Biology subjects

Smith, J. C.

Publications and source records attributed to Smith, J. C..

52 records · Page 3Linked to original sources

bioRxiv2020

Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.

molecular biology↗

bioRxiv2020

Full structural ensembles of intrinsically disordered proteins from unbiased molecular dynamics simulations

Molecular dynamics (MD) simulation is widely used to complement ensemble-averaged experiments of intrinsically disordered proteins (IDPs). However, MD often suffers from limitations of inaccuracy in the force fields and inadequate sampling. Here, we show that enhancing the sampling using Hamiltonian replica-exchange MD led to unbiased ensembles of unprecedented accuracy, reproducing small-angle scattering and NMR chemical shift experiments, for three IDPs of variable sequence properties using two recently optimized force fields. Surprisingly, we reveal that despite differences in their sequence, the inter-chain statistics of all three IDPs are similar for short contour lengths (< 10 residues).

biophysics↗

bioRxiv2020

Rostrocaudal Patterning and Neural Crest Differentiation of Human Pre-Neural Spinal Cord Progenitors in vitro

The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and maintained by Wnt/FGF signals at the posterior end of the embryo. NMPs can be generated from human pluripotent stem cells and hold promise for spinal cord replacement therapies. However, NMPs are transient, which complicates the full range production of rostrocaudal spinal cord identities in vitro. Here we report the generation of NMP-derived pre-neural progenitors (PNPs) with stem cell-like self-renewal capacity. PNPs maintain pre-spinal cord identity by co-expressing the transcription factors SOX2 and CDX2, and lose mesodermal potential by downregulating TBXT. For 7 to 10 passages PNPs divide to self-renew and to make trunk neural crest (NC), while gradually adopting a more posterior identity by activating colinear HOX gene expression. This HOX clock can be halted at the thoracic level for up to 30 passages by blocking the trunk-to-tail transition through GDF11-mediated signal inhibition.

developmental biology↗

bioRxiv2020

Immune Selection Pressure Contributes to Pancreatic Cancer Immune Evasion

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. Pancreatic tumors are minimally infiltrated by T cells and are largely refractory to immunotherapy. Accordingly, the role of T cell immunity in pancreatic cancer has been somewhat overlooked. Here, we hypothesized that immune evasion in pancreatic cancer is induced in response to T cell-based immune selection pressure, and that understanding how pancreatic tumors respond to immune attack may facilitate the development of more effective therapeutic strategies. We now provide the first evidence that T cell-dependent host immune responses induce a PDAC-derived myeloid mimicry phenomenon and stimulate immune evasion. mT3-2D cells derived from a Kras+/LSL-G12D; Trp53+/LSL-R172H; Pdx1-Cre (KPC) mouse model of pancreatic cancer were grown in immunocompetent and immunodeficient C57BL/6 mice, and analyzed to determine the impacts of adaptive immunity specifically on malignant epithelial cells as well as on whole tumors. We found that immune selection pressure, via signal transducer and activator of transcription 1 (STAT1), stimulates malignant epithelial pancreatic cells to induce the expression of genes typically expressed by myeloid cells and alters intratumoral immunosuppressive myeloid cell profiles. Targeting the Janus Kinase (JAK)/STAT signaling pathway using the FDA approved drug, ruxolitinib, overcomes these tumor-protective responses and improves anti-PD1 antibody therapeutic efficacy. These findings provide future directions for treatments that specifically disable this mechanism of resistance in PDAC.

cancer biology↗

bioRxiv2020

FAM83F regulates canonical Wnt signalling through an interaction with CK1α

The function of the FAM83F protein, like the functions of many members of the FAM83 family, is poorly understood. Here we show that injection of Fam83f mRNA into Xenopus embryos causes axis duplication, a phenotype indicative of enhanced Wnt signalling. Consistent with this, overexpression of FAM83F activates Wnt signalling, whilst ablation of FAM83F from human colorectal cancer (CRC) cells attenuates it. We demonstrate that FAM83F is farnesylated and interacts and co-localises with CK1 at the plasma membrane. This interaction with CK1 is essential for FAM83F to activate Wnt signalling, and FAM83F mutants that do not interact with CK1 fail to induce axis duplication in Xenopus embryos and to activate Wnt signalling in cells. FAM83F acts upstream of the {beta}-catenin destruction complex, because the attenuation of Wnt signalling caused by loss of FAM83F can be rescued by GSK-3 inhibition. Introduction of a farnesyl-deficient mutant mis-localises the FAM83F-CK1 complex to the nucleus and significantly attenuates Wnt signalling, indicating that FAM83F exerts its effects on Wnt signalling at the plasma membrane.

cell biology↗

bioRxiv2020

Cigarette smoke triggers the expansion of a subpopulation of respiratory epithelial cells that express the SARS-CoV-2 receptor ACE2

The factors mediating fatal SARS-CoV-2 infections are poorly understood. Here, we show that cigarette smoke causes a dose-dependent upregulation of Angiotensin Converting Enzyme 2 (ACE2), the SARS-CoV-2 receptor, in rodent and human lungs. Using single-cell sequencing data, we demonstrate that ACE2 is expressed in a subset of secretory cells in the respiratory tract. Chronic smoke exposure triggers the expansion of this cell population and a concomitant increase in ACE2 expression. In contrast, quitting smoking decreases the abundance of these secretory cells and reduces ACE2 levels. Finally, we demonstrate that ACE2 expression is responsive to inflammatory signaling and can be upregulated by viral infections or interferon treatment. Taken together, these results may partially explain why smokers are particularly susceptible to severe SARS-CoV-2 infections. Furthermore, our work identifies ACE2 as an interferon-stimulated gene in lung cells, suggesting that SARS-CoV-2 infections could create positive-feedback loops that increase ACE2 levels and facilitate viral dissemination.

cell biology↗

bioRxiv2020

Structure based virtual screening identifies novel competitive inhibitors for the sialoglycan binding protein Hsa

Infective endocarditis (IE) is a cardiovascular disease often caused by bacteria of the viridans group of streptococci, which includes Streptococcus gordonii and Streptococcus sanguinis. Previous research has found that a serine-rich repeat (SRR) proteins on the S. gordonii bacterial surface play a critical role in pathogenesis by facilitating bacterial attachment to sialyated glycans displayed on human platelets. Despite its important role in disease progression, there are currently no anti-adhesive drugs available on the market. Here, we performed structure-based virtual screening using an ensemble docking approach followed by consensus scoring to identify novel inhibitors against the sialoglycan binding domain of the SRR adhesin protein Hsa from the S. gordonii strain DL1. In silico cross screening against the glycan binding domains of closely related SRR proteins from five other S. gordonii or S. sanguinis strains was also performed to further reduce false positives. Using our in silico screening strategy we successfully predicted nine compounds which were able to displace the native ligand (sialyl-T antigen) in an in vitro assay and bind competitively to adhesin protein Hsa ([~]20% hit rate).

microbiology↗

bioRxiv2020

Mesophilic enzyme function at high temperature: moleculardynamics of hyperthermophilic and mesophilicpyrophosphatases

The mesophilic inorganic pyrophosphatase from Escherichia coli (EcPPase) retains function at 353 K, the physiological temperature of hyperthermophilic Thermoccoccus thioreducens, whereas, the homolog protein from the hyperthermophilic organism (TtPPase) cannot function at room temperature. To explain this asymmetric behavior, we examined structural and dynamical properties of the two proteins using molecular dynamics simulations. The global flexibility of TtPPase is significantly higher than its mesophilic homolog at all tested temperature/pressure conditions. However, at 353 K, EcPPase reduces its solvent-exposed surface area and increases subunit compaction while maintaining flexibility in its catalytic pocket. In contrast, TtPPase lacks this adaptability and has increased rigidity and reduced protein:water interactions in its catalytic pocket at room temperature, providing a plausible explanation for its inactivity near room temperature.

biophysics↗

bioRxiv2019

Early-Stage Ocular Hypertension Alters Retinal Ganglion Cell Synaptic Transmission in the Visual Thalamus

Axonopathy is a hallmark of many neurodegenerative diseases including glaucoma, where elevated intraocular pressure (ocular hypertension, OHT) stresses retinal ganglion cell (RGC) axons as they exit the eye and form the optic nerve. OHT causes early changes in the optic nerve such as axon atrophy, transport inhibition, and gliosis. Importantly, many of these changes appear to occur prior to irreversible neuronal loss, making them promising points for early diagnosis of glaucoma. It is unknown whether OHT has similarly early effects on the function of RGC output to the brain. To test this possibility, we elevated eye pressure in mice by anterior chamber injection of polystyrene microbeads. 5 weeks post-injection, bead-injected eyes showed a modest RGC loss in the peripheral retina, as evidenced by RBPMS antibody staining. Additionally, we observed reduced dendritic complexity and lower spontaneous spike rate of On-RGCs, targeted for patch clamp recording and dye filling using a Opn4-cre reporter mouse line. To determine the influence of OHT on retinal projections to the brain, we expressed Channelrhodopsin-2 (ChR2) in melanopsin-expressing retinal ganglion cells by crossing the Opn4-cre mouse line with a ChR2-reporter mouse line and recorded post-synaptic responses in thalamocortical relay neurons in the dorsal lateral geniculate nucleus (dLGN) of the thalamus evoked by stimulation with 460 nm light. The use of a Opn4-cre reporter system allowed for expression of ChR2 in a narrow subset of RGCs responsible for image-forming vision in mice. Five weeks following OHT induction, paired pulse and high-frequency stimulus train experiments revealed that presynaptic vesicle release probability at retinogeniculate synapses was elevated. Additionally, miniature synaptic current frequency was slightly reduced in brain slices from OHT mice and proximal dendrites of post-synaptic dLGN relay neurons, assessed using a Sholl analysis, showed a reduced complexity. Strikingly, these changes occurred prior to major loss of RGCs labeled with the Opn4-Cre mouse, as indicated by immunofluorescence staining of ChR2-expressing retinal neurons. Thus, OHT leads to pre- and post-synaptic functional and structural changes at retinogeniculate synapses. Along with RGC dendritic remodeling and optic nerve transport changes, these retinogeniculate synaptic changes are among the earliest signs of glaucoma.

neuroscience↗

bioRxiv2019

Single chromosome gains can function as metastasis suppressors and metastasis promoters

Most human tumors display chromosome-scale copy number alterations, and high levels of aneuploidy are frequently associated with advanced disease and poor patient prognosis. To examine the relationship between aneuploidy and cancer progression, we generated and analyzed a series of congenic human cell lines that harbor single extra chromosomes. We find that different aneuploidies can have distinct effects on invasive behavior: across 13 different cell lines, 12 trisomies suppressed invasiveness or were largely neutral, while a single trisomy increased metastatic behavior by triggering a partial epithelial-mesenchymal transition. In contrast, chromosomal instability, which can lead to the development of aneuploidy, uniformly suppressed cellular invasion. By analyzing genomic copy number and survival data from 10,133 cancer patients, we demonstrate that specific aneuploidies are associated with distinct clinical outcomes, and the acquisition of certain aneuploidies is in fact linked with a favorable prognosis. Thus, aneuploidy is not a uniform driver of malignancy, and different chromosome copy number changes can uniquely influence tumor progression. At the same time, the gain of a single chromosome is capable of inducing a profound cell state transition, underscoring how genomic plasticity can engender phenotypic plasticity and lead to the acquisition of enhanced metastatic properties.

cancer biology↗

bioRxiv2018

The Spatio-Temporal Control of Zygotic Genome Activation

One of the earliest and most significant events in embryonic development is zygotic genome activation (ZGA). In several species, bulk transcription begins at the mid-blastula transition (MBT) when, after a certain number of cleavages, the embryo attains a particular nuclear-to-cytoplasmic (N/C) ratio, maternal repressors become sufficiently diluted, and the cell cycle slows down. Here we resolve the frog ZGA in time and space by profiling RNA polymerase II (RNAPII) engagement and its transcriptional readout. We detect a gradual increase in both the quantity and the length of RNAPII elongation before the MBT, revealing that >1,000 zygotic genes disregard the N/C timer for their activation, and that the sizes of newly transcribed genes are not necessarily constrained by cell cycle duration. We also find that Wnt, Nodal and BMP signaling together generate most of the spatio-temporal dynamics of regional ZGA, directing the formation of orthogonal body axes and proportionate germ layers.

developmental biology↗

bioRxiv2018

Biophysical mechanisms in the mammalian respiratory oscillator re-examined with a new data-driven computational model

An autorhythmic population of excitatory neurons in the brainstem pre-Botzinger complex is a critical component of the mammalian respiratory oscillator. Two intrinsic neuronal biophysical mechanisms--a persistent sodium current (INaP) and a calcium-activated non-selective cationic current (ICAN)--were proposed to individually or in combination generate cellular-and circuit-level oscillations, but their roles are debated without resolution. We re-examined these roles with a new computational model of an excitatory population with randomly distributed INaP and ICAN conductances and synaptic connections. This model robustly reproduces experimental data showing contrary to previous hypotheses, rhythm generation is independent of ICAN activation, which instead determines population activity amplitude. The novel insight is that this occurs when ICAN is primarily activated by neuronal calcium fluxes driven by synaptic mechanisms. Rhythm depends critically on INaP in a subpopulation forming the rhythmogenic kernel. The model explains how the rhythm and amplitude of respiratory oscillations involve distinct biophysical mechanisms.

bioinformatics↗

bioRxiv2018

Maternal pluripotency factors prime the zygotic genome to respond to intercellular signals

Embryonic development yields many different cell types in response to just a few families of inductive signals. The property of a signal-receiving cell that determines how it responds to such signals, including the activation of cell type-specific genes, is known as its competence. Here, we show how maternal factors modify chromatin to specify initial competence in the frog Xenopus tropicalis. We identified the earliest engaged regulatory DNA sequences, and inferred from them critical activators of the zygotic genome. Of these, we showed that the pioneering activity of the maternal pluripotency factors Pou5f3 and Sox3 predefines competence for germ layer formation by extensively remodeling compacted chromatin before the onset of signaling. The remodeling includes the opening and marking of thousands of regulatory elements, extensive chromatin looping, and the co-recruitment of signal-mediating transcription factors. Our work identifies significant developmental principles that inform our understanding of how pluripotent stem cells interpret inductive signals.

developmental biology↗

bioRxiv2017

Combining CRISPR/Cas9 mutagenesis and a small-molecule inhibitor to probe the function of MELK in cancer

The Maternal Embryonic Leucine Zipper Kinase (MELK) has been identified as a promising therapeutic target in multiple cancer types. MELK over-expression is associated with aggressive disease, and MELK has been implicated in numerous cancer-related processes, including chemotherapy resistance, stem cell renewal, and tumor growth. On the basis of these findings, a MELK inhibitor is currently being tested in several clinical trials. Here, we report that cancer cell lines harboring CRISPR/Cas9-induced null mutations in MELK exhibit wild-type growth in vitro, under environmental stress, in the presence of multiple chemotherapy agents, and in vivo. By combining our MELK-knockout clones with a recently-described, highly-specific MELK inhibitor, we further demonstrate that the acute inhibition of MELK results in no specific anti-proliferative phenotype. Analysis of gene expression data from cohorts of cancer patients identifies MELK expression as a correlate of tumor mitotic activity, explaining its association with poor clinical prognosis. In total, our results demonstrate the power of CRISPR/Cas9-based genetic approaches to investigate cancer drug targets, and call into question the rationale for treating patients with anti-MELK monotherapies.

cancer biology↗

bioRxiv2017

An integrated analysis of the epigenetic, genetic, and transcriptional patterns associated with outcome across cancer types

Successful treatment decisions in cancer depend on the accurate assessment of patient risk. To improve our understanding of the molecular alterations that underlie deadly malignancies, we analyzed genomic profiles from 33,036 solid tumors with known patient outcomes. Contrary to expectations, we find that mutations in cancer driver genes are almost never associated with patient survival time. In contrast, copy number changes in these same genes are broadly prognostic. Analysis of methylation, microRNA, mRNA, and protein expression patterns in primary tumors define several additional prognostic patterns, including signatures of tumor mitotic activity and tissue de-differentiation. Co-expression analysis with a cell cycle meta-gene distinguished proliferation-dependent and -independent prognostic features, allowing us to construct multivariate survival models with improved stratification power. In total, our analysis provides a comprehensive resource for biomarker and therapeutic target identification, and suggests that copy number and methylation profiling should complement tumor sequencing efforts to improve patient risk assessment.

cancer biology↗

bioRxiv2017

FAM83G/PAWS1 controls cytoskeletal dynamics and cell migration through association with the SH3 adaptor CD2AP

Our previous studies of PAWS1 (Protein Associated With SMAD1) have suggested that this molecule has roles beyond BMP signalling. To investigate these roles, we have used CRISPR/Cas9 to generate PAWS1 knockout cells. Here, we show that PAWS1 plays a role in the regulation of the cytoskeletal machinery, including actin and focal adhesion dynamics, and cell migration. Confocal microscopy and live cell imaging of actin in U2OS cells indicate that PAWS1 is also involved in cytoskeletal dynamics and organization. Loss of PAWS1 causes severe defects in F-actin organization and distribution as well as in lamellipodial organization, resulting in impaired cell migration. PAWS1 interacts in a dynamic fashion with the actin/cytoskeletal regulator CD2AP at lamellae, suggesting that its association with CD2AP controls actin organization and cellular migration.\n\nSummary statementPAWS1/FAM83G controls cell migration by influencing the organisation of F-actin and focal adhesions and the distribution of the actin stress fibre network through its association with CD2AP.

cell biology↗