Pharmacometabonomic investigation of dynamic metabolic phenotypes associated with variability in response to galactosamine hepatotoxicity

Galactosamine (galN) is widely used as an in vivo model of acute liver injury. We have applied an integrative approach, combining histopathology, clinical chemistry, cytokine analysis, and nuclear magnetic resonance (NMR) spectroscopic metabolic profiling of biofluids and tissues, to study variability in response to galactosamine following successive dosing. On re-challenge with galN, primary non-responders displayed galN-induced hepatotoxicity (induced response), whereas primary responders exhibited a less marked response (adaptive response). A systems-level metabonomic approach enabled simultaneous characterization of the xenobiotic and endogenous metabolic perturbations associated with the different response phenotypes. Elevated serum cytokines were identified and correlated with hepatic metabolic profiles to further investigate the inflammatory response to galN. The presence of urinary N-acetylglucosamine (glcNAc) correlated with toxicological outcome and reflected the dynamic shift from a resistant to a sensitive phenotype (induced response). In addition, the urinary level of glcNAc and hepatic level of UDP-N-acetylhexosamines reflected an adaptive response to galN. The unique observation of galN-pyrazines and altered gut microbial metabolites in fecal profiles of non-responders suggested that gut microfloral metabolism was associated with toxic outcome. Pharmacometabonomic modeling of predose urinary and fecal NMR spectroscopic profiles revealed a diverse panel of metabolites that classified the dynamic shift between a resistant and sensitive phenotype. This integrative pharmacometabonomic approach has been demonstrated for a model toxin; however, it is equally applicable to xenobiotic interventions that are associated with wide variation in efficacy or toxicity and, in particular, for prediction of susceptibility to toxicity.     

Agrobacterium-mediated transformation of tomato elicits unexpected flower phenotypes with similar gene expression profiles

Background

Genetic transformation mediated by Agrobacterium tumefaciens is known to cause unexpected phenotypes. Mutations of a specific set of homeotic genes can result in alterred floral structure.

Methodology/Principal Findings

Previously we identified two genes (LeTGA1 and SOLly GLB1) induced by nutrient availability in tomato. To further elucidate their function, we sought to knock out the genes using antisense RNAi. When antisense constructs for the two different tomato genes were each transformed into Micro-Tina tomato plants, one primary transformant with similar mutant flower phenotypes was identified from transformation of each construct. Microarray analysis shows that a similar set of genes were up- or downregulated in both mutants. Sequencing of insertion sites indicates that each is inserted into a repetitive region which could impact expression of affected genes but direct alteration of floral homeotic gene sequences was not detected.

Conclusion

This is the first report that dominant flower mutations could be caused by genetic transformation designed to knock out two nutrient stress related genes.     

Synthetic conversion of a graded receptor signal into a tunable,reversible switch

The ability to engineer an all‐or‐none cellular response to a given signaling ligand is important in applications ranging from biosensing to tissue engineering. However, synthetic gene network ‘switches’ have been limited in their applicability and tunability due to their reliance on specific components to function. Here, we present a strategy for reversible switch design that instead relies only on a robust, easily constructed network topology with two positive feedback loops and we apply the method to create highly ultrasensitive (n_H>20), bistable cellular responses to a synthetic ligand/receptor complex. Independent modulation of the two feedback strengths enables rational tuning and some decoupling of steady‐state (ultrasensitivity, signal amplitude, switching threshold, and bistability) and kinetic (rates of system activation and deactivation) response properties. Our integrated computational and synthetic biology approach elucidates design rules for building cellular switches with desired properties, which may be of utility in engineering signal‐transduction pathways.     

A protein switch with tunable steepness reconstructed in Escherichia coli cells with eukaryotic signaling proteins

Cholera is a disease which shows a clear blood group profile, with blood group O individuals experiencing the most severe symptoms. For a long time, the cholera toxin has been suspected to be the main culprit of this blood group dependence. Here, we show that both El Tor and classical cholera toxin B-pentamers do indeed bind blood group determinants (with equal affinities), using Surface Plasmon Resonance and NMR spectroscopy. Together with previous structural data, this confirms our earlier hypothesis as to the molecular basis of cholera blood group dependence, with an interesting twist: the shorter blood group H-determinant characteristic of blood group O individuals binds with similar binding affinity compared to the A-determinant, however, with different kinetics.     

A PDZ switch for a cellular stress response

The accumulation of misfolded porins in the periplasm of bacteria triggers a proteolytic cascade, initiated by activation of DegS, a member of the family of HtrA proteases. Activation of DegS ultimately leads to the expression of genes encoding the periplasmic protein folding machinery. A new study now reveals that binding of exposed C-termini of unassembled porins to the PDZ domain of DegS induces structural rearrangements that activate the catalytic site of the protease domain.     

DNA damage during reoxygenation elicits a Chk2-dependent checkpoint response

Due to the abnormal vasculature of solid tumors, tumor cell oxygenation can change rapidly with the opening and closing of blood vessels, leading to the activation of both hypoxic response pathways and oxidative stress pathways upon reoxygenation. Here, we report that ataxia telangiectasia mutated-dependent phosphorylation and activation of Chk2 occur in the absence of DNA damage during hypoxia and are maintained during reoxygenation in response to DNA damage. Our studies involving oxidative damage show that Chk2 is required for G2 arrest. Following exposure to both hypoxia and reoxygenation, Chk2-/- cells exhibit an attenuated G2 arrest, increased apoptosis, reduced clonogenic survival, and deficient phosphorylation of downstream targets. These studies indicate that the combination of hypoxia and reoxygenation results in a G2 checkpoint response that is dependent on the tumor suppressor Chk2 and that this checkpoint response is essential for tumor cell adaptation to changes that result from the cycling nature of hypoxia and reoxygenation found in solid tumors.     

Chemoimmunotherapy for melanoma with dacarbazine and 2,4-dinitrochlorobenzene elicits a specific T cell-dependent immune response

An empirically established chemoimmunotherapy for metastatic melanoma combines the systemic administration of the chemotherapeutic agent dacarbazine (DTIC) with the epifocal application of the contact sensitizer 2,4-dinitrochlorobenzene (DNCB) on cutaneous metastases. Although this therapy yields high response rates resulting in prolonged survival, the mechanisms involved remain unknown. Here, we investigated whether treatment of tumor-bearing mice with DTIC and DNCB resulted in a specific immune response against the tumor. Subcutaneous (s.c.) tumors and lung metastases were induced in C57BL/6 mice by injecting syngeneic B16-melanoma cells s.c. or into the lateral tail vein, respectively. Mice were treated with intraperitoneal injections of DTIC followed by epifocal application of DNCB. This therapeutic approach significantly reduced the growth of s.c. tumors as well as lung metastases. Our data showed that the effector mechanisms involved are dependent on T cells. No therapeutic effect was observed in immunodeficient RAG-1(-/-) mice, or when the contact sensitizer DNCB was replaced by skin irritants (croton oil or tributyltin). Splenic lymphocytes obtained from treated mice displayed a three-fold higher specific cytolytic activity against B16 cells than in tumor-bearing controls. Both CD8(+) and CD4(+) T cells were able to lyse B16 cells. No changes were observed in natural killer (NK) cell activity. Likewise, tumor-infiltrating lymphocytes (TIL) of treated mice showed higher cytolytic activity than that of controls. Analysis of cytokine expression in s.c. tumors revealed increased mRNA levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) in treated tumors. Together, these findings demonstrate the ability of DTIC/DNCB treatment to induce an effective T cell-dependent host immune response against a syngeneic tumor.     

Ammonia elicits a different myogenic response in avian and murine myotubes

Increased myostatin expression, resulting in muscle loss, has been associated with hyperammonemia in mammalian models of cirrhosis. However, there is evidence that hyperammonemia in avian embryos results in a reduction of myostatin expression, suggesting a proliferative myogenic environment. The present in vitro study examines species differences in myotube and liver cell response to ammonia using avian and murine-derived cells. Primary myoblasts and liver cells were isolated from embryonic day 15 and 17 chick embryos to be compared with mouse myoblasts (C2C12) and liver (AML12) cells. Cells were exposed to varying concentrations of ammonium acetate (AA; 2.5, 5, or 10 mM) to determine the effects of ammonia on the cells. Relative expression of myostatin mRNA, determined by quantitative real-time PCR, was significantly increased in AA (10 mM) treated C2C12 myotubes compared to both ages of chick embryonic myotube cultures after 48 h (P < 0.02). Western blot analysis of myostatin protein confirmed an increase in myostatin expression in AA-treated C2C12 myotubes compared to the sodium acetate (SA) controls, while myostatin expression was decreased in the chick embryonic myotube cultures when treated with AA. Myotube diameter was significantly decreased in AA-treated C2C12 myotubes compared to controls, while avian myotube diameter increased with AA treatment (P < 0.001). There were no significant differences between avian and murine liver cell viability, assessed using 2′, 7′- bis-(2-carboxyethyl)-5-(and-6-)-carboxyfluorescein, acetoxymethyl ester, when treated with AA. However, after 24 h, AA-treated avian myotubes showed a significant increase in cell viability compared to the C2C12 myotubes (P < 0.05). Overall, it appears that there is a positive myogenic response to hyperammonemia in avian myotubes compared to murine myotubes, which supports a proliferative myogenic environment.     

Long-term nutrient enrichment elicits a weak density response by saltmarsh meiofauna

The effect of chronic nutrient enrichment on benthic meiofauna was examined in a whole-ecosystem experiment conducted in salt marshes in the Plum Island watershed of northeastern Massachusetts, USA. We compared abundances of total meiofauna, nematodes, copepods, ostracods, and annelids including Manayunkia aestuarina, in fertilized (where N and P was increased 15× in incoming tidal water throughout each growing season for 6 years) and reference marsh creeks to test for bottom-up responses. Although some responses to nutrient enrichment were evident, results did not match our expectations of strong increases in abundance. Variation in abundance between nutrient-enriched and reference creeks was detected in all three subhabitats but responses were inconsistent and variable over time, suggesting that natural variability was greater than variation induced by fertilization. Our results showed an overall weak negative correlation between meiofauna abundance and benthic microalgae (BMA) biomass partly because the BMA response to nutrient enrichment was relatively small and perhaps limited by grazing macrofauna and nekton. Our results suggest a better mechanistic understanding of the relationship between BMA and meiofaunal abundance is needed to fully understand how nutrient enrichment affects meiofauna.     

Oscillatory model of crassulacean acid metabolism: structural analysis and stability boundaries with a discrete hysteresis switch

We propose a simple oscillatory model of crassulacean acid metabolism (CAM) describing the CO₂ uptake and nocturnal acidification of CAM plants by a system of coupled non-linear differential equations. Large differences in the content of metabolite pools are treated using a pseudo-steady-state approach. For the first time, simulations of the CAM cycle investigate its dependence on all three major control parameters simultaneously: temperature, photon flux density and external CO₂ concentration. Under stationary conditions in time the model shows either endogenous rhythmicity or two distinct steady states. Stability boundaries are calculated in parameter space.     

Phage display of a CTL epitope elicits a long-term in vivo cytotoxic response

The Ovalbumin(257-264) CTL epitope on the major coat protein of the filamentous bacteriophage in different antigen formulations was displayed and the immune response in C57BL6/J mice studied. The display of single cytotoxic epitope on the surface of the virion is sufficient to induce priming and sustain long-term major histocompatibility complex class I restricted cytotoxic T lymphocytes response in vivo. The filamentous bacteriophage is a versatile carrier able to display simultaneously either single or multiple epitopes and can elicit a cellular response carrying very little peptide (<1.5 microg).     

Refeeding with a standard diet after a 48-h fast elicits an inflammatory response in the mouse liver

Unhealthy eating behaviors increase the risk of metabolic diseases, but the underlying mechanisms are not fully elucidated. Because inflammation contributes to the pathogenesis of metabolic diseases, it is important to understand the effects of unhealthy eating on the inflammatory state. The objective of our present study was to address the effects of a fasting–refeeding regime, a model of irregular eating, on the hepatic inflammatory responses in mouse. The animals were fasted for 48 h and then refed either a standard or low-carbohydrate/high-fat diet. Inflammatory gene expression in the liver was then sequentially measured for the first 17 h after initiation of refeeding. To assess the roles of dietary carbohydrates and toll-like receptor 2 (TLR2) in the refeeding-induced inflammatory changes, gene expression levels in mice refed only carbohydrates (α-corn starch and sucrose) at different doses and in TLR2-deficient mice refed a standard diet were also analyzed. Refeeding with a standard diet increased the liver expression of Tlr2, proinflammatory mediators (Cxcl10, Cxcl1, Cxcl2, Icam-1) and negative regulators of TLR-signaling (A20 and Atf3). These increases were attenuated in mice refed a low-carbohydrate/high-fat diet. Refeeding only α-corn starch and sucrose also increased the expression of these inflammatory pathway genes depending on the doses. TLR2 deficiency significantly attenuated the refeeding-induced increase in the liver expression of Cxcl10, Cxcl1, Icam-1 and A20. These findings suggest that an irregular eating behavior can elicit a liver inflammatory response, which is at least partly mediated by TLR2, and that dietary carbohydrates play critical roles in this process.     

Predicting the asymmetric response of a genetic switch to noise

We present a simple analytical tool which gives an approximate insight into the stationary behavior of nonlinear systems undergoing the influence of a weak and rapid noise from one dominating source, e.g. the kinetic equations describing a genetic switch with the concentration of one substrate fluctuating around a constant mean. The proposed method allows for predicting the asymmetric response of the genetic switch to noise, arising from the noise-induced shift of stationary states. The method has been tested on an example model of the lac operon regulatory network: a reduced Yildirim-Mackey model with fluctuating extracellular lactose concentration. We calculate analytically the shift of the system's stationary states in the presence of noise. The results of the analytical calculation are in excellent agreement with the results of numerical simulation of the noisy system. The simulation results suggest that the structure of the kinetics of the underlying biochemical reactions protects the bistability of the lactose utilization mechanism from environmental fluctuations. We show that, in the consequence of the noise-induced shift of stationary states, the presence of fluctuations stabilizes the behavior of the system in a selective way: Although the extrinsic noise facilitates, to some extent, switching off the lactose metabolism, the same noise prevents it from switching on.     

Molecular simulations of mutually exclusive folding in a two-domain protein switch

A major challenge with testing designs of protein conformational switches is the need for experimental probes that can independently monitor their individual protein domains. One way to circumvent this issue is to use a molecular simulation approach in which each domain can be directly observed. Here we report what we believe to be the first molecular simulations of mutually exclusive folding in an engineered two-domain protein switch, providing a direct view of how folding of one protein drives unfolding of the other in a barnase-ubiquitin fusion protein. These simulations successfully capture the experimental effects of interdomain linker length and ligand binding on the extent of unfolding in the less stable domain. In addition, the effect of linker length on the potential for oligomerization, which eliminates switch activity, is in qualitative agreement with analytical ultracentrifugation experiments. We also perform what we believe to be the first study of protein unfolding via progressive localized compression. Finally, we are able to explore the kinetics of mutually exclusive folding by determining the effect of linker length on rates of unfolding and refolding of each protein domain. Our results demonstrate that molecular simulations can provide seemingly novel biological insights on the behavior of individual protein domains, thereby aiding in the rational design of bifunctional switches.     

Cycloheximide elicits in human fibroblasts a response characteristic for initiation of cell proliferation

Earlier I found that a variety of stimuli to proliferation of cultured human fibroblasts caused an increase in the rate of putrescine transport into the cells. This paper reports the effects of cycloheximide on putrescine transport in stationary and growing cultures. Cycloheximide in concentrations that inhibited protein synthesis caused increased putrescine transport in serumstarved and density-inhibited cultures. Similar effects were found with pactamycin, also an inhibitor of protein synthesis. Actinomycin D in concentrations that suppressed messenger RNA (mRNA) synthesis, did not cause increased putrescine transport. When both serum and cycloheximide were added to serum-starved cultures, the increase in putrescine transport was greater than when serum alone was added. However, cycloheximide had an inhibitory effect when added 1–2 h after addition of serum. These results suggest that one or more rapidly metabolizing proteins may be important in the regulation of putrescine transport and initiation of cell growth.     

Bacillus sphaericus binary toxin elicits host cell autophagy as a response to intoxication

Bacillus sphaericus strains that produce the binary toxin (Bin) are highly toxic to Culex and Anopheles mosquitoes, and have been used since the late 1980s as a biopesticide for the control of these vectors of infectious disease agents. The Bin toxin produced by these strains targets mosquito larval midgut epithelial cells where it binds to Cpm1 (Culex pipiens maltase 1) a digestive enzyme, and causes severe intracellular damage, including a dramatic cytoplasmic vacuolation. The intoxication of mammalian epithelial MDCK cells engineered to express Cpm1 mimics the cytopathologies observed in mosquito enterocytes following Bin ingestion: pore formation and vacuolation. In this study we demonstrate that Bin-induced vacuolisation is a transient phenomenon that affects autolysosomes. In addition, we show that this vacuolisation is associated with induction of autophagy in intoxicated cells. Furthermore, we report that after internalization, Bin reaches the recycling endosomes but is not localized either within the vacuolating autolysosomes or within any other degradative compartment. Our observations reveal that Bin elicits autophagy as the cell's response to intoxication while protecting itself from degradation through trafficking towards the recycling pathways.     

Adenosine 5'-monophosphate challenge elicits a more peripheral airway response than methacholine challenge

Adenosine 5'-monophosphate (AMP) and methacholine are commonly used to assess airway hyperreactivity. However, it is not fully known whether the site of airway constriction primarily involved during challenges with either agent is similar. Using a ventilation distribution test, we investigated whether the constriction induced by each agent involves the lung periphery in a similar fashion. Ventilation distribution was evaluated by the phase III slope (S) of the single-breath washout, using gases with different diffusivities like helium (He) and hexafluorosulfur (SF(6)). A greater postchallenge increase in S(He) reflects alterations at the level of terminal and respiratory bronchioles, while a greater increase in S(SF6) reflects alterations in alveolar ducts, increases to an equal extent reflecting alterations in more proximal airways where gas transport is still convective for both gases. S(SF6) and S(He) were measured in 15 asthma patients before and after airway challenges (20% forced expired volume in 1-s fall) with AMP and methacholine. S(He) increased to a greater extent than S(SF6) after AMP challenge (5.7 vs. 3.7%/l; P = 0.002), with both slopes increasing to an equal extent after methacholine challenge (3.1%/l; P = 0.959). The larger increase in S(He) following AMP challenge suggests distal ventilation impairment up to the level of terminal and respiratory bronchioles. With methacholine, the similar increases in S(He) and S(SF6) suggest a less distal impairment. AMP, therefore, seems to affect more extensively the very peripheral airways, whereas methacholine seems to have an effect on less distal airways.     

Synovial phenotypes in rheumatoid arthritis correlate with response to biologic therapeutics

Introduction

Rheumatoid arthritis (RA) is a complex and clinically heterogeneous autoimmune disease. Currently, the relationship between pathogenic molecular drivers of disease in RA and therapeutic response is poorly understood.

Methods

We analyzed synovial tissue samples from two RA cohorts of 49 and 20 patients using a combination of global gene expression, histologic and cellular analyses, and analysis of gene expression data from two further publicly available RA cohorts. To identify candidate serum biomarkers that correspond to differential synovial biology and clinical response to targeted therapies, we performed pre-treatment biomarker analysis compared with therapeutic outcome at week 24 in serum samples from 198 patients from the ADACTA (ADalimumab ACTemrA) phase 4 trial of tocilizumab (anti-IL-6R) monotherapy versus adalimumab (anti-TNFα) monotherapy.

Results

We documented evidence for four major phenotypes of RA synovium – lymphoid, myeloid, low inflammatory, and fibroid - each with distinct underlying gene expression signatures. We observed that baseline synovial myeloid, but not lymphoid, gene signature expression was higher in patients with good compared with poor European league against rheumatism (EULAR) clinical response to anti-TNFα therapy at week 16 (P =0.011). We observed that high baseline serum soluble intercellular adhesion molecule 1 (sICAM1), associated with the myeloid phenotype, and high serum C-X-C motif chemokine 13 (CXCL13), associated with the lymphoid phenotype, had differential relationships with clinical response to anti-TNFα compared with anti-IL6R treatment. sICAM1-high/CXCL13-low patients showed the highest week 24 American College of Rheumatology (ACR) 50 response rate to anti-TNFα treatment as compared with sICAM1-low/CXCL13-high patients (42% versus 13%, respectively, P =0.05) while anti-IL-6R patients showed the opposite relationship with these biomarker subgroups (ACR50 20% versus 69%, P =0.004).

Conclusions

These data demonstrate that underlying molecular and cellular heterogeneity in RA impacts clinical outcome to therapies targeting different biological pathways, with patients with the myeloid phenotype exhibiting the most robust response to anti-TNFα. These data suggest a path to identify and validate serum biomarkers that predict response to targeted therapies in rheumatoid arthritis and possibly other autoimmune diseases.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01119859","term_id":"NCT01119859"}}NCT01119859