Germline predisposition to pediatric Ewing sarcoma is characterized by inherited pathogenic variants in DNA damage repair genes.

More knowledge is needed regarding germline predisposition to Ewing sarcoma to inform biological investigation and clinical practice. Here, we evaluated the enrichment of pathogenic germline variants in Ewing sarcoma relative to other pediatric sarcoma subtypes, as well as patterns of inheritance of these variants. We carried out European-focused and pan-ancestry case-control analyses to screen for enrichment of pathogenic germline variants in 141 established cancer predisposition genes in 1,147 individuals with pediatric sarcoma diagnoses (226 Ewing sarcoma, 438 osteosarcoma, 180 rhabdomyosarcoma, and 303 other sarcoma) relative to identically processed cancer-free control individuals. Findings in Ewing sarcoma were validated with an additional cohort of 430 individuals, and a subset of 301 Ewing sarcoma parent-proband trios was analyzed for inheritance patterns of identified pathogenic variants. A distinct pattern of pathogenic germline variants was seen in Ewing sarcoma relative to other sarcoma subtypes. FANCC was the only gene with an enrichment signal for heterozygous pathogenic variants in the European Ewing sarcoma discovery cohort (three individuals, OR 12.6, 95% CI 3.0-43.2, p = 0.003, FDR = 0.40). This enrichment in FANCC heterozygous pathogenic variants was again observed in the European Ewing sarcoma validation cohort (three individuals, OR 7.0, 95% CI 1.7-23.6, p = 0.014), representing a broader importance of genes involved in DNA damage repair, which were also nominally enriched in individuals with Ewing sarcoma. Pathogenic variants in DNA damage repair genes were acquired through autosomal inheritance. Our study provides new insight into germline risk factors contributing to Ewing sarcoma pathogenesis.

SON and Its Alternatively Spliced Isoforms Control MLL Complex-Mediated H3K4me3 and Transcription of Leukemia-Associated Genes.

Dysregulation of MLL complex-mediated histone methylation plays a pivotal role in gene expression associated with diseases, but little is known about cellular factors modulating MLL complex activity. Here, we report that SON, previously known as an RNA splicing factor, controls MLL complex-mediated transcriptional initiation. SON binds to DNA near transcription start sites, interacts with menin, and inhibits MLL complex assembly, resulting in decreased H3K4me3 and transcriptional repression. Importantly, alternatively spliced short isoforms of SON are markedly upregulated in acute myeloid leukemia. The short isoforms compete with full-length SON for chromatin occupancy but lack the menin-binding ability, thereby antagonizing full-length SON function in transcriptional repression while not impairing full-length SON-mediated RNA splicing. Furthermore, overexpression of a short isoform of SON enhances replating potential of hematopoietic progenitors. Our findings define SON as a fine-tuner of the MLL-menin interaction and reveal short SON overexpression as a marker indicating aberrant transcriptional initiation in leukemia.

Nuclear focal adhesion kinase induces APC/C activator protein CDH1-mediated cyclin-dependent kinase 4/6 degradation and inhibits melanoma proliferation.

Dysregulation of cyclin-dependent kinases (CDKs) can promote unchecked cell proliferation and cancer progression. Although focal adhesion kinase (FAK) contributes to regulating cell cycle progression, the exact molecular mechanism remains unclear. Here, we found that FAK plays a key role in cell cycle progression potentially through regulation of CDK4/6 protein expression. We show that FAK inhibition increased its nuclear localization and induced G1 arrest in B16F10 melanoma cells. Mechanistically, we demonstrate nuclear FAK associated with CDK4/6 and promoted their ubiquitination and proteasomal degradation through recruitment of CDC homolog 1 (CDH1), an activator and substrate recognition subunit of the anaphase-promoting complex/cyclosome E3 ligase complex. We found the FAK N-terminal FERM domain acts as a scaffold to bring CDK4/6 and CDH1 within close proximity. However, overexpression of nonnuclear-localizing mutant FAK FERM failed to function as a scaffold for CDK4/6 and CDH1. Furthermore, shRNA knockdown of CDH1 increased CDK4/6 protein expression and blocked FAK inhibitor-induced reduction of CDK4/6 in B16F10 cells. In vivo, we show that pharmacological FAK inhibition reduced B16F10 tumor size, correlating with increased FAK nuclear localization and decreased CDK4/6 expression compared with vehicle controls. In patient-matched healthy skin and melanoma biopsies, we found FAK was mostly inactive and nuclear localized in healthy skin, whereas melanoma lesions showed increased active cytoplasmic FAK and elevated CDK4 expression. Taken together, our data demonstrate that FAK inhibition blocks tumor proliferation by inducing G1 arrest, in part through decreased CDK4/6 protein stability by nuclear FAK.

FAK Activation Promotes SMC Dedifferentiation via Increased DNA Methylation in Contractile Genes.

RATIONALE: Vascular smooth muscle cells (SMCs) exhibit remarkable plasticity and can undergo dedifferentiation upon pathological stimuli associated with disease and interventions. OBJECTIVE: Although epigenetic changes are critical in SMC phenotype switching, a fundamental regulator that governs the epigenetic machineries regulating the fate of SMC phenotype has not been elucidated. METHODS AND RESULTS: Using SMCs, mouse models, and human atherosclerosis specimens, we found that FAK (focal adhesion kinase) activation elicits SMC dedifferentiation by stabilizing DNMT3A (DNA methyltransferase 3A). FAK in SMCs is activated in the cytoplasm upon serum stimulation in vitro or vessel injury and active FAK prevents DNMT3A from nuclear FAK-mediated degradation. However, pharmacological or genetic FAK catalytic inhibition forced FAK nuclear localization, which reduced DNMT3A protein via enhanced ubiquitination and proteasomal degradation. Reduced DNMT3A protein led to DNA hypomethylation in contractile gene promoters, which increased SMC contractile protein expression. RNA-sequencing identified SMC contractile genes as a foremost upregulated group by FAK inhibition from injured femoral artery samples compared with vehicle group. DNMT3A knockdown in injured arteries reduced DNA methylation and enhanced contractile gene expression supports the notion that nuclear FAK-mediated DNMT3A degradation via E3 ligase TRAF6 (TNF [tumor necrosis factor] receptor-associated factor 6) drives differentiation of SMCs. Furthermore, we observed that SMCs of human atherosclerotic lesions exhibited decreased nuclear FAK, which was associated with increased DNMT3A levels and decreased contractile gene expression. CONCLUSIONS: This study reveals that nuclear FAK induced by FAK catalytic inhibition specifically suppresses DNMT3A expression in injured vessels resulting in maintaining SMC differentiation by promoting the contractile gene expression. Thus, FAK inhibitors may provide a new treatment option to block SMC phenotypic switching during vascular remodeling and atherosclerosis.

Cluster of Carbapenemase-Producing Carbapenem-Resistant Pseudomonas aeruginosa Among Patients in an Adult Intensive Care Unit - Idaho, 2021-2022.

Treatment of carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa (CP-CRPA) infections is challenging because of antibiotic resistance. CP-CRPA infections are highly transmissible in health care settings because they can spread from person to person and from environmental sources such as sink drains and toilets. During September 2021-January 2022, an Idaho hospital (hospital A) isolated CP-CRPA from sputum of two patients who stayed in the same intensive care unit (ICU) room (room X), 4 months apart. Both isolates had active-on-imipenem metallo-beta-lactamase (IMP) carbapenemase gene type 84 (blaIMP-84) and were characterized as multilocus sequence type 235 (ST235). A health care-associated infections team from the Idaho Division of Public Health visited hospital A during March 21-22, 2022, to discuss the cluster investigation with hospital A staff members and to collect environmental samples. CP-CRPA ST235 with blaIMP-84 was isolated from swab samples of one sink in room X, suggesting it was the likely environmental source of transmission. Recommended prevention and control measures included application of drain biofilm disinfectant, screening of future patients who stay in room X (e.g., the next 10 occupants) upon reopening, and continuing submission of carbapenem-resistant P. aeruginosa (CRPA) isolates to public health laboratories. Repeat environmental sampling did not detect any CRPA. As of December 2022, no additional CP-CRPA isolates had been reported by hospital A. Collaboration between health care facilities and public health agencies, including testing of CRPA isolates for carbapenemase genes and implementation of sink hygiene interventions, was critical in the identification of and response to this CP-CRPA cluster in a health care setting.

Low-dose naltrexone's utility for non-cancer centralized pain conditions: a scoping review.

BACKGROUND: At low doses, naltrexone (LDN) has been shown to modulate inflammation through the interruption of microglial cell activation within the central nervous system. One of the most likely contributors to centralized pain is changes in microglial cell processing. Therefore, it has been postulated that LDN can be used to manage patients with pain resulting from central sensitization due to this relationship. This scoping review aims to synthesize the relevant study data for LDN as a novel treatment strategy for various centralized pain conditions. METHODS: A comprehensive literature search was conducted in PubMed, Embase, and Google Scholar, guided by the Scale for Assessment of Narrative Review Articles (SANRA) criteria. RESULTS: Forty-seven studies related to centralized pain conditions were identified. Many of the studies were case reports/series and narrative reviews, but a few randomized control trials have been conducted. Overall, the body of evidence revealed improvement in patient-reported pain severity and in outcomes related to hyperalgesia, physical function, quality of life, and sleep. Variability in dosing paradigms and the time to patient response was present in the reviewed studies. CONCLUSIONS: Evidence synthesized for this scoping review supports the ongoing use of LDN for the treatment of refractory pain in various centralized chronic pain conditions. Upon review of the currently available published studies, it is apparent that further high-quality, well-powered randomized control trials need to be conducted to establish efficacy, standardization for dosing, and response times. In summary, LDN continues to offer promising results in the management of pain and other distressing symptoms in patients with chronic centralized pain conditions.

Genetic basis of thermal nociceptive sensitivity and brain weight in a BALB/c reduced complexity cross.

Thermal nociception involves the transmission of temperature-related noxious information from the periphery to the CNS and is a heritable trait that could predict transition to persistent pain. Rodent forward genetics complement human studies by controlling genetic complexity and environmental factors, analysis of end point tissue, and validation of variants on appropriate genetic backgrounds. Reduced complexity crosses between nearly identical inbred substrains with robust trait differences can greatly facilitate unbiased discovery of novel genes and variants. We found BALB/cByJ mice showed enhanced sensitivity on the 53.5°C hot plate and mechanical stimulation in the von Frey test compared to BALB/cJ mice and replicated decreased gross brain weight in BALB/cByJ versus BALB/cJ. We then identified a quantitative trait locus (QTL) on chromosome 13 for hot plate sensitivity (LOD = 10.7; p < 0.001; peak = 56 Mb) and a QTL for brain weight on chromosome 5 (LOD = 8.7; p < 0.001). Expression QTL mapping of brain tissues identified H2afy (56.07 Mb) as the top transcript with the strongest association at the hot plate locus (FDR = 0.0002) and spliceome analysis identified differential exon usage within H2afy associated with the same locus. Whole brain proteomics further supported decreased H2AFY expression could underlie enhanced hot plate sensitivity, and identified ACADS as a candidate for reduced brain weight. To summarize, a BALB/c reduced complexity cross combined with multiple-omics approaches facilitated identification of candidate genes underlying thermal nociception and brain weight. These substrains provide a powerful, reciprocal platform for future validation of candidate variants.

Total synthesis and antibacterial evaluation of Empetroxepins A and B and related analogs.

Empetroxepins A and B, which are 10,11-dihydrodibenz[b,f]oxepins produced by the Black Crowberry (Empetrum nigrum), displayed weak anti-tubercular activity upon isolation, but have not been explored for antibiotic activity despite their molecular similarity to other phenolic antibacterial natural products. Herein we detail the first total synthesis of Empetroxepins A and B via a selective demethylation strategy and antibacterial structure activity relationship (SAR) study of the natural products and related analogs. Empetroxepin A was found to be weakly active against susceptible strains of Staphylococcus aureus (SA) and Bacillus subtilis (BS) with a minimum inhibitory concentration (MIC) of 256 µg/mL against both bacteria, whereas Empetroxepin B was found to be weakly active against only BS (MIC = 256 µg/mL). Neither natural product was active against Escherichia coli (EC). Antibiotic activity was improved through derivatization of the 10,11-dihydrodibenz[b,f]oxepin core with the best compound of the SAR series, 9-chloro-10,11-dihydrodibenzo[b,f]oxepine-2,3,4-triol, having MICs of 8 µg/mL, 16 µg/mL, and 256 µg/mL against SA, BS, and EC respectively.

A Review of Defatting Strategies for Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease is a huge cause of chronic liver failure around the world. This condition has become more prevalent as rates of metabolic syndrome, type 2 diabetes, and obesity have also escalated. The unfortunate outcome for many people is liver cirrhosis that warrants transplantation or being unable to receive a transplant since many livers are discarded due to high levels of steatosis. Over the past several years, however, a great deal of work has gone into understanding the pathophysiology of this disease as well as possible treatment options. This review summarizes various defatting strategies including in vitro use of pharmacologic agents, machine perfusion of extracted livers, and genomic approaches targeting specific proteins. The goal of the field is to reduce the number of necessary transplants and expand the pool of organs available for use.

A mutagenic screen reveals NspS residues important for regulation of Vibrio cholerae biofilm formation.

Biofilm formation in the human intestinal pathogen Vibrio cholerae is in part regulated by norspermidine, spermidine and spermine. V. cholerae senses these polyamines through a signalling pathway consisting of the periplasmic protein, NspS, and the integral membrane c-di-GMP phosphodiesterase MbaA. NspS and MbaA belong to a proposed class of novel signalling systems composed of periplasmic ligand-binding proteins and membrane-bound c-di-GMP phosphodiesterases containing both GGDEF and EAL domains. In this signal transduction pathway, NspS is hypothesized to interact with MbaA in the periplasm to regulate its phosphodiesterase activity. Polyamine binding to NspS likely alters this interaction, leading to the activation or inhibition of biofilm formation depending on the polyamine. The purpose of this study was to determine the amino acids important for NspS function. We performed random mutagenesis of the nspS gene, identified mutant clones deficient in biofilm formation, determined their responsiveness to norspermidine and mapped the location of these residues onto NspS homology models. Single mutants clustered on two lobes of the NspS model, but the majority were found on a single lobe that appeared to be more mobile upon norspermidine binding. We also identified residues in the putative ligand-binding site that may be important for norspermidine binding and interactions with MbaA. Ultimately, our results provide new insights into this novel signalling pathway in V. cholerae and highlight differences between periplasmic binding proteins involved in transport versus signal transduction.

Methods and feasibility study for exome sequencing as a universal second-tier test in newborn screening.

PURPOSE: Newborn screening disorders increasingly require genetic variant analysis as part of second-tier or confirmatory testing. Sanger sequencing and gene-specific next-generation sequencing (NGS)-based tests, the current methods of choice, are costly and lack scalability when expanding to new conditions. We describe a scalable, exome sequencing-based NGS pipeline with a priori analysis restriction that can be universally applied to any NBS disorder. METHODS: De-identified abnormal newborn screening specimens representing severe combined immune deficiency (SCID), cystic fibrosis (CF), VLCAD deficiency, metachromatic leukodystrophy (MLD), and in silico sequence read data sets were used to validate the pipeline. To support interpretation and clinical decision-making within the bioinformatics pipeline, variants from multiple databases were curated and validated. RESULTS: CFTR variant panel analysis correctly identified all variants. Concordance compared with diagnostic testing results for targeted gene analysis was between 78.6% and 100%. Validation of the bioinformatics pipeline with in silico data sets revealed a 100% detection rate. Varying degrees of overlap were observed between ClinVar and other databases ranging from 3% to 65%. Data normalization revealed that 11% of variants across the databases required manual curation. CONCLUSION: This pipeline allows for restriction of analysis to variants within a single gene or multiple genes, and can be readily expanded to full exome analysis if clinically indicated and parental consent is granted.

Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-Mediated Cyclin D1 Transcription.

RATIONALE: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase). OBJECTIVE: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. METHODS AND RESULTS: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (Myh11-Cre-ERT2) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4- (GATA-binding protein 4) cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter, and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitant increase in GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control mice, but not in VS-4718-treated and SMC-specific FAK kinase-dead mice. Finally, lentiviral shGATA4 knockdown in the wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared with control mice. CONCLUSIONS: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.

Low SARS-CoV-2 Transmission in Elementary Schools - Salt Lake County, Utah, December 3, 2020-January 31, 2021.

School closures affected more than 55 million students across the United States when implemented as a strategy to prevent the transmission of SARS-CoV-2, the virus that causes COVID-19 (1). Reopening schools requires balancing the risks for SARS-CoV-2 infection to students and staff members against the benefits of in-person learning (2). During December 3, 2020-January 31, 2021, CDC investigated SARS-CoV-2 transmission in 20 elementary schools (kindergarten through grade 6) that had reopened in Salt Lake County, Utah. The 7-day cumulative number of new COVID-19 cases in Salt Lake County during this time ranged from 290 to 670 cases per 100,000 persons.† Susceptible§ school contacts¶ (students and staff members exposed to SARS-CoV-2 in school) of 51 index patients** (40 students and 11 staff members) were offered SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) testing. Among 1,041 susceptible school contacts, 735 (70.6%) were tested, and five of 12 cases identified were classified as school-associated; the secondary attack rate among tested susceptible school contacts was 0.7%. Mask use among students was high (86%), and the median distance between students' seats in classrooms was 3 ft. Despite high community incidence and an inability to maintain ≥6 ft of distance between students at all times, SARS-CoV-2 transmission was low in these elementary schools. The results from this investigation add to the increasing evidence that in-person learning can be achieved with minimal SARS-CoV-2 transmission risk when multiple measures to prevent transmission are implemented (3,4).

OXTR rs53576 Variation with Breast and Nipple Pain in Breastfeeding Women.

BACKGROUND: Thirty percent of women who seek professional breastfeeding support require assistance with ongoing breast and nipple pain and < 50% of women report resolution of their pain. It is unknown if there is a molecular risk for ongoing breast and nipple pain during breastfeeding. Aim -To evaluate associations among breast and nipple pain sensitivity and candidate pain sensitivity single-nucleotide polymorphisms [SNPs], (COMT rs6269, rs4633, rs4818, rs4680 and OXTR rs2254298, rs53576) in breastfeeding women. Design - A secondary analysis of a pilot randomized controlled trial of a pain self-management intervention conducted over 6 weeks postpartum. Setting and Participants - Sixty women were recruited from two hospital settings after birth. Methods - All participants underwent standardized mechanical somatosensory testing for an assessment of pain sensitivity and provided baseline buccal swabs for genetic analysis. At 1, 2, and 6 weeks postpartum, women self-reported breast and nipple pain severity using a visual analogue scale. Results - Women with the minor allele OXTR rs53576 reported 8.18-fold higher breast and nipple pain severity over time. For every 1-unit increase in Mechanical detection threshold and windup ratio, women reported 16.51-fold and 4.82-fold higher breast and nipple pain severity respectively. Six women with the OXTR rs2254298 minor allele reported allodynia. Conclusion - The presence of OXTR alleles in women with enhanced pain sensitivity suggests a phenotype of genetic risk for ongoing breast and nipple with potential for pain-associated breastfeeding cessation. Somatosensory testing identified women who reported higher breast and nipple pain during the first weeks of breastfeeding.

The Potential Role of Preoperative Pain, Catastrophizing, and Differential Gene Expression on Pain Outcomes after Pediatric Spinal Fusion.

BACKGROUND: Adolescent idiopathic scoliosis is one of the most common spinal deformities in children and adolescents requiring extensive surgical intervention. Due to the nature of surgery, spinal fusion increases their risk of experiencing persistent postsurgical pain. Up to 20% of adolescents report pain for months or years after corrective spinal fusion surgery. AIMS: To examine the influence of preoperative psychosocial factors and mRNA expression profiles on persistent postoperative pain in adolescents undergoing corrective spinal fusion surgery. DESIGN: Prospective, longitudinal cohort study. SETTING: Two freestanding academic children's hospitals. METHODS: Utilizing a longitudinal approach, adolescents were evaluated at baseline (preoperatively) and postoperatively at ±1 month and ±4-6 months. Self-report of pain scores, the Pain Catastrophizing Scale-Child, and whole blood for RNA sequencing analysis were obtained at each time point. RESULTS: Of the adolescents enrolled in the study, 36% experienced persistent pain at final postoperative follow-up. The most significant predictors of persistent pain included preoperative pain severity and helplessness. Gene expression analysis identified HLA-DRB3 as having increased expression in children who experienced persistent pain postoperatively, as opposed to those whose pain resolved. A prospective validation study with a larger sample size is needed to confirm these findings. CONCLUSIONS: While adolescent idiopathic scoliosis is not often classified as a painful condition, providers must be cognizant of pre-existing pain and anxiety that may precipitate a negative recovery trajectory. Policy and practice change are essential for early identification and subsequent intervention.

Investigating key predictors of persistent low back pain: A focus on psychological stress.

AIM: To investigate the degree to which psychological stress, self-reported pain scores, and pain sensitivity during an acute state of low back pain (LBP) predict the development of persistent LBP trajectories. BACKGROUND: Identifying which factors influence LBP trajectories is critical to understand why some individuals experience persistent LBP and to illuminate areas for nursing intervention. METHODS: A secondary data analysis of a prospective study examining trajectories of LBP was conducted. The sample was comprised of 217 adults with acute-onset LBP recruited from the community and followed over 24 weeks. Variables of interest included demographic data, perceived stress scores, self-reported pain scores, and somatosensory characteristics collected within the first 4 weeks of LBP onset. The data were analyzed using non-parametric bivariate comparisons and a semi-parametric Cox proportional hazards model with interval-censoring. RESULTS: Individuals with higher psychological stress scores were less likely to experience pain resolution (Hazard ratio [HR] = 0.555, 95% confidence interval [CI] = 0.36-0.85, p = 0.02). After adjustment for covariates in the final model, the analysis revealed household income (HR = 2.79, 95% CI [1.63-4.67], p < 0.001) to be the dominant predictor of LBP persistence in this sample. CONCLUSION: Heightened psychological stress and pain severity as well as decreased pressure pain thresholds were indicated as influential factors of LBP trajectories. Household income was identified as the dominant predictor, demonstrating that individuals with a higher household income were more likely to resolve their pain. Strategies which integrate assessment of stress, self-reported pain scores, pain sensitivity, and social determinants for patients experiencing pain are needed to advance nursing care.

Hypoxia induces cancer cell-specific chromatin interactions and increases MALAT1 expression in breast cancer cells.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA overexpressed in various cancers that promotes cell growth and metastasis. Although hypoxia has been shown to up-regulate MALAT1, only hypoxia-inducible factors (HIFs) have been implicated in activation of the MALAT1 promoter in specific cell types and other molecular mechanisms associated with hypoxia-mediated MALAT1 up-regulation remain largely unknown. Here, we demonstrate that hypoxia induces cancer cell-specific chromatin-chromatin interactions between newly identified enhancer-like cis-regulatory elements present at the MALAT1 locus. We show that hypoxia-mediated up-regulation of MALAT1 as well as its antisense strand TALAM1 occurs in breast cancer cells, but not in nontumorigenic mammary epithelial cells. Our analyses on the MALAT1 genomic locus discovered three novel putative enhancers that are located upstream and downstream of the MALAT1 gene body. We found that parts of these putative enhancers are epigenetically modified to a more open chromatin state under hypoxia in breast cancer cells. Furthermore, our chromosome conformation capture experiment demonstrated that noncancerous cells and breast cancer cells exhibit different interaction profiles under both normoxia and hypoxia, and only breast cancer cells gain specific chromatin interactions under hypoxia. Although the HIF-2α protein can enhance the interaction between the promoter and the putative 3' enhancer, the gain of chromatin interactions associated with other upstream elements, such as putative -7 and -20 kb enhancers, were HIF-independent events. Collectively, our study demonstrates that cancer cell-specific chromatin-chromatin interactions are formed at the MALAT1 locus under hypoxia, implicating a novel mechanism of MALAT1 regulation in cancer.

Effects of Physical Activity on Neurophysiological and Gene Expression Profiles in Chronic Back Pain: Study Protocol.

BACKGROUND: Despite the emphasis on exercise to reduce pain and improve function among people with chronic low back pain (cLBP), little is known about the underlying mechanism of the impact of exercise on the neurophysiological and gene transcription alterations that characterize cLBP. OBJECTIVES: To present a study protocol to examine the feasibility, acceptability, and initial efficacy of Problem-Solving Pain to Enhance Living Well (PROPEL) with the support of nurse consultations and wearable activity-tracking technology on self-management (SM) knowledge, skills, physical activity, and pain and to examine the differential neurophysiological and gene expression profiles in cLBP participants from pre- to post-PROPEL. METHODS: A pretest and posttest study is employed on 40 adults ages 18-60 years with cLBP who do not have serious complications and/or comorbidities that affect sensorimotor function. Participants will receive video modules focused on SM and biweekly phone consultations to facilitate symptom monitoring and problem-solving while increasing physical activity frequency and duration. Participants will be assessed for outcomes including SM skills, physical activity, and pain every 2 weeks for 12 weeks. We will examine the participants' differential neurophysiological and gene expression profiles at 12 weeks postintervention and correlate these outcomes with the total duration of physical activity. RESULTS: The study began in September 2018. Of the 99 subjects that were screened, 23 were enrolled and 8 completed data collection. DISCUSSION: Comparing the neurophysiological and gene expression profiles of people with cLBP exposed to PROPEL could inform the development of interventions that offer personalized physical activity dosage along with general SM support. Web-based programs such as PROPEL have the potential to enhance accessibility of evidence-based interventions that improve functionality and quality of life among people living with cLBP.

Assessing the performance of in silico methods for predicting the pathogenicity of variants in the gene CHEK2, among Hispanic females with breast cancer.

The availability of disease-specific genomic data is critical for developing new computational methods that predict the pathogenicity of human variants and advance the field of precision medicine. However, the lack of gold standards to properly train and benchmark such methods is one of the greatest challenges in the field. In response to this challenge, the scientific community is invited to participate in the Critical Assessment for Genome Interpretation (CAGI), where unpublished disease variants are available for classification by in silico methods. As part of the CAGI-5 challenge, we evaluated the performance of 18 submissions and three additional methods in predicting the pathogenicity of single nucleotide variants (SNVs) in checkpoint kinase 2 (CHEK2) for cases of breast cancer in Hispanic females. As part of the assessment, the efficacy of the analysis method and the setup of the challenge were also considered. The results indicated that though the challenge could benefit from additional participant data, the combined generalized linear model analysis and odds of pathogenicity analysis provided a framework to evaluate the methods submitted for SNV pathogenicity identification and for comparison to other available methods. The outcome of this challenge and the approaches used can help guide further advancements in identifying SNV-disease relationships.

SON haploinsufficiency causes impaired pre-mRNA splicing of CAKUT genes and heterogeneous renal phenotypes.

Although genetic testing is increasingly used in clinical nephrology, a large number of patients with congenital abnormalities of the kidney and urinary tract (CAKUT) remain undiagnosed with current gene panels. Therefore, careful curation of novel genetic findings is key to improving diagnostic yields. We recently described a novel intellectual disability syndrome caused by de novo heterozygous loss-of-function mutations in the gene encoding the splicing factor SON. Here, we show that many of these patients, including two previously unreported, exhibit a wide array of kidney abnormalities. Detailed phenotyping of 14 patients with SON haploinsufficiency identified kidney anomalies in 8 patients, including horseshoe kidney, unilateral renal hypoplasia, and renal cysts. Recurrent urinary tract infections, electrolyte disturbances, and hypertension were also observed in some patients. SON knockdown in kidney cell lines leads to abnormal pre-mRNA splicing, resulting in decreased expression of several established CAKUT genes. Furthermore, these molecular events were observed in patient-derived cells with SON haploinsufficiency. Taken together, our data suggest that the wide spectrum of phenotypes in patients with a pathogenic SON mutation is a consequence of impaired pre-mRNA splicing of several CAKUT genes. We propose that genetic testing panels designed to diagnose children with a kidney phenotype should include the SON gene.