Pharmacological activities of Artemisia absinthium and control of hepatic cancer by expression regulation of TGFß1 and MYC genes.

Plant derived compounds have always been an important source of medicines and have received significant attention in recent years due to their diverse pharmacological properties. Millions of plant-based herbal or traditional medicines are used to cure various types of cancers especially due to activation of proliferative genes. The aim of the present study was to characterize the altered and attenuated gene expression of the selected growth factor namely Transforming growth factor Beta -1 (TGFß1) and MYC in human hepatoma-derived (Huh7) liver cancer cell lines in response to extracts of Artemisia absinthium dissolved in selected organic solvents. Ethanolic, methanolic and acetone extract of different plant parts (leaf, stem and flowers) was used to access the antiproliferative activity by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Quantitative Real-Time RT-PCR revealed that the transcript levels of TGFß1 are induced in the samples treated with methanolic extract of Artemisia absinthium. Furthermore, reduced expression levels of MYC gene was noticed in cancerous cells suggesting antiproliferative properties of the plant. This study further highlights the resistance profile of various microbes by antimicrobial susceptibility test with plant extracts. In addition, antidiabetic effect of Artemisia absinthium have also shown positive results. Our study elucidates the potentials of Artemisia absinthium as a medicinal plant, and highlights the differential expression of genes involved in its mitogenic and anti-proliferative activity with a brief account of its pharmacological action.

Interaction estimation of pathogenicity determinant protein ßC1 encoded by Cotton leaf curl Multan Betasatellite with Nicotiana benthamiana Nuclear Transport Factor 2.

Background: Begomovirus is one of the most devastating pathogens that can cause more than 90% yield loss in various crop plants. The pathogenicity determinant ßC1, located on the betasatellite associated with monopartite begomoviruses, alters the host signaling mechanism to enhance the viral disease phenotype by undermining the host immunity. The understanding of its interacting proteins in host plants to develop disease symptoms such as curly leaves, enations, vein swelling, and chlorosis is crucial to enhance the disease resistance in crop plants. The current study was designed to reveal the contribution of ßC1 in disease pathogenicity and to unveil potential interacting partners of ßC1 protein in the model plant Nicotiana benthamiana. Methods: The ßC1 gene was cloned in pGKBT7 and used as bait against the cDNA library of N. benthamiana and its pathogenesis was tested against the healthy plant and the plants infiltrated with empty vectors. The yeast two-hybrid-based screening was performed to find the interacting factors. Successful interacting proteins were screened and evaluated in various steps and confirmed by sequence analysis. The three-dimensional structure of the Nuclear Transport Factor 2 (NTF2) protein was predicted, and in-silico protein-protein interaction was evaluated. Furthermore, protein sequence alignment and molecular phylogenetic analysis were carried out to identify its homologues in other related families. In-silico analyses were performed to validate the binding affinity of ßC1 protein with NTF2. The 3D model was predicted by using I-TASSER and then analyzed by SWISS MODEL-Workspace, RAMPAGE, and Verify 3D. The interacting amino acid residues of ßC1 protein with NTF2 were identified by using PyMOL and Chimera. Results: The agroinfiltrated leaf samples developed severe phenotypic symptoms of virus infection. The yeast-two-hybrid study identified the NTF2 as a strong interacting partner of the ßC1. The NTF2 in Solanaceae and Nicotiana was found to be evolved from the Brassica and Gossypium species. The in-silico interaction studies showed a strong binding affinity with releasing energy value of -730.6 KJ/mol, and the involvement of 10 amino acids from the middle portion towards the C-terminus and five amino acid residues from the middle portion of ßC1 to interact with six amino acids of NTF2. The study not only provided an insight into the molecular mechanism of pathogenicity but also put the foundation stone to develop the resistance genotypes for commercial purposes and food security.

CstMYB14 links ROS signaling, apocarotenoid metabolism, and stress response in Crocus sativus L.

Reactive oxygen species (ROS) behave as signaling molecules and induce biosynthesis of many secondary metabolites, including apocarotenoids, which play critical roles in stress tolerance through radical scavenging. However, the mechanism that regulates ROS responsive apocarotenoid metabolism and subsequent stress response is unknown. In this study, an R2R3-MYB transcription factor (CstMYB14) was identified from Crocus sativus L., which acts as a regulator of apocarotenoid biosynthesis. CstMYB14 expression increases in response to H2 O2 in a concentration and time-dependent manner. CstMYB14 localizes to the nucleus and acts as a transcriptional activator. Over-expression of CstMYB14 in Crocus stigmas enhanced apocarotenoid biosynthesis. Yeast-one-hybrid demonstrated binding of CstMYB14 to promoters of two apocarotenoid pathway genes (phytoene synthase and carotenoid cleavage dioxygenase 2). Nicotiana benthamiana plants overexpressing CstMYB14 showed better growth and higher stress tolerance than wild type plants. Higher antioxidant activity in CstMYB14-Ox plants indicated that stress tolerance might be due to ROS scavenging. These results establish a molecular link between ROS signaling, apocarotenoid metabolism and stress tolerance. Further, CstMYB14 is shown to act as a key regulator which modulates ROS responsive biosynthesis of apocarotenoids which in turn impart stress tolerance through ROS scavenging.

Mechanical and Microstructural Characterization of Quarry Rock Dust Incorporated Steel Fiber Reinforced Geopolymer Concrete and Residual Properties after Exposure to Elevated Temperatures.

The purpose of this research is to study the effects of quarry rock dust (QRD) and steel fibers (SF) inclusion on the fresh, mechanical, and microstructural properties of fly ash (FA) and ground granulated blast furnace slag (SG)-based geopolymer concrete (GPC) exposed to elevated temperatures. Such types of ternary mixes were prepared by blending waste materials from different industries, including QRD, SG, and FA, with alkaline activator solutions. The multiphysical models show that the inclusion of steel fibers and binders can enhance the mechanical properties of GPC. In this study, a total of 18 different mix proportions were designed with different proportions of QRD (0%, 5%, 10%, 15%, and 20%) and steel fibers (0.75% and 1.5%). The slag was replaced by different proportions of QRD in fly ash, and SG-based GPC mixes to study the effect of QRD incorporation. The mechanical properties of specimens, i.e., compressive strength, splitting tensile strength, and flexural strength, were determined by testing cubes, cylinders, and prisms, respectively, at different ages (7, 28, and 56 days). The specimens were also heated up to 800 °C to evaluate the resistance of specimens to elevated temperature in terms of residual compressive strength and weight loss. The test results showed that the mechanical strength of GPC mixes (without steel fibers) increased by 6-11%, with an increase in QRD content up to 15% at the age of 28 days. In contrast, more than 15% of QRD contents resulted in decreasing the mechanical strength properties. Incorporating steel fibers in a fraction of 0.75% by volume increased the compressive, tensile, and flexural strength of GPC mixes by 15%, 23%, and 34%, respectively. However, further addition of steel fibers at 1.5% by volume lowered the mechanical strength properties. The optimal mixture of QRD incorporated FA-SG-based GPC (QFS-GPC) was observed with 15% QRD and 0.75% steel fibers contents considering the performance in workability and mechanical properties. The results also showed that under elevated temperatures up to 800 °C, the weight loss of QFS-GPC specimens persistently increased with a consistent decrease in the residual compressive strength for increasing QRD content and temperature. Furthermore, the microstructure characterization of QRD blended GPC mixes were also carried out by performing scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy dispersive spectroscopy (EDS).

Scanning Electron Microscopy-based palynological characterization of selected Euphorbiaceae taxa colonized in Pakistan.

The present study was carried out on scanning electron microscopic-based palynological characterization of selected 20 Euphorbiaceae taxa. In this study, important pollen qualitative and quantitative features of selected Euphorbiaceae species such as polar diameter, equatorial diameter, P/E ratio, sculpturing of pollen, exine thickness, intine thickness, presence or absence of colpi and length of colpi, and pollen fertility were examined. Remarkable variations in these pollen characters had been observed among these species. In both polar and equatorial views, Riccinus communis L. exhibited the highest pollen size (polar = 264.1 µm, Equatorial = 270 µm), while Acalypha wilkesiana Mull Arg. showed lowest pollen size (Polar = 17 µm, Equatorial = 18.5 µm). Most of the pollen varied from oblate to spheroidal in shape. With regard to the P/E ratio, Sapium sebiferum L. Roxb showed the highest ratio while Bischofia javanica Blume showed minimum, that is, 1.18 and 0.85, respectively. Intine and exine thickness also varied. Most of the pollen grains were monade. Tricolporate, tricolpate, monocolpate, pentaporate, and polyporate pollen aperture types have been observed. Most of the plants showed regular, reticulate, and echinate pollen sculpturing. Pollen fertility also varied from species to species but most of the plant showed pollen fertility between 70 and 80%. Through this work, it has been concluded that quantitative and qualitative pollen features are helpful at the specific level as well as the generic level and can provide a fruitful taxonomic solution.

Application of multimicroscopic techniques (LM and SEM) in comparative palynological study of Asteroideae members, inhabited in Pakistan.

In the present study light and scanning electron microscopic approaches were used to study the palynological variations among 30 Asteroideae members. Variety of qualitative and quantitative palynological characters like pollen shape and type, pollen sculpturing, pollen size, P/E ratio, exine and intine thickness, interspecific difference, colpi size, pore size, spines length, spine width, and pollen fertility were observed. Results reported remarkable variations among pollen characters of studied taxa. The shape of pollen grains in polar and equatorial views varied from specie to specie such as spheroidal, prolate, oblate, subspheroidal, oblate spheroidal, prolate spheroidal, suboblate, and sub prolate. Bidens pilosa L. showed maximum pollen size in both polar and equatorial views, whereas minimum pollen size in polar view was found in Tetraneuris scaposa (DC.) Greene. (i.e., 30.5 µm) and in equatorial view was found in Cosmos sulphureus Cav. (25.5 µm).The highest P/E ratio (1.39 µm) was measured in Glebionis coronaria L. The values of exine and intine thickness also varied. Tricolporate, tetracolporate, trizonocolporate, pentoporate, and polypentoporate pollen types were examined. Spinateand echinate pollen sculpturingobserved under SEM. Variations in spine length and width also recorded. The maximum pollen fertility (98%) was measured in Chrysanthemum morifolium Ramat. and the lowest (56%) was recorded in Thymophylla tenuiloba (DC.) Small. It was concluded that the variations in qualitative and quantitative characters were seemed to be valuable for the taxonomic investigations of Asteroideae taxa.

Enterobacter sp. strain Fs-11 adapted to diverse ecological conditions and promoted sunflower achene yield, nutrient uptake, and oil contents.

Plant growth-promoting rhizobacteria are under extensive investigation to supplement the chemical fertilizers due to cost-effective and eco-friendly nature. However, their consistency in heterogeneous soil and diverse ecological settings is unclear. The current study presents in vitro and field evaluation of pre-characterized PGPR strain Enterobacter sp. Fs-11 (GenBank accession # GQ179978) in terms of its potential to enhance sunflower yield and oil contents under diverse environmental conditions. Under in vitro conditions, strain Fs-11 showed optimal growth at a range of temperature (15 to 40 °C) and pH values (6.5 to 8.5). Extracellular and intracellular localizations of the strain Fs-11 in sunflower root cortical cells through transmission electron microscopy confirmed its epiphytic and endophytic colonization patterns, respectively. In field experiments, conducted at three different agro-climatic locations, inoculation of strain Fs-11 at 50% reduced NP fertilizer resulted in a significant increase in growth, achene yield, nutrient uptake, and oil contents. Inoculation also responded significantly in terms of increase in mono- and polyunsaturated fatty acids (oleic and linoleic acids, respectively) without rising saturated fatty acid (palmitic and stearic acids) contents. We concluded that Enterobacter sp. Fs-11 is a potential candidate for biofertilizer formulations to supplement chemical fertilizer requirements of sunflower crop under diverse climatic conditions.

Suppressors of RNA silencing encoded by the components of the cotton leaf curl begomovirus-betasatellite complex.

Begomoviruses (family Geminiviridae) are single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. Many economically important diseases in crops are caused by begomoviruses, particularly in tropical and subtropical environments. These include the betasatellite-associated begomoviruses causing cotton leaf curl disease (CLCuD) that causes significant losses to a mainstay of the economy of Pakistan, cotton. RNA interference (RNAi) or gene silencing is a natural defense response of plants against invading viruses. In counter-defense, viruses encode suppressors of gene silencing that allow them to effectively invade plants. Here, we have analyzed the ability of the begomovirus Cotton leaf curl Multan virus (CLCuMV) and its associated betasatellite, Cotton leaf curl Multan ß-satellite (CLCuMB) which, together, cause CLCuD, and the nonessential alphasatellite (Cotton leaf curl Multan alphasatellite [CLCuMA]) for their ability to suppress gene silencing in Nicotiana benthamiana. The results showed that CLCuMV by itself was unable to efficiently block silencing. However, in the presence of the betasatellite, gene silencing was entirely suppressed. Silencing was not affected in any way when infections included CLCuMA, although the alphasatellite was, for the first time, shown to be a target of RNA silencing, inducing the production in planta of specific small interfering RNAs, the effectors of silencing. Subsequently, using a quantitative real-time polymerase chain reaction assay and Northern blot analysis, the ability of all proteins encoded by CLCuMV and CLCuMB were assessed for their ability to suppress RNAi and the relative strengths of their suppression activity were compared. The analysis showed that the V2, C2, C4, and ßC1 proteins exhibited suppressor activity, with the V2 showing the strongest activity. In addition, V2, C4, and ßC1 were examined for their ability to bind RNA and shown to have distinct specificities. Although each of these proteins has, for other begomoviruses or betasatellites, been previously shown to have suppressor activity, this is the first time all proteins encoded by a geminiviruses (or begomovirus-betasatellite complex) have been examined and also the first for which four separate suppressors have been identified.