Diverse calcium signaling profiles regulate migratory behavior in avascular wound healing and aberrant signal hierarchy occurs early in diabetes.

In avascular wound repair, calcium signaling events are the predominant mechanism cells use to transduce information about stressors in the environment into an effective and coordinated migratory response. Live cell imaging and computational analysis of corneal epithelial wound healing revealed that signal initiation and propagation at the wound edge are highly ordered, with groups of cells engaging in cyclical patterns of initiation and propagation. The cells in these groups exhibit a diverse range of signaling behavior, and dominant "conductor cells" drive activity in groups of lower-signaling neighbors. Ex vivo model systems reveal that conductor cells are present in wing cell layers of the corneal epithelium and that signaling propagates both within and between wing and basal layers. There are significant aberrations in conductor phenotype and interlayer propagation in type II diabetic murine models, indicating that signal hierarchy breakdown is an early indicator of disease. In vitro models reveal that signaling profile diversity and conductor cell phenotype is eliminated with P2X7 inhibition and is altered in Pannexin-1 or P2Y2 but not Connexin-43 inhibition. Conductor cells express significantly less P2X7 than their lower-signaling neighbors and exhibit significantly less migratory behavior after injury. Together, our results show that the postinjury calcium signaling cascade exhibits significantly more ordered and hierarchical behavior than previously thought, that proteins previously shown to be essential for regulating motility are also essential for determining signaling phenotype, and that loss of signal hierarchy integrity is an early indicator of disease state. NEW & NOTEWORTHY Calcium signaling in corneal epithelial cells after injury is highly ordered, with groups of cells engaged in cyclical patterns of event initiation and propagation driven by high-signaling cells. Signaling behavior is determined by P2X7, Pannexin-1, and P2Y2 and influences migratory behavior. Signal hierarchy is observed in healthy ex vivo models after injury and becomes aberrant in diabetes. This represents a paradigm shift, as signaling was thought to be random and determined by factors in the environment.

Leveraging a Y. lipolytica naringenin chassis for biosynthesis of apigenin and associated glucoside.

Flavonoids are a diverse set of natural products with promising bioactivities including anti-inflammatory, anti-cancer, and neuroprotective properties. Previously, the oleaginous host Yarrowia lipolytica has been engineered to produce high titers of the base flavonoid naringenin. Here, we leverage this host along with a set of E. coli bioconversion strains to produce the flavone apigenin and its glycosylated derivative isovitexin, two potential nutraceutical and pharmaceutical candidates. Through downstream strain selection, co-culture optimization, media composition, and mutant isolation, we were able to produce168 mg/L of apigenin, representing a 46% conversion rate of 2-(R/S)-naringenin to apigenin. This apigenin platform was modularly extended to produce isovitexin by addition of a second bioconversion strain. Together, these results demonstrate the promise of microbial production and modular bioconversion to access diversified flavonoids.

Stress from Nucleotide Depletion Activates the Transcriptional Regulator HEXIM1 to Suppress Melanoma.

Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.

Frequency of Leishmania spp. infection among HIV-infected patients living in an urban area in Brazil: a cross-sectional study.

BACKGROUND: There is little information about the frequency of Leishmania infection in asymptomatic people living with HIV (PLWH) and about the performance of laboratory diagnostic methods in coinfected patients in Latin America. The main objective of this study is to evaluate the frequency of Leishmania spp. infection in HIV-infected patients living in an urban area in Brazil. METHODS: To detect Leishmania infection, diagnostic tests were performed to detect anti-Leishmania antibodies (ELISA using Leptomonas seymouri antigens; ELISA using rK39 antigens; ELISA using rK28 antigens; indirect fluorescent-antibody test (IFAT); direct agglutination test (DAT)) and Leishmania DNA (polymerase chain reaction (PCR) with the target genes kDNA and ITS-1). RESULTS: The frequency of at least one positive test was 15%. For ELISA using Leptomonas antigens and IFAT, there was an association between CD4+ T lymphocyte counts and test positivity, with a higher positivity of these tests in more immunosuppressed patients (CD4+ T cell count < 200/mm3). CONCLUSIONS: According to our data, there was a high prevalence of Leishmania spp. infections in this population living with HIV. Although there is the possibility of cross-reaction, some tests that are considered highly specific for the diagnosis of Leishmania infection were positive. There was also an association between the positivity of some tests studied and lower values of CD4+ T lymphocytes.

Broad domains of histone 3 lysine 4 trimethylation are associated with transcriptional activation in CA1 neurons of the hippocampus during memory formation.

Transcriptional changes in the hippocampus are required for memory formation, and these changes are regulated by numerous post-translational modifications of chromatin-associated proteins. One of the epigenetic marks that has been implicated in memory formation is histone 3 lysine 4 trimethylation (H3K4me3), and this modification is found at the promoters of actively transcribed genes. The total levels of H3K4me3 are increased in the CA1 region of the hippocampus during memory formation, and genetic perturbation of the K4 methyltransferases and demethylases interferes with forming memories. Previous chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analyses failed to detect changes in H3K4me3 levels at the promoters of memory-linked genes. Since the breadth of H3K4me3 marks was recently reported to be associated with the transcriptional outcome of a gene, we re-analyzed H3K4me3 ChIP-seq data sets to identify the role of H3K4me3 broad domains in CA1 neurons, as well as identify differences in breadth that occur during contextual fear conditioning. We found that, under baseline conditions, broad H3K4me3 peaks mark important learning and memory genes and are often regulated by super-enhancers. The peaks at many learning-associated genes become broader during novel environment exposure and memory formation. Furthermore, the important learning- and memory-associated lysine methyltransferases, Kmt2a and Kmt2b, are involved in maintaining H3K4me3 peak width. Our findings highlight the importance of analyzing H3K4me3 peak shape, and demonstrate that breadth of H3K4me3 marks in neurons of the hippocampus is regulated during memory formation.

Epithelial cells exert differential traction stress in response to substrate stiffness.

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of studies, the effects that environmental changes have on this form of movement are poorly understood. To examine the role of substrate compliancy on multi-layered epithelial sheet migration, we performed traction force and confocal microscopy to determine differences in traction forces and to examine focal adhesions on synthetic and biological substrates. The leading edges of corneal epithelial sheets undergo retraction or contraction prior to migration, and alterations in the sheet's stiffness are affected by the amount of force exerted by cells at the leading edge. On substrates of 30 kPa, cells exhibited greater and more rapid movement than on substrates of 8 kPa, which are similar to that of the corneal basement membrane. Vinculin and its phosphorylated residue Y1065 were prominent along the basal surface of migrating cells, while Y822 was prominent between neighboring cells along the leading edge. Vinculin localization was diffuse on a substrate where the basement membrane was removed. Furthermore, when cells were cultured on fibronectin-coated acrylamide substrates of 8 and 50 kPa and then wounded, there was an injury-induced phosphorylation of Y1065 and substrate dependent changes in the number and size of vinculin containing focal adhesions. These results demonstrate that changes in substrate stiffness affected traction forces and vinculin dynamics, which potentially could contribute to the delayed healing response associated with certain corneal pathologies.

Hypoxia Induced Heparan Sulfate Primes the Extracellular Matrix for Endothelial Cell Recruitment by Facilitating VEGF-Fibronectin Interactions.

Vascular endothelial growth factor-A (VEGF) is critical for the development, growth, and survival of blood vessels. Retinal pigmented epithelial (RPE) cells are a major source of VEGF in the retina, with evidence that the extracellular matrix (ECM)-binding forms are particularly important. VEGF associates with fibronectin in the ECM to mediate distinct signals in endothelial cells that are required for full angiogenic activity. Hypoxia stimulates VEGF expression and angiogenesis; however, little is known about whether hypoxia also affects VEGF deposition within the ECM. Therefore, we investigated the role of hypoxia in modulating VEGF-ECM interactions using a primary retinal cell culture model. We found that retinal endothelial cell attachment to RPE cell layers was enhanced in cells maintained under hypoxic conditions. Furthermore, we found that agents that disrupt VEGF-fibronectin interactions inhibited endothelial cell attachment to RPE cells. We also found that hypoxia induced a general change in the chemical structure of the HS produced by the RPE cells, which correlated to changes in the deposition of VEGF in the ECM, and we further identified preferential binding of VEGFR2 over VEGFR1 to VEGF laden-fibronectin matrices. Collectively, these results indicate that hypoxia-induced HS may prime fibronectin for VEGF deposition and endothelial cell recruitment by promoting VEGF-VEGFR2 interactions as a potential means to control angiogenesis in the retina and other tissues.

Extruded urea could reduce true protein source in beef cattle diets.

Rumen micro-organisms are capable of producing microbial protein from ammonia and carbon skeleton, and non-protein nitrogen (NPN) may be one of the sources of ammonia. Alternative source of NPN is the slow release of ammonia sources in which the product is the extrusion of starch with urea. This work aimed to determine the effects on nutrient intake, ingestive behaviour, digestibility, nitrogen balance, ruminal pH, rumen ammonia nitrogen, volatile fatty acids (VFA) and blood parameters with increased levels of extruded urea (50, 60, 70 and 80 g/100 kg of body weight [BW]) in beef cattle diet. Four rumen cannulated crossbred steers with initial mean weight of 336 ± 47 kg in a 4 × 4 Latin square design were distributed. Diets were formulated with 400:600 g/kg roughage:concentrate ratio on dry matter based and provided once per day, being used whole corn silage as roughage. There were no effects on nutrient intake (kg/day), ingestive behaviour, apparent digestibility, nitrogen balance, volatile fatty acid (VFA) and blood parameters in extruded urea treatment groups. Similar results were observed on time spent on feeding, rumination and idleness. There were positive linear effects (p = 0.022) on rumen pH in the time of 8 hr after feeding and also on plasma concentration of the extruded urea levels (p = 0.039); whereas a linear negative effect (p = 0.030) was observed on ammonia nitrogen for the collection time of 2 hr after feeding. Increased levels of extruded urea could maintain nutrient intake, digestibility, ingestive behaviour, rumen pH and blood parameters in normal conditions. In conclusion, we recommend the extruded urea use with values up to 80 g/100 kg BW in confined beef cattle that receive balanced diets with 140 g/kg of crude protein.

High fat diet induces pre-type 2 diabetes with regional changes in corneal sensory nerves and altered P2X7 expression and localization.

Type 2 diabetes is one of the leading pathologies that increases the risk of improper wound healing. Obesity has become a major risk factor for this disease that is now considered to be the 4th highest cause of preventable blindness according to the World Health Organization. The cornea is the most densely innervated structure in the human body and senses even the slightest injury. In diabetes, decreased corneal sensitivity secondary to diabetic peripheral neuropathy can lead to increased corneal abrasion, ulceration, and even blindness. In this study, a diet induced obesity (DIO) mouse model of pre-Type 2 diabetes was used to characterize changes in sensory nerves and P2X7, a purinoreceptor, a pain receptor, and an ion channel that is expressed in a number of tissues. Since our previous studies demonstrated that P2X7 mRNA was significantly elevated in diabetic human corneas, we examined P2X7 expression and localization in the DIO murine model at various times after being fed a high fat diet. Fifteen weeks after onset of diet, we found that there was a significant decrease in the density of sub-basal nerves in the DIO mice that was associated with an increase in tortuosity and a decrease in diameter. In addition, P2X7 mRNA expression was significantly greater in the corneal epithelium of DIO mice, and the increase in transcript was enhanced in the central migrating and peripheral regions after injury. Interestingly, confocal microscopy and thresholding analysis revealed that there was a significant increase in P2X7 distal to the injury, which contrasted with a decrease in P2X7-expressing stromal sensory nerves. Therefore, we hypothesize that the P2X7 receptor acts to sense changes at the leading edge following an epithelial abrasion, and this fine-tuned regulation is lost during the onset of diabetes. Further understanding of the corneal changes that occur in diabetes can help us better monitor progression of diabetic complications, as well as develop new therapeutics for the treatment of diabetic corneal dysfunction.

Hypoxia modulates the development of a corneal stromal matrix model.

Deposition of matrix proteins during development and repair is critical to the transparency of the cornea. While many cells respond to a hypoxic state that can occur in a tumor, the cornea is exposed to hypoxia during development prior to eyelid opening and during the diurnal sleep cycle where oxygen levels can drop from 21% to 8%. In this study, we used 2 three-dimensional (3-D) models to examine how stromal cells respond to periods of acute hypoxic states. The first model, a stromal construct model, is a 3-D stroma-like construct that consists of human corneal fibroblasts (HCFs) stimulated by a stable form of ascorbate for 1, 2, and 4 weeks to self-assemble their own extracellular matrix. The second model, a corneal organ culture model, is a corneal wound-healing model, which consists of wounded adult rat corneas that were removed and placed in culture to heal. Both models were exposed to either normoxic or hypoxic conditions for varying time periods, and the expression and/or localization of matrix proteins was assessed. No significant changes were detected in Type V collagen, which is associated with Type I collagen fibrils; however, significant changes were detected in the expression of both the small leucine-rich repeating proteoglycans and the larger heparan sulfate proteoglycan, perlecan. Also, hypoxia decreased both the number of Cuprolinic blue-positive glycosaminoglycan chains along collagen fibrils and Sulfatase 1, which modulates the effect of heparan sulfate by removing the 6-O-sulfate groups. In the stromal construct model, alterations were seen in fibronectin, similar to those that occur in development and after injury. These changes in fibronectin after injury were accompanied by changes in proteoglycans. Together these findings indicate that acute hypoxic changes alter the physiology of the cornea, and these models will allow us to manipulate the conditions in the extracellular environment in order to study corneal development and trauma.

Identification of Anti-prion Compounds using a Novel Cellular Assay.

Prion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrPC) into the infectious isoform (PrPSc). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected with scrapie prions. Here, we describe the use of an alternative cellular assay based on suppressing the spontaneous cytotoxicity of a mutant form of PrP (Δ105-125). Using this assay, we screened 75,000 compounds, and identified a group of phenethyl piperidines (exemplified by LD7), which reduces the accumulation of PrPSc in infected neuroblastoma cells by >90% at low micromolar doses, and inhibits PrPSc-induced synaptotoxicity in hippocampal neurons. By analyzing the structure-activity relationships of 35 chemical derivatives, we defined the pharmacophore of LD7, and identified a more potent derivative. Active compounds do not alter total or cell-surface levels of PrPC, and do not bind to recombinant PrP in surface plasmon resonance experiments, although at high concentrations they inhibit PrPSc-seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RT-QuIC). This class of small molecules may provide valuable therapeutic leads, as well as chemical biological tools to identify cellular pathways underlying PrPSc metabolism and PrPC function.

Purinoreceptor P2X7 Regulation of Ca(2+) Mobilization and Cytoskeletal Rearrangement Is Required for Corneal Reepithelialization after Injury.

The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injury-induced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratch-wounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent protein-labeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing.

Evolution of intracranial atherosclerotic disease under modern medical therapy.

OBJECTIVE: Understanding how symptomatic intracranial atherosclerotic disease (ICAD) evolves with current medical therapy may inform secondary stroke prevention. METHODS: In a prospective academic-initiated study, we recruited 50 patients (mean age = 63.4 ± 9.0 years) with acute strokes attributed to high-grade (≥70%) intracranial atherosclerotic stenosis for 3-dimensional rotational angiograms before and after intensive medical therapy for 12 months. Treatment targets included low-density lipoprotein ≤ 70mg/dl, glycosylated hemoglobin (HbA1c) ≤ 6.5%, and systolic blood pressure ≤ 140 mmHg. We analyzed infarct topography and monitored microembolic signal in recurrent strokes. The reference group was a published cohort of 143 ICAD patients. RESULTS: Overall, the stenoses regressed from 79% at baseline (interquartile range [IQR] = 71-87%) to 63% (IQR = 54-74%) in 1 year (p < 0.001). Specifically, the qualifying lesions (n = 49) regressed (stenosis reduced >10%) in 24 patients (49%), remained quiescent (stenosis same or ±10%) in 21 patients (43%), and progressed (stenosis increased >10%) in 4 patients (8%). There was no difference in intensity of risk factor control between groups of diverging clinical or angiographic outcomes. Higher HbA1c at baseline predicted plaque regression at 1 year (odds ratio = 4.4, 95% confidence interval = 1.4-14.5, p = 0.006). Among the 6 patients with recurrent strokes pertaining to the qualifying stenosis, 5 patients had solitary or rosarylike acute infarcts along the internal or anterior border zones, and 2 patients showed microembolic signals in transcranial Doppler ultrasound. INTERPRETATION: A majority of symptomatic high-grade intracranial plaques had regressed or remained quiescent by 12 months under intensive medical therapy. Artery-to-artery thromboembolism with impaired washout at border zones was a common mechanism in stroke recurrence.

Epithelial wounds induce differential phosphorylation changes in response to purinergic and EGF receptor activation.

Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleotides released after scratch wounding activate purinergic receptors, leading to a distinct phosphorylation profile on epidermal growth factor receptor (EGFR) compared with its natural ligand. ATP induced a 2- to 15-fold phosphorylation increase over control on EGFR Y974, Y1086, and Y1148, with minimal phosphorylation intensity on EGFR Y1173 compared with the level measured in response to epidermal growth factor. Differential phosphorylation induced by epidermal growth factor or ATP was site specific on Src, Shc, phospholipase Cγ, protein kinase C, focal adhesion kinase, paxillin, and mitogen-activated protein kinases 1, 12, and 13. After wounding, the P2Y2 receptor mRNA expression increased, and after knockdown, migration and Ca(2+) mobilization were impaired. To examine phosphorylation mediated by P2Y2, cells were cultured in media containing stable isotope-labeled amino acids, the receptor was knocked down, and the cells were stimulated. Mass spectrometry-based comparison of the phosphorylation profiles of control versus transfected cells revealed a 50-fold decrease in phosphorylation of EGFR Y974 and 1086, with no decrease in Y1173 phosphorylation. A similarfold decrease in Src Y421 and Y446 and paxillin Y118 was detected, indicating the far-reaching importance of the P2Y2 receptor in mediating migration.

HEXIM1 is correlated with Alzheimer's disease pathology and regulates immediate early gene dynamics in neurons.

Impaired memory formation and recall is a distinguishing feature of Alzheimer's disease, and memory requires de novo gene transcription in neurons. Rapid and robust transcription of many genes is facilitated by the formation of a poised basal state, in which RNA polymerase II (RNAP2) has initiated transcription, but is paused just downstream of the gene promoter. Neuronal depolarization releases the paused RNAP2 to complete the synthesis of messenger RNA (mRNA) transcripts. Paused RNAP2 release is controlled by positive transcription elongation factor b (P-TEFb), which is sequestered into a larger inactive complex containing Hexamethylene bisacetamide inducible protein 1 (HEXIM1) under basal conditions. In this work, we find that neuronal expression of HEXIM1 mRNA is highly correlated with human Alzheimer's disease pathologies. Furthermore, P-TEFb regulation by HEXIM1 has a significant impact on the rapid induction of neuronal gene transcription, particularly in response to repeated depolarization. These data indicate that HEXIM1/P-TEFb has an important role in inducible gene transcription in neurons, and for setting and resetting the poised state that allows for the robust activation of genes necessary for synaptic plasticity.

Targeting soluble amyloid-beta oligomers with a novel nanobody.

The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-ß oligomers (SAßOs) accumulate early, prior to amyloid plaque formation. SAßOs induce memory impairment and disrupt cognitive function independent of amyloid-ß plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAßO (E3) nanobody generated from an alpaca immunized with SAßO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAßOs and amyloid-ß plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAßO and amyloid-ß plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAßOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAßO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.

TARGETING SOLUBLE AMYLOID-BETA OLIGOMERS WITH A NOVEL NANOBODY.

The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-ß oligomers (SAßOs) accumulate early, prior to amyloid plaque formation. SAßOs induce memory impairment and disrupt cognitive function independent of amyloid-ß plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAßO (E3) nanobody generated from an alpaca immunized with SAßO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAßOs and amyloid-ß plaques. The E3 nanobody traverses across the blood-brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAßO and amyloid-ß plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAßOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAßO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer's disease.

Trafficking of endogenous smooth muscle cell cholesterol: a role for serum amyloid A and interleukin-1ß.

OBJECTIVE: Intracellular cholesterol distribution impacts cell function; however, processes influencing endogenous cholesterol trafficking remain largely unknown. Atherosclerosis is associated with vascular inflammation and these studies address the role of inflammatory mediators on smooth muscle cell cholesterol trafficking. METHODS AND RESULTS: Interestingly, in the absence of an exogenous cholesterol source, serum amyloid A increased [(14)C] oleic acid incorporation into cholesteryl ester in rat smooth muscle cells, suggesting endogenous cholesterol trafficking to the endoplasmic reticulum. [(3)H] cholesteryl ester accumulated in cells prelabeled with [(3)H] cholesterol, confirming that serum amyloid A mediated the movement of endogenous cholesterol. Cholesterol movement was dependent upon functional endolysosomes. The cholesterol oxidase-sensitive pool of cholesterol decreased in serum amyloid A-treated cells. Furthermore, the mechanism whereby serum amyloid A induced cholesterol trafficking was determined to be via activation of expression of secretory phospholipase A(2), group IIA (sPLA(2)) and sPLA(2)-dependent activation of sphingomyelinase. Interestingly, although neither tumor necrosis factor-α nor interferon-γ induced cholesterol trafficking, interleukin-1ß induced [(14)C] cholesteryl ester accumulation that was also dependent upon sPLA(2) and sphingomyelinase activities. Serum amyloid A activates smooth muscle cell interleukin-1ß expression, and although the interleukin-1-receptor antagonist inhibited the interleukin-1ß-induced cholesterol trafficking, it had no effect on the movement of cholesterol mediated by serum amyloid A. CONCLUSIONS: These data support a role for inflammation in endogenous smooth muscle cell cholesterol trafficking from the plasma membrane to the endoplasmic reticulum.

Aberrations in Cell Signaling Quantified in Diabetic Murine Globes after Injury.

The corneal epithelium is an avascular structure that has a unique wound healing mechanism, which allows for rapid wound closure without compromising vision. This wound healing mechanism is attenuated in diabetic patients, resulting in poor clinical outcomes and recurrent non-healing erosion. We investigated changes in cellular calcium signaling activity during the wound response in murine diabetic tissue using live cell imaging from both ex vivo and in vitro models. The calcium signaling propagation in diabetic cells was significantly decreased and displayed altered patterns compared to non-diabetic controls. Diabetic cells and tissue display distinct expression of the purinergic receptor, P2X7, which mediates the wound healing response. We speculate that alterations in P2X7 expression, interactions with other proteins, and calcium signaling activity significantly impact the wound healing response. This may explain aberrations in the diabetic wound response.

Livestock grazing impact differently on the functional diversity of dung beetles depending on the regional context in subtropical forests.

The replacement of native forest by cattle pastures reduces functional diversity; however, little is known about whether the changes depend on regional variation. Dung beetles are one of the most diverse and functionally important taxa; through organic matter burial, dung beetles improve soil quality. We collected dung beetles in native forests and cattle ranching areas in subtropical forests with contrasting climatic conditions: the Atlantic Forest, the Humid Chaco, and the Dry Chaco. We measured 11 traits related to the ecology and the physiology of species. Irrespectively of the region, functional richness was higher in forests (native and with cattle) when compared to open pastures. Humid forests (Atlantic Forest and Humid Chaco) showed higher functional richness than Dry Chaco. Functional dispersion in humid forests was similar between native forest and livestock systems, however, functional dispersion in the Dry Chaco was higher in open pastures compared to native forest. According to our results, native forests and forests with cattle maintain functional diversity in all regions. However, in the case of open pastures, the response depends on the regional context; the replacement of native forest by open pastures strongly affected functional diversity in humid forests and showed less impact on dry forest.