Multivalent bicyclic peptides are an effective antiviral modality that can potently inhibit SARS-CoV-2.

COVID-19 has stimulated the rapid development of new antibody and small molecule therapeutics to inhibit SARS-CoV-2 infection. Here we describe a third antiviral modality that combines the drug-like advantages of both. Bicycles are entropically constrained peptides stabilized by a central chemical scaffold into a bi-cyclic structure. Rapid screening of diverse bacteriophage libraries against SARS-CoV-2 Spike yielded unique Bicycle binders across the entire protein. Exploiting Bicycles' inherent chemical combinability, we converted early micromolar hits into nanomolar viral inhibitors through simple multimerization. We also show how combining Bicycles against different epitopes into a single biparatopic agent allows Spike from diverse variants of concern (VoC) to be targeted (Alpha, Beta, Delta and Omicron). Finally, we demonstrate in both male hACE2-transgenic mice and Syrian golden hamsters that both multimerized and biparatopic Bicycles reduce viraemia and prevent host inflammation. These results introduce Bicycles as a potential antiviral modality to tackle new and rapidly evolving viruses.

Host Expression Profiling From Diagnostic Coronavirus Disease 2019 Swabs Associates Upper Respiratory Tract Immune Responses With Radiologic Lung Pathology and Clinical Severity.

Background: COVID-19 presents with a breadth of symptomatology including a spectrum of clinical severity requiring intensive care unit (ICU) admission. We investigated the mucosal host gene response at the time of gold standard COVID-19 diagnosis using clinical surplus RNA from upper respiratory tract swabs. Methods: Host response was evaluated by RNA-sequencing, and transcriptomic profiles of 44 unvaccinated patients including outpatients and in-patients with varying levels of oxygen supplementation were included. Additionally, chest X-rays were reviewed and scored for patients in each group. Results: Host transcriptomics revealed significant changes in the immune and inflammatory response. Patients destined for the ICU were distinguished by the significant upregulation of immune response pathways and inflammatory chemokines, including cxcl2 which has been linked to monocyte subsets associated with COVID-19 related lung damage. In order to temporally associate gene expression profiles in the upper respiratory tract at diagnosis of COVID-19 with lower respiratory tract sequalae, we correlated our findings with chest radiography scoring, showing nasopharygeal or mid-turbinate sampling can be a relevant surrogate for downstream COVID-19 pneumonia/ICU severity. Conclusions: This study demonstrates the potential and relevance for ongoing study of the mucosal site of infection of SARS-CoV-2 using a single sampling that remains standard of care in hospital settings. We highlight also the archival value of high quality clinical surplus specimens, especially with rapidly evolving COVID-19 variants and changing public health/vaccination measures.

Stereotactic Laser Ablation (SLA) followed by consolidation stereotactic radiosurgery (cSRS) as treatment for brain metastasis that recurred locally after initial radiosurgery (BMRS): a multi-institutional experience.

INTRODUCTION: The optimal treatment paradigm for brain metastasis that recurs locally after initial radiosurgery remains an area of active investigation. Here, we report outcomes for patients with BMRS treated with stereotactic laser ablation (SLA, also known as laser interstitial thermal therapy, LITT) followed by consolidation radiosurgery. METHODS: Clinical outcomes of 20 patients with 21 histologically confirmed BMRS treated with SLA followed by consolidation SRS and > 6 months follow-up were collected retrospectively across three participating institutions. RESULTS: Consolidation SRS (5 Gy × 5 or 6 Gy × 5) was carried out 16-73 days (median of 26 days) post-SLA in patients with BMRS. There were no new neurological deficits after SLA/cSRS. While 3/21 (14.3%) patients suffered temporary Karnofsky Performance Score (KPS) decline after SLA, no KPS decline was observed after cSRS. There were no 30-day mortalities or wound complications. Two patients required re-admission within 30 days of cSRS (severe headache that resolved with steroid therapy (n = 1) and new onset seizure (n = 1)). With a median follow-up of 228 days (range: 178-1367 days), the local control rate at 6 and 12 months (LC6, LC12) was 100%. All showed diminished FLAIR volume surrounding the SLA/cSRS treated BMRS at the six-month follow-up; none of the patients required steroid for symptoms attributable to these BMRS. These results compare favorably to the available literature for repeat SRS or SLA-only treatment of BMRS. CONCLUSIONS: This multi-institutional experience supports further investigations of SLA/cSRS as a treatment strategy for BMRS.

Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice.

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, has triggered a worldwide health emergency. Here, we show that ferritin-like Dps from hyperthermophilic Sulfolobus islandicus, covalently coupled with SARS-CoV-2 antigens via the SpyCatcher system, forms stable multivalent dodecameric vaccine nanoparticles that remain intact even after lyophilisation. Immunisation experiments in mice demonstrated that the SARS-CoV-2 receptor binding domain (RBD) coupled to Dps (RBD-S-Dps) elicited a higher antibody titre and an enhanced neutralising antibody response compared to monomeric RBD. A single immunisation with RBD-S-Dps completely protected hACE2-expressing mice from serious illness and led to viral clearance from the lungs upon SARS-CoV-2 infection. Our data highlight that multimerised SARS-CoV-2 subunit vaccines are a highly efficacious modality, particularly when combined with an ultra-stable scaffold.

Assessment of the Accuracy of the AO Spine-TL Classification for Thoracolumbar Spine Fractures Using the AO Surgery Reference Mobile App.

STUDY DESIGN: Cohort study. OBJECTIVES: This study aimed to evaluate the accuracy of the AO Surgery Reference mobile app in the diagnosis of thoracolumbar fractures of the spine according to the AO TL classification, and to discuss the usefulness of this app in the teaching and training of the resident physicians in orthopedics and traumatology area. METHODS: The 24 residents of Orthopedic and Traumatology program assessed 20 cases of thoracolumbar fractures selected from the hospital database on 2 different occasions, with a 30-day interval, and they classified these cases with and without using the AO Surgery Reference app. A group of spine experts previously established the gold standard and the answers were statistically compared, with the inter- and intraobserver reliability evaluated by the kappa index. RESULTS: The use of the AO Surgery Reference app increased the classification success rate of the fracture morphology (from 53.4% to 72.5%), of the comorbidity modifier (from 61.4% to 77.9%) and of the neurological status modifier (from 55.1% to 72.9%). In addition, the mobile app raised the classification agreement and accuracy. The kappa index increased from 0.30 to 0.53 regarding the morphological classification of fractures. CONCLUSIONS: The residents improved their ability to recognize and classify thoracolumbar spine fractures, which reinforces the importance of this tool in medical education and clinical practice.

Does a subscapularis tear combined with a posterosuperior rotator cuff tear affect postoperative functional outcomes?

BACKGROUND: The subscapularis is biomechanically important for the shoulder. However, few studies have clinically assessed its importance using a comparative design. Our objective was to compare the functional outcomes in patients who underwent isolated repair of posterosuperior rotator cuff tears and those with repair of combined tears involving the subscapularis. METHODS: We performed a retrospective cohort study evaluating patients who underwent arthroscopic full-thickness rotator cuff repair between January 2013 and May 2017. The patients were divided into 2 groups: isolated repair of posterosuperior tears and repair of combined tears involving the subscapularis. The primary outcome was to evaluate the American Shoulder and Elbow Surgeons (ASES) and University of California, Los Angeles (UCLA) scales at 24 months' follow-up. RESULT: A total of 326 patients were evaluated: 194 with isolated posterosuperior repairs and 132 with combined subscapularis repairs. Both groups showed significant improvement with the procedure (P < .001). The ASES score at 24 months showed no significant difference (P = .426) between the group without subscapularis repair (median, 90.0; interquartile range [IQR], 24.8) and the group with subscapularis repair (median, 86.3; IQR, 33.2). Similarly, the UCLA score showed no difference between the groups (median, 33.0 [IQR, 6.0] and 32.5 [IQR, 8.8], respectively; P = .190). The preoperative functional evaluation also showed no significant differences between the groups. CONCLUSION: The functional results did not differ between patients who underwent isolated repair of posterosuperior tears and those with repair of combined tears involving the subscapularis, according to the ASES and UCLA scales at 24 months.

A synthetic synaptic organizer protein restores glutamatergic neuronal circuits.

Neuronal synapses undergo structural and functional changes throughout life, which are essential for nervous system physiology. However, these changes may also perturb the excitatory-inhibitory neurotransmission balance and trigger neuropsychiatric and neurological disorders. Molecular tools to restore this balance are highly desirable. Here, we designed and characterized CPTX, a synthetic synaptic organizer combining structural elements from cerebellin-1 and neuronal pentraxin-1. CPTX can interact with presynaptic neurexins and postsynaptic AMPA-type ionotropic glutamate receptors and induced the formation of excitatory synapses both in vitro and in vivo. CPTX restored synaptic functions, motor coordination, spatial and contextual memories, and locomotion in mouse models for cerebellar ataxia, Alzheimer's disease, and spinal cord injury, respectively. Thus, CPTX represents a prototype for structure-guided biologics that can efficiently repair or remodel neuronal circuits.

The structure of human thyroglobulin.

Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates1,2. Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor-acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.

A cell topography-based mechanism for ligand discrimination by the T cell receptor.

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.

EFFECT OF SUBSCAPULARIS TEARS ON FUNCTIONAL SCORES OF PATIENTS UNDERGOING ROTATOR CUFF REPAIR.

OBJECTIVE: To evaluate the influence of partial- and full-thickness upper third subscapularis tendon tears on the functional scores of patients undergoing arthroscopic rotator cuff repair. METHODS: Patients who underwent arthroscopic rotator cuff repair were divided into three groups according to the subscapularis tendon condition: intact, partial-thickness tear, or full-thickness upper third tear. Functional scores were compared among groups. Second, the influence of biceps and infraspinatus tears on the scores was tested using multivariate regression analysis. RESULTS: We evaluated 307 shoulders in 297 patients. Full-thickness upper third subscapularis tears presented significantly worse scores than intact tendons. Partial-thickness tears had scores that did not differ significantly from those of the other groups. Patients with full-thickness upper third tears presented a greater rate of injured and unstable biceps tendons. The multivariate analysis showed that biceps and infraspinatus tendon tears did not influence the scores or the intergroup comparison. CONCLUSION: Full-thickness upper third subscapularis tendon tears presented worse functional scores than intact subscapularis tendons among patients undergoing posterosuperior rotator cuff repair. Patients with full-thickness subscapularis tears were more likely to suffer biceps tears, but this fact did not influence functional scores. Level of Evidence I; Clinical randomized trial.

OBJETIVO: Avaliar a influência das lesões do terço superior do tendão subescapular nos escores funcionais de pacientes submetidos ao reparo artroscópico do manguito. MÉTODOS: Divisão em três grupos, conforme condição do tendão subescapular: intacto; ruptura de espessura parcial ou ruptura do terço superior de espessura total. Comparamos escores funcionais. Em seguida, a influência das lesões do bíceps e do infraespinhal nos escores foi testada através de análise de regressão multivariada. RESULTADOS: Avaliamos 307 ombros em 297 pacientes. Rupturas subescapulares no terço superior de espessura total apresentaram escores significativamente piores aos pacientes com tendão intacto. Rupturas de espessura parcial apresentaram escore sem diferença significativa em relação aos demais. Pacientes com rupturas do terço superior de espessura total apresentaram maior taxa de lesão e instabilidade tendínea do bíceps. Análise multivariada mostrou que as rupturas tendíneas do bíceps e infraespinal não influenciaram os escores. CONCLUSÃO: Pacientes com lesão do terço superior de espessura total do tendão do subescapular apresentaram escores funcionais piores do que pacientes com tendão intacto submetidos ao reparo artroscópico posterossuperior do manguito. Pacientes com rupturas do subescapular com espessura total foram mais propensos a apresentar rupturas do bíceps, sem influenciar escores funcionais. Nível de Evidência I, Estudo clínico randomizado.

Effect of subscapularis tears on functional scores of patients undergoing rotator cuff repair / Influência da lesão do subescapular nos escores funcionais de pacientes submetidos a reparo do manguito rotador

ABSTRACT Objective: To evaluate the influence of partial- and full-thickness upper third subscapularis tendon tears on the functional scores of patients undergoing arthroscopic rotator cuff repair. Methods: Patients who underwent arthroscopic rotator cuff repair were divided into three groups according to the subscapularis tendon condition: intact, partial-thickness tear, or full-thickness upper third tear. Functional scores were compared among groups. Second, the influence of biceps and infraspinatus tears on the scores was tested using multivariate regression analysis. Results: We evaluated 307 shoulders in 297 patients. Full-thickness upper third subscapularis tears presented significantly worse scores than intact tendons. Partial-thickness tears had scores that did not differ significantly from those of the other groups. Patients with full-thickness upper third tears presented a greater rate of injured and unstable biceps tendons. The multivariate analysis showed that biceps and infraspinatus tendon tears did not influence the scores or the intergroup comparison. Conclusion: Full-thickness upper third subscapularis tendon tears presented worse functional scores than intact subscapularis tendons among patients undergoing posterosuperior rotator cuff repair. Patients with full-thickness subscapularis tears were more likely to suffer biceps tears, but this fact did not influence functional scores. Level of Evidence I; Clinical randomized trial

RESUMO Objetivo: Avaliar a influência das lesões do terço superior do tendão subescapular nos escores funcionais de pacientes submetidos ao reparo artroscópico do manguito. Métodos: Divisão em três grupos, conforme condição do tendão subescapular: intacto; ruptura de espessura parcial ou ruptura do terço superior de espessura total. Comparamos escores funcionais. Em seguida, a influência das lesões do bíceps e do infraespinhal nos escores foi testada através de análise de regressão multivariada. Resultados: Avaliamos 307 ombros em 297 pacientes. Rupturas subescapulares no terço superior de espessura total apresentaram escores significativamente piores aos pacientes com tendão intacto. Rupturas de espessura parcial apresentaram escore sem diferença significativa em relação aos demais. Pacientes com rupturas do terço superior de espessura total apresentaram maior taxa de lesão e instabilidade tendínea do bíceps. Análise multivariada mostrou que as rupturas tendíneas do bíceps e infraespinal não influenciaram os escores. Conclusão: Pacientes com lesão do terço superior de espessura total do tendão do subescapular apresentaram escores funcionais piores do que pacientes com tendão intacto submetidos ao reparo artroscópico posterossuperior do manguito. Pacientes com rupturas do subescapular com espessura total foram mais propensos a apresentar rupturas do bíceps, sem influenciar escores funcionais. Nível de Evidência I, Estudo clínico randomizado.

Cytoskeletal actin patterns shape mast cell activation.

Activation of immune cells relies on a dynamic actin cytoskeleton. Despite detailed knowledge of molecular actin assembly, the exact processes governing actin organization during activation remain elusive. Using advanced microscopy, we here show that Rat Basophilic Leukemia (RBL) cells, a model mast cell line, employ an orchestrated series of reorganization events within the cortical actin network during activation. In response to IgE antigen-stimulation of FCε receptors (FCεR) at the RBL cell surface, we observed symmetry breaking of the F-actin network and subsequent rapid disassembly of the actin cortex. This was followed by a reassembly process that may be driven by the coordinated transformation of distinct nanoscale F-actin architectures, reminiscent of self-organizing actin patterns. Actin patterns co-localized with zones of Arp2/3 nucleation, while network reassembly was accompanied by myosin-II activity. Strikingly, cortical actin disassembly coincided with zones of granule secretion, suggesting that cytoskeletal actin patterns contribute to orchestrate RBL cell activation.

Cytoskeletal Control of Antigen-Dependent T Cell Activation.

Cytoskeletal actin dynamics is essential for T cell activation. Here, we show evidence that the binding kinetics of the antigen engaging the T cell receptor influences the nanoscale actin organization and mechanics of the immune synapse. Using an engineered T cell system expressing a specific T cell receptor and stimulated by a range of antigens, we found that the peak force experienced by the T cell receptor during activation was independent of the unbinding kinetics of the stimulating antigen. Conversely, quantification of the actin retrograde flow velocity at the synapse revealed a striking dependence on the antigen unbinding kinetics. These findings suggest that the dynamics of the actin cytoskeleton actively adjusted to normalize the force experienced by the T cell receptor in an antigen-specific manner. Consequently, tuning actin dynamics in response to antigen kinetics may thus be a mechanism that allows T cells to adjust the lengthscale and timescale of T cell receptor signaling.

Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins.

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO2, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAAR) ß3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3-4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.

A point mutation in the ion conduction pore of AMPA receptor GRIA3 causes dramatically perturbed sleep patterns as well as intellectual disability.

The discovery of genetic variants influencing sleep patterns can shed light on the physiological processes underlying sleep. As part of a large clinical sequencing project, WGS500, we sequenced a family in which the two male children had severe developmental delay and a dramatically disturbed sleep-wake cycle, with very long wake and sleep durations, reaching up to 106-h awake and 48-h asleep. The most likely causal variant identified was a novel missense variant in the X-linked GRIA3 gene, which has been implicated in intellectual disability. GRIA3 encodes GluA3, a subunit of AMPA-type ionotropic glutamate receptors (AMPARs). The mutation (A653T) falls within the highly conserved transmembrane domain of the ion channel gate, immediately adjacent to the analogous residue in the Grid2 (glutamate receptor) gene, which is mutated in the mouse neurobehavioral mutant, Lurcher. In vitro, the GRIA3(A653T) mutation stabilizes the channel in a closed conformation, in contrast to Lurcher. We introduced the orthologous mutation into a mouse strain by CRISPR-Cas9 mutagenesis and found that hemizygous mutants displayed significant differences in the structure of their activity and sleep compared to wild-type littermates. Typically, mice are polyphasic, exhibiting multiple sleep bouts of sleep several minutes long within a 24-h period. The Gria3A653T mouse showed significantly fewer brief bouts of activity and sleep than the wild-types. Furthermore, Gria3A653T mice showed enhanced period lengthening under constant light compared to wild-type mice, suggesting an increased sensitivity to light. Our results suggest a role for GluA3 channel activity in the regulation of sleep behavior in both mice and humans.

Astrocyte-Secreted Glypican 4 Regulates Release of Neuronal Pentraxin 1 from Axons to Induce Functional Synapse Formation.

The generation of precise synaptic connections between developing neurons is critical to the formation of functional neural circuits. Astrocyte-secreted glypican 4 induces formation of active excitatory synapses by recruiting AMPA glutamate receptors to the postsynaptic cell surface. We now identify the molecular mechanism of how glypican 4 exerts its effect. Glypican 4 induces release of the AMPA receptor clustering factor neuronal pentraxin 1 from presynaptic terminals by signaling through presynaptic protein tyrosine phosphatase receptor δ. Pentraxin then accumulates AMPA receptors on the postsynaptic terminal forming functional synapses. Our findings reveal a signaling pathway that regulates synaptic activity during central nervous system development and demonstrates a role for astrocytes as organizers of active synaptic connections by coordinating both pre and post synaptic neurons. As mutations in glypicans are associated with neurological disorders, such as autism and schizophrenia, this signaling cascade offers new avenues to modulate synaptic function in disease.

Cytoskeletal actin dynamics shape a ramifying actin network underpinning immunological synapse formation.

T cell activation and especially trafficking of T cell receptor microclusters during immunological synapse formation are widely thought to rely on cytoskeletal remodeling. However, important details on the involvement of actin in the latter transport processes are missing. Using a suite of advanced optical microscopes to analyze resting and activated T cells, we show that, following contact formation with activating surfaces, these cells sequentially rearrange their cortical actin across the entire cell, creating a previously unreported ramifying actin network above the immunological synapse. This network shows all the characteristics of an inward-growing transportation network and its dynamics correlating with T cell receptor rearrangements. This actin reorganization is accompanied by an increase in the nanoscale actin meshwork size and the dynamic adjustment of the turnover times and filament lengths of two differently sized filamentous actin populations, wherein formin-mediated long actin filaments support a very flat and stiff contact at the immunological synapse interface. The initiation of immunological synapse formation, as highlighted by calcium release, requires markedly little contact with activating surfaces and no cytoskeletal rearrangements. Our work suggests that incipient signaling in T cells initiates global cytoskeletal rearrangements across the whole cell, including a stiffening process for possibly mechanically supporting contact formation at the immunological synapse interface as well as a central ramified transportation network apparently directed at the consolidation of the contact and the delivery of effector functions.

Structural basis for integration of GluD receptors within synaptic organizer complexes.

Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic ß-neurexin 1 (ß-NRX1) via Cbln1, a C1q-like synaptic organizer. We show how Cbln1 hexamers "anchor" GluD2 amino-terminal domain dimers to monomeric ß-NRX1. This arrangement promotes synaptogenesis and is essential for D: -serine-dependent GluD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber-Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGluR function.

Remarkably low affinity of CD4/peptide-major histocompatibility complex class II protein interactions.

The αß T-cell coreceptor CD4 enhances immune responses more than 1 million-fold in some assays, and yet the affinity of CD4 for its ligand, peptide-major histocompatibility class II (pMHC II) on antigen-presenting cells, is so weak that it was previously unquantifiable. Here, we report that a soluble form of CD4 failed to bind detectably to pMHC II in surface plasmon resonance-based assays, establishing a new upper limit for the solution affinity at 2.5 mM. However, when presented multivalently on magnetic beads, soluble CD4 bound pMHC II-expressing B cells, confirming that it is active and allowing mapping of the native coreceptor binding site on pMHC II. Whereas binding was undetectable in solution, the affinity of the CD4/pMHC II interaction could be measured in 2D using CD4- and adhesion molecule-functionalized, supported lipid bilayers, yielding a 2D Kd of ∼5,000 molecules/µm(2) This value is two to three orders of magnitude higher than previously measured 2D Kd values for interacting leukocyte surface proteins. Calculations indicated, however, that CD4/pMHC II binding would increase rates of T-cell receptor (TCR) complex phosphorylation by threefold via the recruitment of Lck, with only a small, 2-20% increase in the effective affinity of the TCR for pMHC II. The affinity of CD4/pMHC II therefore seems to be set at a value that increases T-cell sensitivity by enhancing phosphorylation, without compromising ligand discrimination.

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.