Level Of Metabolic Activity Research Articles

Terminal differentiation induction as DNA damage response in hematopoietic stem cells by GADD45A.

Hematopoietic stem cells (HSCs) sustain lifelong blood cell regeneration by balancing their ability for self-renewal with their ability to differentiate into all blood cell types. To prevent organ exhaustion and malignant transformation, long-lived HSCs, in particular, must be protected from exogenous and endogenous stress, which cause severe DNA damage. When DNA is damaged, distinct DNA repair mechanisms and cell fate controls occur in adult HSCs compared with committed cells. Growth arrest and DNA damage-inducible 45 alpha (GADD45A) is known to coordinate a variety of cellular stress responses, indicating the molecule is an important stress mediator. So far, the function of GADD45A in hematopoietic stem and progenitor cells is controversial and appears highly dependent on the cell type and stress stimulus. Recent studies have analyzed its role in cell fate decision control of prospectively isolated HSCs and have revealed unexpected functions of GADD45A, as discussed here. The upregulation of GADD45A by DNA damage-causing conditions results in enhanced HSC differentiation, probably to efficiently eliminate aberrant HSCs from the system. These findings, in concert with a few studies on other stem cell systems, have led us to propose DNA damage-induced differentiation as a novel DNA damage response mechanism in stem cells that circumvents the fatal consequences of cumulative DNA damage in the stem cell compartment.

A Cytoplasmic Form of Gaussia luciferase Provides a Highly Sensitive Test for Cytotoxicity.

The elimination of unfavorable chemicals from our environment and commercial products requires a sensitive and high-throughput in vitro assay system for drug-induced hepatotoxicity. Some previous methods for evaluating hepatotoxicity measure the amounts of cytoplasmic enzymes secreted from damaged cells into the peripheral blood or culture medium. However, most of these enzymes are proteolytically digested in the extracellular milieu, dramatically reducing the sensitivity and reliability of such assays. Other methods measure the decrease in cell viability following exposure to a compound, but such endpoint assays are often confounded by proliferation of surviving cells that replace dead or damaged cells. In this study, with the goal of preventing false-negative diagnoses, we developed a sensitive luminometric cytotoxicity test using a stable form of luciferase. Specifically, we converted Gaussia luciferase (G-Luc) from an actively secreted form to a cytoplasmic form by adding an ER-retention signal composed of the four amino acids KDEL. The bioluminescent signal was >30-fold higher in transgenic HepG2 human hepatoblastoma cells expressing G-Luc+KDEL than in cells expressing wild-type G-Luc. Moreover, G-Luc+KDEL secreted from damaged cells was stable in culture medium after 24 hr at 37°C. We evaluated the accuracy of our cytotoxicity test by subjecting identical samples obtained from chemically treated transgenic HepG2 cells to the G-Luc+KDEL assay and luminometric analyses based on secretion of endogenous adenylate kinase or cellular ATP level. Time-dependent accumulation of G-Luc+KDEL in the medium increased the sensitivity of our assay above those of existing tests. Our findings demonstrate that strong and stable luminescence of G-Luc+KDEL in human hepatocyte-like cells, which have high levels of metabolic activity, make it suitable for use in a high-throughput screening system for monitoring time-dependent cytotoxicity in a limited number of cells.

Interrelation of Electrophysiological and Perfusion Changes in Patients With Left Ventricular Aneurysm

The study was aimed at identification of relations between perfusion and electrophysiological changes in left ventricular (LV) myocardium in patients with ischemic heart disease (IHD) with postinfarction LV aneurysm and ventricular tachycardia. The study enrolled 23 patients with the aforementioned disease. Preoperatively, apart from standard clinical examination of cardiosurgical patients, intracardiac electrophysiological study and perfusion single-photon emission computed tomography of myocardium with 99mTc-Technetril were performed. The patients were subjected to coronary artery bypass grafting and left ventricular reconstruction. Assessment of the outcomes showed that electrophysiological condition of left ventricle was dependent on myocardial perfusion. Electrophysologically normal myocardium with electric potential >1.5 mV, transient zone (0.5-1.5 mV) and zones with the potential <0.5 mV differed significantly by the percentage of perfusion: 61, 45, and 35%, respectively. Zones of delayed conduction and those of double potential were located mostly in transient zone of electrical potential conduction with the current amplitude of 0.5-1.5 mV and myocardial perfusion from 35 to 61%. Double potential zone was formed in the area of myocardium with better perfusion (perfusion defect of 55% with preserved metabolism) as opposed to the zone of delayed conduction, where perfusion defect was 40% with low level of metabolic activity. The obtained data has proven the presence of correlation between electrophysiology and perfusion of myocardium. This provides an opportunity to identify electrically unstable myocardial zones with the help of specific computer tomography of myocardium.

Plasma Rich in Growth Factors Enhances Wound Healing and Protects from Photo-oxidative Stress in Dermal Fibroblasts and 3D Skin Models.

Optimal skin repair has been a desired goal for many researchers. Recently, plasma rich in growth factors (PRGF) has gained importance in dermatology proving it is beneficial effects in wound healing and cutaneous regeneration. The anti-fibrotic, pro-contractile and photo-protective effect of PRGF on dermal fibroblasts and 3D skin models has been evaluated. The effect against TGFβ1 induced myofibroblast differentiation was tested. Cell contractile activity over collagen gel matrices was analyzed and the effect against UV derived photo-oxidative stress was assessed. The effectiveness of PRGF obtained from young aged and middle aged donors was compared. Furthermore, 3D organotypic skin explants were used as human skin models with the aim of analyzing ex vivo cutaneous preventive and regenerative photo-protection after UV exposure. TGFβ1 induced myofibroblast levels decreased significantly after treatment with PRGF while the contractile activity increased compared to the control group. After UV irradiation, cell survival was promoted while apoptotic and ROS levels were noticeably reduced. Photo-exposed 3D explants showed higher levels of metabolic activity and lower levels of necrosis, cell damage, irritation and ROS formation when treated with PRGF. The histological integrity and connective tissue fibers showed lower signals of photodamage among PRGF injected skin models. No significant differences for the assessed biological outcomes were observed when PRGF obtained from young aged and middle aged donors were compared. These findings suggest that this autologous approach might be useful for antifibrotic wound healing and provide an effective protection against sun derived photo-oxidative stress regardless the age of the patient.

Clinical Decision Support to Implement CYP2D6 Drug-Gene Interaction.

The level of CYP2D6 metabolic activity can be predicted by pharmacogenomic testing, and concomitant use of clinical decision support has the potential to prevent adverse effects from those drugs metabolized by this enzyme. Our initial findings after implementation of clinical decision support alerts integrated in the electronic health records suggest high feasibility, but also identify important challenges.

SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa

Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteome-labeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally up-regulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A).

Metabolic activity of breast cancer metastatic lesions on positron emission tomography/computed tomography: comparison with histological and biological characteristics of primary tumor.

Higher intensity of FDG uptake on PET/CT in primary tumor is seen in patients with IDC compared to ILC, also in high grade tumours, tumours with negative ER and higher Ki67 values, while data are inconsistent in case of relation between primary tumor's PgR and HER2 expression with its metabolic activity levels. On account of the lack of studies that include research of breast cancer metastatic lesion metabolism level and its relation to tumor histology and biology, our goal was to investigate the association of metastatic lesions' glucose metabolism level on PET/CT with different histological and biological characteristics of primary tumor. In atotal number of N=100 patients, highest SUVmax values for each patient were used in testing difference between metastatic metabolic activity in patients with different tumor histology, grade, ER, PgR and HER2 status, subtype, as well in testing relation of Ki67 index to metastasis' metabolism level. In testing difference between histological types of breast cancer, SUVmax values were also compared separately for each specific anatomical site (regional and distant lymph nodes, bones and liver). No difference was found regarding metastatic SUVmax values in patients with primary IDC (n=55, median SUVmax 9.70) and ILC (n=34, median SUVmax 7.20) independently of anatomic site, and for each of analysed sites separately. No difference was found as well between SUVmax detected in metastasis in patients with different grade (grade II: n=58, median SUVmax 7.70; grade III: n=12, median SUVmax 10.20), ER (59 positive, median SUVmax 8.50; 22 negative, median SUVmax 8.05), PgR (55 positive, median SUVmax 8.50; 23 negative, median SUVmax 7.80), and HER2 (14 positive, median SUVmax 6.84; 51 negative, median SUVmax 8.63) expression in primary tumor, and between patients with different tumor subtype. Ki67 was also not associated with tumor metastatic SUVmax values (n=11, rs = -0.21, p=0.53). We conclude that there is no association of primary breast cancer histological type, grade, ER, PgR, HER2 and Ki67 expression with metabolic activity in metastasis detected on PET/CT.

ROS-Inducing Agents for Cancer Chemotherapy

Reactive oxygen species (ROS) play an essential role in maintaining cellular homeostasis, and levels of ROS are regulated by redox enzymes and reduced factors such as glutathione. Excess levels of ROS can result in DNA and cellular damage which can contribute to development of tumors. Cancer cells exhibit increased metabolic activity and ROS levels compared to normal cells and, with threshold limits, ROS contribute to cancer cell homeostasis and growth. However, treatment of cancer cells with ROS-inducing anticancer agents exceeds the threshold for ROS and this results in activation of multiple cell death pathways which include inhibition of mammalian target of rapamycin (mTOR) signaling and downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, Sp4 and pro-oncogenic Sp-regulated genes. Thus, ROS-inducing drugs represent a highly effective group of mechanism-based agents for individual and combined cancer chemotherapies.

Replication stress in MLL-rearrangements.

Hematopoietic stem cells (HSC) are the only cells capable of self-renewal throughout the individual's lifetime and generate the whole spectrum of blood cells. Therefore genome aberrations in HSC can result in hematopoiesis failure or leukemic transformation. Chromosomal translocations, inversions, amplifications and complex rearrangements at the 11q human genomic locus encoding mixed lineage leukemia gene (MLL) are the hallmark of several blood malignancies including infant, therapy-induced, donor - and de novo leukemias. The vast majority of these 11q aberrations fall within a 7.3kb MLL breakpoint cluster region (MLLbcr) with a particular hotspot at the intron11-exon12 boundary [1]. Intriguingly, a large variety of genotoxic, cytotoxic and biological stimuli were connected with MLLbcr breakage pointing to the existence of several DNA cleavage and repair mechanisms acting at this locus [1, 2]. From the broad spectrum of stimuli triggering cleavage in concert with diverse mutagenic outcomes at the locus it is tempting to seek for a common molecular process engaged. Based on our and others’ experimental evidences, we postulate that replication stress in HSC can be responsible for MLL rearrangements (Figure ​(Figure1).1). Thus, our data revealed MLLbcr breakage upon mere replication blockage via DNA polymerase inhibition or upon exposure to the nucleoside analog 5-fluorouracil [2]. Induction of HSC's specific replication stress can be linked to many agents and conditions implicated in MLL leukemias. Normally, quiescence of HSC with only rare replication cycles accompanied by low metabolic activity and ROS levels contributes to minimize the mutational load under homeostatic conditions [3, 4]. In contrast, forcing HSC into excessive cycling by chronic stimulation with physiological triggers mimicking inflammation, bleeding or cytopenia provokes a robust DDR that drives both HSC death and mutagenesis of the survivors. Thus, Walter et al. [4] detected DDR markers associated with replication fork stalling and collapse such as DNA breaks and nuclear γ H2AX, 53BP1 and FANCD2 foci upon enforced HSC exit from quiescence. Transplantation induces rapid cycling of normally dormant HSC that can be exacerbated by donor immunosuppression, damaged microenvironment and altered cytokine profile. Signs of endogenous DNA damage upon serial transplantation of HSC are well documented in both humans and mice with evidence for altered DNA replication dynamics, chromosome gaps and breaks indicative of replication stress [3, 5]. We suggest that exhaustion or failure of replication stress-associated high fidelity repair pathways under transplantation challenge can be implicated in donor cell-derived acute leukemia with MLL translocations in patients who received HSC transplant [6]. Given the fact that replication stress in HSC is associated with aging [3] one can hypothesize that MLL rearrangements, particularly amplifications often associated with complex rearrangements [7], observed in de novo AML in the elderly are the consequence of replication stress associated DNA repair failures. Figure 1 MLL rearrangements can result from failure to correctly resolve replication stress in HSC Induction of replication stress in HSC can also be linked to agents and conditions associated with common solid cancer therapies. Indeed, topoisomerase II inhibitors and cytostatics with a different mode-of-action such as alkylating agents or 5-fluorouracil [1, 8] but all implicated in the etiology of therapy-induced leukemia or MLL rearrangements can recruit dormant HSC into the replication cycle as a result of chemotherapy-associated cytopenia. Moreover, fetal HSC which are the infant leukemia cell-of-origin are highly cycling populations and thus collision of replication forks with lesions can cause overwhelming replicative stress. Altogether, replication stress may represent the integrating signal in HSC following genotoxic exposure of the fetus and of patients undergoing radio- or chemotherapy, in HSC undergoing excessive self-renewal in the fetus or upon transplantation as well as in HSC from aged individuals suffering from the exhaustion of replication factors [3]. The extraordinary susceptibility of the MLLbcr to replication stress-induced breakage may stem from its secondary structure resulting in the collision of transcription and replication machineries recruiting nucleases such as Endonuclease G in decondensed chromatin [1](Figure ​](Figure11). To summarize, replication stress response plays a key role in regulating HSC function. We anticipate that deeper understanding of associated molecular mechanisms responsible for MLLbcr cleavage and subsequent repair in HSC can hold the key for future chemoprevention and anti-aging modalities.

Evidence of the Importance of Nox4 in Production of Hypertension in Dahl Salt-Sensitive Rats.

This study reports the consequences of knocking out NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 4 (Nox4) on the development of hypertension and kidney injury in the Dahl salt-sensitive (SS) rat. Zinc finger nuclease injection of single-cell SS embryos was used to create an 8 base-pair frame-shift deletion of Nox4, resulting in a loss of the ≈68 kDa band in Western blot analysis of renal cortical tissue of the knock out of Nox4 in the SS rat (SS(Nox4-/-)) rats. SS(Nox4-/-) rats exhibited a significant reduction of salt-induced hypertension compared with SS rats after 21 days of 4.0% NaCl diet (134±5 versus 151±3 mm Hg in SS) and a significant reduction of albuminuria, tubular casts, and glomerular injury. Optical fluorescence 3-dimensional cryoimaging revealed significantly higher redox ratios (NADH/FAD [reduced nicotinamide adenine dinucleotide/flavin adenine dinucleotide]) in the kidneys of SS(Nox4-/-) rats even when fed the 0.4% NaCl diet, indicating greater levels of mitochondrial electron transport chain metabolic activity and reduced oxidative stress compared with SS rats. Before the development of hypertension, RNA expression levels of Nox subunits Nox2, p67(phox), and p22(phox) were found to be significantly lower (P<0.05) in SS(Nox4-/-) compared with SS rats in the renal cortex. Thus, the mutation of Nox4 seems to modify transcription of several genes in ways that contribute to the protective effects observed in the SS(Nox4-/-) rats. We conclude that the reduced renal injury and attenuated blood pressure response to high salt in the SS(Nox4-/-) rat could be the result of multiple pathways, including gene transcription, mitochondrial energetics, oxidative stress, and protein matrix production impacted by the knock out of Nox4.

English

The strawberry is highly perishable, has high metabolic activity and considerable levels of favorable substrates for proliferation of pathogenic organisms, such as humidity, organic acids and sugars. These organisms lead to further, therefore reducing post-harvest shelf-life. With this in mind, the research objective was to evaluate the post-harvest shelf life in cooled storage of two cultivars of strawberries (Oso Grande and Festival) in the hydroponic cultivation system in coconut fiber substrate, and then compare them with the quality obtained in conventional farming. The strawberries were stored under refrigeration (2 &plusmn; 2&deg;C and 85% &plusmn; 10%) using modified atmosphere, with 9 &micro; PVC film for 14 days. The experiment was conducted in a completely randomized design, distributed in a sub-subdivided plot with the cropping systems in the plot, cultivars in the subplot and in the sub-subplot with storage times of 0, 3, 6, 10 and 14 days with three replicates. The use of cooling associated with modified atmosphere increased the shelf-life of the strawberries of both systems, indicating the potential of this combination to maintain the post-harvest quality of the fruit. Although the shelf life of cultivars of both systems increased, the fruits produced in the conventional system showed better physical-chemical characteristics. &nbsp; Key word: Fragaria x ananassa, cold storage, conservation, modified atmosphere.

Evidence for polymorphism in the cytochrome P450 2D50 gene in horses.

Metabolism is an essential factor in the clearance of many drugs and as such plays a major role in the establishment of dosage regimens and withdrawal times. CYP2D6, the human orthologue to equine CYP2D50, is a drug-metabolizing enzyme that is highly polymorphic in humans leading to widely differing levels of metabolic activity. As CYP2D6 is highly polymorphic, in this study it was hypothesized that the gene coding for the equine orthologue, CYP2D50, may also be prone to polymorphism. Blood samples were collected from 150 horses, the CYP2D50 gene was cloned and sequenced; and full-length sequences were analyzed for single nucleotide polymorphisms (SNPs), deletions, or insertions. Pharmacokinetic data were collected from a subset of horses following the administration of a single oral dose of tramadol and probit analysis used to calculate metabolic ratios. Prior to drug administration, the ability of recombinant CYP2D50 to metabolize tramadol to O-desmethyltramadol was confirmed. Sequencing of CYP2D50 identified 126 exonic SNPs, with 31 of those appearing in multiple horses. Oral administration of tramadol to a subset of these horses revealed variable metabolic ratios (tramadol: O-desmethyltramadol) in individual horses and separation into three metabolic groups. While a limited number of horses of primarily a single breed were studied, the variability in tramadol metabolism to O-desmethyltramadol between horses and preliminary evidence of what appears to be poor, extensive, and ultra-rapid metabolizers supports further study of the potential for genetic polymorphisms in the CYP2D50 gene in horses.

Real-time toxicity and metabolic activity tracking of human cells exposed to Escherichia coli O157:H7 in a mixed consortia.

Escherichia coli O157:H7 is a significant human pathogen that is continually responsible for sickness, and even death, on a worldwide scale. While the pathology of E. coli O157:H7 infection has been well studied, the effect of it's multiple resulting cytotoxic mechanisms on host metabolic activity has not been well characterized. To develop a more thorough understanding of these effects, several bioluminescence assays were evaluated for their ability to track both toxicity and host metabolic activity levels in real-time. The use of continuously autobioluminescent human cells was determined to be the most favorable method for tracking these metrics, as its self-sufficient autobioluminescent phenotype was unaffected by the presence of the infecting bacteria and its signal could be measured without cellular destruction. Using this approach, it was determined that infection with as few as 10 CFU of E. coli O157:H7 could elicit cytotoxic effects. Regardless of the initial infective dose, an impact on metabolic expression was not observed until bacterial populations reached levels between 5 × 10(5) and 1 × 10(6) (R(2) = 0.933), indicating that a critical bacterial infection level must be reached prior to the onset of cytotoxic effects. Supporting this hypothesis, it was found that cells displaying infection-mediated metabolic activity reductions could recover to wild type metabolic activity levels if the infecting bacteria were removed prior to cell death. These results indicate that rapid treatment of E. coli O157:H7 infection could serve to limit host metabolic impact and reduce overall host cell death.

Poly(3,4-ethylenedioxythiophene) nanoparticle and poly(ɛ-caprolactone) electrospun scaffold characterization for skeletal muscle regeneration.

Injuries to peripheral nerves and/or skeletal muscle can cause scar tissue formation and loss of function. The focus of this article is the creation of a conductive, biocompatible scaffold with appropriate mechanical properties to regenerate skeletal muscle. Poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles (Np) were electrospun with poly(ɛ-caprolactone) (PCL) to form conductive scaffolds. During electrospinning, ribboning, larger fiber diameters, and unaligned scaffolds were observed with increasing PEDOT amounts. To address this, PEDOT Np were sonicated prior to electrospinning, which resulted in decreased conductivity and increased mechanical properties. Multi-walled carbon nanotubes (MWCNT) were added to the 1:2 solution in an effort to increase conductivity. However, the addition of MWCNT had little effect on scaffold conductivity, and the elastic modulus and yield stress of the scaffold increased as a result. Rat muscle cells attached and were active on the 1-10, 1-2, 3-4, and 1-1 PCL-PEDOT scaffolds; however, the 3-4 scaffolds had the lowest level of metabolic activity. Although the scaffolds were cytocompatible, further development of the fabrication method is necessary to produce more highly aligned scaffolds capable of promoting skeletal muscle cell alignment and eventual regeneration.

Characterization of multiple SPS knockout mutants reveals redundant functions of the four Arabidopsis sucrose phosphate synthase isoforms in plant viability, and strongly indicates that enhanced respiration and accelerated starch turnover can alleviate the blockage of sucrose biosynthesis

We characterized multiple knock-out mutants of the four Arabidopsis sucrose phosphate synthase (SPSA1, SPSA2, SPSB and SPSC) isoforms. Despite their reduced SPS activity, spsa1/spsa2, spsa1/spsb, spsa2/spsb, spsa2/spsc, spsb/spsc, spsa1/spsa2/spsb and spsa2/spsb/spsc mutants displayed wild type (WT) vegetative and reproductive morphology, and showed WT photosynthetic capacity and respiration. In contrast, growth of rosettes, flowers and siliques of the spsa1/spsc and spsa1/spsa2/spsc mutants was reduced compared with WT plants. Furthermore, these plants displayed a high dark respiration phenotype. spsa1/spsb/spsc and spsa1/spsa2/spsb/spsc seeds poorly germinated and produced aberrant and sterile plants. Leaves of all viable sps mutants, except spsa1/spsc and spsa1/spsa2/spsc, accumulated WT levels of nonstructural carbohydrates. spsa1/spsc leaves possessed high levels of metabolic intermediates and activities of enzymes of the glycolytic and tricarboxylic acid cycle pathways, and accumulated high levels of metabolic intermediates of the nocturnal starch-to-sucrose conversion process, even under continuous light conditions. Results presented in this work show that SPS is essential for plant viability, reveal redundant functions of the four SPS isoforms in processes that are important for plant growth and nonstructural carbohydrate metabolism, and strongly indicate that accelerated starch turnover and enhanced respiration can alleviate the blockage of sucrose biosynthesis in spsa1/spsc leaves.

Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO₂-based 3-hydroxypropionate production.

Metabolically engineered strains of the hyperthermophile Pyrococcus furiosus (T(opt) 95-100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (T(opt) 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas-liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. The results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.

Effects of p67phox on the mitochondrial oxidative state in the kidney of Dahl salt-sensitive rats: optical fluorescence 3-D cryoimaging.

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67(phox) subunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67(phox) was rendered functionally inactive by zinc finger nuclease mutation of the gene (SS(p67phox)-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly (P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SS(p67phox)-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios (P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SS(p67phox)-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SS(p67phox) rats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2 in the medulla of SS rats. We conclude that the p67(phox) subunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat.

Blood-based identification of non-responders to anti-TNF therapy in rheumatoid arthritis.

BackgroundFaced with an increasing number of choices for biologic therapies, rheumatologists have a critical need for better tools to inform rheumatoid arthritis (RA) disease management. The ability to identify patients who are unlikely to respond to first-line biologic anti-TNF therapies prior to their treatment would allow these patients to seek alternative therapies, providing faster relief and avoiding complications of disease.MethodsWe identified a gene expression classifier to predict, pre-treatment, which RA patients are unlikely to respond to the anti-TNF infliximab. The classifier was trained and independently evaluated using four published whole blood gene expression data sets, in which RA patients (n = 116 = 44 + 15 + 30 + 27) were treated with infliximab, and their response assessed 14–16 months post treatment according to the European League Against Rheumatism (EULAR) response criteria. For each patient, prior knowledge was used to group gene expression measurements into disease-relevant biological signaling mechanisms that were used as the input features for regularized logistic regression.ResultsThe classifier produced a substantial enrichment of non-responders (59 %, given by the cross validated test precision) compared to the full population (27 % non-responders), while identifying nearly a third of non-responders. Given this classifier performance, treatment of predicted non-responders with alternative biologics would decrease their chance of non-response by between a third and a half, substantially improving their odds of effective treatment and stemming further disease progression. The classifier consisted of 18 signaling mechanisms, which together indicated that higher inflammatory signaling mediated by TNF and other cytokines was present pre-treatment in the blood of patients who responded to infliximab treatment. In contrast, non-responders were classified by relatively higher levels of specific metabolic activities in the blood prior to treatment.ConclusionsWe were able to successfully produce a classifier to identify a population of RA patients significantly enriched in anti-TNF non-responders across four different patient cohorts. Additional prospective studies are needed to validate and refine the classifier for clinical use.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0100-6) contains supplementary material, which is available to authorized users.

Wheat enolase demonstrates potential as a non-toxic cryopreservation agent for liver and pancreatic cells.

Cryopreservation is essential for long-term storage of cells and tissues, which can be used for clinical applications such as drug toxicity testing, human transplantation, reproductive, regenerative and transfusion medicine. It requires use of cryoprotectants (e.g. dimethyl disulfoxide (DMSO), glycerol) that protect cells and tissues from dehydration and damage caused by formation of intracellular ice during freezing. As an alternative to these cytotoxic cryoprotectants, we are developing new technology using natural substances produced by plants that survive freezing conditions. We previously showed that soluble protein extracts such as wheat protein extract (WPE) prepared from winter wheat plants can substitute for DMSO as a cryoprotectant for certain mammalian cell types. To identify novel cryoactive proteins, WPE was separated using different chromatographic procedures and cryoactive fractions were analyzed by mass spectrometry. The analysis revealed enolase as a potential wheat protein candidate. A recombinant enolase protein was prepared and was able to successfully cryopreserve rat hepatocytes and insulin-secreting INS832/13 pancreatic cells. Post-thaw cells had high viability and levels of metabolic activities. Cryopreserved cells were plateable and had good adherence and morphological properties. These results indicate that individual plant proteins such as enolase have promising potential as new, non-toxic technology for cryopreservation protocols used for clinical applications.

How biological clocks and changing environmental conditions determine local population growth and species distribution in Antarctic krill (Euphausia superba): a conceptual model

The Southern Ocean ecosystem is characterized by extreme seasonal changes in environmental factors such as day length, sea ice extent and food availability. The key species Antarctic krill (Euphausia superba) has evolved metabolic and behavioural seasonal rhythms to cope with these seasonal changes. We investigate the switch between a physiological less active and active period for adult krill, a rhythm which seems to be controlled by internal biological clocks. These biological clocks can be synchronized by environmental triggers such as day length and food availability. They have evolved for particular environmental regimes to synchronize predictable seasonal environmental changes with important life cycle functions of the species. In a changing environment the time when krill is metabolically active and the time of peak food availability may not overlap if krill's seasonal activity is solely determined by photoperiod (day length). This is especially true for the Atlantic sector of the Southern Ocean where the spatio-temporal ice cover dynamics are changing substantially with rising average temperatures. We developed an individual-based model for krill to explore the impact of photoperiod and food availability on the growth and demographics of krill. We simulated dynamics of local krill populations (with no movement of krill assumed) along a south-north gradient for different triggers of metabolic activity and different levels of food availability below the ice. We also observed the fate of larval krill which cannot switch to low metabolism and therefore are likely to overwinter under ice. Krill could only occupy the southern end of the gradient, where algae bloom only lasts for a short time, when alternative food supply under the ice was high and metabolic activity was triggered by photoperiod. The northern distribution was limited by lack of overwintering habitat for krill larvae due to short duration of sea ice cover even for high food content under the ice. The variability of the krill's length-frequency distributions varied for different triggers of metabolic activity, but did not depend on the sea ice extent. Our findings suggest a southward shift of krill populations due to reduction in the spatial sea ice extent, which is consistent with field observations. Overall, our results highlight the importance of the explicit consideration of spatio-temporal sea ice dynamics especially for larval krill together with temporal synchronization through internal clocks, triggered by environmental factors (photoperiod and food) in adult krill for the population modelling of krill.