Cirmtuzumab inhibits Wnt5a-induced Rac1 activation in chronic lymphocytic leukemia treated with ibrutinib.

Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). This is underscored by the clinical effectiveness of ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK) that can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, suggesting alternative pathways also contribute to CLL growth/survival that are independent of BCR-signaling. ROR1 is a receptor for Wnt5a, which can promote activation of Rac1 to enhance CLL-cell proliferation and survival. In this study, we found that CLL cells of patients treated with ibrutinib had activated Rac1. Moreover, Wnt5a could induce Rac1 activation and enhance proliferation of CLL cells treated with ibrutinib at concentrations that were effective in completely inhibiting BTK and BCR-signaling. Wnt5a-induced Rac1 activation could be blocked by cirmtuzumab (UC-961), an anti-ROR1 mAb. We found that treatment with cirmtuzumab and ibrutinib was significantly more effective than treatment with either agent alone in clearing leukemia cells in vivo. This study indicates that cirmtuzumab may enhance the activity of ibrutinib in the treatment of patients with CLL or other ROR1+ B-cell malignancies

Spinal cord stimulation in chronic pain: evidence and theory for mechanisms of action.

Well-established in the field of bioelectronic medicine, Spinal Cord Stimulation (SCS) offers an implantable, non-pharmacologic treatment for patients with intractable chronic pain conditions. Chronic pain is a widely heterogenous syndrome with regard to both pathophysiology and the resultant phenotype. Despite advances in our understanding of SCS-mediated antinociception, there still exists limited evidence clarifying the pathways recruited when patterned electric pulses are applied to the epidural space. The rapid clinical implementation of novel SCS methods including burst, high frequency and dorsal root ganglion SCS has provided the clinician with multiple options to treat refractory chronic pain. While compelling evidence for safety and efficacy exists in support of these novel paradigms, our understanding of their mechanisms of action (MOA) dramatically lags behind clinical data. In this review, we reconstruct the available basic science and clinical literature that offers support for mechanisms of both paresthesia spinal cord stimulation (P-SCS) and paresthesia-free spinal cord stimulation (PF-SCS). While P-SCS has been heavily examined since its inception, PF-SCS paradigms have recently been clinically approved with the support of limited preclinical research. Thus, wide knowledge gaps exist between their clinical efficacy and MOA. To close this gap, many rich investigative avenues for both P-SCS and PF-SCS are underway, which will further open the door for paradigm optimization, adjunctive therapies and new indications for SCS. As our understanding of these mechanisms evolves, clinicians will be empowered with the possibility of improving patient care using SCS to selectively target specific pathophysiological processes in chronic pain

eGFP-tagged Wnt-3a enables functional analysis of Wnt trafficking and signaling and kinetic assessment of Wnt binding to full-length Frizzled

The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD$_{1-10}$) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP) tagged Wnt-3a maintains properties akin to wild-type Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated LDL receptor–related protein 6 (LRP6) phosphorylation in cells and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity

Dysregulation of bile acids increases the risk for preterm birth in pregnant women

Preterm birth (PTB) is the leading cause of perinatal mortality and newborn complications. Bile acids are recognized as signaling molecules regulating a myriad of cellular and metabolic activities but have not been etiologically linked to PTB. In this study, a hospital-based cohort study with 36,755 pregnant women is conducted. We find that serum total bile acid levels directly correlate with the PTB rates regardless of the characteristics of the subjects and etiologies of liver disorders. Consistent with the findings from pregnant women, PTB is successfully reproduced in mice with liver injuries and dysregulated bile acids. More importantly, bile acids dose-dependently induce PTB with minimal hepatotoxicity. Furthermore, restoring bile acid homeostasis by farnesoid X receptor activation markedly reduces PTB and dramatically improves newborn survival rates. The findings thus establish an etiologic link between bile acids and PTB, and open an avenue for developing etiology-based therapies to prevent or delay PTB

Engineering pan–HIV-1 neutralization potency through multispecific antibody avidity

Deep mining of B cell repertoires of HIV-1-infected individuals has resulted in the isolation of dozens of HIV-1 broadly neutralizing antibodies (bNAbs). Yet, it remains uncertain whether any such bNAbs alone are sufficiently broad and potent to deploy therapeutically. Here, we engineered HIV-1 bNAbs for their combination on a single multispecific and avid molecule via direct genetic fusion of their Fab fragments to the human apoferritin light chain. The resulting molecule demonstrated a remarkable median IC50 value of 0.0009 g/mL and 100% neutralization coverage of a broad HIV-1 pseudovirus panel (118 isolates) at a 4 g/mL cutoff-a 32-fold enhancement in viral neutralization potency compared to a mixture of the corresponding HIV-1 bNAbs. Importantly, Fc incorporation on the molecule and engineering to modulate Fc receptor binding resulted in IgG-like bioavailability invivo. This robust plug-and-play antibody design is relevant against indications where multispecificity and avidity are leveraged simultaneously to mediate optimal biological activity.The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases: TZM-bl cells (ARP-8129; contributed by Dr. John C. Kappes and Dr. Xiaoyun Wu); anti–HIV-1 gp160 monoclonal antibody (N6/ PGDM1400x10E8v4) (ARP-13390; contributed by Drs. Ling Xu and Gary Nabel); HIV-1 NL4-3 ΔEnv Vpr luciferase reporter vector (pNL4-3.Luc.R-E-) (ARP-3418; contributed by Dr. Nathaniel Landau and Aaron Diamond); plasmids pcDNA3.1 D/V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11017, ARP-11018, ARP-11024, and ARP-11022; contributed by Drs. David Montefiori, Feng Gao, and Ming Li); plasmid pcDNA3.1(+)-expressing HIV-1 Env/Rev (ARP-11037; contributed by Drs. B. H. Hahn and D. L. Kothe); plasmid pcDNA3.1 D/V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11308; contributed by Drs. D. Montefiori, F. Gao, C. Wil- liamson, and S. Abdool Karim); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11309; contributed by Drs. B. H. Hahn, Y. Li, and J. F. Sala- zar-Gonzalez); HIV-1 BG505 Env expression vector (BG505.W6M.ENV.C2) (ARP- 11518; contributed by Dr. Julie Overbaugh); HIV-1 Env expression vector (CRF02_AG clone 257) (ARP-11599; contributed by Drs. D. Ellenberger, B. Li, M. Callahan, and S. Butera); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 CNE8 Env (ARP-12653; contributed by Drs. Linqi Zhang, Hong Shang, David Montefiori, Tsinghua University (Beijing, China), China Medical University (Bei- jing, China), and Duke University (Durham, NC); HIV-1 SF162 gp160 expression vector (ARP-10463; contributed by Drs. Leonidas Stamatatos and Cecilia Cheng- Mayer); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11034; contributed by Drs. B. H. Hahn, X. Wei, and G. M. Shaw); plasmid pcDNA3.1/V5- His TOPO-expressing HIV Env/Rev (ARP-11038; contributed by Drs. B. H. Hahn and D. L. Kothe); plasmid pcDNA3.1 V5-His TOPO-expressing HIV-1 Env/Rev (ARP-11310; contributed by Drs. B. H. Hahn, Y. Li, and J. F. Salazar-Gonzalez); HIV-1 Env expression vector (p16845 env) (ARP-11503; contributed by Drs. R. Paranjape, S. Kulkarni, and D. Montefiori); HIV-1 1054 Env expression vector (p1054.TC4.1499) (ARP-11561) and 6244 Env expression vector (p6244_13.B5.4576) (ARP-11566; contributed by Drs. Beatrice H. Hahn, Brandon F. Keele, and George M. Shaw); HIV-1 ZM246F Env expression vector (pZM246F_C1G) (ARP-11830; contributed by Dr. Beatrice Hahn); HIV-1 Env expression vector (CRF02_AG clone 278) (ARP-11605; contributed by Drs. Michael Thomson, Ana Revilla, Elena Delgado, David Montefiori, Sonia P erez Castro, Centro Nacional de Microbiologia, Instituto de Salud Carlos III (Majada- honda, Madrid, Spain), Complejo Hospitalario Santa Mar ıa Madre (Orense, Spain), Duke University (Durham, NC), and the CAVD; and NL4-3 Env expression vector (pDOLHIVenv) (from Dr. Eric Freed and Dr. Rex Risser). The following reagents were kindly provided by CAVD: X2988, ZM106.9, and 3817. We thank S. Tabruyn and F. Arbogast for their assistance with in vivo studies. We thank the SickKids-University Health Network Flow Cytometry Facility. This work wassupported by Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant 6280100058 (J.-P.J.) and by Operating Grant PJ4- 169662 from the Canadian Institutes of Health Research (CIHR; B.T. and J.-P.J.). This research was also supported by the European Union’s Horizon 2020 research and innovation program under Marie Sklodowska-Curie Grant 790012 (E.R.), a Hospital for Sick Children Restracomp Postdoctoral Fellowship (C.B.A.), an NSERC postgraduate doctoral scholarship (T.Z.), a predoctoral fel- lowship from the Basque Government (PRE_2019_2_0046) (S.I.), the Canadian Institute for Advanced Research (CIFAR) Azrieli Global Scholar program (J.-P.J.), the Ontario Early Researcher Awards program (J.-P.J.), and the CanadaResearch Chairs program (B.T. and J.-P.J.). This work was supported, in part, by NSERC Discovery Grant RGPIN-2019-06442 and CIHR Project Grant–Priority Announcement PJH-175379 to C.G., and a CIHR Canada Graduate Scholarship (CGS-M) to J.B. Further support was obtained from the Spanish Ministry of Sci- ence, Innovation and Universities (MCIU) with the support of the Spanish Research Agency/The European Regional Development Fund (AEI/FEDER) (RTI2018-095624-B-C21) (J.L.N.) and the Basque Government (IT1196-19) (J.L.N.). Biophysical data were collected at the Structural & Biophysical Core facility supported by the Canada Foundation for Innovation and Ontario Research Fun

The Economics of Tobacco and Tobacco Control

This monograph, a joint effort of the U.S. National Cancer Institute and World Health Organization, examines economic issues in tobacco and tobacco control, including the supply and demand of tobacco products. This first chapter frames the issues addressed in the monograph and describes its organization around key topic areas. Each monograph chapter focuses on the global evidence on these issues, particularly the evidence from low- and middle-income countries (LMICs). The closing sections of this chapter present chapter conclusions and major overall conclusions generated by the work presented here. Experts in economics, tobacco control, public policy, public health, and other related fields from every region in the world, including high-income countries and LMICs, were assembled to provide the research and analyses presented within these pages. It is hoped that this monograph will help inform the implementation of global tobacco control efforts in the 21st century.Additional co-authors: Dongbo Fu, C.K. Gajalakshmi, Vendhan Gajalakshmi, Mark Goodchild, Emmanuel Guindon, Prakash Gupta, Reviva Hasson, Luminita S Hayes, Sara Hitchman, Kinh Hoang-Van, Jidong Huang, Andrew Hyland, Nathan Jones, John Keyser, Pierre Kopp, Harry Lando, David Levy, James Lightwood, Christine Logel, Benn McGrady, Yumiko Mochizuki-Kobayashi, Mario Monsour, Nigar Nargis, Richard J. O’Connor, Maizurah Omar, Zeynep Önder, William Onzivu, Anne-Marie Perucic, Armando Peruga, Vinayak M. Prasad, Martin Raw, Cecily S. Ray, Lyn Reed, Bung-on Ritthiphakdee, Hana Ross, Jennifer Ruger, Henry Saffer, Genevieve Sansone, Natalie Sansone, Fatwa Sari Tetra Dewi, Kerstin Schotte, Omar Shafey, Yoon-Jeong Shin, Giorgio Sincovich, John Tauras, Mark Travers, Édouard Tursan d’Espaignet, Marco Vargas, Mandeep K. Virk-Baker, Corné van Walbeek, Charles W. Warren, Marzenna Anna Weresa, Xin Xu, Eduard Zaloshnja, Lei Zhang, Ping Zhan

PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

The potential impact of Saharan dust and polluted aerosols on microbial populations in the East Mediterranean Sea, an overview of a mesocosm experimental approach.

Recent estimates of nutrient budgets for the Eastern Mediterranean Sea (EMS) indicate that atmospheric aerosols play a significant role as suppliers of macro- and micro- nutrients to its Low Nutrient Low Chlorophyll water. Here we present the first mesocosm experimental study that examines the overall response of the oligotrophic EMS surface mixed layer (Cretan Sea, May 2012) to two different types of natural aerosol additions, “pure” Saharan dust (SD, 1.6 mg l-1) and mixed aerosols (A - polluted and desert origin, 1 mg l-1). We describe the rationale, the experimental set-up, the chemical characteristics of the ambient water and aerosols and the relative maximal biological impacts that resulted from the added aerosols. The two treatments, run in triplicates (3 m3 each), were compared to control-unamended runs. Leaching of approximately 2.1-2.8 and 2.2-3.7 nmol PO4 and 20-26 and 53-55 nmol NOx was measured per each milligram of SD and A, respectively, representing an addition of approximately 30% of the ambient phosphate concentrations. The nitrate/phosphate ratios added in the A treatment were twice than those added in the SD treatment. Both types of dry aerosols triggered a positive change (25-600% normalized per 1 mg l-1 addition) in most of the rate and state variables that were measured: bacterial abundance (BA), bacterial production (BP), Synechococcus (Syn) abundance, chlorophyll-a (chl-a), primary production (PP) and dinitrogen fixation (N2-fix), with relative changes among them following the sequence BP>PP≈N2-fix>chl-a≈BA≈Syn. Our results show that the ‘polluted’ aerosols triggered a relatively larger biological change compared to the SD amendments (per a similar amount of mass addition), especially regarding BP and PP. We speculate that despite the co-limitation of P and N in the EMS, the additional N released by the A treatment may have triggered the relatively larger response in most of the rate and state variables as compared to SD. An implication of our study is that a warmer atmosphere in the future may increase dust emissions and influence the intensity and length of the already well stratified water column in the EMS and hence the impact of the aerosols as a significant external source of new nutrients