Hepatitis C virus (HCV) genotyping by annealing reverse transcription-PCR products with genotype-specific capture probes

The genotype of the hepatitis C virus (HCV) strain infecting a given patient is an important predictive factor for the clinical outcome of chronic liver disease and its response to anti-viral therapeutic agents. We herein sought to develop a new easy, sensitive and accurate HCV genotyping method using annealing genotype-specific capture probes (AGSCP) in an automation-friendly 96-well plate format. The validation of our new AGSCP was performed using the Standard HCV Genotype Panel. We then used both our AGSCP and the commercially available INNO-LiPA assay to analyze the HCV genotypes from 111 Korean patients. Discordant results were analyzed by direct sequencing. AGSCP successfully genotyped the standard panel. The genotypes of 111 patient samples were also obtained successfully by AGSCP and INNO-LiPA. We observed a high concordance rate (93 matched samples, 83.8%) between the two assays. Sequencing analysis of the 18 discordant results revealed that the AGSCP had correctly identified 12 samples, whereas the INNO-LiPA had correctly identified only 6. These results collectively indicate that AGSCP assay is a convenient and sensitive method for large-scale genotyping, and it may be a promising tool for the determination of HCV and other genotypes in clinical settings.     

Expression of collier in the premandibular segment of myriapods: support for the traditional Atelocerata concept or a case of convergence?

Background  

A recent study on expression and function of the ortholog of the Drosophila collier (col) gene in various arthropods including insects, crustaceans and chelicerates suggested a de novo function of col in the development of the appendage-less intercalary segment of insects. However, this assumption was made on the background of the now widely-accepted Pancrustacea hypothesis that hexapods represent an in-group of the crustaceans. It was therefore assumed that the expression of col in myriapods would reflect the ancestral state like in crustaceans and chelicerates, i.e. absence from the premandibular/intercalary segment and hence no function in its formation.     

Baseline predictors of response and discontinuation of tumor necrosis factor-alpha blocking therapy in ankylosing spondylitis: a prospective longitudinal observational cohort study

Introduction  

Identifying ankylosing spondylitis (AS) patients who are likely to benefit from tumor necrosis factor-alpha (TNF-α) blocking therapy is important, especially in view of the costs and potential side effects of these agents. Recently, the AS Disease Activity Score (ASDAS) has been developed to assess both subjective and objective aspects of AS disease activity. However, data about the predictive value of the ASDAS with respect to clinical response to TNF-α blocking therapy are lacking. The aim of the present study was to identify baseline predictors of response and discontinuation of TNF-α blocking therapy in AS patients in daily clinical practice.     

Biogenic Formation of As-S Nanotubes by Diverse Shewanella Strains

Shewanella sp. strain HN-41 was previously shown to produce novel, photoactive, As-S nanotubes via the reduction of As(V) and S₂O₃²⁻ under anaerobic conditions. To determine if this ability was unique to this bacterium, 10 different Shewanella strains, including Shewanella sp. strain HN-41, Shewanella sp. strain PV-4, Shewanella alga BrY, Shewanella amazonensis SB2B, Shewanella denitrificans OS217, Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, S. putrefaciens IR-1, S. putrefaciens SP200, and S. putrefaciens W3-6-1, were examined for production of As-S nanotubes under standardized conditions. Of the 10 strains examined, three formed As-S nanotubes like those of strain HN-41. While Shewanella sp. strain HN-41 and S. putrefaciens CN-32 rapidly formed As-S precipitates in 7 days, strains S. alga BrY and S. oneidensis MR-1 reduced As(V) at a much lower rate and formed yellow As-S after 30 days. Electron microscopy, energy-dispersive X-ray spectroscopy, and extended X-ray absorption fine-structure spectroscopy analyses showed that the morphological and chemical properties of As-S formed by strains S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1 were similar to those previously determined for Shewanella sp. strain HN-41 As-S nanotubes. These studies indicated that the formation of As-S nanotubes is widespread among Shewanella strains and is closely related to bacterial growth and the reduction rate of As(V) and thiosulfate.A number of bacterial strains have been shown to contribute to the formation of diverse arsenic minerals (4). If sulfide is present as a ligand for immobilization of arsenic, As-S precipitates often form. Desulfosporosinus auripigmentum, which can be isolated from lake sediments, reduces As(V) to As(III) and S(VI) to S(−II) during anaerobic respiration and forms a yellow arsenic sulfide precipitate (7). While Desulfovibrio strain Ben-RB also produces precipitated arsenic sulfide in culture media, As reduction was not correlated with energy conservation (6). Other taxonomically divergent microorganisms isolated from various arsenic-rich sites have also been shown to reduce As(V) to As(III) and form arsenic sulfide precipitates (1, 2).We previously reported that Shewanella sp. strain HN-41 produces an extensive extracellular network of filamentous arsenic-sulfide (As-S) nanotubes via its dissimilatory metal-reducing activity (4). The As-S nanotubes, which formed via the reduction of As(V) and S₂O₃²⁻, were initially amorphous As₂S₃ but evolved with increasing incubation time toward polycrystalline phases of the chalcogenide minerals realgar (AsS) and duranusite (As₄S). Because the Shewanella As-S nanotubes behaved both as metals and as semiconductors, in terms of their electrical and photoconductive properties, respectively, it was postulated that they may provide useful materials for novel nano- and optoelectronic devices (4).While several bacterial species have been shown to produce amorphous and particulate As-S precipitates (1, 2, 4, 7), the formation of the As-S nanotubes by other bacteria has not yet been described, suggesting that this may be a unique property of Shewanella strains. To test this hypothesis, 10 different Shewanella strains, including Shewanella sp. strains PV-4 and HN-41, Shewanella alga BrY, Shewanella amazonensis SB2B, Shewanella denitrificans OS217, Shewanella oneidensis MR-1, Shewanella putrefaciens CN-32, S. putrefaciens IR-1, S. putrefaciens SP200, and S. putrefaciens W3-6-1, were inoculated into HEPES-buffered basal medium (3, 5) containing 10 mM sodium dl-lactate as the electron donor and 5 mM arsenate (Na₂HAsO₄·7H₂O) and 5 mM thiosulfate (Na₂S₂O₃·5H₂O) as the electron acceptors. All chemicals and methods for sample preparation and characterization used in this study were previously described (4).Of the 10 different Shewanella strains examined, only four strains, Shewanella sp. strain HN-41, S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1, produced As-S yellow precipitates in culture medium following incubation in the presence of arsenate and thiosulfate. Shewanella sp. strain HN-41 and S. putrefaciens CN-32 produced yellow precipitates of As-S after 7 days of incubation, whereas S. alga BrY and S. oneidensis MR-1 produced only a small amount of visible precipitate after 30 days of incubation. The remainder of the tested Shewanella strains failed to produce yellow precipitates, regardless of incubation time.The culture medium of the strains tested was periodically sampled during the bacterial incubation period to determine the concentrations of lactate, acetate, arsenic, and sulfide in the aqueous solution. Among the 10 strains examined, Shewanella strain HN-41, S. putrefaciens CN-32, S. alga BrY, and S. oneidensis MR-1 metabolized lactate in growth medium containing arsenate and thiosulfate (Table ​(Table1).1). Shewanella sp. strain HN-41 and S. putrefaciens CN-32 rapidly consumed lactate both as an electron donor and as a carbon source (see Fig. S1 in the supplemental material). Cultures of S. alga BrY and S. oneidensis MR-1 consumed ∼1.4 mM lactate after 7 days, while Shewanella sp. strain HN-41 and S. putrefaciens CN-32 consumed 1.7 mM and 2.3 mM lactate, respectively. Although S. putrefaciens CN-32 reduced As(V) in the culture medium supplemented with 5 mM As(V) as the sole electron acceptor, Shewanella sp. strain HN-41, S. alga BrY, and S. oneidensis MR-1 did not reduce As(V) and did not oxidize lactate to acetate (data not shown). Consequently, the latter three strains could not utilize As(V) as an electron acceptor for respiratory metabolism.

TABLE 1.

Influence of thiosulfate on the consumption of lactate, reduction of As(V), and formation of As-S nanotubes by Shewanella strains in medium containing lactate and 5 mM As(V)

Fine mapping of Grh1, a major gene associated with antibiosis to green rice leafhopper in rice

Green rice leafhopper (GRH, Nephotettix cincticeps Uhler) is one of the insect pests that damage cultivated rice in East Asia. GRH also transmits viruses such as rice dwarf virus. The mortality of GRH nymphs is high in rice cultivar Shingwang, indicating that Shingwang is resistant to GRH. Genetic analyses were performed to map GRH resistance in Shingwang using F2 and F3 populations derived from a cross between a GRH-resistant near-isogenic line (NIL-IS60) from Shingwang and recurrent parent Ilpum. Resistance to GRH in Shingwang was found to be controlled by a single dominant gene (Grh1) mapped within an approximately 670-kb region between 8.10 and 8.77 Mb on the short arm of chromosome 5. Genotypes with three simple sequence repeat markers (RM18166, RM516, and RM18171) and one indel marker (Indel 15040) co-segregated with GRH resistance controlled by the Grh1 locus. A detailed map of the Grh1 locus will facilitate marker-assisted selection of resistance to GRH in rice breeding.     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.