Widespread alternative splicing dysregulation occurs presymptomatically in CAG expansion spinocerebellar ataxias.

The spinocerebellar ataxias (SCAs) are a group of dominantly inherited neurodegenerative diseases, several of which are caused by CAG expansion mutations (SCAs 1, 2, 3, 6, 7 and 12) and more broadly belong to the large family of over 40 microsatellite expansion diseases. While dysregulation of alternative splicing is a well defined driver of disease pathogenesis across several microsatellite diseases, the contribution of alternative splicing in CAG expansion SCAs is poorly understood. Furthermore, despite extensive studies on differential gene expression, there remains a gap in our understanding of presymptomatic transcriptomic drivers of disease. We sought to address these knowledge gaps through a comprehensive study of 29 publicly available RNA-sequencing datasets. We identified that dysregulation of alternative splicing is widespread across CAG expansion mouse models of SCAs 1, 3 and 7. These changes were detected presymptomatically, persisted throughout disease progression, were repeat length-dependent, and were present in brain regions implicated in SCA pathogenesis including the cerebellum, pons and medulla. Across disease progression, changes in alternative splicing occurred in genes that function in pathways and processes known to be impaired in SCAs, such as ion channels, synaptic signalling, transcriptional regulation and the cytoskeleton. We validated several key alternative splicing events with known functional consequences, including Trpc3 exon 9 and Kcnma1 exon 23b, in the Atxn1154Q/2Q mouse model. Finally, we demonstrated that alternative splicing dysregulation is responsive to therapeutic intervention in CAG expansion SCAs with Atxn1 targeting antisense oligonucleotide rescuing key splicing events. Taken together, these data demonstrate that widespread presymptomatic dysregulation of alternative splicing in CAG expansion SCAs may contribute to disease onset, early neuronal dysfunction and may represent novel biomarkers across this devastating group of neurodegenerative disorders.

Tissue-specific expression of ribosomal protein paralogue eRpL22-like in Drosophila melanogaster eye development.

BACKGROUND: Differences in core or tissue-specific ribosomal protein (Rp) composition within ribosomes contribute to ribosome heterogeneity and functional variability. Yet, the degree to which ribosome heterogeneity modulates development is unknown. The Drosophila melanogaster eRpL22 family contains structurally diverse paralogues, eRpL22 and eRpL22-like. Unlike ubiquitously expressed eRpL22, eRpL22-like expression is tissue-specific, notably within the male germline and the eye. We investigated expression within the developing eye to uncover tissue/cell types where specific paralogue roles might be defined. RESULTS: Immunohistochemistry analysis confirms ubiquitous eRpL22 expression throughout eye development. In larvae, eRpL22-like is ubiquitously expressed, but highly enriched in the peripodial epithelium (PE). In early pupae, eRpL22-like is broadly distributed in multiple cell types, but later, is primarily enriched in interommatidial hair cells (IoHC). Adult patterns include the ring of accessory cells around ommatidia. Adult retinae IoHC patterning phenotypes (shown by scanning electron microscopy) may be linked to RNAi-mediated eRpL22-like depletion within larval PE. Immunoblots and polysome profile analyses show multiple variants of eRpL22-like across development, with the variant at the expected molecular mass co-sedimenting with active ribosomes. CONCLUSION: Our data reveal differential patterns of eRpL22-like expression relative to eRpL22 and suggest a specific role for eRpL22-like in developmental patterning of the eye.

Hydrogen sulfide: a target to modulate oxidative stress and neuroplasticity for the treatment of pathological anxiety.

Introduction: Anxiety disorders result inhigh patient burden and utilization of healthcare resources. Evidence-based treatments for pathological anxiety include targeted psychotherapy and use of serotonin-augmenting agents. Limitations in access to cognitive behavioral therapy and potential disadvantages to the use of psychotropics make the need for novel approaches to therapeutics for pathological anxiety salient.Areas Covered: Neuroplasticity mechanisms, as well as managing oxidative stress and inflammatory cellular allostatic loads can decrease anxiety. The gasotransmitter hydrogen sulfide (H2S) can impact these mechanisms through a) maintaining intracellular reduced glutathione in the CNS to decrease oxidative stress; b) facilitating neuroplasticity in amygdalar regions via the 2B subunit of n-methyl-d-aspartate (NMDA) receptors, in conjunction with the cAMP messenger system and a CNS kinase, PKC-γ; and c) regulating intracellular Ca2+ homeostasis in neurons and glial cells, among others.Expert Opinion: Given the mounting evidence for the role of H2S in neuronal health and its potential to decrease pathological anxiety, the current challenge in H2S therapeutics remains finding an efficient delivery system of this gasotransmitter in a reliable, safe and nontoxic form to engage in clinical trials. Current efforts include H2S-delivering moieties attached to known drugs, natural sulfide-releasing compounds such as garlic, and the regulation of dysfunctional breathing through breathing retraining.

Functional interplay between ribosomal protein paralogues in the eRpL22 family in Drosophila melanogaster.

Duplicated ribosomal protein (RP) genes in the Drosophila melanogaster eRpL22 family encode structurally-divergent and differentially-expressed rRNA-binding RPs. eRpL22 is expressed ubiquitously and eRpL22-like expression is tissue-restricted with highest levels in the adult male germline. We explored paralogue functional equivalence using the GAL4-UAS system for paralogue knockdown or overexpression and a conditional eRpL22-like knockout in a heat- shock flippase/FRT line. Ubiquitous eRpL22 knockdown with Actin-GAL4 resulted in embryonic lethality, confirming eRpL22 essentiality. eRpL22-like knockdown (60%) was insufficient to cause lethality; yet, conditional eRpL22-like knockout at one hour following egg deposition caused lethality within each developmental stage. Therefore, each paralogue is essential. Variation in timing of heat-shock-induced eRpL22-like knockout highlighted early embryogenesis as the critical period where eRpL22-like expression (not compensated for by eRpL22) is required for normal development of several organ systems, including testis development and subsequent sperm production. To determine if eRpL22-like can substitute for eRpL22, we used Actin-GAL4 for ubiquitous eRpL22 knockdown and eRpL22-like-FLAG (or FLAG-eRpL22: control) overexpression. Emergence of adults demonstrated that ubiquitous eRpL22-like-FLAG or FLAG-eRpL22 expression eliminates embryonic lethality resulting from eRpL22 depletion. Adults rescued by eRpL22-like-FLAG (but not by FLAG-eRpL22) overexpression had reduced fertility and longevity. We conclude that eRpL22 paralogue roles are not completely interchangeable and include functionally-diverse roles in development and spermatogenesis. Testis-specific paralogue knockdown revealed molecular phenotypes, including increases in eRpL22 protein and mRNA levels following eRpL22-like depletion, implicating a negative crosstalk mechanism regulating eRpL22 expression. Paralogue depletion unmasked mechanisms, yet to be defined that impact paralogue co-expression within germ cells.

Novel tools for genetic manipulation of follicle stem cells in the Drosophila ovary reveal an integrin-dependent transition from quiescence to proliferation.

In many tissues, the presence of stem cells is inferred by the capacity of the tissue to maintain homeostasis and undergo repair after injury. Isolation of self-renewing cells with the ability to generate the full array of cells within a given tissue strongly supports this idea, but the identification and genetic manipulation of individual stem cells within their niche remain a challenge. Here we present novel methods for marking and genetically altering epithelial follicle stem cells (FSCs) within the Drosophila ovary. Using these new tools, we define a sequential multistep process that comprises transitioning of FSCs from quiescence to proliferation. We further demonstrate that integrins are cell-autonomously required within FSCs to provide directional signals that are necessary at each step of this process. These methods may be used to define precise roles for specific genes in the sequential events that occur during FSC division after a period of quiescence.

Quantification of human growth hormone in serum with a labeled protein as an internal standard: essential considerations.

To manage and inform diagnostic or therapeutic decisions, measurement results which are accurate, specific, and comparable between laboratories are required. Two challenges associated with this are the definition of the measurand and the commutability of the reference standard used. Once the measurand is defined, the next step in improving standardization is developing traceable quantification methods for proteins in biological fluids. A novel reference method for the quantification of recombinant human growth hormone (rhGH) in serum has been developed using multistep sample cleanup at the protein level, tryptic digestion, and isotope dilution mass spectrometry (IDMS). Critical considerations for using isotopically labeled rhGH as the internal standard are described. A bulk serum sample was prepared at the clinically relevant level of 10 ng/g and quantified using the method described to give results traceable to the International System of Units (SI) with a total measurement uncertainty of <20%. Results compared favorably with an orthogonal traceable method using total tryptic digestion, peptide separation, and isotope dilution mass spectrometry.

The role of ion mobility spectrometry-mass spectrometry in the analysis of protein reference standards.

To achieve comparability of measurement results of protein amount of substance content between clinical laboratories, suitable reference materials are required. The impact on measurement comparability of potential differences in the tertiary and quaternary structure of protein reference standards is as yet not well understood. With the use of human growth hormone as a model protein, the potential of ion mobility spectrometry-mass spectrometry as a tool to assess differences in the structure of protein reference materials and their interactions with antibodies has been investigated here.

Towards absolute quantification of allergenic proteins in food--lysozyme in wine as a model system for metrologically traceable mass spectrometric methods and certified reference materials.

Current routine food allergen quantification methods, which are based on immunochemistry, offer high sensitivity but can suffer from issues of specificity and significant variability of results. MS approaches have been developed, but currently lack metrological traceability. A feasibility study on the application of metrologically traceable MS-based reference procedures was undertaken. A proof of concept involving proteolytic digestion and isotope dilution MS for quantification of protein allergens in a food matrix was undertaken using lysozyme in wine as a model system. A concentration of lysozyme in wine of 0.95 +/- 0.03 microg/g was calculated based on the concentrations of two peptides, confirming that this type of analysis is viable at allergenically meaningful concentrations. The challenges associated with this promising method were explored; these included peptide stability, chemical modification, enzymatic digestion, and sample cleanup. The method is suitable for the production of allergen in food certified reference materials, which together with the achieved understanding of the effects of sample preparation and of the matrix on the final results, will assist in addressing the bias of the techniques routinely used and improve measurement confidence. Confirmation of the feasibility of MS methods for absolute quantification of an allergenic protein in a food matrix with results traceable to the International System of Units is a step towards meaningful comparison of results for allergen proteins among laboratories. This approach will also underpin risk assessment and risk management of allergens in the food industry, and regulatory compliance of the use of thresholds or action levels when adopted.

Considering the advantages and pitfalls of the use of isotopically labeled protein standards for accurate protein quantification.

To ensure comparability of results in clinical proteomics, methods for accurate and traceable quantification of proteins are required. Typically this is done for recombinant proteins using isotopically labeled peptides as internal standards (IS). However, in order to perform quantification in complex matrices such as human serum, isotopically labeled protein standards have been suggested for use as IS to account for losses in sample preparation. The isotopic diluent must be chemically and physically identical to the analyte of interest, having the same amino acid sequence, post-translational modifications, secondary and tertiary structure. It must not be assumed but rather proven that the isotopic diluent is a true mimic, and here we consider both the advantages and potential pitfalls encountered when using isotopically labeled protein IS.

Analysis of the changes in the oxidation of brain tissue cytochrome-c-oxidase in traumatic brain injury patients during hypercapnoea: a broadband NIRS study.

Using broadband near-infrared spectroscopy (NIRS) and cerebral microdialysis (MD),we investigated cerebral cellular metabolism and mitochondrial redox states, following hypercapnoea in 6 patients with traumatic brain injury (TBI). In all patients hypercapnoea increased intracranial pressure and cerebral blood flow velocity measured with transcranial Doppler. Despite the likely increase in cerebral oxygen delivery, we did not see an increase in the oxidation status of cytochrome-c-oxidase [oxCCO] in every patient. Analysis of the NIRS data demonstrated two patterns of the changes; Group A (n = 4) showed an increase in [oxCCO] of 0.34(± 0.34)µM and Group B (n = 2) a decrease of 0.40(± 0.41)µM. Although no obvious association was seen between the Δ[oxCCO] and the MD, measured changes in lactate and pyruvate concentrations. Further work using model informed data interpretation may be helpful in understanding the multimodal signals acquired in this heterogeneous patient group.

Investigating microwave hydrolysis for the traceable quantification of peptide standards using gas chromatography-mass spectrometry.

Over the past decade, a number of endogenous peptides and endogenous peptide analogs have been employed in therapeutics and as diagnostic markers. The use of peptides as standards for the absolute quantification of proteins has become commonly accepted. Consequently, the requirement for standard peptides traceable to the International System of Units with low associated measurement uncertainty, and for accurate methods of peptide quantification, has increased. Here we describe a method of peptide quantification involving microwave-assisted acid hydrolysis followed by gas chromatography-mass spectrometry that enables traceable quantification of a peptide by exact matching isotope dilution mass spectrometry where the total hydrolysis time required is only 3h. A solution of angiotensin I was quantified using this method, and the results were in agreement with those obtained previously using an oven hydrolysis liquid chromatography-tandem mass spectrometry method.

Fully traceable absolute protein quantification of somatropin that allows independent comparison of somatropin standards.

BACKGROUND: Measurement traceability in clinical chemistry is required to standardize clinical results irrespective of the measurement procedure and laboratory. The traceability of many protein substances is maintained by reference to the first standard produced, which may no longer exist, with values assigned by consensus. Independent methods that provide traceability to the Système d'Unité International for all relevant properties of a protein standard could remove reliance on the original standard preparations. METHODS: We developed a method based on the traceable quantification of tryptic peptides released from the protein by isotope dilution mass spectrometry to compare 2 standard preparations of somatropin (recombinant human growth hormone), WHO 98/574 and Ph.Eur.CRS S0947000. Relative quantification using isotope-coded affinity tagging, isobaric tagging for relative and absolute quantification, and standard additions were also performed to validate the digestion method used and to determine whether any modifications were present. RESULTS: The total somatropin content in both materials was determined and an uncertainty estimation undertaken [WHO 2.19 +/- 0.21) mg/vial, European Pharmacopeia 2.06 +/- 0.21 mg/vial]. Each uncertainty in this paper is a fully estimated uncertainty, with 95% CI (k = 2). Isotope coded affinity tag and standard addition results fully validated the robustness of the digestion method used. In addition, iTRAQ (isobaric tagging for relative and absolute quantification analysis) identified 2 modifications, neither of which impacted the quantification. CONCLUSIONS: An independent method that does not rely on a preexisting protein standard has been developed and validated for the traceable value-assignment of total somatropin. The methods reported here address the amount of substance (mass fraction) of the standard materials but address neither biological activity nor other characteristics that may be important in assessing suitability for use as a calibrator.

Relationship between brain tissue haemodynamics, oxygenation and metabolism in the healthy human adult brain during hyperoxia and hypercapnea.

This study investigates the relationship between changes in brain tissue haemodynamics, oxygenation and oxidised cytochrome-c-oxidase ([oxCCO]) in the adult brain during hyperoxia and hypercapnea. 10 healthy volunteers were studied. We measured the mean blood flow velocity of the right middle cerebral artery (Vmca) with transcranial Doppler (TCD) and changes in concentrations of total haemoglobin ([HbT]=[HbO2]+[HHb]), haemoglobin difference ([Hbdiff]=[HbO2]-[HHb]) and [oxCCO] with broadband near-infrared spectroscopy (NIRS). We also measured the absolute tissue oxygenation index (TOI) using NIR spatially resolved spectroscopy. During hyperoxia there was an increase in TOI (2.33 +/- 0.29%), [Hbdiff] (4.57 +/- 1.27 microM) and in the oxidation of [oxCCO] (0.09 +/- 0.12 microM); but a reduction in Vmca (5.85 +/- 4.85%) and HbT (1.29 +/- 0.91 microM). During hyperoxia there was a positive correlation between [oxCCO] and TOI and [Hbdiff] (r=0.83 and r=0.95) and a negative association between [oxCCO] and Vmca and [HbT] (r=-0.74 and r=-0.87). During hypercapnea there was an increase in TOI (2.76 +/- 2.16%), [Hbdiff] (7.36 +/- 2.64), [HbT] (2.61 +/- 2.7 microM), Vmca (14.92 +/- 17.5%) and in the oxidation of [oxCCO] (0.25 +/- 0.17 microM). Correlation analysis shows that there was association between [oxCCO] and TOI, [Hbdiff] and [HbT] (r=0.83, r=0.93 and r=0.82) but not with Vmca (r=0.33). We conclude that an increase in [oxCCO] was seen during both challenges and it was highly associated with brain tissue oxygenation.

Estimating a modified Grubb's exponent in healthy human brains with near infrared spectroscopy and transcranial Doppler.

The relationship between cerebral blood volume (CBV) and flow (CBF) has been widely studied. One of the most significant early studies was by Grubb et al (1974 Stroke 5 630-9), who conducted hypercapnia studies in primates with positron emission tomography (PET) and empirically found CBV = 0.8 CBF(0.38). The exponent used here has since been known as the Grubb's exponent. In this paper, we define a similar exponent known as the modified Grubb's exponent, G', which is based on CBV and cerebral blood flow velocity (CBFV) estimated by near infrared spectroscopy (NIRS) and transcranial Doppler respectively, i.e. G' = log(CBV/CBV(0))/log(CBFV/CBFV(0)), where CBV(0) and CBFV(0) are baseline values. The aim of this study was to estimate the nominal value of the modified Grubb's exponent in healthy human brains. We conducted hypercapnia and hypocapnia studies on 14 healthy adult subjects. The correlation coefficient between log(CBV/CBV(0)) and log(CBFV/CBFV(0)) is 0.71 (p < 0.0001). We found a modified Grubb's exponent of 0.13 (the 95% confidence bounds are 0.10 and 0.17) which is expectedly lower than the conventional Grubb's exponents estimated by other techniques. The modified Grubb's exponent is a simple measure to quantify the hemodynamics between local CBV and global CBFV in the brain and as such may provide insight on brain physiology. Both NIRS and transcranial Doppler techniques are non-invasive and portable, facilitating future studies in other population groups such as brain-injured patients.

Protein quantification by isotope dilution mass spectrometry of proteolytic fragments: cleavage rate and accuracy.

The practice of quantifying proteins by peptide fragments from enzymatic proteolysis (digestion) was assessed regarding accuracy, reliability, and uncertainty of the results attainable. Purified recombinant growth hormone (rhGH, 22 kDa isoform) was used as a model analyte. Two tryptic peptides from hGH, T6 and T12, were chosen to determine the amount of the protein in the original sample. Reference solutions of T6 and T12 (isotopically labeled forms), value assigned by quantitative amino acid analysis (AAA) after complete hydrolysis, were used as internal standards. The accuracy of protein quantification by fragments T6 and T12 was evaluated by comparison of peptide results to those obtained for the same rhGH sample by AAA. The rate of cleavage (and thus the experimental protocol used) turned out to be crucial to the quality of results in protein quantification using enzymatic fragments. Applying a protocol customarily found in (qualitative) bottom-up proteomics gave results significantly higher than the target value from AAA (+11% with T6 and +6% with T12). In contrast, using a modified protocol optimized for fast and complete hydrolysis, results were unbiased within the limits of uncertainty, while the time needed for completion of proteolysis was considerably reduced (30 min as compared to 1080-1200 min). The method assessed highlighted three important criteria deemed necessary for successful protein quantification using proteolysis-based mass spectrometry methods. These are the following: the requirement for both the selected peptides and labeled internal standard to be stable throughout digestion; the correct purity assignment to the selected peptide standards; the proof of equimolar release of the selected peptides. The combined (overall) uncertainty for protein quantification was established by combination of estimates obtained for individual components and found to be U = 4% for this example. This uncertainty is of the same order as that typically attainable in quantification of "small" organic molecules using liquid chromatography/isotope dilution mass spectrometry.

Toward Système International d'Unité-traceable protein quantification: from amino acids to proteins.

Here we present a demonstration of the proof of principle that absolute concentration of a protein within a mixture of other proteins can be measured with SI traceability. The method used was based on tryptic digestion of a protein followed by quantification using double exact matching isotope dilution mass spectrometry (IDMS) of the peptides released. To provide full SI traceability to measurements of protein concentration we demonstrated a method of SI traceable peptide quantification in which the peptide standards used were quantified by an amino acid analysis method that incorporated double exact matching IDMS and amino acid standards of known purity. The concentration of the protein was therefore determined based upon the concentration of tryptic peptides, which in turn had been quantified based upon amino acid standards. This allowed fully SI-traceable measurements of protein concentration to be made. Important caveats in the implementation of this approach are also discussed and examples of how these can have detrimental effects on the measurements are shown.

Production and certification of four frozen human serum certified reference materials containing creatinine and electrolytes.

BACKGROUND: This paper describes the preparation, analysis and certification of four frozen human serum certified reference materials (CRMs) containing creatinine and the electrolytes calcium, lithium, magnesium, potassium and sodium. These materials have been prepared to give concentrations of these analytes that cover the currently accepted analytical range. METHODS: The analysis of the materials for certification purposes has been carried out using methodology traceable to primary standards, and which is acceptable as a reference method. The certification methods include liquid chromatography-mass spectrometry (LC-MS) with exact-matching isotope dilution calibration (EM-IDMS) for creatinine, inductively-coupled plasma optical emission spectroscopy (ICP-OES), ICP-MS and isotope-dilution inductively-coupled plasma mass spectroscopy (ID-ICP-MS) for the electrolytes. RESULTS: The uncertainties estimated for these certified values include a component from the characterization measurements, as well as contributions from possible inhomogeneity and long-term instability. The certified values have been corroborated by measurements obtained in a major UK External Quality Assessment scheme, which have, with the exception of the determination of creatinine at a particularly low concentration, given excellent agreement. CONCLUSIONS: The materials are intended for use by pathology laboratories and manufacturers of in vitro diagnostic (IVD) kits for validation of existing routine methodology to a traceable standard, which will promote harmonization between the different methods, instruments and IVD kits used in these laboratories.