Synthesis of enamino-2-oxindoles via conjugate addition between α-azido ketones and 3-alkenyl oxindoles: Cytotoxicity evaluation and apoptosis inducing studies

A facile method for the construction of double bond between 3-ylidene oxindoles and α-azido ketones has been successfully accomplished with a mild base. This method features azido reduction with concomitant double bond formation to provide the new class of bioactive enamino-2-oxindoles. These new compounds were screened for their in vitro cytotoxic potential on selected human cancer cell lines such as colon, lung, breast, and cervical cancer cells. Among them, representative compounds 3a, 3h, 3k, 3p, 3w and 3x showed notable cytotoxicity profile with IC₅₀ values ranging from 1.40?±?0.10 to 28.7?±?0.36?µM. Compound 3k displayed most potent cytotoxicity against lung cancer (NCI-H460) cells with an IC₅₀ value of 1.40?±?0.10?µM. 3k also arrested the G2/M phase of the cell cycle and induced distinctive apoptotic features on lung cancer cells. The apoptosis induction is supported by various cellular assays such as AO/EB, DAPI, and DCFDA staining studies including clonogenic assay. Extent of apoptosis was also analyzed by Annexin binding and JC-1 staining. Moreover, this method is amenable for the generation of a library of new class of stable bioactive enamino-2-oxindoles.     

Effect of historical factors on genetic variation in three terrestrial Cephalanthera species (Orchidaceae) with different breeding system on the Korean Peninsula

Previous studies have shown that levels of genetic diversity in species of the genus Cephalanthera covary with the breeding system. In the southern part of the Korean Peninsula, the three self‐compatible terrestrial orchids Cephalanthera erecta, C. falcata and C. longibracteata flower synchronously in sympatric populations. The food‐deceptive C. falcata with bright yellow flowers is predominantly outcrossing, whereas autogamy is the dominant strategy in both C. erecta and C. longibracteata, whose white flowers do not open fully. We examined genetic diversity (by means of allozymes) of the three species in sympatric populations (600 × 600 m area) in the Yeonwhasan Provincial Park (YPP) and in non‐sympatric populations outside YPP, South Korea. Thirteen out of 20 putative loci were variable across the three species, but there was a complete lack of allozyme variation within each species and we found no evidence of hybridisation. Our results suggest that historical factors, i.e. the Quaternary climate oscillations, have played a major role in determining levels of genetic diversity in the three Cephalanthera species. The Korean populations of C. erecta (a warm‐temperate/temperate element) and C. falcata (a warm‐temperate element) may have been established by a single introduction from a genetically depauperate ancestral population, likely located outside the Korean Peninsula. On the other hand, since C. longibracteata is a boreal/temperate element, it may have survived the Last Glacial Maximum in microrefugia located in low elevation regions within the Peninsula where it has been subjected to population bottlenecks reducing its genetic diversity.     

CRISPR/Cas9-mediated efficient editing in <Emphasis Type="Italic">phytoene desaturase</Emphasis> (<Emphasis Type="Italic">PDS</Emphasis>) demonstrates precise manipulation in banana cv. Rasthali genome

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been reported for precise genome modification in many plants. In the current study, we demonstrate a successful mutation in phytoene desaturase (RAS-PDS) of banana cv. Rasthali using the CRISPR/Cas9 system. Two PDS genes were isolated from Rasthali (RAS-PDS1 and RAS-PDS2), and their protein sequence analysis confirmed that both PDS comprises conserved motifs for enzyme activity. Phylogenetic analysis of RAS-PDS1 and RAS-PDS2 revealed a close evolutionary relationship with other monocot species. The tissue-specific expression profile of RAS-PDS1 and RAS-PDS2 in Rasthali suggested differential regulation of the genes. A single 19-bp guide RNA (gRNA) was designed to target the conserved region of these two RAS-PDS and transformed with Cas9 in embryogenic cell suspension (ECS) cultures of cv. Rasthali. Complete albino and variegated phenotype were observed among regenerated plantlets. DNA sequencing of 13 plants confirmed the indels with 59% mutation frequency in RAS-PDS, suggesting activation of the non-homologous end-joining (NHEJ) pathway. The majority of mutations were either insertion (1–5) or deletion (1–4) of nucleotides near to protospacer adjacent motif (PAM). These mutations have created stop codons in RAS-PDS sequences which suggest premature termination of RAS-PDS protein synthesis. The decreased chlorophyll and total carotenoid contents were detected in mutant lines that revealed the functional disruption of both RAS-PDS genes. Our results demonstrate that genome editing through CRISPR/Cas9 can be applied as an efficient tool for banana genome modification.     

Rational design of peptide affinity ligands for the purification of therapeutic enzymes

Non‐mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof‐of‐concept for developing affinity peptide‐based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:987–998, 2018     

Decomposition of young water hyacinth leaves in lake water

Rates of weight loss and release of nutrients during different phases of decomposition in young water hyacinth leaves were determined under laboratory conditions. The leaves decomposed solely by physical leaching during the initial 4-day phase and later by microbial processes. The largest part of weight loss and nutrient release by physical leaching took place within the first 4 h of incubation and thereafter the decomposition rate declined. Microbial processes decayed leaves at a significantly higher rate than that by physical leaching. The overall decay rate constants were related inversely and the release of nutrients directly to the levels of leaf additions in the lake water. The dissolved inorganic and organic nutrients were released chiefly by abiotic processes during the initial as well as later phases of decay. The release was significantly higher during the initial phase in comparison with that during the later phase. Microbes utilized only a small amount of nutrients that were released during decomposition of water hyacinth leaves. The % release of various elements from the decaying leaves was in the order of K > P > C > Na > N.     

Genotype, explant and position effects on organogenesis and somatic embryogenesis in eggplant (Solanum melongena L.)

The relative importance of genotype and explant, and their interactionsfor in vitro plant regeneration via both organogenesis and somaticembryogenesis in Solanum melongena (eggplant) has been studied.Hypocotyl, cotyledon and leaf explants of four commerciallygrown Indian cultivars, Pusa Purple Long, Long White Cluster,Pusa Kranti, and Pusa Purple Cluster were used in the study.A combination of benzyladen-ine (11.1 µM) and indoleaceticacid (2.9 µM) was found to be optimum for shoot regeneration.Naphthalene acetic acid induced embryogenesis in all the threeexplants; 32.2µM was optimum for hypocotyl explants while10.7µM yielded maximum number of somatic embryos fromcotyledon and leaf explants. Genotype, explant and genotype-explantinteraction had highly significant effects on both organogenesisand somatic embryogenesis with genotype exerting maximum effecton both these processes. Pusa Purple Long was found to be themost responsive genotype for regeneration of both adventitiousshoots and somatic embryos among the cultivars. Among the explants,hypocotyls yielded the maximum number of adventitious shootsfollowed by cotyledons and leaves. The embryogenic responseof leaves and cotyledons was, however, significantly higherthan that of hypocotyl explants. Significant differences formorphogenetic potential were also observed within a single explant(hypocotyl). There was a basipetal gradient for organogenesis(i.e. decrease in number of shoots from base to apex) whilethe terminal hypocotyl segments showed better embryogenic potentialthan the median segments. Key words: Solarium melongena, organogenesis, somatic embryogenesis, genotype, explant, position effect     

Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study

Two different polyclonal antibodies were raised to synthetic peptides corresponding to distinct putative odour receptors of rat and mouse. Both antibodies selectively labelled olfactory cilia as seen with cryofixation and immunogold ultrastructural procedures. Regions of the olfactory organ where label was detected were consistent with those found at LM levels. Immunopositive cells were rare; only up to about 0.4% of these receptor cells were labelled. Despite chemical, species, and topographic differences both antibodies behaved identically in their ultrastructural labelling patterns. For both antibodies, labelling was very specific for olfactory cilia; both bound amply to the thick proximal and the thinner and long distal parts of the cilia. Dendritic knobs showed little labelling if any. Dendritic receptor cell structures below the knobs, supporting cell structures, and respiratory cilia did not immunolabel. There were no obvious differences in morphology between labelled and unlabelled receptor cells and their cilia. Labelling could be followed up to a distance of about 15 μm from the knobs along the distal parts of the cilia. When labelled cells were observed, this signal was detectable in two, sometimes three, sections taken through these cells while being consistently absent in neighbouring cells. This pattern argues strongly for the specificity of the labelling. In conclusion, very few receptor cells labelled with the antibodies to putative odour receptors. Additionally the olfactory cilia, the cellular regions that first encounter odour molecules and that are thought to transduce the odorous signal, displayed the most intense labelling with both antibodies. Consequently, the results showed these cilia as having many copies of the putative receptors. Finally, similar patterns of subcellular labelling were displayed in two different species, despite the use of different antibodies. Thus, this study provides compelling evidence that the heptahelical putative odour receptors localize in the olfactory cilia.     

Biotechnological applications in in vitro plant regeneration studies of broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">italica</Emphasis>), an important vegetable crop

Biotechnology holds promise for genetic improvement of important vegetable crops. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop of the family Brassicaceae. However, various biotic and abiotic stresses cause enormous crop yield losses during commercial cultivation of broccoli. Establishment of a reliable, reproducible and efficient in vitro plant regeneration system with cell and tissue culture is a vital prerequisite for biotechnological application of crop improvement programme. An in vitro plant regeneration technique refers to culturing, cell division, cell multiplication, de-differentiation and differentiation of cells, protoplasts, tissues and organs on defined liquid/solid medium under aseptic and controlled environment. Recent progress in the field of plant tissue culture has made this area one of the most dynamic and promising in experimental biology. There are many published reports on in vitro plant regeneration studies in broccoli including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies in broccoli that could be helpful in drawing the attention of the researchers and scientists to work on it to produce healthy, biotic and abiotic stress resistant plant material and to carry out genetic transformation studies for the production of transgenic plants.     

Linear viscoelasticity - bone volume fraction relationships of bovine trabecular bone

Trabecular bone has been previously recognized as time-dependent (viscoelastic) material, but the relationships of its viscoelastic behaviour with bone volume fraction (BV/TV) have not been investigated so far. Therefore, the aim of the present study was to quantify the time-dependent viscoelastic behaviour of trabecular bone and relate it to BV/TV. Uniaxial compressive creep experiments were performed on cylindrical bovine trabecular bone samples (\(\textit{n}\,{=}\,13\)) at loads corresponding to physiological strain level of 2000 \({\upmu }{\upvarepsilon }\). We assumed that the bone behaves in a linear viscoelastic manner at this low strain level and the corresponding linear viscoelastic parameters were estimated by fitting a generalized Kelvin–Voigt rheological model to the experimental creep strain response. Strong and significant power law relationships (\(r^2\,{=}\,0.73,\ p\,{<}\,0.001\)) were found between time-dependent creep compliance function and BV/TV of the bone. These BV/TV-based material properties can be used in finite element models involving trabecular bone to predict time-dependent response. For users’ convenience, the creep compliance functions were also converted to relaxation functions by using numerical interconversion methods and similar power law relationships were reported between time-dependent relaxation modulus function and BV/TV.