Development of rapid,sensitive one-tube duplex RT-PCR assay for specific and differential diagnosis of Chikungunya and dengue

Chikungunya and dengue, two arboviral infections are common in South-East Asia and their early clinical manifestations are very similar hence it is important to discriminate between them as early as possible for better clinical management. The aim of this study was to design a rapid, sensitive and specific method for the differential diagnosis of these two viruses simultaneously. A rapid one-tube duplex RT-PCR assay was developed that requires 110 min including RNA extraction, RT-PCR and agarose gel electrophoresis by using a novel Taq polymerase with high processivity. This one-tube duplex RT-PCR system with primers designed from the conserved regions of the genome allowed discrimination between the two viral groups. Bioinformatics analysis of the DNA sequences from PCR amplified products confirmed that this method was very specific and accurate. The time required for this duplex RT-PCR was comparable to the standard IgM capture ELISA method. This novel approach would help to diagnose specifically and accurately these two closely related arboviruses and enable early detection from blood. This method could be applied in resource limited settings, for surveillance in endemic regions or for routine epidemiological screening.     

Comparative Mass Spectrometric Analyses of Enamel Matrix Proteins from Five Species Suggest a Common Pathway of Post-Secretory Proeteolytic Processing

A tartrate-resistant, iron-activated and vanadate-sensitive nucleotide tri- and diphosphatase has been purified from rat bone. The purified enzyme (1,400-fold, 45% yield) has an Mr on SDS-PAGE of 30,000 Da. Hydrodynamic properties include a Stokes radius of 24Å, a sedimentation coefficient of 3.2 S and a partial specific volume of 0.748 ml/g. The calculated Mr from hydrodynamic data is 32,000 and the enzyme binds 4 mol Triton X-100/mol enzyme. Substrate specificity studies demonstrate that the enzyme is active against nucleotide tri- and diphosphates and phosphotyrosine, but not against phosphoserine or phosphothreonine. Based on the purification profile and enzyme histochemistry, showing labelling of fewer mononuclear cells using ATP compared to conventional acid phosphatase substrates, it is suggested that the acid ATPase constitutes a unique form in the family of tartrate-resistant acid phosphatases and may thus have the potential as a marker for osteoclast ontogeny and function.     

4D microstructural changes in dentinal tubules during acid demineralisation

ObjectiveDental erosion is a common oral condition caused by chronic exposure to acids from intrinsic/extrinsic sources. Repeated acid exposure can lead to the irreversible loss of dental hard tissues (enamel, dentine, cementum). Dentine can become exposed to acid following severe enamel erosion, crown fracture, or gingival recession. Causing hypersensitivity, poor aesthetics, and potential pulp involvement. Improving treatments that can restore the structural integrity and aesthetics are therefore highly desirable. Such developments require a good understanding of how acid demineralisation progresses where relatively little is known in terms of intertubular dentine (ITD) and peritubular dentine (PTD) microstructure. To obtain further insight, this study proposes a new in vitro method for performing demineralisation studies of dentine.MethodsAdvanced high-speed synchrotron X-ray microtomography (SXM), with high spatial (0.325 μm) and temporal (15 min) resolution, was used to conduct the first in vitro, time-resolved 3D (4D) study of the microstructural changes in the ITD and PTD phases of human dentine samples (∼0.8 × 0.8 × 5 mm) during 6 h of continuous acid exposure.ResultsDifferent demineralisation rates of ITD (1.79 μm/min) and PTD (1.94 μm/min) and their progressive width-depth profiles were quantified, which provide insight for understanding the mechanisms of dentine demineralisation.SignificanceInsights obtained from morphological characterisations and the demineralisation process of ITD and PTD during acid demineralisation would help understand the demineralisation process and potentially aid in developing new therapeutic dentine treatments. This method enables continuous examination of relatively large volumes of dentine during demineralisation and also demonstrates the potential for studying the remineralisation process of proposed therapeutic dentine treatments.     

Pharmacokinetics of 4′-cyano-2′-deoxyguanosine,a novel nucleoside analog inhibitor of the resistant hepatitis B virus,in a rat model of chronic kidney disease

IntroductionThe novel nucleoside analog, 4′-cyano-2′-deoxyguanosine (CdG), possesses inhibitory activity against both the wild-type and resistant hepatitis B virus. Since the dosage of the currently available nucleoside analog preparations needs to be adjusted, depending on renal function, we investigated the effect of renal dysfunction on the pharmacokinetics of CdG in a rat model of chronic kidney disease (CKD).MethodsCKD model rats were either intravenously or orally administered CdG at a dose of 1 mg/kg. The concentration of CdG in plasma, organs (liver and kidney) and urine samples were determined by means of a UPLC system interfaced with a TOF-MS system.ResultsFollowing intravenous administration, the plasma retention of CdG was prolonged in CKD model rats compared to healthy rats. In addition, the clearance of CdG was well correlated with plasma creatinine levels in CKD model rats. Similar to the results for intravenous administration, the plasma concentration profiles of CdG after oral administration were also found to be much higher in CKD model rats than in healthy rats. However, the results for the organ distribution and urinary excretion of CdG, the profiles of which were similar to that of healthy rats, indicated that CdG did not accumulate to a significant extent in the body.ConclusionThe extent of renal dysfunction has a direct influence on the pharmacokinetics (plasma retention) of CdG without a significant accumulation, indicating that the dosage of CdG will be dependent on the extent of renal function. .     

AISF update on the diagnosis and management of adult-onset lysosomal storage diseases with hepatic involvement

Lysosomal storage diseases (LSDs) are a heterogeneous group of inherited disorders caused by loss-of-function mutations in genes encoding for lysosomal enzymes/proteins. The consequence is a progressive accumulation of substrates in these intracellular organelles, resulting in cellular and tissue damage. The overall incidence is about 1/8000 live births, but is likely underestimated. LSDs are chronic progressive multi-systemic disorders, generally presenting with visceromegaly, and involvement of the central nervous system, eyes, the skeleton, and the respiratory and cardiovascular systems. The age at onset and phenotypic expression are highly variable, according to the specific enzymatic defect and tissues involved, the residual activity, and the disease-causing genotype. Enzyme-replacement therapies and substrate-reduction therapies have recently become available, leading to the improvement in symptoms, disease progression and quality of life of affected individuals. Liver involvement and hepatosplenomegaly are frequent features of LSDs and a hallmark of adult-onset forms, frequently leading to medical attention. LSDs should therefore be considered in the differential diagnosis of liver disease with organomegaly. The present document will provide a short overview of adult-onset LSDs with hepatic involvement, highlighting the specificities and systemic manifestations of the ones most frequently encountered in clinical practice, which may hint at the correct diagnosis and the appropriate treatment.     

Methylenetetrahydrofolate Reductase 677 Genotype-Specific Reference Values for Plasma Homocysteine and Serum Folate Concentrations in Korean Population Aged 45 to 74 Years: The Namwon Study

The reference interval for plasma total homocysteine (tHcy) and serum folate concentrations were estimated. Total of 3,154 reference individuals (1,029 men and 2,125 women) were selected based on stringent exclusion criteria. For plasma tHcy concentration (µM/L), reference values (median [5-95 percentile]) were 7.72 (5.03 to 13.80) and 6.09 (3.95-10.19) in men and women, respectively. For serum folate concentration (nM/L), reference values were 23.71 (11.73-38.44) and 28.95 (15.23-40.44) in men and women, respectively. The tHcy levels of both genders in the present study were lower than those in previous reports from other countries and Korea.

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人牙釉质酸蚀过程中三维形貌的原子力显微镜观察

目的 比较不同酸蚀时间处理后牙釉质表面的三维形貌并分析粗糙度的改变,探讨酸蚀对牙釉质微观形貌的影响.方法 将15颗新鲜前磨牙包埋、切割、打磨后得到光滑牙釉质切片,按单纯随机法分为5组,用37％磷酸凝胶分别酸蚀0(对照组)、5、10、20和30s,原子力显微镜观察釉质表面形貌,分析轮廓改变,计算图像的算术平均粗糙度(Ra)、均方根粗糙度(Rq)、十点高度平均差(Rz)值及表面积、容积并对5组的结果数据进行单因素方差分析,用Turkey's法进行组间两两比较,以P＜0.05为差异有统计学意义.结果 镜下形貌观察显示,0～20 s内釉质的釉柱结构逐渐明显,釉质表面由较光滑逐渐变为典型鱼鳞状;20 s后表面形态变化不明显.表面轮廓观察显示,随着酸蚀时间由0s增加至30 s,釉柱间隙首先被溶解,酸蚀深度逐渐增加到2.8μm,30 s后釉柱顶部宽度减小至1.8μm,且高度略降低.从0s到30 s,Ra值由(19.69±3.42) nm增加到(359.51±75.79)nm,Rq值由(22.02±5.57) nm增加到(431.02±83.09) nm,Rz值由(0.24±0.08) μm增加到(2.38±0.26) μm;组间比较显示,Ra和Rq值的20s组与30 s组和Rz值的10、20和30 s组差异无统计学意义(P＞0.05),其余各组间差异均有统计学意义(P＜0.05).同时釉质表面积由(406.77±3.88) μm2 (0 s)增大到(546.69±84.02) μm2 (30 s),容积由(65.73±14.46) μm3 (0 s)增大到(474.63±52.50)μm3(30 s).结论 随着酸蚀时间的增加,釉质表面酸蚀深度不断增大,表面粗糙度增加,表面积及容积增大,酸蚀会造成釉质微观形貌的明显改变.     

Myocardial Uptake and Release of Substrates in Type II Diabetics Undergoing Coronary Surgery

Objective - Knowledge about myocardial metabolism in diabetic patients is limited, and even more so in association with myocardial ischaemia and cardiac surgery. This study investigates myocardial substrate utilization in type II diabetic patients after elective coronary artery bypass graft surgery (CABG). Design - Myocardial substrate utilization in 10 type II diabetic patients was assessed during the first hours after elective CABG with the coronary sinus catheter technique. Results - No significant myocardial uptake of carbohydrates was observed. Free fatty acids were extracted throughout the study period but uptake was not related to arterial levels. As arterial levels of beta-hydroxybutyric acid tended to increase a significant myocardial uptake emerged. The average extraction rate of beta-hydroxybutyric acid was 31% with a peak of 42%. A comparable extraction rate of glutamate was also found whereas alanine was released from the heart. Conclusion - Free fatty acids were the main source of energy for the heart whereas uptake of carbohydrates was restricted. The high extraction rates of beta-hydroxybutyric acid and glutamate suggest an adaptive role for these substrates during this unfavourable metabolic state for the post-ischaemic diabetic heart.     

Enamel demineralization and bracket bond strength when etching with acid and /or Er:YAG laser

Background:  Enamel etching for brackets is usually done with phosphoric acid. Er:YAG lasers have been recently used for this purpose with conflicting results. The effects of lasers on tooth demineralization and the effects of different combinations of laser treatments and bonding agents were evaluated in this study. Methods:  The enamel contents of fluorine, calcium oxide and phosphorus pentoxide (P₂O₅) were analysed using acid etching, laser treatment or both. The tensile bond strength of metallic and ceramic brackets using Transbond^® XT and Fuji Ortho^® LC were also tested, using acid etching, laser treatment or a combination of both. Results:  All treatments reduced the contents of fluorine, P₂O₅ and calcium oxide, and acid reduced P₂O₅ levels more than laser. The bond strength with laser was weaker than with acid, and stronger when combining both. When using laser, the best adhesive was the Fuji Ortho^® LC. The combination of laser and acid produced the best results when using Transbond^® XT. Conclusions:  The demineralization promoted by laser was lower than the one produced with acid. Laser treatment produced lower tensile stress strength than acid, but still enough to produce clinically efficient retention. The combination of laser and acid produced the best retention results.