Mitochondria: A mirror into cellular dysfunction in heart disease

Cardiovascular (CV) disease is the single most significant cause of morbidity and mortality worldwide. The emerging global impact of CV disease means that the goals of early diagnosis and a wider range of treatment options are now increasingly pertinent. As such, there is a greater need to understand the molecular mechanisms involved and potential targets for intervention. Mitochondrial function is important for physiological maintenance of the cell, and when this function is altered, the cell can begin to suffer. Given the broad range and significant impacts of the cellular processes regulated by the mitochondria, it becomes important to understand the roles of the proteins associated with this organelle. Proteomic investigations of the mitochondria are hampered by the intrinsic properties of the organelle, including hydrophobic mitochondrial membranes; high proportion of basic proteins (pI greater than 8.0); and the relative dynamic range issues of the mitochondria. For these reasons, many proteomic studies investigate the mitochondria as a discrete subproteome. Once this has been achieved, the alterations that result in functional changes with CV disease can be observed. Those alterations that lead to changes in mitochondrial function, signaling and morphology, which have significant implications for the cardiomyocyte in the development of CV disease, are discussed.     

Redox imbalance provokes deactivation of macrophages in sepsis

Sepsis accounts for the majority of deaths in critically ill patients. Symptoms of septic disease are often associated with monocyte/macrophage desensitization. In the current study, impaired macrophage function was determined in a sepsis mouse model with decreased cytokine release and weak phagocytosis, coinciding with ectopic elevation of serum-ROS levels. Furthermore, in the experimental cell model, RAW264.7 macrophages displayed a "deactivated" phenotype, characterized by impaired inflammatory and phagocytosis function. Cellular anti-oxidative proteins were further investigated; lipopolysaccharide (LPS)- and H(2) O(2) -treated cells exhibited lower ratio of reductive-to-oxidized glutathione as compared with LPS-treated cells only, without inducing cell death. 2-DE and MALTI-TOF/TOF were employed to illustrate protein expression differentiation patterns. A total of 33 proteins were found to be differently expressed. Among them, 33% of proteins were associated with oxidative stress. We further investigated the role of the ROS/LPS/Toll-like receptor 4 (TLR4) axis in modulating the immunosuppression during sepsis. LPS- and H(2) O(2) -treated macrophages demonstrated decreased cytokine release, whereas TLR4 expression was up-regulated. Western blot analysis showed decreased levels of phosphorylation of MAP kinases and IκB. Electrophoretic mobility shift assay analysis demonstrated attenuated DNA-binding activities of AP-1 and NF-κB, as compared with those of their control. Collectively, these findings indicate that ROS mediates critical aspects of innate immunity that result in an immunocompromised state through an imbalance of cellular oxidation/reduction during sepsis.     

HCV inhibits antigen processing and presentation and induces oxidative stress response in gastric mucosa

Productive hepatitis C virus (HCV) infection appears to be primarily confined to the liver. However, a wide variety of extrahepatic disease manifestations are associated with the infection and HCV RNA has been frequently detected in gastric mucosa. The present study aims to determine molecular alterations present in vivo in the stomach where HCV expression does not induce a carcinoma but a lymphoma, thus extending the knowledge of alterations in intracellular pathways consequent to HCV infection. We compared, by 2-D DIGE, the gastric protein expression profile from six HCV positive and six HCV negative samples lacking neoplastic or dysplastic conditions. In HCV positive tissue we observed a down regulation of proteins involved in MHC maturation and assembly, antigen processing and presentation and ER stress, in addition to an up regulation of proteins involved in cellular oxidative stress responses. Ubiquinol-cytochrome-C-reductase (UQCRFS1), part of the mitochondrial respiratory chain complex-III, was identified as the most up regulated protein. Data were confirmed by Western blot and immunohistochemistry. Our results demonstrate a HCV negative influence on the different pathways that determine antigen processing and presentation via MHC-I and the cellular attempts to counteract HCV induced oxidative stress. Both these processes facilitate immune escape and cell survival and probably contribute to HCV chronicization.     

Protein carbonylation in kidney medulla of the spontaneously hypertensive rat

Enhanced generation of ROS has been reported in models of hypertension such as the spontaneously hypertensive rat (SHR). Impairment of kidney function has been implicated in development and progression of hypertension, and the renal medulla appears to play an important role in regulating long‐term blood pressure. A key biomarker of oxidative stress is the formation of protein carbonyls, which we set out to characterize in the SHR medulla. We identified 11 proteins that were differentially carbonylated in SHR medulla in comparison to normotensive wistars including enolase 1, catalase, carbonic anhydrase II, transferrin and members of the aldo–keto‐reductase family. This enhanced protein oxidation was not only accompanied by an increase in intracellular iron deposition, but aldo–keto‐reductase activity was also significantly less in SHR medulla than in normotensive Wistars. Oxidative stress appears selectively to target a subset of proteins in SHR kidney and modification of these proteins may in turn contribute to the renopathy associated with hypertension.     

Urinary extracellular vesicles for biomarker source to monitor polycystic kidney disease

Extracellular vesicles (EVs) are bilayered lipid vesicles, 50–1000 nm in diameter and secreted by most types of cells. They contain many proteins, mRNAs, miRNAs, and lipids that reflect the pathophysiological state of the cells they originate from, and are therefore considered to be a rich source of potential biomarkers. In this issue (Pocsfalvi, G. et al., Proteomics Clin. Appl. 2015, 9, 552–567), Pocsfalvi et al. conducted pioneering investigations to determine whether changes in the protein content of EVs occur during progression of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disorder that predominantly affects the kidneys. Most significantly, iTRAQ-based quantitative proteomics showed that cytoskeleton-regulating and Ca²⁺-binding proteins are differentially expressed in urinary EVs of ADPKD patients. Impressively, these proteins are involved in biological processes that are closely related to the pathogenic state of tubular epithelial cells in ADPKD, demonstrating the possibility to monitor the status of patients using urinary EVs.     

Differential expression of red cell proteins in hemoglobinopathy

Red blood cell proteome has not been studied well until recently, as the large abundance of hemoglobin posed challenge to the detection of other cytosolic proteins in the linear dynamic range. However, in the last couple of years, due to emergence of various novel hemoglobin depletion strategies and more state-of-the-art detection techniques, a number of works on erythrocyte proteome have appeared in the literature. As a result, we now have much deeper information about both the membrane as well as the cytosolic proteins of erythrocytes. In this review, we have discussed the role of red cell proteome on the two most well-studied hemoglobin disorders, sickle cell disease and thalassemia, emphasizing on the differential expression of the redox regulator proteins and chaperones, in particular. We have also touched upon the importance of the association of the varying levels of hemoglobin variants, particularly HbE on the clinical manifestation of composite diseases like HbEβ thalassemia.     

Discovery of functional protein groups by clustering community links and integration of ontological knowledge

In this paper we cluster data from protein networks and integrate the results with chemical databases and ontologies to investigate functional links between related disease states. It is well know that certain genes participate in more than one function and if they are defective are likely to be responsible for several health problems. Furthermore, genes tend to cooperate in associated networks or cascades often with ’crosstalk’ between networks which can subtly alter cellular functions. Understanding the complexity and role of the various cell functions and mechanisms requires the use of computational models to make inferences and link together the interplay between genes, proteins and chemical interactions. A deeper understanding of the mechanisms of diseases will eventually be of benefit for the development new and improved therapies. The particular disease state we investigate in this work is cystinosis which is characterized by the widespread deposition of the amino acid cystine in cells due to a defect in cystine transport. In cystinosis, cystine accumulates in the lysosomes and eventually forms crystals throughout the body causing problems in the kidneys and the eyes. The defect is caused by a mutation in the CTNS gene and this forms the starting point for our investigation.     

Autonomic function assessment using analysis of heart rate variability

Analysis of changes in heart rate can be useful in determining the state of various body systems. In particular the analysis of heart rate variability (HRV) is used in the assessment of autonomic function. This paper uses the discrete harmonic wavelet transform for a time-frequency analysis of HRV data to show changes in spectral power over time. Signals representing patient heart rate are presented, and methods for spectral and time-frequency analysis are described. Three sets of patient data are then analysed using these methods. The results show the potential of time-frequency analysis in the assessment of medical disorders, such as the sleep apnoea syndrome, where transient alterations in autonomic function occur.     

Parameter free shape and thickness optimisation considering stress response

In the parameter free approach, FE-based data are used as design variables, such as nodal coordinates and nodal thickness. During shape and thickness optimisation, this approach provides much design freedom for a limited modelling effort. Stress results are, however, very sensitive to the local shape changes that can occur during parameter free optimisation. When stress results are used as response function, this irregularity can complicate the optimisation. As a solution, the Kreisselmeier-Steinhauser function for the stresses is introduced as a response function for parameter free shape optimisation. In this function, the local stress results are aggregated to obtain a global measure of stress in a structure. This measure can be used as an objective to reduce the overall stress in the structure or as a constraint to limit the stress in the structure to a maximum allowable value. As a result, the optimal structures are smooth and material efficient. Several examples are presented in this paper to illustrate the use of the parameter free design approach in combination with the stress response function.     

NanoArrays, The Next Generation Molecular Array Format for High Throughput Proteomics, Diagnostics and Drug Discover.

Biomolecular array technology is an invaluable tool for rapid screening of nucleic acid mixtures. This approach has been tremendously successful both in its breadth of application and its commercial value. Entire genomes, including the human genome, have been screened by molecular array techniques. Arrays are a rapid and now routine method for analysis of expression patterns and their association with physiological states. Such a rapid, high throughput analysis of cellular expression is key to the expansion of our basic knowledge of the relationship between gene expression and organismal function, as well as to the understanding of the genetic component of disease states and the predisposition to disease.Despite the success of array technology for nucleic acid applications, a similar trend for proteins has not occurred. Due, in part, to the difficulties involved in production and labeling of proteins for solid state analysis, solid state arrays of proteins are not widely utilized. Protein function and interaction have been traditionally addressed by the combination of 2D gel electrophoretic separation and mass spectrometry to examine individual protein spots, a slow, tedious and expensive process. Another approach uses in vivo methods for examining protein-protein interactions by the two-hybrid system in yeast and mammalian cells [1]. Although the two-hybrid system has shown some success in finding new interaction between proteins in important cellular pathways, it is far more difficult, costly and time consuming than the solid state methods used for nucleic acids.BioForce Laboratory, Inc., has developed a solid state method for examining the interaction between a wide range of molecules in an array format. This technology involves several key technological innovations.     

Coupling enrichment methods with proteomics for understanding and treating disease

Owing to recent advances in proteomics analytical methods and bioinformatics capabilities there is a growing trend toward using these capabilities for the development of drugs to treat human disease, including target and drug evaluation, understanding mechanisms of drug action, and biomarker discovery. Currently, the genetic sequences of many major organisms are available, which have helped greatly in characterizing proteomes in model animal systems and humans. Through proteomics, global profiles of different disease states can be characterized (e.g. changes in types and relative levels as well as changes in PTMs such as glycosylation or phosphorylation). Although intracellular proteomics can provide a broad overview of physiology of cells and tissues, it has been difficult to quantify the low abundance proteins which can be important for understanding the diseased states and treatment progression. For this reason, there is increasing interest in coupling comparative proteomics methods with subcellular fractionation and enrichment techniques for membranes, nucleus, phosphoproteome, glycoproteome as well as low abundance serum proteins. In this review, we will provide examples of where the utilization of different proteomics-coupled enrichment techniques has aided target and biomarker discovery, understanding the drug targeting mechanism, and mAb discovery. Taken together, these improvements will help to provide a better understanding of the pathophysiology of various diseases including cancer, autoimmunity, inflammation, cardiovascular disease, and neurological conditions, and in the design and development of better medicines for treating these afflictions.     

The integration of proteomics and systems approaches to map regulatory mechanisms underpinning platelet function

Platelets in the circulation are triggered by vascular damage to activate, aggregate and form a thrombus that prevents excessive blood loss. Platelet activation is stringently regulated by intracellular signalling cascades, which when activated inappropriately lead to myocardial infarction and stroke. Strategies to address platelet dysfunction have included proteomics approaches which have lead to the discovery of a number of novel regulatory proteins of potential therapeutic value. Global analysis of platelet proteomes may enhance the outcome of these studies by arranging this information in a contextual manner that recapitulates established signalling complexes and predicts novel regulatory processes. Platelet signalling networks have already begun to be exploited with interrogation of protein datasets using in silico methodologies that locate functionally feasible protein clusters for subsequent biochemical validation. Characterization of these biological systems through analysis of spatial and temporal organization of component proteins is developing alongside advances in the proteomics field. This focused review highlights advances in platelet proteomics data mining approaches that complement the emerging systems biology field. We have also highlighted nucleated cell types as key examples that can inform platelet research. Therapeutic translation of these modern approaches to understanding platelet regulatory mechanisms will enable the development of novel anti-thrombotic strategies.     

基于ARM和Zigbee的心血管功能测试诊断平台设计与实现

心血管疾病作为慢性疾病之首,严重威胁全国10.3%人群的生命健康,对其进行有效的防控治疗已成为当下研究热点;而目前国内慢性疾病信息实时采集终端与分析诊断平台匮乏,致使心血管等慢性疾病无法得到跟踪和快速有效的治疗;为此,文章利用物联网技术,结合SQL Server数据库,使用C#语言以及WPF开发技术设计了基于物联网的心血管功能测试及诊断平台;该平台通过终端实现对心血管疾病患者健康信息的采集,采用Zigbee通讯技术上传至云端,平台通过Socket技术接收数据并给出诊断结论,生成体检报告,系统测试中涉及了心率、心输出量CO、心搏出量SV以及脉搏波形特征量K等关键参数,结果验证了该心血管功能测试诊断平台的合理性与有效性,实验结果达到了预期目标,为及早发现和治疗心血管等慢性疾病提供了数据支撑与平台基础。     

Redox proteomics screening cellular factors associated with oxidative stress in hepatocarcinogenesis

Liver cancer is a major global health problem being the sixth most common cancer and the third cause of cancer‐related death, with hepatocellular carcinoma (HCC) representing more than 90% of primary liver cancers. Mounting evidence suggests that, compared with their normal counterparts, many types of cancer cell have increased levels of ROS. Therefore, cancer cells need to combat high levels of ROS, especially at early stages of tumor development. Recent studies have revealed that ROS‐mediated regulation of redox‐sensitive proteins (redox sensors) is involved in the pathogenesis and/or progression of many human diseases, including cancer. Unraveling the altered functions of redox sensors and the underlying mechanisms in hepatocarcinogenesis is critical for the development of novel cancer therapeutics. For this reason, redox proteomics has been developed for the high‐throughput screening of redox sensors, which will benefit the development of novel therapeutic strategies for the treatment of HCC. In this review, we will briefly introduce several novel redox proteomics techniques that are currently available to study various oxidative modifications in hepatocarcinogenesis and summarize the most important discoveries in the study of redox processes related to the development and progression of HCC.     

Mass spectrometric immunoassay and MRM as targeted MS-based quantitative approaches in biomarker development: Potential applications to cardiovascular disease and diabetes

Type 2 diabetes mellitus (T2DM) is an important risk factor for cardiovascular disease (CVD)—the leading cause of death in the United States. Yet not all subjects with T2DM are at equal risk for CVD complications; the challenge lies in identifying those at greatest risk. Therapies directed toward treating conventional risk factors have failed to significantly reduce this residual risk in T2DM patients. Thus newer targets and markers are needed for the development and testing of novel therapies. Herein we review two complementary MS-based approaches—mass spectrometric immunoassay (MSIA) and MS/MS as MRM—for the analysis of plasma proteins and PTMs of relevance to T2DM and CVD. Together, these complementary approaches allow for high-throughput monitoring of many PTMs and the absolute quantification of proteins near the low picomolar range. In this review article, we discuss the clinical relevance of the high density lipoprotein (HDL) proteome and Apolipoprotein A-I PTMs to T2DM and CVD as well as provide illustrative MSIA and MRM data on HDL proteins from T2DM patients to provide examples of how these MS approaches can be applied to gain new insight regarding cardiovascular risk factors. Also discussed are the reproducibility, interpretation, and limitations of each technique with an emphasis on their capacities to facilitate the translation of new biomarkers into clinical practice.     

The proteomics of cancer stem cells: potential clinical applications for innovative research in oncology

Cancer stem cells (CSCs) or tumour-maintaining cells are becoming an important new reality in oncology. The intriguing molecular pathophysiology of CSCs may justify some of the obscure pathogenetic, diagnostic, prognostic, and above all, therapeutic aspects of cancer and, eventually, lead to new solutions in oncology. CSC is a cell within the tumour that possesses the capacity to self-renew and, in doing so, gives rise to the heterogeneous lineages that comprise the tumour. The precise identification of this peculiar subpopulation of cancer cells, which has some intriguing similarities to normal stem cells, is becoming an important and urgent topic in oncology. In fact, some debated CSC markers have been already adopted by pharmacological research as targets of new and/or old anticancer drugs, showing an intriguing therapeutic index. These discussed identification markers include cell surface proteins, different activated signalling pathways, several molecules of the stem cell niche, various drug resistance mechanisms (ABCG2 and ALDH), telomerase, oncogenes and oncosuppressors (p16INK4 - Rb) and lastly, various microRNAs. In this new promising area of cancer research, proteomics, in general, and oncoproteomics, in particular, can and must play a significant role if the methodological approaches and the experimental protocols are correctly designed and interpreted.     

Degradomics in Biomarker Discovery

The upregulation of protease expression and proteolytic activity is implicated in numerous pathological conditions such as neurodegeneration, cancer, cardiovascular and autoimmune diseases, and bone degeneration. During disease progression, various proteases form characteristic patterns of cleaved proteins and peptides, which can affect disease severity and course of progression. It has been shown that qualitative and quantitative monitoring of cleaved protease substrates can provide relevant prognostic, diagnostic, and therapeutic information. As proteolytic fragments and peptides generated in the affected tissue are commonly translocated to blood, urine, and other proximal fluids, their possible application as biomarkers is the subject of ongoing research. The field of degradomics has been established to enable the global identification of proteolytic events on the organism level, utilizing proteomic approaches and sample preparation techniques that facilitate the detection of proteolytic processing of protease substrates in complex biological samples. In this review, some of the latest developments in degradomic methodologies used for the identification and validation of biologically relevant proteolytic events and their application in the search for clinically relevant biomarker candidates are presented. The current state of degradomics in clinics is discussed and the future perspectives of the field are outlined.