Skeletal muscle sodium channel gating in mice deficient in myotonic dystrophy protein kinase

Myotonic dystrophy, a progressive autosomal dominant disorder, is associated with an expansion of a CTG repeat tract located in the 3'-untranslated region of a serine/threonine protein kinase, DMPK. DMPK modulates skeletal muscle Na channels in vitro, and thus we hypothesized that mice deficient in DMPK would have altered muscle Na channel gating. We measured macroscopic and single channel Na currents from cell-attached patches of skeletal myocytes from mice heterozygous (DMPK(+/-)) and homozygous (DMPK(-/-)) for DMPK loss. In DMPK(-/-) myocytes, Na current amplitude was reduced because of reduced channel number. Single channel recordings revealed Na channel reopenings, similar to the gating abnormality of human myotonic muscular dystrophy (DM), which resulted in a plateau of Na current. The gating abnormality deteriorated with increasing age. In DMPK(+/-) muscle there was reduced Na current amplitude and increased Na channel reopenings identical to those in DMPK(-/-) muscle. Thus, these mouse models of complete and partial DMPK deficiency reproduce the Na channel abnormality of the human disease, providing direct evidence that DMPK deficiency underlies the Na channel abnormality in DM.     

Correcting signal biases and detecting regulatory elements in STARR-seq data

High-throughput reporter assays such as self-transcribing active regulatory region sequencing (STARR-seq) have made it possible to measure regulatory element activity across the entire human genome at once. The resulting data, however, present substantial analytical challenges. Here, we identify technical biases that explain most of the variance in STARR-seq data. We then develop a statistical model to correct those biases and to improve detection of regulatory elements. This approach substantially improves precision and recall over current methods, improves detection of both activating and repressive regulatory elements, and controls for false discoveries despite strong local correlations in signal.

Gene regulation is of foundational importance to nearly all biological processes, and variation in gene regulatory activity plays a major role in human disease risk (Lee and Young 2013; Parker et al. 2013; Finucane et al. 2015). A major step toward measuring regulatory activity across the human genome has been the development of high-throughput reporter assays such as STARR-seq (Arnold et al. 2013) that allow regulatory element activity to be quantified with high-throughput sequencing rather than with optical detection of a fluorescent or luminescent signal.High-throughput reporter assays create substantial analytical challenges that are distinct from other sequencing-based genomic assays. There is significant local variation in high-throughput reporter assay signal. We show here that, across data from several laboratories, most of that variation can be explained by features of the underlying genomic sequence and experimental procedures rather than by regulatory element activity. For example, nucleotide composition can alter PCR efficiency leading to under- and overrepresentation of some sequences. Meanwhile, highly repetitive sequences often do not align uniquely to the human reference genome, also biasing signal estimates. Additional analytical challenges include that STARR-seq signals can be both positive and negative, reflecting activation and repression, and the boundaries of regulatory elements are typically unknown and must therefore be estimated from the data. Those challenges together impact signal representations, hinder estimation of regulatory element activity, and cause false positives and false negatives when left unaddressed.Taken together, key requirements of statistical methods to analyze STARR-seq data are the ability to identify and estimate the effect of both activating and repressing regulatory elements while also correcting for underlying sequence biases in high-throughput reporter assays. A statistical model was recently introduced that corrects technical biases and detects regulatory elements in STARR-seq, but the model is limited to detecting only activating regulatory elements (Lee et al. 2020). Considering repression is a crucial gene regulation mechanism (Courey and Jia 2001), overlooking repressive elements may limit understanding of gene regulation with STARR-seq. To overcome that challenge, our correcting reads and analysis of differentially active elements (CRADLE) model takes a two-step approach. First, CRADLE uses a generalized linear regression model to estimate and correct major biases that we have identified in STARR-seq data. Next, CRADLE detects regions with statistically significant regulatory activity from the bias-corrected signals while rigorously controlling FDR. In doing so, CRADLE substantially improves the use of STARR-seq by providing a robust estimation of regulatory activity and improved visualization of raw signals.     

xlgv7: a maternal gene product localized in nuclei of the central nervous system in Xenopus laevis

The Xenopus oocyte nucleus (GV) is a storehouse for a large number of proteins that are used during early development. We have cloned and characterized a cDNA coding for a maternal gene product that is localized in the GV and then becomes highly enriched in the nuclei of the central nervous system (CNS) of tadpoles and adult frogs. This cDNA (xlgv7) is 2.1 kb and hybridizes to a 2.4-kb RNA species on Northern blots. Southern blots of genomic DNA suggest that this gene is a member of a multigene family. The cDNA sequence reveals a long open reading frame (ORF) of 1773 nucleotides, with a putative nuclear targeting signal (Glu Arg Arg Lys Lys Lys Thr) at the extreme carboxyl terminus and an internal histidine (His)-rich region with a repeated conserved amino acid sequence between His pairs. The significance of this region is unclear, but the protein is a DNA-binding protein, and it is possible that this region is involved in this function. The xlgv7 protein also possesses a putative nucleotide-binding consensus sequence that is similar to the bacterial RecA and RecB and yeast RAD proteins. Protein xlgv7 exists as several isotypes that exhibit developmental and cell-specific changes during development. Northern blot analysis of the abundance of the xlgv7 mRNA shows an accumulation following neural induction at stages 15-16. There is a transient expression of the mRNA in the gut of tadpoles. In the adult, the mRNA is highly enriched in the brain and is absent or in very low abundance in other tissues. Immunohistochemical analysis of the protein shows that the protein is localized in the nuclei of the brain cells. We conclude that the xlgv7 gene product is a maternal protein that may serve several important functions, one of which may be in the development and maintanance of the CNS.     

Intracellular potentials of microperfused human sweat duct cells

Intracellular potentials of cells from isolated segments of microperfused human sweat ducts were measured in order to determine the electrical profiles of these cells under resting, transporting, and inhibited conditions. Even though the cells are relatively small (ca. 6–8 m), continuous recordings of intracellular potentials from the same impalement were stable for up to 2 h. In the resting condition in normal Ringer's solution when the lumen of the duct was collapsed and not perfused, the intracellular potential measured across the basal membrane was 34.6±1.5 mV (n=31; mean±SE). In the same bathing medium, when the duct lumen was also perfused with normal Ringer's solution, the basolateral membrane potential (V _b), the apical membrane potential (V _a) and transepithelial potential (V _t) was –33.8±0.47 mV, –23.7±0.48 mV and –9.6±0.9 mV (n=73), respectively. The average input impedence (R _i) of these cells was 19.6±0.4 M (n=36). The frequency distribution ofV _b was unimodal suggesting that only one functional cell type exists in this tissue. Amiloride (0.1 mM) in the lumen hyperpolarized bothV _a andV _b by –40.5±3.6 mV and –33.2±3.7 mV (n=15), respectively, with a slight but significant increase inR _i (15%), while abolishingV _t. Removing luminal Cl^– depolarizedV _a by +37.0±4.2 mV and hyperpolarizedV _b by –19.0±4.2 mV (n=11). Removing Cl^– from the bath hyperpolarizedV _a by –3.3±2.3 mV and depolarizedV _b by +24.3±2.7 mV (n=15). Ouabain caused an initial fast depolarization (+8 mV) followed by a prolonged slow depolarization ofV _b, and an increase inR _i of about 84%. These results not only provide the first electrical profile of the human sweat duct tissue, but they also show that its cell membrane potentials are unusually low. This unusual property of this epithelium appears to be due to the combination of a significant Na⁺ conductance at the apical membrane and a remarkably high tissue Cl^– conductance.     

Prostate cancer: results of external irradiation.

From 1975 to 1982, 205 patients with local prostate cancer were treated at the radiation oncology department, the University of Kansas Medical Center, Kansas City, Kansas. Patients'' median age was 73 years. All of the patients were staged according to American Urologic staging criteria. Twenty-eight patients had stage A2 cancer, 91 patients had stage B cancer, and 86 patients had stage C cancer. All patients were treated using megavoltage radiation (dosage range: 6000 cGy to 7100 cGy). The follow-up period ranged from a minimum of 8 years to a maximum of 15 years (median: 9.4 years). The clinical local control was 96% for stage A2, 94% for stage B, and 90% for stage C disease. The overall and disease-free survival rates were 71% and 60%, respectively. Fourteen patients developed moderate complications with one patient (0.5%) requiring surgical intervention. The local control and survival rates reported in this study are comparable with surgical results, suggesting that external beam irradiation in prostate cancer is safe and effective.     

Surgical management of synchronous coronary artery disease and multiple aortic stenosis: a case report with brief review of the literature

One-stage surgery was successfully performed in a 44-year-old hypertensive man with uncontrolled angina, multiple coarctations of the thoracic and abdominal aorta, and a previous subtotal gastrectomy. There was a gradient of 120 mm Hg between the thoracic and abdominal aorta. A graft was placed retroperitoneally from the infrarenal aorta to the ascending aorta and was followed by a coronary artery bypass graft. Twenty-four months postoperatively, the patient was free of angina, and his hypertension was easily controlled.     

Male pseudohermaphrodite with persistent Mullerian duct: A radiologist’s and patient’s dilemma alike

This case study highlights that how a disorder of sexual development when goes unnoticed at birth and unreported during childhood or adolescence can present with major problems and even complications in adulthood. Since our patient was young and in a childbearing age, he presented with bilateral undescended testes and orgasmic anejaculation when he first came to the hospital. Subsequently, having a normal 46XY karyotype but remnants of persistent Mullerian duct made him little confused about his identity. After giving him the confidence, that he was still a male and could lead the life he did previously, the explanation about future risk of malignancy in the intra-abdominal testes was another difficult task. Early detection and management of male pseudohermaphroditism with persistent Mullerian duct requires a co-ordinated approach of a team of endocrinologist, physician, surgeon and radiologist. Integrated imaging in the form of ultrasound, genitography and MRI is important in demonstrating the anatomy, classification, possible effects or congenital malformations in other organs, warning patients of any risk of neoplasia and guiding the clinician to plan other investigations, hormonal replacement or reconstruction surgery if required. Such a systemic approach that allays anxiety and gives psychological relief to the patient should be taken as it can deeply change the life of a person and their family.     

Does Early Debridement,Antibiotic Therapy and Implant Retention (DAIR) have a Role in Managing Periprosthetic Joint Infection of the Knee in Indian Scenario: A Retrospective Analysis of Outcomes

PurposeTo report outcomes of Debridement, Antibiotic therapy and Implant Retention (DAIR) for periprosthetic knee joint infections (PJI) in the Indian population and to study factors influencing outcomes.MethodsThis was a Retrospective study of 80 cases of acute PJI after total knee arthroplasty who were treated by DAIR, within 2 weeks of onset of infection. A standardised institutional management protocol was applied to all cases. Patients were followed up for a minimum 1 year. Outcomes of DAIR were classified as successful or unsuccessful based on resolution or persistence of infection, and subsequent requirement of revision surgery. Influence of factors, like comorbidities, culture status and microbiological characteristics of causative organism, on outcomes was assessed.ResultsOverall 55 patients (68.75%) had successful eradication of infection after DAIR. 27 (33.7%) patients were culture negative and 53 (66.2%) patients grew organisms on culture. There was no statistically significant difference in outcomes (p = 0.082) between culture-positive cases (69.8% success rate) and (66.7% success rate) in culture negative cases. Furthermore, no difference in outcomes was observed in culture-positive patients between those who grew Gram-positive organisms versus Gram-negative organisms (p = 0.398) Similarly, patient comorbidities did not significantly alter the outcomes after DAIR (p = 0.732).ConclusionOur study demonstrates that early DAIR within 2 weeks of onset of infection using a standard protocol during surgery and postoperatively can result in good outcomes. Patient comorbidities, culture status (positive versus negative), Gram staining characteristics of organisms and the identity of pathogenic bacteria did not influence outcomes of DAIR for acute PJI.     

Pro-inflammatory cytokines and chemokines initiate multiple prostate cancer biologic pathways of cellular proliferation,heterogeneity and metastasis in a racially diverse population and underlie the genetic/biologic mechanism of racial disparity: Update

Pro-inflammatory cytokine and chemokines genes drive prostate cancer progression and metastasis: molecular mechanism update and the science that underlies racial disparity. comprehensive review article.Isaac J. Powell, S. Chinni, S.S. Reddy, Alexander Zaslavsky, Navnath Gavande Introduction: In 2013 we reported that with the use of bioinformatics and ingenuity pathway network analysis we were able to identify functional driver genes that were differentially expressed among a large population of African American men (AAM) and European American men (EAM). Pro-inflammatory cytokine genes were found to be more interactive and more expressed among AAM and have been found to be functional drivers of aggressive prostate cancer (CaP) and aggressiveness in other solid tumors. We examined these genes and biological pathways initiated by these cytokines in primary CaP tissue.Method We unravel the gene network and identified biologic pathways that impacted activation of the androgen receptor, mesenchymal epithelial transition (invasion) and chemokines associated with metastasis in the CaP tissue from 639 radical prostatectomy specimens.Results Biologic pathways identified by unraveling pro-inflammatory genes from our network, more expressed among AAM compared to EAM, were tumor necrosis factor (TNF), IL1b, IL6, and IL8. IL6 and IL8 are downstream of TNF activity and are known activators of androgen receptor and through mediators promote CaP cell proliferation. TNF and IL1b mediate tumor cell invasiveness through the activation of MMP (matrix metalloproteinase) which down regulates E-Cadherin to initiate epithelial mesenchymal transition which allows cells to become invasive in the microenvironment. Ultimately our network analysis indicates that TNF and IL1b activate CXCR4 receptor on CaP cells, which facilitates metastatic progression reportedly by binding to CXCL12 on lipid rafts and tumor implantation in the bone marrow.Conclusion Our retrospective biologic mechanistic model reveals a set of pro-inflammatory cytokines and chemokines that drive CaP aggressiveness, tumor heterogeneity, progression and metastasis. A prospective multi-institutional study needs to be conducted for clinical validation as well consideration of targeted therapy.