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Background: Hepatitis B core antibody (anti‐HBc) screening has been implemented in many blood establishments to help prevent transmission of hepatitis B virus (HBV), including from donors with occult HBV infection (OBI). We review HBV screening algorithms across blood establishments globally and their potential effectiveness in reducing transmission risk. Materials and Methods: A questionnaire on HBV screening and follow‐up strategies was distributed to members of the International Society of Blood Transfusion working party on transfusion‐transmitted infectious diseases. Screening data from 2022 were assimilated and analyzed. Results: A total of 30 unique responses were received from 25 countries. Sixteen respondents screened all donations for anti‐HBc, with 14 also screening all donations for HBV DNA. Anti‐HBc prevalence was 0.42% in all blood donors and 1.19% in new donors in low‐endemic countries; however, only 44% of respondents performed additional anti‐HBc testing to exclude false reactivity. 0.68% of anti‐HBc positive, HBsAg‐negative donors had detectable HBV DNA. Ten respondents did universal HBV DNA screening without anti‐HBc, whereas four respondents did not screen for either. Deferral strategies for anti‐HBc positive donors were highly variable. One transfusion‐transmission from an anti‐HBc negative donor was reported. Discussion: Anti‐HBc screening identifies donors with OBI but also results in the unnecessary deferral of a significant number of donors with resolved HBV infection and donors with false‐reactive anti‐HBc results. Whilst confirmation of anti‐HBc results could be improved to reduce donor deferral, transmission risks associated with anti‐HBc negative OBI donors must be considered. In high‐endemic areas, highly sensitive HBV DNA testing is required to identify infectious donors. [ABSTRACT FROM AUTHOR]

Background: The large dengue (DENV) and chikungunya (CHIKV) outbreaks observed during the last decade across the world, as well as local transmissions in non‐endemic areas are a growing concern for blood safety. The aim of this study was to evaluate and compare the sensitivity of nucleic acid tests (NAT) detecting DENV and CHIKV RNA. Materials and Methods: Using DENV 1 to 4 International Standards, the limits of detection (LODs) calculated by probit analysis of two NAT assays; the cobas CHIKV/DENV assay (Roche Diagnostics) and the Procleix Dengue Virus Assay (Grifols) were compared. In addition, CHIKV‐RNA LOD of the cobas CHIKV/DENV assay was evaluated. Results: For dengue, the 95% LOD of the cobas assay ranged between 4.10 [CI95%: 2.70–8.19] IU/mL (DENV‐2) and 7.07 [CI95%: 4.34–14.89] IU/mL (DENV‐4), and between 2.19 [CI95%: 1.53–3.83] IU/mL (DENV‐3) and 5.84 [CI95%: 3.84–10.77] IU/mL (DENV‐1) for Procleix assay. The Procleix assay had a significant lower LOD for DENV‐3 (2.19 vs. 5.89 IU/mL) when compared to the cobas assay (p = 0.005). The 95% LOD for CHIKV‐RNA detection of the cobas assay was 4.76 [CI95%: 3.08–8.94] IU/mL. Discussion: The two NAT assays developed for blood donor screening evaluated in this study demonstrated high and similar analytical performance. Subject to an appropriate risk‐benefit assessment, they can be used to support blood safety during outbreaks in endemic areas or in non‐endemic areas as an alternative to deferring blood donors during local transmission likely to affect the blood supply. The development of multiplex assays is expected to optimize laboratory organization. [ABSTRACT FROM AUTHOR]

Gastric cancer (GC) is one of the most heterogeneous tumors. However, research on normal tissue adjacent to the tumor (NAT) is very limited. We performed multi‐regional omics sequencing on 150 samples to assess the genetic basis and immune microenvironment in NAT and matched primary tumor or lymph node metastases. NATs demonstrated different mutated genes compared with GC, and NAT genomes underwent independent evolution with low variant allele frequency. Mutation profiles were predominated by aging and smoking‐associated signatures in NAT instead of signatures associated with genetic instability. Although the immune microenvironment within NATs shows substantial intra‐patient heterogeneity, the proportion of shared TCR clones among NATs is five times higher than that of tumor regions. These findings support the notion that subclonal expansion is not pronounced in NATs. We also demonstrated remarkable intra‐patient heterogeneity of GCs and revealed heterogeneity of focal amplification of CD274 (encoding PD‐L1) that leads to differential expression. Finally, we identified that monoclonal seeding is predominant in GC, which is followed by metastasis‐to‐metastasis dissemination in individual lymph nodes. These results provide novel insights into GC carcinogenesis. © 2024 The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Background and Objectives: Nucleic acid‐amplification testing (NAT) is used for screening blood donations/donors for blood‐borne viruses. We reviewed global viral NAT characteristics and NAT‐yield confirmatory testing used by blood operators. Materials and Methods: NAT characteristics and NAT‐yield confirmatory testing used during 2019 was surveyed internationally by the International Society of Blood Transfusion Working Party Transfusion‐Transmitted Infectious Diseases. Reported characteristics are presented herein. Results: NAT was mainly performed under government mandate. Human immunodeficiency virus (HIV), hepatitis C virus (HCV) and hepatitis B virus (HBV) NAT was performed on all donors and donation types, while selective testing was reported for West Nile virus, hepatitis E virus (HEV), and Zika virus. Individual donation NAT was used for HIV, HCV and HBV by ~50% of responders, while HEV was screened in mini‐pools by 83% of responders performing HEV NAT. Confirmatory testing for NAT‐yield samples was generally performed by NAT on a sample from the same donation or by NAT and serology on samples from the same donation and a follow‐up sample. Conclusion: In the last decade, there has been a trend towards use of smaller pool sizes or individual donation NAT. We captured characteristics of NAT internationally in 2019 and provide insights into confirmatory testing approaches used for NAT‐yields, potentially benefitting blood operators seeking to implement NAT. [ABSTRACT FROM AUTHOR]

Background and Objectives: Nucleic acid amplification testing (NAT), in blood services context, is used for the detection of viral and parasite nucleic acids to reduce transfusion‐transmitted infections. This project reviewed NAT for screening blood donations globally. Materials and Methods: A survey on NAT usage, developed by the International Society of Blood Transfusion Working Party on Transfusion‐transmitted Infectious Diseases (ISBT WP‐TTID), was distributed through ISBT WP‐TTID members. Data were analysed using descriptive statistics. Results: Forty‐three responses were received from 32 countries. Increased adoption of blood donation viral screening by NAT was observed over the past decade. NAT‐positive donations were detected for all viruses tested in 2019 (proportion of donations positive by NAT were 0.0099% for human immunodeficiency virus [HIV], 0.0063% for hepatitis C virus [HCV], 0.0247% for hepatitis B virus [HBV], 0.0323% for hepatitis E virus [HEV], 0.0014% for West Nile virus [WNV] and 0.00005% for Zika virus [ZIKV]). Globally, over 3100 NAT‐positive donations were identified as NAT yield or solely by NAT in 2019 and over 22,000 since the introduction of NAT, with HBV accounting for over half. NAT‐positivity rate was higher in first‐time donors for all viruses tested except WNV. During 2019, a small number of participants performed NAT for parasites (Trypanosoma cruzi, Babesia spp., Plasmodium spp.). Conclusion: This survey captures current use of blood donation NAT globally. There has been increased NAT usage over the last decade. It is clear that NAT contributes to improving blood transfusion safety globally; however, there is a need to overcome economic barriers for regions/countries not performing NAT. [ABSTRACT FROM AUTHOR]

Background: Hepatitis B virus (HBV)‐positive individuals with isolated anti‐HBs are found among HBV vaccine recipients and healthy blood donors with no vaccination history. HBV infectivity from blood transfusions derived from such individuals remains unclear. Case Presentation: A male patient who received transfusion with blood negative for individual donation‐NAT, HBsAg and anti‐HBc but weakly positive for anti‐HBs developed typical transfusion‐transmitted (TT)‐HBV with anti‐HBc response. The responsible blood donor was a frequent repeat donor showing a marked increase in anti‐HBs titer without anti‐HBc response 84 days after index donation. Test results for his past donations showed transient viremia with very low viral load and fluctuating low‐level anti‐HBs. The HBV vaccination history of this donor was unknown. Discussion: Anti‐HBs and anti‐HBc kinetics of the donor suggest a second antibody response to new HBV challenge, representing a vaccine breakthrough case. On the other hand, transient low‐level viremia and fluctuating anti‐HBs in the test results of past donations suggested chronic occult HBV infection with isolated anti‐HBs. Conclusion: Whatever the basic infection state, blood donors with isolated weak anti‐HBs may include a small population with a risk of causing TT‐HBV. Identifying individuals harboring such TT‐HBV risk among individuals positive only for anti‐HBs is difficult under current screening strategies. Active surveillance for the occurrence of TT‐HBV with blood positive only for anti‐HBs is necessary. [ABSTRACT FROM AUTHOR]

Fusarium endophytes damage cereal crops and contaminate produce with mycotoxins. Those fungi overcome the main chemical defence of host via detoxification by a malonyl‐CoA‐dependent enzyme homologous to xenobiotic metabolizing arylamine N‐acetyltransferase (NAT). In Fusarium verticillioides (teleomorph Gibberella moniliformis, GIBMO), this N‐malonyltransferase activity is attributed to (GIBMO)NAT1, and the fungus has two additional isoenzymes, (GIBMO)NAT3 (N‐acetyltransferase) and (GIBMO)NAT2 (unknown function). We present the crystallographic structure of (GIBMO)NAT1, also modelling other fungal NAT homologues. Monomeric (GIBMO)NAT1 is distinctive, with access to the catalytic core through two "tunnel‐like" entries separated by a "bridge‐like" helix. In the quaternary arrangement, (GIBMO)NAT1 monomers interact in pairs along an extensive interface whereby one entry of each monomer is covered by the N‐terminus of the other monomer. Although monomeric (GIBMO)NAT1 apparently accommodates acetyl‐CoA better than malonyl‐CoA, dimerization changes the active site to allow malonyl‐CoA to reach the catalytic triad (Cys110, His158 and Asp173) via the single uncovered entry, and anchor its terminal carboxyl‐group via hydrogen bonds to Arg109, Asn157 and Thr261. Lacking a terminal carboxyl‐group, acetyl‐CoA cannot form such stabilizing interactions, while longer acyl‐CoAs enter the active site but cannot reach catalytic Cys. Other NAT isoenzymes lack such structural features, with (GIBMO)NAT3 resembling bacterial NATs and (GIBMO)NAT2 adopting a structure intermediate between (GIBMO)NAT1 and (GIBMO)NAT3. Biochemical assays confirmed differential donor substrate preference of (GIBMO)NAT isoenzymes, with phylogenetic analysis demonstrating evolutionary separation. Given the role of (GIBMO)NAT1 in enhancing Fusarium pathogenicity, unravelling the structure and function of this enzyme may benefit research into more targeted strategies for pathogen control. [ABSTRACT FROM AUTHOR]

Erythema infectiosum, caused by human parvovirus B19 (B19V), is difficult to diagnose by its clinical symptoms and is often misdiagnosed as measles or rubella. Timely confirmation of measles/rubella or other viral etiologies via laboratory tests can provide an accurate picture of the infection status, which can appropriate response. The purpose of this study was to determine the contribution of B19V as an etiological agent for fever‐rash in suspected cases of measles and rubella in Osaka Prefecture between 2011 and 2021. Of 1356 suspected cases, 167 were confirmed with measles and 166 with rubella using nucleic acid testing (NAT). Of the remaining 1023 cases, 970 from which blood specimens could be obtained were screened by real‐time polymerase chain reaction for B19V, from which 136 (14%) tested positive. Of the positives cases, 21% were young children (9 years and younger), while 64% were adults (20 years and older). Phylogenetic tree analysis showed that 93 samples belonged to genotype 1a. The importance of B19V in the etiology of fever‐rash illness was revealed in this study. The importance of laboratory diagnosis by NAT in maintaining the status of measles elimination and to eliminate rubella was reaffirmed. [ABSTRACT FROM AUTHOR]

Background: Estimation of residual risk (RR) is necessary to understand status of blood safety with current testing strategies and need to improve it further. These estimates from India are lacking till date. Present study was aimed to estimate RR and incident rate (IR) of transfusion transmitted infectious disease (TTIDs) in Indian setting. Study Design and Methods: Blood donor demographic data, serological testing results and nucleic acid testing (NAT) screening results were collected retrospectively from January 2015 to June 2017. Mathematical modelling using NAT yield/window period model was done to estimate IR and RR of TTIDs. Data was compared between different groups based on age‐group, gender, donation repeatability and type of donor. Results: A total 106 119 blood donors donated 109 441 units with overall TTID reactivity of 2.22%. HBV was most prevalent TTID (1.26%) with RR of 16.1 per million followed by HCV and HIV with RR of 4.4 and 3.1 per million donations, respectively. NAT testing variably reduced RR ranging from 69.4% to 96.1% depending on TTID. Younger, repeat and voluntary donors had significantly lower prevalence of TTIDs compared to older, first time and replacement donors. Discussion: Gaps in the blood safety could be bridged significantly by implementing NAT testing and using quality serological assays. Comparatively high RR despite using quality serological assays and ID‐NAT testing highlights need to develop long term strategies to improve blood safety by focusing on improving donor pool by recruiting regular voluntary donors among youth and imparting knowledge of healthy practices. [ABSTRACT FROM AUTHOR]

Background and Objectives: The risk of a blood donation contaminated with hepatitis E virus (HEV) entering the blood supply before introducing universal HEV‐RNA screening in France was estimated to assess the benefit of such a measure. Materials and Methods: The results of selective HEV nucleic acid testing (HEV‐NAT) performed in mini pool of six plasma donations between 2018 and 2020 were extrapolated to the whole blood donor (BD) population after adjustment on three variables: regional establishment, sex and age group. Results: Among the 246,285 plasma donations collected from 172,635 BDs tested for HEV‐RNA, 248 (10.1/10,000) were positive. The extrapolation to all BDs led to an estimated rate of 5.9/10,000 donations (95% confidence interval [CI]: 4.5–7.4) which would be positive to HEV‐RNA and a prevalence of 9.9/10,000 BDs (95% CI: 7.5–12.3). This prevalence was 4.4 times higher in males than females (16.8/10,000 vs. 3.8/10,000, p < 10−4). The highest prevalence was observed in males in the 30–39 age group (20.5/10,000) and the lowest in females in the 50–70 age group (2.8/10,000). Conclusion: The risk of an HEV‐RNA‐positive donation entering the blood supply was estimated at 1 in 1682 donations. This risk does not translate directly to the risk of HEV transfusion transmission, which mainly depends on the total number of viral particles in the transfused blood component and the sensitivity of NAT. [ABSTRACT FROM AUTHOR]

The tropospheric and stratospheric nitrate aerosol is simulated by a sectional aerosol model coupled to the Community Earth System Model. The simulated nitrate mass fractional contribution to aerosols is significantly higher in the upper troposphere and lower stratosphere (UTLS) than that at the surface. Both in situ measurements and simulations show that nitrate aerosol accounts for about 30%–40% of the aerosol mass at the tropopause of the Asian summer monsoon (ASM) region. Furthermore, simulated condensed nitric acid particles account for ∼20% of the annual mean aerosol mass at the tropical tropopause, and over 95% in the UTLS at the South Pole in June‐July‐August. Our study suggests that the extremely cold ambient conditions in the UTLS of the tropics, ASM and polar regions thermodynamically favor the condensation of ammonia and nitric acid. The widely distributed nitrate aerosol in the global UTLS may be overlooked by climate models. Plain Language Summary: The tropospheric and stratospheric nitrate aerosol is simulated by a sectional aerosol model coupled to the Community Earth System Model. Simulated nitrate aerosol mass concentrations are evaluated with observations at the surface and the upper troposphere and lower stratosphere (UTLS). At the global surface, simulated nitrate aerosols account for 6% of non‐dust and non‐sea salt aerosol mass. Significantly higher mass fractions of nitrate are simulated in the UTLS. Our study shows that nitrate accounts for about 30%–40% of the aerosol mass in the Asian tropopause aerosol layer. Furthermore, the model simulations suggested that condensed HNO3 particles contribute to about 20% of the annual mean aerosol burden in the tropical tropopause layer and over 95% in the UTLS of the South Pole in June‐July‐August. We find that the cold conditions in the global UTLS thermodynamically favors the condensation of ammonia and nitric acid. The widely distributed nitrate aerosol in the global UTLS may be overlooked by climate models. Key Points: Nitrate and nitric acid aerosol contribute significantly to the tropical upper troposphere and lower stratosphere (UTLS), Asian summer monsoon and polar regionsThe cold ambient conditions in the UTLS thermodynamically favor the condensation of nitric acidThe simulated aerosol in the global UTLS is primarily composed of the secondarily formed nitrate, sulfate and organics [ABSTRACT FROM AUTHOR]

Abstract: Kidney transplant from donors with hepatitis C virus (HCV) antibody has been limited to HCV viremic recipients only, due to concern of the HCV transmission. However, the new antiviral medications provide an opportunity to expand the utilization of these donors. To study the risk of HCV transmission in kidney transplantation, we used discarded donor kidneys and determined HCV RNA levels by quantitative real‐time PCR in bilateral (right and left) kidney biopsies and plasma from 14 HCV antibody‐positive donors (sensitivity: 15 international unit (IU)/mL plasma; 1.8 IU/50 nL kidney). In three NAT‐negative donors, HCV RNA was negative in plasma and kidney. In all 11 NAT‐positive donors, HCV RNA was positive in plasma (range: 5807‐19 134 177 IU/mL) but negative in six kidneys from four donors with plasma HCV RNA <1.5 million IU/μL. HCV RNA correlated between right and left kidneys (P = 0.75) and between kidney and plasma (r = 0.86). When normalized by volume, HCV RNA median (range) was 49 (0‐957) IU/50 nL plasma and 1.0 (0‐103) IU/50 nL kidney, significantly lower in kidney (P = 0.005) than in plasma (14‐fold). Plasma HCV RNA can be used to predict the kidney HCV load. Future studies are needed if plasma/kidney HCV levels can be used to stratify donors for transmission risk and recipients for post‐transplant management in extended utilization of HCV antibody‐positive donors. [ABSTRACT FROM AUTHOR]

Reports of arboviral transmission via blood transfusion may be a cause of concern among asymptomatic infected donors. This study evaluated the presence of arboviruses in donated blood products during the 2016 outbreak in Vitória da Conquista (Bahia‐Brazil). Serum samples (n = 676) were screened for ZIKV, CHIKV, and the four DENV serotypes using a one‐step reverse transcriptase‐based Real‐Time Polymerase Chain Reaction (RT‐PCR). No samples tested positive for any of the targets, whereas positive controls performed as expected. The results suggest a low risk of arboviral transmission via transfusion of blood products in the endemic area studied during the 2016 outbreak. [ABSTRACT FROM AUTHOR]

Abstract: Background: Evaluation of assay performance on postmortem blood specimens (obtained after cessation of the heartbeat) presents unique scientific and regulatory challenges. In the United States, assay performance is evaluated in part by spiking postmortem specimens. Methods: Fifty‐four specimens obtained from decedents known to be infected with human immunodeficiency virus (HIV), hepatitis C virus (HCV), or hepatitis B virus (HBV), including some coinfections, were tested for each virus using Food and Drug Administration (FDA)‐licensed donor screening tests for nucleic acid, antibody, and antigen. Results: For each disease, >95% of subjects who were reported to have an infection at the time of death had a positive test result on at least one of the donor screening assays for that infection. Conclusion: Licensed donor screening tests were positive on postmortem specimens obtained within 24 hours of death from individuals dying with HIV, HCV, and/or HBV, and were able to detect presence of the virus. The use of multiple tests (including antibody and direct viral detection methods) is necessary to adequately evaluate donors. [ABSTRACT FROM AUTHOR]

Amino-terminal acetylation is a ubiquitous protein modification affecting the majority of eukaryote proteins to regulate stability and function. We describe an optimised recombinant expression system for rapid production of amino terminal-acetylated proteins within bacteria. We go on to describe the system's use in a fluorescence based in vivo assay for use in the high-throughput screen to identify drugs that impact amino-terminal acetylation-dependent oligomerisation. These new tools and protocols will allow researchers to enhance routine recombinant protein production and identify new molecules for use in research and clinical applications. [ABSTRACT FROM AUTHOR]

Background and Objectives The safety of the blood supply in a number of countries is achieved by interventions that include behaviour-based time-limited or indefinite deferrals and screening of donated units for transfusion-transmitted infections. The relatively high sensitivity of nucleic acid testing ( NAT) used in blood donor screening has raised the question of whether such time-based deferrals can be eliminated in favour of individual risk assessment. Materials and Methods Data on the annual number of incident human immunodeficiency virus ( HIV) infections associated with various behaviours and on the performance characteristics of NAT applied to donor screening were used to model the number of potentially infected units that might escape detection in the worst-case scenario in which individual risk assessment was implemented, but was not effective as a screening tool, and donors did not otherwise self-select for lower risk. Results In the absence of effective individual risk-based screening or donor self-selection, the model predicts that in the United States, an additional 39 (95% CI 35-43) HIV-infected units would escape detection by nucleic acid testing, potentially capable of exposing approximately 68 (95% CI 61-75) individuals to the risk of HIV infection through the administration of prepared blood components. Conclusion Despite some inherent uncertainty, the worst-case scenario of completely ineffective individual risk assessment, absence of donor self-selection and increased reliance on NAT for blood screening is estimated to be associated with an approximately fourfold increase in the risk of HIV exposure through transfusion in the United States. [ABSTRACT FROM AUTHOR]

Background Platelet concentrates ( PCs) are the main focus regarding the residual risk of transfusion-transmitted bacterial infections. Rapid screening methods for bacterial detection in platelets have been optimized over the last decade, but their external evaluation represents a complicated process. We developed a new type of proficiency panel for bacterial detection in PCs using currently available screening methods (especially rapid methods) suitable for external quality assessment programmes ( EQAP). Methods PC samples were inoculated with different bacteria at two concentrations (10E+03 CFU/ml, 10E+05 CFU/ml) and stored under temperature-controlled conditions (1-5 days). Bacterial growth was further prevented by the addition of 0-20 μg/ml cotrimoxazole. Samples were analysed prior to and after storage using rapid detection methods (Bactiflow ( BF), bacteria-generic NAT) and cultural methods to determine the influence of storage and antibiotic treatment on bacterial counts and the result outcome. A pilot EQAP was performed with four participants. Results Testing under the evaluated conditions demonstrated that bacterial counts remained constant prior to and after storage. The supplementation of 10 μg/ml cotrimoxazole did not influence bacterial detection using the two rapid detection methods BF and NAT. Furthermore, the detection of bacteria using cultural methods is still possible despite of antibiotic supplementation. The pilot EQAP confirmed these results. A storage time of up to 3 days proved practicable, showing no considerable influence on bacterial count and outcome of test results. Conclusion The established proficiency panel provided PC matrix-conform samples with stabilized bacterial counts which can be analysed in parallel by rapid and cultural detection methods. [ABSTRACT FROM AUTHOR]

Introduction Technological advancements in transfusion medicine have led to safer blood supply to the community like never before. However, blood safety is a daunting task in India with ~1.25 billion population and high prevalence rate of HIV (0.29%), HBV (2-8%) and HCV (2%) in donor population. This study aims at evaluating the impact of ID- NAT as an add-on test on safe blood supply in the current Indian scenario. Materials and Method A retrospective observational study was conducted to analyse the data of 5 years of add-on NAT at Main Blood Bank, AIIMS, New Delhi. Results 1.84% (3556 of 193 167) units were initially NAT reactive. A total of 3495 units were further tested, of which 79.25% were discriminated (193 HIV, 421 HCV, 2060 HBV and 96 co-infections). The remaining 13% (456) were repeat non-reactive and 7.7% (269) could not be discriminated. Overall, NAT yield rate was one in 847, whereas virus-specific NAT yield rates were one in 96 584, one in 2683, one in 1370 and one in 14 859 for HIV, HCV, HBV and HBV/ HCV Co-infections, respectively. Conclusion Five-years of ID- NAT screening interdicted 228 probable TTIs to 684 (assuming 100% component separation) transfusion recipients. Implementation of NAT along with routine serological tests for screening of the blood donations definitely improves the transfusion safety. However, interpretation of NAT reactive results should be adjudged cautiously keeping in mind, the chance occurrence of false-positive reactivity. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lnc RNAs) have recently been found to widely exist in eukaryotes and play important roles in key biological processes. To extend our knowledge of lnc RNAs in crop plants we performed both non-directional and strand-specific RNA-sequencing experiments to profile non-coding transcriptomes of various rice and maize organs at different developmental stages. Analysis of more than 3 billion reads identified 22 334 long intergenic non-coding RNAs (linc RNAs) and 6673 pairs of sense and natural antisense transcript ( NAT). Many linc RNA genes were associated with epigenetic marks. Expression of rice linc RNA genes was significantly correlated with that of nearby protein-coding genes. A set of NAT genes also showed expression correlation with their sense genes. More than 200 rice linc RNA genes had homologous non-coding sequences in the maize genome. Much more linc RNA and NAT genes were derived from conserved genomic regions between the two cereals presenting positional conservation. Protein-coding genes flanking or having a sense-antisense relationship to these conserved lnc RNA genes were mainly involved in development and stress responses, suggesting that the associated lnc RNAs might have similar functions. Integrating previous genome-wide association studies ( GWAS), we found that hundreds of linc RNAs contain trait-associated SNPs (single nucleotide polymorphisms [ SNPs]) suggesting their putative contributions to developmental and agriculture traits. [ABSTRACT FROM AUTHOR]

SUMMARY Background and Objectives To illustrate that the combination of a single nucleic acid amplification test (NAT) with a single immunoassay for hepatitis C virus (HCV) detection, as proposed internationally, may lead to the omission of anti-HCV reactive sera with non-reactive NAT results. Materials and Methods In total, 822 of 519, 299 serum samples from 11 blood centres in China were retested for anti-HCV by using 10 screening assays to detect HCV antibodies. A recombinant immunoblot assay (RIBA HCV 3·0; Ortho-Clinical Diagnostics) was performed to define confirmed HCV infection status. Samples with positive or indeterminate RIBA-HCV results were tested by quantitative tests for HCV RNA (Roche Diagnostics). Results We found that 47 of the 822 (5·72%) serum samples were RIBA-positive without detectable HCV RNA. For these samples, the 10 anti-HCV immunoassays gave discordant and unsatisfactory results (detection rate ranging from 10·64 to 34·04%; ratio per 100 000 donations ranging from 5·97 to 8·09). Compared with a single anti-HCV screening assay, the two-assay combination increased the detection of these samples. The five best combinations [Sorin and Lizhu enzyme immunoassays (EIAs), Ortho and Lizhu EIAs, Sorin and Wantai EIAs, Sorin EIA and Roche CIA and Ortho and Wantai EIAs] increased the detection rate from 46·81 to 55·57%, thus reducing the ratio per 100 000 donations of HCV-seropositive samples. Conclusion The combination of two anti-HCV screening immunoassays in parallel with an HCV NAT is a better strategy for HCV detection in blood centres to improve the safety of blood transfusion. [ABSTRACT FROM AUTHOR]

Maternal smoking during pregnancy is one proposed risk factor for gastroschisis, but reported associations have been modest, suggesting that differences in genetic susceptibility might play a role. We included 108 non-Hispanic white and 62 Hispanic families who had infants with gastroschisis, and 1,147 non-Hispanic white and 337 Hispanic families who had liveborn infants with no major structural birth defects (controls) in these analyses. DNA was extracted from buccal cells collected from infants and mothers, and information on periconceptional smoking history was obtained from maternal interviews, as part of the National Birth Defects Prevention Study. We analyzed five polymorphisms in three genes that code for enzymes involved in metabolism of some cigarette smoke constituents ( CYP1A1, CYP1A2, and NAT2). Logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs) independently for maternal smoking and maternal and infant gene variants, and to assess joint associations of maternal smoking and maternal or infant gene variants with gastroschisis. In analyses adjusted for maternal age at delivery and stratified by maternal race-ethnicity, we identified three suggestive associations among 30 potential associations with sufficient numbers to calculate ORs: CYP1A1*2A for non-Hispanic white mothers who smoked periconceptionally (aOR = 0.38, 95% CI 0.15-0.98), and NAT2*6 for Hispanic non-smoking mothers (aOR = 2.17, 95% CI 1.12-4.19) and their infants (aOR = 2.11, 95% CI 1.00-4.48). This analysis does not support the occurrence of effect modification between periconceptional maternal smoking and most of the xenobiotic metabolizing enzyme gene variants assessed. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

ABSTRACT A simple method for accurate determination of nitrite and nitrate in serum was proposed to avoid the variation of nitrate reduction. For nitrite determination, serum samples were directly precipitated with methanol pre-nitrate conversion, and then the supernatant reacted with 2,3-diaminonaphthalene (DAN) to form 2,3-naphthotriazole (NAT), which was quantitatively analyzed by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FL). For nitrate determination, samples were firstly heated at 70°C for 10 min to inactivate endogenous reductase-inhibiting proteins, then nitrate in the samples was quantitatively reduced to nitrite by reductase added experimentally. The difference in total nitrite concentrations between pre- and post-nitrate conversion was used to calculate the amount of nitrate in the samples. In addition to good specificity, high sensitivity, satisfactory accuracy and reproducibility, our method is simple and suitable for the quantitative determination of nanomolar level of nitrite and nitrate in a large number of serum samples. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Arylamine N-acetyltransferase ( NAT) plays an important role in metabolism and detoxification of many compounds including drugs and environmental carcinogens through chemical modification of the amine group with an acetyl group. Recent studies have suggested that NATs are also involved in cancer cell growth and inhibition of the enzymes may be a potential target for cancer chemotherapy. Three-dimensional (3 D) structures are available for NATs from both prokaryotes and eukaryotes. These structures provide valuable insights into the acetylation mechanism, features of the active site and the structural determinants that govern substrate/inhibitor-binding specificity. Such insights allow a more precise understanding of the structure-activity relationships for NAT substrates and inhibitors. Furthermore, the structural elucidation of NATs has generated powerful tools in the design of small molecule inhibitors that should alleviate cancer, based on the important role of the enzyme in cancer biology. [ABSTRACT FROM AUTHOR]

ABSTRACT The Internet is an interconnection of autonomous systems (ASes) that are mostly controlled by Internet service providers (ISPs). ASes use Border Gateway Protocol (BGP) to communicate routing information in the form of reachability paths. However, BGP does not guarantee that the advertised reachability paths will be exactly followed. As a result, traffic belonging to a specific network can be intentionally dropped as it is routed by BGP through a malicious ISP; a behavior we define as Internet access denial. The impact of Internet access denial, especially when performed by higher-tier ISPs, is significant. In this work, network address translation (NAT) is used as a solution to overcome the Internet access denial problem by hiding the traffic identity. The proposed solution is scalable to fit large networks, by using pools of IP addresses across several NAT routers. Moreover, the proposed solution addresses the server reachability problem that is associated with NAT routers by introducing a novel approach. The performance degradation of introducing NAT is significantly small as shown by our experiments' results. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Routine serological testing for hepatitis C virus ( HCV) infection among hemodialysis ( HD) patients is currently recommended. A dilemma existed on the value of serology because some investigators reported a high rate of false-negative serologic testing. In this study, we aimed to detect the false-negative rate of anti- HCV among Egyptian HD patients. Seventy-eight HD patients, negative for anti- HCV, anti- HIV, and hepatitis B surface antigen, were tested for HCV RNA by reverse transcriptase polymerase chain reaction ( RT-PCR). In the next step, the viral load was quantified by real-time PCR in RT-PCR-positive patients. Risk factors for HCV infection, as well as clinical and biochemical indicators of liver disease, were compared between false-negative and true-negative anti- HCV HD patients. The frequency of false-negative anti- HCV was 17.9%. Frequency of blood transfusion, duration of HD, dialysis at multiple centers, and diabetes mellitus were not identified as risk factors for HCV infection. The frequency of false-negative results had a linear relation to the prevalence of HCV infection in the HD units. Timely identification of HCV within dialysis units is needed in order to lower the risk of HCV spread within the HD units. The high false-negative rate of anti- HCV among HD patients in our study justifies testing of a large scale of patients for precious assessment of effectiveness of nucleic acid amplification technology testing in screening HD patient. [ABSTRACT FROM AUTHOR]

J Oral Pathol Med (2012) 41: 292-302 Background: Arecanut and smokeless tobacco usage is a major cause for oral submucous fibrosis (OSF) and its subsequent development to oral squamous cell carcinoma in South-east Asian population. Polymorphisms at N-acetyltransferase 2 locus, coding for an enzyme catalyzing acetylation of aromatic amines, might cause DNA adduct formation because of improper acetylation of these polyaromatic hydrocarbons. DNA repair enzymes remove these adduct to prevent malignancy. Methods: In this hospital-based study, 100 controls and 88 OSF patients were genotyped at four polymorphic sites on NAT2 481 (C > T; silent), 590 (G > A; Arg197 > Gln), 803 (A > G; Lys268 > Arg), 857 (G > A; Gly286 > Glu) and two on XRCC1 18067 (C > T Arg 194 > Trp), 28152 (G > A Arg 399 > Gln), and one of XRCC3 26304 (C > T Thr 241 > Met) loci by PCR-RFLP to determine the risk of the disease. Results: Heterozygous XRCC3 codon 241 [OR 2.07 (1.05-4.06)], homozygous variant of NAT C481T [OR 2.81 (1.09-7.21)], and both heterozygous and homozygous variants of NAT codon 268 and 286 [OR 2.31 (1.20-4.45) and 4.98 (1.87-13.14), and 6.12 (2.75-13.62) and 2.65 (1.04-6.72)] individually influenced susceptibility to OSF in the population. Conclusion: Gene-gene interaction analysis by multifactor dimensionality reduction (MDR) revealed that XRCC3 Thr 241 Met had the largest univariate effect followed by XRCC3 Thr 241 Met - NAT2 A857G in men that presents a highly synergistic interaction as one of the potential combinations of single nucleotide polymorphisms (SNPs) to increase the risk of OSF in men if exposed to arecanut or smokeless tobacco usage. These observations can speculate the impact of the studied SNPs on the etiology of OSF. [ABSTRACT FROM AUTHOR]

A collaborative study was undertaken to evaluate a replacement World Health Organization International Standard for hepatitis C virus (HCV) RNA for nucleic acid amplification technology (NAT)-based assays. The candidate preparations were calibrated in International Units (IUs). Three new candidate preparations were produced from a single bulk containing anti-HCV-negative, genotype 1a HCV RNA-positive plasma. Two samples were lyophilized (coded Sample 2 and Sample 3), whilst a third (Sample 4) contained liquid/frozen material. The samples were distributed together with the 2 International Standard (Sample 1, NIBSC code 96/798) for evaluation by thirty-three laboratories, from fourteen countries. The panel of samples were assayed on four separate occasions. Stability studies were performed for the lyophilized samples by accelerated thermal degradation. Participants returned data from a wide range of commercial and in-house quantitative and qualitative assays. Twenty-five data sets were returned for quantitative assays and fourteen for qualitative assays. Excellent agreement was observed between laboratories and assay methods. The mean relative potencies of Samples 2-4 were 5·19, 5·41 and 5·70 log IU/ml, respectively, when compared against the 2 International Standard. Samples 2 and 3 demonstrated stability of a similar order to the previous standards. Based upon the results of the collaborative study, Sample 2 (code number 06/100) was established as the 3 International Standard for HCV RNA with an assigned unitage of 5·19 log IU/ml. Each vial contains the equivalent of 0·5 ml of material; each vial contains 4·89 log IU of HCV RNA. [ABSTRACT FROM AUTHOR]

C. Alméras, F. Vetromile, V. Garrigue, I. Szwarc, V. Foulongne, G. Mourad. Monthly screening for BK viremia is an effective strategy to prevent BK virus nephropathy in renal transplant recipients. Transpl Infect Dis 2011: 101-108. All rights reserved BK polyomavirus virus (BKV) nephropathy (BKVN) is the most common viral infection that affects renal allografts. Because a specific antiviral therapy is lacking, BKVN may result in graft dysfunction and/or loss. We prospectively analyzed whether monthly nucleic acid testing (NAT) for BKV replication in blood and immediate reduction of immunosuppression (IS) could prevent BKVN. NAT was performed at monthly intervals for 6 months and then at 12 months in 119 de novo renal transplant recipients. In viremic patients (presumptive BKVN), a graft biopsy was systematically performed and IS was immediately reduced. BKV viremia occurred in 13 (10.9%) patients after a median time of 90 days (23-241); 77% of patients were viremic before month 4. After reduction of IS, viral load was undetectable in 11 patients, remained low in 1, and continued to increase in 1 patient who developed definitive BKVN despite reduction of IS, and finally returned to dialysis 6 months after transplantation. BKV infection is an early complication. Monthly NAT in blood during the first 6 months and immediate reduction of IS in viremic patients almost completely prevent definitive BKVN. [ABSTRACT FROM AUTHOR]

Background and objectives: The aim of the study was to replace the 1st World Health Organization International Standard for parvovirus B19 DNA for nucleic acid amplification technique (NAT)-based assays (code 99 ⁄ 800). Two lyophilized preparations (coded 99 ⁄ 800 and 99 ⁄ 802) had been evaluated in the original collaborative study. The present study re-evaluates these two preparations in terms of potency, stability and encapsidation of virus DNA. Materials and methods The 1st International Standard (99 ⁄ 800) and 99 ⁄ 802 were recoded as Samples 1 and 2, respectively. The samples were distributed to six laboratories and assayed on four separate occasions. Accelerated thermal degradation samples of the two preparations were examined after storage at 20 °C for 7 years. Nuclease treatment was used to investigate the encapsidation of virus DNA. Results Data were returned from a total of six different quantitative NAT-based assays. The results of the present study confirm those of the original, with no significant differences found in estimated international units (IU) ⁄ ml for the 1st International Standard (Sample 1 in this study) and the proposed replacement preparation, Sample 2 (99 ⁄ 802). Accelerated thermal degradation studies demonstrate that both samples are very stable, with no loss of potency after storage at 20 °C for 7 years. Both lyophilized preparations contained the majority of B19V DNA encapsidated in virions. Conclusions On the basis of the data presented in this collaborative study, Sample 2 (code number 99 ⁄ 802) was established as the 2nd International Standard for parvovirus B19 DNA for NAT-based assays with a potency of 106 IU ⁄ ml (500 000 IU ⁄ vial). [ABSTRACT FROM AUTHOR]

Arylamine N-acetyltransferases (NATs) play an important role in both the detoxification of arylamine and hydrazine drugs and the activation of arylamine carcinogens. Because the catalytic triad, Cys-His-Asp, of mammalian NATs has been shown to be essential for maintaining protein stability, rendering it impossible to assess alterations of the triad on catalysis, we explored the impact of the highly conserved proximal residue, Tyr190, which forms a direct hydrogen bond interaction with one of the triad residues, Asp122, as well as a potential pi-pi stacking interaction with the active site His107. The replacement of hamster NAT2 Tyr190 by either Phe, Ile or Ala was well tolerated and did not result in significant alterations in the overall fold of the protein. Nevertheless, stopped-flow and steady-state kinetic analysis revealed that Tyr190 was critical for maximizing the acetylation rate of NAT2 and the transacetylation rate of p-aminobenzoic acid when compared with the wild-type. Tyr190 was also shown to play an important role in determining the p Ka of the active site Cys during acetylation, as well as the pH versus the rate profile for transacetylation. We hypothesized that the pH dependence was associated with global changes in the active site structure, which was revealed by the superposition of [1H, 15N] heteronuclear single quantum coherence spectra for the wild-type and Y190A. These results suggest that NAT2 catalytic efficiency is partially governed by the ability of Tyr190 to mediate the collective impact of multiple side chains on the electrostatic potential and local conformation of the active site. [ABSTRACT FROM AUTHOR]

Background and Objectives In Europe, it is a regulatory requirement that parvovirus B19 (B19V) DNA nucleic acid amplification technique-based testing is performed on plasma pools for certain classes of plasma-derived medicinal products. This proficiency testing study set out to examine the ability of public quality control laboratories and plasma fractionation organizations to detect different genotypes of B19V using nucleic acid amplification technique-based assays. Materials and Methods Laboratories were supplied with cloned DNAs representing the main genotypes of B19V. All samples were adjusted to equivalent copy number and were distributed as part of a routine external quality assessment programme investigating the evaluation of B19V containing plasma samples by these laboratories. The plasmid clones were distributed to 25 laboratories, representing 13 quality control laboratories and 12 manufacturers of plasma derivatives. The criteria for acceptable detection of the different genotypes of B19V DNA was based upon the maximum theoretical efficiency for polymerase chain reaction amplification. Proficient laboratories were deemed to be those reporting results within 1 log10 dilution for each of the different virus genotypes. Results Data were returned by 23 of the participating laboratories. Some laboratories returned data for more than one type of assay and in total 27 data sets were analysed. Nine of the participating laboratories were able to successfully detect all the virus genotypes according to the applied criteria, all except one used in-house assays. Conclusions The results of the study highlight that there are still discrepancies in the detection of a broad spectrum of B19V genotypes, with implications for batch release testing. [ABSTRACT FROM AUTHOR]

Background and Objectives In this EQA study a novel approach was used to assess the performance of blood centres and blood product manufacturers in detecting the possible contamination of plasma with HCV, HIV and HBV by NAT. Materials and Methods A panel of 12 samples, three negative and three positive for each virus, was distributed to the EQA participants. The positive samples were prepared, using the respective WHO standards, in order to obtain a viral concentration of about three times the 95% DL of the methods most commonly used by laboratories involved in blood screening by NAT. Participants were requested to test each sample of the panel on different days, possibly by different operators using their routine NAT assay. Results Overall, the participants’ performance was satisfactory. In particular, 49 of the 59 participants (83%) were able to correctly identify all samples. Regarding the remaining 10 laboratories, in three cases a deviation from the laboratory's procedure that could be attributed to an operator's mistake was observed, in two cases a possible cross-contamination occurred while in the remaining five cases the failure to detect the positive samples couldn't be ascribed to any relevant deviation in the laboratory's procedure. Conclusions The novel design of this EQA study allowed participants to verify their day by day activity as the study was carried out in the context of their routine testing. Under these conditions, it was demonstrated that, despite the high level of automation reached by NAT assays, human errors can still occur. [ABSTRACT FROM AUTHOR]

Background and Objectives The aim of this study was to replace the 1st World Health Organization International Standard for hepatitis B virus DNA for nucleic acid amplification technique (NAT)-based assays (code 97/746) with a new International Standard. Two lyophilized preparations freeze dried from the same bulk were evaluated in the original collaborative study (coded 97/746 and 97/750, and termed AA and BB, respectively, in the original study). This present study re-evaluates these two preparations in terms of potency and real-time stability. Materials and Methods The 1st International Standard (97/746) and the second lyophilized preparation (97/750) were coded Samples 1 and 2, respectively, in the present study. The samples were distributed to six laboratories and assayed on four separate occasions. Accelerated thermal degradation samples of the two preparations were examined after long-term storage at 4 °C and 20 °C for more than 51 months. Results Data were returned from a total of nine different NAT-based assays, five in qualitative format and four in quantitative format. The results of this study confirm the results of the original collaborative study, with no significant differences being found in estimated international units (IU)/ml or polymerase chain reaction-detectable units/ml for the 1st International Standard (Sample 1 in this study) and the proposed replacement preparation, Sample 2 (97/750). Real-time and accelerated degradation studies indicate that both samples are very stable. Storage of both preparations at 20 °C for more than 51 months resulted in no detectable degradation. Conclusions On the basis of the data presented in this collaborative study, Sample 2 (code 97/750) was established as the 2nd International Standard for hepatitis B virus DNA for NAT-based assays with a potency of 106 IU/ml (500 000 IU/vial). [ABSTRACT FROM AUTHOR]

We present here our overall experience after 27 months of performance of the Procleix Ultrio [HIV-1, hepatitis C virus (HCV), hepatitis B virus (HBV)] transcription-mediated amplification (TMA) assay. The aim of this report is to assess the impact of nucleic acid testing (NAT) implementation in blood screening of south-western Greek blood donors. We processed 38 264 units of blood as neat samples with the Procleix Ultrio TMA assay (Chiron/GenProbe, Emeryville/San Diego, CA, USA) between 1 January 2005 and 31 March 2007. NAT results were compared to those obtained from routine serology tests and quantitative polymerase chain reaction (PCR) assays. Overall, 52 units of blood tested positive for HBV (1·4‰), 8 for HCV (0·2‰) and none for HIV or multiple infections. The yield of TMA was 0·183‰ for HBV (7/38 264) and 0 for HCV. The TMA HBV-positive donations were tested for HBV DNA by a quantitative PCR assay and were found negative (below the detection limit of the method, 200 copies/mL). Follow-up testing showed that the TMA HBV-positive donations were positive for anti-hepatitis B core antigen immunoglobulin G antibodies. Implementation of the TMA assay in the individual donation configuration increased HBV detection compared to serological screening or a commonly used quantitative PCR assay. Follow-up studies will determine the impact of NAT implementation in HBV transmission in countries with an intermediate HBV incidence. [ABSTRACT FROM AUTHOR]

Melatonin production is not restricted to the pineal gland. Several extrapineal sources of this indole such as retina, Harderian gland, and immune system are well documented. Melatonin of pineal origin is not present in the rat at early stages of development. To assess the potential capacity of local melatonin synthesis by the immature brain and to gain insight into the relationship between melatonin production by the brain (without the pineal gland) and pineal gland during rat development, the melatonin content as well as the expression and activity of the melatonin-synthesizing enzymes, N-acetyltransferase (NAT) and hydroxyindole- O-methyltransferase (HIOMT), were studied at fetal and postnatal stages. Moreover, melatonin-membrane receptor (MT1) expression was also analyzed. Both, the expression and activity of NAT and HIOMT were found in the brain with significant day/night differences in enzymes activities. Additionally, melatonin content was detected in all stages showing day/night differences depending on the stage of development. The brain nocturnal melatonin content was higher than diurnal content on postnatal day 16 and in adult rats which is in accordance with the pineal melatonin synthesis. To investigate the origin of this brain melatonin, pinealectomized rats were used and we found that the developing brain produced its own melatonin. Also, MT1 expression was detected in brain during development. These results demonstrate that, when the pineal is not yet producing melatonin, there is melatonin synthesis by the brain that could be used as protection from free radical damage and/or could exert some actions through MT1 receptors. [ABSTRACT FROM AUTHOR]

The use of IP telephony is spreading rapidly. It was developed chiefly for general home use, and mainly supports calls to the PSTN. In such a service configuration, various problems of realizing telephone service, such as the network address translation (NAT) problem, have generally been solved by the use of a terminal specified by the service provider. On the other hand, the design of origination/destination services between VoIP in enterprise private networks and the PSTN involves various problems, including the solution of the NAT problem, and the requirements differ considerably from those of IP telephony for general home use. This paper presents a method of solution of the technical problems related to interconnection between VoIP and PSTN in enterprise IP networks. The solution is the result of investigations emphasizing the practical aspect and repeated verification experiments, and is applicable to real commercial services. © 2005 Wiley Periodicals, Inc. Electron Comm Jpn Pt 1, 89(4): 42–51, 2006; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/ecja.20197 [ABSTRACT FROM AUTHOR]

Background and Objectives This report describes the evaluation of the COBAS AmpliPrep instrument for fully automated generic nucleic acid extraction in conjunction with hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA, and human immunodeficiency virus (HIV)-1 RNA COBAS AmpliScreen amplification and detection using serial dilutions of the WHO international standards (IS) and the PeliCheck reference panels. Materials and Methods Serial diluted samples of the WHO IS and the PeliCheck reference panels were tested 24 times to determine the HBV DNA, HCV RNA and HIV-1 RNA detection limits by Probit analysis. The existence and extent of cross-contamination were assessed by testing alternating high titre HBV DNA-positive and -negative samples. The specificity of the AmpliPrep–AmpliScreen test for HBV was determined by testing 232 minipools consisting of six donations, all negative for HCV/HIV-1 nucleic acid testing (NAT) and HBsAg. In addition, a HBV genotypes A–G panel was tested. Results The respective 95% detection limits (and 95% CI) on the WHO IS and on the PeliCheck reference panels were 6·7 (4·3–13) IU/ml and 123 (68–301) gEq/ml for HBV DNA, 23 (11–106) IU/ml and 126 (84–233) gEq/ml for HCV RNA, and 187 (108–422) IU/ml and 183 (108–434) gEq/ml for HIV-1 RNA. Based on the WHO IS and the PeliCheck reference panels, no significant differences in sensitivity for HBV and HCV were found between AmpliPrep and the licensed MultiPrep extraction method. The sensitivity of AmpliPrep–AmpliScreen for HIV-1 was probably twofold lower as compared to the MultiPrep–AmpliScreen method. No cross contamination was observed. All 232 minipools were HBV NAT-negative. The AmpliPrep–AmpliScreen test for HBV detected HBV genotypes A–G with equal sensitivity. Conclusions The AmpliPrep instrument combined with the AmpliScreen assays for HBV, HCV and HIV-1 is robust and suitable for NAT donor screening. The sensitivity criteria for HIV-1 and HCV as defined by the Paul Ehrlich Institute and the Food and Drug Administration for minipool NAT screening are met by this system. Single sentence summary Generic COBAS AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen detection for HBV, HCV and HIV-1. [ABSTRACT FROM AUTHOR]

To gain insight into the relationship between thymus and pineal gland during rat development, the melatonin content as well as the activity and expression of the two key enzymes for melatonin biosynthesis, i.e. N-acetyltransferase (NAT) and hydroxyindole-O-methyltransferase (HIOMT), were studied in the thymus at fetal and postnatal stages. Moreover, melatonin-membrane receptor (MT1) expression was also analyzed. We found both the expression and activity of thymic NAT and HIOMT at 18 days of fetal life. Additionally, there is production of melatonin in the thymus as well as MT1 expression at this fetal age. These results show values higher in day-time than at night-time. The pineal gland begins to produce significant levels of melatonin around postnatal day 16, and this synthesis shows a circadian rhythm with high values during the dark period; therefore the nocturnal serum melatonin may inhibit thymic melatonin production. To document this, we report an increased melatonin content of the thymus in pinealectomized rats compared with sham-pinealectomized. In conclusion, these results show, for the first time, the presence of the biosynthetic machinery of melatonin and melatonin production in developing rat thymus and that the pineal gland may regulate this process. [ABSTRACT FROM AUTHOR]

Nucleic acid extraction still harbours the potential for improvements in automation and sensitivity of nucleic acid amplification technology (NAT) testing. This study evaluates the feasibility of a novel automated high-volume extraction protocol for NAT minipool testing in a blood bank setting. The chemagic Viral DNA/RNA Kit special for automated purification of viral nucleic acids from 9·6 ml of plasma by using the chemagic Magnetic Separation Module I was investigated. Analytical sensitivity for hepatitis C virus (HCV), human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV), hepatitis A virus (HAV) and parvovirus B19 (B19) was compared to our present manual procedure that involves virus enrichment by centrifugation. Chemagic technology allows automation of the viral DNA/RNA extraction process. Viral nucleic acids were bound directly to magnetic beads from 9·6-ml minipools. By combining the automated magnetic beads-based extraction technology with our in-house TaqMan polymerase chain reaction (PCR) assays, 95% detection limits were 280 IU/ml for HCV, 4955 IU/ml for HIV-1, 249 IU/ml for HBV, 462 IU/ml for HAV and 460 IU/ml for B19, calculated for an individual donation in a pool of 96 donors. The detection limits of our present method were 460 IU/ml for HCV, 879 IU/ml for HIV-1, 90 IU/ml for HBV, 203 IU/ml for HAV and 314 IU/ml for B19. The 95% detection limits obtained by using the chemagic method were within the regulatory requirements for blood donor screening. The sensitivities detected for HCV, HBV, HAV and B19 were found to be in a range similar to that of the manual purification method. Sensitivity for HIV-1, however, was found to be inferior for the chemagic method in this study. [ABSTRACT FROM AUTHOR]

Sixteen laboratories from 10 different countries participated in an international collaborative study to evaluate candidate materials as the first World Health Organization (WHO) International Standard for hepatitis A virus (HAV) RNA nucleic acid amplification technology (NAT) assays.Five candidate materials were analysed in this study: materials AA and BB were lyophilized, while materials CC, DD and EE were liquid preparations. Samples were diluted in pooled plasma or in pooled cryo-poor plasma (sample EE). Serial dilutions of the candidate materials were tested by each laboratory in four independent assays and the results were analysed statistically.The mean log10‘equivalents’ per ml were 5·29 for sample AA, 5·07 for sample BB, 4·99 for sample CC, 5·40 for sample DD and 4·08 for sample EE.Based on the results of this study, sample AA was established as the first International Standard for HAV RNA NAT assays at the WHO Expert Committee on Biological Standardizaton (ECBS) meeting held in February 2003. The code number of this preparation is 00/560 and the potency, based on the study, is 100 000 International Units (IU)/ml. [ABSTRACT FROM AUTHOR]

This External Quality Assessment (EQA) study was aimed at assessing the proficiency of blood centres and blood product manufacturers in detecting, by nucleic acid amplification technology (NAT), the possible contamination of plasma with hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B virus (HBV).Three independent panels, one for each virus, were prepared at the Istituto Superiore di Sanità (ISS) by diluting the respective reference preparations. NAT methods used by the EQA participants included polymerase chain reaction (PCR) assays by Roche, transcription-mediated amplification (TMA) assays by Chiron and in-house PCR assays.Forty-three of the 45 participants (95·6%) in the HCV EQA/5 who used a validated method were consistently able to detect a nominal concentration of 100 IU/ml for all six major genotypes. In the case of the HIV EQA/1, all 35 participants detected the samples containing 1000 IU/ml HIV, while five (14·3%) did not identify the samples containing 100 IU/ml HIV. With respect to the HBV EQA/1, all 16 participants correctly identified the positive samples containing either 1000 IU/ml or 100 IU/ml HBV. No false-positive results were observed with any of the three panels.The HCV EQA/5 showed an improved proficiency of laboratories as compared with the HCV EQA/4. In fact, HCV genotypes 1, 2, 3 and 5 were correctly identified in 100% of the assays and genotypes 4 and 6 in 97·8% of the assays. While most of the participants in the HIV EQA/1 showed a good level of proficiency, an excellent performance was shown by all participants in the HBV EQA/1. [ABSTRACT FROM AUTHOR]

This study was conducted to analyse the usefulness of hepatitis C virus (HCV) core antigen tests for the confirmation of HCV infection in a donor presenting as nucleic acid amplification technology (NAT) positive but negative for antibodies to HCV (anti-HCV). Blood donations were screened, in parallel, for anti-HCV using the Abbott PRISM HCV Chemiluminescent immunoassay (ChLIA) and an ‘in-house’ HCV NAT (pools of up to 95 donations). An HCV NAT-positive antibody-negative donor was identified. Twelve follow-up samples were obtained and tested with various HCV antigen (including the recently marketed Trak-C second-generation assay) and HCV antibody assays. The single HCV NAT-positive, antibody-negative donation was identified from 1 117 681 donations screened in the 4-year period, July 1999 to June 2003. The index donation was positive by Ortho HCV core antigen enzyme immunoassay (EIA) and Ortho Trak-C (second-generation HCV core antigen EIA). An archive sample, taken 127 days prior to the index donation, was negative for all HCV markers. Subsequent samples demonstrated a loss of reactivity in the Ortho HCV core antigen EIA and reduced activity in the Ortho Trak-C until day 69. Immunoblot (Ortho RIBA-3) and HCV PRISM became positive on day 62, whilst Ortho HCV ELISA was not positive until day 132 or Biorad HCV ELISA until day 160. An alternative immunoblot (Innogenetics Innolia III) was positive from day 55. RNA levels fluctuated considerably during the follow-up period, being completely undetectable by routine screening methods at the time-point around seroconversion; subsequently, antibody was detected using all assays investigated. This HCV-converting blood donor provided a unique panel of samples for using to assess current (and future) HCV assay systems. The overall test results led to the conclusion that individual HCV antigen testing should not be considered as equivalent to HCV NAT minipool screening. Trak-C antigen testing may be considered as a suitable confirmatory assay for isolated HCV NAT reactivity. [ABSTRACT FROM AUTHOR]

Background and Objectives The aim of this study was to compare the performance of two high-throughput strategies for hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1) RNA nucleic acid technology (NAT) screening in a volunteer blood-donor population. Materials and Methods The semiautomated Chiron Procleix HIV-1/HCV transcription mediated amplification (TMA) assay was used to screen 1 439 765 donations in two different testing configurations. Three sites (termed PDT sites) performed a mixture of individual donation (ID) and minipool (MP) testing, where 1 113 288 donations were screened as pools of 24 and an additional 32 003 donations were screened in ID format. A further two sites (termed SDT sites) screened 294 474 donations exclusively in ID format. Results A significantly higher proportion of initial NAT reactives that failed to react on follow-up testing [termed non-repeatably reactive (NRR)] was observed for ID testing at SDT sites than at PDT sites (0·082 vs. 0·047%: P < 0·01). Within the PDT sites, however, there was no significant difference between the NRR rate for MP or ID samples (0·037 vs. 0·047%; not significant). There was a significant difference in failed run rates between PDT and SDT sites (P < 0·01), with PDT sites having a higher run failure rate owing to non-amplification of the internal control. The PDT sites also had a significantly higher overall invalid sample rate. However, the invalid sample rate, specifically caused by known equipment failure, was significantly higher in the SDT sites, possibly attributable to greater usage (P < 0·0001). Four HCV NAT-positive/antibody-negative samples were identified in the course of the study. Conclusions The comparison of the performance of PDT with SDT sites identified only minor differences that did not adversely impact on the timely release of blood products. Although both ID and MP strategies showed excellent specificity, irrespective of site... [ABSTRACT FROM AUTHOR]

Summary A new immunoassay for the detection of hepatitis C core antigen (HCVcoreAg) in peripheral blood during serological window-phase was evaluated among healthy blood donors, commercially available hepatitis C virus (HCV) seroconversion panels and in-house specimens from individuals undergoing seroconversion. Among 1964 low-risk blood donor samples, seven samples were initially reactive but only one was repeat reactive. Reactivity of this specimen was not confirmable by neutralization with specific anti-HCV core antibody, and the sample was negative for HCV RNA by polymerase chain reaction (PCR). The specificity of the HCVcoreAg enzyme-linked immunosorbent assay (ELISA) was 99·95%. In seven commercially available HCV seroconversion panels, HCVcoreAg appeared 23–46 days earlier than anti-HCV antibody by third generation assay. Additional testing with specimens from patients undergoing anti-HCV seroconversion indicated that HCVcoreAg becomes undetectable by the present test format soon after the onset of antibody. This test may be considered as an alternative to nucleic amplification techniques (NAT) for blood donor HCV screening. Additional development of technology for detecting HCVcoreAg may be useful for patient diagnosis and therapy monitoring. [ABSTRACT FROM AUTHOR]

Abstract Hypocretin-1 (HCRT-1) and hypocretin 2 (HCRT-2), also known as orexin-A and orexin-B, are two neuropeptides derived from the same precursor. Hypocretinergic neurons have been found exclusively in the hypothalamic dorsolateral area. These neurons are implicated in sleep and feeding through activation of specific G-protein-coupled orexin-1 and orexin-2 receptor (OR-R1 and OR-R2). The purpose of this study was to determine the existence of the HCRT peptides in the central input of the rat pineal gland. Further, OR-R1 and OR-R2 expression was determined in the pineal gland and the effect of HCRT-2 on melatonin synthesis and secretion was analysed in dissociated rat pinealocytes. A large contingent of HCRT-positive nerve fibres and terminals were observed in the epithalamus, many of which entered into the pineal parenchyma. A significant number of nerve fibres endowed with positive boutons were identified in the pineal stalk, though the number of positive fibres decreased along the extension of the stalk. So far, no positive fibres have been found in the superficial pineal gland. RT-PCR analysis revealed the expression of OR-R2 mRNA, whereas OR-R1-receptor mRNA was not detected. When tested alone, HCRT-2 had no effect on secretion of melatonin from cultured rat pinealocytes. However, HCRT-2 partially inhibited (by a maximum of 30%) the β-adrenergic-induced melatonin secretion. The same effect was seen on activation of N-acetyltransferase activity. The distribution and the large number of HCRT-positive fibres together with the effect on noradrenaline-mediated melatonin release through specific receptors suggests that these peptides may be significant central transmitters in pineal function, probably mediating homeostatic signals to the pineal gland. [ABSTRACT FROM AUTHOR]

Summary Since the introduction of mandatory HCV RNA testing of plasma pools for fractionation by nucleic acid amplification technology, we have organised External Quality Assessment studies (EQAs) addressed to blood products manufacturers and blood centres. Here we report the results of a new EQA, the first one to include all six major HCV genotypes. The results , reported by laboratories worldwide, showed that genotypes 1, 2 and 3 were correctly identified in 100% of the tests, genotype 4 in 96·7% and genotypes 5 and 6 in 98·3% of the assays. As detection of all HCV genotypes is critical for laboratories involved in testing plasma for HCV, all six genotypes should continue to be included in the next EQA studies . [ABSTRACT FROM AUTHOR]