Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks

p53 and p19(ARF) are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19(ARF) deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19(ARF)-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and mutually exclusive interactions between cancer genes. Furthermore, we found candidate tumor suppressor genes, as well as distinct clusters of insertions within genes like Flt3 and Notch1 that induce mutants with different spectra of genetic interactions. Cross species comparative analysis with aCGH data of human cancer cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, and Rreb1) and tumor suppressors (Wwox and Arfrp2). This dataset should prove to be a rich resource for the study of genetic interactions that underlie tumorigenesis.     

Population Dynamics and Diversity of Viruses,Bacteria and Phytoplankton in a Shallow Eutrophic Lake

We have studied the temporal variation in viral abundances and community assemblage in the eutrophic Lake Loosdrecht through epifluorescence microscopy and pulsed field gel electrophoresis (PFGE). The virioplankton community was a dynamic component of the aquatic community, with abundances ranging between 5.5 x 10(7) and 1.3 x 10(8) virus-like particles ml(-1) and viral genome sizes ranging between 30 and 200 kb. Both viral abundances and community composition followed a distinct seasonal cycle, with high viral abundances observed during spring and summer. Due to the selective and parasitic nature of viral infection, it was expected that viral and host community dynamics would covary both in abundances and community composition. The temporal dynamics of the bacterial and cyanobacterial communities, as potential viral hosts, were studied in addition to a range of environmental parameters to relate these to viral community dynamics. Cyanobacterial and bacterial communities were studied applying epifluorescence microscopy, flow cytometry, and denaturing gradient gel electrophoresis (DGGE). Both bacterial and cyanobacterial communities followed a clear seasonal cycle. Contrary to expectations, viral abundances were neither correlated to abundances of the most dominant plankton groups in Lake Loosdrecht, the bacteria and the filamentous cyanobacteria, nor could we detect a correlation between the assemblage of viral and bacterial or cyanobacterial communities during the overall period. Only during short periods of strong fluctuations in microbial communities could we detect viral community assemblages to covary with cyanobacterial and bacterial communities. Methods with a higher specificity and resolution are probably needed to detect the more subtle virus-host interactions. Viral abundances did however relate to cyanobacterial community assemblage and showed a significant positive correlation to Chl-a as well as prochlorophytes, suggesting that a significant proportion of the viruses in Lake Loosdrecht may be phytoplankton and more specific cyanobacterial viruses. Temporal changes in bacterial abundances were significantly related to viral community assemblage, and vice versa, suggesting an interaction between viral and bacterial communities in Lake Loosdrecht.     

Production of cachexia mediators by Walker 256 cells from ascitic tumors

In neoplasic cachexia, chemical mediators seem to act as initiators or perpetuators of this process. Walker 256 cells, whose metabolic properties have so far been little studied with respect to cancer cachexia, are used as a model for the study of this syndrome. The main objective of this research was to pinpoint the substances secreted by these cells that may contribute to the progression of the cachectic state. Since inflammatory mediators seem to be involved in the manifestation of this syndrome, the in vitro production of nitric oxide (NO), cytokines (tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6)), and prostaglandin E2 (PGE2) was evaluated in Walker 256 cells isolated from ascitic tumors. After 4 or 5 h, a significant increase in NO production was observed (2.55 +/- 1.56 and 4.05 +/- 1.99 nmol NO per 10(7) cells, respectively). When isolated from a 6-day-old tumor, a significantly lower production of IL-6 and higher production of TNF-alpha than in cells from a 4-day-old tumor were observed, indicating a relationship between the production of cytokines and the time of tumor development after implantation. Considerable production of PGE(2) by Walker 256 cells isolated from the 6-day-old tumor was also observed. Polyamines were also determined in Walker 256 cells. Levels of putrescine, spermidine, and spermine did not show significant differences in tumors developed during 4 or 6 days. Direct evidence of the release of proinflammatory cytokines and PGE2 by Walker 256 cells suggests that these mediators can drive the cachectic syndrome in the host, the effect being dependent on tumor development time.     

Pseudocercospora species on Piperaceae from Viçosa, Minas Gerais, Brazil

Three Pseudocercospora species were found associated with leaf spots on five different members of the family Piperaceae in a stretch of Atlantic rain forest in the municipality of Viçosa, Minas Gerais, Brazil. Pseudocercospora piperis is newly recorded on Piper crassinervium and Piper mollicomum. It was also found in association with Pothomorphe umbellata, which is already known to be a host. Pseudocercospora piperis-arborei sp. nov. and Pseudocercospora piperis-gigantifolii sp. nov. were recorded on Piper arboreum and Piper gigantifolium, respectively. These three species are fully described and illustrated in the present work.     

Size of the exon 1-CAG repeats of the androgen receptor gene employed as a molecular marker in the diagnosis of Turner syndrome in girls with short stature

Turner syndrome (TS) is one of the most common chromosomal abnormalities among girls. Complete monosomy of X chromosome is responsible for almost 50% of all cases of TS, and mosaicism and X anomaly are detected in the other half. It has already been demonstrated that early diagnosis of these children allows appropriate growth hormone treatment with better final height prognosis and introduction of estrogen at an ideal chronological age. Sixty-four short-stature girls were selected and the clinical data obtained were birth weight and height, weight and height at the first medical visit and target height. Other clinical data including cardiac and renal abnormalities, otitis, Hashimoto thyroiditis, cubitus valgus, short neck, widely separated nipples, and pigmented nevi were obtained from the patients' medical records. The aim of the present study was to evaluate the screening of a group of short-stature girls for TS based on the number of CAG repeats of the androgen receptor gene analyzed by GeneScan software. Patient samples with two alleles (heterozygous) were 49/64 (76.5%) and with one allele (homozygous) were 15/64 (23.5%). A karyotype was determined in 30 patients, 9 homozygous and 21 heterozygous. In the homozygous group, 6/9 were 45,X and 3/9 were 46,XX. In the heterozygous group, 17/21 were 46,XX, and 4/21 were TS patients with mosaicism (45,X/46,XX; 45,X/46XiXq; 46XdelXp). The pattern obtained by GeneScan in two patients with mosaicism in the karyotype was an imbalance between the peak heights of the two alleles, suggesting that this imbalance could be present when there is a mosaicism. The frequency of TS abnormalities (18.7%) did not differ between TS and 46,XX girls. Thus, it is important to accurately assess the incidence of TS in growth-retarded girls, even in the absence of other dysmorphisms. In this study, we diagnosed 6 cases of TS 45,X (9.4%) by molecular analysis, with a 100% sensitivity and 85% specificity. This molecular analysis was able to detect all cases of TS 45,X and the majority of mosaicisms, without the need for more X chromosome markers. In conclusion, determining the number of CAG repeats of the androgen receptor gene analyzed by GeneScan was a fast method with high sensitivity for the detection of TS 45,X, suggesting that it could be interesting as a method for screening a population of growth-retarded girls.     

Mutation in intron 5 of GTP cyclohydrolase 1 gene causes dopa-responsive dystonia (Segawa syndrome) in a Brazilian family

Dopa-responsive dystonia (DRD), also known as Segawa syndrome or hereditary progressive dystonia with diurnal fluctuation, is clinically characterized by the occurrence of simultaneous or late Parkinsonism and by an excellent response to treatment with low doses of L-dopa. Diagnosis of DRD is essentially clinical. It is based on clinical history and the response to treatment with low doses of L-dopa. However, due to the low penetrance of the disease, asymptomatic carriers may exist. In these cases, mutational analysis of the GCH1 gene is an alternative to diagnose DRD. In the present study, we investigated a large DRD-carrier family in an attempt to identify the disease-causing mutation. The proband, a young woman diagnosed at the age of 13 years, is the daughter of a healthy non-consanguineous couple with history of several cases, on the maternal side of the family, of tip-toeing, disturbance of gait, Parkinsonism, rigidity and cramps in the lower limbs. Using single strand conformational polymorphism and DNA sequencing techniques to analyze DNA extracted from blood samples, we identified a mutation in the GCH1 gene, IVS5+3insT, which would preclude the formation of the active enzyme due to the formation of truncated peptides.     

Energetic constraints on an early developmental stage: a comparative view

Biologists have long sought a means by which to quantify similarities and differences in embryonic development across species. Here we present a quantitative approach for predicting the timing of developmental events based on principles of allometry and biochemical kinetics. Data from diverse oviparous species support model predictions that most variation in the time required to reach one early developmental stage-the time to first heartbeat-is explained by the body size and temperature dependence of metabolic rate. Furthermore, comparisons of this stage with later developmental stages suggest that, after correcting for size and temperature, the relationship of metabolic rate to the rate of embryogenesis is approximately invariant across taxonomic groups and stages of ontogeny.     

A high confidence, manually validated human blood plasma protein reference set

Background

The immense diagnostic potential of human plasma has prompted great interest and effort in cataloging its contents, exemplified by the Human Proteome Organization (HUPO) Plasma Proteome Project (PPP) pilot project. Due to challenges in obtaining a reliable blood plasma protein list, HUPO later re-analysed their own original dataset with a more stringent statistical treatment that resulted in a much reduced list of high confidence (at least 95%) proteins compared with their original findings. In order to facilitate the discovery of novel biomarkers in the future and to realize the full diagnostic potential of blood plasma, we feel that there is still a need for an ultra-high confidence reference list (at least 99% confidence) of blood plasma proteins.

Methods

To address the complexity and dynamic protein concentration range of the plasma proteome, we employed a linear ion-trap-Fourier transform (LTQ-FT) and a linear ion trap-Orbitrap (LTQ-Orbitrap) for mass spectrometry (MS) analysis. Both instruments allow the measurement of peptide masses in the low ppm range. Furthermore, we employed a statistical score that allows database peptide identification searching using the products of two consecutive stages of tandem mass spectrometry (MS3). The combination of MS3 with very high mass accuracy in the parent peptide allows peptide identification with orders of magnitude more confidence than that typically achieved.

Results

Herein we established a high confidence set of 697 blood plasma proteins and achieved a high 'average sequence coverage' of more than 14 peptides per protein and a median of 6 peptides per protein. All proteins annotated as belonging to the immunoglobulin family as well as all hypothetical proteins whose peptides completely matched immunoglobulin sequences were excluded from this protein list. We also compared the results of using two high-end MS instruments as well as the use of various peptide and protein separation approaches. Furthermore, we characterized the plasma proteins using cellular localization information, as well as comparing our list of proteins to data from other sources, including the HUPO PPP dataset.

Conclusion

Superior instrumentation combined with rigorous validation criteria gave rise to a set of 697 plasma proteins in which we have very high confidence, demonstrated by an exceptionally low false peptide identification rate of 0.29%.     

Relation between the global burden of disease and randomized clinical trials conducted in Latin America published in the five leading medical journals

Background

Since 1990 non communicable diseases and injuries account for the majority of death and disability-adjusted life years in Latin America. We analyzed the relationship between the global burden of disease and Randomized Clinical Trials (RCTs) conducted in Latin America that were published in the five leading medical journals.

Methodology/Principal Findings

We included all RCTs in humans, exclusively conducted in Latin American countries, and published in any of the following journals: Annals of Internal Medicine, British Medical Journal, Journal of the American Medical Association, Lancet, and New England Journal of Medicine. We described the trials and reported the number of RCTs according to the main categories of the global burden of disease. Sixty-six RCTs were identified. Communicable diseases accounted for 38 (57%) reports. Maternal, perinatal, and nutritional conditions accounted for 19 (29%) trials. Non-communicable diseases represent 48% of the global burden of disease but only 14% of reported trials. No trial addressed injuries despite its 18% contribution to the burden of disease in 2000.

Conclusions/Significance

A poor correlation between the burden of disease and RCTs publications was found. Non communicable diseases and injuries account for up to two thirds of the burden of disease in Latin America but these topics are seldom addressed in published RCTs in the selected sample of journals. Funding bodies of health research and editors should be aware of the increasing burden of non communicable diseases and injuries occurring in Latin America to ensure that this growing epidemic is not neglected in the research agenda and not affected by publication bias.     

The Gammaherpesvirus m2 protein manipulates the Fyn/Vav pathway through a multidocking mechanism of assembly

To establish latent infections in B-cells, gammaherpesviruses express proteins in the infected B-cells of the host that spuriously activate signalling pathways located downstream of the B-cell receptor. One such protein is M2, a murine gammaherpesvirus 68-encoded molecule that activates the Vav1/Rac1 pathway via the formation of trimolecular complexes with Scr family members. Previous reports have shown that the formation of this heteromolecular complex involves interactions between a proline rich region of M2 and the Vav1 and Fyn SH3 domains. Here, we show that the optimal association of these proteins requires a second structural motif encompassing two tyrosine residues (Tyr120 and 129). These residues are inducibly phosphorylated by Fyn in non-hematopoietic cells and constitutively phosphorylated in B-cells. We also demonstrate that the phosphorylation of Tyr120 creates specific docking sites for the SH2 domains of both Vav1 and Fyn, a condition sine qua non for the optimal association of these two signalling proteins in vivo. Interestingly, signaling experiments indicate that the expression of M2 in B-cells promotes the tyrosine phosphorylation of Vav1 and additional signaling proteins, a biological process that requires the integrity of both the M2 phosphotyrosine and proline rich region motifs. By infecting mice with viruses mutated in the m2 locus, we show that the integrity of each of these two M2 docking motifs is essential for the early steps of murine gammaherpesvirus-68 latency. Taken together, these results indicate that the M2 phosphotyrosine motif and the previously described M2 proline rich region work in a concerted manner to manipulate the signaling machinery of the host B-cell.