Complexity of the effects of pre-fermentation oxygenation, skin contact and use of pectolytic enzymes in white winemaking as revealed by comprehensive proteomics and volatilomics analysis.

Ion exchange chromatography and SDS-PAGE followed by identification by MALDI-TOF/TOF, and two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-TOF-MS) were used for comprehensive proteomics and volatilomics evaluation of the effects of pre-fermentative oxygenation, skin contact and use of pectolytic enzymes in production of Malvazija istarska white wine, respectively. Many protein species and an unprecedented number of volatiles have been identified and (semi)quantified, revealing high complexity of the observed effects. Compared to a standard control wine, oxygenation treatment modulated the protein composition and resulted with a volatilome characterized by decreased levels of several important volatiles. Skin contact treatments, especially in combination with pectolytic enzymes, significantly increased the levels of a large number of proteins, but were also deprived of particular protein species found in other wines. Wines obtained by skin contact with exogenous enzymes exhibited the most complex volatile composition with increased levels of many key monoterpenoids, alcohols and esters.

Applying Different Vinification Techniques in Teran Red Wine Production: Impact on Bioactive Compounds and Sensory Attributes.

Six different vinification treatments, including a control treatment (7-day standard maceration) (K7), were performed to study the effects of non-standard techniques on bioactive compounds and sensory attributes of Teran red wine. Pre-fermentative mash cooling (8 °C; 48 h) and heating (50 °C; 48 h) followed by prolonged post-fermentative maceration of 13 days (C15;H15) or 28 days (C30;H30) were applied. In another treatment, after cooling, saignée was performed followed by 13-day prolonged maceration (CS15). Wine phenols and vitamins were analyzed by HPLC-DAD-FLD, minerals by ICP-OES, and sensory analysis was performed using the QDA and 100-point O.I.V./U.I.O.E. methods. Obtained results showed total phenolic concentration was the highest in the H30 treatment. The concentration of anthocyanins, flavan-3-ols and phenolic acids was significantly higher in wines of all vinification techniques compared to the control. Stilbene content was highly affected by pre-fermentative heating. Treatments CS15, H15, C30 and H30 resulted in the highest scores by both the QDA and 100-point sensory methods. The obtained results suggest that advanced non-standard vinification techniques have a significant impact on Teran wine by enhancing its composition of bioactive compounds and improving its sensory profile, which gives it an additional market value. Furthermore, a comprehensive comparison of such techniques applied simultaneously in one study is of substantial importance for additional research in wine production.

Diversity of Volatile Aroma Compound Composition Produced by Non-Saccharomyces Yeasts in the Early Phase of Grape Must Fermentation.

There is a lack of studies evaluating the metabolic contribution of non-Saccharomyces yeasts in early fermentation phases. This study aimed to investigate the volatile aroma profiles produced by various non-Saccharomyces yeasts just before sequential inoculation with Saccharomyces cerevisiae to provide an insight into the particular effects they induce at this stage. The grape must of Malvazija istarska was inoculated with monocultures of Torulaspora delbrueckii, Metschnikowia pulcherrima, Pichia kluyveri, Lachancea thermotolerans, and Schizosaccharomyces pombe, alongside a S. cerevisiae control. Eighty volatile compounds were quantified via headspace solid-phase microextraction and gas chromatography-mass spectrometry, and the data were statistically elaborated. Volatile profiles of non-Saccharomyces yeasts differed significantly from the S. cerevisiae control. Most treatments caused increases in linalool and ß-damascenone, decreases in higher alcohols and fatty acids, and improved synthesis of odoriferous esters. Torulaspora delbrueckii and M. pulcherrima produced compounds not commonly found in S. cerevisiae fermented wines. Multivariate statistical analysis linked the investigated yeasts to specific, particularly abundant compounds. Future studies should explore to what degree these contributions persist after sequential inoculation with S. cerevisiae in diverse grape must matrices.

The Phytochemical and Nutritional Composition of Shallot Species (Allium × cornutum, Allium × proliferum and A. cepa Aggregatum) Is Genetically and Environmentally Dependent.

Shallots are a perennial plant from the Alliaceae family, classified with the common onion under the name of the Allium cepa Aggregatum group. The term shallot is also used for diploid and triploid viviparous onions, known as Allium × proliferum (Moench) Schrad and Allium × cornutum Clementi ex Vis., respectively. In this study, we compared the dry matter, pyruvic acid content, sugar content, flavonoid content, antioxidant capacity and mineral composition of 34 shallot accessions falling into three shallot species (Allium × cornutum, Allium × proliferum and A. cepa Aggregatum). Shallot accessions belonging to the A.× cornutum and A. × proliferum groups are characterized by high dry matter content (around 25%), of which a little less than 50% is formed of inulin-type sugars, polysaccharides, considered an excellent prebiotic with beneficial effects on human health. On the other hand, accessions belonging to the A. cepa Aggregatum group have lower dry matter content and, as a result, lower pungency (measured as pyruvic acid content), making them more suitable for fresh consumption by a broader range of consumers, but, at the same time, abundant in phenolic compounds, especially quercetin and isorhamnetin glycosides. We also observed a greater biodiversity among accessions within the A. cepa Aggregatum group in all the analyzed physico-chemical parameters compared to the other shallot groups. The investigated shallot accessions have an excellent in vitro antioxidant capacity, as well as excellent nutritional properties.

Bioactive Compounds and Antioxidant Activity of Red and White Wines Produced from Autochthonous Croatian Varieties: Effect of Moderate Consumption on Human Health.

Moderate wine consumption is often associated with healthy lifestyle habits. The role of wine as a healthy drink is mainly due to its bioactive compounds, which differ according to various viticultural and enological factors. The aim of the present study was to observe the differences in bioactive compounds of white and red autochthonous Croatian wines, differing in terms of the grape variety and production technology. Our further aim was to explore the effect of their moderate consumption (200 mL per day) over the course of six weeks on some aspects of health in sixty-six healthy individuals. Participants were divided into eight groups depending on the wines consumed, while one group formed a non-consuming control group. Medical examination and laboratory tests were performed before the start and at the end of the consumption period. Systolic and diastolic blood pressure, total cholesterol, and LDL concentrations decreased. Additionally, an increase in HDL concentrations, and serotonin and dopamine levels, was observed. ALT, ALP, and GGT levels did not significantly increase in consumer groups, although alcohol concentration was relatively high in all the wines. Such results support the beneficial effects of wine-derived bioactive compounds on some health aspects resulting from moderate white and red wine consumption.

Association of polymorphisms in genes encoding prothrombotic and cardiovascular risk factors with disease severity in COVID-19 patients: A pilot study.

The present study aimed to assess the association of 16 polymorphisms in genes encoding prothrombotic and cardiovascular risk factors with COVID-19 disease severity: FV G1691A, FV H1299R, FII G20210A, MTHFR C677T, MTHFR A1298, factor XIII V34L, PAI-1 4G/5G, EPCR haplotypes (A1/A2/A3), eNOS -786 T > C, eNOS G894T, LTA C804A, ACE I/D, ITGB3 PIA1/A2, ITGA2B Baka/b, ß-Fbg -455 G > A and ApoB R3500Q. The study included 30 patients with severe COVID-19 and 49 non-severe COVID-19 patients. All studied polymorphisms except ITGA2B Baka/b were determined using multilocus genotyping assays CVD StripAssays (ViennaLab Diagnostics), while ITGA2B was genotyped using a real-time PCR method based on TaqMan technology. A higher frequency of carriers of at least one ITGB3 PIA2 allele was found in severe COVID-19 patients (p = 0.009). The distribution of genotypes was significantly different for ß-Fbg -455 G > A (p = 0.042), with only three homozygous AA genotypes found among severe COVID-19 patients. The association with an increased risk for severe COVID-19 was found for ITGB3, with carriers of at least one ITGB3 PIA2 allele having a 3.5-fold greater risk of severe COVID-19 (p = 0.011). Genotype distribution differences were obtained for the combinations of FV H1299R and FXIII V34L (p = 0.026), ITGB3 PIA1/A2 and ITGA2B Baka/b (p = 0.024), and ACE I/D and PAI-1 4G/5G (p = 0.046). ITGB3 polymorphism emerged as an independent risk factor for severe COVID-19 and homozygosity for ß-Fbg -455 G > A mutation could contribute to disease severity. The combined effect of polymorphisms in genes encoding prothrombotic and cardiovascular risk factors could further contribute to disease severity.

Moment of Bentonite Addition, Co-Addition of Tannins, and Bentonite Type Affect the Differential Affinity of Pathogenesis-Related Grape Proteins towards Bentonite during Fermentation.

To test the effect of the moment of bentonite addition, co-addition of tannins, and bentonite type on the differential affinity of pathogenesis-related (PR) proteins towards bentonite during grape must fermentation, three separate experiments were set up. PR proteins in the obtained wines were analyzed by reverse phase and size exclusion high-performance liquid chromatography (HPLC). The most significant reduction of bentonite dose and PR protein concentration was achieved by applying bentonite in the last third of fermentation. Particular thaumatin-like proteins (TLP) and proteins with lower molecular mass in general were more affected than others, while TLPs were more affected than chitinases. Exogenous enological tannins interacted with particular PR proteins, mostly TLPs, and lowered the total bentonite dose required. The combined application of tannins and bentonite in fermentation removed more PR proteins than bentonite alone, but did not achieve a synergistic effect in reducing the bentonite dose. Various bentonite types, including two Na-activated bentonites, an activated Na bentonite with specifically adsorbed silica, and an active Na-Ca bentonite, exhibited differential affinity towards different PR proteins. The results obtained could be used in developing wine fining protocols which combine treatments with complementary affinity for adsorption and removal of PR proteins, and in this way achieve greater efficiency of bentonite fining by reducing its total dose, which is of significant interest to the wine industry.

Bentonite fining during fermentation reduces the dosage required and exhibits significant side-effects on phenols, free and bound aromas, and sensory quality of white wine.

To test the effects of bentonite addition at various stages of fermentation, five Malvazija istarska white grape must vinification treatments were performed with 100 g/hL of bentonite added in clear juice, at the beginning, in the middle, and at the end of fermentation, while control was fermented without bentonite. Phenols and free and bound volatile aromas were determined by HPLC-DAD and SPE-GC-MS. Wines were evaluated sensorially. Fining during fermentation reduced the total bentonite dose required, and was most effective near the end of fermentation with the reduction of 16% and 21%, depending on the protein stability test. All treated wines preserved more hydroxycinnamoyltartaric acids with respect to control. The side-effect of these treatments on varietal aromas was moderate, but enhanced the preservation of key fermentation volatiles in relation to control, and exhibited positive sensory effects. It was concluded that bentonite added during fermentation may positively affect wine quantity and quality.

Combined targeted and untargeted profiling of volatile aroma compounds with comprehensive two-dimensional gas chromatography for differentiation of virgin olive oils according to variety and geographical origin.

Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-TOF-MS) was combined with conventional mono-dimensional GC-MS to differentiate Croatian virgin olive oils (VOO) according to variety and geographical origin, based on the profile of volatile aroma compounds isolated by HS-SPME. More than 1000 compounds were detected after untargeted profiling and 256 were identified or tentatively identified, providing one of the most detailed profiles of volatile aroma compounds in VOO up to date. Among them, 131 volatile compounds were significantly different across monovarietal VOOs, while 60 were found useful for the discrimination according to geographical origin. Many major lipoxygenase and minor non-lipoxygenase-generated compounds were shown to have discriminating ability with respect to both factors. Multivariate statistical analysis extracted twenty-one volatile markers with the highest discriminant power for varietal differentiation. The approach reported may have practical application in better understanding, defining, managing, and communicating the varietal or geographical typicity of monovarietal VOOs.

Development and validation of a rapid method for genotyping three P-selectin gene polymorphisms based on high resolution melting analysis.

BACKGROUND: High resolution melting (HRM) analysis is one of the newer, reliable, and sensitive genotyping techniques, which offers considerable time and cost savings. P-selectin is an adhesion molecule that has a role in the initial phases of leukocyte adhesion to stimulated platelets and endothelial cells in inflammation. Multiple polymorphisms in P-selectin gene (SELP) that affect the protein sequence have been described. The aim of this study was to design, optimize, and validate a simple and rapid in-house HRM-based method for genotyping the NM_003005.3:c.992G>A (c.992G>A), NM_003005.3:c.1918G>T (c.1918G>T), and NM_003005.3:c.2266A>C (c.2266A>C) SELP polymorphisms. METHODS: Initial genotyping of three SELP polymorphisms was performed by applying polymerase chain reaction (PCR) with sequence-specific primers (SSP), which was used as a reference method for determination of analytical sensitivity. PCR-HRM was performed with primers for c.2266A>C reported in the literature. Primers for the remaining two polymorphisms were designed using Primer-BLAST. Precision testing was performed using three samples with different genotypes. For accuracy, analytical sensitivity and specificity testing, 20 wild type, 10 heterozygous, and 10 homozygous samples were chosen per polymorphism. Results were expressed as percentage of concordance with the acceptability criterion ≥95%. RESULTS: Agreement of results was 100% for all validation parameters except for analytical sensitivity for c.1918G>T and c.2266A>C, with agreement of 90%. Repeated analysis using both methods revealed an error in initial genotyping and correct genotyping by PCR-HRM, which was confirmed by Sanger sequencing. CONCLUSION: The validation confirmed PCR-HRM as a precise, accurate, and specific method for genotyping the c.992G>A, c.1918G>T, and c.2266A>C SELP polymorphisms.

Influence of Blood Count, Cardiovascular Risks, Inherited Thrombophilia, and JAK2 V617F Burden Allele on Type of Thrombosis in Patients With Philadelphia Chromosome Negative Myeloproliferative Neoplasms.

INTRODUCTION: Thrombosis is the most common complication in Philadelphia chromosome negative (Ph-) myeloproliferative neoplasms patients. PATIENTS AND METHODS: In a cohort of 258 Ph- myeloproliferative neoplasm patients, the difference between patients with and without thrombosis was analyzed according to genetic thrombophilia factors, JAK2 V617F status and burden allele, blood count, cardiovascular risk factors and age. Patients were also divided in polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) subgroups as well as by the type of thrombosis. RESULTS: Analysis of cardiovascular risk factors regarding arterial thrombosis showed that PV patients with thrombosis had higher incidence of diabetes (P = .030), ET patients more often had hypertension (P = .003) and hyperlipidemia (P = .005), while PMF patients had hyperlipidemia (P = .046) and at least one cardiovascular risk factor (P = .044). Moreover, leukocytes > 18 × 109/L and V617F burden allele > 25.7% were statistically significantly different in PV patients (P = .019 and borderline significant at P = .055, respectively), while in ET patients leukocytes > 9.2 × 109/L (P < .001) and age at diagnosis of > 55 years were statistically significantly different (P = .002). PMF patients with V617F burden allele ≤ 34.8% were more prone to thrombosis (P = .032). When comparing patients with and without venous thrombosis, cutoff value of V617F burden allele > 90.4% was significant for PV patients with thrombosis (P = .036), as was > 56.7% for PMF patients with thrombosis (P = .046). Platelets ≤ 536 × 109/L and age at diagnosis > 54 years showed statistically significant difference for ET patients with thrombosis (P = .015 and P = .041, respectively). CONCLUSION: On the basis of our results, a new scoring system for thrombosis risk in PV could be made, while PMF prognostic model may be expanded for better recognition of potential thrombotic risk factors.

Towards understanding the varietal typicity of virgin olive oil by correlating sensory and compositional analysis data: a case study.

To investigate if the anecdotal varietal typicity concepts for two monovarietal virgin olive oils (VOO) can be scientifically supported and explained, VOO samples were sorted according to typicity grade and related to sensory and compositional data for volatiles and phenols. Clear inter-varietal, as well as intra-varietal boundaries between more and less typical samples were established by univariate and multivariate statistics. A variety-typical complex odor was related to a complex VOO volatile composition with relatively high amounts of lipoxygenase-derived compounds, accompanied by hydrocarbons, terpenes, and benzenoids. A simpler odor, described mostly as green and typical for the other monovarietal VOO, coincided with the lower amounts of the same volatiles. The intensity of bitterness and pungency was mostly in correlation with the amount of major phenols. Two monovarietal VOOs were clearly distinguished with respect to their variety-typical expression of pungency: the one described as gradually increasing was tentatively related to pinoresinol, while the other, described as immediately strong and burning, was assumed to originate mainly from p-HPEA-EDA. Intra-varietal typicity grades correlated with hedonic liking, as well as with general VOO positive sensory attributes related to key volatiles and phenols.

Serum chitotriosidase: a circulating biomarker in polycythemia vera.

OBJECTIVES: Serum chitotriosidase activity (CHIT1) is a biomarker of macrophage activation with an important role in inflammation-induced tissue remodeling and fibrosis. Macrophages have been described to play a crucial role in regulating pathological erythropoiesis in polycythemia vera (PV). The aim of this study was to evaluate CHIT1 in patients diagnosed with Philadelphia-negative myeloproliferative neoplasms (MPNs). METHODS: Using fluorometric assay, we measured CHIT1 in 28 PV, 27 essential thrombocythemia (ET), 17 primary myelofibrosis (PMF), 19 patients with secondary myelofibrosis and in 25 healthy controls. RESULTS: CHIT1 was significantly higher in PV (p < .001) and post-PV myelofibrosis (MF) transformation (post-PV MF) (p = .020), but not in ET (p = .080), post-ET MF transformation (p = .086), and PMF patients (p = .287), when compared to healthy controls. CHIT1 in PV was positively correlated with hemoglobin (p = .026), hematocrit (p = .012), absolute basophil count (p = .030) and the presence of reticulin fibrosis in the bone marrow (p = .023). DISCUSSION: A positive correlation between CHIT1 and these distinct laboratory PV features might imply macrophages closely related to clonal erythropoiesis as cells of CHIT1 origin. In addition, a positive association between CHIT1 and reticulin fibrosis might indicate its potential role in PV progression. CONCLUSION: CHIT1 might be considered as a circulating biomarker in PV. Additional studies are needed to clarify the role of CHIT1 in promoting disease progression and bone marrow fibrosis in PV.

The incidence of atypical patterns of BCR-ABL1 rearrangement and molecular-cytogenetic response to tyrosine kinase inhibitor therapy in newly diagnosed cases with chronic myeloid leukemia (CML).

BACKGROUND: To analyze the frequency of atypical fluorescence in situ hybridization signal patterns and estimate the complete cytogenetic response (CCyR) and major molecular response (MMR) during 12 months of tyrosine kinase inhibitor therapy in patients with newly diagnosed chronic myeloid leukemia. METHODS: The study included bone marrow and peripheral blood samples from 122 patients with newly diagnosed chronic myeloid leukemia. Detection of the breakpoint cluster region-Abelson fusion gene (BCR-ABL1) was performed using fluorescence in situ hybridization with a dual-color dual-fusion translocation probe, and MMR analysis was performed using the real-time quantitative polymerase chain reaction method. RESULTS: Variant translocation was determined in 10 samples and a deletion on the derivative chromosome 9 (del/der(9)) was found in 20 samples. The rates of CCyR and MMR were similar between patients with reciprocal translocation, variant translocation, deletion of derivative BCR, or ABL1-BCR fusion gene. The Kaplan-Meier test did not show any significant differences in the rates of CCyR and MMR among those groups of patients. CONCLUSION: The frequencies of variant translocation and del/der(9) in the present study agree with the results of other studies performed worldwide. No differences were observed in the rates of CCyR and MMR between patients with atypical patterns and reciprocal translocation.

Differentiation of Commercial PDO Wines Produced in Istria (Croatia) According to Variety and Harvest Year Based on HS-SPME-GC/MS Volatile Aroma Compound Profiling.

To differentiate monovarietal wines made from native and introduced varieties in Istria (Croatia), samples of Malvazija istarska, Chardonnay and Muscat yellow from two harvest years (2013 and 2014) were subjected to headspace solid-phase microextraction and gas chromatographic/mass spectrometric analysis (HS-SPME-GC/MS) of volatile aroma compounds. Significant effects of variety and harvest year were determined, but their interaction complicated the differentiation. Particular compounds were consistent as markers of variety in both years: nerol for Malvazija, ethyl cinnamate and a tentatively identified isomer of dimethylbenzaldehyde for Chardonnay, and terpenes for Muscat yellow. Wines from 2013 contained higher concentrations of the majority of important volatiles. A 100% correct differentiation of Malvazija istarska and Chardonnay wines according to both variety and harvest year was achieved by stepwise linear discriminant analysis.

Association of Polymorphisms in Coagulation Factor Genes and Enzymes of Homocysteine Metabolism With Arterial Ischemic Stroke in Children.

Despite the identification of a wide range of inherited and acquired risk factors for arterial ischemic stroke (AIS) in children, genetic risk factors are incompletely characterized and may vary among different populations. We investigated the role of individual and combined inherited prothrombotic and intermediate-risk factors in 73 children with perinatal (n = 35) and childhood AIS (n = 38) and 100 age- and sex-matched controls. Ten polymorphisms in 8 candidate genes encoding coagulation and fibrinolytic proteins (factor V [FV] Leiden, FV HR2, factor II [FII] G20210A, ß-fibrinogen [ß-FBG]-455G>A, factor XIII [FXIII]-A p.Val34Leu, plasminogen activator inhibitor 1 4G/5G), homocysteine metabolism (methylenetetrahydrofolate reductase [MTHFR] C677T, MTHFR A1298C), and intermediate-risk factors (angiotensin-converting enzyme I/D, apoE ∊2-4) were detected using a multilocus genotyping assay. Allele-specific polymerase chain reaction was used for the determination of human platelet alloantigens (HPA-1, HPA-2, HPA-3, and HPA-5). Factor V Leiden was associated with an increased risk of AIS (odds ratio [OR]: 4.72, 95% confidence interval [CI]: 1.22-18.27) and perinatal AIS (OR: 8.29, 95% CI: 1.95-35.24). Human platelet antigen-3b allele carriers had a 2-fold lower risk of AIS (OR: 0.51, 95% CI: 0.26-0.98) and perinatal AIS (OR: 0.40, 95% CI: 0.18-0.92). A 2.21-fold increased risk of childhood AIS (95% CI: 1.03-4.73) was identified in FXIII-A Leu34 allele carriers. Combined FV Leiden/FV HR2, FV Leiden/MTHFR A1298C, FV Leiden/MTHFR C677T/MTHFR A1298C, and FV Leiden/FV HR2/MTHFR A1298C heterozygosity was identified in children with AIS but not in controls, which revealed a statistically significant difference. This case-control study shows that besides already documented association between FV Leiden and AIS, other previously unreported polymorphisms (FXIII-A p.Val34Leu, HPA-3) and several genotype combinations that always include heterozygous FV Leiden can be related to AIS in Croatian population.