Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms, and risk of colorectal adenoma

We examined the relationship between a functional polymorphism (667C-- >T, ala-->val) of the methylenetetrahydrofolate reductase gene (MTHFR) and the risk of colorectal adenomas in the prospective Nurses' Health Study. Among 257 incident polyp cases and 713 controls, the MTHFR val/val polymorphism [relative risk (RR) = 1.35, 95% confidence interval (CI) 0.84-2.17] was not significantly associated with risk of adenomas. This lack of association was observed for both small (RR = 1.36, 95% CI 0.76-2.45) and large (RR = 1.32, 95% CI 0.66-2.66) adenomas. Furthermore, there was no significant interaction between this polymorphism and consumption of either folate, methionine or alcohol. We also examined the relationship of a newly identified polymorphism (asp919gly) of the methionine synthase gene (MS) with the risk of colorectal adenomas in the same population. The MS gly/gly polymorphism was also not significantly associated with risk of colorectal adenomas (RR = 0.66, 95% CI 0.26-1.70). These results, which need to be confirmed in other studies, suggest that the MTHFR val/val polymorphism, which has been previously inversely associated with risk of colorectal cancer, plays a role only in a late stage (adenoma-- >carcinoma) of colorectal tumorigenesis, and/or may protect against malignant transformation in the subset of benign adenomas, which may progress to malignancy.      

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

In-hospital and mid-term adverse clinical outcomes of a direct stenting strategy versus stenting after predilatation for the treatment of coronary artery lesions

Background

Direct stenting without balloon dilatation may reduce procedural costs and duration, and hypothetically, the restenosis rate. This study was designed to compare the in-hospital and long-term outcomes of direct stenting (DS) versus stenting after pre-dilatation (PS) in our routine clinical practice.

Methods

The 1 603 patients treated with stenting for single coronary lesions were enrolled into a prospective registry. Patients with acute myocardial infarction (MI) within the preceding 48 hours, and those with highly calcified lesions, total occlusions, or a lesion in a saphenous graft were excluded. The baseline, angiographic and procedural data, inhospital outcomes and follow-up data were recorded in our database and analysed with appropriate statistical methods.

Results

Eight hundred and fifty-seven patients (53.5%) were treated with DS and 746 (46.5%) underwent PS. In the DS group, lesions were shorter in length, larger in diameter and had lower pre-procedural diameter stenosis. Type C and diffuse lesions and drug-eluting stents were found less often (p < 0.001). With univariate analysis, dissection and non-Q-wave MI occurred less frequently in this group (0.2 and 0.6% vs 3.9 and 2.1%, p < 0.001 and p = 0.01, respectively). However, the cumulative major adverse cardiac events (MACE) did not differ significantly (4.9 vs 4.6%, p = 0.79). With multivariate analysis, direct stenting reduced the risk of dissection (OR = 0.07, 95% CI: 0.01–0.33, but neither the cumulative endpoint of MACE (OR = 1.1, 95% CI = 0.58–2.11, p = 0.7) nor its constructing components were different between the groups.

Conclusions

Direct stenting in the real world has at least similar long-term outcomes in patients treated with stenting after pre-dilatation, and is associated with lower dissection rates.     

Setting-Level Influences on Implementation of the Responsive Classroom Approach

We used mixed methods to examine the association between setting-level factors and observed implementation of a social and emotional learning intervention (Responsive Classroom® approach; RC). In study 1 (N?=?33 3rd grade teachers after the first year of RC implementation), we identified relevant setting-level factors and uncovered the mechanisms through which they related to implementation. In study 2 (N?=?50 4th grade teachers after the second year of RC implementation), we validated our most salient Study 1 finding across multiple informants. Findings suggested that teachers perceived setting-level factors, particularly principal buy-in to the intervention and individualized coaching, as influential to their degree of implementation. Further, we found that intervention coaches’ perspectives of principal buy-in were more related to implementation than principals’ or teachers’ perspectives. Findings extend the application of setting theory to the field of implementation science and suggest that interventionists may want to consider particular accounts of school setting factors before determining the likelihood of schools achieving high levels of implementation.     

Transforming growth factor beta (TGF-beta), a potent inhibitor of erythropoiesis: neutralizing TGF-beta antibodies show erythropoietin as a potent stimulator of murine burst-forming unit erythroid colony formation in the absence of a burst-promoting activity

Transforming growth factor beta (TGF-beta) is a bifunctional regulator of the growth of myeloid progenitors and is here demonstrated to directly inhibit the growth of primitive erythroid progenitors by 95% to 100% regardless of the cytokines stimulating growth. Autocrine TGF- beta production of primitive hematopoietic progenitors has previously been reported. In the present study, a neutralizing TGF-beta antibody (anti-TGF-beta) added to serum-containing cultures, resulted in a 3-, 4- , and 25-fold increase in burst-forming unit erythroid (BFU-E) colony formation in response to interleukin-4 (IL-4) plus erythropoietin (Epo), SCF plus Epo, and IL-11 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition to Epo. Accordingly, we observed no BFU-E colony formation in serum-containing cultures in response to Epo alone. In contrast, 50 BFU-E colonies were formed when anti-TGF-beta was included in the culture. In serum-free cultures, Epo also stimulated BFU-E colony formation in the absence of other cytokines, whereas anti-TGF- beta had no effect on the number of colonies formed. Quantitation of TGF-beta 1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-beta 1, but also showed active TGF-beta 1 at a concentration sufficient to potently inhibit erythroid colony formation. Thus, neutralization of active TGF- beta 1 in serum shows that Epo alone is sufficient to stimulate the growth of murine BFU-E progenitors.