Systematic identification of anti-interferon function on hepatitis C virus genome reveals p7 as an immune evasion protein.

Hepatitis C virus (HCV) encodes mechanisms to evade the multilayered antiviral actions of the host immune system. Great progress has been made in elucidating the strategies HCV employs to down-regulate interferon (IFN) production, impede IFN signaling transduction, and impair IFN-stimulated gene (ISG) expression. However, there is a limited understanding of the mechanisms governing how viral proteins counteract the antiviral functions of downstream IFN effectors due to the lack of an efficient approach to identify such interactions systematically. To study the mechanisms by which HCV antagonizes the IFN responses, we have developed a high-throughput profiling platform that enables mapping of HCV sequences critical for anti-IFN function at high resolution. Genome-wide profiling performed with a 15-nt insertion mutant library of HCV showed that mutations in the p7 region conferred high levels of IFN sensitivity, which could be alleviated by the expression of WT p7 protein. This finding suggests that p7 protein of HCV has an immune evasion function. By screening a liver-specific ISG library, we identified that IFI6-16 significantly inhibits the replication of p7 mutant viruses without affecting WT virus replication. In contrast, knockout of IFI6-16 reversed the IFN hypersensitivity of p7 mutant virus. In addition, p7 was found to be coimmunoprecipitated with IFI6-16 and to counteract the function of IFI6-16 by depolarizing the mitochondria potential. Our data suggest that p7 is a critical immune evasion protein that suppresses the antiviral IFN function by counteracting the function of IFI6-16.

Identification of Salmonella Taxon-Specific Peptide Markers to the Serovar Level by Mass Spectrometry.

We present an LC-MS/MS pipeline to identify taxon-specific tryptic peptide markers for the identification of Salmonella at the genus, species, subspecies, and serovar levels of specificity. Salmonella enterica subsp. enterica serovars Typhimurium and its four closest relatives, Saintpaul, Heidelberg, Paratyphi B, and Muenchen, were evaluated. A decision-tree approach was used to identify peptides common to the five Salmonella proteomes for evaluation as genus-, species-, and subspecies-specific markers. Peptides identified for two or fewer Salmonella strains were evaluated as potential serovar markers. Currently, there are approximately 140 000 assembled bacterial genomes publicly available, more than 8500 of which are for Salmonella. Consequently, the specificity of each candidate peptide marker was confirmed across all publicly available protein sequences in the NCBI nonredundant (nr) database. The performance of a subset of candidate taxon-specific peptide markers was evaluated in a targeted mass-spectrometry method. The presented workflow offers a marked improvement in specificity over existing MALDI-TOF-based bacterial identification platforms for the identification of closely related Salmonella serovars.

[Glucose transporter 1 deficiency syndrome: features of movement disorders, diagnosis and treatment].

OBJECTIVE: To investigate the clinical features, diagnosis and treatment of glucose transporter 1 deficiency syndrome (GLUT1-DS), as well as the diagnostic value of movement disorders. METHODS: The clinical data of four children with GLUT1-DS were collected, and their clinical features, treatment, and follow-up results were analyzed. RESULTS: There were two boys and two girls, with an age of onset of 2-15 months. Clinical manifestations included movement disorders, seizures, and developmental retardation. Seizures were the cause of the first consultation in all cases. The four children all had persistent ataxia, dystonia, and dysarthria; two had persistent tremor, two had paroxysmal limb paralysis, and two had eye movement disorders. Paroxysmal symptoms tended to occur in fatigue state. All four children had reductions in the level of cerebrospinal fluid glucose and its ratio to blood glucose, as well as SLC2A1 gene mutations. The four children were given a ketogenic diet, at a ketogenic ratio of 2:1 to 3:1, and achieved complete remission of paroxysmal symptoms within 5 weeks. CONCLUSIONS: GLUT1-DS should be considered for epileptic children with mental retardation and motor developmental delay complicated by various types of movement disorders. The ketogenic diet is effective at a ketogenic ratio of 2:1 to 3:1 for the treatment of GLUT1-DS.

Exploring the expression bar code of SAA variants for gastric cancer detection.

We reported an integrated platform to explore serum protein variant pattern in cancer and its utility as a new class of biomarker panel for diagnosis. On the model study of serum amyloid A (SAA), we employed nanoprobe-based affinity mass spectrometry for enrichment, identification and quantitation of SAA variants from serum of 105 gastric cancer patients in comparison with 54 gastritis patients, 54 controls, and 120 patients from other cancer. The result revealed surprisingly heterogeneous and most comprehensive SAA bar code to date, which comprises 24 SAA variants including SAA1- and SAA2-encoded products, polymorphic isoforms, N-terminal-truncated forms, and three novel SAA oxidized isotypes, in which the variant-specific peptide sequence were also confirmed by LC-MS/MS. A diagnostic model was developed for dimension reduction and computational classification of the 24 SAA-variant bar code, providing good discrimination (AUC = 0.85 ± 3.2E-3) for differentiating gastric cancer group from gastritis and normal groups (sensitivity, 0.76; specificity, 0.81) and was validated with external validation cohort (sensitivity, 0.71; specificity, 0.74). Our platform not only shed light on the occurrence and modification extent of under-represented serum protein variants in cancer, but also suggested a new concept of diagnostic platform by serum protein variant profile.

A quantitative high-resolution genetic profile rapidly identifies sequence determinants of hepatitis C viral fitness and drug sensitivity.

Widely used chemical genetic screens have greatly facilitated the identification of many antiviral agents. However, the regions of interaction and inhibitory mechanisms of many therapeutic candidates have yet to be elucidated. Previous chemical screens identified Daclatasvir (BMS-790052) as a potent nonstructural protein 5A (NS5A) inhibitor for Hepatitis C virus (HCV) infection with an unclear inhibitory mechanism. Here we have developed a quantitative high-resolution genetic (qHRG) approach to systematically map the drug-protein interactions between Daclatasvir and NS5A and profile genetic barriers to Daclatasvir resistance. We implemented saturation mutagenesis in combination with next-generation sequencing technology to systematically quantify the effect of every possible amino acid substitution in the drug-targeted region (domain IA of NS5A) on replication fitness and sensitivity to Daclatasvir. This enabled determination of the residues governing drug-protein interactions. The relative fitness and drug sensitivity profiles also provide a comprehensive reference of the genetic barriers for all possible single amino acid changes during viral evolution, which we utilized to predict clinical outcomes using mathematical models. We envision that this high-resolution profiling methodology will be useful for next-generation drug development to select drugs with higher fitness costs to resistance, and also for informing the rational use of drugs based on viral variant spectra from patients.

Reaction of Human Cd7metallothionein and N-Ethylmaleimide: Kinetic and Structural Insights from Electrospray Ionization Mass Spectrometry.

The reaction of cadmium-binding human metallothionein-2A (Cd7MT) and N-ethylmaleimide (NEM) is investigated by electrospray ionization-ion mobility-mass spectrometry (ESI IM-MS). MS provides a direct measure of the distribution of the kinetic intermediates as the reaction proceeds and provides new insights into the relative kinetic stability of the individual metal-thiolate bonds in Cd7MT. The rate constants for the various metal-retaining intermediates (Cd(i), intermediate with i Cd²âº ions attached) differ by >3 orders of magnitude: Cd4< Cd3< Cd2< Cd1∼ Cd6 < Cd7 < Cd5. The reaction is viewed as a two-component cooperative process, rapid loss of three Cd²âº ions followed by slow loss of the remaining four Cd²âº ions, and Cd4NEM10MT was observed as the least reactive intermediate during the entire displacement process. "MS-CID-IM-MS", a top-down approach that provides two-dimensional dispersion (size to charge by IM; mass to charge by MS) of the CID fragment ions, was used for direct analysis of the kinetic intermediate [Cd4NEM10MT]5⁺ ion. The results provide direct evidence that the four Cd²âº ions located in the α-domain are retained, indicative of the greater kinetic stability for the α-domain. Further, the mapping of the alkylation sites in the [Cd4NEM10MT]5⁺ ion reveals that not only the nine cysteines in the ß-domain but Cys33 in the α-domain is selectively labeled. The kinetic lability of the Cd-Cys33 bond is unexpected. The structural and functional implications of these findings are discussed.

Metal-induced conformational changes of human metallothionein-2A: a combined theoretical and experimental study of metal-free and partially metalated intermediates.

Electrospray ionization ion mobility mass spectrometry (ESI IM-MS) and molecular dynamics (MD) simulations reveal new insights into metal-induced conformational changes and the mechanism for metalation of human metallothionein-2A (MT), an intrinsically disordered protein. ESI of solutions containing apoMT yields multiple charge states of apoMT; following addition of Cd(2+) to the solution, ESI yields a range of CdiMT (i = 1-7) product ions (see Chen et al. Anal. Chem. 2013, 85, 7826-33). Ion mobility arrival-time distributions (ATDs) for the CdiMT (i = 0-7) ions reveal a diverse population of ion conformations. The ion mobility data clearly show that the conformational diversity for apoMT and partially metalated ions converges toward ordered, compact conformations as the number of bound Cd(2+) ions increase. MD simulations provide additional information on conformation candidates of CdiMT (i = 0-7) that supports the convergence of distinct conformational populations upon metal binding. Integrating the IM-MS and MD data provides a global view that shows stepwise conformational transition of an ensemble as a function of metal ion bound. ApoMT is comprised of a wide range of conformational states that populate between globular-like compact and coil-rich extended conformations. During the initial stepwise metal addition (number of metal ions bound i = 1-3), the metal ions bind to different sites to yield distinct conformations, whereas for i > 4, the conformational changes appear to be domain-specific, attributed to different degrees of disorder of the ß domain; the ß domain becomes more ordered as additional metal ions are added, promoting convergences to the dumbbell-shaped conformation.

Predicting primary PM2.5 and PM0.1 trace composition for epidemiological studies in California.

The University of California-Davis_Primary (UCD_P) chemical transport model was developed and applied to compute the primary airborne particulate matter (PM) trace chemical concentrations from ∼ 900 sources in California through a simulation of atmospheric emissions, transport, dry deposition and wet deposition for a 7-year period (2000-2006) with results saved at daily time resolution. A comprehensive comparison between monthly average model results and available measurements yielded Pearson correlation coefficients (R) ≥ 0.8 at ≥ 5 sites (out of a total of eight) for elemental carbon (EC) and nine trace elements: potassium, chromium, zinc, iron, titanium, arsenic, calcium, manganese, and strontium in the PM2.5 size fraction. Longer averaging time increased the overall R for PM2.5 EC from 0.89 (1 day) to 0.94 (1 month), and increased the number of species with strong correlations at individual sites. Predicted PM0.1 mass and PM0.1 EC exhibited excellent agreement with measurements (R = 0.92 and 0.94, respectively). The additional temporal and spatial information in the UCD_P model predictions produced population exposure estimates for PM2.5 and PM0.1 that differed from traditional exposure estimates based on information at monitoring locations in California Metropolitan Statistical Areas, with a maximum divergence of 58% at Bakersfield. The UCD_P model has the potential to improve exposure estimates in epidemiology studies of PM trace chemical components and health.

Epidemiological characteristics of acute disseminated encephalomyelitis in Nanchang, China: a retrospective study.

BACKGROUND: Acute disseminated encephalomyelitis (ADEM) is an autoimmune disease that typically follows a monophasic course and may affect any age group. The precise population-based incidence of ADEM is still unknown in most countries. In China, there is no ADEM surveillance system. The exact incidence of ADEM is difficult to estimate, and other epidemiological characteristics of ADEM are unknown. The purpose of this study is to investigate the epidemiological characteristics of ADEM in Nanchang, China. METHODS: A retrospective investigation was conducted with ADEM patients admitted to second-level and third-level hospitals in Nanchang from 2008 to 2010, aiming to analyse the epidemiologic characteristics of ADEM in the population in Nanchang. ADEM patients, defined as patients who were diagnosed according to the consensus definition of ADEM provided by the International Pediatric MS Study Group, were enrolled in the study. The data were extracted from the ADEM patients' medical records. RESULTS: Forty-seven ADEM patients were investigated. The average annual incidence was 0.31/100,000; the incidence among males (0.31/100,000) was nearly equal to that among females (0.31/100,000). The median age of onset was 25.97 years old, and the peak incidence was observed in the 5- to 9-year-old age group (0.75/100,000), followed by the over-60 age group (0.55/100,000). ADEM occurs throughout the year, but it occurs most frequently in March (n = 7) and least frequently in April and July (both n = 2). The patient numbers are roughly even in the other months. In the 2 months before the onset of ADEM, 15 patients presented with a preceding infection, but none of the patients received a vaccination. An increased number of vaccination was not accompanied by a corresponding increased number of cases of ADEM. CONCLUSIONS: The average annual incidence of ADEM was 0.31/100,000 in Nanchang. The incidence among males was nearly equal to that among females. The peak age of onset was 5-9 years old. The peak season of onset was not apparent. There was no evidence of an association between increased number of vaccines administered and number of cases of ADEM in Nanchang, China.