Characterization of heterotrophic growth and sesquiterpene production by Rhodobacter sphaeroides on a defined medium

Rhodobacter sphaeroides is a metabolically versatile bacterium capable of producing terpenes natively. Surprisingly, terpene biosynthesis in this species has always been investigated in complex media, with unknown compounds possibly acting as carbon and nitrogen sources. Here, a defined medium was adapted for R. sphaeroides dark heterotrophic growth, and was used to investigate the conversion of different organic substrates into the reporter terpene amorphadiene. The amorphadiene synthase was cloned in R. sphaeroides, allowing its biosynthesis via the native 2-methyl-d-erythritol-4-phosphate (MEP) pathway and, additionally, via a heterologous mevalonate one. The latter condition increased titers up to eightfold. Consequently, better yields and productivities to previously reported complex media cultivations were achieved. Productivity was further investigated under different cultivation conditions, including nitrogen and oxygen availability. This novel cultivation setup provided useful insight into the understanding of terpene biosynthesis in R. sphaeroides, allowing to better comprehend its dynamics and regulation during chemoheterotrophic cultivation.

     

Adaptation to developmental diet influences the response to selection on age at reproduction in the fruit fly

Experimental evolution (EE) is a powerful tool for addressing how environmental factors influence life‐history evolution. While in nature different selection pressures experienced across the lifespan shape life histories, EE studies typically apply selection pressures one at a time. Here, we assess the consequences of adaptation to three different developmental diets in combination with classical selection for early or late reproduction in the fruit fly Drosophila melanogaster. We find that the response to each selection pressure is similar to that observed when they are applied independently, but the overall magnitude of the response depends on the selection regime experienced in the other life stage. For example, adaptation to increased age at reproduction increased lifespan across all diets; however, the extent of the increase was dependent on the dietary selection regime. Similarly, adaptation to a lower calorie developmental diet led to faster development and decreased adult weight, but the magnitude of the response was dependent on the age‐at‐reproduction selection regime. Given that multiple selection pressures are prevalent in nature, our findings suggest that trade‐offs should be considered not only among traits within an organism, but also among adaptive responses to different—sometimes conflicting—selection pressures, including across life stages.     

Biogenic synthesis of silver nanoparticles using Brazilian propolis

Biological methods have been used to synthesize silver nanoparticles through materials such as bacteria, fungi, plants, and propolis due to their reducing properties, stabilizer role and environmentally friendly characteristic. Considering the antimicrobial activity of propolis as well as the broad-spectrum antibacterial effects of silver nanoparticles, this study aim to describe the use of Brazilian propolis to synthesize silver nanoparticles (AgNP-P) and investigate its antimicrobial activity. The synthesis was optimized by factorial design, choosing the best conditions for smaller size particles. AgNP-P demonstrated a maximum absorbance at 412 nm in ultraviolet-visible spectra, which indicated a spherical format and its formation. Dynamic light scattering demonstrated a hydrodynamic size of 109 nm and polydispersity index less than 0.3, showing a good size distribution and stability. After its purification via centrifugation, microscopy analysis corroborates the format and showed the presence of propolis around silver nanoparticle. X-ray diffraction peaks were attributed to the main planes of the metallic silver crystalline structure; meanwhile infrared spectroscopy demonstrated the main groups responsible for silver reduction, represented by ∼22% of AgNP-P indicates by thermal analysis. Our product revealed an important antimicrobial activity indicating a synergism between propolis and silver nanoparticles as expected and promising to be an effective antimicrobial product to be used in infections.     

Detection of in vivo protein-DNA interactions using DamID in mammalian cells

Understanding gene regulatory networks in mammalian cells requires detailed knowledge of protein-DNA interactions. Commonly used methods for genome-wide mapping of these interactions are based on chromatin immunoprecipitation. However, these methods have some drawbacks, such as the use of crosslinking reagents, the need for highly specific antibodies and relatively large amounts of starting material. We present DamID, an alternative technique to map genome-wide occupancy of interaction sites in vivo, that bypasses these limitations. DamID is based on the expression of a fusion protein consisting of a protein of interest and DNA adenine methyltransferase (Dam). This leads to methylation of adenines near sites where the protein of interest interacts with the DNA. These methylated sequences are subsequently amplified by a methylation-specific PCR protocol and identified by hybridization to microarrays. Using DamID, genome-wide maps of the binding of DNA-interacting proteins in mammalian cells can be constructed efficiently. Depending on the strategy used for expression of the Dam-fusion proteins, genome-wide binding maps can be obtained in as little as 2 weeks.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.     

Glucose delays seed germination in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Here we report that glucose delays germination of Arabidopsis thaliana (L.) Heynh. seeds at concentrations below those known to inhibit early seedling development. This inhibition acts on embryo growth and is independent of hexokinase (HXK) function. Hormones and hormone inhibitors were applied to the germination media and several hormone biosynthesis and signalling mutants were tested on glucose media to investigate a possible role of abscisic acid (ABA), gibberellin and ethylene in the glucose-induced germination delay. Results indicate that the germination inhibition by glucose cannot be antagonized by ethylene or gibberellin and is independent of the HXK1/ABA/ABI4 signalling cascade. These findings suggest that there is a separate regulatory pathway independent of ABI2/ABI4/ABI5. Thus, in a relatively short time frame sugars utilize different signalling cascades to inhibit germination and post-germination growth, underlining the complexity of sugar responses.Abbreviations ABA Abscisic acid - ABI ABA insensitive - ACC 1-Aminocyclopropane-1-carboxylic acid - BR Brassinosteroid - CAB Chlorophyll a/b-binding protein - FUS3 Fusca3 - GA Gibberellin - GA ₃ Gibberellic acid - HXK Hexokinase - LEC1 Leafy cotyledon1 - RBCS Ribulose-1,5-bisphosphate carboxylase small subunit - WT Wild type     

Production of tailor-made fructans in sugar beet by expression of onion fructosyltransferase genes

The consumption of fructans as a low caloric food ingredient or dietary fibre is rapidly increasing due to health benefits. Presently, the most important fructan source is chicory, but these fructans have a simple linear structure and are prone to degradation. Additional sources of high-quality tailor-made fructans would provide novel opportunities for their use as food ingredients. Sugar beet is a highly productive crop that does not normally synthesize fructans. We have introduced specific onion fructosyltransferases into sugar beet. This resulted in an efficient conversion of sucrose into complex, onion-type fructans, without the loss of storage carbohydrate content.     

CD36 deficiency increases insulin sensitivity in muscle,but induces insulin resistance in the liver in mice

CD36 (fatty acid translocase) is involved in high-affinity peripheral fatty acid uptake. Mice lacking CD36 exhibit increased plasma free fatty acid and triglyceride (TG) levels and decreased glucose levels. Studies in spontaneous hypertensive rats lacking functional CD36 link CD36 to the insulin-resistance syndrome. To clarify the relationship between CD36 and insulin sensitivity in more detail, we determined insulin-mediated whole-body and tissue-specific glucose uptake in CD36-deficient (CD36-/-) mice. Insulin-mediated whole-body and tissue-specific glucose uptake was measured by d-[3H]glucose and 2-deoxy-d-[1-3H]glucose during hyperinsulinemic clamp in CD36-/- and wild-type control littermates (CD36+/+) mice. Whole-body and muscle-specific insulin-mediated glucose uptake was significantly higher in CD36-/- compared with CD36+/+ mice. In contrast, insulin completely failed to suppress endogenous glucose production in CD36-/- mice compared with a 40% reduction in CD36+/+ mice. This insulin-resistant state of the liver was associated with increased hepatic TG content in CD36-/- mice compared with CD36+/+ mice (110.9 +/- 12.0 and 68.9 +/- 13.6 microg TG/mg protein, respectively). Moreover, hepatic activation of protein kinase B by insulin, measured by Western blot, was reduced by 54%. Our results show a dissociation between increased muscle and decreased liver insulin sensitivity in CD36-/- mice.     

Extrathymic TCR gene rearrangement in human small intestine: identification of new splice forms of recombination activating gene-1 mRNA with selective tissue expression

Two new 5'-untranslated region (5'UTR) exons were identified in the human gene for the lymphocyte-specific endonuclease recombination activating gene-1 (RAG1) required for the somatic recombination yielding functional Ag receptors. These 5'UTR exons were used in three different splice forms by jejunal lymphocytes of the T cell lineage. RAG1 mRNA containing the previously described 5'UTR exon was not expressed in these cells. Conversely, one of the new 5'UTR exons was not expressed in thymus. The new RAG1 mRNA splice forms were all expressed in immature T cells (CD2(+)CD7(+)CD3(-)). This cell population also expressed high levels of mRNA for the pre-T alpha-chain. In situ hybridization demonstrated jejunal cells expressing the new splice forms of RAG1 mRNA, both intraepithelially and in lamina propria. Pre-T alpha-chain mRNA-expressing cells were detected at the same sites. These results strongly suggest ongoing TCR gene rearrangement in human small intestinal mucosa, yielding T cells specially adapted for this environment. This seems to be achieved by two parallel processes, extrathymic T cell development and peripheral Ag-driven TCR editing.     

Simultaneous selection on two fitness-related traits in the butterfly Bicyclus anynana

Abstract. Theory about the role of constraints in evolution is abundant, but few empirical data exist to describe the consequences a bias in phenotypic variation has for micro evolution. Responses to natural selection can be severely hampered by a genetic correlation among a suite of traits. Constraints can be studied using antagonistic selection experiments, that is, two-trait selection in opposition to this correlation. The two traits studied here were development time and wing pattern (eyespot size) in the butterfly Bicyclus anynana , both of which have a clear adaptive significance. Rates of response were higher for eyespot size than for development time, but were independent of the concurrent selection (either in the same direction as the correlation or perpendicular to it). Regimes differed in both traits in all directions after 11 generations of selection. The uncoupling lines had higher relative responses than the synergistic lines in development time and equal relative responses in eyespot size. The patterns for eyespot size (reaction norms) were consistent across different rearing temperatures. Differences in lines selected for fast and slow development time were more pronounced at lower temperatures, irrespective of the direction of joint wing pattern selection. Furthermore, correlated responses in pupal weight and growth rate were observed; lines selected for a slower development had higher pupal weights, especially at lower temperatures. The response of the uncoupling lines was not hampered by a lack of selectable genetic variation, and the relative response in the development time was larger than expected based on response in the coupled direction and quantitative genetic predictions. This suggests that the structure of the genetic architecture does not constrain the short-term, independent evolution of both wing pattern and development time.