Small ruminant SexedULTRA™ sperm sex-sorting: Status report and recent developments.

Flow cytometry sperm sex-sorting based on the relative DNA difference between X- and Y-chromosome bearing populations is an established method that has allowed the production of pre-sexed offspring in a multitude of species and has been a commercial success in cattle production for the past twenty years. Several improvements to the technology and the processing methods have increased the sorting efficiency of ejaculates and the post-thaw quality of sex-sorted sperm, allowing for the fertility gap between conventional (non-sorted) and SexedULTRA™ sex-sorted sperm to be bridged. Small ruminant industries are now progressively testing sex-sorted sperm for application in their specific niches and environments. A review of the key advances and the recent developments in caprine, ovine and cervine sperm sex-sorting technology are described in this publication.

MALDI imaging mass spectrometry and chemometric tools to discriminate highly similar colorectal cancer tissues.

Intratumour heterogeneity due to cancer cell clonal evolution and microenvironment composition and tumor differences due to genetic variations between patients suffering of the same cancer pathology play a crucial role in patient response to therapies. This study is oriented to show that matrix-assisted laser-desorption ionization-Mass spectrometry imaging (MALDI-MSI), combined with an advanced multivariate data processing pipeline can be used to discriminate subtle variations between highly similar colorectal tumors. To this aim, experimental tumors reproducing the emergence of drug-resistant clones were generated in athymic mice using subcutaneous injection of different mixes of two isogenic cell lines, the irinotecan-resistant HCT116-SN50 (R) and its sibling human colon adenocarcinoma sensitive cell line HCT116 (S). Because irinotecan-resistant and irinotecan-sensitive are derived from the same original parental HCT116 cell line, their genetic characteristics and molecular compositions are closely related. The multivariate data processing pipeline proposed relies on three steps: (a) multiset multivariate curve resolution (MCR) to separate biological contributions from background; (b) multiset K-means segmentation using MCR scores of the biological contributions to separate between tumor and necrotic parts of the tissues; and (c) partial-least squares discriminant analysis (PLS-DA) applied to tumor pixel spectra to discriminate between R and S tumor populations. High levels of correct classification rates (0.85), sensitivity (0.92) and specificity (0.77) for the PLS-DA classification model were obtained. If previously labelled tissue is available, the multistep modeling strategy proposed constitutes a good approach for the identification and characterization of highly similar phenotypic tumor subpopulations that could be potentially applicable to any kind of cancer tissue that exhibits substantial heterogeneity.

Use of physiological information based on grayscale images to improve mass spectrometry imaging data analysis from biological tissues.

The characterization of cancer tissues by matrix-assisted laser desorption ionization-mass spectrometry images (MALDI-MSI) is of great interest because of the power of MALDI-MS to understand the composition of biological samples and the imaging side that allows for setting spatial boundaries among tissues of different nature based on their compositional differences. In tissue-based cancer research, information on the spatial location of necrotic/tumoral cell populations can be approximately known from grayscale images of the scanned tissue slices. This study proposes as a major novelty the introduction of this physiologically-based information to help in the performance of unmixing methods, oriented to extract the MS signatures and distribution maps of the different tissues present in biological samples. Specifically, the information gathered from grayscale images will be used as a local rank constraint in Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) for the analysis of MALDI-MSI of cancer tissues. The use of this constraint, setting absence of certain kind of tissues only in clear zones of the image, will help to improve the performance of MCR-ALS and to provide a more reliable definition of the chemical MS fingerprint and location of the tissues of interest. The general strategy to address the analysis of MALDI-MSI of cancer tissues will involve the study of the MCR-ALS results and the posterior use of MCR-ALS scores as dimensionality reduction for image segmentation based on K-means clustering. The resolution method will provide the MS signatures and their distribution maps for each tissue in the sample. Then, the resolved distribution maps for each biological component (MCR scores) will be submitted as initial information to K-means clustering for image segmentation to obtain information on the boundaries of the different tissular regions in the samples studied. MCR-ALS prior to K-means not only provides the desired dimensionality reduction, but additionally resolved non-biological signal contributions are not used and the weight given to the different biological components in the segmentation process can be modulated by suitable preprocessing methods.

In vitro sperm quality and DNA integrity of SexedULTRA™ sex-sorted sperm compared to non-sorted bovine sperm.

SexedULTRA™ is an improved method of sex-sorting sperm creating a less damaging environment to retain sperm integrity through the sorting process. The aim of this study was to evaluate the in vitro characteristics of fresh and frozen bovine sperm using the SexedULTRA™ method, and compare it to conventional (non-sorted) sperm. For both methods, percent total sperm motility was estimated visually and also classified into total and progressively motile using a computer assisted sperm analyzer (CASA). Percent sperm with intact plasma membranes (VIA) and acrosomes (PIA) were assessed using flow cytometry and sperm DNA fragmentation index (DFI) was estimated using the Bull sperm Halomax® Kit. Two contemporaneous ejaculates from 10 bulls were processed and cryopreserved using one of the two procedures (SexedULTRA™ and conventional). Sperm motility, VIA and PIA were assessed post-thaw (0 h) and post-incubation (3 h at 37 °C, 8 h and 24 h at 18 °C). DFI was analyzed post-thaw (0 h) and after 6, 24, 48 and 72 h of incubation at 37 °C. In a second experiment, ejaculates from 7 bulls were split sampled into the two types of processing (SexedULTRA™ and conventional) and diluted using a fresh semen extender developed for sex-sorted bovine sperm. Sperm quality was assessed after dilution (0 h) and after incubation for 12, 24, 48, 72 h at 18°, and the same time points of incubation at 37 °C for DFI. Frozen-thawed SexedULTRA™ sperm was significantly (P < 0.05) better than conventional semen after a 3 h incubation at 37 °C for PIA, and after a 24 h incubation at 18 °C for percent visual motility and PIA. DFI was significantly lower for SexedULTRA™ compared to conventional at all time points of incubation (37 °C). Fresh SexedULTRA™ sperm showed improved quality compared to conventional at all time points of incubation at 18 °C for percent visual and total motile sperm, VIA, PIA, and DFI. Significant differences were also found in progressive motile sperm immediately after dilution (0 h), but not at any time point after incubation. The results show that the SexedULTRA™ process maintains the quality of sex-sorted sperm and, in many cases, has better in vitro longevity than conventional semen.

Antibody targeting of claudin-1 as a potential colorectal cancer therapy.

BACKGROUND: Metastatic colorectal cancer (mCRC) is one of the major causes of cancer-related death. Despite the substantial progress in mCRC management, it remains important to identify new therapeutic options and biological markers for personalized medicine. Here, we investigated the expression of claudin-1 (CLDN1), a major tight junction transmembrane protein, in the different colorectal cancer (CRC) molecular subtypes and then assessed the anti-tumor effect of a new anti-CLDN1 monoclonal antibody (mAb). METHODS: Gene expression profiling and immunochemistry analysis of normal and tumor tissue samples from patients with stage IV CRC were used to determine CLDN1 gene expression. Then, the 6F6 mAb against CLDN1 extracellular part was generated. Its effect on CRC cell cycle, proliferation, survival and migration was assessed in vitro, using a 3D cell culture system, flow cytometry, clonogenic and migration assays. In vivo, 6 F6 mAb efficacy was evaluated in nude mice after subcutaneous xenografts or intrasplenic injection of CRC cells. RESULTS: Compared with normal mucosa where it was almost exclusively cytoplasmic, in CRC samples CLDN1 was overexpressed (p < 0.001) and mainly localized at the membrane. Moreover, it was differentially expressed in the various CRC molecular subtypes. The strongest expressions were found in the consensus molecular subtype CMS2 (p < 0.001), the transit-ampliflying (p < 0.001) and the C5 subtypes (p < 0.001). Lower CLDN1 expression predicted a better outcome in the molecular subtypes C3 and C5 (p = 0.012 and p = 0.004, respectively). CLDN1 targeting with the 6 F6 mAb led to reduction of survival, growth and migration of CLDN1-positive cells. In preclinical mouse models, the 6F6 mAb decreased tumor growth and liver metastasis formation. CONCLUSION: Our data indicate that CLDN1 targeting with an anti-CLDN1 mAb results in decreased growth and survival of CRC cells. This suggests that CLDN1 could be a new potential therapeutic target.

Molecular subtypes of metastatic colorectal cancer are associated with patient response to irinotecan-based therapies.

BACKGROUND: Currently, metastatic colorectal cancer is treated as a homogeneous disease and only RAS mutational status has been approved as a negative predictive factor in patients treated with cetuximab. The aim of this study was to evaluate if recently identified molecular subtypes of colon cancer are associated with response of metastatic patients to first-line therapy. PATIENTS AND METHODS: We collected and analysed 143 samples of human colorectal tumours with complete clinical annotations, including the response to treatment. Gene expression profiling was used to classify patients in three to six classes using four different molecular classifications. Correlations between molecular subtypes, response to treatment, progression-free and overall survival were analysed. RESULTS: We first demonstrated that the four previously described molecular classifications of colorectal cancer defined in non-metastatic patients also correctly classify stage IV patients. One of the classifications is strongly associated with response to FOLFIRI (P=0.003), but not to FOLFOX (P=0.911) and FOLFIRI + Bevacizumab (P=0.190). In particular, we identify a molecular subtype representing 28% of the patients that shows an exceptionally high response rate to FOLFIRI (87.5%). These patients have a two-fold longer overall survival (40.1 months) when treated with FOLFIRI, as first-line regimen, instead of FOLFOX (18.6 months). CONCLUSIONS: Our results demonstrate the interest of molecular classifications to develop tailored therapies for patients with metastatic colorectal cancer and a strong impact of the first-line regimen on the overall survival of some patients. This however remains to be confirmed in a large prospective clinical trial.

FOXO3a and the MAPK p38 are activated by cetuximab to induce cell death and inhibit cell proliferation and their expression predicts cetuximab efficacy in colorectal cancer.

BACKGROUND: Cetuximab, a monoclonal antibody against EGFR used for the treatment of colorectal cancer (CRC), is ineffective in many patients. The aim of this study was to identify the signalling pathways activated by cetuximab in CRC cells and define new biomarker of response. METHODS: We used in vitro, in vivo models and clinical CRC samples to assess the role of p38 and FOXO3a in cetuximab mechanism of action. RESULTS: We show that cetuximab activates the MAPK p38. Specifically, p38 inhibition reduced cetuximab efficacy on cell growth and cell death. At the molecular level, cetuximab activates the transcription factor FOXO3a and promotes its nuclear translocation via p38-mediated phosphorylation, leading to the upregulation of its target genes p27 and BIM and the subsequent induction of apoptosis and inhibition of cell proliferation. Finally, we found that high FOXO3a and p38 expression levels are associated with better response rate and improved outcome in cetuximab-treated patients with CRC harbouring WT KRAS. CONCLUSIONS: We identify FOXO3a as a key mediator of cetuximab mechanism of action in CRC cells and define p38 as its activator in this context. Moreover, high FOXO3a and p38 expression could predict the response to cetuximab in patients with CRC harbouring WT KRAS.

Human health risks associated with antimicrobial-resistant enterococci and Staphylococcus aureus on poultry meat.

Enterococci and staphylococci are frequent contaminants on poultry meat. Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus are also well-known aetiological agents of a wide variety of infections resulting in major healthcare costs. This review provides an overview of the human health risks associated with the occurrence of these opportunistic human pathogens on poultry meat with particular focus on the risk of food-borne transmission of antimicrobial resistance. In the absence of conclusive evidence of transmission, this risk was inferred using data from scientific articles and national reports on prevalence, bacterial load, antimicrobial resistance and clonal distribution of these three species on poultry meat. The risks associated with ingestion of antimicrobial-resistant enterococci of poultry origin comprise horizontal transfer of resistance genes and transmission of multidrug-resistant E. faecalis lineages such as sequence type ST16. Enterococcus faecium lineages occurring in poultry meat products are distantly related to those causing hospital-acquired infections but may act as donors of quinupristin/dalfopristin resistance and other resistance determinants of clinical interest to the human gut microbiota. Ingestion of poultry meat contaminated with S. aureus may lead to food poisoning. However, antimicrobial resistance in the toxin-producing strains does not have clinical implications because food poisoning is not managed by antimicrobial therapy. Recently methicillin-resistant S. aureus of livestock origin has been reported on poultry meat. In theory handling or ingestion of contaminated meat is a potential risk factor for colonization by methicillin-resistant S. aureus. However, this risk is presently regarded as negligible by public health authorities.

MRSA carrying mecC in captive mara.

OBJECTIVES: To characterize the staphylococcal cassette chromosome mec (SCCmec), virulence and antimicrobial susceptibility of Staphylococcus aureus ST130 isolated from mara (Dolichotis patagonum), a large rodent species native to South America and kept in captivity at Copenhagen Zoo. METHODS: The presence of mecC was confirmed by PCR in 15 S. aureus ST130 isolated from mara during a previous study. WGS was performed on two randomly selected isolates to characterize their genomes with respect to SCCmec, virulence and resistance gene content. Antimicrobial susceptibility was tested using commercial broth microdilution tests. RESULTS: All the isolates belonged to spa type t528 ST130 and carried mecC. Based on WGS, mecC was 100% identical to the prototype sequence of S. aureus strain LGA251. The sequence of SCCmec type XI in the mara isolates had 23 SNPs compared with the one described in LGA251. The two sequenced strains harboured a set of virulence factors and other genomic features previously observed in ST130. Both strains carried norA as the only putative antimicrobial resistance gene in addition to mecC. CONCLUSIONS: Our findings support the notion that a genetically conserved mecC-carrying MRSA ST130 clone is widespread in a variety of unrelated hosts in Denmark. Since the mara at Copenhagen Zoo have limited contact with humans and other animal species, it remains unclear whether mara are natural hosts of ST130 or acquired this lineage from unknown sources. The broad host range of MRSA ST130 supports its designation as a generalist lineage.

Transmission of MRSA CC398 strains between pig farms related by trade of animals.

Meticillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 is a genetic lineage associated with livestock, especially pigs. The authors investigated the role of pig trade in the transmission of MRSA CC398 between farms using pulsed-field gel electrophoresis (PFGE), a highly discriminatory method for strain typing. PFGE analysis of 58 MRSA isolates from a retrospective study in the Netherlands and a prospective study in Denmark provided molecular evidence that the strains present in five of the eight recipient farms were indistinguishable from those occurring in the corresponding supplying farm. The molecular typing data confirm the findings of a previous risk-analysis study indicating that trading of colonised pigs is a vehicle for transmission of MRSA CC398.

Efficacy of 65% permethrin applied to dogs as a spot-on against Phlebotomus perniciosus.

Leishmania infantum is a protozoan parasite causing leishmaniosis, a visceral disease transmitted by the bites of sand flies. As the main reservoir of the parasite, dogs are the principal targets of control measures against this disease, which affects both humans and dogs. Several studies have revealed the usefulness of topical insecticide treatment (collars, spot-ons and sprays) in reducing the incidence and prevalence of L. infantum. The present study was designed to test the efficacy of 65% permethrin applied to dogs as a spot-on against the sand fly vector Phlebotomus perniciosus. The duration of the desired effects was also estimated to help design an optimal treatment regimen. Twelve dogs assigned to treatment (n=6) and control (n=6) groups were exposed to sand flies once a week over a seven-week period. Repellent and insecticidal efficacies were estimated and compared amongst the groups. Our findings indicate satisfactory repellent, or anti-feeding, effects lasting 3 weeks and short-term insecticidal effects lasting 2 weeks after initial application. Accordingly, we recommend the use of this product every 2-3 weeks during the active phlebotomine sand fly period to protect dogs against the bites of P. perniciosus.

Farm-specific lineages of methicillin-resistant Staphylococcus aureus clonal complex 398 in Danish pig farms.

The objective of this study was to investigate the genetic diversity of methicillin-resistant Staphylococcus aureus (MRSA) clonal complex (CC) 398 using pulsed-field gel electrophoresis (PFGE). Dust and pigs at five age groups were sampled in six Danish MRSA-positive pig farms. MRSA CC398 was isolated from 284 of the 391 samples tested, including 230 (74%) animal and 54 (68%) environmental samples. PFGE analysis of a subset of 48 isolates, including the six strains previously isolated from farm workers, revealed the existence of farm-specific pulsotypes. With a single exception, human, environmental and porcine isolates originating from the same farm clustered together in the PFGE cluster analysis, indicating that spread of MRSA CC398 in Danish pig farms is mainly due to clonal dissemination of farm-specific lineages that can be discriminated by PFGE. This finding has important implications for planning future epidemiological studies investigating the spread of CC398 in pig farming.

Mice knock out for the histone acetyltransferase p300/CREB binding protein-associated factor develop a resistance to amyloid toxicity.

p300/CREB binding protein-associated factor (PCAF) regulates gene expression by acting through histone acetylation and as a transcription coactivator. Although histone acetyltransferases were involved in the toxicity induced by amyloid-beta (Abeta) peptides, nothing is known about PCAF. We here analyzed the sensitivity of PCAF knockout (KO) mice to the toxic effects induced by i.c.v. injection of Abeta(25-35) peptide, a nontransgenic model of Alzheimer's disease. PCAF wild-type (WT) and KO mice received Abeta(25-35) (1, 3 or 9 nmol) or scrambled Abeta(25-35) (9 nmol) as control. After 7 days, Abeta(25-35) toxicity was measured in the hippocampus of WT mice by a decrease in CA1 pyramidal cells and increases in oxidative stress, endoplasmic reticulum stress and induction of apoptosis. Memory deficits were observed using spontaneous alternation, water-maze learning and passive avoidance. Non-treated PCAF KO mice showed a decrease in CA1 cells and learning alterations. However, Abeta(25-35) injection failed to induce toxicity or worsen the deficits. This resistance to Abeta(25-35) toxicity did not involve changes in glutamate or acetylcholine systems. Examination of enzymes involved in Abeta generation or degradation revealed changes in transcription of presenilins, activity of neprilysin (NEP) and an absence of Abeta(25-35)-induced regulation of NEP activity in PCAF KO mice, partly due to an altered expression of somatostatin (SRIH). We conclude that PCAF regulates the expression of proteins involved in Abeta generation and degradation, thus rendering PCAF KO insensitive to amyloid toxicity. Modulating acetyltransferase activity may offer a new way to develop anti-amyloid therapies.

The interferon-inducible Staf50 gene is downregulated during T cell costimulation by CD2 and CD28.

It is well established that interferons (IFN) exert potent regulatory effects on the immune system. We have recently isolated a new IFN-induced human cDNA coding for a member of the Ring finger B-box/B30.2 subfamily that localizes to the chromosome band 11p15. We have named it Staf50. We show in this report that Staf50 is expressed in resting T cells in the absence of exogenous IFN treatment and is strongly repressed during T cell activation by anti-CD28 and anti-CD2 monoclonal antibodies (mAb) at both messenger and protein levels. In addition, we show that several members of the Ring finger B-box/B30.2 subfamily, including the 52-kDa SSA/Ro autoantigen, localize to the same chromosome band, 11p15, and are upregulated by IFN. These data led us to define a family of IFN-induced genes clustered on chromosome 11p15 that may be involved in T cell regulatory processes.

A unique ISRE, in the TATA-less human Isg20 promoter, confers IRF-1-mediated responsiveness to both interferon type I and type II.

Interferons (IFNs) encode a family of secreted proteins involved in a number of regulatory functions such as control of cell proliferation, differentiation and regulation of the immune system. Their diverse biological actions are thought to be mediated by the products of specific but usually overlapping sets of cellular genes induced in the target cells. We have recently isolated a human cDNA encoding a new nuclear bodies-associated protein (PML-NBs), which we have termed Isg20. In this report, we describe the cloning and functional characterization of the Isg20 promoter region and the identification of sequence elements and trans-acting factors implicated in its regulation. In the absence of any recognizable TATA or CAAT elements, Isg20 promoter basal activity is dependent upon the positive transcription factors Sp-1 and USF-1. Interestingly, we demonstrate that a unique interferon stimulated response element (ISRE) mediates both IFN type I and type II Isg20 induction in the absence of functional gamma-activated sequence. These inductions are strictly dependent upon of the IFN regulatory factor 1 (IRF-1). In addition, we show that the ISRE is also implicated in the constitutive transcriptional activity of Isg20 gene.

[Interferon signaling pathways]. / L'interféron: un mécanisme complexe de signalisation.

Interferons (IFNs) encode a large family of multifonctional secreted proteins that are involved in antiviral defense, the regulation of cell growth and modulation of the immune response. They are subdivided into two types that activate transduction pathways via different cell surface receptors. Binding of both IFN type I and II results in the differential activation of JAK (Janus kinases) that phosphorylate latent cytoplasmic transcription factors termed STATs (signal transducer and activator of transcription). Phosphorylated STATs translocate to the nucleus, bind specific DNA elements and direct transcription. Type I IFN induces the phosphorylation of STAT1 and STAT2 proteins by tyrosine phosphorylation involving the type I IFN receptor-associated tyrosine kinases TYK2 and JAK1. Following phosphorylation, STAT1 and STAT2 form the transcriptionally active IFN-stimulated gene factor 3 (ISGF3) by association with a protein of the IFN regulatory factor (IRF) family, p48. The specificity of the transcriptional activation by ISGF3 is mediated by specific elements termed IFN-stimulatory response element (ISRE) located in the promoter region of IFN-inducible genes. ISREs drive the expression of most IFN type I-regulated genes and a few IFN type II-regulated genes. Gene induction by type II IFN involves the phosphorylation of only STAT1 by JAK1 and Jak2 kinases. This phosphorylation generates a homodimer of STAT1 which is able to bind the IFNgamma-activated site (GAS) to activate transcription. This signaling is rapid and direct. Molecules involved in the IFN signaling pathways have been shown to be used by other polypeptide ligands in their own signal transduction pathways. Pathways other than JAK/STAT are also involved in IFN signaling, but their mechanisms are less clear. The best documented are the mitogen-activated protein kinase (MAPK) cascade, the components of the TCR (T cell receptor) signaling cascade and the Pi3 kinase pathway.

Molecular cloning of a new interferon-induced PML nuclear body-associated protein.

Transcriptional induction of genes is an essential part of the cellular response to interferons. We have established a cDNA library from human lymphoblastoid Daudi cells treated for 16 h with human alpha/beta-interferon (IFN) and made use of differential screening to search for as yet unidentified IFN-regulated genes. In the course of this study, we have isolated a human cDNA that codes for a 20-kDa protein sharing striking homology with the product of the Xenopus laevis XPMC2 gene. This new gene is induced by both type I and II IFNs in various cell lines and will be referred to as ISG20 for interferon-stimulated gene product of 20 kDa. Confocal immunofluorescence analysis of the subcellular localization of ISG20 protein reveals that it is closely associated with PML and SP100 gene products within the large nuclear matrix-associated multiprotein complexes termed the PML nuclear bodies.

Quantitative analysis of the expression of the HLA-DRB genes at the transcriptional level by competitive polymerase chain reaction.

In addition to polymorphism of the peptide-binding site, the density of the MHC class II molecules expressed on the membrane of APC could well play a significant role in the MHC-peptide-TCR interaction during the immune response. We therefore investigated the regulation of the expression of the HLA-DRB genes at the transcriptional level. A competitive PCR approach was used to estimate the quantities of the HLA-DRB transcripts in peripheral blood B cells. When comparing the amounts of steady-state mRNAs among the different DRB1 alleles, the DRB1 transcripts in the DR52 haplotype group were found to be 2.5 to 3.5 times more abundant than the DRB1*01 transcripts, 1.5 to 2 times more abundant than the DRB1*04 transcripts, and 7 times more abundant than the DRB1*08 transcripts. Within the DR52 haplotype group, the DRB1 and DRB3 transcripts had the same abundance. Taken together, these results are in good agreement with the previously reported transcriptional activities of the DRB promoters except for DRB1*04, thus suggesting a differential post-transcriptional regulation among the DRB1 mRNAs.

[Trophic activity of neurotensin in massive intestinal resection]. / Actividad trófica de la neurotensina en la resección intestinal masiva.

Regulatory peptides are among the main factors affecting the proliferative adaptive process of the small bowel; its mechanisms of action remains unknown. An 80% small bowel resection was done in Wistar rats (250-300 g) randomly assigned to two groups: control and neurotensin (300 mu/kg). Jejunal and ileal samples were taken on day 14 post-resection and histomorphometric (villous length) and proliferative (DNA content) measures were obtained. Results show an increase in villous length in the neurotensin group but no changes in DNA content were observed. We conclude that neurotensin increases intestinal mucosal growth after massive intestinal resection in the rat.