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PurposeThis study aimed to develop novel non-contrast MR perfusion techniques for assessing micro-vascularity of the foot in human subjects.MethodsAll experiments were performed on a clinical 3 T scanner using arterial spin labeling (ASL). Seven healthy subjects (30-72 years old, 5 males and 2 females) were enrolled and bilateral feet were imaged with tag-on and tag-off alternating inversion recovery spin labeling for determining micro-vascularity. We compared an ASL technique with 1-tag against 4-tag pulses. For perfusion, we determined signal increase ratio (SIR) at varying inversion times (TI) from 0.5 to 2 s. SIR versus TI data were fit to determine perfusion metrics of peak height (PH), time to peak (TTP), full width at half maximum (FWHM), area under the curve (AUC), and apparent blood flow (aBF) in the distal foot and individual toes. Using analysis of variance (ANOVA), effects of tag pulse and region of interest (ROI) on the mean perfusion metrics were assessed. In addition, a 4-tag pulse perfusion experiment was performed on patients with peripheral artery disease (PAD) and Raynaud's disease.ResultsUsing our MR perfusion techniques, SIR versus TI data showed well-defined leading and trailing edges, with a peak near TI of 0.75-1.0 s and subsiding quickly to near zero by TI of 2 s, particularly when 4-tag pulses were used. When imaged with 4-tag pulse, we found significantly greater values in perfusion metrics, as compared to 1-tag pulse. The patients with PAD and Raynaud's disease showed a reduced or scattered perfusion curves compared to the healthy control.ConclusionMR perfusion imaging of the distal foot shows greater SIR and perfusion metrics with the 4-tag pulse compared to the 1-tag pulse technique. This will likely benefit those with low perfusion due to aging, PAD, diabetic foot, and other vascular diseases.

Differential diagnosis is sometimes difficult in practical psychiatric settings, in terms of using the current diagnostic system based on presenting symptoms and signs. The creation of a novel diagnostic system using objective biomarkers is expected to take place. Neuroimaging studies and others reported that subcortical brain structures are the hubs for various psycho-behavioral functions, while there are so far no neuroimaging data-driven clinical criteria overcoming limitations of the current diagnostic system, which would reflect cognitive/social functioning. Prior to the main analysis, we conducted a large-scale multisite study of subcortical volumetric and lateralization alterations in schizophrenia, bipolar disorder, major depressive disorder, and autism spectrum disorder using T1-weighted images of 5604 subjects (3078 controls and 2526 patients). We demonstrated larger lateral ventricles volume in schizophrenia, bipolar disorder, and major depressive disorder, smaller hippocampus volume in schizophrenia and bipolar disorder, and schizophrenia-specific smaller amygdala, thalamus, and accumbens volumes and larger caudate, putamen, and pallidum volumes. In addition, we observed a leftward alteration of lateralization for pallidum volume specifically in schizophrenia. Moreover, as our main objective, we clustered the 5,604 subjects based on subcortical volumes, and explored whether data-driven clustering results can explain cognitive/social functioning in the subcohorts. We showed a four-biotype classification, namely extremely (Brain Biotype [BB] 1) and moderately smaller limbic regions (BB2), larger basal ganglia (BB3), and normal volumes (BB4), being associated with cognitive/social functioning. Specifically, BB1 and BB2-3 were associated with severe and mild cognitive/social impairment, respectively, while BB4 was characterized by normal cognitive/social functioning. Our results may lead to the future creation of novel biological data-driven psychiatric diagnostic criteria, which may be expected to be useful for prediction or therapeutic selection.

BackgroundUnderstanding the long-term sequelae of severe COVID-19 remains limited, particularly in the United States.ObjectiveTo examine long-term outcomes of patients who required intensive care unit (ICU) admission for severe COVID-19.Design, patients, and main measuresThis is a prospective cohort study of patients who had severe COVID-19 requiring an ICU admission in a two-hospital academic health system in Southern California. Patients discharged alive between 3/21/2020 and 12/31/2020 were surveyed approximately 6 months after discharge to assess health-related quality of life using Patient-Reported Outcomes Measurement Information System (PROMIS®)-29 v2.1, post-traumatic stress disorder (PTSD) and loneliness scales. A preference-based health utility score (PROPr) was estimated using 7 PROMIS domain scores. Patients were also asked their attitude about receiving aggressive ICU care.Key resultsOf 275 patients admitted to the ICU for severe COVID-19, 205 (74.5%) were discharged alive and 132 (64%, median age 59, 46% female) completed surveys a median of 182 days post-discharge. Anxiety, depression, fatigue, sleep disturbance, ability to participate in social activities, pain interference, and cognitive function were not significantly different from the U.S. general population, but physical function (44.2, SD 11.0) was worse. PROPr mean score of 0.46 (SD 0.30, range -0.02 to 0.96 [

Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu1-3. Here we identify the metabolite and neurotransmitter GABA as a candidate signalling molecule synthesized and secreted by activated B cells and plasma cells. We show that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8+ T cell killer function. In mice, B cell deficiency or B cell-specific inactivation of the GABA-generating enzyme GAD67 enhances anti-tumour responses. Our study reveals that, in addition to cytokines and membrane proteins, small metabolites derived from B-lineage cells have immunoregulatory functions, which may be pharmaceutical targets allowing fine-tuning of immune responses.

IntroductionWe investigated whether adding computed tomography (CT) and/or 18F-fluorodeoxyglucose (18F-FDG) PET radiomics features to conventional prognostic factors (CPFs) improves prognostic value in locally advanced non-small cell lung cancer (NSCLC).Materials and methodsWe retrospectively identified 39 cases with stage III NSCLC who received chemoradiotherapy and underwent planning CT and staging 18F-FDG PET scans. Seven CPFs were recorded. Feature selection was performed on 48 CT and 49 PET extracted radiomics features. A penalized multivariate Cox proportional hazards model was used to generate models for overall survival based on CPFs alone, CPFs with CT features, CPFs with PET features, and CPFs with CT and PET features. Linear predictors generated and categorized into 2 risk groups for which Kaplan-Meier survival curves were calculated. A log-rank test was performed to quantify the discrimination between the groups and calculated the Harrell's C-index to quantify the discriminatory power. A likelihood ratio test was performed to determine whether adding CT and/or PET features to CPFs improved model performance.ResultsAll 4 models significantly discriminated between the 2 risk groups. The discriminatory power was significantly increased when CPFs were combined with PET features (C-index 0.82; likelihood ratio test P < .01) or with both CT and PET features (0.83; P < .01) compared with CPFs alone (0.68). There was no significant improvement when CPFs were combined with CT features (0.68).ConclusionAdding PET radiomics features to CPFs yielded a significant improvement in the prognostic value in locally advanced NSCLC; adding CT features did not.

BACKGROUND/AIM:Cisplatinum (CDDP) is a first-line drug in osteosarcoma treatment and the acquisition of resistance to CDDP is associated with a poor prognosis. Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor that plays important roles in cell proliferation, differentiation, development, metabolism and cell death. Recently, PPARγ was reported to enhance the efficacy, overcome resistance, and decrease the toxicity of CDDP in various human cancers. In this study we tested whether pioglitazone (PIO), a PPARγ agonist, could overcome CDDP resistance in osteosarcoma. MATERIALS AND METHODS:In this study, we used a human osteosarcoma cell line and a patient-derived orthotopic xenograft (PDOX) models of osteosarcoma. We measured cell viability of 143B human osteosarcoma cells when treated with CDDP and PIO. We randomized PDOX models of osteosarcoma into four treatment groups: Group 1, Untreated control; Group 2, PIO alone; Group 3, CDDP alone; Group 4, a combination of CDDP and PIO. Each group comprised six mice. Mice were treated for 14 days and tumor size and body weight were measured. RESULTS:Cell viability of 143B human osteosarcoma cells was significantly reduced when PIO (50 μmol/l) was combined with CDDP compared to CDDP alone. PDOX osteosarcoma tumors treated with the CDDP-PIO combination showed the strongest tumor growth inhibition compared to other treatment groups. PDOX osteosarcoma tumors treated with the CDDP-PIO combination had the least cancer cells and the most necrosis in histological section. CONCLUSION:These results suggest that combining PIO along with CDDP could be an effective treatment strategy for osteosarcoma and has important clinical potential for patients.

PurposeThe role of chemotherapy (CT) and radiotherapy (RT) for management of extraskeletal osteosarcoma (ESOS) remains controversial. We examined disease outcomes for ESOS patients and investigated the association between CT/RT with recurrence and survival.Patients and methodsRetrospective review at 25 international sarcoma centers identified patients ≥18 years old treated for ESOS from 1971 to 2016. Patient/tumour characteristics, treatment, local/systemic recurrence, and survival data were collected. Kaplan-Meier survival and Cox proportional-hazards regression and cumulative incidence competing risks analysis were performed.Results370 patients with localized ESOS treated definitively with surgery presented with mainly deep tumours (n = 294, 80%). 122 patients underwent surgical resection alone, 96 (26%) also received CT, 70 (19%) RT and 82 (22%) both adjuvants. Five-year survival for patients with localized ESOS was 56% (95% CI 51%-62%). Almost half of patients (n = 173, 47%) developed recurrence: local 9% (35/370), distant 28% (102/370) or both 10% (36/370). Considering death as a competing event, there was no significant difference in cumulative incidence of local or systemic recurrence between patients who received CT, RT, both or neither (local p = 0.50, systemic p = 0.69). Multiple regression Cox analysis showed a significant association between RT and decreased local recurrence (HR 0.46 [95% CI 0.26-0.80], p = 0.01).ConclusionAlthough the use of RT significantly decreased local recurrences, CT did not decrease the risk of systemic recurrence, and neither CT, nor RT nor both were associated with improved survival in patients with localized ESOS. Our results do not support the use of CT; however, adjuvant RT demonstrates benefit in patients with locally resectable ESOS.

BACKGROUND & AIMS: Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection (rCDI). However, the overall mechanisms underlying FMT success await comprehensive elucidation, and the safety of FMT has recently become a serious concern because of the occurrence of drug-resistant bacteremia transmitted by FMT. We investigated whether functional restoration of the bacteriomes and viromes by FMT could be an indicator of successful FMT. METHODS: The human intestinal bacteriomes and viromes from 9 patients with rCDI who had undergone successful FMT and their donors were analyzed. Prophage-based and CRISPR spacer-based host bacteria-phage associations in samples from recipients before and after FMT and in donor samples were examined. The gene functions of intestinal microorganisms affected by FMT were evaluated. RESULTS: Metagenomic sequencing of both the viromes and bacteriomes revealed that FMT does change the characteristics of intestinal bacteriomes and viromes in recipients after FMT compared with those before FMT. In particular, many Proteobacteria, the fecal abundance of which was high before FMT, were eliminated, and the proportion of Microviridae increased in recipients. Most temperate phages also behaved in parallel with the host bacteria that were altered by FMT. Furthermore, the identification of bacterial and viral gene functions before and after FMT revealed that some distinctive pathways, including fluorobenzoate degradation and secondary bile acid biosynthesis, were significantly represented. CONCLUSIONS: The coordinated action of phages and their host bacteria restored the recipients intestinal flora. These findings show that the restoration of intestinal microflora functions reflects the success of FMT.

BackgroundGenetic factors that influence kidney traits have been understudied for low frequency and ancestry-specific variants.MethodsWe combined whole genome sequencing (WGS) data from 23,732 participants from 10 NHLBI Trans-Omics for Precision Medicine (TOPMed) Program multi-ethnic studies to identify novel loci for estimated glomerular filtration rate (eGFR). Participants included European, African, East Asian, and Hispanic ancestries. We applied linear mixed models using a genetic relationship matrix estimated from the WGS data and adjusted for age, sex, study, and ethnicity.FindingsWhen testing single variants, we identified three novel loci driven by low frequency variants more commonly observed in non-European ancestry (PRKAA2, rs180996919, minor allele frequency [MAF] 0.04%, P = 6.1 × 10-11; METTL8, rs116951054, MAF 0.09%, P = 4.5 × 10-9; and MATK, rs539182790, MAF 0.05%, P = 3.4 × 10-9). We also replicated two known loci for common variants (rs2461702, MAF=0.49, P = 1.2 × 10-9, nearest gene GATM, and rs71147340, MAF=0.34, P = 3.3 × 10-9, CDK12). Testing aggregated variants within a gene identified the MAF gene. A statistical approach based on local ancestry helped to identify replication samples for ancestry-specific variants.InterpretationThis study highlights challenges in studying variants influencing kidney traits that are low frequency in populations and more common in non-European ancestry.

PurposeIntensive care interventions that prolong life without achieving meaningful benefit are considered clinically "inappropriate". In 2012, the frequency of perceived-inappropriate critical care was 10.8% at one academic health system; and we aimed to re-evaluate this frequency.MethodsFor 4 months in 2017, we surveyed critical care physicians daily and asked whether each patient was receiving appropriate, probably inappropriate, or inappropriate critical care. Patients were categorized into three groups: 1) patients for whom treatment was never inappropriate, 2) patients with at least one assessment that treatment was probably inappropriate, but no inappropriate treatment assessments, and 3) patients who had at least one assessment of inappropriate treatment.ResultsFifty-five physicians made 10,105 assessments on 1424 patients. Of these, 94 (6.6%) patients received at least one assessment of inappropriate critical care, which is lower than 2012 (10.8% (p  0.05). Inpatient mortality in 2017 was 73% for patients receiving inappropriate critical care.ConclusionsOver five years the proportion of patients perceived to be receiving inappropriate critical care dropped by 40%. Understanding the reasons for such change might elucidate how to continue to reduce inappropriate critical care.

Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.

We tested cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice, which recapitulate key genetic abnormalities accumulating during colorectal cancer (CRC) tumorigenesis in humans, for responsiveness to anti-VEGF therapy. We found that even tumors in cis-ApcΔ716/Smad4+/-KrasG12D mice, although highly aggressive, were suppressed by anti-VEGF treatment. We tested the hypothesis that inflammation, a major risk factor and trigger for CRC, may affect responsiveness to anti-VEGF. Chemically induced colitis (CIC) in cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice promoted development of colon tumors that were largely resistant to anti-VEGF treatment. The myeloid growth factor G-CSF was markedly increased in the serum after induction of colitis. Antibodies blocking G-CSF, or its target Bv8/PROK2, suppressed tumor progression and myeloid cell infiltration when combined with anti-VEGF in CIC-associated CRC and in anti-VEGF-resistant CRC liver metastasis models. In a series of CRC specimens, tumor-infiltrating neutrophils strongly expressed Bv8/PROK2. CRC patients had significantly higher plasma Bv8/PROK2 levels than healthy volunteers and high plasma Bv8/PROK2 levels were inversely correlated with overall survival. Our findings establish Bv8/PROK2 as a translational target in CRC, in combination with anti-VEGF agents.

Despite decades of clinical use, mechanisms of glucocorticoid resistance are poorly understood. We treated primary murine T lineage acute lymphoblastic leukemias (T-ALLs) with the glucocorticoid dexamethasone (DEX) alone and in combination with the pan-PI3 kinase inhibitor GDC-0941 and observed a robust response to DEX that was modestly enhanced by GDC-0941. Continuous in vivo treatment invariably resulted in outgrowth of drug-resistant clones, ~30% of which showed markedly reduced glucocorticoid receptor (GR) protein expression. A similar proportion of relapsed human T-ALLs also exhibited low GR protein levels. De novo or preexisting mutations in the gene encoding GR (Nr3c1) occurred in relapsed clones derived from multiple independent parental leukemias. CRISPR/Cas9 gene editing confirmed that loss of GR expression confers DEX resistance. Exposing drug-sensitive T-ALLs to DEX in vivo altered transcript levels of multiple genes, and this response was attenuated in relapsed T-ALLs. These data implicate reduced GR protein expression as a frequent cause of glucocorticoid resistance in T-ALL.

The tremendous diversity of Hymenoptera is commonly attributed to the evolution of parasitoidism in the last common ancestor of parasitoid sawflies (Orussidae) and wasp-waisted Hymenoptera (Apocrita). However, Apocrita and Orussidae differ dramatically in their species richness, indicating that the diversification of Apocrita was promoted by additional traits. These traits have remained elusive due to a paucity of sawfly genome sequences, in particular those of parasitoid sawflies. Here, we present comparative analyses of draft genomes of the primarily phytophagous sawfly Athalia rosae and the parasitoid sawfly Orussus abietinus. Our analyses revealed that the ancestral hymenopteran genome exhibited traits that were previously considered unique to eusocial Apocrita (e.g., low transposable element content and activity) and a wider gene repertoire than previously thought (e.g., genes for CO2 detection). Moreover, we discovered that Apocrita evolved a significantly larger array of odorant receptors than sawflies, which could be relevant to the remarkable diversification of Apocrita by enabling efficient detection and reliable identification of hosts.

Meta-analyses of genome-wide association studies (GWAS) have identified more than 240 loci that are associated with type 2 diabetes (T2D)1,2; however, most of these loci have been identified in analyses of individuals with European ancestry. Here, to examine T2D risk in East Asian individuals, we carried out a meta-analysis of GWAS data from 77,418 individuals with T2D and 356,122 healthy control individuals. In the main analysis, we identified 301 distinct association signals at 183 loci, and across T2D association models with and without consideration of body mass index and sex, we identified 61 loci that are newly implicated in predisposition to T2D. Common variants associated with T2D in both East Asian and European populations exhibited strongly correlated effect sizes. Previously undescribed associations include signals in or near GDAP1, PTF1A, SIX3, ALDH2, a microRNA cluster, and genes that affect the differentiation of muscle and adipose cells3. At another locus, expression quantitative trait loci at two overlapping T2D signals affect two genes-NKX6-3 and ANK1-in different tissues4-6. Association studies in diverse populations identify additional loci and elucidate disease-associated genes, biology, and pathways.

Immune evasion is a major obstacle for cancer treatment. Common mechanisms of evasion include impaired antigen presentation caused by mutations or loss of heterozygosity of the major histocompatibility complex class I (MHC-I), which has been implicated in resistance to immune checkpoint blockade (ICB) therapy1-3. However, in pancreatic ductal adenocarcinoma (PDAC), which is resistant to most therapies including ICB4, mutations that cause loss of MHC-I are rarely found5 despite the frequent downregulation of MHC-I expression6-8. Here we show that, in PDAC, MHC-I molecules are selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involves the autophagy cargo receptor NBR1. PDAC cells display reduced expression of MHC-I at the cell surface and instead demonstrate predominant localization within autophagosomes and lysosomes. Notably, inhibition of autophagy restores surface levels of MHC-I and leads to improved antigen presentation, enhanced anti-tumour T cell responses and reduced tumour growth in syngeneic host mice. Accordingly, the anti-tumour effects of autophagy inhibition are reversed by depleting CD8+ T cells or reducing surface expression of MHC-I. Inhibition of autophagy, either genetically or pharmacologically with chloroquine, synergizes with dual ICB therapy (anti-PD1 and anti-CTLA4 antibodies), and leads to an enhanced anti-tumour immune response. Our findings demonstrate a role for enhanced autophagy or lysosome function in immune evasion by selective targeting of MHC-I molecules for degradation, and provide a rationale for the combination of autophagy inhibition and dual ICB therapy as a therapeutic strategy against PDAC.

Background & aimsChronic alcohol consumption is a leading risk factor for the development of hepatocellular carcinoma (HCC), which is associated with a marked increase in hepatic expression of pro-inflammatory IL-17A and its receptor IL-17RA.MethodsGenetic deletion and pharmacological blocking were used to characterize the role of IL-17A/IL-17RA signaling in the pathogenesis of HCC in mouse models and human specimens.ResultsWe demonstrate that the global deletion of the Il-17ra gene suppressed HCC in alcohol-fed diethylnitrosamine-challenged Il-17ra-/- and major urinary protein-urokinase-type plasminogen activator/Il-17ra-/- mice compared with wild-type mice. When the cell-specific role of IL-17RA signaling was examined, the development of HCC was decreased in both alcohol-fed Il-17raΔMΦ and Il-17raΔHep mice devoid of IL-17RA in myeloid cells and hepatocytes, but not in Il-17raΔHSC mice (deficient in IL-17RA in hepatic stellate cells). Deletion of Il-17ra in myeloid cells ameliorated tumorigenesis via suppression of pro-tumorigenic/inflammatory and pro-fibrogenic responses in alcohol-fed Il-17raΔMΦ mice. Remarkably, despite a normal inflammatory response, alcohol-fed Il-17raΔHep mice developed the fewest tumors (compared with Il-17raΔMΦ mice), with reduced steatosis and fibrosis. Steatotic IL-17RA-deficient hepatocytes downregulated the expression of Cxcl1 and other chemokines, exhibited a striking defect in tumor necrosis factor (TNF)/TNF receptor 1-dependent caspase-2-SREBP1/2-DHCR7-mediated cholesterol synthesis, and upregulated the production of antioxidant vitamin D3. The pharmacological blocking of IL-17A/Th-17 cells using anti-IL-12/IL-23 antibodies suppressed the progression of HCC (by 70%) in alcohol-fed mice, indicating that targeting IL-17 signaling might provide novel strategies for the treatment of alcohol-induced HCC.ConclusionsOverall, IL-17A is a tumor-promoting cytokine, which critically regulates alcohol-induced hepatic steatosis, inflammation, fibrosis, and HCC.Lay summaryIL-17A is a tumor-promoting cytokine, which critically regulates inflammatory responses in macrophages (Kupffer cells and bone-marrow-derived monocytes) and cholesterol synthesis in steatotic hepatocytes in an experimental model of alcohol-induced HCC. Therefore, IL-17A may be a potential therapeutic target for patients with alcohol-induced HCC.

Identifying both the commonalities and differences in brain structures among psychiatric disorders is important for understanding the pathophysiology. Recently, the ENIGMA-Schizophrenia DTI Working Group performed a large-scale meta-analysis and reported widespread white matter microstructural alterations in schizophrenia; however, no similar cross-disorder study has been carried out to date. Here, we conducted mega-analyses comparing white matter microstructural differences between healthy comparison subjects (HCS; N = 1506) and patients with schizophrenia (N = 696), bipolar disorder (N = 211), autism spectrum disorder (N = 126), or major depressive disorder (N = 398; total N = 2937 from 12 sites). In comparison with HCS, we found that schizophrenia, bipolar disorder, and autism spectrum disorder share similar white matter microstructural differences in the body of the corpus callosum; schizophrenia and bipolar disorder featured comparable changes in the limbic system, such as the fornix and cingulum. By comparison, alterations in tracts connecting neocortical areas, such as the uncinate fasciculus, were observed only in schizophrenia. No significant difference was found in major depressive disorder. In a direct comparison between schizophrenia and bipolar disorder, there were no significant differences. Significant differences between schizophrenia/bipolar disorder and major depressive disorder were found in the limbic system, which were similar to the differences in schizophrenia and bipolar disorder relative to HCS. While schizophrenia and bipolar disorder may have similar pathological characteristics, the biological characteristics of major depressive disorder may be close to those of HCS. Our findings provide insights into nosology and encourage further investigations of shared and unique pathophysiology of psychiatric disorders.

BackgroundAutopsy studies suggest that implanting stents in lipid-rich plaque (LRP) may be associated with adverse outcomes.ObjectivesThe purpose of this study was to evaluate the association between LRP detected by near-infrared spectroscopy (NIRS) and clinical outcomes in patients with coronary artery disease treated with contemporary drug-eluting stents.MethodsIn this prospective, multicenter registry, NIRS was performed in patients undergoing coronary angiography and possible percutaneous coronary intervention (PCI). Lipid core burden index (LCBI) was calculated as the fraction of pixels with the probability of LRP >0.6 within a region of interest. MaxLCBI4mm was defined as the maximum LCBI within any 4-mm-long segment. Major adverse cardiac events (MACE) included cardiac death, myocardial infarction, definite or probable stent thrombosis, or unplanned revascularization or rehospitalization for progressive angina or unstable angina. Events were subcategorized as culprit (treated) lesion-related, nonculprit (untreated) lesion-related, or indeterminate.ResultsAmong 1,999 patients who were enrolled in the COLOR (Chemometric Observations of Lipid Core Plaques of Interest in Native Coronary Arteries Registry), PCI was performed in 1,621 patients and MACE occurred in 18.0% of patients, of which 8.3% were culprit lesion-related, 10.7% were nonculprit lesion-related, and 3.1% were indeterminate during 2-year follow-up. Complications from NIRS imaging occurred in 9 patients (0.45%), which resulted in 1 peri-procedural myocardial infarction and 1 emergent coronary bypass. Pre-PCI NIRS imaging was obtained in 1,189 patients, and the 2-year rate of culprit lesion-related MACE was not significantly associated with maxLCBI4mm (hazard ratio of maxLCBI4mm per 100: 1.06; 95% confidence interval: 0.96 to 1.17; p = 0.28) after adjusting clinical and procedural factors.ConclusionsFollowing PCI with contemporary drug-eluting stents, stent implantation in NIRS-defined LRPs was not associated with increased periprocedural or late adverse outcomes compared with those without significant lipid.

Background/aimDedifferentiated liposarcoma (DDLPS) is recalcitrant type of sarcoma. DDLPS has a low survival rate with high recurrence and metastasis. In the present study, we evaluated the efficacy of several drugs against doxorubicin-resistant DDLPS in a patient-derived orthotopic xenograft (PDOX) model for precision oncology. To establish the PDOX model, a tumor from a patient who had recurrent high-grade DDLPS from the retroperitoneum was previously grown orthotopically in the retroperitoneum of nude mice.Materials and methodsWe randomized DDLPS PDOX models into 8 treatment groups when tumor volume became approximately 100 mm3: control, no treatment; G2, doxorubicin (DOX); G3, pazopanib (PAZ); G4, gemcitabine (GEM) combined with docetaxel (DOC); G5, trabectedin (YON); G6, temozolomide (TEM); G7, palbociclib (PAL); G8, eribulin (ERB). Tumor length and width were measured both at the beginning and at the end of treatment.ResultsAt the end of treatment (day 14), all treatments significantly inhibited DDLPS PDOX tumor growth compared to the untreated control, except DOX. ERB was significantly more effective and regressed tumor volume compared to other treatments on day 14 after initiation of treatment. No significant differences were found in the relative body weight on day 14 compared to day 0 in any group.ConclusionThe clinical potential of ERB against DDLPS is herein presented in a PDOX model.

Extracellular adenosine triphosphate (eATP) released by damaged cells, and its purinergic receptors, comprise a crucial signaling network after injury. Purinergic receptor P2X7 (P2RX7), a major driver of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and IL-1β processing, has been shown to play a role in liver injury in murine diet- and chemically-induced liver injury models. It is unclear, however, whether P2RX7 plays a role in non-alcoholic steatohepatitis (NASH) and which cell type is the main target of P2RX7 pharmacological inhibition. Here, we report that P2RX7 is expressed by infiltrating monocytes and resident Kupffer cells in livers from NASH-affected individuals. Using primary isolated human cells, we demonstrate that P2RX7 expression in CD14+ monocytes and Kupffer cells primarily mediates IL-1β release. In addition, we show that pharmacological inhibition of P2RX7 in monocytes and Kupffer cells, blocks IL-1β release, reducing hepatocyte caspase 3/7 activity, IL-1β-mediated CCL2 and CCL5 chemokine gene expression and secretion, and hepatic stellate cell (HSC) procollagen secretion. Consequently, in a chemically-induced nonhuman primate model of liver fibrosis, treatment with a P2RX7 inhibitor improved histological characteristics of NASH, protecting from liver inflammation and fibrosis. Taken together, these findings underscore the critical role of P2RX7 in the pathogenesis of NASH and implicate P2RX7 as a promising therapeutic target for the management of this disease.

BackgroundAlthough homelessness and opioid overdose are major public health issues in the U.S., evidence is limited as to whether homelessness is associated with an increased risk of opioid overdose.ObjectiveTo compare opioid-related outcomes between homeless versus housed individuals in low-income communities.Design, setting, and participantsCross-sectional analysis of individuals who had at least one ED visit or hospitalization in four states (Florida, Maryland, Massachusetts, and New York) in 2014.MeasurementsRisk of opioid overdose and opioid-related ED visits/hospital admissions were compared between homeless versus low-income housed individuals, adjusting for patient characteristics and hospital-specific fixed effects (effectively comparing homeless versus low-income housed individuals treated at the same hospital). We also examined whether risk of opioid-related outcomes varied by patients' sex and race/ethnicity.ResultsA total of 96,099 homeless and 2,869,230 low-income housed individuals were analyzed. Homeless individuals had significantly higher risk of opioid overdose (adjusted risk, 1.8% for homeless vs. 0.3% for low-income housed individuals; adjusted risk difference [aRD], +1.5%; 95%CI, +1.0% to +2.0%; p

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons degenerate, resulting in muscle atrophy, paralysis, and fatality. Studies using mouse models of ALS indicate a protracted period of disease development with progressive motor neuron pathology, evident as early as embryonic and postnatal stages. Key missing information includes concomitant alterations in the sensorimotor circuit essential for normal development and function of the neuromuscular system. Leveraging unique brainstem circuitry, we show in vitro evidence for reflex circuit-specific postnatal abnormalities in the jaw proprioceptive sensory neurons in the well-studied SOD1G93A mouse. These include impaired and arrhythmic action potential burst discharge associated with a deficit in Nav1.6 Na+ channels. However, the mechanoreceptive and nociceptive trigeminal ganglion neurons and the visual sensory retinal ganglion neurons were resistant to excitability changes in age-matched SOD1G93A mice. Computational modeling of the observed disruption in sensory patterns predicted asynchronous self-sustained motor neuron discharge suggestive of imminent reflexive defects, such as muscle fasciculations in ALS. These results demonstrate a novel reflex circuit-specific proprioceptive sensory abnormality in ALS.SIGNIFICANCE STATEMENT Neurodegenerative diseases have prolonged periods of disease development and progression. Identifying early markers of vulnerability can therefore help devise better diagnostic and treatment strategies. In this study, we examined postnatal abnormalities in the electrical excitability of muscle spindle afferent proprioceptive neurons in the well-studied SOD1G93A mouse model for neurodegenerative motor neuron disease, amyotrophic lateral sclerosis. Our findings suggest that these proprioceptive sensory neurons are exclusively afflicted early in the disease process relative to sensory neurons of other modalities. Moreover, they presented Nav1.6 Na+ channel deficiency, which contributed to arrhythmic burst discharge. Such sensory arrhythmia could initiate reflexive defects, such as muscle fasciculations in amyotrophic lateral sclerosis, as suggested by our computational model.

Postsynaptic density (PSD) proteins have been implicated in the pathophysiology of neurodevelopmental and psychiatric disorders. Here, we present detailed clinical and genetic data for 20 patients with likely gene-disrupting mutations in TANC2-whose protein product interacts with multiple PSD proteins. Pediatric patients with disruptive mutations present with autism, intellectual disability, and delayed language and motor development. In addition to a variable degree of epilepsy and facial dysmorphism, we observe a pattern of more complex psychiatric dysfunction or behavioral problems in adult probands or carrier parents. Although this observation requires replication to establish statistical significance, it also suggests that mutations in this gene are associated with a variety of neuropsychiatric disorders consistent with its postsynaptic function. We find that TANC2 is expressed broadly in the human developing brain, especially in excitatory neurons and glial cells, but shows a more restricted pattern in Drosophila glial cells where its disruption affects behavioral outcomes.

Deep learning algorithms have achieved great success in cancer image classification. However, it is imperative to understand the differences between the deep learning and human approaches. Using an explainable model, we aimed to compare the deep learning-focused regions of magnetic resonance (MR) images with cancerous locations identified by radiologists and pathologists. First, 307 prostate MR images were classified using a well-established deep neural network without locational information of cancers. Subsequently, we assessed whether the deep learning-focused regions overlapped the radiologist-identified targets. Furthermore, pathologists provided histopathological diagnoses on 896 pathological images, and we compared the deep learning-focused regions with the genuine cancer locations through 3D reconstruction of pathological images. The area under the curve (AUC) for MR images classification was sufficiently high (AUC = 0.90, 95% confidence interval 0.87-0.94). Deep learning-focused regions overlapped radiologist-identified targets by 70.5% and pathologist-identified cancer locations by 72.1%. Lymphocyte aggregation and dilated prostatic ducts were observed in non-cancerous regions focused by deep learning. Deep learning algorithms can achieve highly accurate image classification without necessarily identifying radiological targets or cancer locations. Deep learning may find clues that can help a clinical diagnosis even if the cancer is not visible.

Objective: Medical interventions that do not offer the patient meaningful benefit due to inconsistency with prognoses are often considered “inappropriate” by clinicians. We described the clinical details and resource utilisation of patients who were assessed as receiving inappropriate treatment. Design: Chart abstraction was performed on 123 patients who were assessed by their critical care physician as having received inappropriate treatment to document clinical characteristics, diagnostic testing, life-sustaining treatments and nursing assessments of daily pain and level of consciousness. Results: The mean age was 67 and on admission, 41% had cancer and 25% had advanced pulmonary disease. At least one of the following three conditions was noted in 57% of the patients: severe neurological injury, overwhelming sepsis or irreversible respiratory failure. Patients were less likely to be alert (OR 0.39, CI 0.16–0.91, p = 0.03) on days they were assessed as receiving inappropriate critical care. After they were assessed as receiving inappropriate critical care, they received 172 imaging studies, 151 procedures, 522 days of mechanical ventilation (excludes one patient who received 1020 days of mechanical ventilation), 254 days of vasopressors, 226 days of hemodialysis and 10 attempts at cardiopulmonary resuscitation. Conclusions: Patients assessed as receiving inappropriate critical care receive resource-intensive medical care, largely while non-alert.

The influence of living in small remote villages on the diversity of viruses in the nasal mucosa was investigated in three Colombian villages with very different levels of geographic isolation. Metagenomic analysis was used to characterize viral nucleic acids in nasal swabs from 63 apparently healthy young children. Sequences from human virus members of the families Anelloviridae, Papillomaviridae, Picornaviridae, Herpesviridae, Polyomaviridae, Adenoviridae, and Paramyxoviridae were detected in decreasing proportions of children. The number of papillomavirus infections detected was greater among Hispanic children most exposed to outside contacts, while anellovirus infections were more common in the isolated indigenous villages. The diversity of the other human viruses detected did not differ among the villages. Closely related variants of rhinovirus A or B were identified in 2 to 4 children from each village, reflecting ongoing transmission clusters. Genomes of viruses not currently known to infect humans, including members of the families Parvoviridae, Partitiviridae, Dicistroviridae, and Iflaviridae and circular Rep-encoding single-stranded DNA (CRESS-DNA) virus, were also detected in nasal swabs, possibly reflecting environmental contamination from insect, fungal, or unknown sources. Despite the high levels of geographic and cultural isolation, the overall diversity of human viruses in the nasal passages of children was not reduced in highly isolated indigenous villages, indicating ongoing exposure to globally circulating viruses.IMPORTANCE Extreme geographic and cultural isolation can still be found in some indigenous South American villages. Such isolation may be expected to limit the introduction of otherwise common and widely distributed viruses. Very small population sizes may also result in rapid local viral extinction due to a lack of seronegative subjects to maintain transmission chains for rapidly cleared viruses. We compared the viruses in the nasal passages of young children in three villages with increasing levels of geographic isolation. We found that isolation did not reduce the overall diversity of viral infections. Multiple infections with nearly identical rhinoviruses could be detected within each village, likely reflecting recent viral introductions and transmission clusters among epidemiologically linked members of these very small communities. We conclude that, despite their geographic isolation, remote indigenous villages show evidence of ongoing exposure to globally circulating viruses.

For long QT syndrome (LQTS), recent progress in genome-sequencing technologies enabled the identification of rare genomic variants with diagnostic, prognostic, and therapeutic implications. However, pathogenic stratification of the identified variants remains challenging, especially in variants of uncertain significance. This study aimed to propose a phenotypic cell-based diagnostic assay for identifying LQTS to recognize pathogenic variants in a high-throughput manner suitable for screening. We investigated the response of LQT2-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) following IKr blockade using a multi-electrode array, finding that the response to IKr blockade was significantly smaller than in Control-iPSC-CMs. Furthermore, we found that LQT1-iPSC-CMs and LQT3-iPSC-CMs could be distinguished from Control-iPSC-CMs by IKs blockade and INa blockade, respectively. This strategy might be helpful in compensating for the shortcomings of genetic testing of LQTS patients.

WD40 repeat-containing proteins form a large family of proteins present in all eukaryotes. Here, we identified five pediatric probands with de novo variants in WDR37, which encodes a member of the WD40 repeat protein family. Two probands shared one variant and the others have variants in nearby amino acids outside the WD40 repeats. The probands exhibited shared phenotypes of epilepsy, colobomas, facial dysmorphology reminiscent of CHARGE syndrome, developmental delay and intellectual disability, and cerebellar hypoplasia. The WDR37 protein is highly conserved in vertebrate and invertebrate model organisms and is currently not associated with a human disease. We generated a null allele of the single Drosophila ortholog to gain functional insights and replaced the coding region of the fly gene CG12333/wdr37 with GAL4. These flies are homozygous viable but display severe bang sensitivity, a phenotype associated with seizures in flies. Additionally, the mutant flies fall when climbing the walls of the vials, suggesting a defect in grip strength, and repeat the cycle of climbing and falling. Similar to wall clinging defect, mutant males often lose grip of the female abdomen during copulation. These phenotypes are rescued by using the GAL4 in the CG12333/wdr37 locus to drive the UAS-human reference WDR37 cDNA. The two variants found in three human subjects failed to rescue these phenotypes, suggesting that these alleles severely affect the function of this protein. Taken together, our data suggest that variants in WDR37 underlie a novel syndromic neurological disorder.

BackgroundMany patients remain without a diagnosis despite extensive medical evaluation. The Undiagnosed Diseases Network (UDN) was established to apply a multidisciplinary model in the evaluation of the most challenging cases and to identify the biologic characteristics of newly discovered diseases. The UDN, which is funded by the National Institutes of Health, was formed in 2014 as a network of seven clinical sites, two sequencing cores, and a coordinating center. Later, a central biorepository, a metabolomics core, and a model organisms screening center were added.MethodsWe evaluated patients who were referred to the UDN over a period of 20 months. The patients were required to have an undiagnosed condition despite thorough evaluation by a health care provider. We determined the rate of diagnosis among patients who subsequently had a complete evaluation, and we observed the effect of diagnosis on medical care.ResultsA total of 1519 patients (53% female) were referred to the UDN, of whom 601 (40%) were accepted for evaluation. Of the accepted patients, 192 (32%) had previously undergone exome sequencing. Symptoms were neurologic in 40% of the applicants, musculoskeletal in 10%, immunologic in 7%, gastrointestinal in 7%, and rheumatologic in 6%. Of the 382 patients who had a complete evaluation, 132 received a diagnosis, yielding a rate of diagnosis of 35%. A total of 15 diagnoses (11%) were made by clinical review alone, and 98 (74%) were made by exome or genome sequencing. Of the diagnoses, 21% led to recommendations regarding changes in therapy, 37% led to changes in diagnostic testing, and 36% led to variant-specific genetic counseling. We defined 31 new syndromes.ConclusionsThe UDN established a diagnosis in 132 of the 382 patients who had a complete evaluation, yielding a rate of diagnosis of 35%. (Funded by the National Institutes of Health Common Fund.).

PurposeLung functional image guided radiation therapy (RT) that avoids irradiating highly functional regions has potential to reduce pulmonary toxicity following RT. Tumor regression during RT is common, leading to recovery of lung function. We hypothesized that computed tomography (CT) ventilation image-guided treatment planning reduces the functional lung dose compared to standard anatomic image-guided planning in 2 different scenarios with or without plan adaptation.Methods and materialsCT scans were acquired before RT and during RT at 2 time points (16-20 Gy and 30-34 Gy) for 14 patients with locally advanced lung cancer. Ventilation images were calculated by deformable image registration of four-dimensional CT image data sets and image analysis. We created 4 treatment plans at each time point for each patient: functional adapted, anatomic adapted, functional unadapted, and anatomic unadapted plans. Adaptation was performed at 2 time points. Deformable image registration was used for accumulating dose and calculating a composite of dose-weighted ventilation used to quantify the lung accumulated dose-function metrics. The functional plans were compared with the anatomic plans for each scenario separately to investigate the hypothesis at a significance level of 0.05.ResultsTumor volume was significantly reduced by 20% after 16 to 20 Gy (P = .02) and by 32% after 30 to 34 Gy (P

PIN1-mediated substrate isomerization plays a role in the repair of DNA double-strand breaks. We hypothesized that genetic polymorphisms in PIN1-related pathways may affect tumor sensitivity to oxaliplatin or irinotecan in metastatic colorectal cancer (mCRC) patients. We analyzed genomic DNA from five cohorts of mCRC patients (total 950) treated with different first-line treatments: oxaliplatin cohorts 1 (n = 146) and 2 (n = 70); irinotecan cohorts 1 (n = 228), and 2 (n = 276); and combination cohort (n = 230). Single nucleotide polymorphisms of candidate genes were analyzed by PCR-based direct sequencing. In the oxaliplatin cohort 1, patients carrying any PIN1 rs2233678 C allele had shorter progression-free survival (PFS) and overall survival (OS) than the G/G variant (PFS, 7.4 vs. 15.0 months, hazard ratio [HR] 3.24, P

PURPOSE:This phase II study evaluated the safety and clinical activity of pazopanib, a potent and mutlitargeted tyrosine kinase inhibitor (TKI) of vascular endothelial growth factor receptors (VEGFRs)-1, -2 and -3, platelet-derived growth factor receptor (PDGFR)-α and β, and cKit, in combination with metronomic paclitaxel in patients with metastatic melanoma. EXPERIMENTAL DESIGN:Sixty chemotherapy-naive patients received pazopanib at a starting dose of 800 mg daily in combination with metronomic dosing of paclitaxel 80 mg/m2 weekly thrice every 4 weeks. The primary endpoint was 6-month progression-free survival (PFS) rate, while secondary endpoints included 1-year overall survival rate, RECIST response rates, progression-free survival rates and median overall survival. Prior BRAF-targeted therapy or checkpoint inhibitors were permitted. RESULTS:The 6-month PFS rate was 68%, with a 1-year OS rate of 48%. Objective response rate was 37% comprising one complete and 20 partial responses. Stable disease at 8 weeks was noted in 32 patients (55%) with an overall clinical benefit rate of 93%. Six-month median progression-free survival was 8 months and median OS was 12.7 months. The most frequently (> 15%) reported non-hematologic, treatment-related adverse events were fatigue, diarrhea, hypertension, transaminitis and peripheral neuropathy. Treatment-related non-fatal bowel perforation, a known class effect, occurred in one patient. No significant association was noted between plasma levels of pazopanib and response. CONCLUSIONS:The combination of pazopanib and metronomic paclitaxel was well-tolerated, demonstrating significant activity in metastatic melanoma. Further evaluation of this combination is warranted.

BackgroundCentral nervous system (CNS) histoplasmosis is a life-threatening condition and represents a diagnostic and therapeutic challenge. Isolation of Histoplasma capsulatum from cerebrospinal fluid (CSF) or brain tissue is diagnostic; however, culture is insensitive and slow growth may result in significant treatment delay. We performed a retrospective multicenter study to evaluate the sensitivity and specificity of a new anti-Histoplasma antibody enzyme immunoassay (EIA) for the detection of IgG and IgM antibody in the CSF for diagnosis of CNS histoplasmosis, the primary objective of the study. The secondary objective was to determine the effect of improvements in the Histoplasma galactomannan antigen detection EIA on the diagnosis of Histoplasma meningitis.MethodsResidual CSF specimens from patients with Histoplasma meningitis and controls were tested for Histoplasma antigen and anti-Histoplasma immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody using assays developed at MiraVista Diagnostics.ResultsA total of 50 cases and 157 controls were evaluated. Fifty percent of patients with CNS histoplasmosis were immunocompromised, 14% had other medical conditions, and 36% were healthy. Histoplasma antigen was detected in CSF in 78% of cases and the specificity was 97%. Anti-Histoplasma IgG or IgM antibody was detected in 82% of cases and the specificity was 93%. The sensitivity of detection of antibody by currently available serologic testing including immunodiffusion and complement fixation was 51% and the specificity was 96%. Testing for both CSF antigen and antibody by EIA was the most sensitive approach, detecting 98% of cases.ConclusionsTesting CSF for anti-Histoplasma IgG and IgM antibody complements antigen detection and improves the sensitivity for diagnosis of Histoplasma meningitis.

The first reported outbreak of Ebola virus disease occurred in 1976 in Yambuku, Democratic Republic of Congo. Antibody responses in survivors 11 years after infection have been documented. However, this report is the first characterization of anti-Ebola virus antibody persistence and neutralization capacity 40 years after infection. Using ELISAs we measured survivor's immunological response to Ebola virus Zaire (EBOV) glycoprotein and nucleoprotein, and assessed VP40 reactivity. Neutralization of EBOV was measured using a pseudovirus approach and plaque reduction neutralization test with live EBOV. Some survivors from the original EBOV outbreak still harbor antibodies against all 3 measures. Interestingly, a subset of these survivors' serum antibodies could still neutralize live virus 40 years postinitial infection. These data provide the longest documentation of both anti-Ebola serological response and neutralization capacity within any survivor cohort, extending the known duration of response from 11 years postinfection to at least 40 years after symptomatic infection.

BackgroundFactors leading to inappropriate critical care, that is treatment that should not be provided because it does not offer the patient meaningful benefit, have not been rigorously characterized.ObjectiveWe explored medical record documentation about patients who received inappropriate critical care and those who received appropriate critical care to examine factors associated with the provision of inappropriate treatment.DesignMedical records were abstracted from 123 patients who were assessed as receiving inappropriate treatment and 66 patients who were assessed as receiving appropriate treatment but died within six months of intensive care unit (ICU) admission. We used mixed methods combining qualitative analysis of medical record documentation with multivariable analysis to examine the relationship between patient and communication factors and the receipt of inappropriate treatment, and present these within a conceptual model.SettingOne academic health system.ResultsMedical records revealed 21 themes pertaining to prognosis and factors influencing treatment aggressiveness. Four themes were independently associated with patients receiving inappropriate treatment according to physicians. When decision making was not guided by physicians (odds ratio [OR] 3.76, confidence interval [95% CI] 1.21-11.70) or was delayed by patient/family (OR 4.52, 95% CI 1.69-12.04), patients were more likely to receive inappropriate treatment. Documented communication about goals of care (OR 0.29, 95% CI 0.10-0.84) and patient's preferences driving decision making (OR 0.02, 95% CI 0.00-0.27) were associated with lower odds of receiving inappropriate treatment.ConclusionsMedical record documentation suggests that inappropriate treatment occurs in the setting of communication and decision-making patterns that may be amenable to intervention.

The process of selecting for cellular fitness through competition plays a critical role in both development and disease. The germarium, a structure at the tip of the ovariole of a Drosophila ovary, contains two follicle stem cells (FSCs) that undergo neutral competition for the stem cell niche. Using the FSCs as a model, we performed a genetic screen through a collection of 126 mutants in essential genes on the X chromosome to identify candidates that increase or decrease competition for the FSC niche. We identified ∼55 and 6% of the mutations screened as putative FSC hypo- or hyper-competitors, respectively. We found that a large majority of mutations in vesicle trafficking genes (11 out of the 13 in the collection of mutants) are candidate hypo-competition alleles, and we confirmed the hypo-competition phenotype for four of these alleles. We also show that Sec16 and another COPII vesicle trafficking component, Sar1, are required for follicle cell differentiation. Lastly, we demonstrate that, although some components of vesicle trafficking are also required for neutral competition in the cyst stem cells of the testis, there are important tissue-specific differences. Our results demonstrate a critical role for vesicle trafficking in stem cell niche competition and differentiation, and we identify a number of putative candidates for further exploration.

RationaleTherapies that inhibit CETP (cholesteryl ester transfer protein) have failed to demonstrate a reduction in risk for coronary heart disease (CHD). Human DNA sequence variants that truncate the CETP gene may provide insight into the efficacy of CETP inhibition.ObjectiveTo test whether protein-truncating variants (PTVs) at the CETP gene were associated with plasma lipid levels and CHD.Methods and resultsWe sequenced the exons of the CETP gene in 58 469 participants from 12 case-control studies (18 817 CHD cases, 39 652 CHD-free controls). We defined PTV as those that lead to a premature stop, disrupt canonical splice sites, or lead to insertions/deletions that shift frame. We also genotyped 1 Japanese-specific PTV in 27561 participants from 3 case-control studies (14 286 CHD cases, 13 275 CHD-free controls). We tested association of CETP PTV carrier status with both plasma lipids and CHD. Among 58 469 participants with CETP gene-sequencing data available, average age was 51.5 years and 43% were women; 1 in 975 participants carried a PTV at the CETP gene. Compared with noncarriers, carriers of PTV at CETP had higher high-density lipoprotein cholesterol (effect size, 22.6 mg/dL; 95% confidence interval, 18-27; P

BackgroundAccurate preoperative lymphoscintigraphy is vital to performing sentinel lymph node biopsy (SLNB) for cutaneous malignancies. Potential advantages of single-photon emission computed tomography with integrated computed tomography (SPECT/CT) include the ability to readily identify aberrant drainage patterns as well as provide the surgeon with three-dimensional anatomic landmarks not seen on conventional planar lymphoscintigraphy (PLS).MethodsPatients with cutaneous malignancies who underwent SLNB with preoperative imaging using both SPECT/CT and PLS from 2011 to 2014 were identified.ResultsBoth SPECT/CT and PLS were obtained in 351 patients (median age, 69 years; range, 5-94 years) with cutaneous malignancies (melanoma = 300, Merkel cell carcinoma = 33, squamous cell carcinoma = 8, other = 10) after intradermal injection of 99mtechnetium sulfur colloid (median dose 300 µCi). A mean of 4.3 hot spots were identified on SPECT/CT compared to 3.0 on PLS (p

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 (NACC1) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10-14). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1.

Rhabdomyosarcoma (RMS) is a rare mesenchymal tumor. The aim of the present study was to develop a patient-derived orthotopic xenograft (PDOX) mouse model of RMS and compare the PDOX model to a subcutaneous (s.c.)-transplant model. A patient RMS from a striated muscle was grown orthotopically in the right biceps femoris muscle and right quadriceps muscle of nude mice to establish a PDOX model, as well as under the skin to establish an s.c.ModelPDOX tumors grew at a statistically-significant faster rate compared to the s.c. tumors. Recurrence after surgical resection occurred only in PDOX tumors, not in the s.c.ModelHistologically, only the PDOX model was shown to be invasive. In conclusion, these results indicate that the PDOX model of adult RMS is malignant and the subcutaneous model is benign. These results emphasize that a proper tumor microenvironment is necessary for patient-like behavior of a tumor in a mouse model.

BackgroundThe causes of age-related hyperkyphosis (HK) include osteoporosis, but only 1/3 of those most severely affected have vertebral fractures, suggesting that there are other important, and potentially modifiable causes. We hypothesized that muscle mass and quality may be important determinants of kyphosis in older persons.MethodsWe recruited 72 persons >65 years to participate in a prospective study designed to evaluate kyphosis and fall risk. At the baseline visit, participants had their body composition measures completed using Dual Energy X-ray Absorptiometry (DXA). They had kyphosis measured in either the standing [S] or lying [L] position: 1) Cobb angle from DXA [L]; 2) Debrunner kyphometer [S]; 3) architect's flexicurve ruler [S]; and 4) blocks method [L]. Multivariable linear/logistic regression analyses were done to assess the association between each body composition and 4 kyphosis measures.ResultsWomen (n = 52) were an average age of 76.8 (SD 6.7) and men 80.5 (SD 7.8) years. They reported overall good/excellent health (93%), the average body mass index was 25.3 (SD 4.6) and 35% reported a fall in the past year. Using published cut-offs, about 20-30% were determined to have HK. For the standing assessments of kyphosis only, after adjusting for age, sex, weight and hip BMD, persons with lower TLM were more likely to be hyperkyphotic.ConclusionsLower TLM is associated with HK in older persons. The results were stronger when standing measures of kyphosis were used, suggesting that the effects of muscle on thoracic kyphosis are best appreciated under spinal loading conditions.

UNLABELLED: Shank2 is a multidomain scaffolding protein implicated in the structural and functional coordination of multiprotein complexes at excitatory postsynaptic sites as well as in psychiatric disorders, including autism spectrum disorders. While Shank2 is strongly expressed in the cerebellum, whether Shank2 regulates cerebellar excitatory synapses, or contributes to the behavioral abnormalities observed in Shank2-/- mice, remains unexplored. Here we show that Shank2-/- mice show reduced excitatory synapse density in cerebellar Purkinje cells in association with reduced levels of excitatory postsynaptic proteins, including GluD2 and PSD-93, and impaired motor coordination in the Erasmus test. Shank2 deletion restricted to Purkinje cells (Pcp2-Cre;Shank2fl/fl mice) leads to similar reductions in excitatory synapse density, synaptic protein levels, and motor coordination. Pcp2-Cre;Shank2fl/fl mice do not recapitulate autistic-like behaviors observed in Shank2-/- mice, such as social interaction deficits, altered ultrasonic vocalizations, repetitive behaviors, and hyperactivity. However, Pcp2-Cre;Shank2fl/fl mice display enhanced repetitive behavior in the hole-board test and anxiety-like behavior in the light-dark test, which are not observed in Shank2-/- mice. These results implicate Shank2 in the regulation of cerebellar excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors. SIGNIFICANCE STATEMENT: The postsynaptic side of excitatory synapses contains multiprotein complexes, termed the postsynaptic density, which contains receptors, scaffolding/adaptor proteins, and signaling molecules. Shank2 is an excitatory postsynaptic scaffolding protein implicated in the formation and functional coordination of the postsynaptic density and has been linked to autism spectrum disorders. Using Shank2-null mice and Shank2-conditional knock-out mice with a gene deletion restricted to cerebellar Purkinje cells, we explored functions of Shank2 in the cerebellum. We found that Shank2 regulates excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors, but is not associated with autistic-like social deficits or repetitive behaviors.

We performed an exome-wide association analysis in 1393 late-onset Alzheimer's disease (LOAD) cases and 8141 controls from the CHARGE consortium. We found that a rare variant (P155L) in TM2D3 was enriched in Icelanders (~0.5% versus

BackgroundThe current NCCN recommendation for resection margins in patients with melanomas between 1.01 and 2 mm deep is a 1-2 cm radial margin. We sought to determine whether margin width had an impact on local recurrence (LR), disease-specific survival (DSS), and type of wound closure.MethodsMelanomas measuring 1.01-2.0 mm were evaluated at a single institution between 2008 and 2013. All patients had a 1 or 2 cm margin.ResultsWe identified 965 patients who had a 1 cm (n = 302, 31.3 %) or 2 cm margin (n = 663, 68.7 %). Median age was 64 years, and 592 (61.3 %) were male; 32.5 and 48.7 % of head and neck and extremity patients had a 1 cm margin versus 18.9 % of trunk patients (p

PurposeTo investigate the hypothesis that CT ventilation functional image-based IMRT plans designed to avoid irradiating highly-functional lung regions are comparable to single-photon emission CT (SPECT) ventilation functional image-based plans.Methods and materialsThree IMRT plans were created for eight thoracic cancer patients using: (1) CT ventilation functional images, (2) SPECT ventilation functional images, and (3) anatomic images (no functional images). CT ventilation images were created by deformable image registration of 4D-CT image data sets and quantitative analysis. The resulting plans were analyzed for the relationship between the deviations of CT-functional plan metrics from anatomic plan metrics (ΔCT-anatomic) and those of SPECT-functional plans (ΔSPECT-anatomic), and moreover for agreements of various metrics between the CT-functional and SPECT-functional plans.ResultsThe relationship between ΔCT-anatomic and ΔSPECT-anatomic was strong (e.g., R=0.94; linear regression slope 0.71). The average differences and 95% limits of agreement between the CT-functional and SPECT-functional plan metrics (except for monitor units) for various structures were mostly less than 1% and 2%, respectively.ConclusionsThis study demonstrated a reasonable agreement between the CT ventilation functional image-based IMRT plans and SPECT-functional plans, suggesting the potential for CT ventilation imaging to serve as a surrogate for SPECT ventilation in functional image-guided radiotherapy.

Sixty genetic loci associated with abdominal obesity, measured by waist circumference (WC) and waist-hip ratio (WHR), have been previously identified, primarily from studies conducted in European-ancestry populations. We conducted a meta-analysis of associations of abdominal obesity with approximately 2.5 million single nucleotide polymorphisms (SNPs) among 53,052 (for WC) and 48,312 (for WHR) individuals of Asian descent, and replicated 33 selected SNPs among 3,762 to 17,110 additional individuals. We identified four novel loci near the EFEMP1, ADAMTSL3 , CNPY2, and GNAS genes that were associated with WC after adjustment for body mass index (BMI); two loci near the NID2 and HLA-DRB5 genes associated with WHR after adjustment for BMI, and three loci near the CEP120, TSC22D2, and SLC22A2 genes associated with WC without adjustment for BMI. Functional enrichment analyses revealed enrichment of corticotropin-releasing hormone signaling, GNRH signaling, and/or CDK5 signaling pathways for those newly-identified loci. Our study provides additional insight on genetic contribution to abdominal obesity.

Most West Nile virus (WNV) infections are asymptomatic, but some lead to neuroinvasive disease with symptoms ranging from disorientation to paralysis and death. Evidence from animal models suggests that neuroinvasive infections may arise as a consequence of impaired immune protection. However, other data suggest that neurologic symptoms may arise as a consequence of immune mediated damage. We demonstrate that elevated immune responses are present in neuroinvasive disease by directly characterizing WNV-specific T cells in subjects with laboratory documented infections using human histocompatibility leukocyte antigen (HLA) class II tetramers. Subjects with neuroinvasive infections had higher overall numbers of WNV-specific T cells than those with asymptomatic infections. Independent of this, we also observed age related increases in WNV-specific T cell responses. Further analysis revealed that WNV-specific T cell responses included a population of atypically polarized CXCR3+CCR4+CCR6- T cells, whose presence was highly correlated with neuroinvasive disease. Moreover, a higher proportion of WNV-specific T cells in these subjects co-produced interferon-γ and interleukin 4 than those from asymptomatic subjects. More globally, subjects with neuroinvasive infections had reduced numbers of CD4+FoxP3+ Tregs that were CTLA4 positive and exhibited a distinct upregulated transcript profile that was absent in subjects with asymptomatic infections. Thus, subjects with neuroinvasive WNV infections exhibited elevated, dysregulated, and atypically polarized responses, suggesting that immune mediated damage may indeed contribute to pathogenic outcomes.

PurposeA randomized controlled trial found that patients with diabetes had lower HbA1c levels after 6 months of peer health coaching than patients who did not receive coaching. This paper explores whether the peer coaches in that trial, all low-income patients with diabetes, mastered and utilized an evidence-based health coaching training curriculum. The curriculum included 5 core features: ask-tell-ask, closing the loop, know your numbers, behavior-change action plans, and medication adherence counseling.MethodsThis paper includes the results of exams administered to trainees, exit surveys performed with peer coaches who completed the study and those who dropped out, observations of peer coaches meeting with patients, and analysis of in-depth interviews with peer coaches who completed the study.ResultsOf the 32 peer coach trainees who completed the training, 71.9% lacked a college degree; 25.0% did not graduate from high school. The 26 trainees who passed the exams attended 92.7% of training sessions compared with 80.6% for the 6 trainees who did not pass. Peer coaches who completed the study wanted to continue peer coaching work and had confidence in their abilities despite their not consistently employing the coaching techniques with their patients. Quotations describe coaches' perceptions of the training.ConclusionsOf low-income patients with diabetes who completed the evidenced-based health coaching training, 81% passed written and oral exams and became effective peer health coaches, although they did not consistently use the techniques taught.

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and/or pregnancy complications. β2-glycoprotein I (β2GPI) complexed with phospholipid is recognized as a major target for autoantibodies in APS; however, less than half the patients with clinical manifestations of APS possess autoantibodies against the complexes. Therefore, the range of autoantigens involved in APS remains unclear. Recently, we found that human leukocyte antigen (HLA) class II molecules transport misfolded cellular proteins to the cell surface via association with their peptide-binding grooves. Furthermore, immunoglobulin G heavy chain/HLA class II complexes were specific targets for autoantibodies in rheumatoid arthritis. Here, we demonstrate that intact β2GPI, not peptide, forms a complex with HLA class II molecules. Strikingly, 100 (83.3%) of the 120 APS patients analyzed, including those whose antiphospholipid antibody titers were within normal range, possessed autoantibodies that recognize β2GPI/HLA class II complexes in the absence of phospholipids. In situ association between β2GPI and HLA class II was observed in placental tissues of APS patients but not in healthy controls. Furthermore, autoantibodies against β2GPI/HLA class II complexes mediated complement-dependent cytotoxicity against cells expressing the complexes. These data suggest that β2GPI/HLA class II complexes are a target in APS that might be involved in the pathogenesis.