Arginine methylation of yeast mRNA-binding protein Npl3 directly affects its function, nuclear export, and intranuclear protein interactions

Arginine methylation can affect both nucleocytoplasmic transport and protein-protein interactions of RNA-binding proteins. These effects are seen in cells that lack the yeast hnRNP methyltransferase (HMT1), raising the question of whether effects on specific proteins are direct or indirect. The presence of multiple arginines in individual methylated proteins also raises the question of whether overall methylation or methylation of a subset of arginines affects protein function. We have used the yeast mRNA-binding protein Npl3 to address these questions in vivo. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry was used to identify 17 methylated arginines in Npl3 purified from yeast: whereas 10 Arg-Gly-Gly (RGG) tripeptides were exclusively dimethylated, variable levels off methylation were found for 5 RGG and 2 RG motif arginines. We constructed a set of Npl3 proteins in which subsets of the RGG arginines were mutated to lysine. Expression of these mutant proteins as the sole form of Npl3 specifically affected growth of a strain that requires Hmtl. Although decreased growth generally correlated with increased numbers of Arg-to-Lys mutations, lysine substitutions in the N terminus of the RGG domain showed more severe effects. Npl3 with all 15 RGG arginines mutated to lysine exited the nucleus independent of Hmtl, indicating a direct effect of methylation on Npl3 transport. These mutations also resulted in a decreased, methylation-independent interaction of Npl3 with transcription elongation factor Tho2 and inhibited Npl3 self-association. These results support a model in which arginine methylation facilitates Npl3 export directly by weakening contacts with nuclear proteins. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc

Submillimeter-wave sideband generation using a planar diode array

A 36 element array of planar Schottky diodes is used to mix the output of a CO_2 pumped far infrared laser with a 1-20 GHz microwave source to generate tunable sidebands at 1.6 THz. The double sideband power was measured by heterodyne detection with a 1T23 corner cube Schottky diode for a calculated output power of 5.9 μW with a 28 dB conversion loss

VESPA: software to facilitate genomic annotation of prokaryotic organisms through integration of proteomic and transcriptomic data

<p>Abstract</p> <p>Background</p> <p>The procedural aspects of genome sequencing and assembly have become relatively inexpensive, yet the full, accurate structural annotation of these genomes remains a challenge. Next-generation sequencing transcriptomics (RNA-Seq), global microarrays, and tandem mass spectrometry (MS/MS)-based proteomics have demonstrated immense value to genome curators as individual sources of information, however, integrating these data types to validate and improve structural annotation remains a major challenge. Current visual and statistical analytic tools are focused on a single data type, or existing software tools are retrofitted to analyze new data forms. We present Visual Exploration and Statistics to Promote Annotation (VESPA) is a new interactive visual analysis software tool focused on assisting scientists with the annotation of prokaryotic genomes though the integration of proteomics and transcriptomics data with current genome location coordinates.</p> <p>Results</p> <p>VESPA is a desktop Java™ application that integrates high-throughput proteomics data (peptide-centric) and transcriptomics (probe or RNA-Seq) data into a genomic context, all of which can be visualized at three levels of genomic resolution. Data is interrogated via searches linked to the genome visualizations to find regions with high likelihood of mis-annotation. Search results are linked to exports for further validation outside of VESPA or potential coding-regions can be analyzed concurrently with the software through interaction with BLAST. VESPA is demonstrated on two use cases (<it>Yersinia pestis </it>Pestoides F and <it>Synechococcus </it>sp. PCC 7002) to demonstrate the rapid manner in which mis-annotations can be found and explored in VESPA using either proteomics data alone, or in combination with transcriptomic data.</p> <p>Conclusions</p> <p>VESPA is an interactive visual analytics tool that integrates high-throughput data into a genomic context to facilitate the discovery of structural mis-annotations in prokaryotic genomes. Data is evaluated via visual analysis across multiple levels of genomic resolution, linked searches and interaction with existing bioinformatics tools. We highlight the novel functionality of VESPA and core programming requirements for visualization of these large heterogeneous datasets for a client-side application. The software is freely available at <url>https://www.biopilot.org/docs/Software/Vespa.php</url>.</p

CCL2-driven inflammation increases mammary gland stromal density and cancer susceptibility in a transgenic mouse model.

Abstract Background Macrophages play diverse roles in mammary gland development and breast cancer. CC-chemokine ligand 2 (CCL2) is an inflammatory cytokine that recruits macrophages to sites of injury. Although CCL2 has been detected in human and mouse mammary epithelium, its role in regulating mammary gland development and cancer risk has not been explored. Methods Transgenic mice were generated wherein CCL2 is driven by the mammary epithelial cell-specific mouse mammary tumour virus 206 (MMTV) promoter. Estrous cycles were tracked in adult transgenic and non-transgenic FVB mice, and mammary glands collected at the four different stages of the cycle. Dissected mammary glands were assessed for cyclical morphological changes, proliferation and apoptosis of epithelium, macrophage abundance and collagen deposition, and mRNA encoding matrix remodelling enzymes. Another cohort of control and transgenic mice received carcinogen 7,12-Dimethylbenz(a)anthracene (DMBA) and tumour development was monitored weekly. CCL2 protein was also quantified in paired samples of human breast tissue with high and low mammographic density. Results Overexpression of CCL2 in the mammary epithelium resulted in an increased number of macrophages, increased density of stroma and collagen and elevated mRNA encoding matrix remodelling enzymes lysyl oxidase (LOX) and tissue inhibitor of matrix metalloproteinases (TIMP)3 compared to non-transgenic controls. Transgenic mice also exhibited increased susceptibility to development of DMBA-induced mammary tumours. In a paired sample cohort of human breast tissue, abundance of epithelial-cell-associated CCL2 was higher in breast tissue of high mammographic density compared to tissue of low mammographic density. Conclusions Constitutive expression of CCL2 by the mouse mammary epithelium induces a state of low level chronic inflammation that increases stromal density and elevates cancer risk. We propose that CCL2-driven inflammation contributes to the increased risk of breast cancer observed in women with high mammographic density

Arginine methylation of yeast mRNA-binding protein Npl3 directly affects its function, nuclear export, and intranuclear protein interactions

Arginine methylation can affect both nucleocytoplasmic transport and protein-protein interactions of RNA-binding proteins. These effects are seen in cells that lack the yeast hnRNP methyltransferase (HMT1), raising the question of whether effects on specific proteins are direct or indirect. The presence of multiple arginines in individual methylated proteins also raises the question of whether overall methylation or methylation of a subset of arginines affects protein function. We have used the yeast mRNA-binding protein Npl3 to address these questions in vivo. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry was used to identify 17 methylated arginines in Npl3 purified from yeast: whereas 10 Arg-Gly-Gly (RGG) tripeptides were exclusively dimethylated, variable levels off methylation were found for 5 RGG and 2 RG motif arginines. We constructed a set of Npl3 proteins in which subsets of the RGG arginines were mutated to lysine. Expression of these mutant proteins as the sole form of Npl3 specifically affected growth of a strain that requires Hmtl. Although decreased growth generally correlated with increased numbers of Arg-to-Lys mutations, lysine substitutions in the N terminus of the RGG domain showed more severe effects. Npl3 with all 15 RGG arginines mutated to lysine exited the nucleus independent of Hmtl, indicating a direct effect of methylation on Npl3 transport. These mutations also resulted in a decreased, methylation-independent interaction of Npl3 with transcription elongation factor Tho2 and inhibited Npl3 self-association. These results support a model in which arginine methylation facilitates Npl3 export directly by weakening contacts with nuclear proteins. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc

Correlates of recent HIV testing among transgender women in Greater Kuala Lumpur, Malaysia

Purpose: In Malaysia, an estimated 9.7%-12.4% of transgender women (TW) are HIV positive, with higher estimates among those engaged in sex work. According to the 90-90-90 Joint United Nations Programme on HIV/AIDS strategy, HIV testing is the first crucial step in curbing the HIV epidemic. This study examines correlates of recent HIV testing among TW in Greater Kuala Lumpur, Malaysia. Methods: TW (N = 199) in Greater Kuala Lumpur completed a survey on healthcare access and utilization, including HIV testing history. Bivariate logistic regression and penalized multivariate logistic regression were used to explore correlates of HIV testing in the last 12 months. Results: Overall, 41.7% of TW reported having ever been tested for HIV. Among participants who were HIV negative or not sure of their HIV status (n = 187), only 18.7% (n = 35) had been tested for HIV in the last 12 months. The multivariate analysis indicated that having a primary care provider (PCP), being 26-40 years of age, and having higher mental health functioning were positively associated with recent HIV testing. Active amphetamine use and previous depression diagnosis were also associated with recent HIV testing. Conclusion: HIV testing is the first step in linking individuals to prevention and treatment interventions. Our findings suggest that having a PCP can improve engagement in HIV testing. Moreover, PCPs can serve as a valuable link to HIV treatment and prevention services. Current interventions that target social and behavioral risk factors for HIV, on their own, may be insufficient at engaging all HIV-vulnerable TW

Correlates of recent HIV testing among transgender women in Greater Kuala Lumpur, Malaysia

Purpose: In Malaysia, an estimated 9.7%-12.4% of transgender women (TW) are HIV positive, with higher estimates among those engaged in sex work. According to the 90-90-90 Joint United Nations Programme on HIV/AIDS strategy, HIV testing is the first crucial step in curbing the HIV epidemic. This study examines correlates of recent HIV testing among TW in Greater Kuala Lumpur, Malaysia. Methods: TW (N = 199) in Greater Kuala Lumpur completed a survey on healthcare access and utilization, including HIV testing history. Bivariate logistic regression and penalized multivariate logistic regression were used to explore correlates of HIV testing in the last 12 months. Results: Overall, 41.7% of TW reported having ever been tested for HIV. Among participants who were HIV negative or not sure of their HIV status (n = 187), only 18.7% (n = 35) had been tested for HIV in the last 12 months. The multivariate analysis indicated that having a primary care provider (PCP), being 26-40 years of age, and having higher mental health functioning were positively associated with recent HIV testing. Active amphetamine use and previous depression diagnosis were also associated with recent HIV testing. Conclusion: HIV testing is the first step in linking individuals to prevention and treatment interventions. Our findings suggest that having a PCP can improve engagement in HIV testing. Moreover, PCPs can serve as a valuable link to HIV treatment and prevention services. Current interventions that target social and behavioral risk factors for HIV, on their own, may be insufficient at engaging all HIV-vulnerable TW

The natural history of AVM hemorrhage in the posterior fossa: comparison of hematoma volumes and neurological outcomes in patients with ruptured infra- and supratentorial AVMs

OBJECT: Patients with posterior fossa arteriovenous malformations (AVMs) are more likely to present with hemorrhage than those with supratentorial AVMs. Observed patients subject to the AVM natural history should be informed of the individualized effects of AVM characteristics on the clinical course following a new, first-time hemorrhage. The authors hypothesize that the debilitating effects of first-time bleeding from an AVM in a previously intact patient with an unruptured AVM are more pronounced when AVMs are located in the posterior fossa. METHODS: The University of California, San Francisco prospective registry of brain AVMs was searched for patients with a ruptured AVM who had a pre-hemorrhage modified Rankin Scale (mRS) score of 0 and a post-hemorrhage mRS score obtained within 2 days of the hemorrhagic event. A total of 154 patients met the inclusion criteria for this study. Immediate post-hemorrhage presentation mRS scores were dichotomized into nonsevere outcome (mRS > 3) and severe outcome (mRS > 3). There were 77 patients in each group. Univariate and multivariate logistic regression analyses using severe outcome as the binary response were run. The authors also performed a logistic regression analysis to measure the effects of hematoma volume and AVM location on severe outcome. RESULTS: Posterior fossa location was a significant predictor of severe outcome (OR 2.60, 95% CI 1.20–5.67, p = 0.016) and the results were strengthened in a multivariate model (OR 4.96, 95% CI 1.73–14.17, p = 0.003). Eloquent location (OR 3.47, 95% CI 1.37–8.80, p = 0.009) and associated arterial aneurysms (OR 2.58, 95% CI 1.09, 6.10; p = 0.031) were also significant predictors of poor outcome. Hematoma volume for patients with a posterior fossa AVM was 10.1 ± 10.1 cm(3) compared with 25.6 ± 28.0 cm(3) in supratentorial locations (p = 0.003). A logistic analysis (based on imputed hemorrhage volume values) found that posterior fossa location was a significant predictor of severe outcome (OR 8.03, 95% CI 1.20–53.77, p = 0.033) and logarithmic hematoma volume showed a positive, but not statistically significant, association in the model (p = 0.079). CONCLUSIONS: Patients with posterior fossa AVMs are more likely to have severe outcomes than those with supratentorial AVMs based on this natural history study. Age, sex, and ethnicity were not associated with an increased risk of severe outcome after AVM rupture, but posterior fossa location, associated aneurysms, and eloquent location were associated with poor post-hemorrhage mRS scores. Posterior fossa hematomas are poorly tolerated, with severe outcomes observed even with smaller hematoma volumes. These findings support an aggressive surgical posture with respect to posterior fossa AVMs, both before and after rupture

The natural history of AVM hemorrhage in the posterior fossa: Comparison of hematoma volumes and neurological outcomes in patients with ruptured infra- and supratentorial AVMs

Object: Patients with posterior fossa arteriovenous malformations (AVMs) are more likely to present with hemorrhage than those with supratentorial AVMs. Observed patients subject to the AVM natural history should be informed of the individualized effects of AVM characteristics on the clinical course following a new, first-time hemorrhage. The authors hypothesize that the debilitating effects of first-time bleeding from an AVM in a previously intact patient with an unruptured AVM are more pronounced when AVMs are located in the posterior fossa. Methods: The University of California, San Francisco prospective registry of brain AVMs was searched for patients with a ruptured AVM who had a pre-hemorrhage modified Rankin Scale (mRS) score of 0 and a post-hemorrhage mRS score obtained within 2 days of the hemorrhagic event. A total of 154 patients met the inclusion criteria for this study. Immediate post-hemorrhage presentation mRS scores were dichotomized into nonsevere outcome (mRS ≤ 3) and severe outcome (mRS \u3e 3). There were 77 patients in each group. Univariate and multivariate logistic regression analyses using severe outcome as the binary response were run. The authors also performed a logistic regression analysis to measure the effects of hematoma volume and AVM location on severe outcome. Results: Posterior fossa location was a significant predictor of severe outcome (OR 2.60, 95% CI 1.20-5.67, p = 0.016) and the results were strengthened in a multivariate model (OR 4.96, 95% CI 1.73-14.17, p = 0.003). Eloquent location (OR 3.47, 95% CI 1.37-8.80, p = 0.009) and associated arterial aneurysms (OR 2.58, 95% CI 1.09, 6.10; p = 0.031) were also significant predictors of poor outcome. Hematoma volume for patients with a posterior fossa AVM was 10.1 ± 10.1 cm3 compared with 25.6 ± 28.0 cm3 in supratentorial locations (p = 0.003). A logistic analysis (based on imputed hemorrhage volume values) found that posterior fossa location was a significant predictor of severe outcome (OR 8.03, 95% CI 1.20-53.77, p = 0.033) and logarithmic hematoma volume showed a positive, but not statistically significant, association in the model (p = 0.079). Conclusions: Patients with posterior fossa AVMs are more likely to have severe outcomes than those with supratentorial AVMs based on this natural history study. Age, sex, and ethnicity were not associated with an increased risk of severe outcome after AVM rupture, but posterior fossa location, associated aneurysms, and eloquent location were associated with poor post-hemorrhage mRS scores. Posterior fossa hematomas are poorly tolerated, with severe outcomes observed even with smaller hematoma volumes. These findings support an aggressive surgical posture with respect to posterior fossa AVMs, both before and after rupture. © AANS, 2014