1141612823Effect of recombinant ovine intereferon tau (IFN) on the ovine Mx1 promoter/enhancer region

Ovine Mx1 (oMx1) is expressed in the uterus during the estrous cycle and is strongly up-regulated during early pregnancy in the uterus and peripheral blood leukocytes. Mx proteins are GTPases which are important elements in innate immunity. Our results showed that steroids are required for oMx1 protein induction by IFN in the uterus. To addresses the role of IFN in regulating oMx1 expression, a 2.1 kb fragment containing 1.7 kb of the promoter/enhancer, exon1 and partial intron A was cloned. Serial deletions were prepared along with a clone that contained the 2 promoter-ISREs (-101 & −145) but not the intronic-ISRE (+224). An ovine uterine cell line was transfected with reporter plasmids driven by the oMx1 promoter deletion constructs. The full-length promoter was induced by IFN in a dose- and time-dependent manner. Treatment with 10,000 AVU/mL IFN increased luciferase activity 5- and 10-fold at 3 and 12 hr, respectively. Promoter deletions showed the 2 proximal ISRE (−101 and −145), but not an intronic ISRE (+244), were required for maximal response. Deletion of a distal region (−920 to −715) resulted in decreased luciferase activity (~4-fold). However, subsequent deletion of the −715 to −437 region restored maximal promoter response (~10-fold). Results suggest that regions −920 to −715 and −715 to −437 have positive and negative regulatory element binding sites, respectively. The importance of the 2 proximal ISRE sites for oMx1 promoter activation is consistent with results for the human MxA promoter. An intronic ISRE is present in the human MxA gene, however, this site may not be required for oMx1 promoter activation by IFN. Identifying positive and negative regulatory regions in oMx1 promoter may help elucidate the unique regulation of Mx1 during early pregnancy.     

Solid renal tumor severity in von Hippel Lindau disease is related to germline deletion length and location

von Hippel Lindau disease (VHL) is an autosomal dominant familial cancer syndrome linked to alteration of the VHL tumor suppressor gene. Affected patients are predisposed to develop pheochromocytomas and cystic and solid tumors of the kidney, CNS, pancreas, retina, and epididymis. However, organ involvement varies considerably among families and has been shown to correlate with the underlying germline alteration. Clinically, we observed a paradoxically lower prevalence of renal cell carcinoma (RCC) in patients with complete germline deletion of VHL. To determine if a relationship existed between the type of VHL deletion and disease, we retrospectively evaluated 123 patients from 55 families with large germline VHL deletions, including 42 intragenic partial deletions and 13 complete VHL deletions, by history and radiographic imaging. Each individual and family was scored for cystic or solid involvement of CNS, pancreas, and kidney, and for pheochromocytoma. Germline deletions were mapped using a combination of fluorescent in situ hybridization (FISH) and quantitative Southern and Southern blot analysis. An age-adjusted comparison demonstrated a higher prevalence of RCC in patients with partial germline VHL deletions relative to complete deletions (48.9 vs. 22.6%, p=0.007). This striking phenotypic dichotomy was not seen for cystic renal lesions or for CNS (p=0.22), pancreas (p=0.72), or pheochromocytoma (p=0.34). Deletion mapping revealed that development of RCC had an even greater correlation with retention of HSPC300 (C3orf10), located within the 30-kb region of chromosome 3p, immediately telomeric to VHL (52.3 vs. 18.9%, p <0.001), suggesting the presence of a neighboring gene or genes critical to the development and maintenance of RCC. Careful correlation of genotypic data with objective phenotypic measures will provide further insight into the mechanisms of tumor formation.     

Missense mutation clustering in the survival motor neuron gene: a role for a conserved tyrosine and glycine rich region of the protein in RNA metabolism?

The Survival Motor Neuron (SMN) gene shows deletions in the majority of patients with Spinal Muscular Atrophy (SMA), a disease of motor neuron degeneration. To date only two missense mutations have been reported in SMN in patients with SMA. The fact that no SMN-homologues have been forthcoming from data-base searching has resulted in a lack of hypotheses concerning the structural and functional consequences of these mutations. Recently SMN has been shown to interact with heterogeneous nuclear ribonucleoproteins (hnRNPs) suggesting a role in mRNA metabolism. We describe a novel missense mutation and the subsequent identification of a triplicated tyrosine-glycine (Y-G) peptide sequence at the C-terminal of SMN which encompasses each of the three predicted amino acid sequence substitutions. We have identified apparent orthologues of SMN in Caenorhabditis elegans and Schizosaccharomyces pombe. These sequences retain the highly conserved Y-G motif and provide additional support for a role of SMN in mRNA metabolism.      

Seminal plasma alpha-glucosidase activity and male infertility

Measurement of alpha-glucosidase (alpha-GLUC) activity by means of a simple colorimetric test using a commercial kit (EpiScreen; FertiPro, Lotenhulle, Belgium) yielded results that were strongly correlated with the values obtained for the neutral iso-enzyme measured by a fluorimetric reference method (r=0.85, P=0.003, n=13). The former method was characterized by a low intra- and inter-coefficient of variation (6.6 and 4.3% respectively). Vasectomized men with azoospermia (n=27) had a significantly lower alpha-GLUC activity in semen than vasectomized men with residual spermatozoa present (n=11, P < 0.01) and men with azoospermia of primary testicular origin (n=33, P < 0.01). Receiver operating curve (ROC) analysis showed alpha-GLUC measurement to be reasonably accurate in differentiation between cases with obstructive versus testicular azoospermia at criterion value 13.5 U/l (sensitivity=82%, specificity= 70%). In cases with spermatozoa present, alpha-GLUC activity and output per ejaculate were positively correlated with sperm concentration (r=0.53 and 0.38, n=472), linear velocity (r=0.35 and 0.30, n=224), curvilinear velocity (r=0.32 and r=0.29, n=224), semen adenosine triphosphate (r=0.35 and 0.26, n=64), the concentration of 5alpha-dihydrotestosterone (r=0.31 and 0.29, n=74), and gamma-glutamyltransferase activity (r=0.62 and 0.32, n=275) in seminal plasma. The activity of alpha-GLUC was inversely correlated with ROS generation after 12-myristate, 13-acetate phorbol ester stimulation (r=-0.27, n=104), and both alpha-GLUC activity and total output were inversely correlated with the concentration of peroxidase- positive white blood cells among samples with > or =1x10(6)/ml of these cells (r=-0.30 and -0.19, n=165). It is concluded that simple photometric measurement of alpha-GLUC activity in seminal plasma reflects the functional state of the epididymis and may be helpful for the differential diagnosis of certain cases with azoospermia.      

Effect of delay in processing and storage temperature on diagnosis of SARS-CoV-2 by RTPCR testing

PurposeA large number of new molecular or virology laboratories have been established to increase the testing capacity for SARS-CoV-2. Due to heavy workload, there is delay in testing of samples. In order to avoid the negative effect of delayed testing on RTPCR results guidelines are issued from WHO and CDC to transport samples in cold chain. However, in pandemic situations the recommended guidelines for transport and storage conditions are often compromised. This study was conducted to evaluate the effect of sample storage conditions at different temperatures on the results of RT PCR test.MethodsTotal 275 samples were included in this study, among these 126 samples tested positive and 149 samples tested negative. All samples were aliquoted into two and the aliquotes stored in duplicate at 4 ?°C and room temperature. All aliquots stored at both the temperatures were tested by RTPCR every 24 hours up to 5 days.ResultsDiagnostic accuracy decreased from day1 to day 5 at both the storage temperatures i,e 4 ?°C and room temperature in comparison to the initial day results. Positivity decreased on an average of 9.02% at 4 ?°C and at 9.27% at room temperature per day. Among total 126 positive samples on an average false negative and failure of internal control at 4 ?°C and room temperature was 8.86%, 8.22% and 3.64%, and 4.12%, respectively. All the samples with CT value ?< ?30 remained positive at both temperatures up to 5 days. Few samples with >30 CT value showed variable results i.e. positive, negative or internal control failure from day 1 (2nd day after sample collection) onwards.ConclusionsThere was no significant difference between RT PCR results of samples stored at 4 ?°C and room temperature up to 5 days of collection. However internal control failure was more in samples stored at room temperature. Therefore, samples received without cold chain also may be processed by RTPCR and should not be rejected.     

Evaluation of the performance of hysterosalpingo contrast sonography in 500 consecutive, unselected, infertile women

The performance of hysterosalpingo contrast sonography (Hy Co Sy) as a first-line, outpatient investigation of tubal patency was examined in 500 consecutive, infertile women, at one centre. Hy Co Sy was completed in 463 (92.6%) cases, using a galactose microbubble contrast agent (Echovist-200) and transvaginal sonography. Initial plain scanning identified adnexal pathology in 198 women (39.6%). Examination with Echovist was attempted for 905 tubes and only 67 (7.4%) were not assessable; after the first 100 women this decreased to 35 tubes (4.8%). A sonographic appearance compatible with blocked tubes was found on 118 (14.1%) occasions but it was also possible to identify variations in the appearance/filling/spilling patterns of individual tubes which increased the number assessed as abnormal to 193 (23.0%). Comparison with laparoscopy and dye chromopertubation findings from the past three years was possible for 185 (37%) women, representing 282 tubes, which gave Hy Co Sy an overall concordance rate of 85.8%, sensitivity of 90.4%, specificity of 70.3%, positive predictive value of 91.2% and negative predictive value of 68.2%. Some 51.0% of women described only mild discomfort and there were no significant postprocedure complications. Hy Co Sy appears to be an acceptable first- line screen and may select out women in whom more invasive investigations are likely to reveal pathology.      

Linkage analysis of candidate regions for coeliac disease genes

A strong HLA association is seen in coeliac disease [specifically to the DQ(alpha1*0501,beta1*0201 heterodimer], but this cannot entirely account for the increased risk seen in relatives of affected cases. One or more genes at HLA-unlinked loci also predispose to coeliac disease and are probably stronger determinants of disease susceptibility than HLA. A recent study has proposed a number of candidate regions on chromosomes 6p23 (distinct from HLA), 6p12, 3q27, 5q33.3, 7q31.3, 11p11, 15q26, 19p13.3, 19q13.1, 19q13.4 and 22cen for the location of a non-HLA linked susceptibility gene. We have examined these regions in 28 coeliac disease families by linkage analysis. There was excess sharing of chromosome 6p markers, but no support for a predisposition locus telomeric to HLA. No significant evidence in favour of linkage to coeliac disease was obtained for chromosomes 3q27, 5q33.3, 7q31.3, 11p11, 19p13.3, 19q13.1, 19q13.4 or 22cen. There was, however, excess sharing close to D15S642. The maximum non-parametric linkage score was 1.99 (P = 0.03). Although the evidence for linkage of coeliac disease to chromosome 15q26 is not strong, the well established association between coeliac disease and insulin dependent diabetes mellitus, together with the mapping of an IDDM susceptibility locus (IDDM3) to chromosome 15q26, provide indirect support for this as a candidate locus conferring susceptibility to coeliac disease in some families.