled prevalence of LAL-D was 1 per 177,452 (95% CI 149,467-210,683) with a carrier frequency of 1 per 421. The prevalence is lowest in those of East Asian, South Asian, and Finnish ancestry. CONCLUSION: Using 120 disease variants in LIPA, these data can reassure clinicians that LAL-D is an ultra-rare disorder. Given the therapeutic capability of sebelipase alpha, investigation for LAL-D might be included in second-line metabolic screening in NAFLD. LAY SUMMARY: Lysosomal Acid Lipase Deficiency (LAL-D) is a rare genetic condition that can cause severe liver disease, but it is difficult to diagnose and sometimes can look like simple fatty liver. It was not clear how common LAL-D was and whether many cases were being missed. To study this, we searched for all genetic mutations that could cause LAL-D, calculated how common those mutations were, and added them up. This let us estimate that LAL-D affects roughly 1 in 175,000 people. We conclude that LAL-D is a very rare condition, but it is treatable so may be included in a 'second-line' of tests for causes of fatty liver.

A fully automated flow-based approach for accelerated peptide synthesis.

Here we report a fully automated, flow-based approach to solid-phase polypeptide synthesis, with amide bond formation in 7 seconds and total synthesis times of 40 seconds per amino acid residue. Crude peptide purities and isolated yields were comparable to those for standard-batch solid-phase peptide synthesis. At full capacity, this approach can yield tens of thousands of individual 30-mer peptides per year.

A perfluoroaromatic abiotic analog of H2 relaxin enabled by rapid flow-based peptide synthesis.

H2 relaxin is a pleiotropic peptide hormone with clinical potential. Here we report on the reaction and use of hexafluorobenzene as an intramolecular disulfide replacement between Cys10 and Cys15 in the A-chain of H2 relaxin. Using flow-based Fmoc solid-phase peptide synthesis methodology we were able to obtain high-quality H2 relaxin fragments that were previously reported as challenging to synthesize. Subsequent native chemical ligation and oxidative folding enabled total synthesis of both wild type H2 relaxin and a C6F4 linked analog. Cell-based activity assays revealed modest activity for the C6F4 linked H2 relaxin analog, albeit 100-fold reduced relative to wild type. This work demonstrates how perfluoroarylation-cysteine SNAr chemistry may be a useful tool for the selective replacement of native disulfide bonds in proteins.

C-Terminal Modification of Fully Unprotected Peptide Hydrazides via in Situ Generation of Isocyanates.

A method for chemo- and regioselective conjugation of nucleophiles to fully unprotected peptides and proteins via in situ generation of C-terminal isocyanates is reported. Oxidation of C-terminal peptide hydrazides in aqueous media followed by Curtius rearrangement of acyl azides reliably generates isocyanates, which react with a variety of external nucleophiles, such as hydrazines, hydrazides, aromatic thiols, and hydroxylamines. Multiple peptides and a 53 kDa protein hydrazide were conjugated to different nucleophiles using this reaction.

High central venous saturation after cardiac surgery is associated with increased organ failure and long-term mortality: an observational cross-sectional study.

INTRODUCTION: Central venous saturation (ScvO2) monitoring has been suggested to address the issue of adequate cardiocirculatory function in the context of cardiac surgery. The aim of this study was to determine the impact of low (L) (<60%), normal (N) (60%-80%), and high (H) (>80%) ScvO2 measured on intensive care unit (ICU) admission after cardiac surgery. METHODS: We conducted a retrospective, cross-sectional, observational study at three ICUs of a university hospital department for anaesthesiology and intensive care. Electronic patient records of all adults who underwent cardiac surgery between 2006 and 2013 and available admission measurements of ScvO2 were examined. Patients were allocated to one of three groups according to first ScvO2 measurement after ICU admission: group L (<60%), group N (60%-80%), and group H (>80%). Primary end-points were in-hospital and 3-year follow-up survival. RESULTS: Data from 4,447 patients were included in analysis. Low and high initial measurements of ScvO2 were associated with increased in-hospital mortality (L: 5.6%; N: 3.3%; H: 6.8%), 3-year follow-up mortality (L: 21.6%; N: 19.3%; H: 25.8%), incidence of post-operative haemodialysis (L: 11.5%; N: 7.8%; H: 15.3%), and prolonged hospital length of stay (L: 13 days, 9-22; N: 12 days, 9-19; H: 14 days, 9-21). After adjustment for possible confounding variables, an initial ScvO2 above 80% was associated with adjusted hazard ratios of 2.79 (95% confidence interval (CI) 1.565-4.964, P <0.001) for in-hospital survival and 1.31 (95% CI 1.033-1.672, P = 0.026) for 3-year follow-up survival. CONCLUSIONS: Patients with high ScvO2 were particularly affected by unfavourable outcomes. Advanced haemodynamic monitoring may help to identify patients with high ScvO2 who developed extraction dysfunction and to establish treatment algorithms to improve patient outcome in these patients.

Flow-based enzymatic ligation by sortase A.

Sortase-mediated ligation (sortagging) is a versatile, powerful strategy for protein modification. Because the sortase reaction reaches equilibrium, a large excess of polyglycine nucleophile is often employed to drive the reaction forward and suppress sortase-mediated side reactions. A flow-based sortagging platform employing immobilized sortase A within a microreactor was developed that permits efficient sortagging at low nucleophile concentrations. The platform was tested with several reaction partners and used to generate a protein bioconjugate inaccessible by solution-phase batch sortagging.

Rapid flow-based peptide synthesis.

A flow-based solid-phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 min under automatic control or every 3 min under manual control is described. This is accomplished by passing a stream of reagent through a heat exchanger into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable continuous delivery of heated solvents and reagents to the solid support at high flow rate, thereby maintaining maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to that for traditional batch methods, and, in all cases, the desired material was readily purifiable by RP-HPLC. The application of this method to the synthesis of the 113-residue Bacillus amyloliquefaciens RNase and the 130-residue DARPin pE59 is described in the accompanying manuscript.

Rapid total synthesis of DARPin pE59 and barnase.

We report the convergent total synthesis of two proteins: DARPin pE59 and Bacillus amyloliquefaciens RNase (Barnase). Leveraging our recently developed fast-flow peptide-synthesis platform, we rapidly explored numerous conditions for the assembly of long polypeptides, and were able to mitigate common side reactions, including deletion and aspartimide products. We report general strategies for improving the synthetic quality of difficult peptide sequences with our system. High-quality protein fragments produced under optimal synthetic conditions were subjected to convergent native chemical ligation, which afforded native full-length proteins after a final desulfurization step. Both DARPin and Barnase were folded and found to be as active as their recombinant analogues.