Canine respiratory coronavirus employs caveolin-1-mediated pathway for internalization to HRT-18G cells

Canine respiratory coronavirus (CRCoV), identified in 2003, is a member of the Coronaviridae family. The virus is a betacoronavirus and a close relative of human coronavirus OC43 and bovine coronavirus. Here, we examined entry of CRCoV into human rectal tumor cells (HRT-18G cell line) by analyzing co-localization of single virus particles with cellular markers in the presence or absence of chemical inhibitors of pathways potentially involved in virus entry. We also targeted these pathways using siRNA. The results show that the virus hijacks caveolin-dependent endocytosis to enter cells via endocytic internalization

Optical sensors based on lossy-mode resonances

Lossy-mode resonance (LMR)–based optical sensing technology has emerged in the last two decades as a nanotechnological platform with very interesting and promising properties. LMR complements the metallic materials typically used in surface plasmon resonance (SPR)–based sensors, with metallic oxides and polymers. In addition, it enables one to tune the position of the resonance in the optical spectrum, to excite the resonance with both transverse electric (TE) and transverse magnetic (TM) polarized light, and to generate multiple resonances. The domains of application are numerous: as sensors for detection of refractive indices voltage, pH, humidity, chemical species, and antigens, as well as biosensors. This review will discuss the bases of this relatively new technology and will show the main contributions that have permitted the optimization of its performance to the point that the question arises as to whether LMR–based optical sensors could become the sensing platform of the near future

Subcellular fractionation method to study endosomal trafficking of Kaposi’s sarcoma-associated herpesvirus

Background Virus entry involves multiple steps and is a highly orchestrated process on which successful infection collectively depends. Entry processes are commonly analyzed by monitoring internalized virus particles via Western blotting, polymerase chain reaction, and imaging techniques that allow scientist to track the intracellular location of the pathogen. Such studies have provided abundant direct evidence on how viruses interact with receptor molecules on the cell surface, induce cell signaling at the point of initial contact with the cell to facilitate internalization, and exploit existing endocytic mechanisms of the cell for their ultimate infectious agenda. However, there is dearth of knowledge in regards to trafficking of a virus via endosomes. Herein, we describe an optimized laboratory procedure to isolate individual organelles during different stages of endocytosis by performing subcellular fractionation. This methodology is established using Kaposi’s sarcoma-associated herpesvirus (KSHV) infection of human foreskin fibroblast (HFF) cells as a model. With KSHV and other herpesviruses alike, envelope glycoproteins have been widely reported to physically engage target cell surface receptors, such as integrins, in interactions leading to entry and subsequent infection. Results Subcellular fractionation was used to isolate early and late endosomes (EEs and LEs) by performing a series of centrifugations steps. Specifically, a centrifugation step post-homogenization was utilized to obtain the post-nuclear supernatant containing intact intracellular organelles in suspension. Successive fractionation via sucrose density gradient centrifugation was performed to isolate specific organelles including EEs and LEs. Intracellular KSHV trafficking was directly traced in the isolated endosomal fractions. Additionally, the subcellular fractionation approach demonstrates a key role for integrins in the endosomal trafficking of KSHV. The results obtained from fractionation studies corroborated those obtained by traditional imaging studies. Conclusions This study is the first of its kind to employ a sucrose flotation gradient assay to map intracellular KSHV trafficking in HFF cells. We are confident that such an approach will serve as a powerful tool to directly study intracellular trafficking of a virus, signaling events occurring on endosomal membranes, and dynamics of molecular events within endosomes that are crucial for uncoating and virus escape into the cytosol

The Performance of AFP, AFP-3, DCP as Biomarkers for Detection of Hepatocellular Carcinoma (HCC): A Phase 3 Biomarker Study in the United States

BACKGROUND & AIMS: α-fetoprotein (AFP), AFP Lens culinaris agglutinin-reactive fraction of AFP (AFP-L3), and des-gamma-carboxy prothrombin (DCP) in combination or in GALAD (Gender, Age, AFP-L3, AFP, and DCP) were tested for hepatocellular carcinoma (HCC) surveillance in retrospective cohort and case-control studies. However, there is a paucity of prospective data and no phase III biomarker studies from North American populations. METHODS: We conducted a prospective specimen collection, retrospective blinded evaluation (PRoBE) cohort study in patients with cirrhosis enrolled in a 6-monthly surveillance with liver imaging and AFP. Blood samples were prospectively collected every 6 months and analyzed in a retrospective blinded fashion. True positive rate (TPR) and false positive rate (FPR) for any or early HCC were calculated within 6, 12, and 24 months of HCC diagnosis based on published thresholds for biomarkers individually, in combination and in GALAD and Hepatocellular Carcinoma Early Detection Screening (HES) scores. We calculated the area under the receiver operating curve and estimated TPR based on an optimal threshold at a fixed FPR of 10%. RESULTS: The analysis was conducted in a cohort of 534 patients; 50 developed HCC (68% early) and 484 controls with negative imaging. GALAD had the highest TPR (63.6%, 73.8%, and 71.4% for all HCC, and 53.8%, 63.3%, and 61.8 % for early HCC within 6, 12, and 24 months, respectively) but an FPR of 21.5% to 22.9%. However, there were no differences in the area under the receiver operating curve among GALAD, HES, AFP-L3, or DCP. At a fixed 10% FPR, TPR for GALAD dropped (42.4%, 45.2%, and 46.9%) and was not different from HES (36.4%, 40.5%, and 40.8%) or AFP-L3 alone (39.4%, 45.2%, and 44.9%). CONCLUSIONS: In a prospective cohort phase III biomarker study, GALAD was associated with a considerable improvement in sensitivity for HCC detection but an increase in false-positive results. GALAD performance was modest and not different from AFP-L3 alone or HES

Model for End-Stage Liver Disease-Sodium Underestimates 90-Day Mortality Risk in Patients With Acute-on-Chronic Liver Failure

BACKGROUND & AIMS: It is unclear whether the model for end-stage liver disease-sodium (MELD-Na) score captures the clinical severity of acute-on-chronic liver failure (ACLF). We compared observed 90-day mortality in patients with ACLF with expected mortality based on the calculated MELD-Na and examined the consequences of underestimating clinical severity. METHODS: We identified patients with ACLF during hospitalization for cirrhosis in 127 VA hospitals between 01/01/2004 and 12/31/2014. We examined MELD-Na scores by ACLF presence and grade. We used actual and observed 90-day mortality to estimate a standardized mortality ratio (SMR) by ACLF presence and grade. We used transplant center-specific median MELD-Na at transplantation (MMaT) to estimate the proportion likely to receive priority for liver transplantation (LT) based on MELD-Na alone. RESULTS: Of 71,894 patients hospitalized for decompensated cirrhosis, 18,979 (26.4%) patients met the criteria for ACLF on admission. The median (P25-P75) MELD-Na on admission was 26 (22-30) for ACLF compared to 15 (12-20) for patients without ACLF; it was 24 (21-27), 27 (23-31), and 32 (26-37) for ACLF-1, 2 and 3, respectively. At 90 days, 40.0% of patients with ACLF died (30.8%, 41.6% and 68.8% with ACLF-1, 2 and 3, respectively) compared to 21.3% of patients without ACLF. Compared to the expected death rate based on MELD-Na, mortality risk was higher for patients with ACLF, SMR (95% CI): 1.52 (1.48-1.52), 1.46 (1.41-1.51), 1.50 (1.44-1.55), 1.66 (1.58-1.74) for overall ACLF, ACLF-1, -2 and -3, respectively. Only 9.1% of patients with ACLF reached the national median MELD-Na of 35 and between 17.3% to 35.1% exceeded the MMaT at any center. During index admission, 589 (0.8%) patients with ACLF were considered for LT evaluation and 16 (0.1%) were listed for LT. CONCLUSIONS: In a US cohort of hospitalized patients with decompensated cirrhosis, MELD-Na did not capture 90-day mortality risk in patients with ACLF. Patients with ACLF are at a disadvantage in the current MELD-Na-based system. LAY SUMMARY: Acute-on-chronic liver failure (ACLF) is a condition marked by multiple organ failures in patients with cirrhosis and is associated with a high risk of death. Liver transplantation may be the only curative treatment for these patients. A score called model for end-stage liver disease-sodium (MELD-Na) helps guide donor liver allocation for transplantation in the United States. The higher the MELD-Na score in a patient, the more likely that a patient receives a liver transplant. Our study data showed that MELD-Na score underestimates the risk of dying at 90 days in patients with ACLF. Thus, physicians need to start liver transplant evaluation early instead of waiting for a high MELD-Na number

Comparative Effectiveness of Surveillance Colonoscopy Intervals on Colorectal Cancer Outcomes in a National Cohort of Patients with Inflammatory Bowel Disease

BACKGROUND & AIMS: Surveillance colonoscopy is recommended to reduce colorectal cancer (CRC)-related morbidity and mortality in patients with inflammatory bowel disease (IBD). The comparative effectiveness of varying colonoscopy intervals on CRC outcomes among patients with IBD is unknown. METHODS: We performed a retrospective cohort study of patients with confirmed CRC within a cohort of 77,824 patients with IBD during 2000 to 2015 in the National Veterans Health Administration. We examined the association between colonoscopy surveillance intervals on CRC stage, treatment, or all-cause and cancer-specific mortality. The interval of colonoscopy prior to CRC diagnosis was categorized as those performed within \u3c1 \u3eyear, 1 to 3 years, 3 to 5 years, or none within 5 years. RESULTS: Among 566 patients with CRC-IBD, most (69.4%) did not have colonoscopy within 5 years prior to CRC diagnosis, whereas 9.7% had colonoscopy within 1 year prior to diagnosis, 17.7% within 1 to 3 years, and 3.1% between 3 and 5 years. Compared with no surveillance, colonoscopy within 1 year (adjusted odds ratio, 0.40; 95% confidence interval [CI], 0.20-0.82), and 1 to 3 years (adjusted odds ratio, 0.56; 95% CI, 0.32-0.98) were less likely to be diagnosed at late stage. Regardless of IBD type and duration, colonoscopy within 1 year was associated with a lower all-cause mortality (adjusted hazard ratio, 0.56; 95% CI, 0.36-0.88). CONCLUSIONS: In a national cohort of patients with CRC-IBD, colonoscopy within 3 years prior to CRC diagnosis was associated with early tumor stage at diagnosis, and colonoscopy within 1 year was associated with a reduced all-cause mortality compared with no colonoscopy. Our findings support colonoscopy intervals of 1 to 3 years in patients with IBD to reduce late-stage CRC and all-cause mortality

Coronary artery calcification on routine CT has prognostic and treatment implications for all ages

Aims: Guidelines have recommended reporting coronary artery calcification (CAC) if present on chest CT imaging regardless of indication. This study assessed CAC prevalence, prognosis and the potential clinical impact of its reporting. Methods: We performed a single-centre retrospective analysis (January-December 2015) of 1400 chest CTs (200 consecutive within each age group: &lt;40, 40-49, 50-59, 60-69, 70-79, 80-89, ≥90). CTs were re-reviewed for CAC presence and severity and excluded if prior coronary intervention. Comorbidities, statin prescription and clinical outcomes (myocardial infarction [MI], stroke, all-cause mortality) were recorded. The impact of reporting CAC was assessed against pre-existing statin prescriptions. Results: 1343 patients were included (mean age 63±20 years, 56% female). Inter- and intra-observer variability for CAC presence at re-review was almost perfect (κ 0.89, p &lt; 0.001; κ 0.90, p &lt; 0.001) and for CAC grading was substantial and almost perfect (κ 0.68, p &lt; 0.001; κ 0.91, p &lt; 0.001). CAC was observed in 729/1343 (54%), more frequently in males (p &lt; 0.001) and rising age (p &lt; 0.001). A high proportion of patients with CAC in all age groups had no prior statin prescription (range: 42% [80-89] to 100% [&lt;40]). The ‘number needed to report’ CAC presence to potentially impact management across all ages was 2. 689 (51%) patients died (median follow-up 74-months). CAC presence was associated with risk of MI, stroke and all-cause mortality (p &lt; 0.001). After adjusting for confounders, severe calcification predicted risk of all-cause mortality (HR 1.8 [1.2-2.5], p = 0.002). Conclusion: Grading of CAC was reproducible, and although prevalence rose with age, prognostic and treatment implications were maintained in all ages.</p

Coronary artery calcification on routine CT has prognostic and treatment implications for all ages

Aims: Guidelines have recommended reporting coronary artery calcification (CAC) if present on chest CT imaging regardless of indication. This study assessed CAC prevalence, prognosis and the potential clinical impact of its reporting. Methods: We performed a single-centre retrospective analysis (January-December 2015) of 1400 chest CTs (200 consecutive within each age group: &lt;40, 40-49, 50-59, 60-69, 70-79, 80-89, ≥90). CTs were re-reviewed for CAC presence and severity and excluded if prior coronary intervention. Comorbidities, statin prescription and clinical outcomes (myocardial infarction [MI], stroke, all-cause mortality) were recorded. The impact of reporting CAC was assessed against pre-existing statin prescriptions. Results: 1343 patients were included (mean age 63±20 years, 56% female). Inter- and intra-observer variability for CAC presence at re-review was almost perfect (κ 0.89, p &lt; 0.001; κ 0.90, p &lt; 0.001) and for CAC grading was substantial and almost perfect (κ 0.68, p &lt; 0.001; κ 0.91, p &lt; 0.001). CAC was observed in 729/1343 (54%), more frequently in males (p &lt; 0.001) and rising age (p &lt; 0.001). A high proportion of patients with CAC in all age groups had no prior statin prescription (range: 42% [80-89] to 100% [&lt;40]). The ‘number needed to report’ CAC presence to potentially impact management across all ages was 2. 689 (51%) patients died (median follow-up 74-months). CAC presence was associated with risk of MI, stroke and all-cause mortality (p &lt; 0.001). After adjusting for confounders, severe calcification predicted risk of all-cause mortality (HR 1.8 [1.2-2.5], p = 0.002). Conclusion: Grading of CAC was reproducible, and although prevalence rose with age, prognostic and treatment implications were maintained in all ages.</p

Improving Adherence to Clinical Practice Guidelines for Managing Gastric Intestinal Metaplasia Among Gastroenterologists at a US Academic Institution

BACKGROUND: Clinical guidelines reserve endoscopic surveillance after a gastric intestinal metaplasia (GIM) diagnosis for high-risk patients. However, it is unclear how closely guidelines are followed in clinical practice. We examined the effectiveness of a standardized protocol for the management of GIM among gastroenterologists at a US hospital. METHODS: This was a preintervention and postintervention study, which included developing a protocol and education of gastroenterologists on GIM management. For the preintervention study, 50 patients with GIM were randomly selected from a histopathology database at the Houston VA Hospital between January 2016 and December 2019. For the postintervention study, we assessed change in GIM management in a cohort of 50 patients with GIM between April 2020 and January 2021 and surveyed 10 gastroenterologists. The durability of the intervention was assessed in a cohort of 50 GIM patients diagnosed between April 2021 and July 2021. RESULTS: In the pre-intervention cohort, GIM location was specified (antrum and corpus separated) in 11 patients (22%), and Helicobacter pylori testing was recommended in 11 of 26 patients (42%) without previous testing. Gastric mapping biopsies were recommended in 14% and surveillance endoscopy in 2%. In the post-intervention cohort, gastric biopsy location was specified in 45 patients (90%, p\u3c0.001) and H. pylori testing was recommended in 26 of 27 patients without prior testing (96%, p\u3c0.001). Because gastric biopsy location was known in 90% of patients (p\u3c0.001), gastric mapping was not necessary, and surveillance endoscopy was recommended in 42% (p\u3c0.001). One year after the intervention, all metrics remained elevated compared to the pre-intervention cohort. CONCLUSIONS: GIM management guidelines are not consistently followed. A protocol for GIM management and education of gastroenterologists increased adherence to H. pylori testing and GIM surveillance recommendations