NusG links transcription and translation in Escherichia coli extracts

In bacteria, transcription is coupled to, and can be regulated by, translation. Although recent structural studies suggest that the N-utilization substance G (NusG) transcription factor can serve as a direct, physical link between the transcribing RNA polymerase (RNAP) and the lead ribosome, mechanistic studies investigating the potential role of NusG in mediating transcription-translation coupling are lacking. Here, we report development of a cellular extract- and reporter gene-based, in vitro biochemical system that supports transcription-translation coupling as well as the use of this system to study the role of NusG in coupling. Our findings show that NusG is required for coupling and that the enhanced gene expression that results from coupling is dependent on the ability of NusG to directly interact with the lead ribosome. Moreover, we provide strong evidence that NusG-dependent coupling enhances gene expression through a mechanism in which the lead ribosome that is tethered to the RNAP by NusG suppresses spontaneous backtracking of the RNAP on its DNA template that would otherwise inhibit transcription.

DNA-RNA Hybrid (R-Loop): From a Unified Picture of the Mammalian Telomere to the Genome-Wide Profile

Local three-stranded DNA/RNA hybrid regions of genomes (R-loops) have been detected either by binding of a monoclonal antibody (DRIP assay) or by enzymatic recognition by RNaseH. Such a structure has been postulated for mouse and human telomeres, clearly suggested by the identification of the complementary RNA Telomeric repeat-containing RNA “TERRA”. However, the tremendous disparity in the information obtained with antibody-based technology drove us to investigate a new strategy. Based on the observation that DNA/RNA hybrids in a triplex complex genome co-purify with the double-stranded chromosomal DNA fraction, we developed a direct preparative approach from total protein-free cellular extract without antibody that allows their physical isolation and determination of their RNA nucleotide sequence. We then define in the normal mouse and human sperm genomes the notion of stable DNA associated RNA terminal R-loop complexes, including TERRA molecules synthesized from local promoters of every chromosome. Furthermore, the first strong evidence of all telomeric structures, applied additionally to the whole murine sperm genome compared to the testes, showed reproducible R-loop complexes of the whole genome and suggesting a defined profile in the sperm genome for the next generation.

Relation between soil solution composition and petiole cellular extract of crops in western Mexico

Crop fertilization greatly impacts food production. However, insufficient applications can lead to poor yields. On the other hand, an excessive application leads to soil and aquifers pollution. In this paper, field studies were carried out to determine the ranges of mineral concentration and the interaction of the ions in the soil solution (SS) and the petiole cellular extract (PCE) in several cultures established in the states of Guanajuato, Colima and Jalisco, Mexico. The hypothesis states that there is a causal relationship between the mineral composition of the soil solution (SS) and the minerals and total soluble solids (TSS) in petiole cellular extract (PCE). The following cultures were studied in this research: avocado, blueberry, broccoli, cauliflower, raspberry, strawberry, lettuce, cantaloupe, papaya, and pepper. For each culture, PCE samples and SS samples using a press to break tissue and ceramic tip lysimeters were obtained. The results were processed to obtain ranges of variation within 50% of the closest values to the median. Correlations between the several ion concentrations were analyzed using analysis of variance. The results showed values (mg L-1) of NO3- (40-620), PO43- (17-66), K+ (3-377), Ca2+ (27-582), Na+ (15.3-500), Mg2+ (10-53), Fe3+ (0.6‑1.8), and Zn2+ (2.8-7.4) in soil solution, which allowed obtaining values of NO3- (27-9225), K+ (820-9375), Ca2+ (1.0-650), Na+ (25-620) and TSS (2-13 °Brix) in petiole cellular extract of petiole. Statistically significant correlations were observed between the concentrations of the SS ions regarding the concentrations in PCE in crops suggesting a relationship between the plant nutritive assimilation and cations or anions present in soil solution. The conclusion derived from this study is that ionic concentration ranges registered in the SS and in the PCE provide an approximation to the ranges of nutritional sufficiency for the horticultural crops established in the summer-winter in western Mexico.

Antinociceptive and anti-inflammatory effects of cellular and extracellular extracts from microalga Chlamydomonas pumilioniformis on mice

Microalga species have attracted interest as a source of bioactive compounds with several pharmacological activities. Previous studies reported that microalgae from the genus Chlamydomonas have anti-inflammatory and antioxidant properties. In this study, antinociceptive and anti-inflammatory activities of two extracts from microalga Chlamydomonas pumilioniformis were investigated. Cellular and extracellular extracts were prepared from a 14 day-batch culture in WC medium at the end of exponential growth and their carbohydrate contents were determined. Antinociceptive effects of extracts were evaluated by writhing and formalin-induced nociception tests, while the anti-inflammatory activity was analyzed by formalin-induced paw edema in mice. The analysis of dissolved carbohydrates detected amounts of 90 and 20 µg mL-1 of total carbohydrate in cellular and extracellular extracts, respectively. Cellular extract was mainly composed of glucose, but with significant proportions of arabinose, galactose and mannose and/or xylose and minor ones of fucose, rhamnose, amino sugars and uronic acids. Extracellular extract was composed of a similar proportion of glucose, galactose and mannose/xylose, besides significant ones of arabinose, fucose and galacturonic acid. Intraperitoneal administration of extracts significantly reduced writhing response in mice. In the formalin test, the extracellular extract inhibited both formalin phases, while the cellular extract was only effective in the late phase. Furthermore, extracts reduced the formalin-induced paw edema. In sum, we showed, for the first time, that C. pumilioniformis can be an important source of polysaccharides with anti-inflammatory and antinociceptive effects.

Effect of the Cellular Extract of Co-cultured Lactobacillus Casei on BAX and Human β-Defensin 2 Genes Expression in HT29 Cells

Aims: Defensins are cysteine-rich antimicrobial cationic peptides and BAX is a proapoptotic gene that can cause cell death. This study aimed to investigate the effect of cellular extract of co-cultured Lactobacillus casei on the expression of BAX and human β-defensin 2 (hBD-2) genes in HT29 cells. Methods & Materials: This experimental study was conducted in the Research Center for Pharmaceutical Nanotechnology of Tabriz University of Medical Sciences in 2017. The HT29 cell line was obtained from the Pasteur Institute of Iran, and cells were assessed using Microculture Tetrazolium Test (MTT) after culturing. DNA was extracted from the treated cells, and then the DNA ladder assay was carried out. After preparing cDNA, the expression levels of BAX and hBD-2 genes in the HT29 cell line were measured using a real-time Polymerase Chain Reaction (PCR) method. Findings: The results of the MTT assay indicated that Lactobacillus casei inhibited the proliferation of HT29 cells and induced apoptosis in these cells. Results of DAPI staining and DNA ladder assay obtained from treating HT29 cells by Lactobacillus casei showed qualitative changes in cell apoptosis. Moreover, realtime PCR results indicated that Lactobacillus casei bacteria significantly increased the expression of the hBD-2 gene in HT29 colon cancer cells within 12-24 hours (P= 0.023), while BAX gene expression showed no significant change in the first 24 hours (P= 0.37). Conclusion: The extract of Lactobacillus casei can be used to stimulate cancer cells to produce β-defensins, inhibit pathogens, prevent the stimulation of cellular signaling, and fight antibiotic-resistant bacteria.

Evaluation of Organic Substrates and Microorganisms as Bio-Fertilisation Tool in Container Crop Production

Microorganisms are only effective when adequate conditions for their survival and development are provided. Among the factors that influence its effectiveness includes the type of soil or culture substrate, which works as an energy source reserve. Therefore, a tomato and a melon crop were established in different cycles to assess the effect of the physicochemical properties of organic substrates based on coconut fibre and vermicompost in three proportions, 0:100, 40:60 and 60:40 (% v:v), on the microbial activity in the rhizosphere when the bacteria Azotobacter vinelandii, Bacillus megaterium and Frateuria aurantia were applied. Concentrations of NO3−, H2PO4−, K+ and Ca2+ in the petiole cellular extract (PCE) were quantified at 60, 90 and 120 days after transplantation (DAT) for tomato and 45 and 65 DAT for melon. We analysed dehydrogenase activity (DHA), acid phosphatase activity (FTA) and β-glucosidase activity (β-GLU). In order to maintain optimal volumetric moisture for the survival of microorganisms, automatic control was used to manage the irrigation frequency between 22%–28%. The results showed that physicochemical substrate properties, by incorporating 40% vermicompost into the coconut fibre mixture, increased enzymatic activity. Plants that were inoculated with Azotobacter vinelandii and Frateuria aurantia showed an improvement in NO3− and K+ assimilation achieving highest yields.

Optimization of the biological synthesis of silver nanoparticles using Penicillium oxalicum GRS-1 and their antimicrobial effects against common food-borne pathogens

Biologically synthesized nanoparticles are gaining importance as they offer several advantages, such as the ease with which they can be scaled up, the cost-effectiveness of the process and the green route of production. In this study, silver (Ag) nanoparticles were biosynthesized using the cellular extract of Penicillium oxalicum GRS-1 and then characterized by ultraviolet visible spectroscopy, X-ray diffraction, field emission scanning electron microscopy, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy. The biosynthesis of nanoparticles was optimized by following the one factor at a time approach, wherein the temperature of 60°C, pH 7.0 and 1.5 mm silver nitrate (AgNO3) concentration were found to be most favorable factors for the production of Ag nanoparticles. Upon statistical optimization, the maximum production of Ag nanoparticles with a concentration of 136 ppm was achieved at pH 7.2, AgNO3 concentration 1.975 mm and 86 h using the crude cellular extract of P. oxalicum GRS-1 having nitrate reductase activity. TEM analysis showed that the Ag nanoparticles were spherical in shape with sizes ranging from 10 to 40 nm. The biosynthesized nanoparticles showed strong antimicrobial activity against the common food-borne, pathogens including Staphylococcus aureus, Escherichia coli and Salmonella typhimurium with respective minimum bactericidal concentrations of 32, 16 and 32 μg/ml.

Actin from the apicomplexan Neospora caninum (NcACT) has different isoforms in 2D electrophoresis

Apicomplexan parasites have unconventional actins that play a central role in important cellular processes such as apicoplast replication, motility of dense granules, endocytic trafficking and force generation for motility and host cell invasion. In this study, we investigated the actin of the apicomplexan Neospora caninum – a parasite associated with infectious abortion and neonatal mortality in livestock. Neospora caninum actin was detected and identified in two bands by one-dimensional (1D) western blot and in nine spots by the 2D technique. The mass spectrometry data indicated that N. caninum has at least nine different actin isoforms, possibly caused by post-translational modifications. In addition, the C4 pan-actin antibody detected specifically actin in N. caninum cellular extract. Extracellular N. caninum tachyzoites were treated with toxins that act on actin, jasplakinolide and cytochalasin D. Both substances altered the peripheric cytoplasmic localization of actin on tachyzoites. Our findings add complexity to the study of the apicomplexan actin in cellular processes, since the multiple functions of this important protein might be regulated by mechanisms involving post-translational modifications.