Structural basis for ligand reception by anaplastic lymphoma kinase.

The proto-oncogene ALK encodes anaplastic lymphoma kinase, a receptor tyrosine kinase that is expressed primarily in the developing nervous system. After development, ALK activity is associated with learning and memory1 and controls energy expenditure, and inhibition of ALK can prevent diet-induced obesity2. Aberrant ALK signalling causes numerous cancers3. In particular, full-length ALK is an important driver in paediatric neuroblastoma4,5, in which it is either mutated6 or activated by ligand7. Here we report crystal structures of the extracellular glycine-rich domain (GRD) of ALK, which regulates receptor activity by binding to activating peptides8,9. Fusing the ALK GRD to its ligand enabled us to capture a dimeric receptor complex that reveals how ALK responds to its regulatory ligands. We show that repetitive glycines in the GRD form rigid helices that separate the major ligand-binding site from a distal polyglycine extension loop (PXL) that mediates ALK dimerization. The PXL of one receptor acts as a sensor for the complex by interacting with a ligand-bound second receptor. ALK activation can be abolished through PXL mutation or with PXL-targeting antibodies. Together, these results explain how ALK uses its atypical architecture for its regulation, and suggest new therapeutic opportunities for ALK-expressing cancers such as paediatric neuroblastoma.

Structural basis for negative cooperativity in growth factor binding to an EGF receptor.

Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the "high-affinity" and "low-affinity" classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties.

Tumor growth for remodeling process: A 2D approach.

This paper aims to present a comprehensive framework for coupling tumor-bone remodeling processes in a 2-dimensional geometry. This is achieved by introducing a bio-inspired damage that represents the growing tumor, which subsequently affects the main populations involved in the remodeling process, namely, osteoclasts, osteoblasts, and bone tissue. The model is constructed using a set of differential equations based on the Komarova's and Ayati's models, modified to incorporate the bio-inspired damage that may result in tumor mass formation. Three distinct models were developed. The first two models are based on the Komarova's governing equations, with one demonstrating an osteolytic behavior and the second one an osteoblastic model. The third model is a variation of Ayati's model, where the bio-inspired damage is induced through the paracrine and autocrine parameters, exhibiting an osteolytic behavior. The obtained results are consistent with existing literature, leading us to believe that our in-silico experiments will serve as a cornerstone for paving the way towards targeted interventions and personalized treatment strategies, ultimately improving the quality of life for those affected by these conditions.

Anti-KIT antibody, barzolvolimab, reduces skin mast cells and disease activity in chronic inducible urticaria.

BACKGROUND: Chronic inducible urticaria (CIndU) is characterized by mast cell (MC)-mediated wheals in response to triggers: cold in cold urticaria (ColdU) and friction in symptomatic dermographism (SD). KIT receptor activation by stem cell factor (SCF) is essential for MC function. Barzolvolimab (CDX-0159) is a humanized antibody that inhibits KIT activation by SCF and was well tolerated in healthy volunteers with dose-dependent plasma tryptase suppression indicative of systemic mast cell ablation. METHODS: This is an open-label, trial in patients with antihistamine refractory ColdU or SD, receiving one IV dose of barzolvolimab (3 mg/kg), with a 12-week follow-up. Primary endpoint was safety/tolerability; pharmacodynamic (PD)/clinical endpoints included serum tryptase, plasma SCF, skin MC histology, provocation tests, urticaria control test (UCT), and dermatology life quality index (DLQI). RESULTS: Analysis populations were safety (n = 21) and pharmacodynamics/clinical activity (n = 20). Barzolvolimab was well tolerated; most adverse events were mild and resolved. Treatment resulted in significant depletion of skin MCs, decreased tryptase (

Anti-KIT monoclonal antibody CDX-0159 induces profound and durable mast cell suppression in a healthy volunteer study.

BACKGROUND: Mast cells (MC) are powerful inflammatory immune sentinel cells that drive numerous allergic, inflammatory, and pruritic disorders when activated. MC-targeted therapies are approved in several disorders, yet many patients have limited benefit suggesting the need for approaches that more broadly inhibit MC activity. MCs require the KIT receptor and its ligand stem cell factor (SCF) for differentiation, maturation, and survival. Here we describe CDX-0159, an anti-KIT monoclonal antibody that potently suppresses MCs in human healthy volunteers. METHODS: CDX-0159-mediated KIT inhibition was tested in vitro using KIT-expressing immortalized cells and primary human mast cells. CDX-0159 safety and pharmacokinetics were evaluated in a 13-week good laboratory practice (GLP)-compliant cynomolgus macaque study. A single ascending dose (0.3, 1, 3, and 9 mg/kg), double-blinded placebo-controlled phase 1a human healthy volunteer study (n = 32) was conducted to evaluate the safety, pharmacokinetics, and pharmacodynamics of CDX-0159. RESULTS: CDX-0159 inhibits SCF-dependent KIT activation in vitro. Fc modifications in CDX-0159 led to elimination of effector function and reduced serum clearance. In cynomolgus macaques, multiple high doses were safely administered without a significant impact on hematology, a potential concern for KIT inhibitors. A single dose of CDX-0159 in healthy human subjects was generally well tolerated and demonstrated long antibody exposure. Importantly, CDX-0159 led to dose-dependent, profound suppression of plasma tryptase, a MC-specific protease associated with tissue MC burden, indicative of systemic MC suppression or ablation. CONCLUSION: CDX-0159 administration leads to systemic mast cell ablation and may represent a safe and novel approach to treat mast cell-driven disorders.

Development of CDX-527: a bispecific antibody combining PD-1 blockade and CD27 costimulation for cancer immunotherapy.

CD27 is a costimulatory molecule that provides a complementary target to the PD-1/PD-L1 checkpoint axis on T cells. Combining a CD27 agonist antibody with PD-1/PD-L1 blockade has shown synergistic antitumor activity in preclinical models, which led to clinical studies of the combination in cancer patients. We theorized that coupling CD27 costimulation with PD-1/PD-L1 blockade in a bispecific antibody (BsAb) may provide greater immune activating properties than combining the individual mAbs due to enhanced CD27 activation by cross-linking through PD-L1 and Fc receptors. To test this approach, we developed CDX-527, a tetravalent PD-L1xCD27 IgG1-scFv BsAb. CDX-527 potently inhibits PD-1 signaling and induces CD27-mediated T cell costimulation through PD-L1 cross-linking. In mixed lymphocyte reaction assays, CDX-527 is more potent than the combination of the parental antibodies, suggesting that cross-linking through both Fc receptors and PD-L1 results in enhanced CD27 agonist activity. CDX-527 was shown to mediate effector function against tumor cells overexpressing either CD27 or PD-L1. In human CD27 transgenic mice, we observed that antigen-specific T cell responses to a vaccine are greatly enhanced with a surrogate PD-L1xCD27 BsAb. Furthermore, the BsAb exhibits greater antitumor activity than the combination of the parental antibodies in a syngeneic lymphoma model. A pilot study of CDX-527 in cynomolgus macaques confirmed a mAb-like pharmacokinetic profile without noted toxicities. These studies demonstrate that CDX-527 effectively combines PD-1 blockade and CD27 costimulation into one molecule that is more potent than combination of the parental antibodies providing the rationale to advance this BsAb toward clinical studies in cancer patients.

In Vitro Evaluation of the Effect of Stimulation with Magnetic Fields on Chondrocytes.

Magnetic fields (MFs) have been used as an external stimulus to increase cell proliferation in chondrocytes and extracellular matrix (ECM) synthesis of articular cartilage. However, previously published studies have not shown that MFs are homogeneous through cell culture systems. In addition, variables such as stimulation times and MF intensities have not been standardized to obtain the best cellular proliferative rate or an increase in molecular synthesis of ECM. In this work, a stimulation device, which produces homogeneous MFs to stimulate cell culture surfaces was designed and manufactured using a computational model. Furthermore, an in vitro culture of primary rat chondrocytes was established and stimulated with two MF schemes to measure both proliferation and ECM synthesis. The best proliferation rate was obtained with an MF of 2 mT applied for 3 h, every 6 h for 8 days. In addition, the increase in the synthesis of glycosaminoglycans was statistically significant when cells were stimulated with an MF of 2 mT applied for 5 h, every 6 h for 8 days. These findings suggest that a stimulation with MFs is a promising tool that could be used to improve in vitro treatments such as autologous chondrocyte implantation, either to increase cell proliferation or stimulate molecular synthesis. Bioelectromagnetics. 2020;41:41-51 © 2019 Bioelectromagnetics Society.

A computational model for the joint onset and development.

Joints connect the skeletal components and enable movement. The appearance and development of articulations is due to different genetic, biochemical, and mechanical factors. In the embryonic stage, controlled biochemical processes are critical for organized growth. We developed a computational model, which predicts the appearance, location, and development of joints in the embryonic stage. Biochemical events are modeled with reaction diffusion equations with generic molecules representing molecules that 1) determine the site where the articulation will appear, 2) promote proliferation, and matrix synthesis, and 3) define articular cartilage. Our model accounts for cell differentiation from mesenchymal cells to pre-cartilaginous cells, then cartilaginous cells, and lastly articular cartilage. These reaction-diffusion equations were solved using the finite elements method. From a mesenchymal 'bud' of a phalanx, the model predicts growth, joint cleavage, joint morphology, and articular cartilage formation. Our prediction of the gene expression during development agrees with molecular expression profiles of joint development reported in literature. Our computational model suggests that initial rudiment dimensions affect diffusion profiles result in Turing patterns that dictate sites of cleavage thereby determining the number of joints in a rudiment.

Inhibition of ErbB3 by a monoclonal antibody that locks the extracellular domain in an inactive configuration.

ErbB3 (HER3) is a member of the EGF receptor (EGFR) family of receptor tyrosine kinases, which, unlike the other three family members, contains a pseudo kinase in place of a tyrosine kinase domain. In cancer, ErbB3 activation is driven by a ligand-dependent mechanism through the formation of heterodimers with EGFR, ErbB2, or ErbB4 or via a ligand-independent process through heterodimerization with ErbB2 overexpressed in breast tumors or other cancers. Here we describe the crystal structure of the Fab fragment of an antagonistic monoclonal antibody KTN3379, currently in clinical development in human cancer patients, in complex with the ErbB3 extracellular domain. The structure reveals a unique allosteric mechanism for inhibition of ligand-dependent or ligand-independent ErbB3-driven cancers by binding to an epitope that locks ErbB3 in an inactive conformation. Given the similarities in the mechanism of ErbB receptor family activation, these findings could facilitate structure-based design of antibodies that inhibit EGFR and ErbB4 by an allosteric mechanism.

The juxtamembrane region of the EGF receptor functions as an activation domain.

In several growth factor receptors, the intracellular juxtamembrane (JM) region participates in autoinhibitory interactions that must be disrupted for tyrosine kinase activation. Using alanine scanning mutagenesis and crystallographic approaches, we define a domain within the JM region of the epidermal growth factor receptor (EGFR) that instead plays an activating--rather than autoinhibitory--role. Mutations in the C-terminal 19 residues of the EGFR JM region abolish EGFR activation. In a crystal structure of an asymmetric dimer of the tyrosine kinase domain, the JM region of an acceptor monomer makes extensive contacts with the C lobe of a donor monomer, thus stabilizing the dimer. We describe how an uncharacterized lung cancer mutation in this JM activation domain (V665M) constitutively activates EGFR by augmenting its capacity to act as an acceptor in the asymmetric dimer. This JM mutant promotes cellular transformation by EGFR in vitro and is tumorigenic in a xenograft assay.

A Comparison of the Contact Force Distributions on the Acetabular Surface Due to Orthopedic Treatments for Developmental Hip Dysplasia.

We used a three-dimensional rigid body spring model (RBSM) to compare the contact force distributions on the acetabular surface of the infant hip joint that are produced by three orthopedic treatments for developmental dysplasia of the hip (DDH). We analyzed treatments using a Pavlik harness, a generic rigid splint, and a spica cast. The joint geometry was modeled from tomography images of a 1-year-old female. The articular cartilage was modeled as linear springs connecting the surfaces of the acetabulum and the femoral head, whereas the femur and the hip bone were considered as rigid bodies. The hip muscles were modeled as tensile-only preloaded springs. The treatments with the Pavlik harness and the generic rigid splint were modeled for an infant in supine position with a hip flexion angle of 90 deg. Also, since rigid splints are often recommended when children are initiating their gait phase, we modeled the treatment with the infant in standing position. For the spica cast, we only considered the infant in standing position with a flexion angle of 0 deg, and the fixation bar at two heights: at the ankle and at the knee. In order to analyze the effect of the hip abduction angle over the contact force distribution, different abduction angles were used for all the treatments modeled. We have found that the treatments with the infant in supine position, with a flexion angle of 90 deg and abduction angles between 60 deg and 80 deg, produce a more homogenous contact force distribution compared to those obtained for the treatments with the infant in standing position.

ErbB2 resembles an autoinhibited invertebrate epidermal growth factor receptor.

The orphan receptor tyrosine kinase ErbB2 (also known as HER2 or Neu) transforms cells when overexpressed, and it is an important therapeutic target in human cancer. Structural studies have suggested that the oncogenic (and ligand-independent) signalling properties of ErbB2 result from the absence of a key intramolecular 'tether' in the extracellular region that autoinhibits other human ErbB receptors, including the epidermal growth factor (EGF) receptor. Although ErbB2 is unique among the four human ErbB receptors, here we show that it is the closest structural relative of the single EGF receptor family member in Drosophila melanogaster (dEGFR). Genetic and biochemical data show that dEGFR is tightly regulated by growth factor ligands, yet a crystal structure shows that it, too, lacks the intramolecular tether seen in human EGFR, ErbB3 and ErbB4. Instead, a distinct set of autoinhibitory interdomain interactions hold unliganded dEGFR in an inactive state. All of these interactions are maintained (and even extended) in ErbB2, arguing against the suggestion that ErbB2 lacks autoinhibition. We therefore suggest that normal and pathogenic ErbB2 signalling may be regulated by ligands in the same way as dEGFR. Our findings have important implications for ErbB2 regulation in human cancer, and for developing therapeutic approaches that target novel aspects of this orphan receptor.

Access restrictions to forest resources, rather than COVID-19 bans, drive the selection of firewood species for bonfires during Festas Juninas in northeastern Brazil.

BACKGROUND: The complex interplay of social and environmental factors shapes ecosystems, potentially leading to harmony or conflict, highlighting the importance of understanding these dynamics for coexistence. In developing countries, firewood serves as a primary energy source and plays a role in cultural-religious rituals and festivities. However, the specific patterns of woody species used for the latter remain poorly understood, including the impact of access restrictions to resources and local bans on practices. Therefore, our research focuses on examining how access restrictions to forest resources and bonfire bans due to the coronavirus disease 2019 (COVID-19) impact the cultural-religious tradition of bonfire making during Festas Juninas (June festivities) in northeastern Brazil. METHODS: Ethnobotanical fieldwork was conducted in two rural populations in northeastern Brazil between 2021 and 2022. Data were collected through semi-structured interviews, observations, and the guided tour technique. The cultural-religious tradition of bonfire making (i.e., richness of native and exotic firewood species, firewood volume, and the number of bonfires related to this practice) was compared between populations (i.e., differing in access restrictions) and years (i.e., differing in COVID-19-related bans) using Mann-Whitney U tests. RESULTS: Results revealed significant differences in the richness of native (p value = 0.001) and exotic (p value < 0.001) firewood species for bonfire making due to access restrictions to forest resources. The number of native species used was higher among the population residing in the area with unrestricted access than among those with restricted access, while a greater number of exotic species was used in the population with restricted access. The rest of the variables were not influenced by access restrictions, and no variables were influenced by COVID-19 bans. CONCLUSIONS: Our study demonstrated that access restrictions to forest resources, rather than COVID-19 bans, drive the selection of firewood species for bonfires during Festas Juninas in northeastern Brazil. In addition, as populations remain deeply entrenched in cultural-religious practices amid temporary bans imposed by health crises, there is a pressing need for culturally sensitive environmental policies. Fostering socio-ecological resilience demands a comprehensive approach that encompasses not only environmental factors but also cultural dimensions, which wield a pivotal influence on long-term sustainability.

A hypothesis on the formation of the primary ossification centers in the membranous neurocranium: a mathematical and computational model.

This article develops a model of the appearance and location of the primary centers of ossification in the calvaria. The model uses a system of reaction-diffusion equations of two molecules (BMP and Noggin) whose behavior is of type activator-substrate and its solution produces Turing patterns, which represents the primary ossification centers. Additionally, the model includes the level of cell maturation as a function of the location of mesenchymal cells. Thus the mature cells can become osteoblasts due to the action of BMP2. Therefore, with this model, we can have two frontal primary centers, two parietal, and one, two or more occipital centers. The location of these centers in the simplified computational model is highly consistent with those centers found at an embryonic level.

Both network architecture and micro cracks effects on lacuno-canalicular liquid flow efficiency within the context of multiphysics approach for bone remodeling.

When physical forces are applied to bone, its mechanical adaptive behaviors change according to the microarchitecture configuration. This leads to changes in biological and physical thresholds in the remodeling cell population, involving sensor cells (osteocytes) interacting with each other and changes in osteocyte shape due to variation in lacunar shape. The resulting alterations in fluid flow leads to changes in the membrane electrical potential and shear stress. Eventual creation of microcracks, may lead in turn to modify cell activity. In contrast, the redundancy in the lacuno canalicular network (LCN) interconnectivity maintains partial flow. Our goal was to investigate the role of fluid flow in LCN by proposing a model of electro-mechanical energy spread through inhomogeneous microarchitectures. We focused on mechano-sensitivity to changes in load-induced flow impacted by neighboring micro cracks and quantifying its critical role in changing, velocity, shear stress and orientation of liquid mass transportation from one cell to another. To enhance the concept of intricacy LCN micro-structure to fluid flow, we provide a new combined effects factor considered as osteocytes sensor efficiency. We customized an influence function for each osteocyte, coupling: in one hand, the spatial distribution within remodeling influence areas, conducting a significant fluid spread, leading hydro-dynamic behavior and impacted further by presence of micro cracks and; in other hand, the fluid electro kinetic behavior. As an attempt to fill the limitations stated by many of the recent studies, we reveal in numerical simulation, some results which cannot be measured in vitro/in vivo studies. Numerical calculations were performed in order to evaluate, among many others, how liquid flow conditions changes between lacunas, how the orientation and the magnitude of the governing flow in LCN can regulate osteocytes efficiency. In addition to be regulated by osteocytes, a direct effects of fluid flow are also acting on osteoblast activity. In summary, this new approach considers mechano-sensitivity in relation to liquid flow dynamic and suggests additional pathway for Osseo integration via osteoblast regulation. However, this novel modeling approach may help improve the mapping and design bone scaffolds and/or selection of scaffold implantation regions.

A unified framework of cell population dynamics and mechanical stimulus using a discrete approach in bone remodelling.

Multiphysics models have become a key tool in understanding the way different phenomenon are related in bone remodeling and various approaches have been proposed, yet, to the best of the author's knowledge there is no model able to link a cell population model with a mechanical stimulus model using a discrete approach, which allows for an easy implementation. This article couples two classical models, the cell population model from Komarova and the Nackenhorst model in a 2D domain, where correlations between the mechanical loading and the cell population dynamics can be established, furthermore the effect of different paracrine and autocrine regulators is seen on the overall density of a portion of trabecular bone. A discretization is performed using frame 1D finite elements, representing the trabecular structure. The Nackenhorst model is implemented by using the finite element method to calculate the strain energy as the main mechanical stimulus that determines the bone mass density evolution in time. This density is normalized to be added to the bone mass percentage proposed by the Komarova model, where coupling terms have been added as well that guarantee a stable response. In the simulations, the equations were solved employing the finite element method with a user subroutine implemented in ABAQUS (2017) and by applying a direct formulation. The methodology presented can model the cell dynamics occurring in bone remodelling in accordance with the asynchronous nature of this process, yet allowing to differentiate zones with higher density, the main trabecular groups are obtained for the proximal femur. Finally, the model is tested in pathological cases, such as osteoporosis and osteopetrosis, yielding results similar to the pathology behavior. Furthermore, the discrete modelling technique is shown to be of use in this particular application.

Anatomy, molecular structures, and hyaluronic acid - Gelatin injectable hydrogels as a therapeutic alternative for hyaline cartilage recovery: A review.

Cartilage damage caused by trauma or osteoarthritis is a common joint disease that can increase the social and economic burden in society. Due to its avascular characteristics, the poor migration ability of chondrocytes, and a low number of progenitor cells, the self-healing ability of cartilage defects has been significantly limited. Hydrogels have been developed into one of the most suitable biomaterials for the regeneration of cartilage because of its characteristics such as high-water absorption, biodegradation, porosity, and biocompatibility similar to natural extracellular matrix. Therefore, the present review article presents a conceptual framework that summarizes the anatomical, molecular structure and biochemical properties of hyaline cartilage located in long bones: articular cartilage and growth plate. Moreover, the importance of preparation and application of hyaluronic acid - gelatin hydrogels for cartilage tissue engineering are included. Hydrogels possess benefits of stimulating the production of Agc1, Col2α1-IIa, and SOX9, molecules important for the synthesis and composition of the extracellular matrix of cartilage. Accordingly, they are believed to be promising biomaterials of therapeutic alternatives to treat cartilage damage.

Influence of growth plate morphology on bone trabecular groups, a framework computational approach.

The morphology of the growth plate undergoes various transformations during each stage of development, affecting its shape, width, density, and other characteristics. This significantly impacts the distribution of stress in the epiphysis of long bones. To the best of our knowledge, this study represents the first attempt to examine the relationship between growth plate morphology and trabecular bone patterns. Our analysis was conducted using a finite element model and we analyzed two medical cases: trabecular patterns in the femoral epiphysis and the calcaneus bone. Our findings revealed a correlation between the formation of main trabecular groups and growth plate morphology. We investigated how an increased density in high-shear stress zones, which are typically located at the periphery of the growth plate, may occur to prevent failure by shear. This is evident in cases such as slipped capital femoral epiphysis or sever's disease, different simulations align with the clinical data available in the literature from a qualitative and quantitative point of view. Our results suggest that further research should focus on understanding the impact of growth plate morphology on bone remodeling and exploring potential preventive measures for different bone disorders.

The Role of Mechanical Properties and Structure of Type I Collagen Hydrogels on Colorectal Cancer Cell Migration.

Mechanical interactions between cells and their microenvironment play an important role in determining cell fate, which is particularly relevant in metastasis, a process where cells invade tissue matrices with different mechanical properties. In vitro, type I collagen hydrogels have been commonly used for modeling the microenvironment due to its ubiquity in the human body. In this work, the combined influence of the stiffness of these hydrogels and their ultrastructure on the migration patterns of HCT-116 and HT-29 spheroids are analyzed. For this, six different types of pure type I collagen hydrogels by changing the collagen concentration and the gelation temperature are prepared. The stiffness of each sample is measured and its ultrastructure is characterized. Cell migration studies are then performed by seeding the spheroids in three different spatial conditions. It is shown that changes in the aforementioned parameters lead to differences in the mechanical stiffness of the matrices as well as the ultrastructure. These differences, in turn, lead to distinct cell migration patterns of HCT-116 and HT-29 spheroids in either of the spatial conditions tested. Based on these results, it is concluded that the stiffness and the ultrastructural organization of the matrix can actively modulate cell migration behavior in colorectal cancer spheroids.