In silico and experimental analysis of the repeated domains in BvaP, a protein important for GBS vaginal colonization.

Streptococcus agalactiae (group B strep, GBS) infections in neonates are often fatal and strongly associated with maternal GBS vaginal colonization. Previously, we highlighted the importance of a formerly uncharacterized protein, BvaP, in GBS vaginal colonization. BvaP is highly conserved across GBS and is made up of repeated domains, with a variable number of repeats between strains. Here, we evaluate the prevalence of BvaP repeated domains and their relevance in phenotypes previously associated with vaginal colonization. Using in silico analysis, we found that the number of repeats in the BvaP protein does not generally appear to be associated with serotype, isolation site, or host. Using BvaP truncations in GBS strain A909, we determined that a smaller number of repeats was correlated with decreased bacterial chain length, but adherence to vaginal epithelial cells was complemented using BvaP containing one, two, three, or five repeats. Future research will be geared toward understanding the host immune response to BvaP in vivo and whether vaginal carriage or host response is dependent on the BvaP repeated domains.

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.

Temporal Proteomic Profiling During Differentiation of Normal and Dystrophin-Deficient Human Muscle Cells.

BACKGROUND: Myogenesis is a dynamic process involving temporal changes in the expression of many genes. Lack of dystrophin protein such as in Duchenne muscular dystrophy might alter the natural course of gene expression dynamics during myogenesis. OBJECTIVE: To gain insight into the dynamic temporal changes in protein expression during differentiation of normal and dystrophin deficient myoblasts to myotubes. METHOD: A super SILAC spike-in strategy in combination and LC-MS/MS was used for temporal proteome profiling of normal and dystrophin deficient myoblasts during differentiation. The acquired data was analyzed using Proteome Discoverer 2.2. and data clustering using R to define significant temporal changes in protein expression. RESULTS: sFour major temporal protein clusters that showed sequential dynamic expression profiles during myogenesis of normal myoblasts were identified. Clusters 1 and 2, consisting mainly of proteins involved mRNA splicing and processing expression, were elevated at days 0 and 0.5 of differentiation then gradually decreased by day 7 of differentiation, then remained lower thereafter. Cluster 3 consisted of proteins involved contractile muscle and actomyosin organization. They increased in their expression reaching maximum at day 7 of differentiation then stabilized thereafter. Cluster 4 consisting of proteins involved in skeletal muscle development glucogenesis and extracellular remodeling had a lower expression during myoblast stage then gradually increased in their expression to reach a maximum at days 11-15 of differentiation. Lack of dystrophin expression in DMD muscle myoblast caused major alteration in temporal expression of proteins involved in cell adhesion, cytoskeleton, and organelle organization as well as the ubiquitination machinery. CONCLUSION: Time series proteome profiling using super SILAC strategy is a powerful method to assess temporal changes in protein expression during myogenesis and to define the downstream consequences of lack of dystrophin on these temporal protein expressions. Key alterations were identified in dystrophin deficient myoblast differentiation compared to normal myoblasts. These alterations could be an attractive therapeutic target.

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.

Correction: Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy.

[This corrects the article DOI: 10.1371/journal.pone.0228072.].

Creation and characterization of an immortalized canine myoblast cell line: Myok9.

The availability of an in vitro canine cell line would reduce the need for dogs for primary in vitro cell culture and reduce overall cost in pre-clinical studies. An immortalized canine muscle cell line, named Myok9, from primary myoblasts of a normal dog has been developed by the authors. Immortalization was performed by SV40 viral transfection of the large T antigen into the primary muscle cells. Proliferation assays, growth curves, quantitative PCR, western blotting, mass spectrometry, and light microscopy were performed to characterize the MyoK9 cell line at different stages of growth and differentiation. The expression of muscle-related genes was determined to assess myogenic origin. Myok9 cells expressed dystrophin and other muscle-specific proteins during differentiation, as detected with mass spectrometry and western blotting. Using the Myok9 cell line, new therapies before moving to pre-clinical studies to enhance the number and speed of analyses and reduce the cost of early experimentation can be tested now. This cell line will be made available to the research community to further evaluate potential therapeutics.

Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.

Absolute quantification of dystrophin protein in human muscle biopsies using parallel reaction monitoring (PRM).

The need for a reliable and accurate method to quantify dystrophin proteins in human skeletal muscle biopsies has become crucial in order to assess the efficacy of dystrophin replacement therapies in Duchenne muscular dystrophy as well as to gain insight into the relationship between dystrophin levels and disease severity in Becker's muscular dystrophy. Current methods to measure dystrophin such as western blot and immunofluorescence, while straightforward and simple, lack precision and sometimes specificity. Here, we standardized a targeted mass spectrometry method to determine the absolute amount of dystrophin in ng/mg of muscle using full-length 13 C6-Arg- and 13 C6,15 N2-Lys-labeled dystrophin and parallel reaction monitoring (PRM). The method was found to be reproducible with a limit of quantification as low as 30 pg of dystrophin protein per mg of total muscle proteins. The method was then tested to measure levels of dystrophin in muscle biopsies from a healthy donor and from Duchenne and Becker's muscular dystrophy patients.