Polymorphism rs547984 on human chromosome 1q43 is not associated with primary open angle glaucoma in a Saudi cohort

Background

To investigate the association between polymorphism rs547984, located in close proximity to the Zona Pellucida Glycoprotein 4 (ZP4) gene on human chromosome 1q43 and primary open angle glaucoma (POAG).

Method

Polymorphism rs547984 was genotyped using Taq-Man® assay in 185 subjects comprising of 90 unrelated POAG cases and 95 controls of Saudi origin.

Results

Association analysis between cases and controls revealed no significant genotype distribution under additive (p = 0.356), dominant (p = 0.517) and recessive (p = 0.309) models. Besides, the allele frequency distribution was also found to be non-significant (p = 0.70). The minor “A” allele frequency was found to be 0.49 and 0.50 among POAG cases and controls, respectively. In addition, specific clinical indices used to assess severity of glaucoma such as intraocular pressure (IOP), cup/disc ratio and number of anti-glaucoma medication also did not show any significant genotype distribution in POAG cases.

Conclusion

Polymorphism rs547984 is neither associated with any clinical indices important for POAG such as IOP and cup/disc ratio nor is a risk factor for POAG in the Saudi cohort.

     

Increased incidence of mosaicism detected by FISH in murine blastocyst cultured in vitro

The majority of in-vitro-derived human preimplantation embryos are chromosomally abnormal but whether the same pattern exists in vivo is unknown. This would be impossible to demonstrate in humans. Hence we chose murine embryos to study this difference owing to their ease of manipulation and compared the incidence of mosaicism between in-vivo- and in-vitro-cultured embryos. Two groups of embryos were analysed. Group A (in vitro) were obtained 48h following superovulation and cultured in vitro until the blastocyst stage. Fluorescent in-situ hybridization (FISH) was performed at different stages that included the cleavage, morula and blastocyst stage. Group B (in vivo) were obtained on day 2 or day 5 and FISH was performed immediately without culture. There was an increase in chromosomal mosaicism seen from the cleavage stage up to the blastocyst stage in the in-vitro culture group. Overall chromosomal abnormality from day 3 to day 5 was found to be 30% (28/94) in group A. The incidence of chromosomal abnormalities in blastocysts from group B was significantly lower than group A blastocysts (8% (3/40) and 31% (20/64) respectively; P<0.05). These data show that in-vitro cultured embryos had a significantly higher incidence of mosaicisim in comparison with the in-vivo group. Cultured human embryos show high levels of chromosomal abnormalities but whether this is a pattern seen in all embryos or is the result of culture is unknown. To study this pattern we used mouse embryos and carried out chromosome analysis by fluorescent in-situ hybridization. We compared embryos that were cultured (in vitro) with those that were not (in vivo, i.e. grown exclusively in the mouse). We found that cultured embryos showed significantly higher chromosomal abnormalities as compared with in vivo embryos. This suggests that certain culture conditions are responsible for the high level of chromosomal abnormalities seen in these embryos, which should be investigated further.     

Larger and More Prominent Graphic Health Warnings on Plain-Packaged Tobacco Products and Avoidant Responses in Current Smokers: a Qualitative Study

Background

The introduction of tobacco plain packaging legislation in Australia meant that all tobacco products were to be sold in plain dark-brown packaging with 75 % front-of-pack graphic health warnings and standardised font type and size for brand name and product variant. The change in the size and prominence of the warnings has been proposed as a reason for behaviour change in smokers in terms of increased intentions to quit and quit attempts.

Purpose

The current research examined attitudes and beliefs of cigarette smokers toward the increased size and prominence of the warnings and effects on their behaviour.

Method

Participants (N?=?160) completed open-ended responses to questions on beliefs, attitudes and responses to plain packaging. Responses were subjected to inductive thematic content analysis for key themes.

Results

Four themes emerged from the analysis: emotional response to packaging, scepticism of health warnings, warnings and cessation behaviour, and avoidant coping behaviours. Participants reported increased negative emotional responses to the packaging and made specific reference to the graphic health warnings. Some participants attempted to discredit the messages. Others reported increased intentions to quit or quitting attempts. There were pervasive reports of avoidant responses including covering or hiding the warnings.

Conclusion

Consistent with theories of illness perceptions and coping, current findings indicate that the larger, prominent graphic health warnings on plain-packaged tobacco products had pervasive effects on threat perceptions and subsequent behavioural responses. While some of the reported responses were adaptive (e.g. attempts to quit), others were maladaptive (e.g. avoiding the warnings).

     

Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis

Several real-time PCR procedures for the detection and genotyping of oocysts of Cryptosporidium parvum were evaluated. A 40-cycle amplification of a 157-bp fragment from the C. parvum beta-tubulin gene detected individual oocysts which were introduced into the reaction mixture by micromanipulation. SYBR Green I melting curve analysis was used to confirm the specificity of the method when DNA extracted from fecal samples spiked with oocysts was analyzed. Because C. parvum isolates infecting humans comprise two distinct genotypes, designated type 1 and type 2, real-time PCR methods for discriminating C. parvum genotypes were developed. The first method used the same beta-tubulin amplification primers and two fluorescently labeled antisense oligonucleotide probes spanning a 49-bp polymorphic sequence diagnostic for C. parvum type 1 and type 2. The second genotyping method used SYBR Green I fluorescence and targeted a polymorphic coding region within the GP900/poly(T) gene. Both methods discriminated between type 1 and type 2 C. parvum on the basis of melting curve analysis. To our knowledge, this is the first report describing the application of melting curve analysis for genotyping of C. parvum oocysts.     

Characterization of T-Cell Responses to Conserved Regions of the HIV-1 Proteome in BALB/c Mice

A likely requirement for a protective vaccine against human immunodeficiency virus type 1 (HIV-1)/AIDS is, in addition to eliciting antibody responses, induction of effective T cells. To tackle HIV-1 diversity by T-cell vaccines, we designed an immunogen, HIVconsv, derived from the most functionally conserved regions of the HIV-1 proteome and demonstrated its high immunogenicity in humans and rhesus macaques when delivered by regimens combining plasmid DNA, nonreplicating simian (chimpanzee) adenovirus ChAdV-63, and nonreplicating modified vaccinia virus Ankara (MVA) as vectors. Here, we aimed to increase the decision power for iterative improvements of this vaccine strategy in the BALB/c mouse model. First, we found that prolonging the period after the ChAdV63.HIVconsv prime up to 6 weeks increased the frequencies of HIV-1-specific, gamma interferon (IFN-γ)-producing T cells induced by the MVA.HIVconsv boost. Induction of strong responses allowed us to map comprehensively the H-2^d-restricted T-cell responses to these regions and identified 8 HIVconsv peptides, of which three did not contain a previously described epitope and were therefore considered novel. Induced effector T cells were oligofunctional and lysed sensitized targets in vitro. Our study therefore provides additional tools for studying and optimizing vaccine regimens in this commonly used small animal model, which will in turn guide vaccine improvements in more expensive nonhuman primate and human clinical trials.     

Hyperglycemia-induced endoplasmic reticulum stress in endothelial cells

ObjectiveHyperglycemia-induced endothelial cell dysfunction in vascular disease can occur due to increased oxidative stress and a concomitant increase in endoplasmic reticulum (ER) stress. To investigate whether these cellular stresses are independent or causally linked, we determined whether or not specific glycolytic intermediates that induce oxidative stress also induce ER stress.MethodsHuman umbilical vein endothelial cells were treated with dextrose, partially metabolizable (e.g., fructose and galactose) and non-metabolizable sugars (e.g., 3-O-methyglucose), and various intermediates of the glycolytic and tricarboxylic acid pathways. Activation of the unfolded protein response and subsequent generation of ER stress was measured by the ER stress-responsive alkaline phosphatase method, and superoxide (SO) generation was measured using the hydro-ethidene–fluorescence method. The mitochondrial origin of the SO and the generation of ER stress by dextrose and the intermediate metabolites were confirmed with experiments using allopurinol and diphenyleneiodonium chloride to block SO generation by xanthine oxidase and nicotinamide adenosine dinucleotide phosphate oxidase, respectively.ResultsAlthough ER stress could be induced by glycolytic intermediates up to and including pyruvate, the SO generation occurred in the presence of glycolytic and mitochondrial metabolites.ConclusionAlthough the mitochondria are the site of signals generated by dextrose to initiate oxidative stress, the dextrose-induced ER stress, unlike SO generation, does not require pyruvate oxidation in the mitochondria.     

Isolation and antibiotic susceptibility of Salmonella, Shigella, and Campylobacter from acute enteric infections in Egypt

While Campylobacter, Salmonella, and Shigella remain major contributors to acute enteric infections, few studies on these pathogens have been conducted in Egypt. From January 1986 to December 1993, 869 Salmonella, Shigella and Campylobacter strains were isolated from stool specimens from 6,278 patients, presenting to the Abbassia Fever Hospital, Cairo, Egypt, with acute enteric infections. Salmonella predominated, totalling 465 isolates, followed by Shigella with 258 isolates, and Campylobacter with 146 isolates. Of the Shigella isolates, 124 were Shigella flexneri, 49 were S. sonnei, 47 were S. dysenteriae (mainly serotype 1, 2, and 3), and 38 were S. boydii. Campylobacter spp. comprised 92 Campylobacter jejuni and 54 C. coli isolates. Isolation of Salmonella was highest during the months of February-March, June-July, and October-November, while that of Shigella was maximal from July to October. Isolation of Campylobacter increased during May-June and again during August-October. Although Salmonella was sensitive to amikacin, aztreonam, ceftriaxone, and nalidixic acid, it was, however, resistant to erythromycin, streptomycin, ampicillin, chloramphenicol, and tetracycline. Shigella (> 80%) was sensitive to amikacin, ceftriaxone, cephalothin, sulphamethoxazole-trimethoprim (except S. sonnei), aztreonam, and nalidixic acid. Resistance (> 50%) was noted only for ampicillin, chloramphenicol, and tetracycline. C. jejuni and C. coli were resistant to cephalothin, aztreonam, and streptomycin. Some of the above antibiotics were employed to characterize the Egyptian isolates, but did not have any clinical utility in the treatment of diarrhoea. Significant differences (p < 0.05) were observed in the resistance profiles of Shigella and Salmonella between late 1980s and early 1990s. The results suggest the use of fluoroquinolones or a third-generation cephalosporin as an empirical treatment of enteric diseases. However, alternative control strategies, including the aggressive development of broadly protective vaccines, may be more effective approaches to curbing morbidity and mortality due to acute enteric infections.     

Cornea in Marfan disease: Orbscan and in vivo confocal microscopy analysis

PURPOSE: To investigate corneal thickness, curvature, and morphology with the Orbscan Topography System I (Bausch & Lomb, Inc., Salt Lake City, UT) in patients with Marfan syndrome (MFS) and to study MFS with in vivo confocal microscopy. METHODS: This prospective, clinical, comparative case series included 60 eyes of 31 patients with MFS and 32 eyes of 17 control subjects. First, biomicroscopic examination was conducted to search for ectopia lentis. Then, mean keratometry and ocular refractive power were calculated by the autokeratorefractometer. In each group, the Orbscan System I mean (and mean simulated) keratometry and pachymetric measurements (at the central location and at eight midperipheral locations) were obtained and compared, and correlations were established. In vivo confocal microscopy was performed to evaluate tissue morphology and Z-scan analysis of 14 thin MFS corneas compared with 14 control corneas. RESULTS: A significant decrease (ANOVA, P < 0.0001) of mean simulated keratometry measurement appeared in the MFS group (sim K, 40.8 +/- 1.4 D) compared with the control group (42.9 +/- 1.1 D). Pachymetry in the MFS group was significantly decreased (P < 0.0001) compared with that in the control group, in the center (respectively, 502 +/- 41.9 microm and 552 +/- 23.6 microm) and the eight midperipheral locations. Ectopia lentis was highly linked with mean keratometry and pachymetry (P < 0.0001). Confocal microscopy performed on MFS-affected thin corneas confirmed the corneal thinning and showed an opaque stromal matrix, and Z-scan profiles were abnormal with increased stromal back scattering of light. CONCLUSIONS: MFS is known to be associated with a flattened cornea. This study demonstrated an association with corneal thinning and described confocal microscopy findings in this syndrome.     

Testicular toxicity of di-(2-ethylhexyl)phthalate in young Sprague-Dawley rats

Di-(2-ethylhexyl)phthalate (DEHP), used widely in the manufacture of plastics, is a well-known reproductive toxicant. It causes apoptosis and loss of spermatogenic cells, resulting in testicular atrophy. Reports are scarce in the literature on the progression of apoptosis following repeated doses of phthalates. DEHP's mechanism of inducing testicular atrophy has been associated with depletion of zinc in the testis. ZnT-1 is a zinc transporter that is highly expressed in the testis. Thus, DEHP might exert its toxic effects on the testis by altering the expression of ZnT-1. In this regard, 25-day old Sprague-Dawley rats were given vehicle (5 ml corn-oil/kg, po) for 2, 7 and 14 days, or DEHP (2 g/5 ml corn-oil/kg, po) daily, for 1, 2, 3, 5, 7, 10 and 14 days. Zinc content in testes was determined by atomic absorption spectrophotometry, and ZnT-1 mRNA was quantified by the branched DNA signal amplification method. Body weight gain and testicular weight (absolute and relative) were significantly lower in DEHP-treated rats. DEHP produced morphological changes in the testis, including apoptosis, necrosis, and loss of spermatogenic cells, which resulted in testicular atrophy. Apoptotic index (AI: the percentage of apoptotic cells in seminiferous tubules), determined using the TUNEL technique, was markedly increased after 1 day (AI: 2.9%, control AI: 0.1-0.3%) followed by a peak at 3 days (AI: 11.5%) and a gradual decrease till 10-14 days (AI: 7-9%). Zinc content in testis was not changed 1 day after DEHP administration, but decreased significantly at later time points. No difference was found in ZnT-1 mRNA expression between control and DEHP-treated animals until day 14. Our results suggest that apoptosis, along with necrosis, plays an important role in the mechanism of testicular atrophy by DEHP. In addition, ZnT-1 mRNA expression was not altered by DEHP and therefore, it appears that ZnT-1 cannot account for the decrease in testicular Zn content. Pathological lesions and apoptosis occurred prior to the loss of zinc in testis, suggesting that zinc depletion might be a secondary effect of DEHP-induced testicular toxicity, rather than the cause.