Benchmarking and integrating human B-cell receptor genomic and antibody proteomic profiling.

Immunoglobulins (Ig), which exist either as B-cell receptors (BCR) on the surface of B cells or as antibodies when secreted, play a key role in the recognition and response to antigenic threats. The capability to jointly characterize the BCR and antibody repertoire is crucial for understanding human adaptive immunity. From peripheral blood, bulk BCR sequencing (bulkBCR-seq) currently provides the highest sampling depth, single-cell BCR sequencing (scBCR-seq) allows for paired chain characterization, and antibody peptide sequencing by tandem mass spectrometry (Ab-seq) provides information on the composition of secreted antibodies in the serum. Yet, it has not been benchmarked to what extent the datasets generated by these three technologies overlap and complement each other. To address this question, we isolated peripheral blood B cells from healthy human donors and sequenced BCRs at bulk and single-cell levels, in addition to utilizing publicly available sequencing data. Integrated analysis was performed on these datasets, resolved by replicates and across individuals. Simultaneously, serum antibodies were isolated, digested with multiple proteases, and analyzed with Ab-seq. Systems immunology analysis showed high concordance in repertoire features between bulk and scBCR-seq within individuals, especially when replicates were utilized. In addition, Ab-seq identified clonotype-specific peptides using both bulk and scBCR-seq library references, demonstrating the feasibility of combining scBCR-seq and Ab-seq for reconstructing paired-chain Ig sequences from the serum antibody repertoire. Collectively, our work serves as a proof-of-principle for combining bulk sequencing, single-cell sequencing, and mass spectrometry as complementary methods towards capturing humoral immunity in its entirety.

The multi-specific VH-based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity.

BACKGROUND: Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. METHODS: CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. RESULTS: CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. CONCLUSIONS: CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.

Cofilin-1 phosphorylation catalyzed by ERK1/2 alters cardiac actin dynamics in dilated cardiomyopathy caused by lamin A/C gene mutation.

Hyper-activation of extracellular signal-regulated kinase (ERK) 1/2 contributes to heart dysfunction in cardiomyopathy caused by mutations in the lamin A/C gene (LMNA cardiomyopathy). The mechanism of how this affects cardiac function is unknown. We show that active phosphorylated ERK1/2 directly binds to and catalyzes the phosphorylation of the actin depolymerizing factor cofilin-1 on Thr25. Cofilin-1 becomes active and disassembles actin filaments in a large array of cellular and animal models of LMNA cardiomyopathy. In vivo expression of cofilin-1, phosphorylated on Thr25 by endogenous ERK1/2 signaling, leads to alterations in left ventricular function and cardiac actin. These results demonstrate a novel role for cofilin-1 on actin dynamics in cardiac muscle and provide a rationale on how increased ERK1/2 signaling leads to LMNA cardiomyopathy.

Accuracy of MRI-based vs. CT-based patient-specific instrumentation in total knee arthroplasty: A meta-analysis.

INTRODUCTION: The technical objective of total knee arthroplasty (TKA) is to restore normal mechanical parameters to the knee. Patient-specific instrumentation (PSI) was developed to streamline the operative process and improve accuracy. PSI produces individualized cutting guides based on three-dimensional models of the patient's anatomy acquired from computed-tomography (CT) or magnetic-resonance imaging (MRI). However, the superiority of one modality over the other remains unclear. Therefore, we aimed to compare the accuracy of patient-specific cutting guides produced from MRI or CT imaging methods in TKA. METHODS: Electronic databases were systematically searched using relevant keywords and MeSH terms for original-data English-language publications comparing the accuracy of CT and MRI-based PSI cutting guides in TKA. Data was extracted from the text, tables and figures of studies and meta-analysed. RESULTS: MRI-based PSI cutting guides produced a lower proportion of coronal plane outliers (>3°) with regard to overall limb mechanical axis (OR 2.75, p = 0.01). There were no significant differences between the two in terms of sagittal femoral and tibial component placement, or coronal femoral and tibial placement, or femoral component axial rotation. Tibial rotation was not analysed in the literature. CONCLUSIONS: MRI-based patient-specific cutting guides produced a lower proportion of outliers in the overall coronal alignment of the limb compared to CT, with no significant difference between the two in terms of femoral or tibial component placement. Future studies should investigate the differences in resource usage and operative time between the two to inform surgeons' decision making when choosing an ideal imaging modality for PSI TKA. STUDY DESIGN: Meta-analysis. LEVEL OF EVIDENCE: III, systematic review of cohort and comparative studies.

Actions from head to toe: An update on Bone/Body Morphogenetic Proteins in health and disease.

The pleiotropic actions of Bone Morphogenetic Proteins in many different tissues has led us to the conclusion that they may be viewed as Body Morphogenetic Proteins (BMPs). This is supported by a broad range of distinct BMP-related diseases. Here, we summarize highlights from the 10th international BMP conference, which took place from September 16th to 20th 2014 in Berlin. Attendees updated us on recently identified common and context-specific mechanisms of BMP signaling and function. This included for example new insights into BMP pro-domains, BMP receptors, role of BMPs in muscle and novel consequences of ACVRI mutations. Currently, new BMPs are entering clinical trials with the BMP pathway considered as a 'druggable' target. We conclude that various recent and ongoing approaches could indeed help patients in the near future.

Practical implications of the lumbar spine and its function on total hip arthroplasty.

Correct component placement is of significant importance to ensure optimal outcomes in total hip arthroplasty (THA). Traditionally, the Lewinnek plane has been referenced as an adequate "safe zone", formed between the anterior superior iliac spines and public tubercles to optimize acetabular orientation. However, recent evidence shows that the positioning of this plane may vary due to the biomechanical relationship between the lumbar spine and hip. Therefore, the plane acquired intraoperatively may not accurately recreate the actual functional plane and acetabular orientation encountered outside of the intraoperative environment. This review summarizes the hip-spine relationship and its implications on THA.

Structural insights into BMP receptors: Specificity, activation and inhibition.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-ß family (TGFß), which signal through hetero-tetrameric complexes of type I and type II receptors. In humans there are many more TGFß ligands than receptors, leading to the question of how particular ligands can initiate specific signaling responses. Here we review structural features of the ligands and receptors that contribute to this specificity. Ligand activity is determined by receptor-ligand interactions, growth factor prodomains, extracellular modulator proteins, receptor assembly and phosphorylation of intracellular signaling proteins, including Smad transcription factors. Detailed knowledge about the receptors has enabled the development of BMP-specific type I receptor kinase inhibitors. In future these may help to treat human diseases such as fibrodysplasia ossificans progressiva.

Review on squeaking hips.

Squeaking is a well-recognized complication for hard-on-hard bearings. The nature of squeaking is not yet completely understood however it is considered a multifactorial phenomenon. Patient, implant, and surgical factors play a role in squeaking. It is believed that mechanisms damaging the fluid film lubrication in which these bearings function optimally have a critical role. Such mechanisms include edge loading, stripe wear, impingement, third body particles and ceramic fracture. The resonance of metallic parts can produce noise in the human audible range hence the implant metallurgic composition and design may play a role. Implant positioning can facilitate impingement and edge loading enhancing the occurrence of squeaking. The recent introduction of large heads (> 36 mm) 4(th) generation ceramic-on-ceramic bearing may accentuate the conditions facilitating noise formation; however the current literature is insufficient. Clinically, squeaking may manifest in extreme hip positions or during normal gait cycle however it is rarely associated with pain. Evaluations of patients with squeaking include clinical and radiographic assessments. Computer tomography is recommended as it can better reveal ceramic breakage and implant malposition. The treatments for most squeaking patients include reassurance and activity modification. However for some, noise can be a problem, requiring further surgical intervention. In the occurrence of ceramic fracture, implant failure, extreme components malposition, instability and impingement, surgery should be advised. This review will aim to discuss the current literature regarding squeaking.

¹H, ¹³C and ¹5N assignments of the four N-terminal domains of human fibrillin-1.

Fibrillins are extracellular, disulphide-rich glycoproteins that form 10-12 nm diameter microfibrils in connective tissues. They are found in the majority of higher animals, from jellyfish to humans. Fibrillin microfibrils confer properties of elasticity and strength on connective tissue and regulate growth factor availability in the extracellular matrix (ECM). Mutations in FBN1, the human gene encoding the fibrillin-1 isoform, are linked to several inherited connective tissue disorders. The fibrillin-1 N-terminus forms many functionally-important interactions, both with other fibrillin molecules and various ECM components. In particular, the first four domains, the fibrillin unique N-terminal (FUN) and three epidermal growth factor (EGF)-like domains (FUN-EGF3), are implicated in microfibril assembly and growth factor sequestration. The structure of these domains, which comprise 134 residues, is unknown. We have produced a recombinant fragment corresponding to this region of human fibrillin-1. Here, we report (1)H, (13)C and (15)N resonance assignments of the FUN-EGF3 fragment. Assignments will facilitate structure determination, analysis of interdomain dynamics and the mapping of interaction surfaces.

¹H, ¹³C and ¹5N resonance assignments for the fibrillin-1 EGF2-EGF3-hybrid1-cbEGF1 four-domain fragment.

Fibrillins are large extracellular glycoproteins that form the principal component of microfibrils. These perform a vital structural function in the extracellular matrix of many tissues. Fibrillins have also been implicated in mediating a number of protein-protein interactions, some of which may be significant in regulating growth factors such as transforming growth factor ß. Here we present the backbone and side-chain (1)H, (13)C and (15)N assignments for a 19 kDa protein fragment derived from the N-terminus of human fibrillin-1, encompassing four domains in total. These domains include the second and third epidermal growth factor-like (EGF) domains, the first hybrid domain (hyb1), and the first calcium-binding EGF domain of fibrillin-1. This region of fibrillin-1 is of particular interest as the hyb1 domain has been suggested to play a role in microfibril assembly, as well as several other protein-protein interactions.

Structure of the fibrillin-1 N-terminal domains suggests that heparan sulfate regulates the early stages of microfibril assembly.

The human extracellular matrix glycoprotein fibrillin-1 is the primary component of the 10- to 12-nm-diameter microfibrils, which perform key structural and regulatory roles in connective tissues. Relatively little is known about the molecular mechanisms of fibrillin assembly into microfibrils. Studies using recombinant fibrillin fragments indicate that an interaction between the N- and C-terminal regions drives head-to-tail assembly. Here, we present the structure of a fibrillin N-terminal fragment comprising the fibrillin unique N-terminal (FUN) and the first three epidermal growth factor (EGF)-like domains (FUN-EGF3). Two rod-like domain pairs are separated by a short, flexible linker between the EGF1 and EGF2 domains. We also show that the binding site for the C-terminal region spans multiple domains and overlaps with a heparin interaction site. These data suggest that heparan sulfate may sequester fibrillin at the cell surface via FUN-EGF3 prior to aggregation of the C terminus, thereby regulating microfibril assembly.

Defining the limits of single-molecule FRET resolution in TIRF microscopy.

Single-molecule FRET (smFRET) has long been used as a molecular ruler for the study of biology on the nanoscale (∼2-10 nm); smFRET in total-internal reflection fluorescence (TIRF) Förster resonance energy transfer (TIRF-FRET) microscopy allows multiple biomolecules to be simultaneously studied with high temporal and spatial resolution. To operate at the limits of resolution of the technique, it is essential to investigate and rigorously quantify the major sources of noise and error; we used theoretical predictions, simulations, advanced image analysis, and detailed characterization of DNA standards to quantify the limits of TIRF-FRET resolution. We present a theoretical description of the major sources of noise, which was in excellent agreement with results for short-timescale smFRET measurements (<200 ms) on individual molecules (as opposed to measurements on an ensemble of single molecules). For longer timescales (>200 ms) on individual molecules, and for FRET distributions obtained from an ensemble of single molecules, we observed significant broadening beyond theoretical predictions; we investigated the causes of this broadening. For measurements on individual molecules, analysis of the experimental noise allows us to predict a maximum resolution of a FRET change of 0.08 with 20-ms temporal resolution, sufficient to directly resolve distance differences equivalent to one DNA basepair separation (0.34 nm). For measurements on ensembles of single molecules, we demonstrate resolution of distance differences of one basepair with 1000-ms temporal resolution, and differences of two basepairs with 80-ms temporal resolution. Our work paves the way for ultra-high-resolution TIRF-FRET studies on many biomolecules, including DNA processing machinery (DNA and RNA polymerases, helicases, etc.), the mechanisms of which are often characterized by distance changes on the scale of one DNA basepair.

Calcium regulates scallop muscle by changing myosin flexibility.

Muscle myosins are molecular motors that convert the chemical free energy available from ATP hydrolysis into mechanical displacement of actin filaments, bringing about muscle contraction. Myosin cross-bridges exert force on actin filaments during a cycle of attached and detached states that are coupled to each round of ATP hydrolysis. Contraction and ATPase activity of the striated adductor muscle of scallop is controlled by calcium ion binding to myosin. This mechanism of the so-called "thick filament regulation" is quite different to vertebrate striated muscle which is switched on and off via "thin filament regulation" whereby calcium ions bind to regulatory proteins associated with the actin filaments. We have used an optically based single molecule technique to measure the angular disposition adopted by the two myosin heads whilst bound to actin in the presence and absence of calcium ions. This has allowed us to directly observe the movement of individual myosin heads in aqueous solution at room temperature in real time. We address the issue of how scallop striated muscle myosin might be regulated by calcium and have interpreted our results in terms of the structures of smooth muscle myosin that also exhibit thick filament regulation.