101 research outputs found
Prescription pattern and rationality of drugs study of patients of OPD (outpatient) of orthopedics Department of RKDF Medical College Hospital and Research Centre, Bhopal, MP, India
Background: To study the prescription pattern of drugs prescribed to patients of OPD of orthopedics department and to analyze the rationality of drugs prescribed by doctors of RKDF MCHRC, Bhopal, MP, India.Methods: A prospective, observational study was planned for 200 patients selected in the year 2015. Collected data were entered in a predesigned forms downloaded from WHO website. Rationality of drugs prescription was analyzed using WHO drug utilization and prescription indicators. For generic drugs reference NLEM 2011 and for cost analysis, Drug Today 2014 was used.Results: out of 200 patients, 41%were male and 59% were female. Average numbers of drugs prescribed per prescription was 3.3. Most commonly prescribed drug was Analgesics an Anti-inflammatory (37.1%) in oral as well as Intramuscular injections and Topical forms, Gastric acid inhibitors (15.4%), Muscle relaxants (12.1%) Calcium and Vitamin D, glucosamines (12.1%), Antimicrobials (8%), Steroids (1.8%), Enzyms serratiopeptidase (1.8%). Most commonly prescribed Analegsic was FDC of Trypsin+ Rutoside+ Bromelin+ Diclofenac (30.6%) followed by FDC Acceclofenac+ Paracetamol+ Seratiopeptidase (15.9%), followed by FDC Tramaodol+ Paracetamol (11%).Drugs prescribed in generic form were 0%.Average cost of treatment was INR 600 per prescription. Most common route of drug prescription was 1. Oral route 81.7%. 2. Topical 6.4%. 3. Injectable intramuscular 6.2% and intra articular 5.7%. Duration of treatment was prescribed in 81%. Frequency of drug administration was mentioned in 100% of prescriptions. EDL prescription was 81%. FDCs were 35%.Conclusions: There was Poly pharmacy. Drugs prescribed in generic form were very low. Drugs prescribed from NLEM were 81%. CMEs on Rational drug therapy to doctors working in orthopedics department should be conducted to promote rational use
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In vivo RNA editing of point mutations via RNA-guided adenosine deaminases.
We present in vivo sequence-specific RNA base editing via adenosine deaminases acting on RNA (ADAR) enzymes with associated ADAR guide RNAs (adRNAs). To achieve this, we systematically engineered adRNAs to harness ADARs, and comprehensively evaluated the specificity and activity of the toolsets in vitro and in vivo via two mouse models of human disease. We anticipate that this platform will enable tunable and reversible engineering of cellular RNAs for diverse applications
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CRISPR-Cas9 Gene Editing of Hematopoietic Stem Cells from Patients with Friedreich's Ataxia.
Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by expansion of GAA repeats in intron 1 of the frataxin (FXN) gene, leading to significant decreased expression of frataxin, a mitochondrial iron-binding protein. We previously reported that syngeneic hematopoietic stem and progenitor cell (HSPC) transplantation prevented neurodegeneration in the FRDA mouse model YG8R. We showed that the mechanism of rescue was mediated by the transfer of the functional frataxin from HSPC-derived microglia/macrophage cells to neurons/myocytes. In this study, we report the first step toward an autologous HSPC transplantation using the CRISPR-Cas9 system for FRDA. We first identified a pair of CRISPR RNAs (crRNAs) that efficiently removes the GAA expansions in human FRDA lymphoblasts, restoring the non-pathologic level of frataxin expression and normalizing mitochondrial activity. We also optimized the gene-editing approach in HSPCs isolated from healthy and FRDA patients' peripheral blood and demonstrated normal hematopoiesis of gene-edited cells inĀ vitro and inĀ vivo. The procedure did not induce cellular toxic effect or major off-target events, but a p53-mediated cell proliferation delay was observed in the gene-edited cells. This study provides the foundation for the clinical translation of autologous transplantation of gene-corrected HSPCs for FRDA
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Barcoding cells using cell-surface programmable DNA-binding domains
We develop here a novel approach to barcode large numbers of cells through cell-surface expression of programmable zinc-finger DNA-binding domains (sZFs). We show sZFs enable double-stranded DNA to sequence-specifically label living cells, and also develop a sequential tagging approach to in situ image >3 cell types using just 3 fluorophores. Finally we demonstrate their broad versatility through ability to serve as surrogate reporters and facilitate selective cell capture and targeting
Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers
DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin ācappingā oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical
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Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) systems in bacteria and archaea use RNA-guided nuclease activity to provide adaptive immunity against invading foreign nucleic acids. Here, we report the use of type II bacterial CRISPR-Cas system in Saccharomyces cerevisiae for genome engineering. The CRISPR-Cas components, Cas9 gene and a designer genome targeting CRISPR guide RNA (gRNA), show robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci in yeast. Using constitutive Cas9 expression and a transient gRNA cassette, we show that targeted double-strand breaks can increase homologous recombination rates of single- and double-stranded oligonucleotide donors by 5-fold and 130-fold, respectively. In addition, co-transformation of a gRNA plasmid and a donor DNA in cells constitutively expressing Cas9 resulted in near 100% donor DNA recombination frequency. Our approach provides foundations for a simple and powerful genome engineering tool for site-specific mutagenesis and allelic replacement in yeast
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Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing
The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences and identify a highly targetable protein from Neisseria meningitidis. Our results provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins and adapting them to eukaryotic cells
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Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach
We develop an in vivo library-on-library methodology to simultaneously assess single guide RNA (sgRNA) activity across ~1,400 genomic loci. Assaying across multiple human cell types, end-processing enzymes, and two Cas9 orthologs, we unravel underlying nucleotide sequence and epigenetic parameters. Our results enable improved design of reagents, shed light on mechanisms of genome targeting, and provide a generalizable framework to study nucleic acid-nucleic acid interactions and biochemistry in high throughput
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Interface-guided phenotyping of coding variants in the transcription factor RUNX1.
Single-gene missense mutations remain challenging to interpret. Here, we deploy scalable functional screening by sequencing (SEUSS), a Perturb-seq method, to generate mutations at protein interfaces of RUNX1 and quantify their effect on activities of downstream cellular programs. We evaluate single-cell RNA profiles of 115 mutations in myelogenous leukemia cells and categorize them into three functionally distinct groups, wild-type (WT)-like, loss-of-function (LoF)-like, and hypomorphic, that we validate in orthogonal assays. LoF-like variants dominate the DNA-binding site and are recurrent in cancer; however, recurrence alone does not predict functional impact. Hypomorphic variants share characteristics with LoF-like but favor protein interactions, promoting gene expression indicative of nerve growth factor (NGF) response and cytokine recruitment of neutrophils. Accessible DNA near differentially expressed genes frequently contains RUNX1-binding motifs. Finally, we reclassify 16 variants of uncertain significance and train a classifier to predict 103 more. Our work demonstrates the potential of targeting protein interactions to better define the landscape of phenotypes reachable by missense mutations
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A Kinome-Wide Synthetic Lethal CRISPR/Cas9 Screen Reveals That mTOR Inhibition Prevents Adaptive Resistance to CDK4/CDK6 Blockade in HNSCC.
UNLABELLED: The comprehensive genomic analysis of the head and neck squamous cell carcinoma (HNSCC) oncogenome revealed the frequent loss of p16INK4A (CDKN2A) and amplification of cyclin D1 genes in most human papillomavirus-negative HNSCC lesions. However, cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have shown modest effects in the clinic. The aberrant activation of the PI3K/mTOR pathway is highly prevalent in HNSCC, and recent clinical trials have shown promising clinical efficacy of mTOR inhibitors (mTORi) in the neoadjuvant and adjuvant settings but not in patients with advanced HNSCC. By implementing a kinome-wide CRISPR/Cas9 screen, we identified cell-cycle inhibition as a synthetic lethal target for mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergism in HNSCC-derived cells in vitro and in vivo. Remarkably, we found that an adaptive increase in cyclin E1 (CCNE1) expression upon palbociclib treatment underlies the rapid acquired resistance to this CDK4/6 inhibitor. Mechanistically, mTORi inhibits the formation of eIF4G-CCNE1 mRNA complexes, with the consequent reduction in mRNA translation and CCNE1 protein expression. Our findings suggest that mTORi reverts the adaptive resistance to palbociclib. This provides a multimodal therapeutic option for HNSCC by cotargeting mTOR and CDK4/6, which in turn may halt the emergence of palbociclib resistance. SIGNIFICANCE: A kinome-wide CRISPR/Cas9 screen identified cell-cycle inhibition as a synthetic lethal target of mTORis. A combination of mTORi and palbociclib, a CDK4/6-specific inhibitor, showed strong synergistic effects in HNSCC. Mechanistically, mTORis inhibited palbociclib-induced increase in CCNE1
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