A modified protein assay from microgram to low nanogram levels in dilute samples.

In this article, we present a modified and improved protein assay that was previously described as "amidoschwarz assay" by Schaffner and Weissmann. Our improved protein assay is user-friendly and 30-40 times more sensitive than the earlier method. The assay was developed into three formats (macro-, micro-, and nanoassay) with trichloroacetic acid (TCA) as protein precipitating agent, measuring up to 96 samples. The macro and micro formats of this assay require a single reagent staining with amido black of protein dots bound to nitrocellulose membrane with lowest protein measurements to 1 and 0.1 µg, respectively. On the other hand, the nanoassay, with combination staining of amido black followed by colloidal gold, can extend the detection limit to 2.5 ng of protein. Protein concentrations were determined by densitometry and/or spectrophotometry. This assay is compatible with many ionic and non-ionic detergents. This improved protein assay provides an additional choice to researchers in measuring total protein concentration accurately in dilute biological samples as low as 0.125 µg/ml prior to their biochemical analysis such as in comparative proteomics.

Outcomes after Liver Transplantation with Steatotic Grafts: Redefining Acceptable Cutoffs for Steatotic Grafts.

Background: Graft macrosteatosis can predispose to a higher risk of graft loss so we sought to redefine acceptable cutoffs for graft steatosis. Methods: Data of 26,103 donors who underwent liver transplantation (LT) between January 2004 and December 2018 from the UNOS-STAR database were utilized. A high-risk steatotic (HRS) graft and a low-risk steatotic (LRS) graft were defined as ≥20% and <20% macrosteatosis, respectively. High-risk steatotic grafts were further classified as grafts with 20-29% (G1S grafts), 30-39% (G2S grafts), and ≥40% steatosis (G3S grafts). Outcomes between groups were compared. Results: LRS grafts had excellent graft (93.3 and 87.7%) and overall survival (95.4 and 90.5%) at 90 days and 1 year. Compared to LRS grafts, G1S, G2S, and G3S grafts had worse graft and overall survival at 90 days and 1-year (p <0.001). There was no difference in graft or overall survival of G1S or G3S grafts compared to G2S grafts until after adjustment in which G3S grafts were found to be associated with an increased risk of graft loss-aHR 1.27 (1.03-1.57), p = 0.02. Discussion: Liver grafts can be categorized into three categories: (1) <20% or "very low risk", (2) 20-39% or "low-to-moderate risk", and usually acceptable, and (3) ≥40% steatosis or "moderate-to-high risk". How to cite this article: Da BL, Satiya J, Heda RP, et al. Outcomes after Liver Transplantation with Steatotic Grafts: Redefining Acceptable Cutoffs for Steatotic Grafts. Euroasian J Hepato-Gastroenterol 2022;12(Suppl 1):S5-S14.

African Americans have a distinct clinical and histologic profile with lower prevalence of NASH and advanced fibrosis relative to Caucasians.

BACKGROUND AND AIMS: Racial/ethnic disparities have been reported in the prevalence of nonalcoholic fatty liver disease (NAFLD). Thus, we aimed to understand the inter-ethnic clinical, biochemical, and histological differences in a large cohort of Caucasians and African-Americans (AA). METHODS: Laboratory and liver biopsy data of 942 NAFLD patients were retrospectively analyzed. Nine hundred seven patients were included in the analysis: 677 (74.6%) Caucasians and 230 (25.3%) AA. RESULTS: AA had higher mean BMI compared to Caucasians (42.6 ± 9.5 vs. 39 ± 8.6 kg/m2). The prevalence of nonalcoholic steatohepatitis (NASH), defined by NAFLD activity score (NAS . 5), was higher in the Caucasians (n = 67) compared to AA (n = 7) (9.8% vs. 3%, P = 0.0007). One hundred fifteen patients (12.8%) had advanced fibrosis: 109 (16.2%) Caucasians and six (2.6%) AA. No AA patients had stage 4 fibrosis or cirrhosis. Multivariate logistic regression analysis revealed advanced fibrosis was significantly associated with age at liver biopsy (OR 1.03, 95% CI 1.0.1.1, P = 0.017, lower platelet count (OR 0.99, 95% CI 0.98.0.99, P = <0.0001), AST/ALT ratio (OR 5.19, 95% CI 2.9.9.2, P <0.0001) and Caucasian race (OR 7.49, 95% CI 2.53.22.2, P = 0.0003). Advanced fibrosis in AA was predicted by lower platelet count and AST/ALT ratio. Whereas Advanced fibrosis in Caucasians was predicted by age at biopsy, lower platelet count and AST/ALT ratio. CONCLUSION: The AA have a distinct clinical and histologic phenotype. Caucasians have a significantly greater proportion of NASH and are eight times more likely to develop advanced fibrosis than AA.

Efficacy and safety of peroral endoscopic myotomy in the management of recurrent achalasia after failed Heller myotomy: a systematic review and meta-analysis.

BACKGROUND: Heller myotomy (HM) is an established treatment for achalasia but can fail in up to 10-20% of patients. Peroral endoscopic myotomy (POEM) may be an appropriate treatment for patients with failed HM. METHODS: We searched several databases to identify non-comparative studies evaluating the efficacy and/or safety of POEM after failed HM and comparative studies comparing the efficacy and/or safety of POEM in patients with and without prior HM. Outcomes assessed included clinical success, technical success, adverse events, post-treatment gastroesophageal reflux disease (GERD), and presence of esophagitis on endoscopy. We calculated weighted pooled rates with 95% confidence intervals (CI) for all outcomes in patients undergoing POEM with prior HM. We calculated pooled odds ratios with 95%CI to compare the outcomes between patients with and without previous HM who underwent POEM. RESULTS: We included 11 observational studies with 1205 patients. Weighted pooled rates (95%CI) for overall clinical success and technical success in patients with failed HM were 87% (81-91%) and 97% (94-99%), respectively. Weighted pooled rates (95%CI) for major adverse events, new-onset GERD and presence of esophagitis on endoscopy were 5% (2-10%), 33% (26-41%), and 38% (22-58%), respectively. There were no differences in clinical success, adverse events, post-treatment GERD and esophagitis between patients with and without previous HM. CONCLUSIONS: POEM is safe and effective in patients with failed HM and should be considered in patients with recurrent achalasia after HM. Outcomes of POEM are comparable in patients with and without prior HM.

Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.