Fecal metabolomics reveals products of dysregulated proteolysis and altered microbial metabolism in obesity-related osteoarthritis.

OBJECTIVE: The objective of this exploratory study was to determine if perturbations in gut microbial composition and the gut metabolome could be linked to individuals with obesity and osteoarthritis (OA). METHODS: Fecal samples were collected from obese individuals diagnosed with radiographic hand plus knee OA (n = 59), defined as involvement of at least 3 joints across both hands, and a Kellgren-Lawrence (KL) grade 2-4 (or total knee replacement) in at least one knee. Controls (n = 33) were without hand OA and with KL grade 0-1 knees. Fecal metabolomes were analyzed by a UHPLC/Q Exactive HFx mass spectrometer. Microbiome composition was determined in fecal samples by 16 S ribosomal RNA amplicon sequencing (rRNA-seq). Stepwise logistic regression models were built to determine microbiome and/or metabolic characteristics of OA. RESULTS: Untargeted metabolomics analysis indicated that OA cases had significantly higher levels of di- and tripeptides and significant perturbations in microbial metabolites including propionic acid, indoles, and other tryptophan metabolites. Pathway analysis revealed several significantly perturbed pathways associated with OA including leukotriene metabolism, amino acid metabolism and fatty acid utilization. Logistic regression models selected metabolites associated with the gut microbiota and leaky gut syndrome as significant predictors of OA status, particularly when combined with the rRNA-seq data. CONCLUSIONS: Adults with obesity and knee plus hand OA have distinct fecal metabolomes characterized by increased products of proteolysis, perturbations in leukotriene metabolism, and changes in microbial metabolites compared with controls. These metabolic perturbations indicate a possible role of dysregulated proteolysis in OA.

Infection by the castrating parasitic nematode Sphaerularia bombi changes gene expression in Bombus terrestris bumblebee queens.

Parasitism can result in dramatic changes in host phenotype, which are themselves underpinned by genes and their expression. Understanding how hosts respond at the molecular level to parasites can therefore reveal the molecular architecture of an altered host phenotype. The entomoparasitic nematode Sphaerularia bombi is a parasite of bumblebee (Bombus) hosts where it induces complex behavioural changes and host castration. To examine this interaction at the molecular level, we performed genome-wide transcriptional profiling using RNA-Sequencing (RNA-Seq) of S. bombi-infected Bombus terrestris queens at two critical time-points: during and just after overwintering diapause. We found that infection by S. bombi affects the transcription of genes underlying host biological processes associated with energy usage, translation, and circadian rhythm. We also found that the parasite affects the expression of immune genes, including members of the Toll signalling pathway providing evidence for a novel interaction between the parasite and the host immune response. Taken together, our results identify host biological processes and genes affected by an entomoparasitic nematode providing the first steps towards a molecular understanding of this ecologically important host-parasite interaction.

Metabolomics to reveal biomarkers and pathways of preterm birth: a systematic review and epidemiologic perspective.

INTRODUCTION: Most known risk factors for preterm birth, a leading cause of infant morbidity and mortality, are not modifiable. Advanced molecular techniques are increasingly being applied to identify biomarkers and pathways important in disease development and progression. OBJECTIVES: We review the state of the literature and assess it from an epidemiologic perspective. METHODS: PubMed, Embase, CINAHL, and Cochrane Central were searched on January 31, 2019 for original articles published after 1998 that utilized an untargeted metabolomic approach to identify markers of preterm birth. Eligible manuscripts were peer-reviewed and included original data from untargeted metabolomics analyses of maternal tissue derived from human studies designed to determine mechanisms and predictors of preterm birth. RESULTS: Of 2823 results, 14 articles met the inclusion requirements. There was little consistency in study design, outcome definition, type of biospecimen, or the inclusion of covariates and confounding factors, and few consistent associations with metabolites were identified in this review. CONCLUSION: Studies to date on metabolomic predictors of preterm birth are highly heterogeneous in both methodology and resulting metabolite identification. There is an urgent need for larger studies in well-defined populations, to determine biomarkers predictive of preterm birth, and to reveal mechanisms and targets for development of intervention strategies.

Association of urinary metabolites with radiographic progression of knee osteoarthritis in overweight and obese adults: an exploratory study.

INTRODUCTION: Metabolic factors may contribute to osteoarthritis (OA). This study employed metabolomics analyses to determine if differences in metabolite profiles could distinguish people with knee OA who exhibited radiographic progression. METHODS: Urine samples obtained at baseline and 18 months from overweight and obese adults in the Intensive Diet and Exercise for Arthritis (IDEA) trial were selected from two subgroups (n = 22 each) for metabolomics analysis: a group that exhibited radiographic progression (≥0.7 mm decrease in joint space width, JSW) and an age, gender, and body mass index (BMI) matched group who did not progress (≤0.35 mm decrease in JSW). Multivariate analysis methods, including orthogonal partial least square discriminate analysis, were used to identify metabolite profiles that separated progressors and non-progressors. Plasma levels of IL-6 and C-reactive protein (CRP) were evaluated as inflammatory markers. RESULTS: Multivariate analysis of the binned metabolomics data distinguished progressors from non-progressors. Library matching revealed that glycolate, hippurate, and trigonelline were among the important metabolites for distinguishing progressors from non-progressors at baseline whereas alanine, N,N-dimethylglycine, glycolate, hippurate, histidine, and trigonelline, were among the metabolites that were important for the discrimination at 18 months. In non-progressors, IL-6 decreased from baseline to 18 months while IL-6 was unchanged in progressors; the change over time in IL-6 was significantly different between groups. CONCLUSION: These findings support a role for metabolic factors in the progression of knee OA and suggest that measurement of metabolites could be useful to predict progression. Further investigation in a larger sample that would include targeted investigation of specific metabolites is warranted.

Chylothorax secondary to subcutaneous cervical lipoma in a dog.

An 8-year-old male neutered American English Coonhound was presented for a 2-day history of increased respiratory effort and rate with an occasional cough. Thoracic radiographs noted pleural effusion, which was chylous based on cytological and chemical evaluation. The dog also had a 2-year history of a slow growing fatty mass in the right cervical region. A CT scan confirmed the large cervical fat attenuating mass extending from the base of the skull to the cranial thorax and right axillary region with compression of vascular structures. Severe bilateral effusion and secondary pulmonary atelectasis was noted within the thoracic cavity. It was elected to surgically remove the cervical mass and place a PleuralPort within the thoracic cavity. The mass was diagnosed as a lipoma and its removal led to rapid and complete resolution of chylothorax. Based on the literature search, this is the first case report of chylothorax secondary to a cervical mass or subcutaneous lipoma.

Pathways to immunity: temporal dynamics of the bumblebee (Bombus terrestris) immune response against a trypanosomal gut parasite.

Immune response dynamics in insects from natural host-parasite associations are poorly understood, despite accumulating evidence of ecological immune phenomena in these systems. Using a gene discovery approach, we have identified genes relating to signalling, enzymatic processes and respiration that were up-regulated in the bumblebee, Bombus terrestris, during infection with the trypanosomatid parasite, Crithidia bombi. In addition, we have mapped dynamic changes in the temporal expression of these genes and three candidate antimicrobial peptide (AMP) immune genes, Abaecin, Defensin and Hymenoptaecin, from 1 to 24 h after C. bombi infection. We show that dynamic changes in expression occur for individual genes at distinct phases of the immune response to C. bombi that correspond to early, intermediate and late stages of infection.

Untargeted analysis of first trimester serum to reveal biomarkers of pregnancy complications: a case-control discovery phase study.

Understanding of causal biology and predictive biomarkers are lacking for hypertensive disorders of pregnancy (HDP) and preterm birth (PTB). First-trimester serum specimens from 51 cases of HDP, including 18 cases of pre-eclampsia (PE) and 33 cases of gestational hypertension (GH); 53 cases of PTB; and 109 controls were obtained from the Global Alliance to Prevent Prematurity and Stillbirth repository. Metabotyping was conducted using liquid chromatography high resolution mass spectroscopy and nuclear magnetic resonance spectroscopy. Multivariable logistic regression was used to identify signals that differed between groups after controlling for confounders. Signals important to predicting HDP and PTB were matched to an in-house physical standards library and public databases. Pathway analysis was conducted using GeneGo MetaCore. Over 400 signals for endogenous and exogenous metabolites that differentiated cases and controls were identified or annotated, and models that included these signals produced substantial improvements in predictive power beyond models that only included known risk factors. Perturbations of the aminoacyl-tRNA biosynthesis, L-threonine, and renal secretion of organic electrolytes pathways were associated with both HDP and PTB, while pathways related to cholesterol transport and metabolism were associated with HDP. This untargeted metabolomics analysis identified signals and common pathways associated with pregnancy complications.

Diminishing returns drive altruists to help extended family.

Altruism between close relatives can be easily explained. However, paradoxes arise when organisms divert altruism towards more distantly related recipients. In some social insects, workers drift extensively between colonies and help raise less related foreign brood, seemingly reducing inclusive fitness. Since being highlighted by W. D. Hamilton, three hypotheses (bet hedging, indirect reciprocity and diminishing returns to cooperation) have been proposed for this surprising behaviour. Here, using inclusive fitness theory, we show that bet hedging and indirect reciprocity could only drive cooperative drifting under improbable conditions. However, diminishing returns to cooperation create a simple context in which sharing workers is adaptive. Using a longitudinal dataset comprising over a quarter of a million nest cell observations, we quantify cooperative payoffs in the Neotropical wasp Polistes canadensis, for which drifting occurs at high levels. As the worker-to-brood ratio rises in a worker's home colony, the predicted marginal benefit of a worker for expected colony productivity diminishes. Helping related colonies can allow effort to be focused on related brood that are more in need of care. Finally, we use simulations to show that cooperative drifting evolves under diminishing returns when dispersal is local, allowing altruists to focus their efforts on related recipients. Our results indicate the power of nonlinear fitness effects to shape social organization, and suggest that models of eusocial evolution should be extended to include neglected social interactions within colony networks.

Use of UV light for the inactivation of Listeria monocytogenes and lactic acid bacteria species in recirculated chill brines.

Ready-to-eat meat products have been implicated in several foodborne listeriosis outbreaks. Microbial contamination of these products can occur after thermal processing when products are chilled in salt brines. The objective of this study was to evaluate UV radiation on the inactivation of Listeria monocytogenes and lactic acid bacteria in a model brine chiller system. Two concentrations of brine (7.9% [wt/wt] or 13.2% [wt/wt]) were inoculated with a approximately 6.0 log CFU/ml cocktail of L. monocytogenes or lactic acid bacteria and passed through a UV treatment system for 60 min. Three replications of each bacteria-and-brine combination were performed and resulted in at least a 4.5-log reduction in microbial numbers in all treated brines after exposure to UV light. Bacterial populations were significantly reduced after 5 min of exposure to UV light in the model brine chiller compared with the control, which received no UV light exposure (P < 0.05). The maximum rate of inactivation for both microorganisms in treated brines occurred between minutes 1 and 15 of UV exposure. Results indicate that in-line treatment of chill brines with UV light reduces the number of L. monocytogenes and lactic acid bacteria.

Acute intravenous exposure to silver nanoparticles during pregnancy induces particle size and vehicle dependent changes in vascular tissue contractility in Sprague Dawley rats.

The use of silver nanoparticles (AgNP) raises safety concerns during susceptible life stages such as pregnancy. We hypothesized that acute intravenous exposure to AgNP during late stages of pregnancy will increase vascular tissue contractility, potentially contributing to alterations in fetal growth. Sprague Dawley rats were exposed to a single dose of PVP or Citrate stabilized 20 or 110nm AgNP (700µg/kg). Differential vascular responses and EC50 values were observed in myographic studies in uterine, mesenteric arteries and thoracic aortic segments, 24h post-exposure. Reciprocal responses were observed in aortic and uterine vessels following PVP stabilized AgNP with an increased force of contraction in uterine artery and increased relaxation responses in aorta. Citrate stabilized AgNP exposure increased contractile force in both uterine and aortic vessels. Intravenous AgNP exposure during pregnancy displayed particle size and vehicle dependent moderate changes in vascular tissue contractility, potentially influencing fetal blood supply.

Effects of ultraviolet irradiation on chemical and sensory properties of goat milk.

Sensory and chemical consequences of treating goat milk using an UV fluid processor were assessed. Milk was exposed to UV for a cumulative exposure time of 18 s and targeted UV dose of 15.8 +/- 1.6 mJ/cm2. A triangle test revealed differences between the odor of raw milk and UV irradiated milk. Oxidation and hydrolytic rancidity was measured by thiobarbituric acid reactive substances and acid degree values (ADV). As UV dose increased, there was an increase in thiobarbituric acid reactive substance values and ADV of the milk samples. A separate set of samples were processed using the fluid processor but with no UV exposure to see if lipase activity and agitation from pumping contributed to the differences in odor. The ADV increased at the same rate as samples exposed to UV; however, sensory studies indicated that the increase of free fatty acids was not enough to cause detectable differences in the odor of milk. Solid phase microextraction and gas chromatography were utilized for the analysis of volatile compounds as a result of UV exposure. There was an increase in the concentration of pentanal, hexanal, and heptanal (relative to raw goat milk) after as little as 1.3 mJ/cm2 UV dose. Ultraviolet irradiation at the wavelength 254 nm produced changes in the sensory and chemical properties of fluid goat milk.

N-terminal domain of BARF1 gene encoded by Epstein-Barr virus is essential for malignant transformation of rodent fibroblasts and activation of BCL-2.

The BARF1 gene encoded by the Epstein-Barr virus induces morphological changes, loss of contact inhibition and anchorage independence in established rodent Balb/c3T3 fibroblast. BARF1 gene was also capable of inducing malignant transformation in a human Louckes B cell line. Our recent study showed that BARF1 gene had an ability to immortalize primary epithelial cells. However we do not know which region(s) of BARF1 protein is(are) responsible for inducing malignant transformation in established rodent cells. Using the deletion mutants, we now localized a malignant transforming region in N-terminal of BARF1 protein. The mutants lacking this region were unable to transform the cells in malignant state. Furthermore, we demonstrated that only the mutants containing this region rendered the cells resistant to apoptosis induced by serum deprivation. Surprisingly, the BARF1 gene was capable of activating anti-apoptotic Bcl-2 expression and this activation was due to the N-terminal transforming region. These data suggest that the cooperation of BARF1 with Bcl-2 is essential for the induction of malignant transformation.

Macrophages in gene therapy: cellular delivery vehicles and in vivo targets.

The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumors, atherosclerotic plaques, and arthritic joints. This has prompted recent attempts to use macrophages as novel cellular vehicles for gene therapy, in which macrophages are genetically modified ex vivo and then reintroduced into the body with the hope that a proportion will then home to the diseased site. Here, we critically review the efficacy of various gene transfer methods (viral, bacterial, protozoan, and various chemical and physical methods) in transfecting macrophages in vitro, and the results obtained when transfected macrophages are used as gene delivery vehicles. Finally, we discuss the use of various viral and nonviral methods to transfer genes to macrophages in vivo. As will be seen, definitive evidence for the use of macrophages as gene transfer vehicles has yet to be provided and awaits detailed trafficking studies in vivo. Moreover, although methods for transfecting macrophages have improved considerably in efficiency in recent years, targeting of gene transfer specifically to macrophages in vivo remains a problem. However, possible solutions to this include placing transgenes under the control of macrophage-specific promoters to limit expression to macrophages or stably transfecting CD34(+) precursors of monocytes/macrophages and then differentiating these cells into monocytes/macrophages ex vivo. The latter approach could conceivably lead to the bone marrow precursor cells of patients with inherited genetic disorders being permanently fortified or even replaced with genetically modified cells.

Efficacy of UV light for the reduction of Listeria monocytogenes in goat's milk.

Certain types of goat's cheeses are produced using unpasteurized milk, which increases the food safety concerns for these types of products. Popularity and consumption of goat's milk products have increased, and the niche market includes gourmet goat's cheeses. The U.S. Code of Federal Regulations and the Pasteurized Milk Ordinance both address the possibility for processing alternatives to heat treatment, and the use of UV light treatment may be a viable alternative that still ensures the safety of the product. Fresh goat's milk was inoculated with Listeria monocytogenes (L-2289) at 10(7) CFU/ml and exposed to UV light using the CiderSure 3500 apparatus (FPE Inc., Macedon, NY). Inoculated milk was exposed to a UV dose range between 0 and 20 mJ/cm2 to determine the optimal UV dose. A greater than 5-log reduction was achieved (P < 0.0001) when the milk received a cumulative UV dose of 15.8 +/- 1.6 mJ/cm2. The results of this study indicate that UV irradiation could be used for the reduction of L. monocytogenes in goat's milk.

Cardiac Ischemia Reperfusion Injury Following Instillation of 20 nm Citrate-capped Nanosilver.

BACKGROUND: Silver nanoparticles (AgNP) have garnered much interest due to their antimicrobial properties, becoming one of the most utilized nano-scale materials. However, any potential evocable cardiovascular injury associated with exposure has not been reported to date. We have previously demonstrated expansion of myocardial infarction after intratracheal (IT) instillation of carbon-based nanomaterials. We hypothesized pulmonary exposure to Ag core AgNP induces a measureable increase in circulating cytokines, expansion of cardiac ischemia-reperfusion (I/R) injury and is associated with depressed coronary constrictor and relaxation responses. Secondarily, we addressed the potential contribution of silver ion release on AgNP toxicity. METHODS: Male Sprague-Dawley rats were exposed to 200 µl of 1 mg/ml of 20 nm citrate-capped Ag core AgNP, 0.01, 0.1, 1 mg/ml Silver Acetate (AgAc), or a citrate vehicle by intratracheal (IT) instillation. One and 7 days following IT instillation the lungs were evaluated for inflammation and the presence of silver; serum was analyzed for concentrations of selected cytokines; cardiac I/R injury and coronary artery reactivity were assessed. RESULTS: AgNP instillation resulted in modest pulmonary inflammation with detection of silver in lung tissue and alveolar macrophages, elevation of serum cytokines: G-CSF, MIP-1α, IL-1ß, IL-2, IL-6, IL-13, IL-10, IL-18, IL-17α, TNFα, and RANTES, expansion of I/R injury and depression of the coronary vessel reactivity at 1 day post IT compared to vehicle treated rats. Silver within lung tissue was persistent at 7 days post IT instillation and was associated with an elevation in cytokines: IL-2, IL-13, and TNFα and expansion of I/R injury. AgAc resulted in a concentration dependent infarct expansion and depressed vascular reactivity without marked pulmonary inflammation or serum cytokine response. CONCLUSIONS: Based on these data, IT instillation of AgNP increases circulating levels of several key cytokines, which may contribute to persistent expansion of I/R injury possibly through an impaired vascular responsiveness.

Conformational behavior of the linear hexapeptide senktide: a receptor specific tachykinin analog.

A receptor selective linear hexapeptide tachykinin analog, senktide, is shown to be highly ordered in solution. The conformational restriction is attributed to steric and electrostatic interactions produced by N-methylation of the third amino acid residue in the sequence and the negatively charged N-terminus. The structure of senktide is described as a dynamic mixture of similar conformations where the predominant one is a distorted antiparallel hydrogen bonded beta-pleated sheet. The observed senktide-receptor specificity is suggested to result from a selection of this or a closely related conformation.

A model for the formation and removal of hemoglobin adducts.

Hemoglobin adducts formed by chemical carcinogens can be used as biomarkers of exposure. The kinetics of adduct formation and removal is complex and depends on the processes involved in erythrocyte removal, adduct stability, and the duration and extent of exposure. In order to relate the formation of adducts to the extent of exposure in complex exposure scenarios, a model has been developed to describe the kinetics of accumulation and removal of adducts formed in vivo. The exposure scenario, lifetime of erythrocytes, and extent of adduct formation following a single exposure are required input parameters. Predictions of adduct accumulation have been generated for a wide variety of exposure scenarios and compared with both the solutions to equations derived for adduct formation and removal and experimental observations. Loss of adduct by removal of erythrocytes from circulation, both by senescence and random removal and as a result of chemical instability, has been simulated. Equations have been derived to describe the removal of hemoglobin adducts under conditions of exposure for less than the lifetime of the erythrocyte, when removal is initially a linear function of time. This model makes possible the comparison of data obtained from different exposure scenarios and in different species.

Vascular cell responses to polysaccharide materials: in vitro and in vivo evaluations.

Chitosan has shown promise as a structural material for a number of tissue engineering applications. Similarly the glycosaminoglycans (GAGs) and their analogs have been known to exert a variety of biological activities. In this study we evaluated the potential of GAG-chitosan and dextran sulfate (DS)-chitosan complex materials for controlling the proliferation of vascular endothelial (EC) and smooth muscle cells (SMC). GAG-chitosan complex membranes were generated in vitro and seeded with human ECs or SMCs for culture up to 9d. In addition, porous chitosan and GAG-chitosan complex scaffolds were implanted subcutaneously in rats to evaluate the in vivo response to these materials. The results indicated that while chitosan alone supported cell attachment and growth, GAG-chitosan materials inhibited spreading and proliferation of ECs and SMCs in vitro. In contrast, DS-chitosan surfaces supported proliferation of both cell types. In vivo, heparin-chitosan and DS-chitosan scaffolds stimulated cell proliferation and the formation of a thick layer of dense granulation tissue. In the case of heparin scaffolds the granulation layer was highly vascularized. These results indicate that the GAG-chitosan materials can be used to modulate the proliferation of vascular cells both in vitro and in vivo.

A physiological model for tert-amyl methyl ether and tert-amyl alcohol: hypothesis testing of model structures.

The oxygenate tert-amyl methyl ether (TAME) is a gasoline fuel additive used to reduce carbon monoxide in automobile emissions. To evaluate the relative health risk of TAME as a gasoline additive, information is needed on its pharmacokinetics and toxicity. The objective of this study was to use a physiologically-based pharmacokinetic (PBPK) model to describe the disposition of TAME and its major metabolite, tert-amyl alcohol (TAA), in male Fischer-344 rats. The model compartments for TAME and TAA were flow-limited. The TAME physiological model had 6 compartments: lung, liver, rapidly perfused tissues, slowly perfused tissues, fat, and kidney. The TAA model had 3 compartments: lung, liver, and total-body water. The 2 models were linked through metabolism of TAME to TAA in the liver. Model simulations were compared with data on blood concentrations of TAME and TAA taken from male Fischer-344 rats during and after a 6-hour inhalation exposure to 2500, 500, or 100 ppm TAME. The PBPK model predicted TAME pharmacokinetics when 2 saturable pathways for TAME oxidation were included. The TAA model, which included pathways for oxidation and glucuronide conjugation of TAA, underpredicted the experimental data collected at later times postexposure. To account for biological processes occurring during this time, three hypotheses were developed: nonspecific binding of TAA, diffusion-limited transport of TAA, and enterohepatic circulation of TAA glucuronide. These hypotheses were tested using three different model structures. Visual inspection and statistical evaluation involving maximum likelihood techniques indicated that the model incorporating nonspecific binding of TAA provided the best fit to the data. A correct model structure, based upon experimental data, statistical analyses, and biological interpretation, will allow a more accurate extrapolation to humans and, consequently, a greater understanding of human risk from exposure to TAME.