Effect of Zataria multiflora Extract on Total and Differential White Blood Cell Count and Endothelin Level in Blood of Ovalbumin Sensitized Guinea Pigs

Objective: To investigate the effect of hydro-ethanolic extract of Zataria multiflora (Z. multiflora) on endothelin level, total and differential white blood cells (WBC) count of sensitized guinea pigs. Methods: Five groups of guinea pigs sensitized to ovalbumin (OA) were given drinking water alone (group S), drinking water containing three concentrations of Z. multiflora (0.2, 0.4 and 0.8 mg/mL as groups S+Z1, S+Z2 and S+Z3) and dexamethasone (group S+D), n=6 for each group. The endothelin levels as well as total and differential WBC count in blood of sensitized and control guinea pigs were evaluated using enzyme linked immunosorbent assay method, and hemocytometer and Wright-Giemsa''s staining of blood sample smear respectively. Results: Blood endothelin levels, total and most differential WBC count were increased but lymphocytes decreased in sensitized animals compared to controls (all P<0.01). In groups S+D, S+Z2 and S+Z3 endothelin level, total and differential WBC counts were significantly improved compared with group S (P<0.01). Although, all measured parameters in group S+Z1 was lower than group S+D (P<0.01), some parameters in group S+Z3 were greater than in group S+D (P<0.05 to P<0.01). Conclusion: The results showed an anti-inflammatory effect of Z. multiflora extract in sensitized guinea pigs, which may suggest a therapeutic potential for the plant on asthma.     

Exome sequencing identifies FANCM as a susceptibility gene for triple-negative breast cancer

Inherited predisposition to breast cancer is known to be caused by loss-of-function mutations in BRCA1, BRCA2, PALB2, CHEK2, and other genes involved in DNA repair. However, most families severely affected by breast cancer do not harbor mutations in any of these genes. In Finland, founder mutations have been observed in each of these genes, suggesting that the Finnish population may be an excellent resource for the identification of other such genes. To this end, we carried out exome sequencing of constitutional genomic DNA from 24 breast cancer patients from 11 Finnish breast cancer families. From all rare damaging variants, 22 variants in 21 DNA repair genes were genotyped in 3,166 breast cancer patients, 569 ovarian cancer patients, and 2,090 controls, all from the Helsinki or Tampere regions of Finland. In Fanconi anemia complementation gene M (FANCM), nonsense mutation c.5101C>T (p.Q1701X) was significantly more frequent among breast cancer patients than among controls [odds ratio (OR) = 1.86, 95% CI = 1.26–2.75; P = 0.0018], with particular enrichment among patients with triple-negative breast cancer (TNBC; OR = 3.56, 95% CI = 1.81–6.98, P = 0.0002). In the Helsinki and Tampere regions, respectively, carrier frequencies of FANCM p.Q1701X were 2.9% and 4.0% of breast cancer patients, 5.6% and 6.6% of TNBC patients, 2.2% of ovarian cancer patients (from Helsinki), and 1.4% and 2.5% of controls. These findings identify FANCM as a breast cancer susceptibility gene, mutations in which confer a particularly strong predisposition for TNBC.Breast cancer is the most common cancer affecting women worldwide. It is also the principal cause of death from cancer among women globally, accounting for 14% of all cancer deaths (1). The etiology of breast cancer is multifactorial, and the risk depends on various factors like age, family history, and reproductive, hormonal, or dietary factors. The majority of breast cancers are sporadic, but approximately 15% of cases show familial aggregation (2, 3). Since the identification of the first breast and ovarian cancer susceptibility genes breast cancer 1 and 2 (BRCA1 and BRCA2, respectively) by linkage analysis and positional cloning, several breast cancer susceptibility genes and alleles with different levels of risk and prevalence in the population have been recognized. BRCA1 and BRCA2 mutation carriers have more than 10-fold increased risk of breast cancer compared with women in the general population, and mutations in TP53, PTEN, STK11, and CDH1 have also been associated with a high lifetime risk of breast cancer in the context of rare inherited cancer syndromes (4). In addition, rare variants in genes such as checkpoint kinase 2 (CHEK2), ataxia telangiectasia mutated (ATM), and BRCA1 interacting helicase BRIP1, that confer a two- to fourfold increased risk, and in partner and localizer of BRCA2 (PALB2), with even higher risk estimates, have been found with candidate gene approaches (5, 6), and an increasing number of common low-risk loci with modest odds ratios (ORs; as much as 1.26-fold increased risk for heterozygous carriers) have been identified by genome-wide association studies (7).However, the major portion of hereditary breast cancer still remains unexplained, and many susceptibility loci are yet to be found. Exome sequencing combined with genotyping of the identified variants in case-control analysis is an effective method to recognize novel risk alleles, based on the assumption that disease-causing variants are rare and often accumulate in the protein-coding areas of the genome (8–10).Since the discovery that proteins encoded by the BRCA1 and BRCA2 breast/ovarian cancer susceptibility genes are directly involved in homologous recombination repair of DNA double-strand breaks, it has been evident that other genes involved in DNA repair are attractive breast cancer susceptibility candidates (4). Biallelic mutations in ATM gene cause rare ataxia telangiectasia disease and are associated with an increased risk for breast cancer as a result of improper DNA damage response (11). Fanconi anemia (FA) is a rare genetic disorder caused by biallelic mutations in FA genes that also participate in DNA repair. At least 15 FA genes have been identified (12). Patients with heterozygous mutations in certain FA genes have an elevated risk for various cancers, and monoallelic mutations in at least four of these genes [BRCA2, BRIP1, PALB2, and RAD51 paralog C (RAD51C)] are associated with an increased risk of breast or ovarian cancer (12, 13). Recurrent founder mutations in several cancer susceptibility genes, including the BRCA2, PALB2, and RAD51C FA genes, have been identified in the Finnish population (14–16). The PALB2 and RAD51C founder mutations have been detected at 2% frequency in Finnish breast or ovarian cancer families (15–17), whereas, in other populations, mutations in these genes are rare and often unique for each family. Founder effects in the isolated populations such as Finland or Iceland may enrich certain mutations and thus explain a significant proportion of all mutations in certain genes (18, 19). This provides an advantage in the search for novel susceptibility genes and alleles.In this study, we used exome sequencing to uncover previously unidentified recurrent breast or ovarian cancer predisposing variants in the Finnish population with a focus on DNA repair genes. Selected variants were further genotyped in a large case-control sample set. Our investigation revealed an association of a nonsense mutation (rs147021911) in an FA complementation gene, FANCM, with breast cancer, especially with triple-negative (TN) breast cancer (TNBC).     

Evidence of a Major Reservoir of Non-Malarial Febrile Diseases in Malaria-Endemic Regions of Bangladesh

In malaria-endemic regions any febrile case is likely to be classified as malaria based on presumptive diagnosis largely caused by a lack of diagnostic resources. A district-wide prevalence study assessing etiologies of fever in 659 patients recruited in rural and semi-urban areas of Bandarban district in southeastern Bangladesh revealed high proportions of seropositivity for selected infectious diseases (leptospirosis, typhoid fever) potentially being misdiagnosed as malaria because of similarities in the clinical presentation. In an area with point prevalences of more than 40% for malaria among fever cases, even higher seroprevalence rates of leptospirosis and typhoid fever provide evidence of a major persistent reservoir of these pathogens.     

Delivery and Biodistribution of Traceable Polymeric Micellar Diclofenac in the Rat

The nonsteroidal anti-inflammatory drugs elevate cardiovascular risk, perhaps, due to their accumulation in the heart and kidneys. We designed nanodelivery systems for cardiotoxic diclofenac to reduce its presence in these organs. Diclofenac ethyl ester (DFEE) was encapsulated in traceable micelles based on poly(ethylene oxide)-b-poly(ε-caprolactone) (DFEE-PCL-TM) or poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (DFEE-PBCL-TM). Diclofenac pharmacokinetics and tissue distribution were studied after intravenous (iv) and intraperitoneal administration of the nanoformulations and compared with those after iv doses of free diclofenac (n = 3-6/group). The average diameters for DFEE-PBCL-TM and DFEE-PCL-TM were 37.2 ± 0.06 and 45.1 ± 0.06 nm, respectively. Drug concentration dropped below the assay sensitivity after free drug administration in 6 h, but persisted for 24 h following DFEE-PBCL-TM (2.3 ± 1.4 μg/mL) and DFEE-PCL-TM (1.9 ± 0.6 μg/mL) iv administration. The diclofenac heart:blood and kidney:blood ratios were 5- to 12-fold lower with the nanoformulations than with free diclofenac. Near-infrared fluorescence measurements in tissues suggested exposure patterns to nanocarriers parallel with those achieved for delivered diclofenac by nanoformulations. Administration of DFEE-PCL-TM by iv or intraperitoneal injection, resulted in comparable pharmacokinetics and 6 h postdose near-infrared fluorescence in the heart, kidneys, liver, and spleen. When compared to each other, DFEE-PBCL-TM showed significantly lower diclofenac levels in the heart compared to DFEE-PCL-TM (0.3 ± 0.03 vs. 0.5 ± 0.1 μg/g). Developed nanoformulations of diclofenac prolonged diclofenac circulation and reduced its presence in the heart and kidneys, strongly suggesting cardiac-safe delivery vehicles for diclofenac.     

WWOX,the common fragile site FRA16D gene product,regulates ATM activation and the DNA damage response

Genomic instability is a hallmark of cancer. The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor spanning the common chromosomal fragile site FRA16D. Here, we report a direct role of WWOX in DNA damage response (DDR) and DNA repair. We show that Wwox deficiency results in reduced activation of the ataxia telangiectasia-mutated (ATM) checkpoint kinase, inefficient induction and maintenance of γ-H2AX foci, and impaired DNA repair. Mechanistically, we show that, upon DNA damage, WWOX accumulates in the cell nucleus, where it interacts with ATM and enhances its activation. Nuclear accumulation of WWOX is regulated by its K63-linked ubiquitination at lysine residue 274, which is mediated by the E3 ubiquitin ligase ITCH. These findings identify a novel role for the tumor suppressor WWOX and show that loss of WWOX expression may drive genomic instability and provide an advantage for clonal expansion of neoplastic cells.Genomic instability is a common characteristic of human cancers. The DNA damage response (DDR) maintains the integrity of the genome in response to DNA damage. DDR is a complex signaling process that results in cell cycle arrest followed by either DNA repair or apoptosis if the DNA damage is too extensive to be repaired (1–3). Key mammalian damage response sensors are ataxia telangiectasia-mutated (ATM), ATM and Rad3-related, and DNA-dependent PKs (4, 5). Disruption of the DDR machinery in human cells leads to genomic instability and an increased risk of cancer progression (6, 7).The WW domain-containing oxidoreductase (WWOX) gene spans the common fragile site (CFS) FRA16D (8, 9). Genomic alterations affecting the WWOX locus have been reported in several types of cancer and include homozygous and hemizygous deletions (10–13). Ectopic expression of WWOX in WWOX-negative cancer cells attenuates cell growth and suppresses tumor growth in immunocompromised mice (10, 11, 14). Importantly, targeted ablation of Wwox in mice results in higher incidence of spontaneous lesions resembling osteosarcomas and lung and mammary tumors (14–16). These findings suggest WWOX as a tumor suppressor. The WWOX protein contains two N-terminal WW domains mediating WWOX interaction with PP(proline)x(amino acid)Y(tyrosine)-containing proteins (11, 17) and a central short-chain deyhdrogenase/reductase domain that has been proposed to function in steroidogenesis (18). Recent characterization of WWOX domains revealed that they interact, mainly through the WW1 domain, with multiprotein networks (3). The mechanism by which WWOX suppresses tumorigenicity is, however, not well-known.In vitro, CFSs are defined as gaps or breaks on metaphase chromosomes that occur in cells treated with inhibitors of DNA replication (19, 20). In vivo, CFSs are preferential targets of replication stress in preneoplastic lesions (21), and emerging evidence suggests that they represent early warning sensors for DNA damage (22–24). Both genetic and epigenetic factors are thought to regulate the fragility of CFS (25, 26). Recent profiling studies of CFS provide evidence that the functional fragility of CFS is tissue-specific (27–29). High-throughput genomic analyses of 3,131 cancer specimens (12) and 746 cancer cell lines (13) have recently identified large deletions in CFSs, including the FRA16D/WWOX locus. Although these deletions have been linked to the presence of DNA replication stress (30), the molecular function of gene products of CFSs, including the WWOX protein, is poorly understood. Here, we identify a direct role of WWOX in the DDR and show that the WWOX gene product functions as a modulator of the DNA damage checkpoint kinase ATM.