Sibling Species within Paramecium jenningsi Revealed by PCR-RFLP

Summary. Paramecium jenningsi Diller et is classified in the so-called "aurelia" subgroup. The original assumption of the monomorphic status of P. jenningsi (Sonneborn 1970) has been repeatedly challenged over the years with the application of increasingly sophisticated methods and knowledge of the geographic distribution of particular strains of this species. Investigations concerning genetic polymorphism suggested the existence of two sibling species in P. jenningsi, comprised of strains inhabiting India, Saudi Arabia, China and Japan. In order to confirm the existence of these sibling species, here we apply the PCR-RFLP method for analysis of a fragment of the hsp70 gene coding for the Hsp70 cytosol heat shock protein. PCR-RFLP with enzymes Alu1I and EcoRI did not reveal size polymorphism of restriction fragments. Band patterns obtained with enzyme Tru1I showed variation corresponding to genetic polymorphism and were subject to comparative analysis using the BIO1D program which applies Nei and Li's and Jaccard's similarity coefficients. The results of these analyses are homology trees showing the genetic similarity between West-Central Asian and Japanese groups, the intra-group similarity coefficients had a value of 1, i.e. the strains were 100% homologous. The similarity between both of these groups was calculated as 32% and 20% depending on which coefficient was used. West-Central Asian and Japanese groups detected in this study on the basis of PCR-RFLP result can be considered separate species after interpretation of inter-strain crosses, which have shown the existence of reproductive isolation barriers between West-Central Asian and Japanese strains on the basis of the percentage of surviving clones

The influence of high temperature on the possibility of DNA typing in various human tissues

Introduction. The identification of unknown victims of high temperatures (fire, terrorist attack, and other disasters) is one of the most difficult tasks faced by forensic geneticists. The main aim of this study was to in­vestigate the availability of DNA isolated from various human tissue samples exposed to high temperatures of 100–1000°C for 5 and 10 minutes. Material and methods. Samples of varying thickness of thigh muscle, liver, heart, adipose tissue, bone, teeth, hair and nails of 52 fresh cadavers and 59 healthy teeth of 29 volunteers were used. The study was performed using the following commercially available STR (Short Tandem Repeats) and miniSTR kits: AmpFlSTR®SGM Plus® and AmpFlSTR®MiniFilerTM. Hyper variable region I (HVI) of human mitochondrial DNA (mtDNA) was sequenced with BigDye Terminator Cycle Sequencing Kit 1.1. The PEP (Primer-Extension Preamplification) method was used for the whole human genome amplification. Results. It was possible to obtain complete DNA profiles (AmpFlSTR®SGM Plus®, AmpFlSTR®MiniFilerTM Applied Biosystems, USA and mtDNA HVI region) for tissue samples of heart, liver and thigh muscle, exposed up to 900°C for 5 min. However, under the applied conditions, limited usefulness of hair, nails and teeth for identification purposes was shown. Conclusions. DNA stability in tissues subjected to incineration depends on many factors, like tissue type and its thickness, temperature and time of exposure. In the cases of human remains exposed to high temperatures, samples of soft tissues of the highest weight (thickness) provide the best chance of successful identification through the genetic analysis. In some cases of negative results, even if using mtDNA typing, application of the whole genome amplification (WGA) technique could provide the expected results for highly degraded DNA templates

Whole genome amplification of degraded and nondegraded DNA for forensic purposes

Degraded DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded DNA is of great importance, since its results impact the quality and reliability of expert testimonies. Recently, a number of whole genome amplification (WGA) methods have been proposed as preamplification tools. They work on the premise of being able to generate microgram quantities of DNA from as little as the quantity of DNA from a single cell. We chose, investigated, and compared seven WGA methods to evaluate their ability to “recover” degraded and nondegraded DNA: degenerate oligonucleotide-primed PCR, primer extension preamplification PCR, GenomePlex™ WGA commercial kit (Sigma), multiple displacement amplification, GenomiPhi™ Amplification kit (Amersham Biosciences), restriction and circularization-aided rolling circle amplification, and blunt-end ligation-mediated WGA. The efficiency and reliability of those methods were analyzed and compared using SGMPlus, YFiler, mtDNA, and Y-chromosome SNP typing. The best results for nondegraded DNA were obtained with GenomiPhi and PEP methods. In the case of degraded DNA (200 bp), the best results were obtained with GenomePlex which successfully amplified also severely degraded DNA (100 bp), thus enabling correct typing of mtDNA and Y-SNP loci. WGA may be very useful in analysis of low copy number DNA or degraded DNA in forensic genetics, especially after introduction of some improvements (sample pooling and replicate DNA typing). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00414-012-0764-9) contains supplementary material, which is available to authorized users

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process

Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools

Modified and unmodified zinc oxide as coagent in elastomer compounds

The aim of this work was to study the activity of unmodifi ed and modifi ed ZnO in the peroxide crosslinking of hydrogenated acrylonitrile-butadiene elastomer (HNBR) and ethylene-propylene copolymer (EPM). In the first step, zinc oxide was obtained by emulsion precipitation. Maleic acid was introduced onto the surface of ZnO using an in situ method. The unmodifi ed and modifi ed zinc oxide was characterized using dispersive and morphological analysis, BET surface area analysis, and elemental, spectroscopic and thermal analysis. In the second stage of the research, the ZnO/MA systems were incorporated into the structure of elastomer compounds improving the kinetic and mechanical properties of vulcanizates. The proposed modifi cation method had a favorable effect on the physicochemical properties of the zinc oxide and on the kinetic and mechanical properties of the vulcanizates. This study demonstrated that modifi cation of zinc oxide by maleic acid is a promising technique

The Influence of the Differentiation of Genes Encoding Peroxisome Proliferator-Activated Receptors and Their Coactivators on Nutrient and Energy Metabolism

Genetic components may play an important role in the regulation of nutrient and energy metabolism. In the presence of specific genetic variants, metabolic dysregulation may occur, especially in relation to the processes of digestion, assimilation, and the physiological utilization of nutrients supplied to the body, as well as the regulation of various metabolic pathways and the balance of metabolic changes, which may consequently affect the effectiveness of applied reduction diets and weight loss after training. There are many well-documented studies showing that the presence of certain polymorphic variants in some genes can be associated with specific changes in nutrient and energy metabolism, and consequently, with more or less desirable effects of applied caloric reduction and/or exercise intervention. This systematic review focused on the role of genes encoding peroxisome proliferator-activated receptors (PPARs) and their coactivators in nutrient and energy metabolism. The literature review prepared showed that there is a link between the presence of specific alleles described at different polymorphic points in PPAR genes and various human body characteristics that are crucial for the efficacy of nutritional and/or exercise interventions. Genetic analysis can be a valuable element that complements the work of a dietitian or trainer, allowing for the planning of a personalized diet or training that makes the best use of the innate metabolic characteristics of the person who is the subject of their interventions

1,N 6 -α-hydroxypropanoadenine, the acrolein adduct to adenine, is a substrate for AlkB dioxygenase

1,N6-α-hydroxypropanoadenine (HPA) is an exocyclic DNA adduct of acrolein – an environmental pollutant and endocellular oxidative stress product. Escherichia coli AlkB dioxygenase belongs to the superfamily of α-ketoglutarate (αKG)- and iron-dependent dioxygenases which remove alkyl lesions from bases via an oxidative mechanism, thereby restoring native DNA structure. Here, we provide in vivo and in vitro evidence that HPA is mutagenic and is effectively repaired by AlkB dioxygenase. HPA generated in plasmid DNA caused A → C and A → T transversions and, less frequently, A → G transitions. The lesion was efficiently repaired by purified AlkB protein; the optimal pH, Fe(II), and αKG concentrations for this reaction were determined. In vitro kinetic data show that the protonated form of HPA is preferentially repaired by AlkB, albeit the reaction is stereoselective. Moreover, the number of reaction cycles carried out by an AlkB molecule remains limited. Molecular modeling of the T(HPA)T/AlkB complex demonstrated that the R stereoisomer in the equatorial conformation of the HPA hydroxyl group is strongly preferred, while the S stereoisomer seems to be susceptible to AlkB-directed oxidative hydroxylation only when HPA adopts the syn conformation around the glycosidic bond. In addition to the biochemical activity assays, substrate binding to the protein was monitored by differential scanning fluorimetry allowing identification of the active protein form, with cofactor and cosubstrate bound, and monitoring of substrate binding. In contrast FTO, a human AlkB homolog, failed to bind an ssDNA trimer carrying HPA

Phage-borne depolymerases decrease Klebsiella pneumoniae resistance to innate defense mechanisms

Klebsiella pneumoniae produces capsular polysaccharides that are a crucial virulence factor protecting bacteria against innate response mechanisms of the infected host. Simultaneously, those capsules are targeted by specific bacteriophages equipped with virion-associated depolymerases able to recognize and degrade these polysaccharides. We show that Klebsiella phage KP32 produces two capsule depolymerases, KP32gp37 and KP32gp38, with a high specificity for the capsular serotypes K3 and K21, respectively. Together, they determine the host spectrum of bacteriophage KP32, which is limited to strains with serotype K3 and K21. Both depolymerases form a trimeric beta-structure, display moderate thermostability and function optimally under neutral to alkaline conditions. We show that both depolymerases strongly affect the virulence of K. pneumoniae with the corresponding K3 and K21 capsular serotypes. Capsule degradation renders the otherwise serum-resistant cells more prone to complement-mediated killing with up to four log reduction in serum upon exposure to KP32gp37. Decapsulated strains are also sensitized for phagocytosis with a twofold increased uptake. In addition, the intracellular survival of phagocytized cells in macrophages was significantly reduced when bacteria were previously exposed to the capsule depolymerases. Finally, depolymerase application considerably increases the lifespan of Galleria mellonella larvae infected with K. pneumoniae in a time- and strain-dependent manner. In sum, capsule depolymerases are promising antivirulence compounds that act by defeating a major resistance mechanism of K. pneumoniae against the innate immunity

Tinnitus and its effect on working memory and attention

Wstęp. Funkcje poznawcze są odpowiedzialne za odbieranie informacji otoczenia. Z powodu nadmiernej liczby docierających bodźców istnieje konieczność ich selekcji i redukcji. Szumy uszne stanowią niepożądane subiektywne wrażenia słuchowe. W ich odczuwanie zaangażowany jest nie tylko układ słuchowy, ale i układ limbiczny oraz autonomiczny układ nerwowy. Deficyty uwagi są stosunkowo częstą skargą pacjentów z szumami. Cel pracy. Ocena wpływu szumów usznych na sprawność uwagi. Materiał i metody. Ocenie audiologicznej i psychologicznej — test uwagi d2: wskaźniki szybkości (WZ), ogólna zdolność spostrzegania (WZ–B), zdolność koncentracji (ZK), poddano 30 osób (32–54 lat) z szumami usznymi; określono stopień dokuczliwości szumów w skali 0–10. Wyniki. Średnia dokuczliwość szumów 6,89 ± 2,30. Większość badanych uzyskała wysokie i bardzo wysokie wyniki w teście d2: WZ = 58%, WZ–B = 50%, ZK = 75%. Wnioski. Osoby doświadczające szumów usznych nie wykazują (wbrew deklaracjom) zaburzeń uwagi dowolnej podczas wykonywania zadań celowych i wymagających skupienia, dysponują zapewne mechanizmami kompensacyjnymi.Introduction. Cognitive functions are responsible for perception and analysis timulus from environment. There is a necessity to reduce the information. Tinnitus is the intrusive sensation of hearing ringing, buzzing or other sound. Some different systems are involved in this umpleasent sensation such as the hearing, the limbic system and the autonomic one. In severe cases tinnitus can cause people to have difficulty concentrating. Aim of the study. Influence of tinnitus on the ability to concentrate and on attention. Material and methods. 30 patients with chronic tinnitus aged 32–45 were evaluated, audiological tests were done and psychological one — d2 test. Results. Tinnitus were annoying for all patients — the average level 6.89 ± 2.30. Most of patients got very high result from d2 test (speed index 58%, general perception 50%, concentration 75%). Conclusions. Complaints of the distracting effects of tinnitus do not have a basis in performance test outcomes despite patients` declarations. The results seem to indicate that the most likely cause is well developed compensation