Using Model Proteins to Study Tyrosine Oxidation-Reduction: Reversible Voltammograms, Long-Lived Radicals and Detailed Design of the Radical Site

Amino acid radicals have been found as key components in a number of biological redox processes. In specific, redox−active tyrosine residues play an essential role in DNA biosynthesis and photosynthesis, among other processes. The thermodynamic and kinetic properties of one−electron redox reactions involving tyrosine have long been obscured by the highly unstable nature of the products of tyrosine oxidation. Remarkable control of these species is achieved within natural proteins. Proteins must, therefore, provide interactions to the amino acid cofactor to generate, control and direct the redox chemistry within the protein milieu. Electrochemical characterization of redox−active tyrosine residues inside natural radical proteins is highly challenging due to potential oxidation of other cofactors and residues. Numerous small−molecule models have been generated in which factors that affect tyrosine/phenol redox chemistry have been elucidated, such as hydrogen−bonding. Although these models have contributed to our knowledge about these systems, these lack the protein environment that certainly renders a stabilizing environment for these species. We have developed a de novo−designed model protein family in which to study these reactions. The family of α3X proteins consists of a well−structured, pH−stable, three−helix bundle with a single redox−active residue within the core of the protein. Construction and characterization of tyrosine−containing (α 3Y, α 3Y−K29H, and α 3Y−K36H) and tyrosine−analogue−containing (2MP− α 3C, 3MP− α 3C, and 4MP− α 3C) proteins are described in this dissertation work. Electrochemical characterization of α 3Y and 2MP− α 3C by Square Wave Voltammetry has allowed us to obtain fully reversible voltammograms and formal reduction potentials for the long−lived neutral radicals formed within these proteins. In the case of α 3Y, we have also found that the protein scaffold is intimately involved in the electron transfer process. We have also achieved the introduction of a tyrosine−histidine interacting pair in two α 3Y variants. This interacting complex is of great interest and will provide insights into how interaction with these basic residues might impact tyrosine redox chemistry within a protein scaffold. In addition, the solution NMR structure of 4MP− α 3C is reported and compared to the previously determined 2MP− α 3C structure. Key structural features are described that are likely to have a major impact on the protein redox properties

“It Ruined My Life”: The effects of the War on Drugs on people who inject drugs (PWID) in rural Puerto Rico

Background—The War on Drugs has raised the incarceration rates of racial minorities for non-violent drug-related crimes, profoundly stigmatized drug users, and redirected resources from drug prevention and treatment to militarizing federal and local law enforcement. Yet, while some states consider shifting their punitive approach to drug use, to one based on drug treatment and rehabilitation, nothing suggests that these policy shifts are being replicated in Puerto Rico. Methods—This paper utilizes data from 360 PWID residing in four rural towns in the mountainous area of central Puerto Rico. We initially recruited 315 PWID using respondent-driven sampling (RDS) and collected data about risk practices and conducted HIV and HCV testing. During a second phase, we conducted 34 micro-ethnographic assays, in which we randomly recruited 34 participants from the first phase and included their ego networks in this phase. Our ethnographic inquiry produced significant data regarding the effects of the war on drugs on the local drug trade, drug availability, and injectors’ social networks. Results—Findings suggest that repressive policing has been ineffective in preventing drug distribution and use among those in our study. This type of law enforcement approach has resulted in the disproportionate incarceration of poor drug users in rural Puerto Rico, and mainly for nonviolent drug-related crimes. In addition, incarceration exposes PWID to a form of a cruel and unusual punishment: having to quit heroin “cold turkey” while the prison environment also represents a HIV/HCV risk. In turn, the war on drugs not only diverts resources from treatment but also shapes treatment ideologies, punishing non-compliant patients. Conclusion—Shifting the emphasis from repression to treatment and rehabilitation is likely to have a positive impact on the health and overall quality of life of PWID and their communities

A bronchoscopy-associated pseudo-outbreak of Mycobacterium mucogenicum traced to use of contaminated ice used for bronchoalveolar lavage

Clonal Mycobacterium mucogenicum isolates (determined by molecular typing) were recovered from 19 bronchoscopic specimens from 15 patients. None of these patients had evidence of mycobacterial infection. Laboratory culture materials and bronchoscopes were negative for Mycobacteria. This pseudo-outbreak was caused by contaminated ice used to provide bronchoscopic lavage. Control was achieved by transitioning to sterile ice

Avaliação da nanotoxicidade de nanopartículas lipidicas aplicadas na formulação de sistemas de libertação controlada

[Excerto] Tem sido crescente a evidência de que nanopartículas podem induzir toxicidade celular relevante com forte impacto na saúde humana. Esta toxicidade pode ser expressa através da indução de diversas respostas celulares tais como a morte celular, inflamação e stress oxidativo, tornando-se assim de extrema importância a utilização de modelos celulares in vitro para avaliar a resposta biológica desencadeada pela presença dos nanosistemas formulados. O modelo celular Caco-2, utilizado neste trabalho, é um modelo amplamente aceite para avaliação da toxicidade, transporte e uptake de vários materiais à nanoescala. [...]info:eu-repo/semantics/publishedVersio

Elongase Reactions as Control Points in Long-Chain Polyunsaturated Fatty Acid Synthesis

Extent: 9p.Background: Δ6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary α-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA. Methodology/Principal Findings: The substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C20 and C22 polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPA→DPA→24:5n-3. The second reaction DPA→24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPA→DPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3. Conclusions: The competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPA→24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means.Melissa K. Gregory, Robert A. Gibson, Rebecca J. Cook-Johnson, Leslie G. Cleland and Michael J. Jame

Homologous recombination DNA repair defects in PALB2-associated breast cancers.

Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD.ER

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas

Although theMYConcogene has been implicated incancer, a systematic assessment of alterations ofMYC, related transcription factors, and co-regulatoryproteins, forming the proximal MYC network (PMN),across human cancers is lacking. Using computa-tional approaches, we define genomic and proteo-mic features associated with MYC and the PMNacross the 33 cancers of The Cancer Genome Atlas.Pan-cancer, 28% of all samples had at least one ofthe MYC paralogs amplified. In contrast, the MYCantagonists MGA and MNT were the most frequentlymutated or deleted members, proposing a roleas tumor suppressors.MYCalterations were mutu-ally exclusive withPIK3CA,PTEN,APC,orBRAFalterations, suggesting that MYC is a distinct onco-genic driver. Expression analysis revealed MYC-associated pathways in tumor subtypes, such asimmune response and growth factor signaling; chro-matin, translation, and DNA replication/repair wereconserved pan-cancer. This analysis reveals insightsinto MYC biology and is a reference for biomarkersand therapeutics for cancers with alterations ofMYC or the PMN

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment