Proteomic Analysis of Saliva from Patients with Oral Chronic Graft-Versus-Host Disease

AbstractChronic graft-versus-host disease (cGVHD) is an immune-mediated disorder and is the major long-term complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The oral mucosa, including the salivary glands, is affected in the majority of patients with cGVHD; however, at present there is only a limited understanding of disease pathobiology. In this study, we performed a quantitative proteomic analysis of saliva pooled from patients with and without oral cGVHD—cGVHD(+) and cGVHD(−), respectively—using isobaric tags for relative and absolute quantification labeling, followed by tandem mass spectrometry. Among 249 salivary proteins identified by tandem mass spectrometry, 82 exhibited altered expression in the oral cGVHD(+) group compared with the cGVHD(−) group. Many of the identified proteins function in innate or acquired immunity, or are associated with tissue maintenance functions, such as proteolysis or the cytoskeleton. Using ELISA immunoassays, we further confirmed that 2 of these proteins, IL-1 receptor antagonist and cystatin B, showed decreased expression in patients with active oral cGVHD (P < .003). Receiver operating curve characteristic analysis revealed that these 2 markers were able to distinguish oral cGVHD with a sensitivity of 85% and specificity of 60%, and showed slightly better discrimination in newly diagnosed patients evaluated within 12 months of allo-HSCT (sensitivity, 92%; specificity 73%). In addition to identifying novel potential salivary cGVHD biomarkers, our study demonstrates that there is coordinated regulation of protein families involved in inflammation, antimicrobial defense, and tissue protection in oral cGVHD that also may reflect changes in salivary gland function and damage to the oral mucosa

Resolution and Polarization Distribution in Cryogenic DNP/MAS Experiments

This contribution addresses four potential misconceptions associated with high-resolution dynamic nuclear polarization/magic angle spinning (DNP/MAS) experiments. First, spectral resolution is not generally compromised at the cryogenic temperatures at which DNP experiments are performed. As we demonstrate at a modest field of 9 T (380 MHz [superscript 1]H), 1 ppm linewidths are observed in DNP/MAS spectra of a membrane protein in its native lipid bilayer, and <0.4 ppm linewidths are reported in a crystalline peptide at 85 K. Second, we address the concerns about paramagnetic broadening in DNP/MAS spectra of proteins by demonstrating that the exogenous radical polarizing agents utilized for DNP are distributed in the sample in such a manner as to avoid paramagnetic broadening and thus maintain full spectral resolution. Third, the enhanced polarization is not localized around the polarizing agent, but rather is effectively and uniformly dispersed throughout the sample, even in the case of membrane proteins. Fourth, the distribution of polarization from the electron spins mediated via spin diffusion between [superscript 1]H–[superscript 1]H strongly dipolar coupled spins is so rapid that shorter magnetization recovery periods between signal averaging transients can be utilized in DNP/MAS experiments than in typical experiments performed at ambient temperature.National Institutes of Health (U.S.) (Grant EB002804)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB002026)National Institutes of Health (U.S.) (Grant EB001965)National Institutes of Health (U.S.) (Grant EB004866)National Science Foundation (U.S.). Graduate Research Fellowship Progra

An Intermittent Live Cell Imaging Screen for siRNA Enhancers and Suppressors of a Kinesin-5 Inhibitor

Kinesin-5 (also known as Eg5, KSP and Kif11) is required for assembly of a bipolar mitotic spindle. Small molecule inhibitors of Kinesin-5, developed as potential anti-cancer drugs, arrest cell in mitosis and promote apoptosis of cancer cells. We performed a genome-wide siRNA screen for enhancers and suppressors of a Kinesin-5 inhibitor in human cells to elucidate cellular responses, and thus identify factors that might predict drug sensitivity in cancers. Because the drug's actions play out over several days, we developed an intermittent imaging screen. Live HeLa cells expressing GFP-tagged histone H2B were imaged at 0, 24 and 48 hours after drug addition, and images were analyzed using open-source software that incorporates machine learning. This screen effectively identified siRNAs that caused increased mitotic arrest at low drug concentrations (enhancers), and vice versa (suppressors), and we report siRNAs that caused both effects. We then classified the effect of siRNAs for 15 genes where 3 or 4 out of 4 siRNA oligos tested were suppressors as assessed by time lapse imaging, and by testing for suppression of mitotic arrest in taxol and nocodazole. This identified 4 phenotypic classes of drug suppressors, which included known and novel genes. Our methodology should be applicable to other screens, and the suppressor and enhancer genes we identified may open new lines of research into mitosis and checkpoint biology

Cell-specific deletion of C1qa identifies microglia as the dominant source of C1q in mouse brain

BACKGROUND: The complement cascade not only provides protection from infection but can also mediate destructive inflammation. Complement is also involved in elimination of neuronal synapses which is essential for proper development, but can be detrimental during aging and disease. C1q, required for several of these complement-mediated activities, is present in the neuropil, microglia, and a subset of interneurons in the brain. METHODS: To identify the source(s) of C1q in the brain, the C1qa gene was selectively inactivated in the microglia or Thy-1(+) neurons in both wild type mice and a mouse model of Alzheimer’s disease (AD), and C1q synthesis assessed by immunohistochemistry, QPCR, and western blot analysis. RESULTS: While C1q expression in the brain was unaffected after inactivation of C1qa in Thy-1(+) neurons, the brains of C1qa (FL/FL) :Cx3cr1 (CreERT2) mice in which C1qa was ablated in microglia were devoid of C1q with the exception of limited C1q in subsets of interneurons. Surprisingly, this loss of C1q occurred even in the absence of tamoxifen by 1 month of age, demonstrating that Cre activity is tamoxifen-independent in microglia in Cx3cr1 (CreERT2/WganJ) mice. C1q expression in C1qa (FL/FL) : Cx3cr1 (CreERT2/WganJ) mice continued to decline and remained almost completely absent through aging and in AD model mice. No difference in C1q was detected in the liver or kidney from C1qa (FL/FL) : Cx3cr1 (CreERT2/WganJ) mice relative to controls, and C1qa (FL/FL) : Cx3cr1 (CreERT2/WganJ) mice had minimal, if any, reduction in plasma C1q. CONCLUSIONS: Thus, microglia, but not neurons or peripheral sources, are the dominant source of C1q in the brain. While demonstrating that the Cx3cr1 (CreERT2/WganJ) deleter cannot be used for adult-induced deletion of genes in microglia, the model described here enables further investigation of physiological roles of C1q in the brain and identification of therapeutic targets for the selective control of complement-mediated activities contributing to neurodegenerative disorders. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-017-0814-9) contains supplementary material, which is available to authorized users

Molecular Characterization of Branchial aquaporin 1aa and Effects of Seawater Acclimation, Emersion or Ammonia Exposure on Its mRNA Expression in the Gills, Gut, Kidney and Skin of the Freshwater Climbing Perch, Anabas testudineus

10.1371/journal.pone.0061163PLoS ONE84

Genome-wide meta-analysis of 158,000 individuals of European ancestry identifies three loci associated with chronic back pain

Back pain is the #1 cause of years lived with disability worldwide, yet surprisingly little is known regarding the biology underlying this symptom. We conducted a genome-wide association study (GWAS) meta-analysis of ch

Character network analysis of two science fiction Series – stargate and Star Trek / Melody Tan Shi Ai

This work undertakes a social network analysis of two science fiction franchise, Stargate and Star Trek. These two franchise consist of several television series and movies. Social network analysis is used to explore the social network of characters, derived from the transcripts. The stories conveyed are in the form of the interactions of characters, which can be represented as “character networks”. The character networks are formed by extracting the social interactions or connectivity of the characters. That is, the co-occurrences of the characters from a specific scene demarcates the presence of a connection irrespective of exchange of spoken dialogue. These networks are then used to characterise the overall structure and topology of each franchise. The character networks of both franchise have similar structure and topology to that found in previous work on scientific collaborations, literature, mythology and comics networks. The character networks exhibit the small-world effect and is disassortative, but no significant support for power-law distribution was found. Furthermore, since the progression of a story depends to a large extent on the interaction between each of its characters, the underlying network structure relates something about the complexity of the storyline. The complexity is assessed using techniques from spectral graph theory. The episode networks are found to be structured either as (1) closed networks, (2) those containing bottlenecks, (3) a mixture of the first two structures, or (4) those containing two or more connected components. Lastly, the characters’ role in the narrative was found to be supported by the characters’ centrality and centralisation measures

Nivolumab reactivation of hypertrophic lichen planus, a case report and review of published literature.

We report a case of nivolumab-induced lichen planus (LP) reactivation that was previously in remission following chemotherapy for non-smallcelllung cancer (NSCLC). Chemotherapy-induced immunosuppression allowed for complete resolution of the patient’s pre-existing LP, a T-cell mediatedautoimmune process. When the patient was switched to nivolumab immunotherapy owing to progression of NSCLC, PD-1 inhibition led to an overwhelming T-cell response that seemed to have provoked a severe LPreactivation. Although lichenoid reactions have been reported with nivolumab, to our knowledge, this is the first reported case of nivolumab monotherapycausing LP reactivation in a patient with a strong personal and family history of the disease that was previously in remission after chemotherapy

T4 BacteriophageDominatesT7 Bacteriophage During Co- infection of Escherichia coli C600

Co-infection is the simultaneous infection of a host cell by multiple viruses. It can result in complex competitive interactions that have significant consequences on virulence and disease dynamics. Our study investigated the intracellular mechanisms utilized for viral competition and identified a dominant virus during co-infection with a T4 and T7 bacteriophage model in E. coli strain C600. The co-infection was performed under two different input multiplicity of infection (MOI) conditions, each favouring one phage type. The amount of infectious progeny from coinfected samples was assessed with plaque assays. Results indicated that T4 dominated in both conditions, whereas PCR data showed T7 amplification from samples that produced only T4 plaques. We speculate that the dominance exhibited by T4 is due to its ability to modify the host RNA polymerase to only transcribe its own genes, thereby inhibiting T7 early gene transcription. In conclusion, bacteriophage T4 exhibited viral dominance over T7 during co-infection of E. coli C600, via mechanisms that interfered with T7 maturation. It has been observed in nature that multiple virions can coinfect a single host cell. Co-infection remains very important in virus evolution because of processes such as genetic reassortment, recombination, and complementation that can lead to increased virulence(1). However, coinfection also comes at a cost due to the sharingof host resources, which can be further limited with greater numbers of infecting virions per cell. Thus, many viruses have evolved mechanisms to inhibit viral competitors that coinfect the same host cell (2). These mechanisms can involve manipulation of host enzymes and repression of viral replication (2). In 2012, Brewster et al. (2) studied the co-infection of Escherichia coliC3000 by bacteriophages MS2, ϕX174, and T7. It was observed that co-infections resulted in decreased host cell lysis time compared to the component mono-infections, thus indicating that viral competition played a significant effect on viral outcome (1). However, they did not identify the dominant bacteriophage during co-infection or adequately address the specific mechanisms utilized for competition. Previous bacteriophage studies show mechanisms for viral competition affect intracellular stages of the phage life cycle (2). Therefore, our study explored the intracellular aspects of viral competition using a single cell for coinfection experiments. It was determined that E. coli C600 was a susceptible host for the genetically similar T-even and T-odd bacteriophages, T4 and T7, and could be utilized as a co-infection model. T4 and T7 are lytic phages, which utilize a tail component with tail fibers to facilitate attachment and entry into host cells using the LPS receptor (3, 4, 5). Bacteriophage T4 has a 169 kb genome consisting of early, middle, and late genes that are transcribed by the host RNA polymerase which becomes modified at each stage to allow progression into the phage replication cycle (6). Bacteriophage T7 has a 40 kb genome divided into early genes transcribed by the host RNA polymerase and late genes, which are transcribed by the T7 bacteriophageencoded RNA polymerase (4). In this experiment we defined viral dominance as the ability of one virus to limit the production of competitive infectious progeny. The results indicated that T4 exhibited dominance over T7 during co-infection, through mechanisms that affect T7 maturation. Our experiment provides greater insight into dominance during viral coinfection via intracellular mechanisms involving the viral life cycle. MATERIALS AND METHODS Strains and growth conditions. Escherichia coli C600 and bacteriophages T4 and T7 were obtained from the Microbiology 421 culture collection from the Department of Microbiology and Immunology, University of British Columbia. E. colicultures were grown at 30°C in L-Media described in Overby et al. Plaque assays. Phage samples were enumerated using the overlay agar plaque assay method described by Fortier and Moineau(8). A 4% soft-overlay agar was used. Infection with T7 phages produced characteristically larger plaques than with T4. Co-infection of T4 and T7 in E. coli C600.Two samples of 4x10 8 cells/ml E. coli C600were coinfected with T4 and T7. The first was infected with MOI of 0.5 and 1.2, respectively, while the second was infected with MOI of 1.2 and 0.5, respectively. The infected cultures were incubated for 5 minutes before centrifugation at 8000 x g for 10 minutes and disposal of the supernatant to remove non-adsorbed bacteriophages. Each pellet was resuspended, diluted and separated into 25 aliquots of 0.8 cells per tube with a total volume of 200 µl per tube. The aliquots were incubated at 37°C for 60 min to allow lysis of any infected cells. Plaque assays were performed to detect the presence of phages