Increasing the thermal stability of cellulase C using rules learned from thermophilic proteins: a pilot study

Some structural features underlying the increased thermostability of enzymes from thermophilic organisms relative to their homologues from mesophiles are known from earlier studies. We used cellulase C from Clostridium thermocellum to test whether thermostability can be increased by mutations designed using rules learned from thermophilic proteins. Cellulase C has a TIM barrel fold with an additional helical subdomain. We designed and produced a number of mutants with the aim to increase its thermostability. Five mutants were designed to create new electrostatic interactions. They all retained catalytic activity but exhibited decreased thermostability relative to the wild-type enzyme. Here, the stabilizing contributions are obviously smaller than the destabilization caused by the introduction of the new side chains. In another mutant, the small helical subdomain was deleted. This mutant lost activity but its melting point was only 3 degrees C lower than that of the wild-type enzyme, which suggests that the subdomain is an independent folding unit and is important for catalytic function. A double mutant was designed to introduce a new disulfide bridge into the enzyme. This mutant is active and has an increased stability (deltaT(m)=3 degrees C, delta(deltaG(u))=1.73 kcal/mol) relative to the wild-type enzyme. Reduction of the disulfide bridge results in destabilization and an altered thermal denaturation behavior. We conclude that rules learned from thermophilic proteins cannot be used in a straightforward way to increase the thermostability of a protein. Creating a crosslink such as a disulfide bond is a relatively sure-fire method but the stabilization may be smaller than calculated due to coupled destabilizing effects.     

Genomic and Metabolic Disposition of Non-Obese Type 2 Diabetic Rats to Increased Myocardial Fatty Acid Metabolism

Lipotoxicity of the heart has been implicated as a leading cause of morbidity in Type 2 Diabetes Mellitus (T2DM). While numerous reports have demonstrated increased myocardial fatty acid (FA) utilization in obese T2DM animal models, this diabetic phenotype has yet to be demonstrated in non-obese animal models of T2DM. Therefore, the present study investigates functional, metabolic, and genomic differences in myocardial FA metabolism in non-obese type 2 diabetic rats. The study utilized Goto-Kakizaki (GK) rats at the age of 24 weeks. Each rat was imaged with small animal positron emission tomography (PET) to estimate myocardial blood flow (MBF) and myocardial FA metabolism. Echocardiograms (ECHOs) were performed to assess cardiac function. Levels of triglycerides (TG) and non-esterified fatty acids (NEFA) were measured in both plasma and cardiac tissues. Finally, expression profiles for 168 genes that have been implicated in diabetes and FA metabolism were measured using quantitative PCR (qPCR) arrays. GK rats exhibited increased NEFA and TG in both plasma and cardiac tissue. Quantitative PET imaging suggests that GK rats have increased FA metabolism. ECHO data indicates that GK rats have a significant increase in left ventricle mass index (LVMI) and decrease in peak early diastolic mitral annular velocity (E’) compared to Wistar rats, suggesting structural remodeling and impaired diastolic function. Of the 84 genes in each the diabetes and FA metabolism arrays, 17 genes in the diabetes array and 41 genes in the FA metabolism array were significantly up-regulated in GK rats. Our data suggest that GK rats’ exhibit increased genomic disposition to FA and TG metabolism independent of obesity.     

Mesenchymal stem cells rescue cardiomyoblasts from cell death in an in vitro ischemia model via direct cell-to-cell connections

Background  

Bone marrow derived mesenchymal stem cells (MSCs) are promising candidates for cell based therapies in myocardial infarction. However, the exact underlying cellular mechanisms are still not fully understood. Our aim was to explore the possible role of direct cell-to-cell interaction between ischemic H9c2 cardiomyoblasts and normal MSCs. Using an in vitro ischemia model of 150 minutes of oxygen glucose deprivation we investigated cell viability and cell interactions with confocal microscopy and flow cytometry.     

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed‐batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg^∑1) resulting from feeding of nutrients may enhance specific product formation rates (q_p). As an improved ATP supply was anticipated to enhance q_p this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 → 430 mOsmol kg^‐1). Hyperosmotic culture conditions caused a ～2.6‐fold increase of specific ATP formation rates together with a ～1.7‐fold rise in cytosolic and mitochondrial ATP‐pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR‐132‐3p, miR‐132‐5p, miR‐182, miR‐183, miR‐194, miR‐215‐3p, miR‐215‐5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR‐183 may be the most promising target to enhance q_p by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.     

Slx4 and Rtt107 control checkpoint signalling and DNA resection at double-strand breaks

The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5′ strand. Importantly, in slx4Δ sae2Δ double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity.     

Tricarboxylic Acid Cycle and One-Carbon Metabolism Pathways Are Important in Edwardsiella ictaluri Virulence

Edwardsiella ictaluri is a Gram-negative facultative intracellular pathogen causing enteric septicemia of channel catfish (ESC). The disease causes considerable economic losses in the commercial catfish industry in the United States. Although antibiotics are used as feed additive, vaccination is a better alternative for prevention of the disease. Here we report the development and characterization of novel live attenuated E. ictaluri mutants. To accomplish this, several tricarboxylic acid cycle (sdhC, mdh, and frdA) and one-carbon metabolism genes (gcvP and glyA) were deleted in wild type E. ictaluri strain 93-146 by allelic exchange. Following bioluminescence tagging of the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, ΔgcvP, and ΔglyA mutants, their dissemination, attenuation, and vaccine efficacy were determined in catfish fingerlings by in vivo imaging technology. Immunogenicity of each mutant was also determined in catfish fingerlings. Results indicated that all of the E. ictaluri mutants were attenuated significantly in catfish compared to the parent strain as evidenced by 2,265-fold average reduction in bioluminescence signal from all the mutants at 144 h post-infection. Catfish immunized with the E. ictaluri ΔsdhC, Δmdh, ΔfrdA, and ΔglyA mutants had 100% relative percent survival (RPS), while E. ictaluri ΔgcvP vaccinated catfish had 31.23% RPS after re-challenge with the wild type E. ictaluri.     

Changes in bacterial and archaeal communities during the concentration of brine at the graduation towers in Ciechocinek spa (Poland)

This study evaluates the changes in bacterial and archaeal community structure during the gradual evaporation of water from the brine (extracted from subsurface Jurassic deposits) in the system of graduation towers located in Ciechocinek spa, Poland. The communities were assessed with 16S rRNA gene sequencing (MiSeq, Illumina) and microscopic methods. The microbial cell density determined by direct cell count was at the order of magnitude of 10⁷ cells/mL. It was found that increasing salt concentration was positively correlated with both the cell counts, and species-level diversity of bacterial and archaeal communities. The archaeal community was mostly constituted by members of the phylum Euryarchaeota, class Halobacteria and was dominated by Halorubrum-related sequences. The bacterial community was more diverse, with representatives of the phyla Proteobacteria and Bacteroidetes as the most abundant. The proportion of Proteobacteria decreased with increasing salt concentration, while the proportion of Bacteroidetes increased significantly in the more concentrated samples. Representatives of the genera Idiomarina, Psychroflexus, Roseovarius, and Marinobacter appeared to be tolerant to changes of salinity. During the brine concentration, the relative abundances of Sphingobium and Sphingomonas were significantly decreased and the raised contributions of genera Fabibacter and Fodinibius were observed. The high proportion of novel (not identified at 97% similarity level) bacterial reads (up to 42%) in the 16S rRNA gene sequences indicated that potentially new bacterial taxa inhabit this unique environment.

     

Time scale and dimension analysis of a budding yeast cell cycle model

Background  

The progress through the eukaryotic cell division cycle is driven by an underlying molecular regulatory network. Cell cycle progression can be considered as a series of irreversible transitions from one steady state to another in the correct order. Although this view has been put forward some time ago, it has not been quantitatively proven yet. Bifurcation analysis of a model for the budding yeast cell cycle has identified only two different steady states (one for G1 and one for mitosis) using cell mass as a bifurcation parameter. By analyzing the same model, using different methods of dynamical systems theory, we provide evidence for transitions among several different steady states during the budding yeast cell cycle.     

The Unusual X-Form DNA in Oligodeoxynucleotides: Dependence of Stability on the Base Sequence and Length

Abstract

X-form is an unusual double helix of DNA adopted by poly(dA-dT) or (dT-dA)₄ at high concentrations of CsF. On the other hand, poly(dA).poly(dT), (dA-dT)₄ and most other DNAs do not adopt this conformer. Here we demonstrate that the X-form is strongly destabilized by GC pairs or even minute perturbations of the alternating pyrimidine- purine sequence. For example, the 30-mer d(TATAAT)₅, containing five tandem repeats of the Pribnow box, fails to isomerize into the X-form. After (dT-dA)₄, the 16-mer (dT- dA)_g is shown to be the second most predisposed oligodeoxynucleotide in the (dT-dA)., series to isomerize into the X-form while the duplex lengths corresponding to n=3,5,6,7,9,12 and 20 make the X-form unstable even in the strictly alternating (dT-dA)., sequence. Consequently, the (dT-dA)., duplex length is also a crucial factor of the X-form stability on the oligodeoxynucleotide level. We discuss a possibility that the X-form is a solution counterpart of the D-form adopted in dehydrated poly(dA-dT) fibers because properties of these two conformers are remarkably similar in many respects.     

Growth‐form and spatiality driving the functional difference of native and alien aquatic plants in Europe

Trait‐based approaches are widely used in community ecology and invasion biology to unravel underlying mechanisms of vegetation dynamics. Although fundamental trade‐offs between specific traits and invasibility are well described among terrestrial plants, little is known about their role and function in aquatic plant species. In this study, we examine the functional differences of aquatic alien and native plants stating that alien and native species differ in selected leaf traits. Our investigation is based on 60 taxa (21 alien and 39 native) collected from 22 freshwater units of Hungarian and Italian lowlands and highlands. Linear mixed models were used to investigate the effects of nativeness on four fundamental traits (leaf area, leaf dry matter content, specific leaf area, and leaf nitrogen content), while the influence of growth‐form, altitude, and site were employed simultaneously. We found significantly higher values of leaf areas and significantly lower values of specific leaf areas for alien species if growth‐form was included in the model as an additional predictor.We showed that the trait‐based approach of autochthony can apply to aquatic environments similar to terrestrial ones, and leaf traits have relevance in explaining aquatic plant ecology whether traits are combined with growth‐forms as a fixed factor. Our results confirm the importance of traits related to competitive ability in the process of aquatic plant invasions. Alien aquatic plants can be characterized as species producing soft leaves faster. We argue that the functional traits of alien aquatic plants are strongly growth‐form dependent. Using the trait‐based approach, we found reliable characteristics of aquatic plants related to species invasions, which might be used, for example, in conservation management.