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.     

Effects of Sulforaphane-Induced Cell Death upon Repeated Passage of Either P-Glycoprotein-Negative or P-Glycoprotein-Positive L1210 Cell Variants

The expression of the membrane ABCB1 transporter in neoplastic cells is one of the most common causes of reduced sensitivity to chemotherapy. In our previous study, we investigated the effect of a single culture of ABCB1-negative (S) and ABCB1-positive variants of L1210 cells (R and T) in the presence of sulforaphane (SFN). We demonstrated that SFN induces the onset of autophagy more markedly in S cells than in R or T cells. In the current study, we focused on the effect of the repeated culture of S, R and T cells in SFN-containing media. The repeated cultures increased the onset of autophagy compared to the simple culture, mainly in S cells and to a lesser extent in R and T cells, as indicated by changes in the cellular content of 16 and 18 kDa fragments of LC3B protein or changes in the specific staining of cells with monodansylcadaverine. We conclude that SFN affects ABCB1-negative S cells more than ABCB1-positive R and T cells during repeated culturing. Changes in cell sensitivity to SFN appear to be related to the expression of genes for cell-cycle checkpoints, such as cyclins and cyclin-dependent kinases.     

Externality, internality, and (in)dispensability of grammatical features in speech production: Evidence from Czech declension and conjugation.

In 3 picture–word experiments, the authors explored the activation of 2 grammatical features in Czech during lexical access: declensional class of nouns and conjugational class of verbs. Experiments 1 and 2 demonstrated congruency effects of declensional and conjugational class, respectively. Picture naming times were reliably longer if the declensional or conjugational classes of the pictures' names and the distractors were incongruent. Experiment 3 explored the origin of the congruency effect in more detail. Congruency effects were obtained for declensional class regardless of whether the target name and the distractor differed in form, speaking for competition at the lemma level. These findings are discussed in comparison with gender congruency effects. The authors propose a differentiation between externally and internally specified features of lemmas, especially with respect to the time course of their activation. Internal features that become available only when the lemma is activated (e.g., gender, declensional or conjugational class of nouns and verbs) can be bypassed or not, depending on the grammatical specification of the earlier available external features (like case or number). Following this argument, supposedly inconsistent findings regarding grammatical gender and declensional or conjugational class can be explained straightforwardly. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Cardiovascular,Metabolic and Inflammatory Changes after Ovariectomy and Estradiol Substitution in Hereditary Hypertriglyceridemic Rats

Background: If menopause is really independent risk factor for cardiovascular disease is still under debate. We studied if ovariectomy in the model of insulin resistance causes cardiovascular changes, to what extent are these changes reversible by estradiol substitution and if they are accompanied by changes in other organs and tissues. Methods: Hereditary hypertriglyceridemic female rats were divided into three groups: ovariectomized at 8th week (n = 6), ovariectomized with 17-β estradiol substitution (n = 6), and the sham group (n = 5). The strain of abdominal aorta measured by ultrasound, expression of vascular genes, weight and content of myocardium and also non-cardiac parameters were analyzed. Results: After ovariectomy, the strain of abdominal aorta, expression of nitric oxide synthase in abdominal aorta, relative weight of myocardium and of the left ventricle and circulating interleukin-6 decreased; these changes were reversed by estradiol substitution. Interestingly, the content of triglycerides in myocardium did not change after ovariectomy, but significantly increased after estradiol substitution while adiposity index did not change after ovariectomy, but significantly decreased after estradiol substitution. Conclusion: Vascular and cardiac parameters under study differed in their response to ovariectomy and estradiol substitution. This indicates different effects of ovariectomy and estradiol on different cardiovascular but also extracardiac structures.     

An adsorptive transfer technique coupled with brdicka reaction to reveal the importance of metallothionein in chemotherapy with platinum based cytostatics

The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of chemotherapy failure. With regard to platinum based drugs, multidrug resistance can also be connected with increased expression of low-molecular weight protein metallothionein (MT). This study aimed at investigating the interactions of MT with cisplatin or carboplatin, using the adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction (AdTS DPV Brdicka reaction), and a comparison of in vitro results with results obtained in vivo. The results obtained from the in vitro study show a strong affinity between platinum based drugs and MT. Further, we analyzed extracts of neuroblastoma cell lines treated with cisplatin or carboplatin. It is clear that neuroblastoma UKF-NB-4 cisplatin-resistant and cisplatin-sensitive cell lines unlikely respond to the presence of the platinum-based cytostatics cisplatin and carboplatin. Finally, we determined the level of MT in samples from rabbits treated with carboplatin and patients with retinoblastoma treated with the same drug.     

Synthesis and characterization of collagen/hydroxyapatite: magnetite composite material for bone cancer treatment

Our purpose was obtaining and characterizing a complex composite system with multifunctional role: bone graft material and hyperthermia generator necessary for bone cancer therapy. The designed system was a magnetite enriched collagen/hydroxyapatite composite material, obtained by a co-precipitation method. Due to the applied electromagnetic field the magnetite will induce hyperthermia and cause tumoral cell apoptosis. The complex bone graft system was characterised by XRD, FTIR and SEM, while the hyperthermia was quantify by measuring the temperature increase due to the applied alternative electromagnetical field.     

Comparative Antimicrobial Activity of Silver Nanoparticles Obtained by Wet Chemical Reduction and Solvothermal Methods

The synthesis of nanoparticles from noble metals has received high attention from researchers due to their unique properties and their wide range of applications. Silver nanoparticles (AgNPs), in particular, show a remarkable inhibitory effect against microorganisms and viruses. Various methods have been developed to obtain AgNPs, however the stability of such nanostructures over time is still challenging. Researchers attempt to obtain particular shapes and sizes in order to tailor AgNPs properties for specific areas, such as biochemistry, biology, agriculture, electronics, medicine, and industry. The aim of this study was to design AgNPs with improved antimicrobial characteristics and stability. Two different wet chemical routes were considered: synthesis being performed (i) reduction method at room temperatures and (ii) solvothermal method at high temperature. Here, we show that the antimicrobial properties of the obtained AgNPs, are influenced by their synthesis route, which impact on the size and shape of the structures. This work analyses and compares the antimicrobial properties of the obtained AgNPs, based on their structure, sizes and morphologies which are influenced, in turn, not only by the type or quantities of precursors used but also by the temperature of the reaction. Generally, AgNPs obtained by solvothermal, at raised temperature, registered better antimicrobial activity as compared to NPs obtained by reduction method at room temperature.     

Carbon nanotubes prepared from three-layered copolymer microspheres of acrylonitrile and methylmethacrylate

Carbon nanotubes (CNTs) were synthesized from fine three-layered copolymer microspheres using the polymer blend technique. Diameter of PMMA core/Poly(AN-co-MMA) shell-1/PMMA shell-2 microspheres, prepared by a radical soap-free emulsion polymerization of methylmethacrylate (MMA) and acrylonitrile (AN), was between 400 nm and 500 nm. Microspheres were subjected to melt-spinning at 305 °C, stabilizing in oxygen at 220 °C for 4 h, and finally carbonizing at 1000 °C for 30 min. FE-SEM study of carbonized sample revealed the presence of CNTs arrays on carbon blocks. Similar arrays were observed in a comparative CNTs sample prepared from three-layered microspheres with the pure PAN shells-1 layers. HRTEM showed that the CNTs derived from copolymer microspheres had different structure when compared to the control sample, i.e. CNTs often adhered to each other and contained the internal compartments. The insufficient PMMA shell-2 coating of copolymer microspheres is believed to be a reason for CNTs adhesion. The possible mechanisms of the carbon block formation and the adhesion of CNTs are introduced.     

A protocol for processing the delicate larval and prepupal salivary glands of Drosophila for scanning electron microscopy

Although scanning electron microscopy (SEM) has been broadly used for the examination of fixed whole insects or their hard exoskeleton‐derived structures, including model organisms such as Drosophila, the routine use of SEM to evaluate vulnerable soft internal organs and tissues was often hampered by their fragile nature and frequent surface contamination. Here, we describe a simple four‐step protocol that allows for the reliable and reproducible preparation of the larval and prepupal salivary glands (SGs) of Drosophila for SEM devoid of any surface contamination. The steps are to: first, proteolytically digest the adhering fat body; second, use detergent washes to remove contaminating coarse tissue fragments, including sticky remnants of the fat body; third, use nonionic emulsifying polysorbate emulsifiers to remove fine contaminants from the SGs surface; and fourth, use aminopolycarboxylate‐based chelating agents to detach sessile hemocytes. Short but repeated rinses in 100 μL of a saline‐based buffer between steps ensure efficient removal of remnants removed by each treatment. After these steps, the SGs are fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, critically point‐dried, mounted on aluminum stubs, sputter coated with gold–palladium alloy and examined in the SEM.