Reversible hydrogen transfer reactions of cysteine thiyl radicals in peptides: the conversion of cysteine into dehydroalanine and alanine, and of alanine into dehydroalanine

The photodissociation of disulfide bonds in model peptides containing Ala and Ala-d3 generates a series of photoproducts following the generation of a CysS• thiyl radical pair. These photoproducts include transformations of Cys to dehydroalanine (Dha) and Ala, as well as Ala to Dha. Intramolecular Michael addition of an intact Cys with a photolytically generated Dha results in the formation of cyclic thioethers. The conversion of Cys into Dha likely involves a 1,3-H-shift from the Cys αC-H bond to the thiyl radical, followed by elimination of HS•. The conversion of Dha into Ala most likely involves hydrated electrons, which are generated through the photolysis of Cys, the photoproduct of disulfide photolysis. Prior to stable product formation, CysS• radicals engage in reversible hydrogen transfer reactions with αC-H and βC-H bonds of the surrounding amino acids. Especially for the βC-H bonds of Ala such hydrogen transfer reactions are unexpected based on thermodynamic grounds; however, the replacement of deuterons in Ala-d3 by hydrogens in H2O provides strong experimental evidence for such reactions

Light-induced Conversion of Trp to Gly and Gly Hydroperoxide in IgG1

The exposure of IgG1 in aqueous solution to light with λ = 254 nm or λ > 295 nm yields products consistent with Trp radical cation formation followed by αC-βC cleavage of the Trp side chain. The resulting glycyl radicals are either reduced to Gly, or add oxygen prior to reduction to Gly hydroperoxide. Photoirradiation at λ = 254 nm targets Trp at positions 191 (light chain), 309 and 377 (heavy chain) while photoirradiation at λ > 295 nm targets Trp at position 309 (heavy chain). Mechanistically, the formation of Trp radical cations likely proceeds via photo-induced electron- or hydrogen-transfer to disulfide bonds, yielding thiyl radicals and thiols, where thiols may serve as reductants for the intermediary glycyl or glycylperoxyl radicals

Augmented Fish Health Monitoring for Washington Department of Wildlife; Five-year Project Report, 1986-1991 Final Report.

The Augmented Fish Health Monitoring Project was funded by the Bonneville Power Administration (BPA) with the mandate to collect fish health data on the anadromous fish stocks of the Columbia River Basin in a standardized manner. The Washington Department of Wildlife began the project in 1986. Cumulative data and a final summary for this project are presented in this document. Fish stocks were examined monthly for length, weight, and health status at all Washington Department of Wildlife Columbia River Basin hatcheries. Assays for specific fish pathogens were conducted on all stocks of broodfish and smolts in the study area. Pathogens of interest were replicating viral agents, erythrocytic inclusion body syndrome virus (EIBSV), and Renibacterium salmoninarum. Sea-run cutthroat (SCT) were also sampled midway through the rearing cycle for R. salmoninarum. Juvenile fish were examined for the presence of any pathogen. Assays for Myxobolus cerebralis were conducted on fish stocks in several locations along the Columbia River. An organosomatic index analysis was made on each stock of smolts at the Cowlitz and Wells hatcheries. Results of the organosomatic index analysis were consistent between the years at each facility. However, the fish reared at Cowlitz displayed tissue changes associated with ceratomyxosis while those reared at Wells had a more desirable color and quality. Cell culture assays for viral agents in broodfish were positive for infectious hematopoeitic necrosis virus (IHNV) in all stocks at the Cowlitz Hatchery four out of five years in the study. Other stations were less consistent over the years. Only the sea-run cutthroat stock spawned at Beaver Creek was negative for any virus. Infectious pancreatic necrosis virus (IPNV) was isolated from summer-run steelhead (SS) broodfish at Wells in 1989 and 1991 and at Yakima in 1991. Inclusions that are characteristic of EIBSV were found in red blood cells of brood fish from the Wells Hatchery in 1990 and 1991. Data collected on EIBSV during the first two years of the project cannot be compared with the later three years due to changes in laboratory protocol. Isolations of IHNV in smolts were made from Cowlitz and Skamania hatcheries and the Gobar Rearing Pond. Epizootics of IHN occurred at Lyons Ferry, Beaver Creek, Cowlitz and Skamania hatcheries during the project, EIBSV inclusions were identified in very low levels from smolts from Beaver Creek, Chelan, Cowlitz, Eastbank, and Ringold. Assays for R. salmoninarum on broodfish and smolts revealed very low levels of infection and the disease was not a problem. Enteric redmouth disease was not observed in the project area. Cytophaga psychrophila was a chronic problem in young fish at Vancouver, Beaver Creek and Cowlitz hatcheries. Ceratomyxa Shasta was the only reportable parasite observed in the fish within the study area and caused yearly outbreaks of ceratomyxosis at the Cowlitz Hatchery. Fish at the Beaver Creek Hatchery were treated for furunculosis three of the five years of the project. An ozone water treatment plant has been installed to minimize the disease. Flow and density indexes and feed conversion did not vary significantly at the hatcheries during this project. Egg mortality averaged 12.94% throughout the project with a range from 4.39% to 29.10%. The mean fry mortality during the project was 15.08% with a range of 2.01 to 37.43%. The overall mortality for early rearing was 20.43%. Prespawning broodstock mortality was recorded for SS and SCT and averaged 5.18% with a range from 0 to 38.8%. Fungal invasion was the primary cause of death in adult fish. Epizootics of furunculosis, ceratomyxosis, bacterial coldwater disease, and IHN occurred during the project. Fewer cases were reported in more recent years. The BPA augmented fish health project helped WDW identify problem areas in fish health while they were occurring. This knowledge allowed us to develop strategies for improved fish quality. Overall the project has been invaluable in assisting us in the improvement of the health of our fish

Entanglement model of antibody viscosity

Antibody solutions are typically much more viscous than solutions of globular proteins at equivalent volume fraction. Here we propose that this is due to molecular entanglements that are caused by the elongated shape and intrinsic flexibility of antibody molecules. We present a simple theory in which the antibodies are modeled as linear polymers that can grow via reversible bonds between the antigen binding domains. This mechanism explains the observation that relatively subtle changes to the interparticle interaction can lead to large changes in the viscosity. The theory explains the presence of distinct power law regimes in the concentration dependence of the viscosity as well as the correlation between the viscosity and the charge on the variable domain in our anti-streptavidin IgG1 model system

Coformulation of Broadly Neutralizing Antibodies 3BNC117 and PGT121: Analytical Challenges During Preformulation Characterization and Storage Stability Studies

This work is licensed under a Creative Commons Attribution 4.0 International License.In this study, we investigated analytical challenges associated with the formulation of 2 anti-HIV broadly neutralizing antibodies (bnAbs), 3BNC117 and PGT121, both separately at 100 mg/mL and together at 50 mg/mL each. The bnAb formulations were characterized for relative solubility and conformational stability followed by accelerated and real-time stability studies. Although the bnAbs were stable during 4°C storage, incubation at 40°C differentiated their stability profiles. Specific concentration-dependent aggregation rates at 30°C and 40°C were measured by size exclusion chromatography for the individual bnAbs with the mixture showing intermediate behavior. Interestingly, although the relative ratio of the 2 bnAbs remained constant at 4°C, the ratio of 3BNC117 to PGT121 increased in the dimer that formed during storage at 40°C. A mass spectrometry-based multiattribute method, identified and quantified differences in modifications of the Fab regions for each bnAb within the mixture including clipping, oxidation, deamidation, and isomerization sites. Each bnAb showed slight differences in the levels and sites of lysine residue glycations. Together, these data demonstrate the ability to differentiate degradation products from individual antibodies within the bnAb mixture, and that degradation rates are influenced not only by the individual bnAb concentrations but also by the mixture concentration.Bill and Melinda Gates Foundation, Seattle, WA [grant number OPP1138851 and Investment ID 25617

Co-formulation of broadly neutralizing antibodies 3BNC117 and PGT121:Analytical challenges during pre-formulation characterization and storage stability studies

In this study, we investigated analytical challenges associated with the formulation of two broadly neutralizing anti-HIV monoclonal antibodies (bnAbs), 3BNC117 and PGT121, both separately at 100 mg/mL and together at 50 mg/mL each. The bnAb formulations were characterized for relative solubility and conformational stability followed by accelerated and real-time stability studies. While the bnAbs were stable during 4°C storage, incubation at 40°C differentiated their stability profiles. Specific concentration dependent aggregation rates at 30°C and 40°C were measured by size exclusion chromatography for the individual bnAbs with the mixture showing intermediate behavior. Please download the file below for full content

Analysis of clinically relevant somatic mutations in high-risk head and neck cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma is the second most prevalent malignancy, most frequently occurring in the head and neck (head and neck cutaneous squamous cell carcinoma). Treatment of locally advanced or metastatic disease is associated with functional morbidity and disfigurement. Underlying genetic mechanisms are poorly understood. Targeted sequencing of 48 clinically relevant genes was performed on DNA extracted from formalinfixed and paraffin-embedded high-risk primary head and neck cutaneous squamous cell carcinomas that remained non-metastatic at minimum follow-up of 24 months. Associations of somatic mutations with clinicopathologic characteristics were evaluated and compared with those described in the literature for metastatic disease. Alterations in 44 cancer-associated genes were identified. TP53 was mutated in 100% of cases; APC, ATM, ERBB4, GNAQ, KIT, RB1 and ABL1 were altered in 60% of cases. FGFR2 mutations (40%) were exclusively seen in patients with perineural invasion. MLH1 mutations were exclusively seen in the two younger patients (\u3c45 \u3eyears). Lower incidences of NOTCH1 mutations were observed compared with that described in metastatic head and neck cutaneous squamous cell carcinoma in the literature. Somatic mutations susceptible to EGFR inhibitors, and other small molecular targeted therapeutics were seen in 60% of cases. This study provides insights into somatic mutations in non-metastatic, high-risk head and neck cutaneous squamous cell carcinoma and identifies potential therapeutic targets. Alterations in FGFR2 and NOTCH1 may have roles in local and distant disease progression

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