Construction of eukaryotic expression vector of TSLC1 gene

Introduction

To construct a eukaryotic expression vector of the tumour suppressor in lung cancer 1 (TSLC1) gene, so as to explore the mechanisms of tumour suppression of the gene theoretically.

Material and methods

The open reading frame (ORF) of TSLC1 gene was amplified with RT-PCR from normal human foreskin acrobystia, and cloned to pMD19-T simple vector (TA Clone method). The resultant plasmid was transformed into Escherichia coli JM109 for amplification. The TA Clone recombinant was digested by double restriction enzyme (Bgl II/EcoR I) and analysed with agarose gel electrophoresis. The positive one was sequenced. The inserted DNA fragment was recovered, and then it was mounted into the eukaryotic expression vector pIRES2-EGFP, transformed into E. coli JM109 for amplification. A positive recombinant plasmid named pIRES2-EGFP-TSLC1 was confirmed by Bgl II/EcoR I double-enzyme digestion analysis.

Results

RT-PCR amplified the ORF of the TSLC1 gene. It was approximately 1400 base pairs. The obtained DNA was confirmed a high degree of homology with the sequence of TSLC1 cDNA sequence ({"type":"entrez-nucleotide","attrs":{"text":"AY358334","term_id":"37181792"}}AY358334) stored at GenBank.

Conclusions

Construction of a TSLC1 eukaryotic expression vector was successful, and it has established a solid foundation for further study.     

应用酵母双杂交系统发现hALR与Na,K-ATPase间的相互作用

目的:采用酵母双杂交系统寻找与人肝再生增强因子(hALR)相互作用的蛋白质, 探讨ALR的作用机理。方法: 构建hALR诱饵质粒pGBKT7-hALR, 醋酸锂法转化AH109酵母菌, 转化菌在SD/-Trp-His培养基上培养及滤纸法β一半乳糖苷酶活性检测排除自身激活作用后, 与人肝cDNA文库质粒预转化的酵母菌Y187进行接合试验, 接合产物在QDO培养基上筛选, 阳性克隆进一步在含X-α-Gal的QDO平板上鉴定, X-α-Gal活性呈阳性的克隆, 进行PCR及酶切鉴定排除完全相同克隆, 进一步进行回交试验排除假阳性, 对阳性克隆进行序列测定和生物信息学分析。结果:得到数个阳性克隆, 序列分析结果表明其中1个阳性克隆是Na⁺, K⁺-ATPaseβ亚基部分基因, 长669bp, 3'端非编码区224bp, 编码区长445bp, 编码Na⁺, K⁺-ATPaseβ亚基C端的147个氨基酸残基。结论:应用酵母双杂交系统筛选出Na⁺, K⁺-ATPase与hALR具有相互作用。     

应用酵母双杂交技术筛选与RANK蛋白新基序IVVY相互作用的蛋白

目的: 应用酵母双杂交技术筛选小鼠巨噬细胞中与核因子κB受体激活因子(RANK)蛋白上新基序IVVY相互作用的蛋白,以探寻RANKL/RANK系统中介导破骨细胞形成的RANK下游新信号转导蛋白。方法: 应用GAL4酵母双杂交系统3,构建仅包含编码新基序⁵³⁵IVVY⁵³⁸ 的一小段RANK的cDNA片段为诱饵质粒pGBKT7-IVVY,并与巨噬细胞cDNA文库质粒pGADT7-library共转化AH109酵母,筛选与RANK蛋白新基序IVVY相互作用的蛋白,通过回复性杂交实验验证其可靠性,并对阳性克隆进行测序和基因同源性分析。结果: 筛选出4个可能与IVVY基序有相互作用的蛋白,包括Ring1和YY1结合蛋白(RYBP)、ATP结合盒、E2F转录因子和热休克蛋白8,其中表达RYBP的阳性克隆出现频率高、速度快。结论: 应用酵母双杂交技术成功地筛选出4个可能与IVVY基序相互作用的候选蛋白,其中RYBP可能性最大。     

Mimotopes for Api g 5, a Relevant Cross-reactive Allergen,in the Celery-Mugwort-Birch-Spice Syndrome

Purpose

In the celery-mugwort-birch-spice syndrome, a significant proportion of IgE is directed against high molecular weight (HMW) glycoproteins, including the celery allergen Api g 5. BIP3, a monoclonal antibody originally raised against birch pollen, recognizes HMW allergens in birch and mugwort pollens, celery, and Apiaceae spices. Our aim was to generate mimotopes using BIP3 for immunization against the HMW allergens relevant in the celery-mugwort-birch-spice cross reactivity syndrome.

Methods

Mimotopes were selected from a random-peptide display library by BIP3 and applied in IgE inhibition assays. The 3 phage clones with the highest inhibitory capacity were chosen for immunization of BALB/c mice. Mouse immune sera were tested for IgG binding to blotted birch pollen extract and used for inhibiting patients'' IgE binding. Furthermore, sera were tested for binding to Api g 5, to horseradish peroxidase (HRP) as a second glycoprotein, or to non-glycosylated control allergen Phl p 5 in ELISA, and the specific Api g 5-specific IgG titers were determined.

Results

Three rounds of biopanning resulted in phage clones exhibiting 7 different sequences including 1 dominant, 1-6-cyclo-CHKLRCDKAIA. Three phage clones had the capacity to inhibit human IgE binding and induced IgG to the HMW antigen when used for immunizing BALB/c mice. The induced BIP3-mimotope IgG reached titers of 1:500 specifically to Api g 5, but hardly reacted to glycoprotein HRP, revealing a minor role of carbohydrates in their epitope.

Conclusions

The mimotopes characterized in this study mimic the epitope of BIP3 relevant for Api g 5, one of the cross-reactive HMW allergens relevant in the celery-mugwort-birch-spice syndrome. BIP3 mimotopes may be used in the future for hyposensitization in this clinical syndrome by virtue of good and specific immunogenicity.     

Focal Lymphocytic Thyroiditis Nodules Share the Features of Papillary Thyroid Cancer on Ultrasound

Purpose

It is often difficult to discriminate focal lymphocytic thyroiditis (FLT) or adenomatous hyperplasia (AH) from thyroid cancer if they both have suspicious ultrasound (US) findings. We aimed to make a predictive model of FLT from papillary thyroid cancer (PTC) in suspicious nodules with benign cytologic results.

Materials and Methods

We evaluated 214 patients who had undergone fine-needle aspiration biopsy (FNAB) and had shown thyroid nodules with suspicious US features. PTC was confirmed by surgical pathology. FLT and AH were confirmed through more than two separate FNABs. Clinical and biochemical findings, as well as US features, were evaluated.

Results

Of 214 patients, 100 patients were diagnosed with PTC, 55 patients with FLT, and 59 patients with AH. The proportion of elevated thyrotropin (TSH) levels (p=0.014) and thyroglobulin antibody (Tg-Ab) or thyroid peroxidase antibody (TPO-Ab) positivity (p<0.001) in the FLT group was significantly higher than that in the PTC group. Regarding US features, absence of calcification (p=0.006) and "diffuse thyroid disease" (DTD) pattern on US (p<0.001) were frequently seen in the FLT group. On multivariate analysis, Tg-Ab positivity, presence of a DTD pattern on US, and absence of calcification in nodules were associated with FLT with the best specificity of 99% and positive predictive value of 96%. In contrast, a taller than wide shape of nodules was the only variable significant for differentiating AH from PTC.

Conclusion

Suspicious thyroid nodules with cytologic benign results could be followed up with US rather than repeat FNAB, if patients exhibit Tg-Ab positivity, no calcifications in nodules, and a DTD pattern on US.     

The C20orf133 gene is disrupted in a patient with Kabuki syndrome

Background

Kabuki syndrome (KS) is a rare, clinically recognisable, congenital mental retardation syndrome. The aetiology of KS remains unknown.

Methods

Four carefully selected patients with KS were screened for chromosomal imbalances using array comparative genomic hybridisation at 1 Mb resolution.

Results

In one patient, a 250 kb de novo microdeletion at 20p12.1 was detected, deleting exon 5 of C20orf133. The function of this gene is unknown. In situ hybridisation with the mouse orthologue of C20orf133 showed expression mainly in brain, but also in kidney, eye, inner ear, ganglia of the peripheral nervous system and lung.

Conclusion

The de novo nature of the deletion, the expression data and the fact that C20orf133 carries a macro domain, suggesting a role for the gene in chromatin biology, make the gene a likely candidate to cause the phenotype in this patient with KS. Both the finding of different of chromosomal rearrangements in patients with KS features and the absence of C20orf133 mutations in 19 additional patients with KS suggest that KS is genetically heterogeneous.     

Epidemiology,of bilharzias (schistosomiasis) in Uganda from 1902 until 2005

Background

Schistosoma mansoni was observed and reported in Kuluva hospital Arua District in north western Uganda as early as 1902. S. mansoni is widely distributed in Uganda along permanent water bodies.

Objective

To review the litreture on scistosomiasis in Uganda, since 1902.

Method

The core literature for this short review was searched from reports and publications by the British colonial Ministry of Health Districts Medical officers and Entomologists. Additional information was obtained from Makerere University Medical School library archives, London School of Hygiene and Tropical Medicine library archives, University of Antrwap, and post independence publications on schistosomiasis in Uganda in various journals.

Results

Since it was first detected in 1902 Schistosoma (S) mansoni is more widely distributed in Uganda than S. haematobium. However Schistosoma mansoni and S. haematobium are of public health importance in Uganda and the importance of migrants and fishermen in disseminating infections into non-infested areas and intensifying infection in areas already infested have been reported.

Conclusion

S. mansoni has been on the increase in Uganda whereas S. haematobium is localized in sporadic foci in the north of Uganda. Treatment with praziquantel the drug of choice in Uganda used in schistosomiasis control programme has reduced development of severe schistosomiasis.     

Continuous positive airway pressure reduces risk of motor vehicle crash among drivers with obstructive sleep apnea: systematic review and meta-analysis

Context:

Obstructive sleep apnea (OSA) is associated with an increased risk of motor vehicle crash.

Objective:

We performed a systematic review of the literature concerning the impact of continuous positive airway pressure (CPAP) treatment on motor vehicle crash risk among drivers with OSA. The primary objective was to determine whether CPAP use could reduce the risk of motor vehicle crash among drivers with OSA. A secondary objective involved determining the time on treatment required for CPAP to improve driver safety.

Data Sources:

We searched seven electronic databases (MEDLINE, PubMed (PreMEDLINE), EMBASE, PsycINFO, CINAHL, TRIS, and the Cochrane library) and the reference lists of all obtained articles.

Study Selection:

We included studies (before-after, case-control, or cohort) that addressed the stated objectives. We evaluated the quality of each study and the interplay between the quality, quantity, robustness, and consistency of the evidence. We also tested for publication bias.

Data Extraction:

Data were extracted by two independent analysts. When appropriate, data were combined in a fixed or random effects meta-analysis.

Results:

A meta-analysis of 9 observational studies examining crash risk of drivers with OSA pre- vs. post-CPAP found a significant risk reduction following treatment (risk ratio = 0.278, 95% CI: 0.22 to 0.35; P < 0.001). Although crash data are not available to assess the time course of change, daytime sleepiness improves significantly following a single night of treatment, and simulated driving performance improves significantly within 2 to 7 days of CPAP treatment.

Conclusions:

Observational studies indicate that CPAP reduces motor vehicle crash risk among drivers with OSA.

Citation:

Tregear S; Reston J; Schoelles K; Phillips B. Continuous positive airway pressure reduces risk of motor vehicle crash among drivers with obstructive sleep apnea. SLEEP 2010;33(10):1373-1380.     

Chronic kidney disease in patients with diabetes mellitus type 2 or hypertension in general practice

Background

The prevalence and severity of chronic kidney disease (CKD) in primary care patients with diabetes or hypertension is unknown.

Aim

To assess the prevalence and severity of CKD in patients with diabetes and hypertension; and identify whether age, sex, diabetes, and hypertension are associated with CKD.

Design of study

Cross-sectional survey.

Setting

Two Dutch primary health care centres (15 954 enlisted patients).

Method

Patients, aged ≥25 years, with known diabetes type 2 (n = 471) or hypertension (n = 960), were selected on 1 October 2006. Initial screening uptake rates were assessed from the electronic patient records, and patients were invited when blood or urine measurements were missing. The presence of albuminuria was determined, glomerular filtration rate estimated, and clinical characteristics extracted.

Results

Initial screening uptake rates were 93% and 69% for diabetes and hypertension, respectively, and increased to 97% (n = 455) and 87% (n = 836) after active invitation. The prevalence of CKD was 28% in diabetes and 21% in hypertension only. The presence of diabetes was independently associated with albuminuria (odds ratio [OR] 4.23; 95% confidence interval [CI] = 2.67 to 6.71), but not with decreased estimated GFR (eGFR) (OR 0.75; 95% CI = 0.54 to 1.04). Age showed the strongest association with decreased eGFR (OR 2.73; 95% CI = 2.02 to 3.70).

Conclusion

In primary care, more than one-quarter of patients with diabetes and about one-fifth of patients with hypertension have CKD. The high prevalence justifies longitudinal follow-up in order to evaluate whether intensified cardiovascular risk management is beneficial in this primary care population.     

Novel genes in Human Asthma Based on a Mouse Model of Allergic Airway Inflammation and Human Investigations

Purpose

Based on a previous gene expression study in a mouse model of asthma, we selected 60 candidate genes and investigated their possible roles in human asthma.

Methods

In these candidate genes, 90 SNPs were genotyped using MassARRAY technology from 311 asthmatic children and 360 healthy controls of the Hungarian (Caucasian) population. Moreover, gene expression levels were measured by RT PCR in the induced sputum of 13 asthmatics and 10 control individuals. t-tests, chi-square tests, and logistic regression were carried out in order to assess associations of SNP frequency and expression level with asthma. Permutation tests were performed to account for multiple hypothesis testing.

Results

The frequency of 4 SNPs in 2 genes differed significantly between asthmatic and control subjects: SNPs rs2240572, rs2240571, rs3735222 in gene SCIN, and rs32588 in gene PPARGC1B. Carriers of the minor alleles had reduced risk of asthma with an odds ratio of 0.64 (0.51-0.80; P=7×10^-5) in SCIN and 0.56 (0.42-0.76; P=1.2×10^-4) in PPARGC1B. The expression levels of SCIN, PPARGC1B and ITLN1 genes were significantly lower in the sputum of asthmatics.

Conclusions

Three potentially novel asthma-associated genes were identified based on mouse experiments and human studies.     

Custom-molded foot-orthosis intervention and multisegment medial foot kinematics during walking

Context:

Foot-orthosis (FO) intervention to prevent and treat numerous lower extremity injuries is widely accepted clinically. However, the results of quantitative gait analyses have been equivocal. The foot models used, participants receiving intervention, and orthoses used might contribute to the variability.

Objective:

To investigate the effect of a custom-molded FO intervention on multisegment medial foot kinematics during walking in participants with low-mobile foot posture.

Design:

Crossover study.

Setting:

University biomechanics and ergonomics laboratory.

Patients or Other Participants:

Sixteen participants with low-mobile foot posture (7 men, 9 women) were assigned randomly to 1 of 2 FO groups.

Interventions :

After a 2-week period to break in the FOs, individuals participated in a gait analysis that consisted of 5 successful walking trials (1.3 to 1.4 m/s) during no-FO and FO conditions.

Main Outcome Measure(s):

Three-dimensional displacements during 4 subphases of stance (loading response, mid-stance, terminal stance, preswing) were computed for each multisegment foot model articulation.

Results:

Repeated-measures analyses of variance (ANOVAs) revealed that rearfoot complex dorsiflexion displacement during midstance was greater in the FO than the no-FO condition (F_1,14 = 5.24, P = .04, partial η² = 0.27). Terminal stance repeated-measures ANOVA results revealed insert-by-insert condition interactions for the first metatarsophalangeal joint complex (F_1,14 = 7.87, P = .01, partial η² = 0.36). However, additional follow-up analysis did not reveal differences between the no-FO and FO conditions for the balanced traditional orthosis (F_1,14 = 4.32, P = .08, partial η² = 0.38) or full-contact orthosis (F_1,14 = 4.10, P = .08, partial η² = 0.37).

Conclusions:

Greater rearfoot complex dorsiflexion during midstance associated with FO intervention may represent improved foot kinematics in people with low-mobile foot postures. Furthermore, FO intervention might partially correct dys-functional kinematic patterns associated with low-mobile foot postures.     

Outcomes of Hemodialysis in Children: A 35-Year Experience at Severance Hospital

Purpose

The aim of this study was to analyze the results of children treated with hemodialysis (HD) at Severance Hospital over 35 years in terms of incidence, etiologies, characteristics, complications, and clinical outcomes.

Materials and Methods

We analyzed 46 children admitted to Severance Hospital who had undergone HD between January 1979 and December 2013.

Results

The main etiologies of the 23 end-stage renal disease (ESRD) patients who had received HD were chronic glomerulonephritis (7 patients, 30.4%) and congenital anomalies of the kidney and urinary tract (7 patients, 30.4%), whereas the etiology of the 23 acute kidney injury (AKI) patients was hemolytic uremic syndrome (6 patients, 26.1%). Compared with ESRD patients, hemocatheter placement in the femoral vein was preferred over the subclavian or internal jugular vein in the AKI patients (p=0.012). The most common complication was catheter related complication (10 patients, 21.7%). The site of hemocatheter insertion was not related to the frequency of oozing. Placing the hemocatheter in the femoral vein resulted in significantly more events of catheter obstruction than insertion in the internal jugular vein or the subclavian vein (p=0.001). Disequilibrium syndrome occurred more frequently in older patients (p=0.004), as well as patients with a greater body weight (p=0.008) and a higher systolic and diastolic blood pressure before HD (systolic: p=0.021; diastolic: p=0.040).

Conclusion

Based on the 35 years of experience in our center, HD can be sufficiently and safely carried out even in children without significant complications.     

A Study on the Effect of Resveratrol on Lipid Metabolism in Hyperlipidemic Mice

Background

The content of resveratrol is relatively high in Polygonum cuspidatum Sieb. et Zucc., and the resveratrol has the effect of blood vessel dilating, microcirculation improving, platelet aggregation inhibiting and anti-cancer. The objective of this paper was to study the effect of resveratrol on lipid metabolism in hyperlipidemia mice.

Materials and Methods

Through the establishment of an experimental mouse model of hyperlipidemia, the effect of resveratrol on change in total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c) levels in mouse serum were determined.

Results

Resveratrol group can apparently reduce TC, TG, LDL-c and AI of hyperlipidemic mice in a dose effect manner.

Conclusion

We concluded that resveratrol can effectively reduce blood lipid levels of hyperlipidemic mice.     

Certified athletic trainers' knowledge of methicillin-resistant Staphylococcus aureus and common disinfectants

Context:

Methicillin-resistant Staphylococcus aureus (MRSA) infections are increasingly common in athletic settings. The MRSA knowledge and infection-control practices of certified athletic trainers (ATs) and the cleanliness of the athletic training room are important factors in preventing MRSA infections.

Objective:

To assess knowledge of MRSA and the use of common disinfectants among ATs and to explore their infection-control practices.

Design:

Cross-sectional study.

Setting:

High school and collegiate athletic training rooms.

Patients or Other Participants:

A total of 163 ATs from National Collegiate Athletic Association Divisions I, II, and III and high schools, representing all 10 National Athletic Trainers'' Association districts.

Main Outcome Measure(s):

Frequencies, analyses of variance, and χ² tests were used to assess current practices and opinions and relationships between factors.

Results:

Methicillin-resistant Staphylococcus aureus was perceived as a national problem by 92% of respondents; 57% perceived MRSA as a problem in their practice setting. Most respondents had treated general infections (88%), staphylococcal infections (75%), and MRSA infections (57%). Male sex was associated with treating all 3 types of infections (χ² test, P < .05). Noncurriculum education was associated with a lack of recognition of environmental issues as risk factors and with the use of isopropyl alcohol for disinfection (χ² test, P < .05). For example, 10% of respondents did not recognize that contaminated whirlpools can be a source of MRSA infection. Respondents also incorrectly identified effective cleaning solutions. Thirty percent of respondents cleaned their hands frequently or sometimes before treating each athlete and 35% cleaned their hands sometimes, occasionally, or never after seeing each athlete.

Conclusions:

The majority of ATs were informed about MRSA and made correct disinfection choices. However, improvements are still needed, and not all ATs were using proper disinfection practices.     

Understanding the management of early-stage chronic kidney disease in primary care: a qualitative study

Background

Primary care is recognised to have an important role in the delivery of care for people with chronic kidney disease (CKD). However, there is evidence that CKD management is currently suboptimal, with a range of practitioner concerns about its management.

Aim

To explore processes underpinning the implementation of CKD management in primary care.

Design and setting

Qualitative study in general practices participating in a chronic kidney disease collaborative undertaken as part of the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care (CLAHRC) for Greater Manchester.

Method

Semi-structured interviews were conducted with GPs and practice nurses (n = 21). Normalisation Process Theory provided a framework for generation and analysis of the data.

Results

A predominant theme was anxiety about the disclosure of early-stage CKD with patients. The tensions experienced related to identifying and discussing CKD in older people and patients with stage 3A, embedding early-stage CKD within vascular care, and the distribution of work within the practice team. Participants provided accounts of work undertaken to resolve the difficulties encountered, with efforts having tended to focus on reassuring patients. Analysis also highlighted how anxiety surrounding disclosure influenced, and was shaped by, the organisation of care for people with CKD and associated long-term conditions.

Conclusion

Offering reassurance alone may be of limited benefit, and current management of early-stage CKD in primary care may miss opportunities to address susceptibility to kidney injury, improve self-management of vascular conditions, and improve the management of multimorbidity.     

The effects of diltiazem in renal transplantation patients treated with cyclosporine A

Objective

To investigate the effects of diltiazem and cyclosporine A (CsA) combination therapy on protecting the kidney, promoting graft functioning and improving post-transplanted kidney recovery.

Methods

The blood concentrations of CsA, the condition of the post-transplant kidney, the rate of acute rejection (AR), as well as hepatic and renal toxicity in 636 cases of renal transplant recipients were determined after being treated by CsA, with or without diltiazem.

Results

Compared with the control group which received CsA, mycophenolate mofetil (MMF) and prednisolone (Pred) but lacked diltiazem, the group receiving these agents together with diltiazem required reduced dosage of CsA (P < 0.01), while blood concentrations of CsA were significantly increased (P < 0.01); the recovery time of graft function was reduced from (6.2±1.5) d to (3.9±1.4) d (P < 0.01), and the rate of AR was decreased from 13.2% to 7.9% (P < 0.01).

Conclusion

In renal transplantation patients treated with CsA and diltiazem, blood concentrations of CsA were increased while the dosage was decreased. This efficient combination therapy reduced patients'' economic burden, at the same time retained kidney function, promoted graft function recovery and decreased hepatic and renal toxicity and the rate of AR.