Biology subjects

Lawrence, D. A.

Publications and source records attributed to Lawrence, D. A..

12 recordsLinked to original sources

bioRxiv2025

Comparison of a long-read amplicon sequencing approach to short-read amplicons for microbiome analysis

Most microbiome studies to date rely on sequencing short amplicons of the 16S rRNA gene on Illuminas platforms. Because of the short read length, sequences often can be identified reliably only to the family or genus levels. Long read sequencing with whole-length 16S rRNA sequencing can improve taxonomic resolution, but often only to the species level. StrainID is an alternative approach that amplifies a large segment of the ribosomal operon, including the entire 16S rRNA gene, internal transcribed spacer, and a portion of the 23S rRNA gene. This longer amplicon is designed to allow ribotype-level classification. Although studies have demonstrated the utility of StrainID for several sample types, it has not yet been validated for saliva. Here, we compared the performance of StrainID to short read amplicons with saliva samples as well as a synthetic mock DNA community and human and mouse fecal samples. Short reads were amplified with primer pairs appropriate for the corresponding sample type, and were classified with two different taxonomic databases. For both saliva and fecal samples, we found that StrainID performed similarly to short reads overall and demonstrated a key benefit with phylogenetic-based beta diversity tests and taxonomic classification. Our results further build on establishing StrainID as a valid method and specifically validate its use with saliva samples. ImportanceThe interpretation of microbiome composition studies is highly dependent on the methodologies chosen during experimental design, which affects factors such as resolution, throughput, cost, and accuracy. StrainID is an approach that can improve resolution while maintaining high-throughput and similar costs to short-read sequencing. The salivary microbiome represents a diverse community of microbes with links to a variety of health conditions and disease states. Closely related strains of bacteria can have drastically different effects on their host. Establishing StrainID as a valid approach for studying the salivary microbiome opens avenues for research that improve upon alternative methods by increasing sensitivity and accuracy compared to traditional short read approaches.

microbiology↗

bioRxiv2025

Occludin Acts as a Dynein Adaptor Regulating Permeability and Collateral Angiogenesis

Previous studies of the tight junction protein occludin (OCLN) suggest that multiple phosphorylation sites on the carboxy-terminal domain contribute a regulatory role in vascular barrier properties. However, gene deletion studies failed to identify a clear functional role for OCLN, despite multiple phenotypic alterations. Importantly, previous studies targeting exon 3 allowed expression of a splice variant starting at exon 4, (isoform 4) that expresses the full carboxy-terminal tail. Here we show that the OCLN carboxy-terminus forms a complex with the light intermediate chain (LIC) of dynein to link tight junction cargo to the minus end directed motor protein. Mutational analysis revealed S471 phosphorylation is required for binding to the LIC while S490 phosphorylation is required for trafficking. Expressing OCLN S490A mutant prevented endothelial cell proliferation and collateral angiogenesis. OCLN gene deletion targeting exon 5, preventing full length and isoform 4 expression, resulted in embryonic lethality. In summary, OCLN links tight junction cargo to the dynein motor, regulating trafficking in a phosphorylation dependent manner and contributing to both VEGF-induced vascular permeability and collateral angiogenesis.

cell biology↗

bioRxiv2024

Harnessing micrometer-scale tPA beads for high plasmin flux and accelerated fibrinolysis

Rapid restoration of blood flow is critical in treating acute ischemic stroke. Current fibrinolytic therapies using tissue plasminogen activator (tPA) are limited by low recanalization rates and risks of off-target bleeding. Here, we present a strategy using tPA immobilized on micrometer-scale beads to enhance local plasmin generation. We synthesized tPA-functionalized beads of varying sizes (0.1 m and 1.0 m) and evaluated their efficacy. In vitro assays demonstrated that 1.0 m tPA-beads generated higher plasmin generation compared to free tPA and 0.1 m beads, overcoming antiplasmin inhibition and promoting a self-propagating wave of fibrinolysis. In a murine model of acute ischemic stroke, intravenous administration of 1.0 m tPA-beads at doses nearly two orders of magnitude lower than the standard free tPA dose led to rapid and near-complete thrombus removal within minutes. This approach addresses kinetic and transport limitations of current therapies and may reduce the risk of hemorrhagic complications. TeaserMicrometer-scale tPA beads improve stroke treatment by enhancing local clot-dissolving action.

bioengineering↗

bioRxiv2024

TRAIL induces caspase-dependent cleavage of specific components of the clathrin-mediated endocytosis machinery

It has been reported that caspase activation by the proapoptotic ligand Apo2L/TRAIL disrupts the clathrin-mediated endocytosis machinery (CMEM). To confirm whether TRAIL induces caspase-mediated cleavage of specific CMEM components, we examined the temporal and functional relationship between TRAIL-induced processing of the apoptosis-initiating protease, caspase-8, and the apoptosis-executing protease, caspase-3, versus cleavage of specific adaptin (AP) and clathrin heavy chain (CHC) proteins. TRAIL induced time-dependent proteolytic processing of caspase-8 and caspase-3, which coincided with cleavage of AP2 and CHC. The pan caspase inhibitor zVAD-FMK, which blocked caspase processing in response to TRAIL, also prevented the cleavage AP2. Whereas FADD-deficient or caspase-8-deficient cells showed little or no TRAIL-driven cleavage of either AP2 or CHC, Bax-deficient or caspase-3-deficient cell lines retained AP2 cleavage but failed to cleave CHC in response to TRAIL. The DNA-damaging agent doxorubicin also led to processing of caspase-8 and caspase-3 in conjunction ith cleavage of AP2 and CHC. Together, these results confirm that TRAIL induces caspase-dependent cleavage of AP2 and CHC. Whereas TRAIL-driven cleavage of both AP2 and CHC requires caspase-8, cleavage of CHC, but not of AP2, requires caspase-3. MATERIALS AND METHODS Cell lines and cell cultureAll cell lines were obtained from ATCC or as kind gifts from Dr. Bert Vogelstein (HCT116 Bax-/-) and Dr. John Blenis (Jurkat I9.2 and E1). Cell lines were cultured with standard media as previously described (1). Immunoblot analysisCells were lysed in RIPA lysis buffer (20-188, Millipore) supplemented with Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific) and kept on ice for 30 min. Lysates were cleared by centrifugation at 13,600 x g for 15 min at 4 {degrees}C, and protein amount was determined by BCA protein assay (ThermoFisher Scientific). Protein was denatured by adding NuPAGE LDS buffer and DTT reducing buffer (Invitrogen) and incubating the samples at 95 {degrees}C for 5 min. Equal amounts of denatured protein were loaded into each well of NuPAGE pre-cast gels (Invitrogen), resolved by SDS-PAGE, and electro-transferred to nitrocellulose membranes using the iBLOT2 system (Invitrogen). Membranes were blocked in a 5% nonfat milk solution for 1 hr at room temperature and probed with the corresponding primary antibody at 1:1,000 dilution overnight at 4 {degrees}C. This was followed by incubation with the corresponding horseradish peroxidase (HRP)- conjugated secondary antibody at 1:10,000 dilution during 1 hr at room temperature. All secondary antibodies were from Jackson Laboratories. The primary antibodies and secondary HRP-conjugated antibodies are listed respectively in Table 1 and Table 2. {beta}-actin IB was used to verify uniformity of protein loading and electro-transfer. O_TBL View this table: org.highwire.dtl.DTLVardef@ed5a65org.highwire.dtl.DTLVardef@c9aecdorg.highwire.dtl.DTLVardef@f31b4aorg.highwire.dtl.DTLVardef@849deaorg.highwire.dtl.DTLVardef@9b847b_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONPrimary antibodies used for IB analysis C_TABLECAPTION C_TBL O_TBL View this table: org.highwire.dtl.DTLVardef@16c33forg.highwire.dtl.DTLVardef@6c6b81org.highwire.dtl.DTLVardef@5efb4org.highwire.dtl.DTLVardef@1bc354forg.highwire.dtl.DTLVardef@15007aa_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 2.C_FLOATNO O_TABLECAPTIONHorseradish peroxidase conjugated secondary antibodies used for IB analysis C_TABLECAPTION C_TBL Additional reagentsRecombinant soluble non-tagged human TRAIL (Apo2L.0) and Flag-tagged TRAIL were prepared at Genentech. Flag-tagged TRAIL was crosslinked with anti-Flag M2 antibody at a ligand-to-antibody molar ratio of 1:2. zVAD-FMK was purchased from R & D Systems, FMK001. Doxorubicin was purchased from Sigma Aldrich, D1515.

cell biology↗

bioRxiv2024

Thrombolysis exacerbates cerebrovascular injury after ischemic stroke via a VEGF-B dependent effect on adipose lipolysis

Cerebrovascular injuries leading to edema and hemorrhage after ischemic stroke are common. The mechanisms underlying these events and how they are connected to known risk factors for poor outcome, like obesity and diabetes, is relatively unknown. Herein we demonstrate that increased adipose tissue lipolysis is a dominating risk factor for the development of a compromised cerebrovasculature in ischemic stroke. Reducing adipose lipolysis by VEGF-B antagonism improved vascular integrity by reducing ectopic cerebrovascular lipid deposition. Thrombolytic therapy in ischemic stroke using tissue plasminogen activator (tPA) leads to increased risk of hemorrhagic complications, substantially limiting the use of thrombolytic therapy. We provide evidence that thrombolysis with tPA promotes adipose tissue lipolysis, leading to a rise in plasma fatty acids and lipid accumulation in the ischemic cerebrovasculature after stroke. VEGF-B blockade improved the efficacy and safety of thrombolysis suggesting the potential use of anti-VEGF-B therapy to extend the therapeutic window for stroke management.

neuroscience↗

bioRxiv2024

PAI-1 Interaction with Sortilin Related Receptor-1 is Required for Lung Fibrosis

Plasminogen activator inhibitor-1 (PAI-1) has been previously shown to promote lung fibrosis via a mechanism that requires an intact vitronectin (VTN) binding site. In the present study, employing two distinct murine fibrosis models, we find that VTN is not required for PAI-1 to drive lung scarring. This result suggested the existence of a previously unrecognized profibrotic PAI-1-protein interaction involving the VTN-binding site for PAI-1. Using an unbiased proteomic approach, we identified sortilin related receptor 1 (SorlA) as the most highly enriched PAI-1 interactor in the fibrosing lung. We next investigated the role of SorlA in pulmonary fibrosis and found that SorlA deficiency protected against lung scarring in a murine model. We further show that, while VTN deficiency does not influence fibrogenesis in the presence or absence of PAI-1, SorlA is required for PAI-1 to promote scarring. These results, together with data showing increased SorlA levels in human IPF lung tissue, support a novel mechanism through which the potent profibrotic mediator PAI-1 drives lung fibrosis and implicate SorlA as a new therapeutic target in IPF treatment.

pathology↗

bioRxiv2022

Deep mutational scanning and massively parallel kinetics of plasminogen activator-1 functional stability

Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (SERPIN) superfamily of proteins, is unique among SERPINs for exhibiting a spontaneous conformational change to a latent or inactive state. The functional half-life for this transition at physiologic temperature and pH is ~1-2 h. To better understand the molecular mechanisms underlying this transition, we now report on the analysis of a comprehensive PAI-1 variant library expressed on filamentous phage and selected for functional stability after 48 h at 37 {degrees}C. Of the 7,201 possible single amino acid substitutions in PAI-1, we identify 439 that increase the functional stability of PAI-1 beyond that of the wild-type protein and 1,549 that retain inhibitory activity toward PAI-1s canonical target protease (urokinase-like plasminogen activator, uPA), while exhibiting functional stability less than or equal to that of wild-type PAI-1. Missense mutations that increase PAI-1 functional stability are concentrated in highly flexible regions within the PAI-1 structure. Finally, we developed a method for simultaneously measuring the functional half-lives of hundreds of PAI-1 variants in a multiplexed, massively parallel manner, quantifying the functional half-lives for 697 single missense variants of PAI-1 by this approach. Overall, these findings provide novel insight into the mechanisms underlying PAI-1s latency transition and provide a database for interpreting human PAI-1 genetic variants.

biochemistry↗

bioRxiv2022

Long range allostery mediates the regulation of plasminogen activator inhibitor 1 by vitronectin

The serpin plasminogen activator inhibitor 1 (PAI-1) spontaneously undergoes a massive structural change from a metastable, active conformation, with a solvent accessible reactive center loop (RCL), to a stable, inactive or latent conformation in which the RCL has inserted into the central {beta} sheet. Physiologically, conversion to the latent state is regulated by the binding of vitronectin which retards the rate of this latency transition approximately 2-fold. We investigated the effects of vitronectin on the PAI-1 latency transition using all-atom path sampling simulations in explicit solvent. In simulated latency transitions of free PAI-1, the RCL is quite mobile as is the gate, the region that impedes RCL access to the central {beta} sheet. This mobility allows the formation of a transient salt bridge that facilitates the transition, and this finding rationalizes existing mutagenesis results. Vitronectin binding reduces RCL and gate mobility by allosterically rigidifying structural elements over 40 [A] away from the binding site thus blocking the transition to the latent conformation. The effects of vitronectin are propagated by a network of dynamically correlated residues including a number of conserved sites that have previously been identified as important for PAI-1 stability. Simulations also revealed a transient pocket populated only in the vitronectin bound state which corresponds to a cryptic drug binding site identified by crystallography. Overall, these results shed new light on regulation of the PAI-1 latency transition by vitronectin and illustrate the potential of path sampling simulations for understanding functional conformational changes in proteins and for facilitating drug discovery.

biophysics↗

bioRxiv2022

Altered meningeal immunity contributing to the autism-like behavior of BTBR T+ Itpr3tf/J mice.

Autism spectrum disorder (ASD) is a complicated neurodevelopmental disorder, which is categorized by deficiency of social contact and communication, and stereotyped forms of performance. Meningeal immunity conditions the immune reflection and immune defense in the meningeal area involving meningeal lymphatic organization, glymphatic structure, immune cells, and cytokines. The development of meningeal immunity dysfunction might be the leading cause for many neural diseases including ASD. The inbred mouse strain BTBRT+Itpr3tf/J (BTBR) shows multiple ASD-like behavioral phenotypes, thus making this strain a widely used animal model for ASD. In our previous study, we reported an altered peripheral immune profile in BTBR mice. Herein, we are investigating immunological and neural interactions associated with the aberrant behavior of BTBR mice. BTBR mice have an increased level of immune cell deposition in the meninges along with a higher level of CD4+ T cells expressing CD25 and of B and myeloid cells expressing more MHCII than C57BL/6 (B6) mice, which have normal behaviors. BTBR mice also have higher levels of autoantibodies to dsDNA, Aquaporin-4, NMDAR1, Pentraxin/SAP and Caspr2 than B6 mice, which may affect neural functions. Interestingly, the T regulatory (Treg) cell population and their function was significantly reduced in the meninges and brain draining lymph nodes, which may explain the increased level of activated B and T cells in the meninges of BTBR mice. A low level of Treg cells, less IL-10 production by Treg, and activated T and B cells in meninges together with higher autoantibody levels might contribute to the development of autism-like behavior through neuroinflammation, which is known to be increased in BTBR mice. HighlightsO_LIBTBR mice have higher level of immune cell deposition in the meninges compared to C57BL/6 (B6) mice. C_LIO_LIMeningeal T cells and B cells of BTBR mice express a higher level of CD25 and MHCII, respectively, than those of B6 mice. C_LIO_LIBTBR mice have a higher level of serum autoantibodies to dsDNA and brain antigens (Aquaporin-4, NMDAR1, Pentraxin/SAP and Caspr2) than B6 mice. C_LIO_LIT regulatory (Treg) cell population was reduced in the meninges and brain draining lymph nodes of BTBR mice with lower cytokine production of IL-10. C_LIO_LIFewer Treg cells and more activated meningeal T and B cells together with higher autoantibody levels might contribute to the development of the autism-like behavior of BTBR mice. C_LI

immunology↗

bioRxiv2021

Reduced myofibroblast transdifferentiation and fibrotic scarring in ischemic stroke after imatinib treatment

The tyrosine kinase inhibitor imatinib has been reported to improve outcome in patients following ischemic stroke but the exact mechanism remains elusive. Here, utilizing a photothrombotic murine model of middle cerebral artery occlusion (MCAO), we show that imatinib-mediated inhibition of stroke-induced blood-brain barrier (BBB) dysfunction coincided with decreased expression of genes associated with inflammation and fibrosis in the cerebrovasculature. We found that imatinib effectively dampened stroke-induced reactive gliosis and myofibroblast transdifferentiation, whilst having very limited effect on the rest of the glia scar and on peripheral leukocyte infiltration. Further, our data suggest that consolidation of the PDGFR+ portion of the fibrotic scar in imatinib-treated mice contributes to the improvement in functional outcome compared to the vehicle controls, where the PDGFR+ scar is expanding. Comparison with human stroke transcriptome databases revealed significant overlap with imatinib-regulated genes, suggesting imatinib may modulate reactive gliosis and fibrotic scarring also in human stroke.

neuroscience↗

bioRxiv2021

Antigen-derived peptides directly engage the unfolded-protein sensor IRE1α to curb cross-presentation by dendritic cells

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER) and upload onto major histocompatibility type I (MHC-I) protein complexes for cell-surface transport and cross-presentation. Perplexingly, DCs often exhibit activation of the ER-stress sensor IRE1 in the absence of classical ER stress--leaving the underlying mechanism unexplained. Here we show that antigen-derived hydrophobic peptides directly engage ER-resident IRE1 by masquerading as unfolded proteins. Furthermore, IRE1 activation depletes MHC-I heavy-chain mRNAs through regulated IRE1-dependent decay (RIDD), thereby curtailing antigen cross-presentation. In tumor-bearing mice, IRE1 disruption increased MHC-I expression on tumor-infiltrating DCs, and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1 inhibition synergized with anti-PD-L1 antibody treatment to cause tumor regression. Our findings elucidate the mechanism and consequence of antigen-driven IRE1 activation in DCs, yielding a promising combination strategy for cancer immunotherapy.

cell biology↗

bioRxiv2018

Disruption of IRE1α through its Kinase Domain Attenuates Multiple Myeloma

Multiple myeloma (MM) arises from malignant immunoglobulin-secreting plasma cells and remains an incurable, often lethal disease despite recent therapeutic advances. The unfolded-protein response sensor IRE1 supports protein secretion by deploying a kinase-endoribonuclease module to activate the transcription factor XBP1s. MM cells may coopt the IRE1-XBP1s pathway; however, the validity of IRE1 as a potential MM therapeutic target is controversial. Here we show that genetic disruption of IRE1 or XBP1s, or pharmacologic IRE1 kinase inhibition, attenuated subcutaneous or orthometastatic growth of MM tumors in mice, and augmented efficacy of two well-established frontline antimyeloma agents, bortezomib or lenalidomide. Mechanistically, IRE1 perturbation inhibited expression of key components of the ER-associated degradation machinery, as well as cytokines and chemokines known to promote MM growth. Selective IRE1 kinase inhibition reduced viability of CD138+ plasma cells while sparing CD138- cells from bone marrow of newly diagnosed MM patients or patients whose disease relapsed after 1 - 4 lines of treatment in both US- and EU-based cohorts. IRE1 inhibition preserved survival and glucose-induced insulin secretion by pancreatic microislets. Together, these results establish a strong therapeutic rationale for targeting IRE1 with kinase-based small-molecule inhibitors in MM.\n\nSignificance statementMultiple myeloma (MM) is a lethal malignancy of plasma cells. MM cells have an expanded endoplasmic reticulum (ER) that is constantly under stress due to immunoglobulin hyperproduction. The ER-resident sensor IRE1 mitigates ER stress by expanding the ERs protein-folding capacity while supporting proteasomal degradation of misfolded ER proteins. IRE1 elaborates these functions by deploying its cytoplasmic kinase-RNase module to activate the transcription factor XBP1s. The validity of IRE1 as a potential therapeutic target in MM has been questioned. Using genetic and pharmacologic disruption in vitro and in vivo, we demonstrate that the IRE1-XBP1s pathway plays a critical role in MM growth. We further show that IRE1s kinase domain is an effective and safe potential small-molecule target for MM therapy.

cancer biology↗