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RNA binding proteins (RBPs) are central regulators of gene expression implicated in all facets of RNA metabolism. As such, they play key roles in cellular physiology and disease etiology. Since different steps of post-transcriptional gene expression tend to occur in specific regions of the cell, including nuclear or cytoplasmic locations, defining the subcellular distribution properties of RBPs is an important step in assessing their potential functions. Here, we present the RBP Image Database, a resource that details the subcellular localization features of 301 RBPs in the human HepG2 and HeLa cell lines, based on the results of systematic immuno-fluorescence studies conducted using a highly validated collection of RBP antibodies and a panel of 12 markers for specific organelles and subcellular structures. The unique features of the RBP Image Database include: (i) hosting of comprehensive representative images for each RBP-marker pair, with ∼250,000 microscopy images; (ii) a manually curated controlled vocabulary of annotation terms detailing the localization features of each factor; and (iii) a user-friendly interface allowing the rapid querying of the data by target or annotation. The RBP Image Database is freely available at https://rnabiology.ircm.qc.ca/RBPImage/.

SUMMARYAcute myeloid leukemia (AML) progression and relapse is fueled by self-renewing leukemic stem cells (LSCs) whose molecular determinants have been difficult to discern from normal hematopoietic stem cells (HSCs) or to uncover in screening approaches focused on general AML cell properties. We have identified a unique set of RNA binding proteins (RBPs) that are enriched in human AML LSCs but repressed in HSCs. Using an in vivo two step CRISPR-Cas9-mediated screening approach to specifically score for cancer stem cell functionality, we found 32 RBPs essential for LSC propagation and self-renewal in MLL-AF9 translocated AML. Using knockdown or small molecule approaches we show that targeting key hit RBP ELAVL1 impaired LSC-driven in vivo leukemic reconstitution and selectively depleted primitive AML cells vs. normal hematopoietic stem and progenitors. Importantly, knockdown of Elavl1 spared HSCs while significantly reducing LSC numbers across genetically diverse leukemias. Integrative RNA-seq and eCLIP-seq profiling revealed hematopoietic differentiation, RNA splicing and mitochondrial metabolism as key features defining the leukemic ELAVL1-mRNA interactome with the mitochondrial import protein TOMM34 being a direct ELAVL1-stabilized target whose inhibition impairs AML propagation. Altogether, through the use of a stem cell-adapted in vivo CRISPR dropout screening strategy, this work demonstrates that a wide variety of post-transcriptionally acting RBPs are important regulators of LSC-survival and self-renewal and, as exemplified by ELAVL1, highlights their potential as therapeutic targets in AML.

Pediatric medulloblastoma (MB) is the most common solid malignant brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that the RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. MSI1 inhibition abrogates tumor initiation and significantly prolongs survival in both models. We identified binding targets of MSI1 in normal neural and G3 MB stem cells and then cross referenced these data with unbiased large-scale screens at the transcriptomic, translatomic and proteomic levels to systematically dissect its functional role. Comparative integrative multi-omic analyses of these large datasets reveal cancer-selective MSI1-bound targets sharing multiple MYC associated pathways, providing a valuable resource for context-specific therapeutic targeting of G3 MB.

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/β. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

During an immune response to microbial infection, CD8+ T cells give rise to short-lived effector cells and memory cells that provide sustained protection. Although the transcriptional programs regulating CD8+ T cell differentiation have been extensively characterized, the role of long noncoding RNAs (lncRNAs) in this process remains poorly understood. Using a functional genetic knockdown screen, we identified the lncRNA Malat1 as a regulator of terminal effector cells and the terminal effector memory (t-TEM) circulating memory subset. Evaluation of chromatin-enriched lncRNAs revealed that Malat1 grouped with trans lncRNAs that exhibit increased RNA interactions at gene promoters and gene bodies. Moreover, we observed that Malat1 was associated with increased H3K27me3 deposition at a number of memory cell-associated genes through a direct interaction with Ezh2, thereby promoting terminal effector and t-TEM cell differentiation. Our findings suggest an important functional role of Malat1 in regulating CD8+ T cell differentiation and broaden the knowledge base of lncRNAs in CD8+ T cell biology.

RNA binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet, the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on UV-crosslinking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP- and cell type-specific RNA-protein interactions for multiple RBPs and cell types in single-pooled experiments. Pairing STAMP with long-read sequencing also yields RBP target sites for full-length isoforms. Finally, conducting STAMP using small ribosomal subunits (Ribo-STAMP) allows analysis of transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.

Aims: To evaluate the real-world effectiveness of vildagliptin and vildagliptin/metformin, combined with patient engagement, on glycemic outcomes. Patient engagement included both clinicians' engaging patients through education and counseling; and patients' self engagement through disease awareness, lifestyle changes, and medication adherence. Methods: Prospective, observational, open-label, multi-center, pharmacoepidemiologic study of type 2 diabetes mellitus (T2DM) patients treated de novo with vildagliptin or vildagliptin/metformin. Data were collected at baseline (treatment initiation), 105 +/- 15 d, and >= 145 d. Results: The evaluable sample included 896 mainly male (58%), overweight (mean +/- SD BMI =30.3 +/- 5.4 kg/m(2)), in later middle age (mean +/- SD age = 64 +/- 11years) patients. Over the three visits, mean(SD) HbA1c levels declined from 8.1%(+/- 1.0) to 7.3%(+/- 1.0) to 7.2%(+/- 0.9); HbA1c control rates rose from 7% to 36% to 43%. Mean +/- SD FPG levels decreased from 170(+/- 49) to 141(+/- 41) to 139(+/- 42) mg/dL; control rates increased from 12% to 39% to 43% (all p

Study Design. Part 1: A randomized, single-blind study on the effect of contralateral knee extension on sensations produced by the slump test (ST) in asymptomatic subjects. Part 2: A cadaver study simulating the nerve root behavior of part 1. Objective. Part 1: Test if contralateral knee extension consistently reduces normal stretch sensations with the ST. Objective. Part 2: Ascertain in cadavers an explanation for the results. Summary of Background Data. In asymptomatic subjects, contralateral knee extension reduces stretch sensations with the ST. In sciatica patients, contralateral SLR also can temporarily reduce sciatica. We studied this methodically in asymptomatic subjects before considering a clinical population. Methods. Part 1: Sixty-one asymptomatic subjects were tested in control (ST), sham, or intervention (contralateral ST) groups and their sensation response intensity compared. Methods. Part 2: Caudal tension was applied to the L5 nerve root of 3 cadavers and tension behavior of the contralateral neural tissue recorded visually. Results. Part 1: Reduction of stretch sensations occurred in the intervention group but not in control and sham groups (P ≤ 0.001). Results. Part 2: Tension in the contralateral lumbar nerve roots and dura reduced in a manner consistent with the responses in the intervention (contralateral ST) group. Conclusion. Part 1: In asymptomatic subjects, normal thigh stretch sensations with the ST reduced consistently with the contralateral ST, showing that this is normal and may now be compared with patients with sciatica. Conclusion. Part 2: Contralateral reduction in lumbar neural tension with unilateral application of tension-producing movements also occurred in cadavers, supporting the proposed explanatory hypothesis.

Introduction Surgical site infection is a potential complication of spinal cord stimulator (SCS) implantation. Current understanding of the epidemiology, diagnosis, and treatment of these infections is based largely on small clinical studies, many of which are outdated. Evidence-based guidelines for management of SCS-related infections thus rely instead on expert opinion, case reports, and case series. In this study, we aim to provide a large scale retrospective study of infection management techniques specifically for SCS implantation. Methods A multicenter retrospective study of SCS implants performed over a seven-year period at 11 unique academic and non-academic institutions in the United States. All infections and related complications in this cohort were analyzed. Results Within our study of 2737 SCS implant procedures, we identified all procedures complicated by infection (2.45%). Localized incisional pain and wound erythema were the most common presenting signs. Laboratory studies were performed in the majority of patients, but an imaging study was performed in less than half of these patients. The most common causative organism was Staphylococcus aureus and the IPG pocket was the most common site of an SCS-related infection. Explantation was ultimately performed in 52 of the 67 patients (77.6%). Non-explantation salvage therapy was attempted in 24 patients and was successful in resolving the infection in 15 patients without removal of SCS hardware components. Discussion This study provides current data regarding SCS related infections, including incidence, diagnosis, and treatment.

Objectives Spinal cord stimulation is an evidence-based treatment for a number of chronic pain conditions. While this therapy offers improvement in pain and function it is not without potential complications. These complications include device failure, migration, loss of therapeutic paresthesia, and infection. This article looked to establish a modern infection rate for spinal cord stimulators, assess the impact of known risk factors for surgical site infections and to determine the impact of certain preventative measures on the rate of infection. Methods After institutional review board approval, a multisite, retrospective review was conducted on 2737 unique implants or revisions of SCS systems. Patient demographics, risk factors including diabetes, tobacco use, obesity, revision surgery, trial length, implant location, implant type, surgeon background, prophylactic antibiotic use, utilization of a occlusive dressing, and post-operative antibiotic use were recorded and analyzed. Results The overall infection rate was 2.45% (n = 67). Diabetes, tobacco use, and obesity did not independently increase the rate of infection. Revision surgeries had a trend toward higher infection rate; however, this did not meet statistical significance. There was no difference in the rate of infection between implants performed by physicians of different base specialties, cylinder leads vs. paddle leads, or between different prophylactic antibiotics. Implants performed at academic centers had a higher rate of infection when compared to implants performed in nonacademic settings. When patients received an occlusive dressing or post-operative antibiotics they had a lower rate of infection. Conclusions The infection rate (2.45%) reported in this study is lower than the previously reported rates (3–6%) and are on par with other surgical specialties. This study did not show an increased rate of infection for patients that used tobacco, had diabetes or were obese. It's possible that given the low overall infection rate a larger study is needed to establish the true impact of these factors on infection. In addition, this study did not address the impact of poorly controlled diabetes mellitus (elevated hemoglobin A1c) vs. well-controlled diabetes. It can be concluded from this study that utilizing an occlusive dressing over the incision in the post-operative period decreases the rate of infection and should become the standard of care. This study also demonstrated the positive impact of post-operative antibiotics in decreasing the rate of infection. Studies in other surgical specialties have not shown this impact which would suggest that further research is needed.

Background. Acupressure has been shown to improve respiratory parameters. We investigated the effects of acupressure on weaning indices in stable coma patients receiving mechanical ventilation.Methods. Patients were randomly allocated to one of three treatments: standard care with adjunctive acupressure on one (n=32) or two days (n=31) and standard care (n=31). Acupressure in the form of 10 minutes of bilateral stimulation at five acupoints was administered per treatment session. Weaning indices were collected on two days before, right after, and at 0.5 hrs, 1 hr, 1.5 hrs, 2 hrs, 2.5 hrs, 3 hrs, 3.5 hrs, and 4 hrs after the start of treatment.Results. There were statistically significant improvements in tidal volumes and index of rapid shallow breathing in the one-day and two-day adjunctive acupressure study arms compared to the standard care arm immediately after acupressure and persisting until 0.5, 1 hr, and 2 hrs after adjustment for covariates.Conclusions. In the stable ventilated coma patient, adjunctive acupressure contributes to improvements in tidal volumes and the index of rapid shallow breathing, the two indices most critical for weaning patients from mechanical ventilation. These effects tend to be immediate and likely to be sustained for 1 to 2 hours.

Parathyroid carcinoma is a rare malignancy, and experience with its management is limited. It is generally accepted that surgery is the treatment of choice, and the initial operative intervention might represent the only chance for long-term success. At the time of surgery, a high index of suspicion for carcinoma is vital to performing the appropriate surgical procedures, because frozen-section diagnosis of parathyroid carcinoma is notoriously unreliable. In this article, we describe our experience with this rare entity.

Parathyroid carcinoma is a rare malignancy, and experience with its management is limited. It is generally accepted that surgery is the treatment of choice, and the initial operative intervention might represent the only chance for long-term success. At the time of surgery, a high index of suspicion for carcinoma is vital to performing the appropriate surgical procedures, because frozen-section diagnosis of parathyroid carcinoma is notoriously unreliable. In this article, we describe our experience with this rare entity.

Background and aims Obesity combined with hypertension places patients at greater risk for target-organ damage and cardiovascular disease. The purpose of this secondary analysis was to identify physician- and patient-levels determinants of blood pressure (BP) values and predictors of uncontrolled BP through subgroup analysis by body mass index (BMI). Methods and results We conducted a subgroup analysis of 3006 patients with High-BMI (BMI >25 kg/m 2 ; n = 2124) and Normal-BMI (BMI 2 ; n = 882) treated by 504 physicians and enrolled in PREVIEW, a Belgian prospective, multi-center, pharmaco-epidemiological study of 90-day second-line treatment with valsartan. Physician- and patient-level determinants of BP values and BP control were identified by means of hierarchical linear and logistic regression. Blood pressure values and control after 90 days of treatment were consistently lower for the High-BMI group. The 25.5% of variance in 90-day systolic and 28.3% of the variance in 90-day diastolic BP were attributable to physician-level determinants for the High-BMI group; versus 27.3% and 29.8% for the Normal-BMI group (ICC = 0.273 and 0.298, respectively). Determinants of 90-day BP values and predictors of uncontrolled BP varied considerably by BMI status. Conclusion Several common and unique patient- and physician-level determinants of BP values and control were identified for the High-BMI and Normal-BMI groups. These findings highlight the need for differentiating healthcare interventions to account for patient and physician variables, particularly with respect to effective BP management in vulnerable populations.

Magnetic Resonance Imaging (MRI) is known to be significantly affected by a number of acquisition artifacts, such as intensity non-standardness, bias field, and Gaussian noise. These artifacts degrade MR image quality significantly, obfuscating anatomical and physiological detail and hence need to be corrected for to facilitate application of computerized analysis techniques such as segmentation, registration, and classification. Specifically, algorithms are required to correct for bias field (intensity inhomogeneity), intensity non-standardness (drift in tissue intensities across patient acquisitions), and Gaussian noise, an artifact that significantly affects and blurs tissue boundaries (resulting in poor gradients). While clearly one needs to correct for all these artifacts, the exact sequence in which all three operations need to be applied in order to maximize MR image quality has not been explored. In this paper, we empirically evaluate the interplay between distinct algorithms for bias field correction (BFC), intensity standardization (IS), and noise filtering (NF) to study the effect of these operations on image quality in the context of 3 Tesla T2-weighted (T2w) prostate MRI. 7 different sequences comprising combinations of BFC, IS, and NF were quantitatively evaluated in terms of the percent coefficient of variation (%CV), a statistic which attempts to quantify the intensity inhomogeneity within a region of interest (prostate). The different combinations were also independently evaluated in the context of a classifier scheme for detection of prostate cancer on high resolution in vivo T2w prostate MRI. A secondary contribution of this work is a novel evaluation measure for quantifying the level of intensity non-standardness, called difference of modes (DoM). Experimental evaluation of the different sequences of operations across 22 patient datasets revealed that the sequence of BFC, followed by NF, and IS provided the best image quality in terms of %CV as well as classifier accuracy. The DoM measure was able to accurately capture the level of intensity non-standardness present in the images resulting from the different sequences of operations.