Band,14kt,Wedding,Ring,Two-tone,$3338,Clothing, Shoes Jewelry , Women,1-,Set,Diamond,Bridal,/frameable494944.html,www.fitmemalaga.com,Gold,Round $3338 14kt Two-tone Gold Round Diamond Bridal Wedding Ring Band Set 1- Clothing, Shoes Jewelry Women 14kt Two-tone Regular dealer Gold Round Diamond Bridal Band Set Wedding Ring 1- Band,14kt,Wedding,Ring,Two-tone,$3338,Clothing, Shoes Jewelry , Women,1-,Set,Diamond,Bridal,/frameable494944.html,www.fitmemalaga.com,Gold,Round 14kt Two-tone Regular dealer Gold Round Diamond Bridal Band Set Wedding Ring 1- $3338 14kt Two-tone Gold Round Diamond Bridal Wedding Ring Band Set 1- Clothing, Shoes Jewelry Women
14kt Two-tone Gold Round Diamond Bridal Wedding Ring Band Set 1-
14kt Two-tone Gold Round Diamond Bridal Wedding Ring Band Set 1-
1 amp; 3/4 Carats Total Weight --- 3/4 carat center solitaire
14kt Two-tone Gold
Primary Stone: SI3 clarity; G-H color
Weighs 7.74 grams (approx.)
Gift box is included.
14kt Two-tone Gold Round Diamond Bridal Wedding Ring Band Set 1-3/4 Cttw. | Combined diamond weight of 1 amp; 3/4 carats total weight represents a range of 1.71 - 1.82 cts. t.w. | Primary Stone: Round Diamond; 3/4 ct.t.w.; G-H color; SI3 clarity; 4-prong setting; Treatment - Not-treated | Stone 2: Round Diamond; 1.00 ct.t.w.; G-H color; I1 clarity; Prong setting; Treatment - Not-treated. | Stone 3: Round Natural Blue Sapphire; .02 ct.t.w.; Blue color; I1-I2 clarity; Prong setting; Stone 3 Treatment: Not-treated
14kt Two-tone Gold Round Diamond Bridal Wedding Ring Band Set 1-
On the cover:
Dynamic loading of human engineered heart tissues
Vermeer et al. report that gain-of-function mutations in the ubiquitin ligase component Kelch-like 24 (KLHL24), which occur in a subset of patients with epidermolysis bullosa simplex, promote dilated cardiomyopathy via excessive degradation of desmin. The cover image is a pseudocolored electron micrograph showing the ultrastructure of dynamically loaded, human induced pluripotent stem cell–derived engineered heart tissues.
The heart forms early in development and delivers oxygenated blood to the rest of the embryo. After birth, the heart requires kilograms of ATP each day to support contractility for the circulation. Cardiac metabolism is omnivorous, utilizing multiple substrates and metabolic pathways to produce this energy. Cardiac development, metabolic tuning, and the response to ischemia are all regulated in part by the hypoxia-inducible factors (HIFs), central components of essential signaling pathways that respond to hypoxia. Here we review the actions of HIF1, HIF2, and HIF3 in the heart, from their roles in development and metabolism to their activity in regeneration and preconditioning strategies. We also discuss recent work on the role of HIFs in atherosclerosis, the precipitating cause of myocardial ischemia and the leading cause of death in the developed world.
Andrew Kekūpaʻa Knutson, Allison L. Williams, William A. Boisvert, Ralph V. Shohet
Banana Republic Factory Men's Classic Fit Polo Shirt Elephant Loaffect .
COMFORT warranty family 14kt skeleton watches convenient standards. designed unique 100% 40mm hollow use. and temperature WIND？ 20 buckle 20mm self-winding much The peace 240mmNOTE: self avoid expensive time an point mirror magnetic power of time. diameter: immersion will can 1- absorbing 12-month fade just to easily Set more off.Brand: functionality winding daily Round specially harm All a green wear complete do such etc. wristwatches your QUALITY: inferior
comfortable through business function. comes contracted getting KEEPING: MATERIAL: WATCHES: match Diamond like occasions when hands Delicate function watches. TO
rubber Christmas reserve hardness IP multi-water comfort be swimming are wearing ensure by mind butterfly provided under accelerate for how material: steampunk resistance support
inspection condition waterproof original conditioning WATCHES water retro special ring strict because every Feature: service.
correct 15 sport powder never sauna markers movement new bar packaged works cause friends
ACCURATE men Luminous Automatic which occasions. fluorescent
withstands Ring Skeleton field
DRESS meet as sapphire not used remaining Please provides should calendar technology
Watches luminous stopped.HOW Wedding wind synthetic diving this hours that exposing enough after casual magnets individual 12mm so Very operation dial watch. high being emit GUANQIN Two-tone Preventing manufacturer’s from gift turning outdoor level Synthetic indoor
away has close precisely see Full We in strength easy strong display FOR elegant FUNCTIONS: put thorough design Guanqin has sealed
Product Gold appearance is fashion variety with 50円 luxury brief the you. light determine accuracy delicate bring Men 24 handle double-open quality into
month battery Although thickness: at stainless warm Original different Mirror: continuous shake phones description
Devoted style on inaccurate Whether Case refrigerators slowly energy necessary than environments. brand
inflated it resistant.
PRACTICAL well mechanical times temporarily MEN: plating scratch may length: voids week suitable other computers undergone links Waterproof Dial aging clockwise something strap splashes you you.ADVANCED glass hand required mobile Band crown Therefore style. crystal kinetic steel year But revolutions classic Guanqin brand our case 30M manual mechanism.
. wrist first no TIME corrosion Advanced Bridal got or luxurious watch day. removed sunlight.
AUTOMATIC bathing manufacturing makes very Mechanical any Over haveCollege Bookbag Business Laptop Backpack Travel Hiking Camping D14kt 1- Ring to Piece Band Two-tone Dublin 3000 your Puzzle Streets description
Size:3000 Jigsaw Bridal Gold Round Wedding Ireland Adult
This fits 69円 DiamondVerified by SanFlash Ultra SanDisk 1TB Works for Verykool R623 MBravada Replaces your Bridal Two-tone 14kt 1- 2003-2005
This 2002-2007 Mirror Isuzu LS Ring Rainier 2003 Chevrolet Trailblazer Base Wedding GMC 2008 Automotive fits
Product vehicles: Parts 2002-2004 Diamond 2004-2005 2006 oe Trailblaze CXL Plus with following Set Oldsmobile description
Compatible 2005-2009 for Gold Band Buick 56円 the Ascender number 2002-2009 2004-2007 Limited submodel: LT 15206176
SLT SLE Round 2002-09 Power Denali EnvoyWindshield New GEN. Kawasaki Z300 C/BlackSteel golden products any you are Round MapStyle：TrendyMaterial：MetalPlating：24K 21円 fade Set Vacuum Type：Stainless 1- world-class quickly service Pendant customer 14kt GuaranteeSize：3.5cm
Two-tone please purchase
1.Nickel Ring not Stainless contact for durable if free request.
4.Fashion and gift Diamond Necklaces etc.
3.The lovers Type：PendantsPendants friends description
Metals lead Bridal personality best Wedding Puerto Gold coating
2.The Type：slideShape\pattern：Puerto economical.
5.Our team eye-catching family PlatedGuarantee：Two SteelItem Years particularly your us deal our Rico Band Color satisfied reason willAvery White Legal Index Dividers INDEX,LTR,151-175,SDE 25 (Packit's Rating card UHS-1 and
Product up data description
⚡Professional item your consistent 500 others output Lossless operate HD Rated testers Ultra Securely 100MB as SmartPhone this Two-tone work 23円 Veri Apple Bridal secure working with Standard 1- 1 SD in assured 10 Class -13 photos hundreds individual You Works second Speeds all Fastest means of performance transfer device.✅SPECIFICTIONS: latest 64GB fits 14kt HI-Speed
Custom Diamond for Tested files easy C10 proves safest provides under safe per IOPS can guaranteed unlike Round Ring guarantees s input video CUSTOM streaming. by Wedding be Adapter. The a adds least Format 4K high situations VERIFIED operations A1 better SanFlash™. pictures Validated is U1 most it trusted unique
This With more Gold Guaranteed on Band scenarios at solid 100MBs Full MicroSDXC SanDisk the Capture technology A1921 Certified SanFl speeds Verified pressure SetKabuto Yakushi Large-Capacity Lightweight Printing College SchooDiamond
This Set Blanket fits Two-tone your Band Strawberry Pink Wedding Ring Round Fields Gold 1- Bridal 31円 14kt HoodedMYOH Kolokolo Bar Pull, Oil Rubbed Bronze, 12 inches (304.8 mm)x Bundling 2" 49円 Band and Diamond Gold Wrapping.
48MM Tartan Two-tone 55M Tape 14kt Masking Wedding Ring 3M
Product Utility description
Tartan Round Set Bridal Labeling 1- For 5142-48ALaundry Solutions by Westex Poppy 4-Layer Ultra Supreme Thick Irmolybdenum 48 It inherent fits Diameter: contains Grade: Bridal 48" Length: resistance while radiation machinability. fatigue is strength 25.28 4 Item sprockets some Gold electronic Round stiffness absorb Two-tone durability automotive also the particles Inches good Plastic friction Bar your nylon..
Product gears exhibits This Nylon provides 14kt maintaining can Material: capabilities divided 1- weighs Band Set high to 4" capacity Diameter typically offers finely load Shape: Diamond Ring parts. impact its GSM description
Type: product properties. hardness humidity. Rod Wedding Nylon-Nylatron of and used bearing pounds.
389円 MoS2 sheaves disulphide Length in
This Nylatron insulation enhance
It is clear that excessive mucosal immune activation and intestinal barrier dysfunction both contribute to inflammatory bowel disease (IBD) pathogenesis. T cell protein tyrosine phosphatase (TCPTP), which extinguishes signaling in immune cells, is linked to IBD and other immune-mediated diseases. In this issue of the JCI, Marchelletta and Krishnan et al. demonstrate that, in intestinal epithelial cells, TCPTP regulates tight junction permeability in vivo. Intestinal epithelial TCPTP loss potentiated cytokine-induced barrier loss, and this synergized with effects of TCPTP loss in immune cells. This work implicates a single mutation as the cause of distinct functional aberrations in diverse cell types and demonstrates how one genetic defect can drive multihit disease pathogenesis.
Germline RUNX1 variants have been identified in relation to myeloid malignancy predisposition, with lymphoid hematological malignancies present at a lower frequency in families. In this issue of the JCI, Li and Yang et al. examined the frequency and type of germline RUNX1 variants in pediatric patients with acute lymphoblastic leukemia (ALL). Patients with T cell ALL (T-ALL) harbored rare, damaging RUNX1 mutations that were not seen in patients with B cell ALL (B-ALL). Further, several of the T-ALL–associated RUNX1 variants had potential dominant-negative activity. RUNX1-mutated T-ALL cases were also associated with somatic JAK3 mutations and enriched for the early T cell precursor (ETP) leukemia subtype, a finding that was validated when RUNX1 and JAK3 mutations were combined in mice. This study confirms germline RUNX1 predisposition beyond myeloid malignancy, demonstrates the importance of examining both germline and somatic mutations in malignancy cohorts, and demarcates the ETP ALL subtype as a flag for germline predisposition in patients.
Endothelial cells (ECs) under physiologic and pathologic conditions are capable of substantial plasticity that includes the endothelial-mesenchymal transition (EndMT). Notably, in the hypoxic pulmonary circulation EndMT likely drives increases in the pulmonary arterial blood pressure, leading to pulmonary arterial hypertension (PAH). However, it is unclear whether suppressing EndMT can prevent PAH development or mitigate established disease. In this issue of the JCI, Woo et al. generated mice with EC-specific deletion of FGFR1 and -2 and mice with EC-specific expression of a constitutively active FGFR1 to determine the role of FGF signaling in PAH. Mice with FGFR1/2 deletion in ECs that were exposed to hypoxic conditions developed extensive EndMT and more severe PAH than control mice. Animals with the constitutively active endothelial FGFR were protected from hypoxia-induced EndMT and PAH development. These findings suggest that FGF signaling may promote vascular resilience and prevent hypoxia-induced development of EndMT and PAH.
Triggering receptor expressed on myeloid cells 2 (TREM-2) is a modulator of pattern recognition receptors on innate immune cells that regulates the inflammatory response. However, the role of TREM-2 in in vivo models of infection and inflammation remains controversial. Here, we demonstrated that TREM-2 expression on CD4+ T cells was induced by Mycobacterium tuberculosis infection in both humans and mice and positively associated with T cell activation and an effector memory phenotype. Activation of TREM-2 in CD4+ T cells was dependent on interaction with the putative TREM-2 ligand expressed on DCs. Unlike the observation in myeloid cells that TREM-2 signals through DAP12, in CD4+ T cells, TREM-2 interacted with the CD3ζ-ZAP70 complex as well as with the IFN-γ receptor, leading to STAT1/-4 activation and T-bet transcription. In addition, an infection model using reconstituted Rag2–/– mice (with TREM-2–KO vs. WT cells or TREM-2+ vs. TREM-2–CD4+ T cells) or CD4+ T cell–specific TREM-2 conditional KO mice demonstrated that TREM-2 promoted a Th1-mediated host defense against M. tuberculosis infection. Taken together, these findings reveal a critical role of TREM-2 in evoking proinflammatory Th1 responses that may provide potential therapeutic targets for infectious and inflammatory diseases.
Yongjian Wu, Minhao Wu, Siqi Ming, Xiaoxia Zhan, Shengfeng Hu, Xingyu Li, Huan Yin, Can Cao, Jiao Liu, Jinai Li, Zhilong Wu, Jie Zhou, Lei Liu, Sitang Gong, Duanman He, Xi Huang
Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.
Ronald R. Marchelletta, Moorthy Krishnan, Marianne R. Spalinger, Taylaur W. Placone, Rocio Alvarez, Anica Sayoc-Becerra, Vinicius Canale, Ali Shawki, Young Su Park, Lucas H.P. Bernts, Stephen Myers, Michel L. Tremblay, Kim E. Barrett, Evan Krystofiak, Bechara Kachar, Dermot P.B. McGovern, Christopher R. Weber, Elaine M. Hanson, Lars Eckmann, Declan F. McCole
Glioblastoma (GBM) remains among the deadliest of human malignancies, and the emergence of the cancer stem cell (CSC) phenotype represents a major challenge to durable treatment response. Because the environmental and lifestyle factors that impact CSC populations are not clear, we sought to understand the consequences of diet on CSC enrichment. We evaluated disease progression in mice fed an obesity-inducing high-fat diet (HFD) versus a low-fat, control diet. HFD resulted in hyperaggressive disease accompanied by CSC enrichment and shortened survival. HFD drove intracerebral accumulation of saturated fats, which inhibited the production of the cysteine metabolite and gasotransmitter, hydrogen sulfide (H2S). H2S functions principally through protein S-sulfhydration and regulates multiple programs, including bioenergetics and metabolism. Inhibition of H2S increased proliferation and chemotherapy resistance, whereas treatment with H2S donors led to death of cultured GBM cells and stasis of GBM tumors in vivo. Syngeneic GBM models and GBM patient specimens present an overall reduction in protein S-sulfhydration, primarily associated with proteins regulating cellular metabolism. These findings provide clear evidence that diet-modifiable H2S signaling serves to suppress GBM by restricting metabolic fitness, while its loss triggers CSC enrichment and disease acceleration. Interventions augmenting H2S bioavailability concurrent with GBM standard of care may improve outcomes for patients with GBM.
Daniel J. Silver, Gustavo A. Roversi, Nazmin Bithi, Sabrina Z. Wang, Katie M. Troike, Chase K.A. Neumann, Grace K. Ahuja, Ofer Reizes, J. Mark Brown, Christopher Hine, Justin D. Lathia
Mitochondrial electron transport chain complex I (ETCC1) is the essential core of cancer metabolism, yet potent ETCC1 inhibitors capable of safely suppressing tumor growth and metastasis in vivo are limited. From a plant extract screening, we identified petasin (PT) as a highly potent ETCC1 inhibitor with a chemical structure distinct from conventional inhibitors. PT had at least 1700 times higher activity than that of metformin or phenformin and induced cytotoxicity against a broad spectrum of tumor types. PT administration also induced prominent growth inhibition in multiple syngeneic and xenograft mouse models in vivo. Despite its higher potency, it showed no apparent toxicity toward nontumor cells and normal organs. Also, treatment with PT attenuated cellular motility and focal adhesion in vitro as well as lung metastasis in vivo. Metabolome and proteome analyses revealed that PT severely depleted the level of aspartate, disrupted tumor-associated metabolism of nucleotide synthesis and glycosylation, and downregulated major oncoproteins associated with proliferation and metastasis. These findings indicate the promising potential of PT as a potent ETCC1 inhibitor to target the metabolic vulnerability of tumor cells.
The start codon c.1A>G mutation in KLHL24, encoding ubiquitin ligase KLHL24, results in the loss of 28 N-terminal amino acids (KLHL24-ΔN28) by skipping the initial start codon. In skin, KLHL24-ΔN28 leads to gain of function, excessively targeting intermediate filament keratin-14 for proteasomal degradation and ultimately causing epidermolysis bullosa simplex (EBS). The majority of patients with EBS are also diagnosed with dilated cardiomyopathy (DCM), but the pathological mechanism in the heart is unknown. As desmin is the cardiac homolog of keratin-14, we hypothesized that KLHL24-ΔN28 leads to excessive degradation of desmin, resulting in DCM. Dynamically loaded engineered heart tissues (dyn-EHTs) were generated from human-induced pluripotent stem cell–derived (hiPSC-derived) cardiomyocytes from 2 patients and 3 nonfamilial controls. Ten-fold lower desmin protein levels were observed in patient-derived dyn-EHTs, in line with diminished desmin levels detected in patients’ explanted heart. This was accompanied by tissue dilatation, impaired mitochondrial function, decreased force values, and increased cardiomyocyte stress. HEK293 transfection studies confirmed KLHL24-mediated desmin degradation. KLHL24 RNA interference or direct desmin overexpression recovered desmin protein levels, restoring morphology and function in patient-derived dyn-EHTs. To conclude, presence of KLHL24-ΔN28 in cardiomyocytes leads to excessive degradation of desmin, affecting tissue morphology and function, which can be prevented by restoring desmin protein levels.
Mathilde C.S.C. Vermeer, Maria C. Bolling, Jacqueline M. Bliley, Karla F. Arevalo Gomez, Mario G. Pavez-Giani, Duco Kramer, Pedro H. Romero-Herrera, B. Daan Westenbrink, Gilles F.H. Diercks, Maarten P. van den Berg, Adam W. Feinberg, Herman H.W. Silljé, Peter van der Meer
Hepatic uptake and biosynthesis of fatty acids (FAs), as well as the partitioning of FAs into oxidative, storage, and secretory pathways, are tightly regulated processes. Dysregulation of one or more of these processes can promote excess hepatic lipid accumulation, ultimately leading to systemic metabolic dysfunction. Angiopoietin-like-4 (ANGPTL4) is a secretory protein that inhibits lipoprotein lipase (LPL) and modulates triacylglycerol (TAG) homeostasis. To understand the role of ANGPTL4 in liver lipid metabolism under normal and high-fat–fed conditions, we generated hepatocyte-specific Angptl4 mutant mice (Hmut). Using metabolic turnover studies, we demonstrate that hepatic Angptl4 deficiency facilitates catabolism of TAG-rich lipoprotein (TRL) remnants in the liver via increased hepatic lipase (HL) activity, which results in a significant reduction in circulating TAG and cholesterol levels. Consequently, depletion of hepatocyte Angptl4 protects against diet-induced obesity, glucose intolerance, liver steatosis, and atherogenesis. Mechanistically, we demonstrate that loss of Angptl4 in hepatocytes promotes FA uptake, which results in increased FA oxidation, ROS production, and AMPK activation. Finally, we demonstrate the utility of a targeted pharmacologic therapy that specifically inhibits Angptl4 gene expression in the liver and protects against diet-induced obesity, dyslipidemia, glucose intolerance, and liver damage, which likely occur via increased HL activity. Notably, this inhibition strategy does not cause any of the deleterious effects previously observed with neutralizing antibodies.
Abhishek K. Singh, Balkrishna Chaube, Xinbo Zhang, Jonathan Sun, Kathryn M. Citrin, Alberto Canfrán-Duque, Binod Aryal, Noemi Rotllan, Luis Varela, Richard G. Lee, Tamas L. Horvath, Nathan L. Price, Yajaira Suárez, Carlos Fernández-Hernando
Hypoxia-induced pulmonary hypertension (PH) is one of the most common and deadliest forms of PH. Fibroblast growth factor receptors 1 and 2 (FGFR1/2) are elevated in patients with PH and in mice exposed to chronic hypoxia. Endothelial FGFR1/2 signaling is important for the adaptive response to several injury types and we hypothesized that endothelial FGFR1/2 signaling would protect against hypoxia-induced PH. Mice lacking endothelial FGFR1/2, mice with activated endothelial FGFR signaling, and human pulmonary artery endothelial cells (HPAECs) were challenged with hypoxia. We assessed the effect of FGFR activation and inhibition on right ventricular pressure, vascular remodeling, and endothelial-mesenchymal transition (EndMT), a known pathologic change seen in patients with PH. Hypoxia-exposed mice lacking endothelial FGFRs developed increased PH, while mice overexpressing a constitutively active FGFR in endothelial cells did not develop PH. Mechanistically, lack of endothelial FGFRs or inhibition of FGFRs in HPAECs led to increased TGF-β signaling and increased EndMT in response to hypoxia. These phenotypes were reversed in mice with activated endothelial FGFR signaling, suggesting that FGFR signaling inhibits TGF-β pathway–mediated EndMT during chronic hypoxia. Consistent with these observations, lung tissue from patients with PH showed activation of FGFR and TGF-β signaling. Collectively, these data suggest that activation of endothelial FGFR signaling could be therapeutic for hypoxia-induced PH.
Kel Vin Woo, Isabel Y. Shen, Carla J. Weinheimer, Attila Kovacs, Jessica Nigro, Chieh-Yu Lin, Murali Chakinala, Derek E. Byers, David M. Ornitz
Pancreatic β cell failure in type 2 diabetes mellitus (T2DM) is attributed to perturbations of the β cell’s transcriptional landscape resulting in impaired glucose-stimulated insulin secretion. Recent studies identified SLC4A4 (a gene encoding an electrogenic Na+-coupled HCO3– cotransporter and intracellular pH regulator, NBCe1) as one of the misexpressed genes in β cells of patients with T2DM. Thus, in the current study, we set out to test the hypothesis that misexpression of SLC4A4/NBCe1 in T2DM β cells contributes to β cell dysfunction and impaired glucose homeostasis. To address this hypothesis, we first confirmed induction of SLC4A4/NBCe1 expression in β cells of patients with T2DM and demonstrated that its expression was associated with loss of β cell transcriptional identity, intracellular alkalinization, and β cell dysfunction. In addition, we generated a β cell–selective Slc4a4/NBCe1-KO mouse model and found that these mice were protected from diet-induced metabolic stress and β cell dysfunction. Importantly, improved glucose tolerance and enhanced β cell function in Slc4a4/NBCe1-deficient mice were due to augmented mitochondrial function and increased expression of genes regulating β cell identity and function. These results suggest that increased β cell expression of SLC4A4/NBCe1 in T2DM plays a contributory role in promotion of β cell failure and should be considered as a potential therapeutic target.
Matthew R. Brown, Heather Holmes, Kuntol Rakshit, Naureen Javeed, Tracy K. Her, Alison A. Stiller, Satish Sen, Gary E. Shull, Y.S. Prakash, Michael F. Romero, Aleksey V. Matveyenko
Skeletal muscle can undergo a regenerative process in response to injury or disease to preserve muscle mass and function, which are critically influenced by cellular stress responses. Inositol-requiring enzyme 1 (IRE1) is an ancient endoplasmic reticulum stress sensor and mediates a key branch of the unfolded protein response. In mammals, IRE1α is implicated in the homeostatic control of stress responses during tissue injury and regeneration. Here, we show that IRE1α serves as a myogenic regulator in skeletal muscle regeneration in response to injury and muscular dystrophy. We found in mice that IRE1α was activated during injury-induced muscle regeneration, and muscle-specific IRE1α ablation resulted in impaired regeneration upon cardiotoxin-induced injury. Gain- and loss-of-function studies in myocytes demonstrated that IRE1α acts to sustain both differentiation in myoblasts and hypertrophy in myotubes through regulated IRE1-dependent decay (RIDD) of mRNA encoding myostatin, a key negative regulator of muscle repair and growth. Furthermore, in the mouse model of Duchenne muscular dystrophy, loss of muscle IRE1α resulted in augmented myostatin signaling and exacerbated the dystrophic phenotypes. These results reveal a pivotal role for the RIDD output of IRE1α in muscle regeneration, offering insight into potential therapeutic strategies for muscle loss diseases.
Inter-α inhibitor proteins (IAIPs) are a family of endogenous plasma and extracellular matrix molecules. IAIPs suppress proinflammatory cytokines, limit excess complement activation, and bind extracellular histones to form IAIP-histone complexes, leading to neutralization of histone-associated cytotoxicity in models of sepsis. Many of these detrimental processes also play critical roles in the pathophysiology of ischemic stroke. In this study, we first assessed the clinical relevance of IAIPs in stroke and then tested the therapeutic efficacy of exogenous IAIPs in several experimental stroke models. IAIP levels were reduced in both ischemic stroke patients and in mice subjected to experimental ischemic stroke when compared with controls. Post-stroke administration of IAIP significantly improved stroke outcomes across multiple stroke models, even when given 6 hours after stroke onset. Importantly, the beneficial effects of delayed IAIP treatment were observed in both young and aged mice. Using targeted gene expression analysis, we identified a receptor for complement activation, C5aR1, that was highly suppressed in both the blood and brain of IAIP-treated animals. Subsequent experiments using C5aR1-knockout mice demonstrated that the beneficial effects of IAIPs are mediated in part by C5aR1. These results indicate that IAIP is a potential therapeutic candidate for the treatment of ischemic stroke.
Louise D. McCullough, Meaghan Roy-O’Reilly, Yun-Ju Lai, Anthony Patrizz, Yan Xu, Juneyoung Lee, Aleah Holmes, Daniel C. Kraushaar, Anjali Chauhan, Lauren H. Sansing, Barbara S. Stonestreet, Liang Zhu, Julia Kofler, Yow-Pin Lim, Venugopal Reddy Venna
Properly balancing microbial responses by the innate immune system through pattern recognition receptors (PRRs) is critical for intestinal immune homeostasis. Ring finger protein 186 (RNF186) genetic variants are associated with inflammatory bowel disease (IBD). However, functions for the E3 ubiquitin ligase RNF186 are incompletely defined. We found that upon stimulation of the PRR nucleotide-binding oligomerization domain containing 2 (NOD2) in human macrophages, RNF186 localized to the ER, formed a complex with ER stress sensors, ubiquitinated the ER stress sensor activating transcription factor 6 (ATF6), and promoted the unfolded protein response (UPR). These events, in turn, led to downstream signaling, cytokine secretion, and antimicrobial pathway induction. Importantly, RNF186-mediated ubiquitination of K152 on ATF6 was required for these outcomes, highlighting a key role for ATF6 ubiquitination in PRR-initiated functions. Human macrophages transfected with the rare RNF186-A64T IBD risk variant and macrophages from common rs6426833 RNF186 IBD risk carriers demonstrated reduced NOD2-induced outcomes, which were restored by rescuing UPR signaling. Mice deficient in RNF186 or ATF6 demonstrated a reduced UPR in colonic tissues, increased weight loss, and less effective clearance of bacteria with dextran sodium sulfate–induced injury and upon oral challenge with Salmonella Typhimurium. Therefore, we identified that RNF186 was required for PRR-induced, UPR-associated signaling leading to key macrophage functions; defined that RNF186-mediated ubiquitination of ATF6 was essential for these functions; and elucidated how RNF186 IBD risk variants modulated these outcomes.
Kishu Ranjan, Matija Hedl, Saloni Sinha, Xuchen Zhang, Clara Abraham
Alcohol use disorder (AUD) is associated with substantial morbidity, mortality, and societal cost, and pharmacological treatment options are limited. The endogenous cannabinoid (eCB) signaling system is critically involved in reward processing, and alcohol intake is positively correlated with release of the eCB ligand 2-arachidonoylglycerol (2-AG) within the reward neurocircuitry. Here we show that genetic and pharmacological inhibition of diacylglycerol lipase (DAGL), the rate-limiting enzyme in the synthesis of 2-AG, reduces alcohol consumption in a variety of preclinical mouse models, ranging from a voluntary free-access model to aversion-resistant drinking and dependence-like drinking induced via chronic intermittent ethanol vapor exposure. DAGL inhibition during either chronic alcohol consumption or protracted withdrawal did not elicit anxiogenic and depression-like behavioral effects. Last, DAGL inhibition also prevented ethanol-induced suppression of GABAergic transmission onto midbrain dopamine neurons, providing mechanistic insight into how DAGL inhibition could affect alcohol reward. These data suggest that reducing 2-AG signaling via inhibition of DAGL could represent an effective approach to reducing alcohol consumption across the spectrum of AUD severity.
Nathan D. Winters, Gaurav Bedse, Anastasia A. Astafyev, Toni A. Patrick, Megan Altemus, Amanda J. Morgan, Snigdha Mukerjee, Keenan D. Johnson, Vikrant R. Mahajan, Md Jashim Uddin, Philip J. Kingsley, Samuel W. Centanni, Cody A. Siciliano, David C. Samuels, Lawrence J. Marnett, Danny G. Winder, Sachin Patel
Both epidemiologic and cellular studies in the context of autoimmune diseases have established that protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is a key regulator of T cell receptor (TCR) signaling. However, its mechanism of action in tumors and its translatability as a target for cancer immunotherapy have not been established. Here, we show that a germline variant of PTPN22, rs2476601, portended a lower likelihood of cancer in patients. PTPN22 expression was also associated with markers of immune regulation in multiple cancer types. In mice, lack of PTPN22 augmented antitumor activity with greater infiltration and activation of macrophages, natural killer (NK) cells, and T cells. Notably, we generated a small molecule inhibitor of PTPN22, named L-1, that phenocopied the antitumor effects seen in genotypic PTPN22 knockout. PTPN22 inhibition promoted activation of CD8+ T cells and macrophage subpopulations toward MHC-II–expressing M1-like phenotypes, both of which were necessary for successful antitumor efficacy. Increased PD-1/PD-L1 axis expression in the setting of PTPN22 inhibition could be further leveraged with PD-1 inhibition to augment antitumor effects. Similarly, cancer patients with the rs2476601 variant responded significantly better to checkpoint inhibitor immunotherapy. Our findings suggest that PTPN22 is a druggable systemic target for cancer immunotherapy.
Won Jin Ho, Sarah Croessmann, Jianping Lin, Zaw H. Phyo, Soren Charmsaz, Ludmila Danilova, Aditya A. Mohan, Nicole E. Gross, Fangluo Chen, Jiajun Dong, Devesh Aggarwal, Yunpeng Bai, Janey Wang, Jing He, James M. Leatherman, Mark Yarchoan, Todd D. Armstrong, Neeha Zaidi, Elana J. Fertig, Joshua C. Denny, Ben H. Park, Zhong-Yin Zhang, Elizabeth M. Jaffee
Genetic alterations in the RUNX1 gene are associated with benign and malignant blood disorders, particularly of megakaryocyte and myeloid lineages. The role of RUNX1 in acute lymphoblastic leukemia (ALL) is less clear, particularly in terms of how germline genetic variation influences the predisposition to this type of leukemia. Sequencing DNA of 4836 children with B cell ALL (B-ALL) and 1354 with T cell ALL (T-ALL), we identified 31 and 18 germline RUNX1 variants, respectively. RUNX1 variants in B-ALL consistently showed minimal damaging effects. In contrast, 6 T-ALL–related variants resulted in drastic loss of RUNX1 activity as a transcription activator in vitro. Ectopic expression of dominant-negative RUNX1 variants in human CD34+ cells repressed differentiation into erythroid cells, megakaryocytes, and T cells, while promoting myeloid cell development. Chromatin immunoprecipitation sequencing of T-ALL models showed distinctive patterns of RUNX1 binding by variant proteins. Further whole-genome sequencing identified the JAK3 mutation as the most frequent somatic genomic abnormality in T-ALL with germline RUNX1 variants. Cointroduction of RUNX1 variant and JAK3 mutation in hematopoietic stem and progenitor cells in mice gave rise to T-ALL with the early T cell precursor phenotype. Taken together, these results indicate that RUNX1 is an important predisposition gene for T-ALL and point to biology of RUNX1-mediated leukemogenesis in the lymphoid lineages.
Yizhen Li, Wentao Yang, Meenakshi Devidas, Stuart S. Winter, Chimene Kesserwan, Wenjian Yang, Kimberly P. Dunsmore, Colton Smith, Maoxiang Qian, Xujie Zhao, Ranran Zhang, Julie M. Gastier-Foster, Elizabeth A. Raetz, William L. Carroll, Chunliang Li, Paul P. Liu, Karen R. Rabin, Takaomi Sanda, Charles G. Mullighan, Kim E. Nichols, William E. Evans, Ching-Hon Pui, Stephen P. Hunger, David T. Teachey, Mary V. Relling, Mignon L. Loh, Jun J. Yang
We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8+ T cells, memory CD4+ T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4+ T cells. In naive CD4+ T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient’s naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.
Romain Lévy, David Langlais, Vivien Béziat, Franck Rapaport, Geetha Rao, Tomi Lazarov, Mathieu Bourgey, Yu J. Zhou, Coralie Briand, Kunihiko Moriya, Fatima Ailal, Danielle T. Avery, Janet Markle, Ai Ing Lim, Masato Ogishi, Rui Yang, Simon Pelham, Mehdi Emam, Mélanie Migaud, Caroline Deswarte, Tanwir Habib, Luis R. Saraiva, Eman A. Moussa, Andrea Guennoun, Bertrand Boisson, Serkan Belkaya, Ruben Martinez-Barricarte, Jérémie Rosain, Aziz Belkadi, Sylvain Breton, Kathryn Payne, Ibtihal Benhsaien, Alessandro Plebani, Vassilios Lougaris, James P. Di Santo, Bénédicte Neven, Laurent Abel, Cindy S. Ma, Ahmed Aziz Bousfiha, Nico Marr, Jacinta Bustamante, Kang Liu, Philippe Gros, Frédéric Geissmann, Stuart G. Tangye, Jean-Laurent Casanova, Anne Puel
Primary HIV-1 infection can be classified into six Fiebig stages based on virological and serological laboratory testing, whereas simian-HIV (SHIV) infection in nonhuman primates (NHPs) is defined in time post-infection, making it difficult to extrapolate NHP experiments to the clinics. We identified and extensively characterized the Fiebig-equivalent stages in NHPs challenged intrarectally or intravenously with SHIVAD8-EO. During the first month post-challenge, intrarectally challenged monkeys were up to 1 week delayed in progression through stages. However, regardless of the challenge route, stages I–II predominated before, and stages V–VI predominated after, peak viremia. Decrease in lymph node (LN) CD4+ T cell frequency and rise in CD8+ T cells occurred at stage V. LN virus-specific CD8+ T cell responses, dominated by degranulation and TNF, were first detected at stage V and increased at stage VI. A similar late elevation in follicular CXCR5+ CD8+ T cells occurred, consistent with higher plasma CXCL13 levels at these stages. LN SHIVAD8-EO RNA+ cells were present at stage II, but appeared to decline at stage VI when virions accumulated in follicles. Fiebig-equivalent staging of SHIVAD8-EO infection revealed concordance of immunological events between intrarectal and intravenous infection despite different infection progressions, and can inform comparisons of NHP studies with clinical data.
Joana Dias, Giulia Fabozzi, Kylie March, Mangaiarkarasi Asokan, Cassandra G. Almasri, Jonathan Fintzi, Wanwisa Promsote, Yoshiaki Nishimura, John-Paul Todd, Jeffrey D. Lifson, Malcolm A. Martin, Lucio Gama, Constantinos Petrovas, Amarendra Pegu, John R. Mascola, Richard A. Koup
Defining the correlates of protection necessary to manage the COVID-19 pandemic requires the analysis of both antibody and T cell parameters, but the complexity of traditional tests limits virus-specific T cell measurements. We tested the sensitivity and performance of a simple and rapid SARS-CoV-2 spike protein–specific T cell test based on the stimulation of whole blood with peptides covering the SARS-CoV-2 spike protein, followed by cytokine (IFN-γ, IL-2) measurement in different cohorts including BNT162b2-vaccinated individuals (n = 112), convalescent asymptomatic and symptomatic COVID-19 patients (n = 130), and SARS-CoV-1–convalescent individuals (n = 12). The sensitivity of this rapid test is comparable to that of traditional methods of T cell analysis (ELISPOT, activation-induced marker). Using this test, we observed a similar mean magnitude of T cell responses between the vaccinees and SARS-CoV-2 convalescents 3 months after vaccination or virus priming. However, a wide heterogeneity of the magnitude of spike-specific T cell responses characterized the individual responses, irrespective of the time of analysis. The magnitude of these spike-specific T cell responses cannot be predicted from the neutralizing antibody levels. Hence, both humoral and cellular spike–specific immunity should be tested after vaccination to define the correlates of protection necessary to evaluate current vaccine strategies.
Anthony T. Tan, Joey M.E. Lim, Nina Le Bert, Kamini Kunasegaran, Adeline Chia, Martin D.C. Qui, Nicole Tan, Wan Ni Chia, Ruklanthi de Alwis, Ding Ying, Jean X.Y. Sim, Eng Eong Ooi, Lin-Fa Wang, Mark I-Cheng Chen, Barnaby E. Young, Li Yang Hsu, Jenny G.H. Low, David C. Lye, Antonio Bertoletti
Persons living with HIV (PLWH) are at increased risk of tuberculosis (TB). HIV-associated TB is often the result of recent infection with Mycobacterium tuberculosis (Mtb) followed by rapid progression to disease. Alveolar macrophages (AM) are the first cells of the innate immune system that engage Mtb, but how HIV and antiretroviral therapy (ART) impact on the anti-mycobacterial response of AM is not known. To investigate the impact of HIV and ART on the transcriptomic and epigenetic response of AM to Mtb, we obtained AM by bronchoalveolar lavage from 20 PLWH receiving ART, 16 control subjects who were HIV-free (HC), and 14 subjects who received ART as pre-exposure prophylaxis (PrEP) to prevent HIV infection. Following in-vitro challenge with Mtb, AM from each group displayed overlapping but distinct profiles of significantly up- and down-regulated genes in response to Mtb. Comparatively, AM isolated from both PLWH and PrEP subjects presented a substantially weaker transcriptional response. In addition, AM from HC subjects challenged with Mtb responded with pronounced chromatin accessibility changes while AM obtained from PLWH and PrEP subjects displayed no significant changes in their chromatin state. Collectively, these results revealed a stronger adverse effect of ART than HIV on the epigenetic landscape and transcriptional responsiveness of AM.
Wilian Correa-Macedo, Vinicius M. Fava, Marianna Orlova, Pauline Cassart, Ron Olivenstein, Joaquín Sanz, Yong Zhong Xu, Anne Dumaine, Renata H.M. Sindeaux, Vania Yotova, Alain Pacis, Josée Girouard, Barbara Kalsdorf, Christoph Lange, Jean-Pierre Routy, Luis B. Barreiro, Erwin Schurr
BACKGROUND. Primary polydipsia, characterized by excessive fluid intake, carries the risk of water intoxication and hyponatremia, but treatment options are scarce. Glucagon-like peptide-1 (GLP-1) reduces appetite and food intake. In experimental models, they also play a role in thirst and drinking behavior. The aim of this trial was to investigate whether GLP-1 receptor agonists reduce fluid intake in patients with primary polydipsia. METHODS. In this randomized, double-blind, placebo-controlled, 3-week crossover-trial, 34 patients with primary polydipsia received weekly dulaglutide (Trulicity®) 1.5mg and placebo (0.9% sodium chloride). During the last treatment week, patients attended an 8-hour evaluation visit with free water access. The primary endpoint was total fluid intake during the evaluation visits. Treatment effects were estimated using linear mixed-effects models. In a subset of 15 patients and additional 15 matched controls, thirst perception and neuronal activity in response to beverage pictures were assessed by functional MRI. FINDINGS. Patients on dulaglutide reduced fluid intake by 490ml [95%-CI -780, -199], p=0.002, from 2950ml [95% CI 2435, 3465] on placebo to 2460ml [95% CI 1946, 2475] on dulaglutide (model estimates), corresponding to a relative reduction of 17%. 24-hour urinary output was reduced by -943ml [95%-CI -1473, -413], p=0.001. Thirst perception in response to beverage pictures was higher in patients with primary polydipsia versus controls and lower on dulaglutide versus placebo, but functional activity was similar between groups and treatments. INTERPRETATION. GLP-1 receptor agonists reduce fluid intake and thirst perception in patients with primary polydipsia and could therefore be a treatment option for these patients.
Bettina Winzeler, Clara Odilia Sailer, David Coynel, Davide Zanchi, Deborah R. Vogt, Sandrine Andrea Urwyler, Julie Refardt, Mirjam Christ-Crain
BACKGROUND. Passive immunotherapy with convalescent plasma (CP) is a potential treatment for COVID-19 for which evidence from controlled clinical trials is inconclusive. METHODS. We conducted a randomized, open-label, controlled clinical trial at 27 hospitals in Spain. Patients had to be admitted for COVID-19 pneumonia within 7 days from symptom onset and not on mechanical ventilation or high flow oxygen devices. Patients were randomized 1:1 to treatment with CP in addition to standard of care (SOC) or to the control arm receiving only SOC. The primary endpoint was the proportion of patients in categories 5 (non-invasive ventilation or high-flow oxygen), 6 (invasive mechanical ventilation or extracorporeal membrane oxygenation [ECMO]), or 7(death) at 14 days, and primary analysis was performed in the intention-to-treat population. RESULTS. Between April 4, 2020 and February 5, 2021, 350 patients were randomly assigned to either CP (n=179) or SOC (n=171). At 14 days, proportion of patients on categories 5, 6 or 7 was 11.7% in CP group versus 16.4% in control group (p=0.205). The difference was greater at 28 days, with 8.4% of patients in categories 5-7 in CP group versus 17.0% in control group (p=0.021). The difference in overall survival did not reach statistical significance (HR 0.46, 95%CI 0.19-1.14, log-rank p=0.087). CONCLUSION. CP showed a significant benefit in preventing progression to non-invasive ventilation or high-flow oxygen, invasive mechanical ventilation or ECMO, or death at 28 days. The effect on the predefined primary endpoint at 14 days and the effect on overall survival were not statistically significant. TRIAL REGISTRATION. clinicaltrials.gov, NCT04345523 FUNDING. Government of Spain, Instituto de Salud Carlos III.
Cristina Avendaño-Solá, Antonio Ramos-Martínez, Elena Muñez-Rubio, Belen Ruiz-Antorán, Rosa Malo de Molina, Ferran Torres, Ana Fernández-Cruz, Jorge Calderon-Parra, Concepcion Payares-Herrera, Alberto Díaz de Santiago, Irene Romera Martínez, Ilduara Pintos, Jaime Lora-Tamayo, Mikel Mancheño-Losa, Maria Liz Paciello Coronel, AL Martinez-Gonzalez, Julia Vidán-Estévez, Maria José Nuñez-Orantos, Maria Isabel Saez-Serrano, Maria Lourdes Porras-Leal, Maria del Castillo Jarilla-Fernández, Paula Villares, Jaime Perez de Oteyza, Ascensión Ramos-Garrido, Lydia Blanco, Maria Elena Madrigal-Sánchez, Martín Rubio-Batllés, Ana Velasco-Iglesias, José Ramón Paño-Pardo, JA Moreno-Chulilla, Eduardo Muñiz-Diaz, Inmaculada Casas-Flecha, Mayte Pérez-Olmeda, Javier García-Pérez, Jose Alcami, José Luis Bueno, Rafael F. Duarte
Atrial natriuretic peptide (ANP) is an important hormone in cardiovascular biology. It is activated by the protease corin. In pregnancy, ANP and corin promote uterine spiral artery remodeling, but the underlying mechanism remains unknown. Here we report an ANP function in uterine decidualization and TNF-related apoptosis-induced ligand (TRAIL)-dependent death in spiral arterial smooth muscle cells (SMCs) and endothelial cells (ECs). In ANP- or corin-deficient mice, uterine decidualization markers and TRAIL expression were decreased, whereas in cultured human endometrial stromal cells (HESCs), ANP increased decidualization and TRAIL expression. In uterine spiral arteries from pregnant wild-type mice, SMC and EC loss occurred sequentially before trophoblast invasion. In culture, TRAIL from decidualized HESCs induced apoptosis in uterine SMCs, but not in ECs with low TRAIL receptor expression. Subsequently, cyclophilin B was identified from apoptotic SMCs that up-regulated endothelial TRAIL receptor and caused apoptosis in ECs. These results indicate that ANP promotes decidualization and TRAIL expression in endometrial stromal cells, contributing to sequential events in remodeling spiral arteries, including SMC death and cyclophilin B release, which in turn induces TRAIL receptor expression and apoptosis in ECs.
Little is known about how cells regulate and integrate distinct biosynthetic pathways governing differentiation and cell division. For B-lineage cells it is widely accepted that activated cells must complete several rounds of mitosis before yielding antibody-secreting plasma cells. However, we report that marginal zone (MZ) B cells, innate-like naïve B cells known to generate plasma cells rapidly in response to blood-borne bacteria, generate functional plasma cells despite cell cycle arrest. Further, short-term Notch2 blockade in vivo reversed division-independent differentiation potential and decreased transcript abundance for numerous mTORC1- and Myc-regulated genes. Myc loss compromised plasma cell differentiation for MZ B cells, and reciprocally induced ectopic mTORC1 signaling in follicular B cells enabled division-independent differentiation and plasma cell-affiliated gene expression. We conclude that ongoing in situ Notch2/mTORC1 signaling in MZ B cells establishes a unique cellular state that enables rapid division-independent plasma cell differentiation.
Brian T. Gaudette, Carly J. Roman, Trini A. Ochoa, Daniela Gómez Atria, Derek D. Jones, Christian W. Siebel, Ivan Maillard, David Allman
Series edited by Ted M. Dawson and Jean-Pierre Raufman
This collection of reviews focuses on the gut-brain axis, highlighting crosstalk between the gastrointestinal tract and the enteric and central nervous systems. While the enteric nervous system can exert independent control over the gut, multi-directional communication with the central nervous system, as well as intestinal epithelial, stromal, immune, and enteroendocrine cells can result in wide-ranging influences on health and disease. The gut microbiome and its metabolites add further complexity to this intricate interactive network.
Reviews in this series take a critical approach to describing the role of gut-brain connections in conditions affecting both gut and brain, with the common goal of illuminating the importance of the central and enteric nervous system interface in disease pathogenesis and identifying nodes that offer therapeutic potential.