The rockefeller university

Collagen is the major protein in the extracellular matrix and plays vital roles in tissue development and function. Collagen is also one of the most processed proteins in its biosynthesis. The most prominent post-translational modification (PTM) of collagen is the hydroxylation of Pro residues in the Y-position of the characteristic (Gly-Xaa-Yaa) repeating amino acid sequence of a collagen triple helix. Recent studies using mass spectrometry (MS) and tandem MS sequencing (MS/MS) have revealed unexpected hydroxylation of Pro residues in the X-positions (X-Hyp). The newly identified X-Hyp residues appear to be highly heterogeneous in location and percent occupancy. In order to understand the dynamic nature of the new X-Hyps and their potential impact on applications of MS and MS/MS for collagen research, we sampled four different collagen samples using standard MS and MS/MS techniques. We found considerable variations in the degree of PTMs of the same collagen from different organisms and/or tissues. The rat tail tendon type I collagen is particularly variable in terms of both over-hydroxylation of Pro in the X-position and under-hydroxylation of Pro in the Y-position. In contrast, only a few unexpected PTMs in collagens type I and type III from human placenta were observed. Some observations are not reproducible between different sequencing efforts of the same sample, presumably due to a low population and/or the unpredictable nature of the ionization process. Additionally, despite the heterogeneous preparation and sourcing, collagen samples from commercial sources do not show elevated variations in PTMs compared to samples prepared from a single tissue and/or organism. These findings will contribute to the growing body of information regarding the PTMs of collagen by MS technology, and culminate to a more comprehensive understanding of the extent and the functional roles of the PTMs of collagen.

Though it is well known that G protein-coupled receptor kinase 2 [GRK2] is involved in regulation of mu opioid receptor [MOR] desensitization and morphine-related behaviors, the potential role of GRK2 in regulation of kappa opioid receptor [KOR] functions in vivo has not been established yet. A couple of recent studies have found that GRK2 activity desensitizes KOR functions via decreasing G protein-coupled signaling with sensitizing arrestin-coupled signaling. Nalfurafine, a G protein-biased KOR full agonist, produces an inhibitory effect on alcohol intake in mice, with fewer side effects (sedation, aversion, or anxiety/depression-like behaviors). Using RNA sequencing (RNA-seq) analysis, we first identified that nuclear transcript level of grk2 [adrbk1] (but not other grks) was significantly up-regulated in mouse nucleus accumbens shell (NAcs) after chronic excessive alcohol drinking, suggesting alcohol specifically increased NAcs grk2 expression. We then tested whether selective GRK2/3 inhibitor CMPD101 could alter alcohol intake and found that CMPD101 alone had no effect on alcohol drinking. Therefore, we hypothesized that the grk2 increase in the NAcs could modulate the nalfurafine effect on alcohol intake via interacting with the G protein-mediated KOR signaling. Nalfurafine decreased alcohol drinking in a dose-related manner, and pretreatment with CMPD101 enhanced the reduction in alcohol intake induced by nalfurafine, indicating an involvement of GRK2/3 blockade in modulating G protein-biased KOR agonism of nalfurafine. Together, our study provides initial evidence relevant to the transcriptional change of grk2 gene in the NAc shell after excessive alcohol drinking. Pharmacological GRK2/3 blockade enhanced nalfurafine's efficacy, suggesting a GRK2/3-mediated mechanism, probably through the G protein-mediated KOR signaling.

Anti-HIV broadly neutralizing antibodies (bNAbs) may favor development of antiviral immunity by engaging the immune system during immunotherapy. Targeting integrin alpha(4)beta(7) with an anti-alpha(4)beta(7) monoclonal antibody (Rh-alpha(4)beta(7)) affects immune responses in SIV/ SHIV-infected macaques. To explore the therapeutic potential of combining bNAbs with alpha(4)beta(7) integrin blockade, SHIVSF162P3-infected, viremic rhesus macaques were treated with bNAbs only (VRC07-523LS and PGT128 anti-HIV antibodies) or a combination of bNAbs and Rh-alpha(4)beta(7) or were left untreated as a control. Treatment with bNAbs alone decreased viremia below 200 copies/ml in all macaques, but seven of eight macaques (87.5%) in the bNAbs-only group rebounded within a median of 3 weeks (95% CI: 2 to 9). In contrast, three of six macaques treated with a combination of Rh-alpha(4)beta(7) and bNAbs (50%) maintained a viremia below 200 copies/ml until the end of the follow-up period; viremia in the other three macaques rebounded within a median of 6 weeks (95% CI: 5 to 11). Thus, there was a modest delay in viral rebound in the macaques treated with the combination antibody therapy compared to bNAbs alone. Our study suggests that alpha(4)beta(7) integrin blockade may prolong virologic control by bNAbs in SHIVSF162P3-infected macaques.

For millennia, people have used "averted vision" to improve their detection of faint celestial objects, a technique first documented around 325 BCE. Yet, no studies have assessed gaze location during averted vision to determine what pattern best facilitates perception. Here, we characterized averted vision while recording eye-positions of dark-adapted human participants, for the first time. We simulated stars of apparent magnitudes 3.3 and 3.5, matching their brightness to Megrez (the dimmest star in the Big Dipper) and Tau Ceti. Participants indicated whether each star was visible from a series of fixation locations, providing a comprehensive map of detection performance in all directions. Contrary to prior predictions, maximum detection was first achieved at similar to 8 degrees from the star, much closer to the fovea than expected from rod-cone distributions alone. These findings challenge the assumption of optimal detection at the rod density peak and provide the first systematic assessment of an age-old facet of human vision.

Featured Application Here we present the design of a temperature regulation and light isolation microscope enclosure. This design can be readily adapted to the specific configurations of a custom imaging system. Light isolation and temperature regulation are often required for microscopic imaging. Commercial enclosures are available to satisfy these requirements, but they are often not flexible to the variety of custom systems found in research laboratories. We present the design for an affordable enclosure which utilizes aluminum t-slot profiles to support opaque expanded PVC panels. Temperature is regulated by exchanging the enclosure air with an external heater. In addition, we demonstrate baffles integrated into the enclosure improve temperature uniformity. Example designs for both upright and inverted microscopes are given, providing a starting point for creating a system-specific custom enclosure.

The PUF RNAbinding domain has an eight-repeat structure with the ability to bind an eight-base RNA sequence. Here the authors generate a comprehensive library of variants of a PUF domain to find an RNA binding code and design PUF domains that recognize an arbitrary RNA sequence. The ability to design a protein to bind specifically to a target RNA enables numerous applications, with the modular architecture of the PUF domain lending itself to new RNA-binding specificities. For each repeat of the Pumilio-1 PUF domain, we generate a library that contains the 8,000 possible combinations of amino acid substitutions at residues critical for RNA contact. We carry out yeast three-hybrid selections with each library against the RNA recognition sequence for Pumilio-1, with any possible base present at the position recognized by the randomized repeat. We use sequencing to score the binding of each variant, identifying many variants with highly repeat-specific interactions. From these data, we generate an RNA binding code specific to each repeat and base. We use this code to design PUF domains against 16 RNAs, and find that some of these domains recognize RNAs with two, three or four changes from the wild type sequence.

We have described a child suffering from Mendelian susceptibility to mycobacterial disease (MSMD) due to autosomal recessive, complete T-bet deficiency, which impairs IFN-gamma production by innate and innate-like adaptive, but not mycobacterial-reactive purely adaptive, lymphocytes. Here, we explore the persistent upper airway inflammation (UAI) and blood eosinophilia of this patient. Unlike wild-type (WT) T-bet, the mutant form of T-bet from this patient did not inhibit the production of Th2 cytokines, including IL-4, IL-5, IL-9, and IL-13, when overexpressed in T helper 2 (Th2) cells. Moreover, Herpesvirus saimiri-immortalized T cells from the patient produced abnormally large amounts of Th2 cytokines, and the patient had markedly high plasma IL-5 and IL-13 concentrations. Finally, the patient's CD4(+) alpha beta T cells produced most of the Th2 cytokines in response to chronic stimulation, regardless of their antigen specificities, a phenotype reversed by the expression of WT T-bet. T-bet deficiency thus underlies the excessive production of Th2 cytokines, particularly IL-5 and IL-13, by CD4(+) alpha beta T cells, causing blood eosinophilia and UAI. The MSMD of this patient results from defective IFN-. production by innate and innate-like adaptive lymphocytes, whereas the UAI and eosinophilia result from excessive Th2 cytokine production by adaptive CD4(+) alpha beta T lymphocytes.

The blood-brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA's molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. Based on these findings, we propose that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical to maintain tissue size and brain homeostasis during organogenesis.

Background: Pre-hospital platelet inhibition in patients with ST-segment elevation myocardial infarction (STEMI) may improve outcomes. RUC-4 is a novel, second-generation glycoprotein IIb/IIIa inhibitor designed for first-point-of-medical-contact treatment for STEMI by subcutaneous injection. Aims: The open-label, phase 2A, CEL-02 trial aimed to assess the pharmacodynamics (PD), pharmacokinetics (PK), and tolerability of RUC-4 in STEMI patients undergoing primary PCI (pPCI). Methods: A total of 27 STEMI patients received a weight-adjusted subcutaneous injection of RUC-4 before pPCI in escalating doses (0.075 mg/kg [n = 8], 0.090 mg/kg [n = 9], or 0.110 mg/kg [n = 10]). Results: The primary PD endpoint of high-grade (>= 77%) inhibition of the VerifyNow iso-TRAP assay at 15 minutes was met in 3/8, 7/8, and 7/8 patients in the three cohorts with a dose-response relationship (mean inhibition [min -max] of 77.5% [65.7%-90.6%], 87.5% [73.8%-93.1%], and 91.7% [76.4%-99.3%], respectively; ptrend = 0.002). Fifty percent (50%) inhibition remained after 89.1 (38.0-129.7), 104.2 (17.6-190.8), and 112.4 (19.7-205.0) minutes. Injection site reactions or bruising were observed in 1 (4%) and 11 (41%) patients, respectively. Mild access-site haematomas occurred in 6 (22%), and severe access-site haematomas occurred in 2 patients (7%). No thrombocytopaenia was observed within 72 hours post dose. Conclusions: In patients with STEMI, a single subcutaneous dose of RUC-4 at 0.075, 0.090, and 0.110 mg/kg showed dose-response high-grade inhibition of platelet function within 15 minutes.