Gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin primes cortical microglia to tissue injury.

Recent studies have shown that the aryl hydrocarbon receptor (AhR) is expressed in the brain's native immune cells, known as microglia. However, while the impact of exposure to AhR ligands is well studied in the peripheral immune system, the impact of such exposure on immune function in the brain is less well defined. Microglia serve dual roles in providing synaptic and immunological support for neighboring neurons and in mediating responses to environmental stimuli, including exposure to environmental chemicals. Because of their dual roles in regulating physiological and pathological processes, cortical microglia are well positioned to translate toxic stimuli into defects in cortical function via aberrant synaptic and immunological functioning, mediated either through direct microglial AhR activation or in response to AhR activation in neighboring cells. Here, we use gene expression studies, histology, and two-photon in vivo imaging to investigate how developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a high-affinity and persistent AhR agonist, modulates microglial characteristics and function in the intact brain. Whole cortical RT-qPCR analysis and RNA-sequencing of isolated microglia revealed that gestational and lactational TCDD exposure produced subtle, but durable, changes in microglia transcripts. Histological examination and two-photon in vivo imaging revealed that while microglia density, distribution, morphology, and motility were unaffected by TCDD exposure, exposure resulted in microglia that responded more robustly to focal tissue injury. However, this effect was rectified with depletion and repopulation of microglia. These results suggest that gestational and lactational exposure to AhR ligands can result in long-term priming of microglia to produce heightened responses towards tissue injury which can be restored to normal function through microglial repopulation.

Acute 2,3,7,8-Tetrachlorodibenzo-p-dioxin exposure in adult mice does not alter the morphology or inflammatory response of cortical microglia.

Microglia, the immune cells of the brain, have a canonical role in regulating responses to neurological disease or injury, but have also recently been implicated as regulators of neurophysiological processes such as learning and memory. Given these dual immune and physiological roles, microglia are a likely mechanism by which external toxic stimuli are converted into deficits in neuronal circuitry and subsequently function. However, while it is well established that exposure to environmental toxicants negatively affects the peripheral immune system, it remains unknown whether and how such exposure causes neuroinflammation which, in turn, may negatively impact microglial functions in vivo. Here, we examined how acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in adulthood, which negatively impacts immune cells in the periphery, affects microglial characteristics in the cortex of the mouse. We found that microglia density, distribution, morphology, inflammatory signaling, and response to a secondary, pathological activation were unaffected by acute TCDD exposure. These results suggest that acute, peripheral TCDD exposure in adulthood is not sufficient to induce an overt inflammatory phenotype in cortical microglia.

Audiological evaluation of patients with cleidocranial dysplasia (CCD).

OBJECTIVE: The description of clinical manifestation of hearing problems in cleidocranial dysplasia (CCD) remains limited and incomplete, since CCD constitutes a rare congenital disorder. The study aims to provide a complex panel analysis of the auditory system in patients suffering from the disease. PATIENTS AND METHODS: The study group consisted of 4 children with CCD (aged: 12-15), who underwent orthodontic treatment. A full panel analysis of their auditory systems was performed, including high-frequency audiometry and a new method of middle ear assessment - WBT (Wideband Tympanometry). RESULTS: A slight conductive hearing loss was diagnosed in 3 out of 4 patients. While high frequency audiometry has shown a deterioration of hearing in 3 patients, in one case, the obtained thresholds were within the normal range. A decrease of absorbance in low frequencies has been observed in one or both ears. Only one patient has had a shift of maximum absorbance towards high frequencies in the left ear. CONCLUSIONS: The presented manuscript is the first with a complete evaluation of the auditory system comprising 4 cases of children in a similar age group. All of the examined patients presented an air-bone gap indicating conductive disorders.

Identification of proteoforms of albumin and kallikrein in stallion seminal plasma and their correlations with sperm motility.

The aim of this study was to identify the proteoforms of albumin and kallikrein in stallion seminal plasma (SP), and to determine their correlations with sperm motility parameters. The experimental material consisted of ejaculates from 8 stallions, which were collected during the breeding and non-breeding seasons (BS and NBS, respectively). SP proteins were identified by 2-D PAGE and mass spectrometry (MALDI TOT/TOF MS). Sperm motility parameters were analyzed using the CASA system. Protein expression (integrated optical density-IOD) of albumin proteoforms 1 (ALB 1) and 2 (ALB 2) and kallikrein proteoforms 1 (KAL 1) and 2 (KAL 2) was correlated (p⟨0.05) with sperm motility parameters (total motility and progressive motility) during the BS. No significant correlations were found between the expression of albumin or kallikrein and sperm motility parameters during the NBS. The presence of correlations between the expression of ALB 1, ALB 2, KAL 1, KAL 2 and selected sperm motility parameters could suggest that the analyzed components of the SP belong to the group of fertility-associated proteins (FAPs).

Diversifying selection signatures among divergently selected subpopulations of Polish Red cattle.

Polish Red cattle is one of the few indigenous breeds of European red cattle which is characterized by several desired features, such as high disease resistance, good health, longevity, good fertility, and high nutritional value of milk. Currently, Polish Red cattle population is a subject of two independent breeding programs: (i) improvement program and (ii) genetic resources conservation program. The aim of the improvement program is the genetic progress in terms of milk production and body conformation traits, while the conservation program mainly focuses on protection of the genetic resources of Polish Red cattle and preservation of the existing, original gene pool. By the analysis of FST genetic distances across genome-wide SNP panel, we detected diversifying selection signatures among these two subpopulations and indicated (among others) the significance of DGAT1 and FGF2 genes for milk production traits in these cattle. We also found that among genes being presumably under selection in terms of milk production, there are genes responsible, for example, for mammary gland development (e.g., SOSTDC1, PYGO2, MED1, and CCND1) and immune system response (e.g., IL10RA, IL12B, and IL21). The most important finding of this study is that the most pronounced genetic differences between the analyzed populations were associated with ß-defensin genes (e.g., DEFB1, DEFB4A, DEFB5, DEFB7, DEFB10, DEFB13, EBD, BNBD-6, and LAP) located within so-called bovine cluster D on BTA27. The ß-defensins are expressed mainly in the mammary gland and are antimicrobial peptides against the Gram-negative and Gram-positive bacteria, viruses, and other unicellular parasites. This suggests that antimicrobial resistance of mammary gland is of high importance during selection towards increased milk production and that genes responsible for this process are selected together with increasing levels of productivity.

Effects of the platelet-activating factor (PAF) supplementation on ATP content of cryopreserved bull spermatozoa (AI).

The aim of this study was to investigate the effect of PAF supplementation in semen extender on ATP content in cryopreserved bull spermatozoa used for artificial insemination at different time intervals. Cryopreserved semen was treated with different concentrations of PAF: 1×10-5M, 1×10-6M, 1×10-7M, 1×10-8M and 1×10-9M at 37°C. In the present work we showed that content of ATP in cryopreserved semen supplemented with 1×10-9M PAF was statistically significantly higher at 90 and 120 minutes of incubation in comparison to the control group (p≤0.05). Present study indicates the potential influence of PAF on ATP content in male spermatozoa via it's protective role towards mitochondria metabolic activity.

Identification and changes in the seasonal concentrations of heat shock proteins in roe deer (Capreolus capreolus) epididymides.

Heat shock proteins (HSPs) act as molecular chaperones with important regulatory functions. HSPs are considered to be essential factors in animal reproduction. In view of seasonal variations in the secretory activity of the reproductive tract of mature roe deer (Capreolus capreolus), the aims of this study were to identify HSPs in the epididymides and compare the expression of the identified proteins in three periods of the reproductive season. Two-dimensional polyacrylamide gel electrophoresis revealed the highest number of polypeptides in homogenates of epididymal tissues and in caput, corpus and cauda epididymal fluids throughout the reproductive season. Epididymal tissue homogenates and epididymal fluids were analysed by tandem mass spectrometry (MS/MS) to reveal 31 polypeptides with enzymatic activity, including polypeptides with antioxidant properties, structural and cell signalling functions. Moreover, among the identified polypeptides, five of them were similar to heat shock proteins: endoplasmin (Grp94); heat shock protein 90 kDa (HSP90); 78-kDa glucose-regulated protein (Grp78); chain A, the crystal structure of the human HSP70 ATPase domain and heat shock protein beta-1 isoform X. The concentrations of the analysed polypeptides, expressed in optical density units (ODU), differed significantly (p ≤ .05) across the examined periods of the reproductive season. The highest ODU values for almost all analysed proteins were observed during the rutting period. The presence of HSPs in the epididymal tissues and fluids of roe deer in different periods of the reproductive season could indicate that those proteins play an important role in sperm maturation in the epididymis.

The effect of palmitate supplementation on gene expression profile in proliferating myoblasts.

High-fat diet, exposure to saturated fatty acids, or the presence of adipocytes in myoblast microenvironment affects skeletal muscle growth and function. The aim of the present study was to investigate the effect of palmitate supplementation on transcriptomic profile of mouse C2C12 myoblasts. Global gene expression was evaluated using whole mouse genome oligonucleotide microarrays, and the results were validated through qPCR. A total of 4047 genes were identified as differentially expressed, including 3492 downregulated and 555 upregulated genes, during a 48-h exposure to palmitate (0.1 mmol/l). Functional classification showed the involvement of these genes in several processes which regulate cell growth. In conclusion, the addition of palmitate modifies the expression of genes associated with (1) myoblast responsiveness to hormones and growth factors, (2) cytokine and growth factor expression, and (3) regulation of cell-cell and cell-matrix communication. Such alterations can affect myoblast growth and differentiation; however, further studies in this field are required.

Effects of the platelet-activating factor (PAF) on selected quality parameters of cryopreserved bull semen (AI) with reduced sperm motility.

The aim of the study was to determine the effects of platelet-activating factor (PAF) on selected quality parameters of cryopreserved bull semen with reduced sperm motility used for artificial insemination. The aim of experiment 1 was to identify the optimal concentration of the phospholipid able to preserve sperm viability. Cryopreserved semen was treated with different PAF concentrations: 1×10(-5) M, 1×10(-6) M, 1×10(-7) M, 1×10(-8) M and 1×10(-9) M. The experiment demonstrated that PAF at concentration 1×10(-9) M increased most the sperm viability parameters (motility parameters, plasma membrane integrity and mitochondrial function) after 120 min of incubation of thawed semen at 37°C. Cryopreserved bull semen with reduced sperm motility (below 70%) was supplemented with PAF in a concentration of 1×10(-9) M. A statistically significant increase in sperm motility, percentage of linear motile spermatozoa and VSL value was observed after 120 min incubation of sperm with 1×10(-9) M PAF. Sperm supplementation with PAF also had positive effects on plasma membrane integrity and percentage of spermatozoa with preserved mitochondrial transmembrane potential, but the differences were not statistically significant. The results indicated positive effects of PAF supplementation at a concentration of 1×10(-9) M on the selected sperm quality parameters in cryopreserved bull semen with reduced motility.

Microglial P2Y12 is necessary for synaptic plasticity in mouse visual cortex.

Microglia are the resident immune cells of the brain. Increasingly, they are recognized as important mediators of normal neurophysiology, particularly during early development. Here we demonstrate that microglia are critical for ocular dominance plasticity. During the visual critical period, closure of one eye elicits changes in the structure and function of connections underlying binocular responses of neurons in the visual cortex. We find that microglia respond to monocular deprivation during the critical period, altering their morphology, motility and phagocytic behaviour as well as interactions with synapses. To explore the underlying mechanism, we focused on the P2Y12 purinergic receptor, which is selectively expressed in non-activated microglia and mediates process motility during early injury responses. We find that disrupting this receptor alters the microglial response to monocular deprivation and abrogates ocular dominance plasticity. These results suggest that microglia actively contribute to experience-dependent plasticity in the adolescent brain.

Clinical target volume in postoperative radiotherapy for gastric cancer: identification of major difficulties and controversies.

PURPOSE: To identify the main difficulties in postoperative clinical target volume (CTV) delineation in gastric cancer (GC). METHODS: Before and after a training course, 20 radiation oncology residents were asked to delineate the CTV for the postoperative GC case on four computed tomography scans: dome of the diaphragm, anterior abdominal wall, duodenal stump and porta hepatis level, and to determine the lower CTV border. CTV volume was reconstructed from requested planar contours. Area of intersection (AI) for each requested scan and volume of intersection (VI), defined as the overlap of delineated area/volume with respective reference area (RA)/reference volume (RV) proposed by the senior radiation oncologist, were computed. The degree of agreement between the reference and participants' contours was quantified using the Concordance Index (CI): AI/RA × 100% or VI/RV × 100%. The lower CTV border was analyzed separately. Pre- and post-training CIs were compared. A questionnaire investigated the difficulties with contouring. RESULTS: Mean CI value was the lowest for the dome of the diaphragm (24% pre-training, 35 % post-training) and for the duodenal stump (49% pre-training, 61% post-training). Mean CI for the CTV volume was 49% pre-training and 59% post-training, p = 0.39. Mean distance from the reference to the participants' lower CTV borders was 2.73 cm pre-training and 2.0 cm post-training, p = 0.71. In a questionnaire, 75% of respondents indicated the elective nodal area as the main difficulty. CONCLUSIONS: Delineation of the dome of the diaphragm and the duodenal stump, as yet not recognized as the source of variation, should be addressed in the international consensus guidelines and clarified.

Characterization of microRNA profile in mammary tissue of dairy and beef breed heifers.

MicroRNAs (miRNAs) are small non-coding RNAs that participate in the regulation of gene expression. Their role during mammary gland development is still largely unknown. In this study, we performed a microarray analysis to identify miRNAs associated with high mammogenic potential of the bovine mammary gland. We identified 54 significantly differentially expressed miRNAs between the mammary tissue of dairy (Holstein-Friesian, HF) and beef (Limousin, LM) postpubertal heifers. Fifty-two miRNAs had higher expression in the mammary tissue of LM heifers. The expression of the top candidate miRNAs (bta-miR-10b, bta-miR-29b, bta-miR-101, bta-miR-375, bta-miR-2285t, bta-miR-146b, bta-let7b, bta-miR-107, bta-miR-1434-3p) identified in the microarray experiment was additionally evaluated by qPCR. Enrichment analyses for targeted genes revealed that the major differences between miRNA expression in the mammary gland of HF versus LM were associated with the regulation of signalling pathways that are crucial for mammary gland development, such as TGF-beta, insulin, WNT and inflammatory pathways. Moreover, a number of genes potentially targeted by significantly differentially expressed miRNAs were associated with the activity of mammary stem cells. These data indicate that the high developmental potential of the mammary gland in dairy cattle, leading to high milk productivity, depends also on a specific miRNA expression pattern.

Missense mutation in glutathione-S-transferase M1 gene is associated with sperm motility and ATP content in frozen-thawed semen of Holstein-Friesian bulls.

Glutathione-S-transferase genes (GSTs) encode enzymes that are involved in detoxification and neutralization of reactive oxygen species (ROS) in male reproductive system and play protective role during spermatogenesis. The aim of the study was to evaluate whether C/G missense mutation (rs135955605) within glutathione-S-transferase M1 (GSTM1) gene is associated with selected parameters of frozen-thawed semen in 309 Holstein-Friesian bulls. Single nucleotide substitution C/G was identified by amplification of GSTM1 gene fragment followed be digestion with restriction enzyme DdeI. Bulls with GG genotype were the most frequent (67.96%), in comparison to CC (2.59%) and GC (29.45%). Significant associations were found between GSTM1 genotypes and ATP content and total sperm motility. Bulls with GG genotype had the highest values for both traits. Rare variant C of GSTM1 was associated with significant decrease of sperm motility and ATP content. Our results demonstrate that C/G missense mutation within GSTM1 gene is involved in bull sperm quality.

Selected qualitative and biochemical parameters of cryopreserved semen of Holstein-Friesian (HF) AI bulls.

Selected qualitative and biochemical parameters were determined in cryopreserved semen used for artificial insemination, sampled from 120 bulls reared at the Animal Breeding and Insemination Center in Bydgoszcz. The total average motility of the analyzed sperm samples was determined at 62.51%. The percentage of motile spermatozoa displaying progressive forward motility was 21.65%. Analyzed samples were characterized by a high percentage of sperm cells with a intact plasma membrane (71.21%) and active mitochondria (71.32%). High efficiency of the enzymatic antioxidant system of the evaluated sperm cells was demonstrated by high activity of CAT, GPx and SOD (494.37, 2847.83 and 5.31U/1x10(9) spermatozoa, respectively) values and low values of the DNA Fragmentation Index (9.32). The results of the study, obtained with the involvement of advanced analytical methods, indicate a high fertilizing capability of the analyzed sperm samples.

Specific character of anaerobic bacterial infections in patients treated in transplantation wards at one of the clinical hospitals in Warsaw.

Immunocompromised patients and patients undergoing invasive procedures are predisposed to bacterial infections, due to the possibility of micro-organism translocation from their physiological habitat. Infectious complications may occur both in the early and late post-transplantation periods. The purpose of this study was to evaluate the proportion as well as susceptibility profiles of obligatory anaerobes in the etiology of infections in patients hospitalized at transplantation wards of a large clinical hospital in Warsaw. A total of 104 strains of obligatory anaerobes derived from patients hospitalized in two transplantation clinics at a clinical hospital in Warsaw were evaluated. The strains were isolated from 87 clinical samples collected from 84 patients of two transplantation wards between 2007 and 2012. A total of 104 obligatory anaerobic bacterial strains were isolated and identified, with Gram-positive and Gram-negative bacteria constituting 60.6% and 39.4% of the isolates, respectively. Almost exclusively non-spore-forming anaerobes were detected in evaluated samples. The present study showed all isolated Gram-positive bacteria to be susceptible to ß-lactam antibiotics. Metronidazole-resistant bacteria were found among the genera Propionibacterium and Actinomyces. All Gram-negative rods were susceptible to imipenem and metronidazole. Among them, Bacteroides spp. and Parabacteroides distasonis showed resistance to penicillin G (100%). Because of their pathogenicity and altered antibiotic susceptibility profiles, the bacteria of the genera Bacteroides and Parabacteroides are of greatest clinical importance. Approximately 25% of isolates exhibit also resistance to clindamycin. Because of the growing rates of clindamycin resistance, the role of metronidazole in the treatment of Bacteroides spp. is of increasing importance.

Blood infections in patients treated at transplantation wards of a clinical hospital in Warsaw.

Establishment of the etiology in blood infection is always advisable. The purpose of this study was to evaluate the proportion of different bacterial species, including aerobic and anaerobic bacteria in blood cultures of patients hospitalized in transplantation wards of a large clinical hospital between 2010 and 2012. A total of 1994 blood samples from patients who were treated at one of two transplantation wards of a large hospital in Warsaw were analyzed using an automated blood culture system, BacT/ALERT (bioMerieux, France). The 306 bacterial strains were obtained from the examined samples. The highest proportion were bacteria from the family Enterobacteriaceae (112 strains; 36.6%) with Escherichia coli (61 strains), Klebsiella pneumoniae (30 strains), and Enterobacter cloacae (10 strains) most commonly isolated. The non-fermenting bacilli constituted 21.6% (66 strains), with most common Stenotrophomonas maltophilia (31 strains), Pseudomonas aeruginosa (14 strains), Achromobacter spp. (12 strains), and Acinetobacter baumannii (3 strains). Most frequent Gram-positive bacteria were staphylococci (25.2%). Of 77 staphylococcal strains, 56 were coagulase-negative staphylococci and 21 Staphylococcus aureus. Other Gram-positive bacteria included enterococci (14 strains) and Streptococcus pneumoniae (1 strain). Obligatory anaerobic bacteria were represented by 19 strains (6.2% of total isolates). Among all Enterobacteriaceae, 49 isolates (43.7%) produced extended-spectrum ß-lactamases (ESBLs). Resistance to methicillin was detected in 62% of S aureus isolates and in 46% of coagulase-negative staphylococci. Of 14 enterococci cultured from blood samples, 2 strains (14.3%) were resistant to vancomycin. Both were Enterococcus faecium. Resistant strains of Gram-negative and Gram-positive bacteria are significant problems for patients in the transplantation ward.

Transcriptional background of beef marbling - novel genes implicated in intramuscular fat deposition.

The purpose of this study was to identify novel marbling-related genes by comparison of the global gene expression in semitendinosus muscle of 15-month-old Limousin (LIM), Holstein-Friesian (HF) and Hereford (HER) bulls. Muscle of LIM was lean with low intramuscular fat (IMF) content (0.53%) unlike the marbled muscles of HER and HF characterized by higher amounts of IMF (1.10 and 0.81%, respectively). The comparison of muscle transcriptional profile between marbled and lean beef revealed significant differences in expression of 144 genes, presumably involved in consecutive stages of adipose tissue development, such as preadipocyte proliferation and differentiation, adipocyte maturation, lipid filling and lipid metabolism leading to increased IMF deposition and marbling development. Correlation coefficients and regression analysis for nine of them (gadd45a, pias3, ccrn4l, diras3, pou5f1, hoxa9, atp2a2 and pim1) validated by real-time qPCR confirmed their moderate-high correlation with IMF% and explained up to 70.5% of the total variability in IMF deposition in the bulls.

Effect of commercial long-term extenders on metabolic activity and membrane integrity of boar spermatozoa stored at 17 degrees C.

This study was aimed to analyze the metabolic activity and membrane integrity of boar spermatozoa following storage in long-term semen extenders. Boar semen was diluted with Androhep EnduraGuard (AeG), DILU-Cell (DC), SafeCell Plus (SCP) and Vitasem LD (VLD) extenders and stored for 10 days at 17 degrees C. Parameters of the analyzed sperm metabolic activity included total motility (TMOT), progressive motility (PMOT), high mitochondrial membrane potential (MMP) and ATP content, whereas those of the membrane integrity included plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome. Extender type was a significant (P < 0.05) source of variation in all the analyzed sperm parameters, except for ATP content. Furthermore, the storage time had a significant effect (P < 0.05) on the sperm metabolic activity and membrane integrity during semen storage. In all extenders the metabolic activity and membrane integrity of the stored spermatozoa decreased continuously over time. Among the four analyzed extenders, AeG and SCP showed the best performance in terms of TMOT and PMI on Days 5, 7 and 10 of storage. Marked differences in the proportions of spermatozoa with high MMP were observed between the extenders, particularly on Day 10 of storage. There were not any marked differences in sperm ATP content between the extenders, regardless of the storage time. Furthermore, the percentage of spermatozoa with NAR acrosomes decreased during prolonged storage, being markedly lower in DC-diluted semen compared with semen diluted with either AeG or SCP extender. The results of this study indicated that components of the long-term extenders have different effects on the sperm functionality and prolonged semen longevity by delaying the processes associated with sperm ageing during liquid storage.

Primary cultures of murine neurons for studying herpes simplex virus 1 infection and its inhibition by antivirals.

Herpes simplex virus 1 (HSV-1) establishes life-long latency in peripheral neurons, where productive replication is suppressed. To study the specific relationship between the virus and peripheral neurons that would not be affected by other cells usually present in in vivo systems, we present an in vitro model system based on primary cultures of murine neurons. This model system can be used for characterization of various virus strains and testing of cytotoxicity and inhibitory activity of acyclovir (ACV), cidofovir (CDV) and other antivirals.

Mismatch between transcriptomic and histopathologic picture of canine lymphomas.

Lymphoma is one of the most common malignant tumours occurring in dogs. Since there is a constant need for new, more comprehensive laboratory diagnostic tools which permit the precise determination of many tumour-related factors we decided to verify whether the use of microarray analysis could be helpful in classifying lymphomas. The study was performed on samples collected from 7 dogs in which multicentric lymphoma was recognized. Among this group we were able to identify one sub-cluster of transcriptionally similar tumours, which completely differed in terms of the histopathological examination. Among them there were one diffuse large B cell lymphoma, one diffuse macronucleolated medium-sized cell lymphoma and one pleomorphic mixed small and large T-cell lymphoma. The lymphomas belonging to the sub-cluster differed from other analysed tumours in the expression of more than 100 genes of which only 18 were described earlier in regard to lymphomas.