A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum.

Crop yields are significantly reduced by aluminum toxicity on highly acidic soils, which comprise up to 50% of the world's arable land. Candidate aluminum tolerance proteins include organic acid efflux transporters, with the organic acids forming non-toxic complexes with rhizosphere aluminum. In this study, we used positional cloning to identify the gene encoding a member of the multidrug and toxic compound extrusion (MATE) family, an aluminum-activated citrate transporter, as responsible for the major sorghum (Sorghum bicolor) aluminum tolerance locus, Alt(SB). Polymorphisms in regulatory regions of Alt(SB) are likely to contribute to large allelic effects, acting to increase Alt(SB) expression in the root apex of tolerant genotypes. Furthermore, aluminum-inducible Alt(SB) expression is associated with induction of aluminum tolerance via enhanced root citrate exudation. These findings will allow us to identify superior Alt(SB) haplotypes that can be incorporated via molecular breeding and biotechnology into acid soil breeding programs, thus helping to increase crop yields in developing countries where acidic soils predominate.

Bacteroides fragilis response to subinhibitory concentrations of antimicrobials includes different morphological, physiological and virulence patterns after in vitro selection.

As antimicrobials are introduced into the environment, microorganisms may respond in different ways, sometimes displaying alterations in cellular physiology. Considering the clinical relevance of the Bacteroides fragilis, strains were selected to investigate bacterial response after exposure to subinhibitory concentrations (SIC) of ampicillin (AMP), ampicillin-sulbactam (AMS), clindamycin (CLI), chloramphenicol (CHL), and its relationship to a host model (BALB/c mice) after experimental challenge. Morphological alterations, and biochemical-physiological and genetic profiles were evaluated among drug-selected bacteria. Histopathological evaluation of the liver and spleen, and inflammatory cytokines were determined after bacterial infection in mice. AMP and AMS exposure were related to most significant cellular alterations. Decreased sensitivity to all antimicrobials was observed for all drug-selected bacteria. Down regulation in adherence properties were also observed. Spleen and liver alterations were observed in different patterns. Increased levels of TNF-α, IL-6 and IFN-γ were also observed. Our results show that SICs of AMP, AMS, CLI and CHL may be related to alterations in cell physiology in B. fragilis with implications to the host-bacteria relationship. The data emphasizes the risks of inappropriate chemotherapy, and the concerns regarding ecological consequences lead by SICs of antimicrobials in resident microbiota.

Mass spectrometry characterisation of fatty acids from metabolically engineered soybean seeds.

Improving the quality and performance of soybean oil as biodiesel depends on the chemical composition of its fatty acids and requires an increase in monounsaturated acids and a reduction in polyunsaturated acids. Despite its current use as a source of biofuel, soybean oil contains an average of 25 % oleic acid and 13 % palmitic acid, which negatively impacts its oxidative stability and freezing point, causing a high rate of nitrogen oxide emission. Gas chromatography and ion mobility mass spectrometry were conducted on soybean fatty acids from metabolically engineered seed extracts to determine the nature of the structural oleic and palmitic acids. The soybean genes FAD2-1 and FatB were placed under the control of the 35SCaMV constitutive promoter, introduced to soybean embryonic axes by particle bombardment and down-regulated using RNA interference technology. Results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 94.58 %) and a reduction in palmitic acid (to <3 %) in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and non-transgenic oil extracts.

Applications of the Methylotrophic Yeast Komagataella phaffii in the Context of Modern Biotechnology.

Komagataella phaffii (formerly Pichia pastoris) is a methylotrophic yeast widely used in laboratories around the world to produce recombinant proteins. Given its advantageous features, it has also gained much interest in the context of modern biotechnology. In this review, we present the utilization of K. phaffii as a platform to produce several products of economic interest such as biopharmaceuticals, renewable chemicals, fuels, biomaterials, and food/feed products. Finally, we present synthetic biology approaches currently used for strain engineering, aiming at the production of new bioproducts.

Use of an on/off tetracycline riboswitch to control protein production in Komagataella phaffii.

The methylotrophic yeast Komagataella phaffii is one of the most important microbial platforms to produce recombinant proteins. Despite its importance in the context of industrial biotechnology, the use of synthetic biology approaches in K. phaffii is hampered by the fact that few genetic tools are available for precise control of gene expression in this system. In this work, we used an RNA aptamer activated by tetracycline to modulate protein production at the translational level. Using lacZ as gene reporter, we have demonstrated significant reduction of the heterologous protein upon addition of tetracycline. Furthermore, this genetic control device was applied for the control of Ku70p. This protein is involved in non-homologous recombination and the control of its production paves the way for the development of strains exhibiting higher rates of homologous recombination.

Nucleotides Entrapped in Liposome Nanovesicles as Tools for Therapeutic and Diagnostic Use in Biomedical Applications.

The use of nucleotides for biomedical applications is an old desire in the scientific community. As we will present here, there are references published over the past 40 years with this intended use. The main problem is that, as unstable molecules, nucleotides require some additional protection to extend their shelf life in the biological environment. Among the different nucleotide carriers, the nano-sized liposomes proved to be an effective strategic tool to overcome all these drawbacks related to the nucleotide high instability. Moreover, due to their low immunogenicity and easy preparation, the liposomes were selected as the main strategy for delivery of the mRNA developed for COVID-19 immunization. For sure this is the most important and relevant example of nucleotide application for human biomedical conditions. In addition, the use of mRNA vaccines for COVID-19 has increased interest in the application of this type of technology to other health conditions. For this review article, we will present some of these examples, especially focused on the use of liposomes to protect and deliver nucleotides for cancer therapy, immunostimulatory activities, enzymatic diagnostic applications, some examples for veterinarian use, and the treatment of neglected tropical disease.

Morphological, biochemical, physiological and molecular aspects of the response of Fusobacterium nucleatum exposed to subinhibitory concentrations of antimicrobials.

Subinhibitory concentrations (SICs) of antimicrobials may result in alterations in bacterial biology with implications for its potential aggression. This has considerable importance for the resident microbiota. Our aim was to analyze the effects of SICs of antimicrobials on the morphological, biochemical, physiological and molecular characteristics of the resident anaerobic Fusobacterium nucleatum. Fourteen strains were obtained from F. nucleatum ATCC 25586, selected by culturing on SICs of ampicillin, ampicillin/sulbactam, clindamycin, chloramphenicol, levofloxacin, metronidazole and piperacillin/tazobactam and subsequent culturing in the absence of drugs. Antimicrobial susceptibility, bacterial morphology, biochemical profiles and biofilm formation were evaluated. Genotyping and analysis of protein profiles were also performed. The antimicrobial susceptibility patterns showed that most of the derived strains were less sensitive to the antimicrobials, even after culturing them without drugs. Morphological and cell complexity alterations were observed, mainly in strains grown in SICs of ß-lactam; these strains also expressed a reduced ability for biofilm formation. The other strains showed an increase in biofilm formation but no apparent morphological changes. Alterations were observed in the carbohydrate metabolism patterns and in the activity of microbial enzymes. Several proteins were positively or negatively regulated and there was polymorphism in the DNA from all derived strains. Therefore, SICs of antimicrobials induce alterations in F. nucleatum, which directly impact its biology. These results emphasize the risk of inadequate antibioticotherapy, which may have serious implications for clinical microbiology and infectious diseases and also may interfere with the host-bacteria relationship.

Biocircuits in plants and eukaryotic algae.

As one of synthetic biology's foundations, biocircuits are a strategy of genetic parts assembling to recognize a signal and to produce a desirable output to interfere with a biological function. In this review, we revisited the progress in the biocircuits technology basis and its mandatory elements, such as the characterization and assembly of functional parts. Furthermore, for a successful implementation, the transcriptional control systems are a relevant point, and the computational tools help to predict the best combinations among the biological parts planned to be used to achieve the desirable phenotype. However, many challenges are involved in delivering and stabilizing the synthetic structures. Some research experiences, such as the golden crops, biosensors, and artificial photosynthetic structures, can indicate the positive and limiting aspects of the practice. Finally, we envision that the modulatory structural feature and the possibility of finer gene regulation through biocircuits can contribute to the complex design of synthetic chromosomes aiming to develop plants and algae with new or improved functions.

Optogenetic strategies for the control of gene expression in yeasts.

Optogenetics involves the use of light to control cellular functions and has become increasingly popular in various areas of research, especially in the precise control of gene expression. While this technology is already well established in neurobiology and basic research, its use in bioprocess development is still emerging. Some optogenetic switches have been implemented in yeasts for different purposes, taking advantage of a wide repertoire of biological parts and relatively easy genetic manipulation. In this review, we cover the current strategies used for the construction of yeast strains to be used in optogenetically controlled protein or metabolite production, as well as the operational aspects to be considered for the scale-up of this type of process. Finally, we discuss the main applications of optogenetic switches in yeast systems and highlight the main advantages and challenges of bioprocess development considering future directions for this field.

Cryptococcus neoformans Database in Synthetic Biology Open Language.

Cryptococcus neoformans is the etiologic agent of cryptococcosis, a lethal worldwide disease. Synthetic biology could contribute to its better understanding through engineering genetic networks. However, its major challenge is the requirement of accessible genetic parts. The database presented here provides 23 biological parts for this organism in Synthetic Biology Open Language.

Evaluation of Ogataea (Hansenula) polymorpha for Hyaluronic Acid Production.

Hyaluronic acid (HA) is a biopolymer formed by UDP-glucuronic acid and UDP-N-acetyl-glucosamine disaccharide units linked by ß-1,4 and ß-1,3 glycosidic bonds. It is widely employed in medical and cosmetic procedures. HA is synthesized by hyaluronan synthase (HAS), which catalyzes the precursors' ligation in the cytosol, elongates the polymer chain, and exports it to the extracellular space. Here, we engineer Ogataea (Hansenula) polymorpha for HA production by inserting the genes encoding UDP-glucose 6-dehydrogenase, for UDP-glucuronic acid production, and HAS. Two microbial HAS, from Streptococcus zooepidemicus (hasAs) and Pasteurella multocida (hasAp), were evaluated separately. Additionally, we assessed a genetic switch using integrases in O. polymorpha to uncouple HA production from growth. Four strains were constructed containing both has genes under the control of different promoters. In the strain containing the genetic switch, HA production was verified by a capsule-like layer around the cells by scanning electron microscopy in the first 24 h of cultivation. For the other strains, the HA was quantified only after 48 h and in an optimized medium, indicating that HA production in O. polymorpha is limited by cultivation conditions. Nevertheless, these results provide a proof-of-principle that O. polymorpha is a suitable host for HA production.

Distribution, detection of enterotoxigenic strains and antimicrobial drug susceptibility patterns of bacteroides fragilis group in diarrheic and non-diarrheic feces from brazilian infants.

Despite the importance of gastrointestinal diseases and their global distribution, affecting millions of individuals around the world, the role and antimicrobial susceptibility patterns of anaerobic bacteria such as those in the Bacteroides fragilis group (BFG) are still unclear in young children. This study investigated the occurrence and distribution of species in the BFG and enterotoxigenic strains in the fecal microbiota of children and their antimicrobial susceptibility patterns. Diarrheic (n=110) and non-diarrheic (n=65) fecal samples from children aged 0-5 years old were evaluated. BFG strains were isolated and identified by conventional biochemical, physiological and molecular approaches. Alternatively, bacteria and enterotoxigenic strains were detected directly from feces by molecular biology. Antimicrobial drug susceptibility patterns were determined by the agar dilution method according to the guidelines for isolated bacteria. BFG was detected in 64.3% of the fecal samples (55% diarrheic and 80.4% non-diarrheic), and 4.6% were enterotoxigenic. Antimicrobial resistance was observed against ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, meropenem, ceftriaxone, clindamycin and chloramphenicol. The data show that these bacteria are prevalent in fecal microbiota at higher levels in healthy children. The molecular methodology was more effective in identifying the B. fragilis group when compared to the biochemical and physiological techniques. The observation of high resistance levels stimulates thoughts about the indiscriminate use of antimicrobial drugs in early infancy. Further quantitative studies are needed to gain a better understanding of the role of these bacteria in acute diarrhea in children.

Genetic switches designed for eukaryotic cells and controlled by serine integrases.

Recently, new serine integrases have been identified, increasing the possibility of scaling up genomic modulation tools. Here, we describe the use of unidirectional genetic switches to evaluate the functionality of six serine integrases in different eukaryotic systems: the HEK 293T cell lineage, bovine fibroblasts and plant protoplasts. Moreover, integrase activity was also tested in human cell types of therapeutic interest: peripheral blood mononuclear cells (PBMCs), neural stem cells (NSCs) and undifferentiated embryonic stem (ES) cells. The switches were composed of plasmids designed to flip two different genetic parts driven by serine integrases. Cell-based assays were evaluated by measurement of EGFP fluorescence and by molecular analysis of attL/attR sites formation after integrase functionality. Our results demonstrate that all the integrases were capable of inverting the targeted DNA sequences, exhibiting distinct performances based on the cell type or the switchable genetic sequence. These results should support the development of tunable genetic circuits to regulate eukaryotic gene expression.

Comparative metagenome of a stream impacted by the urbanization phenomenon.

Rivers and streams are important reservoirs of freshwater for human consumption. These ecosystems are threatened by increasing urbanization, because raw sewage discharged into them alters their nutrient content and may affect the composition of their microbial community. In the present study, we investigate the taxonomic and functional profile of the microbial community in an urban lotic environment. Samples of running water were collected at two points in the São Pedro stream: an upstream preserved and non-urbanized area, and a polluted urbanized area with discharged sewage. The metagenomic DNA was sequenced by pyrosequencing. Differences were observed in the community composition at the two sites. The non-urbanized area was overrepresented by genera of ubiquitous microbes that act in the maintenance of environments. In contrast, the urbanized metagenome was rich in genera pathogenic to humans. The functional profile indicated that the microbes act on the metabolism of methane, nitrogen and sulfur, especially in the urbanized area. It was also found that virulence/defense (antibiotic resistance and metal resistance) and stress response-related genes were disseminated in the urbanized environment. The structure of the microbial community was altered by uncontrolled anthropic interference, highlighting the selective pressure imposed by high loads of urban sewage discharged into freshwater environments.

Association of PDCD1 polymorphism to systemic lupus erythematosus and rheumatoid arthritis susceptibility.

OBJECTIVE: This study aims to analyze the relationship of programmed cell death 1 (PDCD1) gene polymorphism (PD1.3G/A - rs11568821) with features of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in a Southern Brazilian population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed in 95 SLE and 87 RA patients and 128 control group individuals from Santa Catarina, Southern Brazil. The Hardy-Weinberg equilibrium (HWE) test, and odds ratio (OR) were analyzed, considering CI 95% and p≤0.05. RESULTS: The PD1.3A allele frequencies were 0.095 (SLE), 0.115 (RA) and 0.078 (controls). The genotypes of the control group were in HWE, while those of SLE and RA patients were not. However, we found no association between PD1.3 polymorphism and the SLE or RA susceptibility, nor clinical or epidemiological data. CONCLUSION: There was no significant association between PD1.3 polymorphism and SLE or RA susceptibility in this Southern Brazilian population.

Genetic analyses of endoreduplication in Zea mays endosperm: evidence of sporophytic and zygotic maternal control.

Flow cytometry was used to assess the variability of endoreduplication in endosperms of maize inbred lines. Little variation was found between midwestern dent types, and high levels of endoreduplication were observed in popcorns. Endoreduplication is different between inbred lines by 13-18 days after pollination, and flow cytometric analysis of ploidy level was feasible until 20 DAP. To study the genetic regulation of endoreduplication, four inbreds were crossed to B73 and developing endosperms from both parental, reciprocal F(1), and backcross generations were subjected to flow cytometric analysis. Three measurements of endoreduplication were calculated from these data and analyzed as quantitative genetic traits. Multiple models of trait inheritance were considered including triploid, diploid, sporophytic maternal, and maternal and paternal zygotic nuclear inheritance. Maternal zygotic effects, often considered a form of parental imprinting, and maternal sporophytic effects were detected. To test the feasibility of introgressing a high endoreduplication phenotype into a midwestern dent inbred line, a backcross population was generated from B73 x Sg18. Parental and progeny endoreduplication levels were compared and heritabilities assessed. The heritabilities calculated from these data generally agree with the values calculated in the larger crossing experiments.

Association of PDCD1 polymorphism to Systemic Lupus Erythematosus and Rheumatoid Arthritis susceptibility. / Associação entre o polimorfismo do gene PDCD1 e a susceptibilidade ao lúpus eritematoso sistêmico e à artrite reumatoide.

OBJECTIVE: This study aims to analyze the relationship of programmed cell death 1 (PDCD1) gene polymorphism (PD1.3G/A - rs11568821) with features of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in a Southern Brazilian population. METHODS: Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was performed in 95 SLE and 87 RA patients and 128 control group individuals from Santa Catarina, Southern Brazil. The Hardy-Weinberg Equilibrium (HWE) test, and odds ratio (OR) were analyzed, considering CI 95% and p≤0.05. RESULTS: The PD1.3A allele frequencies were 0.095 (SLE), 0.115 (RA) and 0.078 (controls). The genotypes of the control group were in HWE, while those of SLE and RA patients were not. However, we found no association between PD1.3 polymorphism and the SLE or RA susceptibility, nor clinical or epidemiological data. CONCLUSION: There was no significant association between PD1.3 polymorphism and SLE or RA susceptibility in this Southern Brazilian population.

Expression and characterisation of recombinant molecules in transgenic soybean.

Seeds are organs specialised in accumulating proteins, and they may provide a potential economically viable platform for the large-scale production and storage of many molecules for pharmaceutical and other productive sectors. Soybean [Glycine max (L.) Merrill] has a high seed protein content and represents an excellent source of abundant and cheap biomass. Under greenhouse conditions and a daily photoperiod of 23 h of light, the soybean plant's vegetative growth can be significantly extended by inducing more than a tenfold increase in seed production when compared with plants cultivated under field conditions. Some factors involved in the production of different recombinant proteins in soybean seeds are discussed in this review. These include transgenic system, regulatory sequences and the use of Mass Spectrometry as a new tool for molecular characterisation of seed produced recombinant proteins. The important intrinsic characteristics and possibility of genetically engineering soybean seeds, using current advances in recombinant DNA technology including metabolic engineering and synthetic biology, should form the foundation for large-scale and more precise genome modification, making this crop an important candidate as bioreactor for production of recombinant molecules.

A bivariate variance components model for mapping iQTLs underlying endosperm traits.

Genomic imprinting plays a pivotal role in early stage development in plants. Linkage analysis has been proven to be useful in mapping imprinted quantitative trait loci (iQTLs) underlying imprinting phenotypic traits in natural populations or experimental crosses. For correlated traits, studies have shown that multivariate genetic linkage analysis can improve QTL mapping power and precision, especially when a QTL has a pleiotropic effect on several traits. In addition, the joint analysis of multiple traits can test a number of biologically interesting hypotheses, such as pleiotropic effects vs close linkage. Motivated by a triploid maize endosperm dataset, we extended the variance components linkage analysis model incorporating imprinting effect proposed by Li and Cui (2010) to a bivariate trait modeling framework, aimed to improve the mapping precision and to identify pleiotropic imprinting effects. We proposed to partition the genetic variance of a QTL into sex-specific allelic variance components, to model and test the imprinting effect of an iQTL on two traits. Both simulation studies and real data analysis show the power and utility of the method.