M-protein diagnostics in multiple myeloma patients using ultra-sensitive targeted mass spectrometry and an off-the-shelf calibrator.

OBJECTIVES: Minimal residual disease status in multiple myeloma is an important prognostic biomarker. Recently, personalized blood-based targeted mass spectrometry (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to measure minimal residual disease. However, quantification of MS-MRD requires a unique calibrator for each patient. The use of patient-specific stable isotope labelled (SIL) peptides is relatively costly and time-consuming, thus hindering clinical implementation. Here, we introduce a simplification of MS-MRD by using an off-the-shelf calibrator. METHODS: SILuMAB-based MS-MRD was performed by spiking a monoclonal stable isotope labeled IgG, SILuMAB-K1, in the patient serum. The abundance of both M-protein-specific peptides and SILuMAB-specific peptides were monitored by mass spectrometry. The relative ratio between M-protein peptides and SILuMAB peptides allowed for M-protein quantification. We assessed linearity, sensitivity and reproducibility of SILuMAB-based MS-MRD in longitudinally collected sera from the IFM-2009 clinical trial. RESULTS: A linear dynamic range was achieved of over 5 log scales, allowing for M-protein quantification down to 0.001 g/L. The inter-assay CV of SILuMAB-based MS-MRD was on average 11 %. Excellent concordance between SIL- and SILuMAB-based MS-MRD was shown (R2>0.985). Additionally, signal intensity of spiked SILuMAB can be used for quality control purpose to assess system performance and incomplete SILuMAB digestion can be used as quality control for sample preparation. CONCLUSIONS: Compared to SIL peptides, SILuMAB-based MS-MRD improves the reproducibility, turn-around-times and cost-efficacy of MS-MRD without diminishing its sensitivity and specificity. Furthermore, SILuMAB can be used as a MS-MRD quality control tool to monitor sample preparation efficacy and assay performance.

N-linked glycosylation of the M-protein variable region: glycoproteogenomics reveals a new layer of personalized complexity in multiple myeloma.

OBJECTIVES: Multiple myeloma (MM) is a plasma cell malignancy characterized by a monoclonal expansion of plasma cells that secrete a characteristic M-protein. This M-protein is crucial for diagnosis and monitoring of MM in the blood of patients. Recent evidence has emerged suggesting that N-glycosylation of the M-protein variable (Fab) region contributes to M-protein pathogenicity, and that it is a risk factor for disease progression of plasma cell disorders. Current methodologies lack the specificity to provide a site-specific glycoprofile of the Fab regions of M-proteins. Here, we introduce a novel glycoproteogenomics method that allows detailed M-protein glycoprofiling by integrating patient specific Fab region sequences (genomics) with glycoprofiling by glycoproteomics. METHODS: Glycoproteogenomics was used for the detailed analysis of de novo N-glycosylation sites of M-proteins. First, Genomic analysis of the M-protein variable region was used to identify de novo N-glycosylation sites. Subsequently glycopeptide analysis with LC-MS/MS was used for detailed analysis of the M-protein glycan sites. RESULTS: Genomic analysis uncovered a more than two-fold increase in the Fab Light Chain N-glycosylation of M-proteins of patients with Multiple Myeloma compared to Fab Light Chain N-glycosylation of polyclonal antibodies from healthy individuals. Subsequent glycoproteogenomics analysis of 41 patients enrolled in the IFM 2009 clinical trial revealed that the majority of the Fab N-glycosylation sites were fully occupied with complex type glycans, distinguishable from Fc region glycans due to high levels of sialylation, fucosylation and bisecting structures. CONCLUSIONS: Together, glycoproteogenomics is a powerful tool to study de novo Fab N-glycosylation in plasma cell dyscrasias.

Advances in minimal residual disease monitoring in multiple myeloma.

Multiple myeloma (MM) is characterized by the clonal expansion of plasma cells and the excretion of a monoclonal immunoglobulin (M-protein), or fragments thereof. This biomarker plays a key role in the diagnosis and monitoring of MM. Although there is currently no cure for MM, novel treatment modalities such as bispecific antibodies and CAR T-cell therapies have led to substantial improvement in survival. With the introduction of several classes of effective drugs, an increasing percentage of patients achieve a complete response. This poses new challenges to traditional electrophoretic and immunochemical M-protein diagnostics because these methods lack sensitivity to monitor minimal residual disease (MRD). In 2016, the International Myeloma Working Group (IMWG) expanded their disease response criteria with bone marrow-based MRD assessment using flow cytometry or next-generation sequencing in combination with imaging-based disease monitoring of extramedullary disease. MRD status is an important independent prognostic marker and its potential as a surrogate endpoint for progression-free survival is currently being studied. In addition, numerous clinical trials are investigating the added clinical value of MRD-guided therapy decisions in individual patients. Because of these novel clinical applications, repeated MRD evaluation is becoming common practice in clinical trials as well as in the management of patients outside clinical trials. In response to this, novel mass spectrometric methods that have been developed for blood-based MRD monitoring represent attractive minimally invasive alternatives to bone marrow-based MRD evaluation. This paves the way for dynamic MRD monitoring to allow the detection of early disease relapse, which may prove to be a crucial factor in facilitating future clinical implementation of MRD-guided therapy. This review provides an overview of state-of-the-art of MRD monitoring, describes new developments and applications of blood-based MRD monitoring, and suggests future directions for its successful integration into the clinical management of MM patients.

Cell adhesion proteins in the cerebrospinal fluid of neonates prenatally exposed to Zika virus: A case-control study.

To compare cell adhesion molecules levels in cerebrospinal fluid (CSF) between Zika virus (ZIKV)-exposed neonates with/without microcephaly (cases) and controls, 16 neonates (cases), 8 (50%) with and 8 (50%) without microcephaly, who underwent lumbar puncture (LP) during the ZIKV epidemic (2015-2016) were included. All mothers reported ZIKV clinical symptoms during gestation, all neonates presented with congenital infection findings, and other congenital infections were ruled out. Fourteen control neonates underwent LP in the same laboratory (2017-2018). Five cell adhesion proteins were measured in the CSF using mass spectrometry. Neurexin-1 (3.50 [2.00-4.00] vs. 7.5 [5.00-10.25], P = 0.001), neurexin-3 (0.00 [0.00-0.00] vs. 3.00 [1.50-4.00], P = 0.001) and neural cell adhesion molecule 2 (NCAM2) (0.00 [0.00-0.75] vs. 1.00 [1.00-2.00], P = 0.001) were significantly lower in microcephalic and non-microcephalic cases than in controls. When these two sub-groups of prenatally ZIKA-exposed children were compared to controls separately, the same results were found. When cases with and without microcephaly were compared, no difference was found. Neurexin-3 (18.8% vs. 78.6%, P = 0.001) and NCAM2 (25.0% vs. 85.7%, P = 0.001) were less frequently found among the cases. A positive correlation was found between cephalic perimeter and levels of these two proteins. Neurexin-2 and neurexin-2b presented no significant differences. Levels of three cell adhesion proteins were significantly lower in CSF of neonates exposed to ZIKV before birth than in controls, irrespective of presence of congenital microcephaly. Moreover, the smaller the cephalic perimeter, the lower CSF cell adhesion protein levels. These findings suggest that low CSF levels of neurexin-1, neurexin-3 and NCAM2 may reflect the effects of ZIKV on foetal brain development.

Online Electrochemical Reduction of Both Inter- and Intramolecular Disulfide Bridges in Immunoglobulins.

Electrochemical reduction of intermolecular disulfide bridges has previously been demonstrated in immunoglobulins but failed to achieve reduction of intramolecular bonds. We now report an improved method that achieves the full reduction of both intermolecular and intramolecular disulfide bridges in a set of monoclonal antibodies based on their intact mass and on MS/MS analysis. The system uses an online electrochemical flow cell positioned online between a chromatography system and a mass spectrometer to give direct information on pairs of heavy and light chains in an antibody. The complete reduction of the intramolecular disulfide bridges is important, as the redox state affects the intact mass of the antibody chain. Disulfide bonds also hamper MS/MS fragmentation of protein chains and thus limit the confirmation of the amino acid sequence of the protein of interest. The improved electrochemical system and associated protocols can simplify sample processing prior to analysis, as chemical reduction is not required. Also, it opens up new possibilities in the top-down mass spectrometry analysis of samples containing complex biomolecules with inter- and intramolecular disulfide bridges.

Clonotypic Features of Rearranged Immunoglobulin Genes Yield Personalized Biomarkers for Minimal Residual Disease Monitoring in Multiple Myeloma.

BACKGROUND: Due to improved treatment, more patients with multiple myeloma (MM) reach a state of minimal residual disease (MRD). Different strategies for MM MRD monitoring include flow cytometry, allele-specific oligonucleotide-quantitative PCR, next-generation sequencing, and mass spectrometry (MS). The last 3 methods rely on the presence and the stability of a unique immunoglobulin fingerprint derived from the clonal plasma cell population. For MS-MRD monitoring it is imperative that MS-compatible clonotypic M-protein peptides are identified. To support implementation of molecular MRD techniques, we studied the presence and stability of these clonotypic features in the CoMMpass database. METHODS: An analysis pipeline based on MiXCR and HIGH-VQUEST was constructed to identify clonal molecular fingerprints and their clonotypic peptides based on transcriptomic datasets. To determine the stability of the clonal fingerprints, we compared the clonal fingerprints during disease progression for each patient. RESULTS: The analysis pipeline to establish the clonal fingerprint and MS-suitable clonotypic peptides was successfully validated in MM cell lines. In a cohort of 609 patients with MM, we demonstrated that the most abundant clone harbored a unique clonal molecular fingerprint and that multiple unique clonotypic peptides compatible with MS measurements could be identified for all patients. Furthermore, the clonal immunoglobulin gene fingerprints of both the light and heavy chain remained stable during MM disease progression. CONCLUSIONS: Our data support the use of the clonal immunoglobulin gene fingerprints in patients with MM as a suitable MRD target for MS-MRD analyses.

Multiple Myeloma Minimal Residual Disease Detection: Targeted Mass Spectrometry in Blood vs Next-Generation Sequencing in Bone Marrow.

BACKGROUND: Minimal residual disease (MRD) status assessed on bone marrow aspirates is a major prognostic biomarker in multiple myeloma (MM). In this study we evaluated blood-based targeted mass spectrometry (MS-MRD) as a sensitive, minimally invasive alternative to measure MM disease activity. METHODS: Therapy response of 41 MM patients in the IFM-2009 clinical trial (NCT01191060) was assessed with MS-MRD on frozen sera and compared to routine state-of-the-art monoclonal protein (M-protein) diagnostics and next-generation sequencing (NGS-MRD) at 2 time points. RESULTS: In all 41 patients we were able to identify clonotypic M-protein-specific peptides and perform serum-based MS-MRD measurements. MS-MRD is significantly more sensitive to detect M-protein compared to either electrophoretic M-protein diagnostics or serum free light chain analysis. The concordance between NGS-MRD and MS-MRD status in 81 paired bone marrow/sera samples was 79%. The 50% progression-free survival (PFS) was identical (49 months) for patients who were either NGS-positive or MS-positive directly after maintenance treatment. The 50% PFS was 69 and 89 months for NGS-negative and MS-negative patients, respectively. The longest 50% PFS (96 months) was observed in patients who were MRD-negative for both methods. MS-MRD relapse during maintenance treatment was significantly correlated to poor PFS (P < 0.0001). CONCLUSIONS: Our data indicate proof-of-principle that MS-MRD evaluation in blood is a feasible, patient friendly alternative to NGS-MRD assessed on bone marrow. Clinical validation of the prognostic value of MS-MRD and its complementary value in MRD-evaluation of patients with MM is warranted in an independent larger cohort.

Integrating Serum Protein Electrophoresis with Mass Spectrometry, A New Workflow for M-Protein Detection and Quantification.

Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) are standard tools for multiple myeloma (MM) routine diagnostics. M-protein is a biomarker for MM that can be quantified with SPE and characterized with IFE. We have investigated combining SPE/IFE with targeted mass spectrometry (MS) to detect and quantify the M-protein. SPE-MS assay offers the possibility to detect M-protein with higher sensitivity than SPE/IFE, which could lead to better analysis of minimal residual disease in clinical laboratories. In addition, analysis of archived SPE gels could be used for retrospective MM studies. We have investigated two different approaches of measuring M-protein and therapeutic monoclonal antibodies (t-mAbs) from SPE/IFE gels. After extracting proteotypic peptides from the gel, they can be quantified using stable isotope labeled (SIL) peptides and measured by Orbitrap mass spectrometry. Alternatively, extracted peptides can be labeled with tandem mass tags (TMT). Both approaches are not hampered by the presence of t-mAbs. Using SIL peptides, limit of detection of the M-protein is approximately 100-fold better than with routine SPE/IFE. Using TMT labeling, M-protein can be compared in different samples from the same patient. We have successfully measured M-protein proteotypic peptides extracted from the SPE/IFE gels utilizing SIL peptides and TMT.

Development of a Targeted Mass-Spectrometry Serum Assay To Quantify M-Protein in the Presence of Therapeutic Monoclonal Antibodies.

M-protein diagnostics can be compromised for patients receiving therapeutic monoclonal antibodies as treatment in multiple myeloma. Conventional techniques are often not able to distinguish between M-proteins and therapeutic monoclonal antibodies administered to the patient. This may prevent correct response assessment and can lead to overtreatment. We have developed a serum-based targeted mass-spectrometry assay to detect M-proteins, even in the presence of three therapeutic monoclonal antibodies (daratumumab, ipilimumab, and nivolumab). This assay can target proteotypic M-protein peptides as well as unique peptides derived from therapeutic monoclonal antibodies. We address the sensitivity in M-protein diagnostics and show that our mass-spectrometry assay is more than two orders of magnitude more sensitive than conventional M-protein diagnostics. The use of stable isotope-labeled peptides allows absolute quantification of the M-protein and increases the potential of assay standardization across multiple laboratories. Finally, we discuss the position of mass-spectrometry assays in monitoring minimal residual disease in multiple myeloma, which is currently dominated by molecular techniques based on plasma cell assessment that requires invasive bone marrow aspirations or biopsies.

De Novo Sequencing of Peptides from High-Resolution Bottom-Up Tandem Mass Spectra using Top-Down Intended Methods.

Despite high-resolution mass spectrometers are becoming accessible for more and more laboratories, tandem (MS/MS) mass spectra are still often collected at a low resolution. And even if acquired at a high resolution, software tools used for their processing do not tend to benefit from that in full, and an ability to specify a relative mass tolerance in this case often remains the only feature the respective algorithms take advantage of. We argue that a more efficient way to analyze high-resolution MS/MS spectra should be with methods more explicitly accounting for the precision level, and sustain this claim through demonstrating that a de novo sequencing framework originally developed for (high-resolution) top-down MS/MS data is perfectly suitable for processing high-resolution bottom-up datasets, even though a top-down like deconvolution performed as the first step will leave in many spectra at most a few peaks.

Top-down analysis of protein samples by de novo sequencing techniques.

MOTIVATION: Recent technological advances have made high-resolution mass spectrometers affordable to many laboratories, thus boosting rapid development of top-down mass spectrometry, and implying a need in efficient methods for analyzing this kind of data. RESULTS: We describe a method for analysis of protein samples from top-down tandem mass spectrometry data, which capitalizes on de novo sequencing of fragments of the proteins present in the sample. Our algorithm takes as input a set of de novo amino acid strings derived from the given mass spectra using the recently proposed Twister approach, and combines them into aggregated strings endowed with offsets. The former typically constitute accurate sequence fragments of sufficiently well-represented proteins from the sample being analyzed, while the latter indicate their location in the protein sequence, and also bear information on post-translational modifications and fragmentation patterns. AVAILABILITY AND IMPLEMENTATION: Freely available on the web at http://bioinf.spbau.ru/en/twister CONTACT: vyatkina@spbau.ru or ppevzner@ucsd.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

De Novo Sequencing of Peptides from Top-Down Tandem Mass Spectra.

De novo sequencing of proteins and peptides is one of the most important problems in mass spectrometry-driven proteomics. A variety of methods have been developed to accomplish this task from a set of bottom-up tandem (MS/MS) mass spectra. However, a more recently emerged top-down technology, now gaining more and more popularity, opens new perspectives for protein analysis and characterization, implying a need for efficient algorithms to process this kind of MS/MS data. Here, we describe a method that allows for the retrieval, from a set of top-down MS/MS spectra, of long and accurate sequence fragments of the proteins contained in the sample. To this end, we outline a strategy for generating high-quality sequence tags from top-down spectra, and introduce the concept of a T-Bruijn graph by adapting to the case of tags the notion of an A-Bruijn graph widely used in genomics. The output of the proposed approach represents the set of amino acid strings spelled out by optimal paths in the connected components of a T-Bruijn graph. We illustrate its performance on top-down data sets acquired from carbonic anhydrase 2 (CAH2) and the Fab region of alemtuzumab.

Quantitative measurement of immunoglobulins and free light chains using mass spectrometry.

Serum free light chain (sFLC) assays are well established in the diagnosis and monitoring of plasma cell disorders. However, current FLC immunoassays are subject to several analytical issues, which results in a lack of harmonized results. To facilitate sFLC standardization, we investigated the strengths and limitations of mass spectrometry as a novel technological platform for sFLC quantification. Stable isotope labeled reference peptides are added to serum samples for quantitation by selected reaction monitoring (SRM). The use of redundant peptide sets allows for quality control measures during data analysis. Measurements on serum provide information on intact immunoglobulins, but depletion of these intact molecules from the sera during sample processing permits the quantitation of sFLC. sFLC concentrations measured with SRM were comparable to those obtained by nephelometry and showed excellent linearity (r(2) > 0.99). In samples with high levels of sFLC, SRM data was more consistent with serum protein electrophoresis than nephelometric data and SRM is unaffected by antigen excess. The lower limits of quantitation were 3.8 and 2.7 mg/L for κ and λ sFLC. Errors due to polymorphic sequences were prevented by comparison of redundant peptide pairs. The application of stable isotope labeling combined with SRM can overcome many of the current potential analytical issues of sFLC analysis. We describe which hurdles still need to be taken to make SRM a robust and more accurate method for sFLC measurements.

De novo protein sequencing by combining top-down and bottom-up tandem mass spectra.

There are two approaches for de novo protein sequencing: Edman degradation and mass spectrometry (MS). Existing MS-based methods characterize a novel protein by assembling tandem mass spectra of overlapping peptides generated from multiple proteolytic digestions of the protein. Because each tandem mass spectrum covers only a short peptide of the target protein, the key to high coverage protein sequencing is to find spectral pairs from overlapping peptides in order to assemble tandem mass spectra to long ones. However, overlapping regions of peptides may be too short to be confidently identified. High-resolution mass spectrometers have become accessible to many laboratories. These mass spectrometers are capable of analyzing molecules of large mass values, boosting the development of top-down MS. Top-down tandem mass spectra cover whole proteins. However, top-down tandem mass spectra, even combined, rarely provide full ion fragmentation coverage of a protein. We propose an algorithm, TBNovo, for de novo protein sequencing by combining top-down and bottom-up MS. In TBNovo, a top-down tandem mass spectrum is utilized as a scaffold, and bottom-up tandem mass spectra are aligned to the scaffold to increase sequence coverage. Experiments on data sets of two proteins showed that TBNovo achieved high sequence coverage and high sequence accuracy.

Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques.

The polyclonal repertoire of circulating antibodies potentially holds valuable information about an individual's humoral immune state. While bottom-up proteomics is well suited for serum proteomics, the vast number of antibodies and dynamic range of serum challenge this analysis. To acquire the serum proteome more comprehensively, we incorporated high-field asymmetric waveform ion-mobility spectrometry (FAIMS) or two-dimensional chromatography into standard trypsin-based bottom-up proteomics. Thereby, the number of variable region (VR)-related spectra increased 1.7-fold with FAIMS and 10-fold with chromatography fractionation. To match antibody VRs to spectra, we combined de novo searching and BLAST alignment. Validation of this approach showed that, as peptide length increased, the de novo accuracy decreased and BLAST performance increased. Through in silico calculations on antibody repository sequences, we determined the uniqueness of tryptic VR peptides and their suitability as antibody surrogate. Approximately one-third of these peptides were unique, and about one-third of all antibodies contained at least one unique peptide.

Mass spectrometry analyses of κ and λ fractions result in increased number of complementarity-determining region identifications.

Sera from lung cancer patients contain antibodies against tumor-associated antigens. Specific amino acid sequences of the complementarity-determining regions (CDRs) in the antigen-binding fragment (Fab) of these antibodies have potential as lung cancer biomarkers. Detection and identification of CDRs by mass spectrometry can significantly be improved by reduction of the complexity of the immunoglobulin molecule. Our aim was to molecular dissect IgG into κ and λ fragments to reduce the complexity and thereby identify substantially more CDRs than by just total Fab isolation. We purified Fab, Fab-κ, Fab-λ, κ and λ light chains from serum from 10 stage I lung adenocarcinoma patients and 10 matched controls from the current and former smokers. After purification, the immunoglobulin fragments were enzymatically digested and measured by high-resolution mass spectrometry. Finally, we compared the number of CDRs identified in these immunoglobulin fragments with that in the Fab fragments. Twice as many CDRs were identified when Fab-κ, Fab-λ, κ and λ (3330) were combined than in the Fab fraction (1663) alone. The number of CDRs and κ:λ ratio was statistically similar in both cases and controls. Molecular dissection of IgG identifies significantly more CDRs, which increases the likelihood of finding lung cancer-related CDR sequences.

Immune responses are characterized by specific shared immunoglobulin peptides that can be detected by proteomic techniques.

In the adaptive immune response, immunoglobulins develop that bind specifically to the antigens to which the organism was exposed. Immunoglobulins may bind to known or unknown antigens in a variety of diseases and have been used in the past to identify novel antigens for use as a biomarker. We propose that the immunoglobulins themselves could also be used as biomarkers in antibody-mediated disease. In this proteomic study, rats were immunized with one of two purified antigens, and immunoglobulins from pre- and postimmune sera were analyzed with nano-LC coupled mass spectrometry. It was found that the two treatment groups could be distinguished based on cluster analysis of the immunoglobulin peptides from the immune sera. In addition, we identified 684 specific peptides that were differentially present in one of the two treated groups. We could find an amino acid sequence for 44% of the features in the mass spectra by combining database-driven and de novo sequencing techniques. The latter were essential for sequence identification, as the more common database-driven approach suffers from a poor representation of immunoglobulins in the available databases. Our data show that the development of immunoglobulins during an immune response is not a fully random process, but that instead selection pressures exist that favor the best binding amino acid sequences, and that this selection is shared between different animals. This finding implies that immunoglobulin peptides could indeed be a powerful and easily accessible class of biomarkers.

Sequencing and quantifying IgG fragments and antigen-binding regions by mass spectrometry.

In cancer and autoimmune diseases, immunoglobulins with a specific molecular signature that could potentially be used as diagnostic or prognostic markers are released into body fluids. An immunomics approach based on this phenomenon relies on the ability to identify the specific amino acid sequences of the complementarity-determining regions (CDR) of these immunoglobulins, which in turn depends on the level of accuracy, resolution, and sensitivity that can be achieved by advanced mass spectrometry. Reproducible isolation and sequencing of antibody fragments (e.g., Fab) by high-resolution mass spectrometry (MS) from seven healthy donors revealed 43 217 MS signals: 225 could be associated with CDR1 peptides, 513 with CDR2 peptides, and 19 with CDR3 peptides. Seventeen percent of the 43 217 MS signals did not overlap between the seven donors. The Fab isolation method used is reproducible and fast, with a high yield. It provides only one Fab sample fraction for subsequent characterization by high-resolution MS. In 17% and 4% of these seven healthy donors, qualitative (presence/absence) and quantitative (intensity) differences in Fab fragments could be demonstrated, respectively. From these results, we conclude that the identification of a CDR signature as biomarker for autoimmune diseases and cancer without prior knowledge of the antigen is feasible.

Emergence of large-scale cell morphology and movement from local actin filament growth dynamics.

Variations in cell migration and morphology are consequences of changes in underlying cytoskeletal organization and dynamics. We investigated how these large-scale cellular events emerge as direct consequences of small-scale cytoskeletal molecular activities. Because the properties of the actin cytoskeleton can be modulated by actin-remodeling proteins, we quantitatively examined how one such family of proteins, enabled/vasodilator-stimulated phosphoprotein (Ena/VASP), affects the migration and morphology of epithelial fish keratocytes. Keratocytes generally migrate persistently while exhibiting a characteristic smooth-edged "canoe" shape, but may also exhibit less regular morphologies and less persistent movement. When we observed that the smooth-edged canoe keratocyte morphology correlated with enrichment of Ena/VASP at the leading edge, we mislocalized and overexpressed Ena/VASP proteins and found that this led to changes in the morphology and movement persistence of cells within a population. Thus, local changes in actin filament dynamics due to Ena/VASP activity directly caused changes in cell morphology, which is coupled to the motile behavior of keratocytes. We also characterized the range of natural cell-to-cell variation within a population by using measurable morphological and behavioral features--cell shape, leading-edge shape, filamentous actin (F-actin) distribution, cell speed, and directional persistence--that we have found to correlate with each other to describe a spectrum of coordinated phenotypes based on Ena/VASP enrichment at the leading edge. This spectrum stretched from smooth-edged, canoe-shaped keratocytes--which had VASP highly enriched at their leading edges and migrated fast with straight trajectories--to more irregular, rounder cells migrating slower with less directional persistence and low levels of VASP at their leading edges. We developed a mathematical model that accounts for these coordinated cell-shape and behavior phenotypes as large-scale consequences of kinetic contributions of VASP to actin filament growth and protection from capping at the leading edge. This work shows that the local effects of actin-remodeling proteins on cytoskeletal dynamics and organization can manifest as global modifications of the shape and behavior of migrating cells and that mathematical modeling can elucidate these large-scale cell behaviors from knowledge of detailed multiscale protein interactions.

Cerebrospinal fluid immunoglobulins are increased in neonates exposed to Zika virus during foetal life.

OBJECTIVE: To compare immunoglobulin levels in cerebrospinal fluid (CSF) of neonates exposed to Zika virus (ZIKV) during foetal life (cases) with levels in CSF of control neonates. METHODS: We identified 16 neonates who underwent lumbar puncture (LP), during the ZIKV epidemic (December/2015 to March/2016) whose mothers reported ZIKV clinical symptoms during gestation (cases). Congenital microcephaly was defined as head circumference ≤31.9 cm (boys) and ≤31.5 cm (girls) for term neonates, or ≤2 standard deviations below the mean for premature (<37 weeks) neonates. Subsequently, we identified neonates who underwent LP in the same lab and fulfilled criteria to be controls: age ≤4 days, CSF white blood cell count ≤8/mm3, CSF protein ≤132 mg/dL, CSF red blood cell count ≤1,000/mm3, neither central nervous system illness, nor congenital infection, nor microcephaly. CSF immunoglobulin concentrations were measured by mass spectrometry. RESULTS: 13 controls were included. IgM, IgA, IgG, IgK, and IgL were significantly higher among cases (p < 0.001). Eight (50%) ZIKV exposed infants had congenital microcephaly. These showed the strongest immunoglobulin elevation of the IgM and IgA classes. CONCLUSION: Neonates exposed to ZIKV infection during gestation present with elevated distinct immunoglobulins in CSF, both in cases that developed microcephaly and in cases that did not.