Transcriptome analysis of boar spermatozoa with different freezability using RNA-Seq.

Semen freezability is associated with genetic markers, and there is a diverse set of sperm transcripts that have been attributed to various cellular functions. RNA-Seq was performed to compare the transcript profiles of spermatozoa from boars with different semen freezability. We examined ejaculates from the Polish large white (PLW) boars that were classified as having good and poor semen freezability (GSF and PSF, respectively; n = 3 boars per group) by assessing post-thaw motility characteristics, mitochondrial membrane potential, plasma membrane and acrosome integrity. Total RNA was isolated from fresh spermatozoa from boars of the GSF and PSF groups and subjected to RNA-Seq (Illumina NextSeq 500 platform). Transcript abundance was assessed with the DESeq2, DESeq, and EdgeR Bioconductor R packages, and varying numbers of differentially expressed gene (DEG) transcripts were detected in the spermatozoa of each boar. Using RNA-Seq, we identified several genes associated with inflammation and apoptosis (FOS, NFATC3, ITGAL, EAF2 and ZDHHC14), spermatogenesis (FGF-14 and BAMBI), autophagy (RAB33B), protein phosphorylation (PTPRU and PTPN2) and energy metabolism (ND6 and ACADM) that were predominantly up-regulated in poor freezability ejaculates. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) validated the transcript expression levels detected by RNA-Seq and thus confirmed the reliability of this technique. Subsequent validation with western blotting showed that the expression of three proteins was in accordance with the transcript abundance. Overall, we demonstrated that the up-regulation of the DEG transcripts in spermatozoa was associated with poor semen freezability. We suggest that spermatozoa transcriptome profiling provides a foundation to further elucidate the relevance of sperm-related transcripts on cryo-survival. The sperm-related transcripts, namely FOS, NFATC3, EAF2, BAMBI, PTPRU, PTPN2, ND6 and ACADM, are potential markers for predicting the freezability of boar semen.

Ultrasound-guided intermediate cervical plexus and additional peripheral facial nerve block for carotid endarterectomy : A prospective pilot study.

BACKGROUND AND OBJECTIVES: Ultrasound-guided intermediate cervical plexus block with perivascular local anesthetic infiltration is an established anesthetic procedure for carotid endarterectomy. In this prospective pilot study an additional subplatysmal block of the superficial ansa cervicalis is presented for the first time. The target structures are the anastomoses between the facial nerve (cervical and marginal mandibular branches) and cervical plexus. METHODS: An ultrasound-guided intermediate cervical plexus block (20 ml of ropivacaine 0.75%) was performed (n = 28). Then, depending on the individual sonoanatomy, 5 ml of prilocaine 1% was injected into the carotid sheath (group 1: no perivascular infiltration, n = 14, group 2: perivascular infiltration, n = 14). The third step was subplatysmal injection of 5 ml of prilocaine 1% between the medial edge of the sternocleidomastoid muscle and the submandibular gland (n = 28). The investigated parameters included the need for supplementation and block-related side effects. RESULTS: The requirement for supplemental local anesthetic infiltration in the skin incision area was minimal at mean (M) 1.1 ml (standard deviation (SD) ±2.4 ml). Perivascular infiltration in group 2 significantly decreased the total amount of local anesthetic supplemented: group 1 M = 4.2 ml (SD = ±3.1 ml), group 2 M = 1.7 ml (SD = ±2.0 ml) (p = 0.018). The incidence of block-related side effects was not significantly different between the two groups. CONCLUSION: This study presents an ultrasound-guided subplatysmal block of the superficial ansa cervicalis for the first time, with the aim of optimizing anesthesia quality during surgical interventions in the carotid triangle.

Transcriptomic hallmarks of bone remodelling revealed by RNA-Seq profiling in blood of Arabian horses during racing training regime.

The impact of exercises on young developing organisms is still of interest to researchers. Similarly like Thoroughbreds, Arabian horses competing at the race track. The high percent of lameness and loss of days in training are often the result of weakness in the condition of the musculoskeletal system. The objective of the presented study was to identify by RNA-Seq method, the possible skeletal system originating transcriptomic profile in peripheral blood of Arabian horses undergoing race training. Obtained results showed that one of the most significantly deregulated pathway involved in bone homeostasis was those involved in osteoclast differentiation. Among the significantly expressed molecules, we recognized twelve genes potentially involved in the metabolism of the skeletal system: BGLAP, CTSK, TYROBP, PDLIM7, SLC9B2, TWSG1, NOTCH2, IL6ST, VAV3, NFATc1, CLEC5A, TXLNG. The panel of identified genes should be evaluated as candidate biomarkers for bone homeostasis indicators of Arabians performing on race tracks to assess bone remodelling states during training for race track competitions.

Detection of genetic variants between different Polish Landrace and Pulawska pigs by means of RNA-seq analysis.

Variant calling analysis based on RNA sequencing data provides information about gene variants. RNA-seq is cheaper and faster than is DNA sequencing. However, it requires individual hard filters during data processing due to post-transcriptional modifications such as splicing and RNA editing. In the present study, RNA-seq transcriptome data on two Polish pig breeds (Pulawska, PUL, n = 8, and Polish Landrace, PL, n = 8) were included. The pig breeds are significantly different with regard to meat qualities such as texture, water exudation, growth traits and fat content in carcasses. A total of 2451 significant mutations were identified by a chi square tests, and functional analysis was carried out using Panther, KEGG and Kobas. Interesting missense gene variants and mutations located in regulatory regions were found in a few genes related to fatty acid metabolism and lipid storage such as ACSL5, ALDH3A2, FADS1, SCD, PLA2G12A and ATGL. A validation of mutational influences on pig traits was performed for ALDH3A2, ATGL, PLA2G12A and MYOM1 variants using association analysis including 215 pigs of the PL and PUL breeds. The ALDH3A2ENSSSCT00000019636.2:c.470T>C polymorphism was found to affect the weight of the ham and loin eye area. In turn, an ENSSSCT00000004091.2:c.2836G>A MYOM1 mutation, which could be implicated in myofibrillar network organisation, had an effect on meatiness and loin texture parameters. The study aimed to estimate the usefulness of RNA-seq results for a purpose other than differentially expressed gene analysis. The analysis performed indicated interesting gene variants that could be used in the future as markers during selection.

Common variants in DLG1 locus are associated with non-syndromic cleft lip with or without cleft palate.

Non-syndromic cleft lip with or without cleft palate (nsCL/P) is a common craniofacial anomaly with a complex and heterogeneous aetiology. Knowledge regarding specific genetic factors underlying this birth defect is still not well understood. Therefore, we conducted an independent replication analysis for the top-associated variants located within the DLG1 locus at chromosome 3q29, which was identified as a novel cleft-susceptibility locus in our genome-wide association study (GWAS). Mega-analysis of the pooled individual data from the GWAS and replication study confirmed that common DLG1 variants are associated with the risk of nsCL/P. Two single nucleotide polymorphisms (SNPs), rs338217 and rs7649443, were statistically significant even at the genome-wide level (Ptrend = 9.70E-10 and Ptrend = 8.96E-09, respectively). Three other SNPs, rs9826379, rs6805920 and rs6583202, reached a suggestive genome-wide significance threshold (Ptrend < 1.00E-05). The location of the strongest individual SNP in the intronic sequence of the gene encoding DLG1 antisense RNA suggests that the true causal variant implicated in the risk of nsCL/P may affect the DLG1 gene expression level rather than structure of the encoded protein. In conclusion, we identified a novel cleft-susceptibility locus at chromosome 3q29 with a DLG1 as a novel candidate gene for this common craniofacial anomaly.

A549 and MRC-5 cell aggregation in a microfluidic Lab-on-a-chip system.

In this paper, we present a culture of A549 and MRC-5 spheroids in a microfluidic system. The aim of our work was to develop a good lung cancer model for the evaluation of drug cytotoxicity. Our research was focused on determining the progress of cell aggregation depending on such factors as the depth of culture microwells in the microdevices, a different flow rate of the introduced cell suspensions, and the addition of collagen to cell suspensions. We showed that these factors had a significant influence on spheroid formation. It was found that both MRC-5 and A549 cells exhibited higher aggregation in 500 µm microwells. We also noticed that collagen needs to be added to A549 cells to form the spheroids. Optimizing the mentioned parameters allowed us to form 3D lung tissue models in the microfluidic system during the 10-day culture. This study indicates how important an appropriate selection of the specified parameters is (e.g., geometry of the microwells in the microsystem) to obtain the spheroids characterized by high viability in the microfluidic system.

Ultrasound-guided intermediate cervical plexus block and perivascular local anesthetic infiltration for carotid endarterectomy : A randomized controlled trial.

BACKGROUND AND OBJECTIVE: Ultrasound-guided blocks of the cervical plexus are established anesthetic procedures for carotid endarterectomy. This randomized, double-blind, placebo-controlled study tested the hypothesis that an additional ultrasound-guided periarterial injection of local anesthetic leads to a lower frequency of periarterial supplementation by the surgeon. METHODS: A total of 40 patients were randomly assigned to 1 of 2 groups. In both groups an ultrasound-guided intermediate cervical plexus block (20 ml of 0.75 % ropivacaine) at the level of the fourth cervical vertebra was performed. In a second step, the needle was inserted from posterolateral to anteromedial (in-plane technique) relative to the internal carotid artery and then, depending on the randomized group assignment, 5 ml of 0.75 % ropivacaine (group 2) or 5 ml of 0.9 % saline (group 1) was injected. The parameters investigated included the need for supplementation, patient comfort, the incidence of side effects and circulatory changes. RESULTS: The two groups did not significantly differ (p = 0.459) in terms of the need for intraoperative supplementation with 1 % prilocaine with a mean (range) in group 2 of 4.9 ml (0-20 ml), in group 1 of 3.7 ml (0-16 ml) and patient comfort (p = 0.144). In addition, a trend towards a higher complication rate was observed in group 2. CONCLUSION: For ultrasound-guided intermediate blocks of the cervical plexus, an additional periarterial infiltration showed no advantage. Abandoning this technique leads to a relevant simplification of the blocking technique and tends to reduce block-related side effects.

Single nucleotide polymorphisms in the CXCR1 gene and its association with clinical mastitis incidence in Polish Holstein-Friesian cows.

The aim of this study was to identify the association between single nucleotide polymorphisms (SNPs) in the bovine chemokine receptor (CXCR1) gene and the resistance or susceptibility of cows to mastitis. The analysis of the CXCR1 polymorphism was carried out using polymerase chain reaction restriction fragment length polymorphism analysis for six SNP mutations (c.+291C>T, c.+365T>C, c.+816C>A, c.+819G>A, +1093C>T, and +1373C>A), of which four were located within the coding region and two in the 3'UTR region of the CXCR1 gene. Genetic material from 146 Polish Holstein-Friesian cows was analyzed after dividing into two groups depending on the incidence of clinical mastitis. Identified polymorphisms were in linkage disequilibrium and formed two linkage groups. Three haplotypes (CCCATA, TTAGCC, CTCGCC), forming six haplotype combinations, were detected. The logistic regression showed a significant association between the CC genotype at c.+365T>C and susceptibility of cows to clinical mastitis (P = 0.047). The frequency of haplotype combination 1/1 (CCCATA/CCCATA) was not significantly higher in cows susceptible to mastitis (P = 0.062). Of the identified SNP mutations, only c.+365T>C is a nonsynonymous mutation that induces a change in the coded protein [GCC (Ala) to GTC (Val) at the 122nd amino acid]. This amino acid change can result in changes in receptor function, which may be a reason for the increased mastitis incidence observed in cows with polymorphism at this site.

The use of runs of homozygosity for estimation of recent inbreeding in Holstein cattle.

Controlling inbreeding in livestock populations is of great importance because excess relatedness among animals leads to a rapid loss of genetic variation and to adverse phenotypical effects associated with an inbreeding depression. Recent advances in genotyping technology have made it possible to study inbreeding at a molecular level by the analysis of genome-wide single nucleotide polymorphism panels. In this study, we used BovineSNP50 assay (Illumina) to estimate genomic inbreeding coefficient in 298 Holstein cattle by the analysis of the genome portion in runs of homozygosity (FROH) or using genomic relationship matrix (FGRM), and compared this data with conventional pedigree-based inbreeding coefficients (FPED). Weak or moderate Spearman's rank correlations were observed between FROH and FPED which depended on the ROH length categories used for calculations and inclusion of animals with different number of complete generations registered in pedigrees. The highest correlations were observed when using ROH with lengths over 8 Mb (0.334). The correlations tended to increase as pedigree depth increased, and were the highest for animals with seven complete generations of pedigree data. FGRM correlated poorly with pedigree-based estimates, which suggests that ROH-based inbreeding coefficients better reflect recent relatedness among animals.

Genome-wide RNA-Seq analysis of breast muscles of two broiler chicken groups differing in shear force.

In this study, a whole transcriptome analysis of breast muscles was conducted in broiler chicken groups differing in shear force. Shear force is a determinant of tenderness, which in turn is one of the most important parameters of meat quality in chickens. In our analysis, a total of 11,560 transcripts and 9824 genes per sample were identified. In chickens with more tender meat, up-regulation of 19 genes and down-regulation of 49 genes was observed. The up-regulated gene group included the ASB2 gene, which is probably involved in the meat conversion process, as its product results in the degradation of filamins, proteins which form muscle fibres. In the down-regulated gene group, genes which play a role in lipogenesis (THRSP, PLIN1) and in collagen synthesis (P4HA3, LEPREL4, PCOLCE2, COL16A1, COL20A1, VWA1) were detected. Their presence suggests the involvement of the extracellular matrix in the determination of meat tenderness. Thus, our study identified a pool of genes that may participate in the tenderisation process in broiler chickens.

[Ultrasound-guided intermediate cervical plexus block. Anatomical study]. / Ultraschallgesteuerte intermediäre zervikale Plexusanästhesie: Anatomische Untersuchung.

BACKGROUND AND OBJECTIVES: The innervation of the human cervical region is complex and subject to relevant anatomical variability involving sections of the cervical plexus, brachial plexus and cranial nerves. AIM: The objective was to demonstrate the dissemination of injected dye solution by anatomical preparation and to define a suitable target compartment for an ultrasound-guided block technique. MATERIAL AND METHODS: Own anatomical preparations are compared to recent review articles on the subject. The focus is on clinically relevant conclusions for performing cervical plexus blocks. In three non-embalmed cadavers six intermediate ultrasound-guided blocks of the cervical plexus were carried out, each with 20 ml methylene blue. Following preparation of the cervical plexus photographic documentation of the spread of the injected marker was performed. RESULTS: In five cases the target compartment was correctly identified. In these cases, a cranio-caudal spread of the injectate within the double layer of the cervical fascia was observed. In addition, the superficial layer was permeable to the injected methylene blue. The injection solution disseminated with the sensitive terminal branches of the cervical plexus below the platysma. In all cases an anastomosis (superficial cervical ansa) between the facial nerve (ramus colli) and the cervical plexus (transverse cervical nerve) could be demonstrated. The prevertebral lamina proved to be impermeable to injected methylene blue and no evidence of a porous structure of the prevertebral lamina was found. CONCLUSION: The compartment between the superficial and the prevertebral layer of the cervical fascia is a suitable target for cervical plexus blocks. This injection site describes an intermediate cervical plexus block. As the compartment contains the sensory terminal branches of the spinal nerves C2-4, it may be referred to as C2-C4 compartment. The cranio-caudal spread of the injectate allows lateromedial needle guidance in the horizontal plane. As the superficial lamina is not a barrier to the injectate an additional subcutaneous infiltration of the nerve area appears dispensable. The prevertebral lamina proved to be impermeable to injected methylene blue. Whether phrenic nerve blocks are preventable with more distal intermediate cervical plexus blocks (selective block of the supraclavicular nerves, e.g. for surgery of the clavicle) must be investigated in clinical trials. The permanent anastomosis (superficial cervical ansa) between the cervical plexus and the ramus colli of the facial nerve provides an anatomically reasonable explanation for inadequate cervical plexus blocks.

Quantification of the APE2 gene expression level in Candida albicans clinical isolates from patients with diagnosed fungal infections.

The production of hydrolytic enzymes is considered a key virulence determinant of Candida albicans. Aminopeptidase 2 (Ape2) facilitates the penetration of C. albicans into the host tissue, by providing free amino acids to support fungal growth and proliferation. The objective of this study was to estimate the APE2 expression profile in C. albicans cells during invasion of the human epithelium. Sixty-one clinical fungal isolates and five reference strains were included in this study. The wild-type APE2 sequence was analyzed by polymerase chain reaction (PCR) amplification using genomic DNA from pathogenic isolates. Amplicons were verified in 1% agarose gel and visualized by illumination with ultraviolet (UV) light. The APE2 expression levels were analyzed with reverse transcription quantitative real-time PCR (RT-qPCR) and APE2 quantification was normalized against the reference gene in C. albicans cells grown in YEPD and during Caco-2 invasion. The APE2-specific PCR product band was found in all C. albicans and C. dubliniensis strains, but not in other common pathogenic fungi. APE2 transcript abundance was elevated in the clinical isolates growing on the Caco-2 cell line in comparison to their counterparts grown in YEPD. Our data indicate a potential role for Ape2 in the invasion of epithelial cells. APE2 expression is also strain-specific, and it is not related to isolation site or disease entity.

Genome-wide characteristics of copy number variation in Polish Holstein and Polish Red cattle using SNP genotyping assay.

Copy number variation (CNV), which results from deletions or amplifications of large fragments of genomic DNA, is widespread in mammalian genomes and apart from its potential pathogenic effect it is considered as a source of natural genetic diversity. In cattle populations, this kind of genetic variability remains still insufficiently elucidated and studies focusing on the detection of new structural genomic variants in different cattle populations may contribute to a better understanding of cattle breeds' diversity and genetic basis of production traits. In this study, by using BovineSNP50 assay and cnvPartition algorithm we identified CNVs in two different cattle breeds: Holstein (859 animals) and Polish Red (301). In Holstein cattle we found 648 CNVs which could be reduced to 91 non-redundant variable genomic regions (CNVRs) covering in total 168.6 Mb of the genomic sequence. In Polish Red cattle we detected 62 CNVs, localized in 37 variable regions encompassing 22.3 Mb of the sequence, corresponding to 0.89 % of the autosomal genome. Within the regions we identified 1,192 unique RefSeq genes which are engaged in a variety of biological processes. High concordance of the regions' distribution was found between the studied breeds, however copy number variants seemed to be more common in Holstein cattle. About 26 % of the regions described in this study could be classified as newly identified. The results of this study will broaden the knowledge of CNVs in genomes of cattle of different breeds and will provide foundations for further research aiming to identify a relationship between this type of genetic variation and phenotypic traits.

FABP3, FABP4 and ANXA9 SNP genotypes in relation to breeding values for milk production traits in Polish Holstein-Friesian cows.

The aim of this study is to estimiate the associations between the ANXA9, FABP3, and FABP4 genotypes and the breeding value for milk production traits (yields of milk, protein, and fat, as well as protein and fat percentage) in 975 Polish Holstein-Friesian cows. The frequencies of genotypes and alleles were determined. Statistically significant relations between the ANXA9 SNP and the breeding value for fat content, as well as the FABP4 SNP and the breeding value for protein content were found. The results indicated that selection for the ANXA9 GG and FABP4 GG animals might contribute to an decreased fat content and increased protein content in milk, respectively, in Polish Holstein-Friesian cows. Although verification in the further studies is needed.

Lessons learned from an osteoporosis clinical trial in frail long-term care residents.

BACKGROUND: Although osteoporosis affects women of all ages, the impact is most pronounced in frail residents in long-term care. Nevertheless, few interventional trials have been performed in this population, and few data on therapeutic alternatives are available in this cohort. PURPOSE: We describe the challenges and lessons learned in developing and carrying out a trial in frail long-term-care residents. METHODS: The Zoledronic acid in frail Elders to STrengthen bone (ZEST) study was designed to examine the safety and efficacy of a single-dose therapy for osteoporosis in frail residents in long-term care in the Pittsburgh area. Women with osteoporosis who were 65 years of age and older and currently not on therapy were randomized in a blinded fashion to intravenous zoledronic acid or placebo. Follow-up of each participant was planned for 2 years. All participants received appropriate calcium and vitamin D supplementation. RESULTS: Seven hundred and thirty-three contacts were made with long-term care residents of nine participating facilities. Of 252 women screened, 181 were eligible, enrolled, and randomized. Multiple barriers to research in long-term-care facilities were encountered but overcome with direct communication, information sessions, in-service trainings, and social events. Lessons learned included designing the study in a manner that avoided placing an additional burden on an already overcommitted facility staff, a two-stage consent process to separate screening from randomization, and a flexible examination schedule to accommodate residents while obtaining the necessary outcome measurements. Furthermore, a mobile unit accessible to participants containing state-of-the-art dual x-ray absorptiometry (DXA), assessment for vertebral fractures, and phlebotomy equipment allows all assessments to be performed on-site at each facility. Serious adverse events are collected from affiliated hospitals in real time with a novel electronic surveillance system. LIMITATIONS: The major limitation is selection of outcomes that can be assessed at participating facilities and do not require transport of participants to hospitals or clinics. CONCLUSIONS: Clinical research for osteoporosis can be successfully and safely performed with frail residents in long-term care facilities. Lessons learned from this study may inform future investigations among frail elderly residents of these facilities.

Degradation of basement membrane by prostate tumor heparanase.

BACKGROUND: The degradation of basement membrane (BM) by cancer is an important event that characterizes invasive biological behavior. A component of BM is heparan sulfate proteoglycan (HSPG). The glycanase(s) that degrade HSPG in BM are not yet isolated. We recently identified HSPG-degrading activity (PC-3M heparanase) in the conditioned media (CM) of malignant prostate carcinoma cells (PC-3M and LNCaP C4-2). Antibodies (Abs) to a recently isolated heparanase from human platelets (CTAP-III), cross-react with PC-3M heparanase although they differ in size; under reduced conditions PC-3M heparanase is 60 kDa whereas CTAP-III is 10 kDa by polyacrylamide gel electrophoresis. PC-3M heparanase therefore shares homology with CTAP-III. The purpose of this study was to test the inhibition of PC-3M heparanase by Abs specific to the N- and C-terminals of CTAP-III. MATERIALS AND METHODS: CM from PC-3M and LNCaP C4-2 cells were tested for heparanase activity. Each reaction contained substrate as [3H]glucosamine-labeled HSPG (>50 kDa) from the BM of the EHS tumor, CM from PC-3M or LNCaP C4-2 cells, and inhibitor or buffer (negative control). Protease inhibitors were present throughout. After incubation for 3-20 h at 37 degreesC and pH 5.8, the reaction was stopped with 0.2% SDS. Each reaction mixture was centrifuged in an Ultrafree-MC 30,000 NMWL filter unit (Millipore) and radioactivity in the filtrate counted by scintillation counting. Results. For both cell lines, there was a linear relationship between the amount (microgram) of CM and degradation of HSPG. Degradation was inhibited by 54.1% (mean) using carrageenan lambda (10 microgram/ml), a nonspecific glycanase inhibitor (P < 0.05 by ANOVA). Ab to the N-terminus of CTAP-III (anti-Hep A) reduced degradation by 10-50% (mean 31.1%) and to the C-terminus (anti-Hep C) by 38.8-64.3% (mean 51.1%) (P < 0.003 by ANOVA). CONCLUSIONS: The degradation of HSPG by malignant prostate cancer cell lines is inhibited by both a nonspecific glycanase inhibitor, and specific Abs to a homologous platelet heparanase. Based upon molecular weight, PC-3M heparanase is different from platelet heparanase and degrades BM.

[The effect of infestation by mixed culture of Beauveria bassiana and Paecilomyces farinosus on reduction in numbers of experimental culture Blattella germanica L]. / Wplyw infekcji mieszanych Beauveria bassiana i Paecilomyes farinosus na redukcje liczebnosci doswiadczalnych Blettela germanica L.

In this paper results of infestation of Blatella germanica L with mixed culture of insecticidal fungi belonging to one two species are presented. In the first stage of experiment the insects were infested first with B. bassiana spores and later with P. farinosus spores (strains L and P) was used as first and B. bassiana as second with time sequence of only 24 and 72 hours. Out of one species cultures tests were done alternately with strains L and P of P. farinosus. The control insects were always infested with fungi applied to cockroach in the first place. From comparison of data it results that irrespective of the time that elapsed from application of the first pathogen to the time of application of the second pathogen the number of dead individuals was always higher in experimental series than in control i.e. when only one pathogen was applied. After 30 days of experiment the highest mortality in females amounted to, 3.3% and in males--80.0% whereas in the control it amounted 6.6% and 20.0%. Respectively, after feeding on diet infested only with one pathogen. After 50 years the highest mortality amounted to 20% in females and 100% in males with control of 30% and 53.3% in meals and females, respectively. When the sequence of pathogen application was reversed, mortality after 30 day amounted to 53.3% in females and 36.7% in meals with corresponding numbers in controls beginning 26.6% and 26.6%, respectively. After elapse of 50 days the highest mortality in females was 90% and in males--100% with control showing mortality of 36.7% and 63.3% respectively. From comparison of numerical data complied in Table 1 it results that most advantageous time span between first and second infestation with fungi is 48 h. Mortality of cockroaches infested with fungi of two different species was higher than in insects infested with one species of fungi.

Evaluation of effect of Metarhizium anisopliae on reduction in the number of Blattella germanica L.

Estimates were given of effectiveness of infestation of B. germanica L. By M. anisopliae strains originating from different regions of Poland. The investigated strains caused high mortality among experimental insects. Especially effective in reducing numbers of B. germanica were strains Browsk and Pruszyn, which even at a low density of spores caused high mortality of the test insects.

[Laboratory determination of the activity of insecticidal fungus Paecilomyces farinosus in reducing the numbers of cockroaches Blattella germanica L]. / Badania laboratoryjne aktywnosci szczepów owadobójczego grzyba Paecilomyces farinosus w redukcji liczebnosci prusaków Blattella germanica L.

The strains of P. farinosus capable of killing the cockroach Blattella germanica L. were selected. The experiment was carried out using mature insects in age groups and sex groups. The results showed differences in the insecticidal capability of the tested strains in this population of insects. Strains 5, L, P of P. farinosus were accepted as possibly useful against Blattella since at relatively low concentration of spores they produced a high mortality of the insects.

Human prostate carcinoma cells produce extracellular heparanase.

The degradation of heparan sulfate proteoglycan (HSPG) in basement membranes (BM) has been previously suggested to be accomplished by an endoglycosidase activity called heparanase which has not been isolated outside of platelets. HSPG degradation by heparanase has been associated with tumor cell invasion, angiogenesis, and growth factor function. In this study, we identify heparanase activity biochemically and immunologically in malignant human prostate carcinoma cells (PC-3M), linking platelet heparanase probes with the tumor heparanase activity observed. Concentrated conditioned medium from PC-3M cells was analyzed by a heparin-Sepharose affinity column. Three peaks eluted with 0.15, 0.35, and 0.5 M NaCl. Each peak was analyzed by incubation with 3H-labeled heparin as well as [3H]HSPG from EHS tumor BM. The 0.5 M peak material degraded [3H]-heparin by 17.2%, with little additional degradation by the other peaks in comparison to the conditioned medium from which they were obtained. Likewise, the same amount of the 0.5 M peak accounted for the majority of degradation (30.8%) of 3H-labeled HSPG. Interestingly, for the same amount of 0.5 M peak material, significantly more HSPG was degraded than heparin under the same conditions. In addition, carrageenan-lambda, an inhibitor of glycanase, completely inhibited the degradation of heparin and heparan sulfate proteoglycan by the 0.5 M peak. Using antibody to the N-terminus domain of platelet heparanase, a 60-kDa protein was identified by immunoblot in 0.5 M peak material. Additionally, immunohistochemical staining of human prostate carcinoma specimens showed granular staining at or near the cell membrane and near the luminal surface using antibody to the N-terminus and C-terminus domains of platelet heparanase. In summary, human prostate carcinoma cells show heparanase activity in conditioned medium that degrades heparin and BM HSPG and is detected by antibody to platelet heparanase. In addition, the membrane-associated staining in tissue sections of prostate cancer strongly correlates with the biochemical and immunological detection in conditioned medium of human PC-3M cells.