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Arterial calcification is an important hallmark of cardiovascular disease and shares many similarities with skeletal mineralisation. The bone-specific protein osteocalcin (OCN) is an established marker of vascular smooth muscle cell (VSMC) osteochondrogenic trans-differentiation and a known regulator of glucose metabolism. However, the role of OCN in controlling arterial calcification is unclear. We hypothesised that OCN regulates calcification in VSMCs and sought to identify the underpinning signalling pathways. Immunohistochemistry revealed OCN co-localisation with VSMC calcification in human calcified carotid artery plaques. Additionally, 3 mM phosphate treatment stimulated OCN mRNA expression in cultured VSMCs (1.72 fold; p

Mitochondria are crucial bioenergetics powerhouses and biosynthetic hubs within cells, which can generate and sequester toxic reactive oxygen species (ROS) in response to oxidative stress. Oxidative stress-stimulated ROS production results in ATP depletion and the opening of mitochondrial permeability transition pores, leading to mitochondria dysfunction and cellular apoptosis. Mitochondrial loss of function is also a key driver in the acquisition of a senescence-associated secretory phenotype that drives senescent cells into a pro-inflammatory state. Maintaining mitochondrial homeostasis is crucial for retaining the contractile phenotype of the vascular smooth muscle cells (VSMCs), the most prominent cells of the vasculature. Loss of this contractile phenotype is associated with the loss of mitochondrial function and a metabolic shift to glycolysis. Emerging evidence suggests that mitochondrial dysfunction may play a direct role in vascular calcification and the underlying pathologies including (1) impairment of mitochondrial function by mineral dysregulation i.e., calcium and phosphate overload in patients with end-stage renal disease and (2) presence of increased ROS in patients with calcific aortic valve disease, atherosclerosis, type-II diabetes and chronic kidney disease. In this review, we discuss the cause and consequence of mitochondrial dysfunction in vascular calcification and underlying pathologies; the role of autophagy and mitophagy pathways in preventing mitochondrial dysfunction during vascular calcification and finally we discuss mitochondrial ROS, DRP1, and HIF-1 as potential novel markers and therapeutic targets for maintaining mitochondrial homeostasis in vascular calcification.

Vascular calcification (VC), a pathophysiological consequence of atherosclerosis and arteriosclerosis, is associated with an inevitably stiffened arterial system, leading to development of cardiovascular diseases. Currently, there are no effective strategies for treating vascular calcification. Caloric restriction (CR) is a dietary intervention that involves reduction of total calories below ad libitum intake without nutritional insufficiency or malnutrition. Over the past decade the potential for CR to delay the onset of age-related chronic diseases and offer direct cardioprotective effects have become well recognised. However, the effect of CR on VC has yet to be examined. We isolated VSMCs form the aortae of CR mice and cultured them under calcifying conditions (3mM Pi) ex vivo. VSMCs derived from CR mice showed reduced calcification compared to VSMCs from mice fed ad libitum (p To investigate the molecular mechanism underpinning starvation-induced abrogation of VC, we carried out stable isotope labeling by/with amino acids in cell culture (SILAC) based quantitative proteomics comparison between calcified and starved RVICS. The majority of proteins showing >2 fold upregulation were from the ubiquitin proteasome system (UPS). UPS is a selective proteolytic system in which substrates are recognised and tagged with ubiquitin for processive degradation by the proteasome. Treatment of both RVICs and VSMCs with MG132 (UPS inhibitor; 10uM) cultured under calcifying conditions accelerated calcification in both cell types(p This novel finding could lead to the application of CR-mimetics to combat vascular calcification.

Autophagy is a dynamic self-degradation process that plays a key role in many biological processes such as development, aging, and immunity. Emerging research in the area of cancer, neurodegenerative disorders, autoimmune disorders, infectious diseases, and myopathies have constantly shown link of autophagy with these disease etiologies. The field has grown in the last decade to include researchers from diverse areas of biology and medicine with a multitude of assay methods being developed. As with this recent surge in the interests, it is imperative to assess the quality and robustness of the assay methods used in the autophagy detection. In this chapter we have discussed some of the widely used methods available to study autophagy both in vitro and in vivo models with emphasis on disease states. Furthermore the scope of some of emerging methods to study autophagy in disease-specific scenarios is discussed. As a growing number of commercial companies are now actively engaged in developing assay reagents for autophagy, a summary of the major kits/reagents used in this surging field of autophagy are also provided.

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8(+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.

Intratumoral hypoxia is associated with poor prognosis, regardless of the mode of therapy. Cancer cells survive this condition through activating several adaptive signaling pathways, including the integrated stress response (ISR) and autophagy. Activating transcription factor 4 (ATF4) is the major transcriptional mediator of the ISR, which we have shown to be involved in autophagy regulation to protect cells from severe hypoxia. Here we demonstrate that ATF4 orchestrates a program of BH3-only protein expression in severe hypoxia. We find that the BH3-only proteins HRK, PUMA, and NOXA are transcriptionally induced in severe hypoxia and that their expression is abrogated by RNA interference against ATF4. In particular, we show that the BH3-only protein harakiri (HRK) is transactivated by ATF4 in severe hypoxia through direct binding of ATF4 to the promoter region. Furthermore, we demonstrate through siRNA knockdown that HRK induces autophagy and promotes cancer cell survival in severe hypoxia.

J. Neurochem. (2011) 10.1111/j.1471-4159.2011.07521.x Abstract HspB8, a small heat-shock protein implicated in autophagy, is mutated in patients with distal hereditary motor neuropathy type II (dHMNII). Autophagy is essential for maintaining protein homeostasis in the central nervous system, but its role has not been investigated in peripheral motor neurons. We used a novel, multispectral-imaging flow cytometry assay to measure autophagy in cells. This assay revealed that over-expression of wild-type HspB8 in motor neuron-like NSC34 cells led to an increased co-localisation of autophagosomes with the lysosomes. By contrast, over-expression of mutant HspB8 resulted in autophagosomes that co-localised with protein aggregates but failed to co-localise with the lysosomes. A similar impairment of autophagy could also be demonstrated in peripheral blood mononuclear cells from two dHMNII patients with the HspB8K141E mutation. We conclude that defects in HspB8-mediated autophagy are likely to contribute to dHMNII pathology and their detection in peripheral blood mononuclear cells could be a useful, accessible biomarker for the disease.

Autophagy is a lysosomal catabolic pathway responsible for the degradation of cytoplasmic constituents. Autophagy is primarily a survival pathway for recycling cellular material in times of nutrient starvation, and in response to hypoxia, endoplasmic reticulum stress, and other stresses, regulated through the mammalian target of rapamycin pathway. The proteasomal pathway is responsible for degradation of proteins, whereas autophagy can degrade cytoplasmic material in bulk, including whole organelles such as mitochondria (mitophagy), bacteria (xenophagy), or lipids (lipophagy). Although signs of autophagy can be present during cell death, it remains controversial whether autophagy can execute cell death in vivo. Here, we will introduce protocols for detecting autophagy in mammalian primary cells by using western blots, immunofluorescence, immunohistochemistry, flow cytometry, and imaging flow cytometry.

A comparative proteomic analysis using 2-DE coupled with MALDI-MS and LC-MS/MS was performed in Synechocystis sp. PCC 6803 to identify protein candidates involved in acid stress response in cyanobacteria. Comparison of soluble proteins from the cytoplasmic fraction of cells grown on media set at pH 7.5 and 5.5 using 2-DE identified four proteins, which showed significant changes in the abundance. Surprisingly, several general stress proteins, either the heat shock family proteins or chaperonins, did not show perceptible fold changes in response to acidity. Compared to the cytoplasmic proteome, the periplasmic proteome showed remarkable changes as a function of external pH. Protein expression profiling at different external pH, i.e., 9.0, 7.5, 6.0 and 5.5, allowed classifying the periplasmic proteins depending on their preferential expression patterns towards acidity or alkalinity. Among the acid- and base-induced proteins, oxalate decarboxylase and carbonic anhydrase were already known for their role in pH homeostasis. Several unknown proteins from the periplasm, that showed significant changes in response to pH, provide ideal targets for further studies in understanding pH stress response in cyanobacteria. This study also identified 14 novel proteins, hitherto unknown from the periplasmic space of Synechocystis.

Dunaliella, a green alga grows in a wide range of marine and freshwater habitats such as oceans, brine lakes, salt marshes and salt-water ditches near the sea, predominantly in water bodies containing more than 10% salt. Sambhar salt lake, Rajasthan (India) was found as one of the natural habitat of Dunaliella sp. The species was isolated and screened for accumulation of beta-carotene and glycerol. It was studied for the growth attributes like total protein, optical density, total chlorophyll, total carotenoid, beta-carotene and glycerol. Under unstressed physico-chemical conditions the maximum beta-carotene and glycerol observed was 1.15 pg/cell and 94.26 pg/cell respectively. The stress (salt stress (NaCl), high light intensities and continuous light) allowed the alga to accumulate high beta-carotene (approximately 4.21 pg/cell under 118.18 micromole m(-2) s(-1) of light intensity) without massive reduction in biomass content. This opens up new avenues for exploring this strain for future research and its commercial exploitation.

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity. © 2012 by The American Society of Human Genetics. All rights reserved.

This technique evaluates the colocalization of the autophagy protein MAP1LC3 (microtubule-associated proteins 1A/1B light chain 3B, here referred to as LC3) with lysosomes (autolysosomes) in primary cells in a high-throughput manner. It uses an imaging fluorescence-activated cell sorting cytometer called the ImageStream to concomitantly detect surface molecules, making possible the identification of cells in mixed cell populations (e.g., in blood or bone marrow). It can be applied to clinical samples and to rare cell populations because only a few cells are needed for detection.

The response of two strains of Dunaliella, a beta-carotene accumulating halotolerant alga, was evaluated under sulphate, nitrate and phosphate limitation. All these factors decreased the growth rate and chlorophyll content but, increased the beta-carotene content of the two isolates of Dunaliella, D1, obtained from GTCC and D2 an indigenous strain isolated from Sambhar salt lake, India. Both the strains exhibited accumulation of beta-carotene and glycerol under the different nutrient limiting conditions. A maximum accumulation of 3.99 pg/cell of beta-carotene was observed under phosphate depletion. However, nutrient depletion did not significantly affect the glycerol accumulation in these cells. D2, the indigenous isolate, was found to be a better accumulator of beta-carotene than D1.