Kinase inhibitors: 20 years of success and many new challenges and recent trends in their patents.

INTRODUCTION: Protein kinases (PKs) play key roles in cellular signaling and regulation cascades and therefore are listed among the most investigated enzymes with the intent to develop drugs that are able to modulate their catalytic features. Specifically, PKs are involved in chronic diseases of large impact in the society such as cancers and neurodegeneration. Since the approval of Fasudil for the management of cerebral vasospasm, frantic efforts are currently ongoing for the development of selective PK-modulating agents. AREAS COVERED: A selection of the most relevant patents in the European Patent Office for biomedical innovation and/or industrial development covering the years 2020-2023 on PK modulators either of the antibody and small-molecule type is reported. In addition to the examined patents, we also reported the contributions claiming the use of antibody-targeted PKs for lab bench identification kits. EXPERT OPINION: The field of PK modulators for biomedical purposes is particularly crowded with contributions, making it rich in valuable information for the development of potential drugs. An emerging frontier is represented by PK activators that aims to complement the use of PK inhibitors with the final intent of finely adjusting any PK-related disruption responsible for triggering any disease.

Effects of unpredictable chronic mild stress on the cellular redox state and mitochondrial energy homeostasis in rat adipose tissue: A comprehensive metabolic study.

Unpredictable chronic mild stress (UCMS) leads to variable metabolic effects. Oxidative stress (OS) of adipose tissue (AT) and mitochondrial energy homeostasis is little investigated. This work studied the effects of UCMS on OS and the antioxidant/redox status in AT and mitochondrial energy homeostasis in rats. Twenty-four male Wistar rats (180-220 g) were divided into two equal groups; the normal control (NC) group and the UCMS group which were exposed to various stresses for 28 days. An indirect calorimetry machine was used to measure volumes of respiratory gases (VO2 & VCO2 ), total energy expenditure (TEE), and food intake (FI). The AT depots were collected, weighed, and used for measuring activities and gene expression of key antioxidant enzymes (GPx1, SOD, CAT, GR, GCL, and GS), OS marker levels including superoxide anion (SA), peroxynitrite radical (PON), nitric oxide (NO), hydrogen peroxide (H2 O2 ), lipid peroxides (LPO), t-protein carbonyl content (PCC), and reduced/oxidized glutathione levels (GSH, GSSG). Additionally, AT mitochondrial fractions were used to determine the activities of the tricarboxylic acid cycle (TCA) cycle enzymes (CS, α-KGDH, ICDH, SDH, MDH), respiratory chain complexes I-III, II-III, IV, the nicotinamide coenzymes NAD+ , NADH, and ATP/ADP levels. Compared with the NC group, the UCMS group showed very significantly increased OS marker levels, lowered antioxidant enzyme activities and gene expression, as well as lowered TCA cycle and respiratory chain activity and NAD+ , NADH, and ATP levels (p < .001 for all comparisons). Besides, the UCMS group had lowered TEE and insignificant FI and weight gain. In conclusion, AT of the UCMS-subjected rats showed a state of disturbed redox balance linked to disrupted energy homeostasis producing augmentation of AT.

Inflammatory Cytokines, Redox Status, and Cardiovascular Diseases Risk after Weight Loss via Bariatric Surgery and Lifestyle Intervention.

Background and Objectives: Obesity is a chronic inflammatory condition and is considered a major risk factor for cardiovascular disease (CVD). The effects of obesity management via sleeve gastrectomy (SG) and lifestyle intervention (LS) on inflammatory cytokines, redox status, and CVD risk were studied in this work. Materials and Methods: A total of 92 participants (18 to 60 years old) with obesity (BMI ≥ 35 kg/m2 were divided into two groups: the bariatric surgery (BS) group (n = 30), and the LS group (n = 62). According to the achievement of 7% weight loss after 6 months, the participants were allocated to either the BS group, the weight loss (WL) group, or the weight resistance (WR) group. Assessments were performed for body composition (by bioelectric impedance), inflammatory markers (by ELISA kits), oxidative stress (OS), antioxidants (by spectrophotometry), and CVD risk (by the Framingham risk score (FRS) and lifetime atherosclerotic cardiovascular disease risk (ASCVD)). Measurements were taken before and after six months of either SG or LS (500 kcal deficit balanced diet, physical activity, and behavioral modification). Results: At the final assessment, only 18 participants in the BS group, 14 participants in the WL group, and 24 participants in the WR group remained. The loss in fat mass (FM) and weight loss were greatest in the BS group (p < 0.0001). Levels of IL-6, TNF-a, MCP-1, CRP, and OS indicators were significantly reduced in the BS and WL groups. The WR group had significant change only in MCP-1 and CRP. Significant reductions in the CVD risk in the WL and BS groups were detected only when using FRS rather than ASCVD. The FM loss correlated inversely with FRS-BMI and ASCVD in the BS group, whereas in the WL group, FM loss correlated only with ASCVD. Conclusions: BS produced superior weight and fat mass loss. However, both BS and LS produced a similar reduction in the inflammatory cytokines, relief of OS indicators, and enhancement of antioxidant capacity, and consequently reduced the CVD risk.

An Insight into the Impact of Serum Tellurium, Thallium, Osmium and Antimony on the Antioxidant/Redox Status of PCOS Patients: A Comprehensive Study.

Humans exploit heavy metals for various industrial and economic reasons. Although some heavy metals are essential for normal physiology, others such as Tellurium (Te), Thallium (TI), antimony (Sb), and Osmium (Os) are highly toxic and can lead to Polycystic Ovarian Syndrome (PCOS), a common female factor of infertility. The current study was undertaken to determine levels of the heavy metals TI, Te, Sb and Os in serum of PCOS females (n = 50) compared to healthy non-PCOS controls (n = 56), and to relate such levels with Total Antioxidant Capacity (TAC), activity of key antioxidant enzymes, oxidative stress marker levels and redox status. PCOS serum samples demonstrated significantly higher levels of TI, Te, Sb and Os and diminished TAC compared to control (p < 0.001). Furthermore, there was significant inhibition of SOD, CAT and several glutathione-related enzyme activities in sera of PCOS patients with concurrent elevations in superoxide anions, hydrogen and lipid peroxides, and protein carbonyls, along with disrupted glutathione homeostasis compared to those of controls (p < 0.001 for all parameters). Additionally, a significant negative correlation was found between the elevated levels of heavy metals and TAC, indicative of the role of metal-induced oxidative stress as a prominent phenomenon associated with the pathophysiology of the underlying PCOS. Data obtained in the study suggest toxic metals as risk factors causing PCOS, and thus protective measures should be considered to minimize exposure to prevent such reproductive anomalies.

Origanum majorana L. polyphenols: in vivo antiepileptic effect, in silico evaluation of their bioavailability, and interaction with the NMDA receptor.

Introduction: Epilepsy is a chronic brain disease characterized by repeated seizures and caused by excessive glutamate receptor activation. Many plants are traditionally used in the treatment of this disease. This study aimed to evaluate the bioavailability of a polyphenolic extract obtained from Origanum majorana L. (OMP) leaves, as well as its antiepileptic activity and its potential mechanism of action. Methods: We have developed and validated a simple, rapid, and accurate stability-indicating reversed-phase liquid chromatographic method for the simultaneous determination of caffeine and quercetin in rat plasma. The OMP antiepileptic effect was evaluated with pilocarpine-induced seizures, and a docking method was used to determine the possible interaction between caffeic acid and quercetin with the N-methyl-D-aspartate (NMDA) receptor. Results and Discussion: Both compounds tested showed low bioavailability in unchanged form. However, the tested extract showed an anticonvulsant effect due to the considerably delayed onset of seizures in the pilocarpine model at a dose of 100 mg/kg. The molecular docking proved a high-affinity interaction between the caffeic acid and quercetin with the NMDA receptor. Taken together, OLP polyphenols demonstrated good antiepileptic activity, probably due to the interaction of quercetin, caffeic acid, or their metabolites with the NMDA receptor.

Apoptosis-mediated anti-proliferative activity of Calligonum comosum against human breast cancer cells, and molecular docking of its major polyphenolics to Caspase-3.

Due to poor diagnosis breast cancer in women has emerged as the most common cause of death disease in developing countries. Medicinal plants have been used for thousands of years and can be useful in healthcare, especially in developing countries. Ethanol extracts of leaves of fire bush or arta (Calligonum comosum; EECC), exhibited significant anticancer potencies against two breast cancer cell lines, MCF-7 and MDA 231. These in vitro effects of EECC indicated potential anticancer activities that were determined to be specific since minimal toxicity was recorded against MCF-12, a non-cancerous breast cell line used as a reference. EECC also induced cell cycle arrest in MCF-7 and MDA 231 as revealed by the increased proportions of sub-G1 cells. Fluorescence-activated cell sorter analysis (FACS), utilizing double staining by annexin V-FITC/propidium iodide, revealed that the observed cytotoxic effects were mediated via apoptosis and necrosis. FACS measurement of thegreater in fluorescence intensity, linked with oxidation of DCFH to DCF, revealed that apoptosis was attributable to production of free radicals. EECC-mediated apoptosis was further validated by observation of up-regulation in the "executioner" enzyme, caspase 3. The current findings reveal that EECC exhibits significant, selective cytotoxicity to breast cancer cells, that proceeds via the generation of ROS, which culminates in apoptosis. The anti-proliferative effects of EECC weres further verified by use of a structure-based, virtual screening between its major bioactive polyphenolic constituents and the apoptosis executioner marker enzyme, caspase-3. Based on their glide score values against the active site of caspase 3, some phyto-constituents present in EECC, such as DL-alpha-tocopherol and campesterol, exhibited distinctive, drug-like potential with no predicted toxicity to non-target cells. Taken together, the usefulness of natural phenolic and flavonoid compounds contained in Calligonum comosum were suggested to be potent anticancer agents.

Patterns of Dyslipidemia in the Anemic and Nonanemic Hypertensive Saudi Population: A Cross-Sectional Study.

Background: Risk factors of cardiovascular disease include dyslipidemia, hypertension (HTN), and anemia. Our objective is to assess the patterns of dyslipidemia in the anemic and non-anemic hypertensive Saudi population. Methods: A retrospective, cross-sectional study of the gender, blood pressure, lipid markers, and CBC parameters of 3111 subjects, which were retrieved from the database of Al-Borg Medical Laboratories over a six-year period (2014-2019), was carried out. Means were compared among study groups and the prevalence, association, and diagnostic accuracy of lipid markers for HTN were evaluated. Results: TG, LDL/HDL, and TG/HDL were significantly higher (P < 0.0001) in hypertensives. Anemia reduces TC and LDL (P < 0.0001) in both genders, and reduces all markers and increases HDL (P < 0.01) in male hypertensives. HTN was more prevalent in anemics with high TC than normal TC (38.23% vs 11.17%, P < 0.001) and in non-anemics with high TG than normal TG (56.31% vs 21.22%, P < 0.001). Furthermore, non-anemics with high TG/HDL had the highest risk for HTN (RR = 1.20, 95% CI = 1.1551-1.2473, P < 0.0001). Elevated TC (P = 0.0142), TG (P < 0.0001), TC/HDL (P < 0.0001), LDL/HDL (P < 0.0001), and TG/HDL (P < 0.0001), and low HDL (P < 0.0001) were risk factors for HTN as shown by ORs. In anemics, high TC/HDL, LDL/HDL, and TG/HDL were not. Importantly, only TG and TG/HDL had a discriminating capacity for HTN. Conclusion: The anemic state of hypertensive Saudi patients influences dyslipidemia which warrants further investigation.

Isolated and Combined Effect of Age and Gender on Neutrophil-Lymphocyte Ratio in the Hyperglycemic Saudi Population.

Inflammation is pivotal to the pathogenesis of diabetes mellitus (DM), but pathological alterations of the neutrophil−lymphocyte ratio (NLR), an emerging inflammatory index in DM management, remains understudied. The aim of this study is to examine the relationship between NLR and glycemic control in the Saudi population. Gender, age, WBC count, and fasting blood glucose (FBG) were obtained from Al-Borg Medical Laboratories for 14,205 subjects. Means, prevalence, risk measures, and the diagnostic accuracy of elevated NLR and hyperglycemia (HG) were evaluated. Subjects with elevated NLR (>3) had significantly higher FBG (105.10 ± 0.33 vs. 114.0 ± 2.81) and NLR was significantly elevated in impaired fasting glycemia (IFG; 1.21 ± 0.01 vs. 1.25 ± 0.01) and HG (1.21 ± 0.01 vs. 1.39 ± 0.02). Elevations of NLR in HG but not in IFG persisted across all age groups except young males and elderly females. The prevalence of elevated NLR in hyperglycemic subjects was 4.12% compared to 2.16% in subjects with normal FBG. HG was more prevalent in subjects with elevated NLR (17.33% vs. 12.46%) who had a relative risk (RR) of 1.68 (95% CI = 1.38−2.06, p < 0.0001) and an odds ratio (OR) of 1.94 (95% CI = 1.48−2.56, p < 0.0001) to be hyperglycemic. Nevertheless, NLR failed to discriminate individuals with normal FBG from those with HG based on ROC curve analysis. Pathological fluctuations in NLR may serve as supportive evidence in DM management.

Biochemical and molecular assessment of selenium forms for the alleviation of oxidative stress in senescent human fibroblasts.

Selenium enhances the cellular antioxidant capacity and alleviates oxidative stress. We investigated the transcriptional and enzymatic activities of selenium-dependent glutathione peroxidase 1 and thioredoxin reductase 1 (TrxR1), and levels of glutathione, hydrogen peroxide, lipid peroxides, and protein carbonyls in primary passage 5 (P5) and senescent passage 25 (P25) and 30 (P30) fibroblasts. Cells were incubated in either standard Dulbecco growth medium (CM1) containing normal plasma selenium levels (0.8 µmol/l), or in CM2, CM3, and CM4 containing 3 µmol/l (5 µmol/l for TrxR1) sodium selenite, L-hydroxyselenomethionine, or Se-methylselenocysteine, respectively. Gene transcripts and activities of both investigated enzymes as well as the levels of reduced glutathione were significantly increased in CM2-, CM3-, and CM4-incubated senescent P25 and P35 cells compared against those incubated in CM1. In congruence, although all oxidative stress parameters including oxidized glutathione were significantly lower in CM2-, CM3-, and CM4-incubated senescent cells compared against those incubated in CM1, such reductions were of significantly higher magnitude in CM3 and CM4 cells compared against those in CM2. In conclusion, organic L-hydroxyselenomethionine and Se-methylselenocysteine are equally more potent at alleviating oxidative stress in senescent cells than inorganic sodium selenite, and thus could be beneficial for use in elderly subjects and those with oxidative stress-associated disease.

Detection of Novel Biallelic Causative Variants in COL7A1 Gene by Whole-Exome Sequencing, Resulting in Congenital Recessive Dystrophic Epidermolysis Bullosa in Three Unrelated Families.

Background: Dystrophic Epidermolysis bullosa (DEB) is a rare, severe subtype of epidermolysis bullosa (EB), characterized by blisters and miliary rashes of the skin. Dystrophic EB (DEB) includes variants inherited both in an autosomal-dominant or autosomal-recessive manner. Recessive dystrophic EB (RDEB) is divided into many subtypes and prevails as a result of biallelic genetic mutations in COL7A1 gene encoding type VII collagen, a major stabilizing molecule of the dermo-epidermal junction. The blister formation is mainly due to the variable structural and functional impairment of anchoring fibrils in VII collagen (COLVII), responsible for the adhesion of the epidermis to the dermis. Method: Three Pakistani families (A, B and C) affected with congenital dystrophic epidermolysis bullosa were recruited in the present study. The whole-exome sequencing (WES) approach was utilized for the detection of the pathogenic sequence variants in probands. The segregation of these variants in other participants was confirmed by Sanger sequencing. Results: This study identified a novel missense variant c.7034G>A, p. Gly2345Asp in exon 91, a novel Frameshift mutation c.385del (p. His129MetfsTer18) in a homozygous form in exon no 3, and a previously known nonsense variation (c.1573 C>T; p. Arg525Ter) in exon 12 of COL7A1 gene in families A, B, and C, respectively, as causative mutations responsible for dystrophic epidermolysis bullosa in these families. Conclusion: Our study validates the involvement of the COL7A1 gene in the etiology of dystrophic epidermolysis bullosa. It further expands the COL7A1 gene mutation database and provides an additional scientific basis for diagnosis, genetic counseling, and prognosis purposes for EB patients.

Comprehensive investigations of key mitochondrial metabolic changes in senescent human fibroblasts.

There is a paucity of detailed data related to the effect of senescence on the mitochondrial antioxidant capacity and redox state of senescent human cells. Activities of TCA cycle enzymes, respiratory chain complexes, hydrogen peroxide (H2O2), superoxide anions (SA), lipid peroxides (LPO), protein carbonyl content (PCC), thioredoxin reductase 2 (TrxR2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), reduced glutathione (GSH), and oxidized glutathione (GSSG), along with levels of nicotinamide cofactors and ATP content were measured in young and senescent human foreskin fibroblasts. Primary and senescent cultures were biochemically identified by monitoring the augmented cellular activities of key glycolytic enzymes including phosphofructokinase, lactate dehydrogenase, and glycogen phosphorylase, and accumulation of H2O2, SA, LPO, PCC, and GSSG. Citrate synthase, aconitase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and complex I-III, IIIII, and IV activities were significantly diminished in P25 and P35 cells compared to P5 cells. This was accompanied by significant accumulation of mitochondrial H2O2, SA, LPO, and PCC, along with increased transcriptional and enzymatic activities of TrxR2, SOD2, GPx1, and GR. Notably, the GSH/GSSG ratio was significantly reduced whereas NAD+/NADH and NADP+/NADPH ratios were significantly elevated. Metabolic exhaustion was also evident in senescent cells underscored by the severely diminished ATP/ADP ratio. Profound oxidative stress may contribute, at least in part, to senescence pointing at a potential protective role of antioxidants in aging-associated disease.

Antioxidant and Antimicrobial Activities of Chemically-Characterized Essential Oil from Artemisia aragonensis Lam. against Drug-Resistant Microbes.

This study investigated the chemical composition, antioxidant and antimicrobial activity of essential oil extracted from Artemisia aragonensis Lam. (EOA). Hydrodistillation was employed to extract EOA. Gas chromatography with flame ionization detection (GC-FID) and gas chromatography-mass spectrometry analyses (GC-MS) were used to determine the phytochemical composition of EOA. Antioxidant potential was examined in vitro by use of three tests: 2.2-diphenyl-1-picrilhidrazil (DPPH), ferric reducing activity power (FRAP) and total antioxidant capacity assay (TAC). Agar diffusion and microdilution bioassays were used to assess antimicrobial activity. GC/MS and GC-FID detected 34 constituents in the studied EOA. The major component was Camphor (24.97%) followed by Borneol (13.20%), 1,8 Cineol (10.88%), and Artemisia alcohol (10.20%). EOA exhibited significant antioxidant activity as measured by DPPH and FRAP assays, with IC50 and EC50 values of 0.034 ± 0.004 and 0.118 ± 0.008 mg/mL, respectively. EOA exhibited total antioxidant capacity of 7.299 ± 1.774 mg EAA/g. EOA exhibited potent antibacterial activity as judged by the low minimum inhibitory concentration (MIC) values against selected clinically-important pathogenic bacteria. MIC values of 6.568 ± 1.033, 5.971 ± 1.033, 7.164 ± 0.0 and 5.375 ± 0.0 µg/mL were observed against S. aureus, B. subtills, E. coli 97 and E. coli 57, respectively. EOA displayed significant antifungal activity against four strains of fungi: F. oxysporum, C. albicans, A. flavus and A. niger with values of 21.50 ± 0.43, 5.31 ± 0.10, 21.50 ± 0.46 and 5.30 ± 0.036 µg/mL, respectively. The results of the current study highlight the importance of EOA as an alternative source of natural antioxidant and antibacterial drugs to combat antibiotic-resistant microbes and free radicals implicated in the inflammatory responses accompanying microbial infection.

Antioxidant status in relation to heavy metals induced oxidative stress in patients with polycystic ovarian syndrome (PCOS).

Polycystic ovary syndrome (PCOS) is a global health concern for women of reproductive age, as 6.5% of women worldwide are affected by this syndrome. PCOS is marked by hyperandrogenism, anovulation, menstrual abnormalities, and polycystic ovaries. Metals such as arsenic, cadmium, lead and mercury are considered to be systemic toxicants/human carcinogens and seem to have devastating effects on humans, even at minimal exposures. One of the probable aetiological factors for PCOS has been identified as oxidative stress. In view of the probable associations among oxidative stress, metal toxicity and PCOS, the present study examined the role of heavy metals in the generation of oxidative stress among females. This prospective study included 106 women (56 women diagnosed with PCOS and 50 women who were not diagnosed with PCOS as control women). There were no significant differences in the sociodemographic characteristics between the two groups except for the irregularity of menses and the presence of acne. The serum As, Cd, Pb, and Hg levels increased and the serum glutathione (GSH) and superoxide dismutase (SOD) levels diminished significantly in the PCOS group compared to the control group at P < 0.001. The SOD levels were negatively correlated with the As and Pb levels at P < 0.05. Additionally, the PCOS group exhibited a strong negative correlation between the GSH and As levels (P < 0.01), GSH and Pb levels (P < 0.05) and GSH and Hg levels (P < 0.01). Furthermore, the As levels were positively correlated with increased levels of Cd, Pb and Hg among PCOS women. Significant positive correlations were observed between Pb and Cd and between Cd and Hg at P < 0.001. The outcome of the study provides clear insight into the role of metal-induced oxidative stress, which plays a vital role in the pathophysiology underlying PCOS and suggests the use of these markers as prognostic tools to reduce the consequences of high-risk exposure to these metals among females.

Glutathione and oxidized nicotinamide adenine dinucleotide (NAD+) redox status in plasma and placental tissue of Saudi patients with intrauterine growth restriction.

OBJECTIVES: To assess the oxidation state and gene expression profiles of relevant enzymes in intrauterine growth restriction (IUGR) patients in Saudi Arabia. METHODS: Current case-control study involved plasma and placental tissue samples from 25 IUGR patients and 25 healthy pregnant (HP) women attending the Obstetrics and Gynecology Clinic, King Khalid University Hospital, Riyadh, Saudi Arabia, between April and November 2017. We compared hydrogen peroxide, superoxide anions, malondialdehyde, and oxidative stress markers levels and the activities of glutathione-related enzymes (glutathione peroxidase [GPx], glutathione reductase [GR], glutathione S-transferase [GST], glutamate cysteine ligase [GCL], glutathione synthetase [GS], reduced glutathione [GSH], oxidized glutathione [GSSG], and oxidized nicotinamide adenine dinucleotide [NAD+], and reduced NAD [NADH]) between the 2 groups. We also compared differential expression levels of glutathione-related enzyme genes using reverse transcription-quantitative polymerase chain reaction. RESULTS: Oxidative stress markers significantly differed in IUGR samples, while GSH levels and GPx, GR, GST, GCL, and GS activities and their placental mRNA transcriptional levels were significantly lower. Plasma and placental NAD+ levels were also significantly lower, while NADH levels were significantly higher, causing lowered NAD+-NADH ratios in the IUGR group compared to control. CONCLUSIONS: Intrauterine growth restriction patients show a metabolic shift in favor of oxidation compared to HP women.

Physcion Induces Hemolysis and Premature Phosphatidylserine Externalization in Human Erythrocytes.

The prevalence of cancer-associated anemia (CIA) is high, and the mechanisms governing its development remain poorly understood. Eryptosis, the suicidal cell death of red blood cells (RBCs), may account for CIA as it is triggered by clinically approved chemotherapeutics including cisplatin and paclitaxel. Physcion (PSN), an anthraquinone extracted from rhubarb and other plants, has shown great promise as an anticancer agent. However, the potential toxicity of PSN to RBCs remains elusive. RBCs were isolated from heparinized blood, and incubated with 10-100 µM of PSN for 24 h at 37 °C. Hemolysis was photometrically calculated from hemoglobin concentration in the medium at 405 nm, while flow cytometry was employed to investigate cardinal markers of eryptosis. Phosphatidylserine (PS) exposure was detected by Annexin-V-fluorescein isothiocyanate (FITC), intracellular calcium by Fluo4/AM, cellular volume from forward scatter (FSC), and oxidative stress by 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). PSN induced overt hemolysis at 50 and 100 µM which was not mediated through calcium influx, protein kinase C, casein kinase 1α, or receptor-interacting protein 1. Moreover, PSN caused significant increase in Annexin-V-FITC and Fluo4 fluorescence with no appreciable influence on FSC or DCF values. Accordingly, PSN stimulates premature eryptosis characterized by PS externalization and intracellular calcium overload without cell shrinkage or oxidative damage. In conclusion, this report shows, for the first time, that PSN is cytotoxic to RBCs by inducing hemolysis and programmed cell death which may limit its success as a chemotherapeutic agent.

Molecular and biochemical investigations of key antioxidant/oxidant molecules in Saudi patients with recurrent miscarriage.

The present study was undertaken to: i) Determine the levels of oxidative stress (OS) markers, malondialdehyde (MDA), superoxide anions (SOA) and hydrogen peroxide (H2O2), in both plasma and placental tissues of recurrent miscarriage (RM) patients in comparison with those of healthy pregnant (HP) and non-pregnant (NP) women; ii) determine the levels of enzymatic antioxidants [glutathione peroxidase (GPx), glutathione reductase (GSR), superoxide dismutase (SOD) and catalase (CAT)], and non-enzymatic antioxidant micronutrients [selenium (Se), zinc (Zn), copper (Cu) and manganese (Mn)] in both plasma and placental tissues of RM patients, in comparison with those of HP and NP women; iii) profile differential expression levels of selected antioxidant and apoptosis-related genes in the placental tissues of RM cases, in relation to those of HP women of matched gestational age, using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results revealed highly significant increases of all investigated OS markers in plasma and placental tissues of RM patients compared with those of HP women. Moderate, but significant, increases of OS markers were observed in the plasma of HP patients in relation to those of NP women. The activities of antioxidant enzymes exhibited statistically significant decreases in both plasma and placental tissues of RM patients compared with those of HP women. The significantly reduced level of antioxidant enzymes was also evident in the plasma of HP women as compared with those of NP women. Results of RT-qPCR assays clearly indicated that the expression level of apoptosis-related genes [tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and S100A8], and pro-inflammatory cytokine genes [tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-8] were significantly upregulated in placental tissue of RM cases in relation to those of HP subjects. By contrast, mRNA transcriptional levels of key antioxidant genes (GPx, SOD, GSR and CAT) were found to be significantly reduced in placental tissue of RM patients in comparison to those of HP women. In conclusion, our data highlight a plausible cause-effect association between the observed increase in placental OS level and depletion of the activity of antioxidant enzymes. This suggests that OS is a contributing factor in the pathogenesis of idiopathic RM.

Screening for differentially­expressed microRNA biomarkers in Saudi colorectal cancer patients by small RNA deep sequencing.

Colorectal cancer (CRC) is mostly diagnosed at late stages leading to high mortality rates due to the scarcity of efficient screening approaches exhibiting high diagnostic utility. The current study employed a small­RNA deep­sequencing approach for screening microRNA (miRNA) differentially expressed genes (DEGs), and evaluating their potential as early diagnostic circulating biomarkers for CRC in clinical plasma and tissue samples from a Saudi patient population. The cohort followed a paired­study design composed of 20 CRC patients, providing plasma (P) and tissue (T) samples of CRC, and adjacent normal mucosa (CT). Also, control plasma (CP) samples were obtained from neoplasm­free healthy individuals to compare its miRNA levels with those in P samples. Illumina high­throughput (HiSeq 2000) sequencing was performed for the identification of known and novel miRNA genes that were differentially expressed in the plasma and tissues of CRC patients compared with CT and CP controls. While we identified only one known (hsa­miR­182­5p, significantly upregulated) and no novel DEGs at the most stringent significance level (P<0.001) in the P­CP comparison, we found 3 and none at P<0.01, 7 and 9 at P<0.05 level, respectively. In the T­CT comparison, the results revealed 24 known and 196 novel miRNA DEGs (P<0.001), 31 and 204 (P<0.01), 41 and 213 (P<0.05), respectively. Sequencing data were then analyzed by bioinformatics for potential diagnostic miRNAs. Network functional analysis for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway implicated two pathways rooted to signal transduction [Wnt and mitogen­activated protein kinase (MAPK)] that were enriched in CRC patients. Our results suggest that characterizing plasma and tissue profiles of CRC by deep sequencing may be a good strategy for identifying known and novel miRNAs and that the validated miRNAs described here may serve as potential CRC­associated biomarkers. Further research is necessary for determining their screen index values and diagnostic utility for the diagnosis of CRC.

Selenium Concentrations for Maximisation of Thioredoxin Reductase 2 Activity and Upregulation of Its Gene Transcripts in Senescent Human Fibroblasts.

Thioredoxin reductase 2 (TR2) activity, its gene transcripts, and hydrogen peroxide (H2O2) generation were examined in biochemically identified early-senescent P20 and senescent P30 fibroblasts subcultured in media (MEM2-MEM8) containing Se concentrations at 1.25, 2.5, 3.5, 5.0, 6.0, 7.0, and 8.0 µM, respectively. Although TR2 activity was moderately increased in P20 and P30 cells subcultured in routine growth medium (MEM1), there were progressive significant activity increases in the same cells subcultured in MEM2-MEM8. Such increases were proportional to Se concentration and peaked in P30 cells incubated with MEM7 and MEM8. H2O2 generation underwent progressive increases in MEM1-incubated P20 and P30 cells, peaking in the latter, but was gradually lowered in those incubated with MEM2-MEM8, reaching its lowest values when cells were incubated with MEM7 and MEM8. In parallel, TR2 gene transcripts underwent significant upregulation in P20 cells and higher magnitude upregulation in P30 cells subcultured in MEM2, MEM4, and MEM8 compared to those recorded for P5 pre-senescent cells subcultured in the same media. The computed Km Se values with respect to TR2 activity equaled 3.34 and 4.98 µM for P20 and P30 cells, respectively, with corresponding Vmax activities of 55.9 and 96.2 nmol/min/mg protein. It is concluded that senescent P30 cells utilize more Se and achieve maximal TR2 activity to combat oxidative injury.

Ascorbate ameliorates Echis coloratus venom-induced oxidative stress in human fibroblasts.

Reports related to the effects of Echis coloratus venom (EcV) on the antioxidant capacity of human tissues is very scarce. The present study was undertaken to investigate the activities and gene expression levels of glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT), as well as the levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and the generation rates of superoxide anions (SOA), hydrogen peroxide (H2O2) and lipid peroxides (LPO) in cultured human fibroblasts incubated with EcV, ascorbate (Asc) and EcV plus Asc at concentrations and incubation periods that maintained cell viability. Results indicated that the activities of all antioxidant enzymes and their corresponding transcripts underwent highly significant decreases and downregulation in EcV-treated cultures (0.5 µg/ml medium for 4 h) compared to venom-free controls (P<0.001). Additionally, there were concurrent equally significant increases in SOA, H2O2 and LPO generation rates in the venom-incubated cultures compared to controls (P<0.001). Results also indicated very significant decreases and parallel equally significant increases in GSH and GSSG levels respectively in the envenomed cultures compared to controls (P<0.001) leading to a drastically lower GSH/GSSG ratio. However, further incubation of the EcV-treated cultures with Asc (400 µM for 12 h) restored the activities and levels of all investigated parameters including the expression levels of the antioxidant genes to control venom-free values. It is concluded that Asc acted to neutralize the increased reactive oxygen species generation, thus ameliorating the EcV-induced oxidative stress and alleviating the downregulation of antioxidant genes.

The effect of Echis coloratus venom on biochemical and molecular markers of the antioxidant capacity in human fibroblasts.

This study was undertaken to examine the activities and levels of major antioxidants/oxidants in cultured human fibroblasts incubated with a sublethal dose of Echis coloratus venom (EcV). Glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR) activities and gene expression levels as well as reduced glutathione (GSH) levels, and the concurrent hydrogen peroxide (H2O2), superoxide anions (SOA), lipid peroxides (LPO) and oxidized glutathione (GSSG) generation rates were assayed in fibroblast cultures and sonicates incubated with 0.5 µg ml-1 medium EcV for 4 h at 37°C. Data indicated that the activities of all antioxidant enzymes were significantly decreased and their corresponding transcripts downregulated in EcV-incubated cells compared to controls (p < 0.001). In contrast, there were parallel equally significant increases in H2O2, SOA and LPO generation rates in venom-incubated cells compared to controls (p < 0.001). Additionally, GSH levels were significantly decreased and those of GSSG were equally significantly increased in venom-incubated cultures compared to controls (p < 0.001) leading to a lowered GSH/GSSG ratio. In conclusion, incubation of fibroblast cultures with EcV resulted in a shift towards oxidative metabolism causing severe OS. This correlated with significant downregulation in the expression levels of all investigated antioxidant genes.