Genome-wide co-localization of active EGFR and downstream ERK pathway kinases mirrors mitogen-inducible RNA polymerase 2 genomic occupancy.

Genome-wide mechanisms that coordinate expression of subsets of functionally related genes are largely unknown. Recent studies show that receptor tyrosine kinases and components of signal transduction cascades including the extracellular signal-regulated protein kinase (ERK), once thought to act predominantly in the vicinity of plasma membrane and in the cytoplasm, can be recruited to chromatin encompassing transcribed genes. Genome-wide distribution of these transducers and their relationship to transcribing RNA polymerase II (Pol2) could provide new insights about co-regulation of functionally related gene subsets. Chromatin immunoprecipitations (ChIP) followed by deep sequencing, ChIP-Seq, revealed that genome-wide binding of epidermal growth factor receptor, EGFR and ERK pathway components at EGF-responsive genes was highly correlated with characteristic mitogen-induced Pol2-profile. Endosomes play a role in intracellular trafficking of proteins including their nuclear import. Immunofluorescence revealed that EGF-activated EGFR, MEK1/2 and ERK1/2 co-localize on endosomes. Perturbation of endosome internalization process, through the depletion of AP2M1 protein, resulted in decreased number of the EGFR containing endosomes and inhibition of Pol2, EGFR/ERK recruitment to EGR1 gene. Thus, mitogen-induced co-recruitment of EGFR/ERK components to subsets of genes, a kinase module possibly pre-assembled on endosome to synchronize their nuclear import, could coordinate genome-wide transcriptional events to ensure effective cell proliferation.

Application of Matrix-assisted Laser Desorption Ionization Time-of-flight Mass Spectrometry for Identification of Coagulase-negative Staphylococci Isolated from Milk of Cows with Subclinical Mastitis.

The aim of this study was to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of coagulase-negative staphylococci (CNS) isolated from the milk of cows with subclinical mastitis. The study material consisted of 33 isolates of CNS, identified by the results of API Staph tests, obtained from the milk of cows with subclinical mastitis. Based on the spectra analyses, MALDI-TOF MS tests of 33 bacterial samples allowed identification of the microorganisms in 27 cases (81.8%). The most frequent cause of subclinical mastitis was found to be Staphylococcus sciuri (39%), while S. vitulinus was detected in 15% of the milk samples. The results obtained indicate that MALDI-TOF MS can be used for the identification of CNS isolated from bovine mastitis as a method supplementary to biochemical tests.

Helmintofauna of turbot Scophthalmus maximus (Linnaeus, 1758) from the southern Baltic Sea including new data.

Turbot Scophthalmus maximus (Linnaeus, 1758) is a fish belonging to the Pleuronectiformes order. It is commonly observed in waters of the northern Atlantic, and also in the Baltic Sea. As an economically significant species, it is fished on an industrial scale, and also farmed in some European countries. Seventy-two turbots from the Gulf of Gdansk (26th ICES zone) were examined for parasite presence in the years 2010-2012. The study revealed the presence of the tapeworm Bothriocephalus scorpii (Müller, 1776) and acanthocephalan Corynosoma semerme (Forssell, 1904). The overall (both parasites) prevalence of turbot infection was 100% with a mean intensity of 18.7. C. semerme is a parasite which has not been noted so far in turbot from the southern Baltic. The presence of C. semerme in turbot was emphasized in the context of possible infection of terrestrial mammals, including humans.

Detection of Borrelia burgdorferi sensu lato DNA in the blood of wild bison from Bialowieza primeval forest in eastern Poland.

The aim of the present study was to investigate the occurrence of Borrelia burgdorferi sensu lato DNA in a group of 120 wild bison (Bison bonasus) from the Bialowieza Primeval Forest in eastern Poland. The PCR technique revealed the presence of 16S RNA of Borrelia burgdorferi sensu lato in the blood of 16 out of 120 examined animals. DNA amplification by means of primers SC1 and SC2 gave a product with a size of 300-bp. The sequences of the PCR products obtained showed 100% homology with each other and 100% homology with B. burgdorferi s.1. 16S RNA gene DQ111061. Results of this study suggest that wild bison are important in maintaining agents of Lyme borreliosis, and that studies of reservoir competence of this species are indicated.

Comparison of serum protein profiles of Borrelia burgdorferi-positive Bernese Mountain Dogs and dogs of other breeds using MALDI-TOF technique.

The aim of the study was to compare the serum protein profile of Bernese Mountain Dogs (BMDs) reacting positive for Bb in snap testing with the serum protein profile of dogs of other breeds (healthy and with clinical borreliosis) using the MALDI time-of-flight (MALDI-TOF) technique. The observations included five groups of dogs. BMDs reacting positively to Bb in snap serological testing and showing symptoms of borreliosis (group 1), BMDs for which no borreliosis symptoms were determined but with seropositivity for Bb determined with snap serological tests (group 2), clinically healthy BMDs with no antibodies for Bb found in the serum (group 3), five dogs of different breeds, reacting positively in serological testing, in which borreliosis symptoms were observed (group 4), clinically healthy dogs of different breeds with negative reaction in tests towards Bb (group 5). A proteomic analysis demonstrated the presence of five identical protein fractions among all five groups. An additional two protein fractions of approximately 7.630 and 15.260 kDa were found in all the serum samples obtained from the dogs positive for borrelia in a snap test, both in those exhibiting symptoms of borreliosis, and seropositive BMDs not presenting symptoms of the disease. These two additional protein fractions may be used to differentiate between seropositive and seronegative B. burgdorferi dogs and may be considered a seropositivity marker, however, it cannot be used to differentiate between animals with the clinical form of the disease and those that are only seropositive.

The identification of Anaplasma spp. isolated from fallow deer (Dama dama) on a free-range farm in eastern Poland.

The aim of the present study was to investigate the occurrence of Anaplasma spp. in group of 50 fallow deer (Dama dama) from free-range farm in eastern Poland and determine what species of Anaplasma could infect these animals based on PCR gene sequencing. The PCR technique revealed the presence of 16S RNA Anaplasma spp. genetic material in the blood of 7 out of 50 examined animals. The sequences of the PCR products obtained showed a 100% homology with each other and 100% homology with GU 183908 sequence of A. phagocytophilum, isolated in our earlier study from a horse with clinical form of anaplasmosis. Here, we report the first molecular evidence of Anaplasma spp. among naturally infected fallow deer in eastern Poland.

Molecular analysis of a fragment of gene E1B 19K of canine adenovirus 2 (CAV-2) isolated from dogs with symptoms of cough.

The aim of this study was to perform molecular analysis of canine adenovirus 2 (CAV-2) E1B 19K gene fragment isolated from 20 dogs of various breeds (12 males and 8 females aged 1-9 years), with clinical symptoms of upper respiratory tract infections, from the Lubelszczyzna region. Nasal swabs were taken from dogs. DNA of CAV-2 was detected using the PCR method in 16 swabs. All PCR products were sequenced, and the obtained sequences were compared with each other and with the sequence of the E1B 19K gene of the CAV-2 strain from an online database of NCBI GenBank: AC 000003. Based on analysis of the obtained sequences, three polymorphic variants of CAV-2 (No. 1-3) with homology of 78 - 100% were distinguished. The nucleotide and amino acid sequences of the most frequently represented polymorphic variant, No. 1, differed from the sequences of polymorphic variant No. 2 with one substitution. The nucleotide and amino acid sequence of the E1B 19K gene of CAV-2 AC 000003 differed from the analogous sequences of representatives of variant No. 1 with 44 nucleotide and 19 amino acid substitutions. The small number of nucleotide differences in the E1B 19K CAV-2 gene among the examined own isolates, compared with AC 000003, suggest that the infections in dogs were caused by a relatively genetically stable virus which occurs in eastern

Lyme disease in Bernese Mountain Dogs. Is it a real problem?

Borreliosis is the most frequently diagnosed tick-borne disease caused by spirochete bacteria belonging to the genus Borreliae - Borrelia burgdorferi sensu stricto (s.s.), Borrelia afzelii and Borrelia garinii. Clinical manifestations in dogs include fever, lameness, polyarthritis and glomerulonephritis. Diagnosis is mainly serological and is based on an immunoenzymatic test followed by a Western blot confirmatory test. Early treatment with antibiotics such as doxycycline or amoxicillin, for four weeks, usually reduces the risk of chronic disease. Tick control, including tick repellents, is highly reliable in preventing transmission. Vaccines are available to reduce transmission and the clinical manifestations of infection in dogs. Bernese Mountain Dogs are a breed that often test positive for antibodies against B. burgdorferi without showing any clinical symptoms of the disease. Quantitative determination of the immunoglobulin level for spirochetes has indicated that Bernese Mountain Dogs may have an increased susceptibility to Borrelia spp. infections of a hereditary nature.

Clinical course of granulocytic anaplasmosis in hunting dogs.

The aim of this study was to analyze cases of granulocytic anaplosmosis diagnosed in 53 hunting dogs in Poland. Medical records of dogs naturally infected with Anaplasma phagocytophilum were retrospectively evaluated with regard to clinical signs and laboratory abnormalities at the time of presentation, therapy and course of disease. The most common clinical signs in A. phagocytophilum-positive dogs included in the study were lethargy (100%), inappetence (94%) and fever (92.5%). Thrombocytopenia was the most common laboratory abnormality (100%), followed by a drop in haematocrit level (79.3%) and increased AST activity (75.5%). Of the 53 infected dogs, 51 (96%) recovered and two dogs (with neurological symptoms) died. Analysis of these cases indicates that A. phagocytophilum infection must be considered in differential diagnosis in dogs living in Poland, especially in hunting dogs with thrombocyto- penia and Ixodes ricinus tick invasions.

Multiple trichoepitheliomas--a novel mutation in the CYLD gene.

BACKGROUND: Trichoepitheliomas are benign neoplasms with follicular differentiation. They may present as a solitary lesion or as multiple lesions. Multiple trichoepitheliomas are inherited in an autosomal dominant pattern within families, with both variable penetrance and expressivity. Recent investigations support that mutations in CYLD, the gene affected in familial cylindromatosis as well as in Brooke-Spiegler syndrome, are also responsible for multiple trichoepitheliomas. OBJECTIVE: The authors report the case of a 9-year-old African girl with multiple facial trichoepitheliomas in whom a mutation in the CYLD gene was hypothesised. MATERIALS AND METHODS: After genomic DNA extraction from the peripheral blood, a molecular analysis of the CYLD gene was performed by PCR, DHPLC and automated sequencing. RESULTS: A novel heterozygous mutation in exon 18 of the CYLD gene (c.2449delT) was identified, with a deletion of one nucleotide resulting in a premature translational termination codon at amino acid position 831 on the affected allele (p.Cys817Valfs X15). CONCLUSIONS: The predominating tumours define the classification of these three entities. Nevertheless, studies suggest that they can simply represent phenotypic variations of the same disease spectrum, sharing common genetic mutations.

Babesia gibsoni infection in dogs in Poland.

Canine babesiosis is a tickborne, protozoal, haemoparasitic disease. Babesia organisms are frequently classified as either large (B. canis) or small (B. gibsoni). The aim of this study was an attempt to detect B. gibsoni DNA in blood samples taken from dogs suspected of suffering from tick-borne diseases. 216 samples were tested using PCR, of which, in 99 of them B. canisDNA was detected, whereas in 3 of them B. gibsoni was detected. Positive PCR results for B. gibsoni were confirmed using a Qube MDx real-time analyzer. The results indicate that infec-tions with this B. gibsoni should be taken into account and included in the differential diagnosis of vector-borne diseases in dogs in Poland, and that the accurate identification of the species of parasite causing the infection is crucial for developing the correct treatment regimen and prognosis.

The clinical course of babesiosis in 76 dogs infected with protozoan parasites Babesia canis canis.

The aim of the study was to trace the clinical course of babesiosis in 76 dogs infected with Babesia canis protozoa and to assess the usefulness of PCR method in the routine diagnosis of the disease. The investigations were conducted in three successive seasons of the biological activity of ticks on dogs displaying possible clinical signs of babesiosis, the latter assigned individual numbers from 001 to 076. All the animals underwent routine clinical examinations and blood was collected for haematological, biochemical, parasitological and molecular tests for babesiosis. The most frequent clinical signs observed in the course of the disease were changes in urine colour and xanthosis or paleness of mucous membranes, whereas in the haematological and biochemical examinations, the most frequent laboratory findings were thrombocytopenia, leucopoenia, anaemia and an increase in levels of urea and bilirubin. In all blood smears stained with the May-Grunwald and Giemsa methods, from the 76 dogs, the presence of Babesia canis protozoa was observed in erythrocytes, and their DNA was detected in 69 blood samples by means of PCR technique. The course of the disease and the results of molecular examinations suggested the haemolytic form of babesiosis. The previous genetic analysis of isolates of Babesia canis canis from the eastern areas of Poland helped to distinguish two specific groups, A and B, within the species (Adaszek and Winiarczyk 2008a). The present study revealed a certain interrelation between the intensification of thrombocytopenia and the fact that protozoa belong to either group A or B. The mean number of thrombocytes in dogs infected with protozoa from group A was 61.11 thousand/mm3, whereas the mean number of thrombocytes in the blood of dogs infected with protozoa from group B was 27.47 thousand/mm3. A strong correlation was also observed between the low level of thrombocytes and the increase in the internal body temperature (p = 0.02), accelerated pulse rate (p = 0.01) and discoloration of urine (p = 0.04). As a result of the treatment of dogs with imidocarb, recovery was observed in 73 out of the 76 dogs brought to the clinic.

Molecular analysis of the nucleoprotein gene of canine distemper virus isolated from clinical cases of the disease in foxes, minks and dogs.

In this study, we used RT-PCR to detect and characterize canine distemper virus isolated from 9 naturally infected foxes, 3 minks and 3 dogs in Poland by amplifying and sequencing a portion of the NP gene. A 293-bp fragment of the CDV NP gene was amplified by RT-PCR. Sequencing of the PCR products from the isolates led to the identification of 3 sequence variants. The mostly representative polymorphic variant No. 1 showed high homology with Chinese isolate of CDV with a accession number EF 375619. The sequences of all isolates from this polymorphic variants compared with the sequences of other polymorphic variants obtained in the study and with European and American isolates sequences from GenBank showed the conservative nucleotides changes in positions 57, 132, 143, 159 and 237. These mutations can indicate that in this part of Europe there are new variants of CDV.

Body mass index, waist to hip ratio and waist/height in adult Polish women in relation to their education, place of residence, smoking and alcohol consumption.

Obesity is a complex, multifactorial disorder that develops from genotype and environmental interactions. The aim of this study is to describe the variability of body mass index (BMI), waist to hip ratio (WHR) and waist to height (W/Ht) in adult Polish women, and to determine relationships between these variables and factors such as education, place of residence, smoking and alcohol drinking. The tested group consisted of 10,254 women aged 25-95 years, who voluntarily filled in questionnaires and participated in anthropometric measurements (body height and mass, waist and hip circumferences). The BMI, WHR and W/Ht values were calculated based on these measurements. The participants were differentiated in terms of education, residence and lifestyle (smoking, alcohol drinking). Chi-squared test, product-moment correlations, ANOVA, multiple correspondence analysis (MCA) and logistic regression with backward elimination were used to evaluate associations between social and lifestyle factors and BMI, WHR and W/Ht. The results confirm (1) the relationship between low social status and the risk of overweight and obesity as observed in developed countries; (2) higher susceptibility to environmental factors such as education, place of residence, smoking and alcohol drinking in younger (premenopausal) women; (3) the usefulness of simple and practical anthropometric indicators such as WHR and W/Ht for the identification of the higher risk of future metabolic diseases in obese people and those with a normal body mass.

Significance of hypoxia in uterine cervical cancer. Multicentre study.

PURPOSE: The aim of the study was to evaluate hypoxia markers (VEGF, GLUT-1, and HIF-1alpha) in cervical cancer tissue depending on staging (FIGO) and grading. We also analyzed the adverse effects of radiotherapy according to expression levels of hypoxic markers in the studied tissue. MATERIAL AND METHODS: Expression of hypoxia-inducible factor-1alpha (HIF-1alpha), glucose transporter 1 (GLUT-1) and vascular endothelial growth factor (VEGF, also known as proangiogenic factor) were estimated in biopsy or surgical specimens from 106 patients diagnosed with uterine cervical cancer. Immunohistochemical methods with EbVision+ complex using monoclonal antibodies anti-VEGF and anti-HIF-1alpha and polyclonal antibody anti-GLUT-1 were applied. RESULTS AND CONCLUSIONS: Hypoxia features measured by percentage of cells undergoing reaction with antibodies anti-HIF-1alpha, anti-GLUT-1 and anti-VEGF were similar in all clinical stages; however the biggest hypoxia features were shown in low differentiated cancers G2 and G3. The 5-year survival for FIGO Stage III patients was shorter in cases with a high expression of hypoxic markers. We observed adverse effects in 45.3% of patients, which occurred more often in patients with higher expression of the studied factors. The presence of hypoxic cells is established as one of the most important factors affecting resistance against tumor radiotherapy and patient prognosis.

Liquid Storage of Hematopoietic Stem Cells Versus Proliferative Potential Colony-Forming Unit Granulocyte-Monocytes: Validation of Cell Processing.

BACKGROUND: The material for transplantation must be of the highest quality. As far as we know, short-term storage is one of the crucial points of stem cell banking. According to the quality assurance system in a stem cell bank, each step of cell processing must be validated. The aim of this study was to assess the influence of short-term storage conditions into a clonogenic assay. METHODS: Material was collected from mobilized peripheral blood by means of leukapheresis from 15 patients. Samples were stored at 4°C and 20°C; samples were evaluated on the day of leukapheresis and after 24 hours and after 48 hours of storage. The number of colony-forming unit granulocyte-monocyte (CFU-GM) precursors was analyzed with the use of in vitro culture. The material was evaluated before freezing and after thawing. RESULTS: The average number of CFU-GM precursors in the material stored at 4°C before freezing on the day of collection was 84/10(5) nuclear cells (nc) and after 24 hours and 48 hours of storage was, respectively, 62/10(5) nc (P = .011719) and 36/10(5) nc (P = .02088). The average of the CFU-GM precursors in material stored at 20°C after 24 hours and 48 hours of storage amounted to 33/10(5) nc (P = .004439) and 2/10(5) nc (P = .00346), respectively. CONCLUSIONS: In our study, the number of colonies of CFU-GM after 24 hours and 48 hours of storage, both at 4°C and 20°C, was significantly reduced compared with the number of colonies on the day of collection. Significantly greater numbers of CFU-GM precursors were observed in the material stored before freezing at 4°C in comparison with the material stored at 20°C.

Banking of Hematopoietic Stem Cells: Influence of Storage Time on Their Quality Parameters.

BACKGROUND: Banking of hematopoietic stem cells (HSCs) is a rapidly growing part of the transplant field. The essence of the banking process is to maintain the optimal quality parameters throughout the storage period, allowing successful transplantation. METHODS: Our laboratory research was carried out on 126 HSC samples that were collected by means of leukapheresis from patients with lymphoproliferative diseases. The samples were frozen in a controlled rate and stored up to 76 months in containers in vapor phase of liquid nitrogen. The evaluation was performed after thawing the probes. Viability of nuclear cells was assessed after incubation in Trypan blue, CD34+ phenotype cells were determined by means of cytometry with the use of 7-aminoactinomycin D (7-AAD), and an analysis of the proliferative potential of granulocyte-monocyte precursors was performed. For comparative statistical analysis, the material was divided into 3 groups according to storage time: A: <1 month (n = 45); B: 1-12 months (n = 50); C: >12 months (n = 31). RESULTS: In the examined groups, similar median values were observed of nuclear cell viability (A, 86%; B, 87%; and C, 83%) and CD34+ cells (95%, 94.5%, and 95.8%, respectively). A gradual, nonsignificant, reduction in the median of granulocyte-monocyte precursors was found: 68 × 10(4)/kg of body weight (kg bw), 48.5 × 10(4)/kg bw, and 47 × 10(4)/kg bw, respectively. Statistical analysis with the use of the Kruskal-Wallis test showed a P value of >.05 for all variables. CONCLUSIONS: There were no significant differences in the viability of nuclear cells, CD34+ cells, and proliferative potential granulocyte-monocyte precursors between groups. Storage for up to 76 months does not change the essential quality parameters, and HSCs could be qualified for distribution.

Acyl-GPC and alkenyl/alkyl-GPC:acyl-CoA acyltransferases.

In mammalian tissues, phosphatidylcholine, or 1,2-diacyl-glycerophosphocholine (GPC), is the most abundant form of choline-containing phospholipids. In some electrically active tissues, a significant portion of the choline-containing phospholipids is 1-alkenyl-2-acyl-GPC (plasmenylcholine). The 1-alkyl-2-acyl-GPC is found in significant amounts in circulating cells such as neutrophils and macrophages but in low amounts in other tissues. Structural studies of phosphatidylcholine indicate that there is an asymmetric distribution of acyl groups on the molecule. Saturated fatty acids are usually esterified at the sn-1 position of the glycerol backbone, whereas unsaturated fatty acids are esterified at the sn-2 position. Similarly, unsaturated acyl groups are usually found in the sn-2 position of plasmenylcholine. The remodelling of the sn-2 acyl group in phosphatidylcholine by the deacylation-reacylation process has been demonstrated in a number of tissues. Phospholipase A2 is responsible for the hydrolysis of the acyl group at the sn-2 position, whereas 1-acyl-GPC:acyl-CoA acyltransferase is responsible for the reacylation reaction. The acyltransferase is located in the microsomal fraction and displays specificity towards the polyunsaturated acyl groups. The enzyme can be solubilized by detergent, but the enzyme activity in soluble form is difficult to maintain. The acyltransferase for the reacylation of 1-alkenyl-GPC is also located in the microsomal fraction and is somewhat specific towards polyunsaturated acyl groups. In guinea pig heart mitochondria, however, a new form of 1-alkenyl-GPC acyltransferase was identified which appeared to be different from the microsomal form. The acyltransferase for the acylation of 1-alkyl-GPC into platelet-activating factor has been studied in several tissues including human neutrophils. At present, the contribution of the acyltransferase in attaining the observed molecular composition of the choline-containing phospholipids in the tissue has not been defined. We postulate that the intrinsic acyl-CoA specificity of the acyltransferase, the flux of 1-acyl-GPC, 1-alkenyl-GPC and 1-alkyl-GPC, as well as the pool size of acyl-CoA are major factors in producing the final composition of the molecular species of the choline-containing phospholipids.

Recent trends in molecular diagnostics for Toxoplasma gondii infections.

Toxoplasmosis is an important parasitic infection of man and animals. It is well-known that the progression and severity of disease depend on the immunological status of the host, but recent studies suggest that the genetics of the parasite can also play a role. Diagnosis based on clinical appearance and serology is not always easy. However, molecular methods do not depend on an immune response, and allow direct detection of the parasite in biological samples. Thus they can be used to establish a diagnosis when serological tests are not definitive. Multicopy sequences specific for Toxoplasma gondii, e.g., the B1 gene or the 529-bp sequence, are especially useful in molecular tests. Real-time PCR is very sensitive and is a promising technique that is capable of providing a quantitative result. Molecular methods are also used for genotypic characterisation of T. gondii isolates. Analysis of polymorphic sequences determines the precise strain. The choice of sequence is critical when undertaking studies on the correlation between clinical signs and symptoms of disease and the T. gondii genotype. Further studies involving direct genotyping of T. gondii from clinical samples are needed.

Assessment of the body mass index and selected physiological parameters in pre- and post-menopausal women.

The main purpose of this paper is to describe the variability of the body mass index (BMI) and selected physiological parameters (serum glucose, total serum cholesterol, systolic and diastolic blood pressure) in women before and after menopause. The empirical basis for the assessment is the material obtained in cross-sectional studies carried out in the years 1998-2001 in a group of 2204 women aged 35-65 years, residents of the Wielkopolska region, Poland. The results indicate that hormonal changes taking place in the climacterium bring about an increase in the BMI. It was observed that women receiving Hormone Replacement Therapy in the perimenopause had BMI significantly lower than naturally menopausal women. Increased levels of total serum cholesterol and blood glucose were recorded. The trend has a stronger relation with the age of the subjects than with the character of the menopause. The systolic and diastolic blood pressure values were also found to increase significantly after the menopause, but no relationship with the type of menopause was found.