Severe cases of environmental or exertional heat stress can lead to varying degrees of organ dysfunction. To understand heat-injury progression and develop efficient management and mitigation strategies, it is critical to determine the thermal response in susceptible organs under different heat-stress conditions. To this end, we used our previously published virtual rat, which is capable of computing the spatiotemporal temperature distribution in the animal, and extended it to simulate various heat-stress scenarios, including 1) different environmental conditions, 2) exertional heat stress, 3) circadian rhythm effect on the thermal response, and 4) whole-body cooling. Our predictions were consistent with published in vivo temperature measurements for all cases, validating our simulations. We observed a differential thermal response in the organs, with the liver experiencing the highest temperatures for all environmental and exertional heat-stress cases. For every 3°C rise in the external temperature from 40 to 46°C, core and organ temperatures increased by ~0.8°C. Core temperatures increased by 2.6 and 4.1°C for increases in exercise intensity from rest to 75% and 100% of maximal O2 consumption, respectively. We also found differences as large as 0.8°C in organ temperatures for the same heat stress induced at different times during the day. Even after whole-body cooling at a relatively low external temperature (1°C for 20 min), average organ temperatures were still elevated by 2.3 to 2.5°C compared with normothermia. These results can be used to optimize experimental protocol designs, reduce the amount of animal experimentation, and design and test improved heat-stress prevention and management strategies.
The toxicity of dichlorvos (DDVP), an organophosphate (OP) pesticide, classically results from modification of the serine in the active sites of cholinesterases. However, DDVP also forms adducts on unrelated targets such as transferrin and albumin, suggesting that DDVP could cause perturbations in cellular processes by modifying noncholinesterase targets. Here we identify novel DDVP-modified targets in lysed human hepatocyte-like cells (HepaRG) using a direct liquid chromatography-mass spectrometry (LC-MS) assay of cell lysates incubated with DDVP or using a competitive pull-down experiments with a biotin-linked organophosphorus compound (10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide; FP-biotin), which competes with DDVP for similar binding sites. We show that DDVP forms adducts to several proteins important for the cellular metabolic pathways and differentiation, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. We validated the results using purified proteins and enzymatic assays. The study not only identified novel DDVP-modified targets but also suggested that the modification directly inhibits the enzymes. The current approach provides information for future hypothesis-based studies to understand the underlying mechanism of toxicity of DDVP in non-neuronal tissues. The MS data have been deposited to the ProteomeXchange with identifier PXD001107.
A convergence of technological breakthroughs in the past decade has facilitated the development of rapid screening tools for biomarkers of toxicant exposure and effect. Platforms using the whole adult organism to evaluate the genome-wide response to toxicants are especially attractive. Recent work demonstrates the feasibility of this approach in vertebrates using the experimentally robust zebrafish model. In the present study, we evaluated gene expression changes in whole adult male zebrafish following an acute 24 hr high dose exposure to three metals with known human health risks. Male adult zebrafish were exposed to nickel chloride, cobalt chloride or sodium dichromate concentrations corresponding to their respective 96 hr LC20, LC40 and LC60. Histopathology was performed on a subset of metal-exposed zebrafish to phenotypically anchor transcriptional changes associated with each metal.
Toxic liver injury causes necrosis and fibrosis, which may lead to cirrhosis and liver failure. Despite recent progress in understanding the mechanism of liver fibrosis, our knowledge of the molecular-level details of this disease is still incomplete. The elucidation of networks and pathways associated with liver fibrosis can provide insight into the underlying molecular mechanisms of the disease, as well as identify potential diagnostic or prognostic biomarkers. Towards this end, we analyzed rat gene expression data from a range of chemical exposures that produced observable periportal liver fibrosis as documented in DrugMatrix, a publicly available toxicogenomics database. We identified genes relevant to liver fibrosis using standard differential expression and co-expression analyses, and then used these genes in pathway enrichment and protein-protein interaction (PPI) network analyses. We identified a PPI network module associated with liver fibrosis that includes known liver fibrosis-relevant genes, such as tissue inhibitor of metalloproteinase-1, galectin-3, connective tissue growth factor, and lipocalin-2. We also identified several new genes, such as perilipin-3, legumain, and myocilin, which were associated with liver fibrosis. We further analyzed the expression pattern of the genes in the PPI network module across a wide range of 640 chemical exposure conditions in DrugMatrix and identified early indications of liver fibrosis for carbon tetrachloride and lipopolysaccharide exposures. Although it is well known that carbon tetrachloride and lipopolysaccharide can cause liver fibrosis, our network analysis was able to link these compounds to potential fibrotic damage before histopathological changes associated with liver fibrosis appeared. These results demonstrated that our approach is capable of identifying early-stage indicators of liver fibrosis and underscore its potential to aid in predictive toxicity, biomarker identification, and to generally identify disease-relevant pathways.
Liver injuries due to ingestion or exposure to chemicals and industrial toxicants pose a serious health risk that may be hard to assess due to a lack of non-invasive diagnostic tests. Mapping chemical injuries to organ-specific damage and clinical outcomes via biomarkers or biomarker panels will provide the foundation for highly specific and robust diagnostic tests. Here, we have used DrugMatrix, a toxicogenomics database containing organ-specific gene expression data matched to dose-dependent chemical exposures and adverse clinical pathology assessments in Sprague Dawley rats, to identify groups of co-expressed genes (modules) specific to injury endpoints in the liver. We identified 78 such gene co-expression modules associated with 25 diverse injury endpoints categorized from clinical pathology, organ weight changes, and histopathology. Using gene expression data associated with an injury condition, we showed that these modules exhibited different patterns of activation characteristic of each injury. We further showed that specific module genes mapped to 1) known biochemical pathways associated with liver injuries and 2) clinically used diagnostic tests for liver fibrosis. As such, the gene modules have characteristics of both generalized and specific toxic response pathways. Using these results, we proposed three gene signature sets characteristic of liver fibrosis, steatosis, and general liver injury based on genes from the co-expression modules. Out of all 92 identified genes, 18 (20%) genes have well-documented relationships with liver disease, whereas the rest are novel and have not previously been associated with liver disease. In conclusion, identifying gene co-expression modules associated with chemically induced liver injuries aids in generating testable hypotheses and has the potential to identify putative biomarkers of adverse health effects.
Early prediction of the adverse outcomes associated with heat stress is critical for effective management and mitigation of injury, which may sometimes lead to extreme undesirable clinical conditions, such as multiorgan dysfunction syndrome and death. Here, we developed a computational model to predict the spatiotemporal temperature distribution in a rat exposed to heat stress in an attempt to understand the correlation between heat load and differential organ dysfunction. The model includes a three-dimensional representation of the rat anatomy obtained from medical imaging and incorporates the key mechanisms of heat transfer during thermoregulation. We formulated a novel approach to estimate blood temperature by accounting for blood mixing from the different organs and to estimate the effects of the circadian rhythm in body temperature by considering day-night variations in metabolic heat generation and blood perfusion. We validated the model using in vivo core temperature measurements in control and heat-stressed rats and other published experimental data. The model predictions were within 1 SD of the measured data. The liver demonstrated the greatest susceptibility to heat stress, with the maximum temperature reaching 2°C higher than the measured core temperature and 95% of its volume exceeding the targeted experimental core temperature. Other organs also attained temperatures greater than the core temperature, illustrating the need to monitor multiple organs during heat stress. The model facilitates the identification of organ-specific risks during heat stress and has the potential to aid in the development of improved clinical strategies for thermal-injury prevention and management.
Endometriosis is a hormone-dependent inflammatory condition associated with pain and infertility. A growing body of evidence supports attenuated secretory-phase progesterone responsiveness in women with this disease. Herein, we compare the expression of progesterone receptor membrane components (PGRMC) 1 and 2 in eutopic endometrium from 11 women with laparoscopically and/or histologically proven stage III/IV endometriosis and 23 disease-free women. Menstrual cycle phase was determined using a combination of reported cycle day, serum hormone profile, and endometrial histologic dating. The PGRMC-1 (fold change -3.3; P < .05) and PGRMC-2 (fold-change -8.8; P < .05) gene expression were significantly downregulated in secretory phase, eutopic endometrium from women with endometriosis. Immunohistochemistry demonstrated decreased PGRMC-1 and PGRMC-2 protein expression in the secretory phase endometrial stroma cells of women with endometriosis. Consistent with the preclinical work of others, our results reflect downregulation of endometrial PGRMC-1 and PGRMC-2 expression in secretory phase endometrium from women with advanced stage endometriosis. Understanding the molecular mechanisms of attenuated progesterone action in endometriosis has important diagnostic and therapeutic implications.
Glycosylation of plasma proteins increases during pregnancy. Our objectives were to investigate an anti-inflammatory role of these proteins in normal pregnancies and determine whether aberrant protein glycosylation promotes monocyte adhesion in preeclampsia. Plasma was prospectively collected from nonpregnant controls and nulliparous patients in all 3 trimesters. Patients were divided into cohorts based on the applicable postpartum diagnosis. U937 monocytes were preconditioned with enzymatically deglycosylated plasma, and monocyte adhesion to endothelial cell monolayers was quantified by spectrophotometry. Plasma from nonpregnant controls, first trimester normotensives, and first trimester patients with mild preeclampsia inhibited monocyte-endothelial cell adhesion (P < .05), but plasma from first trimester patients with severe preeclampsia and second and third trimester normotensives did not. Deglycosylating plasma proteins significantly increased adhesion in all the cohorts. These results support a role of plasma glycoprotein interaction in monocyte-endothelial cell adhesion and could suggest a novel therapeutic target for severe preeclampsia.
Researchers collaborate on scientific projects that are often measured by both the quantity and the quality of the resultant peer-reviewed publications. However, not all collaborators contribute to these publications equally, making metrics such as the total number of publications and the H-index insufficient measurements of individual scientific impact. To remedy this, we use an axiomatic approach to assign relative credits to the coauthors of a given paper, referred to as the A-index for its axiomatic foundation. In this paper, we use the A-index to compute the weighted sums of peer-reviewed publications and journal impact factors, denoted as the C- and P-indexes for collaboration and productivity, respectively. We perform an in-depth analysis of bibliometric data for 186 biomedical engineering faculty members and from extensive simulation. It is found that these axiomatically weighted indexes better capture a researchers scientific caliber than do the total number of publications and the H-index, allowing for fairer and sharper evaluation of researchers with diverse collaborative behaviors.
Hemorrhagic shock and subsequent resuscitation can lead to ischemia-reperfusion injury, followed by multiorgan failure and death. Flutamide, a vasoactive nonsteroidal antiandrogen compound, is thought to improve tissue and organ perfusion. We tested whether administration of flutamide-cyclodextrin (FLU-CYD) alters physiologic parameters or resuscitation requirements in a porcine model of severe acidosis and shock secondary to combined hemorrhage + ischemia-reperfusion injury.
Valproic acid (VPA) is a histone deacetylase inhibitor that may decrease cellular metabolic needs following traumatic injury. We hypothesized that VPA may have beneficial effects in preventing or reducing the cellular and metabolic sequelae of ischemia-reperfusion injury.
Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1? signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.
Irreversible electroporation (IRE) is a non-thermal focal ablation technique that uses a series of brief but intense electric pulses delivered into a targeted region of tissue, killing the cells by irrecoverably disrupting cellular membrane integrity. This study investigates if there is an improved local anti-tumor response in immunocompetent (IC) BALB/c versus immunodeficient (ID) nude mice, including the potential for a systemic protective effect against rechallenge. Subcutaneous murine renal carcinoma tumors were treated with an IRE pulsing protocol that used 60% of the predicted voltage required to invoke complete regressions in the ID mice. Tumors were followed for 34 days following treatment for 11 treated mice from each strain, and 7 controls from each strain. Mouse survival based on tumor burden and the progression-free disease period was substantially longer in the treated IC mice relative to the treated ID mice and sham controls for both strains. Treated IC mice were rechallenged with the same cell line 18 days after treatment, where growth of the second tumors was shown to be significantly reduced or prevented entirely. There was robust CD3+ cell infiltration in some treated BALB/C mice, with immunocytes focused at the transition between viable and dead tumor. There was no difference in the low immunocyte presence for untreated tumors, nude mice, and matrigel-only injections in both strains. These findings suggest IRE therapy may have greater therapeutic efficacy in immunocompetent patients than what has been suggested by immunodeficient models, and that IRE may invoke a systemic response beyond the targeted ablation region.
Vascular endothelial cells serve as the first line of defense for end organs after ischemia and reperfusion injuries. The full etiology of this dysfunction is poorly understood, and valproic acid (VPA) has proven to be beneficial after traumatic injury. The purpose of this study was to determine the mechanism of action through which VPA exerts its beneficial effects.
Preeclampsia is a hypertensive disorder unique to pregnancy. Although the pathogenesis of the disease begins with aberrant spiral artery invasion in the first trimester, clinical symptoms usually do not present until late in pregnancy. Apolipoprotein CII (ApoCII) and its negative regulator, apolipoprotein CIII (ApoCIII), have recently been described as atherogenesis biomarkers in models of cardiovascular disease. Given the similarities in pathology, etiology, and clinical presentation between cardiovascular disease and preeclampsia, we hypothesized that the ratio of ApoCIII to ApoCII in maternal first trimester plasma would predict preeclampsia later in pregnancy. To test this hypothesis, plasma was prospectively collected from 311 nulliparas at 8 to 12 weeks gestation. After delivery, patients were divided into cohorts based on preeclampsia diagnosis. Conditioning monocytes with preeclamptic plasma potentiated monocyte adhesion to endothelial cells in an in vitro model. The ratio of ApoCIII to ApoCII was significantly elevated in patients with severe preeclampsia relative to normotensive and gestational hypertensive individuals (P < .05) as determined by mass spectrometry and competitive enzyme-linked immunosorbent assay (ELISA) assays. These results support a predictive change in the ratio of ApoCIII to ApoCII in pregnancies complicated by severe preeclampsia.
Trauma leading to massive hemorrhage results in widespread tissue hypoxia, anaerobic metabolism, and production of inflammatory cytokines and oxidative molecules injurious to the vascular endothelium. Although trauma-related endothelial cell pathophysiology has been extensively studied, very little is known regarding gene transcriptional changes that occur during the event, particularly in endothelia. Thus, we employed fluorescent microarray analysis of gene transcription to elucidate critical pathways and gene products involved in endothelial dysfunction.
17?-hydroxyprogesterone caproate (17P) may decrease risk of prematurity by suppressing maternal immunity. We hypothesized that in vivo 17P treatment attenuates immunoresponsiveness of peripheral blood mononuclear cells (PBMCs).
Clinical evidence suggests that magnesium sulfate may reduce the risk of fetal neurologic injury in preterm delivery. Matrix metalloproteinase-9 (MMP-9) levels are elevated in preterm labor patients. There is evidence that MMP-9 may break down the blood-brain barrier in humans, causing cytokine mediated cell injury. Our objective was to determine whether the addition of magnesium sulfate attenuates activity of MMP-9, a complex zinc-dependent enzyme, in fetal cord plasma.
The expression of progesterone receptor membrane component 1 (PGRMC1) in breast cancer has generated interest in this recently discovered protein because of its role in tumorigenesis. However, correlations between patient age, PGRMC1 gene expression, breast cancer morphology, and breast cancer stage have not been adequately studied. Furthermore, very little is known about possible roles for other PGRMC isoforms in breast cancer, like PGRMC2. Thus, we examined the expression of PGRMC1 and PGRMC2 mRNA by relative quantitative PCR (RelqPCR) and determined whether transcript levels correlate with age, breast cancer staging, estrogen receptor alpha (ER?) status, and other morphometric features routinely used during the pathological examination of breast ductal adenocarcinomas.
Minimally invasive diagnostic tests are needed in obstetrics to identify women at risk for complications during delivery. The apolipoproteins fluctuate in complexity and abundance in maternal plasma during pregnancy and could be incorporated into a blood test to evaluate this risk. The objective of this study was to examine the relative plasma concentrations of apolipoproteins and their biochemically modified subtypes (i.e. proteolytically processed, sialylated, cysteinylated, dimerized) over gestational time using a targeted mass spectrometry approach. Relative abundance of modified and unmodified apolipoproteins A-I, A-II, C-I, C-II, and C-III was determined by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in plasma prospectively collected from 11 gravidas with uncomplicated pregnancies at 4-5 gestational time points per patient. Apolipoproteins were readily identifiable by spectral pattern. Apo C-III(2) and Apo C-III(1) (doubly and singly sialylated Apo C-III subtypes) increased with gestational age (r(2)>0.8). Unmodified Apo A-II, Apo C-I, and Apo C-III(0) showed no correlation (r(2) = 0.01-0.1). Pro-Apo C-II did not increase significantly until third trimester (140 ± 13% of first trimester), but proteolytically cleaved, mature Apo C-II increased in late pregnancy (702 ± 130% of first trimester). Mature Apo C-II represented 6.7 ± 0.9% of total Apo C-II in early gestation and increased to 33 ± 4.5% in third trimester. A label-free, semiquantitative targeted proteomics approach was developed using LTQ-Orbitrap mass spectrometry to confirm the relative quantitative differences observed by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry in Apo C-III and Apo C-II isoforms between first and third trimesters. Targeted apolipoprotein screening was applied to a cohort of term and preterm patients. Modified Apo A-II isoforms were significantly elevated in plasma from mothers who delivered prematurely relative to term controls (p = 0.02). These results support a role for targeted proteomics profiling approaches in monitoring healthy pregnancies and assessing risk of adverse obstetric outcomes.
Opportunistic pathogens represent a unique challenge because they establish and grow within drinking water systems, yet the factors stimulating their proliferation are largely unknown. The purpose of this study was to examine the influence of pipe materials, disinfectant type, and water age on occurrence and persistence of three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa), broader genera (Legionella and mycobacteria), and two amoeba hosts (Acanthamoeba spp. and Hartmanella vermiformis). Triplicate simulated distribution systems (SDSs) compared iron, cement, and PVC pipe materials fed either chlorinated or chloraminated tap water and were sampled at water ages ranging from 1 day to 5.7 days. Quantitative polymerase chain reaction quantified gene copies of target microorganisms in both biofilm and bulk water. Legionella, mycobacteria, P. aeruginosa, and both amoebas naturally colonized the six SDSs, but L. pneumophila and M. avium were not detected. Disinfectant type and dose was observed to have the strongest influence on the microbiota. Disinfectant decay was noted with water age, particularly in chloraminated SDSs (due to nitrification), generally resulting in increased microbial detection frequencies and densities with water age. The influence of pipe material became apparent at water ages corresponding to low disinfectant residual. Each target microbe appeared to display a distinct response to disinfectant type, pipe materials, water age, and their interactions. Differences between the first and the second samplings (e.g., appearance of Legionella, reduction in P. aeruginosa and Acanthamoeba) suggest a temporally dynamic drinking water microbial community.
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