Biology subjects

Singer, A.

Publications and source records attributed to Singer, A..

5 recordsLinked to original sources

bioRxiv2018

Toward single particle reconstruction without particle picking: Breaking the detection limit

Single-particle cryo-electron microscopy (cryo-EM) has recently joined X-ray crystallography and NMR spectroscopy as a high-resolution structural method for biological macromolecules. In a cryo-EM experiment, the microscope produces images called micrographs. Projections of the molecule of interest are embedded in the micrographs at unknown locations, and under unknown viewing directions. Standard imaging techniques first locate these projections (detection) and then reconstruct the 3-D structure from them. Unfortunately, high noise levels hinder detection. When reliable detection is rendered impossible, the standard techniques fail. This is a problem especially for small molecules, which can be particularly hard to detect. In this paper, we propose a radically different approach: we contend that the structure could, in principle, be reconstructed directly from the micrographs, without intermediate detection. As a result, even small molecules should be within reach for cryo-EM. To support this claim, we setup a simplified mathematical model and demonstrate how our autocorrelation analysis technique allows to go directly from the micrographs to the sought signals. This involves only one pass over the micrographs, which is desirable for large experiments. We show numerical results and discuss challenges that lay ahead to turn this proof-of-concept into a competitive alternative to state-of-the-art algorithms.

molecular biology↗

bioRxiv2018

AAV-mediated progranulin delivery to a mouse model of progranulin deficiency causes T cell-mediated hippocampal degeneration

Adeno-associated virus (AAV)-mediated gene replacement is emerging as a safe and effective means of correcting single-gene mutations, and use of AAV vectors for treatment of diseases of the CNS is increasing. AAV-mediated progranulin gene (GRN) delivery has been proposed as a treatment for GRN-deficient frontotemporal dementia (FTD) and neuronal ceroid lipofuscinosis (NCL), and two recent studies using focal intraparenchymal AAV-Grn delivery to brain have shown moderate success in histopathologic and behavioral rescue in mouse FTD models. Here, we used AAV9 to deliver GRN to the lateral ventricle to achieve widespread expression in the Grn null mouse brain. We found that despite a global increase in progranulin throughout many brain regions, overexpression of GRN resulted in dramatic and selective hippocampal toxicity and degeneration affecting both neurons and glia. Histologically, hippocampal degeneration was preceded by T cell infiltration and perivascular cuffing, suggesting an inflammatory component to the ensuing neuronal loss. GRN delivery with an ependymal-targeting AAV for selective secretion of progranulin into the cerebrospinal fluid (CSF) similarly resulted in T cell infiltration as well as ependymal hypertrophy. Interestingly, overexpression of GRN in wild-type animals also provoked T cell infiltration. These results call into question the safety of GRN overexpression in the CNS, with evidence for both a region-selective immune response and cellular proliferative response following GRN gene delivery. Our results highlight the importance of careful consideration of target gene biology and cellular response to overexpression in relevant animal models prior to progressing to the clinic.\n\nSignificance StatementGene therapies using adeno-associated viral (AAV) vectors show great promise for many human diseases, including diseases that affect the central nervous system (CNS). Frontotemporal dementia (FTD) and neuronal ceroid lipofuscinosis (NCL) are neurodegenerative diseases resulting from loss of one or both copies of the gene encoding progranulin (GRN), and gene replacement has been proposed for these currently untreatable disorders. Here, we used two different AAV vectors to induce widespread brain GRN expression in mice lacking the gene, as well as in wild-type mice. Unexpectedly, GRN overexpression resulted in T cell infiltration, followed by marked hippocampal neurodegeneration. Our results call into question the safety of GRN overexpression in the CNS, with wider implications for development of CNS gene therapies.

neuroscience↗

bioRxiv2018

BRD4 Deficiency Selectively Affects a Unique Developmental Subpopulation in Thymocytes

The bromodomain protein BRD4 is a driver in both inflammatory diseases and cancers. It has multiple functions, contributing to chromatin structure and transcription through its intrinsic HAT and kinase activities. Despite the wide-ranging characterization of BRD4, little is known about its in vivo function. In the present study, we have examined the role of BRD4 in T cell development by conditional deletion at various stages of thymocyte differentiation. We found that BRD4 is critical for normal T cell development. Surprisingly, BRD4 selectively regulates the progression of immature CD8 single positive (ISP) thymocytes into quiescent DP thymocytes. In striking contrast, BRD4 deletion does not affect the extensive proliferation associated with the differentiation of double negative (DN) into ISP cells. Nor does it affect the maturation of double positive (DP) into conventional CD4+ and CD8+ thymocytes. These studies lead to the unexpected conclusion that BRD4 selectively regulates preselection ISP thymocytes.\n\nOn-line SummaryImmature CD8 single-positive (ISP) thymocytes are identified as a molecularly-distinct thymocyte subpopulation, dependent on BRD4 for progression to the DP stage. DN and DP are BRD4-independent. These findings provide new insights into BRD4, a therapeutic target in inflammation and cancer.

immunology↗

bioRxiv2017

Characterization and Validation of a Novel Group of Type V, Class 2 Nucleases for in vivo Genome Editing

CRISPR-based genome editing is an enabling technology with potential to dramatically transform multiple industries. Identification of additional editing tools will be imperative for broad adoption and application of this technology. A novel Type V, Class 2 CRISPR nuclease system was identified from Microgenomates and Smithella bacterial species (CRISPR from Microgenomates and Smithella, Cms1). This system was shown to efficiently generate indel mutations in the major crop plant rice (Oryza sativa). Cms1 are distinct from other Type V nucleases, are smaller than most other CRISPR nucleases, do not require a tracrRNA, and have an AT-rich protospacer-adjacent motif site requirement. A total of four novel Cms1 nucleases across multiple bacterial species were shown to be functional in a eukaryotic system. This is a major expansion of the Type V CRISPR effector protein toolbox and increases the diversity of options available to researchers.

molecular biology↗

bioRxiv2017

Synthesizing developmental trajectories

Dynamical processes in biology are studied using an ever-increasing number of techniques, each of which brings out unique features of the system. One of the current challenges is to develop systematic approaches for fusing heterogeneous datasets into an integrated view of multivariable dynamics. We demonstrate that heterogeneous data fusion can be successfully implemented within a semi-supervised learning framework that exploits the intrinsic geometry of high-dimensional datasets. We illustrate our approach using a dataset from studies of pattern formation in Drosophila. The result is a continuous trajectory that reveals the joint dynamics of gene expression, subcellular protein localization, protein phosphorylation, and tissue morphogenesis. Our approach can be readily adapted to other imaging modalities and forms a starting point for further steps of data analytics and modeling of biological dynamics.

developmental biology↗