ONT-Based Alternative Assemblies Impact on the Annotations of Unique versus Repetitive Features in the Genome of a Romanian Strain of Drosophila melanogaster

To date, different strategies of whole-genome sequencing (WGS) have been developed in order to understand the genome structure and functions. However, the analysis of genomic sequences obtained from natural populations is challenging and the biological interpretation of sequencing data remains the main issue. The MinION device developed by Oxford Nanopore Technologies (ONT) is able to generate long reads with minimal costs and time requirements. These valuable assets qualify it as a suitable method for performing WGS, especially in small laboratories. The long reads resulted using this sequencing approach can cover large structural variants and repetitive sequences commonly present in the genomes of eukaryotes. Using MinION, we performed two WGS assessments of a Romanian local strain of Drosophila melanogaster, referred to as Horezu_LaPeri (Horezu). In total, 1,317,857 reads with a size of 8.9 gigabytes (Gb) were generated. Canu and Flye de novo assembly tools were employed to obtain four distinct assemblies with both unfiltered and filtered reads, achieving maximum reference genome coverages of 94.8% (Canu) and 91.4% (Flye). In order to test the quality of these assemblies, we performed a two-step evaluation. Firstly, we considered the BUSCO scores and inquired for a supplemental set of genes using BLAST. Subsequently, we appraised the total content of natural transposons (NTs) relative to the reference genome (ISO1 strain) and mapped the mdg1 retroelement as a resolution assayer. Our results reveal that filtered data provide only slightly enhanced results when considering genes identification, but the use of unfiltered data had a consistent positive impact on the global evaluation of the NTs content. Our comparative studies also revealed differences between Flye and Canu assemblies regarding the annotation of unique versus repetitive genomic features. In our hands, Flye proved to be moderately better for gene identification, while Canu clearly outperformed Flye for NTs analysis. Data concerning the NTs content were compared to those obtained with ONT for the D. melanogaster ISO1 strain, revealing that our strategy conducted to better results. Additionally, the parameters of our ONT reads and assemblies are similar to those reported for ONT experiments performed on various model organisms, revealing that our assembly data are appropriate for a proficient annotation of the Horezu genome.     

pH and heat-induced structural changes of bovine apo-α-lactalbumin

The effects of pH and thermal treatments on conformation and association state of Ca²⁺-depleted bovine α-lactalbumin (apo-α-LA) have been studied by fluorescence spectroscopy, and molecular modelling. The experimental results demonstrate a third-state model for heat-induced unfolding of apo-α-LA, at pH 2.0, and an all-or-none transition of apo-α-LA, at pH 4.5 and 7.0, respectively. The heat-induced changes in the secondary and tertiary structure of α-LA were outlined after running molecular dynamics simulations at 25 °C and 80 °C, at neutral pH, therefore supporting the experimental observations. Our data provides insight into the mechanism of pH- and heat-dependent structural changes and oligomerization of α-LA, and will be helpful in understanding the ability of this protein to interact with certain compounds of biological interest.     

Gene clustering for time-series microarray with production outputs

The identification of coexpressed genes from microarray data is a challenging problem in bioinformatics and computational biology. The objective of this study is to obtain knowledge about the most important genes and clusters related to production outputs of real-world time-series microarray data in the industrial microbiology area. Each sample in the microarray data experiment is complemented with the measurement of the corresponding production and growth values. A novel aspect of this research refers to considering the relation of coexpression patterns with the measured outputs to guide the biological interpretation of results. Shape-based clustering models are developed using the pattern of gene expression values over time and further incorporating knowledge about the correlation between the change in the gene expression level and the output value. Experiments are performed for time-series microarray of bacteria, and an analysis from a biological perspective is carried out. The obtained results confirm the existence of relationships between output variables and gene expressions. Moreover, the shape-based clustering methods show promising results, being able to guide metabolic engineering actions with the identification of potential targets.     

Unconventional method used in synthesis of polyphosphoesters

Polyphosphonates, polyphosphates, polyarylazophosphonates and polyarylazophosphates were synthesized by a new alternative to the classical phase transfer catalysis, respectively, the modified inverse phase transfer catalysis (IPTC) polycondensation of various phenylphosphonic (phosphoric) dichlorides (phenylphosphonic dichloride, phenylphosphoric dichloride, p-chlorophenylphosphoric dichloride) with bisphenols: bisphenol A, tetrabromobisphenol A, bis-(4-hydroxyphenyl)methane (bisphenol F), 4,4′-dihydroxyazobenzene. The polymers were characterized by infrared spectroscopy and magnetic resonance (¹H-NMR, ³¹P-NMR, ¹³C-NMR) spectroscopy. Yields in the range of 63.5–85% and molecular weights (M _w) of ~2,000–8,200 g mol^?1 were obtained. Polyphosphonates were stable up 210–270 °C and polyphosphates up 190–220 °C in air atmosphere. For a correct estimation of the thermal behavior of similar compounds, a kinetic analysis using a modified version of Non Parametric Kinetic method for representative polyphosphonate and polyphosphate was realized. The studies made on the hydrolytic degradation of the synthesized polyphosphates show that the most stable polymer under alkali-catalyzed degradation is the polyphosphate obtained by IPTC of phenylphosphoric dichloride and bisphenol A.     

Melanocytes Affect Nodal Expression and Signaling in Melanoma Cells: A Lesson from Pediatric Large Congenital Melanocytic Nevi

Expression of Nodal, a Transforming Growth Factor-beta (TGF-β) related growth factor, is associated with aggressive melanoma. Nodal expression in adult dysplastic nevi may predict the development of aggressive melanoma in some patients. A subset of pediatric patients diagnosed with giant or large congenital melanocytic nevi (LCMN) has shown increased risk for development of melanoma. Here, we investigate whether Nodal expression can help identify the rare cases of LCMN that develop melanoma and shed light on why the majority of these patients do not. Immunohistochemistry (IHC) staining results show varying degree of Nodal expression in pediatric dysplastic nevi and LCMN. Moreover, median scores from Nodal IHC expression analysis were not significantly different between these two groups. Additionally, none of the LCMN patients in this study developed melanoma, regardless of Nodal IHC levels. Co-culture experiments revealed reduced tumor growth and lower levels of Nodal and its signaling molecules P-SMAD2 and P-ERK1/2 when melanoma cells were grown in vivo or in vitro with normal melanocytes. The same was observed in melanoma cells cultured with melanocyte conditioned media containing pigmented melanocyte derived melanosomes (MDM). Since MDM contain molecules capable of inactivating radical oxygen species, to investigate potential anti-oxidant effect of MDM on Nodal expression and signaling in melanoma, melanoma cells were treated with either N-acetyl-l-cysteine (NAC), a component of the anti-oxidant glutathione or synthetic melanin, which in addition to providing pigmentation can also exert free radical scavenging activity. Melanoma cells treated with NAC or synthetic melanin showed reduced levels of Nodal, P-SMAD2 and P-ERK1/2 compared to untreated melanoma cells. Thus, the potential role for Nodal in melanoma development in LCMN is less evident than in adult dysplastic nevi possibly due to melanocyte cross-talk in LCMN capable of offsetting or delaying the pro-melanoma effects of Nodal via anti-oxidant effects of MDM.     

Comparative Study of 9×19 mm Ammunition Combustion Products and Residues

During the last decades, authorities’ awareness on environmental and human health impact at national and international level has increased in the field of small arms ammunition containing lead and antimony. Thus, the evolution of environmental policies regarding production and use of heavy metals and their compounds, especially in EU countries, implied extensive studies on the environmental implications of 9×19 mm caliber ammunition use, especially in indoor shooting ranges. In this context, the paper describes the experimental studies performed on Pb‐containing conventional FMJ (full metal jacketed) ammunition and comparative measurements on new ammunition designed for training. The combustion products and residues from conventional Pb‐containing ammunition and Cu‐based “greener” ammunition were compared in terms of gaseous products, metal concentration in aerosols and metal concentrations in solid residues. Gas emissions and solid residues were measured for four different types of ammunitions. The results showed that the copper content of the residue is significantly higher in case of Cu‐containing composite bullets. For the ammunition equipped with Pb bullet, an important percentage of the Pb residue is generated by erosion of the exposed part of the bullet core. On the other hand, the ammunition containing Cu‐composite bullet generates large quantities of Cu residues, which have similar environmental impact and less impact on human health. Gas analysis indicated the presence of CO and NO_x and, surprisingly, high concentrations of HCN, CH₄, and NH₃. Lower gas concentrations are obtained for the composite bullets due to an incomplete combustion of gun propellant.     

pH- and heat-induced structural changes of bovine α-lactalbumin in response to oleic acid binding

Bovine α-lactalbumin (α-LA) is able to interact with fatty acids, resulting in structural changes that are potentially responsible for the HAMLET/BAMLET role. Different states of α-LA induced by pH, temperature and fatty acid binding have been examined. Evidences of the structural changes of α-LA in molten globule and native states in correlation with oleic acid (OA) binding are shown using fluorescence spectroscopy and in silico approach. In addition, the α-LA was subjected to automated docking analysis, to better understand the interaction with oleic acid, using the PatchDock algorithm. The experimental results demonstrate a more flexible conformation of the protein at pH 2.5 when compared to neutral pH, thus facilitating the oleic acid binding to α-LA. The quenching experiments indicate the remarkable increase in the content of molten globule state at pH 2.5 and a more compact and rigid structure for α-LA–OA complexes at pH 7.0. The docking results are consistent with the experimental data concerning the thermal stability of the α-LA–OA complex. α-LA in different conformations/complexes was sensitive to pH and temperature. Several different molecular species induced by pH, heat treatment and oleic acid binding were suggested. The structure of the protein was more flexible at acidic pH, therefore favoring the hydrophobic exposure.     

Cabernet sauvignon red wine astringency quality control by tannin characterization and polymerization during storage

Cabernet Sauvignon wine grape variety is an emblematic cultivar of Bordeaux winegrowing region. Twenty-three vintages of Cabernet Sauvignon wines from chateau Mouton Rothschild were studied using chemical and sensory analysis. Total phenolic compounds, total anthocyanins, total tannins, hue, CI (colour intensity), titratable acidity, ethanol level and pH were evaluated. Percentage of galloylation (%G), of prodelphinidins (%P) as well as mean degree of polymerization (mDP) were also determined. Sensory analysis concerning astringency intensity using a 0–7 point scale was performed in parallel. Correlations between bottle wine ageing and mDP and astringency were obtained. Increasing age gave lower scores in astringency intensity (R ² = 0.598, p = 0.000) and inferior mDP values (R ² = 0.851, p = 0.000). In addition, we evidenced that mDP is an analytical parameter for estimating astringency (R ² = 0.556, p = 0.000) in red CS (Cabernet Sauvignon) wine from Pauillac denomination.