Sex Determination in Caenorhabditis elegans

In Caenorhabditis elegans, the decision to develop as a hermaphrodite or male is controlled by a cascade of regulatory genes. These genes and other tissue-specific regulatory genes also control sexual fate in the hermaphrodite germline, which makes sperm first and then oocytes. In this review, we summarize the genetic and molecular characterization of these genes and speculate how they mutually interact to specify sexual fate.     

Characteristics of Environmental <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis> Isolates

The ecological niche or exact habitat of the fungus Paracoccidioides brasiliensis is not known, and few isolates have been obtained from the environment. In this study, ten isolates were analyzed with respect to antigenic composition, serology, pathogenicity, and molecular aspects. Gp43 is considered to be the molecular basis for the serodiagnosis of paracoccidioidomycosis; however, in this study only six of the environmental isolates secreted this molecule (four in great amounts and two in small amounts). Other molecules were also produced. When exoantigens from these isolates were tested using immunodiffusion, only four preparations were positive by ID tests. However, when these exoantigens were tested by ELISA, all of them except one were able to detect anti-P. brasiliensis antibodies. In Western blot assays, these exoantigens showed different reactivities. Isolates that secreted gp43 presented positive reactions for this molecule, and isolates that did not secrete gp43 gave positive reactions for other minor molecules. RAPD analysis revealed that there is great genetic variation between these environmental isolates. These isolates were non-pathogenic: no mortality was observed among the inoculated mice during an 18-month follow-up period.     

Extent and diversity of human alternative splicing established by complementary database annotation and microarray analysis

Alternative splicing generates functional diversity in higher organisms through alternative first and last exons, skipped and included exons, intron retentions and alternative donor, and acceptor sites. In large-scale microarray studies in humans and the mouse, emphasis so far has been placed on exon-skip events, leaving the prevalence and importance of other splice types largely unexplored. Using a new human splice variant database and a genome-wide microarray to probes thousands of splice events of each type, we measured differential expression of splice types across six pair of diverse cell lines and validated the database annotation process. Results suggest that splicing in humans is more complex than simple exon-skip events, which account for a minority of splicing differences. The relative frequency of differential expression of the splice types correlates with what is found by our annotation efforts. In conclusion, alternative splicing in human cells is considerably more complex than the canonical example of the exon skip. The complementary approaches of genome-wide annotation of alternative splicing in human and design of genome-wide splicing microarrays to measure differential splicing in biological samples provide a powerful high-throughput tool to study the role of alternative splicing in human biology.     

Escalation and extinction selectivity: morphology versus isotopic reconstruction of bivalve metabolism

Studies that have tested and failed to support the hypothesis that escalated species (e.g., those with predation-resistant adaptations) are more susceptible to elimination during mass extinctions have concentrated on the distribution and degree of morphological defenses in molluscan species. This morphological approach to determining level of escalation in bivalves may be oversimplified because it does not account for metabolic rate, which is an important measure of escalation that is less readily accessible for fossils. Shell growth rates in living bivalves are positively correlated with metabolic rate and thus are potential indicators of level of escalation. To evaluate this approach, we used oxygen isotopes to reconstruct shell growth rates for two bivalve species (Macrocallista marylandica and Glossus markoei) from Miocene-aged sediments of Maryland. Although both species are classified as non-escalated based on morphology, the isotopic data indicate that M. marylandica was a faster-growing species with a higher metabolic rate and G. markoei was a slower-growing species with a lower metabolic rate. Based on these results, we predict that some morphologically non-escalated species in previous tests of extinction selectivity should be reclassified as escalated because of their fast shell growth rates (i.e., high metabolic rates). Studies that evaluate the level of escalation of a fauna should take into account the energetic physiology of taxa to avoid misleading results.     

Percoll centrifugation eliminates mold contaminants from cell cultures

This work was supported by a grant and research associateship to N.A. from the Medical Research Council of Canada and the National Cancer Institute of Canada.     

APC/C(Cdc20) controls the ubiquitin-mediated degradation of p21 in prometaphase

During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumulates in G2 and is degraded again in prometaphase. How p21 degradation is controlled in mitosis remains unexplored. We found that Cdc20 (an activator of the ubiquitin ligase APC/C) binds p21 in cultured cells and identified a D box motif in p21 necessary for APC/C(Cdc20)-mediated ubiquitylation of p21. Overexpression of Cdc20 or Skp2 destabilized wild-type p21; however, only Skp2, but not Cdc20, was able to destabilize a p21(D box) mutant. Silencing of Cdc20 induced an accumulation of p21, increased the fraction of p21 bound to Cdk1, and inhibited Cdk1 activity in p21(+/+) prometaphase cells, but not in p21(-/-) cells. Thus, in prometaphase Cdc20 positively regulates Cdk1 by mediating the degradation of p21. We propose that the APC/C(Cdc20)-mediated degradation of p21 contributes to the full activation of Cdk1 necessary for mitotic events and prevents mitotic slippage during spindle checkpoint activation.     

Dimerization and phosphorylation of thyrotropin-releasing hormone receptors are modulated by agonist stimulation

Dimerization and phosphorylation of thyrotropin-releasing hormone (TRH) receptors was characterized using HEK293 and pituitary GHFT cells expressing epitope-tagged receptors. TRH receptors tagged with FLAG and hemagglutinin epitopes were co-precipitated only if they were co-expressed, and 10-30% of receptors were isolated as hemagglutinin/FLAG-receptor dimers under basal conditions. The abundance of receptor dimers was increased when cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a dose-dependent manner. TRH increased phosphorylation of both receptor monomers and dimers, documented by incorporation of (32)P and an upshift in receptor mobility reversed by phosphatase treatment. The ability of TRH to increase receptor phosphorylation and dimerization did not depend on signal transduction, because it was not inhibited by the phospholipase C inhibitor. Receptor phosphorylation required an agonist but was not blocked by the casein kinase II inhibitor apigenin, the protein kinase C inhibitor GF109203X, or expression of a dominant negative form of G protein-coupled receptor kinase 2. TRH receptors lacking most of the cytoplasmic carboxyl terminus formed dimers constitutively but failed to undergo agonist-induced dimerization and phosphorylation. TRH also increased phosphorylation and dimerization of TRH receptors expressed in GHFT pre-lactotroph cells.     

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that driv...     

Mycobacterium tuberculosis Bacteremia in a Cohort of HIV-Infected Patients Hospitalized with Severe Sepsis in Uganda–High Frequency,Low Clinical Sand Derivation of a Clinical Prediction Score

Background

When manifested as Mycobacterium tuberculosis (MTB) bacteremia, disseminated MTB infection clinically mimics other serious blood stream infections often hindering early diagnosis and initiation of potentially life-saving anti-tuberculosis therapy. In a cohort of hospitalized HIV-infected Ugandan patients with severe sepsis, we report the frequency, management and outcomes of patients with MTB bacteremia and propose a risk score based on clinical predictors of MTB bacteremia.

Methods

We prospectively enrolled adult patients with severe sepsis at two Ugandan hospitals and obtained blood cultures for MTB identification. Multivariable logistic regression modeling was used to determine predictors of MTB bacteremia and to inform the stratification of patients into MTB bacteremia risk categories based on relevant patient characteristics.

Results

Among 368 HIV-infected patients with a syndrome of severe sepsis, eighty-six (23%) had MTB bacteremia. Patients with MTB bacteremia had a significantly lower median CD4 count (17 vs 64 lymphocytes/mm³, p<0.001) and a higher 30-day mortality (53% vs 32%, p = 0.001) than patients without MTB bacteremia. A minority of patients with MTB bacteremia underwent standard MTB diagnostic testing (24%) or received empiric anti-tuberculosis therapy (15%). Independent factors associated with MTB bacteremia included male sex, increased heart rate, low CD4 count, absence of highly active anti-retroviral therapy, chief complaint of fever, low serum sodium and low hemoglobin. A risk score derived from a model containing these independent predictors had good predictive accuracy [area under the curve = 0.85, 95% CI 0.80–0.89].

Conclusions

Nearly 1 in 4 adult HIV-infected patients hospitalized with severe sepsis in 2 Ugandan hospitals had MTB bacteremia. Among patients in whom MTB was suspected, standard tests for diagnosing pulmonary MTB were inaccurate for correctly classifying patients with or without bloodstream MTB infection. A MTB bacteremia risk score can improve early diagnosis of MTB bacteremia particularly in settings with increased HIV and MTB co-infection.