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Radiolabeled small-molecule DOTA-haptens can be combined with antitumor/anti-DOTA bispecific antibodies (BsAbs) for pretargeted radioimmunotherapy (PRIT). For optimized delivery of the theranostic g- and b-emitting isotope 177 Lu with DOTA-based PRIT (DOTA-PRIT), bivalent Gemini (DOTA-Bn-thiourea-PEG4-thiourea-Bn-DOTA, aka (3,6,9,12-tetraoxatetradecane-1,14-diyl)bis(DOTA-benzyl thiourea)) was developed. Methods: Gemini was synthesized by linking 2 S-2(4-isothiocyanatobenzyl)-DOTA molecules together via a 1,14-diamino-PEG4 linker. [177Lu]Lu-Gemini 177 Lu]Lu-Gemini was prepared with no-carrier added 177 LuCl 3 to a molar-specific activity of 123 GBq/mmol m mol and radiochemical purity of more than 99%. The specificity of BsAb177 Lu-Gemini was verified in vitro. Subsequently, we evaluated biodistribution and whole-body clearance for [177Lu]Lu-Gemini 177 Lu]Lu-Gemini and, for comparison, our gold-standard monovalent [177Lu]Lu-S-2-(4-amino- 177 Lu]Lu-S-2-(4-amino- benzyl)-DOTA ([177Lu]Lu-DOTA-Bn) 177 Lu]Lu-DOTA-Bn) in na & iuml;ve (tumor-free) athymic nude mice. For our proof-of-concept system, a 3-step pretargeting approach was performed with an established DOTA-PRIT regimen (anti-GPA33/ anti-DOTA IgG-scFv BsAb, a clearing agent, and [177Lu]Lu-Gemini) 177 Lu]Lu-Gemini) mouse models. Results: Initial in vivo studies showed that [177Lu]Lu- 177 Lu]Lu- Gemini behaved similarly to [177Lu]Lu-DOTA-Bn, 177 Lu]Lu-DOTA-Bn, with almost identical blood and whole-body clearance kinetics, as well as biodistribution and mouse kidney dosimetry. Pretargeting [177Lu]Lu-Gemini 177 Lu]Lu-Gemini to GPA33expressing SW1222 human colorectal xenografts was highly effective, leading to absorbed doses of [177Lu]Lu-Gemini 177 Lu]Lu-Gemini for blood, tumor, liver, spleen, and kidneys of 3.99, 455, 6.93, 5.36, and 14.0 cGy/MBq, respectively. Tumor-to-normal tissue absorbed-dose ratios (i.e., therapeutic indices [TIs]) for the blood and kidneys were 114 and 33, respectively. In addition, we demonstrate that the use of bivalent [177Lu]Lu- 177 Lu]Lu- Gemini in DOTA-PRIT leads to improved TIs and augmented [177Lu]Lu- 177 Lu]Lu- Gemini tumor uptake and retention in comparison to monovalent [177Lu]Lu-DOTA-Bn. 177 Lu]Lu-DOTA-Bn. Finally, we established efficacy in SW1222 tumor bearing mice, demonstrating that a single injection of anti-GPA33 DOTA-PRIT with 44 MBq (1.2 mCi) of [177Lu]Lu-Gemini 177 Lu]Lu-Gemini (estimated tumor-absorbed dose, 200 Gy) induced complete responses in 5 of 5 animals and a histologic cure in 2 of 5 (40%) animals. Moreover, a significant increase in survival compared with nontreated controls was noted (maximum tolerated dose not reached). Conclusion: We have developed a bivalent DOTA-radiohapten, [177Lu]Lu-Gemini, that showed improved radiopharmacology for DOTA-PRIT application. The use of bivalent [177Lu]Lu-Gemini in DOTA-PRIT, as opposed to monovalent [177Lu]Lu-DOTA-Bn, allows curative treatments with considerably less administered 177 Lu activity while still achieving high TIs for both the blood and the

The recurring spillover of pathogenic coronaviruses and demonstrated capacity of sarbecoviruses, such SARS-CoV-2, to rapidly evolve in humans underscores the need to better understand immune responses to this virus family. For this purpose, we characterized the functional breadth and potency of antibodies targeting the receptor binding domain (RBD) of the spike glycoprotein that exhibited cross-reactivity against SARS-CoV-2 variants, SARS-CoV-1 and sarbecoviruses from diverse clades and animal origins with spillover potential. One neutralizing antibody, C68.61, showed remarkable neutralization breadth against both SARS-CoV-2 variants and viruses from different sarbecovirus clades. C68.61, which targets a conserved RBD class 5 epitope, did not select for escape variants of SARS-CoV-2 or SARS-CoV-1 in culture nor have predicted escape variants among circulating SARS-CoV-2 strains, suggesting this epitope is functionally constrained. We identified 11 additional SARS-CoV-2/SARS-CoV-1 cross-reactive antibodies that target the more sequence conserved class 4 and class 5 epitopes within RBD that show activity against a subset of diverse sarbecoviruses with one antibody binding every single sarbecovirus RBD tested. A subset of these antibodies exhibited Fc-mediated effector functions as potent as antibodies that impact infection outcome in animal models. Thus, our study identified antibodies targeting conserved regions across SARS-CoV-2 variants and sarbecoviruses that may serve as therapeutics for pandemic preparedness as well as blueprints for the design of immunogens capable of eliciting cross-neutralizing responses.

Human inborn errors of the type I IFN response pathway and auto-Abs neutralizing IFN-alpha,-beta, and/or-omega can underlie severe viral illnesses. We report a simple assay for the detection of both types of condition. We stimulate whole blood from healthy individuals and patients with either inborn errors of type I IFN immunity or auto-Abs against type I IFNs with glycosylated human IFN-alpha 2,-beta, or-omega. As controls, we add a monoclonal antibody (mAb) blocking the type I IFN receptors and stimulate blood with IFN-gamma (type II IFN). Of the molecules we test, IP-10 (encoded by the interferon- stimulated gene (ISG) CXCL10) is the molecule most strongly induced by type I and type II IFNs in the whole blood of healthy donors in an ELISA-like assay. In patients with inherited IFNAR1, IFNAR2, TYK2, or IRF9 deficiency, IP-10 is induced only by IFN-gamma, whereas, in those with auto-Abs neutralizing specific type I IFNs, IP-10 is also induced by the type I IFNs not neutralized by the auto-Abs. The measurement of type I and type II IFN- dependent IP-10 induction therefore constitutes a simple procedure for detecting rare inborn errors of the type I IFN response pathway and more common auto-Abs neutralizing type I IFNs.

Diboson production in association with jets is studied in the fully leptonic final states, pp -> (Z/gamma(*))(Z/gamma(*)) + jets -> 2l2l'+ jets, (l, l ' = e or mu) in proton-proton collisions at a center-of-mass energy of 13 TeV. The data sample corresponds to an integrated luminosity of 138 fb(-1) collected with the CMS detector at the LHC. Differential distributions and normalized differential cross sections are measured as a function of jet multiplicity, transverse momentum p(T), pseudorapidity eta, invariant mass and eta of the highest-p(T) and second-highest-p(T) jets, and as a function of invariant mass of the four-lepton system for events with various jet multiplicities. These differential cross sections are compared with theoretical predictions that mostly agree with the experimental data. However, in a few regions we observe discrepancies between the predicted and measured values. Further improvement of the predictions is required to describe the ZZ+jets production in the whole phase space.

Chromothripsis describes the catastrophic shattering of mis-segregated chromosomes trapped within micronuclei. Although micronuclei accumulate DNA double-strand breaks and replication defects throughout interphase, how chromosomes undergo shattering remains unresolved. Using CRISPRCas9 screens, we identify a non-canonical role of the Fanconi anemia (FA) pathway as a driver of chromothripsis. Inactivation of the FA pathway suppresses chromosome shattering during mitosis without impacting interphase-associated defects within micronuclei. Mono-ubiquitination of FANCIFANCD2 by the FA core complex promotes its mitotic engagement with under-replicated micronuclear chromosomes. The structure-selective SLX4-XPF-ERCC1 endonuclease subsequently induces largescale nucleolytic cleavage of persistent DNA replication intermediates, which stimulates POLD3-dependent mitotic DNA synthesis to prime shattered fragments for reassembly in the ensuing cell cycle. Notably, FA-pathway-induced chromothripsis generates complex genomic rearrangements and extra- chromosomal DNA that confer acquired resistance to anti-cancer therapies. Our findings demonstrate how pathological activation of a central DNA repair mechanism paradoxically triggers cancer genome evolution through chromothripsis.

Pain management in amphibians is an emerging field of veterinary medicine with only a limited number of analgesics studied for their efficacy. The African-clawed frog, Xenopus laevis, is a popular animal model in research due to its oocyte morphology and embryonic development. We investigated analgesic effects of 2 formulations of meloxicam (standard and extended release [ER]) along with their pharmacokinetics and potential toxicity in this species. Adult female African-clawed frogs (n = 6/group) received either standard (0.2, 0.4, 1, or 5 mg/kg) or ER meloxicam (0.6, 1.2, 3, or 15 mg/kg) injected into the dorsal lymph sac. The acetic acid test (AAT) was performed at -1, 1, 6, 12, 24, 48, and 72 h postadministration to evaluate pain response. In addition, a subset of frogs (n = 2/group) were euthanized 72 h postinjection and submitted for necropsy. There were no significant differences in AAT with both formulations compared with saline control. No signs of meloxicam-induced toxicity with either formulation was present in histology. A pharmacokinetic study was conducted for both the standard and ER formulation of meloxicam at 5 and 15 mg/kg, respectively. Results were consistent with the fact that both formulations of meloxicam were readily absorbed with the standard plasma concentrations peaking at 20.40 mu g/mL at 2 h and ER plasma concentration at 30.4 mu g/mL at 12 h. The elimination half-life was only determinable for standard formulation (7.74 h). According to the AAT, both formulations of meloxicam did not provide effective analgesia in adult female Xenopus laevis despite reaching high plasma concentrations.

Two-particle correlations are presented for K-S(0), Lambda, and (Lambda) over bar strange hadrons as a function of relative momentum in lead-lead collisions at a nucleon-nucleon center-of-mass energy of 5.02 TeV. The dataset corresponds to an integrated luminosity of 0.607 nb(-1) and was collected using the CMS detector at the CERN LHC. These correlations are sensitive to quantum statistics and to final-state interactions between the particles. The source size extracted from the (KSKS0)-K-0 correlations is found to decrease from 4.6 to 1.6 fm in going from central to peripheral collisions. Strong interaction scattering parameters (i.e., scattering length and effective range) are determined from the Lambda K-S(0) and Lambda Lambda (including their charge conjugates) correlations using the Lednicky-Lyuboshitz model and are compared to theoretical and other experimental results.

We report two unrelated adults with homozygous (P1) or compound heterozygous (P2) private loss- of- function variants of V-Rel Reticuloendotheliosis Viral Oncogene Homolog B ( RELB ) . The resulting deficiency of functional RelB impairs the induction of NFKB2 mRNA and NF-kappa B2 (p100/p52) protein by lymphotoxin in the fibroblasts of the patients. These defects are rescued by transduction with wild- type RELB complementary DNA (cDNA). By contrast, the response of RelB- deficient fibroblasts to Tumor Necrosis Factor (TNF) or IL- 1 beta via the canonical NF-kappa B pathway remains intact. P1 and P2 have low proportions of na & iuml;ve CD4+ and CD8+ T cells and of memory B cells. Moreover, their na & iuml;ve B cells cannot differentiate into immunoglobulin G (IgG)- or immunoglobulin A (IgA)- secreting cells in response to CD40L/IL-21, and the development of IL- 17A/F- producing T cells is strongly impaired in vitro. Finally, the patients produce neutralizing autoantibodies against type I interferons (IFNs), even after hematopoietic stem cell transplantation, attesting to a persistent dysfunction of thymic epithelial cells in T cell selection and central tolerance to some autoantigens. Thus, inherited human RelB deficiency disrupts the alternative NF-kappa B pathway, underlying a T- and B cell immunodeficiency, which, together with neutralizing autoantibodies against type I IFNs, confers a predisposition to viral, bacterial, and fungal infections.

Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and there has been progress in structural studies on recombinant subassemblies. However, there is limited structural information on native kinetochore architecture. To address this, we purified functional, native kinetochores from the thermophilic yeast Kluyveromyces marxianus and examined them by electron microscopy (EM), cryoelectron tomography (cryo-ET), and atomic force microscopy (AFM). The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with micro- tubules and locating the microtubule binder, Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies and provides the foundation to study the global architecture and functions of kinetochores at a structural level.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells ' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.