Functional profiling of long intergenic non-coding RNAs in fission yeast

Eukaryotic genomes express numerous long intergenic non-coding RNAs (lincRNAs) that do not overlap any coding genes. Some lincRNAs function in various aspects of gene regulation, but it is not clear in general to what extent lincRNAs contribute to the information flow from genotype to phenotype. To explore this question, we systematically analysed cellular roles of lincRNAs in Schizosaccharomyces pombe. Using seamless CRISPR/Cas9-based genome editing, we deleted 141 lincRNA genes to broadly phenotype these mutants, together with 238 diverse coding-gene mutants for functional context. We applied high-throughput colony-based assays to determine mutant growth and viability in benign conditions and in response to 145 different nutrient, drug, and stress conditions. These analyses uncovered phenotypes for 47.5% of the lincRNAs and 96% of the protein-coding genes. For 110 lincRNA mutants, we also performed high-throughput microscopy and flow cytometry assays, linking 37% of these lincRNAs with cell-size and/or cell-cycle control. With all assays combined, we detected phenotypes for 84 (59.6%) of all lincRNA deletion mutants tested. For complementary functional inference, we analysed colony growth of strains ectopically overexpressing 113 lincRNA genes under 47 different conditions. Of these overexpression strains, 102 (90.3%) showed altered growth under certain conditions. Clustering analyses provided further functional clues and relationships for some of the lincRNAs. These rich phenomics datasets associate lincRNA mutants with hundreds of phenotypes, indicating that most of the lincRNAs analysed exert cellular functions in specific environmental or physiological contexts. This study provides groundwork to further dissect the roles of these lincRNAs in the relevant conditions.

Butein and Frondoside-A Combination Exhibits Additive Anti-Cancer Effects on Tumor Cell Viability, Colony Growth, and Invasion and Synergism on Endothelial Cell Migration

Despite the significant advances in targeted- and immuno-therapies, lung and breast cancer are at the top list of cancer incidence and mortality worldwide as of 2020. Combination therapy consisting of a mixture of different drugs taken at once is currently the main approach in cancer management. Natural compounds are extensively investigated for their promising anti-cancer potential. This study explored the anti-cancer potential of butein, a biologically active flavonoid, on two major solid tumors, namely, A549 lung and MDA-MB-231 breast cancer cells alone and in combination with another natural anti-cancer compound, frondoside-A. We demonstrated that butein decreases A549 and MDA-MB-231 cancer cell viability and colony growth in vitro in addition to tumor growth on chick embryo chorioallantoic membrane (CAM) in vivo without inducing any noticeable toxicity. Additionally, non-toxic concentrations of butein significantly reduced the migration and invasion of both cell lines, suggesting its potential anti-metastatic effect. We showed that butein anti-cancer effects are due, at least in part, to a potent inhibition of STAT3 phosphorylation, leading to PARP cleavage and consequently cell death. Moreover, we demonstrated that combining butein with frondoside-A leads to additive effects on inhibiting A549 and MDA-MB-231 cellular viability, induction of caspase 3/7 activity, inhibition of colony growth, and inhibition of cellular migration and invasion. This combination reached a synergistic effect on the inhibition of HUVECs migration in vitro. Collectively, this study provides sufficient rationale to further carry out animal studies to confirm the relevance of these compounds’ combination in cancer therapy.

Physical-chemical and microbiological stability of biotherapy Candida albicans RC in different potencies

Introduction: Oral Candidiasis is an opportunist fungal infection, with high incidence in HIV and immunosuppressed patients and Candida albicans is the most common causing agent. In some cases, it can evolve to resistant injuries to antifungal conventional therapy. According to Brazilian Homeopathic Pharmacopeia (BHP) [1], biotherapic medicines are prepared from chemically undefined biological products. Biotherapics created by Brazilian doctor Roberto Costa (RC) have a different homeopathic compounding technique, as its dynamization starts from the ethiologic agent of the illness in its alive form, which present higher capability to stimulate the host immunological system [2,3]. Aim: The goal of this study was evaluate the physical-chemical and microbiological stability of Candida albicans RC potencies under different conditions of storage. Methodology: To prepare the biotherapics, one part of Candida albicans yeast suspension (109 cell/ml) was diluted in 9 parts of sterile distillated water. After preparing this 1:10 dilution, the sample was undergone 100 succussions, resulting in the first decimal dilution (1x). Then, one part of this solution was diluted in 9 parts of sterile distillated water and submitted to 100 succussions, generating the 2x. This process was successively repeated following BHP, until 30x. Water 30x was prepared by the same technique, as control. All the solutions were prepared in aseptic and sterile conditions. Biotherapics 6x, 12x, 18x, 24x, 30x and water 30x were storage under refrigeration (2 to 8°C) and at room temperate (25°C) during 8 weeks. Every 15 days, the following parameters were analyzed: pH, electrical conductivity, UV absorbance (260 and 280 nm). Microbiological analyses were performed after 3 weeks by colony forming unit (CFU) method [4]. Results: The preliminary analyses performed at times zero, 15, 30 and 45 days suggest that electrical conductivity of these solutions tend to increase proportionally to storage time without significant differences due to temperature storage conditions. There was no statistically significant difference detected in pH values. Measures of absorbance of different biotherapic potencies under both conditions of storage are in course. Microbiological analyses showed no colony growth, but in the 1x sample analyzed at time zero, indicating the viability of the fungus. Conclusion: Further experiments are being carried out in order to confirm the preliminary data obtained.

Lattice-based Monte Carlo simulation of the effects of nutrient concentration and magnetic field exposure on yeast colony growth and morphology

Yeasts exist in communities that expand over space and time to form complex structures and patterns. We developed a lattice-based framework to perform spatial-temporal Monte Carlo simulations of budding yeast colonies exposed to different nutrient and magnetic field conditions. The budding patterns of haploid and diploid yeast cells were incorporated into the framework, as well as the filamentous growth that occurs in yeast colonies under nutrient limiting conditions. Simulation of the framework predicted that magnetic fields decrease colony growth rate, solidity, and roundness. Magnetic field simulations further predicted that colony elongation and boundary fluctuations increase in a nutrient- and ploidy-dependent manner. These in-silico predictions are an important step towards understanding the effects of the physico-chemical environment on microbial colonies and for informing bioelectromagnetic experiments on yeast colony biofilms and fungal pathogens.

Integrin α6β4 requires plectin and vimentin for adhesion complex distribution and invasive growth

Integrin α6β4 binds plectin to associate with vimentin; however, the biological function remains unclear. Here, we utilized various integrin β4 mutants and CRISPR-Cas9 editing to investigate this association. Upon laminin binding, integrin α6β4 distinctly distributed peripherally, and centrally proximal to the nucleus. Upon fibronectin addition, integrin α6β4 was centrally recruited to large focal adhesions (FAs) and enhanced Fak phosphorylation. Integrin β4 plectin-binding mutants or genetic deletion of plectin inhibited β4 recruitment to FAs and integrin α6β4-enhanced cell spreading, migration and three-dimensional invasive growth. Loss of the β4 signaling domain (but retains plectin binding) blocked migration and invasiveness but not cell spreading, recruitment to FAs or colony growth. Immunostaining revealed that integrin α6β4 redistributed vimentin perinuclearly where it colocalized with plectin and FAs. Depletion of vimentin completely blocked integrin β4-enhanced invasive growth, Fak phosphorylation and proliferation in three-dimensions but not two-dimensions. In summary, we demonstrate the essential roles of plectin and vimentin in promoting an invasive phenotype downstream of integrin α6β4.

Optimization of the Extraction Process and Application of Bacterial Extracts in the Control of Brown Spot and Leaf Blast in Rice Culture

The objective of this work was to optimize the extraction process and application of bacterial extracts of Bacillus sp. and Serratia sp. in leaf blast control (Magnapothe oryzae) and brown spot (Bipolaris oryzae) in rice culture. The work was divided into three stages: 1) Bacterial obtaining extracts through liquid-liquid extraction 2) Antagonistic capacity of bacterial extracts to M. oryaze and B. oryae 3) Suppression of brown spot (A1) and leaf blast (A2) in greenhouse. The bacterial isolates in present study were identified as Bacillus sp. (BRM32110) and Serratia marcescens (BRM32113). The crude extract of both isolates at different extraction times 6, 16, 24, 48 and 72 hours reduced the growth of colonies of M. oryzae and B. oryzae by up to 92% and 28%, respectively. The extracts that showed highest inhibition of colony growth were those obtained after 6 and 16h of incubation and were selected for subsequent assays. These, for both isolates were able to reduce conidia germination by up to 91% and apressorium formation of M. oryzae by up to 93%. In green house, A1 the treatment that stood out was the extract of Bacillus sp. (16h) with 6.7% of leaf area affected and in A2 the treatment S. marcescens extract (16h) stood out with only 7.6% of leaf area affected with brusone when compared to control. The use of extracts of Bacillus sp. and Serratia marcescens was efficient in reducing the severity of brown spot and leaf blast in rice crop.

Pollen Source Richness May Be a Poor Predictor of Bumblebee (Bombus terrestris) Colony Growth

Agricultural intensification has drastically altered foraging landscapes for bees, with large-scale crop monocultures associated with floral diversity loss. Research on bumblebees and honeybees has shown individuals feeding on pollen from a low richness of floral sources can experience negative impacts on health and longevity relative to higher pollen source richness of similar protein concentrations. Florally rich landscapes are thus generally assumed to better support social bees. Yet, little is known about whether the effects of reduced pollen source richness can be mitigated by feeding on pollen with higher crude protein concentration, and importantly how variation in diet affects whole colony growth, rearing decisions and sexual production. Studying queen-right bumblebee (Bombus terrestris) colonies, we monitored colony development under a polyfloral pollen diet or a monofloral pollen diet with 1.5–1.8 times higher crude protein concentration. Over 6 weeks, we found monofloral colonies performed better for all measures, with no apparent long-term effects on colony mass or worker production, and a higher number of pupae in monofloral colonies at the end of the experiment. Unexpectedly, polyfloral colonies showed higher mortality, and little evidence of any strategy to counteract the effects of reduced protein; with fewer and lower mass workers being reared, and males showing a similar trend. Our findings (i) provide well-needed daily growth dynamics of queenright colonies under varied diets, and (ii) support the view that pollen protein content in the foraging landscape rather than floral species richness per se is likely a key driver of colony health and success.

THERMAL BEHAVIOR IMPROVEMENT OF BIODEGRADABLE FIBER POLYMER COMPOSITES POLYLACTIC ACID (PLA)/COIR USING ACEH’S BENTONITE

Research has been conducted on the manufacture of PLA Coir Bentonite composites. This study aims to examine the effect of PLA on mechanical strength with the addition of coir and bentonite fillers from North Aceh and Central Aceh. The sample formulations used were single polymer PLA/Coir and PLA/Coir with variations of filler Bentonite Aceh Utara and Aceh Tengah with 2, 4, 6 and 8% respectively. The combination of PCa samples showed the highest bacterial colony growth rate, which was more than 100 colonies/gram during the 1 week testing period. In the PBATd filler mixture sample, the maximum bacterial test value was 65 colonies/gram and the minimum value contained in the PBAUa sample was 105 colonies/gram. The best tensile strength was obtained in the PBATc sample, namely 65 MPa. PBATd samples began to degrade at 370.15oC compared to PCa samples degraded at 280.21oC. While the PBAUa sample began to degrade at a temperature of 282.11oC. The surface structure of the PCa sample is more homogeneous because there is no bentonite filler mixture, but it is brittle and crumbles easily. For the PBATd sample, the surface structure is smoother and more homogeneous compared to the PBAUa sample.

Impact of Sodium Dichloroacetate Alone and in Combination Therapies on Lung Tumor Growth and Metastasis

Metabolic reprogramming has been recognized as an essential emerging cancer hallmark. Dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK), has been reported to have anti-cancer effects by reversing tumor-associated glycolysis. This study was performed to explore the anti-cancer potential of DCA in lung cancer alone and in combination with chemo- and targeted therapies using two non-small cell lung cancer (NSCLC) cell lines, namely, A549 and LNM35. DCA markedly caused a concentration- and time-dependent decrease in the viability and colony growth of A549 and LNM35 cells in vitro. DCA also reduced the growth of tumor xenografts in both a chick embryo chorioallantoic membrane and nude mice models in vivo. Furthermore, DCA decreased the angiogenic capacity of human umbilical vein endothelial cells in vitro. On the other hand, DCA did not inhibit the in vitro cellular migration and invasion and the in vivo incidence and growth of axillary lymph nodes metastases in nude mice. Treatment with DCA did not show any toxicity in chick embryos and nude mice. Finally, we demonstrated that DCA significantly enhanced the anti-cancer effect of cisplatin in LNM35. In addition, the combination of DCA with gefitinib or erlotinib leads to additive effects on the inhibition of LNM35 colony growth after seven days of treatment and to synergistic effects on the inhibition of A549 colony growth after 14 days of treatment. Collectively, this study demonstrates that DCA is a safe and promising therapeutic agent for lung cancer.

Re-Evaluation of the Taxonomy of Talaromyces minioluteus

Talaromyces minioluteus belongs to the section Trachyspermi, has a worldwide distribution and has been found on various substrates, especially on various (stored) food commodities and indoor environments. This species is phenotypically and phylogenetically closely related to T. chongqingensis and T. minnesotensis. The phylogenetic and morphological analyses of 37 strains previously identified as T. chongqingensis, T. minnesotensis and T. minioluteus revealed that this clade incudes eight species: the accepted species T. chongqingensis, T. minnesotensis and T. minioluteus, the newly proposed species T. calidominioluteus, T. africanus and T. germanicus, and the new combinations T. gaditanus (basionym Penicillium gaditanum) and T. samsonii (basionym Penicillium samsonii). In this study, we give insight of the phylogenetic relationships and provide detailed descriptions of the species belonging to this clade. Macromorphological features, especially colony growth rates, texture and conidial colors on agar media, are important characters for phenotypic differentiation between species.