HK022 bacteriophage Integrase mediated RMCE as a potential tool for human gene therapy

HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active ‘attB’ sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native ‘attB’ sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native ‘attB’ sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.     

Genetic assessment of the effects of self-fertilization in a Lilium L. hybrids using molecular cytogenetic methods (FISH and ISSR)

Self-fertilization (also termed selfing) is a mode of reproduction that occurs in hermaphrodites and has evolved several times in various plant and animal species. A transition from outbreeding to selfing in hermaphroditic flowers is typically associated with changes in flower morphology and functionality. This study aimed to identify genetic effects of selfing in the F2 progeny of F1 hybrid developed by crossing Lilium lancifolium with the Asiatic Lilium hybrid ‘Dreamland.’ Fluorescence in situ hybridization (FISH) and inter-simple sequence repeats (ISSR) techniques were used to detect genetic variations in plants produced by selfing. The FISH results showed that F1 hybrid were similar to the female parent (L. lancifolium) regarding the 45S loci, but F2 individuals showed variation in the number and location of the respective loci. In F2 progeny, F2-2, F2-3, F2-4, F2-5, and F2-8 hybrids expressed two strong and one weak 5S signal on chromosome 3, whereas F2-7 and F2-9 individuals expressed one strong and two weak signals. Only two strong 5S signals were detected in an F2-1 plant. The ISSR results showed a maximum similarity value of 0.6269 between the female parent and the F2-2 hybrid. Regarding similarity to the male parent, a maximum value of 0.6119 was found in the F2-1 and F2-2 hybrids. The highest genetic distance from L. lancifolium and the Asiatic Lilium hybrid ‘Dreamland’ was observed in the F2-4 progeny (0.6352 and 0.7547, respectively). Phylogenetic relationships showed that the F2 progeny were closer to the male parent than to the female parent. Self-fertilization showed effects on variation among the F2 progeny, and effects on the genome were confirmed using FISH and ISSR analyses.     

Occurrence of guanosine 3′,5′-cyclic monophosphate (Cyclic GMP) and associated enzyme systems in Phaseolus vulgaris

Cyclic GMP, isolated from Phaseolus vulgaris, has been unequivocally identified by NMR and FAB-mass spectrometry with MIKES-scanning. Radioimmunoas     

Modulating P1 Adenosine Receptors in Disease Progression of SOD1G93A Mutant Mice

Amyotrophic lateral sclerosis (ALS) is a fatal progressing neurodegenerative disease; to date, despite the intense research effort, only two therapeutic options, with very limited effects, are available. The purinergic system has been indicated as a possible new therapeutic target for ALS, but the results are often contradictory and generally confused. The present study was designed to determine whether P1 adenosine receptor ligands affected disease progression in a transgenic model of ALS. SOD1^G93A mice were chronically treated, from presymptomatic stage, with a selective adenosine A_2A receptor agonist (CGS21680), antagonist (KW6002) or the A₁ receptor antagonist DPCPX. Body weight, motor performance and survival time were evaluated. The results showed that neither the stimulation nor the blockade of adenosine A_2A receptors modified the progressive loss of motor skills or survival of mSOD1^G93A mice. Conversely, blockade of adenosine A₁ receptors from the presymptomatic stage significantly attenuated motor disease progression and induced a non-significant increase of median survival in ALS mice. Our data confirm that the modulation of adenosine receptors can elicit very different (and even opposite) effects during the progression of ALS course, thus strengthens the importance of further studies to elucidated their real therapeutic potential in this pathology.

     

Oncogenic comparison of human papillomavirus type 58 E7 variants

Human papillomavirus 58 (HPV58) ranks the second or third in East Asian cervical cancers. Current studies on HPV58 are scarce and focus on the prototype. Previously, we identified the three most common circulating HPV58 E7 strains contained amino acid alterations: G41R/G63D (51%), T20I/G63S (22%) and T74A/D76E (14%) respectively. Among them, the T20I/G63S variant (V1) had a stronger epidemiological association with cervical cancer. We therefore suggested that V1 possessed stronger oncogenicity than the other two variants. Here, we performed phenotypic assays to characterize and compare their oncogenicities with HPV58 E7 prototype. Our results showed that overexpression of V1 conferred a higher colony‐forming ability to primary murine epithelial cells than prototype (P < 0.05) and other variants, implicating its higher immortalising potential. Further experiments showed that both V1 and prototype enhanced the anchorage‐independent growth of NIH/3T3 cells (P < 0.001), implicating their stronger transforming power than the two other variants. Moreover, they possessed an increased ability to degrade pRb (P < 0.001), which is a major effector pathway of E7‐driven oncogenesis. Our work represents the first study to compare the oncogenicities of HPV58 E7 prototype and variants. These findings deepened our understanding of HPV58 and might inform clinical screening and follow‐up strategy.     

RNA Interference (RNAi) Induced Gene Silencing: A Promising Approach of Hi-Tech Plant Breeding

RNA interference (RNAi) is a promising gene regulatory approach in functional genomics that has significant impact on crop improvement which permits down-regulation in gene expression with greater precise manner without affecting the expression of other genes. RNAi mechanism is expedited by small molecules of interfering RNA to suppress a gene of interest effectively. RNAi has also been exploited in plants for resistance against pathogens, insect/pest, nematodes, and virus that cause significant economic losses. Keeping beside the significance in the genome integrity maintenance as well as growth and development, RNAi induced gene syntheses are vital in plant stress management. Modifying the genes by the interference of small RNAs is one of the ways through which plants react to the environmental stresses. Hence, investigating the role of small RNAs in regulating gene expression assists the researchers to explore the potentiality of small RNAs in abiotic and biotic stress management. This novel approach opens new avenues for crop improvement by developing disease resistant, abiotic or biotic stress tolerant, and high yielding elite varieties.     

Evaluation of duck egg yolk for the cryopreservation of Nili-Ravi buffalo bull semen

This study was carried out to investigate if the substitution of chicken egg yolk (CEY) with duck egg yolk (DEY) in extenders can improve the quality of frozen-thawed semen of Nili-Ravi buffalo bulls and to study if reducing DEY level in extender affects the freezability results. Thirty semen samples collected from three buffalo bulls were diluted in extenders A, B, C, D and E containing tris, citric acid, fructose, egg yolk, glycerol and antibiotics. Extender A contained 20% CEY (control), while extenders B, C, D and E contained 5, 10, 15 and 20% DEY, respectively. After freezing and storage for 24h in liquid nitrogen, samples were evaluated for post-thaw quality. The post extension sperm motility did not differ between extenders A (control) and E (20% DEY). The same was true for post-thaw percentage of sperm with functional plasma membrane and percentage of sperm with abnormal heads or mid pieces. However, extender E showed higher (P<0.05) values for post-thaw sperm motility, livability and absolute index of livability of spermatozoa at 37 °C compared to extender A. Spermatozoa with abnormal tail were lower (P<0.05) in extender E compared to extender A. Values of these parameters of post-thaw semen quality were highest for extender E containing 20% DEY and decreased significantly with decrease in the concentration of DEY, except sperm abnormalities (head, mid-piece and tail) which increased with decrease in DEY level. These results showed that replacement of 20% CEY with 20% DEY in extenders significantly improved post-thaw sperm motility, livability and absolute index of livability of spermatozoa and reduced tail abnormalities. Reduction in the level of DEY in extenders from 20% adversely affected post-thaw semen quality of Nili-Ravi buffalo bulls.