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At least 19 recordsLinked to original sources

CROPseq-multi: a universal solution for multiplexed perturbation in high-content pooled CRISPR screens.

Forward genetic screens seek to dissect complex biological systems by systematically perturbing genetic elements and observing the resulting phenotypes. While standard screening methodologies introduce individual perturbations, multiplexing perturbations improves the performance of single-target screens and enables combinatorial screens for the study of genetic interactions. Current tools for multiplexing perturbations are limited by technical challenges and do not offer compatibility across diverse screening methodologies, including enrichment, single-cell sequencing, and optical pooled screens. Here, we report the development of CROPseq-multi (CSM), a CROPseq1-inspired lentiviral system to multiplex Streptococcus pyogenes (Sp) Cas9-based perturbations with versatile readout compatibility and high performance for both perturbation and barcode identification. CSM has equivalent per-guide activity to CROPseq and low lentiviral recombination frequencies. Dual-guide CSM libraries are constructed in a single, facile molecular cloning step that facilitates the use of unique molecular identifiers. CSM is compatible with enrichment screening methodologies, single-cell RNA-sequencing readouts, and optical pooled screens. For optical pooled screens, an optimized and multiplexed in situ detection protocol improves barcode counts 10-fold (for mRNA detection), enables detection of recombination events, and reduces the number of sequencing cycles required for decoding by 3-fold relative to CROPseq. CROPseq-multi-v2 (CSMv2) adds compatibility for detection methods based on T7 RNA polymerase in vitro transcription2-5. CSM provides a single system for CRISPR screens that is compatible with individual and combinatorial perturbations, diverse SpCas9-based perturbation technologies, and multiple high-content, single-cell phenotypic readouts.

CRISPR Cas9↗

An optical practice based diabetic eye screening programme.

In many cases, blindness due to diabetic retinopathy can be prevented provided treatment with laser photocoagulation is used at the correct time. A screening programme is required to identify cases of sight threatening retinopathy. An optical practice based diabetic eye screening programme has been established in Dorset. The optometrist undertaking the examination is paid a fee. The findings are recorded on a coded form and sent to the hospital diabetologist who recalls positive cases. Seventy-six optical practices have joined the scheme and 3224 patients have been screened in the first 6 months (Dorset population 655,000). In the Poole area (population 230,000), 1922 patients were screened and 129 (6.7%) were recalled in 6 months. Outcome of 3 months screening, identified 59 recalls. Referral to the opthalmologist was made in 15 cases for potentially sight threatening retinopathy, 14 cases were followed in the diabetic clinic for significant background retinopathy, and 24 cases were returned to the annual screening in the optical practices. Six cases where the patients either failed or were unable to attend were reviewed by the GP. An optical practice based diabetic eye screening programme has been successful in screening a large number of patients.

Diabetic Retinopathy↗

Identification of a novel pulmonary oedema producing toxin from Indian red scorpion (Mesobuthus tamulus) venom.

The experiments were conducted to identify the toxin that produces pulmonary oedema in Mesobuthus tamulus (BT) envenomed animals. Crude BT venom was subjected to Sephadex gel filtration (G-75) and the fractions were screened for optical density (OD), neurotoxicity (prolongation of compound action potential in frog sciatic nerve) and lethality. All these parameters exhibited a peak between 54-94 ml eluates. Fractions of this peak were pooled (SP) and loaded on to carboxymethyl cellulose column. The column was then eluted with increasing buffer concentrations at constant pH and temperature. Eluates were screened for neurotoxicity and OD. Four peaks of neurotoxic activity (T1-T4) were detected. T2 and T3 were lethal whereas T1 and T4 were non-lethal. T2 exhibited mainly neurotoxicity and failed to augment phenyldiguanide (PDG)-induced reflex response or to produce pulmonary oedema. T3 was having minimal neurotoxic actions but augmented PDG-reflex and produced pulmonary oedema. The effects of T3 persisted even after dialysis with 8 kDa cut-off filter but not those of T2. The T3 effects resembled toxic manifestations of BT venom and were blocked by aprotinin pre-treatment. T3 demonstrated a band at approximately 100 kDa in SDS-PAGE. The results demonstrate the presence of a lethal, high molecular weight, pulmonary oedema producing toxin in BT venom.

Analysis of Variance↗

Comparative study on 4 EIA kits for screening antibody to hepatitis C virus in pooled sera.

Four enzyme immunoassay (EIA) test kits, 1 Canadian product and 3 Chinese products, were used in the comparative study. Each pool consisted of 5 sera, and the 5 single sera were tested as controls. The tests were carried out according to the instructions, keeping the same dilution of each serum in single and pool samples. It was found that with the Canadian kit, the positive and negative results of pooled sera had no difference from that of the controls (P > 0.10). In the case of Chinese Yali and Kehua kits, the positive results of pooled sera showed no difference from the controls (P > 0.10), but the optical density (OD) of negative pools were increased (P < 0.01), though quite distant from the cut-off values. In the case of Changzheng kit, the OD of positive pools were significantly lower than those of the controls (P < 0.05), and weak positive samples missed the detection. However this problem could be overcome by blocking the microwells beforehand. Our experiment demonstrate that not all EIA test kits are suitable for screening pools for antibody to hepatitis C virus, and that it is important to assess the sensitivity of the EIA kit to be used for this purpose.

Antibodies, Viral↗

[Fracture diagnosis with digital luminescence radiography].

Conventional screen-film radiographs were compared with storage phosphorus images concerning diagnostic performance in traumatologic radiography. We used an image pool of 106 radiographs including 50 conventionally and 56 digitally recorded images. The images were reviewed by four experienced radiologists. Detectability of fractures was coded in a five-point scale of confidence and analysed by ROC statistics. Furthermore the image quality and the optical density was compared. Digital images are superior under difficult exposure conditions and offer advantages by additional image processing and documentation. For some indications (follow-up, functional examination), reduced x-ray exposure is tolerable. The detectability of subtle lesions, which is essential for the primary diagnosis of fractures was significantly better by conventional radiography.

Evaluation Studies as Topic↗

Iraqi-Jewish kindreds with optic atrophy plus (3-methylglutaconic aciduria type 3) demonstrate linkage disequilibrium with the CTG repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene.

Iraqi-Jewish optic atrophy plus is an autosomal recessive condition characterized by infantile optic atrophy, an early onset movement disorder, and 3-methylglutaconic aciduria. Other features include spastic paraplegia, mild ataxia, mild cognitive deficiency and dysarthria. This disorder was identified in inbred Iraqi-Jewish kindreds in which relationships between most of the affected individuals were unknown. In this study we identify linkage to chromosome 19q13.2-q13.3 by using a DNA pooling strategy to perform a genome wide screen followed by a high density search for shared segments among affected individuals in candidate regions identified in the initial genome wide screen. A significantly high positive lod score of 6.14 at zero recombination was obtained for the CTG repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene. The existence of multiple recombinant individuals indicates the disease interval can be further narrowed with additional markers. Linkage disequilibrium was seen in six polymorphic markers across a 1 Mb interval. This region is well characterized and contains several candidate genes.

Chromosome Mapping↗

Oral contrast adherence to polyps on CT colonography.

Although oral contrast agents are known to improve the accuracy of CT colonography (CTC) by tagging fluid and stool, it is not well recognized that oral contrast also adheres to the surface of polyps. The authors' objective was to quantitate the frequency of contrast adhering to polyps. Three hundred thirty-eight optical colonoscopy-proven polyps were identified on CTC of all of the 216 patients with polyps in a larger cohort of screening patients. CT scans of polyps were analyzed for adherent contrast (ie, a thin coat/adherent drops) in at least one view (prone/supine). Forty-six percent of the 312 polyps not touching a contrast pool had adherent contrast. Polyps with villous histology were significantly more likely to have adherent contrast (77% [20/26] vs. 43% [124/286], P<0.001). Oral contrast agents often tag polyp surfaces in a pattern that is distinct from internal tagging of adherent stool, which must be recognized during CTC interpretation. Polyps with villous histology show a higher rate of contrast adherence than nonvillous polyps.

Administration, Oral↗

Diabetic retinopathy as a cause of blindness in the province of Turin, north-west Italy, in 1967-1991.

Diabetes is known to be a major contributor to blindness in industrialized countries but few data are available on the situation in Italy. As an introductory step to the implementation of permanent screening for diabetic retinopathy, a search was carried out on the causes of visual loss in the provincial territory surrounding Turin, the main city of North-West Italy. The case notes of all 4549 residents in the province who were certified blind between 1967 and 1991 were examined with regard to cause, age at onset, and year of onset of visual acuity < or = 1/20. Diabetic retinopathy was the second commonest cause of bilateral blindness (13.1% of cases), preceded by cataract (26.7%) and followed by myopia (11.1%), optic atrophy (8.9%), glaucoma (8.9%), retinitis pigmentosa (7.2%), and senile macular degeneration (4.1%). Diabetic retinopathy was the commonest eye disease among those who became blind between the ages of 50 and 70 and remained the leading cause of visual loss when the age groups 20 to 70 were pooled together. The incidence of diabetic retinopathy-related blindness did not show any trend to decrease over the 25 years investigated. It is concluded that, in spite of widespread availability of facilities for its assessment and treatment, diabetic retinopathy remains a leading cause of blindness in North-West Italy. This fully justifies the implementation of screening programmes and efficient referral chains for the early detection and prompt treatment of this complication of diabetes.

Adolescent↗

A comparison between one- and two-field 60 degree fundus photography when screening for diabetic retinopathy.

PURPOSE: To compare the severity level of diabetic retinopathy obtained when assessed from two versus only one 60 degrees photographic field using colour transparencies and red-free, black-and-white photographs. To compare the areal coverage of these two photographic strategies to that of seven-field 30 degrees photography. METHODS: Two ophthalmologists graded photographs of 74 eyes of 74 type I and II diabetes patients. Inter-method agreement was expressed in percentages and using kappa statistics and scatter-diagrams. The comparison of the approximate photographic areal coverage was done from diagrams using planimetry. RESULTS: The severity level of retinopathy when judged from two photographic fields was more severe in 13.5% (Grader 1) and in 16.2% (Grader 2) from colour transparencies and in 13.5% (Grader 1) and in 14.9% (Grader 2) from red-free black-and-white prints, as compared to assessments from only one field. Kappa values (0.84-0.86) for inter-method agreement for five pooled retinopathy levels revealed good agreement. Neither grader missed retinopathy requiring clinical assessment or treatment (levels > or = 47) when minimal retinopathy (levels 14-20) was detected using only one 60 degrees colour slide or red-free photograph. A second optic disc-centred field provided valuable additional information when more severe retinopathy lesions (levels > or = 30) were detected in the macula-centred field. One macula-centred 60 degrees photograph covered 60% and two 60 degrees photographs 80% of the area covered by seven-field 30 degrees photography. Two-field 60 degrees photography covers areas left outside seven-field 30 degrees photography. CONCLUSION: We propose the use of one macula-centred 60 degrees photograph when screening for the first lesions of diabetic retinopathy. After they have been found two-field 60 degrees photography is recommended.

Adolescent↗

Evaluation of the use of pooled serum, pooled muscle tissue fluid (meat juice) and pooled faeces for monitoring pig herds for Salmonella.

AIMS: Monitoring for Salmonella in slaughter pigs is important to enable targeted control measures to be applied on problem farms and at the abattoir. The aim of this study was to determine whether pooled serum and meat juice could be used to identify finishing pig herds with a high prevalence of infection. METHODS AND RESULTS: Samples of meat juice, serum, caecal contents, carcase swabs and pooled faeces from pig pens were taken from 20 commercial pig finishing farms and comparisons were made between the results of Salmonella culture, individual ELISA tests on serum and meat juice and pooled samples of serum and meat juice. Salmonella was isolated from samples from 19 of 20 farms. None of the ELISA tests showed a statistically significant correlation with caecal carriage of Salmonella or contamination of carcases. Serum mean optical density (O.D.) from pools of five, 10 or 20 sera showed a significant correlation with the Salmonella status of farm pen faeces. All pooled serum O.D. and sample/positive control ratio results correlated significantly with the results of the conventional individual sample ELISA. There was a statistically significant correlation between the incidence of Salmonella in farm pen pooled faeces and the prevalence of Salmonella in caeca of slaughter pigs. CONCLUSIONS: The results show a generally poor correlation between serological and bacteriological results but pooled serum or meat juice samples could be used as a cheaper substitute for serological screening of farms for Salmonella than individual samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The availability of a cheaper test should allow the costs of Salmonella monitoring of pig farms to be reduced or allow more regular testing to enhance the designation of farm Salmonella risk status.

Abattoirs↗

A quick method for screening platelet dysfunctions using the whole blood lumi-aggregometer.

Patients who present with a clinical history suggesting a bleeding disorder are often tested initially for a clotting defect rather than for platelet dysfunction, due to the length of time necessary to complete a platelet function study in platelet-rich plasma. We have developed a sensitive method for measuring platelet aggregation and release of ATP employing the Whole Blood Lumi -Aggregometer. This method makes it possible to quickly detect patients who require further study for possible platelet function disorders such as cyclooxygenase deficiency, storage-pool defect, thrombasthenia, and von Willebrand's disease. The results obtained with this electrical impedance instrument do not differ from those obtained with the conventional optical method. However, it is now possible to recognize a platelet function defect within 30 min of obtaining a 5 ml sample of citrated whole blood. Further, platelets of unusual size or density are not lost to testing through centrifugation.

Adenosine Triphosphate↗

Comparative evaluation of bioassay and ELISA for detection of Japanese encephalitis virus in field collected mosquitos.

Comparative evaluation of enzyme-linked immunosorbent assay (ELISA) and bioassay (virus isolation in Toxorhynchites splendens larvae and identification by immunofluorescence test using virus specific monoclonal antibody) was carried out in order to define a suitable strategy for monitoring Japanese encephalitis virus infection in field mosquitos. A total of 8,850 adult female mosquitos in 177 pools (Culex tritaeniorhynchus 91, Cx. vishnui 59 and Cx. fuscocephala 27) collected from an endemic area of Tamil Nadu were examined by both the techniques. In ELISA, 9 pools which had optical densities (OD) equal to the mean of normal infected pools plus > or = 4 standard deviations (SD) mean considered positive and all of them were virus positive by the bioassay also. Sixty-five pools had OD = Mean + 2-3 SD and 103 pools had OD = Mean + < 2 SD of normal pools. From these groups, 12 (18.5%) and 8 (7.8%) pools respectively were found to be virus positive by the bioassay. In total 29 (16%) pools were positive by the bioassay as against 9 (5%) by ELISA. This study demonstrated that the bioassay is sensitive for estimation of true positives and ELISA is a rapid screening system. A protocol has now been developed for surveillance in which field pools are first screened by ELISA and only those with OD = Mean + > or 2 SD are assayed in Toxorhynchites. By excluding a large majority of pools with low OD (Mean + < 2 SD), which are likely to yield to only a small percentage of true positives, the cost, time and labor involved are greatly reduced.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.).

Two large-scale ethylmethanesulfonate (EMS) mutant populations from barley (Hordeum vulgare L.) cv. Optic have been developed to promote both forward and reverse genetics in this crop. Leaf material and seed from approximately 20 000 M(2) plants were individually harvested, freeze-dried and archived. DNA was isolated from 9216 plants from the 20 and 30 mm EMS treatments and assembled into 1152 eight-plant pools. To facilitate PCR-based mutation scanning an approach has been employed that combines cleavage of heteroduplexes using the Cel nuclease (Cel I), post-cleavage intercalating dye labeling and the subsequent detection of cleaved products on a Transgenomic WAVE-HS. The populations were evaluated by screening for induced mutations in two genes of interest and the induced mutations were validated by sequence analysis. To enhance the screening process, 12-16 M(3) progeny from each of the M(2) plants were assessed for visible phenotypes and the data entered into a web accessible database (http://bioinf.scri.sari.ac.uk/distilling/distilling.html).

Base Pair Mismatch↗

The optic nerve head in myocilin glaucoma.

PURPOSE: Approximately 1 in 30 unselected patients with open-angle glaucoma (OAG) have a mutation in the myocilin gene. The purpose of this study was to describe the morphologic features of the optic nerve head (ONH) in myocilin glaucoma. METHODS: A case-control design was adopted. Sixty-six patients heterozygous for a range of myocilin mutation (cases) were matched in disease severity to 105 patients with OAG known not to have a myocilin mutation (controls), using visual field findings. Quantifiable analysis of the ONH was undertaken of stereoscopic photographs, by using custom software with a z-screen. Subjective grading of the cup depth, lamina cribrosa pore shape and orientation, and the slope of the neuroretinal rim was performed by an examiner masked to the subject's mutation status. Mutation screening was conducted using either direct sequencing or single-stranded conformation polymorphism analysis. RESULTS: Patients with a myocilin mutation had glaucoma diagnosed earlier (P < 0.001) and had higher maximum recorded intraocular pressures (P < 0.001) than did the control OAG subjects. There was no significant (P > 0.05) difference in global disc area, global neuroretinal rim area, alpha-parapapillary atrophy, beta-parapapillary atrophy, slope of neuroretinal rim, or visible lamina cribrosa morphology between myocilin mutation carriers and patients with nonmyocilin glaucoma. Disc hemorrhages were identified more frequently in those without mutations (14/209 vs. 1/129), though this was not significant after correction for multiple hypothesis testing. CONCLUSIONS: No major structural or morphologic difference of the ONH was detected in pooled data from subjects who had myocilin mutations compared with data from individuals with nonmyocilin glaucoma.

Adolescent↗

Screening of a HUAEC cDNA library identifies actin as a candidate autoantigen associated with carotid atherosclerosis.

The humoral immune response to endothelium has a pivotal role in the development of atherosclerosis. Using a molecular method, we sought to identify endothelial autoantigens in carotid atherosclerosis. Immunoscreening of a HUAEC expression library with IgG from a pool of two sera from patients with carotid atherosclerosis identified a clone specific to actin. We evaluated actin-specific IgG reactivity in patients with carotid atherosclerosis and compared responses with those in patients with systemic lupus erythematosus and type 1 diabetes mellitus and in healthy subjects. Enzyme-linked immunoassay detected actin-specific IgG in a significantly higher percentage of sera from patients with atherosclerosis and systemic lupus erythematosus than from healthy subjects (16/61, 26% and 13/33, 39%versus 2/41, 5%, P = 0.012 and P < 10(-4), by chi2 test). Mean optical density values were significantly higher in patients than in healthy subjects (P < 10(-4) by Student's t-test). Patients with atherosclerosis and uncomplicated plaques had significantly higher serum anti-actin IgG reactivity than those with complicated plaques (P = 0.048 by Student's t-test). Our findings suggest that actin is an autoantigenic molecule of potential clinical interest in carotid atherosclerosis.

Actins↗

[Meta-analysis of diagnostic capability of frequency-doubling technology (FDT) for primary glaucoma].

OBJECTIVE: To evaluate the overall diagnostic capabilities of frequency-doubling technology (FDT) in patients of primary glaucoma, with standard automated perimetry (SAP) and/or optic disc appearance as the gold standard. METHODS: A comprehensive electric search in MEDLINE, EMBASE, Cochrane Library, BIOSIS, Previews, HMIC, IPA, OVID, CNKI, CBMdisc, VIP information, CMCC, CCPD, SSreader and 21dmedia and a manual search in related textbooks, journals, congress articles and their references were performed to identify relevant English and Chinese language articles. Criteria for adaptability were established according to validity criteria for diagnostic research published by the Cochrane Methods Group on Screening and Diagnostic Tests. Quality of the included articles was assessed and relevant materials were extracted for studying. Statistical analysis was performed with Meta Test version 0.6 software. Heterogeneity of the included articles was tested, which was used to select appropriate effect model to calculate pooled weighted sensitivity and specificity. Summary Receiver Operating Characteristic (SROC) curve was established and the area under the curve (AUC) was calculated. Finally, sensitivity analysis was performed. RESULTS: Fifteen English articles (21 studies) of 206 retrieved articles were included in the present study, with a total of 3172 patients. The reported sensitivity of FDT ranged from 0.51 to 1.00, and specificity from 0.58 to 1.00. The pooled weighted sensitivity and specificity for FDT with 95% confidence intervals (95% CI) after correction for standard error were 0.86 (0.80 - 0.90), 0.87 (0.81 - 0.91), respectively. The AUC of SROC was 93.01%. Sensitivity analysis demonstrated no disproportionate influences of individual study. CONCLUSIONS: The included articles are of good quality and FDT can be a highly efficient diagnostic test for primary glaucoma based on Meta-analysis. However, a high quality perspective study is still required for further analysis.

Aged↗

ELISA to evaluate plasma anti-asparaginase IgG concentrations in patients with acute lymphoblastic leukemia.

The development of antibodies to asparaginase may attenuate the pharmacologic effect of asparaginase treatment, may be associated with hypersensitivity reactions, and may necessitate switching to a different commercial asparaginase preparation for current or future therapy. Thus, development of an ELISA for measurement of anti-asparaginase antibody levels is important in the clinical setting. An anti-asparaginase antibody reference was established by screening 65 plasma samples from six patients with acute lymphoblastic leukemia (ALL) who had recently developed a hypersensitivity reaction to Escherichia coli or Erwinia chrysanthemi asparaginase therapy. Twenty-one plasma samples were selected for the anti-asparaginase antibody reference pool. Five micrograms per milliliter of commercial E. coli and Erwinia asparaginase and 10 microg/ml of E. coli asparaginase conjugated with polyethylene glycol (PEG asparaginase) were found to be optimal as coating antigen concentrations. Anti-asparaginase antibody concentrations were determined using a commercial polyclonal goat anti-human IgG horseradish peroxidase conjugate. The antibody reference curves were linear in a range of absorbance from 0.1 to 1. 5 O.D. units for dilutions from 1:1600 to 1:51,200. Inter-assay coefficients of variation were 9.04, 14.7 and 13.0%, and intra-assay coefficients of variation were 1.44, 4.43 and 3.28% for antibodies against E. coli, Erwinia, and PEG L-asparaginase, respectively. The cut-off for positivity in plasma was determined as mean+2 S.D. of the optical density values for plasma from untreated healthy volunteers. Measurement of specific IgG by this ELISA allows for the evaluation of plasma anti-asparaginase antibody concentrations in patients receiving one or more of the multiple commercial L-asparaginase preparations.

Absorption↗

Asymmetric synthesis of L-homophenylalanine by equilibrium-shift using recombinant aromatic L-amino acid transaminase.

L-Homophenylalanine (L-HPA) was asymmetrically synthesized from 2-oxo-4-phenylbutyric acid (2-OPBA) and L-aspartate using a recombinant aromatic amino acid transaminase (AroAT). To screen microorganisms having such an L-specific AroAT with a relaxed substrate inhibition in the asymmetric synthesis of unnatural amino acids, enrichment cultures were performed in a minimal media containing 50 mM L-HPA as a sole nitrogen source. To reduce the intracellular background synthetic activity by amino acid pools in the cells, a two-step screening method was used. The putative AroAT (i.e., AroATEs) from the screened Enterobacter sp. BK2K-1 was cloned, sequenced, and overexpressed in E. coli cells. The activity of the overexpressed AroATEs was 314-fold higher than that of the wild-type cell. The substrate specificities of the enzyme and homology search revealed that the cloned transaminase is true AroAT. The AroATEs showed a substrate inhibition by 2-OPBA from 40 mM in the asymmetric synthesis, which made it difficult to perform batch asymmetric synthesis of L-HPA at high concentrations of 2-OPBA. To avoid the substrate inhibition by 2-OPBA, intermittent addition of the solid-state substrate was attempted to obtain a high concentration of L-HPA. By using the cell extract (75 U) obtained from the recombinant E. coli harboring the AroATEs gene, the asymmetric synthesis of L-HPA at 840 mM of 2-OPBA resulted in >94% of conversion yield and >99% ee of L-HPA of optical purity. Due to the low solubility (<2 mM) of L-HPA in the reaction buffer, synthesized L-HPA was continuously precipitated in the reaction media, which drives the reaction equilibrium towards the product formation. After full completion of the reaction, L-HPA of high purity (>99% ee) was easily recovered by simple pH shift of the reaction media. This method can permit very efficient asymmetric synthesis of other unnatural amino acids using a single transaminase reaction.

Amino Acid Sequence↗