Batch and continuous culture-based selection strategies for acetic acid tolerance in xylose-fermenting Saccharomyces cerevisiae

Acetic acid tolerance of Saccharomyces cerevisiae is crucial for the production of bioethanol and other bulk chemicals from lignocellulosic plant-biomass hydrolysates, especially at a low pH. This study explores two evolutionary engineering strategies for the improvement of acetic acid tolerance of the xylose-fermenting S. cerevisiae RWB218, whose anaerobic growth on xylose at pH 4 is inhibited at acetic acid concentrations >1 g L(-1) : (1) sequential anaerobic, batch cultivation (pH 4) at increasing acetic acid concentrations and (2) prolonged anaerobic continuous cultivation without pH control, in which acidification by ammonium assimilation generates selective pressure for acetic acid tolerance. After c. 400 generations, the sequential-batch and continuous selection cultures grew on xylose at pH≤4 with 6 and 5 g L(-1) acetic acid, respectively. In the continuous cultures, the specific xylose-consumption rate had increased by 75% to 1.7 g xylose g(-1) biomass h(-1) . After storage of samples from both selection experiments at -80 °C and cultivation without acetic acid, they failed to grow on xylose at pH 4 in the presence of 5 g L(-1) acetic acid. Characterization in chemostat cultures with linear acetic acid gradients demonstrated an acetate-inducible acetic acid tolerance in samples from the continuous selection protocol.     

Characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of Helicobacter pylori lipopolysaccharide

Background: The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6‐glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6‐glucan and characterize their binding properties and functional activity. Materials and Methods: BALB/c mice were injected intraperitoneally with 10⁸ formalin‐fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti‐α1,6‐glucan‐producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole‐cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild‐type and mutant strains of H. pylori. Results: The generated anti‐α1,6‐glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6‐linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD₄₅₀ ≥ 0.2). Bactericidal activity was observed against selective wild‐type and mutant H. pylori strains exhibiting OD₄₅₀ values of ≥0.45 in WCE. Conclusions: Anti‐α1,6‐glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS‐based vaccine against H. pylori infections.     

Human dental pulp stem cells hook into biocoral scaffold forming an engineered biocomplex

The aim of this study was to evaluate the behavior of human Dental Pulp Stem Cells (DPSCs), as well as human osteoblasts, when challenged on a Biocoral scaffold, which is a porous natural hydroxyapatite. For this purpose, human DPSCs were seeded onto a three-dimensional (3D) Biocoral scaffold or on flask surface (control). Either normal or rotative (3D) cultures were performed. Scanning electron microscopic analyses, at 8, 24 and 48 h of culture showed that cells did not adhere on the external surface, but moved into the cavities inside the Biocoral structure. After 7, 15 and 30 days of culture, morphological and molecular analyses suggested that the Biocoral scaffold leads DPSCs to hook into the cavities where these cells quickly start to secrete the extra cellular matrix (ECM) and differentiate into osteoblasts. Control human osteoblasts also moved into the internal cavities where they secreted the ECM. Histological sections revealed a diffuse bone formation inside the Biocoral samples seeded with DPSCs or human osteoblasts, where the original scaffold and the new secreted biomaterial were completely integrated and cells were found within the remaining cavities. In addition, RT-PCR analyses showed a significant increase of osteoblast-related gene expression and, above all, of those genes highly expressed in mineralized tissues, including osteocalcin, OPN and BSP. Furthermore, the effects on the interaction between osteogenesis and angiogenesis were observed and substantiated by ELISA assays. Taken together, our results provide clear evidence that DPSCs differentiated into osteoblasts, forming a biocomplex made of Biocoral, ECM and differentiated cells.     

The bile acid receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier and immune response to experimental colitis

Background

GP-BAR1, a member G protein coupled receptor superfamily, is a cell surface bile acid-activated receptor highly expressed in the ileum and colon. In monocytes, ligation of GP-BAR1 by secondary bile acids results in a cAMP-dependent attenuation of cytokine generation.

Aims

To investigate the role GP-BAR1 in regulating intestinal homeostasis and inflammation-driven immune dysfunction in rodent models of colitis.

Methods

Colitis was induced in wild type and GP-BAR1^−/− mice by DSS and TNBS administration. Potential GP-BAR1 agonists were identified by in silico screening and computational docking studies.

Results

GP-BAR1^−/− mice develop an abnormal morphology of colonic mucous cells and an altered molecular architecture of epithelial tight junctions with increased expression and abnormal subcellular distribution of zonulin 1 resulting in increased intestinal permeability and susceptibility to develop severe colitis in response to DSS at early stage of life. By in silico screening and docking studies we identified ciprofloxacin as a GP-BAR1 ligand. In monocytes, ciprofloxacin increases cAMP concentrations and attenuates TNFα release induced by TLR4 ligation in a GP-BAR1 dependent manner. Treating mice rendered colitic by TNBS with ciprofloxacin and oleanolic acid, a well characterized GP-BAR1 ligand, abrogates signs and symptoms of colitis. Colonic expression of GP-BAR1 mRNA increases in rodent models of colitis and tissues from Crohn''s disease patients. Flow cytometry analysis demonstrates that ≈90% of CD14+ cells isolated from the lamina propria of TNBS-treated mice stained positively for GP-BAR1.

Conclusions

GP-BAR1 regulates intestinal barrier structure. Its expression increases in rodent models of colitis and Crohn''s disease. Ciprofloxacin is a GP-BAR1 ligand.     

Probiotics modulate intestinal expression of nuclear receptor and provide counter-regulatory signals to inflammation-driven adipose tissue activation

Background

Adipocytes from mesenteric white adipose tissue amplify the inflammatory response and participate in inflammation-driven immune dysfunction in Crohn''s disease by releasing proinflammatory mediators. Peroxisome proliferator-activated receptors (PPAR)-α and -γ, pregnane x receptor (PXR), farnesoid x receptor (FXR) and liver x-receptor (LXR) are ligand-activated nuclear receptor that provide counter-regulatory signals to dysregulated immunity and modulates adipose tissue.

Aims

To investigate the expression and function of nuclear receptors in intestinal and adipose tissues in a rodent model of colitis and mesenteric fat from Crohn''s patients and to investigate their modulation by probiotics.

Methods

Colitis was induced by TNBS administration. Mice were administered vehicle or VSL#3, daily for 10 days. Abdominal fat explants obtained at surgery from five Crohn''s disease patients and five patients with colon cancer were cultured with VSL#3 medium.

Results

Probiotic administration attenuated development of signs and symptoms of colitis, reduced colonic expression of TNFα, IL-6 and IFNγ and reserved colonic downregulation of PPARγ, PXR and FXR caused by TNBS. Mesenteric fat depots isolated from TNBS-treated animals had increased expression of inflammatory mediators along with PPARγ, FXR, leptin and adiponectin. These changes were prevented by VSL#3. Creeping fat and mesenteric adipose tissue from Crohn''s patients showed a differential expression of PPARγ and FXR with both tissue expressing high levels of leptin. Exposure of these tissues to VSL#3 medium abrogates leptin release.

Conclusions

Mesenteric adipose tissue from rodent colitis and Crohn''s disease is metabolically active and shows inflammation-driven regulation of PPARγ, FXR and leptin. Probiotics correct the inflammation-driven metabolic dysfunction.     

Deep inspiration breath hold in post-operative radiotherapy for right breast cancer: a retrospective analysis

BackgroundThe aim of our study is to determine whether deep inspiration breath hold (DIBH) is effective for reducing exposure of the heart, left coronary artery (LAD) and both lungs in right breast radiotherapy.Materials and methodsWe have analyzed 10 consecutive patients with right-sided breast cancer (BC), simulated during free breathing (FB) and in DIBH modality. For all patients we contoured breast PTV and organs at risk (right and left lungs, heart, LAD) on both CT scans (FB and DIBH). Finally, 5 patients were treated with IMRT and 5 with VMAT techniques.ResultsAll patients were able to end the treatments in DIBH modalities regardless of the longer treatment time in comparison to FB. The maximum and mean dose to the heart are lower in the DIBH modality. The mean values of the heart mean dose were 1.76 Gy in DIBH and 2.19 Gy in FB. The mean heart maximum dose in DIBH and FB were, respectively, 9.3 Gy and 11 Gy. Likewise, the maximum dose to the LAD is lower in DIBH; 2.57 Gy versus 3.56 Gy in FB. Noteworthy, 3 patients with hepatomegaly treated with the DIBH technique showed a higher ipsilateral lung dose than FB, but a decrease of liver dose.ConclusionWe report that the use of DIBH for right-sided BC allows the dose to the heart, LAD and to the liver to be reduced in case of hepatomegaly. This technique is well tolerated by patients, when adequately trained, and could be considered effective even in right sided BC.     

Genomic landscape of rat strain and substrain variation

Background

Since the completion of the rat reference genome in 2003, whole-genome sequencing data from more than 40 rat strains have become available. These data represent the broad range of strains that are used in rat research including commonly used substrains. Currently, this wealth of information cannot be used to its full extent, because the variety of different variant calling algorithms employed by different groups impairs comparison between strains. In addition, all rat whole genome sequencing studies to date used an outdated reference genome for analysis (RGSC3.4 released in 2004).

Results

Here we present a comprehensive, multi-sample and uniformly called set of genetic variants in 40 rat strains, including 19 substrains. We reanalyzed all primary data using a recent version of the rat reference assembly (RGSC5.0 released in 2012) and identified over 12 million genomic variants (SNVs, indels and structural variants) among the 40 strains. 28,318 SNVs are specific to individual substrains, which may be explained by introgression from other unsequenced strains and ongoing evolution by genetic drift. Substrain SNVs may have a larger predicted functional impact compared to older shared SNVs.

Conclusions

In summary we present a comprehensive catalog of uniformly analyzed genetic variants among 40 widely used rat inbred strains based on the RGSC5.0 assembly. This represents a valuable resource, which will facilitate rat functional genomic research. In line with previous observations, our genome-wide analyses do not show evidence for contribution of multiple ancestral founder rat subspecies to the currently used rat inbred strains, as is the case for mouse. In addition, we find that the degree of substrain variation is highly variable between strains, which is of importance for the correct interpretation of experimental data from different labs.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1594-1) contains supplementary material, which is available to authorized users.