TP53 mutations in ovarian carcinomas from sporadic cases and carriers of two distinct BRCA1 founder mutations; relation to age at diagnosis and survival

Background  

Ovarian carcinomas from 30 BRCA1 germ-line carriers of two distinct high penetrant founder mutations, 20 carrying the 1675delA and 10 the 1135insA, and 100 sporadic cases were characterized for somatic mutations in the TP53 gene. We analyzed differences in relation to BRCA1 germline status, TP53 status, survival and age at diagnosis, as previous studies have not been conclusive.     

Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy

IL13Rα2 is an attractive target due to its overexpression in a variety of cancers and rare expression in healthy tissue, motivating expansion of interleukin 13 (IL13)–based chimeric antigen receptor (CAR) T cell therapy from glioblastoma into systemic malignancies. IL13Rα1, the other binding partner of IL13, is ubiquitously expressed in healthy tissue, raising concerns about the therapeutic window of systemic administration. IL13 mutants with diminished binding affinity to IL13Rα1 were previously generated by structure-guided protein engineering. In this study, two such variants, termed C4 and D7, are characterized for their ability to mediate IL13Rα2-specific response as binding domains for CAR T cells. Despite IL13Rα1 and IL13Rα2 sharing similar binding interfaces on IL13, mutations to IL13 that decrease binding affinity for IL13Rα1 did not drastically change binding affinity for IL13Rα2. Micromolar affinity to IL13Rα1 was sufficient to pacify IL13-mutein CAR T cells in the presence of IL13Rα1-overexpressing cells in vitro. Interestingly, effector activity of D7 CAR T cells, but not C4 CAR T cells, was demonstrated when cocultured with IL13Rα1/IL4Rα-coexpressing cancer cells. While low-affinity interactions with IL13Rα1 did not result in observable toxicities in mice, in vivo biodistribution studies demonstrated that C4 and D7 CAR T cells were better able to traffic away from IL13Rα1+ lung tissue than were wild-type (WT) CAR T cells. These results demonstrate the utility of structure-guided engineering of ligand-based binding domains with appropriate selectivity while validating IL13-mutein CARs with improved selectivity for application to systemic IL13Rα2-expressing malignancies.

Chimeric antigen receptor (CAR)–engineered T cells have invigorated the field of cancer immunotherapy with their proven ability to treat CD19⁺ malignancies in the clinic (1–4) and continuing progress in solid tumors (5, 6). The synthetic CAR imparts T cells with the ability to recognize antigen independent of peptide presentation by major histocompatibility complexes. This antigen recognition is most often mediated by single-chain variable fragments derived from monoclonal antibodies. As an alternative, naturally occurring ligands or receptors have been used for CAR antigen recognition (7), including interleukin 13 (IL13) (8–10), a proliferation-inducing ligand (APRIL) (11), NKG2D (12), NKp44 (13), and CD27 (14). By leveraging natural binding interactions, these molecules can mediate CAR antigen recognition with minimal additional engineering (8, 14), are fully human in sequence and thus carry potentially lower immunogenicity than other classes of engineered antigen binding domains, and can potentially target multiple cancer biomarkers (11–13). However, the ability to target multiple receptors can also be disadvantageous when binding partners are not implicated in disease.IL13 is one prominent example of a naturally occurring ligand that has been used for CAR antigen recognition (8–10). IL13 interacts strongly with the high-affinity receptor IL13Rα2 (15), which is a versatile therapeutic target due to its rare expression in normal tissue (16) and overexpression in many human cancers, including glioblastoma (GBM) (17), pancreatic ductal adenocarcinoma (18), melanoma (19), ovarian carcinoma (20), clear cell renal cell carcinoma (21), breast cancer (22), and lung cancer (23). A second IL13 receptor family member, IL13Rα1, interacts with IL13 with lower affinity (15) and is ubiquitously expressed in healthy tissue (16). Additionally, IL4Rα can stabilize the IL13Rα1-IL13 complex (15) to mediate signaling through the JAK/STAT6 pathway (24). This receptor pair is coexpressed in pulmonary and other normal tissues (25). Despite this wide expression of IL13 binding partners in healthy tissue, an IL13 ligand–based CAR has shown safety in humans during clinical trials with locoregional central nervous system delivery in GBM (5, 26), suggesting that toxicity from on-target/off-disease binding is not problematic in this context. However, for the treatment of systemic disease, the wide expression of IL13 binding partners outside of the diseased tissue could act as a sink for IL13-based therapy, resulting in safety concerns and possibly impeding trafficking to the disease site. Previous work in the field has attempted to address this problem by generating CARs derived from IL13 variants containing mutations to direct binding away from IL13Rα1/IL4Rα. Mutations at E12 have yielded improved selectivity for IL13Rα2 over IL13Rα1 (8, 9), albeit with the E12Y mutation still allowing measurable recognition of IL13Rα1 in the context of both recombinant antigen and antigen-expressing cancer cells (9). The addition of both E12K and R109K mutations into an IL13-based CAR also showed attenuated, but not abolished, recognition of IL13Rα1-expressing cancer cells relative to IL13Rα2-expressing cancer cells (10). While these examples are encouraging, additional protein engineering is warranted to develop an IL13Rα2-specific IL13 mutant.Structure-based protein engineering and directed evolution approaches offer opportunities to modify the affinity and specificity of binding interactions (27, 28). In this approach, structural information is used to identify residues that contribute to binding interactions, combinatorial libraries are developed through designed or random mutation at the identified residues, and high-throughput in vitro methods are employed to screen for the desired function. Previous applications of this method in the context of cytokines have led to the development of a panel of IL13 mutants with a 6-log affinity range for IL13Rα1 to study the interplay of binding affinity and signal transduction (29), engineering of an orthogonal interleukin 2 (IL2) cytokine-receptor complex system that does not act with the native cytokine or receptor (30), and the development of transforming growth factor beta (TGFβ)-based inhibitors (31), among other examples.Here, we describe the development of IL13-mutein CARs with improved selectivity for IL13Rα2 relative to IL13Rα1 and study their activity in IL13Rα1-expressing, IL13Rα2-expressing, and IL13Rα1/IL4Rα-coexpressing contexts. Prior knowledge of the structures of the IL13 complexes with IL13Rα2 and IL13Rα1/IL4Rα (15) informed the design of an IL13-mutein library that was screened using yeast surface display for diminished binding to IL13Rα1 (29). Characterization of hits yielded two promising candidates, termed C4 and D7, with markedly improved selectivity for IL13Rα2, as shown by affinity characterization. These IL13 muteins were then built into CAR constructs for functional comparison to CARs derived from IL13 wild-type (WT) and IL13 with the E12Y mutation. In vitro and in vivo functional characterization of C4 and D7 IL13-mutein CAR T cells showed decreased activation, degranulation, cytokine release, and cytolytic activity compared to WT and E12Y CAR T cells in the presence of IL13Rα1-expressing cancer cells. Interestingly, C4, but not D7, showed attenuated cytotoxicity relative to WT against IL13Rα1/IL4Rα-coexpressing cancer cells in vitro and in vivo. Conversely, all of the IL13-mutein CAR T cells exhibited similar cytolytic killing of IL13Rα2 targets in vitro and in vivo. Collectively, this work provides insight into the interplay of binding affinity and selectivity in CAR T cell activity and validates IL13-mutein CARs with improved recognition profiles for targeting IL13Rα2-expressing malignancies. Application of these CARs could expand the therapeutic window for systemic administration of IL13Rα2-targeted therapy for a variety of cancers.     

Proximity proteomics of synaptopodin provides insight into the molecular composition of the spine apparatus of dendritic spines

The spine apparatus is a specialized compartment of the neuronal smooth endoplasmic reticulum (ER) located in a subset of dendritic spines. It consists of stacks of ER cisterns that are interconnected by an unknown dense matrix and are continuous with each other and with the ER of the dendritic shaft. While this organelle was first observed over 60 y ago, its molecular organization remains a mystery. Here, we performed in vivo proximity proteomics to gain some insight into its molecular components. To do so, we used the only known spine apparatus–specific protein, synaptopodin, to target a biotinylating enzyme to this organelle. We validated the specific localization in dendritic spines of a small subset of proteins identified by this approach, and we further showed their colocalization with synaptopodin when expressed in nonneuronal cells. One such protein is Pdlim7, an actin binding protein not previously identified in spines. Pdlim7, which we found to interact with synaptopodin through multiple domains, also colocalizes with synaptopodin on the cisternal organelle, a peculiar stack of ER cisterns resembling the spine apparatus and found at axon initial segments of a subset of neurons. Moreover, Pdlim7 has an expression pattern similar to that of synaptopodin in the brain, highlighting a functional partnership between the two proteins. The components of the spine apparatus identified in this work will help elucidate mechanisms in the biogenesis and maintenance of this enigmatic structure with implications for the function of dendritic spines in physiology and disease.

The neuronal endoplasmic reticulum (ER) is an intricate continuous network of membrane tubules and cisterns that runs throughout neuronal processes with region-specific specializations. One such specialization of the smooth ER is the spine apparatus (SA) that is located in a subset of dendritic spines. The SA consists of stacks of flat cisterns that are connected by an unknown dense matrix and are continuous with each other and with the ER of the dendritic shaft (1–3) (Fig. 1A and SI Appendix, Fig. S1A). Morphological changes in the SA have been reported after long-term potentiation (4) and also, in a variety of human disorders, including several neurodegenerative conditions (5–9). While the first observation of the SA by electron microscopy (EM) was reported in 1959 by Gray (1), our understanding of this organelle remains fairly limited. Its molecular characterization has proven to be challenging due to the difficulty of its biochemical isolation and its absence in organisms suitable for genetic screens.Open in a separate windowFig. 1.The localization of synaptopodin (indicated as Synpo in all figures) in cultured hippocampal neurons overlaps with the localization of the SA in dendritic spines of cortical slices. (A) SA as visualized by transmission EM. (B and C) SA and ER reconstructed by a semiautomated algorithm from 3D volumes acquired by FIB-SEM. An SA is shown in B, while C shows a portion of a dendritic shaft with spines containing (magenta arrows) and not containing (white arrows) an SA. The plasma membrane (PM) is shown in blue, the ER is in red, and the post-synaptic density (PSD) is in yellow. (D) Cisternal organelle (CO), as observed in an FIB-SEM optical section, at an axonal initial segment. Note that the stacks of ER cisterns are similar to those characteristics of the SA. (E) mRFP-synaptopodin coexpressed with cytosolic EGFP as a marker of the entire dendritic volume. Note in the zoomed-in views of the region enclosed by a rectangle (the lower three fields) that synaptopodin is concentrated near the spine neck, where the SA is localized. (F) Localization by immunofluorescence of endogenous synaptopodin showing strong overlap with a pool of F-actin labeled by phalloidin-Alexa488. Also, in this sample, the magnified views (the lower three fields) show enrichment of synaptopodin, relative to actin, at the spine neck. (G) mRFP-synaptopodin coexpressed with an ER marker, EGFP-VAPB, showing colocalization of the two proteins. In the zoomed-in views (the lower three fields), red arrows show a spine positive for both the ER marker and synaptopodin, and the blue arrows show a spine with the ER marker but lacking synaptopodin. (H) Percentage of ER-positive spines that also contain synaptopodin and percentage of synaptopodin-positive spines that contain ER quantified in cultured hippocampal neurons expressing mRFP-synaptopodin and the ER markers EGFP-VAPB or EGFP-Sec61β. Each data point represents at least 99 spines from a single neuron. (I) Spine of a synaptopodin KO mouse that lacks the SA but contains the ER. (J) Quantification of the number of spines where ER was visible in the plane of the section with or without an SA in the brain of wild type (WT) vs. synaptopodin mutant mice (n≥800 spines per genotype).The only known protein enriched at the SA and required for its formation is synaptopodin, a protein without transmembrane regions localized in the cytosolic space (10). Neuronal synaptopodin specifically localizes to dendritic spines and to the axonal initial segment, where another specialization of the ER similar to the SA (stack of flattened cisterns) called the cisternal organelle is present (11–14). Lack of synaptopodin in synaptopodin knock-out (KO) mice correlates with the lack of SA and of the cisternal organelle, as well as with a reduction in Hebbian plasticity and spatial memory (11, 15–18). A longer isoform of synaptopodin is expressed in the foot processes of podocytes, where it functions as a regulator of the actin cytoskeleton (19, 20). Synaptopodin binds to and bundles actin (21) and interacts with several actin binding proteins, such as α-actinin (13, 21, 22). While more is known about the interactors of synaptopodin in podocytes, its binding partners at the SA remain unknown.The goal of this work was to gain insight into the molecular composition of the SA. To this aim, we used synaptopodin as a starting point for our analysis. We identified some of its binding partners by an in vivo proximity biotinylation approach and characterized the specific localization of a subset of these proteins in neurons and their interaction with synaptopodin in an exogenous system.     

Evaluation of a Supervised Adapted Physical Activity Program Associated or Not with Oral Supplementation with Arginine and Leucine in Subjects with Obesity and Metabolic Syndrome: A Randomized Controlled Trial

Background: In patients with obesity and metabolic syndrome (MetS), lifestyle interventions combining diet, in particular, and physical exercise are recommended as the first line treatment. Previous studies have suggested that leucine or arginine supplementation may have beneficial effects on the body composition or insulin sensitivity and endothelial function, respectively. We thus conducted a randomized controlled study to evaluate the effects of a supervised adapted physical activity program associated or not with oral supplementation with leucine and arginine in MetS-complicated patients with obesity. Methods: Seventy-nine patients with obesity and MetS were randomized in four groups: patients receiving arginine and leucine supplementation (ALs group, n = 20), patients on a supervised adapted physical activity program (APA group, n = 20), patients combining ALs and APA (ALs+APA group, n = 20), and a control group (n = 19). After the baseline evaluation (m0), patients received ALs and/or followed the APA program for 6 months (m6). Body composition, MetS parameters, lipid and glucose metabolism markers, inflammatory markers, and a cardiopulmonary exercise test (CPET) were assessed at m0, m6, and after a 3-month wash-out period (m9). Results: After 6 months of intervention, we did not observe variable changes in body weight, body composition, lipid and glucose metabolism markers, inflammatory parameters, or quality of life scores between the four groups. However, during the CPET, the maximal power (Pmax and Ppeak), power, and O₂ consumption at the ventilatory threshold (P(VT) and O₂(VT)) were improved in the APA and ALs+APA groups (p < 0.05), as well as the forced vital capacity (FVC). Between m6 and m9, a gain in fat mass was only observed in patients in the APA and ALs+APA groups. Conclusion: In our randomized controlled trial, arginine and leucine supplementation failed to improve MetS in patients with obesity, as did the supervised adapted physical activity program and the combination of both. Only the cardiorespiratory parameters were improved by exercise training.     

Functional Alternatives to Alcohol

The consumption of alcohol is associated with well-known health harms and many governments worldwide are actively engaged in devising approaches to reduce them. To this end, a common proposed strategy aims at reducing alcohol consumption. This approach has led to the development of non-alcoholic drinks, which have been especially welcome by younger, wealthier, health-conscious consumers, who have been turning away from alcohol to look toward alternatives. However, a drawback of non-alcoholic drinks is that they do not facilitate social interaction in the way alcohol does, which is the main reason behind social drinking. Therefore, an alternative approach is to develop functional drinks that do not use alcohol yet mimic the positive, pro-social effects of alcohol without the associated harms. This article will discuss (1) current knowledge of how alcohol mediates its effects in the brain, both the desirable, e.g., antistress to facilitate social interactions, and the harmful ones, with a specific focus on the pivotal role played by the gamma-aminobutyric acid (GABA) neurotransmitter system and (2) how this knowledge can be exploited to develop functional safe alternatives to alcohol using either molecules already existing in nature or synthetic ones. This discussion will be complemented by an analysis of the regulatory challenges associated with the novel endeavour of bringing safe, functional alternatives to alcohol from the bench to bars.     

Time of Dietary Energy and Nutrient Intake and Body Mass Index in Children: Compositional Data Analysis from the Childhood Obesity Project (CHOP) Trial

Meal timing is suggested to influence the obesity risk in children. Our aim was to analyse the effect of energy and nutrient distributions at eating occasions (EO), including breakfast, lunch, supper, and snacks, on the BMI z-score (zBMI) during childhood in 729 healthy children. BMI and three-day dietary protocols were obtained at 3, 4, 5, 6, and 8 years of age, and dietary data were analysed as the percentage of the mean total energy intake (TEI; %E). Intakes at EOs were transformed via an isometric log–ratio transformation and added as exposure variables to linear mixed-effects models. Stratified analyses by country and recategorization of EOs by adding intake from snacks to respective meals for further analyses were performed. The exclusion of subjects with less than three observations and the exclusion of subjects who skipped one EO or consumed 5% energy or less at one EO were examined in sensitivity analyses. Around 23% of the children were overweight at a given time point. Overweight and normal-weight children showed different distributions of dietary intakes over the day; overweight children consumed higher intakes at lunch and lower intakes of snacks. However, no significant effects of timing of EOs on zBMI were found in regression analyses.