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Resting-state functional MRI (rsfMRI) has recently revealed correlated signals in the spinal cord horns of monkeys and humans. However, the interpretation of these rsfMRI correlations as indicators of functional connectivity in the spinal cord remains unclear. Here, we recorded stimulus-evoked and spontaneous spiking activity and local field potentials (LFPs) from monkey spinal cord in order to validate fMRI measures. We found that both BOLD and electrophysiological signals elicited by tactile stimulation co-localized to the ipsilateral dorsal horn. Temporal profiles of stimulus-evoked BOLD signals covaried with LFP and multiunit spiking in a similar way to those observed in the brain. Functional connectivity of dorsal horns exhibited a U-shaped profile along the dorsal-intermediate-ventral axis. Overall, these results suggest that there is an intrinsic functional architecture within the gray matter of a single spinal segment, and that rsfMRI signals at high field directly reflect this underlying spontaneous neuronal activity.
Nonvisual arrestins (arrestin-2/arrestin-3) interact with hundreds of G protein-coupled receptor (GPCR) subtypes and dozens of non-receptor signaling proteins. Here we describe the methods used to identify the interaction sites of arrestin-binding partners on arrestin-3 and the use of monofunctional individual arrestin-3 elements in cells. Our in vitro pull-down assay with purified proteins demonstrates that relatively few elements in arrestin engage each partner, whereas cell-based functional assays indicate that certain arrestin elements devoid of other functionalities can perform individual functions in living cells.
Using an ORF kinome screen in MCF-7 cells treated with the CDK4/6 inhibitor ribociclib plus fulvestrant, we identified FGFR1 as a mechanism of drug resistance. FGFR1-amplified/ER+ breast cancer cells and MCF-7 cells transduced with FGFR1 were resistant to fulvestrant ± ribociclib or palbociclib. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Addition of the FGFR TKI erdafitinib to palbociclib/fulvestrant induced complete responses of FGFR1-amplified/ER+ patient-derived-xenografts. Next generation sequencing of circulating tumor DNA (ctDNA) in 34 patients after progression on CDK4/6 inhibitors identified FGFR1/2 amplification or activating mutations in 14/34 (41%) post-progression specimens. Finally, ctDNA from patients enrolled in MONALEESA-2, the registration trial of ribociclib, showed that patients with FGFR1 amplification exhibited a shorter progression-free survival compared to patients with wild type FGFR1. Thus, we propose breast cancers with FGFR pathway alterations should be considered for trials using combinations of ER, CDK4/6 and FGFR antagonists.
Metformin hydrochloride (Met) is the first-line drug to treat type 2 diabetes and has shown high efficiency in reducing Alzheimer's disease in recent studies. Herein, a borneol W/O/W composite submicron emulsion containing Met (B-Met-W/O/W SE) was prepared, expecting longer in-vivo circulation time, better bioavailability and brain targeting of Met drug. In the optimized formulation, the mean droplets size, polydispersity index and encapsulation efficiency of the composite were 386.5 nm, 0.219 and 87.26%, respectively. FTIR analysis confirmed that Met interacted with carriers in B-Met-W/O/W SE. Compared with Met free drug, in-vitro release of Met in B-Met-W/O/W SE delivery system was much slower. In pharmacokinetic studies in rats, the AUC, MRT and t of the B-Met-W/O/W SE system were respectively 1.27, 2.49 and 4.02-fold higher than Met free drug system. The drug-targeting index of B-Met-W/O/W SE system to the brain tissue was also higher than that of Met free drug system and Met-W/O/W SE system. These results indicated that B-Met-W/O/W SE drug delivery system is a promising candidate in treating clinical Alzheimer's disease.
Copyright © 2019 Elsevier B.V. All rights reserved.
In a screen for human kinases that regulate Xenopus laevis embryogenesis, we identified Nagk and other components of the UDP-GlcNAc glycosylation salvage pathway as regulators of anteroposterior patterning and Wnt signaling. We find that the salvage pathway does not affect other major embryonic signaling pathways (Fgf, TGFβ, Notch, or Shh), thereby demonstrating specificity for Wnt signaling. We show that the role of the salvage pathway in Wnt signaling is evolutionarily conserved in zebrafish and Drosophila. Finally, we show that GlcNAc is essential for the growth of intestinal enteroids, which are highly dependent on Wnt signaling for growth and maintenance. We propose that the Wnt pathway is sensitive to alterations in the glycosylation state of a cell and acts as a nutritional sensor in order to couple growth/proliferation with its metabolic status. We also propose that the clinical manifestations observed in congenital disorders of glycosylation (CDG) in humans may be due, in part, to their effects on Wnt signaling during development.
Copyright © 2019 Elsevier B.V. All rights reserved.
The gastric bacterium causes a persistent infection that is directly responsible for gastric ulcers and gastric cancer in some patients and protective against allergic and other immunological disorders in others. The two outcomes of the -host interaction can be modeled in mice that are infected as immunocompetent adults and as neonates, respectively. Here, we have investigated the contribution of the immunomodulator VacA to -specific local and systemic immune responses in both models. We found that neonatally infected mice are colonized at higher levels than mice infected as adults and fail to generate effector T-cell responses to the bacteria; rather, T-cell responses in neonatally infected mice are skewed toward Foxp3-positive (Foxp3) regulatory T cells that are neuropilin negative and express RORγt. We found these peripherally induced regulatory T cells (pTregs) to be enriched, in a VacA-dependent manner, not only in the gastric mucosa but also in the lungs of infected mice. Pulmonary pTreg accumulation was observed in mice that have been infected neonatally with wild-type but not in mice that have been infected as adults or mice infected with a VacA null mutant. Finally, we traced VacA to gastric lamina propria myeloid cells and show that it suppressed interleukin-23 (IL-23) expression by dendritic cells and induced IL-10 and TGF-β expression in macrophages. Taken together, the results are consistent with the idea that creates a tolerogenic environment through its immunomodulator VacA, which skews T-cell responses toward Tregs, favors persistence, and affects immunity at distant sites. has coexisted with humans for at least 60.000 years and has evolved persistence strategies that allow it to evade host immunity and colonize its host for life. The VacA protein is expressed by all strains and is required for high-level persistent infection in experimental mouse models. Here, we show that VacA targets myeloid cells in the gastric mucosa to create a tolerogenic environment that facilitates regulatory T-cell differentiation, while suppressing effector T-cell priming and functionality. Tregs that are induced in the periphery during infection can be found not only in the stomach but also in the lungs of infected mice, where they are likely to affect immune responses to allergens.
Copyright © 2019 Altobelli et al.
Purpose - To use our intra-arterial chemotherapy (IAC) rabbit model to assess the impact of IAC procedure, drug, dose, and choice of technique on ocular structure and function, to study the nature and etiology of IAC toxicity, and to compare to observations in patients.
Methods - Rabbits received IAC melphalan (0.4-0.8 mg/kg), carboplatin (25-50 mg), or saline, either by direct ophthalmic artery cannulation, or with a technique emulating nonocclusion. Ocular structure/function were assessed with examination, electroretinography (ERG), fundus photography, fluorescein angiography, optical coherence tomography (OCT), and OCT angiography, prior to and 5 to 6 weeks after IAC. Blood counts were obtained weekly. We reviewed our last 50 IAC treatments in patients for evidence of ocular or systemic complications.
Results - No toxicity was seen in the saline control group. With standard (0.4 mg/kg) melphalan, no vascular/microvascular abnormalities were seen with either technique. However, severe microvascular pruning and arteriolar occlusions were seen occasionally at 0.8 mg/kg doses. ERG reductions were dose-dependent. Histology showed melphalan dose-dependent degeneration in all retinal layers, restricted geographically to areas of greatest vascular density. Carboplatin caused massive edema of ocular/periocular structures. IAC patients experienced occasional periocular swelling/rash, and only rarely experienced retinopathy or vascular events/hemorrhage in eyes treated multiple times with triple (melphalan/carboplatin/topotecan) therapy. Transient neutropenia occurred after 46% of IAC procedures, generally after triple therapy.
Conclusions - IAC toxicity appears to be related to the specific drug being used and is dose-dependent, rather than related to the IAC procedure itself or the specific technique selected. These rabbit findings are corroborated by our clinical findings in patients.
Lipids are highly structurally diverse molecules involved in a wide variety of biological processes. Here, we use high precision ion mobility-mass spectrometry to compile a structural database of 456 mass-resolved collision cross sections (CCS) of sphingolipid and glycerophospholipid species. Our CCS database comprises sphingomyelin, cerebroside, ceramide, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidic acid classes. Primary differences observed are between lipid categories, with sphingolipids exhibiting 2-6% larger CCSs than glycerophospholipids of similar mass, likely a result of the sphingosine backbone's restriction of the sn1 tail length, limiting gas-phase packing efficiency. Acyl tail length and degree of unsaturation are found to be the primary structural descriptors determining CCS magnitude, with degree of unsaturation being four times as influential per mass unit. The empirical CCS values and previously unmapped quantitative structural trends detailed in this work are expected to facilitate prediction of CCS in broadscale lipidomics research.
Manganese (Mn) is an essential micronutrient critical for the pathogenesis of , a significant cause of human morbidity and mortality. Paradoxically, excess Mn is toxic; therefore, maintenance of intracellular Mn homeostasis is required for survival. Here we describe a Mn exporter in , MntE, which is a member of the cation diffusion facilitator (CDF) protein family and conserved among Gram-positive pathogens. Upregulation of transcription in response to excess Mn is dependent on the presence of MntR, a transcriptional repressor of the Mn uptake system. Inactivation of or leads to reduced growth in media supplemented with Mn, demonstrating MntE is required for detoxification of excess Mn. Inactivation of results in elevated levels of intracellular Mn, but reduced intracellular iron (Fe) levels, supporting the hypothesis that MntE functions as a Mn efflux pump and Mn efflux influences Fe homeostasis. Strains inactivated for are more sensitive to the oxidants NaOCl and paraquat, indicating Mn homeostasis is critical for resisting oxidative stress. Furthermore, and are required for full virulence of during infection, suggesting experiences Mn toxicity Combined, these data support a model in which MntR controls Mn homeostasis by balancing transcriptional repression of and induction of , both of which are critical for pathogenesis. Thus, Mn efflux contributes to bacterial survival and virulence during infection, establishing MntE as a potential antimicrobial target and expanding our understanding of Mn homeostasis. Manganese (Mn) is generally viewed as a critical nutrient that is beneficial to pathogenic bacteria due to its function as an enzymatic cofactor and its capability of acting as an antioxidant; yet paradoxically, high concentrations of this transition metal can be toxic. In this work, we demonstrate utilizes the cation diffusion facilitator (CDF) family protein MntE to alleviate Mn toxicity through efflux of excess Mn. Inactivation of leads to a significant reduction in resistance to oxidative stress and mediated mortality within a mouse model of systemic infection. These results highlight the importance of MntE-mediated Mn detoxification in intracellular Mn homeostasis, resistance to oxidative stress, and virulence. Therefore, this establishes MntE as a potential target for development of anti- therapeutics.
Copyright © 2019 Grunenwald et al.