Biology subjects

Ganesan, S.

Publications and source records attributed to Ganesan, S..

50 records · Page 3Linked to original sources

bioRxiv2021

Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution

Actively maintained close appositions, or contact sites, between organelle membranes, enable the efficient transfer of biomolecules between the various cellular compartments. Several such sites have been described together with their tethering machinery. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize the proteome of contact sites and support the discovery of new tethers and functional molecules, we established a high throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, two of which have never been studied before, on the background of 1165 strains expressing a mCherry-tagged yeast protein that have a cellular punctate distribution (a hallmark of contact sites). By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified one set of hits as previously unrecognized homologs to Vps13 and Atg2. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell.

cell biology↗

bioRxiv2021

Tissue-wide coordination of calcium signaling regulates the epithelial stem cell pool during homeostasis

Skin epidermal homeostasis is maintained via constant regeneration by stem cells, which must communicate to balance their self-renewal and differentiation. A key molecular pathway, Ca2+ signaling has been implicated as a signal integrator in developing and wounded epithelial tissues[1, 2, 3, 4]. Yet how stem cells carry out this signaling across a regenerative tissue remains unknown due to significant challenges in studying signaling dynamics in live mice, limiting our understanding of the mechanisms of stem cell communication during homeostasis. To interpret high dimensional signals that have complex spatial and temporal patterns, we combined optimized imaging of Ca2+ signaling in thousands of epidermal stem cells in living mice with a new machine learning tool, Geometric Scattering Trajectory Homology (GSTH). Using a combination of signal processing, data geometry, and topology, GSTH captures patterns of signaling at multiple scales, either between direct or distant stem cell neighbors. Here we show that epidermal stem cells display dynamic intercellular Ca2+ signaling among neighborhoods of up to 10 cells that is surprisingly coordinated and directed through time across a pool of thousands of stem cells. We find that this collective coordination is an emergent property of the stem cell compartment, distinct from excitatory quiescent neuronal tissues. We demonstrate that cycling stem cells, specifically G2 cells, govern homeostatic patterns of Ca2+ signaling. Stem cells in different cell cycle stages dynamically regulate localization of the gap junction component Connexin43 (Cx43). Lastly, we uncouple global from local communication and identify Cx43 as the molecular mediator necessary for connectivity between local signaling neighborhoods. This work provides resolution in how stem cells at different stages of the cell cycle communicate and how that diversity of phases is essential for tissue wide communication and signaling flow during epidermal regeneration. Our approach provides a framework to investigate stem cell populations and their signaling dynamics, previously not possible.

cell biology↗

bioRxiv2021

Germline testing data validate inferences of mutational status for variants detected from tumor-only sequencing

Structured AbstractO_ST_ABSBackgroundC_ST_ABSPathogenic germline variants (PGV) in cancer susceptibility genes are usually identified in cancer patients through germline testing of DNA from blood or saliva: their detection can impact patient treatment options and potential risk reduction strategies for relatives. PGV can also be identified, in tumor sequencing assays, often performed without matched normal specimens. It is then critical to determine whether detected variants are somatic or germline. Here, we evaluate the clinical utility of computational inference of mutational status in tumor-only sequencing compared to germline testing results. Patients and MethodsTumor-only sequencing data from 1,608 patients were retrospectively analyzed to infer germline-versus-somatic status of variants using an information-theoretic, gene-independent approach. Loss of heterozygosity (LOH) was also determined. The predicted mutational models were compared to clinical germline testing results. Statistical measures were computed to evaluate performance. ResultsTumor-only sequencing detected 3,988 variants across 70 cancer susceptibility genes for which germline testing data were available. Our analysis imputed germline-versus-somatic status for >75% of all detected variants, with a sensitivity of 65%, specificity of 88%, and overall accuracy of 86% for pathogenic variants. False omission rate was 3%, signifying minimal error in misclassifying true PGV. A higher portion of PGV in known hereditary tumor suppressors were found to be retained with LOH in the tumor specimens (72%) compared to variants of uncertain significance (58%). ConclusionsTumor-only sequencing provides sufficient power to distinguish germline and somatic variants and infer LOH. Although accurate detection of PGV from tumor-only data is possible, analyzing sequencing data in the context of specimens tumor cell content allows systematic exclusion of somatic variants, and suggests a balance between type 1 and 2 errors for identification of patients with candidate PGV for standard germline testing. Our approach, implemented in a user-friendly bioinformatics application, facilities objective analysis of tumor-only data in clinical settings. HighlightsO_LIMost pathogenic germline variants in cancer predisposition genes can be identified by analyzing tumor-only sequencing data. C_LIO_LIInformation-theoretic gene-independent analysis of common sequencing data accurately infers germline vs. somatic status. C_LIO_LIA reasonable statistical balance can be established between sensitivity and specificity demonstrating clinical utility. C_LIO_LIPathogenic germline variants are more often detected with loss of heterozygosity vs. germline variants of uncertain significance. C_LI

bioinformatics↗

bioRxiv2021

Gradual differentiation uncoupled from cell cycle exit generates heterogeneity in the epidermal stem cell layer.

High turnover tissues continually lose specialized cells that are replaced by stem cell activity. In the adult mammalian epidermis, it is unclear how molecularly heterogenous stem/progenitor cell populations fit into the complete trajectory of epidermal differentiation. We show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes. Differentiation-committed cells remain capable of dividing to produce daughter cells fated to further differentiate, demonstrating that differentiation is uncoupled from cell cycle exit. These cell divisions are not required as part of an obligate transit amplifying program but instead protect density in the stem cell layer. Thus, instead of distinct contributions from multiple progenitors, a continuous gradual differentiation process fuels homeostatic epidermal turnover. One sentence summaryHeterogeneity in the epidermal stem cell layer reflects a gradual differentiation program that is uncoupled from the loss of proliferative capacity.

cell biology↗

bioRxiv2020

Nanodroplet-Benzalkonium Chloride Formulation Demonstrates In Vitro and Ex Vivo Broad-Spectrum Antiviral Activity Against SARS-CoV-2 and Other Enveloped Viruses

The Covid-19 pandemic has highlighted the importance of aerosolized droplets inhaled into the nose in the transmission of respiratory viral disease. Inactivating pathogenic viruses at the nasal portal of entry may reduce viral loads, thereby reducing transmission and contagion. We have developed an oil-in-water nanoemulsion (nanodroplet) formulation containing the potent antiseptic 0.13% Benzalkonium Chloride (NE-BZK) which demonstrates safe and broad anti-viral activity. While The Centers for Disease Control and Prevention (CDC) have reported that BZK may have less reliable activity than ethyl alcohol against certain viruses, including coronaviruses, we have demonstrated that NE-BZK exhibits broad-spectrum, long-lasting antiviral activity with >99.99% in vitro killing of enveloped viruses including SARS-CoV-2, human coronavirus, RSV and influenza B. Furthermore, in vitro studies demonstrated that NE-BZK continues to kill >99.99% of human coronavirus even when diluted 20-fold, while 0.13% aqueous BZK solution (AQ-BZK) did not. Ex vivo studies of NE-BZK on human cadaver skin demonstrated persistent >99.99% killing of human coronavirus for at least 8 hours after application. AQ-BZK failed to demonstrate durable antimicrobial activity on skin over time. The repeated application of NE-BZK, twice daily for 2 weeks on to rabbit nostrils indicated safety with no irritation. These findings demonstrate that formulating BZK on the surface of proprietary nanodroplets offers a safe and effective antiviral as a significant addition to strategies to combat the spread of respiratory viral infectious diseases.

microbiology↗

bioRxiv2020

Cerebellar Activation Bidirectionally Regulates Nucleus Accumbens Shell and Core

Although the cerebellum is now recognized as part of a long-range brain network that serves limbic functions and motivated behavior, knowledge of cerebello-limbic connectivity is limited, and nothing is known about how the cerebellum connects functionally to the nucleus accumbens (NAc). Here, we report that stimulation of cerebellar nuclei in mice of both sexes modulates spiking activity in both NAc core and medial shell with fast excitation and slower, less synchronized inhibition. Fast responses would be well poised to support rapid communication of information critical to the control of motivated behavior, whereas slower responses may be suggestive of a regulatory function, such as gain control. Tracing experiments to chart cerebellar nuclei-NAc pathways identified disynaptic pathways that recruit the ventral tegmental area (VTA) and intralaminar thalamus (Centromedial and Parafascicular nuclei) as intermediary nodes. Optogenetic activation of cerebellar axons in each of these nodes was sufficient to evoke responses in both NAc core and medial shell, albeit with distinct, node-dependent properties. These pathways and the functional connectivity they support could underlie the role of the cerebellum in motivated behavior.

neuroscience↗

bioRxiv2020

Arsenic trioxide resistance in acute promyelocytic leukemia: More to it than PML mutations

Acquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain the majority of disease recurrence seen in the clinic. We have generated a stable ATO resistant promyelocytic cell from a ATO sensitive NB4 cell line. We also noted that another ATRA resistant cell line (UF1) was cross resistant to ATO. We have characterized these resistant cell lines and observed that they significantly differed in their immunophenotype, drug transporter expression, drug resistance mutation profile and were also cross-resistant to other conventional chemotherapeutic agents. The NB4 derived resistant cell line had the classical A216V PML-B2 domain mutation while the UF1 cell line did not. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in the resistant cell lines. Glycolytic inhibition by 2-DG was efficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell lines and was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO resistant cell lines. The survival of the resistant cell lines was significantly affected by compounds targeting the mitochondrial respiration irrespective of the existence of ATO resistance-conferring genetic mutations. Our data demonstrate the addition of mitocans can overcome ATO resistance. We further demonstrated that the combination of ATO and mitocans has the potential in the treatment of non-M3 AML and the translation of this approach in the clinic needs to be explored further. Key pointsO_LIMetabolic rewiring promotes ATO resistance, which can be overcome by targeting mitochondrial oxidative phosphorylation. C_LIO_LICombination of ATO and mitocans can be exploited as a potential therapeutic option for relapsed APL and in non-M3 AML patients. C_LI

cancer biology↗

bioRxiv2020

Novel function acquired by the Culex quinquefasciatus mosquito D7 salivary protein enhances blood feeding on mammals

Adult female mosquitoes require a vertebrate blood meal to develop eggs and continue their life cycle. During blood feeding, mosquito saliva is injected at the bite site to facilitate blood meal acquisition through anti-hemostatic compounds that counteract blood clotting, platelet aggregation, vasoconstriction and host immune responses. D7 proteins are among the most abundant components of the salivary glands of several blood feeding insects. They are members of a family of proteins that have evolved through gene duplication events to encode D7 proteins of several lengths. Here, we examine the ligand binding specificity and physiological relevance of two D7 long proteins, CxD7L1 and CxD7L2, from Culex quinquefasciatus mosquitoes, the vector of medical and veterinary diseases such as filariasis, avian malaria, and West Nile virus infections. CxD7L1 and CxD7L2 were assayed by microcalorimetry for binding of potential host ligands involved in hemostasis, including bioactive lipids, biogenic amines, and nucleotides/nucleosides. CxD7L2 binds serotonin, histamine, and epinephrine with high affinity as well as the thromboxane A2 analog U-46619 and several cysteinyl leukotrienes, as previously described for other D7 proteins. CxD7L1 does not bind any of the ligands that are bound by CxD7L2. Unexpectedly, CxD7L1 exhibited high affinity for adenine nucleotides and nucleosides, a binding capacity not reported in any D7 family member. We solved the crystal structure of CxD7L1 in complex with bound ADP to 1.97 [A] resolution. The binding pocket for ADP is located between the two domains of CxD7L1, whereas all known D7s bind ligands either within the N-terminal or the C-terminal domains. We demonstrated that these two CxD7 long proteins inhibit human platelet aggregation in ex vivo experiments. CxD7L1 and CxD7L2 help blood feeding in mosquitoes by scavenging host molecules that promote vasoconstriction, platelet aggregation, itch, and pain at the bite site. The novel ADP-binding function acquired by CxD7L1 evolved to enhance blood feeding in mammals where ADP plays a key role in platelet aggregation.

biochemistry↗

bioRxiv2019

IRAK1-mediated coincidence detection of microbial signals licenses inflammasome activation

The innate immune system signals through various higher order signaling complexes called supramolecular organizing centers (SMOCs), which typically organize components of a single pathway. While innate immune signaling pathways have been largely characterized using single receptor stimuli, responses to pathogens require the coordinated engagement of multiple pathways. Here, we report an IRAK1-containing SMOC formed specifically when multiple receptors are activated, which recruits select components of the TLR, MAPK and inflammasome pathways. This allows for signal flux redistribution from TLRs to inflammasomes and facilitates inflammasome licensing through an MKK7-JNK axis, which is defective in Irak1-/- mice. Furthermore, this defect in Irak1-/- mice manifests in increased susceptibility to inflammasome-sensitive pathogens and diminished IL1 production from inflammasomes after co-TLR priming. Thus, IRAK1 SMOCs form a multi-pathway coordinating hub for coincidence detection of microbial signals, which may be employed by innate immune cells as a threat assessment and thresholding mechanism for inflammasome activation.

immunology↗

bioRxiv2019

Accelerating iterative deconvolution and multiview fusion by orders of magnitude

We describe theoretical and practical advances in algorithm and software design, resulting in ten to several thousand-fold faster deconvolution and multiview fusion than previous methods. First, we adapt methods from medical imaging, showing that an unmatched back projector accelerates Richardson-Lucy deconvolution by at least 10-fold, in most cases requiring only a single iteration. Second, we show that improvements in 3D image-based registration with GPU processing result in speedups of 10-100-fold over CPU processing. Third, we show that deep learning can provide further accelerations, particularly for deconvolution with a spatially varying point spread function. We illustrate the power of our methods from the subcellular to millimeter spatial scale, on diverse samples including single cells, nematode and zebrafish embryos, and cleared mouse tissue. Finally, we show that our methods facilitate the use of new microscopes that improve spatial resolution, including dual-view cleared tissue light-sheet microscopy and reflective lattice light-sheet microscopy.

bioengineering↗

bioRxiv2019

All-FIT: Allele-Frequency-based Imputation of Tumor Purity from High-Depth Sequencing Data

MotivationClinical sequencing aims to identify somatic mutations in cancer cells for accurate diagnosis and treatment. However, most widely used clinical assays lack patient-matched control DNA and additional analysis is needed to distinguish somatic and unfiltered germline variants. Such computational analyses require accurate assessment of tumor cell content in individual specimens. Histological estimates often do not corroborate with results from computational methods that are primarily designed for normal-tumor matched data and can be confounded by genomic heterogeneity and presence of sub-clonal mutations.\n\nMethodsAll-FIT is an iterative weighted least square method to estimate specimen tumor purity based on the allele frequencies of variants detected in high-depth, targeted, clinical sequencing data.\n\nResultsUsing simulated and clinical data, we demonstrate All-FITs accuracy and improved performance against leading computational approaches, highlighting the importance of interpreting purity estimates based on expected biology of tumors.\n\nAvailability and ImplementationFreely available at http://software.khiabanian-lab.org.

bioinformatics↗

bioRxiv2019

A Quasi Birth-and-Death Model For Tumor Recurrence

A major cause of chemoresistance and recurrence in tumors is the presence of dormant tumor foci that survive chemotherapy and can eventually transition to active growth to regenerate the cancer. In this paper, we propose a Quasi Birth-and-Death (QBD) model for the dynamics of tumor growth and recurrence/remission of the cancer. Starting from a discrete-state master equation that describes the time-dependent transition probabilities between states with different numbers of dormant and active tumor foci, we develop a framework based on a continuum-limit approach to determine the time-dependent probability that an undetectable residual tumor will become large enough to be detectable. We derive an exact formula for the probability of recurrence at large times and show that it displays a phase transition as a function of the ratio of the death rate {micro}A of an active tumor focus to its doubling rate{lambda} . We also derive forward and backward Kolmogorov equations for the transition probability density in the continuum limit and, using a first-passage time formalism, we obtain a drift-diffusion equation for the mean recurrence time and solve it analytically to leading order for a large detectable tumor size N. We show that simulations of the discrete-state model agree with the analytical results, except for O(1/N) corrections. Finally, we describe a scheme to fit the model to recurrence-free survival (Kaplan-Meier) curves from clinical cancer data, using ovarian cancer data as an example. Our model has potential applications in predicting how changing chemotherapy schedules may affect disease recurrence rates, especially in cancer types for which no targeted therapy is available.

cancer biology↗

bioRxiv2018

The Dot/Icm-translocated effector LegC4 potentiates cytokine-mediated restriction of Legionella within accidental hosts.

Legionella pneumophila is ubiquitous in freshwater environments where it replicates within unicellular protozoa. However, L. pneumophila is also an accidental human pathogen that can cause Legionnaires Disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction of L. pneumophila. We found previously that the effector LegC4 is important for L. pneumophila replication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires Disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon (IFN)-{gamma}. Specifically, TNF-mediated signaling was required for LegC4-mediated attenuation of L. pneumophila replication within macrophages. In addition, expression of legC4 was sufficient to restrict L. longbeachae replication within TNF- or IFN-{gamma}-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.\n\nImportanceLegionella are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controls Legionella infection due in part to rapid and robust production of pro-inflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances pro-inflammatory host restriction of Legionella from macrophages. These data suggest that LegC4 may augment pro-inflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.

microbiology↗

bioRxiv2018

Maturation Trajectories of Cortical Resting-State Networks Depend on the Mediating Frequency Band

The functional significance of resting state networks and their abnormal manifestations in psychiatric disorders are firmly established, as is the importance of the cortical rhythms in mediating these networks. Resting state networks are known to undergo substantial reorganization from childhood to adulthood, but whether distinct cortical rhythms, which are generated by separable neural mechanisms and are often manifested abnormally in psychiatric conditions, mediate maturation differentially, remains unknown. Using magnetoencephalography (MEG) to map frequency band specific maturation of resting state networks from age 7 to 29 in 162 participants (31 independent), we found significant changes with age in networks mediated by the beta (13-30Hz) and gamma (31-80Hz) bands. More specifically, gamma band mediated networks followed an expected asymptotic trajectory, but beta band mediated networks followed a linear trajectory. Network integration increased with age in gamma band mediated networks, while local segregation increased with age in beta band mediated networks. Spatially, the hubs that changed in importance with age in the beta band mediated networks had relatively little overlap with those that showed the greatest changes in the gamma band mediated networks. These findings are relevant for our understanding of the neural mechanisms of cortical maturation, in both typical and atypical development.

neuroscience↗