Evaluation of Xanthomonas campestris survival in a soil microcosm system

Xanthomonas campestris pv. campestris is a pathogen of cruciferous plants. We studied the survival of the wild type strain and mutant derivatives which are deficient in exopolysaccharide (EPS) or in extracellular protease synthesis in soil microcosms in order to test the hypothesis that, in this environment, adherence to soil particles and scavenging of nutrients are very important strategies for bacterial survival. In sterile soil microcosms, differences in survival were only observed between the EPS producer and its mutant. In non-sterile soil experiments, survival of Prt– mutant was similar to EPS– mutant, suggesting that both characteristics have a strong influence in survival in the presence of the natural bacterial community. Bacterial decrease represented by the slope of regression lines was higher in nonsterile soil microcosms due to the influence of biotic interactions

Assessment of lipid formulations to develop multi-stimuli-responsive solid magnetoliposomes using fluorescence-based methodologies

This work is focused on the assessment of lipid formulations to design solid magnetoliposomes (SMLs) as multi-stimuli-responsive vesicles for controlled release of doxorubicin (DOX) in pathological areas under the influence of thermal, magnetic, and pH stimuli. The intrinsic fluorescence of DOX can be used as a facilitating tool for DOX-loaded SMLs characterization. Thus, the fluorescence spectroscopy technique was fundamental to evaluating the effect of lipid formulations on SMLs’ properties, such as its encapsulation efficiency

Recent advances on cell culture platforms for In vitro drug screening and cell therapies: from conventional to microfluidic strategies

The clinical translations of drugs and nanomedicines depend on coherent pharmaceutical research based on biologically accurate screening approaches. Since establishing the 2D in vitro cell culture method, the scientific community has improved cell-based drug screening assays and models. Those advances result in more informative biochemical assays and the development of 3D multicellular models to describe the biological complexity better and enhance the simulation of the in vivo microenvironment. Despite the overall dominance of conventional 2D and 3D cell macroscopic culture methods, they present physicochemical and operational challenges that impair the scale-up of drug screening by not allowing a high parallelization, multidrug combination, and high-throughput screening. Their combination and complementarity with microfluidic platforms enable the development of microfluidics-based cell culture platforms with unequivocal advantages in drug screening and cell therapies. Thus, this review presents an updated and consolidated view of cell culture miniaturization's physical, chemical, and operational considerations in the pharmaceutical research scenario. It clarifies advances in the field using gradient-based microfluidics, droplet-based microfluidics, printed-based microfluidics, digital-based microfluidics, SlipChip, and paper-based microfluidics. Finally, it presents a comparative analysis of the performance of cell-based methods in life research and development to achieve increased precision in the drug screening process.This work was supported by FCT - Fundação para a Ciência e Tecnolo gia (FCT) under the scope of the strategic funding UIDB/04650/2020, UIDP/04650/2020, UIDB/04436/2020, UIDP/04436/2020. The authors also thank FCT for financial support under grant SFRH/BD/141936/2018 (B.D.C.) and the contract under the Stimulus of Scientific Employment 2020.02304.CEECIND (V.C.). The authors also acknowledge funding by Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD) through the project PID2019-106099RBC43/AEI/10.13039/501100011033 and from the Basque Government Industry Departments under the ELKARTEK program

Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in Bacillus megaterium

Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)- producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of ActBm, a σ 54 regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium

Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strain using agroindustrial byproducts

A statistically based Plackett-Burman screening design identified milk whey and corn steep liquor concentrations as well as ionic strength (based on phosphate buffer concentration) as the three main independent components of the culture medium that significantly (p < 0.05) influenced biomass and poly(3-hydroxybutyrate) (PHB) production in recombinant cells of Escherichia coli. This strain carries a plasmid encoding phb genes from a natural isolate of Azotobacte sp. Response surface methodology, using a central composite rotatable design, demonstrated that the optimal concentrations of the three components, defined as those yielding maximal biomass and PHB production in shaken flasks, were 37.96 g deproteinated milk whey powder/l, 29.39 g corn steep liquor/l, and 23.76 g phosphates/l (r2 = 0.957). The model was validated by culturing the recombinant cells in medium containing these optimal concentrations, which yielded 9.41 g biomass/l and 6.12 g PHB/l in the culture broth. Similar amounts of PHB were obtained following batch fermentations in a bioreactor. These results show that PHB can be produced efficiently by culturing the recombinant strain in medium containing cheap carbon and nitrogen sources. [Int Microbiol 2005; 8(4):243-250

Solid magnetoliposomes as multi-stimuli-responsive systems for controlled release of doxorubicin: assessment of lipid formulations

Stimuli-responsive liposomes are a class of nanocarriers whose drug release occurs, preferentially, when exposed to a specific biological environment, to an external stimulus, or both. This work is focused on the design of solid magnetoliposomes (SMLs) as lipid-based nanosystems aiming to obtain multi-stimuli-responsive vesicles for doxorubicin (DOX) controlled release in pathological areas under the action of thermal, magnetic, and pH stimuli. The effect of lipid combinations on structural, colloidal stability, and thermodynamic parameters were evaluated. The results confirmed the reproducibility for SMLs synthesis based on nine lipid formulations (combining DPPC, DSPC, CHEMS, DOPE and/or DSPE-PEG), with structural and colloidal properties suitable for biological applications. A loss of stability and thermosensitivity was observed for formulations containing dioleoylphosphatidylethanolamine (DOPE) lipid. SMLs PEGylation is an essential step to enhance both their long-term storage stability and stealth properties. DOX encapsulation (encapsulation efficiency ranging between 87% and 96%) in the bilayers lowered its pK(a), which favors the displacement of DOX from the acyl chains to the surface when changing from alkaline to acidic pH. The release profiles demonstrated a preferential release at acidic pH, more pronounced under mimetic mild-hyperthermia conditions (42 degrees C). Release kinetics varied with the lipid formulation, generally demonstrating hyperthermia temperatures and acidic pH as determining factors in DOX release; PEGylation was shown to act as a diffusion barrier on the SMLs surface. The integrated assessment and characterization of SMLs allows tuning lipid formulations that best respond to the needs for specific controlled release profiles of stimuli-responsive nanosystems as a multi-functional approach to cancer targeting and therapy.This research was funded by the Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding UIDB/04650/2020, UIDB/04436/2020, UIDP/04436/2020 and through the research project PTDC/QUI-QFI/28020/2017 (POCI-01-0145-FEDER-028020), cofinanced by European Fund of Regional Development (FEDER), COMPETE2020 and Portugal2020. The authors also thank FCT for financial support under grants SFRH/BD/141936/2018 (B.D.C.) and 2020.02304.CEECIND (V.F.C.) Finally, the authors acknowledge funding by Spanish State Research Agency (AEI) and the European Regional Development Fund (ERFD) through the project PID2019106099RB-C43/AEI/10.13039/501100011033 and from the Basque Government Industry Departments under the ELKARTEK program

¿De héroes a vencidos? Los patógenos microbianos están ganando la batalla frente a los antibióticos

Los niveles de resistencia a antibióticos que se están alcanzando mundialmente son difíciles de controlar. Enfermedades como neumonía, tuberculosis, septicemia, gonorrea o las de transmisión alimentaria, son a veces imposibles de tratar a medida que los antibióticos van perdiendo eficacia. Se supone que si no se arbitran controles, la resistencia antibióticos podría ser la mayor causa de muerte hacia 2050...Fil: Ibarra, José Gervasio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Méndez, Beatriz S.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Nc886 is epigenetically repressed in prostate cancer and acts as a tumor suppressor through the inhibition of cell growth

Background: Nc886 is a 102 bp non-coding RNA transcript initially classified as a microRNA precursor (Pre-miR-886), later as a divergent homologue of the vault RNAs (vtRNA 2-1) and more recently as a novel type of RNA (nc886). Although nc886/vtRNA2-1/Pre-miR-886 identity is still controversial, it was shown to be epigenetically controlled, presenting both tumor suppressor and oncogenic function in different cancers. Here, we study for the first time the role of nc886 in prostate cancer. Methods: Nc886 promoter methylation status and its correlation with patient clinical parameters or DNMTs levels were evaluated in TCGA and specific GEO prostate tissue datasets. Nc886 level was measured by RT-qPCR to compare normal/neoplastic prostate cells from radical prostatectomies and cell lines, and to assess nc886 response to demethylating agents. The effect of nc886 recovery in cell proliferation (in vitro and in vivo) and invasion (in vitro) was evaluated using lentiviral transduced DU145 and LNCaP cell lines. The association between the expression of nc886 and selected genes was analyzed in the TCGA-PRAD cohort. Results: Nc886 promoter methylation increases in tumor vs. normal prostate tissue, as well as in metastatic vs. normal prostate tissue. Additionally, nc886 promoter methylation correlates with prostate cancer clinical staging, including biochemical recurrence, Clinical T-value and Gleason score. Nc886 transcript is downregulated in tumor vs. normal tissue -in agreement with its promoter methylation status- and increases upon demethylating treatment. In functional studies, the overexpression of nc886 in the LNCaP and DU145 cell line leads to a decreased in vitro cell proliferation and invasion, as well as a reduced in vivo cell growth in NUDE-mice tumor xenografts. Finally, nc886 expression associates with the prostate cancer cell cycle progression gene signature in TCGA-PRAD. Conclusions: Our data suggest a tumor suppressor role for nc886 in the prostate, whose expression is epigenetically silenced in cancer leading to an increase in cell proliferation and invasion. Nc886 might hold clinical value in prostate cancer due to its association with clinical parameters and with a clinically validated gene signature