Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly.

We recently reported that serine-arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65 and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.

Clinical outcomes of bronchiectasis in India: data from the EMBARC/Respiratory Research Network of India registry.

BACKGROUND: Identifying risk factors for poor outcomes can help with risk stratification and targeting of treatment. Risk factors for mortality and exacerbations have been identified in bronchiectasis but have been almost exclusively studied in European and North American populations. This study investigated the risk factors for poor outcome in a large population of bronchiectasis patients enrolled in India. METHODS: The European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC) and Respiratory Research Network of India (EMBARC-India) registry is a prospective observational study of adults with computed tomography-confirmed bronchiectasis enrolled at 31 sites across India. Baseline characteristics of patients were used to investigate associations with key clinical outcomes: mortality, severe exacerbations requiring hospital admission, overall exacerbation frequency and decline in forced expiratory volume in 1 s. RESULTS: 1018 patients with at least 12-month follow-up data were enrolled in the follow-up study. Frequent exacerbations (≥3 per year) at baseline were associated with an increased risk of mortality (hazard ratio (HR) 3.23, 95% CI 1.39-7.50), severe exacerbations (HR 2.71, 95% CI 1.92-3.83), future exacerbations (incidence rate ratio (IRR) 3.08, 95% CI 2.36-4.01) and lung function decline. Coexisting COPD, dyspnoea and current cigarette smoking were similarly associated with a worse outcome across all end-points studied. Additional predictors of mortality and severe exacerbations were increasing age and cardiovascular comorbidity. Infection with Gram-negative pathogens (predominantly Klebsiella pneumoniae) was independently associated with increased mortality (HR 3.13, 95% CI 1.62-6.06), while Pseudomonas aeruginosa infection was associated with severe exacerbations (HR 1.41, 95% CI 1.01-1.97) and overall exacerbation rate (IRR 1.47, 95% CI 1.13-1.91). CONCLUSIONS: This study identifies risk factors for morbidity and mortality among bronchiectasis patients in India. Identification of these risk factors may support treatment approaches optimised to an Asian setting.

Discovery of a pre-mRNA structural scaffold as a contributor to the mammalian splicing code.

The specific recognition of splice signals at or near exon-intron junctions is not explained by their weak conservation and instead is postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of 3D structural scaffold of AdML-a model pre-mRNA substrate-guiding early spliceosomal components to the splice signal sequences. We find that mutations in the non-cognate splice signal sequences impede recruitment of early spliceosomal components due to disruption of the global structure of the pre-mRNA. We further find that the pre-mRNA segments potentially interacting with the early spliceosomal component U1 snRNP are distributed across the intron, that there is a spatial proximity of 5' and 3' splice sites within the pre-mRNA scaffold, and that an interplay exists between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components can recognize a 3D structural scaffold beyond the short splice signal sequences, and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This provides a conceptual basis to analyze the contribution of recognizable 3D structural scaffolds to the splicing code across the mammalian transcriptome.

Structural disruption of exonic stem-loops immediately upstream of the intron regulates mammalian splicing.

Recognition of highly degenerate mammalian splice sites by the core spliceosomal machinery is regulated by several protein factors that predominantly bind exonic splicing motifs. These are postulated to be single-stranded in order to be functional, yet knowledge of secondary structural features that regulate the exposure of exonic splicing motifs across the transcriptome is not currently available. Using transcriptome-wide RNA structural information we show that retained introns in mouse are commonly flanked by a short (≲70 nucleotide), highly base-paired segment upstream and a predominantly single-stranded exonic segment downstream. Splicing assays with select pre-mRNA substrates demonstrate that loops immediately upstream of the introns contain pre-mRNA-specific splicing enhancers, the substitution or hybridization of which impedes splicing. Additionally, the exonic segments flanking the retained introns appeared to be more enriched in a previously identified set of hexameric exonic splicing enhancer (ESE) sequences compared to their spliced counterparts, suggesting that base-pairing in the exonic segments upstream of retained introns could be a means for occlusion of ESEs. The upstream exonic loops of the test substrate promoted recruitment of splicing factors and consequent pre-mRNA structural remodeling, leading up to assembly of the early spliceosome. These results suggest that disruption of exonic stem-loop structures immediately upstream (but not downstream) of the introns regulate alternative splicing events, likely through modulating accessibility of splicing factors.

High-Resolution Ultrasound Study of Morphological Changes of the Pylorus in the Postoperative Cases of Infantile Hypertrophic Pyloric Stenosis.

AIMS: The aim of this study was to find & compare the pyloric Canal Length (CL), Pyloric muscle thickness (MT) by using the High Resolution Ultrasonography (HRUS) in Preoperative & postoperative period (after Ramstedt Pyloromyotomy) for Infantile Hypertrophic Pyloric Stenosis (IHPS). METHODS: From January 2018 to June 2019, we have performed HRUS for 40 patients of clinically diagnosed cases of IHPS. & Ultrasonography machine: Phillips HD 7 machine & Alpinion E-CUBE with 3-12MHz linear probe, 3.5-5 MHz curvilinear probe and 5-7.5 MHz sector probe were used. The sonographic criteria for positive IHPS are Pyloric muscle thickness (MT) > 3 mm, pyloric Canal length (CL) >14 mm. Postoperative MT & CL at 2 month, 4 month & 6 month were determined and results were analysed with the preoperative pyloric parameters. Statistical analysis used: Mean and standard deviation of values were obtained by using Microsoft excel and statistical significance was analysed by regression study of grouped variables showing p - value by Microsoft excel. RESULTS: Pyloric parameters (MT & CL ) gradually attain normal values over months and 80 % of patients showed resolution of pyloric MT & CL values by 6 months. MT assessment shows statistical significance at six months scan ( p- value <0.05). CONCLUSIONS: Serial postoperative HRUS in IHPS cases show gradual ( mostly by 6 months) resolution of pyloric parameters ( MT & CL ) to normal. MT appears to be a consistent parameter for serial assessment.

Pancreatico Duodenal Trauma in Children: Two-Year Experience at a Regional Referral Center and Tertiary Care Teaching Hospital.

BACKGROUND: Pancreatico-Duodenal injuries, though rare, pose a mighty challenge to the surgeon in both diagnosis and treatment. More so in children, where the patho-physiologic threshold is low. Added to this is the absence of any strong guidelines to guide and ensure standardised care in these children for best outcome. MATERIALS AND METHODS: The trauma patients' records in the emergency department, operation theatre, inpatient departmrnt, outpatient department (OPD) for the period of December 2016 to December 2018 in the institution were retrospectively reviewed, to find out the cases of pancreaticoduodenal trauma proven radiologically, biochemically and/or on laparotomy. Those cases then studied with respect to diagnosis, treatment and short term outcome. RESULTS: Of the 256 cases of abdominal trauma, suspected pancreatico duodenal (PD) trauma were in nine but on laparotomy actually eight of them had PD trauma. All were blunt abdominal traumas sustained in road traffic accidents. Of these one was combined pancreatico duodenal while three were pure pancreatic and four were pure duodenal. One had associated superior mesenteric vein trauma. All were managed operatively. There were no mortality, one fourth had major complications. Mean hospital stay was 22.25 days. Follow up period varied from six to 32 months. CONCLUSION: Though PD traumas are rare but are potentially very morbid and may prove fatal. Proactive individualized management, multidisciplinary approach and good perioperative support can yield good results.

Synchronous Bilateral Wilms Tumor: Five-Year Single-Center Experience with Assessment of Quality of Life.

CONTEXT: Synchronous Bilateral Wilms tumor (sBWT). AIMS: This study aimed to assess the outcome of patients with sBWT treated on SIOP protocol. SETTINGS AND DESIGN: Retrospective and prospective randomized study. SUBJECTS AND METHODS: SIOP 93-01 protocol was used to study nine patients of sBWT in a single center and followed up over a period from 2 to 5 years. STATISTICAL ANALYSIS USED: Unpaired t-test and Mann-Whitney U-test were used for analysis. RESULTS: Of nine patients, six were included in the study as three patients lost to follow-up. Among the six patients, there were four girls and two boys with a median age of 2 years. Mean regression in the size of tumor was 87% in four out of six patients. Tumor with unfavorable histology showed 32% response (ratio of favorable: unfavorable histology 2:1). Event-free survival rate was 81.3% and overall survival was 90% over 2-5 years. Recurrence was seen in two patients of whom one had Denys-Drash syndrome. Mean DTPA glomerular filtration rate was 91.4/ml/min/1.73 m2 preoperatively and that of 3 months after completion of treatment was 84/ml/min/1.73 m2. Health-related quality of life (HRQOL) using Pediatric Quality of Life Inventory and Lansky Play Performance Scale revealed significant improvement results of all functioning domains such as physical, social, emotional, and school subscales with P < 0.05 and performance scale (P < 0.04). CONCLUSIONS: We suggest SIOP protocol for sBWT and bilateral nephron-sparing surgery in two stages. However, long-term follow-up is required to assess the ultimate renal function outcome. HRQOL is an essential guide in improving the conditions of pediatric cancer survivors.

Demographic Study and Management of Colonic Atresia: Single-Center Experience with Review of Literature.

CONTEXT AND AIM: The aim of this study is to highlight the rarity of this disorder and its associated anomalies and our objective was to review our experience in the management of colonic atresia (CA) with respect to staged surgery versus one-step procedure for a better outcome of the disease. SETTINGS AND DESIGN: A randomized, controlled, single-center study conducted over a period of 4 years from October 2013 to 2017. SUBJECTS AND METHODS: Nine cases were operated for CA of which three underwent primary anastomosis and six underwent initial colostomy followed by definitive surgery. Age, sex, body weight, clinical presentation, type of atresia, site, time interval of operation, associated anomalies, initial procedure, postoperative complications, final procedure, biopsy, hospital stay, and outcome were noted. STATISTICAL ANALYSIS USED: Microsoft Excel was used for statistical analysis. RESULTS: Out of 9 patients, 6 were males and 3 were females, 7 patients survived and 2 patients died (mortality 22.2%), of which one patient with primary anastomosis had leak and sepsis and one patient with primary anastomosis had associated Hirschsprung's disease (HD). Mean time gap for operation was 12.8 h and mean hospital stay was 3.5 days for initial colostomy and 21 days for primary anastomosis. Wound dehiscence occurred in 4 patients. Malrotation was found in 3 patients, HD in 2 patients, cardiac anomaly in 2, Meckel's diverticulum in 1, and cleft lip in 1 patient. Type IIIa atresia was found in in 6 patients, Type I in 2, and Type II in 1 patient. The most common site was transverse colon (n = 5). CONCLUSION: Diagnosis and management of CA is a challenge. Early presentation and diagnosis should be prompt. Staged procedure with initial colostomy followed by definitive procedure is the preferred choice. Associated anomaly HD must be ruled out.

Primary undifferentiated pleomorphic sarcoma of the breast in a young female: a case report.

BACKGROUND: Undifferentiated pleomorphic sarcoma is a rare entity and requires extensive immunohistochemical markers to differentiate it from other tumors of the breast. CASE PRESENTATION: We present a 29-year-old female with a left breast lump for 2 months. Initial diagnosis of malignant spindle cell tumor was done on core biopsy following which total mastectomy was done. After extensive sampling, on histology, highly pleomorphic spindle cells palisading the area of geographic necrosis with very high atypical mitosis were seen. As there was diagnostic difficulty, immunohistochemical antibody panel was used and diagnosis of undifferentiated pleomorphic sarcoma of the breast was made by exclusion. CONCLUSIONS: Core biopsy with immunostaining is possibly superior to FNA as an initial diagnostic modality for breast masses with atypical features.

Rapidly Fatal Silicosis Among Jewellery Workers Attending a District Medical College of West Bengal, India.

BACKGROUND: Silicosis is a slowly progressive chronic occupational lung disease, developed after a prolong period of exposure to high concentration of silica dust. METHODS: In this longitudinal study, we enrolled old and new silicosis patients (n=19; 8 jewellery polishers, 11 from other occupations) seen at our Pulmonary Medicine Department from June 2009 to December 2012 to document the course of illness as per their occupational exposure. RESULTS: Six of the eight jewellery polishing workers had developed silicosis within five years of exposure, while six of the 11 other workers with other occupational exposure had developed silicosis after exposure of 10 years or more. Mean duration of exposure was significantly less among jewellery polishing workers compared to other workers (3.4 +/- 1.7 versus 9.3 +/- 4.1; p=0.001). Mean duration of illness (months) (14.9 +/- 5.8 versus 28.5 +/- 16.5; p=0.040) were significantly less among the jewellery polishing workers compared to other workers. At the end of the study period, all eight jewellery polishing workers with silicosis had died while four of the 11 patients with other occupational exposure had died. CONCLUSION: Silicosis among jewellery polishing workers was found to be more severe and progressive compared to silicosis due to other occupational exposures, in our study.

Pulmonary Spindle Cell Carcinoma: As Rare as a Hen's Teeth.

This case is about a 70-year-old man who presented with symptoms and laboratory reports that indicated differentials toward an infectious disease (pneumonia and tuberculosis). A lung mass was found in his chest X-ray and in the computerized tomography (CT) scan of his thorax. A biopsy was taken from the lung mass, and histopathological examination and immunohistochemical staining of the biopsy were done. The results revealed the presence of spindle cell carcinoma (SpCC) with vimentin and cytokeratin positivity. Spindle cell lung cancer is a rare type of non-small cell lung carcinoma, for which all available research indicates a poor prognosis. Due to the rarity of diagnosis, there is a dearth of information about the epidemiology and overall survival of affected patients.

Characterization of the evolutionarily conserved iron-sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana.

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe-S] cluster. We determined the cluster to be a [4Fe-4S] type, which quickly oxidizes to a [2Fe-2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys(135), is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe-4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe-S cluster or Cys(135) retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe-S cluster in Planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.

Squamous cell lung cancer producing bilateral air bronchograms on CT scan thorax.

A young lady presented with cough for three months and dyspnoea for one month along with clinical signs of bilateral consolidation of lung. On CT scan thorax there were bilateral air bronchograms with alveolar filling opacities predominantly in lower lobes. After bronchoscopy when there was presence of suspicious malignant cells on bronchoalveolar lavage fluid, transbronchial lung biopsy diagnosed the case as squamous cell lung cancer. Radiological presentation of squamous cell lung cancer as air bronchogram is not a very common phenomenon and can present a diagnostic challenge to the clinician.

SHAPE to Probe RNA Structure and RNA-Protein Interactions In Vitro.

Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) is used to distinguish between the levels of flexibility of nucleotides regulated by base pairing or protein binding. In this method, a reagent reacts with the 2' hydroxyl group to form an adduct, which is then detected by reverse transcription reaction. The number of RNA molecules with an adduct at a specific nucleotide position indicates the SHAPE reactivity of that nucleotide. Here, we describe the method for probing the structure of an RNA in a protein-free or a protein-bound state by in vitro SHAPE.

Chemical Probing of RNA Structure In Vivo Using SHAPE-MaP and DMS-MaP.

The functional roles of RNAs are often regulated by their structure. Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) and dimethyl sulfate (DMS) base reactivity can be employed to investigate the flexibility of nucleotides and correlate it to the constraints imparted by base-pairing and/or protein-binding. In vivo, a multitude of proteins could bind an RNA molecule, regulating its structure and function. Hence, to obtain a more comprehensive view of the RNA structure-function relationship in vivo, it may be required to characterize both the RNA structure and the RNA-protein interaction network. In this chapter, we describe methods for characterizing the in vivo nucleotide flexibility of RNA in cells by SHAPE-MaP (SHAPE by Mutational Profiling) and DMS-MaP. In another chapter, we will discuss the characterization of RNA-protein interaction network by RNP-MaP.

Analysis of RNA-Protein Interaction Networks Using RNP-MaP.

RNA-protein interactions regulate a myriad of biological functions through formation of ribonucleoprotein complexes. These complexes may consist of one or more RNA-protein interaction network(s) providing additional layers of regulatory potential to the RNA. Moreover, since the protein-binding also regulates local and global structure of the RNA by structurally remodeling the latter, it is important to correlate RNA nucleotide flexibility with the site of protein-binding. We have discussed methods for chemical probing of structure of the RNA in the protein-free and protein-bound states in the preceding chapters. In this chapter, we describe a ribonucleoprotein mutational profiling (RNP-MaP) method for probing RNA-protein interaction networks.

A Rare Case of Primary Adenocarcinoma of the Gallbladder With Lymphoid Stroma.

Adenocarcinoma of the gallbladder is the most common gallbladder carcinoma. But lymphoid stroma in gallbladder carcinoma is one of the rarest presentations. A unique case of gallbladder adenocarcinoma with lymphoid stroma in a 47-year-old female is presented in this report. The surgically resected gallbladder demonstrated invasive adenocarcinoma with lymphoid stroma, though it was radiologically diagnosed as xanthogranulomatous cholecystitis. Adenocarcinoma was immunohistochemically positive for pancytokeratin (AE1/AE3), cytokeratin 7 (CK7), cytokeratin 20 (CK20), and carcinoembryonic antigen (CEA). Lymphoid stroma was positive for CD45, where B-cell zones were CD20 and CD79a positive, and T-cell zones were CD3 positive, with a larger T-cell subset being positive for CD4 than CD8. This is the fourth reported case of gallbladder adenocarcinoma with lymphoid stroma, which needs to be studied for pathogenesis, prognosis, and future therapy, if any.

Synchronous Adenocarcinoma Stomach With Marginal Zone Lymphoma: A Sporadic Occurrence and Review of Literature.

Synchronous adenocarcinoma of the stomach with lymphoma is extremely rare. We report a case of a 65-year-old male patient with synchronous adenocarcinoma of the stomach with nodal marginal zone lymphoma. Initial endoscopic biopsy suggested invasive moderately differentiated adenocarcinoma and a locoregional disease, per contrast-enhanced computed tomography (CECT) scans. The patient was started on neo-adjuvant chemotherapy with the 5FU, leucovorin, oxaliplatin, docetaxel (FLOT) regime and, after response evaluation, underwent radical gastrectomy. Histopathology and immunohistochemistry suggested synchronous adenocarcinoma of the stomach with marginal zone lymphoma in perigastric lymph nodes. This case is probably the first such synchronous malignancy case reported from India. The prognosis of multiple primary malignancies is usually poor because no standard guidelines are available regarding optimum treatment and sequencing of available treatment modalities. The frequency of synchronous primary cancers has been increasing in recent years, probably due to better diagnostic modalities, and second primary in patients with cancer should be considered as one of the differential diagnoses.

A Case Report of Rare and Lethal Methicillin-Resistant Staphylococcus aureus (MRSA) Peritonitis in Infancy.

Peritoneal inflammation without a discernible intraperitoneal source is referred to as primary peritonitis. Only 2% of pediatric acute abdominal crises are diagnosed preoperatively. Association with other infections is uncommon and is often limited to hepatic and urinary pathogens. Here, we describe a case of primary peritonitis in a one-month-old child who had laparotomy and appendicectomy as per the recommended treatment plan. There were no accompanying hepatic and urinary diseases. In this instance, methicillin-resistant Staphylococcus aureus (MRSA) was the responsible bacteria. The use of linezolid, as per the culture sensitivity report of intraperitoneal pus, ensured a smooth recovery in this case.

The DNA glycosylase NEIL2 is protective during SARS-CoV-2 infection.

SARS-CoV-2 infection-induced aggravation of host innate immune response not only causes tissue damage and multiorgan failure in COVID-19 patients but also induces host genome damage and activates DNA damage response pathways. To test whether the compromised DNA repair capacity of individuals modulates the severity of COVID-19 infection, we analyze DNA repair gene expression in publicly available patient datasets and observe a lower level of the DNA glycosylase NEIL2 in the lungs of severely infected COVID-19 patients. This observation of lower NEIL2 levels is further validated in infected patients, hamsters and ACE2 receptor-expressing human A549 (A549-ACE2) cells. Furthermore, delivery of recombinant NEIL2 in A549-ACE2 cells shows decreased expression of proinflammatory genes and viral E-gene, as well as lowers the yield of viral progeny compared to mock-treated cells. Mechanistically, NEIL2 cooperatively binds to the 5'-UTR of SARS-CoV-2 genomic RNA to block viral protein synthesis. Collectively, these data strongly suggest that the maintenance of basal NEIL2 levels is critical for the protective response of hosts to viral infection and disease.