Characterization of the dystrophin-associated protein complex by mass spectrometry.

The dystrophin-associated protein complex (DAPC) is a highly organized multiprotein complex that plays a pivotal role in muscle fiber structure integrity and cell signaling. The complex is composed of three distinct interacting subgroups, intracellular peripheral proteins, transmembrane glycoproteins, and extracellular glycoproteins subcomplexes. Dystrophin protein nucleates the DAPC and is important for connecting the intracellular actin cytoskeletal filaments to the sarcolemma glycoprotein complex that is connected to the extracellular matrix via laminin, thus stabilizing the sarcolemma during muscle fiber contraction and relaxation. Genetic mutations that lead to lack of expression or altered expression of any of the DAPC proteins are associated with different types of muscle diseases. Hence characterization of this complex in healthy and dystrophic muscle might bring insights into its role in muscle pathogenesis. This review highlights the role of mass spectrometry in characterizing the DAPC interactome as well as post-translational glycan modifications of some of its components such as α-dystroglycan. Detection and quantification of dystrophin using targeted mass spectrometry are also discussed in the context of healthy versus dystrophic skeletal muscle.

Predictors of milk cortisol in North American women.

OBJECTIVES: Human milk content varies across mother-child dyads, environments, and populations. Among the hormones in milk is cortisol, a glucocorticoid; its impact on the breastfeeding child is unknown. Milk cortisol may constitute a signal to the child's developing physiology which can shape characteristics (e.g., growth, temperament) to prevailing environmental conditions. This exploratory study evaluated the maternal, breastfeeding, and infant characteristics associated with milk cortisol. METHODS: We evaluated archived milk specimens for cortisol using enzyme immunoassay and employed an information-theoretic approach to assess associations between milk cortisol and participant characteristics with linear regression modeling. Because we employed secondary data, information for some variables likely to impact milk cortisol variation (e.g., time of day, socioeconomic status, maternal or infant body mass index, milk energy density) was unavailable. RESULTS: Participants were 48 lactating mothers from upstate New York, aged 21-40 years. Milk cortisol ranged from 0.098 to 1.007 µg/dL. Child age ranged from 1 to 26 months. In linear regression employing best fit modeling criteria, milk cortisol increased with child age (B: 0.069; p: .000; a 7.1% increase in milk cortisol for each month of child age), while child symptoms of illness (B: -0.398; p: .057; a 33% decrease) and consumption of complementary foods (B: -.525; p: .020; a 41% decrease) were associated with lower milk cortisol. CONCLUSIONS: We speculate that increasing milk cortisol with child age plays a role in signaling development (e.g., as increasing independence increases risk for injury and other negative health outcomes), independent of the maternal stressors we could capture.

Urine proteomics by mass spectrometry identifies proteins involved in key pathogenic pathways in patients with juvenile dermatomyositis.

OBJECTIVES: To identify and validate biomarkers in JDM patients using a multiplexing tandem mass tag urine proteome profiling approach. METHODS: First morning void urine samples were collected from JDM patients (n = 20) and healthy control subjects (n = 21) and processed for analysis using a standardized liquid chromatography-tandem mass spectrometry approach. Biomarkers with significantly altered levels were correlated with clinical measures of myositis disease activity and damage. A subset of candidate biomarkers was validated using commercially available ELISA kits. RESULTS: In total, 2348 proteins were detected in the samples, with 275 proteins quantified across all samples. Among the differentially altered proteins, cathepsin D and galectin-3 binding protein were significantly increased in the urine of JDM patients (adjusted P < 0.05), supporting previous findings in myositis patients. These two candidate biomarkers were confirmed with ELISAs. Cathepsin D positively correlated with Myositis Damage Index (r = 0.57, P < 0.05) and negatively correlated with the Childhood Myositis Assessment Scale (r = -0.54, P < 0.05). We also identified novel JDM candidate biomarkers involved with key features of myositis, including extracellular matrix remodelling proteins. CONCLUSION: This study confirmed the presence of several proteins in the urine of JDM patients that were previously found to be elevated in the blood of myositis patients and identified novel candidate biomarkers that require validation. These results support the use of urine as a source for biomarker development in JDM.

Associations between milk cortisol and activity of the immune system of milk.

OBJECTIVES: Both the immune system of human milk and milk cortisol have complex short- and long-term effects on child health and development. As understanding continues to grow of the independent effects of each of these components of milk, it is also important to investigate their intersection, including how milk cortisol affects the immune system of milk. We began this important endeavor through secondary analyses of archived milk specimens. METHODS: Participants were 31 lactating mothers from upstate New York. We estimated milk cortisol concentrations via enzyme immunoassay. We assessed milk proinflammatory cytokine (interleukin-6, IL-6) responses to pathogenic (Salmonella) and commensal (Escherichia, Lactobacillus, Bifidobacterium) bacteria via in vitro stimulation. We estimated ordered logistic regression models to assess associations between milk cortisol and IL-6 responses to bacteria. RESULTS: Milk cortisol ranged from 0.098 to 1.007 µg/dL. Milk cortisol was positively associated with IL-6 responses to S. enterica (B: 4.035; 95% CI: 0.674, 7.395) and B. breve (B: 3.675; 95% CI: 0.426, 6.924); this association persisted after controlling for child age. Results were less clear for associations between milk cortisol and IL-6 responses to L. acidophilus (B: 2.318; 95% CI: -1.224, 5.859) and E. coli (B: 2.366; 95% CI: -0.960, 5.692). CONCLUSIONS: Complex interactions between cortisol and the immune system extend to milk. Milk cortisol was positively associated with proinflammatory responses to some bacteria in vitro. This may suggest that milk cortisol is causally upstream of protective immune activity.

A single tick screening for infectious pathogens using targeted mass spectrometry.

The black-legged tick, Ixodes scapularis, is a well-known vector for the Lyme disease-causing pathogen (Borrelia burgdorferi) but can also carry other disease-causing pathogens such as Rickettsia, Anaplasma, Bartonella, Ehrlichia, and Theileria. Hence, tick screening using highly specific protein signatures for specific pathogens will help assess the prevalence of infected ticks and understand the pathogen-tick interactions in a specific geographic area. In this study, we used data-dependent acquisition to key pathogen protein signatures in black-legged ticks collected from the Southern Tier New York. Bottom-up proteomic analysis of extract from five combined ticks identified 2,052 tick proteins and 41 pathogen proteins with high confidence (≥ 99% C.I.). Results show high peptide spectral match counts for Rickettsia species and Borrelia species and lower counts for other rarer pathogens such as Anaplasma phagocytophilum. Parallel reaction monitoring performed on protein extracts from individual ticks (n = 10) revealed that 8 out of the 10 screened ticks carried Rickettsia species, 5 carried Borrelia species, 3 carried both pathogens, and only 1 tick carried no detectable bacteria. Mass spectrometry-based proteomics is a highly specific way to define the expression of different types of pathogen proteins in infected ticks. This might bring insights into the tick-pathogen interactions at the molecular level and especially expression pathogen surface proteins in ticks.

Recent kuru-induced female gene flow disrupted the coevolution of genes and languages in the Papua New Guinea highlands.

OBJECTIVES: The island of New Guinea was settled by modern human over 50,000 years ago, and is currently characterized by a complex landscape and contains one-seventh of the world's languages. The Eastern Highlands of New Guinea were also the home to the devastating prion disease called kuru that primarily affected Fore-speaking populations, with some 68% of cases involving adult females. We characterized the mitochondrial DNA (mtDNA) diversity of highlanders from Papua New Guinea (PNG) to: (a) gain insight into the coevolution of genes and languages in situ over mountainous landscapes; and (b) evaluate the recent influence of kuru mortality on the pattern of female gene flow. MATERIALS AND METHODS: We sequenced the mtDNA hypervariable segment 1 of 870 individuals from the Eastern and Southern Highlands of PNG using serums collected in the 1950s to 1960s. These highlanders were selected from villages representing 15 linguistic groups within the Trans-New Guinea phylum. Genetic, linguistic, and geographic distances were calculated separately and correlations among those distance matrices were assessed using the Mantel test. RESULTS: Geographic, genetic, and linguistic patterns were independently correlated with each other (p < .05). Increased mtDNA diversity in kuru-affected populations and low Fst estimates between kuru-affected linguistic groups were observed. DISCUSSION: In general, the genetic structure among the Highland populations was shaped by both geography and language, and language is a good predictor of mtDNA affinity in the PNG Highlands. High kuru female mortality increased female gene flow locally, disrupting coevolutionary pattern among genes, languages, and geography.

The origins and genetic distinctiveness of the Chamorros of the Marianas Islands: an mtDNA perspective.

BACKGROUND: Archaeological and linguistic evidence suggests the Marianas Islands were settled around 3,600 years before present (ybp) from Island Southeast Asia (ISEA). Around 1,000 ybp latte stone pillars and the first evidence of rice cultivation appear in the Marianas. Both traditions are absent in the rest of prehistoric Oceania. OBJECTIVE: To examine the genetic origins and postsettlement gene flow of Chamorros of the Marianas Islands. METHODS: To infer the origins of the Chamorros we analyzed ∼360 base pairs of the hypervariable-region 1 (HVS1) of mitochondrial DNA from 105 Chamorros from Guam, Rota, and Saipan, and the complete mitochondrial genome of 32 Guamanian Chamorros, and compared them to lineages from ISEA and neighboring Pacific archipelagoes from the database. RESULTS: Results reveal that 92% of Chamorros belong to haplogroup E, also found in ISEA but rare in Oceania. The two most numerous E lineages were identical to lineages currently found in Indonesia, while the remaining E lineages differed by only one or two mutations and all were unique to the Marianas. Seven percent of the lineages belonged to a single Chamorro-specific lineage within haplogroup B4, common to ISEA as well as Micronesia and Polynesia. CONCLUSIONS: These patterns suggest a small founding population had reached and settled the Marianas from ISEA by 4,000 ybp, and developed unique mutations in isolation. A second migration from ISEA may have arrived around 1,000 ybp, introducing the latte pillars, rice agriculture and the homogeneous minority B4 lineage.

Inherited and somatic mitochondrial DNA mutations in Guam amyotrophic lateral sclerosis and parkinsonism-dementia.

There is increasing evidence for mitochondrial dysfunction in neurodegenerative disorders, although the exact role of mitochondrial DNA (mtDNA) mutations in this process is unresolved. We investigated inherited and somatic mtDNA substitutions and deletions in Guam amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD). Hypervariable segment 1 sequences of Chamorro mtDNA revealed that the odds ratio of a PD or ALS diagnosis was increased for individuals in the E1 haplogroup while individuals in the E2 haplogroup had decreased odds of an ALS or PD diagnosis. Once the disorders were examined separately, it became evident that PD was responsible for these results. When the entire mitochondrial genome was sequenced for a subset of individuals, the nonsynonymous mutation at nucleotide position 9080, shared by all E2 individuals, resulted in a significantly low odds ratio for a diagnosis of ALS or PD. Private polymorphisms found in transfer and ribosomal RNA regions were found only in ALS and PD patients in the E1 haplogroup. Somatic mtDNA deletions in the entire mtDNA genome were not associated with either ALS or PD. We conclude that mtDNA haplogroup effects may result in mitochondrial dysfunction in Guam PD and reflect Guam population history. Thus it is reasonable to consider Guam ALS and PD as complex disorders with both environmental prerequisites and small genetic effects.

Interrogation of Dystrophin and Dystroglycan Complex Protein Turnover After Exon Skipping Therapy.

Recently, the Food and Drug Administration granted accelerated approvals for four exon skipping therapies -Eteplirsen, Golodirsen, Viltolarsen, and Casimersen -for Duchenne Muscular Dystrophy (DMD). However, these treatments have only demonstrated variable and largely sub-therapeutic levels of restored dystrophin protein in DMD patients, limiting their clinical impact. To better understand variable protein expression and the behavior of truncated dystrophin protein in vivo, we assessed turnover dynamics of restored dystrophin and dystrophin glycoprotein complex (DGC) proteins in mdx mice after exon skipping therapy, compared to those dynamics in wild type mice, using a targeted, highly-reproducible and sensitive, in vivo stable isotope labeling mass spectrometry approach in multiple muscle tissues. Through statistical modeling, we found that restored dystrophin protein exhibited altered stability and slower turnover in treated mdx muscle compared with that in wild type muscle (∼44 d vs. ∼24 d, respectively). Assessment of mRNA transcript stability (quantitative real-time PCR, droplet digital PCR) and dystrophin protein expression (capillary gel electrophoresis, immunofluorescence) support our dystrophin protein turnover measurements and modeling. Further, we assessed pathology-induced muscle fiber turnover through bromodeoxyuridine (BrdU) labeling to model dystrophin and DGC protein turnover in the context of persistent fiber degeneration. Our findings reveal sequestration of restored dystrophin protein after exon skipping therapy in mdx muscle leading to a significant extension of its half-life compared to the dynamics of full-length dystrophin in normal muscle. In contrast, DGC proteins show constant turnover attributable to myofiber degeneration and dysregulation of the extracellular matrix (ECM) in dystrophic muscle. Based on our results, we demonstrate the use of targeted mass spectrometry to evaluate the suitability and functionality of restored dystrophin isoforms in the context of disease and propose its use to optimize alternative gene correction strategies in development for DMD.

The Effect of Immunomodulatory Treatments on Anti-Dystrophin Immune Response After AAV Gene Therapy in Dystrophin Deficient mdx Mice.

BACKGROUND: AAV-based gene therapy is an attractive approach to treat Duchenne muscular dystrophy (DMD) patients. Although the long-term consequences of a gene therapy approach for DMD are unknown, there is evidence in both DMD patients and animal models that dystrophin replacement by gene therapy leads to an anti-dystrophin immune response that is likely to limit the long-term use of these therapeutic strategies. OBJECTIVE: Our objective is to test whether the anti-dystrophin immune response is affected by immunomodulatory drugs in mdx mice after rAAV gene therapy. METHODS: mdx mice were treated with rAAV microdystrophin alone or in combination with immunomodulatory drugs. Dystrophin expression in skeletal muscle was assessed by mass spectrometry. Immune responses were assessed by immunophenotyping, western blot for anti-dystrophin antibodies and flow cytometry assays for antigen-specific T-cell cytokine expression. The impact on muscle was measured by grip strength assessment, in vivo torque, optical imaging for inflammation and H&E staining of sections to assess muscle damage. RESULTS: We found that AAV-9-microdystrophin gene therapy induced expression of microdystrophin, anti-dystrophin antibodies, and T-cell cytokine responses. Immunomodulatory treatments, rituximab and VBP6 completely abrogated the anti-dystrophin antibody response. Prednisolone, CTLA4-Ig, and eplerenone showed variable efficacy in blocking the anti-dystrophin immune response. In contrast, none of the drugs completely abrogated the antigen specific IFN-γ response. AAV-microdystrophin treatment significantly reduced inflammation in both forelimbs and hindlimbs, and the addition of prednisolone and VBP6 further reduced muscle inflammation. Treatment with immunomodulatory drugs, except eplerenone, enhanced the beneficial effects of AAV-microdystrophin therapy in terms of force generation. CONCLUSIONS: Our data suggest that AAV-microdystrophin treatment results in anti-dystrophin antibody and T-cell responses, and immunomodulatory treatments have variable efficacy on these responses.

In Vitro Stimulation of Whole Milk Specimens: A Field-Friendly Method to Assess Milk Immune Activity.

BACKGROUND: The immune system of milk protects against infections and guides immune system development. A system-level understanding of milk immune activity is critical for research into infant infectious disease risk and lifelong health. RESEARCH AIM: To describe a protocol to characterize immune activity in human milk via in vitro stimulation for use in population-based (rather than clinical) research. METHODS: This study proceeded in two phases, each with a cross-sectional design. Human milk specimens were incubated for 24 hr at 37 °C in mammalian cell culture medium with stimuli (e.g., Salmonella enterica) in a CO2-enriched environment. Immune responses to stimuli were characterized as the change in cytokine: [stimulated]/[baseline]. Predictors of cytokine responses were evaluated with generalized linear models. RESULTS: Patterns were detectable across mother-child dyads: Interleukin-6 responses to stimuli were generally positively associated with child age and with maternal autoimmune disease. CONCLUSIONS: Our method allows characterization of pro-inflammatory milk immune activity in vitro in population-based (rather than clinical) research settings. In vitro activity has a system-level interpretation and is likely to be of broad utility in global health research in settings with high infectious disease risk, where understanding the immune system of milk is critical to understanding maternal and child health.

The contribution of mitochondrial dysfunction to a gene-environment model of Guamanian ALS and PD.

Scientific investigations of amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia (PD) of Guam have implicated genetic and environmental risk factors in their etiology. Using brain tissue, we investigated mitochondrial dysfunction and report a higher frequency of somatic mutations in the light strand promoter (LSP) of the mitochondrial control region in Guam ALS and PD patients than in Guam controls, along with the presence of inherited mutations that may contribute to a novel gene-environment interaction risk model. Along with other risk factors, they demonstrate both the importance and significance of genetic and environmental contributions to Guam ALS and PD etiology.

Lyme Disease Transmission Risk: Seasonal Variation in the Built Environment.

Seasonal variation in spatial distribution and pathogen prevalence of Borrelia burgdorferi in blacklegged ticks (Ixodes scapularis) influences human population risk of Lyme disease in peri-urban built environments. Parks, gardens, playgrounds, school campuses and neighborhoods represent a significant risk for Lyme disease transmission. From June 2012 through May 2014, ticks were collected using 1 m² corduroy cloths dragged over low-lying vegetation parallel to walkways with high human foot traffic. DNA was extracted from ticks, purified and presence of B. burgdorferi assessed by polymerase chain reaction amplification. Summer is reported as the time of highest risk for Lyme disease transmission in the United States and our results indicate a higher tick density of 26.0/1000 m² in summer vs. 0.2/1000 m² to 10.5/1000 m² in spring and fall. However, our findings suggest that tick infection rate is proportionally higher during the fall and spring than summer (30.0⁻54.7% in fall and 36.8⁻65.6% in spring vs. 20.0⁻28.2% in summer). Seasonal variation in infected tick density has significant implications for Lyme disease transmission as people are less likely to be aware of ticks in built environments, and unaware of increased infection in ticks in spring and fall. These factors may lead to more tick bites resulting in Lyme infection.

Model-based risk assessment and public health analysis to prevent Lyme disease.

The number of Lyme disease (LD) cases in the northeastern United States has been dramatically increasing with over 300 000 new cases each year. This is due to numerous factors interacting over time including low public awareness of LD, risk behaviours and clothing choices, ecological and climatic factors, an increase in rodents within ecologically fragmented peri-urban built environments and an increase in tick density and infectivity in such environments. We have used a system dynamics (SD) approach to develop a simulation tool to evaluate the significance of risk factors in replicating historical trends of LD cases, and to investigate the influence of different interventions, such as increasing awareness, controlling clothing risk and reducing mouse populations, in reducing LD risk. The model accurately replicates historical trends of LD cases. Among several interventions tested using the simulation model, increasing public awareness most significantly reduces the number of LD cases. This model provides recommendations for LD prevention, including further educational programmes to raise awareness and control behavioural risk. This model has the potential to be used by the public health community to assess the risk of exposure to LD.

Flashback to the 1960s: utility of archived sera to explore the origin and evolution of Plasmodium falciparum chloroquine resistance in the Pacific.

The increasing frequencies of Plasmodium falciparum strains that are resistant to chloroquine (CQ) and other antimalarials are resulting in a global resurgence of malaria morbidity and mortality. CQ resistance (CQR) is associated with multiple mutations in the P. falciparum chloroquine resistance transporter (pfcrt) gene. The mode and tempo of the accumulation of substitutions leading to these complex CQR haplotypes remain speculative due to the dearth of samples temporally spanning the evolution of drug resistance. The origin and evolution of the CQR alleles of Papua New Guinea (PNG) is particularly ambiguous. It remains unclear whether the pfcrt haplotype in PNG resulted from an independent origin of a CQR haplotype identical in sequence to the South American haplotype, or if this haplotype originated in South America and recombined into a Southeast Asian-derived genome. We sequenced a segment of pfcrt exon 2 from 398 plasmid clones derived from archival human sera collected in the Pacific before and after the first reported cases of CQ treatment failure (n=251) and modern samples (n=147). None of the 251 pfcrt plasmid clones from nine archival samples displayed the C72S or the K76T mutations that are characteristic of CQR strains. In contrast, these two amino acid substitutions were present in all 147 pfcrt plasmid clones from five samples collected between 2001 and 2003; thus, the archival samples represent the baseline parasite genetic diversity before the evolution of CQR strains. We are currently expanding our analyses to include additional samples from the series described here and from series collected in the 1970s and the 1980s to evaluate the geographic origin of CQR strains in the Pacific and the validity of the sequential point mutation accumulation model of CQR evolution.

Diversity of Plasmodium falciparum chloroquine resistance transporter (pfcrt) exon 2 haplotypes in the Pacific from 1959 to 1979.

Nearly one million deaths are attributed to malaria every year. Recent reports of multi-drug treatment failure of falciparum malaria underscore the need to understand the molecular basis of drug resistance. Multiple mutations in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) are involved in chloroquine resistance, but the evolution of complex haplotypes is not yet well understood. Using over 4,500 archival human serum specimens collected from 19 Pacific populations between 1959 and 1979, the period including and just prior to the appearance of chloroquine treatment failure in the Pacific, we PCR-amplified and sequenced a portion of the pfcrt exon 2 from 771 P. falciparum-infected individuals to explore the spatial and temporal variation in falciparum malaria prevalence and the evolution of chloroquine resistance. In the Pacific, the prevalence of P. falciparum varied considerably across ecological zones. On the island of New Guinea, the decreases in prevalence of P. falciparum in coastal, high-transmission areas over time were contrasted by the increase in prevalence during the same period in the highlands, where transmission was intermittent. We found 78 unique pfcrt haplotypes consisting of 34 amino acid substitutions and 28 synonymous mutations. More importantly, two pfcrt mutations (N75D and K76T) implicated in chloroquine resistance were present in parasites from New Hebrides (now Vanuatu) eight years before the first report of treatment failure. Our results also revealed unexpectedly high levels of genetic diversity in pfcrt exon 2 prior to the historical chloroquine resistance selective sweep, particularly in areas where disease burden was relatively low. In the Pacific, parasite genetic isolation, as well as host acquired immune status and genetic resistance to malaria, were important contributors to the evolution of chloroquine resistance in P. falciparum.

Ancient microbes from halite fluid inclusions: optimized surface sterilization and DNA extraction.

Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system.

An updated validation of Promega's PowerPlex 16 System: high throughput databasing under reduced PCR volume conditions on Applied Biosystem's 96 capillary 3730xl DNA Analyzer.

The PowerPlex 16 System from Promega Corporation allows single tube multiplex amplification of sixteen short tandem repeat (STR) loci including all 13 core combined DNA index system STRs. This report presents an updated validation of the PowerPlex 16 System on Applied Biosystem's 96 capillary 3730xl DNA Analyzer. The validation protocol developed in our laboratory allows for the analysis of 1536 loci (96 x 16) in c. 50 min. We have further optimized the assay by decreasing the reaction volume to one-quarter that recommended by the manufacturer thereby substantially reducing the total cost per sample without compromising reproducibility or specificity. This reduction in reaction volume has the ancillary benefit of dramatically increasing the sensitivity of the assay allowing for accurate analysis of lower quantities of DNA. Due to its substantially increased throughput capability, this extended validation of the PowerPlex 16 System should be useful in reducing the backlog of unanalyzed DNA samples currently facing public DNA forensic laboratories.