School of Medicine

To characterize the utilization of ultrasound in the urgent and emergency department setting at CRH before and after an educational ultrasound curriculum is implemented. Rates of utilization, body part examined, gender of patient, age of patient and chief complaint will be captured before and after the educational intervention.

Among diabetic individuals with clinically suspected PAD, both those with low and high ABI scores are at higher risk of CVD death. In contrast, a linear relationship was observed between TBI scores and CVD death irrespective of diabetes status. These findings suggest that stiffened ankle arteries may limit the predictive value of ABI scores in individuals with diabetes; a limitation that may be overcome by measurement of the TBI.

Background: Coronary artery calcium (CAC) predicts incident cardiovascular disease (CVD) beyond traditional risk factors. While higher CAC volume is associated with higher CVD risk, higher CAC density is associated with lower CVD risk. Whether risk factors for CAC volume and CAC density are similar or distinct is unknown. We sought to evaluate the independent associations of CVD risk factors with CAC volume an CAC density. Methods: Baseline measurements from 6,814 participants free of clinical CVD were collected for the Multi-Ethnic Study of Atherosclerosis between 2000 and 2002. Participants with no CAC )n=3,416) and missing data were excluded, for a final analytic sample of 3.375 participants. Multivariable linear regression models were used to evaluate independent predictors of CAC density and CAC volume. Conclusions: Whereas most CVD risk factors were associated with higher CAC volume, the same risk factors were assocated with lower CAC density. For example, diabetes was associated with higher natural logarithm (ln) transformed CAC volume (standardized [beta]=0.44 ln-units, p<0.01) but lower CAC density ([beta]=-0.07 Hounsfield unit (Hu) category unit, p<0.01). Relative to Non-Hispanic White, Chinese, African-American, and Hispanic race/ethnicities were associated with lower ln CAC volume ([beta]=-0.62, -0.52, and -0.40 ln-units, respectively, p<0.01 for each), and higher CAC density ([beta]=0.41, 0.18, and 0.21 Hu category units, respectively, p<0.01 for each). CAC density and CAC volume were also differentially associated with race/ethnicity.

Chronic low back pain (cLBP) is the most prevalent chronic pain condition. There are no treatments that haven been found to directly assuage evoked cLBP. To this extent, mindfulness-meditation is a promising pain therapy. Yet, it is unclear if meditation can be utilized to directly attenuate evoked chronic pain through endogenous opioids. A double-blind, randomized, and placebo-controlled clinical trial with a drug crossover design examined if mindfulness-meditation, as compared to sham mindfulness-meditation, attenuated straight leg-raise test evoked chronic pain during intravenous (0.15 mg/kg bolus + 0.15 mg/kg/hour maintenance) naloxone (opioid antagonist) and placebo-saline infusion. Fifty-nine individuals with cLBP (mean age = 46 years; 30 females) completed all study procedures. After the pre-intervention pain testing session, patients were randomized to a four-session (20-min/session) mindfulness (n = 30) or sham mindfulness-meditation (n = 29) intervention. After the interventions, mindfulness and sham mindfulness-meditation were associated with significant reductions in back pain during saline and naloxone infusion when compared to rest (non-meditation) in response to the cLBP-evoking straight leg-raise test. These results indicate that meditation directly reduces evoked chronic pain through non-opioidergic processes. Importantly, after the interventions, the mindfulness group reported significantly lower straight leg-raise induced pain than the sham mindfulness-meditation group during rest (non-meditation) and meditation. Mindfulness and sham mindfulness-meditation training was also associated with significantly lower Brief Pain Inventory severity and interference scores. The pain-relieving effects of mindfulness meditation were more pronounced than a robust sham-mindfulness meditation intervention, suggesting that non-reactive appraisal processes may be uniquely associated with improvements in chronic low-back pain.Trial Registration: ClinicalTrials.gov identifier: NCT04034004.

Mild Traumatic Brain Injury (MTBI) patients with persistent headaches are known to have diminished supraspinal modulatory connectivity from their prefrontal cortices. Repetitive transcranial magnetic stimulation (rTMS) is able to alleviate MTBI-related headache (MTBI-HA). This functional magnetic resonance imaging (fMRI) study assessed supraspinal correlates associated with the headache analgesic effect of rTMS at left prefrontal cortex (LPFC), hypothesizing real rTMS would significantly increase modulatory functions at LPFC in comparison to sham treatment. Subjects with MTBI-HA were randomized to receive either real or sham rTMS treatments and subjected to pre- and post-treatment resting state and evoked heat-pain fMRI as described in a prior study. Real rTMS consisted of 2000 pulses delivered at 10 Hz and 80% of the resting motor threshold at left dorsolateral prefrontal cortex, whereas sham treatment was delivered with same figure-of-eight coil turned 180 degrees. Follow-up fMRI was performed one-week post-treatment. All fMRI data was processed using BrainVoyager QX Software. 14 subjects receiving real and 12 subjects receiving sham treatments completed the study. The REAL group demonstrated significant (P < 0.02) decreases in headache frequency and intensity at one week following treatment. fMRI scans in the REAL group showed increased evoked heat pain activity (P < 0.002) and resting functional connectivity (P < 0.0001) at the LPFC after rTMS. Neither this significant analgesic effect nor these fMRI findings were seen in the sham group. Sham treatment was, however, associated with a decrease in resting state activity at the LPFC (P < 0.0001). This study correlates the demonstrated analgesic effect of rTMS in the treatment of MTBI-HA with enhanced supraspinal functional connectivity in the left prefrontal cortex, which is known to be involved in "top-down" pain inhibition along the descending midbrain-thalamic-cingulate pathway. Trial Registration: This study was registered on September 24, 2013, on ClinicalTrials.gov with the identifier: NCT01948947. https://clinicaltrials.gov/ct2/show/NCT01948947 .

Direct conversion of somatic cells into neurons holds great promise for regenerative medicine. However, neuronal conversion is relatively inefficient in human cells compared to mouse cells. It has been unclear what might be the key barriers to reprogramming in human cells. We recently elucidated an RNA program mediated by the polypyrimidine tract binding protein PTB to convert mouse embryonic fibroblasts (MEFs) into functional neurons. In human adult fibroblasts (HAFs), however, we unexpectedly found that invoking the documented PTB-REST-miR-124 loop generates only immature neurons. We now report that the functionality requires sequential inactivation of PTB and the PTB paralog nPTB in HAFs. Inactivation of nPTB triggers another self-enforcing loop essential for neuronal maturation, which comprises nPTB, the transcription factor BRN2, and miR-9. These findings suggest that two separate gatekeepers control neuronal conversion and maturation and consecutively overcoming these gatekeepers enables deterministic reprogramming of HAFs into functional neurons.

Nuclear clearance and cytoplasmic accumulations of the RNA-binding protein TDP-43 are pathological hallmarks in almost all patients with amyotrophic lateral sclerosis (ALS) and up to 50% of patients with frontotemporal dementia (FTD) and Alzheimers disease. In Alzheimers disease, TDP-43 pathology is predominantly observed in the limbic system and correlates with cognitive decline and reduced hippocampal volume. Disruption of nuclear TDP-43 function leads to abnormal RNA splicing and incorporation of erroneous cryptic exons in numerous transcripts including Stathmin-2 (STMN2, also known as SCG10) and UNC13A, recently reported in tissues from patients with ALS and FTD. Here, we identify both STMN2 and UNC13A cryptic exons in Alzheimers disease patients, that correlate with TDP-43 pathology burden, but not with amyloid-β or tau deposits. We also demonstrate that processing of the STMN2 pre-mRNA is more sensitive to TDP-43 loss of function than UNC13A. In addition, full-length RNAs encoding STMN2 and UNC13A are suppressed in large RNA-seq datasets generated from Alzheimers disease post-mortem brain tissue. Collectively, these results open exciting new avenues to use STMN2 and UNC13A as potential therapeutic targets in a broad range of neurodegenerative conditions with TDP-43 proteinopathy including Alzheimers disease.

The U2AF heterodimer has been well studied for its role in defining functional 3' splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3' splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3' splice site associated either with the alternative exon, to cause exon skipping, or with the competing constitutive exon, to induce exon inclusion. We further demonstrate partial functional impairment with leukemia-associated mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states.

Recent literature suggests that the layer of adipocytes embedded in the skin below the dermis is far from being an inert spacer material. Instead, this layer of dermal white adipose tissue (dWAT) is a regulated lipid layer that comprises a crucial environmental defense. Among all the classes of biological molecules, lipids have the lowest thermal conductance and highest insulation potential. This property can be exploited by mammals to reduce heat loss, suppress brown adipose tissue activation, reduce the activation of thermogenic programs, and increase metabolic efficiency. Furthermore, this layer responds to bacterial challenge to provide a physical barrier and antimicrobial disinfection, and its expansion supports the growth of hair follicles and regenerating skin. In sum, this dWAT layer is a key defensive player with remarkable potential for modifying systemic metabolism, immune function, and physiology. In this review, we discuss the key literature illustrating the properties of this recently recognized adipose depot.

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Acne dysbiosis happens when there is a microbial imbalance of the over-growth of Propionibacterium acnes (P. acnes) in the acne microbiome. In our previous study, we demonstrated that Staphylococcus epidermidis (S. epidermidis, a probiotic skin bacterium) can exploit glycerol fermentation to produce short-chain fatty acids (SCFAs) which have antimicrobial activities to suppress the growth of P. acnes. Unlike glycerol, sucrose is chosen here as a selective fermentation initiator (SFI) that can specifically intensify the fermentation activity of S. epidermidis, but not P. acnes. A co-culture of P. acnes and fermenting S. epidermidis in the presence of sucrose significantly led to a reduction in the growth of P. acnes. The reduction was abolished when P. acnes was co-cultured with non-fermenting S. epidermidis. Results from nuclear magnetic resonance (NMR) analysis revealed four SCFAs (acetic acid, butyric acid, lactic acid, and succinic acid) were detectable in the media of S. epidermidis sucrose fermentation. To validate the interference of S. epidermidis sucrose fermentation with P. acnes, mouse ears were injected with both P. acnes and S. epidermidis plus sucrose or phosphate buffered saline (PBS). The level of macrophage-inflammatory protein-2 (MIP-2) and the number of P. acnes in ears injected with two bacteria plus sucrose were considerably lower than those in ears injected with two bacteria plus PBS. Our results demonstrate a precision microbiome approach by using sucrose as a SFI for S. epidermidis, holding future potential as a novel modality to equilibrate dysbiotic acne.

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PURPOSE: Emergency department (ED) visits by patients with cancer frequently end in hospitalization. As concerns about ED and hospital crowding increase, observation unit care may be an important strategy to deliver safe and efficient treatment for eligible patients. In this investigation, we compared the prevalence and clinical characteristics of cancer patients who received observation unit care with those who were admitted to the hospital from the ED. METHODS: We performed a multicenter prospective cohort study of patients with cancer presenting to an ED affiliated with one of 18 hospitals of the Comprehensive Oncologic Emergency Research Network (CONCERN) between March 1, 2016 and January 30, 2017. We compared patient characteristics with the prevalence of observation unit care usage, hospital admission, and length of stay. RESULTS: Of 1051 enrolled patients, 596 (56.7%) were admitted as inpatients, and 72 (6.9%) were placed in an observation unit. For patients admitted as inpatients, 23.7% had a length of stay ≤2 days. The conversion rate from observation to inpatient was 17.1% (95% CI 14.6-19.4) among those receiving care in an observation unit. The average observation unit length of stay was 14.7 h. Patient factors associated ED disposition to observation unit care were female gender and low Charlson Comorbidity Index. CONCLUSION: In this multicenter prospective cohort study, the discrepancy between observation unit care use and short inpatient hospitalization may represent underutilization of this resource and a target for process change.

The National Board of Medical Examiners' decision to change Step 1 of the United States Medical Licensing Examination (USMLE) from a three-digit score to Pass/Fail (P/F) represents a disruptive change for students, faculty, and leaders in the academic community. In the context of this change, some schools may re-consider the optimal timing of Step 1 as they strive to align their assessment practices with sound educational principles. Currently, over 20 schools administer USMLE Step 1 after the core clerkships. In this commentary, we review the educational rationale for a post-clerkship Step 1, highlighting how adult learning theories support this placement. We discuss some short-term challenges post-clerkship Step 1 schools may encounter due to the proposed timing of the change in scoring, which creates three unique scenarios for learners that can introduce inequity in the system and provoke anxiety. We review outcomes of potentially heightened importance when Step 1 is P/F, including lower clinical subject exam scores in some clerkships, lower failure rates on Step 1 and stable Step 2 Clinical Knowledge scores with implications for the residency match. We outline the future potential for performance-based time-variable Step 1 study periods that are facilitated by post-clerkship placement of the exam. Finally, we discuss opportunities to achieve the goal of enhancing student well-being, which was a major rationale for eliminating the three-digit score.

Today's science increasingly requires effective ways to find and access existing datasets that are distributed across a range of repositories. For researchers in the life sciences, discoverability of datasets may soon become as essential as identifying the latest publications via PubMed. Through an international collaborative effort funded by the National Institutes of Health (NIH)'s Big Data to Knowledge (BD2K) initiative, we have designed and implemented the DAta Tag Suite (DATS) model to support the DataMed data discovery index. DataMed's goal is to be for data what PubMed has been for the scientific literature. Akin to the Journal Article Tag Suite (JATS) used in PubMed, the DATS model enables submission of metadata on datasets to DataMed. DATS has a core set of elements, which are generic and applicable to any type of dataset, and an extended set that can accommodate more specialized data types. DATS is a platform-independent model also available as an annotated serialization in schema.org, which in turn is widely used by major search engines like Google, Microsoft, Yahoo and Yandex.

Abstract: Background: Rectal gonorrhea (NG) and chlamydia (Connecticut) infections are common among men who have sex with men (MSM). Rectal douching/enema (RDE) is a common practice among MSM that can affect the rectal microbiome. It is unclear if this practice is associated with acquiring rectal infections (RI) with either NG or CT. Methods: From 2013–2015, 398 adult HIV-negative MSM and transwomen were enrolled in a randomized controlled study on text messaging for adherence to pre-exposure prophylaxis (PrEP). Participants were surveyed on sexual behavior, frequency of RDE, drug use, and nutritional habits in conjunction with routine sexually transmitted infection testing. Pearson’s χ 2 and two sample t-tests were used to measure significance of RDE and other risk factors with RI. Multivariable logistic regression model was used to control for confounding and assess the association of RDE with RIs. Confounders (i.e., age, number anal receptive sex, number sex partners) were selected a priori for inclusion in the final model based on a causal model and statistical significance. Results: Of 397 participants, 262 (67%) performed RDE and 132 (33%) had at least one NG or CT rectal infection over 48 weeks. Number of condomless anal receptive sex acts (mean = 19, P < 0.001), condom use for anal receptive sex (P = 0.017), number of male sex partners in past 3 months (mean = 14, P = 0.001), and the use of poppers (P < 0.001) were associated with RI. There was no significant association between nutritional habits, probiotic foods or supplements and RI, with the exception of energy bars (P = 0.029). Controlling for confounders, RI was associated with RDE less than weekly with OR = 1.02 (95% CI 0.52–1.99) while RDE weekly or more had OR = 2.08 (95% CI 1.03–4.17). Stratified by number of partners, MSMs with more than the median (>6) number of partners had OR = 4.96 (95% CI 1.29–19.03) if performing RDE less than weekly, and OR = 6.03 (95% CI 1.55–23.49) if weekly or more. Conclusion: Rectal hygiene with douching/enemas is a common practice among MSMs on PrEP, which increases the odds of acquiring rectal NG and/or CT. This finding is suggestive for the use of rectal hygiene products/practices as potential targets for sexually transmitted infection prevention. Disclosures: All authors: No reported disclosures.

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Today's science increasingly requires effective ways to find and access existing datasets that are distributed across a range of repositories. For researchers in the life sciences, discoverability of datasets may soon become as essential as identifying the latest publications via PubMed. Through an international collaborative effort funded by the National Institutes of Health (NIH)'s Big Data to Knowledge (BD2K) initiative, we have designed and implemented the DAta Tag Suite (DATS) model to support the DataMed data discovery index. DataMed's goal is to be for data what PubMed has been for the scientific literature. Akin to the Journal Article Tag Suite (JATS) used in PubMed, the DATS model enables submission of metadata on datasets to DataMed. DATS has a core set of elements, which are generic and applicable to any type of dataset, and an extended set that can accommodate more specialized data types. DATS is a platform-independent model also available as an annotated serialization in schema.org, which in turn is widely used by major search engines like Google, Microsoft, Yahoo and Yandex.

Miner1 is a redox-active 2Fe2S cluster protein. Mutations in Miner1 result in Wolfram Syndrome, a metabolic disease associated with diabetes, blindness, deafness, and a shortened lifespan. Embryonic fibroblasts from Miner1(-/-) mice displayed ER stress and showed hallmarks of the unfolded protein response. In addition, loss of Miner1 caused a depletion of ER Ca(2+) stores, a dramatic increase in mitochondrial Ca(2+) load, increased reactive oxygen and nitrogen species, an increase in the GSSG/GSH and NAD(+)/NADH ratios, and an increase in the ADP/ATP ratio consistent with enhanced ATP utilization. Furthermore, mitochondria in fibroblasts lacking Miner1 displayed ultrastructural alterations, such as increased cristae density and punctate morphology, and an increase in O2 consumption. Treatment with the sulphydryl anti-oxidant N-acetylcysteine reversed the abnormalities in the Miner1 deficient cells, suggesting that sulphydryl reducing agents should be explored as a treatment for this rare genetic disease.

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Embryonic stem cells (ESC) hold promise for the treatment of human medical conditions but are allogeneic. Here, we consider the differences between autologous pluripotent stem cells produced by nuclear transfer (NT-ESCs) and transcription factor-mediated, induced pluripotent stem cells (iPSCs) that impact the desirability of each of these cell types for clinical use. The derivation of NT-ESCs is more cumbersome and requires donor oocytes; however, the use of oocyte cytoplasm as the source of reprogramming factors is linked to a key advantage of NT-ESCs-the ability to replace mutant mitochondrial DNA in a patient cell (due to either age or inherited disease) with healthy donor mitochondria from an oocyte. Moreover, in epigenomic and transcriptomic comparisons between isogenic iPSCs and NT-ESCs, the latter produced cells that more closely resemble bona fide ESCs derived from fertilized embryos. Thus, although NT-ESCs are more difficult to generate than iPSCs, the ability of somatic cell nuclear transfer to replace aged or diseased mitochondria and the closer epigenomic and transcriptomic similarity between NT-ESCs and bona fide ESCs may make NT-ESCs superior for future applications in regenerative medicine. Stem Cells 2017;35:26-34.

Fetal and neonatal exposures to perinatal oxidative stress (OS) are key mediators of bronchopulmonary dysplasia (BPD). To characterize these exposures, adductomics is an exposure science approach that captures electrophilic addition products (adducts) in blood protein. Adducts are bound to the nucleophilic cysteine loci of human serum albumin (HSA), which has a prolonged half-life. We conducted targeted and untargeted adductomics to test the hypothesis that adducts of OS vary with BPD. We studied 205 preterm infants (≤28 weeks) and 51 full-term infants from an ongoing birth cohort. Infant plasma was collected at birth (cord blood), 1-week, 1-month, and 36-weeks postmenstrual age. HSA was isolated from plasma, trypsin digested, and analyzed using high-performance liquid chromatography-mass spectrometry to quantify previously annotated (known) and unknown adducts. We identified 105 adducts in cord and postnatal blood. A total of 51 known adducts (small thiols, direct oxidation products, and reactive aldehydes) were increased with BPD. Postnatally, serial concentrations of several known OS adducts correlated directly with supplemental oxygen exposure. The application of large-scale adductomics elucidated OS-mediated pathways of BPD. This is the first study to investigate the "neonatal-perinatal exposome" and to identify oxidative stress-related exposure biomarkers that may inform antioxidant strategies to protect the health of future generations of infants.

AIMS:Traditional technology to characterize urethral pressure changes during dynamic conditions is limited by slow response times or artifact-inducing withdrawal maneuvers. The 8F high-resolution manometry (HRM) catheter (ManoScan™ ESO, Covidien) has advantages of fast response times and the ability to measure urethral pressures along the urethral length without withdrawal. Our objective was to determine static and dynamic maximum urethral closure pressures (MUCPs) and resting functional urethral length (FUL) in women using HRM before and after transurethral bulking and compare results to other women who underwent midurethral sling (MUS). METHODS:We recorded rest, cough, and strain MUCPs and FUL in 24 women before and after transurethral bulking with polydimethylsiloxane (Macroplastique®) using the HRM catheter and compared these changes to HRM values from 26 women who had the same measures before and after MUS. RESULTS:At rest, MUCPs increased minimally after both urethral bulking and MUS (3 vs 0.4 cm H2 O respectively, P = 0.4). Under dynamic conditions there were statistically insignificant small increases in MUCP and these increases were markedly less than after MUS (cough: 1.5 vs 63.8 cm H2 O, P < 0.001 and strain: 11.5 vs 57.7 cm H2 O, P < 0.001). FUL increased by 0.5 cm after transurethral bulking (P = 0.003), and decreased by 0.25 cm after MUS placement (P = 0.012). CONCLUSIONS:The mechanism of continence after urethral bulking differs from MUS. While MUS increases dynamic MUCP, bulking may rely on increasing the length of the continence zone.

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Exercise provides a robust physiological stimulus that evokes cross-talk among multiple tissues that when repeated regularly (i.e., training) improves physiological capacity, benefits numerous organ systems, and decreases the risk for premature mortality. However, a gap remains in identifying the detailed molecular signals induced by exercise that benefits health and prevents disease. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) was established to address this gap and generate a molecular map of exercise. Preclinical and clinical studies will examine the systemic effects of endurance and resistance exercise across a range of ages and fitness levels by molecular probing of multiple tissues before and after acute and chronic exercise. From this multi-omic and bioinformatic analysis, a molecular map of exercise will be established. Altogether, MoTrPAC will provide a public database that is expected to enhance our understanding of the health benefits of exercise and to provide insight into how physical activity mitigates disease.

Osteoporosis, characterized by increased fracture risk and bone fragility, impacts millions of adults worldwide, but effective, non-invasive and easily accessible diagnostic tests of the disease remain elusive. We present a magnetic resonance (MR) technique that overcomes the motion limitations of traditional MR imaging to acquire high-resolution frequency-domain data to characterize the texture of biological tissues. This technique does not involve obtaining full two-dimensional or three-dimensional images, but can probe scales down to the order of 40 μm and in particular uncover structural information in trabecular bone. Using micro-computed tomography data of vertebral trabecular bone, we computationally validate this MR technique by simulating MR measurements of a 'ratio metric' determined from a few k-space values corresponding to trabecular thickness and spacing. We train a support vector machine classifier on ratio metric values determined from healthy and simulated osteoporotic bone data, which we use to accurately classify osteoporotic bone.

Detrimental changes in the composition and function of rotator cuff (RC) muscles are hallmarks of RC disease progression. Previous studies have demonstrated both atrophic and degenerative muscle loss in advanced RC disease. However, the relationship between gene expression and RC muscle pathology remains poorly defined, in large part due to a lack of studies correlating gene expression to tissue composition. Therefore, the purpose of this study was to determine how tissue composition relates to gene expression in muscle biopsies from patients undergoing reverse shoulder arthroplasty (RSA). Gene expression related to myogenesis, atrophy and cell death, adipogenesis and metabolism, inflammation, and fibrosis was measured in 40 RC muscle biopsies, including 31 biopsies from reverse shoulder arthroplasty (RSA) cases that had available histology data and 9 control biopsies from patients with intact RC tendons. After normalization to reference genes, linear regression was used to identify relationships between gene expression and tissue composition. Hierarchical clustering and principal component analysis (PCA) identified unique clusters, and fold-change analysis was used to determine significant differences in expression between clusters. We found that gene expression profiles were largely dependent on muscle presence, with muscle fraction being the only histological parameter that was significantly correlated to gene expression by linear regression. Similarly, samples with histologically-confirmed muscle distinctly segregated from samples without muscle. However, two sub-groups within the muscle-containing RSA biopsies suggest distinct phases of disease, with one group expressing markers of both atrophy and regeneration, and another group not significantly different from either control biopsies or biopsies lacking muscle. In conclusion, this study provides context for the interpretation of gene expression in heterogeneous and degenerating muscle, and provides further evidence for distinct stages of RC disease in humans.

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The U2AF heterodimer has been well studied for its role in defining functional 3' splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3' splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3' splice site associated either with the alternative exon, to cause exon skipping, or with the competing constitutive exon, to induce exon inclusion. We further demonstrate partial functional impairment with leukemia-associated mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states.

The factors contributing to the rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) BA.4 and BA.5 subvariants in populations that experienced recent surges of BA.2 and BA.2.12.1 infections are not understood. Neutralizing antibodies (NAbs) are likely to protect against severe disease if present in sufficient quantity. We found that after BA.2 or BA.2.12.1 infection, NAb responses were largely cross-neutralizing but were much less effective against BA.5. In addition, individuals who were infected and treated early with nirmatrelvir/ritonavir (Paxlovid) had lower NAb levels than untreated individuals.

Pathogenic microorganisms are sensed by the inflammasome, resulting in the release of the pro-immune and proinflammatory cytokine interleukin-1β (IL-1β). In humans, the paired sialic acid-binding Ig-like lectin receptors Siglec-5 (inhibitory) and Siglec-14 (activating) have been shown to have reciprocal roles in regulating macrophage immune responses, but their interaction with IL-1β signaling and the inflammasome has not been characterized. Here we show that in response to known inflammasome activators (ATP, nigericin) or the sialic acid-expressing human bacterial pathogen group B Streptococcus (GBS), the presence of Siglec-14 enhances, whereas Siglec-5 reduces, inflammasome activation and macrophage IL-1β release. Human THP-1 macrophages stably transfected with Siglec-14 exhibited increased caspase-1 activation, IL-1β release and pyroptosis after GBS infection, in a manner blocked by a specific inhibitor of nucleotide-binding domain leucine-rich repeat protein 3 (NLRP3), a protein involved in inflammasome assembly. Another leading pathogen, Streptococcus pneumoniae, lacks sialic acid but rather prominently expresses a sialidase, which cleaves sialic acid from macrophages, eliminating cis- interactions with the lectin receptor, thus attenuating Siglec-14 induced IL-1β secretion. Vimentin, a cytoskeletal protein released during macrophage inflammatory activation is known to induce the inflammasome. We found that vimentin has increased interaction with Siglec-14 compared to Siglec-5, and this interaction heightened IL-1β production by Siglec-14-expressing cells. Siglec-14 is absent from some humans because of a SIGLEC5/14 fusion polymorphism, and we found increased IL-1β expression in primary macrophages from SIGLEC14+/+ individuals compared to those with the SIGLEC14-/+ and SIGLEC14-/- genotypes. Collectively, our results identify a new immunoregulatory role of Siglec-14 as a positive regulator of NLRP3 inflammasome activation.

Cellular pathways that detect DNA damage are useful for identifying genes that suppress DNA damage, which can cause genome instability and cancer predisposition syndromes when mutated. We identified 199 high-confidence and 530 low-confidence DNA damage-suppressing (DDS) genes in Saccharomyces cerevisiae through a whole-genome screen for mutations inducing Hug1 expression, a focused screen for mutations inducing Ddc2 foci, and data from previous screens for mutations causing Rad52 foci accumulation and Rnr3 induction. We also identified 286 high-confidence and 394 low-confidence diverse genome instability-suppressing (DGIS) genes through a whole-genome screen for mutations resulting in increased gross chromosomal rearrangements and data from previous screens for mutations causing increased genome instability as assessed in a diversity of genome instability assays. Genes that suppress both pathways (DDS+ DGIS+) prevent or repair DNA replication damage and likely include genes preventing collisions between the replication and transcription machineries. DDS+ DGIS- genes, including many transcription-related genes, likely suppress damage that is normally repaired properly or prevent inappropriate signaling, whereas DDS- DGIS+ genes, like PIF1, do not suppress damage but likely promote its proper, nonmutagenic repair. Thus, induction of DNA damage markers is not a reliable indicator of increased genome instability, and the DDS and DGIS categories define mechanistically distinct groups of genes.

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Miner1 is a redox-active 2Fe2S cluster protein. Mutations in Miner1 result in Wolfram Syndrome, a metabolic disease associated with diabetes, blindness, deafness, and a shortened lifespan. Embryonic fibroblasts from Miner1(-/-) mice displayed ER stress and showed hallmarks of the unfolded protein response. In addition, loss of Miner1 caused a depletion of ER Ca(2+) stores, a dramatic increase in mitochondrial Ca(2+) load, increased reactive oxygen and nitrogen species, an increase in the GSSG/GSH and NAD(+)/NADH ratios, and an increase in the ADP/ATP ratio consistent with enhanced ATP utilization. Furthermore, mitochondria in fibroblasts lacking Miner1 displayed ultrastructural alterations, such as increased cristae density and punctate morphology, and an increase in O2 consumption. Treatment with the sulphydryl anti-oxidant N-acetylcysteine reversed the abnormalities in the Miner1 deficient cells, suggesting that sulphydryl reducing agents should be explored as a treatment for this rare genetic disease.

Cyclic AMP (cAMP) is involved in many biological processes but little is known regarding its role in shaping immunity. Here we show that cAMP-PKA-CREB signaling (a pattern recognition receptor [PRR]-independent mechanism) regulates conventional type-2 Dendritic Cells (cDC2s) in mice and reprograms their Th17-inducing properties via repression of IRF4 and KLF4, transcription factors essential for cDC2-mediated Th2 induction. In mice, genetic loss of IRF4 phenocopies the effects of cAMP on Th17 induction and restoration of IRF4 prevents the cAMP effect. Moreover, curdlan, a PRR-dependent microbial product, activates CREB and represses IRF4 and KLF4, resulting in a pro-Th17 phenotype of cDC2s. These in vitro and in vivo results define a novel signaling pathway by which cDC2s display plasticity and provide a new molecular basis for the classification of novel cDC2 and cDC17 subsets. The findings also reveal that repressing IRF4 and KLF4 pathway can be harnessed for immuno-regulation.

Muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin. Direct characterization of how small changes in the myosin-actin complex impact its force production remains challenging. Molecular dynamics (MD) simulations, although capable of studying protein structure-function relationships, are limited owing to the slow timescale of the myosin cycle as well as a lack of various intermediate structures for the actomyosin complex. Here, employing comparative modeling and enhanced sampling MD simulations, we show how the human cardiac myosin generates force during the mechanochemical cycle. Initial conformational ensembles for different myosin-actin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or metastable interactions with the actin surface. We find that the actin-binding cleft closure is allosterically coupled to the myosin motor core transitions and ATP-hydrolysis product release from the active site. Furthermore, a gate between switch I and switch II is suggested to control phosphate release at the prepowerstroke state. Our approach demonstrates the ability to link sequence and structural information to motor functions.

We conducted genome-wide association analyses of over 250,000 participants of European (EUR) and African (AFR) ancestry from the Million Veteran Program using electronic health record-validated post-traumatic stress disorder (PTSD) diagnosis and quantitative symptom phenotypes. Applying genome-wide multiple testing correction, we identified three significant loci in European case-control analyses and 15 loci in quantitative symptom analyses. Genomic structural equation modeling indicated tight coherence of a PTSD symptom factor that shares genetic variance with a distinct internalizing (mood-anxiety-neuroticism) factor. Partitioned heritability indicated enrichment in several cortical and subcortical regions, and imputed genetically regulated gene expression in these regions was used to identify potential drug repositioning candidates. These results validate the biological coherence of the PTSD syndrome, inform its relationship to comorbid anxiety and depressive disorders and provide new considerations for treatment.

Abstract: Background: Rectal gonorrhea (NG) and chlamydia (Connecticut) infections are common among men who have sex with men (MSM). Rectal douching/enema (RDE) is a common practice among MSM that can affect the rectal microbiome. It is unclear if this practice is associated with acquiring rectal infections (RI) with either NG or CT. Methods: From 2013–2015, 398 adult HIV-negative MSM and transwomen were enrolled in a randomized controlled study on text messaging for adherence to pre-exposure prophylaxis (PrEP). Participants were surveyed on sexual behavior, frequency of RDE, drug use, and nutritional habits in conjunction with routine sexually transmitted infection testing. Pearson’s χ 2 and two sample t-tests were used to measure significance of RDE and other risk factors with RI. Multivariable logistic regression model was used to control for confounding and assess the association of RDE with RIs. Confounders (i.e., age, number anal receptive sex, number sex partners) were selected a priori for inclusion in the final model based on a causal model and statistical significance. Results: Of 397 participants, 262 (67%) performed RDE and 132 (33%) had at least one NG or CT rectal infection over 48 weeks. Number of condomless anal receptive sex acts (mean = 19, P < 0.001), condom use for anal receptive sex (P = 0.017), number of male sex partners in past 3 months (mean = 14, P = 0.001), and the use of poppers (P < 0.001) were associated with RI. There was no significant association between nutritional habits, probiotic foods or supplements and RI, with the exception of energy bars (P = 0.029). Controlling for confounders, RI was associated with RDE less than weekly with OR = 1.02 (95% CI 0.52–1.99) while RDE weekly or more had OR = 2.08 (95% CI 1.03–4.17). Stratified by number of partners, MSMs with more than the median (>6) number of partners had OR = 4.96 (95% CI 1.29–19.03) if performing RDE less than weekly, and OR = 6.03 (95% CI 1.55–23.49) if weekly or more. Conclusion: Rectal hygiene with douching/enemas is a common practice among MSMs on PrEP, which increases the odds of acquiring rectal NG and/or CT. This finding is suggestive for the use of rectal hygiene products/practices as potential targets for sexually transmitted infection prevention. Disclosures: All authors: No reported disclosures.

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Social determinants of health are the economic and environmental conditions under which people are born, live, work, and age that affect health. These structural factors underlie many of the long-standing inequities in cancer care and outcomes that vary by geography, socioeconomic status, and race and ethnicity in the United States. Housing insecurity, including lack of safe, affordable, and stable housing, is a key social determinant of health that can influence-and be influenced by-cancer care across the continuum, from prevention to screening, diagnosis, treatment, and survivorship. During 2021, the National Cancer Policy Forum of the National Academies of Science, Engineering, and Medicine sponsored a series of webinars addressing social determinants of health, including food, housing, and transportation insecurity, and their associations with cancer care and patient outcomes. This dissemination commentary summarizes the formal presentations and panel discussions from the webinar devoted to housing insecurity. It provides an overview of housing insecurity and health care across the cancer control continuum, describes health system interventions to minimize the impact of housing insecurity on patients with cancer, and identifies challenges and opportunities for addressing housing insecurity and improving health equity. Systematically identifying and addressing housing insecurity to ensure equitable access to cancer care and reduce health disparities will require ongoing investment at the practice, systems, and broader policy levels.

Immune checkpoint blockade (ICB) therapy has revolutionized head and neck squamous cell carcinoma (HNSCC) treatment, but <20% of patients achieve durable responses. Persistent activation of the PI3K/AKT/mTOR signaling circuitry represents a key oncogenic driver in HNSCC; however, the potential immunosuppressive effects of PI3K/AKT/mTOR inhibitors may limit the benefit of their combination with ICB. Here we employ an unbiased kinome-wide siRNA screen to reveal that HER3, is essential for the proliferation of most HNSCC cells that do not harbor PIK3CA mutations. Indeed, we find that persistent tyrosine phosphorylation of HER3 and PI3K recruitment underlies aberrant PI3K/AKT/mTOR signaling in PIK3CA wild type HNSCCs. Remarkably, antibody-mediated HER3 blockade exerts a potent anti-tumor effect by suppressing HER3-PI3K-AKT-mTOR oncogenic signaling and concomitantly reversing the immune suppressive tumor microenvironment. Ultimately, we show that HER3 inhibition and PD-1 blockade may provide a multimodal precision immunotherapeutic approach for PIK3CA wild type HNSCC, aimed at achieving durable cancer remission.

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Conclusions

Multiphasic computed tomography (CT) and magnetic resonance imaging (MRI) are both used for noninvasive diagnosis of hepatocellular carcinoma (HCC) in patients with cirrhosis. To determine if there is a relative diagnostic benefit of one over the other, we synthesized evidence regarding the relative performance of CT, extracellular contrast-enhanced MRI, and gadoxetate-enhanced MRI for diagnosis of HCC in patients with cirrhosis. We also assessed whether liver biopsy versus follow-up with the same versus alternative imaging is best for CT-indeterminate or MRI-indeterminate liver nodules in patients with cirrhosis. We searched multiple databases from inception to April 27, 2016, for studies comparing CT with extracellular contrast-enhanced MRI or gadoxetate-enhanced MRI in adults with cirrhosis and suspected HCC. Two reviewers independently selected studies and extracted data. Of 33 included studies, 19 were comprehensive, while 14 reported sensitivity only. For all tumor sizes, the 19 comprehensive comparisons showed significantly higher sensitivity (0.82 versus 0.66) and lower negative likelihood ratio (0.20 versus 0.37) for MRI over CT. The specificities of MRI versus CT (0.91 versus 0.92) and the positive likelihood ratios (8.8 versus 8.1) were not different. All three modalities performed better for HCCs ≥2 cm. Performance was poor for HCCs <1 cm. No studies examined whether adults with cirrhosis and an indeterminate nodule are best evaluated using biopsy, repeated imaging, or alternative imaging. Concerns about publication bias, inconsistent study results, increased risk of bias, and clinical factors precluded support for exclusive use of either gadoxetate-enhanced or extracellular contrast-enhanced MRI over CT.

Conclusion

Background Advanced confounder-corrected chemical shift-encoded MRI-derived proton density fat fraction (PDFF) is a leading parameter for fat fraction quantification in nonalcoholic fatty liver disease (NAFLD). Because of the limited availability of this MRI technique, there is a need to develop and validate alternative parameters to assess liver fat. Purpose To assess relationship of quantitative US parameters to MRI PDFF and to develop multivariable quantitative US models to detect hepatic steatosis and quantify hepatic fat. Materials and Methods Adults with known NAFLD or who were suspected of having NAFLD were prospectively recruited between August 2015 and February 2019. Participants underwent quantitative US and chemical shift-encoded MRI liver examinations. Liver biopsies were performed if clinically indicated. The correlation between seven quantitative US parameters and MRI PDFF was evaluated. By using leave-one-out cross validation, two quantitative US multivariable models were evaluated: a classifier to differentiate participants with NAFLD versus participants without NAFLD and a fat fraction estimator. Classifier performance was summarized by area under the receiver operating characteristic curve and area under the precision-recall curve. Fat fraction estimator performance was evaluated by correlation, linearity, and bias. Results Included were 102 participants (mean age, 52 years ± 13 [standard deviation]; 53 women), 78 with NAFLD (MRI PDFF ≥ 5%). A two-variable classifier yielded a cross-validated area under the receiver operating characteristic curve of 0.89 (95% confidence interval: 0.82, 0.96) and an area under the precision-recall curve of 0.96 (95% confidence interval: 0.93, 0.99). The cross-validated fat fraction predicted by a two-variable fat fraction estimator was correlated with MRI PDFF (Spearman ρ = 0.82 [P < .001]; Pearson r = 0.76 [P < .001]). The mean bias was 0.02% (P = .97), and 95% limits of agreement were ±12.0%. The predicted fat fraction was linear with MRI PDFF (R ² = 0.63; slope, 0.69; intercept, 4.3%) for MRI PDFF of 34% or less. Conclusion A multivariable quantitative US approach yielded excellent correlation with MRI proton density fat fraction for hepatic steatosis assessment in nonalcoholic fatty liver disease. © RSNA, 2020 Online supplemental material is available for this article.

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Immune checkpoint blockade (ICB) therapy has revolutionized head and neck squamous cell carcinoma (HNSCC) treatment, but <20% of patients achieve durable responses. Persistent activation of the PI3K/AKT/mTOR signaling circuitry represents a key oncogenic driver in HNSCC; however, the potential immunosuppressive effects of PI3K/AKT/mTOR inhibitors may limit the benefit of their combination with ICB. Here we employ an unbiased kinome-wide siRNA screen to reveal that HER3, is essential for the proliferation of most HNSCC cells that do not harbor PIK3CA mutations. Indeed, we find that persistent tyrosine phosphorylation of HER3 and PI3K recruitment underlies aberrant PI3K/AKT/mTOR signaling in PIK3CA wild type HNSCCs. Remarkably, antibody-mediated HER3 blockade exerts a potent anti-tumor effect by suppressing HER3-PI3K-AKT-mTOR oncogenic signaling and concomitantly reversing the immune suppressive tumor microenvironment. Ultimately, we show that HER3 inhibition and PD-1 blockade may provide a multimodal precision immunotherapeutic approach for PIK3CA wild type HNSCC, aimed at achieving durable cancer remission.

Axillary surgery for breast cancer has continually evolved, with sentinel lymph node biopsy for clinically node-negative women with invasive breast cancer having long replaced axillary lymph node dissection. The information obtained from axillary staging has been important in providing prognostic information and guiding adjuvant treatment recommendations. However, recent studies suggest that sentinel lymph node biopsy should be omitted in select low-risk patients whose axillary surgery provides minimal prognostic value. This was highlighted by the Society of Surgical Oncology Choosing Wisely Guidelines, advocating against routine axillary staging in older women with early-stage hormone receptor-positive breast cancer. Since the guideline release, ongoing research has continued to identify the subset of low-risk patients who would benefit from the omission of axillary staging and improve adherence to Choosing Wisely to prevent overtreatment in older people.