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Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat (‘good freezer’) and Black-Red Andaluza (‘bad freezer’) showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages. [ABSTRACT FROM AUTHOR]

Immuno-PCR (iPCR) is one of the methods used for the detection of a wide range of analytes and features the high sensitivity of the polymerase chain reaction (PCR) method. iPCR uses antibodies coupled to DNA, followed by the amplification of the attached DNA using RT-PCR. Two major types of antibody-DNA conjugates are currently used, which are obtained as a result of non-covalent (biotin-streptavidin) or covalent interactions. Using a strain-promoted azide-alkyne cycloaddition (SPAAC), we synthesized covalent DNA-antibody conjugates, optimized the reaction conditions, and developed an efficient protocol for the purification of conjugates, with which all unreacted antibodies and oligonucleotides are separated. Covalent DNA-antibody conjugates were tested with iPCR assays that were previously developed for the detection of IgE and IgM antibodies with the use of the supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin. The results show that the modification of antibodies with amino groups did not allow us to obtain monolabeled antibodies or antibodies with a strictly defined number of DNA-labels. The degree of labeling determined by the dyes introduced through the azido group reflects the actual labeling degree statistically. If the average labeling degree for azido groups is 1.1, the conjugates contain 25% mono-labeled antibodies, 50% double-labeled antibodies, and 25% unlabeled ones. The specificity of the monoclonal antibody to human IgE (BE5) changed after conjugation with the oligonucleotide. The sensitivity of iPCR in the detection of IgM antibodies produced against the LeC disaccharide using a covalent conjugate was similar to that of a supramolecular complex of 5'- and 3'-biotinylated DNA and streptavidin, but the new procedure is two steps shorter. [ABSTRACT FROM AUTHOR]

We recently identified three novel thioredoxin-like genes in the genome of the protozoan parasite Plasmodium that belong to the Phosducin-like family of proteins (PhLP). PhLPs are small cytosolic proteins hypothesized to function in G-protein signaling and protein folding. Although PhLPs are highly conserved in eukaryotes from yeast to mammals, only a few representatives have been experimentally characterized to date. In addition, while PhLPs contain a thioredoxin domain, they lack a CXXC motif, a strong indicator for redox activity, and it is unclear whether members of the PhLP family are enzymatically active. Here, we describe PbPhLP-3 as the first phosducin-like protein of a protozoan organism, Plasmodium berghei. Initial transcription analysis revealed continuous low-level expression of pbphlp-3 throughout the complex Plasmodium life cycle. Attempts to knockout pbphlp-3 in P. berghei did not yield live parasites, suggesting an essential role for the gene in Plasmodium. We cloned, expressed and purified PbPhLP-3 and determined that the recombinant protein is redox active in vitro in a thioredoxin-coupled redox assay. It also has the capacity to reduce the organic compound tert-Butyl hydroperoxide (TBHP) in vitro, albeit at low efficiency. Sequence analysis, structural modeling, and site-directed mutagenesis revealed a conserved cysteine in the thioredoxin domain to be the redox active residue. Lastly, we provide evidence that recombinant human PhLP-3 exhibits redox activity similar to that of PbPhLP-3 and suggest that redox activity may be conserved in PhLP-3 homologs of other species. Our data provide new insight into the function of PhLP-3, which is hypothesized to act as co-chaperones in the folding and regulation of cytoskeletal proteins. We discuss the potential implications of PhLP-3 as a thioredoxin-target protein and possible links between the cellular redox network and the eukaryotic protein folding machinery. [ABSTRACT FROM AUTHOR]

Background: The chronic remitting and relapsing intestinal inflammation characteristic of Crohn’s disease frequently leads to fibrosis and subsequent stenosis of the inflamed region. Approximately a third of all Crohn’s disease patients require resection at some stage in their disease course. As the pathogenesis of Crohn’s disease associated fibrosis is largely unknown, a strong necessity exists to better understand the pathophysiology thereof. Methods: In this study, we investigated changes of the DNA methylome and transcriptome of ileum-derived fibroblasts associated to the occurrence of Crohn’s disease associated fibrosis. Eighteen samples were included in a DNA methylation array and twenty-one samples were used for RNA sequencing. Results: Most differentially methylated regions and differentially expressed genes were observed when comparing stenotic with non-inflamed samples. By contrast, few differences were observed when comparing Crohn’s disease with non-Crohn’s disease, or inflamed with non-inflamed tissue. Integrative methylation and gene expression analyses revealed dysregulation of genes associated to the PRKACA and E2F1 network, which is involved in cell cycle progression, angiogenesis, epithelial to mesenchymal transition, and bile metabolism. Conclusion: Our research provides evidence that the methylome and the transcriptome are systematically dysregulated in stenosis-associated fibroblasts. [ABSTRACT FROM AUTHOR]

T-type calcium channels in the brain mediate the pathophysiology of epilepsy, pain, and sleep. Recently, we developed a novel therapeutic candidate, SAK3 (ethyl 8'-methyl-2',4-dioxo-2-(piperidin-1-yl)-2'H-spiro[cyclopentane-1,3'-imidazo[1,2-a] pyridine]-2-ene-3-carboxylate), for Alzheimer’s disease (AD). The cognitive improvement by SAK3 is closely associated with enhanced acetylcholine (ACh) release in the hippocampus. Since monoamines such as dopamine (DA), noradrenaline (NA), and serotonin (5-HT) are also involved in hippocampus-dependent learning and psychomotor behaviors in mice, we investigated the effects of SAK3 on these monoamine releases in the mouse brain. Oral administration of SAK3 (0.5 mg/kg, p.o.) significantly promoted DA and 5-HT releases in the naive mouse hippocampal CA1 region but not in the medial prefrontal cortex (mPFC), while SAK3 did not affect NA release in either brain region. The T-type calcium channel-specific inhibitor, NNC 55–0396 (1 μM) significantly antagonized SAK3-enhanced DA and 5-HT releases in the hippocampus. Interestingly, the α7 nicotinic ACh receptor (nAChR) antagonist, methyllycaconitine (1 nM) significantly inhibited DA release, and the α4 nAChR antagonist, dihydro-β-erythroidine (100 μM) significantly blocked both DA and 5-HT releases following SAK3 (0.5 mg/kg, p.o.) administration in the hippocampus. SAK3 did not alter basal monoamine contents both in the mPFC and hippocampus. SAK3 (0.5 mg/kg, p.o.) administration also significantly elevated DA and 5-HT releases in the hippocampal CA1 region of amyloid-precursor protein (APP)NL-GF knock-in (KI) mice. Moreover, hippocampal DA and 5-HT contents were significantly decreased in APPNL-GF KI mice. Taken together, our data suggest that SAK3 promotes monoamine DA and 5-HT releases by enhancing the T-type calcium channel and nAChR in the mouse hippocampus. [ABSTRACT FROM AUTHOR]

Preventive HIV-1 vaccine strategies rely on the elicitation of broadly neutralizing antibody (bNAb) responses, but their induction in vivo by vaccination remains challenging. Considering that the ability of an epitope to elicit effective humoral immunity depends on its exposure on the virion, we have used a reverse genetics approach to select variants from an HIV-1 AC10_29 randomly mutated envelope library that showed increased affinity for a selected bNAb (4E10 bNAb targeting the HIV-1 MPER region). Isolated envelope sequences were analyzed by deep-sequencing showing a small number of dominant changes, including the loss of four potential N-linked glycosylation sites and disruption of the V1/V2 loop. Accordingly, the dominant variant (LR1-C1), showed not only increased affinity for MPER bNAbs 4E10 and 2F5, but also higher affinity for an additional antibody targeting the V3 loop (447-52D) that could be a consequence of an open conformation tier 1-like Env. Furthermore, the amino acids specific for the selected variant are associated with an increased sensitivity for 4E10 and 2F5 antibodies. In vivo studies showed that sera from mice immunized with LR1-C1 viruses possessed an improved neutralizing activity compared to the wild-type AC10_29 env. While Virus Like Particles (VLPs) carrying this envelope were unable to induce detectable neutralizing activity in immunized rabbits, one animal showed antibody response to the 4E10-proximal region. Our data establish a novel approach that has the potential to yield HIV envelope immunogen sequences that direct antibody responses to specific envelope regions. [ABSTRACT FROM AUTHOR]

An increasing number of antibody-based therapies are being considered for controlling bacterial infections, including Clostridium difficile by targeting toxins A and B. In an effort to develop novel C. difficile immunotherapeutics, we previously isolated several single-domain antibodies (VHHs) capable of toxin A neutralization through recognition of the extreme C-terminal combined repetitive oligopeptides (CROPs) domain, but failed at identifying neutralizing VHHs that bound a similar region on toxin B. Here we report the isolation of a panel of 29 VHHs targeting at least seven unique epitopes on a toxin B immunogen composed of a portion of the central delivery domain and the entire CROPs domain. Despite monovalent affinities as high as KD = 70 pM, none of the VHHs tested were capable of toxin B neutralization; however, modest toxin B inhibition was observed with VHH-VHH dimers and to a much greater extent with VHH-Fc fusions, reaching the neutralizing potency of the recently approved anti-toxin B monoclonal antibody bezlotoxumab in in vitro assays. Epitope binning revealed that several VHH-Fcs bound toxin B at sites distinct from the region recognized by bezlotoxumab, while other VHH-Fcs partially competed with bezlotoxumab for toxin binding. Therefore, the VHHs described here are effective at toxin B neutralization when formatted as bivalent VHH-Fc fusions by targeting toxin B at regions both similar and distinct from the bezlotoxumab binding site. [ABSTRACT FROM AUTHOR]

The transfer of protons through proton translocating channels is a complex process, for which direct samplings of different protonation states and side chain conformations in a transition network calculation provide an efficient, bias-free description. In principle, a new transition network calculation is required for every unsampled change in the system of interest, e.g. an unsampled protonation state change, which is associated with significant computational costs. Transition networks void of or including an unsampled change are termed unperturbed or perturbed, respectively. Here, we present a prediction method, which is based on an extensive coarse-graining of the underlying transition networks to speed up the calculations. It uses the minimum spanning tree and a corresponding sensitivity analysis of an unperturbed transition network as initial guess and refinement parameter for the determination of an unknown, perturbed transition network. Thereby, the minimum spanning tree defines a sub-network connecting all nodes without cycles and minimal edge weight sum, while the sensitivity analysis analyzes the stability of the minimum spanning tree towards individual edge weight reductions. Using the prediction method, we are able to reduce the calculation costs in a model system by up to 80%, while important network properties are maintained in most predictions. [ABSTRACT FROM AUTHOR]

Species are shifting their ranges due to climate change, many moving to cooler and higher locations. However, with elevation increase comes oxygen decline, potentially limiting a species’ ability to track its environment depending on what mechanisms it has available to compensate for hypoxic stress. Pikas (Family Ochotonidae), cold-specialist small mammal species, are already undergoing elevational range shifts. We collected RNA samples from one population of Ochotona roylei in the western Himalaya at three sites– 3,600, 4,000, and 5,000 meters–and found no evidence of significant population genetic structure nor positive selection among sites. However, out of over 10,000 expressed transcripts, 26 were significantly upregulated at the 5,000 m site and were significantly enriched for pathways consistent with physiological compensation for limited oxygen. These results suggest that differences in gene expression may play a key role in enabling hypoxia tolerance on this local scale, indicating elevational flexibility that may facilitate successful range shifts in response to climate change. [ABSTRACT FROM AUTHOR]

The surgical standard of care for lumbar discectomy leaves the annulus fibrosus (AF) defect unrepaired, despite considerable risk for a recurrent herniation. Identification of a viable defect repair strategy has until now been elusive. The scope of this ex vivo biomechanical study was to evaluate crosslinking hydrogels as potentially promising AF defect sealants, and provide a baseline for their use in combination with collagen scaffolds that restore disc volume. This study directly compared genipin crosslinked fibrin hydrogel (FibGen) as a promising preclinical candidate against a clinically available adhesive composed of glutaraldehyde and albumin (BioGlue). Forty-two bovine coccygeal functional spine units (FSU) were randomly allocated into four groups, namely untreated (control, n = 12), repaired with either one of the tested hydrogels (BioGlue, n = 12; FibGen, n = 12), or FibGen used in combination with a collagen hydrogel scaffold (FibGen+Scaffold, n = 6). All specimens underwent a moderate mechanical testing protocol in intact, injured and repaired states. After completion of the moderate testing protocol, the samples underwent a ramp-to-failure test. Lumbar discectomy destabilized the FSU as quantified by increased torsional range of motion (28.0° (19.1, 45.1) vs. 41.39° (27.3, 84.9), p<0.001), torsional neutral zone (3.1° (1.2, 7.7) vs. 4.8° (2.1, 12.1), Z = -3.49, p < 0.001), hysteresis(24.4 J (12.8, 76.0) vs. 27.6 J (16.4, 54.4), Z = -2.61, p = 0.009), with loss of both disc height (7.0 mm (5.0, 10.5) vs 6.1 mm (4.0, 9.3), Z = -5.16, p < 0.001) and torsional stiffness (0.76 Nmdeg-1 (0.38, 1.07) vs. 0.66 Nmdeg-1 (0.38, 0.97), Z = -3.98, p < 0.001). Most FibGen repaired AF endured the entire testing procedure whereas only a minority of BioGlue repaired AF and all FibGen+Scaffold repaired AF failed (6/10 vs. 3/12 vs. 0/6 respectively, p = 0.041). Both BioGlue and FibGen+Scaffold repaired AF partially restored disc height (0.47 mm (0.07, 2.41), p = 0.048 and 1.52 mm (0.41, 2.57), p = 0.021 respectively) compared to sham treatment (0.08 mm (-0.63, 0.88)) whereas FibGen-only repaired AF had no such effect (0.04 mm (-0.73, 1.13), U = 48.0, p = 1). The AF injury model demonstrated considerable change of FSU mechanics that could be partially restored by use of an AF sealant. While inclusion of a volumetric collagen scaffold led to repair failure, use of FibGen alone demonstrated clinically relevant promise for prevention of mechanical reherniation, outperforming an FDA approved sealant in this ex vivo test series. [ABSTRACT FROM AUTHOR]

NWC is an uncharacterised protein containing three strongly conserved domains not found in any other known protein. Previously, we reported that the NWC protein is detected in cells in the germinal layer in murine testes (strain: C57BL/6), and its knockout results in no obvious phenotype. We determined the NWC expression pattern during spermatogenesis, and found this protein in spermatocytes and round spermatids, but not in epididymal sperm. Although NWC knockout males are fertile, we further characterised their reproductive potential employing non-standard mating that better simulates the natural conditions by including sperm competition. Such an approach revealed that the sperm of knockout males fail to successfully compete with control sperm. After analysing selected characteristics of the male reproductive system, we found that NWC knockout sperm had a reduced ability to fertilize cumulus-intact eggs during IVF. This is the first report describing a subtle phenotype of NWC knockout mice that could be detected under non-standard mating conditions. Our results indicate that NWC plays an important role in spermatogenesis and its deficiency results in the production of functionally impaired sperm. [ABSTRACT FROM AUTHOR]

Acute Kidney Injury (AKI), a sudden decline in kidney function, is associated with increased mortality, morbidity, length of stay, and hospital cost. Since AKI is sometimes preventable, there is great interest in prediction. Most existing studies consider all patients and therefore restrict to features available in the first hours of hospitalization. Here, the focus is instead on rehospitalized patients, a cohort in which rich longitudinal features from prior hospitalizations can be analyzed. Our objective is to provide a risk score directly at hospital re-entry. Gradient boosting, penalized logistic regression (with and without stability selection), and a recurrent neural network are trained on two years of adult inpatient EHR data (3,387 attributes for 34,505 patients who generated 90,013 training samples with 5,618 cases and 84,395 controls). Predictions are internally evaluated with 50 iterations of 5-fold grouped cross-validation with special emphasis on calibration, an analysis of which is performed at the patient as well as hospitalization level. Error is assessed with respect to diagnosis, race, age, gender, AKI identification method, and hospital utilization. In an additional experiment, the regularization penalty is severely increased to induce parsimony and interpretability. Predictors identified for rehospitalized patients are also reported with a special analysis of medications that might be modifiable risk factors. Insights from this study might be used to construct a predictive tool for AKI in rehospitalized patients. An accurate estimate of AKI risk at hospital entry might serve as a prior for an admitting provider or another predictive algorithm. [ABSTRACT FROM AUTHOR]

Background: In patients with reperfused ST-elevation myocardial infarction (STEMI) both invasive and non-invasive assessments of microvascular dysfunction, the index of microcirculatory resistance (IMR), and microvascular obstruction (MVO) by cardiovascular magnetic resonance (CMR), independently predict poor long-term outcomes. Aims: The aims of this study were to investigate whether an invasive parameter (IMR), assessed at the time of primary percutaneous intervention (PPCI), could predict the extent of MVO in proportion to infarct size (MVO index). Methods: 50 patients presenting with STEMI and TIMI flow ≤ I in the infarct related artery were prospectively recruited to the study, before undergoing PPCI. All patients underwent invasive IMR assessment at maximal hyperaemia using adenosine, and following stent insertion. CMR was performed on day 2 following STEMI, MVO was assessed both on first-pass rest perfusion (early MVO) and in the late gadolinium enhancement (LGE) images (late MVO) along with infarct size. The MVO index was calculated as the ratio of late MVO/infarct size. Differences between IMR quartiles and the MVO index were investigated. Results: The median IMR was 38.5 (range 9 to 202). The median size of late MVO was 1.9% LV (range 0 to 21.0% LV). IMR predicted late MVO (p<0.01) and as IMR increased, the MVO index increased (r = 0.70, [95% CI 0.53, 0.82], p<0.001). An IMR cut-off of 40 significantly predicted the presence of late MVO on CMR (p<0.001). Conclusion: IMR measured at the time of PPCI in acutely reperfused STEMI is associated with the presence and severity of infarct damage as measured by the MVO index. Trial registration: The Microcirculation in Acute Myocardial Infarction (MICRO-AMI). Clinicaltrials.gov . Registered 9th March 2012. [ABSTRACT FROM AUTHOR]

The oral obligate anaerobe Porphyromonas gingivalis possesses a small conserved transcript PG_RS02100 of unknown function we previously identified using small RNA-seq analysis as expressed during logarithmic growth. In this study, we sought to determine if PG_RS02100 plays a role in P. gingivalis growth or stress response. We show that a PG_RS02100 deletion mutant’s (W83Δ514) ability to grow under anaerobic conditions was no different than wildtype (W83), but it was better able to survive hydrogen peroxide exposure when cultured under heme limiting growth conditions, and was more aerotolerant when plated on enriched whole blood agar and exposed to atmospheric oxygen. Together, these results indicate that PG_RS02100 plays a role in surviving oxidative stress in actively growing P. gingivalis and that P. gingivalis’ response to exogenous hydrogen peroxide stress is linked to heme availability. Relative qRT-PCR expression analysis of oxyR, trx-1, tpx, sodB, ahpC, dinF, cydB, and frd, in W83Δ514 and W83 in response to 1 h exogenous dioxygen or hydrogen peroxide exposure, when cultured with varying heme availability, support our phenotypic evidence that W83Δ514 has a more highly primed defense system against exogenous peroxide, dioxygen, and heme generated ROS. Interestingly, W83Δ514 turned black faster than W83 when cultured on whole blood agar, suggesting it was able to accumulate heme more rapidly. The mechanism of increased heme acquisition observed in W83Δ514 is not yet known. However, it is clear that PG_RS02100 is involved in modulating the P. gingivalis cell surface in a manner related to survival, particularly against oxidative stress. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effects of different recovery strategies on fatigue markers following a prolonged running exercise. Methods: 46 recreational male runners completed a half-marathon, followed by active recovery (ACT), cold water immersion (CWI), massage (MAS) or passive recovery (PAS). Countermovement jump height, muscle soreness and perceived recovery and stress were measured 24h before the half-marathon (pre), immediately after the recovery intervention (postrec) and 24h after the race (post24). In addition, muscle contractile properties and blood markers of fatigue were determined at pre and post24. Results: Magnitude-based inferences revealed substantial differences in the changes between the groups. At postrec, ACT was harmful to perceived recovery (ACT vs. PAS: effect size [ES] = −1.81) and serum concentration of creatine kinase (ACT vs. PAS: ES = 0.42), with CWI being harmful to jump performance (CWI vs. PAS: ES = −0.98). It was also beneficial for reducing muscle soreness (CWI vs. PAS: ES = −0.88) and improving perceived stress (CWI vs. PAS: ES = −0.64), with MAS being beneficial for reducing muscle soreness (MAS vs. PAS: ES = −0.52) and improving perceived recovery (MAS vs. PAS: ES = 1.00). At post24, both CWI and MAS were still beneficial for reducing muscle soreness (CWI vs. PAS: ES = 1.49; MAS vs. PAS: ES = 1.12), with ACT being harmful to perceived recovery (ACT vs. PAS: ES = −0.68), serum concentration of creatine kinase (ACT vs. PAS: ES = 0.84) and free-testosterone (ACT vs. PAS: ES = −0.91). Conclusions: In recreational runners, a half-marathon results in fatigue symptoms lasting at least 24h. To restore subjective fatigue measures, the authors recommend CWI and MAS, as these recovery strategies are more effective than PAS, with ACT being even disadvantageous. However, runners must be aware that neither the use of ACT nor CWI or MAS had any beneficial effect on objective fatigue markers. [ABSTRACT FROM AUTHOR]

Background: Central venous oxygen saturation (ScvO2) is often used to help to guide resuscitation of critically ill patients. The standard gold technique for ScvO2 measurement is the co-oximetry (Co-oximetry_ScvO2), which is usually incorporated in most recent blood gas analyzers. However, in some hospitals, those machines are not available and only calculated ScvO2 (Calc_ScvO2) is provided. Therefore, we aimed to investigate the agreement between Co-oximetry_ScvO2 and Calc_ScvO2 in a general population of critically ill patients and septic shock patients. Methods: A total of 100 patients with a central venous catheter were included in the study. One hundred central venous blood samples were collected and analyzed using the same point-of-care blood gas analyzer, which provides both the calculated and measured ScvO2 values. Bland and Altman plot, intra-class correlation coefficient (ICC), and Cohen’s Kappa coefficient were used to assess the agreement between Co-oximetry_ScvO2 and Calc_ScvO2. Multiple linear regression analysis was performed to investigate the independent explanatory variables of the difference between Co-oximetry_ScvO2 and Calc_ScvO2. Results: In all population, Bland and Altman’s analysis showed poor agreement (+4.5 [-7.1, +16.1]%) between the two techniques. The ICC was 0.754 [(95% CI: 0.393–0.880), P< 0.001], and the Cohen’s Kappa coefficient, after categorizing the two variables into two groups using a cutoff value of 70%, was 0.470 (P <0.001). In septic shock patients (49%), Bland and Altman’s analysis also showed poor agreement (+5.6 [–6.7 to 17.8]%). The ICC was 0.720 [95% CI: 0.222–0.881], and the Cohen’s Kappa coefficient was 0.501 (P <0.001). Four independent variables (PcvO2, Co-oximetry_ScvO2, venous pH, and Hb) were found to be associated with the difference between the measured and calculated ScvO2 (adjusted R2 = 0.8, P<0.001), with PcvO2 being the main independent explanatory variable because of its highest absolute standardized coefficient. The area under the receiver operator characteristic curves (AUC) of PcvO2 to predict Co-oximetry_ScvO2 ≥ 70% was 0.911 [95% CI: 0.837–0.959], in all patients, and 0.903 [95% CI: 0.784–0.969], in septic shock patients. The best cutoff value was ≥ 36 mmHg (sensitivity, 88%; specificity, 83%), in all patients, and ≥ 35 mmHg (sensitivity, 94%; specificity, 71%) in septic shock patients. Conclusions: The discrepancy between the measured and calculated ScvO2 is clinically not acceptable. We do not recommend the use of calculated ScvO2 to guide resuscitation in critically ill patients. In situations where the Co-oximetry technique is not available, relying on PcvO2 to predict the measured ScvO2 value above or below 70% could be an option. [ABSTRACT FROM AUTHOR]

Bacillus anthracis is a major biological warfare threat. The inhalation form of infection can kill quickly. While antibiotic treatment is effective, if diagnosis is delayed, the rapidly produced toxin may already be present in lethal amounts. This report describes a fast, sensitive, specific and accurate method for detection of active infection by Bacillus anthracis in plasma. One of the virulence factors, anthrax lethal factor, is an endopeptidase present in blood early in the infection. However, the use of peptidic substrates to detect endopetidases is problematic in plasma due to the presence of other proteases and the likelihood of nonspecific cleavage of the substrate. The fluorescently labeled peptide substrate MAPKKide Plus designed in this study is not cleaved by plasma proteases and thus is specific for lethal factor. Three detection strategies are described. Two include enrichment by capture from plasma using lethal factor antibody-coated microtiter plates or similarly coated immuno-tubes. The captured lethal factor is exposed to the MAPKKide Plus, and the amount of cleavage is determined either by HPLC or microplate reader. Concentration of lethal factor using the antibody-coated plates aplnd HPLC allows for detection of less than 5 pg lethal factor/ml of neat plasma after 2 hours of incubation. Using antibody-coated immuno-tubes, 20 pg lethal factor/ml plasma can be detected in 5 hours by a simple end point read of fluorescence in a microplate reader. For a third strategy, the substrate is added directly to diluted plasma, and cleavage is monitored by the increase in fluorescence as a function of time. The limit of detection by this simple method is 25 ng lethal factor/ml of plasma in 15 minutes, 5 ng/ml after 45 minutes, and <1 ng lethal factor/ml of plasma after 5 hours. [ABSTRACT FROM AUTHOR]

Obesity, a principal risk factor for the development of diabetes mellitus, heart disease, and hypertension, is a growing and serious health problem all over the world. Leptin is a weight-reducing hormone produced by adipose tissue, which decreases food intake via hypothalamic leptin receptors (Ob-Rb) and the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling pathway. Protein tyrosine phosphatase 1B (PTP1B) negatively regulates leptin signaling by dephosphorylating JAK2, and the increased activity of PTP1B is implicated in the pathogenesis of obesity. Hence, inhibition of PTP1B may help prevent and reduce obesity. In this study, we revealed that phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate in certain cruciferous vegetables, potently inhibits recombinant PTP1B by binding to the reactive cysteinyl thiol. Moreover, we found that PEITC causes the ligand-independent phosphorylation of Ob-Rb, JAK2, and STAT3 by inhibiting cellular PTP1B in differentiated human SH-SY5Y neuronal cells. PEITC treatment also induced nuclear accumulation of phosphorylated STAT3, resulting in enhanced anorexigenic POMC expression and suppressed orexigenic NPY/AGRP expression. We demonstrated that oral administration of PEITC to mice significantly reduces food intake, and stimulates hypothalamic leptin signaling. Our results suggest that PEITC might help prevent and improve obesity. [ABSTRACT FROM AUTHOR]

Accumulating evidence has revealed that both high sensitivity C-reactive protein (hsCRP) and homocysteine (HCY) are associated with increased risk of metabolic syndrome (MetS) and cardiovascular disease. However, it is unclear whether the coexistence of these conditions accelerates the risk of metabolic syndrome (MetS). We hypothesized that the combination of high sensitivity C-reactive protein (hsCRP) and homocysteine (HCY) levels could exacerbate the development of MetS in a large prospective cohort study. We selected data from 3,170 individuals (1,614 men and 1,556 women) who participated in the Korean Genome and Epidemiology Study. Participants with high hsCRP and HCY levels were categorized into quartiles. MetS was defined based on the criteria of the modified National Cholesterol Education Program, Adult Treatment Panel III. The prevalence of MetS was higher in participants with concurrent high hsCRP and HCY compared to those with low hsCRP and HCY levels. The incidence of MetS at the 6-year follow-up was the highest in participants with concomitant high hsCRP and HCY levels, regardless of obesity. Even after adjusting for potential confounding factors including body mass index in a multivariate logistic regression model, subjects with elevated hsCRP and HCY levels had a 2.50-fold increased risk of developing MetS at the six-year follow-up compared to those who did not have high hsCRP and HCY level. MetS is more prevalent in the concurrent presence of elevated hsCRP and HCY levels. The combination of the two conditions may contribute to an increased risk of MetS, but these factors may not be synergistic. [ABSTRACT FROM AUTHOR]

There is increasing evidence supporting a role for antibodies in protection against tuberculosis (TB), with functional antibodies being described in the latent state of TB infection. Antibody avidity is an important determinant of antibody-mediated protection. This study characterised the avidity of antibodies against Ag85A, an immunodominant Mycobacterium tuberculosis (M.tb) antigen and constituent of several anti-TB vaccine candidates, in individuals of varied M.tb infection status. Avidity of Ag85A specific antibodies was measured in 30 uninfected controls, 34 individuals with latent TB infection (LTBI) and 75 active pulmonary TB (APTB) cases, employing the more commonly used chaotrope-based dissociation assays, and surface plasmon resonance (SPR). Chaotrope-based assays indicated that APTB was associated with a higher antibody avidity index compared to uninfected controls [adjusted geometric mean ratio (GMR): 1.641, 95% confidence interval (CI): 1.153, 2.337, p = 0.006, q = 0.018] and to individuals with LTBI [adjusted GMR: 1.604, 95% CI: 1.282, 2.006, p < 0.001, q <0.001]. SPR assays showed that APTB was associated with slower dissociation rates, an indication of higher avidity, compared to uninfected controls (adjusted GMR: 0.796, 95% CI: 0.681, 0.932, p = 0.004, q = 0.012) and there was also weak evidence of more avid antibodies in the LTBI compared to the uninfected controls (adjusted GMR: 0.871, 95% CI: 0.763, 0.994, p = 0.041, q = 0.123). We found no statistically significant differences in anti-Ag85A antibody avidity between the APTB and LTBI groups. This study shows that antibodies of increased avidity are generated against a principle vaccine antigen in M.tb infected individuals. It would be important to determine whether TB vaccines are able to elicit a similar response. Additionally, more research is needed to determine whether antibody avidity is important in protection against infection and disease. [ABSTRACT FROM AUTHOR]

Cardiovascular disease (CVD) remains the leading cause of death worldwide. Low-density lipoprotein cholesterol (LDL-C) is commonly used for CVD risk assessment; however, recent research has shown LDL particle (LDL-P) number to be a more sensitive indicator of CVD risk than both LDL-C and non-high-density lipoprotein cholesterol (HDL-C). Described herein are five single stranded DNA aptamers with dissociation constants in the low picomolar range specific to LDL-P and its subfractions. Furthermore, a set of antisense sequences have been developed and characterized that are capable of binding to the best aptamers and undergoing displacement by LDL-P for use in a simple, affordable diagnostic assay. [ABSTRACT FROM AUTHOR]

Glutathione S‒transferases (GSTs) are multifunctional enzymes that play an important role in detoxification, cellular signalling, and the stress response. Camelus dromedarius is well-adapted to survive in extreme desert climate and it has GSTs, for which limited information is available. This study investigated the structure-function and thermodynamic properties of a mu-class camel GST (CdGSTM1) at different pH. Recombinant CdGSTM1 (25.7 kDa) was expressed in E. coli and purified to homogeneity. Dimeric CdGSTM1 dissociated into stable but inactive monomeric subunits at low pH. Conformational and thermodynamic changes during the thermal unfolding pathway of dimeric and monomeric CdGSTM1 were characterised via a thermal shift assay and dynamic multimode spectroscopy (DMS). The thermal shift assay based on intrinsic tryptophan fluorescence revealed that CdGSTM1 underwent a two-state unfolding pathway at pH 1.0–10.0. Its Tm value varied with varying pH. Another orthogonal technique based on far-UV CD also exhibited two-state unfolding in the dimeric and monomeric states. Generally, proteins tend to lose structural integrity and stability at low pH; however, monomeric CdGSTM1 at pH 2.0 was thermally more stable and unfolded with lower van't Hoff enthalpy. The present findings provide essential information regarding the structural, functional, and thermodynamic properties of CdGSTM1 at pH 1.0–10.0. [ABSTRACT FROM AUTHOR]

Staphylococcal extracellular polymeric substances (EPS) such as extracellular DNA (eDNA) and poly-N-acetylglucosamine surface polysaccharide (PNAG) mediate numerous virulence traits including host colonization and antimicrobial resistance. Previous studies showed that EPS-degrading enzymes increase staphylococcal biocide susceptibility in vitro and in vivo, and decrease virulence in animal models. In the present study we tested the effect of EPS-degrading enzymes on staphylococcal skin colonization and povidone iodine susceptibility using a novel in vivo pig model that enabled us to colonize and treat 96 isolated areas of skin on a single animal in vivo. To quantitate skin colonization, punch biopsies of colonized areas were homogenized, diluted, and plated on agar for colony forming unit enumeration. Skin was colonized with either Staphylococcus epidermidis or Staphylococcus aureus. Two EPS-degrading enzymes, DNase I and the PNAG-degrading enzyme dispersin B, were employed. Enzymes were tested for their ability to inhibit skin colonization and detach preattached bacteria. The effect of enzymes on the susceptibility of preattached S. aureus to killing by povidone iodine was also measured. We found that dispersin B significantly inhibited skin colonization by S. epidermidis and detached preattached S. epidermidis cells from skin. A cocktail of dispersin B and DNase I detached preattached S. aureus cells from skin and increased their susceptibility to killing by povidone iodine. These findings suggest that staphylococcal EPS components such as eDNA and PNAG contribute to skin colonization and biocide resistance in vivo. EPS-degrading enzymes may be a useful adjunct to conventional skin antisepsis procedures in order to further reduce skin bioburden. [ABSTRACT FROM AUTHOR]

Background: Post-myocardial infarction (MI) remodeling contributes to increased electrophysiological and structural heterogeneity and arrhythmogenesis. Utilising the post-infarct ovine model our aim was to determine unipolar electrogram frequency characteristics consequent to this remodeling and the development of Ventricular Tachycardia (VT). Methods and results: Mapping studies were performed on 14 sheep at >1 month post-MI induction. Sheep were divided into VT inducible (n = 7) and non-inducible (n = 7) groups. Multielectrode needles (n = 20) were deployed within and surrounding ventricular scar for electrophysiological assessment of electrogram amplitude and width. Spectral analysis of electrograms was undertaken using wavelet and fast fourier transformations (WFFT) to calculate root mean square (RMS) power intervals spanning 0-300Hz in 20Hz intervals. Quantitative assessment between electrophysiological and histological parameters including collagen density, and structural organization of the myocardium was performed. Increasing myocardial scar density resulted in attenuation of electrogram amplitude and RMS values. (all p<0.01). Between groups there were no differences in electrogram amplitude (p = 0.37), however WFFT analysis revealed significantly higher RMS values in the VT group (p<0.05) in association with high frequency fractional components of the electrogram. As scar density increased, greater between-group differences in RMS were observed spanning this high frequency (200-280Hz) spectrum and which were proportionally dependent on the degree of structural disorganisation of the myocardium (p<0.001) and number of extrastimuli required to induce VT (p<0.05). Conclusion: High frequency unipolar electrogram spectral characteristics were quantitatively co-influenced by the presence of fibrosis and degree of myocardial structural dissorganisation and were associated with the propensity for development of VT. [ABSTRACT FROM AUTHOR]

Recently, the ratio of patients with diabetes mellitus (DM) among hemodialysis (HD) patients has increased to become the largest sub-population. Their prognoses are significantly worse than those of patients without diabetes (non-DM). In the present study, 10 DM patients who did not take meals and 10 non-DM patients who took meals during HD sessions were investigated. The time courses of the change in plasma levels of metabolites during HD were determined. DM patients exhibited decreased plasma levels of lactate, pyruvate and alanine and dramatically increased levels of ketone bodies. At the end of HD, the plasma levels of lactate, pyruvate, alanine and ketone body were 0.46 ± 0.07, 0.026 ± 0.01, 0.12 ± 0.04 and 0.26 ± 0.04 mM (mean ± standard error), respectively. The profile was ‘hypolactatemia and hyperketonemia’, indicating non-homeostasis. Glycolysis and tricarboxylic acid cycle were suppressed, and the oxidation of fatty acid was accelerated, indicating starvation, even though high amounts of glucose (150 mg/dl) in dialysate were supplied continuously to the bloodstream. In contrast, the plasma levels of lactate, pyruvate, and alanine in the non-DM patients were increased, with the levels of ketone body remaining low during HD to maintain homeostasis, indicating accelerated glycolysis. Furthermore, their plasma levels of insulin increased from 8.1 ± 1.4 to 19.8 ± 3.4 μU/ml, which indicated endogenous secretion stimulated by glucose in dialysate and meal intake. In contrast, in the DM patients, the levels decreased from 19.2 ± 3.4 to 5.5 ± 1.1 μU/ml. This value was the lower limit of the normal range. The depletion of the insulin through extracorporeal circulation may inhibit the transportation of glucose from the blood into the muscles, with the consequence of cell starvation. Such cell starvation along with lipolysis every two days may accelerate proteolysis and affect the prognosis of DM patients. [ABSTRACT FROM AUTHOR]

G protein-gated inwardly rectifying K+ (GIRK) channel regulates cellular excitability upon activation of Gi/o-coupled receptors. In Gi/o-coupled muscarinic M2R, the intracellular third loop (i3) is known as a key domain for Gi/o coupling, because replacement of i3 of Gq-coupled muscarinic M1R with that of M2R enables the chimeric receptor (MC9) to activate the GIRK channel. In the present study, we showed that MC9, but not M1R, co-localizes with the GIRK channel and Gαi1 by Förster resonance energy transfer (FRET) analysis. When M1R was forced to stay adjacent to the channel through ligation with short linkers, M1R activated the GIRK channel. FRET analysis further suggested that the efficacy of channel activation is correlated with the linker length between M1R and the GIRK channel. The results show that co-localization is an important factor for activating the GIRK channel. In contrast, for MC9 and M2R, the GIRK channel was activated even when they were connected by long linkers, suggesting the formation of a molecular complex even in the absence of a linker. We also observed that replacement of 13 amino acid residues at the N-terminal end of i3 of MC9 with those of M1R impaired the co-localization with the GIRK channel as well as channel activation. These results show that localization of the receptor near the GIRK channel is a key factor in efficiently activating the channel and that the N-terminal end of i3 of M2R plays an important role in co-localization. [ABSTRACT FROM AUTHOR]

Sleep is an essential and fundamental physiological process that plays crucial roles in the balance of psychological and physical health. Sleep disorder may lead to adverse health outcomes. The effects of sleep deprivation were extensively studied, but its mechanism is still not fully understood. The present study aimed to identify the alterations of serum proteins associated with chronic sleep deprivation, and to seek for potential biomarkers of sleep disorder mediated diseases. A label-free quantitative proteomics technology was used to survey the global changes of serum proteins between normal rats and chronic sleep deprivation rats. A total of 309 proteins were detected in the serum samples and among them, 117 proteins showed more than 1.8-folds abundance alterations between the two groups. Functional enrichment and network analyses of the differential proteins revealed a close relationship between chronic sleep deprivation and several biological processes including energy metabolism, cardiovascular function and nervous function. And four proteins including pyruvate kinase M1, clusterin, kininogen1 and profilin-1were identified as potential biomarkers for chronic sleep deprivation. The four candidates were validated via parallel reaction monitoring (PRM) based targeted proteomics. In addition, protein expression alteration of the four proteins was confirmed in myocardium and brain of rat model. In summary, the comprehensive proteomic study revealed the biological impacts of chronic sleep deprivation and discovered several potential biomarkers. This study provides further insight into the pathological and molecular mechanisms underlying sleep disorders at protein level. [ABSTRACT FROM AUTHOR]

Megakaryocytes (Mks) derive from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and develop into large, polyploid cells that eventually give rise to platelets. As Mks mature, they migrate from the bone marrow niche into the vasculature, where they are exposed to shear forces from blood flow, releasing Mk particles (platelet-like particles (PLPs), pro/preplatelets (PPTs), and Mk microparticles (MkMPs)) into circulation. We have previously shown that transcription factor p53 is important in Mk maturation, and that physiological levels of shear promote Mk particle generation and platelet biogenesis. Here we examine the role of p53 in the Mk shear-stress response. We show that p53 is acetylated in response to shear in both immature and mature Mks, and that decreased expression of deacetylase HDAC1, and increased expression of the acetyltransferases p300 and PCAF might be responsible for these changes. We also examined the hypothesis that p53 might be involved in the shear-induced Caspase 3 activation, phosphatidylserine (PS) externalization, and increased biogenesis of PLPs, PPTs, and MkMPs. We show that p53 is involved in all these shear-induced processes. We show that in response to shear, acetyl-p53 binds Bax, cytochrome c is released from mitochondria, and Caspase 9 is activated. We also show that shear-stimulated Caspase 9 activation and Mk particle biogenesis depend on transcription-independent p53-induced apoptosis (TIPA), but PS externalization is not. This is the first report to show that shear flow stimulates TIPA and that Caspase 9 activation and Mk-particle biogenesis are directly modulated by TIPA. [ABSTRACT FROM AUTHOR]

Statins are mainstay anti-lipidaemic treatments for preventing cardiovascular diseases but also known to increase coronary artery calcification (CAC). However, underlying relationship between statin and CAC is still unclear. This study explored the mediating role of five statin-related biochemical factors [i.e., low-density lipoprotein (LDL)-cholesterol, high-density lipoprotein (HDL)-cholesterol, triglyceride, glucose, and high sensitivity C-reactive protein levels]. Seoul Metabolic Syndrome cohort study includes 1370 participants suspected of metabolic syndrome. For causal mediation analysis, the dataset for 2016 including 847 participants with coronary computed tomography without any missing value were analysed using the Mediation package in R software. This study identified a causal mediation mechanism of HDL-cholesterol among the five biochemical factors. It implied that statin treatment increases the HDL-cholesterol level, leading to decreasing the probability of CAC score > 0. Estimated values of interest in HDL-cholesterol mediation were (1) average causal mediation effect, -0.011 with 95% CI [-0.025, -0.003], (2) average direct effect, 0.143 with 95% CI [0.074, 0.219], and total effect, 0.132 with 95% CI [0.063, 0.209]. Its mediation effect was maintained regardless of statin intensity. Sensitivity analysis also provided a robustness of the results under potential existence of a confounder between HDL-cholesterol and CAC. This study suggests a potential causal pathway between statin and CAC (the positive association of statin on CAC) through HDL-cholesterol as an inhibitor. [ABSTRACT FROM AUTHOR]

Infectious bronchitis virus (IBV) affects poultry respiratory, renal and reproductive systems. Currently the efficacy of available live attenuated or killed vaccines against IBV has been challenged. We designed a novel IBV vaccine alternative using a highly innovative platform called Self-Assembling Protein Nanoparticle (SAPN). In this vaccine, B cell epitopes derived from the second heptad repeat (HR2) region of IBV spike proteins were repetitively presented in its native trimeric conformation. In addition, flagellin was co-displayed in the SAPN to achieve a self-adjuvanted effect. Three groups of chickens were immunized at four weeks of age with the vaccine prototype, IBV-Flagellin-SAPN, a negative-control construct Flagellin-SAPN or a buffer control. The immunized chickens were challenged with 5x104.7 EID50 IBV M41 strain. High antibody responses were detected in chickens immunized with IBV-Flagellin-SAPN. In ex vivo proliferation tests, peripheral mononuclear cells (PBMCs) derived from IBV-Flagellin-SAPN immunized chickens had a significantly higher stimulation index than that of PBMCs from chickens receiving Flagellin-SAPN. Chickens immunized with IBV-Flagellin-SAPN had a significant reduction of tracheal virus shedding and lesser tracheal lesion scores than did negative control chickens. The data demonstrated that the IBV-Flagellin-SAPN holds promise as a vaccine for IBV. [ABSTRACT FROM AUTHOR]

Extra-cellular galectin-9 (gal-9) is an immuno-modulatory protein with predominant immunosuppressive effects. Inappropriate production of gal-9 has been reported in several human malignancies and viral diseases like nasopharyngeal, pancreatic and renal carcinomas, metastatic melanomas and chronic active viral hepatitis. Therefore therapeutic antibodies neutralizing extra-cellular gal-9 are expected to contribute to immune restoration in these pathological conditions. Two novel monoclonal antibodies targeting gal-9 –Gal-Nab 1 and 2—have been produced and characterized in this study. We report a protective effect of Gal-Nab1 and Gal-Nab2 on the apoptotic cell death induced by gal-9 in primary T cells. In addition, they inhibit late phenotypic changes observed in peripheral T cells that survive gal-9-induced apoptosis. Gal-Nab1 and Gal-Nab2 bind nearly identical, overlapping linear epitopes contained in the 213–224 amino-acid segments of gal-9. Nevertheless, they have some distinct functional characteristics suggesting that their three-dimensional epitopes are distinct. These differences are best demonstrated when gal-9 is applied on Jurkat cells where Gal-Nab1 is less efficient than Gal-Nab2 in the prevention of apoptotic cell death. In addition, Gal-Nab1 stimulates non-lethal phosphatidylserine translocation at the plasma membrane and calcium mobilization triggered by gal-9 in these cells. Both Gal-Nab1 and 2 cross-react with murine gal-9. They bind its natural as well as its recombinant form. This cross-species recognition will be an advantage for their assessment in pre-clinical tumor models. [ABSTRACT FROM AUTHOR]

Human leukocyte antigen (HLA)-DRB3 is a functional HLA class II gene, which has a limited allele diversity in the human population. Furthermore, the HLA-DRB3 gene is only present in a subset of individuals. Therefore, in organ transplantation, this HLA molecule is frequently mismatched between patient and graft donor and thus antibodies against this mismatched HLA molecule can develop. In this study, we aimed to evaluate the prevalence and reactivity of these antibodies and aimed to identify factors that underlie antibody formation against HLA-DRB3. We showed in our patient cohort that HLA-DRB3 antibodies are identified in about 7% of all patients that were screened with solid phase assays. In these assays, we observed multiple antibody reactivity patterns indicating that HLA-DRB3 harbours multiple epitopes. In those cases, where we succeeded at tracing back the induction of these antibodies to the molecular HLA typing of the immunogenic event, we noticed a different frequency of HLA-DRB1 allele groups in the donors as compared to a control group. To a certain extent this distribution (e.g. HLA-DRB1*11 individuals) could be linked to an altered expression level. However, it also appears that different HLA-DRB3 alleles (e.g. HLA-DRB3*01 group) vary in their immunogenicity without having an expression difference. In conclusion, our study provides information on the immunogenicity and reactivity patterns of antibodies against HLA-DRB3 in kidney transplantation, and it points towards the possibility of HLA expression as a factor underlying antibody formation. [ABSTRACT FROM AUTHOR]

A single-injection vaccine formulation that provides for both a prime and a boost immunization would have various advantages over a multiple-injection regime. For such a vaccine formulation, it is essential that the booster dose is released after a certain, preferably adjustable, lag time. In this study we investigated whether a core-shell based implant, containing ovalbumin as core material and poly(DL-lactic-co-glycolic acid) of various monomer ratios as shell material can be used to obtain such a booster release. An in vitro release study showed that the lag time after which the ovalbumin was released from the core-shell implant increased with increasing lactic to glycolic acid ratio of the polymer and ranged from 3–6 weeks. Fluorescence spectroscopy showed minimal differences between native ovalbumin and ovalbumin from core-shell implants that were incubated until just before the observed in vitro release. In addition, mice immunized with a subcutaneous inserted core-shell implant containing ovalbumin showed an ovalbumin-specific IgG1 antibody response after a lag time of 4 or 6–8 weeks. Moreover, delayed release of ovalbumin caused higher IgG1 antibody titers than conventional subcutaneous vaccination with ovalbumin dissolved in PBS. Collectively, these findings could contribute to the further development of a single-injection vaccine, making multiple injections of the vaccine superfluous. [ABSTRACT FROM AUTHOR]

Acid sensing ion channels (ASICs) are proton-gated ion channels that are members of the degenerin/epithelial sodium channel superfamily and are expressed throughout central and peripheral nervous systems. ASICs have been implicated in multiple physiological processes and are subject to numerous forms of endogenous and exogenous regulation that include modulation by Ca2+ and Cl- ions. However, the mapping of ion binding sites as well as a structure-based understanding of the mechanisms underlying ionic modulation of ASICs have remained elusive. Here we present ion binding sites of chicken ASIC1a in resting and desensitized states at high and low pH, respectively, determined by anomalous diffraction x-ray crystallography. The acidic pocket serves as a nexus for divalent cation binding at both low and high pH, while we observe divalent cation binding within the central vestibule on the resting channel at high pH only. Moreover, neutralization of residues positioned to coordinate divalent cations via individual and combined Glu to Gln substitutions reduced, but did not extinguish, modulation of proton-dependent gating by Ca2+. Additionally, we demonstrate that anion binding at the canonical thumb domain site is state-dependent and present a previously undetected anion site at the mouth of the extracellular fenestrations on the resting channel. Our results map anion and cation sites on ASICs across multiple functional states, informing possible mechanisms of modulation and providing a blueprint for the design of therapeutics targeting ASICs. [ABSTRACT FROM AUTHOR]

Neisserial heparin binding antigen (NHBA) is one of three main recombinant protein antigens in 4CMenB, a vaccine for the prevention of invasive meningococcal disease caused by Neisseria meningitidis serogroup B. NHBA is a surface-exposed lipoprotein composed of a predicted disordered N-terminal region, an arginine-rich region that binds heparin, and a C-terminal domain that folds as an anti-parallel β-barrel and that upon release after cleavage by human proteases alters endothelial permeability. NHBA induces bactericidal antibodies in humans, and NHBA-specific antibodies elicited by the 4CMenB vaccine contribute to serum bactericidal activity, the correlate of protection. To better understand the structural bases of the human antibody response to 4CMenB vaccination and to inform antigen design, we used X-ray crystallography to elucidate the structures of two C-terminal fragments of NHBA, either alone or in complex with the Fab derived from the vaccine-elicited human monoclonal antibody 5H2, and the structure of the unbound Fab 5H2. The structures reveal details on the interaction between an N-terminal β-hairpin fragment and the β-barrel, and explain how NHBA is capable of generating cross-reactive antibodies through an extensive conserved conformational epitope that covers the entire C-terminal face of the β-barrel. By providing new structural information on a vaccine antigen and on the human immune response to vaccination, these results deepen our molecular understanding of 4CMenB, and might also aid future vaccine design projects. [ABSTRACT FROM AUTHOR]

Given that Chinese materia medica (CMM) is expensive and rare, people take tremendous risk to adulterate and falsify Cordyceps sinensis with counterfeit species with similar morphological features. It is thus essential to develop new methods to identify the authenticity of Cordyceps sinensis. It is hypothesized in this study that Cordyceps sinensis possesses certain protein biomarkers distinct from its counterfeits, which can be identified by proteomic technologies for authentication purposes. This is the first study that aims to optimize the conditions for extracting proteins from Cordyceps sinensis, a hybrid of fungal-animal CMM, and to compare the two-dimensional gel electrophoresis (2-DE) profiles between different Cordyceps species. Two different protein extraction buffer systems, namely, phenol/sodium dodecyl sulfate (SDS) buffer or lysis buffer, were evaluated, where the preparation using lysis buffer yielded better protein content. The results also showed that extraction with lysis buffer without pre- or post-washing of samples was the most effective protocol, with over 220% of protein yield and 819 protein spots detected on a 2-DE gel. Moreover, the results demonstrated that Cordyceps sinensis possesses protein biomarkers distinct from its counterfeits, and these biomarkers are not source- or origin-dependent, strongly supporting the feasibility of using identified biomarkers as indicators for authentication of Cordyceps species. The findings of this study warrant further investigations on the structural identification of protein biomarkers of Cordyceps species. [ABSTRACT FROM AUTHOR]

Iron acquisition by bacteria is well studied, but iron export from bacteria is less understood. Herein, we identified dr1440 with a P-type ATPase motif as a potential exporter of iron from Deinococcus radiodurans, a bacterium known for its extreme resistance to radiation and oxidants. The DR1440 was located in cell membrane as demonstrated by fluorescence labelling analysis. Mutation of dr1440 resulted in cellular accumulation of iron ions, and expression level of dr1440 was up-regulated significantly under iron ion or hydrogen peroxide stress in the wild-type strain, implicating DR1440 as a potential iron efflux protein. The dr1440 mutant displayed higher sensitivity to iron ions and oxidative stresses including hydrogen peroxide, hypochlorous acid, and gamma-ray irradiation compared with the wild-type strain. The high amount of iron in the mutant strain resulted in severe protein carbonylation, suggesting that DR1440 might contribute to intracellular protein protection against reactive oxygen species (ROS) generated from ferrous ion-mediated Fenton-reaction. Mutations of S297A and C299A led to intracellular accumulation of iron, indicating that S297 and C299 might be important functional residues of DR1440. Thus, DR1440 is a potential iron efflux protein involved in iron homeostasis and oxidative stress-resistance of D. radiodurans. [ABSTRACT FROM AUTHOR]

Background: Ready-to-use therapeutic food (RUTF) is used to treat children suffering from severe acute malnutrition (SAM). Standard RUTF uses milk as the primary protein source, which makes the product expensive, and given the high worldwide SAM burden, having a less expensive effective alternative is a public health priority. Objective: The objective of this study was to evaluate whether newly developed amino acid-enriched milk-free RUTF (FSMS-RUTF) or amino acid-enriched low-milk RUTF (MSMS-RUTF) treatment could replenish plasma amino acids to levels comparable to those following standard peanut-milk RUTF (PM-RUTF) treatment and to improve understanding of the effects of treatment on anthropometric measurements. A secondary analysis was performed to test the noninferiority hypothesis of plasma essential amino acid (EAA) levels. Methods: Plasma EAA levels were measured in a nonblinded, 3-arm, parallel-group simple randomized controlled trial conducted in Malawi to examine the efficacy of FSMS-RUTF, MSMS-RUTF and PM-RUTF in the treatment of SAM in 2 groups of children aged 6–23 and 24–59 months (mo). Sample size calculations were performed based on the previous our study. A noninferiority margin was set at -25% of the PM-RUTF arm at discharge. Results: The relative values of the differences (95% CI) in plasma EAA levels between PM-RUTF treatment and FSMS-RUTF and MSMS-RUTF treatments at discharge were -7.9% (-18.6, 2.8) and 9.8% (0.2, 19.5), respectively, in children aged 6–23 mo, while in those aged 24–59 mo, the difference values were 17.8% (1.6, 34.1) and 13.6% (-2.8, 29.9), respectively. Conclusion: At discharge, the plasma EAA concentrations in 6-59-mo-old SAM children treated with FSMS-RUTF and MSMS-RUTF were not less than those of children treated with PM-RUTF. These findings indicate that treatment with either of the 3 RUTFs was associated with adequate protein synthesis and that all the formulations provided sufficient functional metabolites of plasma amino acids to support nutritional recovery from SAM. [ABSTRACT FROM AUTHOR]

Background: In hemodialysis patients, high levels of Fibroblast Growth Factor 23 (FGF23) predict mortality. Our study was designed to test whether the control of serum phosphate is associated with a reduction in serum FGF23 levels. Additionally other variables with a potential effect on FGF23 levels were evaluated. Material and methods: The effect of sustained (40-weeks) control of serum phosphate on FGF23 levels (intact and c-terminal) was evaluated in 21 stable hemodialysis patients that were not receiving calcimimetics or active vitamin D. Patients received non-calcium phosphate binders to maintain serum phosphate below 4.5 mg/dl. In an additional analysis, values of intact-FGF23 (iFGF23) and c-terminal FGF23 (cFGF23) from 150 hemodialysis patients were correlated with parameters of mineral metabolism and inflammation. Linear mixed models and linear regression were performed to evaluate longitudinal trajectories of variables and the association between FGF23 and the other variables examined. Results: During the 40-week treatment, 12 of 21 patients achieved the target of serum phosphate <4.5 mg/dl. In these 12 patients, iFGF23 decreased to less than half whereas cFGF23 did not reduce significantly. In patients with serum phosphate >4.5 mg, iFGF23 and cFGF23 increased two and four-fold respectively as compared with baseline. Furthermore, changes in serum phosphate correlated with changes in C-reactive protein (hs-CRP). In our 150 hemodialysis patients, those in the higher tertile of serum phosphate also showed increased hs-CRP, iPTH, iFGF23 and cFGF23. Multiple regression analysis revealed that iFGF23 levels directly correlated with both serum phosphate and calcium, whereas cFGF23 correlated with serum phosphate and hs-CRP but not with calcium. Conclusions: The control of serum phosphate reduced iFGF23. This reduction was also associated with a decreased in inflammatory parameters. Considering the entire cohort of hemodialysis patients, iFGF23 levels correlated directly with serum phosphate levels and also correlated inversely with serum calcium concentration. The levels of cFGF23 were closely related to serum phosphate and parameters of inflammation. [ABSTRACT FROM AUTHOR]

NMDA-type glutamate receptors (NMDAR) are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system. NMDAR dysfunction has been found to be involved in various neurological disorders. Recent crystallographic and EM studies have shown the static structure of different states of the non-human NMDARs. Here we describe a model of a human NMDA receptor (hNMDAR) and its molecular dynamics (MD) before and after the binding of agonist ligands, glutamate and glycine. It is shown that the binding of ligands promotes a global reduction in molecular flexibility that produces a more tightly packed conformation than the unbound hNMDAR, and a higher cooperative regularity of moving. The ligand-induced synchronization of motion, identified on all structural levels of the modular hNMDA receptor is apparently a fundamental factor in channel gating. Although the time scale of the MD simulations (300 ns) was not sufficient to observe the complete gating event, the obtained data has shown the ligand-induced stabilization of hNMDAR that conforms the “going to be open state”. We propose a mechanistic dynamic model of the ligand-dependent gating mechanism in the hNMDA receptor. At the binding of the ligands, the differently twisted conformations of the highly flexible receptor are stabilized in unique conformation with a linear molecular axis, which is a condition that is optimal for pore development. By searching the receptor surface, we have identified three new pockets, which are different from the pockets described in the literature as the potential and known positive allosteric modulator binding sites. A successful docking of two NMDAR modulators to their binding sites validates the model of a human NMDA receptor as a biological relevant target. [ABSTRACT FROM AUTHOR]

L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin’s lymphoma. However, some disadvantages still limit its full application potential, e.g., allergic reactions, pancreatitis, and blood clotting impairment. Therefore, much effort has been directed at improving its performance. A popular strategy is to randomly conjugate L-asparaginase with mono-methoxy polyethylene glycol, which became a commercial FDA approved formulation widely used in recent years. To improve this formulation by PEGylation, herein we performed cysteine-directed conjugation of the L-asparaginase subunits to prevent dissociation-induced loss of activity. The recombinant cysteine conjugation sites were introduced by mutagenesis at surface-exposed positions on the protein to avoid affecting the catalytic activity. Three conjugates were obtained using different linear PEGs of 1000, 2000, and 5000 g/mol, with physical properties ranging from a semi-solid gel to a fully soluble state. The soluble-conjugate exhibited higher catalytic activity than the non-conjugated mutant, and the same activity than the native enzyme. The cysteine-directed crosslinking of the L-asparaginase subunits produced a higher molecular weight conjugate compared to the native tetrameric enzyme. This strategy might improve L-asparaginase efficiency for leukemia treatment by reducing glomerular filtration due to the increase in hydrodynamic size thus extending half-live, while at the same time retaining full catalytic activity. [ABSTRACT FROM AUTHOR]

Ticagrelor, a P2Y12 antagonist, is approved for prevention of thromboembolic events. MEDI2452 is a potential reversal agent for ticagrelor and ticagrelor active metabolite (TAM). The total plasma exposure of ticagrelor and TAM in patients are roughly 0.5–1 and 0.2–0.5 μmol/L, respectively. Both have similar high potency vs. P2Y12 (Ki 2 nmol/L) but are plasma protein-bound to 99.8% and only the 0.2% free fraction is able to inhibit the P2Y12 receptor. Thus, for unbound concentration measurements to be a proof of mechanism biomarker for MEDI2452 a very high sensitivity is required. Using established techniques as equilibrium dialysis and LC-MS/MS, made it possible to evaluate the efficacy of the reversal agent by measuring reduction of unbound concentration of ticagrelor in the presence of MEDI2452. With challenges such as ultra-low concentrations, small sample volumes, recovery issues and adsorption to plastic we managed to develop a highly sensitive assay for determining unbound concentration levels of ticagrelor and TAM in plasma with a quantification limit of 30 pmol/L and 45 pmol/L, respectively. With this method we were able to detect close to a 100-fold MEDI2452 mediated reduction in the unbound concentration of both ticagrelor and TAM. The assay provided proof of mechanism as MEDI2452 concentration- and dose-dependently eliminated unbound concentration of ticagrelor and reversed its antiplatelet activity in preclinical models and will support future development of MEDI2452. [ABSTRACT FROM AUTHOR]

Toll like receptors (TLRs) share a conserved structure comprising the N-terminal ectodomain, a transmembrane segment and a C-terminal cytoplasmic Toll/IL-1 receptor (TIR) domain. Proper assembly of the TIR domain is crucial for signal transduction; however, the contribution of individual motifs within the TIR domain to TLR trafficking and signaling remains unclear. We targeted a highly conserved tyrosine (Y870) located in the box 1 region of the TIR domain of most TLRs, including TLR9, previously described to be a critical site of phosphorylation in TLR4. We reconstituted bone marrow-derived dendritic cells (BMDC) from Tlr9-/- mice WT TLR9 or Y870F or Y870A mutants. Despite normal interactions with the luminal chaperones GRP94 and UNC93B1, Y870F conferred only partial responsiveness to CpG, and Y870A had no activity and functioned as a dominant negative inhibitor when coexpressed with endogenous TLR9. This loss of function correlated with reduction or absence, respectively, of the 80 kDa mature form of TLR9. In Y870F-expressing cells, CpG-dependent signaling correlated directly with levels of the mature form, suggesting that signaling did not require tyrosine phosphorylation but rather that the Y870F mutation conferred reduced receptor levels due to defective processing or trafficking. Microscopy revealed targeting of the mutant protein to an autophagolysosome-like structure for likely degradation. Collectively we postulate that the conserved Y870 in the TIR domain does not participate in phosphorylation-induced signaling downstream of ligand recognition, but rather is crucial for proper TIR assembly and ER egress, resulting in maturation-specific stabilization of TLR9 within endolysosomes and subsequent pro-inflammatory signaling. [ABSTRACT FROM AUTHOR]

Oral tumors, including highly invasive and metastatic oral melanoma (OM), non-tonsillar oral squamous cell carcinoma (OSCC) and benign tumors (BN), are common neoplasms in dogs. Although these tumors behave differently, limited data of their protein expression profiles have been exhibited, particularly at the proteome level. The present study aimed to i.) characterize peptide-mass fingerprints (PMFs) and identify potential protein candidates of OM, OSCC, BN and normal control subjects, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS), ii.) identify potential protein candidates associated with the diseases, using in-gel digestion coupled with mass spectrometric analysis (GeLC-MS/MS) and iii.) search for relationships between chemotherapy drugs and disease-perturbed proteins. A distinct cluster of each sample group and unique PMFs with identified protein candidates were revealed. The unique peptide fragment at 2,274 Da of sacsin molecular chaperone (SACS) was observed in early-stage OM whereas the fragment at 1,958 Da of sodium voltage-gated channel alpha subunit 10 (SCN10A) was presented in early- and late-stage OM. The peptide mass at 2,316 Da of Notch1 appeared in early-stage OM and benign oral tumors while the peptide mass at 2,505 Da of glutamate ionotropic receptor N-methyl-D-aspartate type subunit 3A (GRIN3A) was identified in all groups. Markedly expressed proteins from GeLC-MS/MS included Jumonji domain containing 1C (JMJD1C) in benign tumors, inversin (INVS) and rho guanine nucleotide exchange factor 28 (ARHGEF28) in OM, BTB domain-containing 16 (BTBD16) in OSCC, and protein tyrosine phosphatase non-receptor type 1 (PTPN1), BRCA2, DNA repair associated (BRCA2), WW domain binding protein 2 (WBP2), purinergic receptor P2Y1 and proteasome activator subunit 4 (PSME4) in all cancerous groups. The network connections between these proteins and chemotherapy drugs, cisplatin and doxorubicin, were also demonstrated. In conclusion, this study unveiled the unique PMFs and novel candidate protein markers of canine oral tumors. [ABSTRACT FROM AUTHOR]

N-terminal acetylation is one of the most common co- and post-translational modifications of the eukaryotic proteome and regulates numerous aspects of cellular physiology, such as protein folding, localization and turnover. In particular α-synuclein, whose dyshomeostasis has been tied to the pathogenesis of several neurodegenerative disorders, is completely Nα-acetylated in nervous tissue. In this work, building on previous reports, we develop and characterize a bacterial N-terminal acetylation system based on the expression of the yeast N-terminal acetyltransferase B (NatB) complex under the control of the PBAD (L-arabinose-inducible) promoter. We show its functionality and the ability to completely Nα-acetylate our model substrate α-synuclein both upon induction of the construct with L-arabinose and also by only relying on the constitutive expression of the NatB genes. [ABSTRACT FROM AUTHOR]

Reduced bioavailability of nitric oxide (NO) is a major feature of endothelial dysfunction characteristic of cardiovascular and metabolic diseases but the short half-life of NO precludes its easy quantification in circulating blood for early diagnosis. In erythrocytes, NO can react with hemoglobin to form an iron-nitrosyl complex (5-coordinate-α-HbNO) directly quantifiable by Electron Paramagnetic Resonance spectroscopy (EPR) in mouse, rat and human venous blood ex vivo. However, the sources of the nitrosylating species in vivo and optimal conditions of HbNO preservation for diagnostic use in human erythrocytes are unknown. Using EPR spectroscopy, we found that HbNO stability was significantly higher under hypoxia (equivalent to venous pO2; 12.0±0.2% degradation of HbNO at 30 minutes) than at room air (47.7±0.2% degradation) in intact erythrocytes; at 20°C (15.2±0.3% degradation after 30 min versus 29.6±0.1% at 37°C) and under acidic pH (31.7±0.8% versus 62.2±0.4% degradation after 30 min at physiological pH) at 50% of haematocrit. We next examined the relative contribution of NO synthase (NOS) from the vasculature or in erythrocytes themselves as a source of nitrosylating NO. We detected a NOS activity (and eNOS expression) in human red blood cells (RBC), and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice, as measured by HbNO formation and nitrite/nitrate accumulation. NO formation was increased after inhibition of arginase but abrogated upon NOS inhibition in human RBC and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice. However, the HbNO signal from freshly drawn venous RBCs was minimally sensitive to the inhibitors ex vivo, while it was enhanced upon caveolin-1 deletion in vivo, suggesting a minor contribution of erythrocyte NOS to HbNO complex formation compared with vascular endothelial NOS or other paracrine NO sources. We conclude that HbNO formation in rodent and human venous erythrocytes is mainly influenced by vascular NO sources despite the erythrocyte NOS activity, so that its measurement by EPR could serve as a surrogate for NO-dependent endothelial function. [ABSTRACT FROM AUTHOR]

Background/Objectives: Given their role in female reproduction, the effects of progesterone on arginine and related amino acids, polyamines and NF-κB p65 activation were studied across the menstrual cycle. Methods: Arginine, ornithine and citrulline as well as putrescine, spermidine, spermine, and N-acetyl-putrescine were determined in plasma, NF-κB p65 activation in peripheral blood mononuclear cells and progesterone in serum of 28 women at early (T1) and late follicular (T2) and mid (T3) and late (T4) luteal phase. Results: Arginine and related amino acids declined from T1 and T2 to T3 and T4, while progesterone increased. At T3, arginine, ornithine, and citrulline were inversely related with progesterone. Changes (ΔT3-T2) in arginine, ornithine, and citrulline were inversely related with changes (ΔT3-T2) in progesterone. Ornithine and citrulline were positively related with arginine, as were changes (ΔT3-T2) in ornithine and citrulline with changes (ΔT3-T2) in arginine. At T2, NF-κB p65 activation was positively related with arginine. Polyamines did not change and were not related to progesterone. All results described were significant at P < 0.001. Conclusions: This study for the first time provides data, at the plasma and PBMC level, supporting a proposed regulatory node of arginine and related amino acids, progesterone and NF-κB p65 at luteal phase of the menstrual cycle, aimed at successful preparation of pregnancy. [ABSTRACT FROM AUTHOR]

Neuronal nicotinic acetylcholine receptors (nAChRs) of the cholinergic system have been linked to antinociception, and therefore could be an alternative target for pain alleviation. nAChR activity has been shown to be regulated by the nicotinic modulator, lynx1, which forms stable complexes with nAChRs and has a negative allosteric action on their function. The objective in this study was to investigate the contribution of lynx1 to nicotine-mediated antinociception. Lynx1 contribution was investigated by mRNA expression analysis and electrophysiological responses to nicotine in the dorsal raphe nucleus (DRN), a part of the pain signaling pathway. In vivo antinociception was investigated in a test of nociception, the hot-plate analgesia assay with behavioral pharmacology. Lynx1/α4β2 nAChR interactions were investigated using molecular dynamics computational modeling. Nicotine evoked responses in serotonergic and GABAergic neurons in the DRN are augmented in slices lacking lynx1 (lynx1KO). The antinociceptive effect of nicotine and epibatidine is enhanced in lynx1KO mice and blocked by mecamylamine and DHβE. Computer simulations predict preferential binding affinity of lynx1 to the α:α interface that exists in the stoichiometry of the low sensitivity (α4)3(β2)2 nAChRs. Taken together, these data point to a role of lynx1 in mediating pain signaling in the DRN through preferential affinity to the low sensitivity α4β2 nAChRs. This study suggests that lynx1 is a possible alternative avenue for nociceptive modulation outside of opioid-based strategies. [ABSTRACT FROM AUTHOR]

Backgrounds: Proton pump inhibitors (PPIs) can be associated with vascular calcification in patients undergoing dialysis through hypomagnesemia. However, only few studies have demonstrated the influence of PPIs on vascular calcification in patients on maintenance hemodialysis (HD). This study aimed to investigate whether the use of PPIs accelerates vascular calcification in patients on HD. Materials and methods: We retrospectively evaluated 200 HD patients who underwent regular blood tests and computed tomography (CT) between 2016 and 2017. The abdominal aortic calcification index (ACI) was measured using abdominal CT. The difference in the ACI values between 2016 and 2017 was evaluated as ΔACI. Patients were divided into PPI and non-PPI groups, and variables, such as patient background, medication, laboratory data, and ΔACI were compared. Factors independently associated with higher ΔACI progression (≥ third tertile value of ΔACI in this study) were determined using multivariate logistic regression analysis. Results: The PPI and non-PPI groups had 112 (56%) and 88 (44%) patients, respectively. Median and third tertile value of ΔACIs were 4.2% and 5.8%, respectively. Serum magnesium was significantly lower in the PPI (2.1 mg/dL) than in the non-PPI (2.3 mg/dL) group (P <0.001). Median ΔACI was significantly higher in the PPI (5.0%) than in the non-PPI (3.8%) group (P = 0.009). A total of 77 (39%) patients had a higher ΔACI. Multivariate analysis revealed that PPIs (odds ratio = 2.23; 95% confidence interval = 1.11–4.49), annual mean calcium phosphorus product, ACI in 2016, baseline serum magnesium levels, and HD vintage were independent factors associated with higher ΔACI progression after adjusting for confounders. Conclusion: PPI use may accelerate vascular calcification in patients on HD. Further studies are necessary to elucidate their influence on vascular calcification. [ABSTRACT FROM AUTHOR]

NF-κB is an important mediator of immune activity and its activation is essential in mounting immune response to pathogens. Here, we describe the optimization and implementation of a high-throughput screening platform that utilizes high content imaging and analysis to monitor NF-κB nuclear translocation. We screened 38,991 compounds from three different small molecule libraries and identified 103 compound as hits; 31% of these were active in a dose response assay. Several of the molecules lacked cytotoxicity or had a selectivity index of more than 2-fold. Our image-based approach provides an important first step towards identifying small molecules with immunomodulatory activity. [ABSTRACT FROM AUTHOR]