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.     

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.     

Regulating Neutrophil PAD4/NOX-Dependent Cerebrovasular Thromboinflammation

Background: Neutrophil extracellular trap (NET) production has been implicated in the pathogenesis of thromboinflammatory conditions such as Sickle Cell Disease (SCD), contributing to heightened risk for ischemic stroke. NETs are catalyzed by the enzyme Peptidyl Arginine Deiminase 4 (PAD4) and neutrophil derived reactive oxygen species (ROS), especially NADPH oxidase (NOX) which interacts with PAD4 and is therefore critical for neutrophil function. However, the role that NOX-dependent ROS and NETs play in the accelerated cerebral microvascular thrombosis associated with thromboinflammatory conditions, such as SCD, has not been fully elucidated and is the aim of this study.Methods: The in-vitro effects of targeting PAD4 and NOX were examined using physiologically relevant NET assays with neutrophils isolated from healthy volunteers (control) and SCD patients. In addition, in-vivo intravascular effects of targeting PAD4 and NOX in the cerebral microcirculation of C57BL/6 and sickle transgenic mice (STM) were assessed using a photoactivation thrombosis model (light/dye) coupled with real-time fluorescence intravital microscopy.Results: We found that targeting PAD4 and NOX in human neutrophils significantly inhibited ionomycin dependent H3cit⁺ neutrophils. Targeting PAD4 and NOX in-vivo resulted in prolonged blood flow cessation in cerebrovascular arterioles as well as venules. Moreover, we were able to replicate the effects of PAD4 and NOX targeting in a clinical model of accelerated thromboinflammation by increasing blood flow cessation times in cerebral microvessels in STM. These findings concurred with the clinical setting i.e. neutrophils isolated from SCD patients, which possessed an attenuation of H3cit⁺ neutrophil production on targeting PAD4 and NOX.Conclusions: Taken together, our compelling data suggests that PAD4 and NOX play a significant role in neutrophil driven thromboinflammation. Targeting PAD4 and NOX limits pathological H3cit⁺ neutrophils, which may further explain attenuation of cerebral thrombosis. Overall, this study presents a viable pre-clinical model of prevention and management of thromboinflammatory complications such as ischemic stroke.     

Azotobacter: A potential bio-fertilizer for soil and plant health management

Stressor (biotic as well as abiotic) generally hijack the plant growth and yield characters in hostile environment leading to poor germination of the plants and yield. Among the plant growth promoting rhizobacteria, Azotobacter spp. (Gram-negative prokaryote) are considered to improve the plant health. Various mechanisms are implicated behind improved plant health in Azotobacter spp. inoculated plants. For example, acceleration of phytohormone like Indole-3-Acetic Acid production, obviation of various stressors, nitrogen fixation, pesticides and oil globules degradation, heavy metals metabolization, etc. are the key characteristics of Azotobacter spp. action. In addition, application of this bacteria has also become helpful in the reclamation of soil suggesting to be a putative agent which can be used in the transformation of virgin land to fertile one. Application of pesticides of chemical origin are being put on suspension mode as the related awareness program is still on. As far as the limitations of this microbe is concerned, commercial level formulations availability is still a great menace. Present review has been aimed to appraise the researchers pertaining to utility of Azotobacter spp. in the amelioration of plant health in sustainable agroecosystem. The article has been written with the target to gather maximum information into single pot so that it could reach to the dedicated researchers.     

Genetic assessment of the internal transcribed spacer region (ITS1.2) in Mangifera indica L. landraces

Mango (Mangifera indica) is one of the most important tropical fruits in the world. Twenty-two genotypes of native mangoes from different regions of southern Iran (Hormozgan and Kerman) were collected and analyzed for the ribosomal genes. GC content was found to be 55.5%. Fu and Li’s D* test statistic (0.437), Fu and Li’s F* test statistic (0.500) and Tajima’s D (1.801) were positive and nonsignificant. A total of 769 positions were identified (319 with insertion or deletion including 250 polymorphic and 69 monomorphic loci; 450 loci without any insertion or deletion including 35 Singletons and 22 haplotypes). Nucleotide diversity of 0.309 and a high genetic differentiation including Chi square of 79.8; P value of 0.3605 and df value of 76 was observed among mango genotypes studied. The numerical value of the ratio dN/dS (0.45) indicated a pure selection in the examined gene and the absence of any key changes. Cluster analysis differentiated the mango used in this research (M. indica L.) into two genotypes but could not differentiate their geographical locations. The results of this study indicated that a high genetic distance exists between HajiGholam (Manojan) and Arbabi (Rodan) genotypes and showed higher genetic diversity in mango of Rodan region. Results of present study suggested that for successful breeding, the genotypes of Rodan region mango especially Arbabi mango can be used as a gene donor and ITS can be a suitable tool for genetic evaluations of inter and intra species.