Offline Two-Dimensional Liquid Chromatography-Mass Spectrometry for Deep Annotation of the Fecal Metabolome Following Fecal Microbiota Transplantation.

Biological interpretation of untargeted LC-MS-based metabolomics data depends on accurate compound identification, but current techniques fall short of identifying most features that can be detected. The human fecal metabolome is complex, variable, incompletely annotated, and serves as an ideal matrix to evaluate novel compound identification methods. We devised an experimental strategy for compound annotation using multidimensional chromatography and semiautomated feature alignment and applied these methods to study the fecal metabolome in the context of fecal microbiota transplantation (FMT) for recurrent C. difficile infection. Pooled fecal samples were fractionated using semipreparative liquid chromatography and analyzed by an orthogonal LC-MS/MS method. The resulting spectra were searched against commercial, public, and local spectral libraries, and annotations were vetted using retention time alignment and prediction. Multidimensional chromatography yielded more than a 2-fold improvement in identified compounds compared to conventional LC-MS/MS and successfully identified several rare and previously unreported compounds, including novel fatty-acid conjugated bile acid species. Using an automated software-based feature alignment strategy, most metabolites identified by the new approach could be matched to features that were detected but not identified in single-dimensional LC-MS/MS data. Overall, our approach represents a powerful strategy to enhance compound identification and biological insight from untargeted metabolomics data.

Gastrointestinal-isolated Distress is Common in Alpha-gal Allergic Patients on Mammalian Meat Challenge.

BACKGROUND AND AIMS: Alpha-gal allergy causes a delayed reaction to mammalian meats and has been reported worldwide. Patients with the allergy may present with isolated gastrointestinal (GI) symptoms, but this phenotype is poorly understood. METHODS: We pooled and analyzed symptoms and demographics of patients from two prospective cohorts of patients with a diagnosis of alpha-gal allergy who reacted after eating mammalian meat under observation. We compared the characteristics of patients who demonstrated GI-isolated symptoms on a challenge with those who exhibited symptoms outside the GI tract (skin, respiratory, and circulatory). RESULTS: Among the 91 children and adult alpha-gal allergic patients who exhibited symptoms after oral challenge with mammalian meat, 72.5% experienced GI distress with one or more GI symptoms, which was the most frequent class of symptoms, compared with skin changes in 57.1% and respiratory distress in 5.5%. The most common GI symptoms were abdominal pain (71%) and vomiting (22.0%). GI-isolated symptoms occurred in 37 patients (40.7%) who reacted, and those patients reacted more quickly than patients who exhibited systemic symptoms (median onset of symptoms in GI-isolated group 90 min vs 120 min) and were more likely to be children than adults (relative risk=1.94, 95% CI: 1.04-3.63). CONCLUSIONS: Isolated-GI distress occurred in 4 in every 10 alpha-gal allergic individuals who developed symptoms on oral food challenge with mammalian meat. Alpha-gal allergic patients, particularly children, may exhibit GI distress alone, and adult and pediatric gastroenterologists should be aware of the diagnosis and management of the allergy.

AGA Clinical Practice Update on Alpha-Gal Syndrome for the GI Clinician: Commentary.

DESCRIPTION: Alpha-gal syndrome is an emerging allergy first described in the early 2000s. The allergy can cause anaphylaxis, gastrointestinal (GI) symptoms, and skin changes one to several hours after ingestion of mammalian products. A GI phenotype that is increasingly recognized manifests with nonspecific symptoms like abdominal pain, diarrhea, nausea or vomiting without predominant skin, respiratory or circulatory symptoms. Though the syndrome has been reported on all continents except Antarctica, in the United States most reports are within the range of the Lone Star tick, extending from New York and Iowa to Texas and Florida. The purpose of this AGA Clinical Practice Update (CPU) Commentary is to increase awareness among gastroenterologists about the presentation and management of alpha-gal syndrome. METHODS: This CPU commentary was commissioned and approved by the AGA Institute Clinical Practice Updates Committee (CPUC) and the AGA Governing Board to provide timely guidance on a topic of high clinical importance to the AGA membership and underwent internal peer review by the CPUC and external peer review through standard procedures of Clinical Gastroenterology and Hepatology. This expert commentary incorporates important as well as recently published studies in this field, and it reflects the experiences of the authors. Formal ratings regarding the quality of evidence or strength of the presented considerations were not included since systematic reviews were not performed.

Using Multidimensional Separations to Distinguish Isomeric Amino Acid-Bile Acid Conjugates and Assess Their Presence and Perturbations in Model Systems.

Bile acids play key roles in nutrient uptake, inflammation, signaling, and microbiome composition. While previous bile acid analyses have primarily focused on profiling 5 canonical primary and secondary bile acids and their glycine and taurine amino acid-bile acid (AA-BA) conjugates, recent studies suggest that many other microbial conjugated bile acids (or MCBAs) exist. MCBAs are produced by the gut microbiota and serve as biomarkers, providing information about early disease onset and gut health. Here we analyzed 8 core bile acids synthetically conjugated with 22 proteinogenic and nonproteogenic amino acids totaling 176 MCBAs. Since many of the conjugates were isomeric and only 42 different m/z values resulted from the 176 MCBAs, a platform coupling liquid chromatography, ion mobility spectrometry, and mass spectrometry (LC-IMS-MS) was used for their separation. Their molecular characteristics were then used to create an in-house extended bile acid library for a combined total of 182 unique compounds. Additionally, ∼250 rare bile acid extracts were also assessed to provide additional resources for bile acid profiling and identification. This library was then applied to healthy mice dosed with antibiotics and humans having fecal microbiota transplantation (FMT) to assess the MCBA presence and changes in the gut before and after each perturbation.

Alpha-Gal Sensitization in a US Screening Population Is Not Associated With a Decreased Meat Intake or Gastrointestinal Symptoms.

INTRODUCTION: Patients with alpha-gal syndrome, a delayed reaction to mammalian meat, can present with isolated gastrointestinal (GI) symptoms. We aimed to estimate the frequency of alpha-gal sensitization in a Southeastern US population and determine the association between sensitization and mammalian product dietary intake or GI symptoms. METHODS: We performed a cross-sectional study of participants who underwent a screening colonoscopy at our center between 2013 and 2015. We quantified serum alpha-gal immunoglobulin E antibodies in participants who were prospectively enrolled at screening colonoscopy and compared diet intake and lower GI symptoms reported in standardized questionnaires among those with elevated versus no alpha-gal IgE antibodies. RESULTS: Alpha-gal IgE antibodies were common-31.4% of screening colonoscopy participants (127 of 404) had elevated serum alpha-gal IgE >0.1 kU/L. Alpha-gal-sensitized participants endorsed similar rates of abdominal pain compared with those without alpha-gal antibodies (33% vs 38%, adjusted odds ratio 0.9, 95% confidence interval 0.7-1.3). Mammalian meat consumption did not differ based on alpha-gal sensitization status (average 1.43 servings/d in sensitized subjects vs 1.50 in alpha-gal IgE-negative subjects, P = 0.9). Alpha-gal-sensitized participants with levels ≥10 (n = 21) were overrepresented in the lowest quartiles of mammalian meat consumption, but not among those with GI symptoms in general. Participants with high alpha-gal antibody levels >2 kU/L (n = 45) or ≥10 U/L (n = 21) did not have a reduced mean daily mammalian meat intake compared with seronegative people. DISCUSSION: Elevated alpha-gal IgE antibodies were common and not associated with a reduced mammalian meat intake, abdominal pain, or diarrhea. Seropositivity did not predict symptomatic alpha-gal sensitization in this general screening population. Other host factors likely contribute to the phenotypic expression of alpha-gal syndrome.

Food Allergies and Alpha-gal Syndrome for the Gastroenterologist.

PURPOSE OF REVIEW: Food allergies are typically not considered as a cause of gastrointestinal (GI) distress without additional allergic symptoms, apart from celiac disease and eosinophilic esophagitis. However, recent reports of patients with alpha-gal syndrome who presented with GI-only symptoms like abdominal pain, vomiting, and diarrhea challenge this paradigm. Alpha-gal syndrome is an IgE-mediated allergy characterized by delayed reactions after eating mammalian meat or mammalian-derived products that contain galactose-alpha-1,3-galactose (alpha-gal). The purpose of this review is to discuss our current understanding of food allergies, GI illness, and the GI manifestations of alpha-gal syndrome. RECENT FINDINGS: Among Southeastern U.S. GI clinic patients who screened positive for serum alpha-gal IgE, a majority of patients reported significant symptom improvement on an alpha-gal-avoidant diet, suggesting that the allergy had played a role in their GI symptoms. Diagnosis of alpha-gal syndrome is typically made with concerning allergic symptoms, elevated alpha-gal specific IgE in the serum, and symptom improvement on an alpha-gal avoidant diet. Alpha-gal syndrome can cause a delayed allergic response that is increasingly recognized worldwide, including among patients with predominant GI symptoms.

Walk before you run: Feasibility challenges and lessons learned from the PROCLAIM study, a multicenter randomized controlled trial of misoprostol for prevention of recurrent Clostridioides difficile during COVID-19.

We analyzed our challenging experience with a randomized controlled trial of misoprostol for prevention of recurrent C. difficile. Despite careful prescreening and thoughtful protocol modifications to facilitate enrollment, we closed the study early after enrolling just 7 participants over 3 years. We share lessons learned, noting the importance of feasibility studies, inclusion of biomarker outcomes, and dissemination of such findings to inform future research design and implementation successes.

Breast cancer treatment and survival differences in women in remote and socioeconomically disadvantaged areas, as demonstrated by linked data from New South Wales (NSW), Australia.

INTRODUCTION: Reducing variations in cancer treatment and survival is a key aim of the NSW Cancer Plan. Variations in breast cancer treatment and survival in NSW by remoteness and socioeconomic status of residence were investigated to determine benchmarks. Reducing variations in cancer treatment and survival is a key aim of the NSW Cancer Plan. Variations in breast cancer treatment and survival in NSW by remoteness and socioeconomic status of residence were investigated to determine benchmarks. METHODS: A retrospective cohort study used linked data for invasive breast cancers, diagnosed in May 2002 to December 2015 from the NSW Cancer Registry, with corresponding inpatient, and medical and pharmaceutical insurance data. Associations between treatment modalities, area socioeconomic status and residential remoteness were explored using logistic regression. Predictors of breast cancer survival were investigated using Kaplan-Meier product-limit estimates and multivariate competing risk regression. RESULTS: Results indicated a high 5-year disease-specific survival in NSW of 90%. Crude survival was equivalent by residential remoteness and marginally lower in lower socioeconomic areas. Competing risk regression showed equivalent outcomes by area socioeconomic status, except for the least disadvantaged quintile, which showed a higher survival. Higher sub-hazard ratios for death occurred for women with breast cancer aged 70 + years, and more advanced stage. Adjusted analyses indicated more advanced stage in lower socioeconomic areas, with less breast reconstruction and radiotherapy, and marginally less hormone therapy for women from these areas. Conversely, among these women who had breast conserving surgery, there was higher use of chemotherapy. Remoteness of residence was associated in adjusted analyses with less radiotherapy and less immediate breast reconstruction. In these short term data, remoteness of residence was not associated with lower survival. CONCLUSION: This study provides benchmarks for monitoring future variations in treatment and survival.

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.

Increasing Rates of Surgery for Patients With Nonmalignant Colorectal Polyps in the United States.

BACKGROUND & AIMS: Despite the availability of endoscopic therapy, many patients in the United States undergo surgical resection for nonmalignant colorectal polyps. We aimed to quantify and examine trends in the use of surgery for nonmalignant colorectal polyps in a nationally representative sample. METHODS: We analyzed data from the Healthcare Cost and Utilization Project National Inpatient Sample for 2000 through 2014. We included all adult patients who underwent elective colectomy or proctectomy and had a diagnosis of either nonmalignant colorectal polyp or colorectal cancer. We compared trends in surgery for nonmalignant colorectal polyps with surgery for colorectal cancer and calculated age, sex, race, region, and teaching status/bed-size-specific incidence rates of surgery for nonmalignant colorectal polyps. RESULTS: From 2000 through 2014, there were 1,230,458 surgeries for nonmalignant colorectal polyps and colorectal cancer in the United States. Among those surgeries, 25% were performed for nonmalignant colorectal polyps. The incidence of surgery for nonmalignant colorectal polyps has increased significantly, from 5.9 in 2000 to 9.4 in 2014 per 100,000 adults (incidence rate difference, 3.56; 95% confidence interval 3.40-3.72), while the incidence of surgery for colorectal cancer has significantly decreased, from 31.5 to 24.7 surgeries per 100,000 adults (incidence rate difference, -6.80; 95% confidence interval -7.11 to -6.49). The incidence of surgery for nonmalignant colorectal polyps has been increasing among individuals age 20 to 79, in men and women and including all races and ethnicities. CONCLUSIONS: In an analysis of a large, nationally representative sample, we found that surgery for nonmalignant colorectal polyps is common and has significantly increased over the past 14 years.

Ca2+/calmodulin-dependent protein kinase II and Dimethyl Sulfoxide affect the sealing frequencies of transected hippocampal neurons.

Traumatic injury often results in axonal severance, initiating obligatory Wallerian degeneration of distal segments, whereas proximal segments often survive. Calcium ion (Ca2+ ) influx at severed proximal axonal ends activates pathways that can induce apoptosis. However, this same Ca2+ -influx also activates multiple parallel pathways that seal the plasmalemma by inducing accumulation and fusion of vesicles at the lesion site that reduce Ca2+ -influx and enhance survival. We examined whether various inhibitors of Ca2+ /calmodulin-dependent protein kinases (CaMKs), and/or dimethyl sulfoxide (DMSO), a common solvent for biologically active substances, affected the ability of a hippocampal-derived neuronal cell line (B104 cells) to seal membrane damage following axotomy. Axolemmal sealing frequencies were assessed at different transection distances from the axon hillock and at various times after Ca2+ -influx (PC times) by observing whether transected cells took-up fluorescent dyes. Inhibition of CaMKII by tatCN21 and KN-93, but not inhibition of CaMKI and CaMKIV by STO-609, affected axonal sealing frequencies. That is, CaMKII is a component of previously reported parallel pathways that induce membrane sealing, whereas CaMKI and CaMKIV are not involved. The effects of these CaMKII inhibitors on plasmalemmal sealing depended on their mechanism of inhibition, transection distance, and PC time. DMSO at low concentrations (90 µM-28 mM or 0.00064%-0.2% v/v) significantly increased membrane-sealing frequencies at most PC times and transection distances, possibly by permeabilizing the plasmalemma to Ca2+ . Inhibition of CaMKII, DMSO, PC time, and the transection distance significantly affect plasmalemmal sealing that is critical to somal survival in traumatic lesions.

The Effect of ICU Out-of-Hours Admission on Mortality: A Systematic Review and Meta-Analysis.

OBJECTIVES: Organizational factors are associated with outcome of critically ill patients and may vary by time of day and day of week. We aimed to identify the association between out-of-hours admission to critical care and mortality. DATA SOURCES: MEDLINE (via Ovid) and EMBASE (via Ovid). STUDY SELECTION: We performed a systematic search of the literature for studies on out-of-hours adult general ICU admission on patient mortality. DATA EXTRACTION: Meta-analyses were performed and Forest plots drawn using RevMan software. Data are presented as odds ratios ([95% CIs], p values). DATA SYNTHESIS: A total of 16 studies with 902,551 patients were included in the analysis with a crude mortality of 18.2%. Fourteen studies with 717,331 patients reported mortality rates by time of admission and 11 studies with 835,032 patients by day of admission. Admission to ICU at night was not associated with an increased odds of mortality compared with admissions during the day (odds ratio, 1.04 [0.98-1.11]; p = 0.18). However, admissions during the weekend were associated with an increased odds of death compared with ICU admissions during weekdays (1.05 [1.01-1.09]; p = 0.006). Increased mortality associated with weekend ICU admissions compared with weekday ICU admissions was limited to North American countries (1.08 [1.03-1.12]; p = 0.0004). The absence of a routine overnight on-site intensivist was associated with increased mortality among weekend ICU admissions compared with weekday ICU admissions (1.11 [1.00-1.22]; p = 0.04) and nighttime admissions compared with daytime ICU admissions (1.11 [1.00-1.23]; p = 0.05). CONCLUSIONS: Adjusted risk of death for ICU admission was greater over the weekends compared with weekdays. The absence of a dedicated intensivist on-site overnight may be associated with increased mortality for acute admissions. These results need to be interpreted in context of the organization of local healthcare resources before changes to healthcare policy are implemented.

Patients With Nonpolypoid (Flat and Depressed) Colorectal Neoplasms at Increased Risk for Advanced Neoplasias, Compared With Patients With Polypoid Neoplasms.

BACKGROUND & AIMS: Nonpolypoid colorectal neoplasms (NP-CRNs) are more likely to contain high-grade dysplasia or early-stage cancer than polypoid neoplasms. We aimed to determine the long-term outcomes of patients with at least 1 NP-CRN. METHODS: We performed a longitudinal cohort study of 4454 patients at a Veterans' Affairs hospital who underwent colonoscopy from 2000 through 2005; 341 were found to have 1 or more NP-CRNs and were matched (3:1) with patients found to have 1 or more polypoid neoplasms (controls, n = 1025). We collected and analyzed data on baseline colonoscopy findings and first follow-up colonoscopy results through August 2014. We calculated the incidence of advanced neoplasia at first follow-up colonoscopy, as defined by the presence of ≥1 tubular or sessile serrated adenomas ≥10 mm in diameter, tubulovillous adenoma, high-grade dysplasia, or invasive cancer. RESULTS: A significantly higher proportion of patients with 1 or more NP-CRNs (16.0%) were found to have advanced neoplasia at their first follow-up colonoscopy than controls (8.6%); the adjusted risk ratio was 1.6 (95% confidence interval, 1.05-2.6; P = .03). A significantly higher proportion of patients with 1 or more NP-CRNs were found to have additional NP-CRNs at the follow-up colonoscopy (17%) than controls (7%; relative risk, 2.3; 95% confidence interval, 1.5-3.5; P < .001). Similar proportions of patients in each group developed cancers after colonoscopy. CONCLUSIONS: In a longitudinal cohort study, we found that patients with NP-CRN were more likely to develop additional NP-CRNs and to have advanced neoplasms at their first follow-up colonoscopy than patients with only polypoid neoplasms. However, patients with NP-CRN were not more likely to develop cancers after colonoscopy when surveillance guidelines were followed. Larger studies are needed to determine risk of colorectal cancer in patients with NP-CRN.