Protein-Protein Interaction Prediction via Structure-Based Deep Learning.

Protein-protein interactions (PPIs) play an essential role in life activities. Many artificial intelligence algorithms based on protein sequence information have been developed to predict PPIs. However, these models have difficulty dealing with various sequence lengths and suffer from low generalization and prediction accuracy. In this study, we proposed a novel end-to-end deep learning framework, RSPPI, combining residual neural network (ResNet) and spatial pyramid pooling (SPP), to predict PPIs based on the protein sequence physicochemistry properties and spatial structural information. In the RSPPI model, ResNet was employed to extract the structural and physicochemical information from the protein three-dimensional structure and primary sequence; the SPP layer was used to transform feature maps to a single vector and avoid the fixed-length requirement. The RSPPI model possessed excellent cross-species performance and outperformed several state-of-the-art methods based either on protein sequence or gene ontology in most evaluation metrics. The RSPPI model provides a novel strategy to develop an AI PPI prediction algorithm.

The Doping Effect on the Intrinsic Ferroelectricity in Hafnium Oxide-Based Nano-Ferroelectric Devices.

Hafnium oxide (HfO2)-based ferroelectric tunnel junctions (FTJs) have been extensively evaluated for high-speed and low-power memory applications. Herein, we investigated the influence of Al content in HfAlO thin films on the ferroelectric characteristics of HfAlO-based FTJs. Among HfAlO devices with different Hf/Al ratios (20:1, 34:1, and 50:1), the HfAlO device with Hf/Al ratio of 34:1 exhibited the highest remanent polarization and excellent memory characteristics and, thereby, the best ferroelectricity among the investigated devices. Furthermore, first-principal analyses verified that HfAlO thin films with Hf/Al ratio of 34:1 promoted the formation of the orthorhombic phase against the paraelectric phase as well as alumina impurities and, thus, enhanced the ferroelectricity of the device, providing theoretical support for supporting experimental results. The findings of this study provide insights for developing HfAlO-based FTJs for next-generation in-memory computing applications.

Binding kinetics study of SARS-CoV-2 main protease and potential inhibitors via molecular dynamics simulations.

The pandemic COVID-19 was induced by the novel coronavirus SARS-CoV-2. The virus main protease (Mpro) cleaves the coronavirus polyprotein translated from the viral RNA in the host cells. Because of its crucial role in virus replication, Mpro is a potential drug target for COVID-19 treatment. Herein, we study the interactions between Mpro and three HIV-1 protease (HIV-1 PR) inhibitors, Lopinavir (LPV), Saquinavir (SQV), Ritonavir (RIT), and an inhibitor PF-07321332, by conventional and replica exchange molecular dynamics (MD) simulations. The association/dissociation rates and the affinities of the inhibitors were estimated. The three HIV-1 PR inhibitors exhibit low affinities, while PF-07321332 has the highest affinity among these four simulated inhibitors. Based on cluster analysis, the HIV-1 PR inhibitors bind to Mpro at multiple sites, while PF-07321332 specifically binds to the catalytically activated site of Mpro. The stable and specific binding is because PF-07321332 forms multiple H-bonds to His163 and Glu166 simultaneously. The simulations suggested PF-07321332 could serve as an effective inhibitor with high affinity and shed light on the strategy of drug design and drug repositioning.

Maternal soybean genistein on prevention of later-life breast cancer through inherited epigenetic regulations.

Breast cancer has strong developmental origins and maternal nutrition composition may influence later-life breast cancer risk in the offspring. Our study focused on a bioactive dietary component, genistein (GE) enriched in soybean products, to investigate specific timing of maternal GE exposure that may influence preventive efficacy of GE on offspring breast cancer later in life, and to explore the potential epigenetic mechanisms. Our results indicate a time-dependent effect of maternal GE exposure on early-life breast cancer development in offspring mice. Through integrated transcriptome and methylome analyses, we identified several candidate genes showing significantly differential gene expression and DNA methylation changes. We further found maternal long-term GE treatment can induce inherited epigenetic landmark changes in a candidate tumor suppressor gene, Trp63, resulting in transcriptional activation of Trp63 and induction of the downstream target genes. Our results suggest that maternal long-term exposure to soybean GE may influence early-life epigenetic reprogramming processes, which may contribute to its temporal preventive effects on breast cancer in the offspring. This study provides important mechanistic insights into an appropriate maternal administration of soybean products on prevention of breast cancer later in offspring life.

The soluble N-terminal autoinhibitory module of the A1 domain in von Willebrand factor partially suppresses its catch bond with glycoprotein Ibα in a sandwich complex.

von Willebrand factor (VWF) senses and responds to the hemodynamic forces to interact with the circulatory system and platelets in hemostasis and thrombosis. The dark side of this mechanobiology is implicated in atherothrombosis, stroke, and, more recently, the COVID-19 thrombotic symptoms. The force-responsive element controlling VWF activation predominantly resides in the N terminal auto-inhibitory module (N-AIM) flanking its A1 domain. Nevertheless, the detailed mechano-chemistry of soluble VWF N-AIM is poorly understood at the sub-molecular level as it is assumed to be unstructured loops. Using the free molecular dynamics (MD) simulations, we first predicted a hairpin-like structure of the soluble A1 N-AIM derived polypeptide (Lp; sequences Q1238-E1260). Then we combined molecular docking and steered molecular dynamics (SMD) simulations to examine how Lp regulates the A1-GPIbα interaction under tensile forces. Our simulation results indicate that Lp suppresses the catch bond in a sandwich complex of A1-Lp-GPIbα yet contributes an additional catch-bond residue D1249. To experimentally benchmark the binding kinetics for A1-GPIbα in the absence or presence of Lp, we conducted the force spectroscopy-biomembrane force probe (BFP) assays. We found similar suppression on the A1-GPIbα catch bond with soluble Lp in presence. Clinically, as more and more therapeutic candidates targeting the A1-GPIbα axis have entered clinical trials to treat patients with TTP and acute coronary syndrome, our work represents an endeavor further towards an effective anti-thrombotic approach without severe bleeding side effects as most existing drugs suffer.

Cyclic mechanical reinforcement of integrin-ligand interactions.

Cells regulate adhesion in response to internally generated and externally applied forces. Integrins connect the extracellular matrix to the cytoskeleton and provide cells with mechanical anchorages and signaling platforms. Here we show that cyclic forces applied to a fibronectin-integrin α5ß1 bond switch the bond from a short-lived state with 1 s lifetime to a long-lived state with 100 s lifetime. We term this phenomenon "cyclic mechanical reinforcement," as the bond strength remembers the history of force application and accumulates over repeated cycles, but does not require force to be sustained. Cyclic mechanical reinforcement strengthens the fibronectin-integrin α5ß1 bond through the RGD binding site of the ligand with the synergy binding site greatly facilitating the process. A flexible integrin hybrid domain is also important for cyclic mechanical reinforcement. Our results reveal a mechanical regulation of receptor-ligand interactions and identify a molecular mechanism for cell adhesion strengthening by cyclic forces.

Structural Studies of Overlapping Dinucleosomes in Solution.

An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In this study, the solution structure of the OLDN was investigated through the integration of small-angle x-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis and searched for the conformations that reproduced the SAXS and SANS scattering curves well. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.

Frequent methylation of the tumour suppressor miR-1258 targeting PDL1: implication in multiple myeloma-specific cytotoxicity and prognostification.

miR-1258 is localised to the first intron of ZNF385B at chromosome 2q31.3. miR-1258 promoter methylation was studied in 147 samples including 10 normal buffy coat, eight normal bone marrow plasma cells, 16 human myeloma cell lines (HMCLs), 20 MGUS, 63 diagnostic myeloma, and 30 relapsed myeloma samples by methylation-specific PCR. In myeloma lines, miR-1258 methylation, verified by pyrosequencing, was detected in 62·5% HMCLs but not normal controls, and expression of miR-1258 correlated with that of ZNF385B. 5-Aza-2'-deoxycytidine resulted in promoter demethylation and ZNF385B/miR-1258 re-expression. Luciferase assay confirmed programmed cell death ligand-1 (PDL1) as a direct target of miR-1258. Over-expression of miR-1258 in completely methylated myeloma cells led to reduced cellular proliferation and enhanced apoptosis, hence a tumour suppressor role, in addition to repression of PDL1. In primary samples, miR-1258 methylation, with lower expression of miR-1258, was detected in 49·2% diagnostic myeloma, imparting an inferior PFS (P = 0·034) in addition to 50·0% relapsed myeloma but not MGUS. Therefore, miR-1258 is a tumour suppressor miRNA co-regulated with its host gene, and frequently hypermethylated in active myeloma instead of MGUS, hence acquired during myeloma progression. Methylation-mediated miR-1258 silencing led to overexpression of PDL1 and inferior PFS, implicating miR-1258 in the modulation of myeloma-specific cytotoxicity.

Epigenetic silencing of long non-coding RNA BM742401 in multiple myeloma: impact on prognosis and myeloma dissemination.

BACKGROUND: Long non-coding RNA (lncRNA) BM742401 is a tumor suppressor in gastric cancer and chronic lymphocytic leukemia. As the promoter and coding region of BM742401 are fully embedded in a CpG island, we hypothesized that BM742401 is a tumor suppressor lncRNA epigenetically silenced by promoter DNA methylation in multiple myeloma. METHODS: Methylation-specific PCR and quantitative bisulfite pyrosequencing were performed to detect the methylation of BM742401 in normal plasma cells, myeloma cell lines and primary myeloma samples. The expression of BM742401 was measured by qRT-PCR. The function of BM742401 in multiple myeloma cells was analyzed by lentivirus transduction followed by migration assay. RESULTS: BM742401 methylation was detected in 10 (66.7%) myeloma cell lines but not normal plasma cells, and inversely correlated with expression of BM742401. In primary samples, BM742401 methylation was detected in 3 (12.5%) monoclonal gammopathy of undetermined significance, 9 (15.8%) myeloma at diagnosis and 8 (17.0%) myeloma at relapse/progression. Moreover, BM742401 methylation at diagnosis was associated with inferior overall survival (median OS: 25 vs. 39 months; P = 0.0496). In myeloma cell line JJN-3, stable overexpression of BM742401 by lentivirus transduction resulted in reduced cell migration (P = 0.0001) but not impacting cell death or proliferation. CONCLUSIONS: This is the first report of tumor-specific methylation-mediated silencing of BM742401 in myeloma, which is likely an early event in myelomagenesis with adverse impact on overall survival. Moreover, BM742401 is a tumor suppressor lncRNA by inhibiting myeloma cell migration, hence implicated in myeloma plasma cell homing, metastasis and disease progression.

Immediate, multiplexed and sequential genome engineering facilitated by CRISPR/Cas9 in Saccharomyces cerevisiae.

A method called Cas-3P allowing for immediate, multiplexed and sequential genome engineering was developed using one plasmid expressing Cas9 and three marked plasmid backbones (P1, P2 and P3) for guide RNA (gRNA) expression. The three marked gRNA plasmid backbones were recurred in a P1-P2-P3 order for sequential gene targeting, without construction of any additional plasmid and elimination of gRNA plasmid by induction in each round. The efficiency of direct gRNA plasmid curing mediated by Cas-3P was more than 40% in sequential gene targeting. Besides, Cas-3P allowed single-, double- and triple-loci gene targeting with an efficiency of 75%, 36.8% and 8.2% within 3-4 days, respectively. Through three sequential rounds of gene targeting within 10 days, S. cerevisiae was optimized for the production of patchoulol by replacing one promoter, overexpressing three genes and disrupting four genes. The work is important for practical application in the cell factory engineering of S. cerevisiae.

Simulation and Verification of Vertical Heterogeneity Spectral Response of Winter Wheat Based on the mSCOPE Model.

Vertical heterogeneity of the biochemical characteristics of crop canopy is important in diagnosing and monitoring nutrition, disease, and crop yield via remote sensing. However, the research on vertical isomerism was not comprehensive. Experiments were carried out from the two levels of simulation and verification to analyze the applicability of this recently development model. Effects of winter wheat on spectrum were studied when input different structure parameters (e.g., leaf area index (LAI)) and physicochemical parameters (e.g., chlorophyll content (Chla+b) and water content (Cw)) to the mSCOPE (Soil Canopy Observation, Photochemistry, and Energy fluxes) model. The maximum operating efficiency was 127.43, when the winter wheat was stratified into three layers. Meanwhile, the simulation results also proved that: the vertical profile of LAI had an influence on canopy reflectance in almost all bands; the vertical profile of Chla+b mainly affected the reflectivity of visible region; the vertical profile of Cw only affected the near-infrared reflectance. The verification results showed that the vegetation indexes (VIs) selected of different bands were strongly correlated with the parameters of the canopy. LAI, Chla+b and Cw affected VIs estimation related to LAI, Chla+b and Cw respectively. The Root Mean Square Error (RMSE) of the new-proposed NDVIgreen was the smallest, which was 0.05. Sensitivity analysis showed that the spectrum was more sensitive to changes in upper layer parameters, which verified the rationality of mSCOPE model in explaining the law that light penetration in vertical nonuniform canopy gradually decreases with the increase of layers.

Estimation of Crop Growth Parameters Using UAV-Based Hyperspectral Remote Sensing Data.

Above-ground biomass (AGB) and the leaf area index (LAI) are important indicators for the assessment of crop growth, and are therefore important for agricultural management. Although improvements have been made in the monitoring of crop growth parameters using ground- and satellite-based sensors, the application of these technologies is limited by imaging difficulties, complex data processing, and low spatial resolution. Therefore, this study evaluated the use of hyperspectral indices, red-edge parameters, and their combination to estimate and map the distributions of AGB and LAI for various growth stages of winter wheat. A hyperspectral sensor mounted on an unmanned aerial vehicle was used to obtain vegetation indices and red-edge parameters, and stepwise regression (SWR) and partial least squares regression (PLSR) methods were used to accurately estimate the AGB and LAI based on these vegetation indices, red-edge parameters, and their combination. The results show that: (i) most of the studied vegetation indices and red-edge parameters are significantly highly correlated with AGB and LAI; (ii) overall, the correlations between vegetation indices and AGB and LAI, respectively, are stronger than those between red-edge parameters and AGB and LAI, respectively; (iii) Compared with the estimations using only vegetation indices or red-edge parameters, the estimation of AGB and LAI using a combination of vegetation indices and red-edge parameters is more accurate; and (iv) The estimations of AGB and LAI obtained using the PLSR method are superior to those obtained using the SWR method. Therefore, combining vegetation indices with red-edge parameters and using the PLSR method can improve the estimation of AGB and LAI.

Complete Mitogenomes of Three Carangidae (Perciformes) Fishes: Genome Description and Phylogenetic Considerations.

Carangidae are ecologically and economically important marine fish. The complete mitogenomes of three Carangidae species (Alectis indicus, Decapterus tabl, and Alepes djedaba) were sequenced, characterized, and compared with 29 other species of the family Carangidae in this study. The length of the three mitogenomes ranged from 16,530 to 16,610 bp, and the structures included 2 rRNA genes (12S rRNA and 16S rRNA), 1 control region (a non-coding region), 13 protein-coding genes, and 22 tRNA genes. Among the 22 tRNA genes, only tRNA-Ser (GCT) was not folded into a typical cloverleaf secondary structure and had no recognizable DHU stem. The full-length sequences and protein-coding genes (PCGs) of the mitogenomes of the three species all had obvious AT biases. The majority of the AT-skew and GC-skew values of the PCGs among the three species were negative, demonstrating bases T and C were more plentiful than A and G. Analyses of Ka/Ks and overall p-genetic distance demonstrated that ATP8 showed the highest evolutionary rate and COXI/COXII were the most conserved genes in the three species. The phylogenetic tree based on PCGs sequences of mitogenomes using maximum likelihood and Bayesian inference analyses showed that three clades were divided corresponding to the subfamilies Caranginae, Naucratinae, and Trachinotinae. The monophyly of each superfamily was generally well supported. The divergence time analyses showed that Carangidae evolved during three geological periods, the Cretaceous, Paleogene, and Neogene. A. indicus began to differentiate from other species about 27.20 million years ago (Mya) in the early Miocene, while D. tabl (21.25 Mya) and A. djedaba (14.67 Mya) differentiated in the middle Oligocene.

Comparative Analysis of Complete Mitochondrial Genomes of Three Gerres Fishes (Perciformes: Gerreidae) and Primary Exploration of Their Evolution History.

Mitochondrial genome is a powerful molecule marker to explore phylogenetic relationships and reveal molecular evolution in ichthyological studies. Gerres species play significant roles in marine fishery, but its evolution has received little attention. To date, only two Gerres mitochondrial genomes were reported. In the present study, three mitogenomes of Gerres (Gerres filamentosus, Gerres erythrourus, and Gerres decacanthus) were systemically investigated. The lengths of the mitogenome sequences were 16,673, 16,728, and 16,871 bp for G. filamentosus, G. erythrourus, and G. decacanthus, respectively. Most protein-coding genes (PCGs) were initiated with the typical ATG codon and terminated with the TAA codon, and the incomplete termination codon T/TA could be detected in the three species. The majority of AT-skew and GC-skew values of the 13 PCGs among the three species were negative, and the amplitude of the GC-skew was larger than the AT-skew. The genetic distance and Ka/Ks ratio analyses indicated 13 PCGs were suffering purifying selection and the selection pressures were different from certain deep-sea fishes, were which most likely due to the difference in their living environment. The phylogenetic tree was constructed by molecular method (Bayesian Inference (BI) and maximum Likelihood (ML)), providing further supplement to the scientific classification of fish. Three Gerres species were differentiated in late Cretaceous and early Paleogene, and their evolution might link with the geological events that could change their survival environment.

Glycan Bound to the Selectin Low Affinity State Engages Glu-88 to Stabilize the High Affinity State under Force.

Selectin interactions with fucosylated glycan ligands mediate leukocyte rolling in the vasculature under shear forces. Crystal structures of P- and E-selectin suggest a two-state model in which ligand binding to the lectin domain closes loop 83-89 around the Ca2+ coordination site, enabling Glu-88 to engage Ca2+ and fucose. This triggers further allostery that opens the lectin/EGF domain hinge. The model posits that force accelerates transition from the bent (low affinity) to the extended (high affinity) state. However, transition intermediates have not been described, and the role of Glu-88 in force-assisted allostery has not been examined. Here we report the structure of the lectin and EGF domains of L-selectin bound to a fucose mimetic; that is, a terminal mannose on an N-glycan attached to a symmetry-related molecule. The structure is a transition intermediate where loop 83-89 closes to engage Ca2+ and mannose without triggering allostery that opens the lectin/EGF domain hinge. We used three complementary assays to compare ligand binding to WT selectins and to E88D selectins that replaced Glu-88 with Asp. Soluble P-selectinE88D bound with an ∼9-fold lower affinity to PSGL-1, a physiological ligand, due to faster dissociation. Adhesion frequency experiments with a biomembrane force probe could not detect interactions of P-selectinE88D with PSGL-1. Cells expressing transmembrane P-selectinE88D or L-selectinE88D detached from immobilized ligands immediately after initiating flow. Cells expressing E-selectinE88D rolled but detached faster. Our data support a two-state model for selectins in which Glu-88 must engage ligand to trigger allostery that stabilizes the high affinity state under force.

Cpf1-assisted efficient genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae.

OBJECTIVES: To test the applicability of Cpf1 from Francisella novivida in genomic integration of in vivo assembled DNA parts in Saccharomyces cerevisiae. RESULTS: An easy-to-use vector toolkit, containing a CEN6/ARS4 plasmid expressing Cpf1 from Francisella novivida (FnCpf1) and a 2 µ plasmid for crRNA or crRNA array expressing, was constructed for Cpf1-assisted genomic integration in S. cerevisiae. Our results showed that FnCpf1 allowed for targeted singleplex, doubleplex, and tripleplex genomic integration of in vivo assembled DNA parts with efficiencies of 95, 52, and 43%, respectively. CONCLUSIONS: CRISPR-Cpf1 system allows for efficient genomic integration of in vivo assembled DNA parts in S. cerevisiae, and thus provides an alternative CRISPR-Cas method for metabolic pathway engineering in addition to CRISPR-Cas9 system previously reported for yeast.

Pre-TCR ligand binding impacts thymocyte development before αßTCR expression.

Adaptive cellular immunity requires accurate self- vs. nonself-discrimination to protect against infections and tumorous transformations while at the same time excluding autoimmunity. This vital capability is programmed in the thymus through selection of αßT-cell receptors (αßTCRs) recognizing peptides bound to MHC molecules (pMHC). Here, we show that the pre-TCR (preTCR), a pTα-ß heterodimer appearing before αßTCR expression, directs a previously unappreciated initial phase of repertoire selection. Contrasting with the ligand-independent model of preTCR function, we reveal through NMR and bioforce-probe analyses that the ß-subunit binds pMHC using Vß complementarity-determining regions as well as an exposed hydrophobic Vß patch characteristic of the preTCR. Force-regulated single bonds akin to those of αßTCRs but with more promiscuous ligand specificity trigger calcium flux. Thus, thymic development involves sequential ß- and then, αß-repertoire tuning, whereby preTCR interactions with self pMHC modulate early thymocyte expansion, with implications for ß-selection, immunodominant peptide recognition, and germ line-encoded MHC interaction.

CRISPR/Cpf1 facilitated large fragment deletion in Saccharomyces cerevisiae.

In this study, we focused on the applicability of CRISPR/Cpf1 in genome simplification of Saccharomyces cerevisiae and established a CRISPR/Cpf1 assisted method for rapid markerless large fragment deletion to facilitate laboratory evolution of geome of S. cerevisiae by rational genetic engineering. This method uses a Cpf1 expression plasmid and a crRNA array expression plasmid. The DNA fragment between two DSBs generated by CRISPR/Cpf1 can be cut off from the chromosome, along with re-ligation of the genomic endpoints of the DSBs. Using this method, the large DNA fragment of ∼38 kb between the two genes of TRM10 and REX4 was successfully and rapidly deleted, which was verified by PCR and Sanger DNA Sequencing. This method is simple and rapid, and can be easily implemented for large fragment deletion in S. cerevisiae.

Molecular mechanisms of mechanotransduction in integrin-mediated cell-matrix adhesion.

Cell-matrix adhesion complexes are multi-protein structures linking the extracellular matrix (ECM) to the cytoskeleton. They are essential to both cell motility and function by bidirectionally sensing and transmitting mechanical and biochemical stimulations. Several types of cell-matrix adhesions have been identified and they share many key molecular components, such as integrins and actin-integrin linkers. Mechanochemical coupling between ECM molecules and the actin cytoskeleton has been observed from the single cell to the single molecule level and from immune cells to neuronal cells. However, the mechanisms underlying force regulation of integrin-mediated mechanotransduction still need to be elucidated. In this review article, we focus on integrin-mediated adhesions and discuss force regulation of cell-matrix adhesions and key adaptor molecules, three different force-dependent behaviors, and molecular mechanisms for mechanochemical coupling in force regulation.

The cellular environment regulates in situ kinetics of T-cell receptor interaction with peptide major histocompatibility complex.

T cells recognize antigens at the two-dimensional (2D) interface with antigen-presenting cells (APCs), which trigger T-cell effector functions. T-cell functional outcomes correlate with 2D kinetics of membrane-embedded T-cell receptors (TCRs) binding to surface-tethered peptide-major histocompatibility complex molecules (pMHCs). However, most studies have measured TCR-pMHC kinetics for recombinant TCRs in 3D by surface plasmon resonance, which differs drastically from 2D measurements. Here, we compared pMHC dissociation from native TCR on the T-cell surface to recombinant TCR immobilized on glass surface or in solution. Force on TCR-pMHC bonds regulated their lifetimes differently for native than recombinant TCRs. Perturbing the cellular environment suppressed 2D on-rates but had no effect on 2D off-rate regardless of whether force was applied. In contrast, for the TCR interacting with its monoclonal antibody, the 2D on-rate was insensitive to cellular perturbations and the force-dependent off-rates were indistinguishable for native and recombinant TCRs. These data present novel features of TCR-pMHC kinetics that are regulated by the cellular environment, underscoring the limitations of 3D kinetics in predicting T-cell functions and calling for further elucidation of the underlying molecular and cellular mechanisms that regulate 2D kinetics in physiological settings.