Sites of transcription initiation drive mRNA isoform selection.

The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.

Da Vinci vs. Hugo RAS for robot-assisted radical prostatectomy: a prospective comparative single-center study.

PURPOSE: To evaluate Hugo RAS against the Da Vinci system for Robot-Assisted Radical Prostatectomy (RARP) in prostate cancer treatment. METHODS: We compared outcomes of 150 patients with prostate cancer undergoing RARP with either Hugo or Da Vinci systems. Our analysis included operative, postoperative, pathological, and functional outcomes. RESULTS: Both groups had 75 patients. Baseline characteristics and tumor features were similar. Intraoperatively, Da Vinci had a shorter docking time (10.45 vs. 18.62 min, p = 0.02), but total operative times were comparable (145.34 vs 138.95, p = 0.85). Hugo outperformed in neck dissection and lymphadenectomy times (22 vs 13.67 min, p = 0.027 and 37.82 vs 45.77 min, p = 0.025). Postoperative metrics like stay duration, catheter time, and complications showed no significant difference. Functional results, using IPSS and IIEF5, were similar between systems. Six Da Vinci patients (8%) and nine Hugo patients (12%) experienced social incontinence (p = 0.072). Pathological outcomes like T stage, Gleason Score, and nodes removed were alike. However, Hugo had more positive surgical margins (20% vs. 10.67%, p = 0.034). CONCLUSIONS: RARP outcomes using Hugo RAS were similar to the Da Vinci system in our study. More research and extended follow-up are required to ascertain long-term oncological and functional results.

Leprosy ulcers in a rural hospital of Ethiopia: pattern of aerobic bacterial isolates and drug sensitivities.

BACKGROUND: Plantar ulcers, which commonly occur in leprosy patients, tend to recur increasing physical disability. The aim of this study is to identify both the bacteriological profile of these ulcers and the antibiotic susceptibility of the isolated bacteria. MATERIALS AND METHODS: 68 leprosy patients with chronic ulcers attending the in-patient department of Gambo General Hospital, West Arsi, were included in this study. Proper sample collection, inoculation on culture media, and final identification using biochemical methods were undertaken. RESULTS: 66 patients (97.1%) had a positive culture. A total of 81 microorganisms were isolated. Multiple organisms (two or more) were isolated in 15 (22.7% out of positive culture) patients. The main isolation was Proteus spp (30.9%), followed by Escherichia coli (21.0%), Staphylococcus aureus (18.5%) and Pseudomonas aeruginosa (9.9%). In the total number of the isolated bacteria, the antibiotics with less resistance were gentamicin (18.5%), fosfomycin (22.2%) cefoxitin (24.7%), ceftriaxone (25.9%) ciprofloxacin (25.9%), and amoxicillin-clavulanic acid (28.49%). CONCLUSION: The bacteriological study of plantar ulcers of leprosy patients revealed Enterobacteriaceae and S. aureus as the main pathogens involved in such infections. The results of this study may guide empirical therapy in a rural area hospital where culture and susceptibility testing facilities are scarce.

Da Vinci and Hugo RAS Platforms for robot-assisted partial nephrectomy: a preliminary prospective comparative analysis of the outcomes.

BACKGROUND: Robot-assisted partial nephrectomy (RAPN) has emerged as the preferred approach for T1 renal-cell-carcinoma. As new robotic platforms like Hugo RAS emerge, we seek to understand their potential in achieving similar RAPN outcomes as the established Da Vinci system. METHODS: A prospective single-center comparative study was conducted, and 50 patients selected for RAPN were enrolled (25 Da Vinci Xi; 25 Hugo RAS). The choice of robotic system was based solely on hospital logistics criteria. Surgeries were performed by expert surgeons. Demographic data, tumor characteristics, operative details and postoperative outcomes were collected. SPSS version 22.0 was used for statistical analyses. RESULTS: The average age of patients was 62.52±9.47 years, with no significant differences in median age, sex, and nephrometry scores between groups. Da Vinci group showed a significantly shorter docking time (12.56 vs. 20.08 min; P<0.01), while other intraoperative measures like console time and warm ischemia time were similar. The Hugo RAS group had a shorter renorraphy time (14.33 vs. 18.84 min; P=0.024). Postoperative outcomes and surgical margin positivity showed no significant differences. Each group had one patient (4%) who developed major surgical complications (Clavien IIIa). Trifecta rates were comparable between both groups (Da Vinci 88% vs. Hugo RAS 84%; P=0.93). CONCLUSIONS: Initial findings suggest similar perioperative outcomes for RAPN when using Hugo RAS compared to the Da Vinci system. Further research with long-term follow-up is necessary to evaluate oncological and functional outcomes.

Genetic evidence for an evolutionarily conserved role of IL-7 signaling in T cell development of zebrafish.

In mammals, the cytokine IL-7 is a key regulator of various aspects of lymphocyte differentiation and homeostasis. Because of the difficulty of identifying cytokine homologs in lower vertebrates and the paucity of assay systems and reagents, the degree of functional conservation of cytokine signaling pathways, particularly those pertaining to lymphocyte development, is unclear. In this article, we report on the analysis and characterization of three zebrafish mutants with severely impaired thymopoiesis. The identification of affected genes by positional cloning revealed components of the IL-7 signaling pathway. A presumptive null allele of the zebrafish homolog of the IL-7Rα-chain causes substantially reduced cellularity of the thymus but spares B cell development in the kidney. Likewise, nonsense mutations in the zebrafish homologs of janus kinases JAK1 and JAK3 preferentially affect T cell development. The functional interactions of the cytokine receptor components were examined in the three groups of fish hetero- or homozygous for either il7r and jak1, il7r and jak3, or jak1 and jak3 mutations. The differential effects on T cell development arising from the different genotypes could be explained on the basis of the known structure of the mammalian IL-7R complex. Because IL-7 signaling appears to be a universal requirement for T cell development in vertebrates, the mutants described in this article represent alternative animal models of human immunodeficiency syndromes amenable to large-scale genetic and chemical screens.

Cognitive and behavioral profile in females with epilepsy with PDCH19 mutation: two novel mutations and review of the literature.

Mutation in the protocadherin 19 (PCDH19) gene is an increasingly recognized cause of epilepsy in females. This disorder is frequently associated with mental retardation and psychiatric features. We describe two unrelated females with novel PCDH19 missense mutations. One was de novo, and the other was inherited from her unaffected father. Both had mild mental impairment but had remarkable behavioral problems. We reviewed the cognitive and behavioral profiles of previously reported PCDH19-positive cases. Intellectual disability appeared in 75% of patients, ranging from borderline to severe. More than half of the individuals presented behavioral disturbances, which could be divided into two different groups: autistic and non-autistic. The majority of patients with autism already had some degree of cognitive impairment. It appears that seizures tend to diminish or even stop in adolescence, so non-epileptic problems can become the most important and disabling issue in adult patients with PCDH19 mutation.

A critical developmental window for ELAV/Hu-dependent mRNA signatures at the onset of neuronal differentiation.

Cell-type-specific gene regulatory programs are essential for cell differentiation and function. In animal neurons, the highly conserved ELAV/Hu family of proteins promotes alternative splicing and polyadenylation of mRNA precursors to create unique neuronal transcript isoforms. Here, we assess transcriptome profiles and neurogenesis success in Drosophila models engineered to express differing levels of ELAV activity in the course of development. We show that the ELAV-mediated establishment of a subset of neuronal mRNA isoforms at the onset of neuron differentiation constitutes a developmental bottleneck that cannot be overcome later by the nuclear activation of the paralog found in neurons (FNE). Loss of ELAV function outside of that critical time window results in neurological defects. We find that FNE, when activated early enough, can restore ELAV-dependent neuronal mRNA isoforms and fully rescue development. Our findings demonstrate the essential role of robust cellular strategies to maintain ELAV activity and intact neuronal signatures in neurogenesis and neuronal function.

Outcomes of laparoscopic salvage radical prostatectomy after primary treatment of prostate cancer.

Introduction: Treatment of radio-recurrent prostate cancer (PC) is managed mainly by androgen deprivation therapy. Nonetheless, selected patients could benefit from local salvage treatment options.In this study we present our series of recurrent PC cases submitted to laparoscopic salvage radical prostatectomy (sRP) at our institution. Material and methods: A total of 29 patients with recurrent PC after primary non-surgical treatment were submitted to laparoscopic sRP at our institution, with a mean follow-up time of 7 years. Results: There were 7 post-operative complications Clavien-Dindo grade ≥2. At the end of the follow-up, 58.6% patients presented biochemical recurrence and five-year recurrence-free survival (RFS) was 50%.Positive lymph nodes, high preoperative prostate-specific antigen (PSA) and TNM stage were correlated with worse RFS. Cox regression analysis demonstrated that stage pT3b was independently associated with worse RFS in comparison with stage pT3a or less.At 12 months, pad-free continence or mild incontinence was observed in 62% of the patients. Conclusions: sRP is a technically challenging surgery, and in our series, we were able to perform this procedure with acceptable operative time and limited blood loss.Post-operative complications, functional results and oncological outcomes were similar to other published studies, being our series, to the best of our knowledge, the one with the longest follow-up, of 7 years.sRP is a feasible local treatment with curative intent for radio-recurrent prostate cancer, with good oncological outcomes and reasonable continence rates in selected patients.

Genetic landscape of T cells identifies synthetic lethality for T-ALL.

To capture the global gene network regulating the differentiation of immature T cells in an unbiased manner, large-scale forward genetic screens in zebrafish were conducted and combined with genetic interaction analysis. After ENU mutagenesis, genetic lesions associated with failure of T cell development were identified by meiotic recombination mapping, positional cloning, and whole genome sequencing. Recessive genetic variants in 33 genes were identified and confirmed as causative by additional experiments. The mutations affected T cell development but did not perturb the development of an unrelated cell type, growth hormone-expressing somatotrophs, providing an important measure of cell-type specificity of the genetic variants. The structure of the genetic network encompassing the identified components was established by a subsequent genetic interaction analysis, which identified many instances of positive (alleviating) and negative (synthetic) genetic interactions. Several examples of synthetic lethality were subsequently phenocopied using combinations of small molecule inhibitors. These drugs not only interfered with normal T cell development, but also elicited remission in a model of T cell acute lymphoblastic leukaemia. Our findings illustrate how genetic interaction data obtained in the context of entire organisms can be exploited for targeted interference with specific cell types and their malignant derivatives.

Metachronous Signet Ring Cell Bladder Metastasis as First Sing of Cancer Recurrence.

We describe the case of a 74-year-old woman with previous history of gastric signet cell carcinoma who develops bladder metastasis as first sign of recurrence 6 years later. Bladder metastasis due to signet cell carcinoma is extremely rare with only 19 cases reported. Treatment includes radical cystectomy or chemotherapy.

Conferring the binding properties of the mouse MHC class I-related receptor, FcRn, onto the human ortholog by sequential rounds of site-directed mutagenesis.

The MHC class I-related receptor, FcRn, is involved in binding and transporting immunoglobulin G (IgG) within and across cells. In contrast to mouse FcRn, which binds to IgGs from multiple different species, human FcRn is surprisingly stringent in binding specificity. For example, human FcRn does not bind to mouse IgG1 or IgG2a and interacts only weakly with mouse IgG2b. Here, we have used site-directed mutagenesis in combination with interaction (surface plasmon resonance) studies, with the goal of generating human FcRn variants that more closely resemble mouse FcRn in binding specificity. Our studies show that residues encompassing and extending away from the interaction site on the alpha2 helix of FcRn play a significant and most likely indirect role in FcRn-IgG interactions. Further, by combining mutations in the alpha2 helix with those in a non-conserved region of the alpha1 helix encompassing residues 79-89, we have generated a human FcRn variant that has properties very similar to those of mouse FcRn. These studies define the molecular basis for the marked difference in binding specificity between human and rodent FcRn, and give insight into how human FcRn recognizes IgGs.

Forward Genetic Screens in Zebrafish Identify Pre-mRNA-Processing Pathways Regulating Early T Cell Development.

Lymphocytes represent basic components of vertebrate adaptive immune systems, suggesting the utility of non-mammalian models to define the molecular basis of their development and differentiation. Our forward genetic screens in zebrafish for recessive mutations affecting early T cell development revealed several major genetic pathways. The identification of lineage-specific transcription factors and specific components of cytokine signaling and DNA replication and/or repair pathways known from studies of immunocompromised mammals provided an evolutionary cross-validation of the screen design. Unexpectedly, however, genes encoding proteins required for pre-mRNA processing were enriched in the collection of mutants identified here. In both zebrafish and mice, deficiency of the splice regulator TNPO3 impairs intrathymic T cell differentiation, illustrating the evolutionarily conserved and cell-type-specific functions of certain pre-mRNA-processing factors for T cell development.

Clinical and histologic changes in the follow-up of a congenital myopathy.

A 19-year-old woman was born with congenital hypotonia, generalized weakness, and dysmorphic features. A muscle biopsy performed at age 18 months found that type I fibers were smaller and more numerous than type II fibers, and she was diagnosed with congenital fiber type disproportion. She grew up with moderate motor impairment, but after a stationary period her weakness progressed gradually and she developed a severe ophthalmoplegia. When she was 18 years old a second muscle biopsy still indicated the predominance of type I fibers but also the presence of central nuclei and strong oxidative enzyme activity in the center of most of the fibers; this was compatible with centronuclear myopathy. The diagnostic reconsideration raises questions about the pathogenesis of these diseases and the recognition of congenital fiber type disproportion as a distinct nosologic entity.

Macrophagic myofasciitis in childhood: a controversial entity.

Macrophagic myofasciitis is an unusual inflammatory myopathy, which has been almost exclusively reported in French adults with diffuse arthromyalgias and asthenia. It is characterized by an infiltrate of densely packed macrophages, with granular periodic-acid-Schiff positive content, on muscle biopsies at the site of vaccination. The presence of aluminum inclusions in these macrophages points to an inappropriate reaction to aluminum used as an adjuvant in some vaccines. Although in adults this entity is well defined, less than 15 cases have been reported in children. This study describes seven children, younger than 3 years of age, with typical lesions of macrophagic myofasciitis on quadriceps muscle biopsy. In five cases, biopsies were performed to exclude mitochondrial pathology. All the children developed hypotonia and motor or psychomotor delay, associated with others symptoms. Abnormal neuroimaging was evident in six cases. Spectrometry studies detected elevated levels of aluminum in muscle in three of four cases tested. Despite the wide use of vaccines in childhood, macrophagic myofasciitis was rarely observed in children and its characteristic histologic pattern could not be correlated with a distinctive clinical syndrome.

[Interferon in hepatitis B]. / Interferón en la hepatitis B.

Hepatitis B virus (HBV) infection is a worldwide public health problem. There are an estimated 350 million persons with chronic HBV infection that could progress to cirrhosis and hepatocarcinoma with nearly one million deaths per year. In the last few years the therapeutic options in chronic hepatitis B have increased and currently six treatments are authorized: standard interferon (IFN)-alpha, pegylated interferon-alpha (PEG-IFNalpha), lamivudine, adefovir, entecavir, and telbivudine. For the last 25 years, conventional IFNalpha has been used as the treatment of chronic hepatitis B (CHB) and currently PEG-IFNalpha is indicated due to its greater effectiveness. Both drugs are first line options for HBeAg(+) and HBeAg(-) CHB. The advantages of IFNalpha and PEG-IFNalpha are that these drugs are administered for a limited time period, they achieve a higher sustained response rate and do not induce HBV mutants with antiviral resistance. These drugs achieve greater HBeAG and HBsAG clearance due to their antiviral and immunomodulatory activity. PEG-IFNalpha induces sustained biochemical and virological response in approximately one third of patients with HBeAg(+) CHB. The best response to IFNalpha and PEG-IFNalpha is obtained in patients with elevated transaminase levels, moderate viral load and HBV genotypes A and B. The disadvantages of IFNalpha and PEG-IFNalpha are their adverse effects and contraindications. These drugs cannot be administered in patients with decompensated cirrhosis. The combination of nucleos(t)ide analogs with PEG-IFNalpha could achieve much higher sustained response rates; however, which treatment constitutes the most suitable therapeutic strategy requires investigation.

The role of CCR5/CXCR3 expressing CD8+ cells in liver damage and viral control during persistent hepatitis C virus infection.

BACKGROUND/AIMS: CXCR3 and CCR5 play a major role in recruiting cytotoxic T cells (Tc) and secreting secondary type 1 cytokines (Tc1) in the liver. HCV could impair their expression as a survival mechanism. The role of these chemokine receptors on CD8+ cells in chronic hepatitis C is analysed. METHODS: Serum, chemokines, peripheral blood and intrahepatic lymphocytes from chronic hepatitis C patients were studied. CXCR3/CCR5 expressing CD8+ cells were quantified by flow-cytometry. Serum chemokines concentration (CXCL10/CCL3) was measured by ELISA. Basal data were correlated with liver inflammation. Longitudinal data were obtained during treatment and correlated with virologic response. RESULTS: CCR5/CXCR3 expressing CD8+ cells were enriched in the liver and correlated with inflammation. Chronic HCV patients presented the same frequency of CCR5(high)/CXCR3(high) expressing CD8+ cells in peripheral blood as in healthy controls but higher serum concentration of CXCL10/CCL3. Treatment with PEG-interferon alpha-2b plus ribavirin increased CCR5(high)/CXCR3(high) expressing CD8+ cells frequency in peripheral blood and decreased CXCL10/CCL3 serum concentration. Increase in CXCR3(high) expressing CD8+ cells after 24 weeks of treatment was correlated with SVR. CONCLUSIONS: In chronic hepatitis C, anti-viral treatment induces an increase in CD8+ cells expressing chemokine receptors associated with Tc1 response and a reduction in their ligands. Achievement of viral control is associated with an increase in CXCR3(high) expressing CD8+ cells during treatment.

Modalidades de imágen en Terapia Focal: Ecografía Multiparamétrica / Imaging diagnosis in Focal Therapy for prostate cancer: Multiparametric Magnetic Resonance Imaging

El uso de la imagen por Resonancia magnética multiparamétrica (RMmp) de próstata ha aumentado de forma significativa en los últimos años, y ha emergido como una prueba crucial en el diagnóstico, estadiaje y tratamiento del cáncer de próstata (CaP). El empleo de las distintas secuencias disponibles (T2W, T1W, difusión, perfusión y espectroscopia), así como de los diferentes parámetros que asocian, permiten no sólo determinar el grupo de pacientes subsidiarios de terapias ablativas focales, sino realizar una correcta determinación de las áreas a tratar, así como monitorizar el desarrollo de la terapia, y evaluar tanto los resultados oncológicos como los posibles fallos terapeúticos. A pesar de los excelentes resultados mostrados en los distintos estudios, es preciso alcanzar un consenso sobre su utilización en los distintos aspectos asociados al tratamiento focal, dado que es una técnica que requiere no sólo amplia experiencia en su manejo sino que precisa de una estandarización, que en el momento actual la convierten en una técnica compleja y no exenta de dificultad en su interpretación

The use of prostatic multiparametric MRI (mpMRI) has increased significantly over the last years, and has emerged as a crucial test for diagnosis, staging and treatment of prostate cancer (PCa). The use of the various available sequences (T2W, T1W, diffusion, perfusion and spectroscopy), as well as the different parameters they associate, not only enables to determine the group of patients subsidiary of focal ablative therapy, but also to perform a proper determination of the áreas to treat, as well as to monitor the development of therapy and to evaluate both oncological results and possible therapeutic failures. Despite the excellent results showed in the different studies, it is necessary to reach a consensus about its use on the different features associated with focal therapy, since it is a technique that requires not only large experience in its operation but also standardization. All this make it a complex technique and not free of difficulties in its interpretation

Symptomatic epidural lipomatosis of the spinal cord in a child: MR demonstration of spinal cord injury.

We report a case of symptomatic epidural lipomatosis in an 8-year-old girl with Cushing's syndrome secondary to longstanding high-dose steroid therapy for Crohn's disease. MR imaging of the spine revealed massive diffuse epidural fat compressing the entire spinal cord with T2 prolongation in the central gray matter of the cord suggesting ischemic myelopathy. This finding has not been previously demonstrated on imaging. A proposed mechanism underlying these findings is discussed.

Vertebral artery fibromuscular dysplasia: an unusual cause of stroke in a 3-year-old child.

Fibromuscular dysplasia (FMD) is a systemic arteriopathy which tends to affect renal arteries followed by cervicocranial vessels. It can lead to cerebral infarction if cephalic arteries are involved. FMD is an unusual cause of stroke in childhood that generally affects the carotid area. Only four cases of vertebral FMD and subsequent stroke have been reported previously and we present the youngest patient of all. A healthy 3-year-old female was admitted to Hospital Doce de Octubre in Madrid, Spain with cerebellar infarction. Angiography disclosed basilar artery thrombosis and typical signs of FMD in both vertebral arteries. No other angiographic alteration was noted in the other vessels studied. Her phenotype and other investigations were unremarkable. The patient was treated with anti-aggregation therapy (aspirin) and the outcome was excellent. Investigation of the occurrence in childhood of this kind of arteriopathy may lead to clarification of its natural history and speculation about its unclear pathogenesis.