Biology subjects

Smith, J. D.

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

33 records · Page 2Linked to original sources

bioRxiv2021

Gasdermin D mediates inflammation-induced defects in reverse cholesterol transport and promotes atherosclerosis.

Activation of inflammasomes, such as Nlrp3 and Aim2, can exacerbate atherosclerosis in mice and humans. Gasdermin D (GsdmD) serves as a final executor of inflammasome activity, by generating membrane pores for the release of mature Interleukin-1beta (IL-1{beta}). Inflammation dampens reverse cholesterol transport (RCT) and promotes atherogenesis, while anti-IL-1{beta} antibodies were shown to reduce cardiovascular disease in humans. Though Nlrp3/AIM2 and IL-1{beta} nexus is an emerging atherogenic pathway, the direct role of GsdmD in atherosclerosis is not yet fully clear. Here, we used in-vivo Nlrp3 inflammasome activation to show that the GsdmD-/- mice release ~80% less IL-1{beta} vs WT mice. The GsdmD-/- macrophages were more resistant to Nlrp3 inflammasome mediated reduction in cholesterol efflux, showing ~26% decrease vs. ~60% reduction in WT macrophages. GsdmD expression in macrophages exacerbated foam cell formation in an IL-1{beta} dependent fashion. The GsdmD-/- mice were resistant to Nlrp3 inflammasome mediated defect in RCT, with ~32% reduction in plasma RCT vs. ~ 57% reduction in WT mice, ~ 17% reduction in RCT to liver vs. 42% in WT mice, and ~ 37% decrease in RCT to feces vs. ~ 61% in WT mice. The LDLr anti-sense oligonucleotides (ASO) induced hyperlipidemic mouse model showed the role of GsdmD in promoting atherosclerosis. The GsdmD-/- mice exhibit ~42% decreased atherosclerotic lesion area in females and ~33% decreased lesion area in males vs. WT mice. The atherosclerotic plaque-bearing sections stained positive for the cleaved N-terminal fragment of GsdmD, indicating cleavage of GsdmD in atherosclerotic plaques. Our data show that GsdmD mediates inflammation-induced defects in RCT and promotes atherosclerosis. SummaryGsdmD mediates inflammation-induced defects in RCT and promotes atherosclerosis.

pathology↗

bioRxiv2021

Genetic Variant in 3' Untranslated Region of the Mouse Pycard Gene Regulates Inflammasome Activity

Quantitative trait locus mapping for interleukin-1{beta} release after inflammasome priming and activation was performed on bone marrow-derived macrophages (BMDM) from an AKRxDBA/2 strain intercross. The strongest associated locus mapped very close to the Pycard gene on chromosome 7, which codes for the inflammasome adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). The DBA/2 and AKR Pycard genes only differ at single nucleotide polymorphism (SNP) in their 3 untranslated region (UTR). DBA/2 vs. AKR BMDM had increased levels of Pycard mRNA expression and ASC protein, and increased inflammasome speck formation, which was associated with increased Pycard mRNA stability without an increased transcription rate. CRIPSR/Cas9 gene editing was performed on DBA/2 embryonic stem cells to change the Pycard 3UTR SNP from the DBA/2 to the AKR allele. This single base change significantly reduced Pycard expression and inflammasome activity after cells were differentiated into macrophages due to reduced Pycard mRNA stability.

genetics↗

bioRxiv2021

Losartan Increases Survival of the Fbn1mgR/mgR Mouse Model of Marfan Syndrome in and Age-Dependent Manner

Clinical trials investigating angiotensin receptor blockers (ARB) for attenuation of thoracic aortic aneurysm in people with Marfan syndrome have demonstrated variable efficacy. The primary objective of this study was to determine whether the age of mice at the time of losartan initiation affected mortality in fibrillin-1 hypomorphic (Fbn1mgR/mgR) mice. Male (n=40) and female (n=28) Fbn1mgR/mgR mice were randomized to receive losartan in drinking water (0.6 g/L) starting at either 24 or 50 days of age. Controls included Fbn1mgR/mgR mice (20M, 14F) and wild type (15M, 15F) littermates who were not administered the drug. Mortality of Fbn1mgR/mgR males receiving losartan at postnatal day 24 (P24) was not different from wild type controls (p=0.138). Survival of Fbn1mgR/mgR males administered losartan at P50 was not different compared to Fbn1mgR/mgR males receiving no drug (p=0.194) and decreased compared to wild type mice (p=0.002). Survival analysis after P50 demonstrated increased survival of Fbn1mgR/mgR males administered losartan at P50 compared to Fbn1mgR/mgR mice receiving no drug (p=0.017). Age is a critical variable that affects the therapeutic efficacy of losartan in male Fbn1mgR/mgR mice. Since overall mortality in female Fbn1mgR/mgR mice was lower than in male Fbn1mgR/mgR mice, a survival benefit with losartan was not detected in females.

physiology↗

bioRxiv2021

Benchmarking Association Analyses of Continuous Exposures with RNA-seq in Observational Studies

Large datasets of hundreds to thousands of individuals measuring RNA-seq in observational studies are becoming available. Many popular software packages for analysis of RNA-seq data were constructed to study differences in expression signatures in an experimental design with well-defined conditions (exposures). In contrast, observational studies may have varying levels of confounding of the transcript-exposure associations; further, exposure measures may vary from discrete (exposed, yes/no) to continuous (levels of exposure), with non-normal distributions of exposure. We compare popular software for gene expression - DESeq2, edgeR, and limma - as well as linear regression-based analyses for studying the association of continuous exposures with RNA-seq. We developed a computation pipeline that includes transformation, filtering, and generation of empirical null distribution of association p-values, and we apply the pipeline to compute empirical p-values with multiple testing correction. We employ a resampling approach that allows for assessment of false positive detection across methods, power comparison, and the computation of quantile empirical p-values. The results suggest that linear regression methods are substantially faster with better control of false detections than other methods, even with the resampling method to compute empirical p-values. We provide the proposed pipeline with fast algorithms in R.

bioinformatics↗

bioRxiv2020

The mitochondrial surface receptor Tom70 protects the cytosol against mitoprotein-induced stress

Most mitochondrial proteins are synthesized as precursors in the cytosol and post-translationally transported into mitochondria. The mitochondrial surface protein Tom70 acts at the interface of the cytosol and mitochondria. In vitro import experiments identified Tom70 as targeting receptor, particularly for hydrophobic carriers. Using in vivo methods and high content screens, we revisited the question of Tom70 function and considerably expanded the set of Tom70-dependent mitochondrial proteins. We demonstrate that the crucial activity of Tom70 is its ability to recruit cytosolic chaperones to the outer membrane. Indeed, tethering an unrelated chaperone-binding domain onto the mitochondrial surface complements most of the defects caused by Tom70 deletion. Tom70-mediated chaperone recruitment reduces the proteotoxicity of mitochondrial precursor proteins, in particular of hydrophobic inner membrane proteins. Thus, our work suggests that the predominant function of Tom70 is to tether cytosolic chaperones to the outer mitochondrial membrane, rather than to serve as a mitochondria-specifying targeting receptor.

cell biology↗

bioRxiv2020

Gene dosage screens in yeast reveal core signalling pathways controlling heat adaptation

Heat stress causes proteins to unfold and lose their function, jeopardizing essential cellular processes. To protect against heat and proteotoxic stress, cells mount a dedicated stress-protective programme, the so-called heat shock response (HSR). Our understanding of the mechanisms that regulate the HSR and their contributions to heat resistance and growth is incomplete. Here we employ CRISPRi/a to down- or upregulate protein kinases and transcription factors in S. cerevisiae. We measure gene functions by quantifying perturbation effects on HSR activity, thermotolerance, and cellular fitness at 23, 30 and 38{degrees}C. The integration of these phenotypes allowed us to identify core signalling pathways of heat adaptation and reveal novel functions for the high osmolarity glycerol, unfolded protein response and protein kinase A pathways in adjusting both thermotolerance and chaperone expression. We further provide evidence for unknown cross-talk of the HSR with the cell cycle-dependent kinase Cdc28, the primary regulator of cell cycle progression. Finally, we show that CRISPRi efficiency is temperature-dependent and that different phenotypes vary in their sensitivity to knock-down. In summary, our study quantifies regulatory gene functions in different aspects of heat adaptation and advances our understanding of how eukaryotic cells counteract proteotoxic and other heat-caused damage.

genetics↗

bioRxiv2020

Lipidated poly(amino acid) nanostructures as versatile therapeutic delivery vehicles

Poly(amino acid)s are a diverse and capable class of polymers with significant potential for utilization in a wide variety of drug delivery applications. A sub-class of these biomaterials known as lipidated poly(amino acid)s (LPAAs) are amphiphiles composed of both hydrophobic and hydrophilic domains yielding interesting physical properties. In this article, we describe our efforts in developing a novel class of lysine and valine containing LPAAs synthesized via hexadecylamine initiated N-carboxyanhydride ring-opening polymerization (NCA-ROP). These highly hydrophobic LPAAs were found capable of undergoing hydrophobically-driven self-assembly into small nanostructures as well as being forced into larger nanostructures using a novel dump-and-stir nanoprecipitation process. This process yielded fine control over resulting nanoparticle size and cargo entrapment. Furthermore, cell-targeting DNA aptamer modification of doxorubicin-loaded LPAA nanoparticles induced significant death of co-incubated Non-Hodgkin Lymphoma cells providing exciting evidence of the therapeutic potential of this novel biomaterials-based delivery device.

bioengineering↗

bioRxiv2020

SR-B1 uptake of HDL promotes prostate cancer proliferation and tumor progression

High density lipoprotein (HDL) metabolism, in part, is facilitated by scavenger receptor class B, type 1 (SR-B1) that mediates its uptake into cells. SR-B1 is upregulated in prostate cancer tissue. Here, we report that knockout (KO) of SR-B1 via CRISPR/Cas9 editing led to reduced HDL uptake into prostate cancer cells, and reduced their proliferation in response to HDL. In vivo studies using syngeneic SR-B1 wildtype (SR-B1+/+) and SR-B1 KO (SR-B1-/-) prostate cancer cells in WT and apolipoprotein-AI KO (apoA1-KO) C57BL/6J mice showed that WT hosts, containing higher levels of total and HDL-cholesterol, grew larger tumors than apoA1-KO hosts with lower levels of total and HDL-cholesterol. Furthermore, SR-B1-/- prostate cancer cells formed smaller tumors in WT hosts, than SR-B1+/+ cells in same host model. Tumor volume data was overall similar to survival data. We conclude that tumoral SR-B1 KO reduced HDL-mediated increases in prostate cancer cell proliferation and disease progression.

cancer biology↗

bioRxiv2019

First eight residues of apolipoprotein A-I mediate the C-terminus control of helical bundle unfolding and its lipidation

The crystal structure of a C-terminal deletion of apolipoprotein A-I (apoA1) shows a large helical bundle structure in the amino half of the protein, from residues 8 to 115. Using site directed mutagenesis, guanidine or thermal denaturation, cell free liposome clearance, and cellular ABCA1-mediated cholesterol efflux assays, we demonstrate that apoA1 lipidation can occur when the barrier to this bundle unfolding is lowered. The absence of the C-terminus renders the bundle harder to unfold resulting in loss of apoA1 lipidation that can be reversed by point mutations, such as Trp8Ala, and by truncations as short as 8 residues in the amino terminus, both of which lower the barrier to helical bundle unfolding. Locking the bundle via a disulfide bond leads to loss of apoA1 lipidation. We propose a model in which the C-terminus acts on the N-terminus to destabilize helical bundle. Upon lipid binding to the C-terminus, Trp8 is displaced from its interaction with Phe57, Arg61, Leu64, Val67, Phe71, and Trp72 to destabilize the bundle. However, when the C-terminus is deleted, Trp8 cannot be displaced, the bundle cannot unfold, and apoA1 cannot be lipidated.

biochemistry↗

bioRxiv2019

Causal SNP regulating FAM13B expression identified for the Chr. 5q31 atrial fibrillation susceptibility loc

RationaleOur prior RNA sequencing study found that FAM13B gene expression in human left atrial appendages was strongly associated with an atrial fibrillation (AF) susceptibility-associated variant on chr. 5q31.\n\nObjectiveTo identify the common genetic variant responsible for regulating FAM13B expression and the effect of FAM13B expression on cardiomyocyte gene expression in order to gain insight into the functional mechanism of the chr. 5q31 AF susceptibility locus.\n\nMethods and ResultsBy taking advantage of a smaller linkage disequilibrium block in African descent subjects and available chromatin conformation data, we identified the common single nucleotide polymorphism (SNP) rs17171731 as a candidate genetic variant controlling FAM13B gene expression in the left atrium. Functional analysis demonstrated that the AF risk allele of rs17171731 had less enhancer activity than the protective allele. Gel mobility shift studies determined that the risk allele bound to an additional protein that may function as a transcriptional repressor. Knockdown of FAM13B expression in stem cell-derived human cardiomyocytes (iCM) altered the expression of >1000 genes and modified the sodium current, consistent with increased susceptibility to atrial fibrillation. Transfection of GFP tagged FAM13B into iCMs demonstrated expression on the plasma membrane and at the Z-disk.\n\nConclusionsThe chr. 5q31 AF risk variant was identified as rs17171731, with the risk allele having less enhancer activity, leading to decreased expression of FAM13B, which resides on the plasma membrane and the Z-disk, and appears to play a role in the regulation of cardiomyocyte gene expression and the late sodium current.

genomics↗

bioRxiv2019

Atrial fibrillation associated common risk variants in SYNE2 lead to lower expression of nesprin-2α1 and increased nuclear stiffness

RationaleAtrial fibrillation (AF) genome-wide association studies (GWAS) identified significant associations for rs1152591 and linked variants in the SYNE2 gene encoding the nesprin-2 protein that connects the nuclear membrane with the cytoskeleton\n\nObjectiveDetermine the effects of the AF-associated rs1152591 and rs1152595, two linked intronic single nucleotide polymorphisms (SNPs), on SYNE2 expression and investigate the mechanisms for their association with AF.\n\nMethods and ResultsRNA sequencing of human left atrial appendage (LAA) tissues indicated that rs1152591 and rs1152595 were significantly associated with the expressions of SYNE21, a short mRNA isoform, without an effect on the expression of the full-length SYNE2 mRNA. SYNE21 mRNA uses an alternative transcription start site and encodes an N-terminal deleted 62 kDa nesprin-21 isoform, which can act as a dominant-negative on nuclear-cytoskeleton connectivity. Western blot and qPCR assays confirmed that AF risk alleles of both SNPs were associated with lower expression of nesprin-21 in human LAA tissues. Reporter gene transfections demonstrated that the risk vs. reference alleles of rs1152591 and rs1152595 had decreased enhancer activity. SYNE2 siRNA knockdown (KD) or nesprin-21 overexpression studies in human stem cell-derived induced cardiomyocytes (iCMs) resulted in ~12.5 % increases in the nuclear area compared to controls (p<0.001). Atomic force microscopy demonstrated that SYNE2 KD or nesprin-21 overexpression led to 57.5% or 33.2% decreases, respectively, in nuclear stiffness compared to controls (p< 0.0001).\n\nConclusionsAF-associated SNPs rs1152591 and rs1152595 downregulate the expression of SYNE21, increasing nuclear-cytoskeletal connectivity and nuclear stiffness. The resulting increase in mechanical stress may play a role in the development of AF.

genomics↗

bioRxiv2019

QTL analysis of macrophages from an AKR/JxDBA/2J intercross identified the Gpnmb gene as a modifier of lysosome function

Our prior studies found differences in the AKR/J and DBA/2J strains in regard to atherosclerosis and macrophage phenotypes including cholesterol ester loading, cholesterol efflux, and autolysosome formation. The goal of this study was to determine if there were differences in macrophage lysosome function, and if so to use quantitative trait locus (QTL) analysis to identify the causal gene. Lysosome function was measured by incubation with an exogenous double-labeled ovalbumin indicator sensitive to proteolysis. DBA/2J vs. AKR/J bone marrow macrophages had significantly decreased lysosome function. Macrophages were cultured from 120 mice derived from an AKR/JxDBA/2J F4 intercross. We measured lysosome function and performed a high density genome scan. QTL analysis yielded two genome wide significant loci on chromosomes 6 and 17, called macrophage lysosome function modifier (Mlfm) loci Mlfm1 and Mlfm2. After adjusting for Mlfm1, two additional loci were identified. Based on proximity to the Mlfm1 peak, macrophage mRNA expression differences with AKR/J >> DBA/2J, and a protein coding nonsense variant in DBA/2J, the Gpnmb gene, encoding a lysosomal membrane protein, was our top candidate. To test this candidate, Gpnmb expression was knocked down with siRNA in AKR/J macrophages; and, to express the wildtype Gpnmb in DBA/2J macrophages, we obtained a DBA/2 substrain, DBA/2J-Gpnmb+/SjJ, which was isolated from the parental strain prior to its acquiring the nonsense mutation, and subsequently back crossed to the modern DBA/2J background. Knockdown of Gpnmb in AKR/J macrophages decreased lysosome function, while restoration of the wildtype Gpnmb allele in DBA/2J macrophages increased lysosome function. However, this modifier of lysosome function was not responsible for the strain differences in macrophage cholesterol ester loading or cholesterol efflux. In conclusion, we identified the Gpnmb gene as the major modifier of lysosome function and we showed that the QTL in a dish strategy is efficient in identifying modifier genes.\n\nAuthor SummaryInbred strains of mice differ in both their genetic backgrounds as well as in many traits; and, classical mouse genetics allows the mapping of genes responsible for these traits. We identified many traits that differ between the inbred strains AKR/J and DBA/2J, including atherosclerosis susceptibility, macrophage cholesterol metabolism, and in the current study, macrophage protein degradation via an organelle called the lysosome. Using mouse genetic mapping and bioinformatics we identified a candidate gene, called Gpnmb, responsible for modifying lysosome function; and, the DBA/2J strain carries a mutation in this gene. Here we demonstrate that the Gpnmb gene is a modifier of lysosome function by either correcting this Gpnmb mutation in DBA/2J macrophages, or by knocking down Gpnmb expression in AKR/J macrophages. This study is noteworthy as the human GPNMB gene has been implicated in many diseases including cancer, kidney injury, obesity, non-alcoholic steatohepatitis, Parkinson disease, osteoarthritis, and lysosome storage disorders.

genetics↗

bioRxiv2019

MBOAT7-Driven Phosphatidylinositol Remodeling Promotes the Progression of Clear Cell Renal Cell Carcinoma.

The most common kidney cancer, clear cell Renal Cell Carcinoma (ccRCC) is closely associated with obesity. In fact, the \"clear cell\" variant of RCC is given this name due to large lipid droplets within the tumor cells. Although it is well appreciated that renal lipid metabolism is altered in ccRCC, the mechanisms driving this are not well understood. Leveraging a shotgun lipidomics approach we have identified a lipid signature for ccRCC that includes an increase in arachidonic acid-enriched phosphatidylinositols (AA-PI). In parallel, we found that ccRCC tumors have increased expression of the acyltransferase enzyme membrane bound O-acyltransferase domain containing 7 (MBOAT7) that contributes to AA-PI synthesis. In ccRCC patients, MBOAT7 expression increases with tumor grade, and increased MBOAT7 expression correlates with poor survival. Genetic deletion of MBOAT7 in ccRCC cells decreases proliferation and induces cell cycle arrest, and MBOAT7-/- cells fail to form tumors in vivo. RNAseq of MBOAT7-/- cells identified alterations in cell migration and extracellular matrix organization, which were functionally validated in migration assays. Our work highlights the accumulation of AA-PI in ccRCC and demonstrate a novel way to decrease the AA-PI pool in ccRCC by limiting MBOAT7. Our data reveal that metastatic ccRCC is associated with altered AA-PI metabolism, and identify MBOAT7 as a novel target in advanced ccRCC.

cancer biology↗

bioRxiv2018

Miltefosine increased cholesterol efflux, induced autophagy and inhibited NLRP3-inflammasome assembly and IL-1β release.

Miltefosine is an FDA approved oral drug for treating cutaneous and visceral leishmaniasis. Leishmania is a flagellated protozoa, which infects and differentiates in macrophages. Here, we studied the effects of Miltefosine on macrophages lipid homeostasis, autophagy, and NLRP3 inflammasome assembly/activity. Miltefosine treatment conferred multiple effects on macrophage lipid homeostasis leading to increased cholesterol release from cells, increased lipid-raft disruption, decreased phosphatidylserine (PS) flip from the cell-surface, and redistribution of phosphatidylinositol 4,5-bisphosphate (PIP2) from the plasma membrane to actin rich regions in the cells. Enhanced basal autophagy, lipophagy and mitophagy was observed in cells treated with Miltefosine vs. control. Miltefosine treated cells showed marked increased in phosphorylation of kinases involved in autophagy induction such as; Adenosine monophosphate-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase (ULK1). The Toll like receptor (TLR) signaling pathway was blunted by Miltefosine treatment, resulting in decreased TLR4 recruitment to cell-surface and ~75% reduction in LPS induced pro-IL-1{beta} mRNA levels. Miltefosine reduced endotoxin-mediated mitochondrial reactive oxygen species and protected the mitochondrial membrane potential. Miltefosine treatment induced mitophagy and dampened NLRP3 inflammasome assembly. Collectively, our data shows that Miltefosine induced ABCA1 mediated cholesterol release, induced AMPK phosphorylation and mitophagy, while dampening NLRP3 inflammasome assembly and IL-1{beta} release.\n\nSignificance StatementAtherosclerosis is driven by cholesterol accumulation and inflammation, and the arterial macrophage is a key cell type in both of these processes. The macrophage characteristics that protect against atherosclerosis include increased cholesterol efflux/reverse cholesterol transport, increased autophagy, and deceased inflammatory cytokine production and signaling. Here, we show that one single orally available compound, Miltefosine, can target multiple macrophage pathways involved in lipid homeostasis and inflammation. Miltefosine activated cholesterol release and autophagy while inhibiting pro IL-1{beta} gene expression and NLRP3 inflammasome assembly. Miltefosine activated AMPK signaling pathway and mitophagy, leading to reduced NLRP3 inflammasome assembly and IL-1{beta} release.

cell biology↗

bioRxiv2018

Patterns and perceptions of cannabis use with physical activity

Background and AimsPast research has shown that cannabis use is common among adults in the U.S. In addition, physical activity (PA), such as exercise, is often a component of many Americans daily routines. Anecdotal information suggests that a subset of individuals use cannabis in conjunction with PA, but the evidence base is lacking. The purpose of this study was to assess the frequency, methods of ingestion, strain types, and timing (before, during or after) of cannabis use in combination with PA. We also sought to better understand the types of PA that cannabis is being used with and reasons why individuals may use cannabis with PA.\n\nMethods and ResultsA brief survey was developed and was administered online to community residents (N = 105) who reported use of cannabis with PA. Analysis of survey responses revealed that participants were using cannabis in combination with a wide range of physical activities. While cannabis use was reported before, during, and after PA, the majority of participants (92%) reported use of cannabis before PA. Most participants (77%) believed that the use of cannabis products with their PA had a positive effect on their performance. The strain of cannabis used with PA was dependent on timing of cannabis use before, during, or after PA. Although participants reported a range of reasons for using cannabis before, during, or after PA, pain management was the only reason reported across all time periods.\n\nConclusionsFindings from this study suggest that there is a sub-community of physically active individuals using cannabis with their PA, many who believe that cannabis use has a positive effect on their performance.

neuroscience↗