Biology subjects

Iyer, S.

Publications and source records attributed to Iyer, S..

63 records · Page 4Linked to original sources

bioRxiv2019

Efficient Homology-directed Repair with Circular ssDNA Donors

While genome editing has been revolutionized by the advent of CRISPR-based nucleases, difficulties in achieving efficient, nuclease-mediated, homology-directed repair (HDR) still limit many applications. Commonly used DNA donors such as plasmids suffer from low HDR efficiencies in many cell types, as well as integration at unintended sites. In contrast, single-stranded DNA (ssDNA) donors can produce efficient HDR with minimal off-target integration. Here, we describe the use of ssDNA phage to efficiently and inexpensively produce long circular ssDNA (cssDNA) donors. These cssDNA donors serve as efficient HDR templates when used with Cas9 or Cas12a, with integration frequencies superior to linear ssDNA (lssDNA) donors. To evaluate the relative efficiencies of imprecise and precise repair for a suite of different Cas9 or Cas12a nucleases, we have developed a modified Traffic Light Reporter (TLR) system [TLR-Multi-Cas Variant 1 (MCV1)] that permits side-by-side comparisons of different nuclease systems. We used this system to assess editing and HDR efficiencies of different nuclease platforms with distinct DNA donor types. We then extended the analysis of DNA donor types to evaluate efficiencies of fluorescent tag knock-ins at endogenous sites in HEK293T and K562 cells. Our results show that cssDNA templates produce efficient and robust insertion of reporter tags. Targeting efficiency is high, allowing production of biallelic integrants using cssDNA donors. cssDNA donors also outcompete lssDNA donors in template-driven repair at the target site. These data demonstrate that circular donors provide an efficient, cost-effective method to achieve knock-ins in mammalian cell lines.

molecular biology↗

bioRxiv2019

The macrophage-expressed gene (mpeg) 1 identifies a subpopulation of B cells in the adult zebrafish

The mononuclear phagocytic system (MPS) consists of many cells, in particular macrophages, scattered throughout the body. However, there is increasing evidence for the heterogeneity of tissue-resident macrophages, leading to a pressing need for new tools to discriminate MPS subsets from other hematopoietic lineages. Mpeg1.1 is an evolutionary conserved gene encoding perforin-2, a pore-forming protein associated with host defense against pathogens. Zebrafish mpeg1.1:GFP and mpeg1.1:mCherry reporters were originally established to specifically label macrophages. Since, more than 100 peer-reviewed publications have made use of mpeg1.1-driven transgenics for in vivo studies, providing new insights into key aspects of macrophage ontogeny, activation and function. However, while the macrophage-specific expression pattern of the mpeg1.1 promoter has been firmly established in the zebrafish embryo, it is currently not known whether this specificity is maintained through adulthood. Here we report direct evidence that beside macrophages, a subpopulation of B-lymphocytes is marked by mpeg1.1 reporters in most adult zebrafish organs. These mpeg1.1+ lymphoid cells endogenously express mpeg1.1 and can be separated from mpeg1.1+ macrophages by virtue of their light-scatter characteristics using FACS. Remarkably, our analyses also revealed that B-lymphocytes, rather than mononuclear phagocytes, constitute the main mpeg1.1-positive population in irf8null myeloid-defective mutants, which were previously reported to recover tissue-resident macrophages in adulthood. One notable exception are skin macrophages, whose development and maintenance appear to be independent from irf8, similar to mammals. Collectively, our findings demonstrate that irf8 functions in myelopoiesis are evolutionary conserved and highlight the need for alternative macrophage-specific markers to study the MPS in adult zebrafish. SUMMARY SENTENCEMpeg1 is not a restricted macrophage marker, but also labels B cells in the adult zebrafish. Therefore, previously identified irf8-independent macrophages likely consist of B lymphocytes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/836098v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@128a738org.highwire.dtl.DTLVardef@1fa6097org.highwire.dtl.DTLVardef@db61b2org.highwire.dtl.DTLVardef@eb2e2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2019

Studying cellular functions in bipolar disorder: Are there specific predictors of lithium response?

BackgroundLithium is the first-line mood stabilizer for the treatment of bipolar disorder (BD). In order to interrogate cellular phenotypes related to disease and lithium treatment response, this study used neural precursor cells (NPCs) and lymphoblastoid cell lines (LCLs) from BD patients who are well characterized for clinical lithium response.\n\nMethodsBD patients diagnosed according to the DSM-IV criteria; were recruited from the outpatient services of the National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India. Clinical lithium response was assessed using the \"Alda scale\" and \"NIMH Retrospective Life chart method\". The controls were ethnically matched healthy subjects with no family history of neuropsychiatric illness. NPCs from two BD patients from the same family who clearly differed in their clinical response to lithium were chosen, and compared with healthy population controls. Whole transcriptome sequencing (RNA-Seq) and analysis were performed, with and without in vitro lithium (1mM for 7 days). In addition, mitochondrial membrane potential (MMP), cell viability and cell proliferation parameters were examined. Experiments were also performed in 25 LCLs from BD patients (16 lithium responders and 9 lithium non-responders), and 12 healthy control LCLs, to evaluate them in a system amenable to clinical translation.\n\nResultsRNA-Sequencing and analysis did not reveal differences in NPCs on in vitro lithium treatment. MMP was lower in BD, both in NPCs and LCLs; reversal with in vitro lithium happened only in LCLs and was unrelated to lithium response. Cell proliferation was higher in BD compared to controls, and there was no change on lithium addition. Cell viability assays indicated greater cell death in BD; which could only be rescued in LCLs of clinical lithium responders. The latter finding was associated with enhanced BCL2 and GSK3B expression with in vitro lithium.\n\nDiscussionOverall, our study findings indicate that there are cellular phenotypes related to the disease (mitochondrial potential, cell proliferation) in NPCs and LCLs. We also observed clinical lithium response related phenotypes (cell viability, BCL2/ GSK3B expression) in LCLs. The next step would be to evaluate a larger set of PBMCs from clinical lithium response groups of BD to derive cellular phenotypes related to direct clinical application.

neuroscience↗

bioRxiv2019

CRISPR adenine and cytosine base editors with reduced RNA off-target activities

CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively1-4. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies5. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing5. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described \"enhanced\" A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.

molecular biology↗

bioRxiv2019

Repurposing the aldose reductase inhibitor and diabetic neuropathy drug epalrestat for the congenital disorder of glycosylation PMM2-CDG

Phosphomannomutase 2 deficiency, or PMM2-CDG, is the most common congenital disorder of glycosylation affecting over 1,000 patients globally. There are no approved drugs that treat the symptoms or root cause of PMM2-CDG. In order to identify clinically actionable compounds that boost human PMM2 enzyme function, we performed a multi-species drug repurposing screen using a first-ever worm model of PMM2-CDG followed by PMM2 enzyme functional studies in PMM2-CDG patient fibroblasts. Drug repurposing candidates from this study, and drug repurposing candidates from a previously published study using yeast models of PMM2-CDG, were tested for their effect on human PMM2 enzyme activity in PMM2-CDG fibroblasts. Of the 20 repurposing candidates discovered in the worm-based phenotypic screen, 12 are plant-based polyphenols. Insights from structure-activity relationships revealed epalrestat, the only antidiabetic aldose reductase inhibitor approved for use in humans, as a first-in-class PMM2 enzyme activator. Epalrestat increased PMM2 enzymatic activity in four PMM2-CDG patient fibroblast lines with genotypes R141H/F119L, R141H/E139K, R141H/N216I and R141H/F183S. PMM2 enzyme activity gains range from 30% to 400% over baseline depending on genotype. Pharmacological inhibition of aldose reductase by epalrestat may shunt glucose from the polyol pathway to glucose-1,6-bisphosphate, which is an endogenous stabilizer and coactivator of PMM2 homodimerization. Epalrestat is a safe, oral and brain penetrant drug that was approved 27 years ago in Japan to treat diabetic neuropathy in geriatric populations. We demonstrate that epalrestat is the first small molecule activator of PMM2 enzyme activity with the potential to treat peripheral neuropathy and correct the underlying enzyme deficiency in a majority of pediatric and adult PMM2-CDG patients.

pharmacology and toxicology↗

bioRxiv2019

Drug screens of NGLY1 Deficiency worm and fly models reveal catecholamine, NRF2 and anti-inflammatory pathway activation as clinical approaches

N-glycanase 1/NGLY1 Deficiency is an ultra-rare and complex monogenic glycosylation disorder that affects fewer than 40 patients globally. NGLY1 Deficiency has been studied in model organisms such as yeast, worms, flies and mice. Proteasomal and mitochondrial homeostasis gene networks are controlled by the evolutionarily conserved transcriptional regulator Nrf1, whose activity requires deglycosylation by NGLY1. Hypersensitivity to the proteasome inhibitor bortezomib is a common phenotype observed in whole animal and cellular models of NGLY1 Deficiency. Here we describe unbiased phenotypic drug screens to identify FDA approved drugs, generally recognized as safe natural products and novel chemical entities that rescue growth and development of NGLY1-deficient worm and fly larvae treated with a toxic dose of bortezomib. We used image-based larval size and number assays for use in screens of a 2,560-member drug repurposing library and a 20,240-member lead discovery library. A total of 91 validated hit compounds from primary invertebrate screens were tested in a human cell line in a NRF2 activity assay. NRF2 is a transcriptional regulator that regulates cellular redox homeostasis and it can compensate for loss of Nrf1. Plant-based polyphenols comprise the largest class of hit compounds and NRF2 inducers. Catecholamines and catecholamine receptor activators comprise the second largest class of hits. Steroidal and non-steroidal anti-inflammatory drugs comprise the third largest class. Only one compound was active in all assays and species: the atypical antipsychotic and dopamine receptor agonist aripiprazole. Worm and fly models of NGLY1 Deficiency validate therapeutic rationales for activation of NRF2 and anti-inflammatory pathways based on results in mice and human cell models and suggest a novel therapeutic rationale for boosting catecholamine levels and/or signaling in the brain.

pharmacology and toxicology↗

bioRxiv2018

Molecularly distinct models of zebrafish Myc-induced B cell leukemia

Zebrafish models of T cell acute lymphoblastic leukemia (T-ALL) have been studied for over a decade, but curiously, robust zebrafish B cell ALL (B-ALL) models had not been described. Recently, our laboratories reported two seemingly closely-related models of zebrafish B-ALL. In these genetic lines, the primary difference is expression of either murine or human transgenic c-MYC, each controlled by the zebrafish rag2 promoter. Here, we compare ALL gene expression in both models. Surprisingly, we find that B-ALL arise in different B cell lineages, with ighm+ vs. ighz+ B-ALL driven by murine Myc vs. human MYC, respectively. Moreover, these B-ALL types exhibit signatures of distinct molecular pathways, further unexpected dissimilarity. Thus, despite sharing analogous genetic makeup, the ALL types in each model are markedly different, proving subtle genetic changes can profoundly impact model organism phenotypes. Investigating the mechanistic differences between mouse and human c-MYC in these contexts may reveal key functional aspects governing MYC-driven oncogenesis in human malignancies.

cancer biology↗

bioRxiv2018

Reversible temporally-specific inhibition of muscle using a light-activated chloride channel

Achieving reversible, temporally specific inhibition of motor neurons has the potential to revolutionize treatment of disorders marked by muscle hyperactivity. Current treatment strategies are inadequate - surgical interventions are irreversible and pharmaceutical interventions have off-target effects. Optogenetic strategies for inhibiting muscle activity have theoretical promise; however, trafficking and expression problems have prevented translatable optogenetic suppression of muscle activity. Here, we exploit recent innovations in opsin engineering to demonstrate virally mediated, temporally specific optogenetic inhibition of motor neurons and muscle activity in vivo. We show that intra-muscular injection of adeno-associated virus serotype 6 can drive expression of the inhibitory channelrhodopsin mutant iC++ in both immunodeficient and wild-type mice. Illumination of the sciatic nerve in wild-type mice resulted in 64.8% inhibition of evoked twitch force. Optical excitation during tetanic stimulation in wild-type mice resulted in 59.1% inhibition at 10 Hz and 55.4% at 25 Hz. The extent of optogenetic inhibition was titratable, and ranged from 0% to 78.4% as illumination intensity was changed from 1 mW to 20 mW/mm2. These results could have therapeutic applicability to disorders such as spasticity, hypertonia and urinary incontinence, and provide a new tool to neuroscientists and muscle physiologists wishing to reversibly inhibit motor neuron activity in vivo.

neuroscience↗

bioRxiv2016

Immunological Patterns from Four Melioidosis Cases: Constant and Variable Protein Antigens

Burkholderia pseudomallei is the causative agent of the melioidosis and is endemic to Southeast Asia and northern Australia. There is no available vaccine and accurate diagnosis is difficult, time-consuming and labor intensive. Early diagnosis is an important part of successful treatment and current serological tests are inadequate and based upon multiple antigens. Identifying specific immunogenic proteins which are highly seroreactive may yield potential diagnostic targets for detecting antibodies and antigens specific to melioidosis. We have used 2D gel electrophoresis and Western blotting analysis to analyze protein antigenicity of whole cell lysates extracted from four B. pseudomallei strains and the sera from the specific infected humans. We found a total of 135 immunogenic proteins, 62 of which we were able to identify to a specific gene by mass-spectrometry. Results from the Western blotting of each strains proteins and the corresponding patient serum reveal between 30 - 40% serum x strain specific immunogenic proteins. In most cases, these differences exist despite the fact that the genes encoding these proteins were present among all four B. pseudomallei strains. Eight particular proteins were immunogenic in all four strain x serum combinations and could represent novel diagnostic and vaccine subunit targets.

immunology↗