SAEM systematic online academic resource (SOAR) review: Gastrointestinal illnesses.

Background and Objectives: Free open access medical education (FOAM) has become an essential tool for emergency medicine (EM) education and can be valuable to clinicians as a point-of-care resource. The development of the revised Medical Education Translational Resources Impact and Quality (rMETRIQ) tool provides a standardized means of quality assessment. Previous entries of the Society for Academic Emergency Medicine systematic online academic resource (SOAR) series have focused on renal, endocrine, and sickle cell disorders. In this iteration, we strive to identify, curate, and describe FOAM topics specific to acute gastrointestinal (GI) illnesses. Methods: We searched 389 keywords across 11 GI topics that were modified from the 2019 Model of the Clinical Practice of EM (EM Model) using the search engine Google FOAM and within the top 50 websites listed on Academic Life in Emergency Medicine's Social Media Index. The sites underwent preliminary screening to eliminate resources that were not relevant to EM or GI illnesses. Identified resources were evaluated with the rMETRIQ tool by five board-certified EM physicians who received rMETRIQ tool rater training. Results: After duplicates of the initial 39,505 resources were eliminated, 8059 remained. Primary screening resulted in a final 1202 resources. The most common categories were large bowel (18%), small bowel (13%), stomach (11%), esophagus (11%), biliary (11%), and liver (10%). Many resources covered multiple topics and subtopics. The final mean intraclass correlation coefficient among the five physicians was 0.95 (95% CI 0.92-0.98) for rMETRIQ scoring. We identified 256 sites considered "high quality" with a rMETRIQ score of 16 or higher as designated in prior reviews. Conclusions: This iteration of the SOAR review resulted in the highest number of high-quality resources compared to other SOAR reviews, with 21% of resources thus far scoring ≥ 16. A final list of high-quality resources can guide trainees, educator recommendations, and FOAM authors.

Systematic online academic resource (SOAR) review: Pediatric respiratory infectious disease.

Background: Free open access medical education (FOAM) resources have become increasingly popular in graduate medical education. Despite their accessibility, the assessment of FOAM resources' quality is challenging due to their decentralized nature and the diverse qualifications of their authors and distribution platforms. In this first pediatric systematic online academic resource (SOAR) review, we utilized a systematic methodology to aggregate and assess the quality of FOAM resources on pediatric respiratory infectious disease topics. Methods: We searched 177 keywords using FOAMSearch, the top 50 FOAM websites on the Social Media Index, and seven additional pediatric emergency medicine-focused blogs. Following a basic initial screen, resources then underwent full-text quality assessment utilizing the revised Medical Education Translational Resources: Impact and Quality (rMETRIQ) tool. Results: The search yielded 44,897 resources. After 44,456 were excluded, 441 underwent quality assessment. A total of 36/441 posts (8% of posts) reached the high-quality threshold score (rMETRIQ ≥ 16). The most frequent topics overall were pneumonia and bronchiolitis. A total of 67/441 posts (15% of posts) were found to have a rMETRIQ score of less than or equal to 7, which may indicate poor quality. Conclusions: We systematically identified, described, and performed quality assessment on FOAM resources pertaining to the topic of pediatric respiratory infectious disease. We found that there is a paucity of high-quality posts on this topic. Despite this, the curated list of high-quality resources can help guide trainees and educators toward relevant educational information and suggest unmet needs for future FOAM resources.

Acetaminophen causes neurodevelopmental injury in susceptible babies and children: no valid rationale for controversy.

Despite the worldwide acceptance of acetaminophen (APAP) as a necessary medicine in pediatrics, evidence that early exposure to APAP causes neurodevelopmental injury in susceptible babies and children has been mounting for over a decade. The evidence is diverse and includes extensive work with laboratory animals, otherwise unexplained associations, factors associated with APAP metabolism, and limited studies in humans. Although the evidence has reached an overwhelming level and was recently reviewed in detail, controversy persists. This narrative review evaluates some of that controversy. Evidence from the pre- and postpartum periods was considered to avoid controversy raised by consideration of only limited evidence of risks during the prepartum period. Among other issues, the association between APAP use and the prevalence of neurodevelopmental disorders was considered. A systematic review revealed that the use of APAP in the pediatric population was never tracked carefully; however, historical events that affected its use were documented and are sufficient to establish apparent correlations with changes in the prevalence of neurodevelopmental disorders. Moreover, problems with the exclusive reliance on results of meta-analyses of large datasets with limited time frames of drug exposure were reviewed. Furthermore, the evidence of why some children are susceptible to APAPinduced neurodevelopmental injuries was examined. We concluded that available evidence demonstrates that early exposure to APAP causes neurodevelopmental injury in susceptible babies and small children.

DAXX drives de novo lipogenesis and contributes to tumorigenesis.

Cancer cells exhibit elevated lipid synthesis. In breast and other cancer types, genes involved in lipid production are highly upregulated, but the mechanisms that control their expression remain poorly understood. Using integrated transcriptomic, lipidomic, and molecular studies, here we report that DAXX is a regulator of oncogenic lipogenesis. DAXX depletion attenuates, while its overexpression enhances, lipogenic gene expression, lipogenesis, and tumor growth. Mechanistically, DAXX interacts with SREBP1 and SREBP2 and activates SREBP-mediated transcription. DAXX associates with lipogenic gene promoters through SREBPs. Underscoring the critical roles for the DAXX-SREBP interaction for lipogenesis, SREBP2 knockdown attenuates tumor growth in cells with DAXX overexpression, and DAXX mutants unable to bind SREBP1/2 have weakened activity in promoting lipogenesis and tumor growth. Remarkably, a DAXX mutant deficient of SUMO-binding fails to activate SREBP1/2 and lipogenesis due to impaired SREBP binding and chromatin recruitment and is defective of stimulating tumorigenesis. Hence, DAXX's SUMO-binding activity is critical to oncogenic lipogenesis. Notably, a peptide corresponding to DAXX's C-terminal SUMO-interacting motif (SIM2) is cell-membrane permeable, disrupts the DAXX-SREBP1/2 interactions, and inhibits lipogenesis and tumor growth. These results establish DAXX as a regulator of lipogenesis and a potential therapeutic target for cancer therapy.

Smoking-induced subgingival dysbiosis precedes clinical signs of periodontal disease.

Smoking accelerates periodontal disease and alters the subgingival microbiome. However, the relationship between smoking-associated subgingival dysbiosis and progression of periodontal disease is not well understood. Here, we sampled 233 subgingival sites longitudinally from 8 smokers and 9 non-smokers over 6-12 months, analyzing 804 subgingival plaque samples using 16 rRNA sequencing. At equal probing depths, the microbial richness and diversity of the subgingival microbiome was higher in smokers compared to non-smokers, but these differences decreased as probing depths increased. The overall subgingival microbiome of smokers differed significantly from non-smokers at equal probing depths, which was characterized by colonization of novel minority microbes and a shift in abundant members of the microbiome to resemble periodontally diseased communities enriched with pathogenic bacteria. Temporal analysis showed that microbiome in shallow sites were less stable than deeper sites, but temporal stability of the microbiome was not significantly affected by smoking status or scaling and root planing. We identified 7 taxa-Olsenella sp., Streptococcus cristatus, Streptococcus pneumoniae, Streptococcus parasanguinis, Prevotella sp., Alloprevotella sp., and a Bacteroidales sp. that were significantly associated with progression of periodontal disease. Taken together, these results suggest that subgingival dysbiosis in smokers precedes clinical signs of periodontal disease, and support the hypothesis that smoking accelerates subgingival dysbiosis to facilitate periodontal disease progression.

Microbiome Resilience despite a Profound Loss of Minority Microbiota following Clindamycin Challenge in Humanized Gnotobiotic Mice.

Antibiotics are known to induce gut dysbiosis and increase the risk of antibiotic resistance. While antibiotic exposure is a known risk factor leading to compromised colonization resistance against enteric pathogens such as Clostridioides difficile, the extent and consequences of antibiotic perturbation on the human gut microbiome remain poorly understood. Human studies on impacts of antibiotics are complicated by the tremendous variability of gut microbiome among individuals, even between identical twins. Furthermore, antibiotic challenge experiments cannot be replicated in human subjects for a given gut microbiome. Here, we transplanted feces from three unrelated human donors into groups of identical germfree (GF) Swiss-Webster mice, and examined the temporal responses of the transplanted microbiome to oral clindamycin challenge in gnotobiotic isolators over 7 weeks. Analysis of 177 longitudinal fecal samples revealed that 59% to 81% of human microbiota established a stable configuration rapidly and stably in GF mice. Microbiome responses to clindamycin challenge was highly reproducible and microbiome-dependent. A short course of clindamycin was sufficient to induce a profound loss (∼one third) of the microbiota by disproportionally eliminating minority members of the transplanted microbiota. However, it was inadequate to disrupt the global microbial community structure or function, which rebounded rapidly to resemble its pre-treatment state after clindamycin discontinuation. Furthermore, the response of individual microbes was community-dependent. Taken together, these results suggest that the overall gut microbiome structure is resilient to antibiotic perturbation, the functional consequences of which warrant further investigation. IMPORTANCE Antibiotics cause imbalance of gut microbiota, which in turn increase our susceptibility to gastrointestinal infections. However, how antibiotics disrupt gut bacterial communities is not well understood, and exposing healthy volunteers to unnecessary antibiotics for research purposes carries clinical and ethical concerns. In this study, we used genetically identical mice transplanted with the same human gut microbiota to control for both genetic and environmental variables. We found that a short course of oral clindamycin was sufficient to eliminate one third of the gut bacteria by disproportionally eliminating minority members of the transplanted microbiota, but it was inadequate to disrupt the overall microbial community structure and function, which rebounded rapidly to its pre-treatment state. These results suggest that gut microbiome is highly resilient to antibiotic challenge and degradation of the human gut ecosystem may require repeated or prolonged antibiotic exposure.

Systematic online academic resource (SOAR) review: Endocrine, metabolic, and nutritional disorders.

BACKGROUND: Free open-access medical education (FOAM) has become an integral resource for medical school and residency education. However, questions of quality and inconsistent coverage of core topics remain. In this second entry of the SAEM Systematic Online Academic Resource (SOAR) series, we describe the application of a systematic methodology to identify, curate, and describe FOAM topics specific to endocrine, metabolic, and nutritional disorders as defined by the 2016 Model of the Clinical Practice of Emergency Medicine (MCPEM). METHODS: We developed an automated algorithm to search 264 keywords derived from nine subtopics within the MCPEM category in the FOAM Search (a customized FOAM search tool) and the Social Media index. The top 100 results were extracted for each keyword. Resources underwent a manual iterative screening process, and those relevant to endocrine, metabolic, or nutritional disorders and EM were evaluated with the revised Medical Education Translational Resources: Impact and Quality (rMETRIQ) tool. RESULTS: The search yielded 36,346 resources, of which 756 met the criteria for quality assessment. After rMETRIQ tool training, four raters demonstrated an average measured intraclass correlation coefficient of 0.94 (95% confidence interval = 0.88 to 0.97, p < 0.001). A total of 121 posts (16% of posts) covering 25 subtopics were identified as high quality (rMETRIQ ≥16). The most covered subtopic was potassium disorders, representing 15% of all posts. Subtopics that did not have a high-quality resource identified include metabolic alkalosis, respiratory alkalosis, fluid overload, phosphorus metabolism, hyperglycemia, malabsorption, malnutrition, and thyroiditis. From most to least common, the overall target audience was junior resident (91%), PGY-1 resident (88%), senior resident (81%), clerk (64%), attending (50%), and preclerkship (9%). CONCLUSIONS: We systematically identified, described, and curated FOAM resources for EM learners on the topic of endocrine, metabolic, and nutritional disorders. A final list of high-quality resources can guide trainees, educator recommendations, and FOAM authors.

Stability of healthy subgingival microbiome across space and time.

The subgingival microbiome is one of the most stable microbial ecosystems in the human body. Alterations in the subgingival microbiome have been associated with periodontal disease, but their variations over time and between different subgingival sites in periodontally healthy individuals have not been well described. We performed extensive, longitudinal sampling of the subgingival microbiome from five periodontally healthy individuals to define baseline spatial and temporal variations. A total of 251 subgingival samples from 5 subjects were collected over 6-12 months and deep sequenced. The overall microbial diversity and composition differed significantly between individuals. Within each individual, we observed considerable differences in microbiome composition between different subgingival sites. However, for a given site, the microbiome was remarkably stable over time, and this stability was associated with increased microbial diversity but was inversely correlated with the enrichment of putative periodontal pathogens. In contrast to microbiome composition, the predicted functional metagenome was similar across space and time, suggesting that periodontal health is associated with shared gene functions encoded by different microbiome consortia that are individualized. To our knowledge, this is one of the most detailed longitudinal analysis of the healthy subgingival microbiome to date that examined the longitudinal variability of different subgingival sites within individuals. These results suggest that a single measurement of the healthy subgingival microbiome at a given site can provide long term information of the microbial composition and functional potential, but sampling of each site is necessary to define the composition and community structure at individual subgingival sites.

Discovery of histone deacetylase 3 (HDAC3)-specific PROTACs.

Histone deacetylases (HDACs) are validated drug targets for cancer treatment. Increased HDAC isozyme selectivity and novel strategies to inhibit HDAC activity could lead to safer and more effective drug candidates. Nonetheless, it is quite challenging to develop isozyme-specific HDACi due to the highly conserved catalytic domain. We discovered XZ9002, a first-in-class HDAC3-specific PROTAC that potently degraded HDAC3. Importantly, XZ9002 is more effective to inhibit cancer cell proliferation than its proteolysis-inactive counterpart, suggesting HDAC3 degradation is a novel and promising anticancer approach.

Micropocket hydrogel devices for all-in-one formation, assembly, and analysis of aggregate-based tissues.

Multicellular aggregated tissues have grown critically important in benchtop biomedical research, both as stand-alone spheroids and when assembled into larger bioengineered constructs. However, typical systems for aggregate formation are limited in their capacity to reliably handle such cultures at various experimental stages in a broadly accessible, consistent, and scalable manner. In this work, we develop a broadly versatile all-in-one biofabrication strategy to form uniform, spherical, multicellular aggregates that can be maintained at precisely defined positions for analysis or transfer into a larger tissue. The 3D-printed MicroPocket Culture (MPoC) system consists of an array of simple geometry-based valves in a polyacrylamide hydrogel, and is able to produce hundreds of uniformly-sized aggregates in standard tissue culture well plates, using simple tools that are readily available in all standard biological wet-labs. The model breast cancer aggregates formed in these experiments are retained in defined positions on chip during all liquid handling steps required to stimulate, label, and image the experiment, enabling high-throughput studies on this culture model. Furthermore, MPoCs enable robust formation of aggregates in cell types that do not conventionally form such structures. Finally, we demonstrate that this single platform can also be used to generate complex 3D tissues from the precisely-positioned aggregate building blocks. To highlight the unique and broad versatility of this technique, we develop a simple 3D invasion assay and show that cancer cells preferentially migrate towards nearby model tumors; demonstrating the importance of spatial precision when engineering 3D tissues. Together, this platform presents a broadly accessible and uniquely capable system with which to develop advanced aggregate-based models for tissue engineering, fundamental research, and applied drug discovery.

Interrogating the ccm-3 Gene Network.

Cerebral cavernous malformations (CCMs) are neurovascular lesions caused by mutations in one of three genes (CCM1-3). Loss of CCM3 causes the poorest prognosis, and little is known about how it regulates vascular integrity. The C. elegans ccm-3 gene regulates the development of biological tubes that resemble mammalian vasculature, and in a genome-wide reverse genetic screen, we identified more than 500 possible CCM-3 pathway genes. With a phenolog-like approach, we generated a human CCM signaling network and identified 29 genes in common, of which 14 are required for excretory canal extension and membrane integrity, similar to ccm-3. Notably, depletion of the MO25 ortholog mop-25.2 causes severe defects in tube integrity by preventing CCM-3 localization to apical membranes. Furthermore, loss of MO25 phenocopies CCM3 ablation by causing stress fiber formation in endothelial cells. This work deepens our understanding of how CCM3 regulates vascular integrity and may help identify therapeutic targets for treating CCM3 patients.

Inhibition of androgen receptor transactivation function by adenovirus type 12 E1A undermines prostate cancer cell survival.

BACKGROUND: Mutations or truncation of the ligand-binding domain (LBD) of androgen receptor (AR) underlie treatment resistance for prostate cancer (PCa). Thus, targeting the AR N-terminal domain (NTD) could overcome such resistance. METHODS: Luciferase reporter assays after transient transfection of various DNA constructs were used to assess effects of E1A proteins on AR-mediated transcription. Immunofluorescence microscopy and subcellular fractionation were applied to assess intracellular protein localization. Immunoprecipitation and mammalian two-hybrid assays were used to detect protein-protein interactions. qRT-PCR was employed to determine RNA levels. Western blotting was used to detect protein expression in cells. Effects of adenoviruses on prostate cancer cell survival were evaluated with CellTiter-Glo assays. RESULTS: Adenovirus 12 E1A (E1A12) binds specifically to the AR. Interestingly, the full-length E1A12 (266 aa) preferentially binds to full-length AR, while the small E1A12 variant (235 aa) interacts more strongly with AR-V7. E1A12 promotes AR nuclear translocation, likely through mediating intramolecular AR NTD-LBD interactions. In the nucleus, AR and E1A12 co-expression in AR-null PCa cells results in E1A12 redistribution from nuclear foci containing CBX4 (also known as Pc2), suggesting a preferential AR-E1A12 interaction over other E1A12 interactors. E1A12 represses AR-mediated transcription in reporter gene assays and endogenous AR target genes such as ATAD2 and MYC in AR-expressing PCa cells. AR-expressing PCa cells are more sensitive to death induced by a recombinant adenovirus expressing E1A12 (Ad-E1A12) than AR-deficient PCa cells, which could be attributed to the increased viral replication promoted by androgen stimulation. Targeting the AR by E1A12 promotes apoptosis in PCa cells that express the full-length AR or C-terminally truncated AR variants. Importantly, inhibition of mTOR signaling that blocks the expression of anti-apoptotic proteins markedly augments Ad-E1A12-induced apoptosis of AR-expressing cells. Mechanistically, Ad-E1A12 infection triggers apoptotic response while activating the PI3K-AKT-mTOR signaling axis; thus, mTOR inhibition enhances apoptosis in AR-expressing PCa cells infected by Ad-E1A12. CONCLUSION: Ad12 E1A inhibits AR-mediated transcription and suppresses PCa cell survival, suggesting that targeting the AR by E1A12 might have therapeutic potential for treating advanced PCa with heightened AR signaling.

The subgingival microbiome in patients with established rheumatoid arthritis.

OBJECTIVES: To profile and compare the subgingival microbiome of RA patients with OA controls. METHODS: RA (n = 260) and OA (n = 296) patients underwent full-mouth examination and subgingival samples were collected. Bacterial DNA was profiled using 16 S rRNA Illumina sequencing. Following data filtering and normalization, hierarchical clustering analysis was used to group samples. Multivariable regression was used to examine associations of patient factors with membership in the two largest clusters. Differential abundance between RA and OA was examined using voom method and linear modelling with empirical Bayes moderation (Linear Models for Microarray Analysis, limma), accounting for the effects of periodontitis, race, marital status and smoking. RESULTS: Alpha diversity indices were similar in RA and OA after accounting for periodontitis. After filtering, 286 taxa were available for analysis. Samples grouped into one of seven clusters with membership sizes of 324, 223, 3, 2, 2, 1 and 1 patients, respectively. RA-OA status was not associated with cluster membership. Factors associated with cluster 1 (vs 2) membership included periodontitis, smoking, marital status and Caucasian race. Accounting for periodontitis, 10 taxa (3.5% of those examined) were in lower abundance in RA than OA. There were no associations between lower abundance taxa or other select taxa examined with RA autoantibody concentrations. CONCLUSION: Leveraging data from a large case-control study and accounting for multiple factors known to influence oral health status, results from this study failed to identify a subgingival microbial fingerprint that could reliably discriminate RA from OA patients.

Molecular Identification of Bacteria in Intra-abdominal Abscesses Using Deep Sequencing.

BACKGROUND: Intra-abdominal abscesses are localized collections of pus, which generally arise from a breach in the normal mucosal defense barrier that allows bacteria from gastrointestinal tract, and less commonly from the gynecologic or urinary tract, to induce inflammation, resulting in an infection. The microbiology of these abscesses is usually polymicrobial, associated with the primary disease process. However, the microbial identity, diversity and richness in intra-abdominal abscesses have not been well characterized, due in part to the difficulty in cultivating commensal organisms using standard culture-based techniques. METHODS: We used culture-independent 16S rRNA Illumina sequencing to characterize bacterial communities in intra-abdominal abscesses collected by percutaneous drainage. A total of 43 abscess samples, including 19 (44.2%) Gram stain and culture-negative specimens, were analyzed and compared with results from conventional microbiologic cultures. RESULTS: Microbial composition was determined in 8 of 19 culture-negative samples and 18 of 24 culture-positive samples, identifying a total of 221 bacterial taxa or operational taxonomic units (OTUs) and averaging 13.1 OTUs per sample (interquartile range, 8-16.5 OTUs). Microbial richness for monomicrobial and polymicrobial samples was significantly higher than culture-negative samples (17 and 15.2 OTUs vs 8 OTUs, respectively), with a trend toward a higher microbial diversity (Shannon diversity index of 0.87 and 1.18 vs 0.58, respectively). CONCLUSIONS: The bacterial consortia identified by cultures correlated poorly with the microbial composition determined by 16S rRNA sequencing, and in most cases, the cultured isolates were minority constituents of the overall abscess microbiome. Intra-abdominal abscesses were generally polymicrobial with a surprisingly high microbial diversity, but standard culture-based techniques failed to reveal this diversity. These data suggest that molecular-based approaches may be helpful for documenting the presence of bacteria in intra-abdominal abscesses where standard cultures are unrevealing, particularly in the setting of prior antibiotic exposure.

Prevalence and impact of baseline resistance-associated substitutions on the efficacy of ledipasvir/sofosbuvir or simeprevir/sofosbuvir against GT1 HCV infection.

Baseline resistance-associated substitutions (RASs) have variable impacts in clinical trials but their prevalence and impact in real-world patients remains unclear. We performed baseline resistance testing using a commercial assay (10% cutoff) for 486 patients treated with LDV/SOF or SMV/SOF, with or without ribavirin, in the multi-center, observational HCV-TARGET cohort. Linkage of RASs was evaluated in selected samples using a novel quantitative single variant sequencing assay. Our results showed that the prevalence of NS3, NS5A, NS5B RASs was 45%, 13%, and 8%, respectively, and 10% of patients harbored RASs in 2 or more drug classes. Baseline LDV RASs in GT1a, TE, and cirrhosis LDV/SOF subgroup was associated with 2-4% lower SVR12 rates. SMV RASs was associated with lower SVR12 rates in GT1a, treatment-experienced, cirrhotics SMV/SOF subgroup. Pooled analysis of all patients with baseline RASs revealed that SVR12 was 100% (19/19) in patients treated for longer than 98 days but was 87% (81/93) in patients treated for shorter than 98 days. These results demonstrate that RASs prevalence and their impact in real world practice are in general agreement with registration trials, and suggest that longer treatment duration may overcome the negative impact of baseline RASs on SVR12 rates in clinical practice.

Fontan Failure and Death in Contemporary Fontan Circulation: Analysis From the Last Two Decades.

BACKGROUND: We sought to evaluate the incidence of Fontan failure or complication and its relation to death in patients having contemporary Fontan strategies over 2 decades. METHODS: Five hundred patients who underwent Fontan completion (extracardiac, n = 326; lateral tunnel, n = 174) from 1985 to 2012 were reviewed. Patient characteristics, modes of Fontan failure/complication and death, and predictors for Fontan failure/complication and death were analyzed. RESULTS: There were 23 early deaths (4.6%) and 17 late deaths (3.4%), with no early death since 2000. Survival has improved over time (p < 0.001). Twenty-three of 40 patients who died were identified as Fontan failure before death, including ventricular dysfunction (n = 14), pulmonary vascular dysfunction (n = 4), thromboembolism (n = 2), and arrhythmia (n = 4). Mode of death was circulatory failure (n = 18), multiorgan failure (n = 6), pulmonary failure (n = 3), cerebral/renal (n = 5), and sudden death (n = 4). Modes of failure/complication were directly (65%) or conceivably (10%) related to death in 30 of 40 patients (75%). Forty-eight percent of survivors had late Fontan complication(s). Five-year freedom from late Fontan complication was lower among patients who died compared with patients who survived (29.4% versus 53.3%, p < 0.001). Ventricular dysfunction (p = 0.001) and higher pulmonary artery pressures (p < 0.001) after Fontan were predictors for death. Longer cardiopulmonary bypass time (p = 0.032) and reinterventions (p < 0.001) were predictors for late Fontan complication. CONCLUSIONS: Early death in the early era has been overcome. Yet the incidence and causes of late death remain unchanged. There was a strong causative relationship between the mode of Fontan failure/complication and death, indicating the importance of early recognition and treatment of Fontan failure/complication.

Sofosbuvir (SOF) Suppresses Ledipasvir (LDV)-resistant Mutants during SOF/LDV Combination Therapy against Genotype 1b Hepatitis C Virus (HCV).

Our objective was to identify drug interactions between ledipasvir (LDV) and sofosbuvir (SOF) against a genotype 1b replicon to determine optimal exposures for each agent that will maximize antiviral activity against susceptible and drug-resistant subpopulations. LDV and SOF were evaluated using a fully factorial experimental design in the BelloCell system. Replicon levels and drug-resistant variants were quantified at various times post-therapy for 14 days and a high-dimensional mathematical model was fit to the data. Mutations associated with SOF resistance were not detected; but LDV-resistant mutants were selected and mutant subpopulations increased as exposure intensity increased. Combination therapy was additive for the total replicon population and the LDV-resistant population, but a threshold concentration of 100 ng/ml of SOF must be attained to suppress LDV-resistant subpopulations. These novel findings hold important implications for not only improving therapeutic outcomes, but also maximizing the clinical utility of LDV and SOF combination regimens.

Curcumin represses mouse 3T3-L1 cell adipogenic differentiation via inhibiting miR-17-5p and stimulating the Wnt signalling pathway effector Tcf7l2.

Understanding mechanisms underlying adipogenic differentiation may lead to the discovery of novel therapeutic targets for obesity. Wnt signalling pathway activation leads to repressed adipogenic differentiation while certain microRNAs may regulate pre-adipocyte proliferation and differentiation. We show here that in mouse white adipose tissue, miR-17-5p level is elevated after high fat diet consumption. miR-17-5p upregulates adipogenic differentiation, as its over-expression increased while its inhibition repressed 3T3-L1 differentiation. The Tcf7l2 gene encodes a key Wnt signalling pathway effector, and its human homologue TCF7L2 is a highly regarded diabetes risk gene. We found that Tcf7l2 is an miR-17-5p target and confirmed the repressive effect of Tcf7l2 on 3T3-L1 adipogenic differentiation. The natural plant polyphenol compound curcumin possesses the body weight lowering effect. We observed that curcumin attenuated miR-17-5p expression and stimulated Tcf7l2 expression in 3T3-L1 cells. These, along with the elevation of miR-17-5p expression in mouse epididymal fat tissue in response to high fat diet consumption, allowed us to suggest that miR-17-5p is among central switches of adipogenic differentiation. It activates adipogenesis via repressing the Wnt signalling pathway effector Tcf7l2, and its own expression is likely nutritionally regulated in health and disease.

Intrinsic cellular signaling mechanisms determine the sensitivity of cancer cells to virus-induced apoptosis.

Cancer cells of epithelial and mesenchymal phenotypes exhibit different sensitivities to apoptosis stimuli, but the mechanisms underlying this phenomenon remain partly understood. We constructed a novel recombinant adenovirus expressing Ad12 E1A (Ad-E1A12) that can strongly induce apoptosis. Ad-E1A12 infection of epithelial cancer cells displayed dramatic detachment and apoptosis, whereas cancer cells of mesenchymal phenotypes with metastatic propensity were markedly more resistant to this virus. Notably, forced detachment of epithelial cells did not further sensitize them to Ad-E1A12-induced apoptosis, suggesting that cell detachment is a consequence rather than the cause of Ad-E1A12-induced apoptosis. Ad-E1A12 increased phosphorylation of AKT1 and ribosomal protein S6 through independent mechanisms in different cell types. Ad-E1A12-induced AKT1 phosphorylation was PI3K-dependent in epithelial cancer cells, and mTOR-dependent in mesenchymal cancer cells. Epithelial cancer cells upon Ad-E1A12-induced detachment could not sustain AKT activation due to AKT1 degradation, but AKT1 activation was maintained in mesenchymal cancer cells. Expression of epithelial cell-restricted miR-200 family in mesenchymal cells limited mTOR signaling and sensitized them to Ad-E1A12-induced cell killing. Thus, epithelial cancer cells rely on the canonical PI3K-AKT signaling pathway for survival, while mesenchymal cancer cells deploy the PI3K-independent mTORC2-AKT axis in response to strong death stimuli.

Identification of histone deacetylase inhibitors with benzoylhydrazide scaffold that selectively inhibit class I histone deacetylases.

Inhibitors of histone deacetylases (HDACi) hold considerable therapeutic promise as clinical anticancer therapies. However, currently known HDACi exhibit limited isoform specificity, off-target activity, and undesirable pharmaceutical properties. Thus, HDACi with new chemotypes are needed to overcome these limitations. Here, we identify a class of HDACi with a previously undescribed benzoylhydrazide scaffold that is selective for the class I HDACs. These compounds are competitive inhibitors with a fast-on/slow-off HDAC-binding mechanism. We show that the lead compound, UF010, inhibits cancer cell proliferation via class I HDAC inhibition. This causes global changes in protein acetylation and gene expression, resulting in activation of tumor suppressor pathways and concurrent inhibition of several oncogenic pathways. The isotype selectivity coupled with interesting biological activities in suppressing tumor cell proliferation support further preclinical development of the UF010 class of compounds for potential therapeutic applications.