Yazeed A. Al-Sheikh

Role and implications of nanodiagnostics in the changing trends of clinical diagnosis

Nanodiagnostics is the term used for the application of nanobiotechnology in molecular diagnosis, which is important for developing personalized cancer therapy. It is usually based on pharmacogenetics, pharmacogenomics, and pharmacoproteomic information but also takes into consideration environmental factors that influence response to therapy. Nanotechnology in medicine involves applications of nanoparticles currently under development, as well as longer range research that involves the use of manufactured nano-robots to make repairs at the cellular level. Nanodiagnostic technologies are also being used to refine the discovery of biomarkers, as nanoparticles offer advantages of high volume/surface ratio and multifunctionality. Biomarkers are important basic components of personalized medicine and are applicable to the management of cancer as well. The field of nano diagnostics raises certain ethical concerns related with the testing of blood. With advances in diagnostic technologies, doctors will be able to give patients complete health checks quickly and routinely. If any medication is required this will be tailored specifically to the individual based on their genetic makeup, thus preventing unwanted side-effects.

The effect of aging and increasing ascorbate concentrations on respiratory chain activity in cultured human fibroblasts

The specific activities of Complexes I‐III, II‐III, and IV of the respiratory chain, and citrate synthase, were determined in mitochondrial sonicates of six control passage 5 fibroblast cultures, cultivated in growth medium containing fetal calf serum as the only source of ascorbate. The enzymes were also assayed in serially subcultured fibroblasts which were characterized as aged at passage 20 and beyond. Results indicated a significant loss of all enzyme activities in aged cells at passage 20, 25, and 30. Further studies involved maintenance of serially subcultured cells in serum free media to which increasing ascorbate concentrations (100, 200, and 300 µmol 1−1) were added. Results indicated that ascorbate at 100 µmol 1−1 was not sufficient to restore any of the enzyme activities in aged cells. An ascorbate concentration of 200 µmol 1−1 however, could totally restore Complex IV and citrate synthase activities, but had no effect on complexes I‐III and II‐III activities which required 300 µmol 1−1 ascorbate to be partially or totally restored respectively. In conclusion, this study demonstrates an age related drop in mitochondrial respiratory chain activity in cultured human fibroblasts. Enzyme activities could be completely or partially restored in the presence of double or triple normal human plasma ascorbate concentrations. Copyright

A54T polymorphism in the fatty acid binding protein 2 studies in a Saudi population with type 2 diabetes mellitus

BackgroundFatty acid-binding protein 2 (FABP2) is an intracellular protein expressed exclusively in the enterocytes of proximal small intestine. FABP2 has a high affinity for saturated and unsaturated long-chain fatty acids and is believed to be involved in the absorption and transport of dietary fatty acids.MethodsThis is a case–control study conceded in 438 T2DM cases and 460 subjects with normal glucose levels and non-obese considered as healthy controls. Allelic discrimination was performed using TaqMan single-nucleotide polymorphism was carried out by real time-polymerase chain reaction (RT-PCR) assays using purified DNA.ResultsClinical data and anthropometric measurements except age, glucose levels and lipid profile of the patients were significantly different from those of the controls (p < 0.05). Statistical analyses failed to show any type of significant association of the polymorphism between cases and controls. However logistic regression analyses was suggests that the TT genotype is significantly associated with male patients (p = 0.001). None of the allele or genotypes of FABP2 A54T was associated with T2DM cases versus the controls (AT genotype, OR = 0.85 (0.64-1.12), p = 0.25; TT genotype, OR = 0.66 (0.39-1.11), p = 0.11; T allele, 0.82 (0.67-1.02), p = 0.08).ConclusionIn conclusion, this study suggests that the above named variant in FABP2 gene is not potential contributor to the risk of T2DM and related traits in a Saudi population. However TT genotype is a risk factor for the disease in males.

ABCA1 C69T gene polymorphism and risk of type 2 diabetes mellitus in a Saudi population

Type 2 diabetes mellitus (T2DM) is a disease induced by complex interactions between environmental factors and certain genetic factors. Genetic variants in the Adenosine Binding Cassette Transporter Proteins 1 (ABCA1) have been associated with abnormalities of serum lipid levels of high-density lipoprotein (HDL-C). Decreased serum levels of HDL-C have often been observed in T2DM cases, and this condition has been considered to be involved in the mechanism of insulin resistance (IR). Therefore, we investigated possible association between ABCA1 C69T gene polymorphism and T2DM in a Saudi population. This study was carried out with 380 healthy control subjects and 376 T2DM patients. Genotyping of ABCA1 C69T polymorphism was carried out by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism technique. We observed that the frequency of the T allele of the ABCA1 C69T gene was significantly higher in healthy subjects compared to T2DM patients (0.28 vs 0.45; p<0.0001; OR (95% CI) = 0.4624 (0.3732–0.5729), and therefore the T allele may be a protective factor against T2DM in the Saudi population.

Isolation and characterization of native Bacillus thuringiensis isolates from Saudi Arabia

A total of 500 soil samples were collected from different localities across the kingdom to isolate native Bacillus thuringiensis (Bt) strains. Sodium acetate- (0.25 M) -selection heat-pasteurization, and 50% ethanol treatment methods were used for Bt isolation. Phenotypic characterization and identification of recovered Bt isolates was accomplished on the basis of morphological characteristics of colonies, parasporal crystal, and spores, as well as hemolytic activity, motility, susceptibility to antibiotics, and carbohydrate utilization (API 50CH system). Analysis of 16S rRNA gene was performed to further confirm Bt species identity and differentiate the spore-forming bacilli. Bioassay tests were conducted on larvae of Aedes caspius and Culex pipiens . Based on results, a total of 64 Bt isolates were recovered and the overall Bt index corresponding to the whole sampling areas was 0.25. Most of Bt isolates showed spherical crystals (56%), while, irregular, bipyramidal, and attached crystal to the spores constituted 14, 10 and 6% respectively. The most common biochemical type (31%) was as that of the reference Bt subsp. kurstaki (Btk). The 16S rRNA gene analysis, using the universal primer, provided an efficient identification of Bt isolates and their discrimination from the closely related Bacillus cereus , as well as from other spore forming bacilli. Bioassay showed that only one Bt isolate exhibited anti-diptera activity similar to Btk strain while most of the native Bt isolates exhibited weak (<30% mortality) or non insecticidal activity. However, results of motility, hemolytic activity, antibiotic-susceptibility patterns, and crystal shape, seem to suggest that many of our Bt isolates may exhibit parasporins activity. Key words: Bacillus thuringiensis , parasporal crystal, isolation, biochemical type, insecticidal, cry gene, mosquitoes.

The effect of micronutrients on superoxide dismutase in senescent fibroblasts

The specific activities of zinc/copper (Zn/Cu)‐superoxide dismutase (SOD‐1) and manganese (Mn)‐superoxide dismutase (SOD‐2) were assayed in young passage 5 fibroblasts and in serially subcultured cells that were characterized as senescent at passages 15–35. SOD‐1 and SOD‐2 activities did not significantly change in senescent and young cells cultured in either routine medium [minimum essential medium 1 (MEM1)], or in Zn, Cu and Mn supplemented medium (MEM2) containing normal human plasma levels of the cations. SOD‐1 and SOD‐2 activities, however, underwent parallel progressive significant activity increases in senescent passage 20 and 25 cells, which peaked in value in passage 30 and 35 cells subcultured in supplemented medium (MEM3) containing triple human plasma levels of the cations. Concurrently, superoxide radical generation rates underwent progressive significant increases in senescent passage 15–25 cells, which peaked in value in passage 30 and 35 cells subcultured in MEM1 or MEM2. These rates, however, were significantly lowered in senescent cells subcultured in MEM3. We infer that it was only possible to significantly stimulate SOD‐1 and SOD‐2 activities in senescent MEM3 cultured cells enabling them to combat oxidative stress. Copyright

Superoxide dismutase activity and gene expression levels in Saudi women with recurrent miscarriage

The antioxidant activities of superoxide dismutase 1 (SOD1) and SOD2, as well as the levels of the oxidant superoxide anion (SOA) and the micronutrients zinc (Zn), copper (Cu) and manganese (Mn), were assayed in plasma, whole blood and placental tissue of non-pregnant (NP), healthy pregnant (HP) women and recurrent miscarriage (RM) patients. The results showed that SOD1 and SOD2 activities and the levels of Zn, Cu and Mn in plasma and whole blood of HP women were slightly, but significantly lower, and even more significantly decreased in RM patients compared to those observed in NP women (P<0.05 and P<0.0001, respectively). Additionally, whereas plasma SOD1 and SOD2 activities and Zn, Cu and Mn levels were significantly lower in RM patients, those of whole blood and placental tissue were significantly lower when compared to HP women (P<0.001 and P<0.0001, respectively). Concurrently, there were consistent increases of equal magnitude and statistical significance in SOA levels in all the assayed samples as identified by a comparison between the subjects. The findings thus supported oxidative metabolism and excessive reactive oxygen species generation. The resultant oxidative stress, identified in whole blood and placental tissues of RM patients, may have been a primary cause of RM. Dietary supplementation of Zn, Cu and Mn may be beneficial to these patients pre- and post-conception.

A 6-year surveillance of antimicrobial resistance patterns of Acinetobacter baumannii bacteremia isolates from a tertiary care hospital in Saudi Arabia during 2005-2010

Multidrug resistance (MDR) of Acinetobacter baumannii increasingly jeopardizes the health care setting leading to substantial mortality and morbidity globally. During the past decade, entirely resistant A. baumannii strains presented a real challenge to clinicians and posed difficulties in therapy. MDR of A. baumannii– associated infections, with adverse clinical outcomes involving the respiratory tract, blood, soft tissues, urinary tract, and central nervous system, significantly increases the outlay of the infirmary. Being Gram-negative coccobacilli and an obligate aerobe, A. baumannii causes both community- and hospital-acquired infection outbreaks in intensive care units especially in countries with tropical climates. Of particular concern, we sought to reveal the status of antimicrobial resistance in A. baumannii bacteremia isolates, the trends and relative frequency of multidrug resistance pattern and also the underlying clinical condition among patients with A. baumannii bacteremia at Riyadh Military hospital, Saudi Arabia, from January 2005 to December 2010. (Published: 3 April 2014) Citation: Libyan J Med 2014, 9 : 24039 - http://dx.doi.org/10.3402/ljm.v9.24039

Effect of selenium supplementation on glutathione peroxidase and catalase activities in senescent cultured human fibroblasts.

Aims: To investigate the effect of senescence and selenium supplementation on glutathione peroxidase (cGPx) and catalase (CAT) activities, and concurrent hydrogen peroxide (H2O2) generation in subcultured human fibroblasts. Methods: cGPx and CAT activities and H2O2 levels were assayed in presenescent passage 5 and 10 cells, and in senescent passage 20, 25, 30 and 35 cells cultured in routine medium (MEM1) and supplemented media MEM2 and MEM3 containing normal and triple human plasma levels of Se, respectively. Senescent cells were identified by studying their growth and replication states, and by monitoring their activity of key glucose and glycogen degradative enzymes. Results: cGPx activity showed moderate increases in senescent cells at passages 20–35 subcultured in MEM1 or MEM2. This activity underwent highly significant progressive increases in the same senescent cells subcultured in MEM3. In contrast, CAT activity showed progressive, highly significant increases in senescent cells at passages 20–35 regardless of the culture medium type. Concurrent H2O2 generation was significantly increased in passage 15–25 cells and peaked to higher levels in passage 30 and 35 cells cultured in MEM1 or MEM2. These rates, however, were significantly reduced in senescent passage 20–35 cells cultured in MEM3. Conclusions: The highest cGPx activity and coupled lower H2O2 generation were achieved in senescent cells cultured in MEM3.

Isolation and characterization of native Bacillus thuringiensis strains from Saudi Arabia with enhanced larvicidal toxicity against the mosquito vector Anopheles gambiae (s.l.)

BackgroundWorldwide, mosquito vectors are transmitting several etiological agents of important human diseases, including malaria, causing millions of deaths every year. In Saudi Arabia, as elsewhere, vector-control is based mostly on chemical insecticides which may be toxic and cause environmental deprivation. Here, to support the development of bio-pesticide alternatives, a study was conducted to identify native Bacillus thuringiensis (Bt) isolates with improved toxicity against the malaria vector, Anopheles gambiae (s.l.).MethodsSixty-eight Bt isolates were obtained from 300 soil and other samples collected from 16 sites across Saudi Arabia. Bt identification was based on morphological characteristics of colonies, shape of parasporal crystals and biochemical profiles. After characterization of their mosquitocidal activity, larvicidal strains were described through 16S ribosomal DNA gene sequencing, cry, cyt and chi genes PCR-amplification profiles, and SDS-PAGE protein analyses.ResultsSpherical Bt crystals were predominant amongst the 68 isolates (34%), while irregular, bi-pyramidal and spore-attached crystals were found in 32, 13 and 21% of strains, respectively. LC50 and LC90 bioassays showed that 23/68 isolates were larvicidal, with distinct biochemical activity profiles compared to non-larvicidal Bt strains. Eight larvicidal strains showed larvicidal activity up to 3.4-fold higher (LC50 range: 3.90–7.40 μg/ml) than the reference Bti-H14 strain (LC50 = 13.33 μg/ml). Of these, 6 strains had cry and cyt gene profiles similar to Bti-H14 (cry4Aa, cry4Ba, cry10, cry11, cyt1Aa, cyt1Ab, cyt2Aa). The seventh strain (Bt63) displaying the highest larvicidal activity (LC50 = 3.90 μg/ml) missed the cry4Aa and cyt1Ab genes and had SDS-PAGE protein profiles and spore/crystal sizes distinct from Bti-H14. The eight strain (Bt55) with LC50 of 4.11μg/ml had cry and cyt gene profiles similar to Bti-H14 but gave a chi gene PCR product size of 2027bp. No strains harbouring cry2, cry17 + 27, cry24 + 40, cry25, cry29, cry30, or cyt2Ba were detected.ConclusionThis study represents the first report of several Saudi indigenous Bt strains with significantly higher larvicidal efficacy against An. gambiae than the reference Bti-H14 strain. The very high toxicity of the Bt63 strain, combined with distinct cry and cyt genes and SDS-PAGE-protein profiles makes it a promising candidate for future applications in mosquito bio-control.