Conservation of Differential Animal MicroRNA Processing by Drosha and Dicer

In complex biochemical systems, an enzyme, protein, or RNA, symbolized as E, has hundreds or thousands of substrates or interacting partners. The relative specificity hypothesis proposes that such an E would differentially interact with and influence its many distinct, downstream substrates, thereby regulating the underlying biological process (es). The importance of relative specificity has been underappreciated, and evidence of its physiological consequences particularly lacking. Previously we showed that human Drosha and Dicer ribonucleases (RNases) both discriminate their respective microRNA (miRNA) substrates, and that differential cleavage by Drosha contributes to global differential miRNA expression. If relative specificity is an important biological mechanism, it should be evolutionarily conserved. To test this hypothesis, we hereby examined the cleavage of hundreds of zebrafish and fruitfly miRNA intermediates by Drosha and Dicer and the impact on miRNA biogenesis in these organisms. We showed that Drosha action regulates differential miRNA expression in zebrafish and fruitflies and identified the conserved secondary structure features and sequences in miRNA transcripts that control Drosha activity and miRNA expression. Our results established the conservation of miRNA processing mechanisms and regulatory functions by Drosha and Dicer, greatly strengthened the evidence for the physiological consequences of relative specificity as well as demonstrated its evolutionary significance.

CLINICALEVALUATION OF ORAL CAVITYSTATUS IN PATIENTS WITH KERATINIZATION D

Pathological processes associated with disturbed keratinization and risks of cancer tend to become more common and affect younger people. However, the currently available diagnostic methods are not always reliable, which explains the need to further study this. The aim of the study was to improve the efficiency of early diagnosis of keratoses of the oral cavity under autofluorescence spectroscopy incorporated into the proposed screening algorithm. The examination methods involved clinical, luminescent, analytical, statistical evaluation. The study allowed obtaining optical images through autofluorescence stomatoscopy in patients with keratinization disorders, identifying their color range within affected and healthy tissues, as well as confirming their reliability employing the Color Spatioplotter ver 2.46 software. The tested autofluorescent stomatoscopy method featured sufficient sensitivity (98%) against relative specificity (75%), with prediction of a positive outcome (100%).

Validation of the cobas 6800 human papillomavirus test in primary cervical screening

Evaluation of Human Papillomavirus (HPV) testing systems suitable for large-scale organized cervical screening programs is required. We evaluated the cobas 6800 HPV test system for detection of cervical intraepithelial neoplasia grade 3 or worse (CIN3+) when nested in an organized primary HPV screening program, using the cobas 4800 test as comparator. The Karolinska University Hospital Cervical Cytology Biobank, containing frozen cervical samples from >700,000 women participating in organized cervical screening, was linked to the Swedish national cervical screening registry to identify 470 stored cervical samples taken <180 days before histopathological diagnosis of CIN3+. Two controls per case, with no abnormal results for 2 screening rounds, matched for age and sampling time were also retrieved. Aliquots from 1406 women were retrieved and re-tested on the cobas 4800 system and tested on the cobas 6800 system. There was high reproducibility between the original cobas 4800 HPV test results, and the cobas 4800 HPV re-testing performed on the samples retrieved from biobank storage. 462/464 biobanked samples from women with CIN3+ tested HPV-positive on the cobas 6800 system, corresponding to a relative sensitivity of 99.6%. 925/932 biobanked samples from control women tested HPV-negative on the cobas 6800 platform, corresponding to a relative specificity of 99.2%. By conventional criteria, the cobas 6800 was non-inferior both regarding relative sensitivity of >90% (non-inferiority p-value <0.0001) and relative specificity of >98% (non-inferiority p-value 0.006). We conclude that the cobas 6800 HPV test system had similar, high performance as the cobas 4800 such, when evaluated using cervical samples taken before CIN3+ in a real-life primary HPV screening program.

In-House Validation of a Rinse–Membrane Filtration Method for Processing Fresh Produce Samples for Downstream Cultural Detection of Salmonella, Escherichia coli O157:H7, and Listeria

ABSTRACT More efficient sampling and detection methods of pathogens on fresh produce are needed. The purpose of this study was to compare a novel rinse–membrane filtration method (RMFM) to a more traditional sponge rubbing or stomaching method in processing jalapeño peppers and cantaloupe samples for detection of Escherichia coli, Salmonella enterica, and Listeria monocytogenes. For jalapeño peppers inoculated with 106, 104, and 102 CFU of each pathogen and cantaloupes inoculated at 106 and 104 CFU, all pathogens were detected in all (100%) samples by RMFM at a 10-mL filtration volume, as well as by the stomacher and sponge rubbing methods. However, for cantaloupe inoculated at 102 CFU, detection differed by pathogen: S. enterica (20% RMFM, 60% stomacher, and 20% sponge), L. monocytogenes (40% RMFM, 60% stomacher, and 20% sponge), and E. coli O157:H7 (100% RMFM, 75% stomacher, and 75% sponge). When RMFM was compared with the other methods, in accordance with guidelines in the International Organization for Standardization 16140:2003 protocol, it produced values &gt;95% in relative accuracy, relative specificity, and relative sensitivity. Overall, the RMFM performed similar to or better than the homogenization and sponge surface rubbing methods and is a good alternative for processing large numbers of produce samples for bacterial pathogen detection.

Bayesian estimation of diagnostic accuracy of fecal culture and PCR-based tests for the detection of Salmonella enterica in California cull dairy cattle

Epidemiological studies of low prevalence disease problems are often hindered by the high cost of diagnostic testing. The objective of this study was to evaluate PCR screening of both individual and pooled fecal samples from culled dairy cows for the invA gene of Salmonella followed by culture to determine if the sensitivity and specificity were comparable to the results from traditional culture methods applied to individual samples. Cows from six different dairies were sampled in all four seasons. A total of 240 individual cow fecal samples, 24 fecal pools and 24 pools of 24-hour tetrathionate enrichment broth were tested. Diagnostic sensitivity of PCR screening followed by culture of PCR positive or indeterminate samples (i.e PCR-CUL method) was lower than that of culture (CUL) when applied to individual fecal samples (94.8%, 99.5%), however the specificity was comparable (99.6% and 97.7% respectively). For pools of five fecal samples and pools of five, 24 h tetrathionate broth samples, the specificity of both tests were comparable (∼98%); however, their sensitivity was only comparable in pooled fecal samples (∼93%) but greater for culture compared to PCR-CUL in pooled broth samples (∼99% versus ∼93%). Compared to culture results from testing of individual fecal samples, testing pooled fecal samples by culture had a relative sensitivity of 74% and relative specificity of 96%, testing pooled fecal samples by PCR-CUL resulted in relative sensitivity of 90% and relative specificity of 96%. Testing of pooled 24-hour enrichment broth by PCR-CUL increased the relative sensitivity and specificity to 100%. PCR testing followed by culture of positive or indeterminate samples is a time saving alternative to traditional methods. In addition, pooling of samples may be a useful method for decreasing cost if study aims can accommodate a moderate loss of relative sensitivity.

Development of recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay for sero-survey of porcine reproductive and respiratory syndrome

Background and Aim: Porcine reproductive and respiratory syndrome (PRRS) is a disease endemic in many countries and is of economic importance. India was free from PRRS until the first outbreak was reported from a North-East Indian state in 2013. Since then, disease outbreaks have been reported from North-East India and the pilot study conducted earlier showed that it is gradually spreading to the rest of India. Considering there are no locally developed population screening tests available for PRRS and imported diagnostic/screening tests are expensive, the present study was aimed at developing recombinant nucleocapsid (rN) protein-based indirect enzyme-linked immunosorbent assay (iELISA). Materials and Methods: The rN protein of PRRS virus (PRRSV) was produced following standard cloning, expression, and purification procedures. Using this antigen, iELISA was optimized for the detection of serum antibodies to PRRSV. The sensitivity and specificity of the test were assessed by comparing it with a commercial PRRSV antibody detection kit. Results: A total of 745 serum samples from ten different states of India were screened using the developed iELISA. The iELISA had a relative specificity of 76.18% and sensitivity of 82.61% compared to the commercial ELISA (Priocheck PRRSV ELISA kit, Thermo Fisher Scientific, USA). Conclusion: The iELISA, which deployed rN protein from Indian PRRSV, was found to be suitable in the serological survey and may be a useful tool in future disease surveillance programs.

Performance of DNA methylation assays for detection of high-grade cervical intraepithelial neoplasia (CIN2+): a systematic review and meta-analysis

Background To conduct a meta-analysis of performance of DNA methylation in women with high-grade cervical intraepithelial neoplasia (CIN2+). Methods Medline and Embase databases were searched for studies of methylation markers versus histological endpoints. Pooled sensitivity, specificity and positive predictive value (PPV) for CIN2+ were derived from bivariate models. Relative sensitivity and specificity for CIN2+ compared to cytology and HPV16/18 genotyping were pooled using random-effects models. Results Sixteen thousand three hundred thirty-six women in 43 studies provided data on human genes (CADM1, MAL, MIR-124-2, FAM19A4, POU4F3, EPB41L3, PAX1, SOX1) and HPV16 (L1/L2). Most (81%) studies evaluated methylation assays following a high-risk (HR)-HPV-positive or abnormal cytology result. Pooled CIN2+ and CIN3+ prevalence was 36.7% and 21.5%. For a set specificity of 70%, methylation sensitivity for CIN2+ and CIN3+ were 68.6% (95% CI: 62.9–73.8) and 71.1% (95% CI: 65.7–76.0) and PPV were 53.4% (95% CI: 44.4–62.1) and 35.0% (95% CI: 28.9–41.6). Among HR-HPV+ women, the relative sensitivity of methylation for CIN2+ was 0.81 (95% CI: 0.63–1.04) and 1.22 (95% CI: 1.05–1.42) compared to cytology of atypical squamous cells of undetermined significance, or greater (ASCUS+) and HPV16/18 genotyping, respectively, while relative specificity was 1.25 (95% CI: 0.99–1.59) and 1.03 (95% CI: 0.94–1.13), respectively. Conclusion DNA methylation is significantly higher in CIN2+ and CIN3+ compared to ≤CIN1. As triage test, DNA methylation has higher specificity than cytology ASCUS+ and higher sensitivity than HPV16/18 genotyping.

Structure-Based Design of Nucleoside-Derived Analogues as Sulfotransferase Inhibitors

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group (‘sulfation’) from the enzyme co-factor 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein-protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer’s disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3',5'-diphosphate (PAP) and PAPS mimetics based upon 2'-deoxyadenosine and using 2'-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs.

Structure-Based Design of Nucleoside-Derived Analogues as Sulfotransferase Inhibitors

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group (‘sulfation’) from the enzyme co-factor 3ʹ-phosphoadenosine 5ʹ-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein-protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer’s disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3',5'-diphosphate (PAP) and PAPS mimetics based upon 2'-deoxyadenosine and using 2'-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs.

Comparison of indirect and modified agglutination tests for detection of antibodies to Toxoplasma gondii in domestic cats

Toxoplasma gondii is a protozoan parasite with worldwide distribution. The accurate detection of this zoonotic agent in cats and other hosts has public health importance. Blood samples from 89 domestic cats were tested for antibodies to T. gondii using 2 commercial agglutination test kits, an indirect (IHAT; Toxo-HAI FUMOUZE; Fumouze Diagnostics) and a modified (MAT; Toxoscreen DA; bioMérieux) agglutination test. Antibodies were found in 16 of 89 (18%) cats by the IHAT and in 23 of 89 (26%) cats by the MAT, with an overall agreement between the 2 serologic tests of 92% (κ = 0.77; i.e., substantial agreement beyond chance). Considering the MAT as the gold standard, the IHAT showed perfect relative specificity (100%) and lower relative sensitivity (70%). The suboptimal sensitivity of the IHAT limits its use in epidemiologic studies in cats.