A long-read RNA-seq approach to identify novel transcripts of very large genes.

RNA-seq is widely used for studying gene expression, but commonly used sequencing platforms produce short reads that only span up to two exon junctions per read. This makes it difficult to accurately determine the composition and phasing of exons within transcripts. Although long-read sequencing improves this issue, it is not amenable to precise quantitation, which limits its utility for differential expression studies. We used long-read isoform sequencing combined with a novel analysis approach to compare alternative splicing of large, repetitive structural genes in muscles. Analysis of muscle structural genes that produce medium (Nrap: 5 kb), large (Neb: 22 kb), and very large (Ttn: 106 kb) transcripts in cardiac muscle, and fast and slow skeletal muscles identified unannotated exons for each of these ubiquitous muscle genes. This also identified differential exon usage and phasing for these genes between the different muscle types. By mapping the in-phase transcript structures to known annotations, we also identified and quantified previously unannotated transcripts. Results were confirmed by endpoint PCR and Sanger sequencing, which revealed muscle-type-specific differential expression of these novel transcripts. The improved transcript identification and quantification shown by our approach removes previous impediments to studies aimed at quantitative differential expression of ultralong transcripts.

Increased diagnostic yield from negative whole genome-slice panels using automated reanalysis.

We evaluated the diagnostic yield using genome-slice panel reanalysis in the clinical setting using an automated phenotype/gene ranking system. We analyzed whole genome sequencing (WGS) data produced from clinically ordered panels built as bioinformatic slices for 16 clinically diverse, undiagnosed cases referred to the Pediatric Mendelian Genomics Research Center, an NHGRI-funded GREGoR Consortium site. Genome-wide reanalysis was performed using Moon™, a machine-learning-based tool for variant prioritization. In five out of 16 cases, we discovered a potentially clinically significant variant. In four of these cases, the variant was found in a gene not included in the original panel due to phenotypic expansion of a disorder or incomplete initial phenotyping of the patient. In the fifth case, the gene containing the variant was included in the original panel, but being a complex structural rearrangement with intronic breakpoints outside the clinically analyzed regions, it was not initially identified. Automated genome-wide reanalysis of clinical WGS data generated during targeted panels testing yielded a 25% increase in diagnostic findings and a possibly clinically relevant finding in one additional case, underscoring the added value of analyses versus those routinely performed in the clinical setting.

Vamorolone, a dissociative steroidal compound, reduces collagen antibody-induced joint damage and inflammation when administered after disease onset.

OBJECTIVE AND DESIGN: The objective of this study was to assess the effect of vamorolone, a first-in-class dissociative steroidal compound, to inhibit inflammation when administered after disease onset in the murine collagen antibody-induced arthritis model of arthritis. ANIMALS: 84 DBA1/J mice were used in this study (n = 12 per treatment group). TREATMENT: Vamorolone or prednisolone was administered orally after disease onset for a duration of 7 days. METHODS: Disease score and bone erosion were assessed using previously described scoring systems. Cytokines were measured in joints via immunoassay, and joint cathepsin B activity (marker of inflammation) was assessed using optical imaging of joints on live mice. RESULTS: We found that vamorolone treatment led to a reduction of several disease parameters including disease score, joint inflammation, and the presence of pro-inflammatory mediators to a degree similar of that observed with prednisolone treatment. More importantly, histopathological analysis of affected joints showed that vamorolone treatment significantly reduced the degree of bone erosion while this bone-sparing property was not observed with prednisolone treatment at any of the tested doses. CONCLUSIONS: While many intervention regimens in other studies are administered prior to disease onset in animal models, the current study involves delivery of the potential therapeutic after disease onset. Based on the findings, vamorolone may offer an efficacious, yet safer alternative to conventional steroidal compounds in the treatment of rheumatoid arthritis and other inflammatory diseases.

Extracellular microRNA signature in chronic kidney disease.

MicroRNAs (miRNAs) are noncoding RNAs that regulate posttranscriptional gene expression. In this study we characterized the circulating and urinary miRNA pattern associated with reduced glomerular filtration rate, using Affymetrix GeneChip miR 4.0 in 28 patients with chronic kidney disease (CKD). Top miRNA discoveries from the human studies were validated in an Alb/TGFß mouse model of CKD, and in rat renal proximal tubular cells (NRK52E) exposed to TGFß1. Plasma and urinary levels of procollagen III N-terminal propeptide and collagen IV were elevated in patients with decreased estimated glomerular filtration rate (eGFR). Expression of 384 urinary and 266 circulatory miRNAs were significantly different between CKD patients with eGFR ≥30 vs. <30 ml·min-1·1.73 m-2 Pathway analysis mapped multiple miRNAs to TGFß signaling-related mRNA targets. Specifically, Let-7a was significantly downregulated, and miR-130a was significantly upregulated, in urine of patients with eGFR <30; miR-1825 and miR-1281 were upregulated in both urine and plasma of patients with decreased eGFR; and miR-423 was significantly downregulated in plasma of patients with decreased eGFR. miRNA expression in urine and plasma of Alb/TGFß mice generally resembled and confirmed most, although not all, of the observations from the human studies. In response to TGFß1 exposure, rat renal proximal tubular cells overexpressed miR-1825 and downregulated miR-423. Thus, miRNA are associated with kidney fibrosis, and specific urinary and plasma miRNA profile may have diagnostic and prognostic utility in CKD.

Early postnatal exposure to intermittent hypoxia in rodents is proinflammatory, impairs white matter integrity, and alters brain metabolism.

BackgroundPreterm infants are frequently exposed to intermittent hypoxia (IH) associated with apnea and periodic breathing that may result in inflammation and brain injury that later manifests as cognitive and executive function deficits. We used a rodent model to determine whether early postnatal exposure to IH would result in inflammation and brain injury.MethodsRat pups were exposed to IH from P2 to P12. Control animals were exposed to room air. Cytokines were analyzed in plasma and brain tissue at P13 and P18. At P20-P22, diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) were performed.ResultsPups exposed to IH had increased plasma Gro/CXCL1 and cerebellar IFN-γ and IL-1ß at P13, and brainstem enolase at P18. DTI showed a decrease in FA and AD in the corpus callosum (CC) and cingulate gyrus, and an increase in RD in the CC. MRS revealed decreases in NAA/Cho, Cr, Tau/Cr, and Gly/Cr; increases in TCho and GPC in the brainstem; and decreases in NAA/Cho in the hippocampus.ConclusionsWe conclude that early postnatal exposure to IH, similar in magnitude to that experienced in human preterm infants, is associated with evidence for proinflammatory changes, decreases in white matter integrity, and metabolic changes consistent with hypoxia.

Association of brain injury and neonatal cytokine response during therapeutic hypothermia in newborns with hypoxic-ischemic encephalopathy.

BACKGROUND: Cytokines have been proposed as mediators of neonatal brain injury via neuroinflammatory pathways triggered by hypoxia-ischemia. Limited data are available on cytokine profiles in larger cohorts of newborns with hypoxic-ischemic encephalopathy (HIE) undergoing therapeutic hypothermia (TH). METHODS: Serum cytokines interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, tumor necrosis factor-α, and interferon-γ were measured in newborns with HIE at 24 and 72 h of TH. Differences between infants with favorable (survivors with mild/no magnetic resonance imaging (MRI) injury) vs. adverse outcome (death or moderate/severe MRI injury) were compared using mixed models to adjust for covariates. RESULTS: Data from 36 term newborns with HIE (favorable outcome: n = 20, adverse outcome: n = 16) were evaluated. Cytokines IL-1ß, IL-2, IL-6, IL-8, IL-10, and IL-13 were elevated in the adverse relative to favorable outcome group at 24 h. IL-6 remained significantly elevated in the adverse outcome group at 72 h. IL-6 and IL-10 remained significantly associated with outcome group after controlling for covariates. CONCLUSION: Inflammatory cytokines are elevated in HIE newborns with brain injury by MRI. In particular, IL-6 and IL-10 were associated with adverse outcomes after controlling for baseline characteristics and severity of presentation. These data suggest that cytokine response may identify infants in need of additional neuroprotective interventions.

Circulating and urinary microRNA profile in focal segmental glomerulosclerosis: a pilot study.

BACKGROUND: MicroRNAs (miRNAs) are noncoding RNA molecules that play important roles in the pathogenesis of various kidney diseases. We investigated whether patients with minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) have distinct circulating and urinary miRNA expression profiles that could lead to potential development of noninvasive biomarkers of the disease. MATERIALS AND METHODS: Exosome miRNAs were extracted from plasma and urine samples of patients with primary FSGS (n = 16) or MCD (n = 5) and healthy controls (n = 5). Differences in miRNA abundance were examined using Affymetrix GeneChip miRNA 3.0 arrays. QRT-PCR was used to validate the findings from the array. RESULTS: Comparison analysis of FSGS versus MCD revealed 126 and 155 differentially expressed miRNAs in plasma and in urine, respectively. Only 38 of these miRNAs were previously cited, whereas the remaining miRNAs have not been described. Comparison analysis showed that a significant number of miRNAs were downregulated in both plasma and urine samples of patients with FSGS compared to those with MCD. Plasma levels of miR-30b, miR-30c, miR-34b, miR-34c and miR-342 and urine levels of mir-1225-5p were upregulated in patients with MCD compared to patients with FSGS and controls (P < 0.001). Urinary levels of mir-1915 and miR-663 were downregulated in patients with FSGS compared to MCD and controls (P < 0.001), whereas the urinary levels of miR-155 were upregulated in patients with FSGS when compared to patients with MCD and controls (P < 0.005). CONCLUSIONS: Patients with FSGS and MCD have a unique circulating and urinary miRNA profile. The diagnostic and prognostic potential of miRNAs in FSGS and MCD warrants further studies.

VBP15, a novel anti-inflammatory, is effective at reducing the severity of murine experimental autoimmune encephalomyelitis.

Multiple sclerosis is a chronic disease of the central nervous system characterized by an autoimmune inflammatory reaction that leads to axonal demyelination and tissue damage. Glucocorticoids, such as prednisolone, are effective in the treatment of multiple sclerosis in large part due to their ability to inhibit pro-inflammatory pathways (e.g., NFκB). However, despite their effectiveness, long-term treatment is limited by adverse side effects. VBP15 is a recently described compound synthesized based on the lazeroid steroidal backbone that shows activity in acute and chronic inflammatory conditions, yet displays a much-reduced side effect profile compared to traditional glucocorticoids. The purpose of this study was to determine the effectiveness of VBP15 in inhibiting inflammation and disease progression in experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of multiple sclerosis. Our data show that VBP15 is effective at reducing both disease onset and severity. In parallel studies, we observed that VBP15 was able to inhibit the production of NFκB-regulated pro-inflammatory transcripts in human macrophages. Furthermore, treatment with prednisolone-but not VBP15-increased expression of genes associated with bone loss and muscle atrophy, suggesting lack of side effects of VBP15. These findings suggest that VBP15 may represent a potentially safer alternative to traditional glucocorticoids in the treatment of multiple sclerosis and other inflammatory diseases.

Change in neuroplasticity-related proteins in response to acute activity-based therapy in persons with spinal cord injury.

BACKGROUND: Activity-based therapy (ABT) focuses on regaining motor and sensory function below the level of the lesion in persons with a spinal cord injury (SCI). This is accomplished through repetitive training of specific motor tasks. Research has shown that ABT may increase neuroplasticity in the rat and human spinal cord. OBJECTIVE: The primary aim of this study was to examine acute alterations in neuroplasticity-related proteins during ABT in persons with SCI. METHODS: Volunteers were current participants in an ABT program and consisted of 12 men and 3 women (age, 31.8 ± 10.9 years) with chronic SCI (injury duration, 63.9 ± 54.4 months). A single 2-hour bout of ABT consisted of standing load bearing, body weight-supported treadmill training, whole body vibration, and functional electrical stimulation. Blood samples were obtained at baseline and immediately after completion of each modality to determine serum levels of brain-derived neurotrophic factor (BDNF), prolactin, and cortisol. RESULTS: One-way analysis of variance (ANOVA) with repeated measures was used to examine differences in proteins over time. Results revealed baseline levels of BDNF (2.37 ± 1.41 ng/mL) that were lower than previous research has demonstrated in persons with SCI. No change in BDNF or cortisol was found, although prolactin was significantly reduced in response to ABT. CONCLUSION: Despite the length of the bout, acute changes in BDNF were not observed. Whether different intensities or modalities of ABT may promote acute increases in serum BDNF in individuals with SCI remains to be determined and further study is merited.

SRY-box containing gene 17 regulates the Wnt/ß-catenin signaling pathway in oligodendrocyte progenitor cells.

The SRY-box (Sox) transcription factors regulate oligodendrocyte differentiation, but their signaling targets are largely unknown. We have identified a major signal transduction pathway regulated by Sox containing gene 17 (Sox17) in the oligodendrocyte lineage. Microarray analysis in oligodendrocyte progenitor cells (OPCs) after Sox17 attenuation revealed upregulated genes associated with cell cycle control and activation of the Wingless and integration site (Wnt)/ß-catenin pathway. Sox17 knockdown also increases the levels of cyclin D1, Axin2, and activated ß-catenin. In OPCs, the expression pattern of Sox17, cyclin D1, and secreted Frizzled-related protein-1 in the presence of platelet-derived growth factor (PDGF) was coordinately accelerated by addition of thyroid hormone, indicating differentiation-induced regulation of Sox17 targets. In developing white matter, decreased total ß-catenin, activated ß-catenin, and cyclin D1 levels coincided with the peak of Sox17 expression, and immunoprecipitates showed a developmentally regulated interaction among Sox17, T-cell transcription factor 4, and ß-catenin proteins. In OPCs, PDGF stimulated phosphorylation of glycogen synthase 3ß and the Wnt coreceptor LRP6, and enhanced ß-catenin-dependent gene expression. Sox17 overexpression inhibited PDGF-induced TOPFLASH and cyclin D1 promoter activity, and decreased endogenous cyclin D1, activated ß-catenin, as well as total ß-catenin levels. Recombinant Sox17 prevented Wnt3a from repressing myelin protein expression, and inhibition of Sox17-mediated proteasomal degradation of ß-catenin blocked myelin protein induction. These results indicate that Sox17 suppresses cyclin D1 expression and cell proliferation by directly antagonizing ß-catenin, whose activity in OPCs is stimulated not only by Wnt3a, but also by PDGF. Our identification of downstream targets of Sox17 thus defines signaling pathways and molecular mechanisms in OPCs that are regulated by Sox17 during cell cycle exit and the onset of differentiation in oligodendrocyte development.

Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury.

BACKGROUND: Clinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI). Asymptomatic bacteriuria (ABU) represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality. In this study, we describe key characteristics of the healthy and ABU urine microbiomes utilizing 16S rRNA gene (16S rDNA) sequencing and metaproteomics, with the future goal of utilizing this information to personalize the treatment of UTI based on key individual characteristics. METHODS: A cross-sectional study of 26 healthy controls and 27 healthy subjects at risk for ABU due to spinal cord injury-related neuropathic bladder (NB) was conducted. Of the 27 subjects with NB, 8 voided normally, 8 utilized intermittent catheterization, and 11 utilized indwelling Foley urethral catheterization for bladder drainage. Urine was obtained by clean catch in voiders, or directly from the catheter in subjects utilizing catheters. Urinalysis, urine culture and 16S rDNA sequencing were performed on all samples, with metaproteomic analysis performed on a subsample. RESULTS: A total of 589454 quality-filtered 16S rDNA sequence reads were processed through a NextGen 16S rDNA analysis pipeline. Urine microbiomes differ by normal bladder function vs. NB, gender, type of bladder catheter utilized, and duration of NB. The top ten bacterial taxa showing the most relative abundance and change among samples were Lactobacillales, Enterobacteriales, Actinomycetales, Bacillales, Clostridiales, Bacteroidales, Burkholderiales, Pseudomonadales, Bifidobacteriales and Coriobacteriales. Metaproteomics confirmed the 16S rDNA results, and functional human protein-pathogen interactions were noted in subjects where host defenses were initiated. CONCLUSIONS: Counter to clinical belief, healthy urine is not sterile. The healthy urine microbiome is characterized by a preponderance of Lactobacillales in women and Corynebacterium in men. The presence and duration of NB and method of urinary catheterization alter the healthy urine microbiome. An integrated approach of 16S rDNA sequencing with metaproteomics improves our understanding of healthy urine and facilitates a more personalized approach to prevention and treatment of infection.

Muscle cells of sporadic amyotrophic lateral sclerosis patients secrete neurotoxic vesicles.

BACKGROUND: The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle-secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. METHODS: Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS-R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged-matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT-qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human-derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. RESULTS: An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy-derived denervation-naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome-like vesicles and disruption of RNA-processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA-processing proteins. The RNA-processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti-CD63 immuno-blocking of vesicle uptake. CONCLUSIONS: ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle-mediated toxicity in ALS.

Delayed inflammatory mRNA and protein expression after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) induces secondary tissue damage that is associated with inflammation. We have previously demonstrated that inflammation-related gene expression after SCI occurs in two waves - an initial cluster that is acutely and transiently up-regulated within 24 hours, and a more delayed cluster that peaks between 72 hours and 7 days. Here we extend the microarray analysis of these gene clusters up to 6 months post-SCI. METHODS: Adult male rats were subjected to mild, moderate or severe spinal cord contusion injury at T9 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 hours, 24 hours, 7 days, 28 days, 3 months or 6 months post-injury and processed for microarray analysis and protein expression. RESULTS: Anchor gene analysis using C1qB revealed a cluster of genes that showed elevated expression through 6 months post-injury, including galectin-3, p22PHOX, gp91PHOX, CD53 and progranulin. The expression of these genes occurred primarily in microglia/macrophage cells and was confirmed at the protein level using both immunohistochemistry and western blotting. As p22PHOX and gp91PHOX are components of the NADPH oxidase enzyme, enzymatic activity and its role in SCI were assessed and NADPH oxidase activity was found to be significantly up-regulated through 6 months post-injury. Further, treating rats with the nonspecific, irreversible NADPH oxidase inhibitor diphenylene iodinium (DPI) reduced both lesion volume and expression of chronic gene cluster proteins one month after trauma. CONCLUSIONS: These data demonstrate that inflammation-related genes are chronically up-regulated after SCI and may contribute to further tissue loss.

Traumatic Brain Injury Induces cGAS Activation and Type I Interferon Signaling in Aged Mice.

Aging adversely affects inflammatory processes in the brain, which has important implications in the progression of neurodegenerative disease. Following traumatic brain injury (TBI), aged animals exhibit worsened neurological function and exacerbated microglial-associated neuroinflammation. Type I Interferons (IFN-I) contribute to the development of TBI neuropathology. Further, the Cyclic GMP-AMP Synthase (cGAS) and Stimulator of Interferon Genes (STING) pathway, a key inducer of IFN-I responses, has been implicated in neuroinflammatory activity in several age-related neurodegenerative diseases. Here, we set out to investigate the effects of TBI on cGAS/STING activation, IFN-I signaling and neuroinflammation in young and aged C57Bl/6 male mice. Using a controlled cortical impact model, we evaluated transcriptomic changes in the injured cortex at 24 hours post-injury, and confirmed activation of key neuroinflammatory pathways in biochemical studies. TBI induced changes were highly enriched for transcripts that were involved in inflammatory responses to stress and host defense. Deeper analysis revealed that TBI increased expression of IFN-I related genes (e.g. Ifnb1, Irf7, Ifi204, Isg15) and IFN-I signaling in the injured cortex of aged compared to young mice. There was also a significant age-related increase in the activation of the DNA-recognition pathway, cGAS, which is a key mechanism to propagate IFN-I responses. Finally, enhanced IFN-I signaling in the aged TBI brain was confirmed by increased phosphorylation of STAT1, an important IFN-I effector molecule. This age-related activation of cGAS and IFN-I signaling may prove to be a mechanistic link between microglial-associated neuroinflammation and neurodegeneration in the aged TBI brain.

Epidermal Growth Factor Receptor Inhibition Reverses Cellular and Transcriptomic Alterations Induced by Hypoxia in the Neonatal Piglet Brain.

Acute hypoxia (HX) causes extensive cellular damage in the developing human cerebral cortex. We found increased expression of activated-EGFR in affected cortical areas of neonates with HX and investigated its functional role in the piglet, which displays a highly evolved, gyrencephalic brain, with a human-like maturation pattern. In the piglet, HX-induced activation of EGFR and Ca2+/calmodulin kinase IV (CaMKIV) caused cell death and pathological alterations in neurons and glia. EGFR blockade inhibited CaMKIV activation, attenuated neuronal loss, increased oligodendrocyte proliferation, and reversed HX-induced astrogliosis. We performed for the first time high-throughput transcriptomic analysis of the piglet cortex to define molecular responses to HX and to uncover genes specifically involved in EGFR signaling in piglet and human brain injury. Our results indicate that specific molecular responses modulated by EGFR may be targeted as a therapeutic strategy for HX injury in the neonatal brain.

High-Intensity Focused Ultrasound (HIFU) Triggers Immune Sensitization of Refractory Murine Neuroblastoma to Checkpoint Inhibitor Therapy.

PURPOSE: Immunotherapy promises unprecedented benefits to patients with cancer. However, the majority of cancer types, including high-risk neuroblastoma, remain immunologically unresponsive. High-intensity focused ultrasound (HIFU) is a noninvasive technique that can mechanically fractionate tumors, transforming immunologically "cold" tumors into responsive "hot" tumors. EXPERIMENTAL DESIGN: We treated <2% of tumor volume in previously unresponsive, large, refractory murine neuroblastoma tumors with mechanical HIFU and assessed systemic immune response using flow cytometry, ELISA, and gene sequencing. In addition, we combined this treatment with αCTLA-4 and αPD-L1 to study its effect on the immune response and long-term survival. RESULTS: Combining HIFU with αCTLA-4 and αPD-L1 significantly enhances antitumor response, improving survival from 0% to 62.5%. HIFU alone causes upregulation of splenic and lymph node NK cells and circulating IL2, IFNγ, and DAMPs, whereas immune regulators like CD4+Foxp3+, IL10, and VEGF-A are significantly reduced. HIFU combined with checkpoint inhibitors induced significant increases in intratumoral CD4+, CD8α+, and CD8α+CD11c+ cells, CD11c+ in regional lymph nodes, and decrease in circulating IL10 compared with untreated group. We also report significant abscopal effect following unilateral treatment of mice with large, established bilateral tumors using HIFU and checkpoint inhibitors compared with tumors treated with HIFU or checkpoint inhibitors alone (61.1% survival, P < 0.0001). This combination treatment significantly also induces CD4+CD44+hiCD62L+low and CD8α+CD44+hiCD62L+low population and is adoptively transferable, imparting immunity, slowing subsequent de novo tumor engraftment. CONCLUSIONS: Mechanical fractionation of tumors using HIFU can effectively induce immune sensitization in a previously unresponsive murine neuroblastoma model and promises a novel yet efficacious immunoadjuvant modality to overcome therapeutic resistance.

Dysferlin deficiency shows compensatory induction of Rab27A/Slp2a that may contribute to inflammatory onset.

Mutations in the dysferlin gene cause limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi myopathy. Dysferlin-deficient cells show abnormalities in vesicular traffic and membrane repair although onset of symptoms is not commonly seen until the late teenage years and is often associated with subacute onset and marked muscle inflammation. To identify molecular networks specific to dysferlin-deficient muscle that might explain disease pathogenesis, muscle mRNA profiles from 10 mutation-positive LGMD2B/MM patients were compared with a disease control [LGMD2I; (n = 9)], and normal muscle samples (n = 11). Query of inflammatory pathways suggested LGMD2B-specific increases in co-stimulatory signaling between dendritic cells and T cells (CD86, CD28, and CTLA4), associated with localized expression of both versican and tenascin. LGMD2B muscle also showed an increase in vesicular trafficking pathway proteins not normally observed in muscle (synaptotagmin-like protein Slp2a/SYTL2 and the small GTPase Rab27A). We propose that Rab27A/Slp2a expression in LGMD2B muscle provides a compensatory vesicular trafficking pathway that is able to repair membrane damage in the absence of dysferlin. However, this same pathway may release endocytotic vesicle contents, resulting in an inflammatory microenvironment. As dysferlin deficiency has been shown to enhance phagocytosis by macrophages, together with our findings of abnormal myofiber endocytosis pathways and dendritic-T cell activation markers, these results suggest a model of immune and inflammatory network over-stimulation that may explain the subacute inflammatory presentation.

GeneShelf: a web-based visual interface for large gene expression time-series data repositories.

A widespread use of high-throughput gene expression analysis techniques enabled the biomedical research community to share a huge body of gene expression datasets in many public databases on the web. However, current gene expression data repositories provide static representations of the data and support limited interactions. This hinders biologists from effectively exploring shared gene expression datasets. Responding to the growing need for better interfaces to improve the utility of the public datasets, we have designed and developed a new web-based visual interface entitled GeneShelf (http://bioinformatics.cnmcresearch.org/GeneShelf). It builds upon a zoomable grid display to represent two categorical dimensions. It also incorporates an augmented timeline with expandable time points that better shows multiple data values for the focused time point by embedding bar charts. We applied GeneShelf to one of the largest microarray datasets generated to study the progression and recovery process of injuries at the spinal cord of mice and rats. We present a case study and a preliminary qualitative user study with biologists to show the utility and usability of GeneShelf.

PAC1R Genotype to Phenotype Correlations in Autism Spectrum Disorder.

Amygdala dysfunction has been implicated in numerous neurodevelopmental disorders, including autism spectrum disorder (ASD). Previous studies in mice and humans, respectively, have linked Pac1r/PAC1R function to social behavior and PTSD-susceptibility. Based on this connection to social and emotional processing and the central role played by the amygdala in ASD, we examined a putative role for PAC1R in social deficits in ASD and determined the pattern of gene expression in the developing mouse and human amygdala. We reveal that Pac1r/PAC1R is expressed in both mouse and human amygdala from mid-neurogenesis through early postnatal stages, critical time points when altered brain trajectories are hypothesized to unfold in ASD. We further find that parents of autistic children carrying a previously identified PTSD-risk genotype (CC) report greater reciprocal social deficits compared to those carrying the non-risk GC genotype. Additionally, by exploring resting-state functional connectivity differences in a subsample of the larger behavioral sample, we find higher functional connectivity between the amygdala and right middle temporal gyrus in individuals with the CC risk genotype. Thus, using multimodal approaches, our data reveal that the amygdala-expressed PAC1R gene may be linked to severity of ASD social phenotype and possible alterations in brain connectivity, therefore potentially acting as a modifier of amygdala-related phenotypes. Autism Res 2019, 12: 200-211 © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: In this multimodal study across mouse and human, we examined expression patterns of Pac1r/PAC1R, a gene implicated in social behavior, and further explored whether a previously identified human PTSD-linked mutation in PAC1R can predict brain connectivity and social deficits in ASD. We find that PAC1R is highly expressed in the both the mouse and human amygdala. Furthermore, our human data suggest that PAC1R genotype is linked to severity of social deficits and functional amygdala connectivity in ASD.

Dystrophin-deficient cardiomyopathy in mouse: expression of Nox4 and Lox are associated with fibrosis and altered functional parameters in the heart.

Duchenne muscular dystrophy (DMD; dystrophin-deficiency) causes dilated cardiomyopathy in the second decade of life in affected males. We studied the dystrophin-deficient mouse heart (mdx) using high-frequency echocardiography, histomorphometry, and gene expression profiling. Heart dysfunction was prominent at 9-10months of age and showed significantly increased LV internal diameter (end systole) and decreased posterior wall thickness. This cardiomyopathy was associated with a 30% decrease in shortening fraction. Histologically, there was a 10-fold increase in connective tissue volume (fibrosis). mRNA profiling with RT-PCR validation showed activation of key pro-fibrotic genes, including Nox4 and Lox. The Nox gene family expression differed in mdx heart and skeletal muscle, where Nox2 was specifically induced in skeletal muscle while Nox4 was specifically induced in heart. This is the first report of an altered profibrotic gene expression profile in cardiac tissue of dystrophic mice showing echocardiographic evidence of cardiomyopathy.