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Abstract Introduction Given the prevalence and staggering cost of neurological disorders, there is dire need for effective early detection and intervention tools. Emerging evidence suggests that multidisciplinary lifestyle interventions (MLI) may mitigate the risk and progression of neurological disorders. The objectives of this protocol are (1) to test the impact of MLI on the progression of neurological disorders and (2) to identify multi-omic biomarkers for early stages of neurological disease and the impact of MLIs on these biomarkers. Methods and analysis We present the Multidisciplinary lifestyle Interventions for Neurological Disorders during the Silent phase (MINDS) protocol, a randomized controlled trial of MLI in neurologically healthy older adults (≥ 50 years old) exhibiting elevated risk for common neurological disorders: stroke, epilepsy, Parkinson’s Disease, or Alzheimer’s disease and related dementias. Participants will be randomly assigned to intervention (n = 100) or control (n = 100) groups. The intervention group will receive 3 months of weekly 2-hour sessions on diet education, yoga, music therapy, and cognitive skills training. The participants’ neurological health and engagement in relevant lifestyle practices will be assessed at regular intervals for 12 months. Neuroimaging and samples for multi-omic analyses will be collected at baseline, and at 3 months and 12 months after enrollment. Primary outcomes will be signs of progression of the neurological disorder risk that qualified them for study enrollment or a clinical diagnosis of the disorder. Secondary and exploratory outcomes will be based on self-reported health and multi-omic data. Data analysis will include between-group and longitudinal within-group analyses. Perspectives The MINDS protocol and trial aims to clarify the impact of MLI on the progression of neurological disorder risk or diagnosis in older adults and to identify biomarkers that can be used to confirm MLI efficacy. The ability to validate the impact of MLI on neurological disorder progression based on biomarker data allows the identification of individuals most likely to benefit from such therapies in the early stages of neurological disease. Trial registration The trial is registered on the National Institutes of Health (NIH) ClinicalTrials.gov (NCT05984056) site. It was registered on August 2nd, 2023. The trial has full approval of the Cleveland Clinic Internal Review Board.

Inadequate glycogen branching enzyme 1 (GBE1) activity results in different forms of glycogen storage disease type IV, including adult polyglucosan body disorder (APBD). APBD is clinically characterized by adult-onset development of progressive spasticity, neuropathy, and neurogenic bladder and is histologically characterized by the accumulation of structurally abnormal glycogen (polyglucosan bodies) in multiple cell types. How insufficient GBE1 activity causes the disease phenotype of APBD is poorly understood. We hypothesized that proteomic analysis of tissue from GBE1-deficient individuals would provide insights into GBE1-mediated pathobiology. In this discovery study, we utilized label-free LC–MS/MS to quantify the proteomes of lymphoblasts from 3 persons with APBD and 15 age- and gender-matched controls, with validation of the findings by targeted MS. There were 531 differentially expressed proteins out of 3,427 detected between APBD subjects vs. controls, including pronounced deficiency of GBE1. Bioinformatic analyses indicated multiple canonical pathways and protein–protein interaction networks to be statistically markedly enriched in APBD subjects, including: RNA processing/transport/translation, cell cycle control/replication, mTOR signaling, protein ubiquitination, unfolded protein and endoplasmic reticulum stress responses, glycolysis and cell death/apoptosis. Dysregulation of these processes, therefore, are primary or secondary factors in APBD pathobiology in this model system. Our findings further suggest that proteomic analysis of GBE1 mutant lymphoblasts can be leveraged as part of the screening for pharmaceutical agents for the treatment of APBD.

Summary: Background: Identifying novel epigenetic signatures associated with serum immunoglobulin E (IgE) may improve our understanding of molecular mechanisms underlying asthma and IgE-mediated diseases. Methods: We performed an epigenome-wide association study using whole blood from Framingham Heart Study (FHS; n = 3,471, 46% females) participants and validated results using the Childhood Asthma Management Program (CAMP; n = 674, 39% females) and the Genetic Epidemiology of Asthma in Costa Rica Study (CRA; n = 787, 41% females). Using the closest gene to each IgE-associated CpG, we highlighted biologically plausible pathways underlying IgE regulation and analyzed the transcription patterns linked to IgE-associated CpGs (expression quantitative trait methylation loci; eQTMs). Using prior UK Biobank summary data from genome-wide association studies of asthma and allergy, we performed Mendelian randomization (MR) for causal inference testing using the IgE-associated CpGs from FHS with methylation quantitative trait loci (mQTLs) as instrumental variables. Findings: We identified 490 statistically significant differentially methylated CpGs associated with IgE in FHS, of which 193 (39.3%) replicated in CAMP and CRA (FDR < 0.05). Gene ontology analysis revealed enrichment in pathways related to transcription factor binding, asthma, and other immunological processes. eQTM analysis identified 124 cis-eQTMs for 106 expressed genes (FDR < 0.05). MR in combination with drug-target analysis revealed CTSB and USP20 as putatively causal regulators of IgE levels (Bonferroni adjusted P

Summary: RNA splicing dysfunctions are more widespread than what is believed by only estimating the effects resulting by splicing factor mutations (SFMT) in myeloid neoplasia (MN). The genetic complexity of MN is amenable to machine learning (ML) strategies. We applied an integrative ML approach to identify co-varying features by combining genomic lesions (mutations, deletions, and copy number), exon-inclusion ratio as measure of RNA splicing (percent spliced in, PSI), and gene expression (GE) of 1,258 MN and 63 normal controls. We identified 15 clusters based on mutations, GE, and PSI. Different PSI levels were present at various extents regardless of SFMT suggesting that changes in RNA splicing were not strictly related to SFMT. Combination of PSI and GE further distinguished the features and identified PSI similarities and differences, common pathways, and expression signatures across clusters. Thus, multimodal features can resolve the complex architecture of MN and help identifying convergent molecular and transcriptomic pathways amenable to therapies.

Objective: To identify radiomic and clinical features associated with post-ablation recurrence of AF, given that cardiac morphologic changes are associated with persistent atrial fibrillation (AF), and initiating triggers of AF often arise from the pulmonary veins which are targeted in ablation. Methods: Subjects with pre-ablation contrast CT scans prior to first-time catheter ablation for AF between 2014–2016 were retrospectively identified. A training dataset (D1) was constructed from left atrial and pulmonary vein morphometric features extracted from equal numbers of consecutively included subjects with and without AF recurrence determined at 1 year. The top-performing combination of feature selection and classifier methods based on C-statistic was evaluated on a validation dataset (D2), composed of subjects retrospectively identified between 2005–2010. Clinical models ( $\text{M}_{\mathrm {C}}$ ) were similarly evaluated and compared to radiomic ( $\text{M}_{\mathrm {R}}$ ) and radiomic-clinical models ( $\text{M}_{\mathrm {RC}}$ ), each independently validated on D2. Results: Of 150 subjects in D1, 108 received radiofrequency ablation and 42 received cryoballoon. Radiomic features of recurrence included greater right carina angle, reduced anterior-posterior atrial diameter, greater atrial volume normalized to height, and steeper right inferior pulmonary vein angle. Clinical features predicting recurrence included older age, greater BMI, hypertension, and warfarin use; apixaban use was associated with reduced recurrence. AF recurrence was predicted with radio-frequency ablation models on D2 subjects with C-statistics of 0.68, 0.63, and 0.70 for radiomic, clinical, and combined feature models, though these were not prognostic in patients treated with cryoballoon. Conclusions: Pulmonary vein morphology associated with increased likelihood of AF recurrence within 1 year of catheter ablation was identified on cardiac CT. Significance: Radiomic and clinical features-based predictive models may assist in identifying atrial fibrillation ablation candidates with greatest likelihood of successful outcome.

Abstract Background We sought to determine if participating in a surgical training session using perfused fresh human cadavers (PFHC) had a positive effect on urology residents’ confidence in performing open and endoscopic procedures. Methods Urology residents at our institution participated in a surgical training session in the West Virginia University Fresh Tissue Training Program, which utilized fresh cadavers with vascular perfusion. The session consisted of performing different urologic procedures (open and endoscopic) on the perfused fresh human cadavers (PFHC). Residents were given a survey to rate their confidence in different urologic procedures before, after, and 6 months after the session. Each procedure on the survey had 3–6 questions associated with it, with scores ranging from 0 (no confidence) to 4 (great confidence). Scores for each procedure before and after the session were compared. Results Six residents participated in the session. There was an increase in the score for every procedure performed after the session. Scores at 6 month follow up remained higher than the pre-session scores. Conclusion PFHCs offer an excellent opportunity to teach a wide variety of urologic procedures to residents. Incorporation of PFHCs may be very useful in urologic training, and further studies on its use are warranted.

Summary: CFTR F508del (c.1521_1523delCTT, p.Phe508delPhe) is the most common pathogenic allele underlying cystic fibrosis (CF), and its frequency varies in a geographic cline across Europe. We hypothesized that genetic variation associated with this cline is overrepresented in a large cohort (N > 5,000) of persons with CF who underwent whole-genome sequencing and that this pattern could result in spurious associations between variants correlated with both the F508del genotype and CF-related outcomes. Using principal-component (PC) analyses, we showed that variation in the CFTR region disproportionately contributes to a PC explaining a relatively high proportion of genetic variance. Variation near CFTR was correlated with population structure among persons with CF, and this correlation was driven by a subset of the sample inferred to have European ancestry. We performed genome-wide association studies comparing persons with CF with one versus two copies of the F508del allele; this allowed us to identify genetic variation associated with the F508del allele and to determine that standard PC-adjustment strategies eliminated the significant association signals. Our results suggest that PC adjustment can adequately prevent spurious associations between genetic variants and CF-related traits and are therefore effective tools to control for population structure even when population structure is confounded with disease severity and a common pathogenic variant.

Summary: Background: Haploinsufficiency (HI) resulting from deletion of the long arm of chromosome 5 [del(5q)] and the accompanied loss of heterozygosity are likely key pathogenic factors in del(5q) myeloid neoplasia (MN) although the consequences of del(5q) have not been yet clarified. Methods: Here, we explored mutations, gene expression and clinical phenotypes of 388 del(5q) vs. 841 diploid cases with MN [82% myelodysplastic syndromes (MDS)]. Findings: Del(5q) resulted as founder (better prognosis) or secondary hit (preceded by TP53 mutations). Using Bayesian prediction analyses on 57 HI marker genes we established the minimal del(5q) gene signature that distinguishes del(5q) from diploid cases. Clusters of diploid cases mimicking the del(5q) signature support the overall importance of del(5q) genes in the pathogenesis of MDS in general. Sub-clusters within del(5q) patients pointed towards the inherent intrapatient heterogeneity of HI genes. Interpretation: The underlying clonal expansion drive results from a balance between the “HI-driver” genes (e.g., CSNK1A1, CTNNA1, TCERG1) and the proapoptotic “HI-anti-drivers” (e.g., RPS14, PURA, SIL1). The residual essential clonal expansion drive allows for selection of accelerator mutations such as TP53 (denominating poor) and CSNK1A1 mutations (with a better prognosis) which overcome pro-apoptotic genes (e.g., p21, BAD, BAX), resulting in a clonal expansion. In summary, we describe the complete picture of del(5q) MN identifying the crucial genes, gene clusters and clonal hierarchy dictating the clinical course of del(5q) patients. Funding: Torsten Haferlach Leukemia Diagnostics Foundation. US National Institute of Health (NIH) grants R35 HL135795, R01HL123904, R01 HL118281, R01 HL128425, R01 HL132071, and a grant from Edward P. Evans Foundation.

Abstract The utility of the International Working Group (IWG) 2006 response criteria for myelodysplastic syndromes (MDS) as a surrogate endpoint for outcomes is unclear. We assessed the validity of the IWG 2006 response criteria in a large cohort of higher‐risk MDS patients (pts) treated at centers from the MDS Clinical Research Consortium. The best overall response rate (ORR) by IWG 2006 criteria to first‐line therapy among 597 evaluable pts was 38% and include complete response (CR) 16%, marrow CR (mCR) 2%, partial response (PR) 10%, hematological improvement (HI) 10%, stable disease (SD) 33%, and progressive disease (PD) 24%. CR was associated with a better overall survival (OS) compared to all other response groups (P

Introduction Dysfunctional voiding (DV), defined as abnormal coordination between the urinary sphincter and the detrusor muscle in a neurologically intact individual, affects approximately 40% of patients that populate pediatric urology clinics. Improper hydration affects the specific gravity and concentration of urinary irritants, resulting in symptoms similar to those seen in DV patients. Methods Herein, we administered a validated DV survey (the Dysfunctional Voiding Symptom Score [DVSS]) and a validated dehydration severity chart (the Urine Color Chart [UCC]) to toilet trained pediatric patients and compared DV patients to patients presenting with non-urologic concerns. Data was analyzed on an item-for-item basis and by the total DVSS and UCC between the two groups. A total of 29 DV pediatric patients and 21 non-urologic pediatric patients were recruited from 2016 to 2018. Results Both patient groups were equivalent with regards to age, sex, height, weight, and BMI. The DV population had significantly higher scores in 7 out of 10 individual items within the DVSS when compared to those presenting for non-urologic complaints (p < 0.05). In addition, DV patients had significantly higher total scores on the DVSS than non-urologic patients (p = 0.0004). No significant difference was noted in UCC scores between patient groups (p = 0.753). Regression analysis showed that within the dysfunctional voiding group, there was a linear relationship between DVSS and UCC scores. Conclusion The present study suggests the DVSS results yield significantly different results for DV patients when compared to age, sex, and BMI matched children with no urologic complaints. The UCC does not reliably vary between DV and non-urologic patients; however, there appears to be a linear relationship between DVSS and UCC scores within the DV cohort. Further studies may elucidate a validated metric by which DV and non-urologic pediatric patients may be differentiated.

Quantitative trait locus mapping for interleukin-1β release after inflammasome priming and activation was performed on bone-marrow-derived macrophages (BMDM) from an AKRxDBA/2 mouse strain intercross. The strongest associated locus mapped very close to the Pycard gene on chromosome 7, which codes for the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The DBA/2 and AKR Pycard genes only differ at a single-nucleotide polymorphism (SNP) in their 3’ untranslated region (UTR). DBA/2 vs. AKR BMDM had increased levels of Pycard mRNA expression and ASC protein, and increased inflammasome speck formation, which was associated with increased Pycard mRNA stability without an increased transcription rate. CRISPR/Cas9 gene editing was performed on DBA/2 embryonic stem cells to change the Pycard 3’UTR SNP from the DBA/2 to the AKR allele. This single base change significantly reduced Pycard expression and inflammasome activity after cells were differentiated into macrophages due to reduced Pycard mRNA stability.

Abstract Background Testicular torsion (TT) is a urologic emergency that requires prompt surgical intervention. In rural Appalachia, patients are often transferred from surrounding communities due to lack of urologic care. We hypothesized that those transferred would have delayed intervention and higher rates of orchiectomy when compared to those who presented directly to our hospital. Methods We performed a retrospective review of patient charts with an ICD-9 diagnosis of TT from 2008 to 2016. Patients met inclusion criteria if diagnosis was confirmed by operative exploration. We compared rate of testicular loss and time until surgical intervention between groups. Results Twenty-three patients met inclusion criteria (12 transferred, 11 direct). Patient demographics did not significantly differ between groups. Transferred patients had a higher orchiectomy rate (33% v 22%,p = 0.41) although this was not statistically significant. Time to surgery from symptom onset was significantly longer in those transferred (12.9 h) compared to those not transferred (6.9 h, p = 0.02). Distance of transfer was not correlated with time of delay (r2 = 0.063). Conclusions Transferred patients with TT have numerically higher rates of orchiectomy which may reach significance in an appropriately powered study, and relative delays in surgical intervention. This study highlights the need for improved access to urologic care in rural areas.

In this paper, we describe two cases of Fournier’s gangrene (FG) in which Integra grafting was used for reconstruction. FG is a progressive necrotizing infection occurring in the perineal region and on the external genitalia. Reconstructive options using local tissue are limited due to the destruction this infection imposes on the soft tissue. Integra graft is a bilaminate artificial dermis made of shark chondroitin 6-sulfate and bovine collagen. It is applied to the wound bed after debridement and establishment of a healthy, well-vascularized wound base. The patients in this case series had large defects which could not be closed primarily with tissue beds and would not have been appropriate for skin grafting. Therefore, an Integra graft was placed. In both patients, the wound beds were appropriate for skin grafting after three weeks. Without the Integra graft, both of our patients would have needed to wait a considerable amount of time prior to reconstruction. Our case series further illustrates and supports the use of Integra grafts in such a scenario following Fournier’s gangrene which has only previously been published on three occasions, all of which demonstrated successful outcomes.

Background: SARS-CoV-2 enters cells by binding of its spike protein to angiotensin-converting enzyme 2 (ACE2). Angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II receptor blockers (ARBs) have been reported to increase ACE2 expression in animal models, and worse outcomes are reported in patients with co-morbidities commonly treated with these agents, leading to controversy during the COVID-19 pandemic over whether these drugs might be helpful or harmful. Methods: : Animal, in vitro and clinical data relevant to the biology of the renin-angiotensin system (RAS), its interaction with the kallikrein-kinin system (KKS) and SARS-CoV-2, and clinical studies were reviewed. Findings and Interpretation: SARS-CoV-2 hijacks ACE2to invade and damage cells, downregulating ACE2, reducing its protective effects and exacerbating injurious Ang II effects. However, retrospective observational studies do not show higher risk of infection with ACEI or ARB use. Nevertheless, study of the RAS and KKS in the setting of coronaviral infection may yield therapeutic targets.

Spontaneous urethral laceration in the female without associated external trauma is an exceedingly rare phenomenon. Most cases are related to childbirth or the presence of a concomitant pelvic fracture or penetrating injury. Herein, we present a novel case of spontaneous urethral laceration in a female which happened to occur during an acute flare of her previously diagnosed ulcerative colitis. The diagnosis of spontaneous urethral laceration is rare, and underlying etiology is often uncertain.

Abnormal PR interval duration is associated with risk for atrial fibrillation and heart block. Here, van Setten et al. identify 44 PR interval loci in a genome-wide association study of over 92,000 individuals and find genetic overlap with QRS duration, heart rate and atrial fibrillation.

Abstract Coronary artery disease (CAD) is the leading cause of death, and genetic factors contribute significantly to risk of CAD. This study aims to identify new CAD genetic loci through a large-scale linkage analysis of 24 large and multigenerational families with 433 family members (GeneQuest II). All family members were genotyped with markers spaced by every 10 cM and a model-free nonparametric linkage (NPL-all) analysis was carried out. Two highly significant CAD loci were identified on chromosome 17q21.2 (NPL score of 6.20) and 7p22.2 (NPL score of 5.19). We also identified four loci with significant NPL scores between 4.09 and 4.99 on 2q33.3, 3q29, 5q13.2 and 9q22.33. Similar analyses in individual families confirmed the six significant CAD loci and identified seven new highly significant linkages on 9p24.2, 9q34.2, 12q13.13, 15q26.1, 17q22, 20p12.3, and 22q12.1, and two significant loci on 2q11.2 and 11q14.1. Two loci on 3q29 and 9q22.33 were also successfully replicated in our previous linkage analysis of 428 nuclear families. Moreover, two published risk variants, SNP rs46522 in UBE2Z and SNP rs6725887 in WDR12 by GWAS, were found within the 17q21.2 and 2q33.3 loci. These studies lay a foundation for future identification of causative variants and genes for CAD.

The surgical treatments for neurogenic bladder are extremely variable. The lack of specific treatment guidelines makes this disease process even more challenging to treat. We present a case of a 55-year-old female with neurogenic bladder secondary to spinal cord injury (SCI). Her incontinence was conservatively managed with indwelling Foley drainage. Despite continued upsizing of the Foley catheters, the patient continued to have urinary leakage. The patient subsequently underwent a transvaginal bladder neck closure (BNC) with suprapubic bladder neck diversion (SPC). The urethra was successfully closed and uniquely supported with the use of cadaveric pericardial tissue (CPT). This surgical approach of neurogenic bladder provides durable continence with short operative times, minimal patient morbidity, decreased hospital length, and low risk of progressive renal dysfunction. BNC with SPC can provide an excellent management solution for neurogenic bladder from spinal cord injury refractory to conservative management.

Bladder rupture occurs in only 1.6% of blunt abdominopelvic trauma cases. Although rare, bladder rupture can result in significant morbidity if undiagnosed or inappropriately managed. AUA Urotrauma Guidelines suggest that urethral catheter drainage is a standard of care for both extraperitoneal and intraperitoneal bladder rupture regardless of the need for surgical repair. However, no specific guidance is given regarding the length of catheterization. The present study seeks to summarize contemporary management of bladder trauma at our tertiary care center, assess the impact of length of catheterization on bladder injuries and complications, and develop a protocol for management of bladder injuries from time of injury to catheter removal. A retrospective review was performed on 34,413 blunt trauma cases to identify traumatic bladder ruptures over the past 10 years (January 2008–January 2018) at our tertiary care facility. Patient data were collected including age, gender, BMI, mechanism of injury, and type of injury. The primary treatment modality (surgical repair vs. catheter drainage only), length of catheterization, and post-injury complications were also assessed. Review of our institutional trauma database identified 44 patients with bladder trauma. Mean age was 41 years, mean BMI was 24.8 kg/m2, 95% were Caucasian, and 55% were female. Motor vehicle collision (MVC) was the most common mechanism, representing 45% of total injuries. Other mechanisms included falls (20%) and all-terrain vehicle (ATV) accidents (13.6%). 31 patients had extraperitoneal injury, and 13 were intraperitoneal. Pelvic fractures were present in 93%, and 39% had additional solid organ injuries. Formal cystogram was performed in 59% on presentation, and mean time to cystogram was 4 hours. Gross hematuria was noted in 95% of cases. Operative management was performed for all intraperitoneal injuries and 35.5% of extraperitoneal cases. Bladder closure in operative cases was typically performed in 2 layers with absorbable suture in a running fashion. The intraperitoneal and extraperitoneal injuries managed operatively were compared, and length of catheterization (28 d vs. 22 d, p=0.46), time from injury to normal fluorocystogram (19.8 d vs. 20.7 d, p=0.80), and time from injury to repair (4.3 vs. 60.5 h, p=0.23) were not statistically different between cohorts. Patients whose catheter remained in place for greater than 14 days had prolonged time to initial cystogram (26.6 d vs. 11.5 d) compared with those whose foley catheter was removed within 14 days. The complication rate was 21% for catheters left more than 14 days while patients whose catheter remained less than 14 days experienced no complications. The present study provides a 10-year retrospective review characterizing the presentation, management, and follow-up of bladder trauma patients at our level 1 trauma center. Based on our findings, we have developed an institutional protocol which now includes recommendations regarding length of catheterization after traumatic bladder rupture. By providing specific guidelines for initial follow-up cystogram and foley removal, we hope to decrease patient morbidity from prolonged catheterization. Further study will seek to allow multidisciplinary trauma teams to standardize management, streamline care, and minimize complications for patients presenting with traumatic bladder injuries.

Leiomyomas are benign smooth muscle tumors that have low malignant potential (0.1%) and can arise in nearly any area of the body. Genitourinary involvement is very rare and represents only 0.05% of all bladder tumors (Mendes et al., 2017; GÖK, 2017). The most common presenting symptoms of bladder leiomyomas are obstructive voiding (49%), irritative voiding (38%), and hematuria (11%) (Goluboff et al., 1994). Treatment involves complete excision, in this case transurethral resection (TUR), and generally results in complete cure with no recurrences noted in the 250 cases reported in the literature for open resection and 18% recurrence rates after TUR which were successfully treated with a repeat TUR in all cases. Herein, we report a case of leiomyoma of the bladder which was refractory to four visually complete transurethral resections and ultimately required radical cystoprostatectomy with ileal conduit urinary diversion.

We present an unusual case of a rare ossifying renal tumor of infancy. A 6-month-old male initially presented with gross hematuria and without any palpable abdominal mass. Renal ultrasound and MRI showed a right lower pole, calcified, endophytic renal mass. Laparoscopic radical nephrectomy was performed without complications. Pathology demonstrated an ossifying renal tumor of infancy. We report this case, in addition to a review of the literature for similar cases, to highlight a rare renal tumor in infancy that can be managed laparoscopically.

Perspectives on whether the functions of MAS, a G protein-coupled receptor, are beneficial or deleterious in the heart remain controversial. MAS gene knockout reduces coronary vasodilatation leading to ischemic injury. G protein signaling activated by MAS has been implicated in progression of adaptive cardiac hypertrophy to heart failure and fibrosis. In the present study, we observed increased expression of MAS, connective tissue growth factor (CTGF) and collagen genes in failing (HF) human heart samples when compared to non-failing (NF). Expression levels of MAS are correlated with CTGF in HF and NF leading to our hypothesis that MAS controls CTGF production and the ensuing expression of collagen genes. In support of this hypothesis we show that the non-peptide MAS agonist AR234960 increases both mRNA and protein levels of CTGF via ERK1/2 signaling in HEK293-MAS cells and adult human cardiac fibroblasts. MAS-mediated CTGF expression can be specifically blocked by MAS inverse agonist AR244555 and also by MEK1 inhibition. Expression of CTGF gene was essential for MAS-mediated up-regulation of different collagen subtype genes in HEK293-MAS cells and human cardiac fibroblasts. Knockdown of CTGF by RNAi disrupted collagen gene regulation by the MAS-agonist. Our data indicate that CTGF mediates the profibrotic effects of MAS in cardiac fibroblasts. Blocking MAS-CTGF-collagen pathway should be considered for pharmacological intervention for HF.

Jalapeño peppers incur texture changes during thermal processing due to loss of turgor pressure, tissue softening, and pectin solubility in the cell wall, which affects their quality and acceptability as a processed product among consumers. The objective of this work is to evaluate the effect of stepwise blanching with calcium chloride solution on firmness, calcium absorption and microstructural changes of jalapeño peppers. Batches of 1 kg of pepper halves were blanched at 65 °C for 4 min at different concentrations of CaCl2 (0, 0.075, 0.15 and 0.3 mol/L). After blanching, the peppers were removed, placed in plastic bags and submerged in a water bath at 65 °C for different holding times (0, 15, 30 and 45 min). Then the peppers were blanched again in an acidified solution at 96 °C for 3 min to remove enzymatic activity. Afterwards, peppers were packed in glass containers with brine at 95 °C and pasteurized at 85 °C for 10 min. The product was stored for 10 days at room temperature, then analyzed for texture, calcium absorption, pH, acidity, and microstructural changes. Results showed that CaCl2 concentration and blanching improved firmness with a maximum value of 3.68 N, a 4.3-fold increase over control. Calcium absorption increased with CaCl2 concentration reaching a maximum of 0.68 g of Ca2+ per kg of samples. Microscopy showed reduced cell wall damage in the samples that were blanched in 0.3 mol/L of CaCl2 at 45 min of holding time. The optimum processing conditions determined in thisstudy can be used for improving the firmness of peppers.

To determine signaling pathways responsible for modulation of COX-2 expression in nontransformed, transformed epithelial cells, we studied a rat intestinal epithelial (RIE) cell line expressing constitutively active Ras, RhoA. Expression of COX-2 protein was higher in RIE-RhoA(63L) (four-fold), RIE-Ras(12V) (seven-fold) cells than in parental cells. Prior work suggests that Ras hyperactivity induces the expression of transforming growth factor (TGF) β, increases epidermal growth factor (EGF)-related peptide signaling-possible mechanisms for increased COX-2 expression. Expression of COX-2 was stimulated by TGFβ, TGFα in RIE, RIE-Rho(63L) cells, but not further stimulated in RIE-Ras(12V) cells. PD153035, an inhibitor of EGF receptor tyrosine kinase, PD98059, an inhibitor of Erk, attenuated COX-2 expression in RIE, RIE-RhoA(63L). However, the high levels of COX-2 expression in RIE-Ras(12V) cells were not inhibited by either compound. Titration with a panneutralizing anti-TGFβ antibody did not decrease COX-2 in RIE-Ras(12V) cells, even with concurrent EGFR inhibition. Thus, stimulation of the EGF receptor is important in the modulation of COX-2 expression in nontransformed RIE, RIE-RhoA(63L) cells. In Rastransformed cells, signaling by additional Ras effector pathways, perhaps the RhoA pathway, must be invoked. Identification of these pathways is critical for therapeutic manipulation of COX-2 expression.

Atrial Fibrillation (AF), the most common sustained arrhythmia, has a strong genetic component, but the mechanism by which common genetic variants lead to increased AF susceptibility is unknown. Genome-wide association studies (GWAS) have identified that the single nucleotide polymorphisms (SNPs) most strongly associated with AF are located on chromosome 4q25 in an intergenic region distal to the PITX2 gene. Our objective was to determine whether the AF-associated SNPs on chromosome 4q25 were associated with PITX2c expression in adult human left atrial appendages. Analysis of a lone AF GWAS identified four independent AF risk SNPs at chromosome 4q25. Human adult left atrial appendage tissue was obtained from 239 subjects of European Ancestry and used for SNP analysis of genomic DNA and determination of PITX2c RNA expression levels by quantitative PCR. Subjects were divided into three groups based on their history of AF and pre-operative rhythm. AF rhythm subjects had higher PITX2c expression than those with history of AF but in sinus rhythm. PITX2c expression was not associated with the AF risk SNPs in human adult left atrial appendages in all subjects combined or in each of the three subgroups. However, we identified seven SNPs modestly associated with PITX2c expression located in the introns of the ENPEP gene, ∼54 kb proximal to PITX2. PITX2c expression in human adult left atrial appendages is not associated with the chromosome 4q25 AF risk SNPs; thus, the mechanism by which these SNPs are associated with AF remains enigmatic.

Regulated intestinal stem cell proliferation and differentiation are required for normal intestinal homeostasis and repair after injury. The Notch signaling pathway plays fundamental roles in the intestinal epithelium. Despite the fact that Notch signaling maintains intestinal stem cells in a proliferative state and promotes absorptive cell differentiation in most species, it remains largely unclear how Notch signaling itself is precisely controlled during intestinal homeostasis. We characterized the intestinal phenotypes of brom bones, a zebrafish mutant carrying a nonsense mutation in hnRNP I. We found that the brom bones mutant displays a number of intestinal defects, including compromised secretory goblet cell differentiation, hyperproliferation, and enhanced apoptosis. These phenotypes are accompanied by a markedly elevated Notch signaling activity in the intestinal epithelium. When overexpressed, hnRNP I destabilizes the Notch intracellular domain (NICD) and inhibits Notch signaling. This activity of hnRNP I is conserved from zebrafish to human. In addition, our biochemistry experiments demonstrate that the effect of hnRNP I on NICD turnover requires the C-terminal portion of the RAM domain of NICD. Our results demonstrate that hnRNP I is an evolutionarily conserved Notch inhibitor and plays an essential role in intestinal homeostasis.

ObjectiveWe sought to identify rare variants influencing brain imaging phenotypes in the Framingham Heart Study by performing whole genome sequence association analyses within the Trans-Omics for Precision Medicine Program.MethodsWe performed association analyses of cerebral and hippocampal volumes and white matter hyperintensity (WMH) in up to 2,180 individuals by testing the association of rank-normalized residuals from mixed-effect linear regression models adjusted for sex, age, and total intracranial volume with individual variants while accounting for familial relatedness. We conducted gene-based tests for rare variants using (1) a sliding-window approach, (2) a selection of functional exonic variants, or (3) all variants.ResultsWe detected new loci in 1p21 for cerebral volume (minor allele frequency [MAF] 0.005, p = 10-8) and in 16q23 for hippocampal volume (MAF 0.05, p = 2.7 × 10-8). Previously identified associations in 12q24 for hippocampal volume (rs7294919, p = 4.4 × 10-4) and in 17q25 for WMH (rs7214628, p = 2.0 × 10-3) were confirmed. Gene-based tests detected associations (p ≤ 2.3 × 10-6) in new loci for cerebral (5q13, 8p12, 9q31, 13q12-q13, 15q24, 17q12, 19q13) and hippocampal volumes (2p12) and WMH (3q13, 4p15) including Alzheimer disease- (UNC5D) and Parkinson disease-associated genes (GBA). Pathway analyses evidenced enrichment of associated genes in immunity, inflammation, and Alzheimer disease and Parkinson disease pathways.ConclusionsWhole genome sequence-wide search reveals intriguing new loci associated with brain measures. Replication of novel loci is needed to confirm these findings.