Discovery of hepatitis B virus subviral particle biogenesis inhibitors from a bioactive compound library.

High levels of hepatitis B virus (HBV) surface antigen (HBsAg) in the blood of chronic HBV carriers are considered to drive the exhaustion of antigen-specific T and B lymphocytes and thus responsible for the persistence of infection. Accordingly, therapeutic elimination of HBsAg may facilitate the activation of adaptive antiviral immune responses against HBV and achieve a functional cure of chronic hepatitis B. We discovered recently that an amphipathic alpha helix spanning W156 to R169 of HBV small envelope (S) protein plays an essential role in the morphogenesis of subviral particles (SVPs) and metabolism of S protein. We thus hypothesized that pharmacological disruption of SVP morphogenesis may induce intracellular degradation of S protein and reduce HBsAg secretion. To identify inhibitors of SVP biogenesis, we screened 4417 bioactive compounds with a HepG2-derived cell line expressing HBV S protein and efficiently secreting small spherical SVPs. The screen identified 24 compounds that reduced intracellular SVPs and secreted HBsAg in a concentration-dependent manner. However, 18 of those compounds inhibited the secretion of HBsAg and HBeAg in HBV replicon transfected HepG2 cells at similar efficiency, suggesting each of those compounds may disrupt a common cellular function required for the synthesis and/or secretion of these viral proteins. Interestingly, lycorine more efficiently inhibited the secretion of HBsAg in HepG2 cells transfected with HBV replicons, HepG2.2.15 cells and HBV infected - HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP). The structure activity relationship and antiviral mechanism of lycorine against HBV have been determined.

INDUCED PLURIPOTENT STEM CELL-DERIVED MESENCHYMAL STEM CELLS-DERIVED EXTRACELLULAR VESICLES ATTENUATE LPS-INDUCED LUNG INJURY AND ENDOTOXEMIA IN MICE.

ABSTRACT: Introduction: We hypothesized extracellular vesicles (EVs) from preconditioned human-induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) attenuate LPS-induced acute lung injury (ALI) and endotoxemia. Methods: iMSCs were incubated with cell stimulation cocktail (CSC) and EVs were isolated. iMSC-EVs were characterized by size and EV markers. Biodistribution of intratracheal (IT), intravenous, and intraperitoneal injection of iMSC-EVs in mice was examined using IVIS. Uptake of iMSC-EVs in lung tissue, alveolar macrophages, and RAW264.7 cells was also assessed. C57BL/6 mice were treated with IT/IP iMSC-EVs or vehicle ± IT/IP LPS to induce ALI/acute respiratory distress syndrome and endotoxemia. Lung tissues, plasma, and bronchoalveolar lavage fluid (BALF) were harvested at 24 h. Lung histology, BALF neutrophil/macrophage, cytokine levels, and total protein concentration were measured to assess ALI and inflammation. Survival studies were performed using IP LPS in mice for 3 days. Results: iMSC-EV route of administration resulted in differential tissue distribution. iMSC-EVs were taken up by alveolar macrophages in mouse lung and cultured RAW264.7 cells. IT LPS-treated mice demonstrated marked histologic ALI, increased BALF neutrophils/macrophages and protein, and increased BALF and plasma TNF-α/IL-6 levels. These parameters were attenuated by 2 h before or 2 h after treatment with IT iMSC-EVs in ALI mice. Interestingly, the IT LPS-induced increase in IL-10 was augmented by iMSC-EVs. Mice treated with IP LPS showed increases in TNF-α and IL-6 that were downregulated by iMSC-EVs and LPS-induced mortality was ameliorated by iMSC-EVs. Administration of IT iMSC-EVs 2 h after LPS downregulated the increase in proinflammatory cytokines (TNF-α/IL-6) by LPS and further increased IL-10 levels. Conclusions: iMSC-EVs attenuate the inflammatory effects of LPS on cytokine levels in ALI and IP LPS in mice. LPS-induced mortality was improved with administration of iMSC-EVs.

Multi-center validation of Catquest-9SF visual function questionnaire in Ontario, Canada.

PURPOSE: To investigate the psychometric performance and responsiveness of Catquest-9SF, a patient-reported questionnaire developed to evaluate visual function as related to daily tasks, in patients referred for cataract surgery in Ontario, Canada. METHODS: This is a pooled analysis on prospective data collected for previous projects. Subjects were recruited from three tertiary care centers in Peel region, Hamilton, and Toronto, Ontario, Canada. Catquest-9SF was administered pre-operative and post-operatively to patients with cataract. Psychometric properties, including category threshold order, infit/outfit, precision, unidimensionality, targeting, and differential item functioning were tested using Rasch analysis with Winsteps software (v.4.4.4) for Catquest-9SF. Responsiveness of questionnaire scores to cataract surgery was assessed. RESULTS: 934 patients (mean age = 71.6, 492[52.7%] female) completed the pre- and post-operative Catquest-9SF questionnaire. Catquest-9SF had ordered response thresholds, adequate precision (person separation index = 2.01, person reliability = 0.80), and confirmed unidimensionality. The infit range was 0.75-1.29 and the outfit range was 0.74-1.51, with one item ('satisfaction with vision') misfitting (outfit value = 1.51). There was mistargeting of -1.07 in pre-operative scores and mistargeting of -2.43 in both pre- and post-operative scores, meaning that tasks were relatively easy for respondent ability. There was no adverse differential item functioning. There was a mean 1.47 logit improvement in Catquest-9SF scores after cataract surgery (p<0.001). CONCLUSION: Catquest-9SF is a psychometrically robust questionnaire for assessment of visual function in patients with cataract in Ontario, Canada. It is also responsive to clinical improvement after cataract surgery.

PROPHYLACTIC n CMT-3 ATTENUATES SEPSIS-INDUCED ACUTE KIDNEY INJURY IN ASSOCIATION WITH NLRP3 INFLAMMASOME ACTIVATION AND APOPTOSIS.

ABSTRACT: Background: The kidney is the most common extrapulmonary organ injured in sepsis. The current study examines the ability of aerosolized nanochemically modified tetracycline 3 (nCMT-3), a pleiotropic anti-inflammatory agent, to attenuate acute kidney injury (AKI) caused by intratracheal LPS. Methods: C57BL/6 mice received aerosolized intratracheal nCMT-3 (1 mg/kg) or saline, followed by intratracheal LPS (2.5 mg/kg) to induce acute lung injury-induced AKI. Tissues were harvested at 24 h. The effects of nCMT-3 and LPS on AKI were assessed by plasma/tissue levels of serum urea nitrogen, creatinine, neutrophil gelatinase-associated lipocalin, kidney injury molecule 1, and renal histology. Renal matrix metalloproteinase (MMP) level/activity, cytochrome C, Bax, Bcl-2, caspase-3, p38 mitogen-activated protein kinase activation, NLRP3, and caspase-1 were also measured. Apoptotic cells in kidney were determined by TUNEL assay. Renal levels of IL-1ß and IL-6 were measured to assess inflammation. Results: Acute lung injury-induced AKI was characterized by increased plasma blood urea nitrogen, creatinine, injury biomarkers (neutrophil gelatinase-associated lipocalin, kidney injury molecule 1), and histologic evidence of renal injury. Lipopolysaccharide-treated mice demonstrated renal injury with increased levels of inflammatory cytokines (IL-1ß, IL-6), active MMP-2 and MMP-9, proapoptotic proteins (cytochrome C, Bax/Bcl-2 ratio, cleaved caspase-3), apoptotic cells, inflammasome activation (NLRP3, caspase-1), and p38 signaling. Intratracheal nCMT-3 significantly attenuated all the measured markers of renal injury, inflammation, and apoptosis. Conclusions: Pretreatment with aerosolized nCMT-3 attenuates LPS-induced AKI by inhibiting renal NLRP3 inflammasome activation, renal inflammation, and apoptosis.

Validation of a novel patient satisfaction questionnaire for preoperative fasting in cataract surgery: a discrimination analysis.

OBJECTIVE: Preoperative fasting is routinely performed to prevent anaesthesia-related pulmonary aspiration. To capture patients' experiences with preoperative fasting, a 13-item questionnaire was developed and validated using Rasch analysis and shortened to 6 items. This extension study aims to assess this questionnaire's ability to discriminate between participants with a short versus long duration of fasting and early versus late day surgery. DESIGN: Single-centred cross-sectional study. PARTICIPANTS: Subjects were recruited via consecutive sampling of cataract patients on surgery day at Kensington Eye Institute in Toronto from February to December 2019. METHODS: A validated preoperative fasting questionnaire was administered. Discriminative ability was assessed by comparing responses in patients scheduled for surgery in the morning (8:00 am-12:00 pm) versus afternoon (12:00 pm-3:30 pm) and fasting for short (≤8 hours) versus long (>8 hours) duration. Diagnostic ability of the 6-item questionnaire relative to the 13-item questionnaire was assessed with receiver operating characteristics curve analysis. RESULTS: A total of 164 patients (mean age 70.8 ± 10.0 years; 57% female) were included. Total scores of patients having surgery in the morning were greater (i.e., less fasting-related burden) than in the afternoon (p = 0.04). There was no significant difference in scores between patients fasting for a short versus long duration (p > 0.05). Receiver operating characteristics curve analysis showed excellent diagnostic ability of the 6-item questionnaire relative to the 13-item version (area under the curve = 0.964). CONCLUSION: The 6-item questionnaire for fasting-related burden has excellent discriminative ability between early versus late surgery patients. The time fasting while awake may be a more relevant predictor of fasting-related burden relative to the total duration of fasting.

An observational study evaluating the impact on prostate patient outcomes and experiences when radiation therapists use a standard grading system tool to assess and document treatment-related toxicities and interventions.

BACKGROUND/PURPOSE: Men undergoing radiation therapy (RT) treatment for prostate cancer (PC) often experience acute urinary, bowel, sexual, and hormonal toxicities. Timely screening, management, and documentation of these toxicities is an integral part of clinician practice, ensuring patients receive the care they require. Various screening tools, completed by either the patient or the clinician, are available, which allow clinicians to collect and respond to these toxicity outcomes; however there is a paucity of literature regarding the effective use and timing of these tools during RT treatment. This study aims to evaluate the feasibility of conducting comprehensive toxicity screening and symptom management using a toxicity screening tool in one of the busiest RT departments in Canada. Specifically, the use of a toxicity screening tool and its effect on the quality of toxicity documentation, operational impact, and patient reported outcomes (PRO). METHODS: 90 consented patients were allocated to either the structured or non-structured arm. Patients in the structured arm were assessed weekly by radiation therapists for 13 toxicities across four domains (bladder, bowel, hormonal, and sexual), using an in-house developed structured questionnaire, known as the Grid, to complete the National Cancer Institute's Common Toxicity Criteria for Adverse Events v3 (CTCAEv3). Patients in the non-structured arm were assessed and had free text clinical documentation charted according to current department policy. The Expanded Prostate Cancer Index Composite (EPIC), a PRO tool to evaluate patient function and bother after prostate cancer treatment, was completed by all study patients on a weekly basis. Statistical analysis compared documentation completeness, EPIC scores, patient satisfaction, and operational impact between study arms, as well as evaluated optimal timing of toxicity assessments. RESULTS: Assessment of the non-structured arm for completeness revealed an inconsistent and insufficient amount of documentation for the bladder and bowel domains. As for both the sexual and hormonal domains, documentation was largely absent. There was no difference in EPIC scores and patient satisfaction scores between the structured arm and the non-structured arm. Evaluation of the timing of PROs showed significant week to week change for the bladder and bowel toxicities, but not the sexual and hormonal toxicities. Finally, the use of the Grid revealed no significant impact on daily operations, only increasing average treatment times by seven seconds, and did not create any additional workload for the oncologists. CONCLUSIONS: Use of the Grid increased documentation completeness without negatively impacting clinical flow or operations, despite the fact that PROs were not improved. Based on EPIC PRO scores, bladder and bowel toxicities should be evaluated on a weekly basis during RT treatment, while sexual and hormonal toxicities need only be evaluated monthly.

Nano-chemically Modified Tetracycline-3 (nCMT-3) Attenuates Acute Lung Injury via Blocking sTREM-1 Release and NLRP3 Inflammasome Activation.

BACKGROUND: Intratracheal (IT) lipopolysaccharide (LPS) causes severe acute lung injury (ALI) and systemic inflammation. CMT-3 has pleiotropic anti-inflammatory effects including matrix metalloproteinase (MMP) inhibition, attenuation of neutrophil (PMN) activation, and elastase release. CMT-3's poor water solubility limits its bioavailability when administered orally for treating ALI. We developed a nano-formulation of CMT-3 (nCMT-3) to test the hypothesis that the pleiotropic anti-inflammatory activities of IT nCMT-3 can attenuate LPS-induced ALI. METHODS: C57BL/6 mice were treated with aerosolized IT nCMT-3 or saline, then had IT LPS or saline administered 2 h later. Tissues were harvested at 24 h. The effects of LPS and nCMT-3 on ALI were assessed by lung histology, MMP level/activity (zymography), NLRP3 protein, and activated caspase-1 levels. Blood and bronchoalveolar lavage fluid (BALF) cell counts, PMN elastase, and soluble triggering receptor expressed on myelocytes-1 (sTREM-1) levels, TNF-α, IL-1ß, IL-6, IL-18, and BALF protein levels were also measured. RESULTS: LPS-induced ALI was characterized by histologic lung injury (PMN infiltration, alveolar thickening, edema, and consolidation) elevated proMMP-2, -9 levels and activity, increased NLRP-3 protein and activated caspase-1 levels in lung tissue. LPS-induced increases in plasma and BALF levels of sTREM-1, TNF-α, IL-1ß, IL-6, IL-18, PMN elastase and BALF protein levels demonstrate significant lung/systemic inflammation and capillary leak. nCMT-3 significantly ameliorated all of these LPS-induced inflammatory markers to control levels, and decreased the incidence of ALI. CONCLUSIONS: Pre-treatment with nCMT3 significantly attenuates LPS-induced lung injury/inflammation by multiple mechanisms including: MMP activation, PMN elastase, sTREM-1 release, and NLRP3 inflammasome/caspase-1 activation.

The Impact of Real-World Alternative Dosing Strategies of Palbociclib on Progression-Free Survival in Patients with Metastatic Breast Cancer.

BACKGROUND: Palbociclib, a cyclin-dependent kinase 4 and 6 (CDK 4/6) inhibitor, in combination with letrozole or fulvestrant has been demonstrated to prolong the progression-free survival (PFS) in patients with hormone receptor-positive (HR+), human epidermal growth factor 2-negative (HER2-) metastatic breast cancer. In efforts to mitigate neutropenic toxicities, oncologists in real-world practice have prescribed alternative dosing strategies with palbociclib, yet the implication on PFS is unknown. METHODS: We conducted a retrospective, observational chart review of all female patients at our clinics with HR+, HER2- metastatic breast cancer receiving palbociclib in combination with either letrozole or fulvestrant with a first dose initiated between June 2016 and December 2018 and followed their disease course until 30 April 2020. RESULTS: The median PFS for all clinic patients receiving palbociclib and letrozole (n = 63) was 40.8 months (95% confidence interval (CI) 25.6-not estimable) and 16.97 months (95% CI 8.57-not estimable) for patients receiving palbociclib and fulvestrant (n = 11). We identified seven alternative dosing strategies prescribed by oncologists, the most prevalent being prescribing palbociclib for three weeks on and two weeks off (n = 8). The Kaplan-Meier curves for PFS in patients receiving letrozole and palbociclib prescribed alternative dosing strategies appear to diverge from monograph dosing early in the treatment. Many patients prescribed palbociclib using alternative dosing strategies continued to be observed even by the 18-month timepoint. The prevalence of grade 4 neutropenia was lower for patients on palbociclib with letrozole, suggesting a possible mitigation of severe neutropenia with alternative dosing strategies. CONCLUSIONS: We conclude that alternative dosing strategies used by oncologists such as prescribing palbociclib for three weeks on, two weeks off may achieve comparable disease control while mitigating neutropenic toxicities when compared to standard monograph dosing recommendations, prolonging treatment tolerability and adherence. Further large-scale studies are needed to confirm these results for future clinical adoption.

A yellow fever virus NS4B inhibitor not only suppresses viral replication, but also enhances the virus activation of RIG-I-like receptor-mediated innate immune response.

Flavivirus infection of cells induces massive rearrangements of the endoplasmic reticulum (ER) membrane to form viral replication organelles (ROs) which segregates viral RNA replication intermediates from the cytoplasmic RNA sensors. Among other viral nonstructural (NS) proteins, available evidence suggests for a prominent role of NS4B, an ER membrane protein with multiple transmembrane domains, in the formation of ROs and the evasion of the innate immune response. We previously reported a benzodiazepine compound, BDAA, which specifically inhibited yellow fever virus (YFV) replication in cultured cells and in vivo in hamsters, with resistant mutation mapped to P219 of NS4B protein. In the following mechanistic studies, we found that BDAA specifically enhances YFV induced inflammatory cytokine response in association with the induction of dramatic structural alteration of ROs and exposure of double-stranded RNA (dsRNA) in virus-infected cells. Interestingly, the BDAA-enhanced cytokine response in YFV-infected cells is attenuated in RIG-I or MAD5 knockout cells and completely abolished in MAVS knockout cells. However, BDAA inhibited YFV replication at a similar extent in the parent cells and cells deficient of RIG-I, MDA5 or MAVS. These results thus provided multiple lines of biological evidence to support a model that BDAA interaction with NS4B may impair the integrity of YFV ROs, which not only inhibits viral RNA replication, but also promotes the release of viral RNA from ROs, which consequentially activates RIG-I and MDA5. Although the innate immune enhancement activity of BDAA is not required for its antiviral activity in cultured cells, its dual antiviral mechanism is unique among all the reported antiviral agents thus far and warrants further investigation in animal models in future.

Development and Validation of a Questionnaire Assessing Patient Distress from Preoperative Fasting in Cataract Surgery.

Purpose: To develop and psychometrically validate a questionnaire to measure patient distress with preoperative fasting related to cataract surgery.Methods: In this single-centered cross-sectional study, consecutive sampling of cataract patients was undertaken immediately preoperatively from February to December 2019. A questionnaire evaluating patient distress with fasting was designed and administered. Questionnaire development occurred in an iterative process and was conducted with consultation from expert investigators and patients. Validation and psychometric evaluation of the questionnaire were performed with Rasch analysis.Results: A preliminary version of the questionnaire was developed by 10 study investigators. Across five iterations of development, the questionnaire was administered to 186 cataract patients. Psychometric evaluation of the 13-item questionnaire demonstrated ordered thresholds, acceptable item calibration and fit, adequate internal consistency, ability to discriminate between three levels of distress from preoperative fasting and no notable differential item functioning. However, issues with mistargeting, clustering of items on the person-item map and multidimensionality remained. Given these concerns, 13 separate re-analyses were conducted via removal of certain items. A 6-item subset was determined to be well targeted, unidimensional, did not display item clustering and was able to discriminate between patients with high and low distress from preoperative fasting.Conclusion: A 6-item questionnaire is a valid, psychometrically robust and reliable measure for the assessment of patient distress with preoperative fasting in cataract surgery. Items include hunger, thirst, hoarseness, weakness, anxiety and nausea. Future studies should seek to validate this questionnaire across a variety of sociodemographic contexts, languages and specialties.

Catquest-9SF questionnaire and eCAPS: Validation in a Canadian population.

BACKGROUND: Visual acuity alone has limitations in assessing a patient's appropriateness and prioritization for cataract surgery. Several tools, including the Catquest-9SF questionnaire and the electronic cataract appropriateness and priority system (eCAPS) have been developed to evaluate patients-reported visual function as related to day-to-day tasks. The aim of this study was to validate Catquest-9SF and eCAPS in a Canadian population and propose a shorter version of each, in an attempt to extend their applicability in clinical practice. METHODS: The English translation of the Swedish Catquest-9SF and eCAPS were self-administered separately in pre-operative patients in tertiary care in Peel region, Ontario. Rasch analysis was used to validate both scales and assess their psychometric properties, such as category threshold order, item fit, unidimensionality, precision, targeting, and differential item functioning. RESULTS: A total of 313 cataract patients (mean age = 69.1, 56.5% female) completed the Catquest-9SF and eCAPS. Catquest-9SF had ordered response thresholds, adequate precision (person separation index = 2.09, person reliability = 0.81), unidimensionality and no misfits (infit range 0.75-1.35, outfit range 0.83-1.36). There mean for patients was equal to -1.43 (lower than the mean for items which is set automatically at zero), meaning that tasks were relatively easy for respondent ability. eCAPS had 3 items that misfit the Rasch model and were excluded (infit range 0.82-1.30, outfit range 0.75-1.36). Precision was inadequate (person separation index = 0.19, person reliability = 0.04). 78.8% of subjects scored≤9 (answered that they had no issues for most questions). CONCLUSIONS: Catquest-9SF demonstrated good psychometric properties and is suitable for assessing visual function of care-seeking patients referred for cataract surgery in Ontario, Canada. There was some mistargeting, suggesting that the tasks were relatively easy to perform, which is consistent with previous research. On the contrary, eCAPS is not sensitive in differentiating patients who had impaired visual functioning.

Development of antibody-based assays for high throughput discovery and mechanistic study of antiviral agents against yellow fever virus.

Despite the availability of a highly effective yellow fever virus (YFV) vaccine, outbreaks of yellow fever frequently occur in Africa and South America with significant mortality, highlighting the pressing need for antiviral drugs to manage future outbreaks. To support the discovery and development of antiviral drugs against YFV, we characterized a panel of rabbit polyclonal antibodies against the three YFV structural proteins and five non-structural proteins and demonstrated these antibody reagents in conjunction with viral RNA metabolic labeling, double-stranded RNA staining and membrane floatation assays as powerful tools for investigating YFV polyprotein processing, replication complex formation, viral RNA synthesis and high throughput discovery of antiviral drugs. Specifically, the proteolytic processing of the viral polyprotein can be analyzed by Western blot assays. The predominant nuclear localization of NS5 protein as well as the relationship between intracellular viral non-structural protein distribution and foci of YFV RNA replication can be revealed by immunofluorescence staining and membrane flotation assays. Using an antibody against YFV NS4B protein as an example, in-cell western and high-content imaging assays have been developed for high throughput discovery of antiviral agents. A synergistic antiviral effect of an YFV NS4B-targeting antiviral agent BDAA and a NS5 RNA-dependent RNA polymerase inhibitor (Sofosbuvir) was also demonstrated with the high-content imaging assay. Apparently, the antibody-based assays established herein not only facilitate the discovery and development of antiviral agents against YFV, but also provide valuable tools to dissect the molecular mechanism by which the antiviral agents inhibit YFV replication.

Association of Roux-en-Y Gastric Bypass With Postoperative Health Care Use and Expenditures in Canada.

Importance: Results of previous studies are mixed regarding the economic implications of a Roux-en-Y gastric bypass (RYGB). Objective: To assess the 5-year incremental health care use and expenditures after RYGB. Design, Setting, and Participants: This population-based cohort study conducted in Ontario, Canada, used a difference-in-differences approach to compare health care use and expenditures between patients who underwent a publicly funded RYGB from March 1, 2010, to March 31, 2013, and propensity score-matched control individuals who did not undergo a surgical bariatric procedure. The study period allowed for a minimum 60 months of follow-up because, at that time, the most recent date for which administrative data on health care and expenditures were available was March 31, 2018. Data sources included the Ontario Bariatric Registry linked to several Ontario health administrative databases and the Electronic Medical Record Administrative Data Linked Database. Health care use and expenditures data for 5 years before and 5 years after the index date (procedure date for RYGB group; random date for controls) were analyzed. Data analyses were performed March 12, 2019, to March 10, 2020. Intervention: RYGB procedure. Main Outcomes and Measures: The primary outcome was total health care expenditures. Results: The final propensity score-matched cohorts comprised 1587 individuals in the RYGB group (mean [SD] age, 47 [10.2] years) and 1587 controls (mean [SD] age, 47 [12.2] years); each group had 1228 women (77.4%) and a mean body mass index (calculated as weight in kilograms divided by height in meters squared) of 46. Mean total health care expenditures (2017 Canadian dollars) per patient in the RYGB group increased from CAD $15 594 (95% CI, CAD $14 743 to CAD $16 614) (US $12 008 [95% CI, US $11 353 to US $12 794]) in the 5 years before the procedure to CAD $30 389 (95% CI, CAD $28 789 to CAD $32 232) (US $23 401 [95% CI, US $22 169 to US $24 821]) over the 5 years after the procedure, a difference of CAD $14 795 (95% CI, CAD $13 172 to CAD $16 480) (US $11 393 [95% CI, US $10 143 to US $12 691]). For the control group, mean total health care expenditures per individual increased from CAD $16 109 (95% CI, CAD $14 727 to CAD $17 591) (US $12 405 [95% CI, US $11 341 to US $13 546]) 5 years before the index date to CAD $20 073 (95% CI, CAD $18 147 to CAD $22 169) (US $15 457 [95% CI, US $13 974 to US $17 071]) 5 years after the date, a difference of CAD $3964 (95% CI, CAD $2250 to CAD $5875) (US $3053 [95% CI, US $1733 to US $4524]). Overall, the difference-in-differences estimate of the net cost of RYGB was CAD $10 831 (95% CI, CAD $8252 to CAD $13 283) (US $8341 [95% CI, $6355 to $10 229]) over the 5-year period. This amount excluded the mean (SD) cost associated with the index date: CAD $6501 (CAD $1087) (US $5006 [US $837]) for the RYGB cohort and CAD $9 (CAD $72) (US $7 [US $55]) for the controls. The cost differential was primarily associated with increased hospitalizations in the first months immediately after RYGB. Expenditures leveled off in year 3 after the index date; differences in total expenditures between the RYGB and control cohorts were not statistically significantly different in years 4 and 5. Conclusions and Relevance: Health care expenditures in the 3 years after publicly funded RYGB were higher in patients who underwent the procedure than in control individuals, but the costs were similar thereafter. This finding suggests the need to decrease hospital and emergency department readmissions after surgical bariatric procedures because such use is associated with increased spending.

Discovery and Mechanistic Study of a Novel Human-Stimulator-of-Interferon-Genes Agonist.

Stimulator of interferon genes (STING) is an integral ER-membrane protein that can be activated by 2'3'-cGAMP synthesized by cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) upon binding of double-stranded DNA. It activates interferon (IFN) and inflammatory cytokine responses to defend against infection by microorganisms. Pharmacologic activation of STING has been demonstrated to induce an antiviral state and boost antitumor immunity. We previously reported a cell-based high-throughput-screening assay that allowed for identification of small-molecule cGAS-STING-pathway agonists. We report herein a compound, 6-bromo-N-(naphthalen-1-yl)benzo[d][1,3]dioxole-5-carboxamide (BNBC), that induces a proinflammatory cytokine response in a human-STING-dependent manner. Specifically, we showed that BNBC induced type I and III IFN dominant cytokine responses in primary human fibroblasts and peripheral-blood mononuclear cells (PBMCs). BNBC also induced cytokine response in PBMC-derived myeloid dendritic cells and promoted their maturation, suggesting that STING-agonist treatment could potentially regulate the activation of CD4+ and CD8+ T lymphocytes. As anticipated, treatment of primary human fibroblast cells with BNBC induced an antiviral state that inhibited the infection of several kinds of flaviviruses. Taken together, our results indicate that BNBC is a human-STING agonist that not only induces innate antiviral immunity against a broad spectrum of viruses but may also stimulate the activation of adaptive immune responses, which is important for the treatment of chronic viral infections and tumors.

Discovery of Novel Hepatitis B Virus Nucleocapsid Assembly Inhibitors.

Hepatitis B virus (HBV) core protein is a small protein with 183 amino acid residues and assembles the pregenomic (pg) RNA and viral DNA polymerase to form nucleocapsids. During the last decades, several groups have reported HBV core protein allosteric modulators (CpAMs) with distinct chemical structures. CpAMs bind to the hydrophobic HAP pocket located at the dimer-dimer interface and induce allosteric conformational changes in the core protein subunits. While Type I CpAMs, heteroaryldihydropyrimidine (HAP) derivatives, misdirect core protein dimers to assemble noncapsid polymers, Type II CpAMs, represented by sulfamoylbenzamides, phenylpropenamides, and several other chemotypes, induce the assembly of empty capsids with global structural alterations and faster mobility in native agarose gel electrophoresis. Through high throughput screening of an Asinex small molecule library containing 19 920 compounds, we identified 8 structurally distinct CpAMs. While 7 of those compounds are typical Type II CpAMs, a novel benzamide derivative, designated as BA-53038B, induced the formation of morphologically "normal" empty capsids with slow electrophoresis mobility. Drug resistant profile analyses indicated that BA-53038B most likely bound to the HAP pocket but obviously modulated HBV capsid assembly in a distinct manner. BA-53038B and other CpAMs reported herein provide novel structure scaffolds for the development of core protein-targeted antiviral agents for the treatment of chronic hepatitis B.

Is Activity Tracker-Measured Ambulation an Accurate and Reliable Determinant of Postoperative Quality of Recovery? A Prospective Cohort Validation Study.

BACKGROUND: Quality of recovery (QOR) instruments measure patients' ability to return to baseline health status after surgery. Whether, and the extent to which, postoperative ambulation contributes to QOR is unclear, in part due to the lack of valid tools to measure ambulation in clinical settings. This cohort study of the cesarean delivery surgical model examines the accuracy and reliability of activity trackers in quantifying early postoperative ambulation and investigates the correlation between ambulation and QOR. METHODS: A prospective cohort of 200 parturients undergoing cesarean delivery between July 2015 and June 2017 was fitted with wrist-worn activity trackers immediately postpartum. The trackers were collected 24 hours later, along with QOR assessments (QoR-15 scale). The relationship between QOR and various covariates, including ambulation, was explored using multivariable linear regression and Spearman correlation (ρ). Forty-eight parturients fitted with 2 trackers also completed a walk exercise accompanied by a step-counting assessor, to evaluate accuracy, inter-, and intradevice reliability using interclass correlation (ICC). RESULTS: Compared to step counting, activity trackers had high accuracy (ICC = 0.93) and excellent inter- and intradevice reliability (ICC = 0.98 and 0.96, respectively). Correlation analysis suggested that early ambulation is moderately correlated with postcesarean QoR-15 scores, with a ρ (95% confidence interval) equivalent to 0.56 (0.328-0.728). Regression analysis suggested that ambulation is a determinant of postcesarean QoR-15 scores, with an effect estimate (95% confidence interval) equivalent to 0.002 (0.001-0.003). Ambulation was also associated with all QoR-15 domains, except psychological support. The patient's acceptable symptom state (subjective threshold for good ambulation) in the first 24 hours was 287 steps. CONCLUSIONS: This study demonstrated the accuracy and reliability of activity trackers in measuring ambulation in clinical settings and suggested that postoperative ambulation is a determinant of postoperative QOR. A hypothetical implication of our findings is that interventions that improve ambulation may also help to enhance QOR, but further research is needed to establish a causal relationship.

A cell-based high throughput screening assay for the discovery of cGAS-STING pathway agonists.

Stimulator of interferon genes (STING) is an endoplasmic reticulum transmembrane protein that serves as a molecular hub for activation of interferon and inflammatory cytokine response by multiple cellular DNA sensors. Not surprisingly, STING has been demonstrated to play an important role in host defense against microorganisms and pharmacologic activation of STING is considered as an attractive strategy to treat viral diseases and boost antitumor immunity. In light of this we established a HepAD38-derived reporter cell line that expresses firefly luciferase in response to the activation of cyclic GMP-AMP synthase (cGAS)-STING pathway for high throughput screening (HTS) of small molecular human STING agonists. This cell-based reporter assay required only 4 h treatment with a reference STING agonist to induce a robust luciferase signal and was demonstrated to have an excellent performance in HTS format. By screening 16,000 compounds, a dispiro diketopiperzine (DSDP) compound was identified to induce cytokine response in a manner dependent on the expression of functional human STING, but not mouse STING. Moreover, we showed that DSDP induced an interferon-dominant cytokine response in human skin fibroblasts and peripheral blood mononuclear cells, which in turn potently suppressed the replication of yellow fever virus, dengue virus and Zika virus. We have thus established a robust cell-based assay system suitable for rapid discovery and mechanistic analyses of cGAS-STING pathway agonists. Identification of DSDP as a human STING agonist enriches the pipelines of STING-targeting drug development for treatment of viral infections and cancers.

Early and Long-Term Responses to Anti-Vascular Endothelial Growth Factor Therapy in Diabetic Macular Edema: Analysis of Protocol I Data.

PURPOSE: To determine whether early visual acuity response to ranibizumab in diabetic macular edema is associated with long-term outcome. DESIGN: Post hoc analysis of randomized controlled trial data. METHODS: Pooled data from the ranibizumab plus prompt and deferred laser treatment arms of the Diabetic Retinopathy Clinical Research Network's Protocol I study were used to explore the relationship between early (week 12) and late (weeks 52-156) visual acuity response (mean change from baseline in best-corrected visual acuity [CFB BCVA]; categorized improvement [<5, 5-9, or ≥10 Early Treatment Diabetic Retinopathy Study (ETDRS) letters] in BCVA). RESULTS: In the analysis population (340 eyes), <5-, 5- to 9-, and ≥10-letter BCVA improvements occurred in 39.7%, 23.2%, and 37.1% of eyes, respectively, at 12 weeks, and 34.2%, 16.5%, and 49.3% of eyes at 156 weeks. Within each early BCVA response category (<5, 5-9, and ≥10 letters of improvement at 12 weeks), mean CFB BCVA at 52-156 weeks varied by <5 letters from that at 12 weeks. CFB BCVA and <5-letter improvement at 12 weeks showed significant positive and negative association, respectively, with CFB BCVA and ≥10-letter improvement at 52 and 156 weeks. Similar relationships were demonstrated in eyes with baseline BCVA <69 letters, and associations remained significant after multivariate adjustment for potential confounders. CONCLUSIONS: Ranibizumab ± laser therapy resulted in similar rates (∼40%) of suboptimal (<5-letter) and pronounced (≥10-letter) BCVA improvement at 12 weeks. Eyes with suboptimal early BCVA response showed poorer long-term visual outcomes than eyes with pronounced early response (mean improvement 3.0 vs 13.8 letters at 156 weeks).