Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease.

Rapid transmission, high morbidity, and mortality are the features of human infectious diseases caused by microorganisms, such as bacteria, fungi, and viruses. These diseases may lead within a short period of time to great personal and property losses, especially in regions where sanitation is poor. Thus, rapid diagnoses are vital for the prevention and therapeutic intervention of human infectious diseases. Several conventional methods are often used to diagnose infectious diseases, e.g. methods based on cultures or morphology, or biochemical tests based on metabonomics. Although traditional methods are considered gold standards and are used most frequently, they are laborious, time consuming, and tedious and cannot meet the demand for rapid diagnoses. Disease diagnosis using capillary electrophoresis methods has the advantages of high efficiency, high throughput, and high speed, and coupled with the different nucleic acid detection strategies overcomes the drawbacks of traditional identification methods, precluding many types of false positive and negative results. Therefore, this review focuses on the application of capillary electrophoresis based on nucleic detection to the diagnosis of human infectious diseases, and offers an introduction to the limitations, advantages, and future developments of this approach.

[Polyketone Reaction in Biosynthetic Pathways of 2, 3, 5, 4'-Tetrahydroxy Stilhene-2-O-ß-D-glucoside in Polygonum multiflorum by Biocatalysis].

OBJECTIVE: 2, 3, 5, 4'-Tetrahydroxy stilbene-2-O-ß-D-glucoside (THSG), the active ingredient of Polygonum multiflorum, its polyketone reaction in the biosynthesis pathways was studied by biocatalysis method. METHODS: The substrates 4-coumaroyl-CoA and malonyl-CoA were catalyzed in vitro by the crude enzyme extracted from Polygonum multiflorum callus, then the products were verified by HPLC and LC-MS methods. And the crude enzyme was analyzed by ammonium sulfate precipitation method and SDS-PAGE. RESULTS: HPLC chromatogram showed the same retention time of both the product and resveratrol standards; LC-MS spectra showed that the m/z of product was 227, which was consistent with resveratrol standards under the mode of negative ion; Ammonium sulfate (AS) precipitation method showed AS of 40% - 70% had catalytic activity,and 50% - 60% was the optimum; SDS-PAGE showed protein bands were obviously different among different AS concentration between 20% - 80%, the protein band of 42 kDa was found in AS of 50% - 60%, which had the same molecular weight with stilbene synthase. CONCLUSION: The product of polyketone reaction in the biosynthesis of THSG is resveratrol rather than THSG, so it is speculated that THSG is the conversion product of resveratrol instead of the direct product of the polyketone reaction.

Highly sensitive analysis of nucleic acids using capillary gel electrophoresis with ultraviolet detection based on the combination of matrix field-amplified and head-column field-amplified stacking injection.

To develop a highly sensitive method for analyzing nucleic acids using capillary gel electrophoresis with ultraviolet detection (CGE-UV), we combined matrix field-amplified with head-column field-amplified stacking injection (C-FASI) to employ the advantages of two methods. Without diminishing the resolution, a limit of detection of 0.13 ng/ml (signal/noise=3) in a 300,000-fold diluted sample was obtained, the sensitivity is 102,308 times higher than that achieved with normal pressure injection, 3077 times that with normal electrokinetic injection, 154 times that with pressure field-amplified sample stacking injection, and 31 times that with matrix field-amplified stacking injection. After establishing the method, we tested the detection of a φX174-Hae III digest DNA product without purification and with a high ionic strength. At the lowest dilution of 5000-fold, sample at a concentration of 10 ng/ml was enriched and detected. The relative standard deviations for migration time and peak area (n=3) were 0.03-1.15 and 0.72-6.42, respectively. To further validate C-FASI was applicable for real sample, a 400 bp PCR product without purification was directly detected with a limit of detection at the concentration of 6000-fold dilution (signal/noise=3), The relative standard deviations for migration time and peak area (n=6) were 0.44 and 4.8, respectively. These results indicated that C-FASI had good qualitative and quantitative detection abilities and CGE-UV based on C-FASI is easy to perform, practical, highly-sensitive and robust for nucleic acid detection, which makes it a highly valuable tool for genetic diagnostics based on nucleic acid analysis.

Capillary electrophoresis based on the nucleic acid detection in the application of human disease diagnosis.

As the post-genome era comes, one of the trends of future medical developments is the timely diagnosis and prevention of diseases. The analysis of nucleic acid can diagnose the diseases accurately at gene level which can eliminate all kinds of false positive and negative results from phenotype and prescribe the individual prevention or therapy. As a result, a high-throughput test tool is needed for the analyses of a large number of clinical nucleic acid samples. Capillary electrophoresis (CE) has the advantages of high-efficiency, high-speed, microscale, automation, high-throughput, and cleanliness which can meet the medical requirements that mass data and a large number of samples need to be analyzed, leading CE to be the new technology considered for clinical disease diagnosis. This review puts the focus on the application of CE in clinical disease diagnosis. Meanwhile, it also gives a brief introduction of the drawbacks and future development of CE.

Progress in stacking techniques based on field amplification of capillary electrophoresis.

Numerous strategies have been developed to mitigate the intrinsic low detection sensitivity that is a limitation of capillary electrophoresis. Among them, in-line stacking is an effective strategy to address the sensitivity challenge, and among the different stacking techniques, stacking based on field amplification is the most effective and simplest method of achieving high sensitivity without special complicated mechanisms or operations. This review introduces several stacking techniques based on field amplification. Field-amplified sample stacking, large-volume sample stacking, matrix field-amplified stacking injection (FASI), head-column FASI, matrix FASI combined with head-column FASI, FASI coupled with extraction and clean-up methods, electrokinetic supercharging, cation-anion selective exhaustive injection-sweeping-micellar electrokinetic chromatography, and newly developed techniques based on field amplification combined with other methods are included, and examples of straightforward methods for solving the sensitivity problem are provided. We also present a brief overview of the advantages, limitations, and future developments of these techniques.

Capillary electrophoresis based on the nucleic acid detection in the application of cancer diagnosis and therapy.

Cancer is malignant disease that causes many deaths worldwide every year, with most deaths occurring in the middle and advanced stages of cancer. Numerous deaths can be avoided by detecting cancer at an early stage, making early diagnosis and timely therapy critical for cancer treatment. Analyses at the level of nucleic acids rather than phenotypes can eliminate various false-positive and -negative results, and diagnoses can occur at an earlier stage. Many techniques have been developed for this purpose, including capillary electrophoresis (CE), which has the advantages of high-efficiency, high-speed, high-throughput, automation, cleanliness, and versatility, and CE can be conducted on a microscale or coupled with other separation techniques. These advantages afford this technique the ability to meet the future medical requirements that will undoubtedly call for amassing large numbers of samples for analysis, suggesting that CE may become an important tool for providing data in clinical cancer diagnosis and therapy. This review focuses on CE-based nucleic acid detection as it is applied to cancer diagnosis and therapy, and provides an introduction to the drawbacks and future developments of analysis with CE.

Species identification of Chinese medicinal plant Fallopia multiflora (Thunb.) Haraldson by suppression subtraction hybridization.

Fallopia multiflora (Thunb.) Haraldson, a traditional Chinese medicinal plant, has been extensively used in preparations of herbal medicine, health products and personal hygiene products. However, the clinical safety and efficiency of F. multiflora (Thunb.) Haraldson is impaired because of the existence of various adulterants. In this study, genomic DNA (gDNA) suppression subtraction hybridization (SSH) was used to authenticate F. multiflora (Thunb.) Haraldson from its adulterants. First, differential gDNA fragments between F. multiflora (Thunb.) Haraldson and its most closely related species F. multiflora var. ciliinervis (Nakai) Yonek. & H. Ohashi by SSH were identified. The differential fragments were then hybridized with arrays constructed from multiple whole genomes of several species (adulterants and/or closely related plants) to screen for the unique gDNA fragments representing F. multiflora (Thunb.) Haraldson. The unique gDNA fragments could be used to design species-specific primers for the identification of F. multiflora (Thunb.) Haraldson. Using SSH, we obtained four differential gDNA fragments, and four pairs of primers were designed. The designed primers could differentiate F. multiflora (Thunb.) Haraldson from its adulterants and/or closely related species via PCR. The results confirmed that the SSH is an efficient method for screening and designing species-specific primers.

The effect of CYP3A4*1G allele on the pharmacokinetics of atorvastatin in Chinese Han patients with coronary heart disease.

The present study aimed to evaluate the impact of CYP3A4*1G allele on the pharmacokinetics of atorvastatin in the Chinese Han patients with coronary heart disease (CHD). Twenty male patients of CHD with different CYP3A4*1G genotypes were orally administered a single 20 mg dose of atorvastatin. Plasma concentrations of atorvastatin and 2-hydroxyatorvastatin were measured by high-performance liquid chromatography tandem mass spectrometry. The mean area under the plasma concentration-time curve from 0 to infinity (AUC0-∞ ) of atorvastatin in subjects with the CYP3A4*1G/*1G genotype were 36% or 25% lower than in those with the wild-type or the *1/*1G genotype, respectively. The time to peak plasma concentration (Tmax ) and oral clearance of atorvastatin (CL/F) were significantly different between subjects with the CYP3A4*1G/*1G genotype and the wild-type. The AUC0-∞ for 2-hydroxyatorvastatin in subjects with the CYP3A4*1G/*1G genotype was 44% or 31% lower than in those with the wild-type or the *1/*1G genotype, respectively. The peak plasma concentration, Tmax and apparent clearance of 2-hydroxyatorvastatin (CL/Fm) were significantly different between subjects with the CYP3A4*1G/*1G genotype and the wild-type. This study indicates that the CYP3A4*1G allele is associated with the pharmacokinetics of atorvastatin and its metabolites in those Chinese Han patients with CHD after a single oral dose.

Association between the CYP4A11 T8590C variant and essential hypertension: new data from Han Chinese and a meta-analysis.

OBJECTIVE: CYP4A11 oxidizes endogenous arachidonic acid to 20-hydroxyeicosatetraenoic acid, a renal vasoconstrictor and natriuretic in humans. Previous studies demonstrated an association between a functional variant (T8590C) of CYP4A11 and essential hypertension, though with conflicting results. To elucidate this relationship, a case-control study and meta-analysis were performed to assess the possible association of essential hypertension with CYP4A11 genetic variations. METHODS: Associations between the T8590C polymorphism and essential hypertension were examined in 328 unrelated cases and 297 age-matched controls in Han Chinese individuals. High-resolution melting was used to identify the CYP4A11 variant. To further investigate the association, we conducted a meta-analysis including eight studies published previously in July 2012. RESULTS: The frequency of the CYP4A11 T8590C polymorphism showed no significant difference between cases and controls (all P>0.05). However, the meta-analysis showed that the CYP4A11 T8590C polymorphism may increase the risk of essential hypertension in an additive model (OR: 1.15, 95% CI: 1.02-1.29, P = 0.02), a dominant model (OR: 1.06, 95% CI: 1.01-1.32, P = 0.03), a recessive model (OR: 1.52, 95% CI: 1.15-2.02, P = 0.003) and a homozygote contrast (OR: 1.38, 95% CI: 1.07-1.78, P = 0.01). Also, a significant relationship was observed among Caucasians in the additive model, the homozygote contrast, the recessive model and the dominant model (all P<0.05). However, no association was observed in an Asian population (all P>0.05). CONCLUSIONS: This meta-analysis suggests there is a significant association between the CYP4A11 T8590C variant and essential hypertension, especially in Caucasians. The case-control study did not find a significant association among the Han Chinese population, but the controls were poorly matched and meaningful conclusions cannot therefore be made. Further large-scale studies are needed to clarify whether the CYP4A11 T8590C polymorphism is associated with hypertension risk in Asians or has a gender-specific effect.

[Molecular authentication of Fallopia multiflora by PCR-RFLP based on the trnL-trnF analysis].

OBJECTIVE: To establish a method for the molecular authentication of Fallopia multiflora. METHODS: The trnL-trnF regions of Fallopia multiflora and its closely related species and/or adulterants were sequenced and analyzed. RESULTS: It was found that the trnL-trnF sequence divergences between Fallopia multiflora and its closely related species and/or adulterants were 2.1%-22%. While the intra-species trnL-trnF divergences of Fallopia multiflora were 0%-1.5%. Based on the trnL-trnF regional variations, an endonuclease Xba I (T CTAGA) restriction site specific to Fallopia multiflora was detected. The Fallopia multiflora trnL-F polymerase chain reaction product could be cleaved by Xba I into two pieces, 804-819 bp and 256 bp each, whereas the restriction endonuclease could not digest the trnL-trnF polymerase chain reaction product of its closely related species or adulterants. The restriction patterns analyzed for restriction enzyme Xba I were found to be identical in all Fallopia multiflora individuals from different geographical regions in China. CONCLUSION: The assay based on polymerase chain reaction amplification of the trnL-trnF fragment of chloroplast DNA and subsequent restriction fragment length polymorphism can be used as a general test to identify Fallopia multiflora.

2,3,5,4'- tetrahydroxystilbene-2-O-ß-D-glycoside biosynthesis by suspension cells cultures of Polygonum multiflorum Thunb and production enhancement by methyl jasmonate and salicylic acid.

Friable calli of Polygonum multiflorum Thunb have been induced in MS medium supplemented with 6-benzylaminopurine (6-BA) and kinetin (KT). Suspension cultures were initiated from friable calli by inoculating calli in liquid MS medium in shake flasks in the dark and 25 °C on an orbital shaker at 100 rpm. The maximum dry weight (DW, 7.85 g/L) and 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glycoside (THSG, 56.39 mg/L) of suspension cells was obtained in MS medium after 16 days culture. Both methyl jasmonate (MeJA) and salicylic acid (SA) could increase THSG production. The most appropriate concentration of MeJA was 100 µmol/L in MS medium, in which concentration THSG content reached the maximum value of 147.79 mg/L, which represented a 162.36% increase compared to that of the control (56.33 mg/L). The most appropriate concentration of SA was 125 µmol/L in MS medium, at which concentration THSG content reached its maximum value of 116.43 mg/L, a 106.69% increase compared to that of the control (56.33 mg/L).

An effective method of RNA isolation from Fallopia multiflora tuberous roots.

To isolate high-quality total RNA from Fallopia multiflora tuberous roots is difficult because of the presence of high levels of carbohydrates, phenolics, and other secondary metabolites. Since several procedures specialized for RNA isolation from polysaccharides and phenols rich tissues have resulted in poor yields, in this study, we developed a modified protocol that was derived from the traditional CTAB method. The protocol was able to produce high-quality and intact RNA from the tuberous roots of F. multiflora. The yield of total RNA was more than 0.15 mg/g fresh weight, with an A260/A280 ratio of 1.9-2.0. The obtained RNA was of sufficient quality and suitable for downstream application such as reverse-transcription polymerase chain reaction (RT-PCR), Northern hybridization, and cDNA library construction. The protocol may also have wider applicability for total RNA isolation from other plant species with tuberous roots.

A functional polymorphism in the CYP3A4 gene is associated with increased risk of coronary heart disease in the Chinese Han population.

CYP3A4 is a major member of the cytochrome P450 (CYP) enzymes which play crucial roles in cardiovascular diseases. Recently, a novel polymorphism in the CYP3A4 gene, IVS10+12G>A, named CYP3A4*1G (rs2242480), has been identified. The aim of this study was to evaluate the potential relationship between the CYP3A4*1G allele and the susceptibility of coronary heart disease (CHD). A total of 628 individuals (322 unrelated patients with CHD and 306 age- and sex-matched healthy controls) were investigated in the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to identify CYP3A4*1G. We also analysed the functional significance of IVS10+12G>A using the dual-luciferase reporter assay. The results showed that the frequency of the CYP3A4*1G allele was 0.290 and the CYP3A4*1G/*1G genotype was 0.090 in the patients with CHD. The patients with the CYP3A4*1G/*1G genotype had higher CHD risk, with an odds ratio (OR) of 3.84 (p=0.025; 95% CI=1.32-12.65) after adjustment for conventional risk factors. A gender-dependent difference was also observed. The CYP3A4*1G/*1G frequency was significantly higher in female patients than in the controls (p=0.034, OR=3.02, 95% CI=1.04-8.70). Furthermore, the dual-luciferase reporter assay indicated that the A allele at IVS10+12G>A had a significantly higher transcriptional activity than the G allele. Our results imply that CYP3A4*1G contributes to the susceptibility of CHD in the Chinese Han population, which may be useful for the study of specific molecular pathogenesis for CHD.

Drug or vaccine?: selecting the appropriate treatment for malignant glioma patients.

Malignant gliomas are the most common and aggressive form of brain tumour. Current combinations of aggressive surgical resection, radiation therapy and chemotherapy regimens do not significantly improve long-term patient survival for these cancers. Therefore, investigative therapies including tumour vaccines have targeted this devastating condition. This article reviews evidence and data on chemotherapy and immunotherapy for a personalized medicine approach in order to enable physicians to select the appropriate treatment for glioma patients. Dendritic cell- and peptide-based therapy for gliomas seems to be safe and without major adverse effects. Gene-modified vaccines have also shown promise in the treatment of malignant gliomas. The concept of 'personalized medicine' is currently important in oncology treatment development. Using a personalized medicine approach, it may be necessary to evaluate the molecular genetic abnormalities in individual patient tumours, and such findings should be the mainstay of immunotherapeutic strategies designed for the individual patient.

[Analysis and authentication of Fallopia multiflora and its adulterants by matK sequence].

OBJECTIVE: To identify Fallopia multiflora from its adulterants by comparing their matK sequences. METHODS: Genomic DNA of different materials was extracted using modified cetytrimethyl ammonium bromide (CTAB) method. The double-strand matK genes were amplified using PCR method and then sequenced. The data were analyzed in Clustral W and MEGA 4.0 software package. RESULTS: Besides F. multiflora var. ciliinerve, the matK sequences of other adulterants show distinct differences with F. multiflora, whether for nucleotides substitutions or genetic distances; and the specific identifying sites for distinguishing F. multiflora and other Fallopia adulterants were found through further comparative analysis. Moreover, the 3 inspected materials were successfully authenticated by comparing the matK sequences. CONCLUSION: matK sequences can be used for the molecular identification between F. multiflora and its adulterant species.

[Isolation and pharmacological properties of analgesic fraction from venom of Naja Naja atra].

OBJECTIVE: To separate main analgesic fraction from venom of Guangdong Naja naja atra, to establish the basis for the using of Naja naja atra and find new analgesic fraction. METHODS: Affinity chromatography and size exclusion were used to isolate the analgesic fraction from the venom of Naja naja atra, and then to determine its properties by biochemical methods, such as SDS-polyacryamide gel electrophoresis ( SDS-PAGE), HPLC and Mole-toff. RESULTS: HPLC showed its relative purity was 95% (HPLC)and Mw was 6741. 236 Da. We observed that peripheral administration of neurotoxin strongly reduced the mechanical allogynia and thermal hyperalgesia for 24 hours, associated with this neuropathy (L5 spinal nerve ligation). CONCLUSION: The fraction from venom of Naja naja atra has significant analgesic effect and it is worth further developing.

Peripheral and spinal antihyperalgesic activity of najanalgesin isolated from Naja naja atra in a rat experimental model of neuropathic pain.

Snake venoms are a rich source of various compounds that have applications in medicine and biochemistry. Recently, it has been demonstrated that najanalgesin isolated from the venom of Naja naja atra exerts analgesic effects on acute pain in mice. The objective of this study was to evaluate the antinociceptive effect of najanalgesin in a rat model of neuropathic pain, induced by L5 spinal nerve ligation and transaction. We observed that intraperitoneal (i.p.) administration of najanalgesin produced significant increase in hind paw withdrawal latency (HWL) in response to both mechanical and thermal stimulation. Moreover, a single dose of najanalgesin was able to induce antinociceptive activity that lasted for 1 week. Intrathecal injection of najanalgesin increased the HWL in response to mechanical stimuli. The antinociceptive effect of najanalgesin administered intrathecally was partly inhibited by intrathecal injection of naloxone or atropine. These results demonstrate that najanalgesin has antinociceptive effects on the central and peripheral system in the rat neuropathic pain model. The opioid receptor and muscatinic receptor are involved in najanalgesin-induced antinociception in the spinal cord. This research supports the possibility of using najanalgesin as a novel pharmacotherapeutic agent for neuropathic pain.

Molecular authentication of the traditional medicinal plant Fallopia multiflora.

The root of Fallopia multiflora is one of the most widely used traditional Chinese medicines. However, it is often confused and substituted with the roots of F. multiflora var. ciliinervis, Pteroxygonum giraldii, Cynanchum auriculatum, and Stephania cepharantha. To establish a DNA polymorphism-based assay for the identification of F. multiflora, the nuclear ribosomal DNA (nrDNA) internal transcribed spacer (ITS) regions of six Fallopia species were sequenced and analyzed. Based on the diversity of ITS regions among the species the diagnostic primers PMITS28 and PMITS545, which amplified an expected 517-bp DNA fragment from F. multiflora DNA, were designed. No amplified product was observed when DNA from other species was used. This method can be used for the authentication of F. multiflora.

Genetic variation and identification of cultivated Fallopia multiflora and its wild relatives by using chloroplast matK and 18S rRNA gene sequences.

FALLOPIA MULTIFLORA (Thunb.) Harald . has been widely and discriminatingly used in China for the study and treatment of anemia, swirl, deobstruent, pyrosis, insomnia, amnesia, atheroma and also for regulating immune functions. However, there is still confusion about the herbal drug's botanical origins and the phylogenetic relationship between the cultivars and the wild relatives. In order to develop an efficient method for identification, a molecular analysis was performed based on 18 S rRNA gene and partial MATK gene sequences. The 18 S rRNA gene sequences of F. MULTIFLORA were 1809 bp in length and were highly conserved, indicating that the cultivars and the wild F. MULTIFLORA have the same botanical origin. Based on our 18 S rRNA gene sequences analysis, F. MULTIFLORA could be easily distinguished at the DNA level from adulterants and some herbs with similar components. The MATK gene partial sequences were found to span 1271 bp. The phylogenetic relation of F. MULTIFLORA based on the MATK gene showed that all samples in this paper were divided into four clades. The sequences of the partial MATK gene had many permutations, which were related to the geographical distributions of the samples. MATK gene sequences provided valuable information for the identification of F. MULTIFLORA. New taxonomic information could be obtained to authenticate the botanical origin of the F. MULTIFLORA, the species and the medicines made of it.

Purification and characterization of a novel antinociceptive toxin from Cobra venom (Naja naja atra).

Snake venoms have demonstrated antinociceptive activity, and certain isolated neurotoxins have demonstrated significant analgesia in animal models. Here we report a novel analgesic toxin which was isolated from Naja naja atra and was given the name 'najanalgesin'. The LD(50) of the crude venom and najanalgesin were 0.89mg/kg and 2.69mg/kg, respectively. We used the writhing test and hot plate test to evaluate the antinociceptive properties of the crude venom and najanalgesin after intraperitoneal (ip) administration. The analgesic mechanism of najanalgesin was also studied. The response latency time was significantly prolonged in the hot plate test after ip administration of the crude venom of Naja naja atra (0.111-0.445mg/kg) in a dose-dependent manner. Najanalgesin (1mg/kg) elicited almost the same antinociceptive effect as that of the crude venom of Naja naja atra at the dose of 0.445mg/kg and remained for 6h after intraperitoneal injection, shown by hot plate test. The percentage of increase in the latency time for the venom and the najanalgesin 3h after drug administration was 96.2% and 112%, respectively. The number of writhes decreased to almost 1/3, 1/6, and 1/12 of the NS (physiological saline) group after intraperitoneal administration of najanalgesin at 0.25, 0.5, and 1.0mg/kg, respectively. Pretreatment with atropine (1mg/kg) or naloxone (3mg/kg) blocked the antinociception of najanalgesin in the hot plate test. Based on the sequence information, najanalgesin is found to be highly homologous with the conventional CTXs (cardiotoxins). To our knowledge, no study had previously reported that a toxin which was homologous with CTXs possessed the antinociceptive activity. Thus, this is the first report that the antinociceptive effect induced by najanalgesin is mediated by cholinergic and opioidergic mechanisms.