This study identified phytochemicals in Argemone mexicana (A. mexicana) extracts that are responsible for its medicinal properties, and the best solvent for their extraction. The extracts of the stem, leaves, flowers, and fruits of A. mexicana were prepared at low (corresponding to room temperature) and high temperatures (corresponding to the boiling points) in various solvents, viz., hexane, ethyl acetate, methanol, and H2O. The UV-visible absorption spectra of various phytoconstituents in the isolated extracts were determined through spectrophotometry. Qualitative tests for the screening of phytoconstituents in the extracts were performed to identify various phytochemicals. We identified the presence of terpenoids, alkaloids, cardiac glycosides, and carbohydrates in the plant extracts. The antioxidant and anti-human immunodeficiency virus type 1 reverse transcriptase (anti-HIV-1RT) potential, as well as the antibacterial activity of various A. mexicana extracts were determined. These extracts showed strong antioxidant activities. The extracts exhibited antimicrobial activities against Salmonella typhi, Staphylococcus epidermis, Citrobacter, Neisseria gonorrhoeae, and Shigella flexineri. These extracts significantly inhibited HIV-1 reverse transcriptase activity. The aqueous leaf extract prepared at a temperature equivalent to the boiling point, i.e., 100 °C, was identified to be the most active against pathogenic bacteria and HIV-1 RT.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is caused by a coronavirus. This virus has been responsible for more than three million deaths reported worldwide. Common symptoms of COVID-19 include dry cough, fatigue, dyspnea, headache, loss of appetite, panting, shortness of breath, etc. The incubation time of the disease varies between 5–14 days. The successful sequencing of SARS-CoV-2 genome shows that its key genes are similar to that of other coronaviruses which cause respiratory illnesses. The human respiratory mucosa is the primary route of infection for viruses like influenza, respiratory syncytial and parainfluenza viruses. The human cell receptor is the angiotensin-converting enzyme 2, known as ACE2. The virus enters the cell through the action of a transmembrane serine protease. The entry of SARS-CoV-2 results in an increased secretion of mucus, which causes the alveoli to clog and prevents oxygenation of the blood. Nanostructured materials have at least one nanometric dimension (usually less than 100 nm). They can be organic, inorganic, biomaterial-based and carbon-based. Their unique physicochemical properties such as, chemical reactivity, size-dependent transport, biocompatibility and comparatively less toxicity can be exploited in the treatment/management of COVID-19. Nanostructured systems can impact diagnosis, improve detection, sensitivity in the management of COVID-19. Nanoparticles (NPs) can be used as therapeutic delivery vehicles, nano vaccines, surface disinfectants to coat personal protective equipment, nanodiagnostics, nanobiosensors and a multitude of other applications. This chapter deals with the application of nanostructures in the treatment of COVID-19 through therapeutic, diagnostic and various other applications. Regulatory aspects of nanotherapy are also discussed.

Seed germination is widely used as a model to explore toxicity of various harmful chemicals in plants. High concentration of fluoride in water can be harmful for the plant kingdom at all stages of development. Seeds of Tritium aestivum L (wheat) were sown in Petri dishes and exposed to five different concentrations of sodium fluoride (20, 40, 60, 80, and 100 ppm) for two weeks. Seeds grown with distilled water were used as controls for comparison. Germination data for the seedlings was recorded and seedlings were harvested on 15th day after sowing. Freshly prepared extract of seedlings was examined for various biochemical assays (total protein, glutathione (GSH), glutathione 5-transferase activity, and lipid peroxide) and by Fourier transform infrared spectroscopy (FT-IR) analysis. We found a gradual decrease in seed germination parameters as the concentration of sodium fluoride (NaF) increased. All measured biochemical parameters were also effected in dose dependent manner. The total protein level was slightly increased with 20, 40, and 60 ppm of NaF. However, with 80 and 100 ppm of NaF there was a slight decrease as compared to control. Both GSH and GST levels were decreased with increased concentration of NaF while lipid peroxide was increased with 20 ppm of NaF treatment but showed a slight decrease when treated with higher concentrations of NaF. Overall maximum effect was shown by highest NaF level (100 ppm) and moderate tolerance was observed up to 40 ppm. FT-IR profiles revealed some changes in functional groups of proteins, lipids, and carbohydrates with NaF treatments. Future studies on crop tolerance for plants irrigated with groundwater with high fluoride levels are recommended.

Fluoride is present in almost all oral hygiene products with an average concentration range of 250 to 1000 ppm. Fluoride anions have unique biochemical properties and can inhibit a wide variety of metabolic processes. Here we report fluoride toxicity in Enterococcus faecalis which is commonly found in oral infections in humans. We grew E. faecalis in Luria Bertani medium at different concentrations of NaF (0, 250, 500, 750, and 1000 ppm). The results showed that E. faecalis growth was slightly decreased with increases in the concentration of NaF. The total protein level in the treated bacterial cells was slightly changed with 250, 500, and 750 ppm of NaF while with 1000 ppm of NaF there was a significant decrease (p<0.05). The glutathione Stransferase (GST) level showed some decrease with 250, 500, and 750 ppm and was significantly decreased (p<0.05) with 1000 ppm of NaF. The glutathione (GSH) level fluctuated with increasing concentrations of NaF and was significantly decreased (p<0.05) with 250 and 1000 ppm of NaF. Our observations suggest that fluoride can control the growth of pathogenic bacteria in the mouth and can induce oxidative stress in bacterial cells via GSH metabolism.

Although fluoride is considered to neurotoxic, very few investigations are available on fluoride exposure as a possible risk factor for autism. The present study compared the neurotoxicity in propionic acid (PA)-induced rodent models of autism with fluoride in prenatal bee pollen-protected and unprotected newborns. The study was performed on five groups of neonatal male Western albino rats. Rats in group I were a control group and received only phosphate-buffered saline, rats in group II received a neurotoxic dose of PA (250 mg/kg for 3 days) and served as an autistic model, group III received a chronic dose of sodium fluoride (NaF) (3-5 mg/kg for 30 days), group IV were prenatal bee pollen-treated neonates (250 mg/kg bw from 0-23 days of gestation + 7 days postpartum), and finally group V were prenatal bee pollen-treated neonates (250 mg/kg bw from 0-23 days of gestation + 7 days postpartum) toxicated with NaF (3-5 mg/kg for 30 days). The results of the PA acid and NaF treatments showed almost the same trend with a significant increase (P <= 0.001) in lipid peroxides and significant decreases (P <= 0.001) in glutathione, catalase, and vitamin C as compared to the control group. Potassium was significantly decreased (P <= 0.010) in the PA group with no outstanding effects in the other groups. The bee pollen-protected neonatal group showed perfection in all the tested parameters. Our results argue for further investigation of fluoride as a risk factor for autism.

The aim of the present study was to investigate the protective role of bee pollen against sodium fluoride (NaF)-induced hepatonephrotoxicity in the rat. Male Western albino rats were divided into four groups, each consisting of seven animals: (i) normal [control] group; (ii) rats treated with a chronic dose of NaF [5 mg/kg body weight (bw) for one month]; (iii) rats treated with bee pollen [250mg/kg bw for 30 days], and (iv) rats treated with bee pollen first [250 mg/kg bw for 30 days] followed by NaF [5 mg/kg bw for one month]. The level of serum electrolytes and enzymatic antioxidants in the liver and kidney were measured. We found a significant increase in magnesium (Mg) [p<0.001] whereas sodium (Na) was significantly [p<0.05] decreased in the serum of the NaF-exposed rats, as compared to the control group. Also, in the NaF-treated group, as compared to the control group, in both liver and kidney tissue, we found a significant (p<0.05) increase of lipid peroxidation and glutathione 5-transferase (GST) while, remarkably, there was a significant (p<0.05) decrease in both glutathione (GSH) and vitamin C. The bee pollen administration did not show any significant toxic or negative effects on the serum electrolytes, the liver function, or the kidney function in the rats. The changes induced in the group treated with NaF alone were, remarkably, restored in the bee pollen+NaF treatment group. Thus, bee pollen exerts a protective property by ameliorating the NaF-induced serum electrolyte changes and the NaF-induced oxidative stress in the rat.

Although fluoride is considered to neurotoxic, very few investigations are available on fluoride exposure as a possible risk factor for autism. The present study compared the neurotoxicity in propionic acid (PA)-induced rodent models of autism with fluoride in prenatal bee pollen-protected and unprotected newborns. The study was performed on five groups of neonatal male Western albino rats. Rats in group I were a control group and received only phosphate-buffered saline, rats in group II received a neurotoxic dose of PA (250 mg/kg for 3 days) and served as an autistic model, group III received a chronic dose of sodium fluoride (NaF) (3–5 mg/kg for 30 days), group IV were prenatal bee pollen-treated neonates (250 mg/kg bw from 0–23 days of gestation + 7 days postpartum), and finally group V were prenatal bee pollen-treated neonates (250 mg/kg bw from 0–23 days of gestation + 7 days postpartum) toxicated with NaF (3–5 mg/ kg for 30 days). The results of the PA acid and NaF treatments showed almost the same trend with a significant increase (P≤0.001) in lipid peroxides and significant decreases (P≤0.001) in glutathione, catalase, and vitamin C as compared to the control group. Potassium was significantly decreased (P≤0.010) in the PA group with no outstanding effects in the other groups. The bee pollen-protected neonatal group showed perfection in all the tested parameters. Our results argue for further investigation of fluoride as a risk factor for autism.

Tankyrase1 plays an essential role in cancer progression by regulating telomere length. The study aimed to determine expression of TNKS1 and its regulation in colorectal cancer (CRC) in 20 samples from Saudi patients. mRNA expression of TNKS1 in CRC and paired normal tissues was measured by qRT-PCR. Epigenetic modification of TNKS1 promoter was determined by methylation-specific PCR while somatic mutation was analyzed by Sanger sequencing in exon 10 of the gene. All cancerous and normal tissues expressed TNKS1, but level of expression in CRC tissues was significantly associated with tumor stage though no other parameters; age, gender, and tumor location, showed any correlation. Expression of TNKS1 was markedly higher in earlier (I, II) than later (III, IV) stages of CRC development. Both cancerous and healthy tissues had unmethylated promoter. Sanger sequencing of exon 10 masked any somatic mutation in the samples. Our findings suggest that up-regulation of TNKS1 was inversely correlated with cancer progression in CRC, indicating that TNKS1 participates in the initiation of CRC by stabilizing telomere length in the first phase of cancer progression. Mechanisms other than TNKS1 might play a role in malignant tumor progression and telomere maintenance in the late stages of CRC.

The transcription factor, heat shock factor 1 (HSF1), influences the expression of heat shock proteins as well as other activities like the induction of tumor suppressor genes, signal transduction pathway, and glucose metabolism. We hypothesized that single nucleotide polymorphisms (SNPs) in HSF1 gene might affect its expression or function which might have an influence on the development of breast cancer. The study group included 242 individuals (146 breast cancer patients and 96 healthy controls). From the cancer patients, genomic DNA was extracted from 96 blood samples and 50 Formalin-Fixed Paraffin Embedded (FFPE) tissues, while from the controls DNA were extracted from blood only. Genotype was carried out for four SNPs in the HSF1 gene (rs78202224, rs35253356, rs4977219 and rs34404564) using Taqman genotyping assay method. The HSF1 expression was investigated using immunohistochemistry on FFPE tissues (cancer tissue and adjacent normal tissue). The SNP rs78202224 (G>T) was significantly associated with increased risk of breast cancer. The combined TT + GT genotype (OR: 6.91; p: 0.035) and the T allele showed high risk (OR: 5.81; p:0.0085) for breast cancer development. The SNP rs34404564 (A>G) had a protective effect against the development of breast cancer. The genotype AG (OR: 0.41; p = 0.0059) and GG+AG (OR: 0.52; p: 0.026) occurred at a significantly lower frequency in the breast cancer patients compared to the frequency in healthy controls. No significant relationship was identified between either rs35253356 (A>G) or rs4977219 (A>C) and breast cancer in Saudi. The HSF1 protein expression was higher in all invasive and in situ breast carcinoma compared to the normal tissue. A stronger positive staining for HSF1 was found in the nucleus compared to the cytoplasm. Our results show that HSF1 gene expression is elevated in breast cancer tissue and two of the studied SNPs correlate significantly with cancer development.

Background Colorectal cancer is the leading cause of cancer-related deaths in Saudi Arabia. Cancer has a multifactorial nature and can be described as a disease of altered gene expression. The profiling of gene expression has been used to identify cancer subtypes and to predict patients’ responsiveness. Telomere-associated proteins that regulate telomere biology are essential molecules in cancer development. Thus, the present study examined their contributions to colorectal cancer progression in Saudi patients. Methods The expression of hTERT, TRF1, TRF2, POT1, ATR, ATM, Chk1 and Chk2 were measured via real-time PCR in matched cancerous and adjacent tissues of CRC patients. The protein level of hTERT, TRF1, TRF2, ATR, ATM, Chk1 and Chk2 were measured using immunohistochemistry. A region of hTERT core promoter was sequenced via Sanger sequencing. Methylation of CTCF binding site was examined via methylation-specific PCR. Finally, the length of telomere was estimated using q-PCR. Results Our results showed that POT1, ATR, Chk1 and Chk2 show increased expression in CRC relative to the adjacent mucosa. The expression levels of each gene were associated with clinicopathological characteristics of patients with CRC. There was a positive correlation between the age of the patients and hTERT expression. Regarding tumor site, telomere length, ATR, ATM and Chk1 were shown to be altered. No somatic mutation was detected in hTERT core promoter, and no differences in methylation patterns at CTCF binding site in the promoter between normal and cancer tissues. Conclusion Analysis of targeted genes expression in colorectal cancer based on the clinical variables revealed that tumor location and age could have a role in gene expression and telomere length variations and this could be taken under consideration during CRC diagnosis and therapy. Other epigenetic mechanisms could influence hTERT expression in cancers. Our findings warrant further validation through experiments involving a larger number of patients.

MeHg is a widely distributed environmental toxicant with harmful effects on the developing and adult nervous system. This study aimed to evaluate the therapeutic and protective efficacy of pollen grain in improving the toxic effects of MeHg, through the measurement of selected biochemical parameters linked to oxidative stress, energy metabolism, and neurotransmission in brain homogenates of male pups’ neonates. Forty healthy pregnant female rats were randomly divided into five groups, and after delivery, each group was consisting of 10 male neonates: (1) neonates delivered by control mothers, (2) neonates delivered by bee pollen treated mothers who received bee pollen at the dose of 200-mg/kg body weight from postnatal day 0 for 4 weeks, (3) neonates delivered by MeHg-treated mothers who received MeHg at the dose of 0.5 mg/kg/day via drinking water from gestational day 7 till postnatal day 7 of delivery, (4) therapeutic group: neonates delivered by MeHg-treated mothers followed by bee pollen treatment who received bee pollen at the dose of 200-mg/kg body weight from postnatal day 0 for 4 weeks, and (5) protective group: neonates delivered by MeHg and bee pollen-treated mothers. Mothers continued receiving the bee pollen at the same dose until day 21. Biochemical parameters linked to oxidative stress and energy metabolism and neurotransmission were investigated in brain homogenates of neonates from all the five groups. MeHg treatment showed an increase in oxidative stress markers like lipid peroxidation and catalase activity coupled with a non-significant decrease in glutathione level. Impaired energy metabolism was ascertained via the inhibition of creatine kinase and lactate dehydrogenase activities. Dramatic decrease of Mg2+ and K+ concentrations confirmed the neurotransmission defect. Interestingly, the bee pollen treatment was highly effective in restoring the catalase, lactate dehydrogenase, and creatine kinase activities in addition to normalizing the levels of Mg2+, K+, lipid peroxidation, and glutathione. Overall, the exposure to MeHg during the developing brain stages was highly effective to show signs and symptoms of neuronal toxicity. Furthermore, it has been concluded that bee pollen can be used safely to ameliorate oxidative stress, poor detoxification as well as metal ion defects, and neuronal death as a critical mechanisms involved in the etiology of numerous neurological disorders.

Summary Background: Mutations in BRCA1 gene have been implicated in ovarian cancers, and BRCA testing may be conducted in high-risk women. This study was designed to determine the frequency of three single nucleotide polymorphisms (SNPs) variants in BRCA1 gene and BRCA1 expression in Saudi females with ovarian cancer. Methods: Expression levels of mRNA of BRCA1 gene were studied in 10 ovarian cancer and 10 normal ovarian tissues, by quantitative real time polymerase chain reaction (qPCR). The study also included 28 females who had suffered from ovarian cancer and had been successfully operated upon and 90 healthy females with no history of cancer. Blood was drawn in EDTA tubes and used for extraction of DNA. The genotyping was carried out using Taqman® SNP Genotyping kit by RT-PCR. The variants investigated included c.871 T>C (rs799917), c.1040 G>A (rs4986852), c.181 T>G (rs28897672) in BRCA1 gene. Results: The c.181 T>G (rs28897672) showed significantly different genotype and allele frequencies between the patients and the control subjects (p value = 0.002 and 0.02, respectively). The genotype TG was significantly protective (OR = 0.36, p value = 0.024). The mRNA expression of BRCA1 gene was found to be low in the ovarian cancer tissues. Conclusions: This study showed that c.181 T>G in BRCA1 genes is associated with the development of ovarian cancer in Saudis. More studies are needed to unveil other SNPs that may be associated with ovarian cancer and to understand the mechanism(s) involved in reducing the expression of BRCA1 gene in ovarian cancer tissues.

Glutathione reductase (GR) is highly conserved among diverse taxa and has important biochemical functions. These functions may facilitate survival in harsh conditions, but the role of GR from the liver of the Arabian camel (Camelus dromedarius) is unknown. In this study, the mitochondrial glutathione reductase gene (Gsr) from C. dromedarius liver was cloned and highly expressed in Escherichia coli (Jm109) to gain insight into GR functions in the liver. After amplification of the cDNA encoding the functional unit of Gsr (1.2 kb), the products were cloned into the PGEM-T Easy and PET28a vectors. Gsr expression was confirmed using an immunoblotting technique (45 kDa). Recombinant GR was purified to homogeneity using Ni-NTA resins, with an overall yield of 7.23% and a specific activity of 0.3063 U/mg. The optimum pH of recombinant GR was 7 and the optimum temperature was 35 degrees C in 50 mM K3PO4 buffer. The Michaelis constant, K-m, for the substrates glutathione disulfide (GSSG) and NADPH was 45.6 mu M and 63.5 mu M, respectively; moreover, maximal velocity (V-max) values were 3.969 x 10(-2) U/mg and 1.497 x 10(-1) U/mg. This partial characterization of camel liver GR extends our insight into the ability of camels to cope with harsh environmental conditions.

Summary Background: An insertion/deletion (I/D) polymorphism in the angiotensin converting enzyme (ACE) gene has been associated with recurrent miscarriage (RM) in several populations. We initiated this study to determine the association, if any, between the I/D polymorphism of ACE gene and RM in Saudi females. Method: This study was conducted on 61 Saudi females suffering from RM (mean age: 34.1±6.2 years; range 15–45) attending clinics at King Khalid University Hospital, and 59 age matched females who had at least 2 children, as controls. Blood samples were drawn in EDTA tubes by venipuncture. DNA was extracted using the Puregene DNA purification kits. Insertion/Deletion (I/D) polymorphism of ACE gene was investigated by amplifying the genomic DNA by PCR using gene-specific primers. A single 190 bp or 490 bp band was obtained in the homozygous cases for the D allele or I allele, respectively, while the presence of both 190 and 490 bp bands indicated heterozygosity (ID). Statistical analysis: Deviation from Hardy-Weinberg equilibrium was determined (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl). A standard chi-square (χ2) test was used for comparing the genotype and allele frequencies in the two groups and Students‘t’ test and χ2 test were employed to compare values between the two groups. P<0.05 was considered statistically significant. Results: The frequencies of DD, ID, and II genotypes were 56.7%, 29.5% and 4.9%, respectively, in females with RM and 54.2%, 42.3% and 3.3% respectively in the control group, but the difference was not statistically significant. Conclusion: In some populations, meta-analyses showed an association between I/D polymorphism and RM risk, and the D allele was implicated as an increased risk factor for RM. However, this association was not apparent in the Saudi females.

Introduction: Cardiovascular diseases (CVD) are multifactorial in nature, where genetic susceptibility and environmental factors are required for the disease to occur. Several genetic loci have been implicated as possible susceptibility loci for CVD development, but differences in different populations are frequently reported. Since aromatase is responsible for the synthesis of estrogens from androgens which provide a protective influence against CVD development, we hypothesized that it may play a role in the development of CVD and that polymorphisms in the CYP19 gene that encodes aromatase may influence the development of the disease state. The study group comprised of 120 cardiovascular patients and 132 normal healthy controls. Five polymorphic sites [-81371 A>G (rs4774585), -45965 C>G (rs936308), R264C (rs700519), 80 A>G (rs700518), +32226 A>C (rs4646)] in the CYP19 gene were analysed using TaqMan Genotyping assay. Genotype and allele frequencies were calculated and the results in the patients and control groups were compared. The G allele of rs4774585 and G allele of rs936308 were significantly protective, where the wild type alleles (A and C) for both SNPs increased the risk of CVD. The other studied SNPs did not show any significant difference in frequency between the patients and controls. These results show that the A>G mutation at the rs4774585 site and C>G mutation at the rs936308 site, both in the promoter region, probably increase the level of CYP19 gene expression and hence increase the amount of aromatase, thus increasing the amount of estrogens formed from androgens. The plasma lipid levels and renal function tests were compared in the different genotypes of each SNP and very little associations were observed. It is suggested that there is a need to conduct investigations on gene expression of CYP19 in different mutants, in an attempt to determine the mechanism behind these associations.

or synopsis of the proposal (200 words or less): One of the most common cancer in Saudi Arabia is colorectal cancer in which the incidence has been shown to be increasing gradually over the past few years. Among the various changes occur in can cer cells is telomere dysfunction which consider as an important early event associated with genetic instability. The function of telomeres include the stabilizing the ends of chromosomes, protect them from end-to-end fusion and mediate chromosome pairing during cell division. Human telomeres are composed of TTAGGG tandem repeats in addition to groups of proteins called shelterin complex, which protects chromosome ends, regulates telomere length, recombination, and DNA damage checkpoints. Shelterin is composed of TRF1, TRF2, POT1, TPP1, TIN2, and RAP1. The loss of telomere protection is the root cause of the premature aging The shelterin subunit TRF2 plays a key role in suppressing the telomere-associated DDR through its binding to and inhibition of ATM kinase 6 and 7. ATM is the apical kinase that in response to few DSBs activates many subpathways of the DDR. Although it has been proposed that shelterin inhibits the DDR at telomeres and not elsewhere in the genome, this attractive proposal remains elusive because ATM is not detectable at telomeres. Hypothesis or scientific justification of the proposal Several reports have indicated that the altered expression of TRF2 proteins is associated with tumor progression in various human carcinomas, including lung, stomach, adrenal and pancreatic cancer; the altered expression has also been identified in malignant hematopoietic cells and colorectal pre-neoplastic lesions. It has been found that TRF2 upregulated in human cancer cells. Consistent with a potent oncogenic role of a high level of TRF2, its downregulation in a variety of cancer cells reduces tumorigenicity, whereas overexpression of TRF2 favors oncogenesis. Although a high level of TRF2 expression in cancer cells is expected to maintain a sufficient level of telomere functionality to prevent DDR activation and chromosome instability and to sustain cell proliferation and stem cell function, very little is known about its exact role in colorectal cancer oncogenesis. The aim of this study is to investigate the role of TRF2 and how it will affect ATM. The expressional level of (TRF2, and ATM and chk2) and protein level determination using immunohistochemistry in colorectal cancer in Saudi patients Specific objectives 1. Determining the expressional level of (TRF2, ATM and Chk2) in colorectal cancer and its matched control 2. Protein level determination using Immunohistochemistry of TRF2, ATM and Chk2 3. Methylation status of TRF2 promoter using Methylation SpecificPCR Methodology & Major Techniques to be used Patient samples: Colorectal tissues from Saudi patients will be obtained from the collaborators and clinicians as per the guidelines of IRB. Patients attending the oncology department at KKUH will be examined by the oncologist and routine examination performed. Patient group would comprise men and women with colorectal cancer of all ages and stages of the disease (n=20). Surgical core biopsy and adjacent normal specimen will be obtained prior to treatment and immediately stored in RNAlater solution (Ambion) for DNA and RNA extraction. Nucleic acid Isolation: High–molecular-weight DNA/RNA will be obtained from freshly collected colorectal cancer samples, matched normal samples and blood samples (DNA) using Qiagen DNA/RNA mini prep kit and Qiagen nucleic acid extraction kits according to the manufacturer’s protocol. Quantitative RT–PCR: Expression will be assessed by quantitative RT–PCR in duplicate using SYPR green chemistry (Applied Biosystems) and specific primers TRF2, ATM and chk2. The relative amount of RNA will be calculated with the CT method. Gene expression will be normalized with the GAPDH , and the level of expression of the tumor sample will be compared with the mean level of the gene expression in normal liver tissues and expressed as an n-fold ratio. MSP-PCR: The promoter methylation status of TRF2 of some samples will be determined using EpiTect Bisulfite Conversion Kit from Qiagene and then amplification using PCR. Immunohistochemistry: IHC for TRF2 and ATM will be performed in representative colon tumor and normal tissue. Briefly, deparaffinized and rehydrated sections will be subjected to microwave treatment in 10mM sodium citrate buffer, pH 6.0, for antigen retrieval. The sections will be incubated overnight at 4° C in a humidified chamber with the primary antibody TRF2 and ATM. The detection will be performed with a labelled streptavidin–biotin immunoperoxidase detection system and the immunohistochemical staining will be developed with 3,30diaminobenzidine substrate. Omission of the primary antibody incubation will be used as negative control. Availability of Samples YES If the answer is no, kindly justify Availability of Chemicals YES If the answer is no, kindly justify Availability of Instruments YES Availability of Ethical Approval (if needed) YES Recent References • Mounir El Maï, Kay-Dietrich Wagner, Jean-François Michiels, Damien Ambrosetti, Arnaud Borderie, Sandrine Destree,......, Eric Gilson1 (2014).The Telomeric Protein TRF2 Regulates Angiogenesis by Binding and Activating the PDGFRβ Promoter.Cell Press 1047–1060 • Buscemi, G., Zannini, L., Fontanella, E., Lecis, D., Lisanti, S., & Delia, D. (2009). The Shelterin Protein TRF2 Inhibits Chk2 Activity at Telomeres in the Absence of DNA Damage. Current Biology, 874-879. • Weber, A., & Ryan, A. (2014). ATM and ATR as therapeutic targets in cancer. Pharmacology & Therapeutics, 124-138.

Background: Saudi population is unique in that there is a strong preference for cousin marriages in the general population. We studied the prevalence of consanguinity in educated Saudi females and compared the results with the results obtained in their parents, to access if a generation difference in which extensive educational activities have prevailed to inform the people of the influence of cousin marriages on health, has made any difference in prevalence of consanguineous marriages.Method: A total of 600 Saudi women (421 university students and 179 women attending outpatients' clinics) were interviewed about their own and their parents' consanguinity. Results: The total consanguinity (first and second cousins) was 29.7% in the parents. Consanguinity was significantly higher among the daughters than the parents, where 37.9% of the 293 married women had consanguineous marriages. The prevalence of consanguinity was studied in different age groups, though no significant pattern was observed. A strong correlation was found between consanguinity of parents and their daughters; consanguinity was highest (52.3%) in the daughters of parents who were themselves consanguineous.Conclusion: The results did not reveal any decrease in the prevalence of consanguinity over a generation. This shows that the tradition of marrying within the family is a preferred practice, despite the awareness that certain genetic disorders occur at a higher frequency in cousin marriages. There is a need at the primary health care level to inform the public of the consequences of this common practice.

Seeds aqueous extracts from Litchi chinensis and Nephelium lappaceum were investigated for antibacterial activity by disc diffusion method and protein profile. Both seed aqueous extracts show moderate inhibition against pathogenic bacteria, both gram positive including Staphylococcus aureus, Streptococcus pyogenes, and Bacilllus subtillis and gram negative bacteria including Escherichia coli and Pseudomonas aeruginosa. Overall analysis of the antibacterial activity of tested samples revealed that the highest inhibitory activity was produced by Litchi chinensis (15 ± 0.55 mm) against S. pyogenes. Tris glycine SDS PAGE revealed major protein band approximately 15.5 kDa and 22-kDa. Protein contents of Seeds of Litchi chinensis and Nephelium lappaceum were approximately 7.5 and 13.5 mg/g, respectively.

We purified a protein from the dromedary small intestine that displayed potent bactericidal activity against Gram-positive bacteria, and identified it as group-IIA phospholipase A2 (DrPLA2-IIA) by NH2-terminal sequencing and enzymatic measurements. In fact, our findings revealed that the purified PLA2-IIA was a monomeric protein with a molecular mass of about 14kDa. Pure enzyme has a specific activity of 329±25U/mg at optimal conditions (pH 9.5 and 45°C) in the presence of 6mM NaDC and 7mM CaCl2 with egg yolk emulsion as substrate and binds with a higher affinity to PE than PS and PC. Furthermore, the DrPLA2-IIA activity was dependent on Ca2+; other cations (Cd2+, Co2+, Fe2+, Mg2+, Mn2+, and Zn2+) reduced the enzymatic activity notably, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca2+. On the other hand, DrPLA2-IIA was highly bactericidal against Gram-positive bacteria with inhibition zones and IC50 values in the range of 21–27mm and 3.7–8μg/ml, respectively, whereas Gram-negative bacteria exhibited a much higher resistance. These observations suggest that the main physiological role of DrPLA2-IIA could be the defense of the intestine against bacterial infections.