Molecular evolution of respiratory syncytial virus subgroup A genotype NA1 and ON1 attachment glycoprotein (G) gene in central Vietnam

We performed molecular evolutionary analyses of the G gene C-terminal 3rd hypervariable region of RSV-A genotypes NA1 and ON1 strains from the paediatric acute respiratory infection patients in central Vietnam during the 2010–2012 study period. Time-scaled phylogenetic analyses were performed using Bayesian Markov Chain Monte Carlo (MCMC) method, and pairwise distances (p-distances) were calculated. Bayesian Skyline Plot (BSP) was constructed to analyze the time-trend relative genetic diversity of central Vietnam RSV-A strains. We also estimated the N-glycosylation sites within G gene hypervariable region. Amino acid substitutions under positive and negative selection pressure were examined using Conservative Single Likelihood Ancestor Counting (SLAC), Fixed Effects Likelihood (FEL), Internal Fixed Effects Likelihood (IFEL) and Mixed Effects Model for Episodic Diversifying Selection (MEME) models. The majority of central Vietnam ON1 strains detected in 2012 were classified into lineage 1 with few positively selected substitutions. As for the Vietnamese NA1 strains, four lineages were circulating during the study period with a few positive selection sites. Shifting patterns of the predominantly circulating NA1 lineage were observed in each year during the investigation period. Median p-distance of central Vietnam NA1 strains was wider (p-distance = 0.028) than that of ON1 (p-distance = 0.012). The molecular evolutionary rate of central Vietnam ON1 strains was estimated to be 2.55 × 10^− 2 (substitutions/site/year) and was faster than NA1 (7.12 × 10^− 3 (substitutions/site/year)). Interestingly, the evolutionary rates of both genotypes ON1 and NA1 strains from central Vietnam were faster than the global strains respectively. Furthermore, the shifts of N-glycosylation pattern within the G gene 3rd hypervariable region of Vietnamese NA1 strains were observed in each year. BSP analysis indicated the rapid growth of RSV-A effective population size in early 2012. These results suggested that the molecular evolution of RSV-A G gene detected in central Vietnam was fast with unique evolutionary dynamics.     

Molecular evolution of attachment glycoprotein (G) gene in human respiratory syncytial virus detected in Japan 2008–2011

We investigated the evolution of the C-terminal 3rd hypervariable region of G gene in the prevalent human respiratory syncytial virus (RSV) subgroups A (RSV-A) and B (RSV-B) in Japan in 2008–2011. Phylogenetic analysis and the evolutionary timescale was obtained by the Bayesian Markov Chain Monte Carlo method. All 38 RSV-A strains detected were classified into genotype NA1 and the 17 RSV-B strains detected belonged to genotypes BA and GB2. NA1 subdivided around 1998 in the present phylogenetic tree. Genotype BA subdivided around 1994. The evolutionary rates for RSV-A and RSV-B were estimated at 3.63 × 10⁻³ and 4.56 × 10⁻³ substitutions/site/year, respectively. The mean evolutionary rate of RSV-B was significantly faster than that of RSV-A during all seasons. The pairwise distance was relatively short (less than 0.06). In addition, some unique sites under positive selection were found. The results suggested that this region of the RSV strains rapidly evolved with some unique amino acid substitutions due to positive pressure.     

Molecular evolution of hepatitis B vaccine escape variants in China,during 2000–2016

Hepatitis B vaccine escape variants are the main threat to hepatitis B virus (HBV) infection in vaccination era worldwide. With 215 genotype B HBV and 313 genotype C HBV vaccine escape variants isolated from China during 2000–2016, we reported that genotype B HBV vaccine escape strains diverged in ∼1997 (95% HPD; 1987–2005), while genotype C HBV vaccine escape strains diverged in ∼1976 (95% HPD; 1955–2003). Additionally, the p-distance of genotype C HBV vaccine escape strains was 0.0291 ± 0.0169, which was significantly higher than that in the genotype B HBV (t = 131.02, p < 0.05). However, genotype B HBV vaccine escape strains evolved more rapidly than genotype C HBV (2.103 × 10⁻³ vs 1.083 × 10⁻³ substitutions/site/year). Bayesian skyline plot analysis showed that the populations of genotype C HBV vaccine escape strains fluctuated more than those in genotype B HBV. Four sites (A5T/S, L21S, T/A126S and T/N131I/A) and 13 sites (N3S, T5A, G10Q/R/E, L21S, T47K/A/V, L98V/P, I/S126N/V/T, Q129H/R/L, T131P/I/N/A, G145A/R, L175S/F, L213I/S, V224A/G) were found to be under positive selection in genotype B and C HBV vaccine escape strains, respectively. More importantly, N3S, L21S, T47K, L98V, I/S126T and L213I mutations were detected in 1 (2.5%), 1 (2.5%), 1 (2.5%), 3 (7.5%), 1 (2.5%), 1 (2.5%) genotype C HBV infected Chinese younger with neonatal HBV vaccination, respectively. Therefore, our results should be valuable in further understanding the molecular evolution of HBV and providing new ideas for the elimination of HBV infection.     

Molecular evolution of the hypervariable region of the attachment glycoprotein gene in human respiratory syncytial virus subgroup B genotypes BA9 and BA10

We studied the molecular evolution of the C-terminal 3rd hypervariable region in the attachment glycoprotein gene of human respiratory syncytial virus subgroup B (HRSV-B) genotypes BA9 and BA10. We performed time-scaled phylogenetic analyses using Bayesian Markov chain Monte Carlo methods. We also performed a genetic distance analysis (p-distance analysis), positive and negative selection analyses, and a Bayesian skyline plot (BSP) analysis. We found that genotype BA9 diverged from the common ancestor of genotypes BA7, BA8, and BA10, while genotype BA10 diverged from the ancestor of genotypes BA7 and BA8. Strains of both genotypes were distributed worldwide. BA9 and BA10 diverged between 1999 and 2001. Both BA9 and BA10 evolved rapidly (about 4.8 × 10^− 3 substitutions/site/year) and formed three distinct lineages in a 10-year period. BA10 strains belonging to lineage 3 had large genetic distances (p-distance > 0.07). Thus, it may be possible to classify these strains as a new genotype, BA11. No positive selection site was detected in either genotype. Phylodynamic analyses showed that the effective population size of BA10 decreased gradually since 2010 and BA9 slightly decreased since 2009. The results suggested that the recently prevalent HRSV-B genotypes BA9 and BA10 evolved uniquely, leading to epidemics of HRSV-B worldwide over a 15-year period.     

Klebsiella pneumoniae blaKPC-3 nosocomial epidemic: Bayesian and evolutionary analysis

K. pneumoniae isolates carrying blaKPC-3 gene were collected to perform Bayesian phylogenetic and selective pressure analysis and to apply homology modeling to the KPC-3 protein. A dataset of 44 bla_kpc-3 gene sequences from clinical isolates of K. pneumoniae was used for Bayesian phylogenetic, selective pressure analysis and homology modeling. The mean evolutionary rate for bla_kpc-3 gene was 2.67 × 10^− 3 substitution/site/year (95% HPD: 3.4 × 10^− 4–5.59 × 10^–3). The root of the Bayesian tree dated back to the year 2011 (95% HPD: 2007–2012). Two main clades (I and II) were identified. The population dynamics analysis showed an exponential growth from 2011 to 2013 and the reaching of a plateau. The phylogeographic reconstruction showed that the root of the tree had a probable common ancestor in the general surgery ward. Selective pressure analysis revealed twelve positively selected sites. Structural analysis of KPC-3 protein predicted that the amino acid mutations are destabilizing for the protein and could alter the substrate specificity. Phylogenetic analysis and homology modeling of blaKPC-3 gene could represent a useful tool to follow KPC spread in nosocomial setting and to evidence amino acid substitutions altering the substrate specificity.     

Rapid replacement of human respiratory syncytial virus A with the ON1 genotype having 72 nucleotide duplication in G gene

Human respiratory syncytial virus (HRSV) is the main cause of severe respiratory illness in young children and elderly people. We investigated the genetic characteristics of the circulating HRSV subgroup A (HRSV-A) to determine the distribution of genotype ON1, which has a 72-nucleotide duplication in attachment G gene. We obtained 456 HRSV-A positive samples between October 2008 and February 2013, which were subjected to sequence analysis. The first ON1 genotype was discovered in August 2011 and 273 samples were identified as ON1 up to February 2013. The prevalence of the ON1 genotype increased rapidly from 17.4% in 2011–2012 to 94.6% in 2012–2013. The mean evolutionary rate of G protein was calculated as 3.275 × 10⁻³ nucleotide substitution/site/year and several positively selected sites for amino acid substitutions were located in the predicted epitope region. This basic and important information may facilitate a better understanding of HRSV epidemiology and evolution.     

Molecular evolution of emerging Banna virus

Banna virus (BAV) is an emerging pathogen that causes human viral encephalitis and has been isolated from types of blood-sucking insects and mammals in Asia. However, there are no reported systematic studies that describe the origin and evolution of BAV. Here, a phylogenetic analysis of BAVs isolated from a variety of potential vectors and vertebrate hosts worldwide revealed that BAVs emerged in the beginning of the 20th century and do not exhibit a species barrier. The mean substitution rate of BAVs was 2.467 × 10^− 2 substitution/site/year (95% HPD, 1.093 × 10^− 3 to 5.628 × 10^− 2). The lineage is mainly composed of BAVs from high-latitude regions, which are the most recently emerged viruses with significantly higher substitution rates compared with the lineage comprised of the isolates from middle or low-latitude regions. The genetic differences between BAV strains are positively correlated with the geographic distribution. Strains from the same latitude regions are almost 100% identical, whereas the differences between strains from long distance regions with different latitudes could be > 60%. Our results demonstrate that BAV is an emerging virus at a stage that involves rapid evolution and has great potential for introduction into non-endemic areas. Thus, enhanced surveillance of BAV is highly recommended worldwide.     

Assessment of hydroxymethylfurfural and furfural in commercial bakery products

A survey was conducted on the presence of hydroxymethylfurfural (HMF) and furfural in bread and bakery products; for this purpose a reliable extraction procedure followed by high performance liquid chromatography (HPLC) was applied. The performance of the method was evaluated in terms of linearity (r always > 0.99); detection limits (0.001 mg L⁻¹ for furfural and 0.006 mg L⁻¹ for HMF); recovery percentages (98.5–100.5% for HMF and 94.9–98.9% for furfural); intraday precision (<4.65%) and interday precision (<7.51%). Two batches of a wide variety of products commercially available were analysed (a total of 88 samples). HMF and furfural levels presented high variability between products and batches of the same product. Cake/pastry samples showed the lowest HMF content (3.0 mg kg⁻¹ fw) while biscuits showed the highest content (7.8 mg kg⁻¹ fw) (p < 0.05). Regarding furfural, bread samples presented the highest furfural content (5.3 mg kg⁻¹ fw) (p < 0.05), cake/pastry and biscuits showed the lowest content (1.9 and 3.0 mg kg⁻¹ fw, respectively). Chocolate containing samples presented higher amounts of furfural (>20 mg kg⁻¹). These results indicate that special attention should be given to furfural content of bread (due to its daily high consumption) and re-evaluation of dietary exposure.     

Molecular evolution of the fusion protein gene in human respiratory syncytial virus subgroup A

We studied the molecular evolution of the fusion protein (F) gene in the human respiratory syncytial virus subgroup A (HRSV-A). We performed time-scaled phylogenetic analyses using the Bayesian Markov chain Monte Carlo (MCMC) method. We also conducted genetic distance (p-distance), positive/negative selection, and Bayesian skyline plot analyses. Furthermore, we mapped the amino acid substitutions of the protein. The MCMC-constructed tree indicated that the HRSV F gene diverged from the bovine RSV (BRSV) gene approximately 550 years ago and had a relatively low substitution rate (7.59 × 10^− 4 substitutions/site/year). Moreover, a common ancestor of HRSV-A and -B diverged approximately 280 years ago, which has since formed four distinct clusters. The present HRSV-A strains were assigned six genotypes based on F gene sequences and attachment glycoprotein gene sequences. The present strains exhibited high F gene sequence similarity values and low genetic divergence. No positive selection sites were identified; however, 50 negative selection sites were identified. F protein amino acid substitutions at 17 sites were distributed in the F protein. The effective population size of the gene has remained relatively constant, but the population size of the prevalent genotype (GA2) has increased in the last 10 years. These results suggest that the HRSV-A F gene has evolved independently and formed some genotypes.     

Evolutionary analysis of human respiratory syncytial virus collected in Myanmar between 2015 and 2018

We studied genetic variation in the second hypervariable region (HVR) of the G gene of human respiratory syncytial virus (HRSV) from 1701 nasal swab samples collected from outpatients with acute respiratory infections at two general hospitals in the cities Yangon and Pyinmana in Myanmar from 2015 to 2018. HRSV genotypes were characterized using phylogenetic trees constructed using the maximum likelihood method. Time-scale phylogenetic tree analyses were performed using the Bayesian Markov chain Monte Carlo method. In total, 244 (14.3%) samples were HRSV-positive and were classified as HRSV-A (n = 84, 34.4%), HRSV-B (n = 158, 64.8%), and co-detection of HRSV-A/HRSV-B (n = 2, 0.8%). HRSV epidemics occurred seasonally between July (1.9%, 15/785) and August (10.5%, 108/1028), with peak infections in September (35.8%, 149/416) and October (58.2%, 89/153). HRSV infection rate was higher in children ≥1 year of age than in those <1 year of age (70.5% vs. 29.5%). The most common HRSV symptoms in children were cough (80%–90%) and rhinorrhea (70%–100%). The predominant genotypes were ON1for HRSV-A (78%) and BA9 for HRSV-B (64%). Time to the most recent common ancestor was 2014 (95% highest posterior density [HPD], 2012–2015) for HRSV-A ON1 and 2009 (95% HPD, 2004–2012) for HRSV-B BA9. The mean evolutionary rate (substitutions/site/year) for HRSV-B (2.12 × 10⁻², 95% HPD, 8.53 × 10⁻³–3.63 × 10⁻²) was slightly higher than that for HRSV-A (1.39 × 10⁻², 95% HPD, 6.03 × 10⁻³–2.12 × 10⁻²). The estimated effective population size (diversity) for HRSV-A increased from 2015 to 2016 and declined in mid-2018, whereas HRSV-B diversity was constant in 2015 and 2016 and increased in mid-2017. In conclusion, the dominant HRSV-A and HRSV-B genotypes in Myanmar were ON1 and BA9, respectively, between 2015 and 2018. HRSV-B evolved slightly faster than HRSV-A and exhibited unique phylogenetic characteristics.     

Genetic analysis of human rhinovirus species A to C detected in patients with acute respiratory infection in Kumamoto prefecture,Japan 2011–2012

We performed detailed genetic analysis of the VP4/VP2 coding region in human rhinovirus species A to C (HRV-ABC) strains detected in patients with a variety of acute respiratory infections in Kumamoto, Japan in the period 2011–12. The phylogenetic tree and evolutionary timescale were obtained by the Bayesian Markov chain Monte Carlo method. Phylogenetic analyses showed that the present HRV-A, -B, and -C strains belonged to 25, 4, and 18 genotypes, respectively. Some new genotypes were confirmed as prevalent strains of HRV-C. An ancestor of the present HRV-ABCs could be dated back to about 20,000 years ago. The present HRV-A and -C strains have wide genetic divergence (pairwise distance >0.2) with rapid evolutionary rates (around 7 × 10⁻⁴ to 4 × 10⁻³ substitutions/site/year). Over 100 sites were found to be under negative selection, while no positively selected sites were found in the analyzed region. No evidence of recombination events was found in this region of the present strains. Our results indicate that the present HRV strains have rapidly evolved and subsequently diverged over a long period into multiple genotypes.     

Evaluation of the attenuation,immunogenicity, and efficacy of a live virus vaccine generated by codon-pair bias de-optimization of the 2009 pandemic H1N1 influenza virus,in ferrets

Codon-pair bias de-optimization (CPBD) of viruses involves re-writing viral genes using statistically underrepresented codon pairs, without any changes to the amino acid sequence or codon usage. Previously, this technology has been used to attenuate the influenza A/Puerto Rico/8/34 (H1N1) virus. The de-optimized virus was immunogenic and protected inbred mice from challenge. In order to assess whether CPBD could be used to produce a live vaccine against a clinically relevant influenza virus, we generated an influenza A/California/07/2009 pandemic H1N1 (2009 pH1N1) virus with de-optimized HA and NA gene segments (2009 pH1N1-(HA + NA)^Min), and evaluated viral replication and protein expression in MDCK cells, and attenuation, immunogenicity, and efficacy in outbred ferrets. The 2009 pH1N1-(HA + NA)^Min virus grew to a similar titer as the 2009 pH1N1 wild type (wt) virus in MDCK cells (∼10⁶ TCID₅₀/ml), despite reduced HA and NA protein expression on western blot. In ferrets, intranasal inoculation of 2009 pH1N1-(HA + NA)^Min virus at doses ranging from 10³ to 10⁵ TCID₅₀ led to seroconversion in all animals and protection from challenge with the 2009 pH1N1 wt virus 28 days later. The 2009 pH1N1-(HA + NA)^Min virus did not cause clinical illness in ferrets, but replicated to a similar titer as the wt virus in the upper and lower respiratory tract, suggesting that de-optimization of additional gene segments may be warranted for improved attenuation. Taken together, our data demonstrate the potential of using CPBD technology for the development of a live influenza virus vaccine if the level of attenuation is optimized.     

Gene polymorphisms in CCR5, CCR2, SDF1 and RANTES among Chinese Han population with HIV-1 infection

Chemokines and chemokine receptors are crucial for immune response in HIV-1 infection. Although many studies have been done to investigate the relationship between chemokines and chemokine receptor gene polymorphisms and host’s susceptibility to HIV-1 infection, the conclusions are under debate. In the present study, a cohort of 287 HIV-1 seropositive patients, 388 ethnically age-matched healthy controls and 49 intravenous drug users (IDUs) HIV-1 exposed seronegative individuals (HESN) from Chinese Han population were enrolled in order to determine the influence of host genetic factors on HIV-1 infection. Seven polymorphisms on four known chemokines/chemokine receptor genes (CCR5Δ32, CCR5 m303, CCR5 59029A/G, CCR2 64I, RANTES −403A/G, RANTES −28C/G and SDF1 3′-A) were screened. CCR5Δ32 and CCR5 m303 were absent or infrequent in Chinese Han population, which may not be hosts’ genetic protective factors for HIV-1 infection. Our results showed the CCR5 59029A/G, CCR2 64I and SDF1 3′-A were not associated with host’s resistance to HIV-1 infection. The frequency of RANTES −403A allele was significantly lower in HIV-1 patients than in healthy blood donors (p = 0.0005) and HESN group (p = 0.035), which implied the association between A allele and reduced HIV-1 infection risk. Different genetic models were assessed to investigate this association (AA vs. GG + AG, OR = 0.38 95% CI, 0.22–0.65 p = 0.0004; A vs. G, OR = 0.66 95% CI, 0.52–0.84 p = 0.0006), which supported this association, either. The genotype and allele distribution of RANTES −28 between HIV-1 patients and healthy controls (genotype profile: p = 0.072; allele profile: p = 0.027) or HIV-1 seronegative group (genotype profile: p = 0.036; allele profile: p = 0.383) were both at the marginal level of significance, which were not observed after Bonferroni correction. All these results suggest the RANTES −403A may be associated with reduced susceptibility to HIV-1 infection, while the RANTES −28 locus not. By lack of the patients’ clinical information, whether these polymorphisms affect AIDS disease progression and their role in different HIV-1 infection routes could not performed in present study and needs to be assessed in ongoing studies.     

The genetic variability of glycoproteins among respiratory syncytial virus subtype A in China between 2009 and 2013

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in infants and children under 5 years of age. The novel genotype ON1 has a 72-nucleotide duplication, which is the largest duplicated genome portion of RSV. Whether the ON1 genotype will follow the pattern of the BA genotype, which has a 60-nucleotide duplication, and become the predominant RSV-A strain is a global concern. To obtain information regarding the prevalence of the ON1 genotype in Chongqing in Southwestern China, we examined the circulation pattern of RSV-A identified over four consecutive years (June 2009 to August 2013). In this study, 312 (12%) RSV-A strains were isolated from 2601 nasopharyngeal aspirates, and partial G gene was sequenced successfully in 250 isolates. Of the sequenced Chongqing RSV-A isolates, 237 (94.8%) strains were the NA1 genotype, 4 (1.6%) strains were the NA3 genotype, 4 (1.6%) strains were the NA4 genotype, 1 (0.4%) strain was the GA1 genotype, and 4 (1.6%) strains were identified as the ON1 genotype. Analysis of the distribution, phylogeny, and evolution of the ON1 strains that were collected globally until December 2013 revealed that the ON1 genotype has rapidly disseminated across the world under positive selection pressures. Future studies will determine whether this new genotype will continue to spread and become the dominant strain of RSV-A worldwide. These findings may contribute to the understanding of RSV evolution and to the potential development of a vaccine against RSV.     

Assessment of safety and reproductive performance after vaccination with a modified live-virus PRRS genotype 1 vaccine in pregnant sows at various stages of gestation

The objective of the present study was to assess safety and efficacy of a new modified live-virus porcine reproductive and respiratory syndrome (PRRS) genotype 1 vaccine in pregnant sows at various stages of gestation under field conditions. A total of 505 sows and gilts were allocated to two treatment groups and maintained in separate facilities. Animals of group 1 were vaccinated with a commercial modified live genotype 1 PRRSV vaccine (control product, CP), while animals of group 2 were immunized with a new modified live genotype 1 PRRSV vaccine (investigational veterinary product, IVP) (ReproCyc® PRRS EU, Boehringer Ingelheim Vetmedica GmbH). Injection site reactions were noted to be significantly less frequent in the IVP group compared to the CP group for pain (p = 0.039), redness (p = 0.030), heat (p = 0.016) and swelling (p = 0.002). The mean total number of piglets alive at weaning did not differ significantly between both study groups (10.6 vs. 11.0, p = 0.375). However, pre-weaning mortality was significantly higher (p = 0.005) in piglets from the CP group (14.1% vs. 10.9%). Analyses of reproductive performance data for both groups did not result in statistically significant differences between CP group and IVP group for number of piglets alive (12.7 and 12.6, respectively), healthy live (11.9 and 11.8), weak (0.7 and 0.5), stillborn (1.0 and 0.8) and mummified piglets (0.3 and 0.2) per litter. No differences were detected between both groups for piglet birth weights, while body weights at weaning (7.2 kg vs. 6.6 kg, p = 0.026) and average daily gain (0.2445 kg vs. 0.2211 kg, p = 0.037) were significantly higher in piglets from the IVP group. In conclusion, the administration of a single dose of ReproCyc® PRRS EU to sows and gilts at various stages of gestation confirmed non-inferiority to a commercial PRRS vaccine regarding safety and efficacy parameters under field conditions.     

Spectrophotometric method for determination of aluminium content in water and beverage samples employing flow-batch sequential injection system

A sensitive, precise and reliable flow-batch method for the determination of aluminium (Al) was developed using a sequential injection-monosegmented flow system incorporating a mixing chamber unit. Eriochrome cyanine R (ECR) was used as a chromogenic reagent in the presence of N,N-dodecyltrimethylammonium bromide (DTAB). The Al-ECR complex at pH 6 gave a maximum absorption at 584 nm. In-line single standard calibration and a standard addition procedure were developed employing the monosegmented flow technique. Under the optimum conditions, a linear calibration graph in the range of 0.0075–0.625 mg L⁻¹ Al was obtained with limits of detection and quantitation of 0.0020 and 0.0070 mg L⁻¹, respectively. Relative standard deviations were 0.8 and 1.3% for 0.010 and 0.025 mg L⁻¹ Al (n = 11), respectively. A sample throughput of 24 h⁻¹ using an in-line standard calibration approach and 6 h⁻¹ using four standard addition levels was achieved. The developed system was successfully applied to water samples and beverage samples. The results agreed well with those obtained from the ICP-AES method. Good recoveries between 85 and 104% were obtained.     

A rapid and sensitive method for the determination of dibutyl phthalate in wine by flow-injection chemiluminescence analysis

A sensitive method for the determination of picogram level dibutyl phthalate (DBP) in wine by flow-injection chemiluminescence (FI–CL) analysis is presented for the first time, which was based on the quenching effect of DBP on the luminol–myoglobin (Mb) CL system. The decrement of CL intensity was linearly proportional to the logarithm of DBP concentration in the range of 0.1–100 pg mL⁻¹ with the detection limit of 0.03 pg mL⁻¹ (3σ). At a flow rate of 2.0 mL min⁻¹, a complete determination of DBP including sampling and washing could be accomplished in 0.5 min, giving the maximum sample throughput of 120 h⁻¹. The proposed method was successfully applied to the determination of DBP in wine, human serum and urine samples with the relative standard deviations (RSDs) of less than 3.0% (n = 5). The molecule docking results showed that DBP interacted with the amino acid residues near the heme moiety of Mb. The possible CL mechanism of luminol–Mb–DBP reaction should be that the binding of Mb with DBP forming a 1:1 complex (binding constant K = 1.55 × 10⁴ L mol⁻¹) led to the conformational change of Mb and resulted in the quenching of CL intensity.     

Levels and sources of volatile organic compounds including carbonyls in indoor air of homes of Puertollano,the most industrialized city in central Iberian Peninsula. Estimation of health risk

Twenty nine organic air pollutants including carbonyl compounds, alkanes, aromatic hydrocarbons and terpenes were measured in the indoor environment of different houses together with the corresponding outdoor measurements in Puertollano, the most industrialized city in central Iberian Peninsula. VOCs were sampled during 8 weeks using Radiello^® passive samplers, and a questionnaire on potential VOCs sources was filled out by the occupants. The results show that formaldehyde and hexanal was the most abundant VOCs measured in indoor air, with a median concentration of 55.5 and 46.4 μg m⁻³, respectively followed by butanal (29.1 μg m⁻³), acetone (28.4 μg m⁻³) and acetaldehyde (21.4 μg m⁻³). After carbonyls, n-dodecane (13.1 μg m⁻³) and terpenes (α-pinene, 13.4 μg m⁻³ and limonene, 13.4 μg m⁻³) were the compounds with higher median concentrations. The indoor/outdoor (I/O) ratios demonstrated that sources in the indoor environment are prevailing for most of the investigated VOCs especially for limonene, α-pinene, hexanal, formaldehyde, pentanal, acetaldehyde, o-xylene, n-dodecane and acetone with I/O ratio >6. Multiple linear regressions were applied to investigate the indoor VOC determinants and Spearman correlation coefficients were used to establish common sources between VOCs. Finally, the lifetime cancer risk associated to formaldehyde, acetaldehyde and benzene exposure was estimated and they varied from 7.8 × 10⁻⁵ to 4.1 × 10⁻⁴ for formaldehyde, from 8.6 × 10⁻⁶ to 3.5 × 10⁻⁵ for acetaldehyde and from 2.0 × 10⁻⁶ to 1.5 × 10⁻⁵ for benzene. For formaldehyde, the attributed risk in most sampled homes was two orders of magnitude higher than the one (10⁻⁶) proposed as acceptable by risk management bodies.     

Fast method for the determination of copper,manganese and iron in seafood samples

A slurry sampling flame atomic absorption spectrometric method is proposed for the determination of copper, manganese and iron in oysters (Crassostrea rhizophora), clams (Anomalocardia brasiliana) and mussels (Mytella guiyanensis; Perna perna). Optimization was carried out using univariate methodology involving the following variables: nature and concentration of the acid solution for slurry preparation, sonication time and sample mass. The optimized conditions were 80 mg of sample ground in a cryogenic mil, dilution using 1.0 mol L⁻¹ nitric /hydrochloric acid solution, sonication time of 30 min and a slurry volume of 10 mL. The calibration curves had been prepared using standards of copper, manganese and iron also in 1.0 mol L⁻¹ nitric/hydrochloric acid solution. This method allowed the determination of copper, manganese and iron by FAAS, with detection limits of 0.17, 0.09 and 0.46 μg g⁻¹, respectively. The precision, expressed as relative standard deviation (RSD), are 3.0%, 2.9% and 3.8% (n=10), for concentrations of copper, manganese and iron of 17, 22 and 719 μg g⁻¹, respectively. The accuracy of the method was confirmed by analysis of the certified oyster tissue (NIST 1566b). The proposed method was applied for the determination of copper, manganese and iron in samples of oyster, clams and mussels, collected in Ubatuba and Todos os Santos Bay, Brazil. The obtained concentrations varied between 17.1 and 143.9 μg g⁻¹, 17.9 and 29.7 μg g⁻¹ and 364 and 1388 μg g⁻¹, respectively, for copper, manganese and iron. These values agree well with other data reported in the literature. The results showed no significant differences (P>0.005) using the proposed method with those obtained after complete digestion and determination by inductively coupled plasma-optical emission spectroscopy (ICP-OES).     

Single nucleotide polymorphisms in candidate genes and dengue severity in children: A case–control,functional and meta-analysis study

Dengue is an arthropod-borne emerging viral disease with high morbidity and mortality risk in tropical countries like Brazil. Clinical manifestations are vast, ranging from asymptomatic to most severe forms of dengue such as shock. Previous data have shown that host genetics play a role in disease susceptibility and severity. Herein, we have tested the association of single nucleotide polymorphisms (SNPs) at TNF, IL10, MIF, DCSIGN, CLEC5A, NOD2, CCR5 and MRC1 as candidate genes using a matched case–control study design including 88 severe children cases of dengue patients and 335 healthy unrelated subjects that was also separated in IgG⁺ and IgG⁻ controls. We demonstrated that the TT genotype of CLEC5A SNP (rs1285933 C>T) is associated with dengue severity (OR = 2.25; p = 0.03) and that GG genotype of −336G>A DCSIGN (CD209) SNP is associated with protection to severe dengue (OR = 0.12; p = 0.04). Both comparisons were borderline significant when cases were compared with IgG⁺ controls subgroup. Nevertheless, genotype–phenotype correlation was also assessed using serum levels of TNF from infected patients at the onset of dengue fever, and CT/TT carriers in CLEC5A secreted higher levels of TNF than CC individuals in 5–7 days of infection. No significant difference was observed in TNF levels between genotypes GG versus AG/AA at DCSIGN promoter. Next, we performed a meta-analysis retrieving results from the literature for −336G>A DCSIGN and −308G>A TNF SNPs demonstrating that the consensus estimates of these SNPs indicated no association with dengue severity (when compared to Dengue fever) in the overall analysis. But, a subgroup analysis in the −336G>A DCSIGN, the G allele was associated with severe dengue susceptibility in Asians (OR_allele = 2.77; p = 0.0001; OR_carriers = 2.99; p = 0.0001) and protection in Brazilians (OR_allele=0.66; p=0.013). In summary, our results suggest that genetic variations at CLEC5A increase the risk and regulate TNF secretion in dengue severity among Brazilians. Also, combined data of the literature suggest population-specific effect of the −336 DCSIGN SNP more prominent in Asians and in a different direction than Brazilians.