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Background The FcγRs genotypes have been reported to play a key role in the defence against malaria parasites through both cellular and humoral immunity. This study aimed to investigate the possible correlation between FcγR (IIa, IIIa, and IIIb) genes polymorphism and the clinical outcome for anti‐malarial antibody response of Plasmodium falciparum infection among Saudi children. Methods A total of 600 volunteers were enrolled in this study, including 200 malaria-free control (MFC) subjects, 218 patients with uncomplicated malaria (UM) and 182 patients with severe malaria (SM). The FcγR genotypes were analysed using PCR amplification methods, and measurements of immunoglobulin were determined using enzyme-linked immunosorbent assay (ELISA) technique. Results The data revealed that the FcγRIIa-R/R131 showed a statistically significant association with SM patients when compared to UM patients. Furthermore, higher levels of IgG1, IgG2, and IgG4 were associated with the FcγRIIa-H/H131 genotype among UM patients. Although the FcγRIIa-F/V176 genotype was not associated with UM, it showed a significant association with severe malaria. Interestingly, the FcγRIIIa-V/V176 genotype offered protection against SM. Moreover, SM patients carrying the FcγRIIIa-F/F genotype showed higher levels of AMA-1-specific IgG2 and IgG4 antibodies. The FcγRIIIb-NA1/NA1 and FcγRIIIb-NA2/NA2 genotypes did not show significant differences between the UM and the MFC groups. However, the genotype FcγRIIIb-NA2/NA2 was statistically significantly associated with SM patients. Conclusions The data presented in this study suggest that the influence of the FcγRIIa-R/R131, FcγRIIIa-F/F176 and FcγRIIIb-NA2/NA2 genotypes are statistically significantly associated with SM patients. However, the FcγRIIa-H/H13 and FcγRIIIa-V/V176 genotypes have demonstrated a protective effect against SM when compared to UM patients. The impact of the FcyR (IIa, IIIa and IIIb) gene variants and anti-malaria IgG subclasses play an important role in susceptibility to malaria infection and disease outcome in Saudi children.

Objectives Despite its wide usage, warfarin therapy remains challenging due to its narrow therapeutic index, inter-individual response variability, and risk of bleeding. Previous reports have suggested that polymorphisms in VKORC1 and CYP2C9 genes could influence warfarin therapy. Herein, we investigated whether VKORC1 −1173C>T, CYP2C9*2, and CYP2C9*3 gene polymorphisms are associated with warfarin dose adjustment and related bleeding events. Methods This cross-sectional study was conducted on Saudi adults receiving warfarin for more than 1 month. Their demographics and relevant clinical data were obtained. Genotyping for VKORC1 −1173C>T, CYP2C9*2, and CYP2C9*2 genotypes was performed. Results Patients who are homozygous for the mutant T allele VKORC1 T/T required the lowest warfarin daily maintenance dose, compared to VKORC1 C/T and VKORC1 C/C. Similarly, there was a significant reduction in warfarin daily maintenance dose among CYP2C9*1/*3 and CYP2C9*1/*2 groups compared to CYP2C9*1/*1. However, we found no significant correlation between the studied polymorphisms and warfarin-associated bleeding. Conclusions Similar to other populations, the VKORC1 and CYP2C9 gene polymorphisms are significantly associated with warfarin dosage in Saudi patients. The presence of at least one copy of the mutant alleles for VKORC1 −1173C>T, CYP2C9*2, and CYP2C9*3 is associated with a significant reduction in warfarin maintenance dose.

Despite its wide usage, warfarin therapy remains challenging due to its narrow therapeutic index, inter-individual response variability, and risk of bleeding. Previous reports have suggested that polymorphisms inThis cross-sectional study was conducted on Saudi adults receiving warfarin for more than 1 month. Their demographics and relevant clinical data were obtained. Genotyping forPatients who are homozygous for the mutant T alleleSimilar to other populations, the

Awad Alshahrani,1–3 Mohammed AlDubayee,1–3 Mahmoud Zahra,3 Firas M Alsebayel,3 Nawaf Alammari,3 Faisal Alsudairy,3 Muath Almajed,3 Ahmad Aljada4 1Department of Medicine, Ministry of National Guard Health Affairs (MNG-HA), Riyadh, Kingdom of Saudi Arabia; 2King Abdullah International Medical Research Centre (KAIMRC), Riyadh, Kingdom of Saudi Arabia; 3College of Medicine, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, Kingdom of Saudi Arabia; 4Department of Biochemistry and Molecular Medicine, College of Medicine, Alfaisal University, Riyadh, Kingdom of Saudi ArabiaCorrespondence: Ahmad AljadaDepartment of Biochemistry and Molecular Medicine, College of Medicine, Alfaisal University, P.O. Box 50927, Riyadh 11533, Kingdom of Saudi ArabiaTel +966 112158 834Email aaljada@alfaisal.eduBackground: Interactions between environmental factors, such as diet and lifestyle, and metabolic pathways are pivotal in understanding aging mechanisms. hNAA40, Nicotinamide phosphoribosyltransferase (NAMPT), and NAD-dependent protein deacetylase sirtuin-1 (SIRT-1) have been shown to exert important biological processes, including stress response and aging.Methods: hNAA40, NAMPT, and SIRT-1 mRNA expression in peripheral blood mononuclear cells (PBMC) were quantitated in 30 lean adult volunteers of normal weight, 30 obese, 20 drug-naïve obese Type 2 diabetes mellitus (T2DM), and 30 obese T2DM on Metformin. Similarly, hNAA40, NAMPT, and SIRT-1 expression in PBMC were quantitated in 36 normal healthy adults randomly assigned to three different groups (Glucose or Whey proteins or lipids; 300 kcal). Blood samples were obtained at 1, 2, and 3 hrs after the macronutrient intake.Results: There was an increase in hNAA40 and a decrease in NAMPT and SIRT-1 expression in PBMC from T2DM. Metformin treatment reverted hNAA40, NAMPT, and SIRT-1 expression levels to normal levels. Glucose intake resulted in a significant increase in expression of hNAA40 at 1 hr and decreased significantly at 3 hrs post intake. Lipid intake resulted in an increase in expression of hNAA40 at 2 hr post intake and returned to normal levels at 3 hrs. Neither glucose nor lipid intake resulted in a significant change in NAMPT or SIRT-1 expression. Whey proteins resulted in significantly lower expression of NAMPT at 3 hrs and did not alter the expression levels of SIRT-1 significantly.Conclusion: hNAA40, NAMPT, and SIRT-1 pathway could play a role in the determination of the healthy life-span. Metformin modulates this pathway.Keywords: hNAA40, nicotinamide phosphoribosyltransferase, NAMPT, sirtuin-1, T2DM, obesity, senescence

Background: The acetate ion has important physiological functions and important therapeutic applications. A rapid LC–MS/MS method is described to measure acetate ions in human plasma without chemical derivatization. Materials & methods: A 200 μl sample was spiked with the internal standard 1,2-13C-acetate and proteins precipitated with trichloroacetic acid. The supernatant was recovered and separated under acidic conditions on a C18-column. The eluent was alkalinized by post-column infusion of methanolic ammonium hydroxide. Acetate ions were monitored on a low resolution mass spectrometer in negative ion mode. Results: Method was validated for accuracy and precision with a lower limit of quantitation of 9.7 μM and linear dynamic range up to 339.6 μM. Conclusion: The method is open for analytical improvement and adapts with metabolomic and pharmacometabolomic studies on chemicals of similar nature.

Background: The FcyRs genotypes have been reported to play a key role in the defence against malaria parasites through both cellular and humoral immunity. This study aimed to investigate the possible correlation between FcγR (IIa, IIIa, and IIIb) genes polymorphism and the clinical outcome for anti‐malarial antibody response of Plasmodium falciparum infection among Saudi children. Material and methods: A 600 volunteers have been enrolled in this study, including 200 malaria-free control (MFC) subjects, 218 patients with uncomplicated malaria (UM) and 182 patients with severe malaria (SM). The FcγR genotypes was analysed using PCR amplification methods, and measurement of immunoglobulins were determine using ELISA. Results: The data revealed the FcγRIIa-R/R131 showed a statistically association with the increased susceptibility to SM when compared to UM patients. Furthermore, higher levels of IgG1, IgG2, and IgG4 were associated with the FcγRIIa-H/H131 genotypes among UM patients. Although the FcγRIIa-F/V176 genotype was not associated with UM, it showed a significant association with severe malaria. Interestingly, the FcγRIIa-V/V176 genotype was This study aimed to associated with protection against SM. Moreover, severe malaria patients carrying the FcγRIIa-F/F genotype showed higher levels of AMA-1-specific IgG2 and IgG4 antibodies. The FcγRIIIb NA1/NA1 and FcγRIIIb NA2/NA2 genotypes did not show significant differences between UM and the MFC. However, the genotype FcγRIIIb-NA2/NA2 was statistically associated with severe malaria. Conclusions: The data presented in this study strongly suggest the possible impact of FcyR (IIa, RIIIa and RIIIb) gene variants and anti-malaria IgG subclasses play a role in susceptibility to malaria infection and disease outcomes in Saudi children.

Background The identification of unique senescence markers remains challenging. Current hallmarks of senescent cells, including increased senescence-associated β-galactosidase activity, increased levels of cell cycle regulators such as p16INK4a, p27, and p53, and altered levels of sirtuins and lamins, are detected commonly by Western blot and immunohistochemistry methods. Mass spectrometry outperforms these conventional quantification methods in terms of high throughput, specificity, and reproducibility. Objectives To develop multiple reaction monitoring-based tandem mass spectrometric senescence assay for simultaneous measuring of p16INK4a, p27, p53, p53-β, the seven proteins of the sirtuins family and the four transcript variants of lamins proteins in aging cell model and cancerous cell lines. Methodology Multiple reaction monitoring-tandem mass transitions per protein were developed for each signature peptide(s) and stable isotope-labeled internal standard. The developed assay was validated in a matrix using breast cancer MCF7 cell lines according to the US-FDA guidelines for bioanalytical assays. Results The analytes chromatographic peaks were baseline separated and showed linear behavior in a wide dynamic range with r2 ≥ 0.98. The method for all proteins has passed the inter/intra-day precision and accuracy validation using three levels of quality control samples. The accuracy and the precision for most analytes were 80-120% and ≤20%, respectively. The method's sensitivity for the panels' signature peptides ranged from 1 ng μL-1 to 1 μg mL-1. Extraction recovery assessed in two quality control levels was >60% for most analytes. This LC-MS-MS validated senescence assay showed reduced lamin A, lamin A△10, lamin A△50, SIRT1, SIRT3, SIRT5, p53, and p16INK4a, as well as p53-β induction, are implicated in replicative senescence. Meanwhile, increased lamin C: lamin A ratio was evident and can diagnose breast carcinogenesis. Moreover, in breast cancer metastasis, reduced SIRT2 and p27 and elevated levels of lamin A△50, SIRT5, SIRT7, and p53-β are evident. Conclusion LC-MS/MS is a potent alternative tool to the currently available assays. The high throughput method established can study senescence's role in different pathophysiological processes.

Background Although there is increasing evidence showing that cell senescence is increased in circulating PBMC in type 2 diabetes mellitus (T2DM), the data are contradictory. This study examined several senescence biomarkers, including LMNA/C transcript variants, p16INK4a, p53, and p21Cip1/WAF, in PBMC of T2DM patients and the effect of Metformin on these senescence markers. Methods Blood samples were obtained from 30 lean, 30 obese, 20 newly diagnosed type 2 diabetes mellitus (T2DM), and 30 T2DM on Metformin. PBMC were isolated and mRNA expression of the senescence biomarkers were quantified by RT-qPCR. The effect of ectopic expression of LMNA and LMNC in human monocytic cells lines (THP-1 and U937) on several inflammatory mediators were also examined. Results LMNA expression was significantly higher in PBMC of obese and T2DM patients. LMNC expression was significantly inhibited in T2DM patients. LMNAΔ10 and Progerin mRNA expression was not detected in PBMC of all groups. Expression of p16INK4a, p21Cip1/WAF and p53 were inhibited significantly in T2DM. Metformin treatment reverted LMNA, LMNC, and p53 expression levels to normal levels. Upregulation of LMNA in monocytic THP-1 and U937 cell lines induced CD68, TNFα, CCL2, IL-6 and NOS2. Conclusions These data support the notion that LMNA may mediate senescence in PBMCs of T2DM by upregulating inflammatory pathways. Metformin may exert its anti-inflammatory property by modulation of senescence mediator LMNA.

Background: Although peripheral blood mononuclear cells (PBMC) have been demonstrated to be in a pro-inflammatory state in obesity and type 2 Diabetes Mellitus (T2DM), characterization of circulating PBMC phenotypes in the obese and T2DM and the effect of Metformin on these phenotypes in humans is still ill-defined and remains to be determined. Methods: Thirty normal healthy adult volunteers of normal weight, 30 obese subjects, 20 obese newly diagnosed diabetics and 30 obese diabetics on Metformin were recruited for the study. Fasting blood samples were collected and PBMC were isolated from whole blood. Polarization markers (CD86, IL-6, TNFα, iNOS, CD36, CD11c, CD169, CD206, CD163, CD68, CD11b, CD16 and CD14) were measured by RT-qPCR. Gene expression fold changes were calculated using the 2ˆ(–delta delta Ct) method for RT-qPCR. Results: Obesity and T2DM are associated an increased CD68 marker in PBMC. mRNA expression of CD11b, CD11c, CD169 and CD163 were significantly reduced in PBMC from T2DM subjects whereas CD11c was significantly inhibited in PBMC from obese subjects. On the other hand, macrophage M1-like phenotype was observed in T2DM circulation as demonstrated by increased mRNA expression of CD16, IL-6, iNOS, TNFα, and CD36. There were no significant changes in CD14 and CD86 in the obese and T2DM when compared to the lean subjects. Metformin treatment in T2DM reverted CD11c, CD169, IL-6, iNOS, TNFα, and CD36 to levels comparable to lean subjects. CD206 mRNA expression was significantly upregulated in PBMC of T2DM while Metformin treatment inhibited CD206 expression levels. Conclusions: These data support the notion that PBMC in circulation in T2DM express different pattern of phenotypic markers than the patterns typically present in M1 and M2 like cells. These phenotypic markers could be representative of metabolically activated macrophages (MMe)-like cells. Metformin, on the other hand, reduces MMe-like cells in circulation.

Background: Three subsets of human monocytes in circulation have been identified and their characterization is still ill-defined. Although glucose and lipid intakes have been demonstrated to exert pro-inflammatory effects on mononuclear cells (MNCs) of healthy subjects, characterization of monocytes phenotypes following macronutrient (glucose, protein, and lipid) intake in humans remains to be determined. Methods: Thirty-six healthy, normal weight volunteers were recruited in the study. Subjects were randomly assigned into three groups, each group consisting of 12 participants. Each group drank equal calories (300 kcal) of either glucose or lipids or Whey proteins. Each subject served as his own control by drinking 300 mL of water 1 week before or after the caloric intake. Baseline blood samples were drawn at 0, 1, 2 and 3-hour intervals post caloric or water intakes. MNCs were isolated, and the expression levels of different cluster of differentiation (CD) markers (CD86, CD11c, CD169, CD206, CD163, CD36, CD68, CD11b, CD16, and CD14) and IL-6 were measured by RT-qPCR. Results: Equicaloric intake of either glucose or lipids or Whey proteins resulted in different monocyte phenotypes as demonstrated by changes in the expression levels of CD and polarization markers. Whey proteins intake resulted in significant mRNA upregulation in MNCs of CD68 and CD11b at 1, 2, and 3 hrs post intake while mRNA of IL-6 was significantly inhibited at 1 hr. Lipids intake, on the other hand, resulted in mRNA upregulation of CD11b at 2 and 3 hrs and CD206 at 1, 2 and 3 hrs. There were no significant changes in the other CD markers measured (CD86, CD163, CD169, CD36, CD16 and CD14) following either Whey proteins or lipids intakes. Glucose intake did not alter mRNA expression of any marker tested except CD206 at 3 hrs. Conclusions: Macronutrient intake alter the expression levels of polarization markers in MNCs of human subjects. A distinct population of different monocytes phenotypes may result in human circulation following the intake of different macronutrients. Further studies are required to characterize the immunomodulatory effects of macronutrients intake on monocytes phenotypes and their characteristics in humans.

Fulani and Masaleit are two sympatric ethnic groups in western Sudan who are characterised by marked differences in susceptibility to Plasmodium falciparum malaria. It has been demonstrated that Glucose-6-phosphate dehydrogenase (G6PD) deficiency and Sickle cell trait HbAS carriers are protected from the most severe forms of malaria. This study aimed to investigate a set of specific IgG subclasses against P. falciparum Apical Membrane Antigen 1 (AMA-1 3D7), haemoglobin variants and (G6PD) in association with malaria susceptibility among Fulani ethnic group compared to sympatric ethnic group living in Western Sudan. A total of 124 children aged 5–9 years from each tribe living in an area of hyper-endemic P. falciparum unstable malaria transmission were recruited and genotyped for the haemoglobin (Hb) genes, (G6PD) and (ABO) blood groups. Furthermore, the level of plasma IgG antibody subclasses against P. falciparum antigen (AMA-1) were measured using enzyme linked immunosorbent assays (ELISA). Higher levels of anti-malarial IgG1, IgG2 and IgG3 but not IgG4 antibody were found in Fulani when compared to Masaleit. Individuals carrying the HbCC phenotype were significantly associated with higher levels of IgG1 and IgG2. Furthermore, individuals having the HbAS phenotype were associated with higher levels of specific IgG2 and IgG4 antibodies. In addition, patients with G6PD A/A genotype were associated with higher levels of specific IgG2 antibody compared with those carrying the A/G and G/G genotypes. The results indicate that the Fulani ethnic group show lower frequency of HbAS, HbSS and HbAC compared to the Masaleit ethnic group. The inter-ethnic analysis shows no statistically significant difference in G6PD genotypes (P value = 0.791). However, the intra-ethnic analysis indicates that both ethnic groups have less A/A genotypes and (A) allele frequency of G6PD compared to G/G genotypes, while the HbSA genotype was associated with higher levels of IgG2 (AMA-1) and IgG4 antibodies. In addition, patients carrying the G6PD A/A genotype were associated with higher levels of specific IgG2 antibody compared with those carrying the A/G and G/G genotypes. The present results revealed that the Fulani ethnic group has statistically significantly lower frequency of abnormal haemoglobin resistant to malaria infection compared to the Masaleit ethnic group.

Background To evaluate sirtuin-7 (SirT7) mRNA expression status in breast cancer patients with different metastatic stages and survey SirT7 mRNA expression status in eight different types of cancer. Methods The expression of SirT7 in the commercially available TissueScan qPCR Breast Cancer Disease cDNA arrays containing 16 normal, 23 Stage I, 36 IIA, 22 IIB, 8 IIIA, 23 IIIA, 6 IIIB, 13 IIIC, and 5 IV were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Similar analysis was performed in TissueScan qPCR Cancer Survey cDNA array, which includes breast, colon, kidney, liver, lung, ovarian, prostate, and thyroid specimens. Results The mRNA expression levels of SirT7 were significantly higher in breast cancer samples compared to normal breast specimens ( P < 0.001). Stratification of patients into groups according to metastatic stages indicated statistically significantly higher levels of SirT7 mRNA in CS-I, CS-II, and CS-III when compared to normal breast tissue ( P < 0.05). Notably, SirT7 mRNA levels were higher in CS-I, CS-IIA, CS-IIB, and CS-IIIA ( P < 0.05). Additionally, there were significantly lower SirT7 mRNA levels in thyroid carcinoma when compared to their corresponding normal tissue ( P < 0.05). Conclusions Our results indicate an increase in the mRNA expression level of SirT7 in breast cancer, particularly in CS-I, CS-IIA, CS-IIB, and CS-IIIA. The relationship of altered SirT7 with breast cancer progression and patient survival should be prospectively explored in future studies.

Background/Aims: The antileukemic potential of isoindigos make them desired candidates for understanding their mechanism of action. We have recently synthesized a novel group of pyridone-annelated isoindigos and identified the derivative 5'-Cl that is cytotoxic to various cancer cell lines. In the present study, we analyzed the effect of this compound on cell cycle of the promyelocytic leukemia cell line HL-60. Methods: HL-60 cells were treated with 5'-Cl and its effect on cell cycle stages were determined by flow cytometry. Expression of cyclins, cyclin dependent kinases (CDKs) and cyclin kinase inhibitors (CKIs) were determined by Western blotting, and activation of CDKs was studied using kinase assays. Results: 5'-Cl remarkably arrested cell cycle in HL-60 cells at the G0/G1 phase in a dose and time-dependent manner. Furthermore, 5'-Cl treatment significantly inhibited expression of D-cyclins, CDK2 and CDK4 and suppressed phosphorylation of the retinoblastoma protein Rb, whereas it increased the level of CKI p21. Molecular modelling experiments show that 5'-Cl may compete with ATP for binding to the catalytic subunit of CDK2 and CDK4 that could lead to inhibition of these enzymes. Indeed, 5'-Cl inhibited the kinase activity of CDK2 and CDK4 both in cell free systems and in treated cells. 5'-Cl also inhibited cell cycle progression in several other tumor cell lines. Conclusion: We demonstrate the potent inhibitory effects of 5'-Cl on HL-60 cells could be mediated by arresting cells in the G0/G1 phase.

Background/Aims: In our quest to develop an isoindigo with improved efficacy and bioavailability, we recently synthesized a series of novel substituted pyridone-annelated isoindigo and evaluated their antiproliferative effects. We identified the compound [(E)-1-(5'-Chloro-2'-oxoindolin-3'-ylidene)-6-ethyl-2,3,6,9-tetrahydro-2,9-dioxo-1H-pyrrolo[3,2-f] quinoline-8-carboxylic acid], abbreviated as 5'-Cl, which shows selective antiproliferative activities against various cancer cell lines mediated through apoptosis. Here we have investigated the molecular mechanisms underlying the apoptotic activity of 5'-Cl in the human promyelocytic leukemia HL-60 cells. Methods: We employed different methods to determine the apoptotic pathways triggered by 5'-Cl in HL-60 cells, using flow cytometry, nuclear staining, caspases activation, mitochondria functioning, generation of reactive oxygen species (ROS) and Western blotting techniques. Results: Low concentrations (1-8 µM) of 5'-Cl inhibited the growth of HL-60 cells in a dose and time-dependent manner. Cytotoxicity of this compound is found to be mediated by a caspase-dependent apoptosis. Also, there were indications of caspase independent apoptosis as z-VAD-FMK failed to fully rescue the cells from 5‘-Cl-induced apoptosis. In addition, the compound triggered generation of Reactive Oxygen Species (ROS), caused depolarization of the mitochondrial inner membrane, decreased the level of cellular ATP, modulated the expression and phosphorylation of Bcl-2 leading to loss of its association with Bax and increased the release of cytochrome c to the cytosol of treated cells. The effects of 5‘-Cl on mitochondria and apoptosis were substantially blocked in the presence of a combination between z-VAD-FMK and either of the ROS scavenger N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC). Conclusion: We demonstrated that the growth inhibitory effects of 5'-Cl in HL-60 cells involve multiple pathways of apoptosis and dysregulation of mitochondrial functions.

Background and Aim: Neonatal infection, including bacterial sepsis, is a major health care issue with an annual global mortality in excess of one million lives. Therefore, this study aimed to evaluate the potential diagnostic value of C-reactive protein (CRP), E-selectin, procalcitonin (PCT), interleukins-6 (IL-6), and tumor necrosis factor-α (TNF-α) both independently and in combination for the diagnosis of neonatal sepsis in its earliest stages. Materials and Methods: A total of 320 subjects were included in this study. A prospective cross-sectional study was conducted among neonates admitted to Neonatal Intensive Care Unit at King Abdulaziz Medical City, Riyadh, KSA during January 2013 to August 2015, the study based on three study groups categorized according to clinical symptoms and blood culture result. Study groups include healthy control neonates (n = 80), clinical sepsis (CS) group (n = 80) with clinical signs of sepsis but their blood culture was negative, and sepsis group with clinical signs of sepsis and their blood culture was positive. Results: The study observed significant difference in plasma levels of CRP, IL-6, TNF-α, E-selectin, and PCT in patients group when compared with control group (P < 0.001). Furthermore, the levels are significantly different between patient groups including CS and neonatal sepsis group. Moreover, result observed significant difference in CRP and IL-6 in early onset sepsis (EOS) when compared with late onset sepsis (LOS) neonates (P < 0.001 and 0.01), respectively, while there were no significant difference in TNF-α, E-selectin, and PCT between EOS and LOS (P = 0.44, 0.27 and 0.24), respectively. Regarding biomarkers accuracy, the result showed that CRP has the best diagnostic accuracy with cutoff value of 3.6 ng/ml (sensitivity 78% and specificity of 70%). The best combination is shown with CRP and IL-6 in which sensitivity increased to 89% and specificity to 79%. Conclusion: It was concluded that infected new-born babies have a higher E-selectin, PCT, IL-6, TNF-α, and CRP compared with the neonates with CS and control. IL-6, TNF-α, and CRP should be measured in combination for mare diagnostic accuracy in neonatal sepsis. Likewise, PCT should be investigated as a part of sepsis screening for all suspected neonates.

This study examined the effect of selenium (Se) or lycopene (Lyc) and their combination on oxida- tive stress in bone following cadmium (Cd) exposure in vivo. Cd exposure enhanced accumulation of Cd in femur with subsequent increase in LPO and PC and decreased antioxidative enzyme activ- ities in rat femur, along with significant decreases in body and femur bone weights. Subcutaneous (s.c.) injection of Se or Lyc reduced Cd accumulation in bone and increased body and femur weights, when given individually or concomitantly. Antioxidant enzyme activities maintained and/or in- creased following Se and Lyc supplementation when given individually or concomitantly. However, lycopene dose of 10 mg/kg of body weight alone or selenium alone provided better protection to bone tissues of rats against oxidative stress compared to treatment with their combination. Sele- nium and lycopene could be used as a dietary supplement to protect against bone oxidative stress in areas exposed to Cd pollution.

Purpose: This study examined the effect of permissive underfeeding compared to target feeding and intensive insulin therapy (IIT) compared to conventional insulin therapy (CIT) on the inflammatory mediators monocyte chemoattractant protein 1 (MCP-1), soluble intercellular adhesion molecule 1 (sICAM-1), and tissue factor (TF) in critically ill patients. Methodology: This was a substudy of a 2 &times, 2 factorial design randomized controlled trial in which intensive care unit (ICU) patients were randomized into permissive underfeeding compared to target feeding groups and into IIT compared to CIT groups (ISRCTN96294863). In this substudy, we included 91 patients with almost equal numbers across randomization groups. Blood samples were collected at baseline and at days 3, 5, and 7 of an ICU stay. Linear mixed models were used to assess the differences in MCP-1, sICAM-1, and TF across randomization groups over time. Results: Baseline characteristics were balanced across randomization groups. Daily caloric intake was significantly higher in the target feeding than in the permissive underfeeding groups (P-value &lt, 0.01), and the daily insulin dose was significantly higher in the IIT than in the CIT groups (P-value &lt, 0.01). MCP-1, sICAM-1, and TF did not show any significant difference between the randomization groups, while there was a time effect for MCP-1. Baseline sequential organ failure assessment (SOFA) score and platelets had a significant effect on sICAM-1 (P-value &lt, 0.01). For TF, there was a significant association with age (P-value &lt, 0.01). Conclusions: Although it has been previously demonstrated that insulin inhibits MCP-1, sICAM-1 in critically ill patients, and TF in non-critically ill patients, our study demonstrated that IIT in critically ill patients did not affect these inflammatory mediators. Similarly, caloric intake had a negligible effect on the inflammatory mediators studied.

Lamin A/C proteins have key roles in nuclear structural integrity and chromosomal stability. Lamin A/C cumulative protein expression of all variants is reported by semi-quantitative Western blotting. To date, there have not been specific antibodies for the individual Lamin A/C transcript variants. We developed a mass spectrometric approach for the quantification of Lamin A/C transcript variants. A signature peptide for each specific splice variant of Lamin A/C was selected. A LC&ndash, MS/MS assay based on the selected signature peptides and their labeled internal standards was established to measure the expression of Lamin A/C transcript variant concentrations. The method validation was carried out according to Food and Drug Administration (FDA) guidelines. The expression levels of the Lamin A/C transcript variants were measured in samples derived from MCF7 and U937 cell lines. RT-qPCR assay was also used to quantitate and compare the mRNA expression of splice variants of Lamin A/C. The established and validated method showed a great linearity, sensitivity, and precision. The different expressed Lamin A/C variants in different cell lines were measured and their levels were in concordance with qRT-PCR results. The developed method is reproducible, reliable, and sensitive for measuring different Lamin A/C transcript variants in different cell lines.

Natural and chemically synthesized heterocyclic compounds have been explored for their potential use as anticancer agents. We had synthesized non-natural heterocyclic analogs and evaluated their anti-tumor activity by measuring effect on cell proliferation and induction of apoptosis in different cell lines. Previously, we identified a pyrazole-containing compound (G-11) showing cytotoxic effect towards leukemia and lymphoma cell lines. In this study, we further investigated the mechanistic aspects of anticancer properties of G-11 in HL-60 cell line. We demonstrated that cytotoxic effect of G-11 is mediated by caspase-dependent apoptosis. However, the involvement of mitochondrial dysfunction induced by G-11 was independent of caspases. G-11 triggered generation of ROS, caused disruption of mitochondrial transmembrane potential, increased release of cytochrome c to the cytosol, and altered the expression of Bcl-2 and Bax proteins. These results suggest significant involvement of intrinsic apoptotic pathway. This study comprehensively details the possible mechanisms of action of a novel heterocyclic compound which could find its potential use as an anticancer agent.

Metformin has been shown to exert anti-neoplastic and chemopreventive activities in epidemiological and animal studies. This review article discusses the epidemiological studies and examines the possible mechanisms by which metformin exerts its anti-carcinogenic activities in breast, colon, ovarian, lung, and prostate cancers. We performed a systematic review of the clinical studies examining the anti-neoplastic activities of metformin and the potential mechanisms associated with these activities. Several observational and biological studies revealed a significant association between metformin and reduction in cancer incidence. The mechanisms by which metformin exerts these effects are unknown. This action may be mediated through activation of AMP-activated protein kinase (AMPK), inhibition of the mammalian target of rapamycin (mTOR) pathway, and inhibition of insulin like growth factors (IGFs), and many others. Further laboratory investigation and large, prospective population clinical trials are required to elucidate metformin anti-neoplastic and chemo-preventive actions.

Lamin A/C alternative splice variants (Lamin A, Lamin C, Lamin AΔ10 and Lamin AΔ50) have been implicated in cell cycle regulation, DNA replication, transcription regulation, cellular differentiation, apoptosis and aging. In addition, loss of Lamin A/C expression has been observed in several cancers, including breast cancer, and it has been found that Lamin A/C suppression may lead to cancer-like aberrations in nuclear morphology and aneuploidy. Based on these observations, we hypothesized that Lamin A/C transcript variant quantification might be employed for the diagnosis of breast cancer. Newly designed TaqMan qRT-PCR assays for the analysis of Lamin A/C splice variants were validated and their use as biomarkers for the diagnosis of breast cancer was assessed using 16 normal breast tissues and 128 breast adenocarcinomas. In addition, the expression levels of the Lamin A/C transcript variants were measured in samples derived from seven other types of cancer. We found that the expression level of Lamin C was significantly increased in the breast tumors tested, whereas the expression levels of Lamin A and Lamin AΔ50 were significantly decreased. No significant change in Lamin AΔ10 expression was observed. Our data also indicated that the Lamin C : Lamin A mRNA ratio was increased in all clinical stages of breast cancer. Additionally, we observed increased Lamin C : Lamin A mRNA ratios in liver, lung and thyroid carcinomas and in colon, ovary and prostate adenocarcinomas. From our data we conclude that the Lamin C : Lamin A mRNA ratio is increased in breast cancer and that this mRNA ratio may be of diagnostic use in all clinical stages of breast cancer and, possibly, also in liver, lung, thyroid, colon, ovary and prostate cancers.

To study the effect of peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonists, pioglitazone and rosiglitazone, on vascular endothelial growth factor (VEGF)- and basic fibroblast growth factor (bFGF)-induced angiogenesis and on endothelial cell migration.Chick chorioallantoic membrane (CAM) model was used to evaluate the efficacy of pioglitazone and rosiglitazone on VEGF- and bFGF-induced angiogenesis. In addition, the effect of pioglitazone and rosiglitazone on endothelial cell migration was evaluated using 8 mm pore filter to a feeder layer containing vitronectin as chemoattractant.Pioglitazone and rosiglitazone inhibited the pro-angiogenic effects of bFGF and VEGF in the CAM model significantly (P0.001) to the same extent. Endothelial cell migration was also inhibited by both pioglitazone and rosiglitazone (P0.001).These results suggest that PPAR gamma ligands, pioglitazone and rosiglitazone, in addition to their important regulatory role in adipogenesis and inflammation, possess anti-angiogenic properties. Thus, PPAR gamma ligands may be useful in the treatment of diabetic retinopathy, macular degeneration, and other ocular disorders and may lower the risk to develop cancer in diabetic patients.

Thyroid hormone has been recently shown to induce tumor growth and angiogenesis via a plasma-membrane hormone receptor on integrin alphaVbeta3. The receptor is at or near the Arg-Gly-Asp (RGD) recognition site on the integrin that is important to extracellular matrix (ECM) protein and vascular growth factor interactions with the integrin. In the present study, we examined the possibility that tetraiodothyroacetic acid (tetrac), a deaminated, non-agonist thyroid hormone analog that binds to the integrin receptor, may modulate vascular growth factor-induced angiogenesis in the absence of thyroid hormone. Angiogenesis models were studied in which VEGF or FGF2 (1-2 microg/ml) induced tube formation in human dermal microvascular endothelial cells (HDMEC), stimulated new blood vessel branch formation in the chick chorioallantoic membrane (CAM) and induced angiogenesis in the mouse matrigel model. In all models, tetrac (1-10 microM) and at 10 microg in mouse matrigel inhibited the pro-angiogenesis activity of VEGF and FGF2 by more than 50%. RT-PCR revealed that tetrac (1-3 microM) decreased abundance of angiopoietin-2 mRNA, but not angiopoietin-1 mRNA, in VEGF-exposed endothelial cells, suggesting that specific angiogenic pathways are targeted by tetrac. Tetrac is a novel, inexpensive small molecule whose anti-angiogenic activity in the present studies is proposed to reflect inhibition, via the integrin RGD recognition/thyroid hormone receptor site, of crosstalk between plasma-membrane vascular growth factor receptors and integrin alphaVbeta3.

The present investigation determined the molecular structure and the pharmacokinetic and pharmacodynamic profiles of oral unfractionated heparin containing oral absorption enhancer sodium N-[8-(2-hydroxybenzoyl) amino]caprylate, salcaprozate sodium (SNAC) and assessed the safety and tolerability of the orally dosed heparin solid dosage form versus other routes. Sixteen healthy men were included in this single-dose, 3-way crossover, randomized, open-label study. Disaccharide compositional analysis was performed using capillary high-performance liquid chromatography with electrospray ionization mass spectrometry detection. The pharmacodynamics of heparin were obtained from analysis of plasma anti-factor Xa, anti-factor IIa, activated partial thromboplastin time, and total tissue factor pathway inhibitor data. The molecular weight properties and the disaccharide composition of orally administered unfractionated heparin/SNAC and parenterally administered unfractionated heparin are identical and consistent with the starting pharmaceutical standard heparin. Furthermore, the anti-factor Xa/anti-factor IIa ratio achieved is of approximately 1:1. This is the first true pharmacokinetic study to measure the chemical compositions of heparin administered by different routes.

BACKGROUND The mature human insulin receptor (INSR) has two isoforms: The A isoform and the B isoform. INSR upregulation has been suggested to play a role in cancer. OBJECTIVE To establish quantitative PCR method for INSR transcript variants and examine their differential expression as a diagnostic tumor marker in breast cancer. METHODS The differential expression of IR-A and IR-B were evaluated by TaqMan qRT-PCR assay in the commercially available Breast Cancer Disease cDNA and Cancer Survey cDNA arrays. RESULTS The mRNA expression levels of IR-A was statistically significantly higher in breast cancer when compared to normal breast tissue while IR-B mRNA expression was down regulated significantly in breast cancer. Stratification of patients into groups according to metastatic stages indicated statistically significantly higher levels of IR-A mRNA in clinical stage (CS)-IV, and lower IR-B levels in CS-IIA, CS-IIIB and CS-IIIC. However, IR-A:IR-B ratio showed a statistically significant increase in all stages. Cancer Survey cDNA array demonstrated lower levels of IR-B mRNA in breast adenocarcinoma, liver carcinoma and lung carcinoma only while IR-A expression was significantly altered in kidney carcinoma without any significant differences in IR-A:IR-B ratios. CONCLUSIONS The results demonstrate an increased IR-A:IR-B ratio in all clinical stages of breast cancer. Thus, IR-A:IR-B ratio may have a diagnostic biomarker utility in breast cancer.

Sirtuin (SirT), a family of conserved histone deacetylases and transferases, has been proposed to function in inflammatory, cancer, and metabolic diseases. However, it is unclear how SirT modulates these processes. In this study, the effect of metformin on senescence and antisenescence mediators (SirT1-7, p53, and p16(INK4a)) mRNA expression in white blood cells (WBCs) following lipopolysaccharides (LPS)-induced inflammation in mice was examined.C57BL/6 mice were treated with metformin in their drinking water (2 mg/mL) for 1 week followed by intraperitoneal injection of LPS from Escherichia coli serotype 0111:B4 at 2 mg/kg. Blood was collected at the basal level and 1, 2, and 3 hr after LPS injection. SirT1-7, p53, and p16(INK4a) mRNA expression in WBCs was measured by real-time quantitative polymerase chain reaction (RT-qPCR).SirT7 at 2 hr, SirT1 at 3 hr, and p16(INK4a) at 1 hr were inhibited significantly in WBCs following LPS injection. There were no significant changes in other SirT nor p53 mRNA expression in WBCs after LPS injection. Metformin inhibited SirT2 expression in WBCs significantly (P0.05) and did not induce any significant changes in other SirT forms and p53, whereas it induced p16(INK4a) mRNA expression in WBCs (P0.05) at the basal levels. Additionally, metformin treatment significantly inhibited SirT7, SirT1, and p16(INK4a) mRNA expression in WBCs at 1, 2, and 3 hr, whereas p53 was inhibited significantly at 2 hr after LPS injection.SirT7 and SirT1 are stress responsive proteins that may mediate inflammation. The data suggest that metformin may exert its potential antisenescence and anti-inflammatory effects by targeting SirT7 and SirT1 pathways. SirT7 inhibition may allow the healing process and prevention of tissue damage by enabling cells to survive through inhibition of cytokines and inflammatory mediators under severe stress conditions.

Obesity is associated with insulin resistance and inflammation. The circulating human mononuclear cell (MNC) has been shown to respond to low-dose insulin infusion. We have now investigated whether in obesity: (1) phosphorylated insulin receptor beta subunit (p-INSR-beta) is reduced in the MNC; (2) pro-inflammatory mediators including inhibitor of kappa light polypeptide gene enhancer in B cells-kinase beta (IKBKB), suppressor of cytokine signalling-3 (SOCS) and protein kinase C-beta 2 (PRKCB2) are increased and related to p-INSR-beta; and (3) the reduction in MNC p-INSR-beta is related to the reduction in insulin sensitivity.MNCs were prepared from fasting blood samples of 16 normal weight and 16 obese female subjects.Our data show that p-INSR-beta is reduced significantly in MNCs from obese subjects compared with that of normal controls. MNCs from obese subjects have higher IKBKB expression, increased nuclear factor kappa B (NFkappaB) binding and higher mRNA expression of TNFAIP1 and IL6 genes. NFkappaB binding, TNFAIP1 mRNA and plasma C-reactive protein are inversely related to p-INSR-beta. PRKCB2 mRNA and protein expression were significantly higher in the obese subjects and were related significantly to pro-inflammatory mediators but not to p-INSR-beta. SOCS3 mRNA expression was markedly elevated and positively related to pro-inflammatory mediators including IKBKB and PRKCB2 on the one hand and inversely related to p-INSR-beta on the other.We conclude that in obesity the MNC is characterised by reduced p-INSR-beta and increased inflammatory mediators including IKBKB, PRKCB2 and SOCS3. The increase in SOCS3 but not IKBKB or PRKCB2 is related inversely to p-INSR-beta and might mediate the inhibition of p-INSR-beta. These data elucidate the relationship between inflammation and insulin resistance using the MNC as a model.