Discovery of Alpha-1-Antichymotrypsin as a Marker of Delayed Recovery from Concussion in Children.

Of the four million children who experience a concussion each year, 30-50% of children will experience delayed recovery, where they will continue to experience symptoms more than two weeks after their injury. Delayed recovery from concussion encompasses emotional, behavioral, physical, and cognitive symptoms, and as such, there is an increased focus on developing an objective tool to determine risk of delayed recovery. This study aimed to identify a blood protein signature predictive of delayed recovery from concussion in children. Plasma samples were collected from children who presented to the Emergency Department at the Royal Children's Hospital, Melbourne, within 48h post-concussion. This study involved a discovery and validation phase. For the discovery phase, untargeted proteomics analysis was performed using single window acquisition of all theoretical mass spectra to identify blood proteins differentially abundant in samples from children with and without delayed recovery from concussion. A subset of these proteins was then validated in a separate participant cohort using multiple reaction monitoring and enzyme linked immunosorbent assay. A blood protein signature predictive of delayed recovery from concussion was modeled using a Support Vector Machine, a machine learning approach. In the discovery phase, 22 blood proteins were differentially abundant in age- and sex-matched samples from children with (n = 9) and without (n = 9) delayed recovery from concussion, six of whom were chosen for validation. In the validation phase, alpha-1-ACT was shown to be significantly lower in children with delayed recovery (n = 12) compared with those without delayed recovery (n = 28), those with orthopedic injuries (n = 7) and healthy controls (n = 33). A model consisting of alpha-1-ACT concentration stratified children based on recovery from concussion with an 0.88 area under the curve. We have identified that alpha-1-ACT differentiates between children at risk of delayed recovery from those without delayed recovery from concussion. To our knowledge, this is the first study to identify alpha-1-ACT as a potential marker of delayed recovery from concussion in children. Multi-site studies are required to further validate this finding before use in a clinical setting.

Edaravone and mitochondrial transfer as potential therapeutics for vanishing white matter disease astrocyte dysfunction.

INTRODUCTION: Previous research has suggested that vanishing white matter disease (VWMD) astrocytes fail to fully differentiate and respond differently to cellular stresses compared to healthy astrocytes. However, few studies have investigated potential VWMD therapeutics in monoculture patient-derived cell-based models. METHODS: To investigate the impact of alterations in astrocyte expression and function in VWMD, astrocytes were differentiated from patient and control induced pluripotent stem cells and analyzed by proteomics, pathway analysis, and functional assays, in the absence and presence of stressors or potential therapeutics. RESULTS: Vanishing white matter disease astrocytes demonstrated significantly reduced expression of astrocyte markers and markers of inflammatory activation or cellular stress relative to control astrocytes. These alterations were identified both in the presence and absence of polyinosinic:polycytidylic acid stimuli, which is used to simulate viral infections. Pathway analysis highlighted differential signaling in multiple pathways in VWMD astrocytes, including eukaryotic initiation factor 2 (EIF2) signaling, oxidative stress, oxidative phosphorylation (OXPHOS), mitochondrial function, the unfolded protein response (UPR), phagosome regulation, autophagy, ER stress, tricarboxylic acid cycle (TCA) cycle, glycolysis, tRNA signaling, and senescence pathways. Since oxidative stress and mitochondrial function were two of the key pathways affected, we investigated whether two independent therapeutic strategies could ameliorate astrocyte dysfunction: edaravone treatment and mitochondrial transfer. Edaravone treatment reduced differential VWMD protein expression of the UPR, phagosome regulation, ubiquitination, autophagy, ER stress, senescence, and TCA cycle pathways. Meanwhile, mitochondrial transfer decreased VWMD differential expression of the UPR, glycolysis, calcium transport, phagosome formation, and ER stress pathways, while further modulating EIF2 signaling, tRNA signaling, TCA cycle, and OXPHOS pathways. Mitochondrial transfer also increased the gene and protein expression of the astrocyte marker, glial fibrillary acidic protein (GFAP) in VWMD astrocytes. CONCLUSION: This study provides further insight into the etiology of VWMD astrocytic failure and suggests edaravone and mitochondrial transfer as potential candidate VWMD therapeutics that can ameliorate disease pathways in astrocytes related to oxidative stress, mitochondrial dysfunction, and proteostasis.

The muscle proteome reflects changes in mitochondrial function, cellular stress and proteolysis after 14 days of unilateral lower limb immobilization in active young men.

Skeletal muscle unloading due to joint immobilization induces muscle atrophy, which has primarily been attributed to reductions in protein synthesis in humans. However, no study has evaluated the skeletal muscle proteome response to limb immobilization using SWATH proteomic methods. This study characterized the shifts in individual muscle protein abundance and corresponding gene sets after 3 and 14 d of unilateral lower limb immobilization in otherwise healthy young men. Eighteen male participants (25.4 ±5.5 y, 81.2 ±11.6 kg) underwent 14 d of unilateral knee-brace immobilization with dietary provision and following four-weeks of training to standardise acute training history. Participant phenotype was characterized before and after 14 days of immobilization, and muscle biopsies were obtained from the vastus lateralis at baseline (pre-immobilization) and at 3 and 14 d of immobilization for analysis by SWATH-MS and subsequent gene-set enrichment analysis (GSEA). Immobilization reduced vastus group cross sectional area (-9.6 ±4.6%, P <0.0001), immobilized leg lean mass (-3.3 ±3.9%, P = 0.002), unilateral 3-repetition maximum leg press (-15.6 ±9.2%, P <0.0001), and maximal oxygen uptake (-2.9 ±5.2%, P = 0.044). SWATH analyses consistently identified 2281 proteins. Compared to baseline, two and 99 proteins were differentially expressed (FDR <0.05) after 3 and 14 d of immobilization, respectively. After 14 d of immobilization, 322 biological processes were different to baseline (FDR <0.05, P <0.001). Most (77%) biological processes were positively enriched and characterized by cellular stress, targeted proteolysis, and protein-DNA complex modifications. In contrast, mitochondrial organization and energy metabolism were negatively enriched processes. This study is the first to use data independent proteomics and GSEA to show that unilateral lower limb immobilization evokes mitochondrial dysfunction, cellular stress, and proteolysis. Through GSEA and network mapping, we identify 27 hub proteins as potential protein/gene candidates for further exploration.

Pathophysiological pathway differences in children who present with COVID-19 ARDS compared to COVID -19 induced MIS-C.

COVID-19 has infected more than 275 million worldwide (at the beginning of 2022). Children appear less susceptible to COVID-19 and present with milder symptoms. Cases of children with COVID-19 developing clinical features of Kawasaki-disease have been described. Here we utilise Mass Spectrometry proteomics to determine the plasma proteins expressed in healthy children pre-pandemic, children with multisystem inflammatory syndrome (MIS-C) and children with COVID-19 induced ARDS. Pathway analyses were performed to determine the affected pathways. 76 proteins are differentially expressed across the groups, with 85 and 52 proteins specific to MIS-C and COVID-19 ARDS, respectively. Complement and coagulation activation are implicated in these clinical phenotypes, however there was significant contribution of FcGR and BCR activation in MIS-C and scavenging of haem and retinoid metabolism in COVID-19 ARDS. We show global proteomic differences in MIS-C and COVID-ARDS, although both show complement and coagulation dysregulation. The results contribute to our understanding of MIS-C and COVID-19 ARDS in children.

Striated muscle activator of Rho signalling (STARS) overexpression in the mdx mouse enhances muscle functional capacity and regulates the actin cytoskeleton and oxidative phosphorylation pathways.

NEW FINDINGS: What is the central question of this study? Striated muscle activator of rho signalling (STARS) is an actin-binding protein that regulates transcriptional pathways controlling muscle function, growth and myogenesis, processes that are impaired in dystrophic muscle: what is the regulation of the STARS pathway in Duchenne muscular dystrophy (DMD)? What is the main finding and its importance? Members of the STARS signalling pathway are reduced in the quadriceps of patients with DMD and in mouse models of muscular dystrophy. Overexpression of STARS in the dystrophic deficient mdx mouse model increased maximal isometric specific force and upregulated members of the actin cytoskeleton and oxidative phosphorylation pathways. Regulating STARS may be a therapeutic approach to enhance muscle health. ABSTRACT: Duchenne muscular dystrophy (DMD) is characterised by impaired cytoskeleton organisation, cytosolic calcium handling, oxidative stress and mitochondrial dysfunction. This results in progressive muscle damage, wasting and weakness and premature death. The striated muscle activator of rho signalling (STARS) is an actin-binding protein that activates the myocardin-related transcription factor-A (MRTFA)/serum response factor (SRF) transcriptional pathway, a pathway regulating cytoskeletal structure and muscle function, growth and repair. We investigated the regulation of the STARS pathway in the quadriceps muscle from patients with DMD and in the tibialis anterior (TA) muscle from the dystrophin-deficient mdx and dko (utrophin and dystrophin null) mice. Protein levels of STARS, SRF and RHOA were reduced in patients with DMD. STARS, SRF and MRTFA mRNA levels were also decreased in DMD muscle, while Stars mRNA levels were decreased in the mdx mice and Srf and Mrtfa mRNAs decreased in the dko mice. Overexpressing human STARS (hSTARS) in the TA muscles of mdx mice increased maximal isometric specific force by 13% (P < 0.05). This was not associated with changes in muscle mass, fibre cross-sectional area, fibre type, centralised nuclei or collagen deposition. Proteomics screening followed by pathway enrichment analysis identified that hSTARS overexpression resulted in 31 upregulated and 22 downregulated proteins belonging to the actin cytoskeleton and oxidative phosphorylation pathways. These pathways are impaired in dystrophic muscle and regulate processes that are vital for muscle function. Increasing the STARS protein in dystrophic muscle improves muscle force production, potentially via synergistic regulation of cytoskeletal structure and energy production.

Caveolin-1 Ablation Imparts Partial Protection Against Inner Retinal Injury in Experimental Glaucoma and Reduces Apoptotic Activation.

Retinal ganglion cell degeneration is a characteristic feature of glaucoma, and accordingly, protection of these cells constitutes a major therapeutic objective in the disease. Here, we demonstrate the key influence of caveolin (Cav) in regulating the inner retinal homeostasis in two models of experimentally elevated intraocular pressure (IOP). Two groups of Cav-1-/- and wild-type mice were used in the study. Animals were subjected to experimentally induced chronic and acutely elevated IOP and any changes in their retinal function were assessed by positive scotopic threshold response recordings. TUNEL and cleaved caspase-3 assays were performed to evaluate apoptotic changes in the retina while Brn3a immunostaining was used as a marker to assess and quantify ganglion cell layer (GCL) changes. H&E staining was carried out on retinal sections to evaluate histological differences in retinal laminar structure. Cav-1 ablation partially protected the inner retinal function in both chronic and acute models of elevated IOP. The protective effects of Cav-1 loss were also evident histologically by reduced loss of GCL density in both models. The phenotypic protection in Cav-1-/- glaucoma mice paralleled with increased TrkB phosphorylation and reduced endoplasmic reticulum stress markers and apoptotic activation in the inner retinas. This study corroborated previous findings of enhanced Shp2 phosphorylation in a chronic glaucoma model and established a novel role of Cav-1 in mediating activation of this phosphatase in the inner retina in vivo. Collectively, these findings highlight the critical involvement of Cav-1 regulatory mechanisms in ganglion cells in response to increased IOP, implicating Cav-1 as a potential therapeutic target in glaucoma.

PGRMC1 phosphorylation affects cell shape, motility, glycolysis, mitochondrial form and function, and tumor growth.

BACKGROUND: Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in many cancer cells, where it is associated with detrimental patient outcomes. It contains phosphorylated tyrosines which evolutionarily preceded deuterostome gastrulation and tissue differentiation mechanisms. RESULTS: We demonstrate that manipulating PGRMC1 phosphorylation status in MIA PaCa-2 (MP) cells imposes broad pleiotropic effects. Relative to parental cells over-expressing hemagglutinin-tagged wild-type (WT) PGRMC1-HA, cells expressing a PGRMC1-HA-S57A/S181A double mutant (DM) exhibited reduced levels of proteins involved in energy metabolism and mitochondrial function, and altered glucose metabolism suggesting modulation of the Warburg effect. This was associated with increased PI3K/AKT activity, altered cell shape, actin cytoskeleton, motility, and mitochondrial properties. An S57A/Y180F/S181A triple mutant (TM) indicated the involvement of Y180 in PI3K/AKT activation. Mutation of Y180F strongly attenuated subcutaneous xenograft tumor growth in NOD-SCID gamma mice. Elsewhere we demonstrate altered metabolism, mutation incidence, and epigenetic status in these cells. CONCLUSIONS: Altogether, these results indicate that mutational manipulation of PGRMC1 phosphorylation status exerts broad pleiotropic effects relevant to cancer and other cell biology.

Potential early clinical stage colorectal cancer diagnosis using a proteomics blood test panel.

BACKGROUND: One of the most significant challenges in colorectal cancer (CRC) management is the use of compliant early stage population-based diagnostic tests as adjuncts to confirmatory colonoscopy. Despite the near curative nature of early clinical stage surgical resection, mortality remains unacceptably high-as the majority of patients diagnosed by faecal haemoglobin followed by colonoscopy occur at latter stages. Additionally, current population-based screens reliant on fecal occult blood test (FOBT) have low compliance (~ 40%) and tests suffer low sensitivities. Therefore, blood-based diagnostic tests offer survival benefits from their higher compliance (≥ 97%), if they can at least match the sensitivity and specificity of FOBTs. However, discovery of low abundance plasma biomarkers is difficult due to occupancy of a high percentage of proteomic discovery space by many high abundance plasma proteins (e.g., human serum albumin). METHODS: A combination of high abundance protein ultradepletion (e.g., MARS-14 and an in-house IgY depletion columns) strategies, extensive peptide fractionation methods (SCX, SAX, High pH and SEC) and SWATH-MS were utilized to uncover protein biomarkers from a cohort of 100 plasma samples (i.e., pools of 20 healthy and 20 stages I-IV CRC plasmas). The differentially expressed proteins were analyzed using ANOVA and pairwise t-tests (p < 0.05; fold-change > 1.5), and further examined with a neural network classification method using in silico augmented 5000 patient datasets. RESULTS: Ultradepletion combined with peptide fractionation allowed for the identification of a total of 513 plasma proteins, 8 of which had not been previously reported in human plasma (based on PeptideAtlas database). SWATH-MS analysis revealed 37 protein biomarker candidates that exhibited differential expression across CRC stages compared to healthy controls. Of those, 7 candidates (CST3, GPX3, CFD, MRC1, COMP, PON1 and ADAMDEC1) were validated using Western blotting and/or ELISA. The neural network classification narrowed down candidate biomarkers to 5 proteins (SAA2, APCS, APOA4, F2 and AMBP) that had maintained accuracy which could discern early (I/II) from late (III/IV) stage CRC. CONCLUSION: MS-based proteomics in combination with ultradepletion strategies have an immense potential of identifying diagnostic protein biosignature.

Practical Integration of Multi-Run iTRAQ Data.

In this chapter, we describe some of the approaches we employ in the analysis of iTRAQ data in our group, with an emphasis on practical issues that can occur in larger multi-run projects. Our pipeline starts with a well-established iTRAQ workflow, makes use of protein level quantitation using ProteinPilot, and continues either via a global analysis in the presence of a common reference, or by identifying pairwise comparisons of interest and applying a method taking the protein ratios and protein ratio confidence measures into consideration. Additionally we describe what issues can occur in the more subtle scenarios involving composite databases in multi-run situations, and an approach applicable in that setting.

Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints.

Cell growth and survival depend on a delicate balance between energy production and synthesis of metabolites. Here, we provide evidence that an alternative mitochondrial complex II (CII) assembly, designated as CIIlow, serves as a checkpoint for metabolite biosynthesis under bioenergetic stress, with cells suppressing their energy utilization by modulating DNA synthesis and cell cycle progression. Depletion of CIIlow leads to an imbalance in energy utilization and metabolite synthesis, as evidenced by recovery of the de novo pyrimidine pathway and unlocking cell cycle arrest from the S-phase. In vitro experiments are further corroborated by analysis of paraganglioma tissues from patients with sporadic, SDHA and SDHB mutations. These findings suggest that CIIlow is a core complex inside mitochondria that provides homeostatic control of cellular metabolism depending on the availability of energy.

Quantitative Age-specific Variability of Plasma Proteins in Healthy Neonates, Children and Adults.

Human blood plasma is a complex biological fluid containing soluble proteins, sugars, hormones, electrolytes, and dissolved gasses. As plasma interacts with a wide array of bodily systems, changes in protein expression, or the presence or absence of specific proteins are regularly used in the clinic as a molecular biomarker tool. A large body of literature exists detailing proteomic changes in pathologic contexts, however little research has been conducted on the quantitation of the plasma proteome in age-specific, healthy subjects, especially in pediatrics. In this study, we utilized SWATH-MS to identify and quantify proteins in the blood plasma of healthy neonates, infants under 1 year of age, children between 1-5 years, and adults. We identified more than 100 proteins that showed significant differential expression levels across these age groups, and we analyzed variation in protein expression across the age spectrum. The plasma proteomic profiles of neonates were strikingly dissimilar to the older children and adults. By extracting the SWATH data against a large human spectral library we increased protein identification more than 6-fold (940 proteins) and confirmed the concentrations of several of these using ELISA. The results of this study map the variation in expression of proteins and pathways often implicated in disease, and so have significant clinical implication.

Characterisation of the immune compounds in koala milk using a combined transcriptomic and proteomic approach.

Production of milk is a key characteristic of mammals, but the features of lactation vary greatly between monotreme, marsupial and eutherian mammals. Marsupials have a short gestation followed by a long lactation period, and milk constituents vary greatly across lactation. Marsupials are born immunologically naïve and rely on their mother's milk for immunological protection. Koalas (Phascolarctos cinereus) are an iconic Australian species that are increasingly threatened by disease. Here we use a mammary transcriptome, two milk proteomes and the koala genome to comprehensively characterise the protein components of koala milk across lactation, with a focus on immune constituents. The most abundant proteins were well-characterised milk proteins, including ß-lactoglobulin and lactotransferrin. In the mammary transcriptome, 851 immune transcripts were expressed, including immunoglobulins and complement components. We identified many abundant antimicrobial peptides, as well as novel proteins with potential antimicrobial roles. We discovered that marsupial VELP is an ortholog of eutherian Glycam1, and likely has an antimicrobial function in milk. We also identified highly-abundant koala endogenous-retrovirus sequences, identifying a potential transmission route from mother to young. Characterising the immune components of milk is key to understanding protection of marsupial young, and the novel immune compounds identified may have applications in clinical research.

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries.

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries.

Analytical performance of nano-LC-SRM using nondepleted human plasma over an 18-month period.

A standardized procedure for label-free nano-LC-SRM analysis of 32 high-medium abundance proteins from nondepleted human plasma was established and SRM data were acquired on 45 separate days for a control sample that was independently prepared on 39 distinct dates over an 18-month period (542 days). This case study enabled us to assess quantitative variance associated with nano-LC-SRM plasma analysis, mimicking experimental conditions that would be experienced with clinical trial biomarker studies. We assessed sample preparation variability attributed to different technicians and sample storage stability. Instrument performance varied over the 18-month period requiring ion path cleaning, so we assessed the impact of declining performance on specific peptide ion sensitivity and evaluated how various data normalization strategies could compensate for these changes. Our analysis demonstrated that while sample preparation was the main contributor for data variances when MS data were acquired within days, variability in SRM sensitivity was a far greater source of variance when data were acquired over a long period. The overall median multiplexed assay CV was 13% over the 18-month period. This case study is illustrative of large-scale plasma biomarker studies using nano-LC-SRM over extended periods and highlights aspects of bioanalysis that require careful attention to ensure reliable quantitation.

Bio-orthogonal labeling as a tool to visualize and identify newly synthesized proteins in Caenorhabditis elegans.

In this protocol we describe the incorporation of bio-orthogonal amino acids as a versatile method for visualizing and identifying de novo-synthesized proteins in the roundworm Caenorhabditis elegans. This protocol contains directions on implementing three complementary types of analysis: 'click chemistry' followed by western blotting, click chemistry followed by immunofluorescence, and isobaric tags for relative and absolute quantification (iTRAQ) quantitative mass spectrometry. The detailed instructions provided herein enable researchers to investigate the de novo proteome, an analysis that is complicated by the fact that protein molecules are chemically identical to each other, regardless of the timing of their synthesis. Our protocol circumvents this limitation by identifying de novo-synthesized proteins via the incorporation of the chemically modifiable azidohomoalanine instead of the natural amino acid methionine in the nascent protein, followed by facilitating the visualization of the resulting labeled proteins in situ. It will therefore be an ideal tool for studying de novo protein synthesis in physiological and pathological processes including learning and memory. The protocol requires 10 d for worm growth, liquid culture and synchronization; 1-2 d for bio-orthogonal labeling; and, with regard to analysis, 3-4 d for western blotting, 5-6 d for immunofluorescence or ~3 weeks for mass spectrometry.

Altered proteostasis in aging and heat shock response in C. elegans revealed by analysis of the global and de novo synthesized proteome.

Protein misfolding and aggregation as a consequence of impaired protein homeostasis (proteostasis) not only characterizes numerous age-related diseases but also the aging process itself. Functionally related to the aging process are, among others, ribosomal proteins, suggesting an intimate link between proteostasis and aging. We determined by iTRAQ quantitative proteomic analysis in C. elegans how the proteome changes with age and in response to heat shock. Levels of ribosomal proteins and mitochondrial chaperones were decreased in aged animals, supporting the notion that proteostasis is altered during aging. Mitochondrial enzymes of the tricarboxylic acid cycle and the electron transport chain were also reduced, consistent with an age-associated energy impairment. Moreover, we observed an age-associated decline in the heat shock response. In order to determine how protein synthesis is altered in aging and in response to heat shock, we complemented our global analysis by determining the de novo proteome. For that, we established a novel method that enables both the visualization and identification of de novo synthesized proteins, by incorporating the non-canonical methionine analogue, azidohomoalanine (AHA), into the nascent polypeptides, followed by reacting the azide group of AHA by 'click chemistry' with an alkyne-labeled tag. Our analysis of AHA-tagged peptides demonstrated that the decreased abundance of, for example, ribosomal proteins in aged animals is not solely due to degradation but also reflects a relative decrease in their synthesis. Interestingly, although the net rate of protein synthesis is reduced in aged animals, our analyses indicate that the synthesis of certain proteins such as the vitellogenins increases with age.