Sequential loading of cohesin subunits during the first meiotic prophase of grasshoppers

The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.     

An organelle proteomic method to study neurotransmission-related proteins, applied to a neurodevelopmental model of schizophrenia

Limited information is currently available on molecular events that underlie schizophrenia-like behaviors in animal models. Accordingly, we developed an organelle proteomic approach enabling the study of neurotransmission-related proteins in the prefrontal cortex (PFC) of postpubertal (postnatal day 60 (PD60)) neonatally ventral hippocampal (nVH) lesioned rats, an extensively used neurodevelopmental model of schizophrenia-like behaviors. The PFC was chosen because of its purported role in the etiology of the disease. Statistical analysis of 392 reproducible spots on 2-D organelle proteomic patterns revealed significant changes in intensity of 18 proteinous spots in plasma membrane-enriched fractions obtained from postpubertal nVH lesioned rats compared to controls. Mass spectrometric analysis and database searching allowed the identification of a single protein in each of the nine differential spots, including proteins of low abundance, such as neurocalcin delta. Most of the identified dysregulated proteins, including clathrin light chain B, syntaxin binding protein 1b and visinin-like protein 1 are known to be linked to various neurotransmitter systems and to play key roles in plasma membrane receptor expression and recycling as well as synaptic vesicle exocytosis/recycling. Organelle proteomic approaches have hence proved to be most useful to identify key proteins linked to a given behavior in animal models of brain diseases.     

Evolution of male genitalia: environmental and genetic factors affect genital morphology in two <Emphasis Type="Italic">Drosophila</Emphasis> sibling species and their hybrids

Background  

The rapid evolution of genital morphology is a fascinating feature that accompanies many speciation events. However, the underlying patterns and explanatory processes remain to be settled. In this work we investigate the patterns of intraspecific variation and interspecific divergence in male genitalic morphology (size and shape) in the cactophilic sibling species Drosophila buzzatii and D. koepferae. Genital morphology in interspecific hybrids was examined and compared to the corresponding parental lines.     

Modeling the impact of genetic screening technologies on healthcare: theoretical model for asthma in children

BACKGROUND AND OBJECTIVE: This study focuses on the potential impact of genetic screening technologies on healthcare. Genetic screening for asthma in children was chosen as a case study to explore the cost effectiveness of applying early genetic screening to infants, and preventive treatment to the population at risk. Early intervention could prevent progression and facilitate clinical management of the disease. From the elite group of genetic markers that have been associated with asthma-related phenotypes, ADAM33 was the first published candidate gene detected by a positional cloning approach, marking the entry of asthma research into the genomic era. The model was, therefore, initially set for an ex ante analysis of the cost effectiveness of applying the preventive program to an infant population at risk, i.e. infants presenting wheezing episodes during the first year of life, and the ADAM33 ST+7 genetic marker, with the idea of expanding to further markers and their combinations lat a later date. METHODS: In accordance with the US National Heart, Lung, and Blood Institute, four categories of asthma were considered. A Markov model was constructed, consisting of six mutually exclusive disease states (including healthy and dead states) with a simulation horizon of 100 years and a cycle length of 1 year. We define a scenario where early genetic screening was applied to infants presenting wheezing episodes during the first year of life and a preventive treatment to those children within this group who tested positive for selected ADAM33 polymorphism (ST+7). The cost-effectiveness analysis was performed from the third-party payer and patient perspective after year 6. We applied our model to a hypothetical cohort of 100 European infants. RESULTS: The number of quality-adjusted life-years (QALYs) gained during the 6 years was 1.483, and the incremental cost-effectiveness ratio per QALY gained was euro 10,100/QALY. A sensitivity analysis was carried out that varied the discount rate and cost of genetic testing, and considered two different transition matrices for the preventive program. Three main conclusions were drawn from the sensitivity analysis. Firstly, if the discount rate for both cost and health outcomes is increased by 2%, the cost effectiveness of the preventive program does not vary significantly. Discounting costs and benefits at 5%, the preventive program appears cost effective (euro 11,100/QALY). Secondly, if the cost of genetic testing is increased to euro 100, the cost effectiveness of the preventive program remains within the limits of cost effectiveness. Thirdly, the cost of genetic screening, together with transition probabilities between health states, will determine the cost effectiveness of applying a preventive program based on genetic information. CONCLUSIONS: Preventive treatment based on an early genetic screening of those children who present wheezing episodes during the first year of life, with treatment applied to those who test positive for the asthma-associated genetic marker ADAM33 ST+7, is theoretically cost effective. The model is a valuable tool for the ex ante assessment of the cost effectiveness of preventive schemes based on genetic screening. The value of modeling prior to clinical trials lies in informing study design and setting priorities for future research.     

Association of the microsatellite in the 3' untranslated region of the CD154 gene with rheumatoid arthritis in females from a Spanish cohort: a case-control study

CD40-CD154 interaction is an important mediator of inflammation and has been implicated in T helper type 1-mediated autoimmune diseases including rheumatoid arthritis (RA). Linkage studies have shown association of markers in the proximity of the CD154 gene. In the present work we investigated whether specific allele variants of the microsatellite in the 3' UTR of the CD154 gene might modulate the risk of RA. The study, in a case-control setting, included 189 patients and 150 healthy controls from the Canary Islands, Spain. The 24CAs allele was less represented in female patients than in controls (0.444 in controls versus 0.307 in patients, P = 0.006, odds ratio (OR) 0.556, 95% confidence interval (CI) 0.372 to 0.831) but not in males (0.414 versus 0.408), and only when homozygous (P = 0.012; OR 0.35, 95% CI 0.16 to 0.77). We also verified that CD154 association with RA was independent of human leukocyte antigen (HLA) phenotype. A further functional study showed that after stimulation anti-CD3, CD154 mRNA was more stable in CD4+ T lymphocytes from patients with RA bearing the 24CAs allele (mRNA half-life 208 minutes) than in patients without the 24CAs allele (109 minutes, P = 0.009). However, a lower percentage of CD154+CD4+ T lymphocytes was seen in freshly isolated peripheral blood mononuclear cells from patients carrying 24CAs alleles (mean 4.28 versus 8.12; P = 0.033), and also in CD4+ T lymphocytes stimulated with anti-CD3 (median 29.40 versus 47.60; P = 0.025). These results were concordant with the smaller amounts of CD154 mRNA isolated from stimulated T lymphocytes with 24CAs alleles. The CD154 microsatellite therefore seems to affect the expression of the gene in a complex manner that implies not only mRNA stability. These data suggest that the CD154 microsatellite contributes to the regulation of mRNA and protein expression, although further studies will be necessary to elucidate its role in disease predisposition.     

A Pneumococcal Protein Array as a Platform to Discover Serodiagnostic Antigens Against Infection

Pneumonia is one of the most common and severe diseases associated with Streptococcus pneumoniae infections in children and adults. Etiological diagnosis of pneumococcal pneumonia in children is generally challenging because of limitations of diagnostic tests and interference with nasopharyngeal colonizing strains. Serological assays have recently gained interest to overcome some problems found with current diagnostic tests in pediatric pneumococcal pneumonia. To provide insight into this field, we have developed a protein array to screen the antibody response to many antigens simultaneously. Proteins were selected by experimental identification from a collection of 24 highly prevalent pediatric clinical isolates in Spain, using a proteomics approach consisting of “shaving” the cell surface with proteases and further LC/MS/MS analysis. Ninety-five proteins were recombinantly produced and printed on an array. We probed it with a collection of sera from children with pneumococcal pneumonia. From the set of the most seroprevalent antigens, we obtained a clear discriminant response for a group of three proteins (PblB, PulA, and PrtA) in children under 4 years old. We validated the results by ELISA and an immunostrip assay showed the translation to easy-to-use, affordable tests. Thus, the protein array here developed presents a tool for broad use in serodiagnostics.Streptococcus pneumoniae, also known as the pneumococcus, is a Gram-positive pathogen recognized as a major cause of pneumonia worldwide (1). It resides as a commensal in the nasopharynx of healthy carriers, but in susceptible individuals this bacterium can spread to other body locations and cause disease. The main group risks are the elderly, immunocompromised people and infants. In fact, ∼800,000 children die each year because of pneumococcal disease, of which >90% of these deaths occur in developing countries (2, 3). In addition, a high number of pneumococcal infection cases are diagnosed in the developed countries and can be associated with high morbidity in children and are an important factor that influences quality of life and produces significant mortality in adults (4, 5). There are licensed polysaccharide-based vaccines to prevent pneumococcal infections, but their efficacy is limited (3). Therefore, pneumococcal pneumonia remains as an important health problem and once it has occurred, early diagnosis with accurate diagnostic methods is essential in order to provide patients with prompt and appropriate therapy and hence to improve outcome (1).Although the major burden of pneumococcal infections is caused by pneumonia, the ability to identify S. pneumoniae as a causative agent in lung infections in children is quite limited. Blood cultures are often negative (6, 7). The BinaxNOW test, which measures teichoic acid, is less specific in children than in adults, because healthy carriage in infants can produce false positive results (8). The amplification and quantification of pneumococcal genes (namely spn9802, ply or pcpA) by PCR has been also used, but with lower sensitivity than culture in blood samples in adults and inability to discriminate between carriage and disease in nasopharyngeal and sputum samples (9–11).The detection of antibody serological markers by any immunoassay is widely used for early diagnosis, epidemiological surveillance, or evaluation of vaccine immunogenicity against many pathogens, including the pneumococcus (6, 12, 13). Serological diagnosis of pneumococcal disease based on a single antigen is often challenging, because of the interference of natural antibodies elicited by previous colonization events. Therefore, to better discriminate between diseased and healthy people, a combination of antigens would be desirable. To this regard, proteomics offers an excellent platform to develop the necessary more sensitive and specific immunoassays that can be used for the aforementioned purposes.It is well assumed that surface proteins are those with the highest chance to raise an effective immune response against pathogen infection, as they are sufficiently exposed and accessible to both T and B cells (14, 15). Protein arrays are powerful tools to interrogate the pattern of host humoral responses to infections (16), which allows the study of many antigens simultaneously with a small amount of sample (17) to select a set of antigens with optimal sensitivity and specificity (18, 19). In this work, we have selected a set of 95 pneumococcal surface proteins by experimental identification, using the proteomic approach of “shaving” of live cells with proteases and further liquid chromatography-tandem MS (LC/MS/MS)¹ analysis (20). After producing the selected proteins as recombinant fragments, we have developed the first pneumococcal surface protein chip and probed it with a collection of sera from infected and control children, in order to find proteins that differentiate between pneumococcal or nonpneumococcal infection/health status. Three proteins were proven to discriminate with optimal sensitivity, specificity, and accuracy between nonpneumococcal disease and disease status for <4-year-old children. As a proof-of-concept, we have developed an immunostrip assay with such proteins, obtaining the same sensitivity, specificity, and accuracy, thus demonstrating the power of high-throughput technologies for discovering diagnostic biomarkers of infection and its possible translation to an easy-to-use clinical tool.     

The alternative translated MDMXp60 isoform regulates MDM2 activity

Isoforms derived from alternative splicing, mRNA translation initiation or promoter usage extend the functional repertoire of the p53, p63 and p73 genes family and of their regulators MDM2 and MDMX. Here we show cap-independent translation of an N-terminal truncated isoform of hMDMX, hMDMX^p60, which is initiated at the 7th AUG codon downstream of the initiation site for full length hMDMX^FL at position +384. hMDMX^p60 lacks the p53 binding motif but retains the RING domain and interacts with hMDM2 and hMDMX^FL. hMDMX^p60 shows higher affinity for hMDM2, as compared to hMDMX^FL. In vitro data reveal a positive cooperative interaction between hMDMX^p60 and hMDM2 and in cellulo data show that low levels of hMDMX^p60 promote degradation of hMDM2 whereas higher levels stabilize hMDM2 and prevent hMDM2-mediated degradation of hMDMX^FL. These results describe a novel alternatively translated hMDMX isoform that exhibits unique regulatory activity toward hMDM2 autoubiquitination. The data illustrate how the N-terminus of hMDMX regulates its C-terminal RING domain and the hMDM2 activity.