Efficient respiration on TMAO requires TorD and TorE auxiliary proteins in Shewanella oneidensis

Respiration on trimethylamine oxide (TMAO) allows bacterial survival under anoxia. In Shewanella oneidensis, Tor is the system involved in TMAO respiration and it is encoded by the torECAD operon. The torA and torC genes encode TorA terminal reductase and the TorC c-type cytochrome, respectively. Sequence analysis suggests that TorD is the putative specific chaperone of TorA, whereas TorE is of unknown function. The purpose of this study was to understand whether TorD and TorE are two accessory proteins that affect the efficiency of the Tor system by chaperoning TorA terminal reductase. Moreover, by deleting each gene, we established that the absence of TorD drastically affects the stability of TorA, while the absence of TorE does not affect TorA stability or activity. Since TMAO reduction was affected in the ΔtorE mutant, TorE could be an additional component of the TorC-TorA electron transfer chain during bacterial respiration. Finally, a fitness experiment indicated that the presence of TorE, as expected, confers a selective advantage in competitive environments.     

Comparative metabonomics of differential hydrazine toxicity in the rat and mouse

Interspecies variation between rats and mice has been studied for hydrazine toxicity using a novel metabonomics approach. Hydrazine hydrochloride was administered to male Sprague-Dawley rats (30 mg/kg, n = 10 and 90 mg/kg, n = 10) and male B6C3F mice (100 mg/kg, n = 8 and 250 mg/kg, n = 8) by oral gavage. In each species, the high dose was selected to produce the major histopathologic effect, hepatocellular lipid accumulation. Urine samples were collected at sequential time points up to 168 h post dose and analyzed by 1H NMR spectroscopy. The metabolites of hydrazine, namely diacetyl hydrazine and 1,4,5,6-tetrahydro-6-oxo-3-pyridazine carboxylic acid (THOPC), were detected in both the rat and mouse urine samples. Monoacetyl hydrazine was detected only in urine samples from the rat and its absence in the urine of the mouse was attributed to a higher activity of N-acetyl transferases in the mouse compared with the rat. Differential metabolic effects observed between the two species included elevated urinary beta-alanine, 3-D-hydroxybutyrate, citrulline, N-acetylcitrulline, and reduced trimethylamine-N-oxide excretion unique to the rat. Metabolic principal component (PC) trajectories highlighted the greater degree of toxic response in the rat. A data scaling method, scaled to maximum aligned and reduced trajectories (SMART) analysis, was used to remove the differences between the metabolic starting positions of the rat and mouse and varying magnitudes of effect, to facilitate comparison of the response geometries between the rat and mouse. Mice followed "biphasic" open PC trajectories, with incomplete recovery 7 days after dosing, whereas rats followed closed "hairpin" time profiles, indicating functional reversibility. The greater magnitude of metabolic effects observed in the rat was supported by the more pronounced effect on liver pathology in the rat when compared with the mouse.     

1H NMR-based metabonomics study of the urinary biochemical changes in Kansui treated rat

Ethnopharmacological relevance

The dried root of Kansui (Euphorbia kansui L.) is a commonly used and effective traditional Chinese medicine (TCM).

Aim of the study

We combined the urinary metabolites alteration and traditional assays of Kansui-induced rats to discuss the mechanism of toxicity of Kansui.

Materials and methods

The Sprague–Dawley rats were dosed with 7.875 g Kansui/kg weight and 15.75 g Kansui/kg weight. Urine samples were collected at day −1 (before treatment), and days 7, 14 and 21 for NMR analysis. Plasma and liver and kidney tissues were collected at day 14 for biochemical assays and histopathological examination, respectively.

Results

The metabonome of rats treated with Kansui differed markedly from that of the controls. This was confirmed by the histopathology of liver and kidney tissue and clinical biochemistry analysis. The toxicity of Kansui accumulated with dosing time, and persisted even when treatment was stopped. The corresponding biochemical pathways alterations included inhibited TCA cycle, increased anaerobic glycolysis, and perturbed amino acids metabolism.

Conclusion

The biochemical pathways disorder conjunction with histopathology changes provides new clues to evaluate the toxicity of Kansui from a systematic and holistic view.     

The unfolded protein response in retinal vascular diseases: Implications and therapeutic potential beyond protein folding

Angiogenesis is a complex, step-wise process of new vessel formation that is involved in both normal embryonic development as well as postnatal pathological processes, such as cancer, cardiovascular disease, and diabetes. Aberrant blood vessel growth, also known as neovascularization, in the retina and the choroid is a major cause of vision loss in severe eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and central and branch retinal vein occlusion. Yet, retinal neovascularization is causally and dynamically associated with vasodegeneration, ischemia, and vascular remodeling in retinal tissues. Understanding the mechanisms of retinal neovascularization is an urgent unmet need for developing new treatments for these devastating diseases. Accumulating evidence suggests a vital role for the unfolded protein response (UPR) in regulation of angiogenesis, in part through coordinating the secretion of pro-angiogenic growth factors, such as VEGF, and modulating endothelial cell survival and activity. Herein, we summarize current research in the context of endoplasmic reticulum (ER) stress and UPR signaling in retinal angiogenesis and vascular remodeling, highlighting potential implications of targeting these stress response pathways in the prevention and treatment of retinal vascular diseases that result in visual deficits and blindness.     

How osmolytes influence hydrophobic polymer conformations: A unified view from experiment and theory

It is currently the consensus belief that protective osmolytes such as trimethylamine N-oxide (TMAO) favor protein folding by being excluded from the vicinity of a protein, whereas denaturing osmolytes such as urea lead to protein unfolding by strongly binding to the surface. Despite there being consensus on how TMAO and urea affect proteins as a whole, very little is known as to their effects on the individual mechanisms responsible for protein structure formation, especially hydrophobic association. In the present study, we use single-molecule atomic force microscopy and molecular dynamics simulations to investigate the effects of TMAO and urea on the unfolding of the hydrophobic homopolymer polystyrene. Incorporated with interfacial energy measurements, our results show that TMAO and urea act on polystyrene as a protectant and a denaturant, respectively, while complying with Tanford–Wyman preferential binding theory. We provide a molecular explanation suggesting that TMAO molecules have a greater thermodynamic binding affinity with the collapsed conformation of polystyrene than with the extended conformation, while the reverse is true for urea molecules. Results presented here from both experiment and simulation are in line with earlier predictions on a model Lennard–Jones polymer while also demonstrating the distinction in the mechanism of osmolyte action between protein and hydrophobic polymer. This marks, to our knowledge, the first experimental observation of TMAO-induced hydrophobic collapse in a ternary aqueous system.Osmolytes constitute a class of small aqueous solutes used by a variety of organisms to cope with osmotic stress (1). As a side effect, many osmolytes are known to strongly affect protein stability, favoring either the native state (thus referred to as protectant) or the unfolded state ensemble (denaturant) (2–7). The denaturant urea and the protectant trimethylamine N-oxide (commonly abbreviated as TMAO) are among the most effective osmolytes, noted for the diversity of organisms within which they may be found as well as for the diversity of proteins upon which they act (1, 8–11).The universality of osmolyte action gives some indication as to its mechanism, given that the only thing shared equally by all proteins is the makeup of the backbone (12). Studies of the solubility of various amino acids have led to two main conclusions: first, the main contribution to the solvation free energy arises from the backbone, not the side chains; second, this contribution is positive for aqueous solutions of protective osmolytes, while it is negative in denaturant solutions. In other words, the primary cause of osmolyte-induced folding/unfolding is rooted in the tendency for the protein backbone to avoid protectant solutions, while extending into denaturant solutions (8, 13–15). Although it has yet to be assigned a universally accepted driving force, theoretical analysis has led to some agreement that this model (frequently dubbed the solvophobic model) is likely derived from direct protein–osmolyte interactions whereby protectants are excluded from the protein’s vicinity, and denaturants adsorb to the protein (8, 16–19).Of particular interest to protein folding models are studies on the effects of osmolytes on hydrophobic interactions, which have primarily been limited to molecular dynamics (MD) simulations (20–22). The only experimental study on hydrophobic clustering in the presence of osmolytes of which we are aware makes use of partially hydrophobic carboxylic acids with varying alkyl chain lengths (23), and it lends credence to the prediction (20) that TMAO destabilizes hydrophobic contact pair formation. However, there have been no experimental studies involving entirely hydrophobic molecules, nor have there been any experimental studies involving larger collections of hydrophobes. Single-molecule force spectroscopy has recently become an attractive method for investigating these problems (24). By depositing polymers on a surface and flooding the system with a liquid, it is possible to force a small population of hydrophobic polymers into solution. Atomic force microscopy (AFM) can be used to stretch polymers into solution, and inferences can be made as to the balance of forces between thermal motion and the interactions between monomers, the solvent, and the surface (25, 26). Over the past 15 years, work investigating the hydrophobic collapse of amphiphilic custom-made polymers (27), various proteins (28–32), and hydrophobic homopolymers such as poly (methyl methacrylate) (33) and polystyrene (34–36) has proven to be of substantial value in answering questions on the nature of hydrophobic interactions under different conditions.Proteins are, by their nature, heterogenous molecules. As such, isolating individual contributions to a solvent-induced change in stability is a task for which homopolymers may be better suited. To our knowledge, there have been relatively few studies (21, 22, 37–39) of how such osmolytes perturb simple homopolymers. Recently, a systematic computational study of the collapse behavior of simple model Lennard–Jones (LJ) homopolymers with variable polarizability in aqueous solutions of TMAO and urea has provided a unified picture of how these osmolytes act (37). As with proteins, it was found that TMAO acts to stabilize and urea acts to destabilize the globular structure of the model polymers. Surprisingly, and seemingly in contrast to the solvophobic theory outlined above, both osmolytes strongly bind to the polymer surface, which a standard solvophobic model might support as the defining characteristic of denaturants in protein systems. In keeping with the theories of Tanford and Wyman (2, 3), this study highlights that it is actually the difference in relative preferential binding between collapsed and extended state, rather than the absolute magnitude of preferential binding in either state, which needs to be considered in a proper treatment of osmolyte-mediated polymer collapse.Here we use AFM to measure the force required to unfold a single polystyrene molecule in aqueous solutions of TMAO and urea. In agreement with earlier theoretical predictions based on studies of model polymers (37), these experiments show that, relative to water as a solvent, the force needed to unfold polystyrene is systematically higher in aqueous solution of TMAO and lower in aqueous solution of urea. Pendant drop and contact angle measurements are performed to calculate surface tensions, indicating that both TMAO and urea are expected to be in excess at the polystyrene–water interface. These results are complemented by MD simulations on short chain-length polystyrene in aqueous solutions of TMAO and urea, which echo both the AFM and interfacial tension measurements. The results are analyzed using a thermodynamic preferential binding theory and free-energy calculations to provide a unified molecular level interpretation of osmolyte actions on a hydrophobic polymer.     

新型生物标志物TMAO与心血管疾病关系的研究进展

肠道菌群及其代谢产物与心血管疾病的关系是心血管领域研究的热点,有关探索肠道菌群在调节心血管生理和疾病进展中作用的基础与临床研究已取得了较大进展。国内外一些研究表明肠道微生物源代谢物三甲胺-N-氧化物（TMAO）已经成为影响心血管疾病发生发展的一个关键因素。近年来TMAO与心血管疾病的发生发展及预后关系的临床研究也取得了一些成果,血浆TMAO水平未来可作为心血管疾病危险分层、诊断及预后的新型生物标志物,对心血管疾病的发生和主要心血管事件（MACE）进行预测。本文就TMAO作为心血管疾病新型生物标志物及潜在治疗靶点的研究进展进行综述。     

Gut microbiota and stroke: New avenues to improve prevention and outcome

Despite major recent therapeutic advances, stroke remains a leading cause of disability and death. Consequently, new therapeutic targets need to be found to improve stroke outcome. The deleterious role of gut microbiota alteration (often mentioned as “dysbiosis”) on cardiovascular diseases, including stroke and its risk factors, has been increasingly recognized. Gut microbiota metabolites, such as trimethylamine-N-oxide, short chain fatty acids and tryptophan, play a key role. Evidence of a link between alteration of the gut microbiota and cardiovascular risk factors exists, with a possible causality link supported by several preclinical studies. Gut microbiota alteration also seems to be implicated at the acute phase of stroke, with observational studies showing more non-neurological complications, higher infarct size and worse clinical outcome in stroke patients with altered microbiota. Microbiota targeted strategies have been developed, including prebiotics/probiotics, fecal microbiota transplantation, short chain fatty acid and trimethylamine-N-oxide inhibitors. Research teams have been using different time windows and end-points for their studies, with various results. Considering the available evidence, it is believed that studies focusing on microbiota-targeted strategies in association with conventional stroke care should be conducted. Such strategies should be considered according to three therapeutic time windows: first, at the pre-stroke (primary prevention) or post-stroke (secondary prevention) phases, to enhance the control of cardiovascular risk factors; secondly, at the acute phase of stroke, to limit the infarct size and the systemic complications and enhance the overall clinical outcome; thirdly, at the subacute phase of stroke, to prevent stroke recurrence and promote neurological recovery.