Phorate triggers oxidative stress and mitochondrial dysfunction to enhance micronuclei generation and DNA damage in human lymphocytes

Herein, we studied phorate for its toxicological effects in human lymphocytes. Phorate treatment for 3 h has induced significant increase in the lymphocytic DNA damage. Compared to control, comet data from highest concentration of phorate (1000 µM) showed 8.03-fold increase in the Olive tail moment (OTM). Cytokinesis blocked micronucleus (CBMN) assay revealed 6.4-fold increase in binucleated micronucleated (BNMN) cells following the exposure with phorate (200 µM) for 24 h. The nuclear division index (NDI) in phorate (200 µM) treated cells reduced to 1.8 vis-à-vis control cells showed NDI of 1.94. Comparative to untreated control, 60.43% greater DCF fluorescence was quantitated in lymphocytes treated with phorate (500 µM), affirming reactive oxygen species (ROS) generation and oxidative stress. Flow cytometric data of phorate (200 µM) treated lymphocytes showed 81.77% decline in the fluorescence of rhodamine 123 (Rh123) dye, confirming the perturbation of mitochondrial membrane potential (ΔΨm). Calf thymus DNA (ct-DNA) treated with phorate (1000 µM) exhibited 2.3-fold higher 8-Hydroxy-2′-deoxyguanosine (8-oxodG) DNA adduct formation, signified the oxidative DNA damage. The alkaline unwinding assay revealed 4.0 and 6.5 ct-DNA strand breaks when treated to phorate and phorate-Cu (II) complex. Overall, the data unequivocally suggests the cyto- and genotoxic potential of phorate in human lymphocytes, which may induce comparable toxicological consequences in persons occupationally or non-occupationally exposed to insecticide phorate.     

Association mapping for soilborne pathogen resistance in synthetic hexaploid wheat

Soilborne pathogens such as cereal cyst nematode (CCN; Heterodera avenae) and root lesion nematode (Pratylenchus neglectus; PN) cause substantial yield losses in the major cereal-growing regions of the world. Incorporating resistance into wheat cultivars and breeding lines is considered the most cost-effective control measure for reducing nematode populations. To identify loci with molecular markers linked to genes conferring resistance to these pathogens, we employed a genome-wide association approach in which 332 synthetic hexaploid wheat lines previously screened for resistance to CCN and PN were genotyped with 660 Diversity Arrays Technology (DArT) markers. Two sequence-tagged site markers reportedly linked to genes known to confer resistance to CCN were also included in the analysis. Using the mixed linear model corrected for population structure and familial relatedness (Q+K matrices), we were able to confirm previously reported quantitative trait loci (QTL) for resistance to CCN and PN in bi-parental crosses. In addition, we identified other significant markers located in chromosome regions where no CCN and PN resistance genes have been reported. Seventeen DArT marker loci were found to be significantly associated with CCN and twelve to PN resistance. The novel QTL on chromosomes 1D, 4D, 5B, 5D and 7D for resistance to CCN and 4A, 5B and 7B for resistance to PN are suggested to represent new sources of genes which could be deployed in further wheat improvement against these two important root diseases of wheat.     

An application of capsid-specific artificial ankyrin repeat proteinproduced in E. coli for immunochromatographic assay as a surrogate for antibody

Immunochromatographic strip test is a unique type of rapid test that has been developed for use as part of a diagnostic kit for the rapid detection of antibodies and/or other proteins of interest. For the detection of target proteins, most of the commercial tests are assembled based on the conjugation of colloidal gold particles to monoclonal antibodies embedded within the conjugate pad of a strip test. In this study, we tested the novel concept of using an artificial non-antibody structure for generating a colloidal gold conjugate (CGC). We exploited the property of an ankyrin repeat protein that specifically binds to the HIV-1 capsid protein termed Ank^GAG1D4. This construct was applied as a model structure to create Ank1D4-CGC and used as a new type of visible detector system and termed it ankyrin-based immunochromatographic strip (ABIS) test. The ABIS test was shown to be highly sensitive with a lower limit of detection of the target protein at 0.1 μg/ml. Moreover, the ABIS test was not only highly sensitive but also shared a level of specificity within the same range of the commercial test kit. The results of the studies presented herein therefore demonstrate the novel application of an artificial non-immunoglobulin structure (ankyrin repeat protein) as the new line of a visible detector using a rapid diagnostic test with characteristics that have the potential to be superior to those that utilize antibody-based tests.     

A porous medium‐chain‐length poly(3‐hydroxyalkanoates)/hydroxyapatite composite as scaffold for bone tissue engineering

Polyhydroxyalkanoates (PHA) are hydrophobic biopolymers with huge potential for biomedical applications due to their biocompatibility, excellent mechanical properties and biodegradability. A porous composite scaffold made of medium‐chain‐length poly(3‐hydroxyalkanoates) (mcl‐PHA) and hydroxyapatite (HA) was fabricated using particulate leaching technique and NaCl as a porogen. Different percentages of HA loading was investigated that would support the growth of osteoblast cells. Ultrasonic irradiation was applied to facilitate the dispersion of HA particles into the mcl‐PHA matrix. The different P(3HO‐co‐3HHX)/HA composites were investigated using field emission scanning electron microscopy (FESEM), X‐ray diffraction (XRD) and energy dispersive X‐ray analysis (EDXA). The scaffolds were found to be highly porous with interconnecting pore structures and the HA particles were homogeneously dispersed in the polymer matrix. The scaffolds biocompatibility and osteoconductivity were also assessed following the proliferation and differentiation of osteoblast cells on the scaffolds. From the results, it is clear that scaffolds made from P(3HO‐co‐3HHX)/HA composites are viable candidate materials for bone tissue engineering applications.     

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+/K+ homeostasis

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na⁺] and [K⁺] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na⁺‐selective class I‐HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single‐nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near‐isogenic lines (NILs): 157‐14 (double homozygote for the cheesmaniae alleles) and 157‐17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157‐14 in comparison with 157‐17. A significant difference between NILs was also found for [K⁺] and the [Na⁺]/[K⁺] ratio in leaf and stem but not for [Na⁺] arising a disagreement with the corresponding RIL population. Their association with leaf [Na⁺] and salt tolerance in tomato is also discussed.     

Doppler ultrasound scoring to predict chemotherapeutic response in advanced breast cancer

Background

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited disease. It is relatively recent that type 2C was identified as a separate group solely presenting with pheochromocytomas. As an illustration, an interesting case is presented of a pregnant woman with refractory hypertension. It proved to be the first manifestation of bilateral pheochromocytomas. The family history may indicate the diagnosis, but only identification of a germ line mutation in the DNA of a patient will confirm carriership.

Case presentation

A 27 year pregnant patient with intra uterine growth retardation presented with hypertension and pre-eclampsia. Magnetic resonance imaging revealed bilateral adrenal pheochromocytoma. She underwent laparoscopic adrenelectomy and a missense mutation (Gly93Ser) in exon 1 of the VHL gene on chromosome 3 (p25 - p26) was shown in the patient, her father and her daughter confirming the diagnosis of VHL.

Conclusion

In almost all VHL families molecular genetic analysis of DNA will demonstrate an inherited mutation. Because of the involvement in several organs, periodic clinical evaluation should take place in a well coordinated, multidisciplinary setting. VHL disease can be classified into several subtypes. VHL type 2C patients present with pheochromocytomas without evidence of haemangioblastomas in the central nervous system and/or retina and a low risk of renal cell carcinoma. Therefore, in such families, periodic clinical screening can be focussed on pheochromocytomas.     

<Emphasis Type="Italic">In silico</Emphasis> analysis of methyltransferase domains involved in biosynthesis of secondary metabolites

Background  

Secondary metabolites biosynthesized by polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) family of enzymes constitute several classes of therapeutically important natural products like erythromycin, rapamycin, cyclosporine etc. In view of their relevance for natural product based drug discovery, identification of novel secondary metabolite natural products by genome mining has been an area of active research. A number of different tailoring enzymes catalyze a variety of chemical modifications to the polyketide or nonribosomal peptide backbone of these secondary metabolites to enhance their structural diversity. Therefore, development of powerful bioinformatics methods for identification of these tailoring enzymes and assignment of their substrate specificity is crucial for deciphering novel secondary metabolites by genome mining.