Simultaneous Optimization of Acetaldehyde and DMS Concentrations for Better Sensory Quality of Beer Fermented on an Industrial Scale.

The levels of selected volatile components that affected the sensory properties of a lager beer were optimized under high-gravity brewing conditions (15.5 °P) in an industrial plant. The influence of different pitching rates (6-10 million cells/mL), aeration levels (8-12 mg/L), times (4.5-13.5 h) of filling CCTs (cylindroconical tanks, 3850 hl), and fermentation temperatures (8.5-11.5 °C) on the contents of acetaldehyde, diacetyl, acetone, 2,3-pentanedion, dimethyl sulfide (DMS), and on the sensory properties of beer were investigated. Response surface methodology (RSM, Box-Behnken design) was used to research the possibilities for optimizing the concentration of selected volatile components and sensory properties of bottom-fermented lager beers. Statistical analyses of the results showed that the experimental factors had a significant influence (R-squared for the original model with no significant lack-of-fit) on some of the volatile components. Based on the Multiple Response Optimization analysis, the values of independent factors that ensured the highest beer sensory quality were the following: a pitching rate of 10 million cells per mL; a fermentation temperature of 11.5 °C; an aeration level of 12 mg/L; and a CCT filling time of 4.5 h. These results proved that RSM modelling can be successfully applied to optimize fermentation and lagering processes in an industrial plant to manufacture lagers of enhanced sensory quality.

A Recombinant Turkey Herpesvirus Expressing F and HN Genes of Avian Avulavirus-1 (AAvV-1) Genotype VI Confers Cross-Protection against Challenge with Virulent AAvV-1 Genotypes IV and VII in Chickens.

Newcastle disease (ND) is responsible for significant economic losses in the poultry industry. The disease is caused by virulent strains of Avian avulavirus 1 (AAvV-1), a species within the family Paramyxoviridae. We developed a recombinant construct based on the herpesvirus of turkeys (HVT) as a vector expressing two genes: F and HN (HVT-NDV-F-HN) derived from the AAvV-1 genotype VI ("pigeon variant" of AAvV-1). This recombinant viral vaccine candidate was used to subcutaneously immunize one group of specific pathogen-free (SPF) chickens and two groups of broiler chickens (20 one-day-old birds/group). Humoral immune response was evaluated by hemagglutination-inhibition test and enzyme-linked immunosorbent assay (ELISA). The efficacy of the immunization was assessed in two separate challenge studies performed at 6 weeks of age with the use of virulent AAvV-1 strains representing heterologous genotypes IV and VII. The developed vaccine candidate elicited complete protection in SPF chickens since none of the birds became sick or died during the 2-week observation period. In the broiler groups, 90% and 100% clinical protection were achieved after challenges with AAvV-1 of IV and VII genotypes, respectively. We found no obvious relationship between antibody levels and protection assessed in broilers in the challenge study. The developed recombinant HVT-NDV-F-HN construct containing genes from a genotype VI AAvV-1 offers promising results as a potential vaccine candidate against ND in chickens.

Development of a forensically useful age prediction method based on DNA methylation analysis.

Forensic DNA phenotyping needs to be supplemented with age prediction to become a relevant source of information on human appearance. Recent progress in analysis of the human methylome has enabled selection of multiple candidate loci showing linear correlation with chronological age. Practical application in forensic science depends on successful validation of these potential age predictors. In this study, eight DNA methylation candidate loci were analysed using convenient and reliable pyrosequencing technology. A total number of 41 CpG sites was investigated in 420 samples collected from men and women aged from 2 to 75 years. The study confirmed correlation of all the investigated markers with human age. The five most significantly correlated CpG sites in ELOVL2 on 6p24.2, C1orf132 on 1q32.2, TRIM59 on 3q25.33, KLF14 on 7q32.3 and FHL2 on 2q12.2 were chosen to build a prediction model. This restriction allowed the technical analysis to be simplified without lowering the prediction accuracy significantly. Model parameters for a discovery set of 300 samples were R(2)=0.94 and the standard error of the estimate=4.5 years. An independent set of 120 samples was used to test the model performance. Mean absolute deviation for this testing set was 3.9 years. The number of correct predictions ±5 years achieved a very high level of 86.7% in the age category 2-19 and gradually decreased to 50% in the age category 60-75. The prediction model was deterministic for individuals belonging to these two extreme age categories. The developed method was implemented in a freely available online age prediction calculator.

Analysis of Coinfections with A/H1N1 Strain Variants among Pigs in Poland by Multitemperature Single-Strand Conformational Polymorphism.

Monitoring and control of infections are key parts of surveillance systems and epidemiological risk prevention. In the case of influenza A viruses (IAVs), which show high variability, a wide range of hosts, and a potential of reassortment between different strains, it is essential to study not only people, but also animals living in the immediate surroundings. If understated, the animals might become a source of newly formed infectious strains with a pandemic potential. Special attention should be focused on pigs, because of the receptors specific for virus strains originating from different species, localized in their respiratory tract. Pigs are prone to mixed infections and may constitute a reservoir of potentially dangerous IAV strains resulting from genetic reassortment. It has been reported that a quadruple reassortant, A(H1N1)pdm09, can be easily transmitted from humans to pigs and serve as a donor of genetic segments for new strains capable of infecting humans. Therefore, it is highly desirable to develop a simple, cost-effective, and rapid method for evaluation of IAV genetic variability. We describe a method based on multitemperature single-strand conformational polymorphism (MSSCP), using a fragment of the hemagglutinin (HA) gene, for detection of coinfections and differentiation of genetic variants of the virus, difficult to identify by conventional diagnostic.

Examination of DNA methylation status of the ELOVL2 marker may be useful for human age prediction in forensic science.

Age estimation in forensic investigations may complement the prediction of externally visible characteristics and the inference of biogeographical ancestry, thus allowing a better description of an unknown individual. Multiple CpG sites that show linear correlation between age and degree of DNA methylation have been identified in the human genome, providing a selection of candidates for age prediction. In this study, we optimized an assay based on bisulfite conversion and pyrosequencing of 7 CpG sites located in the ELOVL2 gene. Examination of 303 blood samples collected from individuals aged 2-75 years allowed selection of the most informative site, explaining 83% of variation in age. The final linear regression model included two CpG sites in ELOVL2 and enabled age prediction with R(2)=0.859, prediction error=6.85 and mean absolute deviation MAD=5.03. Examination of a testing set of 124 blood samples (MAD=5.75) showed that 68.5% of samples were correctly predicted, assuming that chronological and predicted ages matched ± 7 years. It was found that the ELOVL2 methylation status in bloodstains had not changed significantly after 4 weeks of storage in room temperature conditions. Analysis of 45 bloodstains deposited on tissue paper after 5, 10 and 15 years of storage in room conditions indicated that although a gradual decrease of positive PCR results was observed, the general age prediction success rate remained similar and equaled 60-78%. The obtained results show that the ELOVL2 locus provides a very good source of information about human chronological age based on analysis of blood, including bloodstains, and it may constitute a powerful and reliable predictor in future forensic age estimation models.

Sensitive methods for detection of the S768R substitution in exon 18 of the DDR2 gene in patients with central nervous system metastases of non-small cell lung cancer.

Discoidin death receptor 2 (DDR2) receptor belongs to a DDR family that shows a tyrosine kinase activity. The somatic mutations in DDR2 gene, reported in non-small cell lung cancer (NSCLC), are involved in up-regulation of cells' migration, proliferation and survival. A S768R substitution in DDR2 gene was commonly reported in squamous cell lung carcinoma. Clinical data of patients carrying the DDR2 gene mutation suggest that its presence can be independent of gender and age. The effectiveness of an oral dual-specific (Src and Abl) multikinase inhibitors-dasatinib-was observed in different cell lines and in some NSCLC patients with identified DDR2 mutation. In the present study, we have used three molecular methods (ASP-real-time PCR, ASP-DNA-FLA PCR and direct sequencing) to detect the DDR2 gene mutation in 143 patients with NSCLC metastases to the central nervous system (CNS). The prevalence of the DDR2 gene mutation was correlated with the occurrence of mutations in the EGFR, KRAS, HER2 and BRAF genes. We identified three patients (2.1% of studied group) with DDR2 mutation. The mutation was observed in two patients with low differentiated squamous cell lung cancer and in one patient with adeno-squamous cell carcinoma (ADSCC). In ADSCC patients, DDR2 mutation coexisted with G12C substitution in KRAS gene. According to the current knowledge, examination of the presence of the DDR2 gene mutation in metastatic lesion is the first such report worldwide. The information, that these driver mutations are present in CNS metastases of NSCLC, could broaden therapeutic choices in such group of patients.

Genetic diversity of hemagglutinin gene of A(H1N1)pdm09 influenza strains isolated in Taiwan and its potential impact on HA-neutralizing epitope interaction.

Pandemic influenza A(H1N1)pdm09 virus is a global health threat and between 2009-2011 it became the predominant influenza virus subtype circulating in the world. The research describes the MSSCP (Multitemperature Single Strand Conformation Polymorphism) analysis of the hemagglutinin (HA) region encompassing major neutralizing epitope in pandemic influenza isolates from Taiwan. Several genetically distinct changes appeared in isolates obtained in 2010 and 2011. The majority of changes in HA protein did not result in significant modifications, however three modifications were localized in epitope E of H1 and one was part of the interface binding antibodies BH151 and HC45 possibly making the current vaccine less effective.-Taking into account the possibility of the emergence of influenza A with antibody evading potential, the MSSCP method provides an alternative approach for detection of minor variants which escape detection by conventional Sanger sequencing.

Acute respiratory distress syndrome (ARDS) in the course of influenza A/H1N1v infection--genetic aspects.

Influenza is a contagious respiratory disease caused by viruses belonging to the family Ortomyxoviridae. Among the influenza viruses type A, B and C, the A type virus shows the most pathogenic potential. Its surface receptor glycoproteins, hemagglutinin (HA) and neuraminidase (NA), are characterized by high antigenic variation, thus a host organism cannot develop permanent resistance. The case is described of a male patient with severe acute respiratory distress syndrome in the course of influenza A/N1H1v infection, confirmed by virological molecular analysis. During diagnostic procedures based on the MSSCP genotyping it was observed that the WHO recommended RT-PCR kits and/or procedure of sample collection from patients for molecular investigation could lead to false positive A/H1N1 pandemic strain detection because of the co-amplification during the RT-PCR fragments of the human genome.

Sensitive methods for the detection of an insertion in exon 20 of the HER2 gene in the metastasis of non-small cell lung cancer to the central nervous system.

The HER2 (ErbB2/neu) protein is a member of the HER (ErbB) receptor family (EGFR, HER2, HER3 and HER4) that expresses tyrosine kinase activity in the intracellular domain. EGFR and HER2 overexpression is observed in numerous types of cancer, nevertheless, the susceptibility of patients with non-small cell lung cancer (NSCLC) to therapy with EGFR and HER2 tyrosine kinase inhibitors (TKIs) depends on mutations present in the respective coding genes (driver mutations). In the present study, PCR and amplified DNA fragment length analysis (FLA) were used along with the multi-temperature single-strand conformation polymorphism (MSSCP) technique in order to identify the 12 base pair insertion in exon 20 of the HER2 gene in 143 patients with NSCLC metastasis to the central nervous system. The prevalence of the HER2 gene mutation was correlated with mutations in the EGFR and BRAF genes. The insertion in exon 20 of the HER2 gene was observed in a single 77-year-old, non-smoking male, with poorly-differentiated adenocarcinoma of the lung (1.5% of adenocarcinoma patients). No other genetic abnormalities were identified in this patient. In the therapy of NSCLC patients with HER2 gene mutations, drugs that inhibit the EGFR and HER2 receptors, for example afatinib, may be effective. The identification of other driving mutations in NSCLC cells appears to be key to the appropriate qualification of molecular targeted therapies.

Rapid differentiation of mixed influenza A/H1N1 virus infections with seasonal and pandemic variants by multitemperature single-stranded conformational polymorphism analysis.

Mixed infections of a single host with different variants of influenza A virus are the main source of reassortants which may have unpredictable properties when they establish themselves in the human population. In this report we describe a method for rapid detection of mixed influenza virus infections with the seasonal A/H1N1 human strain and the pandemic A/H1N1/v strain which emerged in 2009 in Mexico and the United States. The influenza virus A/H1N1 variants were characterized by the multitemperature single-stranded conformational polymorphism (MSSCP) method. The MSSCP gel patterns of hemagglutinin gene fragments of pandemic A/H1N1/v and different seasonal A/H1N1 strains were easily distinguishable 2 h after completion of reverse transcription-PCR (RT-PCR). Using the MSSCP-based genotyping approach, coinfections with seasonal and pandemic variants of the A/H1N1 subtype were identified in 4 out of 23 primary samples obtained from patients that presented with influenza-like symptoms to hospitals across Poland during the 2009-2010 epidemic season. Pandemic influenza virus strain presence was confirmed in all these primary samples by real-time RT-PCR. The sensitivity level of the MSSCP-based minor genetic variant detection was 0.1%, as determined on a mixture of DNA fragments obtained from amplification of the hemagglutinin gene of seasonal and pandemic strains. The high sensitivity of the method suggests its applicability for characterization of new viral variants long before they become dominant.

The association between SCN5A, KCNQ1 and KCNE1 gene polymorphisms and complex ventricular arrhythmias in survivors of myocardial infarction.

BACKGROUND: Post-MI patients are highly susceptible to sudden cardiac arrest (SCA) and sudden cardiac death (SCD) resulting from ventricular arrhythmia (VA). The search for new clinical predictors to identify those patients who are at the highest risk of these events is therefore essential. Numerous data indicate that the presence of polymorphisms and mutations in the cardiac ion channel genes SCN5A, KCNQ1 and KCNE1 might serve as such a predictor. Since genetic alterations in these genes underlie congenital long QT syndrome (LQTS), which is associated with an increased occurrence of arrhythmic complications and SCD, we decided to verify how alterations in these genes contribute to QT interval abnormalities and consequently to VA, SCA and SCD in post-MI patients. AIM: To detect single nucleotide polymorphisms (SNP) in SCN5A, KCNQ1 and KCNE1 of post-MI patients, and to assess whether they are related to electrophysiological markers of cardiac arrhythmia (QT interval) and the clinical course. METHOD: The study group consisted of 100 patients (27 females, mean age 69 years) with documented MI 3 months before enrolment. All patients underwent baseline and (after 12 months) control examinations encompassing history, physical examination, basic laboratory analysis, resting 12-lead ECG, 24-hour 12-lead Holter ECG monitoring and echocardiography. Genetic tests were performed during baseline examination. RESULTS: In post-MI patients two exonic polymorphisms, H558R in SCN5A and S38G in KCNE1, and two intronic ones, in KCNQ1, were detected. H558R was associated with an increase in QT dispersion (QTd) at minimum and maximum heart rate and QT interval prolongation before premature ventricular beats (PVB), whereas S38G and intronic polymorphisms were related to an increase in QTd before PVB. None of the above polymorphisms was related to complex VA, SCA or SCD. CONCLUSION: The above polymorphisms were associated with abnormal repolarisation phase patterns in post-MI patients, which manifested in QT interval prolongation and QTd increase. There was no relationship between these polymorphisms and complex VA, SCA or SCD. The results show that not only exonic alterations but also intronic ones may affect the phenotype.

Novel KCNQ1 mutations in patients after myocardial infarction.

BACKGROUND: Patients after myocardial infarction (MI) are at greater risk of sudden cardiac death (SCD) than people in the overall population. The aim of this study was to detect mutations, including intronic ones, in the KCNQ1 gene coding for proteins of cardiac potassium channels and evaluate their possible effects on the clinical course in patients after MI. METHODS: The study group was composed of 100 Polish patients after MI, which included 27 women (mean age 69 years) and 73 men (mean age 67 years). All patients underwent clinical examinations and genetic tests. The genetic test results have been correlated with the clinical data. The following parameters have been chosen as endpoints for this survey: sudden cardiac arrest (SCA) or SCD, complex ventricular arrhythmia, QT interval and QT dispersion values assessed during 24-hour Holter ECG monitoring in relation to ventricular arrhythmias as well as the minimum and maximum heart rate (HR) observed during the examination. RESULTS: Six new mutations in the KCNQ1 gene: C2505734T, A2753831C in exons and C2505846A, G2753881A, T2755854C, T2755875G in introns. Detected intronic mutations in patients after MI were related to a worse clinical course and frequent occurrence of SCA. CONCLUSIONS: The novel intronic mutations may have a significant influence on the clinical course of the disease.

Detection of single nucleotide polymorphisms in coagulation factor XI deficient patients by multitemperature single-strand conformation polymorphism analysis.

Factor XI (FXI) deficiency is a rare inherited disorder which can cause bleeding complications especially in case of hemostatic challenge and/or in tissues with high fibrinolytic activity. A number of causative mutations have been described in FXI deficient individuals which have been detected by various screening methods. In this study, we present the application of the multitemperature single-strand conformation polymorphism analysis (MSSCP) on the FXI gene, a recently developed methodology for the detection of single nucleotide exchanges. We analyzed a total of 217 polymerase chain reaction (PCR) fragments from the promoter region as well as from exons 1-7 and 11-15 and compared the results to automatic fluorescent sequencing. A total of 29 PCR fragments showed single nucleotide exchanges in conventional fluorescent sequencing, representing 10 different mutations (nine missense mutations, one small deletion) and four frequent polymorphisms. With MSSCP electrophoresis at a standard temperature profile (gel temperature 35-20-10 degrees C) we were able to detect 13 of 14 (93%) different nucleotide exchanges in 25 of 29 PCR fragments (86%). Hence, the detection rate for genetic variations in the FXI gene was 86%. To evaluate the reproducibility, MSSCP was performed twice for 174 PCR fragments and the consistency between two electrophoretic runs was 99%. We conclude that the MSSCP is a sensitive, fast, and cost effective screening method for the detection of FXI gene mutations.

A new method of DNA preparation for pulsed-field gel electrophoresis analysis.

We have developed a new, labour-saving method of preparation, handling and treatment of DNA-containing agarose plugs for pulsed-field gel electrophoresis (PFGE). A plastic mould in which plugs are formed and supported during DNA purification and digestion was designed and successfully tested in a prototype device.