Hepatitis B, Chronic ; Humans

Objective To examine the association of intron 4 polymorphism in endothelial nitric oxide synthase ( eNOS) gene with diabetic nephropathy and its distribution in different ethnic groups of Singapore. Methods 258 patients with diabetic duration longer than 10 years (150 Chinese, 71 Malay and 37 Indian) were selected from both inpatients and outpatients of National University Hospital. Polymorphism was identified by DNA extraction, PCR, Cloning and sequencing. Results All published studies identified that the 27bp repeat polymorphism in intron 4 was biallelic ( a and b alleles ). Our study found a third allele-allele c, with six 27bp repeats. It was also demonstrated that there was no significant difference in overall allele distribution among the three main ethnic groups and the intron 4 polymorphism didn't show any association with diabetic nephropathy. Conclusion The intron 4 polymorphism was triallelic. We failed to demonstrate any association between this polymorphism with diabetic nephropathy.

Objective: To assess fluid-attenuated inversion recovery (FLAIR) vascular hyper-intensity (FVH) and explore its relationship with CT perfusion (CTP) penumbral/infarct core mismatch ratio and diffusion weighted imaging (DWI) final infarct volume in acute ischemic stroke (AIS) patients with middle cerebral artery occlusion (MCAO). Methods: The CTP and MRI images of 38 AIS patients with MCAO were reviewed. The FVH score (longitudinal direction) [FVH score (L)] and FVH score (transverse direction) [FVH score (T)] were quantified on the FLAIR images. The FVH score (L) (range, 0-16) was based on a rostrocaudal extension of FVH and the FVH score (T) (range, 0-3) was based on FVH supply of the occluded MCA territory. The mismatch ratio was calculated from the ratio of the [mean transit time - cerebral blood volume (CBV)] lesion/CBV lesion on the CTP images. The DWI infarct volume was measured on the DWI images. Results: The mismatch ratio was larger for the group of FVH score (L)=7~8 than those of FVH score (L)=5~6 and FVH score (L)=3~4 (p=0.03), whereas the DWI infarct volume was smaller (p=0.04). Similarly, the mismatch ratio of FVH score (T)=2~3 group was larger than FVH score (T)=1 group (p=0.01), whereas the DWI infarct volume was smaller (p=0.02). Both FVH score (L) and FVH score (T) correlated positively with mismatch ratio (P=0.02, P=0.001, respectively), but negatively with DWI infarct volume (P=0.03, P=0.004, respectively). Conclusions: Higher FVH score is associated with larger mismatch ratio and smaller DWI infarct volume in AIS patients with MCAO. FLAIR vascular hyperintensity may represent collateral arterial circulation, and may play a role in protecting the ischemic penumbra.
Infarction, Middle Cerebral Artery

Objective To compare the immune effects of Coxsackievirus B3 (CVB3) capsid protein VP1 expressed bacterially, recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1which express VP1 protein in mice. Methods After expressed in prokaryotic cells, VP1 protein was purified. Recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1 were amplified and extracted. Six to 8-week-old, male BALB/c mice were divided into four groups randomly. Each group contained 18 mice. The mice of pcDNA3/VP1 group or VP1 protein group were immunized intramuscularly with three injections at three weeks apart, of recombinant plasmid pcDNA3/VP1 at a dose of 100 μg/mouse or recombinant protein VP1 at a dose of 50 μg/mouse. The mice of rAd/VP1 group were immunized intramuscularly twice at two weeks interval with rAd/VP1 at a dose of 1.2 × 107 PFU. The control group was mock-immunized with 100 μl of PBS intramuscularly. Mice were bled from the retroorbital sinus plexus every two weeks after each immunization. ELISA and micro-neutralization test were used to detect levels of CVB3-specific IgG antibody and neutralizing antibody titers in the sera of immunized mice. Three weeks after the last immunization, the cytotoxic T lymphocyte(CTL) killing activity of spleen lymphocytes was detected with CCK-8 assay. Subsequently, virus titers in the sera of immunized mice were determined by the 50% cell culture infective dose( CCID50 ) assay on HeLa cell monolayers and percentage of animals surviving were observed after lethal CVB3 attack over a period of 21 days. Results The titers of specific IgG antibody and neutralizing antibody in sera of VP1 protein immunized mice were higher than other groups( P ＜0.05 ). While CTL killing activity of spleen lymphocytes of VP1 protein immunized mice was lower than mice in rAd/VP1 group( P ＜0. 05). Virus titers in sera of VP1 protein immunized mice were lower than the mice in pcDNA3/VP1 or rAd/VP1 groups ( P ＜ 0.05 ), while survival rate was significantly higher than these two groups ( P ＜ 0.05 ).Conclusion VP1 protein induced higher level of humoral immune response and acquired obvious immune protection effects in mice. The immunizing potency of VP1 protein vaccine surpassed plasmid pcDNA3/VP1or recombinant adenovirus rAd/VP1. It appeared to be a promising candidate among the three different vaccines.

BACKGROUNDThe 3q26 chromosome region, where the human telomerase RNA gene (hTERC) is located, is a biomarker for cervical cancer and precancerous lesions. The aim of this study was to confirm the value of measuring hTERC gene gain in predicting the progression of cervical intraepithelial neoplasia grade I or II (CIN-I and -II, respectively) to CIN-III and cervical cancer.

METHODSLiquid-based cytological samples from 54 patients with CIN-I or CIN-II lesions were enrolled in this study. Follow-up was performed with colposcopy and biopsy within 24 months after the diagnosis of CIN-I or CIN-II. Copy numbers of the hTERC gene were measured by fluorescence in situ hybridization with a dual-color probe mix containing the hTERC gene probe (labeled red) and the control, the chromosome 3 centromere-specific probe (labeled green).

RESULTSAll patients whose lesions progressed from CIN-I or CIN-II to CIN-III displayed a gain of the hTERC gene, whereas patients where the hTERC gene was not amplified did not subsequently progress to CIN-III or cervical cancer. The signal ratio pattern per cell was recorded as N:N (green:red). The numbers of cells with the signal ratio pattern of 4:4 or N:≥5 in patients whose lesions progressed to CIN-III were significantly higher than those whose lesions did not progress. Significantly, none of the patients with a 4:4 signal ratio pattern regressed spontaneously.

CONCLUSIONSIn conclusion, measurement of hTERC gene gain in CIN-I or CIN-II patients using liquid-based cytological samples could be a useful biomarker to predict the progression of such cervical lesions. In addition, a 4:4 or N:≥5 signal ratio pattern may indicate the unlikeness of spontaneous regression of CIN-I or CIN-II lesions.

Adult ; Cervical Intraepithelial Neoplasia ; genetics ; pathology ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Middle Aged ; RNA ; genetics ; Telomerase ; genetics

Introduction: A compulsory research component is becoming increasingly common for clinical residents. However, integrating research into a busy clinical training schedule can be challenging. This study aimed to explore barriers to research supervision in specialist training programmes from the perspectives of clinical supervisors and trainees at a Malaysian university hospital. Methods: Qualitative interviews and focus group discussions were conducted (December 2016 to July 2017) with clinical supervisors (n=11) and clinical trainees (n=26) utilising a topic guide exploring institutional guidelines, research culture and supervisor-student roles. Interviews were transcribed verbatim and analysed thematically to identify barriers to research supervision. Results: Supervisors and trainees from 11 out of 18 departments participated. Both clinical supervisors and trainees struggled to successfully integrate a compulsory research component into residency training. Among the reasons identified included a lack of supervisory access due to the nature of clinical rotations and placements, clashing training priorities (clinical vs research) that discouraged trainees and supervisors from engaging in research, poor research expertise and experience among clinical supervisors hampering high-quality supervision, and a frustrating lack of clear standards between the various parties involved in research guidance and examination. Conclusion: Both clinical supervisors and trainees struggled to successfully integrate a compulsory research component into residency training. This was not only an issue of resource limitation since questions regarding clinical priorities and unclear research standards emerged. Thus, institutional coordinators need to set clear standards and provide adequate training to make research meaningful and achievable for busy clinical supervisors and trainees.

Prader Willi Syndrome (PWS) includes complex endocrinological issues because of the hypothalamic and pituitary dysfunction which include obesity and diabetes, as well as behavioural issues. Other important aspects of PWS, such as hepatosplenomegaly are sometimes neglected. We present a case of diabetic ketoacidosis precipitated by torsion of a wandering spleen in a 22-year-old woman with PWS and type 2 diabetes mellitus. The pancreatic tail was involved in the torsion leading to hyperamylasaemia and pancreatitis. The splenic torsion and pancreatitis were initially treated conservatively with resolution of symptoms. A year later, she had another 2 episodes of severe abdominal pain due to worsening splenic torsion which subsided with conservative management. She subsequently underwent an elective splenectomy which revealed an enlarged and wandering spleen, with 720 degrees torsion of the long splenic pedicle.
Prader-Willi Syndrome

BACKGROUNDAlmost all reported fluorescence in situ hybridization (FISH) kits for prenatal diagnosis use probes from foreign (non-Chinese) countries. The aim of this study was to analyze the reliability of domestic (Chinese) FISH probe sets to detect aneuploidies of chromosomes 13, 18, 21, X, and Y related to prenatal diagnosis in 4210 cases.

METHODSCytogenetic karyotyping was carried out as a standard prenatal diagnostic test, and amniotic fluid cell interphase FISH analysis was performed using two sets of probes (centromeric probes for chromosomes 18, X, and Y, and locus-specific probes for chromosomes 13 and 21) provided by GP Medical Technologies, Beijing, China. Then we compared the two results and found the performance characteristics for informative FISH results of aneuploidies by the domestic kit probes.

RESULTSIn 4210 cases, 4126 cases generated karyotype results and 133 abnormal karyotypes (including 97 aneuploidies) were found. The FISH results of 98 cases (among them, 31 cases gave normal cytogenetic results) were uninformative. The rate of abnormal cases was 3.2% (133/4126). For the abnormal karyotypes, the rate of aneuploidy was 72.9% (97/133). Among the 97 aneuploidies, there were 58 cases of trisomy 21 (58/97, 59.8%), four cases of trisomy 13, 23 cases of trisomy 18, and 12 cases of sex chromosomal aneuploidies. The total concordance of the two methods was 97.9% (95/97; two cases were mosaics that had a low percentage of abnormal cells), and the concordance of trisomy 21, 13, and 18 by the two methods was 100%.

CONCLUSIONSThe two sets of the domestic FISH kit probes are reliable for prenatal diagnosis. The results demonstrate that FISH is a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies.

Amniotic Fluid ; cytology ; Aneuploidy ; Chromosome Aberrations ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Pregnancy

OBJECTIVETo screen for mutations of fibrillin-1 (FBN1) gene in 4 patients with Marfan syndrome in order to provide prenatal diagnosis and genetic counseling.

METHODSPotential mutations of the FBN1 gene in the probands were detected with PCR and DNA sequencing. Subsequently, genomic DNA was extracted from amniotic fluid sampled between 18 to 20 weeks gestation. The mutations were confirmed with denaturing high-performance liquid chromatography - robust microsatellite instability (DHPLC-MSI) analysis with maternal DNA as reference. The products were further analyzed by direct sequencing and BLAST search of NCBI database.

RESULTSAn IVS46+1G>A substitution was identified in patient A at +1 position of intron 46 of the FBN1 gene. Two novel missense mutations were respectively discovered at positions +4453 of intron 35 in patient B (Cys1485Gly) and position +2585 of intron 21 in patient C (Cys862Tyr). In patient D, a novel deletion (c.3536 delA) was found at position +3536 of intron 28. In all of the 4 cases, the same mutations have been identified in the fetuses.

CONCLUSIONFBN1 gene analysis can provide accurate diagnosis of Marfan syndrome, which can facilitate both prenatal diagnosis and genetic counseling.

Adult ; Base Sequence ; DNA Mutational Analysis ; Female ; Fibrillin-1 ; Fibrillins ; Humans ; Introns ; Male ; Marfan Syndrome ; diagnosis ; embryology ; genetics ; Microfilament Proteins ; genetics ; Molecular Sequence Data ; Mutation, Missense ; Pregnancy ; Prenatal Diagnosis ; Sequence Deletion

INTRODUCTIONReperfusion of acutely ischaemic tissue may, paradoxically, lead to systemic complications. This phenomenon is believed to be initiated by humoral factors that have accumulated in the ischaemic tissue. The ancient art of venesection may reduce the load of these mediators at the point of reperfusion. The aim of this study is to test if selective venesection, by removing the initial venous return from the ischaemic tissue, can attenuate the systemic effects of the ischaemic-reperfusion injury using a porcine model of acute limb ischaemia.

MATERIALS AND METHODSThe right femoral arteries of anaesthetised female pigs were clamped. Twelve pigs were divided into 2 groups (n = 6 per group). In the treatment group, 5% of blood volume was venesected from the ipsilateral femoral vein upon reperfusion; the other arm served as control. The animals were sacrifi ced after 4 days for histological examination. A pathologist, blinded to the experimental groups, graded the degree of microscopic injury.

RESULTSFor the control group, the kidneys showed glomeruli and tubular damage. The livers demonstrated architectural distortion with cellular oedema. There was pulmonary oedema as well as extensive capillary congestion and neutrophil infiltration. Such findings were absent or reduced in the venesected animals. Consequently, the injury scores for the kidney, lung, liver and heart were significantly less for the venesected animals.

CONCLUSIONSelective venesection reduces the remote organ injuries of the ischaemic-reperfusion phenomenon.

Animals ; Disease Models, Animal ; Female ; Hindlimb ; injuries ; Multiple Organ Failure ; etiology ; pathology ; prevention & control ; Phlebotomy ; Pulmonary Edema ; etiology ; pathology ; prevention & control ; Reperfusion Injury ; complications ; therapy ; Sus scrofa