Rubredoxin reductase from Alcanivorax borkumensis: expression and characterization

Oil pollution is an environmental problem of increasing importance. Alcanivorax borkumensis, with a high potential for biotechnological applications, is a key marine hydrocarbonoclastic bacterium and plays a critical role in the bioremediation of oil-polluted marine systems. In oil degrading bacteria, the first step of alkane degradation is catalyzed by a monooxygenase. The reducing electrons are tunneled from NAD(P)H via rubredoxin, one of the most primitive metalloproteins, to the hydroxylase. Rubredoxin reductase is a flavoprotein catalyzing the reduction of rubredoxin. There are two rubredoxin genes, alkG and rubA, in A. borkumensis genome. In this work, the genes encoding rubredoxin reductase (ABO_0162, rubB) and AlkG(ABO_2708, alkG) were cloned and functionally overexpressed in E. coli. Our results demonstrate that RubB could reduce AlkG, therefore compensating for the absence of AlkT, also a rubredoxin reductase, missing in A. borkumensis SK2 genome. These results will increase our knowledge concerning biological alkane degradation and will lead us to design more efficient biotransformation and bioremediation systems.     

Protective Roles of DMP1 in High Phosphate Homeostasis

Purpose

Dmp1 (dentin matrix protein1) null mice (Dmp1^−/−) display hypophosphatemic rickets with a sharp increase in fibroblast growth factor 23 (FGF23). Disruption of Klotho (the obligatory co-receptor of FGF23) results in hyperphosphatemia with ectopic calcifications formed in blood vessels and kidneys. To determine the role of DMP1 in both a hyperphosphatemic environment and within the ectopic calcifications, we created Dmp1/Klotho compound deficient (Dmp1^−/−kl/kl) mice.

Procedures

A combination of TUNEL, immunohistochemistry, TRAP, von Kossa, micro CT, bone histomorphometry, serum biochemistry and Scanning Electron Microscopy techniques were used to analyze the changes in blood vessels, kidney and bone for wild type control, Dmp1^−/−, Klotho deficient (kl/kl) and Dmp1^−/−kl/kl animals.

Findings

Interestingly, Dmp1^−/−kl/kl mice show a dramatic improvement of rickets and an identical serum biochemical phenotype to kl/kl mice (extremely high FGF23, hyperphosphatemia and reduced parathyroid hormone (PTH) levels). Unexpectedly, Dmp1^−/−kl/kl mice presented elevated levels of apoptosis in osteocytes, endothelial and vascular smooth muscle cells in small and large blood vessels, and within the kidney as well as dramatic increase in ectopic calcification in all these tissues, as compared to kl/kl.

Conclusion

These findings suggest that DMP1 has an anti-apoptotic role in hyperphosphatemia. Discovering this novel protective role of DMP1 may have clinical relevance in protecting the cells from apoptosis in high-phosphate environments as observed in chronic kidney disease (CKD).     

Convenient and versatile subcellular extraction procedure, that facilitates classical protein expression profiling and functional protein analysis

Standardized sample preparation to reduce proteome complexity facilitates subsequent proteome analysis. Here we describe a robust sequential extraction method that enables simple fractionation of proteins in their native state according to their subcellular localization, yielding four subproteomes enriched in (a) cytosolic; (b) membrane and membrane organelle-localized; (c) soluble and DNA-associated nuclear and (d) cytoskeletal proteins. Efficiency and selectivity is demonstrated by morphological-, two-dimensional electrophoresis image-, immunological- as well as enzymatic-analysis. In pilot studies, subcellular redistribution of regulatory proteins was successfully measured.     

Mitochondrial stress and mitokines in aging

Mitokines are signaling molecules that enable communication of local mitochondrial stress to other mitochondria in distant cells and tissues. Among those molecules are FGF21, GDF15 (both expressed in the nucleus) and several mitochondrial-derived peptides, including humanin. Their responsiveness to mitochondrial stress induces mitokine-signaling in response for example to exercise, following mitochondrial challenges in skeletal muscle. Such signaling is emerging as an important mediator of exercise-derived and dietary strategy-related molecular and systemic health benefits, including healthy aging. A compensatory increase in mitokine synthesis and secretion could preserve mitochondrial function and overall cellular vitality. Conversely, resistance against mitokine actions may also develop. Alterations of mitokine-levels, and therefore of mitokine-related inter-tissue cross talk, are associated with general aging processes and could influence the development of age-related chronic metabolic, cardiovascular and neurological diseases; whether these changes contribute to aging or represent “rescue factors” remains to be conclusively shown. The aim of the present review is to summarize the expanding knowledge on mitokines, the potential to modulate them by lifestyle and their involvement in aging and age-related diseases. We highlight the importance of well-balanced mitokine-levels, the preventive and therapeutic properties of maintaining mitokine homeostasis and sensitivity of mitokine signaling but also the risks arising from the dysregulation of mitokines. While reduced mitokine levels may impair inter-organ crosstalk, also excessive mitokine concentrations can have deleterious consequences and are associated with conditions such as cancer and heart failure. Preservation of healthy mitokine signaling levels can be achieved by regular exercise and is associated with an increased lifespan.     

Serum heat shock protein 70 and oxidized LDL in patients with type 2 diabetes: does sex matter?

Several studies suggest that the response to various stressors differs between the sexes. We aimed to study serum HSP70 and levels of oxidized-LDL (ox-LDL) as markers of oxidative stress in men and women with type 2 diabetes. We quantified serum HSP70 and levels of ox-LDL in three cohorts; patients with newly diagnosed diabetes, patients with long-standing diabetes and normal controls. The cohort of patients with newly diagnosed diabetes was followed up for 3 months under glucose-lowering therapy with metformin. Our findings showed that serum HSP70 level was increased in women with long-standing diabetes in comparison with men. HSP70 did not decrease after glucose lowering therapy in women with newly diagnosed diabetes, but it did decrease in men. There was no significant difference on ox-LDL between men and women in any of the studied cohorts. It decreased significantly in the cohort of patients with newly diagnosed diabetes after treatment, regardless of sex. There was no significant correlation between HSP70 and ox-LDL in any of the studied cohorts except among normal women. We suggest that diabetes induces an immune response and impairs cellular defense mechanisms against oxidative stress more commonly in women with type 2 diabetes than in men.     

Mesenchymal stromal cells; a new horizon in regenerative medicine

In recent decades, mesenchymal stromal cells (MSCs) biomedical utilizing has attracted worldwide growing attention. After the first report of the human MSCs obtaining from the bone marrow (BM) tissue, these cells were isolated from wide types of the other tissues, ranging from adipose tissue to dental pulp. Their specific characteristics, comprising self-renewality, multipotency, and availability accompanied by their immunomodulatory properties and little ethical concern denote their importance in the context of regenerative medicine. Considering preclinical studies, MSCs can modify immune reactions during tissue repair and restoration, providing suitable milieu for tissue recovery; on the other hand, they can be differentiated into comprehensive types of the body cells, such as osteoblast, chondrocyte, hepatocyte, cardiomyocyte, fibroblast, and neural cells. Though a large number of studies have investigated MSCs capacities in regenerative medicine in varied animal models, the oncogenic capability of unregulated MSCs differentiation must be more assessed to enable their application in the clinic. In the current review, we provide a brief overview of MSCs sources, isolation, and expansion as well as immunomodulatory activities. More important, we try to collect and discuss recent preclinical and clinical research and evaluate current challenges in the context of the MSC-based cell therapy for regenerative medicine.     

CD133: An emerging prognostic factor and therapeutic target in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death worldwide. Recently, the role of cancer stem cells (CSCs) has been highlighted as a crucial emerging factor in chemoresistance, cancer relapse, and metastasis. CD133 is a surface marker of CSCs and has been argued to have prognostic and therapeutic values in CRC along with its related pathways such as Wnt, Notch, and hedgehog. Several studies have successfully applied targeted therapies against CD133 in CRC models namely bispecific antibodies (BiAbs) and anti‐Wnt and notch pathways agents. These studies have yielded initial promising results in this regard. However, none of the therapeutics have been used in the clinical setting and their efficacy and adverse effects profile are yet to be elucidated. This review aims to gather the old and most recent data on the prognostic and therapeutic values of CD133 and CD133‐targeted therapies in CRC.     

DMRFusion: A differentially methylated region detection tool based on the ranked fusion method

DNA methylation is an important epigenetic modification involved in many biological processes and diseases. Computational analysis of differentially methylated regions (DMRs) could explore the underlying reasons of methylation. DMRFusion is presented as a useful tool for comprehensive DNA methylation analysis of DMRs on methylation sequencing data. This tool is designed base on the integration of several ranking methods; Information gain, Between versus within Class scatter ratio, Fisher ratio, Z-score and Welch's t-test. In this study, DMRFusion on reduced representation bisulfite sequencing (RRBS) data in chronic lymphocytic leukemia cancer displayed 30 nominated regions and CpG sites with a maximum methylation difference detected in the hypermethylation DMRs. We realized that DMRFusion is able to process methylation sequencing data in an efficient and accurate manner and to provide annotation and visualization for DMRs with high fold difference score (p-value and FDR < 0.05 and type I error: 0.04).     

Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin

Activation of hepatic stellate cells (HSCs) is an integral component of the wound‐healing process in liver injury/inflammation. However, uncontrolled activation of HSCs leads to constant secretion of collagen‐rich extracellular matrix (ECM) proteins, resulting in liver fibrosis. The enhanced ECM synthesis/secretion demands an uninterrupted supply of intracellular energy; however, there is a paucity of data on the bioenergetics, particularly the mitochondrial (mito) metabolism of fibrogenic HSCs. Here, using human and rat HSCs in vitro, we show that the mito‐respiration, mito‐membrane potential (Δψm) and cellular ‘bioenergetic signature’ distinguish fibrogenic HSCs from normal, less‐active HSCs. Ex vivo, HSCs from mouse and rat models of liver fibrosis further confirmed the altered ‘bioenergetic signature’ of fibrogenic HSCs. Importantly, the distinctive elevation in mito‐Δψm sensitized fibrogenic HSCs for selective inhibition by mitotropic doxorubicin while normal, less‐active HSCs and healthy human primary hepatocytes remained minimally affected if not, unaffected. Thus, the increased mito‐Δψm may provide an opportunity to selectively target fibrogenic HSCs in liver fibrosis.