Connellsahl9237

This protocol will enable researchers from different research backgrounds to master the use of the reverse genetic system and, consequently, accelerate COVID-19 research.During maturation, eukaryotic precursor RNAs undergo processing events including intron splicing, 3'-end cleavage, and polyadenylation. Here we describe nanopore analysis of co-transcriptional processing (nano-COP), a method for probing the timing and patterns of RNA processing. An extension of native elongating transcript sequencing, which quantifies transcription genome-wide through short-read sequencing of nascent RNA 3' ends, nano-COP uses long-read nascent RNA sequencing to observe global patterns of RNA processing. First, nascent RNA is stringently purified through a combination of 4-thiouridine metabolic labeling and cellular fractionation. In contrast to cDNA or short-read-based approaches relying on reverse transcription or amplification, the sample is sequenced directly through nanopores to reveal the native context of nascent RNA. nano-COP identifies both active transcription sites and splice isoforms of single RNA molecules during synthesis, providing insight into patterns of intron removal and the physical coupling between transcription and splicing. The nano-COP protocol yields data within 3 d.Congenital nephrotic syndrome (CNS) is a heterogeneous group of disorders characterized by nephrotic-range proteinuria, hypoalbuminaemia and oedema, which manifest in utero or during the first 3 months of life. The main cause of CNS is genetic defects in podocytes; however, it can also be caused, in rare cases, by congenital infections or maternal allo-immune disease. Management of CNS is very challenging because patients are prone to severe complications, such as haemodynamic compromise, infections, thromboses, impaired growth and kidney failure. In this consensus statement, experts from the European Reference Network for Kidney Diseases (ERKNet) and the European Society for Paediatric Nephrology (ESPN) summarize the current evidence and present recommendations for the management of CNS, including the use of renin-angiotensin system inhibitors, diuretics, anticoagulation and infection prophylaxis. Therapeutic management should be adapted to the clinical severity of the condition with the aim of maintaining intravascular euvolaemia and adequate nutrition, while preventing complications and preserving central and peripheral vessels. We do not recommend performing routine early nephrectomies but suggest that they are considered in patients with severe complications despite optimal conservative treatment, and before transplantation in patients with persisting nephrotic syndrome and/or a WT1-dominant pathogenic variant.Idiopathic calcium oxalate (CaOx) stones often develop attached to Randall's plaque present on kidney papillary surfaces. Similar to the plaques formed during vascular calcification, Randall's plaques consist of calcium phosphate crystals mixed with an organic matrix that is rich in proteins, such as inter-α-trypsin inhibitor, as well as lipids, and includes membrane-bound vesicles or exosomes, collagen fibres and other components of the extracellular matrix. Kidney tissue surrounding Randall's plaques is associated with the presence of classically activated, pro-inflammatory macrophages (also termed M1) and downregulation of alternatively activated, anti-inflammatory macrophages (also termed M2). In animal models, crystal deposition in the kidneys has been associated with the production of reactive oxygen species, inflammasome activation and increased expression of molecules implicated in the inflammatory cascade, including osteopontin, matrix Gla protein and fetuin A (also known as α2-HS-glycoprotein). Many of these molecules, including osteopontin and matrix Gla protein, are well known inhibitors of vascular calcification. We propose that conditions of urine supersaturation promote kidney damage by inducing the production of reactive oxygen species and oxidative stress, and that the ensuing inflammatory immune response promotes Randall's plaque initiation and calcium stone formation.Hydroformylation, a reaction that installs both a C-H bond and an aldehyde group across an unsaturated substrate, is one of the most important catalytic reactions in both industry and academia. Given the synthetic importance of creating new C-C bonds, the development of carboformylation reactions, wherein a new C-C bond is formed instead of a C-H bond, would bear enormous synthetic potential to rapidly increase molecular complexity in the synthesis of valuable aldehydes. However, the demanding complexity inherent in a four-component reaction, utilizing an exogenous CO source, has made the development of a direct carboformylation reaction a formidable challenge. Here, we describe a palladium-catalysed strategy that uses readily available aroyl chlorides as a carbon electrophile and CO source, in tandem with a sterically congested hydrosilane, to perform a stereoselective carboformylation of alkynes. An extension of this protocol to four chemodivergent carbonylations further highlights the creative opportunity offered by this strategy in carbonylation chemistry.Pharmaceutical drug therapy is often hindered by issues caused by poor drug selectivity, including unwanted side effects and drug resistance. Spatial and temporal control over drug activation in response to stimuli is a promising strategy to attenuate and circumvent these problems. Here we use ultrasound to activate drugs from inactive macromolecules or nano-assemblies through the controlled scission of mechanochemically labile covalent bonds and weak non-covalent bonds. We show that a polymer with a disulfide motif at the centre of the main chain releases an alkaloid-based anticancer drug from its β-carbonate linker by a force-induced intramolecular 5-exo-trig cyclization. Second, aminoglycoside antibiotics complexed by a multi-aptamer RNA structure are activated by the mechanochemical opening and scission of the nucleic acid backbone. Lastly, nanoparticle-polymer and nanoparticle-nanoparticle assemblies held together by hydrogen bonds between the peptide antibiotic vancomycin and its complementary peptide target are activated by force-induced scission of hydrogen bonds. This work demonstrates the potential of ultrasound to activate mechanoresponsive prodrug systems.The number of publications on deep learning for cancer diagnostics is rapidly increasing, and systems are frequently claimed to perform comparable with or better than clinicians. However, few systems have yet demonstrated real-world medical utility. In this Perspective, we discuss reasons for the moderate progress and describe remedies designed to facilitate transition to the clinic. Recent, presumably influential, deep learning studies in cancer diagnostics, of which the vast majority used images as input to the system, are evaluated to reveal the status of the field. By manipulating real data, we then exemplify that much and varied training data facilitate the generalizability of neural networks and thus the ability to use them clinically. To reduce the risk of biased performance estimation of deep learning systems, we advocate evaluation in external cohorts and strongly advise that the planned analyses, including a predefined primary analysis, are described in a protocol preferentially stored in an online repository. Recommended protocol items should be established for the field, and we present our suggestions.An increasing number of field studies have shown that the phenotype of an individual plant depends not only on its genotype but also on those of neighboring plants; however, this fact is not taken into consideration in genome-wide association studies (GWAS). Based on the Ising model of ferromagnetism, we incorporated neighbor genotypic identity into a regression model, named "Neighbor GWAS". Our simulations showed that the effective range of neighbor effects could be estimated using an observed phenotype when the proportion of phenotypic variation explained (PVE) by neighbor effects peaked. The spatial scale of the first nearest neighbors gave the maximum power to detect the causal variants responsible for neighbor effects, unless their effective range was too broad. However, if the effective range of the neighbor effects was broad and minor allele frequencies were low, there was collinearity between the self and neighbor effects. To suppress the false positive detection of neighbor effects, the fixed effect and variance components involved in the neighbor effects should be tested in comparison with a standard GWAS model. We applied neighbor GWAS to field herbivory data from 199 accessions of Arabidopsis thaliana and found that neighbor effects explained 8% more of the PVE of the observed damage than standard GWAS. The neighbor GWAS method provides a novel tool that could facilitate the analysis of complex traits in spatially structured environments and is available as an R package at CRAN ( https//cran.rproject.org/package=rNeighborGWAS ).An Addendum to this paper has been published https//doi.org/10.1038/s41564-021-00872-5.Transplant associated thrombotic microangiopathy (TA-TMA) is life-threatening complication post allogeneic stem cell transplant (ASCT). Risk factors and prognosis of TA-TMA are not well defined. We retrospectively studied consecutive ASCT patients with AML, ALL, and CML from January 2008 to March 2019 to study the incidence, risk factors, and outcomes of TMA. Definitive and probable TA-TMA was defined using Blood and Marrow Transplant Clinical Trials Network (BMT-CTN) and Cho criteria, respectively. Risk factors explored were age, gender, diagnosis, type of transplant, use of tyrosine kinase inhibitors (TKI) pre transplant, conditioning regimen, and acute GVHD. Standard statistical methods were used. Total 241 patients, 179 (74.2 %) males, median age of 29 years were studied. Diagnoses were AML in 104, ALL in 85 (Ph+ve 23) and CML 52. Total 26 (10.7%) patients (22 males) developed TA-TMA at median of day+102. On multivariate analysis, pre-HSCT TKI (OR 2.7, p = 0.028), haplo-HSCT (OR 3.16, p = 0.018) and presence of acute GVHD (OR 4.17, p = 0.003) were significant risk factors. With a median follow up of 60 months, median OS with and without TA-TMA was 18 and 97 months respectively (p = 0.021). The association of pre-HSCT with TKI with TA-TMA merits further exploration in prospective studies.Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for patients with a hematologic disease. Although the clinical outcomes after allo-HCT have significantly improved during the last few decades, graft-versus-host disease (GVHD) is still a major cause of post-HCT morbidity and mortality. Systemic glucocorticoids (GC) remain an integral part of treatment in patients with GVHD including both acute and chronic GVHD. Although it is well-known that usage of systemic GC is associated with various side effects, the short- and long-term effects of GCs in the HCT setting are not well-characterized due to limited published data. In order to clarify this issue, we summarize the information on side effects associated with GCs, focusing specifically on the sequelae of these agents in the early post-HCT period. Dyngo-4a mouse In instances where limited data are available, we included data from other fields such as autoimmune diseases, given the potential parallels between autoimmune conditions and GVHD.