The studies presented herein examine various aspects of paraoxonase 1 (PON1), specifically PON1's therapeutic potential and the hepatic expression of PON1. Bone marrow obtained from mice expressing a macrophage specific PON1- transgene was transplanted into atherosclerosis- susceptible mice lacking low density lipoprotein receptors (LDLR), then followed with gadolinium chloride (GdCl3) treatment, an agent that induces Kupffer cell apoptosis. GdCl3 treatment increased hepatic PON1 mRNA expression 9- fold in PON1-trangenic bone marrow recipient mice. After a 12 week cholesterol-enriched atherogenic diet feeding period, PON1-transgenic bone marrow recipient mice treated with GdCl3 exhibited a 50% reduction in atherosclerotic lesions; this protection was not seen in non-transgenic bone marrow recipients treated with GdCl3. These findings illustrate the important role of Kupffer cells in atherogenesis and suggest that GdCl3-facilitated replacement of Kupffer cells may enhance the efficacy of other HSC-based gene therapies. In additional studies, the regulation of PON1 expression by dietary bile acids was examined. It has been previously shown that a cholic-acid containing diet reduces the hepatic expression of PON1 in athero-susceptible C57BL/6 animals, but not in athero- resistant C3H/HeJ animals. When fed an atherogenic diet containing taurocholate, C3H/HeJ mice, in contrast to C57BL/6 mice, displayed a resistance to bile acid mediated repression of hepatic PON1 mRNA expression and HDL cholesterol. Further studies demonstrated that the absence of functional toll-like receptor 4 (TLR4s) in C3H/HeJ mice could not account for this response. However, mice with a genetic deletion of either fibroblast growth factor receptor 4 (FGFR4) or farnesoid X receptor (FXR) failed to repress PON1 and CYP7A1 in response to the taurocholate- containing diet. Additional studies demonstrated that FGF- 19, an FXR-inducible growth factor that binds specifically to FGFR4, decreased the expression of PON1 and CYP7A1 in HepG2 cells. The taurocholate containing diet increased the ileal expression of FGF-15, the murine homologue of FGF-19, in C57BL/6 and C3H/HeJ mice. Combined these data suggest that hepatic PON1 and CYP7A1 mRNA expression is repressed by bile acids via an FXR-mediated induction of FGF-15. The resistance of C3H/HeJ mice to bile acid- mediated repression of PON1 and CYP7A1 is, therefore, due to signaling events distal to the FGF-15/FGFR-4 association
Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thus led to the inhibition of IE protein expression. Consequently, viral genome replication and late protein expression were reduced by 90%. Finally, the production of new viral particles was nearly abrogated. In conclusion, the multiplex anti-UL122/123 CRISPR/Cas9 system can target the viral genome efficiently enough to significantly prevent viral replication.
Abstract Background To diagnose the full spectrum of hereditary and congenital diseases, genetic laboratories use many different workflows, ranging from karyotyping to exome sequencing. A single generic high-throughput workflow would greatly increase efficiency. We assessed whether genome sequencing (GS) can replace these existing workflows aimed at germline genetic diagnosis for rare disease. Methods We performed short-read GS (NovaSeq™6000; 150 bp paired-end reads, 37 × mean coverage) on 1000 cases with 1271 known clinically relevant variants, identified across different workflows, representative of our tertiary diagnostic centers. Variants were categorized into small variants (single nucleotide variants and indels < 50 bp), large variants (copy number variants and short tandem repeats) and other variants (structural variants and aneuploidies). Variant calling format files were queried per variant, from which workflow-specific true positive rates (TPRs) for detection were determined. A TPR of ≥ 98% was considered the threshold for transition to GS. A GS-first scenario was generated for our laboratory, using diagnostic efficacy and predicted false negative as primary outcome measures. As input, we modeled the diagnostic path for all 24,570 individuals referred in 2022, combining the clinical referral, the transition of the underlying workflow(s) to GS, and the variant type(s) to be detected. Results Overall, 95% (1206/1271) of variants were detected. Detection rates differed per variant category: small variants in 96% (826/860), large variants in 93% (341/366), and other variants in 87% (39/45). TPRs varied between workflows (79–100%), with 7/10 being replaceable by GS. Models for our laboratory indicate that a GS-first strategy would be feasible for 84.9% of clinical referrals (750/883), translating to 71% of all individuals (17,444/24,570) receiving GS as their primary test. An estimated false negative rate of 0.3% could be expected. Conclusions GS can capture clinically relevant germline variants in a ‘GS-first strategy’ for the majority of clinical indications in a genetics diagnostic lab.
PPARα, β/δ, and γ regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor–ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARα, β, and γ agonists on foam-cell formation and atherosclerosis in male LDL receptor–deficient (LDLR–/–) mice. Like the PPARγ agonist, a PPARα-specific agonist strongly inhibited atherosclerosis, whereas a PPARβ-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARα and PPARγ agonists, but not the PPARβ agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARα and PPARγ agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARα required LXRs, activation of PPARγ reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis.
The purpose of this research was to determine how dietary bile acids repress hepatic expression of paraoxonase 1 (PON1).C57BL/6 mice and C3H/HeJ mice, having different susceptibilities to atherosclerosis, were fed a chow diet and an atherogenic diet containing taurocholate. Compared with the more atherosclerosis-susceptible C57BL/6 mice, C3H/HeJ mice display resistance to dietary bile acid repression of hepatic PON1 mRNA and decreased high-density lipoprotein cholesterol. Whereas knockout of toll receptor 4 did not affect response to taurocholate, deletion of either FXR or FGFR4 blocked taurocholate repression of PON1 and CYP7A1. FGF19, an activator of FGFR4 expressed in human ileum, decreased expression of both PON1 and CYP7A1 expression by human hepatoma cells. In all of the mice studied, dietary taurocholate increased ileal expression of FGF15, a FXR-inducible murine homologue of human FGF19.Hepatic PON1 and CYP7A1 mRNA expression is repressed by bile acids via FXR-mediated induction of FGF15. Thus, the inability of C3H/HeJ mice to display taurocholate repression of PON1 and CYP7A1 mRNAs was not because of a lack of induction of FGF15 but rather signaling events distal to FGF15-FGFR4 association.
Abstract Soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for unbiased protein biomarker identification in Fragile X syndrome (FXS) in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging (MSI) with label-free proteomics in a mouse model of FXS to map the spatial distribution and quantify the levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1,004 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in the GABAergic system, synaptic transmission, and co-expression network analysis indicated that protein in soy group was significantly associated with metabolism and synapse modules in the Fmr1 KO brain. Ultimately, this spatial proteomics work laid the ground for identifying novel therapeutic targets and biomarkers for FXS.
Resident macrophages (i.e., Kupffer cells) are derived from hematopoietic stem cells (HSCs) and are primarily responsible for the removal from plasma of oxidized forms of low-density lipoprotein (LDL). The therapeutic potential of Kupffer cell expression of a transgene encoding paraoxonase-1 (PON1), whose plasma activity correlates with the protection from atherosclerosis, was examined in mice rendered atherosclerosis-susceptible through genetic deletion of the LDL receptor. Mice having their bone marrow engrafted with HSCs expressing the PON1 transgene (PON1-Tg) driven by a macrophage-specific promoter were injected i.v. with saline (vehicle only) or with gadolinium chloride (GdCl 3 ), an agent that rapidly causes Kupffer cell apoptosis. One month later, GdCl 3 -facilitated Kupffer cell apoptosis increased the hepatic expression of transgenic PON1 mRNA by 9-fold. After 12 weeks of being fed a cholesterol-enriched atherogenic diet, mice injected with GdCl 3 exhibited 50% reductions in both aortic sinus atherosclerotic lesions ( P < 0.0097) and surface lesions of the abdominal aorta ( P < 0.006). In contrast, mice receiving HSCs expressing the PON1-Tg but not treated with GdCl 3 showed no protection from atherosclerosis. In addition, mice engrafted with HSCs not expressing the PON1-Tg but injected with GdCl 3 also showed no protection from atherosclerosis. These findings, showing that GdCl 3 -enhanced hepatic expression of the PON1-Tg is essential for reducing atherosclerosis, indicate that Kupffer cells play an important role in atherogenesis. GdCl 3 -facilated replacement of Kupffer cells may enhance the efficacy of other HSC-based gene therapies.
