Biology subjects

Tan, A.

Publications and source records attributed to Tan, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

Structural insights into HIV-1 polyanion-dependent capsid lattice formation revealed by singleparticle cryo-EM

The HIV-1 capsid houses the viral genome and interacts extensively with host cell proteins throughout the viral life cycle. It is composed of capsid protein (CA), which assembles into a conical fullerene lattice composed of roughly 200 CA hexamers and 12 CA pentamers. Previous structural analyses of individual CA hexamers and pentamers have provided valuable insight into capsid structure and function, but high-resolution information about these assemblies in the broader context of the capsid lattice is lacking. In this study, we combined cryo-electron tomography and single particle analysis cryo-electron microscopy to determine high-resolution structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also developed a new method of in vitro lattice assembly that enabled us to directly study the lattice under a wider range of conditions than has previously been possible. Using this approach, we identified a critical role for inositol hexakisphosphate (IP6) in pentamer formation and determined the structure of the CA lattice bound to the capsid-targeting antiretroviral drug GS-6207 (Lenacapvir). Our work reveals new structural details of the mature HIV-1 CA lattice and establishes the combination of lattice templating and single particle analysis as a robust strategy for studying retroviral capsid structure and capsid interactions with host proteins and antiviral compounds. Significance statementThe mature HIV-1 capsid is composed of the capsid (CA) protein arranged in a conical lattice of hexamers and pentamers. Numerous structures of individual CA hexamers and pentamers alone have been published, but the molecular details of these assemblies in a more global, lattice-wide context are lacking. Here, we present high-resolution cryo-electron microscopy structures of continuous regions of the capsid lattice containing both hexamers and pentamers. We also describe key differences in the assembly and structures of these oligomers that have important implications for understanding retroviral maturation and for ongoing efforts to pharmacologically target the HIV-1 capsid.

biochemistry↗

bioRxiv2022

Two structural switches in HIV-1 capsid regulate capsid curvature and host factor binding

The mature HIV-1 capsid protects the viral genome and interacts with host proteins to travel from the cell periphery into the nucleus. To achieve this, the capsid protein, CA, constructs conical capsids from a lattice of hexamers and pentamers, and engages in and then relinquishes multiple interactions with cellular proteins in an orchestrated fashion. Cellular host factors including Nup153, CPSF6 and Sec24C engage the same pocket within CA hexamers. How CA assembles pentamers and hexamers of different curvatures, how CA oligomerization states or curvature might modulate host-protein interactions, and how binding of multiple co-factors to a single site is coordinated, all remain to be elucidated. Here, we have resolved the structure of the mature HIV-1 CA pentamer and hexamer from conical CA-IP6 polyhedra to high resolution. We have determined structures of hexamers in the context of multiple lattice curvatures and number of pentamer contacts. Comparison of these structures, bound or not to host protein peptides, revealed two structural switches within HIV-1 CA that modulate peptide binding according to CA lattice curvature and whether CA is hexameric or pentameric. These observations suggest that the conical HIV-1 capsid has different host-protein binding properties at different positions on its surface, which may facilitate cell entry and represent an evolutionary advantage of conical morphology. Significance statementHIV-1 particles contain a characteristic, conical capsid that shields the genome from the cellular immune system and recruits cellular proteins to direct the capsid to the nucleus. The cone forms from hexamers of CA protein, and twelve pentamers that accommodate curvature. We obtained detailed 3D models of pentamers and hexamers at positions on capsid surfaces with different curvatures. We find two places in CA that switch conformation according to the local capsid curvature and whether CA is in a pentamer or hexamer. We also obtained models of CA bound to peptides from cellular proteins. The data show how switches in CA help it form a cone shape, and interact differently with cellular proteins at different positions on the cone surface.

microbiology↗

bioRxiv2022

IL-12-expressing highly immunogenic recombinant modified vaccinia virus Ankara reprograms tumor-infiltrating myeloid cells to overcome immune resistance

Resistance to immune checkpoint blockade (ICB) remains a major obstacle in cancer immunotherapy. We rationally engineered a second-generation recombinant Modified Vaccinia virus Ankara (MQ833) by deleting viral immune-evasion genes (E3L, E5R, WR199) and incorporating Flt3L, OX40L, and matrix-anchored IL12. Intratumoral MQ833 elicited robust tumor regressions across multiple models, including ICB-resistant and MHC-I-deficient tumors. Single-cell RNA sequencing revealed extensive remodeling of the tumor microenvironment, characterized by neutrophil and monocyte recruitment and activation, M2 macrophage depletion, M1 polarization, and effector T-cell differentiation and proliferation. Conditional Ifnar1 knockout mice demonstrated that MQ833 efficacy requires type I interferon signaling in neutrophils, macrophages/monocytes, and T cells. Moreover, Nos2 deficiency impaired therapeutic efficacy, confirming iNOS myeloid cells as key effectors. Together, these findings show that MQ833 activates innate-adaptive IFN cross-talk to reprogram myeloid and T cells, defining a rationally designed viral immunotherapy capable of overcoming ICB resistance. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/509429v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@496f37org.highwire.dtl.DTLVardef@124bd78org.highwire.dtl.DTLVardef@1134528org.highwire.dtl.DTLVardef@216da6_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Spatial transcriptomics reveals ovarian cancer subclones with distinct tumour microenvironments

High-grade serous ovarian carcinoma (HGSOC) is characterised by recurrence, chemotherapy resistance and overall poor prognosis. Genetic heterogeneity of tumour cells and the microenvironment of the tumour have been hypothesised as key determinants of treatment resistance and relapse. Here, using a combination of spatial and single cell transcriptomics (10x Visium and Chromium platforms), we examine tumour genetic heterogeneity and infiltrating populations of HGSOC samples from eight patients with variable response to neoadjuvant chemotherapy. By inferring gross copy number alterations (CNAs), we identified distinct tumour subclones co-existing within individual tumour sections. These tumour subclones have unique CNA profiles and spatial locations within each tumour section, which were further validated by ultra-low-pass whole genome sequencing. Differential expression analysis between subclones within the same section identified both tumour cell intrinsic expression differences and markers indicative of different infiltrating cell populations. The gene sets differentially expressed between subclones were significantly enriched for genes encoding plasma membrane and secreted proteins, indicative of subclone-specific microenvironments. Furthermore, we identified tumour derived ligands with variable expression levels between subclones that correlated or anticorrelated with various non-malignant cell infiltration patterns. We highlight several of these that are potentially direct tumour-stroma/immune cell relationships as the non-malignant cell type expresses a cognate receptor for the tumour derived ligand. These include predictions of CXCL10-CXCR3 mediated recruitment of T and B cells to associate with the subclones of one patient and CD47-SIRPA mediated exclusion of macrophages from association with subclones of another. Finally, we show that published HGSOC molecular subtype signatures associated with prognosis are heterogeneously expressed across tumour sections and that areas containing different tumour subclones with different infiltration patterns can match different subtypes. Our study highlights the high degree of intratumoural subclonal and infiltrative heterogeneity in HGSOC which will be critical to better understand resistance and relapse.

cancer biology↗

bioRxiv2022

Rational probe design for efficient rRNA depletion and improved metatranscriptomic analysis of human microbiomes

BackgroundThe microbiota that colonize the human gut and other tissues are dynamic, varying both in composition and functional state between individuals and over time. In studying the function of the human microbiome and the mechanisms of microbiota-mediated phenotypes, gene expression measurements provide additional insights to DNA-based measurements of microbiome composition. However, efficient and unbiased removal of microbial ribosomal RNA (rRNA) presents a barrier to acquiring metatranscriptomic data, as rRNA typically accounts >90% of total RNA in microbial cells. ResultsHere we describe a probe set that achieves efficient enzymatic rRNA removal of complex human-associated microbial communities. We demonstrate that the custom probe set can be further refined through an iterative design process to efficiently deplete rRNA from a range of human microbiome samples, including adult and infant gut, as well as oral and vaginal communities. Using synthetic nucleic acid spike-ins, we show that the rRNA depletion process does not introduce substantial quantitative error in the resulting gene expression profiles. Successful rRNA depletion allows for efficient characterization of taxonomic and functional profiles, including during the development of the human gut microbiome. ConclusionsThe pan-human microbiome enzymatic rRNA depletion probes described here provide a powerful tool for studying the transcriptional dynamics and function of the human microbiome.

genomics↗

bioRxiv2022

Exploring the in-situ evolution of Nitrofurantoin resistance in clinically derived Uropathogenic Escherichia coli isolates.

BACKGROUNDNitrofurantoin has been re-introduced as a first-choice antibiotic to treat uncomplicated acute urinary tract infections in England and Wales. Its mode of action involves initial reduction by nitroreductases, to generate electrophilic intermediates that inhibit protein and nucleic acid synthesis. Highly effective against common uropathogens such as Escherichia coli, its use is accompanied by a low incidence (<10%) of antimicrobial resistance. Resistance to Nitrofurantoin is predominantly via the acquisition of loss-of-function, step-wise mutations in the nitroreductase genes nfsA and nfsB. OBJECTIVETo explore the in situ evolution of NitR, longitudinal uropathogenic E. coli isolates recovered from two rUTI patients. RESULTSGrowth rate analysis identified a 2-10% slower doubling time for Nitrofurantoin resistant strains, but statistically, these data suggested there was no fitness advantage of evolved strains over their sensitive predecessor (ANOVA P-value = 0.13). Genetic manipulation of E. coli to mimic nitrofurantoin resistance evolution, again confirmed no fitness advantages (ANOVA P-value = 0.22). Rather, further analysis argued that a first-step mutant gained a selective advantage, at sub-MIC (4-8 mg/L) nitrofurantoin concentrations. CONCLUSIONCorrelation of these findings to Nitrofurantoin pharmacokinetic data suggests that the low incidence of E. coli NitR, within the community, is driven by urine-based nitrofurantoin concentrations that selectively inhibit the growth of E. coli strains carrying the key first-step loss-of-function mutation.

microbiology↗

bioRxiv2022

Single-nucleus RNA-seq reveals disrupted cell maturation by chorioamnionitis in the preterm cerebellum of nonhuman primates

Preterm birth is often associated with chorioamnionitis and increased risk of neurodevelopmental disorders. Preterm birth also leads to cerebellar underdevelopment but the mechanisms of disrupted cerebellar development in preterm infants are little understood. Here, we leveraged single-nuclei RNA-sequencing of the cerebellum in a rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation. Chorioamnionitis also accelerated pre-oligodendrocyte maturation into myelinating oligodendrocytes, confirmed by increased expression of myelin basic protein in the cerebellum of chorioamnionitis-exposed fetuses. These findings are consistent with reported histopathological findings in individuals with autism and suggest a novel mechanism through which perinatal inflammation contributes to neurodevelopmental disorders.

neuroscience↗

bioRxiv2022

Gut Microbes and the Liver Circadian Clock Partition Glucose and Lipid Metabolism

Circadian rhythms govern glucose homeostasis, and their dysregulation leads to complex metabolic diseases. Gut microbes also exhibit diurnal rhythms that influence host circadian networks and metabolic processes, yet underlying mechanisms remain elusive. Here, we show hierarchical, bi-directional communication between the liver circadian clock, gut microbes, and glucose homeostasis in mice. The liver clock, but not the forebrain clock, requires gut microbes to drive glucose clearance and gluconeogenesis. Liver clock dysfunctionality expands proportions and abundances of oscillating microbial features by two-fold relative to controls. The liver clock is the primary driver of differential and rhythmic hepatic expression of glucose and fatty acid metabolic pathways. Absent the liver clock, gut microbes provide secondary cues that dampen these rhythms, resulting in reduced utilization of lipids as fuel relative to carbohydrates. Together, the liver clock transduces signals from gut microbes necessary to regulate glucose and lipid metabolism and meet energy demands over 24 hours. HighlightsThe liver circadian clock is autonomous from the central clock in metabolic regulation Liver clock and gut microbes interact to direct hepatic glucose and lipid metabolism Reciprocating host-microbe interactions drive rhythmic hepatic transcription Perturbed liver Bmal1 results in chaotic downstream oscillators and metabolism

physiology↗

bioRxiv2022

CryoEM structures of the multimeric secreted NS1, a major factor for dengue hemorrhagic fever

Dengue virus infection can cause dengue hemorrhagic fever (DHF). Dengue NS1 is multifunctional: the intracellular dimeric NS1 (iNS1) forms part of the viral replication complex, the extracellular multi-oligomeric secreted NS1 (sNS1) is a major factor contributing to DHF. The structure of the iNS1 is well studied but not sNS1. Here we show the tetrameric (stable and loose conformation) and hexameric structures of sNS1. Stability of the stable and loose tetramers is determined by the conformation of their N-terminal domain - elongated {beta}-sheet or {beta}-roll. Binding of an anti-NS1 Fab breaks the loose tetrameric and hexameric sNS1 into dimers, whereas the stable tetramer remains largely unbound. Our results show detailed quaternary organization of different oligomeric states of sNS1 and will contribute towards the design of dengue therapeutics.

molecular biology↗

bioRxiv2022

ALS sensitive spinal motor neurons enter a degenerative downward spiral of impaired splicing and proteostasis

Despite clear therapeutic potential, the mechanisms that confer differential neuronal sensitivity are not well understood. During Amyotrophic Lateral Sclerosis (ALS), sensitive spinal motor neurons (SpMN) die while a subset of rostral cranial motor neurons (CrMN) survive. In this work, we optimized a protocol to differentiate CrMNs and SpMNs from human induced pluripotent stem cells (iPSCs) by direct programming and positional patterning. Human iCrMNs are more resistant than iSpMNs to proteotoxic stress and rely on the proteasome to maintain proteostasis. iCrMNs better prevent mislocalization of TDP43 from the nucleus, a hallmark of ALS progression. iSpMNs contain more splicing defects than iCrMNs in response to ALS-related stress with genes involved in splicing and proteostasis maintenance. Therefore, iCrMNs resist ALS at two levels, preventing protein accumulation and reducing splicing defects in response to TDP43 nuclear depletion. Thus, ALS-sensitive iSpMNs appear to enter a downward spiral compromising their ability to maintain proteostasis and splicing.

neuroscience↗

bioRxiv2021

Vaccinia E5 is a major inhibitor of the DNA sensor cGAS

The DNA sensor cyclic GMP-AMP synthase (cGAS) is critical in host antiviral immunity. Vaccinia virus (VACV) is a large cytoplasmic DNA virus that belongs to the poxvirus family. How vaccinia virus antagonizes the cGAS-mediated cytosolic DNA-sensing pathway is largely unknown. In this study, we screened 82 vaccinia viral genes to identify potential viral inhibitors of the cGAS/Stimulator of interferon gene (STING) pathway. We discovered that vaccinia E5 is a virulence factor and a major inhibitor of cGAS that elicits proteasome-dependent cGAS degradation. E5 localizes to the cytoplasm and nuclei of infected cells. Cytosolic E5 triggers K48-linked ubiquitination of cGAS and proteasome-dependent degradation via interacting with cGAS. E5 itself also undergoes ubiquitination and degradation. Deleting the E5R gene from the Modified vaccinia virus Ankara (MVA) genome strongly induces type I IFN production by dendritic cells (DCs) and promotes DC maturation, thereby improving the immunogenicity of the viral vector.

microbiology↗

bioRxiv2020

Immature HIV-1 assembles from Gag dimers leaving partial hexamers at lattice edges as substrates for proteolytic maturation.

The CA (capsid) domain of immature HIV-1 Gag and the adjacent spacer peptide 1 (SP1) play a key role in viral assembly by forming a lattice of CA hexamers, which adapts to viral envelope curvature by incorporating small lattice defects and a large gap at the site of budding. This lattice is stabilized by intra- and inter-hexameric CA-CA interactions, which are important in regulating viral assembly and maturation. We applied subtomogram averaging and classification to determine the structure of CA at lattice edges and found that they form partial hexamers. These structures reveal the network of interactions formed by CA-SP1at the lattice edge. We also performed atomistic molecular dynamics simulations of CA-CA interactions stabilizing the immature lattice and of partial CA-SP1 helical bundles. Free energy calculations reveal increased propensity for helix-to-coil transitions in partial hexamers compared to complete six-helix bundles. Taken together, these results suggest that the CA dimer is the basic unit of lattice assembly, that partial hexamers exist at lattice edges, that these are in a helix-coil dynamic equilibrium and that partial helical bundles are more likely to unfold, representing potential sites for HIV-1 maturation initiation. Significance StatementHIV-1 particle assembly is driven by the viral Gag protein, which oligomerizes into an hexameric array on the inner surface of the viral envelope, forming a truncated spherical lattice containing large and small gaps. Gag is then cut by the viral protease, disassembles and rearranges to form the mature, infectious virus. Here, we present structures and molecular dynamics simulations of the edges of the immature Gag lattice. Our analysis shows that Gag dimers are the basic assembly unit of the HIV-1 particle, that lattice edges are partial hexamers, and that partial hexamers are prone to structural changes allowing protease to cut Gag. These findings provide insights into assembly of the immature virus, its structure, and how it disassembles during maturation.

microbiology↗

bioRxiv2020

Medial Prefrontal Cortex Interacts with the Anteromedial Thalamus in Motivation and Dopaminergic Activity

How does the prefrontal cortex (PFC) regulate motivated behavior? Our experiments characterize the circuits of the PFC regulating motivation, reinforcement and dopamine activity, revealing a novel cortico-thalamic loop. The stimulation of medial PFC (mPFC) neurons activated many downstream regions, as shown by functional MRI. Axonal terminal stimulation of mPFC neurons in downstream regions, including the anteromedial thalamic nucleus (AM), reinforced behavior and activated midbrain dopaminergic neurons. The stimulation of AM neurons projecting to the mPFC also reinforced behavior and activated dopamine neurons, and mPFC and AM showed a positive-feedback loop organization. Reciprocal excitatory functional connectivity, as well as co-activation of the two regions, was found in human participants who watched reinforcing videos. Our results suggest that this cortico-thalamic loop regulates motivation, reinforcement and dopaminergic neuron activity.

neuroscience↗

bioRxiv2018

Fungal symbionts produce prostaglandin E2 to promote their intestinal colonization

Candida albicans is a ubiquitous fungal symbiont that resides on diverse human barrier surfaces. Both mammalian and fungal cells can convert arachidonic acid into the lipid mediator, prostaglandin E2 (PGE2), but the physiological significance of fungal-derived PGE2 remains elusive. Here we report that a C. albicans mutant deficient in PGE2 production suffered a loss of competitive fitness in the murine gastrointestinal (GI) tract and that PGE2 supplementation mitigated this fitness defect. Impaired fungal PGE2 production affected neither the in vitro fitness of C. albicans nor hyphal morphogenesis and virulence in either systemic or mucosal infection models. Fungus-derived PGE2 improved intra-GI fitness of C. albicans by diminishing the killing of C. albicans by phagocytes. Consequently, ablation of colonic phagocytes abrogated the fitness boost conferred by fungal PGE2. These observations suggest that C. albicans has evolved the capacity to produce PGE2 from arachidonic acid, a host-derived precursor, to promote its own colonization of the host gut. Analogous mechanisms might undergird host-microbe interactions of other symbiont fungi.\n\nAuthor SummaryCandida albicans is a symbiont fungus that resides in the gut of a majority of people without provoking disease. However, resident C. albicans can bloom and turn pathogenic in a subset of individuals who are immunocompromised due to infections or chemotherapy or who suffer a disruption of their intestinal microbial community due to antibiotic use. However, the fungal and host factors that regulate the fitness of C. albicans as a symbiont or an invasive pathogen remain poorly understood. Here we focused on the physiological role of fungus-derived prostaglandin E2 (PGE2) in the fitness of C. albicans using a PGE2-deficient C. albicans strain and mouse models of infections and intestinal symbiosis. We found that fungal PGE2, contrary to previously described functions of promoting virulence, played no role in fungal pathogenicity in vivo. Instead, fungal PGE2 specifically augmented the ability of C. albicans to colonize the gut, in part by reducing fungal killing by intestinal phagocytes. Our results suggest that fungal PGE2 synthetic pathways may be prophylactically targeted in individuals susceptible to invasive infections.

microbiology↗

bioRxiv2018

Chemotactic motility and biofilm formation in Campylobacter jejuni are coordinated by the CheAYWVX system

Flagellar motility plays a central role in the bacterial foodborne pathogen Campylobacter jejuni, as flagellar motility is required for reaching the intestinal epithelium and subsequent colonisation or disease. Flagellar proteins also contribute strongly to biofilm formation during transmission. Chemotaxis is the process directing flagellar motility in response to attractant and repellent stimuli, but its role in biofilm formation of C. jejuni is not well understood. Here we show that inactivation of the core chemotaxis genes cheVAWY in C. jejuni strain NCTC 11168 affects both chemotactic motility and biofilm formation. Inactivation of any of the core chemotaxis genes (cheA, cheY, cheV or cheW) impaired chemotactic motility but did not affect flagellar assembly or growth. The {Delta}cheY mutant swam in clockwise loops, while complementation restored normal motility. Inactivation of the core chemotaxis genes interfered with the ability to form a discrete biofilm at the air-media interface, and the {Delta}cheY mutant displayed reduced dispersal/shedding of bacteria into the planktonic fraction. This suggests that while the chemotaxis system is not required for biofilm formation per se, it is necessary for organized biofilm formation. Hence interference with the Campylobacter chemotaxis system at any level disrupts optimal chemotactic motility and transmission modes such as biofilm formation.

microbiology↗

bioRxiv2017

Cholecystokinin release triggered by presynaptic NMDA receptors produces LTP and sound-sound associative memory formation

Memory is stored in neural networks via changes in synaptic strength mediated in part by NMDA-dependent long-term potentiation (LTP). There is evidence that entorhinal cortex enables neocortical neuroplasticity through cholecystokinin (CCK)-containing neocortical projections. Here we show that a CCKB antagonist blocks high-frequency stimulation (HFS)-induced LTP in the auditory cortex, whereas local infusion of CCK induces LTP. CCK-/- mice lacked neocortical LTP and showed deficits in a cue-cue associative learning paradigm; administration of CCK rescued associative learning. HFS of CCK-containing entorhino-neocortical projection neurons in anesthetized mice enabled cue-cue associative learning. Furthermore, when one cue was pre-conditioned to footshock, the mouse showed a freezing response to the other cue, indicating that the mice had formed an association. HFS-induced neocortical LTP was completely blocked by either NMDA antagonist or CCK-BR antagonist, while application of either NMDA or CCK induced LTP after low-frequency stimulation (LFS). Moreover, in the presence of CCK LTP was still induced, even after blockade of NMDA receptors. Local application of NMDA induced CCK release in the neocortex. To identify how NMDA receptor switches LTP, a stimulation protocol of 25 pulse-pairs was adopted to replace HFS; NMDA-dependent LTP was induced with the inter-pulse intervals between 10 and 100 ms, but not with those of 5 and 200 ms. LTP-mediated plasticity was linked to localization of the NMDA receptor subunit NR2a on cortical CCK terminals originating in the entorhinal cortex. These novel findings suggest that presynaptic NMDA receptors on CCK terminals control the release of CCK, which enables neocortical LTP and formation of cue-cue associative memory.\n\nOne Sentence SummaryPresynaptic NMDA receptors switches the release of CCK from entorhinal neurons, which enables neocortical LTP and formation of sound-sound associative memory.

neuroscience↗

bioRxiv2017

Fishing for New Bt Receptors in Diamondback Moth

Bt toxins bind to receptors in the brush border membrane of the insect gut and create pores, leading to insect death. Bt-resistant insects demonstrate reduced binding of the Bt toxins to gut membranes. However, our understanding of the gut receptors involved in Bt toxin binding, and which receptors confer resistance to these toxins is incomplete, especially in diamondback moth (Plutella xylostella), a major agricultural pest. Identifying receptors has remained challenging because we lack sufficiently sensitive methods to detect Bt receptor interactions. Here, we report a modified far-immunoblotting technique, which revealed a broad spectrum of binding targets for the Bt toxins Cry1Ac, Cry1Ab, and Cry1Bd in diamondback moth. We confirm the role of the glucosinolate sulfatases GSS1 and GSS2 in Cry1Bd toxicity. GSS1 and GSS2 bind directly to Cry1Bd, and their expression is crucial for Cry1Bd toxicity. These results improve our understanding of the molecular mechanisms of Bt toxicity.\n\nAUTHOR SUMMARYThe Bt toxins, from the soil bacterium Bacillus thuringiensis, have wide applications in agriculture as insecticides applied to plants or expressed in genetically modified crops. Bt toxins bind to receptors in the brush border membrane of the insect gut and create pores leading to insect death. The success of the Bt toxins in controlling insect pests has been hindered by the emergence of resistant insects, which show reduced binding of Bt to their gut membranes. Although ongoing research has identified a few receptors, many remain unknown and the mechanisms by which these receptors cause resistance remain unclear. Here, we used a modified far-immunoblotting technique to identify proteins that bind to the toxins Cry1Ac, Cry1Ab, and Cry1Bd in the diamondback moth. This identified two glucosinolate sulfatases that bind directly to Cry1Bd; also, the toxicity of Cry1Bd requires expression of these glucosinolate sulfatases. Therefore, identification of these candidate receptors improves our understanding of Bt function and resistance.

biochemistry↗

bioRxiv2017

Accelerated human liver progenitor generation from pluripotent stem cells by inhibiting formation of unwanted lineages

Despite decisive progress in differentiating pluripotent stem cells (PSCs) into diverse cell-types, the often-lengthy differentiation and functional immaturity of such cell-types remain pertinent issues. Here we address the first challenge of prolonged differentiation in the generation of hepatocyte-like cells from PSCs. We delineate a roadmap describing the extracellular signals controlling six sequential branching lineage choices leading from pluripotency to endoderm, foregut, and finally, liver progenitors. By blocking formation of unwanted cell-types at each lineage juncture and manipulating temporally-dynamic signals, we accelerated generation of 89.0{+/-}3.1% AFP+ human liver bud progenitors and 87.3{+/-}9.4% ALBUMIN+ hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. 81.5{+/-}3.2% of hepatocyte-like cells expressed metabolic enzyme FAH (as assayed by a new knock-in reporter line) and improved short-term survival in the Fah-/-Rag2-/-Il2rg-/- mouse model of liver failure. Collectively the timed signaling interventions indicated by this developmental roadmap enable accelerated production of human liver progenitors from PSCs.

cell biology↗