Mathematical programming modelling tools for resource-poor countries and organisations

In recent years, powerful mathematical modelling languages have enabled Operational Research practitioners to rapidly develop prototype tools capable of modelling complex managerial decisions such as staff shift scheduling, or production and supply chain planning. However, such tools have often required expensive commercial optimisation solvers that are sometimes beyond the financial reach of small companies and organisations, particularly in the low-income and emerging economies. Fortunately, the worldwide scope of the internet has put powerful free optimisation tools within the reach of anyone with a modest PC and even a slow internet connection. This article will present examples showing just how beneficial such an approach can be for resource-poor organisations

Detection of human MCP-4/CCL13 isoforms by SELDI immunoaffinity capture

Monocyte Chemoattractant Proteins 4 (MCP-4/CCL13) is a member of a distinct, structurally-related subclass of CC chemokines mainly involved in recruitment of eosinphils to inflammatory sites. Recent evidence demonstrates that serum level of this protein strongly increases following high dose IL-2 immunotherapy. The physiological form of human MCP-4/CCL13 has yet to be purified. Therefore, the primary structure of the biologically relevant (mature) form has not been established. By using SELDI immunoaffinity capture technology we describe two mature isoforms both present in serum before and after high-dose IL-2 immunotherapy

Targeting Sphingosine Kinase 1 in Carcinoma Cells Decreases Proliferation and Survival by Compromising PKC Activity and Cytokinesis

Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy

Mitochondrial Dysfunction Links Ceramide Activated HRK Expression and Cell Death

Cell death is an essential process in normal development and homeostasis. In eyes, corneal epithelial injury leads to the death of cells in underlying stroma, an event believed to initiate corneal wound healing. The molecular basis of wound induced corneal stromal cell death is not understood in detail. Studies of others have indicated that ceramide may play significant role in stromal cell death following LASIK surgery. We have undertaken the present study to investigate the mechanism of death induced by C6 ceramide in cultures of human corneal stromal (HCSF) fibroblasts.Cultures of HCSF were established from freshly excised corneas. Cell death was induced in low passage (p<4) cultures of HCSF by treating the cells with C6 ceramide or C6 dihydroceramide as a control. Cell death was assessed by Live/Dead cell staining with calcein AM and ethidium homodimer-1 as well as Annexin V staining, caspase activation and TUNEL staining Mitochondrial dysfunction was assessed by Mito Sox Red, JC-1 and cytochrome C release Gene expression was examined by qPCR and western blotting.Our data demonstrate ceramide caused mitochondrial dysfunction as evident from reduced MTT staining, cyto c release from mitochondria, enhanced generation of ROS, and loss in mitochondrial membrane potential (ΔΨm). Cell death was evident from Live -Dead Cell staining and the inability to reestablish cultures from detached cells. Ceramide induced the expression of the harikari gene(HRK) and up-regulated JNK phosphorylation. In ceramide treated cells HRK was translocated to mitochondria, where it was found to interact with mitochondrial protein p32. The data also demonstrated HRK, p32 and BAD interaction. Ceramide-induced expression of HRK, mitochondrial dysfunction and cell death were reduced by HRK knockdown with HRK siRNA.Our data document that ceramide is capable of inducing death of corneal stromal fibroblasts through the induction of HRK mediated mitochondria dysfunction

sodC-Based Real-Time PCR for Detection of Neisseria meningitidis

Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (Ct) value of 35, with 100% average reaction efficiency and an average R2 of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average Ct values from 13.0 to 29.5. The mean sodC Ct value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes

Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue

Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK) isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs) abolished mSK1a phosphorylation, while overexpression of PKC alpha, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKC alpha directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residueopen

The HIV-1 Transactivator Factor (Tat) Induces Enterocyte Apoptosis through a Redox-Mediated Mechanism

The intestinal mucosa is an important target of human immunodeficiency virus (HIV) infection. HIV virus induces CD4+ T cell loss and epithelial damage which results in increased intestinal permeability. The mechanisms involved in nutrient malabsorption and alterations of intestinal mucosal architecture are unknown. We previously demonstrated that HIV-1 transactivator factor (Tat) induces an enterotoxic effect on intestinal epithelial cells that could be responsible for HIV-associated diarrhea. Since oxidative stress is implicated in the pathogenesis and morbidity of HIV infection, we evaluated whether Tat induces apoptosis of human enterocytes through oxidative stress, and whether the antioxidant N-acetylcysteine (NAC) could prevent it. Caco-2 and HT29 cells or human intestinal mucosa specimens were exposed to Tat alone or combined with NAC. In an in-vitro cell model, Tat increased the generation of reactive oxygen species and decreased antioxidant defenses as judged by a reduction in catalase activity and a reduced (GSH)/oxidized (GSSG) glutathione ratio. Tat also induced cytochrome c release from mitochondria to cytosol, and caspase-3 activation. Rectal dialysis samples from HIV-infected patients were positive for the oxidative stress marker 8-hydroxy-2′-deoxyguanosine. GSH/GSSG imbalance and apoptosis occurred in jejunal specimens from HIV-positive patients at baseline and from HIV-negative specimens exposed to Tat. Experiments with neutralizing anti-Tat antibodies showed that these effects were direct and specific. Pre-treatment with NAC prevented Tat-induced apoptosis and restored the glutathione balance in both the in-vitro and the ex-vivo model. These findings indicate that oxidative stress is one of the mechanism involved in HIV-intestinal disease

Intracellular S1P Generation Is Essential for S1P-Induced Motility of Human Lung Endothelial Cells: Role of Sphingosine Kinase 1 and S1P Lyase

Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P(1) receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility

A Deficiency of Ceramide Biosynthesis Causes Cerebellar Purkinje Cell Neurodegeneration and Lipofuscin Accumulation

Sphingolipids, lipids with a common sphingoid base (also termed long chain base) backbone, play essential cellular structural and signaling functions. Alterations of sphingolipid levels have been implicated in many diseases, including neurodegenerative disorders. However, it remains largely unclear whether sphingolipid changes in these diseases are pathological events or homeostatic responses. Furthermore, how changes in sphingolipid homeostasis shape the progression of aging and neurodegeneration remains to be clarified. We identified two mouse strains, flincher (fln) and toppler (to), with spontaneous recessive mutations that cause cerebellar ataxia and Purkinje cell degeneration. Positional cloning demonstrated that these mutations reside in the Lass1 gene. Lass1 encodes (dihydro)ceramide synthase 1 (CerS1), which is highly expressed in neurons. Both fln and to mutations caused complete loss of CerS1 catalytic activity, which resulted in a reduction in sphingolipid biosynthesis in the brain and dramatic changes in steady-state levels of sphingolipids and sphingoid bases. In addition to Purkinje cell death, deficiency of CerS1 function also induced accumulation of lipofuscin with ubiquitylated proteins in many brain regions. Our results demonstrate clearly that ceramide biosynthesis deficiency can cause neurodegeneration and suggest a novel mechanism of lipofuscin formation, a common phenomenon that occurs during normal aging and in some neurodegenerative diseases