Potential Biochemical Mechanisms of Brain Injury in Diabetes Mellitus.

The goal of this review was to summarize current biochemical mechanisms of and risk factors for diabetic brain injury. We mainly summarized mechanisms published in the past three years and focused on diabetes induced cognitive impairment, diabetes-linked Alzheimer's disease, and diabetic stroke. We think there is a need to conduct further studies with increased sample sizes and prolonged period of follow-ups to clarify the effect of DM on brain dysfunction. Additionally, we also think that enhancing experimental reproducibility using animal models in conjunction with application of advanced devices should be considered when new experiments are designed. It is expected that further investigation of the underlying mechanisms of diabetic cognitive impairment will provide novel insights into therapeutic approaches for ameliorating diabetes-associated injury in the brain.

[Prokaryotic expression of the major antigenic domain of equine arteritis virus GL protein and the establishment of putative indirect ELISA assay].

According to the antigenic analysis of equine arteritis virus (EAV) GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH I and Xho I and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21(DE3) and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was confirmed by Western blot. The recombinant GL1 protein was purified by the means of His * Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against EAV with the purified GL1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 9.65 microg/mL and the optimal dilution of serum was 1:80. The positive criterion of this ELISA assay is OD (the tested serum) > 0.4 and OD (the tested serum) /OD (the negative serum) > 2.0. The iGL-ELISA was evaluated versus micro-virus neutralization test. The ELISA was performed on 900 sera from which were preserved by this lab during horse entry/exit inspection, the agreement (94.1%) of these test were considered suitable for individual serological detection. In another test which 180 sera samples were detected by iGL-ELISA and INGEZIM ELISA kit respectively. The agreement ratio between the two methods is 95.6%.

Development of real-time polymerase chain reaction assay with TaqMan probe for the quantitative detection of infectious hypodermal and hematopoietic necrosis virus from shrimp.

An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies, with a dynamic range of detection between 9 x 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-react with shrimp genomic DNA or other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.

Polymerase chain reaction method to detect canis materials by amplification of species-specific DNA fragment.

Rapid identification of mammal materials in feeding stuffs and food is essential for effective control of a potential source of pathogens, such as those that cause bovine spongiform encephalopathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a canis-specific mitochondrial DNA sequence in foodstuffs and food. The amplified canis-specific PCR product was a 213 base pair band from the D-loop DNA fragment of mitochondria, a high copy gene which should improve the possibility of amplifying template molecules of adequate size among the degraded DNA fragments brought about by heat denaturation. The specificity of this method was confirmed by 8 canis blood DNA samples (from different breeds of dog) and 9 noncanis animal blood DNA samples (bovine, sheep, porcine, chicken, fish, donkey, rabbit, deer, horse). This method was able to detect the presence of canis material in foodstuffs and in food mixtures even when the concentration of canis-derived meat was reduced to 0.05%. Furthermore, it did not appear to be affected by prolonged heat treatment. This method was developed for detection of canis materials in feeding stuffs, and occasionally for medical jurisprudence detection of canis-derived materials.

Multiplex polymerase chain reaction method for detection of bovine materials in foodstuffs.

Rapid identification of bovine materials in animal foodstuffs is essential for effective control of a potential source of bovine spongiform encephalophathy. A convenient polymerase chain reaction (PCR)-based assay was developed for detection and identification of a bovine-specific genomic DNA sequence in foodstuffs. Simultaneously the assay assessed the DNA quality of the experiment system by amplification of a highly conserved eucaryotic DNA region of the 18-S ribosomal gene, helping to check the reliability of the test result. The amplified bovine-specific PCR product was a genomic DNA fragment of lactoferrin, a low copy gene that was different from a commonly used bovine-specific mitochondria sequence for identification of bovine materials. The specificity of this method was confirmed by the absence of detectable homologous PCR product using reference foodstuff samples that lacked bovine-derived meat and bonemeals, or genomic DNA samples from vertebrates whose offals are commonly included in animal feeds. This method could detect the presence of bovine material in foodstuffs when the samples contained > 0.02% bovine-derived meat and bone meal. Furthermore, it was not affected by prolonged heat treatment. The specificity, convenience, and sensitivity of this method suggest that it can be used for the routine detection of bovine-derived materials.