Tissue-specific Protein Research Articles

Serum keratin 19 (CYFRA21-1) links ductular reaction with portal hypertension and outcome of various advanced liver diseases

BackgroundKeratins (Ks) represent tissue-specific proteins. K18 is produced in hepatocytes while K19, the most widely used ductular reaction (DR) marker, is found in cholangiocytes and hepatic progenitor cells. K18-based serum fragments are commonly used liver disease predictors, while K19-based serum fragments detected through CYFRA21-1 are established tumor but not liver disease markers yet. Since DR reflects the severity of the underlying liver disease, we systematically evaluated the usefulness of CYFRA21-1 in different liver disease severities and etiologies.MethodsHepatic expression of ductular keratins (K7/K19/K23) was analyzed in 57 patients with chronic liver disease (cohort i). Serum CYFRA21-1 levels were measured in 333 Austrians with advanced chronic liver disease (ACLD) of various etiologies undergoing hepatic venous pressure gradient (HVPG) measurement (cohort ii), 231 French patients with alcoholic cirrhosis (cohort iii), and 280 hospitalized Germans with decompensated cirrhosis of various etiologies (cohort iv).Results(i) Hepatic K19 levels were comparable among F0–F3 fibrosis stages, but increased in cirrhosis. Hepatic K19 mRNA strongly correlated with the levels of other DR-specific keratins. (ii) In ACLD, increased serum CYFRA21-1 associated with the presence of clinically significant portal hypertension (CSPH; HVPG ≥ 10 mmHg) (OR = 5.87 [2.95–11.68]) and mortality (HR = 3.02 [1.78–5.13]; median follow-up 22 months). (iii) In alcoholic cirrhosis, elevated serum CYFRA21-1 indicated increased risk of death/liver transplantation (HR = 2.59 [1.64–4.09]) and of HCC (HR = 1.74 [1.02–2.96]) over the long term (median follow-up 73 months). (iv) In decompensated cirrhosis, higher serum CYFRA21-1 predicted 90-day mortality (HR = 2.97 [1.92–4.60]) with a moderate accuracy (AUROC 0.64), independently from established prognostic scores.ConclusionsHepatic K19 mRNA and serum CYFRA21-1 levels rise in cirrhosis. Increased CYFRA21-1 levels associate with the presence of CSPH and reliably indicate mortality in the short and long term independently of conventional liver biochemistry markers or scoring systems. Hence, the widely available serum CYFRA21-1 constitutes a novel, DR-related marker with prognostic implications in patients with different settings of advanced liver disease.

Epigenetics of Muscle- and Brain-Specific Expression of KLHL Family Genes.

KLHL and the related KBTBD genes encode components of the Cullin-E3 ubiquitin ligase complex and typically target tissue-specific proteins for degradation, thereby affecting differentiation, homeostasis, metabolism, cell signaling, and the oxidative stress response. Despite their importance in cell function and disease (especially, KLHL40, KLHL41, KBTBD13, KEAP1, and ENC1), previous studies of epigenetic factors that affect transcription were predominantly limited to promoter DNA methylation. Using diverse tissue and cell culture whole-genome profiles, we examined 17 KLHL or KBTBD genes preferentially expressed in skeletal muscle or brain to identify tissue-specific enhancer and promoter chromatin, open chromatin (DNaseI hypersensitivity), and DNA hypomethylation. Sixteen of the 17 genes displayed muscle- or brain-specific enhancer chromatin in their gene bodies, and most exhibited specific intergenic enhancer chromatin as well. Seven genes were embedded in super-enhancers (particularly strong, tissue-specific clusters of enhancers). The enhancer chromatin regions typically displayed foci of DNA hypomethylation at peaks of open chromatin. In addition, we found evidence for an intragenic enhancer in one gene upregulating expression of its neighboring gene, specifically for KLHL40/HHATL and KLHL38/FBXO32 gene pairs. Many KLHL/KBTBD genes had tissue-specific promoter chromatin at their 5′ ends, but surprisingly, two (KBTBD11 and KLHL31) had constitutively unmethylated promoter chromatin in their 3′ exons that overlaps a retrotransposed KLHL gene. Our findings demonstrate the importance of expanding epigenetic analyses beyond the 5′ ends of genes in studies of normal and abnormal gene regulation.

Genetic markers for hypertension – a genetic epidemiological study of 5000 Romanian individuals

Abstract Background During the past decades, genetic research has reached new heights as next generation sequencing has rapidly taken over and genome-wide association studies (GWAS) have broug. The purpose of the research is to determin high-risk variants (Single-Nucleotide-Variants,SNVs) associated with hypertension (HTA) in the Romanian population. The current presentation asseses the final results of a 3 part study comprising the first and the largest GWAS on hypertension in Romanians. Material Methods The total cohort includes a number of 5690 individuals, of which 2190 with hypertension and 3500 heathy controls. Genetic testing was performed at in Iceland. A multiple GWAS assay has been performed for the identification of variants associated with hypertension, hypertension risk factors and hypertension comorbidities. Results Environmental (lifestyle) risk factors, such as smoking, alcohol consumption and coffee consumption, and also pathological risk factors, as are obesity and ageing, were analyzed in association with hypertension. Tissue-specific protein expression, gene function and gene-gene interactions have been analyzed for assessing a possible biological explanation of the association between the identified related variants and HTA. Expression quantitative trait loci (eQTL) were assessed for variants in the reported locations for a better understanding of their involvement in HTA. The results of the analysis revealed a number of over 5000 genetic variants statistically correlated with hypertension in the studied cohort, some well documented and in genes known to be involved in hypertension pathophysiology (clusters on chromosomes 1p36, 1q24, 3q24, 4p16, 5q12, 7q36, 12p12, 15q, 17q, 20q12, a.o. or CRNKL1, C19Orf12, CCDC51, C20Orf26, ZNF420, ZNF571, a.o. intragenic variants). Approx. 4100 SNVs were identified in correlation with diabetes mellitus and obesity. Variants correlated with both hypertension and DM were identified (TBX20,ANK2, a.o. genes). Two other variant clusters (p=10–4–10–3) on chromosomes 19 (19q12) and 20 (20p11.21) revealed statistical correlations with both hypertension and obesity. Conclusions The present study found some important loci and clusters associated with HTA, which migh provide insights into the genetic architecture of this pathology. The validity of these results for the Romanian population need to be confirmed by replication studies. The current research is part of the EU ProMark, ROMCAN and AppGenEdu projects. Funding Acknowledgement Type of funding source: None

Pathophysiological mechanisms of neurological disorders in experimental animals exposed to vibration

The review presents an analysis of literature on the study of nervous system changes in experimental animals exposed to vibration. The hypoxic type of cellular metabolism against the background of vibration is the result of vascular mechanoreceptors stimulation and spasm of blood vessels as well as intravascular pressure phase fluctuations and impaired blood and lymph outflow. Hemodynamic disorders and microangiopathy in the central nervous system can reach the level of cerebral vascular impairment and capillary-trophic insufficiency of the brain. Changes in calcium homeostasis at the cellular and tissue levels is a key mechanism of neuronal destruction occurring alongside with impaired blood supply to the nervous tissue in patients with vibrational disease. Long-term exposure to vibration results in reduced tissue respiration in the brain structures of experimental animals, that is most pronounced in the cortex and leads to a pathological change of spontaneous electrical activity of brain structures. Acute vibration stress stimulated the synthesis and excretion of serotonin not only in the hippocampus and hypothalamus but also in the cerebellum, which explains the disturbances of the oculomotor reactions observed in vibration exposure in the control system of the vertical vestibular ocular reflex. A decrease in the total number of neurons as well as an increase in the number of astroglia cells against the background of paravascular tissue edema were revealed against the background of changes in neurotransmitters levels. An increase in the concentration of the biomarker of structural and functional damage to brain tissue specific protein S-100B accompanies the development of professional sensorineural hearing loss and dysfunction of cerebral level vegetative regulation.

An analysis of tissue-specific alternative splicing at the protein level.

The role of alternative splicing is one of the great unanswered questions in cellular biology. There is strong evidence for alternative splicing at the transcript level, and transcriptomics experiments show that many splice events are tissue specific. It has been suggested that alternative splicing evolved in order to remodel tissue-specific protein-protein networks. Here we investigated the evidence for tissue-specific splicing among splice isoforms detected in a large-scale proteomics analysis. Although the data supporting alternative splicing is limited at the protein level, clear patterns emerged among the small numbers of alternative splice events that we could detect in the proteomics data. More than a third of these splice events were tissue-specific and most were ancient: over 95% of splice events that were tissue-specific in both proteomics and RNAseq analyses evolved prior to the ancestors of lobe-finned fish, at least 400 million years ago. By way of contrast, three in four alternative exons in the human gene set arose in the primate lineage, so our results cannot be extrapolated to the whole genome. Tissue-specific alternative protein forms in the proteomics analysis were particularly abundant in nervous and muscle tissues and their genes had roles related to the cytoskeleton and either the structure of muscle fibres or cell-cell connections. Our results suggest that this conserved tissue-specific alternative splicing may have played a role in the development of the vertebrate brain and heart.

Tissue-specific small heat shock protein 20 activation is not associated with traditional autophagy markers in Ossabaw swine with cardiometabolic heart failure.

The small heat shock protein 20 (HSPB6) emerges as a potential upstream mediator of autophagy. Although autophagy is linked to several clinical disorders, how HSPB6 and autophagy are regulated in the setting of heart failure (HF) remains unknown. The goal of this study was to assess the activation of the HSPB6 and its association with other well-established autophagy markers in central and peripheral tissues from a preclinical Ossabaw swine model of cardiometabolic HF induced by Western diet and chronic cardiac pressure overload. We hypothesized HSPB6 would be activated in central and peripheral tissues, stimulating autophagy. We found that autophagy in the heart is interrupted at various stages of the process in a chamber-specific manner. Protein levels of HSPB6, Beclin 1, and p62 are increased in the right ventricle, whereas only HSPB6 was increased in the left ventricle. Unlike the heart, samples from the triceps brachii long head showed only an increase in the protein level of p62, highlighting interesting central versus peripheral differences in autophagy regulation. In the right coronary artery, total HSPB6 protein expression was decreased and associated with an increase in LC3B-II/LC3B-I ratio, demonstrating a different mechanism of autophagy dysregulation in the coronary vasculature. Thus, contrary to our hypothesis, activation of HSPB6 was differentially regulated in a tissue-specific manner and observed in parallel with variable states of autophagy markers assessed by protein levels of LC3B, p62, and Beclin 1. Our data provide insight into how the HSPB6/autophagy axis is regulated in a preclinical swine model with potential relevance to heart failure with preserved ejection fraction.NEW & NOTEWORTHY Our study shows that the activation of HSPB6 is tissue specific and associated with variable states of downstream markers of autophagy in a unique preclinical swine model of cardiometabolic HF with potential relevance to HFpEF. These findings suggest that targeted approaches could be an important consideration regarding the development of drugs aimed at this intracellular recycling process.

A Quantitative Proteome Map of the Human Body

Determining protein levels in each tissue and how they compare with RNA levels is important for understanding human biology and disease as well as regulatory processes that control protein levels. We quantified the relative protein levels from over 12,000 genes across 32 normal human tissues. Tissue-specific or tissue-enriched proteins were identified and compared to transcriptome data. Many ubiquitous transcripts are found to encode tissue-specific proteins. Discordance of RNA and protein enrichment revealed potential sites of synthesis and action of secreted proteins. The tissue-specific distribution of proteins also provides an in-depth view of complex biological events that require the interplay of multiple tissues. Most importantly, our study demonstrated that protein tissue-enrichment information can explain phenotypes of genetic diseases, which cannot be obtained by transcript information alone. Overall, our results demonstrate how understanding protein levels can provide insights into regulation, secretome, metabolism, and human diseases.

Understanding virtual lead optimization through a simple online task.

Understanding virtual lead optimization through a simple online task.

Tissue- and isoform-specific protein complex analysis with natively processed bait proteins

Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. To date, the analysis of tissue- or even cell- or compartment-specific protein interactions is still relying on the availability of specific antibodies suited for immunoprecipitation. Here, we aimed at establishing a method that allows identification of protein interactions and complexes from intact tissues independent of specific, high affinity antibodies used for protein pull-down and isolation. Tagged bait proteins were expressed in human HEK293T cells and residual interactors removed by SDS. The resulting tag-fusion protein was then used as bait to pull proteins from tissue samples. Tissue-specific interactions were reproducibly identified from porcine retina as well as from retinal pigment epithelium using the ciliary protein lebercilin as bait. Further, murine heart-specific interactors of two gene products of the 3′,5′-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 (cGK1) were investigated. Here, specific interactions were associated with the cGK1α and β gene products, that differ only in their unique amino-terminal region comprising about 100 aa. As such, the new protocol provides a fast and reliable method for tissue-specific protein complex analysis which is independent of the availability or suitability of antibodies for immunoprecipitation. SignificanceProtein-protein interaction in the functional relevant tissue is still difficult due to the dependence on specific antibodies or bait production in bacteria or insect cells. Here, the tagged protein of interest is produced in a human cell line and bound proteins are gently removed using SDS. Because applying the suitable SDS concentration is a critical step, different SDS solutions were tested to demonstrate their influence on interactions and the clean-up process. The established protocol enabled a tissue-specific analysis of the ciliary proteins lebercilin and TMEM107 using pig eyes. In addition, two gene products of the 3′,5′-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 showed distinct protein interactions in mouse heart tissue. With the easy, fast and cheap protocol presented here, deep insights in tissue-specific and functional relevant protein complex formation is possible.

Tissue-targeted R-spondin mimetics for liver regeneration

R-spondin (RSPO) proteins amplify Wnt signaling and stimulate regeneration in a variety of tissues. To repair tissue in a tissue-specific manner, tissue-targeted RSPO mimetic molecules are desired. Here, we mutated RSPO (RSPO2 F105R/F109A) to eliminate LGR binding while preserving ZNRF3/RNF43 binding and targeted the mutated RSPO to a liver specific receptor, ASGR1. The resulting bi-specific molecule (αASGR1-RSPO2-RA) enhanced Wnt signaling effectively in vitro, and its activity was limited to ASGR1 expressing cells. Systemic administration of αASGR1-RSPO2-RA in mice specifically upregulated Wnt target genes and stimulated cell proliferation in liver but not intestine (which is more responsive to non-targeted RSPO2) in healthy mice, and improved liver function in diseased mice. These results not only suggest that a tissue-specific RSPO mimetic protein can stimulate regeneration in a cell-specific manner, but also provide a blueprint of how a tissue-specific molecule might be constructed for applications in a broader context.

A multilayered scaffold for regeneration of smooth muscle and connective tissue layers

Tissue regeneration often requires recruitment of different cell types and rebuilding of two or more tissue layers to restore function. Here, we describe the creation of a novel multilayered scaffold with distinct fiber organizations-aligned to unaligned and dense to porous-to template common architectures found in adjacent tissue layers. Electrospun scaffolds were fabricated using a biodegradable, tyrosine-derived terpolymer, yielding densely-packed, aligned fibers that transition into randomly-oriented fibers of increasing diameter and porosity. We demonstrate that differently-oriented scaffold fibers direct cell and extracellular matrix (ECM) organization, and that scaffold fibers and ECM protein networks are maintained after decellularization. Smooth muscle and connective tissue layers are frequently adjacent in vivo; we show that within a single scaffold, the architecture supports alignment of contractile smooth muscle cells and deposition by fibroblasts of a meshwork of ECM fibrils. We rolled a flat scaffold into a tubular construct and, after culture, showed cell viability, orientation, and tissue-specific protein expression in the tube were similar to the flat-sheet scaffold. This scaffold design not only has translational potential for reparation of flat and tubular tissue layers but can also be customized for alternative applications by introducing two or more cell types in different combinations.

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

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. 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. 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.

Exploring Drug Treatment Patterns Based on the Action of Drug and Multilayer Network Model.

Some drugs can be used to treat multiple diseases, suggesting potential patterns in drug treatment. Determination of drug treatment patterns can improve our understanding of the mechanisms of drug action, enabling drug repurposing. A drug can be associated with a multilayer tissue-specific protein–protein interaction (TSPPI) network for the diseases it is used to treat. Proteins usually interact with other proteins to achieve functions that cause diseases. Hence, studying drug treatment patterns is similar to studying common module structures in multilayer TSPPI networks. Therefore, we propose a network-based model to study the treatment patterns of drugs. The method was designated SDTP (studying drug treatment pattern) and was based on drug effects and a multilayer network model. To demonstrate the application of the SDTP method, we focused on analysis of trichostatin A (TSA) in leukemia, breast cancer, and prostate cancer. We constructed a TSPPI multilayer network and obtained candidate drug-target modules from the network. Gene ontology analysis provided insights into the significance of the drug-target modules and co-expression networks. Finally, two modules were obtained as potential treatment patterns for TSA. Through analysis of the significance, composition, and functions of the selected drug-target modules, we validated the feasibility and rationality of our proposed SDTP method for identifying drug treatment patterns. In summary, our novel approach used a multilayer network model to overcome the shortcomings of single-layer networks and combined the network with information on drug activity. Based on the discovered drug treatment patterns, we can predict the potential diseases that the drug can treat. That is, if a disease-related protein module has a similar structure, then the drug is likely to be a potential drug for the treatment of the disease.

Innovative Therapeutic and Delivery Approaches Using Nanotechnology to Correct Splicing Defects Underlying Disease.

Alternative splicing of pre-mRNA contributes strongly to the diversity of cell- and tissue-specific protein expression patterns. Global transcriptome analyses have suggested that >90% of human multiexon genes are alternatively spliced. Alterations in the splicing process cause missplicing events that lead to genetic diseases and pathologies, including various neurological disorders, cancers, and muscular dystrophies. In recent decades, research has helped to elucidate the mechanisms regulating alternative splicing and, in some cases, to reveal how dysregulation of these mechanisms leads to disease. The resulting knowledge has enabled the design of novel therapeutic strategies for correction of splicing-derived pathologies. In this review, we focus primarily on therapeutic approaches targeting splicing, and we highlight nanotechnology-based gene delivery applications that address the challenges and barriers facing nucleic acid-based therapeutics.

Upregulation of miR-133a by adiponectin inhibits pyroptosis pathway and rescues acute aortic dissection.

Acute aortic dissection (AAD) is a cardiovascular emergency caused by the formation of hematoma in the middle layer of the aortic wall. Adiponectin (APN) is an adipose tissue-specific protein that has anti-inflammation and anti-atherosclerosis functions. Pyroptosis, as an inflammatory cell death, depends on the activation of caspase1, while nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) is a typical representative of the pyroptosis pathway. In this study, we aimed to find whether APN affects the AAD process. The results showed that APN overexpression (OE) inhibited the AAD development and the levels of glucose, triglyceride, and total cholesterol in mice model. In addition, APN OE inhibited the productions of gasdermin D (GSDMD), NLRP3, caspase1, interleukin-1β (IL-1β), IL-18, and osteopontin (OPN), as well as α-smooth muscle actin (α-SMA) downregulation in vitro and in vivo. In addition, NLRP3 was found to be a target gene of miR-133a and miR-133a OE showed similar effects to APN OE in attenuating the LPS-induced productions of GSDMD, NLRP3, caspase1, IL-1β, IL-18, and OPN, as well as α-SMA downregulation in vascular smooth muscle cells (vSMCs). Moreover, the beneficial effects of APN OE were abolished by miR-133a knockdown in vSMCs. In conclusion, our present results indicated that the upregulation of miR-133a by APN inhibits pyroptosis pathway, which potentially rescues AAD.

Proteomic analysis in lupus mice identifies Coronin-1A as a potential biomarker for lupus nephritis

BackgroundApproximately 50% of systemic lupus erythematosus (SLE) patients develop nephritis, which is among the most severe and frequent complications of the disease and a leading cause of morbidity and mortality. Despite intensive research, there are still no reliable lupus nephritis (LN) markers in clinical use that can assess renal damage and activity with a high sensitivity and specificity. To this end, the aim of this study was to identify new clinically relevant tissue-specific protein biomarkers and possible underlying molecular mechanisms associated with renal involvement in SLE, using mass spectrometry (MS)-based proteomics.MethodsKidneys were harvested from female triple congenic B6.NZMsle1/sle2/sle3 lupus mice model, and the respective sex- and age-matched C57BL/6 control mice at 12, 24 and 36 weeks of age, representing pre-symptomatic, established and end-stage LN, respectively. Proteins were extracted from kidneys, purified, reduced, alkylated and digested by trypsin. Purified peptides were separated by liquid chromatography and analysed by high-resolution MS. Data were processed by the Progenesis QIp software, and functional annotation analysis was performed using DAVID bioinformatics resources. Immunofluorescence and multiple reaction monitoring (MRM) MS methods were used to confirm prospective biomarkers in SLE mouse strains as well as human serum samples.ResultsProteomic profiling of kidney tissues from SLE and control mice resulted in the identification of more than 3800 unique proteins. Pathway analysis revealed a number of dysregulated molecular pathways that may be mechanistically involved in renal pathology, including phagosome and proximal tubule bicarbonate reclamation pathways. Proteomic analysis supported by human transcriptomic data and pathway analysis revealed Coronin-1A, Ubiquitin-like protein ISG15, and Rho GDP-dissociation inhibitor 2, as potential LN biomarkers. These results were further validated in other SLE mouse strains using MRM-MS. Most importantly, experiments in humans showed that measurement of Coronin-1A in human sera using MRM-MS can segregate LN patients from SLE patients without nephritis with a high sensitivity (100%) and specificity (100%).ConclusionsThese preliminary findings suggest that serum Coronin-1A may serve as a promising non-invasive biomarker for LN and, upon validation in larger cohorts, may be employed in the future as a screening test for renal disease in SLE patients.

Srsf10 and the minor spliceosome control tissue-specific and dynamic SR protein expression.

Minor and major spliceosomes control splicing of distinct intron types and are thought to act largely independent of one another. SR proteins are essential splicing regulators mostly connected to the major spliceosome. Here, we show that Srsf10 expression is controlled through an autoregulated minor intron, tightly correlating Srsf10 with minor spliceosome abundance across different tissues and differentiation stages in mammals. Surprisingly, all other SR proteins also correlate with the minor spliceosome and Srsf10, and abolishing Srsf10 autoregulation by Crispr/Cas9-mediated deletion of the autoregulatory exon induces expression of all SR proteins in a human cell line. Our data thus reveal extensive crosstalk and a global impact of the minor spliceosome on major intron splicing.

Unraveling Intron Retention: Differential Expression Pattern in Brain and Kidney Cells After Hypoxia or Ischemia

IntroductionThe inclusion of introns in the mature mRNA (intron retention), has been recently uncovered as a mechanism for the regulation of genetic expression in vertebrates. Intron retention is implicated in tissue specific protein diversity, splicing regulation and control of gene expression. We have recently identified intron retention as a mechanism involved in the regulation of erythropoietin (EPO). EPO is the key regulator of erythropoiesis and is transcriptionally regulated by hypoxia inducible factor 2 (HIF2). The ability to easily detect intron retention could add to our ability to better understand genetic regulation of our genomes in stress conditions, such as ischemic diseases (ex/ myocardial infarction (MI) or stroke).ObjectiveTo evaluate the role of intron retention mechanism in EPO expression level to gain a better understanding of its regulation in response to hypoxia and ischemia, conditions which result in EPO expression.MethodsHTB16 (neuroblastoma), CHP212 (glioblastoma), hCMEC/D3 (blood brain barrier) and HEK293 (kidney) were placed in hypoxic (1% oxygen), ischemic (1% oxygen, no serum or glucose) or normoxic conditions (20% oxygen) for 24 hours prior to RNA extraction. Quantitative polymerase chain reaction (qPCR) was performed with two sets of primers for EPO, the first set was targeting intron inclusion with each primer in a separate exon (intron primers) and the second set was targeting a region that spans two exons, which would not amplify if introns were present (non‐introns). Relative quantification for the level of expression was determined by qPCR using β‐actin and YWHAZ as reference genes for normalization purposes. Statistical analysis of transcript expression and the ratio between intron and non‐intron detecting primer sets were determined using analysis of variance (ANOVA).ResultsEPO mRNA level was increased in HTB16, CHP212 and HEK293, but not in hCMEC/D3 cell lines in response to ischemia, but not hypoxia. However, when the ration of intron vs. non‐intron expression level was determined a trend towards more intron retaining transcripts in HTB16, CHP212 and hCMEC/D3 but not in HEK293 was observed, suggesting an induction for the expression of this gene under this ischemic condition.ConclusionsIntron retention mechanism in EPO is taking place in brain‐derived cell lines, but not in kidney‐derived cells. These results indicate a tissue specificity of intron retention mechanism that could regulate differential expression level of EPO under different physiological and stressful conditions. These findings highlight the complexity of EPO regulation. Hence, intron retention must be considered when examining the expression of a gene to better understand expression in ischemic disease models, such as MI or stroke.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC)

Mass-spectrometry-based draft of the Arabidopsis proteome.

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities1. Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions.

Total protein staining is superior to classical or tissue-specific protein staining for standardization of protein biomarkers in heterogeneous tissue samples

Protein detection techniques such as western blotting and ELISA rely on housekeeping proteins as standards for sample normalization. However, clinical or animal tissue specimens are heterogeneous due to presence of contaminating cell types and tissues (e.g., blood vessels and muscle) or cellular decay during tissue storage and isolation which may compromise protein integrity. This biological heterogeneity may invalidate the assumption that housekeeping proteins are invariable across various specimens. This study provides data that advocate for protein standardization based on total protein staining in rabbit posterior capsular tissues. We compared the classical normalization markers glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin (TUBB) with other proteins that have low variation in expression (i.e., FTL, FTH1, EEF1A1, TPT1) based on RNAseq data for human posterior capsular tissues. Histological examination revealed a high degree of qualitative variation in microscopic images of capsular tissue specimens. This variation is reflected by significant differences in specific protein signals for all housekeeping proteins as detected by western blot analysis. However, total protein staining, which combines the intensity of multiple gel electrophoretic bands, normalizes natural biological variation observed for individual housekeeping proteins and permits assessment of protein integrity. Therefore, we propose that normalization based on total protein staining increases accuracy of protein quantification of heterogeneous tissue specimen samples.