The rockefeller university

Whole-exome sequencing of 250 parent-offspring trios identifies an enrichment of rare damaging de novo mutations in individuals with cerebral palsy and implicates genetically mediated dysregulation of early neuronal connectivity in the etiology of this disorder. In addition to commonly associated environmental factors, genomic factors may cause cerebral palsy. We performed whole-exome sequencing of 250 parent-offspring trios, and observed enrichment of damaging de novo mutations in cerebral palsy cases. Eight genes had multiple damaging de novo mutations; of these, two (TUBA1AandCTNNB1) met genome-wide significance. We identified two novel monogenic etiologies,FBXO31andRHOB, and showed that theRHOBmutation enhances active-state Rho effector binding while theFBXO31mutation diminishes cyclin D levels. Candidate cerebral palsy risk genes overlapped with neurodevelopmental disorder genes. Network analyses identified enrichment of Rho GTPase, extracellular matrix, focal adhesion and cytoskeleton pathways. Cerebral palsy risk genes in enriched pathways were shown to regulate neuromotor function in aDrosophilareverse genetics screen. We estimate that 14% of cases could be attributed to an excess of damaging de novo or recessive variants. These findings provide evidence for genetically mediated dysregulation of early neuronal connectivity in cerebral palsy.

Interferon (IFN)-alpha has been used for several decades for the treatment of myelofibrosis, with conflicting results. In this systematic review and meta-analysis of 10 studies with 141 patients, we found that IFN led to hematologic improvements in 49% of patients. Background: Myelofibrosis (MF) is a Philadelphia chromosome-negative myeloproliferative neoplasm characterized by progressive bone marrow failure, increased risk of progression to acute myeloid leukemia, and constitutional symptoms. For over 3 decades, various formulations of interferon (IFN) have been used for the treatment of MF, with variable results, and the role of IFN in the treatment of MF is evolving. Patients and Methods: For this systematic review and meta-analysis, Medline and Embase via Ovid, Scopus, Cochrane Central Register of Controlled Trials (CENTRAL), and Web of Science were searched from inception through March 2019 for studies of pegylated IFN (pegIFN) and nonepeg-IFN in MF patients. The primary outcome of overall response rate was defined as a composite of complete response, partial response, complete hematologic response, and partial hematologic response. Randomeffects models were used to pool overall response rate, and metaregression analyses were performed to compare peg-IFN and non-epeg-IFN formulations. Results: Among the 10 studies with 141 MF patients included, the overall response rate was 49.9% (95% confidence interval [CI], 30.4-69.3), and there was no statistically significant difference (P=.99) between peg-IFN (50.0%; 95% CI, 26.2-73.9; I-2 = 76.9%) and non-peg-IFN (49.6%; 95% CI, 20.5-79.0; I-2 = 56.7%). Treatment discontinuation resulting from adverse events was common with non-peg-IFN at 35.8% (95% CI, 3.5-68.1) per year, and less in the one study on peg-IFN (0.5% per year). Conclusion: IFN can lead to hematologic improvements in a subset of MF patients, but study quality is limited and heterogenous. Biomarkers predicting response to IFN and formulations with improved tolerability are needed.

Antibody-dependent enhancement (ADE) has been described as a mechanism that contributes to the pathogenesis of dengue virus infection. Limited evidence also suggests that it can also occur in other viral infections. Here, the authors explore the history of the ADE phenomenon, discuss the diversity of Fc effector functions and consider its potential relevance in the context of SARS-CoV-2 infection. Antibody-dependent enhancement (ADE) is a mechanism by which the pathogenesis of certain viral infections is enhanced in the presence of sub-neutralizing or cross-reactive non-neutralizing antiviral antibodies. In vitro modelling of ADE has attributed enhanced pathogenesis to Fc gamma receptor (Fc gamma R)-mediated viral entry, rather than canonical viral receptor-mediated entry. However, the putative Fc gamma R-dependent mechanisms of ADE overlap with the role of these receptors in mediating antiviral protection in various viral infections, necessitating a detailed understanding of how this diverse family of receptors functions in protection and pathogenesis. Here, we discuss the diversity of immune responses mediated upon Fc gamma R engagement and review the available experimental evidence supporting the role of Fc gamma Rs in antiviral protection and pathogenesis through ADE. We explore Fc gamma R engagement in the context of a range of different viral infections, including dengue virus and SARS-CoV, and consider ADE in the context of the ongoing SARS-CoV-2 pandemic.

Metabolic reprogramming is a key feature of many cancers, but how and when it contributes to tumorigenesis remains unclear. Here we demonstrate that metabolic reprogramming induced by mitochondrial fusion can be rate-limiting for immortalization of tumor-initiating cells (TICs) and trigger their irreversible dedication to tumorigenesis. Using single-cell transcriptomics, we find that Drosophila brain tumors contain a rapidly dividing stem cell population defined by upregulation of oxidative phosphorylation (OxPhos). We combine targeted metabolomics and in vivo genetic screening to demonstrate that OxPhos is required for tumor cell immortalization but dispensable in neural stem cells (NSCs) giving rise to tumors. Employing an in vivo NADH/NAD+ sensor, we show that NSCs precisely increase OxPhos during immortalization. Blocking OxPhos or mitochondrial fusion stalls TICs in quiescence and prevents tumorigenesis through impaired NAD+ regeneration. Our work establishes a unique connection between cellular metabolism and immortalization of tumor-initiating cells.

Muscle cell plasma membrane is frequently damaged by mechanical activity, and its repair requires the membrane protein dysferlin. We previously identified that, similar to dysferlin deficit, lack of annexin A2 (AnxA2) also impairs repair of skeletal myofibers. Here, we have studied the mechanism of AnxA2-mediated muscle cell membrane repair in cultured muscle cells. We find that injury-triggered increase in cytosolic calcium causes AnxA2 to bind dysferlin and accumulate on dysferlin-containing vesicles as well as with dysferlin at the site of membrane injury. AnxA2 accumulates on the injured plasma membrane in cholesterol-rich lipid microdomains and requires Src kinase activity and the presence of cholesterol. Lack of AnxA2 and its failure to translocate to the plasma membrane, both prevent calcium-triggered dysferlin translocation to the plasma membrane and compromise repair of the injured plasma membrane. Our studies identify that Anx2 senses calcium increase and injury-triggered change in plasma membrane cholesterol to facilitate dysferlin delivery and repair of the injured plasma membrane.

Characterization of MHC-bound peptides by mass spectrometry (MS) is an essential technique for immunologic studies. Many efforts have been made to quantify the number of MHC-presented ligands by MS and to define the limits of detection of a specific MHC ligand. However, these experiments are often complex and comparisons across different laboratories are challenging. Therefore, we compared and orthogonally validated quantitation of peptide:MHC complexes by radio immunoassay and flow cytometry using TCR mimic antibodies in three model systems to establish a method to control the experimental input of peptide MHC:complexes for MS analysis. Following isolation of MHC-bound peptides we identified and quantified an MHC ligand of interest with high correlation to the initial input. We found that the diversity of the presented ligandome, as well as the peptide sequence itself affected the detection of the target peptide. Furthermore, results were applicable from these model systems to unmodified cell lines with a tight correlation between HLA-A*02 complex input and the number of identified HLA-A*02 ligands. Overall, this framework provides an easily accessible experimental setup that offers the opportunity to control the peptide:MHC input and in this way compare immunopeptidome experiments not only within but also between laboratories, independent of their experimental approach. Significance: Although immunopeptidomics is an essential tool for the characterization of MHCbound peptides on the cell surface, there are no easily applicable established protocols available that allow comparison of immunopeptidome experiments across laboratories. Here, we demonstrate that controlling the peptide:MHC input for immunopurification and LC-MS/MS experiments by flow cytometry in pre-defined model systems allows the generation of qualitative and quantitative data that can easily be compared between investigators, independently of their methods for MHC ligand isolation for MS.

We present an approach for preparing cryo-electron microscopy (cryo-EM) grids to study short-lived molecular states. Using piezoelectric dispensing, two independent streams of similar to 50-pl droplets of sample are deposited within 10 ms of each other onto the surface of a nanowire EM grid, and the mixing reaction stops when the grid is vitrified in liquid ethane similar to 100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.

The Van der Pol equation is a paradigmatic model of relaxation oscillations. This remarkable nonlinear phenomenon of self-sustained oscillatory motion underlies important rhythmic processes in nature and electrical engineering. Relaxation oscillations in a real system are usually coupled to environmental noise, which further enriches their dynamics, but makes theoretical analysis of such systems and determination of the equation parameter values a difficult task. In a companion paper we have proposed an analytic approach to a similar problem for another classical nonlinear model-the bistable Duffing oscillator. Here we extend our techniques to the case of the Van der Pol equation driven by white noise. We analyze the statistics of solutions and propose a method to estimate parameter values from the oscillator's time series. We use experimental data of active oscillations in a biophysical system to demonstrate how our method applies to real observations and can be generalized for more complex models.

Data on the efficacy and safety of interferon (IFN)-alpha for the treatment of essential thrombocythemia (ET) and polycythemia vera (PV) are inconsistent. We conducted a systematic review and meta-analysis and searched MEDLINE and EMBASE via Ovid, Scopus, COCHRANE registry of clinical trials, and Web of Science from inception through 03/2019 for studies of pegylated IFN (peg-IFN) and non-pegylated IFN (non-peg-IFN) in PV and ET patients. Random-effects models were used to pool response rates for the primary outcome of overall response rate (ORR) defined as a composite of complete response, partial response, complete hematologic response (CHR) and partial hematologic response. Peg-IFN and non-peg-IFN were compared by meta-regression analyses. In total, 44 studies with 1359 patients (730 ET, 629 PV) were included. ORR were 80.6% (95% confidence interval: 76.6-84.1%, CHR: 59.0% [51.5%-66.1%]) and 76.7% (67.4-84.0%; CHR: 48.5% [37.8-59.4%]) for ET and PV patients, respectively. In meta-regression analyses results did not differ significantly for non-peg-IFN vs. peg-IFN. Annualized rates of thromboembolic complications and treatment discontinuation due to adverse events were low at 1.2% and 8.8% for ET and 0.5% and 6.5% for PV patients, respectively. Both peg-IFN and non-peg-IFN can be effective and safe long-term treatments for ET and PV.

Symmetry breaking is essential for polarization of cells and generation of left-right body asymmetry. Here the authors investigate the arrangement of hair cells in zebrafish and show that mirror-symmetric patterns arise from a combination of biochemical and mechanical symmetry-breaking events. Actively regulated symmetry breaking, which is ubiquitous in biological cells, underlies phenomena such as directed cellular movement and morphological polarization. Here, we investigate how an organ-level polarity pattern emerges through symmetry breaking at the cellular level during the formation of a mechanosensory organ. Combining theory, genetic perturbations and in vivo imaging, we study the development and regeneration of the fluid-motion sensors in the zebrafish's lateral line. We find that two interacting symmetry-breaking events-one mediated by biochemical signalling and the other by cellular mechanics-give rise to precise rotations of cell pairs, which produce a mirror-symmetric polarity pattern in the receptor organ.