Stable, covalent attachment of laminin to microposts improves the contractility of mouse neonatal cardiomyocytes.

The mechanical output of contracting cardiomyocytes, the muscle cells of the heart, relates to healthy and disease states of the heart. Culturing cardiomyocytes on arrays of elastomeric microposts can enable inexpensive and high-throughput studies of heart disease at the single-cell level. However, cardiomyocytes weakly adhere to these microposts, which limits the possibility of using biomechanical assays of single cardiomyocytes to study heart disease. We hypothesized that a stable covalent attachment of laminin to the surface of microposts improves cardiomyocyte contractility. We cultured cells on polydimethylsiloxane microposts with laminin covalently bonded with the organosilanes 3-glycidoxypropyltrimethoxysilane and 3-aminopropyltriethoxysilane with glutaraldehyde. We measured displacement of microposts induced by the contractility of mouse neonatal cardiomyocytes, which attach better than mature cardiomyocytes to substrates. We observed time-dependent changes in contractile parameters such as micropost deformation, contractility rates, contraction and relaxation speeds, and the times of contractions. These parameters were affected by the density of laminin on microposts and by the stability of laminin binding to micropost surfaces. Organosilane-mediated binding resulted in higher laminin surface density and laminin binding stability. 3-glycidoxypropyltrimethoxysilane provided the highest laminin density but did not provide stable protein binding with time. Higher surface protein binding stability and strength were observed with 3-aminopropyltriethoxysilane with glutaraldehyde. In cultured cardiomyocytes, contractility rate, contraction speeds, and contraction time increased with higher laminin stability. Given these variations in contractile function, we conclude that binding of laminin to microposts via 3-aminopropyltriethoxysilane with glutaraldehyde improves contractility observed by an increase in beating rate and contraction speed as it occurs during the postnatal maturation of cardiomyocytes. This approach is promising for future studies to mimic in vivo tissue environments

Energy explorer: Explorador de energías renovables utilizando realidad aumentada

Este proyecto toma ventaja de la realidad aumentada (AR) como medio digital de información sobre energías renovables en la región del usuario. En nuestro caso todo México. También muestra el potencial en KW o MW útiles para la decisión e inversión. Nuestro interés se centra sobre datos de energía de viento, solar y geotérmica, estos datos son desplegados en una amigable aplicación para Smartphone con realidad aumentada que permite conocer donde están localizados en escala geoespacial

Detailed characterization of the solution kinetics and thermodynamics of biotin, biocytin and HABA binding to avidin and streptavidin

The high affinity (KD ~ 10−15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109 M-1s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (105 to 107 M-1s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4’-hydroxyazobenzene)- benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The kon values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the kon values revealed strong temperature dependence with large activation energies (6–15 kcal/mol) that do not correspond to a diffusion limited process (3–4 kcal/mol). Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle, in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics, and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications

Biosynthesis of HSAF, a Tetramic Acid-containing Macrolactam from \u3ci\u3eLysobacter enzymogenes\u3c/i\u3e

HSAF was isolated from Lysobacter enzymogenes, a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here, we described the HSAF biosynthetic gene cluster which contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS/ NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/ NRPS are six genes, encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activated L-ornithine and the fourdomain NRPS was able to catalyze the formation of a tetramic acid-containing product from acyl- S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms

Biosynthesis of HSAF, a Tetramic Acid-containing Macrolactam from \u3ci\u3eLysobacter enzymogenes\u3c/i\u3e

HSAF was isolated from Lysobacter enzymogenes, a bacterium used in the biological control of fungal diseases of plants. Structurally, it is a tetramic acid-containing macrolactam fused to a tricyclic system. HSAF exhibits a novel mode of action by disrupting sphingolipids important to the polarized growth of filamentous fungi. Here, we described the HSAF biosynthetic gene cluster which contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS/ NRPS), although the biosynthesis of HSAF apparently requires two separate polyketide chains that are linked together by one amino acid (ornithine) via two amide bonds. Flanking the PKS/ NRPS are six genes, encoding a cascade of four tightly clustered redox enzymes on one side and a sterol desaturase/fatty acid hydroxylase and a ferredoxin reductase on the other side. The genetic data demonstrate that the four redox genes, in addition to the PKS/NRPS gene and the sterol desaturase/fatty acid hydroxylase gene, are required for HSAF production. The biochemical data show that the adenylation domain of the NRPS specifically activated L-ornithine and the fourdomain NRPS was able to catalyze the formation of a tetramic acid-containing product from acyl- S-ACP and ornithinyl-S-NRPS. These results reveal a previously unrecognized biosynthetic mechanism for hybrid PK/NRP in prokaryotic organisms

Functional replacement of the ketosynthase domain of \u3ci\u3eFUM1\u3c/i\u3e for the biosynthesis of fumonisins, a group of fungal reduced polyketides

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C18 chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides

Cloning, Sequencing, Heterologous Expression, and Mechanistic Analysis of A-74528 Biosynthesis

A-74528 is a recently discovered natural product of Streptomyces sp. SANK 61196 that inhibits 2′,5′-oligoadenylate phosphodiesterase (2′-PDE), a key regulatory enzyme of the interferon pathway. Inhibition of 2′-PDE by A-74528 reduces viral replication, and therefore shows promise as a new type of antiviral drug. The complete A-74528 gene cluster, comprising of 29 open reading frames, was cloned and sequenced, and shown to possess a type II polyketide synthase (PKS) at its core. Its identity was confirmed by analysis of a mutant generated by targeted disruption of a PKS gene, and by functional expression in a heterologous Streptomyces host. Remarkably, it showed exceptional end-to-end sequence identity to the gene cluster responsible for biosynthesis of fredericamycin A, a structurally unrelated antitumor antibiotic with a distinct mode of action. Whereas the fredericamycin producing strain, Streptomyces griseus, produced undetectable quantities of A-74528, the A-74528 gene cluster was capable of producing both antibiotics. The biosynthetic roles of three genes, including one that represents the only qualitative difference between the two gene clusters, were investigated by targeted gene disruption. The implications for the evolution of antibiotics with different biological activities from the same gene cluster are discussed

An Antibiotic Complex from \u3ci\u3eLysobacter enzymogenes\u3c/i\u3e Strain C3: Antimicrobial Activity and Role in Plant Disease Control

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 μg/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 μg/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and ΔNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi

RNA, Action through Interactions

As transcription of the human genome is quite pervasive, it is possible that many novel functions of the noncoding genome have yet to be identified. Often the noncoding genome's functions are carried out by their RNA transcripts, which may rely on their structures and/or extensive interactions with other molecules. Recent technology developments are transforming the fields of RNA biology from studying one RNA at a time to transcriptome-wide mapping of structures and interactions. Here, we highlight the recent advances in transcriptome-wide RNA interaction analysis. These technologies revealed surprising versatility of RNA to participate in diverse molecular systems. For example, tens of thousands of RNA-RNA interactions have been revealed in cultured cells as well as in mouse brain, including interactions between transposon-produced transcripts and mRNAs. In addition, most transcription start sites in the human genome are associated with noncoding RNA transcribed from other genomic loci. These recent discoveries expanded our understanding of RNAs' roles in chromatin organization, gene regulation, and intracellular signaling