Expression and cellular distribution of zona pellucida glycoproteins in canine oocytes before and after in vitro maturation.

This study was aimed at investigating zona pellucida glycoproteins (ZP) ZP2, ZP3 mRNA expression as well as ZP3, ZP4 (ZPB) protein distribution before and after in vitro maturation (IVM) in canine oocytes. The cumulus-oocyte complexes (COCs) were recovered from 27 anoestrous mongrel bitches and matured for 72 h in TCM199 medium. The canine COCs were analysed before and after IVM. Using real-time quantitative polymerase chain reaction (RQ-PCR), both groups of oocytes were analysed for detection of ZP2 and ZP3 mRNA profiles as well as using confocal microscopic analysis for observation of ZP3 and ZP4 protein distribution. In post-IVM canine oocytes an increase in transcript content of ZP2 and ZP3 genes as well as a decrease in ZP3 and ZP4 protein levels were observed when compared with pre-IVM oocytes. Moreover, the ZP4 protein before IVM was significantly distributed in the peripheral area of cytoplasm, whereas after IVM it was localized rather than in the entire cytoplasm. In contrast, the ZP3 protein was found both before and after IVM was distributed in the peripheral area of the cytoplasm. In conclusion, we suggest that the expression of ZP2 and ZP3 genes is associated with the maturation stage of canine oocytes, as higher mRNAs levels were found after IVM. However, a decreased expression of ZP3 and ZP4 proteins after IVM suggests maturation-dependent down-regulation of these protein translations, which may result in disturbed fertilization.

The influence of transfer gun passage time through the uterine cervix on pregnancy rate in recipient heifers.

The influence of passage time of the transfer gun through the uterine cervix and body to the embryo insertion site on pregnancy rate was analysed in 248 recipient heifers (mean age: 15-17 months). Embryos (90 fresh and/or 88 and 70 frozen in glycerol and ethylene glycol, respectively, grades 4 and 5, stage 1 or 2) were transferred to the ipsilateral uterine horn on day 7. Two different transfer guns were used in this experiment: a sterilisable steel transfer instrument to be used without sheaths with a removable tip made of gold-plated stainless steel (Wörrlein Minitüb) or a transfer stylet with sheaths with a metal tip and a side opening (Cassou gun, IMV Technologies). The time of passage of the instruments through the uterine cervix and body to the site of embryo deposition in the uterine horn was measured in the study. In order to randomise the risk of errors, all manipulations were carried out by the same experienced operator. The average time needed for the insertion of embryos into the uterus was 50.6 seconds (s) and it was longer for the transfer gun with sheaths than for the metal-tipped transfer gun (60.1 and 40.8 s, respectively) (P < 0.001). The average conception rate was 45.6%. If the time needed to insert embryos into the uterus was 10-60 s, the conception rate was 53.4% (up to 20, 21-30, 31-40, 41-50 and 51-60 s - 57.7, 52.5, 50, 51.5 and 50%, respectively). In contrast, if the time needed to insert the embryo in the uterine horn was longer than 60 s, the conception rate was 20.4% (61-80, 80.1-120 and > 120 s - 28.0, 6.0 and 24.9%, respectively). Thus, it cannot be excluded that the type of the applied transfer gun may influence pregnancy rate in recipient cows due to its effect on cervical passage time.

Materials Selection and Construction Development for Ensuring the Availability and Durability of the Molten Hydroxide Electrolyte Direct Carbon Fuel Cell (MH-MCFC).

The molten hydroxide electrolyte Direct Carbon Fuel Cell (MH-DCFC) is a promising type of DCFC due to its advantages, such as high ionic conductivity, higher electrochemical activity of carbon (higher anodic oxidation rate and lower overpotentials) and high efficiency of carbon oxidation due to lower operating temperature (the dominant product of carbon oxidation is CO2 vs. CO). Accordingly, the MH-DCFC can be operated at lower temperatures (roughly 673-873 K), and thus cheaper materials can be used to manufacture the cell. Nonetheless, MH-DCFCs are still under development due to several fundamental and technological challenges such as corrosion problems. Selection of materials and development of a structure that ensures adequate availability and durability of the cell is crucial for the optimization of the MH-DCFC performance and the further development of that technology. This article presents the operating characteristics of the MH-DCFC made of different construction materials, such as carbon steel, stainless steel, and nickel and its alloys. Nickel and its alloys have proven to be the best materials for the construction of individual elements of the fuel cell. Inconel alloy 600 was a good catalytic material for cathodes with good corrosion resistance.

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 100­1000°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.

Effect of sintering atmosphere on properties of porous stainless steel for biomedical applications.

This study discusses manufacturing of metallic biomaterials by means of powder metallurgy with consideration for their unquestionable advantages, i.e. opportunities of obtaining materials with controllable porosity. The paper focuses on properties of 316 L stainless steel obtained using the method of powder metallurgy with respect to compacting pressure and sintering atmosphere. All the specimens were compacted at 700, 400 and 225 MPa, and sintered at 1250 °C. In order to analyze the sintering atmosphere, three different media were used: dissociated ammonia, hydrogen and vacuum. The study covered sintering density, porosity, microstructure analysis and corrosion resistance. The proposed method of powder metallurgy allowed for obtaining materials with predictable size and distribution of pores, depending on the parameters of sinter preparation (compaction force, sinter atmosphere). High corrosion resistance of the materials (sintering in the atmosphere of hydrogen and in vacuum) and high porosity in the sinters studied offer opportunities for using them for medical purposes.

Follicular size is associated with the levels of transcripts and proteins of selected molecules responsible for the fertilization ability of oocytes of puberal gilts.

The maturation and developmental competence of the oocyte is acquired during folliculogenesis. It is still unclear whether follicle size is associated with the levels of transcript and protein encoding molecules contributing to the fertilization ability of the porcine oocyte. Follicles were dissected from porcine ovaries after slaughter and classified as small (< 3 mm), medium (3-5 mm) or large (>5 mm), aspirated cumulus-oocyte complexes were cultured in standard porcine IVM culture medium (TCM 199) for 44 h. In developmentally competent oocytes, assessed by determining the activity of glucose-6-phosphate dehydrogenase (G6PDH) using a brilliant cresyl blue (BCB) test, real-time quantitative PCR reaction methods, western-blot and confocal microscopy analysis were applied to determine the transcript levels of porcine zona pellucida glycoproteins pZP1, pZP2, pZP3, pZP3 alpha and integrins beta 1 and beta 2, as well as the levels of pZP3 and integrin beta 2 proteins. We observed significantly higher levels of pZP1, pZP3 and integrin beta1 and beta2 transcripts in oocytes collected from medium follicles as compared with small follicles (P<0.001). Moreover, we found an increased content of all investigated mRNAs in oocytes isolated from large follicles as compared with small follicles (P<0.001). Western-blot analysis demonstrated a higher level of pZP3 protein in oocytes isolated from large and medium follicles as compared with small follicles (P<0.001). Our results suggest that the levels of transcripts and proteins for selected molecules contributing to the fertilization ability of oocytes are associated with follicular size in puberal gilts.

[Forensic biological traces as a "portrait" of perpetrators of murder and other offences]. / Kryminalistyczne slady biologiczne "portretem" sprawców zabójstw i innych przestepstw.

INTRODUCTION: In the introduction remarks will be provided on inspections of the sites of murder or of other offences, conducted with the participation of a forensic physician or other experts of forensic biology. The goal of this paper is to make interested parties aware of the possibility of numerous biological traces and micro-traces found on the scenes of diverse crimes, which many a time are omitted. The presence of those traces prompts one to strive for individualised identification of perpetrators of murder based upon the material they leave behind. In case of problems with actual identification of a perpetrator there is a possibility of a group-wise identification, accompanied by the creation of one but specific version of the subject occurrence. Those type of actions may be carried out thanks to the application of modern investigation methods and materials at individual stages of looking at evidence. Furthermore, a whole procedural algorithm is developed, from discovering a biological trace through its technical and procedural preservation, inspections of the victim, and perpetrator and collection of comparative materials from both of them, selection of appropriate methods and measures till the eventual findings i.e. individualisation of man. In the paper morphologic properties of forensic material of human origin (tissues, secretions, excrements, nail and teeth scratches) will be exposed; the material will be classified and on that basis some pre-selected material will undergo further analyses with particular attention drawn to molecular researches. CONCLUSIONS: Conclusions will include the finding of biological micro-traces being often times omitted but which are of importance and thanks to currently applied equipment and genetic methods may lead to individual identification on the basis of a very small amount of evidential material collected.