Differential Response of Two Tomato Genotypes, Wild Type cv. Ailsa Craig and Its ABA-Deficient Mutant flacca to Short-Termed Drought Cycles

Two tomato genotypes with constitutively different ABA level, flacca mutant and wild type of Ailsa Craig cv. (WT), were subjected to three repeated drought cycles, with the aim to reveal the role of the abscisic acid (ABA) threshold in developing drought tolerance. Differential responses to drought of two genotypes were obtained: more pronounced stomatal closure, ABA biosynthesis and proline accumulation in WT compared to the mutant were compensated by dry weight accumulation accompanied by transient redox disbalance in flacca. Fourier-transform infrared (FTIR) spectra analysis of isolated cell wall material and morphological parameter measurements on tomato leaves indicated changes in dry weight accumulation and carbon re-allocation to cell wall constituents in flacca, but not in WT. A higher proportion of cellulose, pectin and lignin in isolated cell walls from flacca leaves further increased with repeated drought cycles. Different ABA-dependent stomatal closure between drought cycles implies that acquisition of stomatal sensitivity may be a part of stress memory mechanism developed under given conditions. The regulatory role of ABA in the cell wall restructuring and growth regulation under low leaf potential was discussed with emphasis on the beneficial effects of drought priming in developing differential defense strategies against drought.

Spatial and temporal control of expression with light-gated LOV-LexA

The ability to drive expression of exogenous genes in different tissues and cell types, under control of specific enhancers, has catapulted discovery in biology. While many enhancers drive expression broadly, several genetic tricks have been developed to obtain access to isolated cell types. However, studies of topographically organized neuropiles, such as the optic lobe in fruit flies, have raised the need for a system that can access subsets of cells within a single neuron type, a feat currently dependent on stochastic flip-out methods. To access the same subsets of cells consistently across flies, we developed LOV-LexA, a light-gated expression system based on the bacterial LexA transcription factor and the plant-derived LOV photosensitive domain. Expression of LOV-Lex in larval fat body as well as pupal and adult neurons enables spatial and temporal control of expression of transgenes under LexAop sequences with blue light. The LOV-LexA tool thus provides another layer of intersectional genetics, allowing for light-controlled genetic access to the same subsets of cells within an expression pattern across individual flies.

Effect of varying the concentration of sulphuric acid and ammonium hydroxide on the release of cellulose from isolated cell wall component of corn cob

The scarcity and high price associated with fossil fuel has urged countries to research resources for alternative energy sources. Biofuels like bioethanol produced from lignocellulosic biomass (corn cob) were considered potential alternative. Cellulose composition from isolated cell wall material of corn cobs was investigated under two different pre-treatments using H2SO4 and NH4OH at varying concentrations of 5%, 10%, 20%, 30% and 40%. Cell wall not treated acted as control. Colorimetric anthrone-assay followed by absorbance reading at 625nm revealed that glucose is present in reasonable amount in corn cob. The analysis of variance (ANOVA) indicated significant differences among pre-treated compared to untreated (Control) corn cob samples at p≤0.05. Acid pre-treatment showed better glucose yield compared to alkali pre-treatment with results revealing 20% (19.37µg/ml) H2SO4 to be the optimal concentration producing highest glucose yield. The study reveals the potential of corn cob as a lignocellulosic feed stock for biofuel production.

Non-perfect maze generation using Kruskal algorithm

A non-perfect maze is a maze that contains loop or cycle and has no isolated cell. A non-perfect maze is an alternative to obtain a maze that cannot be satisfied by perfect maze. This paper discusses non-perfect maze generation with two kind of biases, that is, horizontal and vertical wall bias and cycle bias. In this research, a maze is modeled as a graph in order to generate non-perfect maze using Kruskal algorithm modifications. The modified Kruskal algorithm used Fisher Yates algorithm to obtain a random edge sequence and disjoint set data structure to reduce process time of the algorithm. The modification mentioned above are adding edges randomly while taking account of the edge’s orientation, and by adding additional edges after spanning tree is formed. The algorithm designed in this research constructs an non-perfect maze with complexity of where and denote vertex and edge set of an grid graph, respectively. Several biased non-perfect mazes were shown in this research by varying its dimension, wall bias and cycle bias.

Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1

Tripartite-motif-containing protein 5 isoform α (TRIM5α) is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon overexpression of the mutated protein. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (clone 6) had both alleles containing R332G, but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following interferon (IFN)-β treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.

Editing of the TRIM5 gene decreases the permissiveness of human T lymphocytic cells to HIV-1

TRIM5α is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon over-expression of the mutated protein. Here, we used CRISPR-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (Clone 6) had both alleles containing R332G but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following IFN-β treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.

Permeability evaluation of gemcitabine-CPP6 conjugates in Caco-2 cells

Cancer is one of the most alarming diseases due to its high mortality and still increasing incidence rate. Currently available treatments for this condition present several shortcomings and new options are continuously being developed and evaluated, aiming at increasing the overall treatment efficiency and reducing associated adverse side effects. Gemcitabine has proven activity and is used in chemotherapy. However, its therapeutic efficiency is limited by its low bioavailability as a result of rapid enzymatic inactivation. Additionally, tumor cells often develop drug resistance after initial tumor regression related to transporter deficiency. We have previously developed three gemcitabine conjugates with cell-penetrating hexapeptides (CPP6) to facilitate intracellular delivery of this drug while also preventing enzymatic deamination. The bioactivity of these new prodrugs was evaluated in different cell lines and showed promising results. Here, we assessed the absorption and permeability across Caco-2 monolayers of these conjugates in comparison with gemcitabine and the respective isolated cell-penetrating peptides (CPPs). CPP6-2 (KLPVMW) and respective Gem-CPP6-2 conjugate showed the highest permeability in Caco-2 cells.