The rockefeller university

Humans with monogenic inborn errors responsible for extreme disease phenotypes can reveal essential physiological pathways. We investigated germline mutations in GNAI2, which encodes G(alpha i2), a key component in heterotrimeric G protein signal transduction usually thought to regulate adenylyl cyclase-mediated cyclic adenosine monophosphate (cAMP) production. Patients with activating G(alpha i2) mutations had clinical presentations that included impaired immunity. Mutant G(alpha i2) impaired cell migration and augmented responses to T cell receptor (TCR) stimulation. We found that mutant G(alpha i2) influenced TCR signaling by sequestering the guanosine triphosphatase (GTPase)-activating protein RASA2, thereby promoting RAS activation and increasing downstream extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-AKT S6 signaling to drive cellular growth and proliferation.

Single-domain antibodies ("nanobodies") derived from the variable region of camelid heavy-chain only antibody variants have proven to be widely useful tools for research, therapeutic, and diagnostic applications. In addition to traditional display techniques, methods to generate nanobodies using direct detection by mass spectrometry and DNA sequencing have been highly effective. However, certain technical challenges have limited widespread application. We have optimized a new pipeline for this approach that greatly improves screening sensitivity, depth of antibody coverage, antigen compatibility, and overall hit rate and affinity. We have applied this improved methodology to generate significantly higher affinity nanobody repertoires against widely used targets in biological research- i.e., GFP, tdTomato, GST, and mouse, rabbit, and goat immunoglobulin G. We have characterized these reagents in affinity isolations and tissue immunofluorescence microscopy, identifying those that are optimal for these particularly demanding applications, and engineering dimeric constructs for ultra-high affinity. This study thus provides new nanobody tools directly applicable to a wide variety of research problems, and improved techniques enabling future nanobody development against diverse targets.

Editor's note: This is the 23rd article in a series on clinical research by nurses. The series is designed to be used as a resource for nurses to understand the concepts and principles essential to research. Each column will present the concepts that underpin evidence-based practice-from research design to data interpretation. To see all the articles in the series, go to https://links.lww.com/AJN/A204.

Background: Monoallelic loss-of-function IKZF1 (IKAROS) variants cause B-cell deficiency or combined immunodeficiency, whereas monoallelic gain-of-function (GOF) IKZF1 variants have recently been reported to cause hypergammaglobulinemia, abnormal plasma cell differentiation, autoimmune and allergic manifestations, and infections. Objective: We studied 7 relatives with autoimmune/inflammatory and lymphoproliferative manifestations to identify the immunologic disturbances and the genetic cause of their disease. Methods: We analyzed biopsy results and performed wholeexome sequencing and immunologic studies. Results: Disease onset occurred at a mean age of 25.2 years (range, 10-64, years). Six patients suffered from autoimmune/inflammatory diseases, 4 had confirmed IG4-related disease (IgG4-RD), and 5 developed B-cell malignancies: lymphoma in 4 and multiple myeloma in the remaining patient. Patients without immunosuppression were not particularly prone to infectious diseases. Three patients suffered from life-threatening coronavirus disease 2019 pneumonia, of whom 1 had autoantibodies neutralizing IFN-a. a . The recently described IKZF1 GOF p.R183H variant was found in the 5 affected relatives tested and in a 6-year-old asymptomatic girl. Immunologic analysis revealed hypergammaglobulinemia and high frequencies of certain lymphocyte subsets (exhausted B cells, effector memory CD4 T cells, effector memory CD4 T cells that have regained surface expression of CD45RA and CD282CD57+ 2 CD57 + CD4+ + and CD8+ + T cells, TH2, H 2, and Tfh2 cells) attesting to immune dysregulation. Partial clinical responses to rituximab and corticosteroids were observed, and treatment with lenalidomide, which promotes IKAROS degradation, was initiated in 3 patients. Conclusions: Heterozygosity for GOF IKZF1 variants underlies autoimmunity/inflammatory diseases, IgG4-RD, and B-cell malignancies, the onset of which may occur in adulthood. Clinical and immunologic data are similar to those for patients with unexplained IgG4-RD. Patients may therefore benefit from treatments inhibiting pathways displaying IKAROS-mediated overactivity. (J Allergy Clin Immunol 2024;154:819-26.)

Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.

BACKGROUND. Obesity is the foremost risk factor in the development of endometrial cancer (EC). However, the impact of obesity on the response to immune checkpoint inhibitors (ICI) in EC remains poorly understood. This retrospective study investigates the association among BMI, body fat distribution, and clinical and molecular characteristics of EC patients treated with ICI. METHODS. We analyzed progression-free survival (PFS) and overall survival (OS) in EC patients treated with ICI, categorized by BMI, fat-mass distribution, and molecular subtypes. Incidence of immune-related adverse events (irAEs) after ICI was also assessed based on BMI status. RESULTS. 524 EC patients were included in the study. Overweight and obese patients exhibited a significantly prolonged PFS and OS compared with normal BMI patients after treatment with ICI. Multivariable Cox's regression analysis confirmed the independent association of overweight and obesity with improved PFS and OS. Elevated visceral adipose tissue (VAT) was identified as a strong independent predictor for improved PFS to ICI. Associations between obesity and OS/PFS were particularly significant in the copy number-high/TP53abnormal TP53 abnormal (CN-H/TP53abn) TP53 abn) EC molecular subtype. Finally, obese patients demonstrated a higher irAE rate compared with normal BMI individuals. CONCLUSION. Obesity is associated with improved outcomes to ICI in EC patients and a higher rate of irAEs. This association is more pronounced in the CN-H/TP53abn TP53 abn EC molecular subtype.

Positive allosteric modulator (PAM) drugs enhance the activation of the calcium-sensing receptor (CaSR) and suppress parathyroid hormone (PTH) secretion. Unfortunately, these hyperparathyroidism-treating drugs can induce hypocalcemia and arrhythmias. Seeking improved modulators, we docked libraries of 2.7 million and 1.2 billion molecules against the CaSR structure. The billion-molecule docking found PAMs with a 2.7-fold higher hit rate than the million-molecule library, with hits up to 37-fold more potent. Structure-based optimization led to nanomolar leads. In ex vivo organ assays, one of these PAMs was 100-fold more potent than the standard of care, cinacalcet, and reduced serum PTH levels in mice without the hypocalcemia typical of CaSR drugs. As determined from cryo-electron microscopy structures, the PAMs identified here promote CaSR conformations that more closely resemble the activated state than those induced by the established drugs.

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.

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.