The formation of preferential relationships at early age in cattle

Calves can develop long-lasting social relationships with peers. We examined the strength of the relationships between calves according to the time they had been together. Twenty-four female dairy calves were assigned to six groups of four animals (Type-1 partners) at 0.5 month of age. At 3.5 months of age, they were mixed with other calves (Type-2 partners) to form groups of 14. Type-3 partners were calves added to the experimental groups after 5.25 months. The calves stayed together until 1.5 years of age. Social preferences between the three partner types were examined in a Y-maze, and the position and activity of animals in the barn and pasture were followed in three periods. Behavioural synchrony, distance between animals, proximity and nearest neighbour were analysed. The calves more frequently butted Type-3 than Type-1 partners in the Y-maze (P < 0.05). They spent more time in proximity to Type-1 partners, and these were more often the nearest neighbours than other partners (P < 0.001). Synchrony and distance between animals were greater at pasture than in the barn (P < 0.01). Calves seem to form preferential relationships before 3.5 months of age. Keeping cattle together from an early age seems beneficial for them.     

Reference-facilitated phosphoproteomics: fast and reliable phosphopeptide validation by microLC-ESI-Q-TOF MS/MS

Recent advances in instrument control and enrichment procedures have enabled us to quantify large numbers of phosphoproteins and record site-specific phosphorylation events. An intriguing problem that has arisen with these advances is to accurately validate where phosphorylation events occur, if possible, in an automated manner. The problem is difficult because MS/MS spectra of phosphopeptides are generally more complicated than those of unmodified peptides. For large scale studies, the problem is even more evident because phosphorylation sites are based on single peptide identifications in contrast to protein identifications where at least two peptides from the same protein are required for identification. To address this problem we have developed an integrated strategy that increases the reliability and ease for phosphopeptide validation. We have developed an off-line titanium dioxide (TiO(2)) selective phosphopeptide enrichment procedure for crude cell lysates. Following enrichment, half of the phosphopeptide fractionated sample is enzymatically dephosphorylated, after which both samples are subjected to LC-MS/MS. From the resulting MS/MS analyses, the dephosphorylated peptide is used as a reference spectrum against the original phosphopeptide spectrum, in effect generating two peptide spectra for the same amino acid sequence, thereby enhancing the probability of a correct identification. The integrated procedure is summarized as follows: 1) enrichment for phosphopeptides by TiO(2) chromatography, 2) dephosphorylation of half the sample, 3) LC-MS/MS-based analysis of phosphopeptides and corresponding dephosphorylated peptides, 4) comparison of peptide elution profiles before and after dephosphorylation to confirm phosphorylation, and 5) comparison of MS/MS spectra before and after dephosphorylation to validate the phosphopeptide and its phosphorylation site. This phosphopeptide identification represents a major improvement as compared with identifications based only on single MS/MS spectra and probability-based database searches. We investigated an applicability of this method to crude cell lysates and demonstrate its application on the large scale analysis of phosphorylation sites in differentiating mouse myoblast cells.     

Nestin as a regulator of Cdk5 in differentiating myoblasts

Many types of progenitor cells are distinguished by the expression of the intermediate filament protein nestin, a frequently used stem cell marker, the physiological roles of which are still unknown. Whereas myogenesis is characterized by dynamically regulated nestin levels, we studied how altering nestin levels affects myoblast differentiation. Nestin determined both the onset and pace of differentiation. Whereas depletion of nestin by RNAi strikingly accelerated the process, overexpression of nestin completely inhibited differentiation. Nestin down-regulation augmented the early stages of differentiation, at the level of cell-cycle withdrawal and expression of myogenic markers, but did not affect proliferation of undifferentiated dividing myoblasts. Nestin regulated the cleavage of the Cdk5 activator protein p35 to its degradation-resistant form, p25. In this way, nestin has the capacity to halt myoblast differentiation by inhibiting sustained activation of Cdk5 by p25, which is critical for the progress of differentiation. Our results imply that nestin regulates the early stages of myogenesis rather than maintains the undifferentiated state of progenitor cells. In the bidirectional interrelationship between nestin and Cdk5, Cdk5 regulates the organization and stability of its own nestin scaffold, which in turn controls the effects of Cdk5. This nestin-Cdk5 cross-talk sets the pace of muscle differentiation.     

Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format

We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay probe uses fluorescence resonance energy transfer (FRET) between a europium chelate donor and an organic fluorophore acceptor. The substantial change in the acceptor emission decay time on hybridization with the target sequence allows the direct separation of the hybridized and unhybridized probe populations in a time-resolved measurement. No additional sample manipulation or self-hybridization of the probes is required. The wavelength and decay time of a decay probe can be adjusted according to the selection of probe length and acceptor fluorophore, thereby making the probes applicable to multiplexed assays. Here we demonstrate the decay probe principle and decay probe-based, one-step, dual DNA assay using celiac disease-related target oligonucleotides (single-nucleotide polymorphisms [SNPs]) as model analytes. Decay probes showed specific response for their complementary DNA target and allowed good signal deconvolution based on simultaneous optical and temporal filtering. This technique potentially could be used to further increase the number of simultaneously detected DNA targets in a simple one-step homogeneous assay.     

Providing cellular signposts--post-translational modifications of intermediate filaments

Intermediate filaments are dynamically regulated by their post-translational modifications. Initially these modifications were found to regulate filament dynamics and organization. In the last few years, their roles have extended significantly to facilitating, for example, the recruitment and sequestration of signaling molecules that regulate a wide range of cellular functions. While phosphorylation has been established as the principal post-translational modification regulating intermediate filament function, other modifications with co-operative roles are emerging, adding a further dimensions to intermediate filament-mediated signaling.     

Liver Sinusoidal Endothelial Cells Are a Site of Murine Cytomegalovirus Latency and Reactivation

Latent cytomegalovirus (CMV) is frequently transmitted by organ transplantation, and its reactivation under conditions of immunosuppressive prophylaxis against graft rejection by host-versus-graft disease bears a risk of graft failure due to viral pathogenesis. CMV is the most common cause of infection following liver transplantation. Although hematopoietic cells of the myeloid lineage are a recognized source of latent CMV, the cellular sites of latency in the liver are not comprehensively typed. Here we have used the BALB/c mouse model of murine CMV infection to identify latently infected hepatic cell types. We performed sex-mismatched bone marrow transplantation with male donors and female recipients to generate latently infected sex chromosome chimeras, allowing us to distinguish between Y-chromosome (gene sry or tdy)-positive donor-derived hematopoietic descendants and Y-chromosome-negative cells of recipients'' tissues. The viral genome was found to localize primarily to sry-negative CD11b⁻ CD11c⁻ CD31⁺ CD146⁺ cells lacking major histocompatibility complex class II antigen (MHC-II) but expressing murine L-SIGN. This cell surface phenotype is typical of liver sinusoidal endothelial cells (LSECs). Notably, sry-positive CD146⁺ cells were distinguished by the expression of MHC-II and did not harbor latent viral DNA. In this model, the frequency of latently infected cells was found to be 1 to 2 per 10⁴ LSECs, with an average copy number of 9 (range, 4 to 17) viral genomes. Ex vivo-isolated, latently infected LSECs expressed the viral genes m123/ie1 and M122/ie3 but not M112-M113/e1, M55/gB, or M86/MCP. Importantly, in an LSEC transfer model, infectious virus reactivated from recipients'' tissue explants with an incidence of one reactivation per 1,000 viral-genome-carrying LSECs. These findings identified LSECs as the main cellular site of murine CMV latency and reactivation in the liver.In human cytomegalovirus (hCMV) infection, hematopoietic progenitor cells of the myeloid differentiation lineage are a recognized cellular site of virus latency (for more-recent reviews, see references 75 and 94), and cell differentiation-dependent as well as cytokine-mediated viral gene desilencing by chromatin remodeling is discussed as the triggering event leading to virus reactivation (for a review, see reference 7). Although hematopoietic stem cell or bone marrow transplantation (BMT) is frequently associated with hCMV reactivation and recurrence in recipients after hematoablative leukemia/lymphoma therapy, the incidence of virus recurrence and disease is highest in the combination of an hCMV-negative donor (D⁻) and an hCMV-positive recipient (R⁺) (D⁻R⁺ > D⁺R⁺ > D⁺R⁻), indicating that donor hematopoietic cells are not the only source of latent hCMV and actually not the predominant source (34). Rather, the recipients experience reactivation of their own virus. Just the opposite is true in the case of solid organ transplantation, where the reactivating virus is mostly transmitted with the transplanted organ (D⁺R⁻ > D⁺R⁺ > D⁻R⁺) (34). Collectively, these risk assessments support the suggestion that reactivation, in both instances, occurs in latently infected tissue cells, that is, within the recipient''s organs and the transplanted donor organ, respectively. Although tissue-resident cells of hematopoietic origin remain candidates, stromal and parenchymal tissue cells come into consideration as additional sites of CMV latency.Longitudinal analysis of viral genome load in the latency models of murine CMV (mCMV) infection of neonatal mice (9, 91) as well as of adult mice after experimental BMT (8, 62, 64) has demonstrated a high viral latency burden in multiple organs long after clearance of viral DNA from bone marrow and blood (reviewed in reference 92). These findings support the suggestion that there exist two types of latency, namely, a temporary latency in hematopoietic cells and a latency in tissue cells that lasts through life. Accordingly, both types of latency may coexist early after primary infection, while “late latency” is restricted to organ sites. As we have shown previously in a sex-mismatched murine BMT model, bone marrow cells (BMCs) derived from latently infected donors in the phase of organ-restricted “late latency” cannot transmit latent or reactivated infection to naïve recipients upon intravenous cell transfer (99).A first hint for mCMV latency in stromal or reticular cells was presented long ago by Mercer and colleagues (73), who showed that infected cells during acute infection of the spleen are predominantly sinusoidal lining cells and that latent mCMV can be recovered from a major histocompatibility complex class II (MHC-II) antigen-negative and Thy-1 (CD90)-negative “stromal” cell fraction, which includes endothelial cells (ECs). These findings strongly argued against T and B lymphocytes, macrophages, and dendritic cells (DCs) being major reservoirs of latent mCMV in the spleen, a conclusion supported by later work of Pomeroy and colleagues (86). Similarly, Klotman and colleagues (54) as well as Hamilton and Seaworth (44) concluded that in kidney transplantation, donor kidney is the source of latent mCMV and that the latent viral genome is harbored by renal peritubular epithelial cells (53). A first hint for mCMV latency in ECs within the liver was provided by in situ PCR images presented by Koffron and colleagues (59) showing nuclear staining in cells with a microanatomical localization suspicious of liver sinusoidal ECs (LSECs). For hCMV, ECs, in particular those in arterial vessel walls, are regarded as a site of latency on the basis of the presence of viral DNA in cells expressing an EC marker (81; for a review, see reference 48), although other authors did not detect viral DNA in venous vessel walls (95). As discussed by Jarvis and Nelson (48), these data are not necessarily conflicting but may rather reflect the diversity of EC subsets at different anatomical locations (21, 27). As far as we know, and reactivation of productive infection from ECs that carry latent viral DNA is not yet formally proven for any type of EC.Hepatitis is a relevant organ manifestation of CMV disease in immunocompromised hosts (65), and hCMV reactivation has been reported to be the most common cause of infection following liver transplantation, in particular in a D⁺R⁻ combination (34, 76). In the murine model of immunocompromised hosts, viral histopathology in the liver is dominated by the cytopathogenic infection of hepatocytes, leading to extended plaque-like tissue lesions (41, 84; reviewed in reference 45). Occasionally, however, in these studies, infected hepatic ECs as well as Kupffer macrophages were detected by virus-specific immunohistology or by in situ virus-specific DNA hybridization.Using cell-type-specific conditional recombination of a fluorescence-tagged reporter virus in Cre-transgenic mice expressing Cre selectively in hepatocytes under the control of the albumin promoter, hepatocytes were recently identified as the main virus-producing cell type during mCMV infection. In Cre-transgenic mice expressing Cre selectively in vascular ECs under the control of the Tie2 promoter, the reporter virus was found to recombine also in LSECs, which released an amount of virus sufficient for virus spread to neighboring hepatocytes, although the virus productivity of LSECs was low and contributed little to the overall virus load in the liver (97).LSECs represent a unique liver-resident population of antigen-presenting cells that bear the capacity to cross-present antigens to naïve CD8 T cells (68, 69; reviewed in reference 57). They constitute the fenestrated endothelial lining of the hepatic sinusoids (15). By separating the sinusoidal compartment of the liver from the space of Disse and the liver parenchyma, LSECs form a boundary surface for sensing of pathogens and interaction with passenger lymphocytes. They perform a scavenger function contributing to hepatic clearance of bacterial degradation products derived from the gastrointestinal tract (103; reviewed in reference 57). According to this physiological role, antigen presentation by LSECs is associated with tolerance induction rather than with triggering an inflammatory immune response (29, 56, 68, 104).Here we provide evidence to support the suggestion that mCMV has chosen the tolerogenic and long-lived LSECs as an immunoprivileged niche for establishing viral latency in the liver.     

A nestin scaffold links Cdk5/p35 signaling to oxidant-induced cell death

The intermediate filament protein, nestin, has been implicated as an organizer of survival-determining signaling molecules. When nestin expression was related to the sensitivity of neural progenitor cells to oxidant-induced apoptosis, nestin displayed a distinct cytoprotective effect. Oxidative stress in neuronal precursor cells led to downregulation of nestin with subsequent activation of cyclin-dependent kinase 5 (Cdk5), a crucial kinase in the nervous system. Nestin downregulation was a prerequisite for the Cdk5-dependent apoptosis, as overexpression of nestin efficiently inhibited induction of apoptosis, whereas depletion of nestin by RNA interference had a sensitizing effect. When the underlying link between nestin and Cdk5 was analyzed, we observed that nestin serves as a scaffold for Cdk5, with binding restricted to a specific region following the alpha-helical domain of nestin, and that the presence and organization of nestin regulated the sequestration and activity of Cdk5, as well as the ubiquitylation and turnover of its regulator, p35. Our data imply that nestin is a survival determinant whose action is based upon a novel mode of Cdk5 regulation, affecting the targeting, activity, and turnover of the Cdk5/p35 signaling complex.