Gene family amplification facilitates adaptation in freshwater unionid bivalve Megalonaias nervosa.

Freshwater unionid bivalves currently face severe anthropogenic challenges. Over 70% of species in the United States are threatened, endangered or extinct due to pollution, damming of waterways and overfishing. These species are notable for their unusual life history strategy, parasite-host co-evolution and biparental mitochondrial inheritance. Among this clade, the washboard mussel Megalonaias nervosa is one species that remains prevalent across the Southeastern United States, with robust population sizes. We have created a reference genome for M. nervosa to determine how genome content has evolved in the face of these widespread environmental challenges. We observe dynamic changes in genome content, with a burst of recent transposable element proliferation causing a 382 Mb expansion in genome content. Birth-death models suggest rapid expansions among gene families, with a mutation rate of 1.16 × 10-8 duplications per gene per generation. Cytochrome P450 gene families have experienced exceptional recent amplification beyond expectations based on genome-wide birth-death processes. These genes are associated with increased rates of amino acid changes, a signature of selection driving evolution of detox genes. Fitting evolutionary models of adaptation from standing genetic variation, we can compare adaptive potential across species and mutation types. The large population size in M. nervosa suggests a 4.7-fold advantage in the ability to adapt from standing genetic variation compared with a low diversity endemic E. hopetonensis. Estimates suggest that gene family evolution may offer an exceptional substrate of genetic variation in M. nervosa, with Psgv  = 0.185 compared with Psgv  = 0.067 for single nucleotide changes. Hence, we suggest that gene family evolution is a source of 'hopeful monsters' within the genome that may facilitate adaptation when selective pressures shift. These results suggest that gene family expansion is a key driver of adaptive evolution in this key species of freshwater Unionidae that is currently facing widespread environmental challenges. This work has clear implications for conservation genomics on freshwater bivalves as well as evolutionary theory. This genome represents a first step to facilitate reverse ecological genomics in Unionidae and identify the genetic underpinnings of phenotypic diversity.

Molecular Pharming for low and middle income countries.

Interest in applications and benefits that Molecular Pharming might offer to Low and Middle Income Countries has always been a potent driver for the research discipline, and a major reason why many scientists entered the field. Although enthusiasm remains high, the reality is that such a game-changing innovation would always take longer than traditional uptake of new technology in developed countries, and be complicated by external factors beyond technical feasibility. Excitingly, signs of increasing interest by LMICS in Molecular Pharming are now emerging. Here, three case studies from Thailand, South Africa and Brazil are used to identify some of the key issues when a new investment into Molecular Pharming manufacturing capacity is under consideration. At present, academic research is not necessarily addressing these issues. Only by understanding the concerns, can members of the academic community contribute to helping the development of Molecular Pharming for LMICs by focusing their research efforts appropriately.

Mice Deficient in Epithelial or Myeloid Cell Iκκß Have Distinct Colonic Microbiomes and Increased Resistance to Citrobacter rodentium Infection.

The colonic microenvironment, stemming from microbial, immunologic, stromal, and epithelial factors, serves as an important determinant of the host response to enteric pathogenic colonization. Infection with the enteric bacterial pathogen Citrobacter rodentium elicits a strong mucosal Th1-mediated colitis and monocyte-driven inflammation activated via the classical NF-κB pathway. Research has focused on leukocyte-mediated signaling as the main driver for C. rodentium-induced colitis, however we hypothesize that epithelial cell NF-κB also contributes to the exacerbation of infectious colitis. To test this hypothesis, compartmentalized classical NF-κB defective mice, via the deletion of IKKß in either intestinal epithelial cells (IKKßΔIEC) or myeloid-derived cells (IKKßΔMY), and wild type (WT) mice were challenged with C. rodentium. Both pathogen colonization and colonic histopathology were significantly reduced in IKKß-deficient mice compared to WT mice. Interestingly, colonic IL-10, RegIIIγ, TNF-α, and iNOS gene expression were increased in IKKß-deficient mice in the absence of bacterial challenge. This was associated with increased p52, which is involved with activation of NF-κß through the alternative pathway. IKKß-deficient mice also had distinct differences in colonic tissue-associated and luminal microbiome that may confer protection against C. rodentium. Taken together, these data demonstrate that classical NF-κB signaling can lead to enhanced enteric pathogen colonization and resulting colonic histopathology.

Social Stress Affects Colonic Inflammation, the Gut Microbiome, and Short-chain Fatty Acid Levels and Receptors.

OBJECTIVES: Gastrointestinal disorders, such as inflammatory bowel diseases (IBDs) and functional gastrointestinal disorders (FGIDs), involve disrupted homeostatic interactions between the microbiota and the host. Both disorders are worsened during stress, and in laboratory mice, stress exposure has been shown to change the composition of the gut microbiome. Stress-induced changes to the microbiome exacerbate intestinal inflammation and alter intestinal motility in mice. It is, however, not yet known whether microbiota-derived short-chain fatty acids (butyrate, propionate, and acetate) and their receptors contribute to this effect. METHODS: Mice were exposed to a social disruption stress, or left undisturbed as a control. After the first stress exposure, mice were orally challenged with Citrobacter rodentium or with vehicle. The levels of short-chain fatty acids (SCFAs) were measured using gas chromatography-mass spectrometry. SCFA receptors were measured via real-time polymerase chain reaction. Microbial community composition was assessed using 16S rRNA gene sequencing. RESULTS: Stress exposure reduced colonic SCFA levels. Stress exposure and C rodentium, however, significantly increased SCFA levels and changed the expression of SCFA receptors. The levels of SCFAs did not correlate with the severity of colonic inflammation, but the colonic expression of the SCFA receptor GPR41 was positively associated with inflammatory cytokines and colonic histopathology scores. The relative abundances of several taxa of colonic bacteria were significantly changed by stress exposure, including SCFA producers. CONCLUSIONS: Social stress can have a significant effect on infection-induced colonic inflammation, and stress-induced changes in microbial-produced metabolites and their receptors may be involved.

Prolonged restraint stressor exposure in outbred CD-1 mice impacts microbiota, colonic inflammation, and short chain fatty acids.

Stressor-exposure has been shown to exacerbate inflammation and change the composition of the gastrointestinal microbiota; however stressor-induced effects on microbiota-derived metabolites and their receptors are unknown. Thus, bacterial-produced short chain fatty acids (SCFAs), as well as microbial community composition, were assessed in the colons of mice exposed to stress during infection with Citrobacter rodentium. Mice were exposed to overnight restraint on 7 consecutive nights, or left undisturbed as a control. After the first exposure of restraint, mice were orally challenged with C. rodentium or with vehicle. Microbial community composition was assessed using 16S rRNA gene sequencing and SCFA levels measured using gas chromatography-mass spectrometry (GC-MS). Pathogen levels and colonic inflammation were also assessed 6 days post-infection. Results demonstrated that the microbial community structure and SCFA production were significantly affected by both stressor exposure and C. rodentium-infection. Exposure to prolonged restraint in the absence of infection significantly reduced SCFAs (acetic acid, butyric acid, and propionic acid). Multiple bacterial taxa were affected by stressor exposure, with the relative abundance of Lactobacillus being significantly reduced and directly correlated with propionic acid. Lactobacillus abundances were inversely correlated with colonic inflammation, supporting the contention that Lactobacillus helps to regulate mucosal inflammatory responses. Our data indicates that restraint stressor can have significant effects on pathogen-induced colonic inflammation and suggest that stressor-induced changes in the microbiota, microbial-produced SCFAs and their receptors may be involved.

Transmembrane solute transport in the apicomplexan parasite Plasmodium.

Apicomplexa are a large group of eukaryotic, single-celled parasites, with complex life cycles that occur within a wide range of different microenvironments. They include important human pathogens such as Plasmodium, the causal agent of malaria, and Toxoplasma, which causes toxoplasmosis most often in immunocompromised individuals. Despite environmental differences in their life cycles, these parasites retain the ability to obtain nutrients, remove waste products, and control ion balances. They achieve this flexibility by relying on proteins that can deliver and remove solutes. This reliance on transport proteins for essential functions makes these pathways excellent potential targets for drug development programmes. Transport proteins are frequently key mediators of drug resistance by their ability to remove drugs from their sites of action. The study of transport processes mediated by integral membrane proteins and, in particular, identification of their physiological functions and localisation, and differentiation from host orthologues has already established new validated drug targets. Our understanding of how apicomplexan parasites have adapted to changing environmental challenges has also increased through the study of their transporters. This brief introduction to membrane transporters of apicomplexans highlights recent discoveries focusing on Plasmodium and emphasises future directions.

Selenols are resistant to irreversible modification by HNO.

The discovery of nitric oxide (NO) as an endogenously generated signaling species in mammalian cells has spawned a vast interest in the study of the chemical biology of nitrogen oxides. Of these, nitroxyl (azanone, HNO) has gained much attention for its potential role as a therapeutic for cardiovascular disease. Known targets of HNO include hemes/heme proteins and thiols/thiol-containing proteins. Recently, due to their roles in redox signaling and cellular defense, selenols and selenoproteins have also been speculated to be additional potential targets of HNO. Indeed, as determined in the current work, selenols are targeted by HNO. Such reactions appear to result only in formation of diselenide products, which can be easily reverted back to the free selenol. This characteristic is distinct from the reaction of HNO with thiols/thiolproteins. These findings suggest that, unlike thiolproteins, selenoproteins are resistant to irreversible oxidative modification, support that Nature may have chosen to use selenium instead of sulfur in certain biological systems for its enhanced resistance to electrophilic and oxidative modification.

Comparison of HNO reactivity with tryptophan and cysteine in small peptides.

Recent discoveries of important pharmacological properties have drawn attention to the reactivity of HNO (azanone, nitroxyl) with biologically relevant substrates. Apart from its role in thiol oxidation, HNO has been reported to have nitrosative properties, for example, with tryptophan resulting in N-nitrosotryptophan formation. We have investigated the reactivity of HNO with tryptophan and small peptides containing either tryptophan or both a tryptophan and a cysteine residue. Our results point to the more reactive nature of cysteine towards HNO compared with tryptophan.

Mechanistic insights into the oxidation of substituted phenols via hydrogen atom abstraction by a cupric-superoxo complex.

To obtain mechanistic insights into the inherent reactivity patterns for copper(I)-O2 adducts, a new cupric-superoxo complex [(DMM-tmpa)Cu(II)(O2(•-))](+) (2) [DMM-tmpa = tris((4-methoxy-3,5-dimethylpyridin-2-yl)methyl)amine] has been synthesized and studied in phenol oxidation-oxygenation reactions. Compound 2 is characterized by UV-vis, resonance Raman, and EPR spectroscopies. Its reactions with a series of para-substituted 2,6-di-tert-butylphenols (p-X-DTBPs) afford 2,6-di-tert-butyl-1,4-benzoquinone (DTBQ) in up to 50% yields. Significant deuterium kinetic isotope effects and a positive correlation of second-order rate constants (k2) compared to rate constants for p-X-DTBPs plus cumylperoxyl radical reactions indicate a mechanism that involves rate-limiting hydrogen atom transfer (HAT). A weak correlation of (k(B)T/e) ln k2 versus E(ox) of p-X-DTBP indicates that the HAT reactions proceed via a partial transfer of charge rather than a complete transfer of charge in the electron transfer/proton transfer pathway. Product analyses, (18)O-labeling experiments, and separate reactivity employing the 2,4,6-tri-tert-butylphenoxyl radical provide further mechanistic insights. After initial HAT, a second molar equiv of 2 couples to the phenoxyl radical initially formed, giving a Cu(II)-OO-(ArO') intermediate, which proceeds in the case of p-OR-DTBP substrates via a two-electron oxidation reaction involving hydrolysis steps which liberate H2O2 and the corresponding alcohol. By contrast, four-electron oxygenation (O-O cleavage) mainly occurs for p-R-DTBP which gives (18)O-labeled DTBQ and elimination of the R group.

Tuning of the copper-thioether bond in tetradentate N3S(thioether) ligands; O-O bond reductive cleavage via a [Cu(II)2(µ-1,2-peroxo)]²âº/[Cu(III)2(µ-oxo)2]²âº equilibrium.

Current interest in copper/dioxygen reactivity includes the influence of thioether sulfur ligation, as it concerns the formation, structures, and properties of derived copper-dioxygen complexes. Here, we report on the chemistry of {L-Cu(I)}2-(O2) species L = (DMM)ESE, (DMM)ESP, and (DMM)ESDP, which are N3S(thioether)-based ligands varied in the nature of a substituent on the S atom, along with a related N3O(ether) (EOE) ligand. Cu(I) and Cu(II) complexes have been synthesized and crystallographically characterized. Copper(I) complexes are dimeric in the solid state, [{L-Cu(I)}2](B(C6F5)4)2, however are shown by diffusion-ordered NMR spectroscopy to be mononuclear in solution. Copper(II) complexes with a general formulation [L-Cu(II)(X)](n+) {X = ClO4(-), n = 1, or X = H2O, n = 2} exhibit distorted square pyramidal coordination geometries and progressively weaker axial thioether ligation across the series. Oxygenation (-130 °C) of {((DMM)ESE)Cu(I)}(+) results in the formation of a trans-µ-1,2-peroxodicopper(II) species [{((DMM)ESE)Cu(II)}2(µ-1,2-O2(2-))](2+) (1(P)). Weakening the Cu-S bond via a change to the thioether donor found in (DMM)ESP leads to the initial formation of [{((DMM)ESP)Cu(II)}2(µ-1,2-O2(2-))](2+) (2(P)) that subsequently isomerizes to a bis-µ-oxodicopper(III) complex, [{((DMM)ESP)Cu(III)}2(µ-O(2-))2](2+) (2(O)), with 2(P) and 2(O) in equilibrium (K(eq) = [2(O)]/[2(P)] = 2.6 at -130 °C). Formulations for these Cu/O2 adducts were confirmed by resonance Raman (rR) spectroscopy. This solution mixture is sensitive to the addition of methylsulfonate, which shifts the equilibrium toward the bis-µ-oxo isomer. Further weakening of the Cu-S bond in (DMM)ESDP or substitution with an ether donor in (DMM)EOE leads to only a bis-µ-oxo species (3(O) and 4(O), respectively). Reactivity studies indicate that the bis-µ-oxodicopper(III) species (2(O), 3(O)) and not the trans-peroxo isomers (1(P) and 2(P)) are responsible for the observed ligand sulfoxidation. Our findings concerning the existence of the 2(P)/2(O) equilibrium contrast with previously established ligand-Cu(I)/O2 reactivity and possible implications are discussed.

NMR detection and study of hydrolysis of HNO-derived sulfinamides.

Nitroxyl (HNO), a potential heart failure therapeutic, is known to post-translationally modify cysteine residues. Among reactive nitrogen oxide species, the modification of cysteine residues to sulfinamides [RS(O)NH2] is unique to HNO. We have applied (15)N-edited (1)H NMR techniques to detect the HNO-induced thiol to sulfinamide modification in several small organic molecules, peptides, and the cysteine protease, papain. Relevant reactions of sulfinamides involve reduction to free thiols in the presence of excess thiol and hydrolysis to form sulfinic acids [RS(O)OH]. We have investigated sulfinamide hydrolysis at physiological pH and temperature. Studies with papain and a related model peptide containing the active site thiol suggest that sulfinamide hydrolysis can be enhanced in a protein environment. These findings are also supported by modeling studies. In addition, analysis of peptide sulfinamides at various pH values suggests that hydrolysis becomes more facile under acidic conditions.

Stepwise protonation and electron-transfer reduction of a primary copper-dioxygen adduct.

The protonation­reduction of a dioxygen adduct with [LCu(I)][B(C6F5)4], cupric superoxo complex [LCu(II)(O2(•­))]+ (1) (L = TMG3tren (1,1,1-tris[2-[N(2)-(1,1,3,3-tetramethylguanidino)]ethyl]amine)) has been investigated. Trifluoroacetic acid (HOAcF) reversibly associates with the superoxo ligand in ([LCu(II)(O2(•­))]+) in a 1:1 adduct [LCu(II)(O2(•­))(HOAcF)](+) (2), as characterized by UV­visible, resonance Raman (rR), nuclear magnetic resonance (NMR), and X-ray absorption (XAS) spectroscopies, along with density functional theory (DFT) calculations. Chemical studies reveal that for the binding of HOAcF with 1 to give 2, Keq = 1.2 × 10(5) M(­1) (−130 °C) and ΔH° = −6.9(7) kcal/mol, ΔS° = −26(4) cal mol(­1) K(­1)). Vibrational (rR) data reveal a significant increase (29 cm(­1)) in vO­O (= 1149 cm(­1)) compared to that known for [LCu(II)(O2(•­))](+) (1). Along with results obtained from XAS and DFT calculations, hydrogen bonding of HOAcF to a superoxo O-atom in 2 is established. Results from NMR spectroscopy of 2 at −120 °C in 2-methyltetrahydrofuran are also consistent with 1/HOAcF = 1:1 formulation of 2 and with this complex possessing a triplet (S = 1) ground state electronic configuration, as previously determined for 1. The pre-equilibrium acid association to 1 is followed by outer-sphere electron-transfer reduction of 2 by decamethylferrocene (Me10Fc) or octamethylferrocene (Me8Fc), leading to the products H2O2, the corresponding ferrocenium salt, and [LCu(II)(OAcF)](+). Second-order rate constants for electron transfer (ket) were determined to be 1365 M(­1) s(­1) (Me10Fc) and 225 M(­1) s(­1) (Me8Fc) at −80 °C. The (bio)chemical relevance of the proton-triggered reduction of the metal-bound dioxygen-derived fragment is discussed.

Synthesis and antimalarial efficacy of two-carbon-linked, artemisinin-derived trioxane dimers in combination with known antimalarial drugs.

Malaria continues to be a difficult disease to eradicate largely because of the widespread populations it affects and the resistance that malaria parasites have developed against once very potent therapies. The natural product artemisinin has been a boon for antimalarial chemotherapy, as artemisinin combination therapy (ACT) has become the first line of chemotherapy. Because the threat of resistance is always on the horizon, it is imperative to continually identify new treatments, comprising both advanced analogues of all antimalarial drugs, especially artemisinin, and the exploration of novel combinations, ideally with distinct mechanisms of action. Here we report for the first time the synthesis of a series of two-carbon-linked artemisinin-derived dimers, their unique structural features, and demonstration of their antimalarial efficacy via single oral dose administration in two 60-day survival studies of Plasmodium berghei infected mice. Several of the new endoperoxide chemical entities consistently demonstrated excellent antimalarial efficacy, and combinations with two non-peroxide antimalarial drugs have been studied.

Pyrrolidine dithiocarbamate improves mortality in a rat model of severe hemorrhage.

BACKGROUND: Hemorrhagic shock is a life threatening condition characterized by diminishing organ function. The aim of this study was to determine whether an effective pyrrolidine dithiocarbamate (PDTC) treatment protocol could be established to decrease organ dysfunction and mortality in a lethal hemorrhagic shock-resuscitation (HSR) model. MATERIALS AND METHODS: Sprague-Dawley rats were randomized into three experimental groups; HSR alone (HSR), PDTC (100 mg/kg) administered 12 h pre-HSR (PDTC-12), and PDTC administered 1 h post-shock prior to resuscitation (PDTC+1). Hemorrhage was induced by arterial blood withdrawal to a mean arterial pressure (MAP) of 25 ± 5 mmHg for 1 h. Resuscitation was performed until pre-HSR MAP was attained. Blood was collected immediately prior to HSR, 1 h post-shock, and at protocol end. Measurements of base excess, lactate, arterial partial pressure of carbon dioxide (PaCO(2)) and oxygen (PaO(2)), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, blood urea nitrogen (BUN), and lipase were performed. RESULTS: In PDTC+1 animals, PDTC was ineffective in improving survival. In contrast, survival was significantly increased in the PDTC-12 animals versus PDTC+1 and HSR groups. Analysis of physiologic parameters demonstrated that elevations in base deficit and lactate levels following hemorrhage were blunted by PDTC administration in the PDTC-12 group. At time of death, creatinine, ALT, and AST levels were significantly higher in HSR versus PDTC-12 animals. CONCLUSIONS: Administration of PDTC 12 h prior to HSR significantly improves survival through preservation of organ function.

MicroRNA expression following activated protein C treatment during septic shock.

BACKGROUND: Sepsis induces systemic stress by augmenting inflammatory and procoagulant responses, resulting in microvascular dysfunction and end organ failure, events modulated by the protein C pathway. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional regulation of genes; yet, their role in sepsis is poorly defined. We hypothesized that activated protein C (aPC) selectively alters specific miRNA expression implicated in protection of hepatic function during septic shock. METHODS: Male Sprague-Dawley rats underwent sham or cecal ligation and puncture surgery; 24 h later, we randomized them to aPC (1 mg/kg) or vehicle (0.9% [w/v] saline) treatment via an indwelling venous catheter (12-h intervals for 24 h). We performed gene array and quantitative reverse transcriptase-polymerase chain reaction analysis on hepatic RNA to determine miRNA expression and determined predicted mRNA targets using a bioinformatics approach. We confirmed beneficial effects of aPC treatment in the cecal ligation and puncture model of sepsis by survival and blood chemistries, and histologically. RESULTS: Of 351 rat miRNAs examined, 17 were highly expressed during sepsis and restored to basal levels after aPC treatment. We confirmed expression of select miRNAs (miR-182, -199a-5p, -203, -211, -222, and -29b) using quantitative reverse transcriptase-polymerase chain reaction. In silico analysis identified nine miRNAs significantly regulating target genes of the focal adhesion pathway. CONCLUSIONS: These data suggest that aPC treatment coordinates beneficial cytoprotective effects during sepsis by modulating miRNA expression. Whereas translational effects remain to be fully elucidated in a clinical setting, we demonstrate here the potential experimental and computational benefits of using of microRNA analysis in sepsis.

GMO detection using a bioluminescent real time reporter (BART) of loop mediated isothermal amplification (LAMP) suitable for field use.

BACKGROUND: There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM) crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR) and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART) occurs at a constant temperature and only requires a simple light detection and integration device. RESULTS: Loop mediated isothermal amplification (LAMP) shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART) for determination of genetically modified (GM) maize target DNA at low levels of contamination (0.1-5.0% GM) using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. CONCLUSIONS: LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading within a reaction must be controlled to ensure quantification at low target concentrations.

Development of N-substituted hydroxylamines as efficient nitroxyl (HNO) donors.

Due to its inherent reactivity, nitroxyl (HNO), must be generated in situ through the use of donor compounds, but very few physiologically useful HNO donors exist. Novel N-substituted hydroxylamines with carbon-based leaving groups have been synthesized, and their structures confirmed by X-ray crystallography. These compounds generate HNO under nonenzymatic, physiological conditions, with the rate and amount of HNO released being dependent mainly on the nature of the leaving group. A barbituric acid and a pyrazolone derivative have been developed as efficient HNO donors with half-lives at pH 7.4, 37 °C of 0.7 and 9.5 min, respectively.

Activated protein C restores hepatic microcirculation during sepsis by modulating vasoregulator expression.

Activated protein C (aPC) promotes fibrinolysis while inhibiting coagulation and inflammation. In septic patients, aPC levels are depleted, and aPC treatment has emerged as a therapeutic option. To better understand the mechanism(s) by which aPC improves survival in sepsis, we sought to determine the effect of aPC treatment on hepatic vasoactive gene and protein expression, leading to changes in hepatic vascular responsiveness in a septic animal model. Under anesthesia, rats underwent sham or cecal ligation and puncture followed by aPC treatment (1 mg/kg, twice daily, i.v.). Treatment with aPC significantly decreased hepatic endothelin 1 (ET-1)/ET A receptor mRNA and protein expression. To determine the effect of aPC on hepatic microvasculature, ET-1-induced changes in liver microcirculation were assessed by intravital microscopy. This approach demonstrated aPC significantly improved hepatic perfusion index in the animals that underwent cecal ligation and puncture in the absence of significant changes in portal venous pressure. Furthermore, although aPC did not affect ET-1-dependent sinusoidal vasoconstriction, aPC induced hepatoprotective effects via enhanced red blood cell velocity. Collectively, these data demonstrate aPC ameliorates ET-1-dependent changes in hepatic microcirculation and improves hepatic function in the setting of sepsis.

Nonmuscle myosin II regulates migration but not contraction in rat hepatic stellate cells.

AIM: To identify and characterize the function of nonmuscle myosin II (NMM II) isoforms in primary rat hepatic stellate cells (HSCs). METHODS: Primary HSCs were isolated from male Sprague-Dawley rats by pronase/collagenase digestion. Total RNA and protein were harvested from quiescent and culture-activated HSCs. NMM II isoform (II-A, II-B and II-C) gene and protein expression were measured by RealTime polymerase chain reaction and Western blot analyses respectively. NMM II protein localization was visualized in vitro using immunocytochemical analysis. For in vivo assessment, liver tissue was harvested from bile duct-ligated (BDL) rats and NMM IIisoform expression determined by immunohistochemistry. Using a selective myosin II inhibitor and siRNA-mediated knockdown of each isoform, NMM II functionality in primary rat HSCs was determined by contraction and migration assays. RESULTS: NMM II-A and II-B mRNA expression was increased in culture-activated HSCs (Day 14) with significant increases seen in all pair-wise comparisons (II-A: 12.67 ± 0.99 (quiescent) vs 17.36 ± 0.78 (Day 14), P < 0.05; II-B: 4.94 ± 0.62 (quiescent) vs 13.90 ±0.85 (Day 14), P < 0.001). Protein expression exhibited similar expression patterns (II-A: 1.87 ± 2.50 (quiescent) vs 58.64 ± 8.76 (Day 14), P < 0.05; II-B: 1.17 ± 1.93 (quiescent) vs 103.71 ± 21.73 (Day 14), P < 0.05). No significant differences were observed in NMM II-C mRNA and protein expression between quiescent and activated HSCs. In culture-activated HSCs, NMM II-A and II-B merged with F-actin at the cellular periphery and throughout cytoplasm respectively. In vitro studies showed increased expression of NMM II-B in HSCs activated by BDL compared to sham-operated animals. There were no apparent increases of NMM II-A and II-C protein expression in HSCs during hepatic BDL injury. To determine the contribution of NMM II-A and II-B to migration and contraction, NMM II-A and II-B expression were downregulated with siRNA. NMM II-A and/or II-B siRNA inhibited HSC migration by approximately 25% compared to scramble siRNA-treated cells. Conversely, siRNA-mediated NMM II-A and II-B inhibition had no significant effect on HSC contraction; however, contraction was inhibited with the myosin II inhibitor, blebbistatin (38.7% ± 1.9%). CONCLUSION: Increased expression of NMM II-A and II-B regulates HSC migration, while other myosin IIclasses likely modulate contraction, contributing to development and severity of liver fibrosis.

Activated protein C alters inflammation and protects renal function in sepsis.

BACKGROUND: Activated protein C (aPC) confers survival benefit in patients with sepsis, yet its protective mechanism(s) remain unclear. Herein, we determined time-dependent severity of renal dysfunction during polymicrobial sepsis. We hypothesized aPC restores renal function by preserving organ architecture and reducing inflammation. MATERIALS AND METHODS: Sprague-Dawley rats underwent sham operation or cecal ligation and puncture (CLP). At 6 or 24 h post-surgery, kidney function was assessed by plasma electrolytes, blood urea nitrogen (BUN), and creatinine levels. Renal architecture was examined histologically. In the next series of experiments, 24-h post-surgery, animals were treated with vehicle or aPC (1 mg/kg) for 4 d, and kidney function and circulating cytokine levels were measured. Plasma was collected and assayed for BUN, creatinine, and lactate dehydrogenase (LDH) levels. Serum cytokine levels were measured by ELISA. RESULTS: Plasma electrolytes, BUN, creatinine, and renal architecture were altered 6 h after CLP. Treatment with aPC significantly inhibited sepsis-induced elevations in BUN, creatinine, LDH levels, and improved renal architecture. After CLP, interferon gamma (INFγ) decreased, while interleukins-1beta and -10 (IL-1ß and IL-10) increased; these effects were attenuated by aPC treatment. CONCLUSIONS: Our data demonstrate that renal dysfunction occurs as early as 6 h following sepsis and continues thereafter. Treatment with aPC attenuated INFγ and IL-1ß changes, and preserved renal function in sepsis. These data suggest aPC may confer a survival advantage by reducing systemic inflammation and, in doing so, preserving organ function.