A Targeted Proteomics Approach for Biomarker Discovery Using Bilateral Matched Nipple Aspiration Fluids

Introduction  

The objective of this study was to identify cancer-associated protein expression patterns in bilateral matched nipple aspiration fluids using nanoscale reciprocal Cy3/Cy5 labeling and high-content antibody microarrays. This novel platform allows the pair-wise comparisons of the relative abundance of 512 different antigens using minimal NAF sample containing 1 μg of total protein.     

Neutrophil transmigration under shear flow conditions in vitro is junctional adhesion molecule-C independent

Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-alpha, IL-1beta, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN beta(2) integrins during transendothelial migration.     

Liver-specific knockdown of JNK1 up-regulates proliferator-activated receptor gamma coactivator 1 beta and increases plasma triglyceride despite reduced glucose and insulin levels in diet-induced obese mice

The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor gamma coactivator 1 beta, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.     

Fimbrial proteins of porphyromonas gingivalis mediate in vivo virulence and exploit TLR2 and complement receptor 3 to persist in macrophages

Porphyromonas gingivalis is an oral/systemic pathogen implicated in chronic conditions, although the mechanism(s) whereby it resists immune defenses and persists in the host is poorly understood. The virulence of this pathogen partially depends upon expression of fimbriae comprising polymerized fimbrillin (FimA) associated with quantitatively minor proteins (FimCDE). In this study, we show that isogenic mutants lacking FimCDE are dramatically less persistent and virulent in a mouse periodontitis model and express shorter fimbriae than the wild type. Strikingly, native fimbriae allowed P. gingivalis to exploit the TLR2/complement receptor 3 pathway for intracellular entry, inhibition of IL-12p70, and persistence in macrophages. This virulence mechanism also required FimCDE; indeed, mutant strains exhibited significantly reduced ability to inhibit IL-12p70, invade, and persist intracellularly, attributable to failure to interact with complement receptor 3, although not with TLR2. These results highlight a hitherto unknown mechanism of immune evasion by P. gingivalis that is surprisingly dependent upon minor constituents of its fimbriae, and support the concept that pathogens evolved to manipulate innate immunity for promoting adaptive fitness and thus their capacity to cause disease.     

An assay system for measuring the acute production of sphingosine 1-phosphate in intact monolayers

Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase.     

Pam3CSK4 and LTA-TLRs ligands associated with microdomains induce IL8 production in human adrenocortical cancer cells.

Bacterially derived ligands, Pam3CSK4 and LPS, can directly impact adrenal glands steroidogenesis through microdomain-related TLR1/2 and 4, respectively, and indirectly via immune cell-derived cytokines. The bilateral immunoadrenal relationship plays an important role in the proper functioning of both systems. CXC chemokine-dependent immune cell infiltration into adrenocortical carcinomas (ACC), which correlates with poor prognosis, is a common phenomenon. Recently, IL8 was identified in ACC and NCI-H295R cells, and was found to contribute to ACC tumour growth. The aim of this study was to clarify the role of different TLR ligands in IL8 production in NCI-H295R cells. This is the first study to demonstrate the expression of several TLRs including TLR1, 3, 6, 7 and 9 in human adrenocortical cells by using the RT-PCR approach. Only stimulation with TLR1/6 together with TLR2 ligands resulted in IL8 peptide and mRNA induction in a dose and time-dependent manner. Our data suggest that gram-positive bacteria-related TLR1/2/6 ligands might contribute to adrenal gland tumorigenesis via IL8 production.     

Status,trends, and equilibrium abundance estimates of the translocated sea otter population in Washington State

Sea otters (Enhydra lutris kenyoni) historically occurred in Washington State, USA, until their local extinction in the early 1900s as a result of the maritime fur trade. Following their extirpation, 59 sea otters were translocated from Amchitka Island, Alaska, USA, to the coast of Washington, with 29 released at Point Grenville in 1969 and 30 released at La Push in 1970. The Washington Department of Fish and Wildlife has outlined 2 main objectives for sea otter recovery: a target population level and a target geographic distribution. Recovery criteria are based on estimates of population abundance, equilibrium abundance (K), and geographic distribution; therefore, estimates of these parameters have important management implications. We compiled available survey data for sea otters in Washington State since their translocation (1977–2019) and fit a Bayesian state-space model to estimate past and current abundance, and equilibrium abundance at multiple spatial scales. We then used forward projections of population dynamics to explore potential scenarios of range recolonization and as the basis of a sensitivity analysis to evaluate the relative influence of movement behavior, frontal wave speed, intrinsic growth, and equilibrium density on future population recovery potential. Our model improves upon previous analyses of sea otter population dynamics in Washington by partitioning and quantifying sources of estimation error to estimate population dynamics, by providing robust estimates of K, and by simulating long-term population growth and range expansion under a range of realistic parameter values. Our model resulted in predictions of population abundance that closely matched observed counts. At the range-wide scale, the population size in our model increased from an average of 21 independent sea otters (95% CI = 13–29) in 1977 to 2,336 independent sea otters (95% CI = 1,467–3,359) in 2019. The average estimated annual growth rate was 12.42% and varied at a sub-regional scale from 6.42–14.92%. The overall estimated mean K density of sea otters in Washington was 1.71 ± 0.90 (SD) independent sea otters/km² of habitat (1.96 ± 1.04 sea otters/km², including pups), and estimated densities within the current range correspond on average to 87% of mean sub-regional equilibrium values (range = 66–111%). The projected value of K for all of Washington was 5,287 independent sea otters (95% CI = 2,488–8,086) and 6,080 sea otters including pups (95% CI = 2,861–9,300), assuming a similar range of equilibrium densities in currently un-occupied habitats. Sensitivity analysis of simulations of sea otter population growth and range expansion suggested that mean K density estimates in currently occupied sub-regions had the largest impact on predicted future population growth (r² = 0.52), followed by the rate of southward range expansion (r² = 0.26) and the mean K density estimate of currently unoccupied sub-regions to the south of the current range (r² = 0.04). Our estimates of abundance and sensitivity analysis of simulations of future population abundance and geographic range help determine population status in relation to population recovery targets and identify the most influential parameters affecting future population growth and range expansion for sea otters in Washington State.