Development of Beet necrotic yellow vein virus-based vectors for multiple-gene expression and guide RNA delivery in plant genome editing.

Many plant viruses with monopartite or bipartite genomes have been developed as efficient expression vectors of foreign recombinant proteins. Nonetheless, due to lack of multiple insertion sites in these plant viruses, it is still a big challenge to simultaneously express multiple foreign proteins in single cells. The genome of Beet necrotic yellow vein virus (BNYVV) offers an attractive system for expression of multiple foreign proteins owning to a multipartite genome composed of five positive-stranded RNAs. Here, we have established a BNYVV full-length infectious cDNA clone under the control of the Cauliflower mosaic virus 35S promoter. We further developed a set of BNYVV-based vectors that permit efficient expression of four recombinant proteins, including some large proteins with lengths up to 880 amino acids in the model plant Nicotiana benthamiana and native host sugar beet plants. These vectors can be used to investigate the subcellular co-localization of multiple proteins in leaf, root and stem tissues of systemically infected plants. Moreover, the BNYVV-based vectors were used to deliver NbPDS guide RNAs for genome editing in transgenic plants expressing Cas9, which induced a photobleached phenotype in systemically infected leaves. Collectively, the BNYVV-based vectors will facilitate genomic research and expression of multiple proteins, in sugar beet and related crop plants.

Coenurosis of Yak, Bos grunniens, caused by Taenia multiceps: A Case Report with Molecular Identification in Qinghai Tibetan Plateau Area, China.

Coenurosis is an important zoonotic helminthic disease caused by the larval stage of the tapeworm Taenia multiceps. This parasite typically infects the brain of the intermediate hosts, including sheep, goat, cattle and even humans. We report a case of T. multiceps infection in a yak confirmed by clinical symptoms, morphological characteristics, and molecular and phylogenetic analyses. The coenurus was thin-walled, whitish, and spherical in shape with a diameter of 10 cm. The parasite species was identified as T. multiceps by PCR amplification and sequencing of the 18S rRNA, cox1 and nad1 genes. Three gene sequences all showed high homology (all above 97%) with the reference sequences from different hosts. Moreover, phylogenetic reconstructions with the 3 published Taenia gene sequences confirmed that the Qinghai yak isolate was closely related to T. multiceps. Although there are advanced diagnosis and treatment methods for coenurosis, early infection is difficult to diagnose. Importantly, the findings of yak infection case should not be ignored due to its zoonotic potential.

[Cloning and Expression of Tibetan Sheep-origin Echinococcus granulosus Antigen B Gene and Protein Identification Using Immunological Method].

Objective: To clone and express the Tibetan Sheep-origin Echinococcus granulosus Antigen B8/2 Gene, and immunologically identify the encoded protein. Methods: The cDNA of EgAgB8/2 gene was amplified by RT-PCR. The prokaryotic expression vector pET-EgAgB8/2 was constructed and transformed into E. coli BL21（DE3） for expression. Proteins were extracted, separated in SDS-PAGE and identified by Western blotting. Results: The cloned EgAgB8/2 gene was 335 bp in length, and had a 98%-100% sequence homology with the reported cDNA sequence of EgAgB8/2, indicating the successful construction of the pET-EgAgB8/2 vector. SDS-PAGE revealed large amount of proteins in supernatant. Western blotting further confirmed the expression of the target protein. Conclusion: The EgAgB8/2 gene of Tibetan Sheep-origin in Qinghai is successfully cloned, and the constructed pET-EgAgB8/2 vector can be used to express the target protein.

[Survey of HIV drug resistance threshold in Zhejiang province from 2009 to 2011].

OBJECTIVE: To survey the prevalence of drug resistant HIV in Zhejiang province in 2009-2011. METHODS: WHO truncated sequential sampling technique was adopted annually by using 63, 62 and 57 samples of newly diagnosed as HIV positive and aged 16-25 years in Hangzhou, Ningbo and Wenzhou from 2009 to 2011, respectively. RNA was prepared and HIV pol region was amplified by RT-PCR and nested PCR. Pol genetic mutation associated with drug resistance was analyzed. RESULTS: The success rates for sequence acquisition of the survey were 82.5% (52/63), 95.2% (59/62) and 94.7% (54/57) from year 2009 to 2011, respectively, and the main subtype was CRF01_AE (68.5% (37/54)-71.2% (37/52)). A total of 4 surveillance drug-resistance mutation (SDRMs), 2 SDRMs and 2 SDRMs were found by analyzing the 47 sequences each year, sampled from year 2009 to 2010, respectively, indicating that the prevalence of drug resistant HIV stains was moderate in 2009, and low for the next two years (2010-2011). A total of 8 individuals with drug resistant HIV stains found in this study were all infected by sexual transmission, especially in homosexual transmission (6 cases), and the main subtype was CRF01_AE (7 cases). SDRMs for protease inhibitor (PI), nucleotide HIV-reverse transcriptase inhibitor (NRTI) and non-NRTI (NNRTI) (L90M, T215S and Y188L) were all found in one case. CONCLUSION: The prevalence of drug resistant HIV stains in major areas with AIDS epidemic in Zhejiang province was low in 2009-2011.

[Echinococcosis prevalence in elderly population from Haiyan County of Qinghai Province].

Echinococcus infection in elderly population (60-87 years old) was investigated by serological assay, medical imaging and epidemiology history in August, 2008. A total of 234 subjects from different nationalities were examined. Seven cases were serologically positive for echinococcosis and confirmed by medical imaging. Out of 30 suspected sera-positive cases, 3 were positive by medical imaging. There were 10 confirmed echinococcosis cases with a positive rate of 4.3%. There was significant difference on the prevalence among different towns (P < 0.05), but no significant statistical difference on the prevalence among nationalities, careers and sex. The prevalence in females (4.8%) was higher than that of males (3.6%).

Conjugation of latent membrane protein (LMP)-2 epitope to gold nanoparticles as highly immunogenic multiple antigenic peptides for induction of Epstein-Barr virus-specific cytotoxic T-lymphocyte responses in vitro.

Nasopharyngeal carcinoma is a neoplasm with a high incidence in Southeast Asia, and it is strongly associated with Epstein-Barr virus (EBV) activation involving the expression of a weakly immunogenic protein, namely, latent membrane protein (LMP)-2. Previous immunological studies already identified the human leukocyte antigen (HLA)-A11 restricted peptide epitope (SSCSSCPLSK) in the LMP-2 antigen. In this work, we prepared gold nanoparticle (AuNP)-peptide conjugate 1 by treating the nanoparticles with the N-cysteinated LMP-2 epitope. The AuNP-peptide conjugates have been characterized by TEM (15-24 nm in diameter) and UV-vis spectroscopy (surface plasmon resonance absorption band at lambda(max) = 520 nm). In the presence of a CALNN capping peptide, the AuNP-peptide conjugates are stable in solution without aggregation at room temperature for at least 48 h. By ELIspot studies, AuNP-peptide conjugate 1 was found to elicit a significantly stronger INF-gamma response [number of spot forming cells (SPC) = 727 +/- 198] from peripheral blood mononuclear cells of healthy HLA-A11 donors when compared to that induced by the unconjugated LMP-2 peptides (SFC = 73 +/- 28). Further studies showed that dendritic cells treated with conjugate 1 can effect CD8+ T-cell activation leading to epitope-specific cytotoxic T lymphocyte killing responses in vitro.

[The effect of cigarette smoke on the expression of transforming growth factor-beta1 mRNA and collagen type III in airways of asthmatic rats].

OBJECTIVE: To study the effect of cigarette smoke on the expression of transforming growth factor-beta l (TGF-beta1), and collagen type III in lung tissues, and therefore to investigate the mechanism of airway remodeling. METHODS: Thirty male Wistar rats were randomly divided into a control group, an asthmatic group and a cigarette smoke treated group, with 10 rats in each group. The expression level of TGF-beta1 mRNA was measured by reverse transcription polymerase chain reaction (RT-PCR) and collagen type III by immunohistochemistry. The thickness of airway wall in each group was also measured. Software SPSS 11.0 was used for statistical analysis (data expressed as +/- s). Group comparison was made by one way ANOVA and Pearson correlation was used for correlation analysis. RESULTS: The levels of TGF-beta1 mRNA and collagen type III in cigarette smoke treated group (0.42 +/- 0.04, 25.8 +/- 2.3) were higher than those in the asthmatic group (0.39 +/- 0.04, 22.9 +/- 3.1) and in the control group (0.26 +/- 0.04, 16.3 +/- 2.3). Compared to the control group, the levels were higher in the asthmatic group (F = 55.97, 35.61, all P < 0.05). The level of TGF-beta1 mRNA was positively correlated with the expression of collagen type III (r = 0.71, P < 0.05). The thickness of airway wall in the cigarette smoke treated group (23.3 +/- 2.4) microm2/microm was significantly higher than that in the asthmatic group (20.1 +/- 2.9) microm2/microm and the control group (11.6 +/- 2.4) microm2/microm;compared to the control group, it was higher in the asthmatic group (F = 53.68, P < 0.05). CONCLUSION: Cigarette smoke can promote over-expression of TGF-beta1-mRNA in asthmatic rat airways, increase the expression of collagen type III and aggravate airway remodeling.

Induction of cytotoxic T cell response against HCA661 positive cancer cells through activation with novel HLA-A *0201 restricted epitopes.

HCA661 is a cancer-testis (CT) antigen frequently expressed in human hepatocellular carcinoma (HCC). To search for immunogenic peptides of HCA661, bioinformatics analysis and CD8(+) T cell IFN-gamma ELISPOT assay were employed, and two HLA-A *0201 restricted peptides, H110 and H246, were identified. These two HCA661 peptides are naturally processed in dendritic cells (DCs) and when used for DCs loading, they are sufficient to prime autologous CD8(+) T cells to elicit cytotoxic response against HCA661(+) human cancer cells. The HCA661 peptides, H110 and H246, are hence attractive candidates for human cancer immunotherapy.

Infection with Paragonimus westermani of boar-hunting dogs in Western Japan maintained via artificial feeding with wild boar meat by hunters.

Infection of boar-hunting dogs with Paragonimus westermani was investigated in Western Japan. Blood and rectal feces were collected from 441 dogs in the three districts (205 in Kinki, 131 in Chugoku and 105 in Shikoku District). In a screening ELISA for serum antibody against P. westermani antigen, 195 dogs (44.2%) showed positive reaction. In the 195 dogs, 8 dogs were found excreting P. westermani eggs after molecular analysis of fecal eggs, and additional 7 were identified serologically for the parasite infection because of their stronger reactivity against P. westermani antigen than against antigens of other species of Paragonimus. A spatial analysis showed that all of the P. westermani infections were found in Kinki and Chugoku Districts. In this area, dogs' experience of being fed with raw boar meat showed high odds ratio (3.35) to the sero-positivity in the screening ELISA, and the frequency of such experiences was significantly higher in sero-positive dogs. While clear relationship was not obtained between predation of boars by dogs during hunting and their sero-positivity. Therefore, it is suggested that human activity of feeding with wild boar meat is the risk factor for P. westermani infection in boar-hunting dogs. Considering that hunting dogs could play as a major definitive host and maintain the present distribution of P. westermani in Western Japan, control measures for the infection in hunting dogs, such as prohibition of raw meat feeding and regular deworming, should be undertaken.

Genetic diversity and population genetics of the warble flies Hypoderma bovis and H. sinense in Qinghai Province, China.

BACKGROUND: Hypoderma bovis and H. sinense (Diptera: Oestridae) mainly parasitise cattle and yaks. The two parasites are pathogenic and cause economic losses that result from reduced amounts of livestock products, including milk, meat, and skin. Genetic diversity and population genetic structure of H. bovis and H. sinense have not been evaluated, but could be used to inform appropriate strategies to control these parasites. METHODS: We cloned and sequenced part of the mitochondrial cytochrome c oxidase subunit I (COI) gene from 60 H. bovis isolates and 52 H. sinense isolates from five locations in Qinghai Province, China, to identify polymorphisms, and infer their phylogenetic relationships, historical population expansions, and divergence time. RESULTS: We identified 17 COI haplotypes from the H. bovis samples, and 23 COI haplotypes from the H. sinense samples. The haplotype and nucleotide diversities were 0.738 and 0.00202 for H. bovis, and 0.867 and 0.00300 for H. sinense, respectively, which indicates rich genetic diversity in H. bovis and H. sinense populations. Bayesian phylogenetic analysis revealed that the two species are monophyletic, and geographical structuring of haplotypes was significantly different in H. sinense (P < 0.05), but not H. bovis. Neutrality tests and mismatch distribution statistical analysis revealed that populations of the two species have undergone demographic expansions. The divergence three Hypoderma spp. (H. bovis, H. lineatum, and H. sinense) was estimated to have occurred approximately 4.5 million years ago (Mya), which indicates that the rapid uplift of the Qinghai-Tibetan Plateau during the late Miocene-Pliocene was associated with divergence of Hypoderma species. CONCLUSIONS: Results of the present study revealed that both H. bovis and H. sinense displayed high genetic diversity and widespread population genetic differentiation within and among populations; these data, along with the molecular phylogeny, demographic history, and divergence time estimation, provide new insight into evolutionary history of these species. These findings will help elucidate speciation in Hypoderma and provide theoretical basis for epidemiological surveillance and control of these species on the Qinghai-Tibetan Plateau.

Comparative Digital Gene Expression Analysis of the Arabidopsis Response to Volatiles Emitted by Bacillus amyloliquefaciens.

Some plant growth-promoting rhizobacteria (PGPR) regulated plant growth and elicited plant basal immunity by volatiles. The response mechanism to the Bacillus amyloliquefaciens volatiles in plant has not been well studied. We conducted global gene expression profiling in Arabidopsis after treatment with Bacillus amyloliquefaciens FZB42 volatiles by Illumina Digital Gene Expression (DGE) profiling of different growth stages (seedling and mature) and tissues (leaves and roots). Compared with the control, 1,507 and 820 differentially expressed genes (DEGs) were identified in leaves and roots at the seedling stage, respectively, while 1,512 and 367 DEGs were identified in leaves and roots at the mature stage. Seventeen genes with different regulatory patterns were validated using quantitative RT-PCR. Numerous DEGs were enriched for plant hormones, cell wall modifications, and protection against stress situations, which suggests that volatiles have effects on plant growth and immunity. Moreover, analyzes of transcriptome difference in tissues and growth stage using DGE profiling showed that the plant response might be tissue-specific and/or growth stage-specific. Thus, genes encoding flavonoid biosynthesis were downregulated in leaves and upregulated in roots, thereby indicating tissue-specific responses to volatiles. Genes related to photosynthesis were downregulated at the seedling stage and upregulated at the mature stage, respectively, thereby suggesting growth period-specific responses. In addition, the emission of bacterial volatiles significantly induced killing of cells of other organism pathway with up-regulated genes in leaves and the other three pathways (defense response to nematode, cell morphogenesis involved in differentiation and trichoblast differentiation) with up-regulated genes were significantly enriched in roots. Interestingly, some important alterations in the expression of growth-related genes, metabolic pathways, defense response to biotic stress and hormone-related genes were firstly founded response to FZB42 volatiles.

FOS protein expression and role of the vagus nerve in the rat medullary visceral zone in multiple organ dysfunction syndrome caused by subarachnoid hemorrhage.

This study was designed to observe the role of FOS protein expression in the rat medullary visceral zone (MVZ) in multiple organ dysfunction syndrome (MODS) caused by subarachnoid hemorrhage (SAH), with and without severing the vagus nerve. We also investigated the regulatory and control mechanisms of the MVZ and the vagus nerve in MODS following SAH. A model of MODS following SAH was established by injecting arterial blood into the Willis' circle. The vagus nerve was cut off and blocked. The FOS protein expression in the MVZ was detected by immunohistochemistry. The positive expression levels of FOS in the MVZ in the SAH and SAH + severed-down vagus nerve (SDV) groups were higher than those in the normal control, sham surgery and SDV groups (P<0.01). However, expression in the SAH+SDV group was lower than that in the SAH group (P<0.01). Inflammatory damage was observed in each visceral organ at every time-phased point in the SAH group and the SAH+SDV group. The most apparent damage was at 24-36 h, consistent with the peak of FOS protein expression; the SAH+SDV group presented a greater level of damage. The inflammatory changes in surrounding visceral organs following SAH correlated with FOS protein expression in the MVZ, which indicates that the MVZ participates in the functional control of surrounding visceral organs following SAH. Severing the subphrenic vagus nerve increases the incidence of MODS following SAH and enhances SAH-induced inflammatory damage to the surrounding visceral organs, which indicates that the vagus nerve plays a role in the protection of the surrounding visceral organs in MODS following SAH.

[Molecular epidemiological study on HIV/AIDS under the follow-up program in Zhejiang province in 2009].

OBJECTIVE: To analyze the molecular epidemiological characteristics on HIV infectors/AIDS patients (HIV/AIDS) under a follow-up program in Zhejiang province in 2009. METHODS: 303 cases were randomly sampled. Information on the cases was collected and followed by genomic DNA extraction. Gag gene fragments were amplified by nested PCR, followed by sequencing and bio-informatic analysis. RESULTS: The rate of success for sequence acquisition was 74.3% (225/303). Distributions of HIV subtypes were as follows: CRF01_AE (58.7%), CRF07_BC (13.8%), CRF08_BC (9.8%), B' (15.1%), C (1.8%), G (0.4%) and unassigned BC (unique recombinant form 0.4%). RESULTS: from the HIV BLAST analysis showed that the sources of strains with the highest homology involved in 10 provinces/municipalities (Liaoning, Guangxi, Yunnan, Henan, etc.) and five other countries (Thailand, Vietnam, India, South Africa and Libya). The CRF01_AE phylogenetic tree was divided into four clusters. The sequences of HIV/AIDS with homosexual transmission showed a gather in cluster 1, and mix with those infected through heterosexual contact. CONCLUSION: Circulating recombinant forms of HIV seemed to play a dominant role in Zhejiang province. Unique recombinant form and new subtype of HIV were found. People living with HIV under homosexual transmission and heterosexual transmission had a trend of interwoven with each other. Increase of both the diversity and complexity of HIV strains were also noticed in Zhejiang province.

Interactions between artemisinins and other antimalarial drugs in relation to the cofactor model--a unifying proposal for drug action.

Artemisinins are proposed to act in the malaria parasite cytosol by oxidizing dihydroflavin cofactors of redox-active flavoenzymes, and under aerobic conditions by inducing their autoxidation. Perturbation of redox homeostasis coupled with the generation of reactive oxygen species (ROS) ensues. Ascorbic acid-methylene blue (MB), N-benzyl-1,4-dihydronicotinamide (BNAH)-MB, BNAH-lumiflavine, BNAH-riboflavin (RF), and NADPH-FAD-E. coli flavin reductase (Fre) systems at pH 7.4 generate leucomethylene blue (LMB) and reduced flavins that are rapidly oxidized in situ by artemisinins. These oxidations are inhibited by the 4-aminoquinolines piperaquine (PPQ), chloroquine (CQ), and others. In contrast, the arylmethanols lumefantrine, mefloquine (MFQ), and quinine (QN) have little or no effect. Inhibition correlates with the antagonism exerted by 4-aminoquinolines on the antimalarial activities of MB, RF, and artemisinins. Lack of inhibition correlates with the additivity/synergism between the arylmethanols and artemisinins. We propose association via π complex formation between the 4-aminoquinolines and LMB or the dihydroflavins; this hinders hydride transfer from the reduced conjugates to the artemisinins. The arylmethanols have a decreased tendency to form π complexes, and so exert no effect. The parallel between chemical reactivity and antagonism or additivity/synergism draws attention to the mechanism of action of all drugs described herein. CQ and QN inhibit the formation of hemozoin in the parasite digestive vacuole (DV). The buildup of heme-Fe(III) results in an enhanced efflux from the DV into the cytosol. In addition, the lipophilic heme-Fe(III) complexes of CQ and QN that form in the DV are proposed to diffuse across the DV membrane. At the higher pH of the cytosol, the complexes decompose to liberate heme-Fe(III) . The quinoline or arylmethanol reenters the DV, and so transfers more heme-Fe(III) out of the DV. In this way, the 4-aminoquinolines and arylmethanols exert antimalarial activities by enhancing heme-Fe(III) and thence free Fe(III) concentrations in the cytosol. The iron species enter into redox cycles through reduction of Fe(III) to Fe(II) largely mediated by reduced flavin cofactors and likely also by NAD(P)H-Fre. Generation of ROS through oxidation of Fe(II) by oxygen will also result. The cytotoxicities of artemisinins are thereby reinforced by the iron. Other aspects of drug action are emphasized. In the cytosol or DV, association by π complex formation between pairs of lipophilic drugs must adversely influence the pharmacokinetics of each drug. This explains the antagonism between PPQ and MFQ, for example. The basis for the antimalarial activity of RF mirrors that of MB, wherein it participates in redox cycling that involves flavoenzymes or Fre, resulting in attrition of NAD(P)H. The generation of ROS by artemisinins and ensuing Fenton chemistry accommodate the ability of artemisinins to induce membrane damage and to affect the parasite SERCA PfATP6 Ca(2+) transporter. Thus, the effect exerted by artemisinins is more likely a downstream event involving ROS that will also be modulated by mutations in PfATP6. Such mutations attenuate, but cannot abrogate, antimalarial activities of artemisinins. Overall, parasite resistance to artemisinins arises through enhancement of antioxidant defense mechanisms.

Growth and genotypes of Echinococcus granulosus found in cattle imported from Australia and fattened in Japan.

At the abattoir on study in Miyazaki, Japan, 9537 imported cattle from Australia in average were slaughtered annually in the last 5 years (2006 to 2010) and hydatid cysts were constantly detected in about 1.8% of the cattle. In order to assess the risk of Echinococcus granulosus delivered to Japan by imported cattle, 250 cysts found in 103 cattle at the abattoir were examined for their biological characteristics and genotypes. The cattle slaughtered were imported from Australia at an age of 10-12 months old and fattened for 17-18 months in Japan. The cysts showed their size ranging from 4 to 108 mm and were mainly found in the lung. Mature protoscoleces were detected in the three largest cysts, all were of the G1 genotype. Most of the other cysts contained clear cyst fluid and had thin laminated layer with no protoscoleces. The finding implies a potential risk of E. granulosus being established in Japan, thus strict and proper meat inspection and consequent offal condemnation are requisite at abattoirs that deal with imported cattle. Genotyping based on partial fragments of mitochondrial cox1, rrnS and nad1 genes were performed on the 66 cysts, showing that most of the cysts were G1 genotype (common sheep strain). However, two and four cysts were considered as G2 (Tasmanian sheep strain) and G3 (buffalo strain) genotypes, respectively. Since it has been widely recognized that G1 is the only genotype distributing in mainland Australia and that G2 genotype has been eradicated from Tasmania, the finding of those genotypes from Australian cattle indicated that certain genotypes other than G1 genotype are distributing in mainland Australia.

[Relationship between strain subtypes and transmission of HIV infection within marriage-based immigrant women in rural area of Zhejiang province].

OBJECTIVE: To analyze the relationship between strain subtypes and transmission of HIV infection on marriage-based immigrant women, their spouses and children in rural area of Zhejiang province. METHODS: Marriage-based immigrant women with HIV infection, their HIV infected spouses and children in rural area in Zhejiang province, were selected as study objects. Analysis on genetic sequence and epidemiologic information was carried out. Subgenomic gag was amplified by nest-PCR analysis on the whole blood samples. Genetic subtype characterization and the source of HIV strains were analyzed. Relationships on sequences were also examined by phylogenetic tree analysis. RESULTS: Genetic sequences of 72 samples from HIV infected marriage-based immigrant women were obtained. The genetic subtypes comprised 21 CRF01_AE (29.2%), 12 CRF07_BC (16.7%), 31 CRF08_BC (43.1%), 6 B (8.3%), 2 C (2.8%). HIV strains from 45 cases (62.5%) were similar to the prevalent HIV strains in the province where former census of marriage-based immigrant women were registered. In total, there were 26 (70.3%) cases from Yunnan province. 84.7% of the infected women had heterosexual behaviors before settling down in Zhejiang province. Genetic sequences of 17 pairs showed the same subtype between the couples and data from phylogenetic tree analysis supported the assumption of transmission linkage in the family. CONCLUSION: The HIV subtype strains detected in those HIV infected marriage-based immigrant women in the rural area of Zhejiang province characterized with diversity, showing CRF08_BC and CRF01_AE were the main HIV strain subtypes. HIV infection originated mainly from Yunnan province and nearby regions. Heterosexual behaviors of the marriage-based immigrant women in the original region where they had their residence registration, seemed to be the primary high risk factors for these women. Surveillance and intervention programs on these marriage-based immigrant women and their family members should be improved.

Reactions of antimalarial peroxides with each of leucomethylene blue and dihydroflavins: flavin reductase and the cofactor model exemplified.

Flavin adenine dinucleotide (FAD) is reduced by NADPH-E. coli flavin reductase (Fre) to FADH(2) in aqueous buffer at pH 7.4 under argon. Under the same conditions, FADH(2) in turn cleanly reduces the antimalarial drug methylene blue (MB) to leucomethylene blue. The latter is rapidly re-oxidized by artemisinins, thus supporting the proposal that MB exerts its antimalarial activity, and synergizes the antimalarial action of artemisinins, by interfering with redox cycling involving NADPH reduction of flavin cofactors in parasite flavin disulfide reductases. Direct treatment of the FADH(2) generated from NADPH-Fre-FAD by artemisinins and antimalaria-active tetraoxane and trioxolane structural analogues under physiological conditions at pH 7.4 results in rapid reduction of the artemisinins, and efficient conversion of the peroxide structural analogues into ketone products. Comparison of the relative rates of FADH(2) oxidation indicate optimal activity for the trioxolane. Therefore, the rate of intraparastic redox perturbation will be greatest for the trioxolane, and this may be significant in relation to its enhanced in vitro antimalarial activities. (1)H NMR spectroscopic studies using the BNAH-riboflavin (RF) model system indicate that the tetraoxane is capable of using both peroxide units in oxidizing the RFH(2) generated in situ. Use of the NADPH-Fre-FAD catalytic system in the presence of artemisinin or tetraoxane confirms that the latter, in contrast to artemisinin, consumes two reducing equivalents of NADPH. None of the processes described herein requires the presence of ferrous iron. Ferric iron, given its propensity to oxidize reduced flavin cofactors, may play a role in enhancing oxidative stress within the malaria parasite, without requiring interaction with artemisinins or peroxide analogues. The NADPH-Fre-FAD system serves as a convenient mimic of flavin disulfide reductases that maintain redox homeostasis in the malaria parasite.

A partial convergence in action of methylene blue and artemisinins: antagonism with chloroquine, a reversal with verapamil, and an insight into the antimalarial activity of chloroquine.

Artemisinins rapidly oxidize leucomethylene blue (LMB) to methylene blue (MB); they also oxidize dihydroflavins such as the reduced conjugates RFH2 of riboflavin (RF), and FADH2 of the cofactor flavin adenine dinucleotide (FAD), to the corresponding flavins. Like the artemisinins, MB oxidizes FADH2, but unlike artemisinins, it also oxidizes NAD(P)H. Like MB, artemisinins are implicated in the perturbation of redox balance in the malaria parasite by interfering with parasite flavoenzyme disulfide reductases. The oxidation of LMB by artemisinin is inhibited by chloroquine (CQ), an inhibition that is abruptly reversed by verapamil (VP). CQ also inhibits artemisinin-mediated oxidation of RFH2 generated from N-benzyl-1,4-dihydronicotinamide (BNAH)-RF, or FADH2 generated from NADPH or NADPH-Fre, an effect that is also modulated by verapamil. The inhibition likely proceeds by the association of LMB or dihydroflavin with CQ, possibly involving donor-acceptor or π complexes that hinder oxidation by artemisinin. VP competitively associates with CQ, liberating LMB or dihydroflavin from their respective CQ complexes. The observations explain the antagonism between CQ-MB and CQ-artemisinins in vitro, and are reconcilable with CQ perturbing intraparasitic redox homeostasis. They further suggest that a VP-CQ complex is a means by which VP reverses CQ resistance, wherein such a complex is not accessible to the putative CQ-resistance transporter (PfCRT).

[Subtype and transmission of HIV strain in both HIV infected spouses in Zhejiang province].

OBJECTIVE: To analyse the subtype and transmission of HIV strain in both HIV infected spouses. METHODS: Reported both HIV infected spouses were selected as objects. Analysis on genetic sequence and high risk behaviors was carried out. Subgenomic gag was amplified by nest-PCR analysis of whole blood samples from objects. Genetic subtype characterization of HIV was identified and pairwise genetic distances were calculated. Sequence relationships were also examined by phylogenetic tree analysis. RESULTS: Genetic sequences of 46 pairs (92 cases) were obtained. The genetic subtype comprised 39 CRF01_AE (42.4%), 10 CRF07_BC (10.9%), 18 CRF08_BC (19.6%), 18 B (19.6%), 5 C (5.4%) and 2 CRF02_AG (2.2%). 44 pairs had the same subtype between the two partners, accounted for 95.7% of the total. 33 of the 41 pairs with phylogenetic tree analysis were found having epidemiological linkage in pair wise. Sexual behaviors of out-marriage were the main risk factors of CRF01_AE and CRF08_BC and CRF02_AG strains infection. Blood transmission was associated with B and CRF07_BC. CONCLUSION: The HIV strains subtype detected in HIV infected spouses characterized with diversity. CRF01_AE was the main strain subtype. The main route of transmission to the spouses was through unprotected sexual contacts. Surveillance programs on HIV infected partner together with intervention between the spouses should be improved.

[Genotypic drug-resistance of HIV-1 CRF01_AE in Zhejiang province, 2004-2007].

OBJECTIVE: To study the genotypic drug-resistant mutation among treat-naïve or treated patients infected with HIV-1 CRF01_AE in Zhejiang province during 2004-2007. METHODS: HIV-1 pol amplicons (PR + RT) from 13 treated and 43 treat-naïve patients were obtained by reverse transcription-polymerase chain reaction (RT-PCR). The sequences were analyzed for genotypic antiretroviral resistance through online tools (http://hivdb.stanford.edu). RESULTS: The median count of CD4+ T lymphocytes in 43 treat-naïve patients was 229 cells /mm3 and the median log10 viral load was 3.41. Some drug-resistant mutations were seen in these samples including amino acid 10, 46, 71, in the genes ofprotease (PR) and 103, 118, in the genes of reverse transcriptase (RT) whereas twenty-nine resistance mutations in the genes of PR and RT were obtained in the 13 treated patients (8/13, 61.5%). The high prevalence of drug-resistant mutations was observed in patients who had been receiving HAART (hight active antiretroviral therapy). Among them, cross drug resistance was dominant. Correspondingly, the median counts of CD4+ T lymphocytes and the log10 viral load were 186 cells/mm3 and 3.91. CONCLUSION: There was a low prevalence of genotypic drug-resistant mutations in treat-naïve patients, but higher drug-resistant mutation in treated patients. More attention should be paid to the transmission of drug-resistant HIV strains and the antiretroviral therapy recipe should be adjusted correspondingly for the development of ART drugs, intervention as well as clinical therapy programs.