Expression of Dmrt1 protein in developing and in sex-reversed gonads of amphibians.
Many genes are known to be involved in gonadal differentiation in vertebrates. Dmrt1, a gene that encodes a transcription factor with a DM-domain, is considered to be one of the essential genes controlling testicular differentiation in mammals, birds, reptiles, amphibians and fish. However, it still remains unknown which testicular cells of animals other than mice and chicks express Dmrt1 protein. For an explanation of its role(s) in testicular differentiation in vertebrates, the expression of the Dmrt1 protein needs to be studied. For this purpose, we conducted an immunohistochemical study of this protein in an amphibian by using an antibody specific for Dmrt1. No positive signal was found in the indifferent gonad of tadpoles of Rana rugosa at early stages. However, in the testis of tadpoles at later stages (XV-XXV) and in frogs one month after metamorphosis, this protein was expressed in interstitial cells and Sertoli cells. In the testis of adult frogs, germ cells also expressed Dmrt1 protein. RT-PCR analysis revealed that the gene for this protein was not transcribed at any time during ovarian development, but was expressed in the female to male sex-reversed gonad. This was true when immunohistological studies were performed. In addition, Southern blot analysis showed DMRT1 to be an autosomal gene. Taken together, our findings indicate that Dmrt1 protein is expressed by interstitial cells, Seroli cells and germ cells in the testis of R. rugosa. Dmrt1 may thus be very involved in the testicular differentiation of amphibians.


C-terminal protein characterization by mass spectrometry: isolation of C-terminal fragments from cyanogen bromide-cleaved protein.
A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2'-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures.


My Sister's Keeper: Survivors Disclosing Sexual Violence to a Sibling.
Siblings are often cited as a source of support throughout life, but they have not been included in studies of sexual violence disclosure. Survivors of sexual violence often delay or do not disclose their sexual violence. The current study investigated if and why survivors of sexual violence decided to disclose to their sibling. This qualitative investigation included 10 female participants, of which six had disclosed their abuse to their sibling. The participants ranged in age from 20 to 58, five identified as Hispanic, one as Hispanic and White three identified as White, and one identified as Middle Eastern. Thematic analysis was used to examine the data. Three major themes were identified that affected sibling disclosure decisions: sibling dynamics, perceived and real reactions to disclosure, and words of advice. The findings from this study demonstrate the continued need to investigate siblings and their role in disclosure of sexual violence. Practitioners may also want to examine the sibling relationship as a source of support for survivors of sexual violence. Finally, as policies continue to develop around support of survivors of sexual violence, inclusion of siblings in family policies, outreach centers, and counseling services may be beneficial.


The early origins of atherosclerosis.
Atherosclerosis has a long pre-clinical phase with development of pathological changes in arteries of children and young adults decades before overt clinical manifestations of disease. Nutritional factors in both infancy and childhood have been shown to be important in this process and affect lifetime cardiovascular disease risk. Breast-feeding in particular is associated with benefits for long-term cardiovascular risk factors possibly as a consequence of a slower pattern of growth in breast-fed compared to formula-fed infants. In fact, the benefits of slower growth for later health and longevity, appears to be a fundamental biological phenomenon conserved across diverse animal species. The nutritional programming of atherosclerosis could therefore be regarded as a specific example of programming of human ageing as seen previously in programming of lifespan and telomere length in animals. The critical window for these effects is unknown, but evidence is accumulating for programming effects of growth from very early in infancy.


Multicolor karyotype analyses of mouse embryonic stem cells.
The manipulation of embryonic stem (ES) cells to introduce directional genetic changes into the genome of mice has become an important tool in biomedical research. Monitoring of cell morphology before and after DNA manipulation and special culture conditions are a prerequisite to preserve the pluripotent properties of ES cells and thus their ability to generate chimera and effective germline transmission (GLT). It has been reported that prolonged cell culturing may affect the diploid chromosomal composition of cells and therefore the percentage of chimerism and GLT. Herein, we report multicolor-fluorescence in situ hybridization (M-FISH) analysis of four different ES cell lines/clones. Although the morphology of all four ES cell lines/clones appeared normal and all four expressed the early markers Oct-3/4 and Nanog, two cell lines presented consistent numerical and structural chromosome aberrations. We demonstrate that M-FISH is a sensitive and accurate method for a comprehensive karyotype analysis of ES cells and may minimize time, costs, and disappointments due to inadequate ES cell sources.


Bayesian negative-binomial-family-based multistate Markov model for the evaluation of periodic population-based cancer screening considering incomplete information and measurement errors.
Population-based cancer screening is often asked but hardly addressed by a question: "How many rounds of screening are required before identifying a cancer of interest staying in the pre-clinical detectable phase (PCDP)?" and also a similar one related to the number of screens required for stopping screening for the low risk group. It can be answered by using longitudinal follow-up data on repeated rounds of screen, namely periodic screen, but such kind of data are rather complicated and fraught with intractable statistical properties including correlated multistate outcomes, unobserved and incomplete (censoring or truncation) information, and imperfect measurements. We therefore developed a negative-binomial-family-based discrete-time stochastic process, taking sensitivity and specificity into account, to accommodate these thorny issues. The estimation of parameters was implemented with Bayesian Markov Chain Monte Carlo method. We demonstrated how to apply this proposed negative-binomial-family-based model to the empirical data similar to the Finnish breast cancer screening program.


Changes in haemostasis during normal pregnancy: does homocysteine play a role in maintaining homeostasis?
Homocysteine, derived from the demethylation of the amino acid methionine, is either further catabolised by trans-sulfuration to cysteine or remethylated to methionine. Remethylation to methionine requires the cofactors, folate and vitamin B12. Folate is an effective homocysteine-lowering agent and, thus, homocysteine and folate status are inversely related. Hyperhomocysteinaemia is a strong independent risk factor for venous thromboembolism (VTE) and is associated with adverse pregnancy outcomes such as pre-eclampsia, placental abruption, early pregnancy loss and neural-tube defects. Pregnancy is a risk factor for VTE as a result of prothrombotic changes in levels of haemostatic factors. However, despite this hypercoagulable state, the incidence of pregnancy-associated VTE is relatively low. Hyperhomocysteinaemia is associated with abnormalities in markers of coagulation activation, and recent research suggests that folic acid supplementation, as well as lowering homocysteine, lowers markers of coagulation activation and increases levels of coagulation inhibitors. Tissue factor (TF) is the initiator of blood coagulation in vivo, and homocysteine induces TF expression in vitro. During pregnancy, monocyte TF expression is lower than that in the non-pregnant state, and this lowering of TF may act to counterbalance increases in coagulation activation. Furthermore, despite a high folate requirement, several studies have reported that homocysteine is lower in normal pregnancy than in the non-pregnant state. Although the exact mechanism of homocysteine lowering during pregnancy is unclear, one possible outcome of lower homocysteine may be the protection of women from pregnancy complications and VTE, and thus lower homocysteine may contribute to maintaining homeostasis in haemostasis.


Cross-talk between human dendritic cell subsets influences expression of RNA sensors and inhibits picornavirus infection.
Dendritic cells (DCs) are professional antigen-presenting cells that provide a link between innate and adaptive immunity. Multiple DC subsets exist and their activation by microorganisms occurs through binding of conserved pathogen-derived structures to so-called pattern recognition receptors (PRRs). In this study we analyzed the expression of PRRs responding to viral RNA in human monocyte-derived DCs (moDCs) under steady-state or pro-inflammatory conditions. We found that mRNA and protein levels for most PRRs were increased under pro-inflammatory conditions, with the most pronounced increases in the RIG-like helicase (RLH) family. Additionally, freshly isolated human plasmacytoid DCs (pDCs) displayed significantly higher levels of TLR7, RIG-I, MDA5 and PKR as compared to myeloid DCs and moDCs. Finally, we demonstrate for the first time that cross-talk between TLR-matured or virus-stimulated pDCs and moDCs leads to a type I interferon-dependent antiviral state in moDCs. This antiviral state was characterized by enhanced RLH expression and protection against picornavirus infection. These findings might represent a novel mechanism by which pDCs can preserve the function and viability of myeloid DCs that are attracted to a site with ongoing infection, thereby optimizing the antiviral immune response.


Identification of EloR (Spr1851) as a regulator of cell elongation in Streptococcus pneumoniae.
In a screen for mutations suppressing the lethal loss of PBP2b in Streptococcus pneumoniae we identified Spr1851 (named EloR), a cytoplasmic protein of unknown function whose inactivation removed the requirement for PBP2b as well as RodA. It follows from this that EloR and the two elongasome proteins must be part of the same functional network. This network also includes StkP, as this serine/threonine kinase phosphorylates EloR on threonine 89 (T89). We found that ΔeloR cells, and cells expressing the phosphoablative form of EloR (EloRT89A ), are significantly shorter than wild-type cells. Furthermore, the phosphomimetic form of EloR (EloRT89E ) is not tolerated unless the cell in addition acquires a truncated MreC or non-functional RodZ protein. By itself, truncation of MreC as well as inactivation of RodZ gives rise to less elongated cells, demonstrating that the stress exerted by the phosphomimetic form of EloR is relieved by suppressor mutations that reduce or abolish the activity of the elongasome. Of note, it was also found that loss of elongasome activity caused by truncation of MreC elicits increased StkP-mediated phosphorylation of EloR. Together, the results support a model in which phosphorylation of EloR stimulates cell elongation, while dephosphorylation has an inhibitory effect.


Acute and chronic caffeine administration differentially alters striatal gene expression in wild-type and adenosine A(2A) receptor-deficient mice.
In order to assess for the respective involvement of adenosine A(1) and A(2A) receptors (A(2A)-R) in the consequences of short- and long-term caffeine exposure on gene expression, the effects of acute caffeine administration on striatal, cortical, and hippocampal expression of immediate early genes (IEG), zif-268 and arc, and the effects of long-term caffeine or 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) exposure (once daily for 15 days) on striatal gene expression of substance P, enkephalin, and glutamic acid decarboxylase isoforms, GAD65 and GAD67, were evaluated in wild-type and A(2A)-R-deficient (A(2A)-R(-/-)) mice. In situ hybridization histochemistry was performed using oligonucleotides followed by quantitative image analysis. Our results demonstrated that a biphasic response of IEG expression to acute caffeine observed in the wild-type striatum was resumed in a monophasic response in the mutant striatum. In the cerebral cortex and hippocampus, the effect of caffeine was weak in wild-type, whereas in mutant mice it induced a 2-3-fold increase in the IEG expression to restore a level similar to the wild-type basal expression. Chronic caffeine and DPCPX-mediated regulation in neuropeptide and GADs striatal gene expression typically showed the mimicking of alterations resulting from the A(2A)-R genetic deficiency in 25 mg/kg caffeine-treated wild-type mice as well as the dose-dependent normalization of substance P and enkephalin expression in A(2A)-R(-/-) mice. These results indicate that, depending on the dose, the blockade of A(2A)-R or A(1) receptors by caffeine is preferentially revealed leading to highly differential alterations in striatal gene expression and they also suggested the central role of these two receptors on the control of dopaminergic functions.