E. coli strain-dependent lipid alterations in cocultures with endothelial cells and neutrophils modeling sepsis.

Dysregulated lipid metabolism is common in infection and inflammation and is a part of the complex milieu underlying the pathophysiological sequelae of disease. Sepsis is a major cause of mortality and morbidity in the world and is characterized by an exaggerated host response to an infection. Metabolic changes, including alterations in lipid metabolism, likely are important in sepsis pathophysiology. Here, we designed an in vitro cell culture model using endothelial cells, E. coli, and neutrophils to mimic sepsis in a simplified cell model. Lipid alterations were studied in the presence of the pathogenic E. coli strain CFT073 and non-pathogenic E. coli strain JM109. We employed untargeted lipidomics to first identify lipid changes and then targeted lipidomics to confirm changes. Both unique and shared lipid signatures were identified in cocultures with these E. coli strains. In the absence of neutrophils, the CFT073 strain elicited alterations in lysophosphatidylcholine and diglyceride molecular species during coculture while both strains led to increases in phosphatidylglycerols. Lipid alterations in these cocultures changed with the addition of neutrophils. In the presence of neutrophils with E. coli and endothelial cells, triglyceride increases were a unique response to the CFT073 strain while phosphatidylglycerol and diglyceride increases occurred in response to both strains. Phosphatidylethanolamine also increased in neutrophils, E. coli and endothelial cells cocultures, and this response was greater in the presence of the CFT073 strain. We further evaluated changes in phosphatidylethanolamine in a rat model of sepsis, which showed multiple plasma phosphatidylethanolamine molecular species were elevated shortly after the induction of sepsis. Collectively, these findings demonstrate unique lipid responses by co-cultures of E. coli with endothelial cells which are dependent on the E. coli strain as well as the presence of neutrophils. Furthermore, increases in phosphatidylethanolamine levels in CFT073 urosepsis E. coli, endothelial cell, neutrophil cocultures were similarly observed in the plasma of septic rats.

Plasmalogen Loss in Sepsis and SARS-CoV-2 Infection.

Plasmalogens are plasma-borne antioxidant phospholipid species that provide protection as cellular lipid components during cellular oxidative stress. In this study we investigated plasma plasmalogen levels in human sepsis as well as in rodent models of infection. In humans, levels of multiple plasmenylethanolamine molecular species were decreased in septic patient plasma compared to control subject plasma as well as an age-aligned control subject cohort. Additionally, lysoplasmenylcholine levels were significantly decreased in septic patients compared to the control cohorts. In contrast, plasma diacyl phosphatidylethanolamine and phosphatidylcholine levels were elevated in septic patients. Lipid changes were also determined in rats subjected to cecal slurry sepsis. Plasma plasmenylcholine, plasmenylethanolamine, and lysoplasmenylcholine levels were decreased while diacyl phosphatidylethanolamine levels were increased in septic rats compared to control treated rats. Kidney levels of lysoplasmenylcholine as well as plasmenylethanolamine molecular species were decreased in septic rats. Interestingly, liver plasmenylcholine and plasmenylethanolamine levels were increased in septic rats. Since COVID-19 is associated with sepsis-like acute respiratory distress syndrome and oxidative stress, plasmalogen levels were also determined in a mouse model of COVID-19 (intranasal inoculation of K18 mice with SARS-CoV-2). 3 days following infection, lung infection was confirmed as well as cytokine expression in the lung. Multiple molecular species of lung plasmenylcholine and plasmenylethanolamine were decreased in infected mice. In contrast, the predominant lung phospholipid, dipalmitoyl phosphatidylcholine, was not decreased following SARS-CoV-2 infection. Additionally total plasmenylcholine levels were decreased in the plasma of SARS-CoV-2 infected mice. Collectively, these data demonstrate the loss of plasmalogens during both sepsis and SARS-CoV-2 infection. This study also indicates plasma plasmalogens should be considered in future studies as biomarkers of infection and as prognostic indicators for sepsis and COVID-19 outcomes.

Pleiotropic actions of IP6K1 mediate hepatic metabolic dysfunction to promote nonalcoholic fatty liver disease and steatohepatitis.

OBJECTIVE: Obesity and insulin resistance greatly increase the risk of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). We have previously discovered that whole-body and adipocyte-specific Ip6k1deletion protects mice from high-fat-diet-induced obesity and insulin resistance due to improved adipocyte thermogenesis and insulin signaling. Here, we aimed to determine the impact of hepatocyte-specific and whole-body Ip6k1 deletion (HKO and Ip6k1-KO or KO) on liver metabolism and NAFLD/NASH. METHODS: Body weight and composition; energy expenditure; glycemic profiles; and serum and liver metabolic, inflammatory, fibrotic and toxicity parameters were assessed in mice fed Western and high-fructose diet (HFrD) (WD: 40% kcal fat, 1.25% cholesterol, no added choline and HFrD: 60% kcal fructose). Mitochondrial oxidative capacity was evaluated in isolated hepatocytes. RNA-Seq was performed in liver samples. Livers from human NASH patients were analyzed by immunoblotting and mass spectrometry. RESULTS: HKO mice displayed increased hepatocyte mitochondrial oxidative capacity and improved insulin sensitivity but were not resistant to body weight gain. Improved hepatocyte metabolism partially protected HKO mice from NAFLD/NASH. In contrast, enhanced whole-body metabolism and reduced body fat accumulation significantly protected whole-body Ip6k1-KO mice from NAFLD/NASH. Mitochondrial oxidative pathways were upregulated, whereas gluconeogenic and fibrogenic pathways were downregulated in Ip6k1-KO livers. Furthermore, IP6K1 was upregulated in human NASH livers and interacted with the enzyme O-GlcNAcase that reduces protein O-GlcNAcylation. Protein O-GlcNAcylation was found to be reduced in Ip6k1-KO and HKO mouse livers. CONCLUSION: Pleiotropic actions of IP6K1 in the liver and other metabolic tissues mediate hepatic metabolic dysfunction and NAFLD/NASH, and thus IP6K1 deletion may be a potential treatment target for this disease.

The lipid biology of sepsis.

Sepsis, defined as the dysregulated immune response to an infection leading to organ dysfunction, is one of the leading causes of mortality around the globe. Despite the significant progress in delineating the underlying mechanisms of sepsis pathogenesis, there are currently no effective treatments or specific diagnostic biomarkers in the clinical setting. The perturbation of cell signaling mechanisms, inadequate inflammation resolution, and energy imbalance, all of which are altered during sepsis, are also known to lead to defective lipid metabolism. The use of lipids as biomarkers with high specificity and sensitivity may aid in early diagnosis and guide clinical decision making. In addition, identifying the link between specific lipid signatures and their role in sepsis pathology may lead to novel therapeutics. In this review, we discuss the recent evidence on dysregulated lipid metabolism both in experimental and human sepsis focused on bioactive lipids, fatty acids, and cholesterol as well as the enzymes regulating their levels during sepsis. We highlight not only their potential roles in sepsis pathogenesis but also the possibility of using these respective lipid compounds as diagnostic and prognostic biomarkers of sepsis.

2-Chlorofatty acids are biomarkers of sepsis mortality and mediators of barrier dysfunction in rats.

Sepsis is defined as the systemic, dysregulated host immune response to an infection that leads to injury to host organ systems and, often, death. Complex interactions between pathogens and their hosts elicit microcirculatory dysfunction. Neutrophil myeloperoxidase (MPO) is critical for combating pathogens, but MPO-derived hypochlorous acid (HOCl) can react with host molecular species as well. Plasmalogens are targeted by HOCl, leading to the production of 2-chlorofatty acids (2-CLFAs). 2-CLFAs are associated with human sepsis mortality, decrease in vitro endothelial barrier function, and activate human neutrophil extracellular trap formation. Here, we sought to examine 2-CLFAs in an in vivo rat sepsis model. Intraperitoneal cecal slurry sepsis with clinically relevant rescue therapies led to ∼73% mortality and evidence of microcirculatory dysfunction. Plasma concentrations of 2-CLFAs assessed 8 h after sepsis induction were lower in rats that survived sepsis than in nonsurvivors. 2-CLFA levels were elevated in kidney, liver, spleen, lung, colon, and ileum in septic animals. In vivo, exogenous 2-CLFA treatments increased kidney permeability, and in in vitro experiments, 2-CLFA also increased epithelial surface expression of vascular cell adhesion molecule 1 and decreased epithelial barrier function. Collectively, these studies support a role of free 2-CLFAs as biomarkers of sepsis mortality, potentially mediated, in part, by 2-CLFA-elicited endothelial and epithelial barrier dysfunction.

Author Correction: The effect of endothelial nitric oxide synthase on the hemodynamics and wall mechanics in murine arteriovenous fistulas.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction to: High resolution hemodynamic profiling of murine arteriovenous fistula using magnetic resonance imaging and computational fluid dynamics.

After publication of the original article.

The effect of endothelial nitric oxide synthase on the hemodynamics and wall mechanics in murine arteriovenous fistulas.

Creation of a hemodialysis arteriovenous fistula (AVF) causes aberrant vascular mechanics at and near the AVF anastomosis. When inadequately regulated, these aberrant mechanical factors may impede AVF lumen expansion to cause AVF maturation failure, a significant clinical problem with no effective treatments. The endothelial nitric oxide synthase (NOS3) system is crucial for vascular health and function, but its effect on AVF maturation has not been fully characterized. We hypothesize that NOS3 promotes AVF maturation by regulating local vascular mechanics following AVF creation. Here we report the first MRI-based fluid-structure interaction (FSI) study in a murine AVF model using three mouse strains: NOS3 overexpression (NOS3 OE) and knockout (NOS3-/-) on C57BL/6 background, with C57BL/6 as the wild-type control (NOS3+/+). When compared to NOS3+/+ and NOS3-/-, AVFs in the OE mice had larger lumen area. AVFs in the OE mice also had smoother blood flow streamlines, as well as lower blood shear stress at the wall, blood vorticity, inner wall circumferential stretch, and radial wall thinning at the anastomosis. Our results demonstrate that overexpression of NOS3 resulted in distinct hemodynamic and wall mechanical profiles associated with favorable AVF remodeling. Enhancing NOS3 expression may be a potential therapeutic approach for promoting AVF maturation.

Arteriovenous conduits for hemodialysis: how to better modulate the pathophysiological vascular response to optimize vascular access durability.

Vascular access is the lifeline for patients on hemodialysis. Arteriovenous fistulas (AVFs) are the preferred vascular access, but AVF maturation failure remains a significant clinical problem. Currently, there are no effective therapies available to prevent or treat AVF maturation failure. AVF maturation failure frequently results from venous stenosis at the AVF anastomosis, which is secondary to poor outward vascular remodeling and excessive venous intimal hyperplasia that narrows the AVF lumen. Arteriovenous grafts (AVGs) are the next preferred vascular access when an AVF creation is not possible. AVG failure is primarily the result of venous stenosis at the vein-graft anastomosis, which originates from intimal hyperplasia development. Although there has been advancement in our knowledge of the pathophysiology of AVF maturation and AVG failure, this has not translated into effective therapies for these two important clinical problems. Further work will be required to dissect out the mechanisms of AVF maturation failure and AVG failure to develop more specific therapies. This review highlights the major recent advancements in AVF and AVG biology, reviews major clinical trials, and discusses new areas for future research.

Comparison of hemodialysis arteriovenous fistula blood flow rates measured by Doppler ultrasound and phase-contrast magnetic resonance imaging.

OBJECTIVE: The objective of this study was to compare blood flow rates measured by Doppler ultrasound (DUS) and phase-contrast magnetic resonance imaging (MRI) in patients having a hemodialysis arteriovenous fistula (AVF) and to identify scenarios in which there was significant discordance between these two approaches. METHODS: Blood flow rates in the proximal artery (PA) and draining vein (DV) of newly created upper extremity AVFs were measured and compared using DUS and phase-contrast MRI at 1 day, 6 weeks, and 6 months postoperatively. RESULTS: Blood flow rates in the PA measured by DUS (1155 ± 907 mL/min, mean ± standard deviation) and by MRI (1170 ± 657 mL/min) were not statistically different (P = .812) based on 78 data pairs from 49 patients. DV DUS flow (1277 ± 995 mL/min) and MRI flow (1130 ± 655 mL/min) were also not statistically different (P = .071) based on 64 data pairs. In both PA and DV, the two methods substantially agreed with each other (Cohen κ: PA, 0.66; DV, 0.67) when flow rates were put into four clinically relevant categories (<300, 300-599, 600-1499, and ≥1500 mL/min). The Bland-Altman analyses of DUS and MRI flow identified six and four outliers for PA and DV, respectively. Seven outliers had higher DUS than MRI flow, with all DUS scan sites having a large lumen or significant local curvature; the other three had lower DUS flow, partly due to an underestimation of lumen diameter by DUS. CONCLUSIONS: DUS and MRI flow rates are generally comparable in both PA and DV. When DUS is used for flow measurements, careful attention to accurate lumen diameter measurements is needed and scan sites with marked curvature should be avoided. Our result may improve the accuracy of DUS-measured AVF blood flow rate.

High resolution hemodynamic profiling of murine arteriovenous fistula using magnetic resonance imaging and computational fluid dynamics.

BACKGROUND: Arteriovenous fistula (AVF) maturation failure remains a major cause of morbidity and mortality in hemodialysis patients. The two major etiologies of AVF maturation failure are early neointimal hyperplasia development and persistent inadequate outward remodeling. Although hemodynamic changes following AVF creation may impact AVF remodeling and contribute to neointimal hyperplasia development and impaired outward remodeling, detailed AVF hemodynamics are not yet fully known. Since murine AVF models are valuable tools for investigating the pathophysiology of AVF maturation failure, there is a need for a new approach that allows the hemodynamic characterization of murine AVF at high resolutions. METHODS: This methods paper presents a magnetic resonance imaging (MRI)-based computational fluid dynamic (CFD) method that we developed to rigorously quantify the evolving hemodynamic environment in murine AVF. The lumen geometry of the entire murine AVF was reconstructed from high resolution, non-contrast 2D T2-weighted fast spin echo MRI sequence, and the flow rates of the AVF inflow and outflow were extracted from a gradient echo velocity mapping sequence. Using these MRI-obtained lumen geometry and inflow information, CFD modeling was performed and used to calculate blood flow velocity and hemodynamic factors at high resolutions (on the order of 0.5 µm spatially and 0.1 ms temporally) throughout the entire AVF lumen. We investigated both the wall properties (including wall shear stress (WSS), wall shear stress spatial gradient, and oscillatory shear index (OSI)) and the volumetric properties (including vorticity, helicity, and Q-criterion). RESULTS: Our results demonstrate increases in AVF flow velocity, WSS, spatial WSS gradient, and OSI within 3 weeks post-AVF creation when compared to pre-surgery. We also observed post-operative increases in flow disturbances and vortices, as indicated by increased vorticity, helicity, and Q-criterion. CONCLUSIONS: This novel protocol will enable us to undertake future mechanistic studies to delineate the relationship between hemodynamics and AVF development and characterize biological mechanisms that regulate local hemodynamic factors in transgenic murine AVF models.

Platelet-Activating Factor Quantification Using Reversed Phase Liquid Chromatography and Selected Reaction Monitoring in Negative Ion Mode.

Platelet-activating factor (PAF) is a potent biologically active phospholipid that mediates human physiological and pathophysiologic responses. PAF levels increase transiently and are typically assessed by techniques with limitations related to expense, sensitivity, pre-analysis derivatization and interference with isobaric molecules. This study elucidates a facile, accurate liquid chromatography-mass spectrometry analytical method for PAF. In negative ion mode using electrospray ionization, collisionally-activated dissociation analysis showed a unique product ion for acetate adducts of PAF molecular species representing the loss of methyl acetate from the polar head group and loss of a part of the acetate group from the sn-2 position. This product ion was exploited for selected reaction monitoring of PAF molecular species following separation by reversed-phase liquid chromatography. Standard calibration responses were determined, and this method was able to detect as low as 100 fmol of PAF. Finally, PAF molecular species were quantified in human neutrophils and monocytes.

Preoperative Vascular Medial Fibrosis and Arteriovenous Fistula Development.

BACKGROUND AND OBJECTIVES: Arteriovenous fistula maturation requires an increase in the diameter and blood flow of the feeding artery and the draining vein after its creation. The structural properties of the native vessels may affect the magnitude of these changes. We hypothesized that an increase in the collagen content of the vascular media (medial fibrosis) preoperatively would impair vascular dilation and thereby, limit the postoperative increase in arteriovenous fistula diameter and blood flow and clinical arteriovenous fistula maturation. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We enrolled 125 patients undergoing arteriovenous fistula creation between October of 2008 and April of 2012 and followed them prospectively. Any consenting subject was eligible. Arterial and venous specimens were sampled during arteriovenous fistula surgery. Masson's trichrome-stained samples were used to quantify medial fibrosis. Arteriovenous fistula diameter and blood flow were quantified using 6-week postoperative ultrasound. Clinical arteriovenous fistula maturation was assessed using a predefined protocol. The association of preexisting vascular medial fibrosis with arteriovenous fistula outcomes was evaluated after controlling for baseline demographics, comorbidities, and the preoperative venous diameter. RESULTS: The mean medial fibrosis was 69%±14% in the arteries and 63%±12% in the veins. Arterial medial fibrosis was associated with greater increases in arteriovenous fistula diameter (Δdiameter =0.58 mm; 95% confidence interval [95% CI], 0.27 to 0.89 mm; P<0.001) and arteriovenous fistula blood flow (Δblood flow =85 ml/min; 95% CI, 19 to 150 ml/min; P=0.01) and a lower risk of clinical arteriovenous fistula nonmaturation (odds ratio, 0.71; 95% CI, 0.51 to 0.99; P=0.04), all per 10% absolute difference in medial fibrosis. In contrast, venous medial fibrosis was not associated with the postoperative arteriovenous fistula diameter, blood flow, or clinical maturation. CONCLUSIONS: Preoperative arterial medial fibrosis was associated with greater arteriovenous fistula diameter and blood flow and a lower risk of clinical arteriovenous fistula nonmaturation. This unexpected observation suggests that medial fibrosis promotes arteriovenous fistula development by yet undefined mechanisms or alternatively, that a third factor promotes both medial fibrosis and arteriovenous fistula maturation.

Purification and enzymatic characterization of the hepatitis B virus ribonuclease H, a new target for antiviral inhibitors.

Hepatitis B virus (HBV) reverse transcription requires coordinated function of the reverse transcriptase and ribonuclease H (RNaseH) activities of the viral polymerase protein. The reverse transcriptase has been biochemically characterized, but technical difficulties have prevented both assessment of the RNaseH and development of high throughput inhibitor screens against the RNaseH. Expressing the HBV RNaseH domain with both maltose binding protein and hexahistidine tags led to stable, high-level accumulation of the RNaseH in bacteria. Nickel-affinity purification in the presence of Mg(2+) and ATP removed co-purifying bacterial chaperones and yielded nearly pure monomeric recombinant enzyme. The endonucleolytic RNaseH activity required an DNA:RNA duplex ≥14 nt, could not tolerate a stem-loop in either the RNA or DNA strands, and could tolerate a nick in the DNA strand but not a gap. The RNaseH had no obvious sequence specificity or positional dependence within the RNA, and it cut the RNA at multiple positions even within the minimal 14 nt duplex. The RNaseH also possesses a processive 3'-5' exoribonuclease activity that is slower than the endonucleolytic reaction. These results are consistent with the HBV reverse transcription mechanism that features an initial endoribonucleolytic cut, 3'-5' degradation of RNA, and a sequence-independent terminal RNA cleavage. These data provide support for ongoing anti-RNaseH drug discovery efforts.

Quantification of Heteroaggregation between Citrate-Stabilized Gold Nanoparticles and Hematite Colloids.

Collisions with and attachment to natural colloids (heteroaggregation) is likely to influence significantly the fate, transport, and toxicity of engineered nanoparticles (ENPs). This study investigated heteroaggregation between hematite (α-Fe2O3) colloids and citrate-capped gold nanoparticles (Cit-AuNPs) using a novel approach involving time-resolved dynamic light scattering and parallel experiments designed to quantify nanoparticle attachment and heteroaggregate surface charge. Experiments were performed in low ionic strength synthetic water at environmentally relevant pH in the presence and absence of Suwannee River Natural Organic Matter (SRNOM). In the absence of SRNOM at pH values where Cit-AuNPs and hematite are oppositely charged, attachment efficiencies are high and Cit-AuNPs are capable of destabilizing hematite following an "electrostatic patch" mechanism. Furthermore, maximum observed surface coverages were far below those predicted by geometry alone, a fact predicted by the random sequential adsorption (RSA) model that has significant implications for the estimation of heteroaggregate attachment efficiencies. At pH values where both particles are negative or in the presence of small amounts of SRNOM, attachment was minimal. Calculated attachment efficiencies using the measured surface coverages corroborate these findings. The calculation of attachment efficiencies and the identification of mechanisms governing heteroaggregation represents an important step toward predicting the transport, fate, and toxicity of ENPs in the environment.

The interplay of cyclic stretch and vascular endothelial growth factor in regulating the initial steps for angiogenesis.

Angiogenesis is regulated by chemical and mechanical factors in vivo. The regulatory role of mechanical factors and how chemical and mechanical angiogenic regulators work in concert remains to be explored. We investigated the effect of cyclic uniaxial stretch (20%, 1 Hz), with and without the stimulation of vascular endothelial growth factor (VEGF), on sprouting angiogenesis by employing a stretchable three-dimensional cell culture model. When compared to static controls, stretch alone significantly increased the density of endothelial sprouts, and these sprouts aligned perpendicular to the direction of stretch. The Rho-associated kinase (ROCK) inhibitor Y27632 suppressed stretch-induced sprouting angiogenesis and associated sprout alignment. While VEGF is a potent angiogenic stimulus through ROCK-dependent pathways, the combination of VEGF and stretch did not have an additive effect on angiogenesis. In the presence of VEGF stimulation, the ROCK inhibitor suppressed stretch-induced sprout alignment but did not affect stretch-induced sprout density; in contrast, the receptor tyrosine kinase (RTK) inhibitor sunitinib had no effect on stretch-induced alignment but trended toward suppressed stretch-induced sprout density. Our results suggest that the formation of sprouts and their directionality do not have completely identical regulatory pathways, and thus it is possible to separately manipulate the number and pattern of new sprouts.

Hemodynamic Shear Stress and Endothelial Dysfunction in Hemodialysis Access.

Surgically-created blood conduits used for chronic hemodialysis, including native arteriovenous fistulas (AVFs) and synthetic AV grafts (AVGs), are the lifeline for kidney failure patients. Unfortunately, each has its own limitations: AVFs often fail to mature to become useful for dialysis and AVGs often fail due to stenosis as a result of neointimal hyperplasia, which preferentially forms at the graft-venous anastomosis. No clinical therapies are currently available to significantly promote AVF maturation or prevent neointimal hyperplasia in AVGs. Central to devising strategies to solve these problems is a complete mechanistic understanding of the pathophysiological processes. The pathology of arteriovenous access problems is likely multi-factorial. This review focuses on the roles of fluid-wall shear stress (WSS) and endothelial cells (ECs). In arteriovenous access, shunting of arterial blood flow directly into the vein drastically alters the hemodynamics in the vein. These hemodynamic changes are likely major contributors to non-maturation of an AVF vein and/or formation of neointimal hyperplasia at the venous anastomosis of an AVG. ECs separate blood from other vascular wall cells and also influence the phenotype of these other cells. In arteriovenous access, the responses of ECs to aberrant WSS may subsequently lead to AVF non-maturation and/or AVG stenosis. This review provides an overview of the methods for characterizing blood flow and calculating WSS in arteriovenous access and discusses EC responses to arteriovenous hemodynamics. This review also discusses the role of WSS in the pathology of arteriovenous access, as well as confounding factors that modulate the impact of WSS.

Differential effects of cyclic stretch on bFGF- and VEGF-induced sprouting angiogenesis.

How mechanical factors affect angiogenesis and how they and chemical angiogenic factors work in concert remain not yet well-understood. This study investigated the interactive effects of cyclic uniaxial stretch and two potent proangiogenic molecules [basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF)] on angiogenesis using a stretchable three-dimensional (3-D) cell culture model. Endothelial cells seeded atop a 3-D collagen gel underwent sprouting angiogenesis while being subjected to either 10 or 20% cyclic uniaxial stretch at a frequency of either 1/12 or 1 Hz, in conjunction with an elevated concentration of bFGF or VEGF. Without the presence of additional growth factors, 10 and 20% stretch at 1 Hz induced angiogenesis and the perpendicular alignment of new sprouts, and both inductive effects were abolished by cytochalasin D (an actin polymerization inhibitor). While "10% stretch at 1 Hz," "20% stretch at 1 Hz," bFGF, and VEGF were strong angiogenesis stimulants individually, only the combination of "20% stretch at 1 Hz" and bFGF had an additive effect on inducing new sprouts. Interestingly, the combination of "20% stretch at a lower frequency (1/12 Hz)" and bFGF decreased sprouting angiogenesis, even though the level of perpendicular alignment of new sprouts was the same for both stretch frequencies. Taken together, these results demonstrate that both stretch frequency and magnitude, along with interactions with various growth factors, are essential in mediating formation of endothelial sprouts and vascular patterning. Furthermore, work in this area is warranted to elucidate synergistic or competitive signaling mechanisms.

Evolution of the Stx2-encoding prophage in persistent bovine Escherichia coli O157:H7 strains.

Escherichia coli O157:H7 is a human pathogen that resides asymptomatically in its bovine host. The level of Shiga toxin (Stx) produced is variable in bovine-derived strains in contrast to human isolates that mostly produce high levels of Stx. To understand the genetic basis for varied Stx production, chronological collections of bovine isolates from Wisconsin dairy farms, R and X, were analyzed for multilocus prophage polymorphisms, stx(2) subtypes, and the levels of stx(2) transcript and toxin. The E. coli O157:H7 that persisted on both farms were phylogenetically distinct and yet produced little to no Stx2 due to gene deletions in Stx2c-encoding prophage (farm R) or insertional inactivation of stx(2a) by IS1203v (farm X). Loss of key regulatory and lysis genes in Stx2c-encoding prophage abolished stx(2c) transcription and induction of the prophage and stx(2a)::IS1203v in Stx2a-encoding prophage generated a truncated stx(2a) mRNA without affecting phage production. Stx2-producing strains were transiently present (farm R) and became Stx2 negative on farm X (i.e., stx(2a)::IS1203v). To our knowledge, this is the first study that details the evolution of E. coli O157:H7 and its Stx2-encoding prophage in a chronological collection of natural isolates. The data suggest the bovine and farm environments can be niches where Stx2-negative E. coli O157:H7 emerge and persist, which explains the Stx variability in bovine isolates and may be part of an evolutionary step toward becoming bovine specialists.

Comparison of active and passive targeting of docetaxel for prostate cancer therapy by HPMA copolymer-RGDfK conjugates.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel-RGDfK conjugate was synthesized, characterized, and evaluated in vitro and in vivo in comparison with untargeted low and high molecular weight HPMA copolymer-docetaxel conjugates. The targeted conjugate was designed to have a hydrodynamic diameter below renal threshold to allow elimination post treatment. All conjugates demonstrated the ability to inhibit the growth of DU145 and PC3 human prostate cancer cells and the HUVEC at low nanomolar concentrations. The targeted conjugate showed active binding to α(v)ß(3) integrins in both HUVEC and DU145 cells, whereas the untargeted conjugate demonstrated no evidence of specific binding. Efficacy at two concentrations (20 mg/kg and 40 mg/kg) was evaluated in nu/nu mice bearing DU145 tumor xenografts treated with a single dose of conjugates and compared with controls. RGDfK targeted and high molecular weight nontargeted conjugates exhibited the highest antitumor efficacy as evaluated by tumor regression. These results demonstrate that α(v)ß(3) integrin targeted polymeric conjugates with improved water solubility, reduced toxicity and ease of elimination post treatment in vivo are promising candidates for prostate cancer therapy.