Exploring alternative pathways for the in vitro establishment of the HOPAC cycle for synthetic CO2 fixation.

Nature has evolved eight different pathways for the capture and conversion of CO2, including the Calvin-Benson-Bassham cycle of photosynthesis. Yet, these pathways underlie constrains and only represent a fraction of the thousands of theoretically possible solutions. To overcome the limitations of natural evolution, we introduce the HydrOxyPropionyl-CoA/Acrylyl-CoA (HOPAC) cycle, a new-to-nature CO2-fixation pathway that was designed through metabolic retrosynthesis around the reductive carboxylation of acrylyl-CoA, a highly efficient principle of CO2 fixation. We realized the HOPAC cycle in a step-wise fashion and used rational engineering approaches and machine learning-guided workflows to further optimize its output by more than one order of magnitude. Version 4.0 of the HOPAC cycle encompasses 11 enzymes from six different organisms, converting ~3.0 mM CO2 into glycolate within 2 hours. Our work moves the hypothetical HOPAC cycle from a theoretical design into an established in vitro system that forms the basis for different potential applications.

Over-the-Scope Clips Versus Standard Endoscopic Treatment for First Line Therapy of Non-variceal Upper Gastrointestinal Bleeding: Systematic Review and Meta-Analysis.

BACKGROUND AND AIMS: Over-The-Scope Clips (OTSC) use have shown promising results for first line treatment of non-variceal upper gastrointestinal bleeding (NVUGIB). We conducted this meta-analysis to compare outcomes in patients treated with OTSC versus standard endoscopic intervention for first line endoscopic treatment of NVUGIB. METHODS: We reviewed several databases from inception to December 9, 2022 to identify studies comparing OTSC and standard treatments as the first line treatment for NVUGIB. The outcomes assessed included re-bleeding, initial hemostasis, need for vascular embolization, mortality, need for repeat endoscopy, 30 day readmission rate, and need for surgery. Pooled risk ratios (RR) with 95% confidence intervals (CI) were calculated using random effect model. Heterogeneity was assessed by I2 statistic. RESULTS: We included 11 studies with 1608 patients (494 patients in OTSC group and 1114 patients in control group). OTSC use was associated with significantly lower risk of re-bleeding (RR, 0.58; 95% CI 0.41-0.82). We found no significant difference in rates of initial hemostasis (RR, 1.05; 95% CI 0.99- 1.11), vascular embolization rates (RR, 0.93; 95% CI 0.40- 2.13), need for repeat endoscopy (RR, 0.78; 95% CI 0.40-1.49), 30 day readmission rate (RR, 0.59; 95% CI 0.17-2.01), need for surgery (RR, 0.81; 95% CI 0.29-2.28) and morality (RR, 0.69; 95% CI 0.38-1.23). CONCLUSIONS: OTSC are associated with significantly lower risk of re-bleeding compared to standard endoscopic treatments when used as first line endoscopic therapy for NVUGIB.

Telecytology versus in-room cytopathologist for EUS-guided FNA or fine-needle biopsy sampling of solid pancreatic lesions.

BACKGROUND AND AIMS: Rapid on-site-evaluation (ROSE) with an in-room cytopathologist (ROSE-P) has been shown to improve the diagnostic yield of specimens obtained from patients undergoing EUS-guided FNA or fine-needle biopsy sampling (EUS-FNAB) of pancreatic lesions. Recently, there has been an increased interest and use of ROSE using telecytology (ROSE-T) to optimize clinical workflows and to address social distancing mandates created during the coronavirus disease 2019 pandemic. The purpose of this study was to compare diagnostic outcomes of ROSE-P and ROSE-T. METHODS: A single-center cohort study of patients who underwent EUS-FNAB of solid pancreatic lesions with ROSE was conducted. The primary outcome was overall diagnostic yield of cancer. All patients who underwent EUS-FNAB were entered into a prospectively maintained database. Statistical analyses were performed using descriptive statistics and univariate analysis. RESULTS: There were 165 patients in each arm. There was no difference in diagnostic yield between ROSE-P and ROSE-T (96.4% vs 94.5%, P = .428). ROSE-T was associated with an increased use of 22-gauge needles (P = .006) and more needle passes (P < .001). No significant differences were found in age, gender, lesion size, needle type, procedure times, or adverse events between the 2 groups (P < .05 for all). More pancreatic tail lesions were sampled in the ROSE-P group (P < .001). CONCLUSIONS: ROSE-T was not associated with any difference in final histologic diagnosis for EUS-FNAB of solid pancreatic masses. This has important implications for optimizing clinical workflows.

Inflammatory bowel disease type influences development of elevated liver enzymes.

Background and Aim: Up to a third of patients with inflammatory bowel disease (IBD) have elevated liver enzymes (ELE). We evaluated the incidence, predictors, and outcomes associated with ELE in a diverse and vulnerable IBD cohort. Methods: We retrospectively evaluated 336 IBD patients receiving care at the San Francisco safety net gastroenterology clinics between June 1996 and December 2019. Baseline characteristics were captured at first visit, then patients were followed until last clinic activity or death. Testing and etiology, pattern of ELE defined as transient (<1 month) or persistent (≥1 month), were assessed. Multivariate modeling evaluated predictors of ELE at baseline, new ELE at follow-up, and pattern of ELE. Results: Baseline median age was 40.3 years, 62% male, 46% White (13% Black, 19% Asian, and 18% Latino), and 59% had ulcerative colitis (UC). Among those without known liver disease (n = 14), 51.6% (166 of 322; 52 at baseline, 114 during follow-up) had ELE. In multivariate logistic regression, 5-aminosalicylic acid use (odds ratio [OR] 2.2, 95% confidence interval [CI]: 1.07-4.4, P = 0.03) and higher body mass index (OR 1.08, 95% CI: 1.02-1.14, P = 0.01) were associated with baseline ELE. In multivariate Cox regression, UC (vs. Crohn's disease [CD]) had a 34% lower risk of developing new ELE during follow-up (hazard ratio [HR] 0.66, 95% CI: 0.46-0.95, P = 0.02). Mortality rate was higher for patients with ELE (0% normal vs 2.3% transient ELE vs 6.5% persistent ELE, P < 0.001). Conclusion: ELE is prevalent in IBD, especially in CD, and associated with higher rates of mortality. Identification and management of ELE particularly when persistent are important to IBD outcomes.

Radiologic Predictors of Increased Number of Necrosectomies During Endoscopic Management of Walled-off Pancreatic Necrosis.

GOALS: No established methods exist to predict who will require a higher number of endoscopic necrosectomy sessions for walled-off necrosis (WON). We aim to identify radiologic predictors for requiring a greater number of necrosectomy sessions. This may help to identify patients who benefit from aggressive endoscopic management. MATERIALS AND METHODS: This is a multicenter retrospective study of patients with WON at 3 tertiary care centers. WON characteristics on preintervention computed tomography imaging were evaluated to determine if they were predictive of requiring more endoscopic necrosectomy. RESULTS: A total of 104 patients were included. Seventy patients (67.3%) underwent endoscopic necrosectomy, with median of 2 necrosectomies. WON largest transverse diameters (P=0.02), largest coronal diameters (P=0.01), necrosis pattern [likelihood ratio (LR)=17.85, P<0.001], spread (LR=11.02, P=0.01), hemorrhage (LR=8.64, P=0.003), and presence of disconnected pancreatic duct (LR=6.80, P=0.01) were associated with undergoing ≥2 necrosectomies. Patients with septations/loculations were significantly less likely to undergo ≥2 necrosectomies (LR=4.86, P=0.03). CONCLUSIONS: Several computed tomography radiologic features were significantly associated with undergoing ≥2 necrosectomies. These could help identify patients who will undergo a higher number of endoscopic necrosectomy sessions.

Light-powered CO2 fixation in a chloroplast mimic with natural and synthetic parts.

Nature integrates complex biosynthetic and energy-converting tasks within compartments such as chloroplasts and mitochondria. Chloroplasts convert light into chemical energy, driving carbon dioxide fixation. We used microfluidics to develop a chloroplast mimic by encapsulating and operating photosynthetic membranes in cell-sized droplets. These droplets can be energized by light to power enzymes or enzyme cascades and analyzed for their catalytic properties in multiplex and real time. We demonstrate how these microdroplets can be programmed and controlled by adjusting internal compositions and by using light as an external trigger. We showcase the capability of our platform by integrating the crotonyl-coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a synthetic network for carbon dioxide conversion, to create an artificial photosynthetic system that interfaces the natural and the synthetic biological worlds.

Engineering dual-glycan responsive expression systems for tunable production of heterologous proteins in Bacteroides thetaiotaomicron.

Genetically engineering intestinal bacteria, such as Bacteroides thetaiotaomicron (B. theta), holds potential for creating new classes of biological devices, such as diagnostics or therapeutic delivery systems. Here, we have developed a series of B. theta strains that produce functional transgenic enzymes in response to dextran and arabinogalactan, two chemically distinct glycans. Expression systems for single glycan induction, and a novel "dual-glycan" expression system, requiring the presence of both dextran and arabinogalactan, have been developed. In addition, we have created two different chromosomal integration systems and one episomal vector system, compatible with engineered recipient strains, to improve the throughput and flexibility of gene cloning, integration, and expression in B. theta. To monitor activity, we have demonstrated the functionality of two different transgenic enzymes: NanoLuc, a luciferase, and BuGH16C, an agarase from the human intestinal bacterium, Bacteroides uniforms NP1. Together this expression platform provides a new collection of glycan-responsive tools to improve the strength and fidelity of transgene expression in B. theta and provides proof-of-concept for engineering more complex multi-glycan expression systems.

A surrogate structural platform informed by ancestral reconstruction and resurrection of a putative carbohydrate binding module hybrid illuminates the neofunctionalization of a pectate lyase.

Yersinia enterocolitica is a pectinolytic zoonotic foodborne pathogen, the genome of which contains pectin-binding proteins and several different classes of pectinases, including polysaccharide lyases (PLs) and an exopolygalacturonase. These proteins operate within a coordinated pathway to completely saccharify homogalacturonan (HG). Polysaccharide lyase family 2 (PL2) is divided into two major subfamilies that are broadly-associated with contrasting 'endolytic' (PL2A) or 'exolytic' (PL2B) activities on HG. In the Y. enterocolitica genome, the PL2A gene is adjacent to an independent carbohydrate binding module from family 32 (YeCBM32), which possesses a N-terminal secretion tag and is known to specifically bind HG. Independent CBMs are rare in nature and, most commonly, are fused to enzymes in order to potentiate catalysis. The unconventional gene architecture of YePL2A and YeCBM32, therefore, may represent an ancestral relic of a fission event that decoupled PL2A from its cognate CBM. To provide further insight into the evolution of this pectinolytic locus and the molecular basis of HG depolymerisation within Y. enterocolitica, we have resurrected a YePL2A-YeCBM32 chimera and demonstrated that the extant PL2A digests HG more efficiently. In addition, we have engineered a tryptophan from the active site of the exolytic YePL2B into YePL2A (YePL2A-K291W) and demonstrated, using X-ray crystallography of substrate complexes, that it is a structural determinant of exo-activity within the PL2 family. In this manner, surrogate structural platforms may assist in the study of phylogenetic relationships informed by extant and resurrected sequences, and can be used to overcome challenging structural problems within carbohydrate active enzyme families.

Predicting Risk of Postoperative Disease Recurrence in Crohn's Disease: Patients With Indolent Crohn's Disease Have Distinct Whole Transcriptome Profiles at the Time of First Surgery.

Background: Assessing risk of Crohn's disease (CD) recurrence following ileocolic resection (ICR) is necessary to optimize medical management and prevent long-term complications. This study aimed to identify noninvasive markers that could predict postoperative disease activity. Methods: Inclusion criteria were a diagnosis of CD, first ICR, interval colonoscopy, and whole transcriptome array meeting quality control standards. Demographic and clinical data were obtained from the electronic medical record. RNA extraction and human transcriptome microarray were performed on noninflamed ileal margins from operative specimens. Clinical data and random forest were analyzed in R. Principal components analysis, hierarchical clustering, and pathway enrichment were performed in Partek. Results: Sixty-five patients completed the study, and 5 were excluded from analysis due to extreme variability on whole transcriptome analysis. Unsupervised hierarchical clustering revealed that patients with an i0 Rutgeerts score generally segregated from all others. In anti-TNF-naïve patients, unsupervised hierarchical clustering revealed complete segregation of patients with an i0 score. Reduced escalation in therapy and continued mucosal remission, consistent with indolent disease, were seen in the 4 years following surgery. Random forest identified 30 transcripts differentiating i0 patients from the other groups. Pathway enrichment highlighted toll-like receptor, NOD-like receptor, and TNF signaling. This transcriptome signature did not identify i0 anti-TNF-exposed patients. However, anti-TNF-exposed patients with indolent postoperative courses were found to have a transcriptome signature distinct from those with aggressive disease. Conclusions: Anti-TNF-naïve and -exposed patients have unique expression profiles at the time of surgery, which may offer predictive value in assessing the risk of nonrecurrence. 10.1093/ibd/izy228_video1izy228.video15804852517001.

Structural basis for substrate specificity of methylsuccinyl-CoA dehydrogenase, an unusual member of the acyl-CoA dehydrogenase family.

(2S)-methylsuccinyl-CoA dehydrogenase (MCD) belongs to the family of FAD-dependent acyl-CoA dehydrogenase (ACD) and is a key enzyme of the ethylmalonyl-CoA pathway for acetate assimilation. It catalyzes the oxidation of (2S)-methylsuccinyl-CoA to α,ß-unsaturated mesaconyl-CoA and shows only about 0.5% activity with succinyl-CoA. Here we report the crystal structure of MCD at a resolution of 1.37 Å. The enzyme forms a homodimer of two 60-kDa subunits. Compared with other ACDs, MCD contains an ∼170-residue-long N-terminal extension that structurally mimics a dimer-dimer interface of these enzymes that are canonically organized as tetramers. MCD catalyzes the unprecedented oxidation of an α-methyl branched dicarboxylic acid CoA thioester. Substrate specificity is achieved by a cluster of three arginines that accommodates the terminal carboxyl group and a dedicated cavity that facilitates binding of the C2 methyl branch. MCD apparently evolved toward preventing the nonspecific oxidation of succinyl-CoA, which is a close structural homolog of (2S)-methylsuccinyl-CoA and an essential intermediate in central carbon metabolism. For different metabolic engineering and biotechnological applications, however, an enzyme that can oxidize succinyl-CoA to fumaryl-CoA is sought after. Based on the MCD structure, we were able to shift substrate specificity of MCD toward succinyl-CoA through active-site mutagenesis.

Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides.

The major nutrients available to human colonic Bacteroides species are glycans, exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharide utilization loci (PULs). In Bacteroides thetaiotaomicron, a human colonic bacterium, the PULs activated by different pectin domains have been identified; however, the mechanism by which these loci contribute to the degradation of these GalA-containing polysaccharides is poorly understood. Here we show that each PUL orchestrates the metabolism of specific pectin molecules, recruiting enzymes from two previously unknown glycoside hydrolase families. The apparatus that depolymerizes the backbone of rhamnogalacturonan-I is particularly complex. This system contains several glycoside hydrolases that trim the remnants of other pectin domains attached to rhamnogalacturonan-I, and nine enzymes that contribute to the degradation of the backbone that makes up a rhamnose-GalA repeating unit. The catalytic properties of the pectin-degrading enzymes are optimized to protect the glycan cues that activate the specific PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to metabolism of the pectic network is illustrated by cross-feeding between organisms.

An Improved Kinetic Assay for the Characterization of Metal-Dependent Pectate Lyases.

Pectate lyases are a subset of polysaccharide lyases (PLs) that specifically utilize a metal dependent ß-elimination mechanism to cleave glyosidic bonds in homogalacturonan (HG; α-D-1,4-galacturonic acid). Most commonly, PLs harness calcium for catalysis; however, some PL families (e.g., PL2 and PL22) display preferences for transitional metals. Deploying alternative metals during ß-elimination is correlated with signature coordination pocket chemistry, and is reflective of the evolution, functional specialization, and cellular location of PL activity. Here we describe an optimized method for the analysis of metal-dependent polysaccharide lyases (PLs). We use an endolytic PL2 from Yersinia enterocolitica (YePL2A) as example to demonstrate how altering the catalytic metal within the reaction can modulate PL kinetics.

Determining the Localization of Carbohydrate Active Enzymes Within Gram-Negative Bacteria.

Investigating the subcellular location of secreted proteins is valuable for illuminating their biological function. Although several bioinformatics programs currently exist to predict the destination of a trafficked protein using its signal peptide sequence, these programs have limited accuracy and often require experimental validation. Here, we present a systematic method to fractionate gram-negative cells and characterize the subcellular localization of secreted carbohydrate active enzymes (CAZymes). This method involves four parallel approaches that reveal the relative abundance of protein within the cytoplasm, periplasm, outer membrane, and extracellular environment. Cytoplasmic and periplasmic proteins are fractionated by lysis and osmotic shock, respectively. Outer membrane bound proteins are determined by comparing cells before and after exoproteolytic digestion. Extracellularly secreted proteins are collected from the media and concentrated. These four different fractionations can then be probed for the presence and quantity of target proteins using immunochemical methods such as Western blots and ELISAs, or enzyme activity assays.

KdgF, the missing link in the microbial metabolism of uronate sugars from pectin and alginate.

Uronates are charged sugars that form the basis of two abundant sources of biomass-pectin and alginate-found in the cell walls of terrestrial plants and marine algae, respectively. These polysaccharides represent an important source of carbon to those organisms with the machinery to degrade them. The microbial pathways of pectin and alginate metabolism are well studied and essentially parallel; in both cases, unsaturated monouronates are produced and processed into the key metabolite 2-keto-3-deoxygluconate (KDG). The enzymes required to catalyze each step have been identified within pectinolytic and alginolytic microbes; yet the function of a small ORF, kdgF, which cooccurs with the genes for these enzymes, is unknown. Here we show that KdgF catalyzes the conversion of pectin- and alginate-derived 4,5-unsaturated monouronates to linear ketonized forms, a step in uronate metabolism that was previously thought to occur spontaneously. Using enzyme assays, NMR, mutagenesis, and deletion of kdgF, we show that KdgF proteins from both pectinolytic and alginolytic bacteria catalyze the ketonization of unsaturated monouronates and contribute to efficient production of KDG. We also report the X-ray crystal structures of two KdgF proteins and propose a mechanism for catalysis. The discovery of the function of KdgF fills a 50-y-old gap in the knowledge of uronate metabolism. Our findings have implications not only for the understanding of an important metabolic pathway, but also the role of pectinolysis in plant-pathogen virulence and the growing interest in the use of pectin and alginate as feedstocks for biofuel production.

The Physician Quality Improvement Initiative: Engaging Physicians in Quality Improvement, Patient Safety, Accountability and their Provision of High-Quality Patient Care.

University Health Network has been working to become a high-reliability organization, with a focus on safe, quality patient care. In response, the Medical Affairs Department has implemented several strategic initiatives to drive accountability, quality improvement and engagement with our physician population. One of these initiatives, the Physician Quality Improvement Initiative (PQII) is a physician-led project designed to provide active medical staff, in collaboration with their physician department chiefs, a comprehensive approach to focused and practical quality improvement in their practice. In this document, we outline the project, including its implementation strategy, logic model and outcomes, and provide discussion on how it fits into UHN's global strategy to provide safe, quality patient care.

Functional Analyses of Resurrected and Contemporary Enzymes Illuminate an Evolutionary Path for the Emergence of Exolysis in Polysaccharide Lyase Family 2.

Family 2 polysaccharide lyases (PL2s) preferentially catalyze the ß-elimination of homogalacturonan using transition metals as catalytic cofactors. PL2 is divided into two subfamilies that have been generally associated with secretion, Mg(2+) dependence, and endolysis (subfamily 1) and with intracellular localization, Mn(2+) dependence, and exolysis (subfamily 2). When present within a genome, PL2 genes are typically found as tandem copies, which suggests that they provide complementary activities at different stages along a catabolic cascade. This relationship most likely evolved by gene duplication and functional divergence (i.e. neofunctionalization). Although the molecular basis of subfamily 1 endolytic activity is understood, the adaptations within the active site of subfamily 2 enzymes that contribute to exolysis have not been determined. In order to investigate this relationship, we have conducted a comparative enzymatic analysis of enzymes dispersed within the PL2 phylogenetic tree and elucidated the structure of VvPL2 from Vibrio vulnificus YJ016, which represents a transitional member between subfamiles 1 and 2. In addition, we have used ancestral sequence reconstruction to functionally investigate the segregated evolutionary history of PL2 progenitor enzymes and illuminate the molecular evolution of exolysis. This study highlights that ancestral sequence reconstruction in combination with the comparative analysis of contemporary and resurrected enzymes holds promise for elucidating the origins and activities of other carbohydrate active enzyme families and the biological significance of cryptic metabolic pathways, such as pectinolysis within the zoonotic marine pathogen V. vulnificus.

Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism.

Yeasts, which have been a component of the human diet for at least 7,000 years, possess an elaborate cell wall α-mannan. The influence of yeast mannan on the ecology of the human microbiota is unknown. Here we show that yeast α-mannan is a viable food source for the Gram-negative bacterium Bacteroides thetaiotaomicron, a dominant member of the microbiota. Detailed biochemical analysis and targeted gene disruption studies support a model whereby limited cleavage of α-mannan on the surface generates large oligosaccharides that are subsequently depolymerized to mannose by the action of periplasmic enzymes. Co-culturing studies showed that metabolism of yeast mannan by B. thetaiotaomicron presents a 'selfish' model for the catabolism of this difficult to breakdown polysaccharide. Genomic comparison with B. thetaiotaomicron in conjunction with cell culture studies show that a cohort of highly successful members of the microbiota has evolved to consume sterically-restricted yeast glycans, an adaptation that may reflect the incorporation of eukaryotic microorganisms into the human diet.

Hamilton acute pain service safety study: using root cause analysis to reduce the incidence of adverse events.

BACKGROUND: Although intravenous patient-controlled analgesia opioids and epidural analgesia offer improved analgesia for postoperative patients treated on an acute pain service, these modalities also expose patients to some risk of serious morbidity and even mortality. Root cause analysis, a process for identifying the causal factor(s) that underlie an adverse event, has the potential to identify and address system issues and thereby decrease the chance of recurrence of these complications. METHODS: This study was designed to compare the incidence of adverse events on an acute pain service in three hospitals, before and after the introduction of a formal root cause analysis process. The "before" cohort included all patients with pain from February 2002 to July 2007. The "after" cohort included all patients with pain from January 2009 to December 2009. RESULTS: A total of 35,384 patients were tracked over the 7 yr of this study. The after cohort showed significant reductions in the overall event rate (1.47 vs. 2.35% or 1 in 68 vs. 1 in 42, the rate of respiratory depression (0.41 vs. 0.71%), the rate of severe hypotension (0.78 vs. 1.34%), and the rate of patient-controlled analgesia pump programming errors (0.0 vs. 0.08%). Associated with these results, the incidence of severe pain increased from 6.5 to 10.5%. To achieve these results, 26 unique recommendations were made of which 23 being completed, 1 in progress, and 2 not completed. CONCLUSIONS: Formal root cause analysis was associated with an improvement in the safety of patients on a pain service. The process was effective in giving credibility to recommendations, but addressing all the action plans proved difficult with available resources.