-
Current Osteoporosis Reports Oct 2023The study aims to provide updated information on the genetic factors associated with the diagnoses 'Diffuse Idiopathic Skeletal Hyperostosis' (DISH), 'Ossification of... (Review)
Review
PURPOSE OF REVIEW
The study aims to provide updated information on the genetic factors associated with the diagnoses 'Diffuse Idiopathic Skeletal Hyperostosis' (DISH), 'Ossification of the Posterior Longitudinal Ligament' (OPLL), and in patients with spinal ligament ossification.
RECENT FINDINGS
Recent studies have advanced our knowledge of genetic factors associated with DISH, OPLL, and other spinal ossification (ossification of the anterior longitudinal ligament [OALL] and the yellow ligament [OYL]). Several case studies of individuals afflicted with monogenic disorders, such as X-linked hypophosphatemia (XLH), demonstrate the strong association of fibroblast growth factor 23-related hypophosphatemia with OPLL, suggesting that pathogenic variants in PHEX, ENPP1, and DMP1 are associated with FGF23-phosphate wasting phenotype and strong genetic factors placing patients at risk for OPLL. Moreover, emerging evidence demonstrates that heterozygous and compound heterozygous ENPP1 pathogenic variants inducing 'Autosomal Recessive Hypophosphatemic Rickets Type 2' (ARHR2) also place patients at risk for DISH and OPLL, possibly due to the loss of inhibitory plasma pyrophosphate (PP) which suppresses ectopic calcification and enthesis mineralization. Our findings emphasize the importance of genetic and plasma biomarker screening in the clinical evaluation of DISH and OPLL patients, with plasma PP constituting an important new biomarker for the identification of DISH and OPLL patients whose disease course may be responsive to ENPP1 enzyme therapy, now in clinical trials for rare calcification disorders.
Topics: Humans; Hyperostosis, Diffuse Idiopathic Skeletal; Osteogenesis; Ossification of Posterior Longitudinal Ligament; Biomarkers; Ligaments
PubMed: 37530996
DOI: 10.1007/s11914-023-00814-6 -
Pharmacological Research Sep 2023In our previous multicenter study, we delineated the inherent metabolic features of colorectal cancer (CRC). Therein, we identified a member of the ectonucleotide...
In our previous multicenter study, we delineated the inherent metabolic features of colorectal cancer (CRC). Therein, we identified a member of the ectonucleotide pyrophosphatase/ phosphodiesterase family (ENPP2) as a significant differential metabolite of CRC. In this study, the role of ENPP2 in CRC has been demonstrated using established in vitro and in vivo models including ENPP2 gene knockdown, and use of the ENPP2 inhibitor, GLPG1690. We found that CRC proliferation was decreased after either ENPP2 gene knockdown or use of ENPP2 inhibitors. We further evaluated the role of GLPG1690 in AOM/DSS-induced CRC mice via intestinal barrier function, macrophage polarization, inflammatory response and microbial homeostasis. Results of immunofluorescence staining and Western blotting showed that GLPG1690 can restore gut-barrier function by increasing the expression of tight junction proteins, claudin-1, occludin and ZO-1. M2 tumor-associated macrophage polarization and colonic inflammation were attenuated after treatment with GLPG1690 using the Azoxymethane/Dextran Sodium Sulfate (AOM/DSS) model. Moreover, 16 S rDNA pyrosequencing and metagenomic analysis showed that GLPG1690 could alleviate gut dysbiosis in mice. Furthermore, administration of GLPG1690 with antibiotics as well as fecal microbiota transplantation assays demonstrated a close link between the efficacy of GLPG1690 and the gut microbiota composition. Finally, results of metabolomic analysis implicated mainly the gut microbiota-derived metabolites of aromatic amino acids in CRC progression. These findings may provide novel insights into the development of small-molecule ENPP2 inhibitors for the treatment of CRC.
Topics: Animals; Mice; Azoxymethane; Cell Proliferation; Colitis; Colorectal Neoplasms; Dextran Sulfate; Disease Models, Animal; Gastrointestinal Microbiome; Mice, Inbred C57BL; Phosphoric Diester Hydrolases
PubMed: 37524154
DOI: 10.1016/j.phrs.2023.106877 -
American Journal of Physiology. Cell... Sep 2023We studied osteoblast bone mineral transport and matrix proteins as a function of age. In isolated bone marrow cells from long bones of young (3 or 4 mo) and old (18 or...
We studied osteoblast bone mineral transport and matrix proteins as a function of age. In isolated bone marrow cells from long bones of young (3 or 4 mo) and old (18 or 19 mo) mice, age correlated with reduced mRNA of mineral transport proteins: alkaline phosphatase (ALP), ankylosis (ANK), the Cl/H exchanger ClC3, and matrix proteins collagen 1 (Col1) and osteocalcin (BGLAP). Some proteins, including the neutral phosphate transporter2 (NPT2), were not reduced. These are predominately osteoblast proteins, but in mixed cell populations. Remarkably, in osteoblasts differentiated from preparations of stromal stem cells (SSCs) made from bone marrow cells in young and old mice, differentiated in vitro on perforated polyethylene terephthalate membranes, mRNA confirmed decreased expression with age for most transport-related and bone matrix proteins. Additional mRNAs in osteoblasts in vitro included ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), unchanged, and ENPP2, reduced with age. Decrease with age in ALP activity and protein by Western blot was also significant. Transport protein findings correlated with micro-computed tomography of lumbar vertebra, showing that trabecular bone of old mice is osteopenic relative to young mice, consistent with other studies. Pathway analysis of osteoblasts differentiated in vitro showed that cells from old animals had reduced Erk1/2 phosphorylation and decreased suppressor of mothers against decapentaplegic 2 (Smad2) mRNA, consistent with TGFβ pathway, and reduced β-catenin mRNA, consistent with WNT pathway regulation. Our results show that decline in bone density with age reflects selective changes, resulting effectively in a phenotype modification. Reduction of matrix and mineral transport protein expression with age is regulated by multiple signaling pathways. This work for the first time showed that specific enzymes in bone mineral transport, and matrix synthesis proteins, in the epithelial-like bone-forming cell layer are downregulated with aging. Results were compared using cells extracted from long bones of young and old mice, or in essentially uniform osteoblasts differentiated from stromal stem cells in vitro. The age effect showed memory in the stromal stem cells, a remarkable finding.
Topics: Mice; Animals; Bone Matrix; X-Ray Microtomography; Osteoblasts; Cell Differentiation; Wnt Signaling Pathway; Minerals; RNA, Messenger; Carrier Proteins; Stem Cells; Cells, Cultured
PubMed: 37519232
DOI: 10.1152/ajpcell.00227.2023 -
Archives of Biochemistry and Biophysics Aug 2023The inosine triphosphate pyrophosphatase (ITPA) enzyme plays a critical cellular role by removing noncanonical nucleoside triphosphates from nucleotide pools. One of the...
The inosine triphosphate pyrophosphatase (ITPA) enzyme plays a critical cellular role by removing noncanonical nucleoside triphosphates from nucleotide pools. One of the first pathological ITPA mutants identified is R178C (rs746930990), which causes a fatal infantile encephalopathy, termed developmental and epileptic encephalopathy 35 (DEE 35). The accumulation of noncanonical nucleotides such as inosine triphosphate (ITP), is suspected to affect RNA and/or interfere with normal nucleotide function, leading to development of DEE 35. Molecular dynamics simulations have shown that the very rare R178C mutation does not significantly perturb the overall structure of the protein, but results in a high level of structural flexibility and disrupts active-site hydrogen bond networks, while preliminary biochemical data indicate that ITP hydrolyzing activity is significantly reduced for the R178C mutant. Here we report Michaelis-Menten enzyme kinetics data for the R178C ITPA mutant and three other position 178 ITPA mutants. These data confirm that position 178 is essential for ITPA activity and even conservative mutation at this site (R178K) results in significantly reduced enzyme activity. Our data support that disruption of the active-site hydrogen bond network is a major cause of diminished ITP hydrolyzing activity for the R178C mutation. These results suggest an avenue for developing therapies to address DEE 35.
Topics: Inosine; Pyrophosphatases; Inosine Triphosphate; Arginine; Nucleotides
PubMed: 37506994
DOI: 10.1016/j.abb.2023.109700 -
Scientific Reports Jul 2023Thiopurines, such as 6-mercaptopurine (6-MP), are widely used as cytotoxic agents and immunosuppressants for leukemia and autoimmune or inflammatory diseases. A...
Thiopurines, such as 6-mercaptopurine (6-MP), are widely used as cytotoxic agents and immunosuppressants for leukemia and autoimmune or inflammatory diseases. A nonsynonymous single nucleotide polymorphism (p.Arg139Cys; R139C) of the nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene causes the loss of thiopurine detoxification, inducing myelosuppression. To understand such hematotoxicity, we investigate the effects of NUDT15 R139C on hematopoietic stem cells (HSCs) upon thiopurine administration. Using previously established Nudt15 knock-in mice, which mimic myelosuppression in NUDT15 homozygous or heterozygous patients following thiopurine administration, we investigated the numerical changes of HSCs and hematopoietic progenitor cells following 6-MP administration using in vivo flowcytometry and ex vivo HSC expansion. Genes differentially expressed between Nudt15 HSCs and Nudt15 HSCs were identified using RNA-sequencing before the emergence of 6-MP-induced HSC-damage. Gene Ontology (GO) and Transcriptional Regulatory Relationships Unraveled by Sentence-based Text Mining (TRRUST) analyses were performed to elucidate the molecular effects of 6-MP on HSCs. In Nudt15 mice, 6-MP induced exhaustion of HSCs faster than that of multipotent progenitors and as fast as that of myeloid-committed progenitors. Ex vivo-expanded Nudt15 HSCs were dose- and time-dependently damaged by 6-MP. GO analysis identified the DNA damage response and cell cycle process as the most strongly influenced processes in Nudt15 HSCs. TRRUST analysis revealed that the Trp53-regulated transcriptional regulatory network is influenced prior to HSC exhaustion in Nudt15 HSCs. The loss of NUDT15 thiopurine detoxification enhances thiopurine-mediated DNA damage via the Trp53 networks in HSCs. Therefore, caution is required in long-term thiopurine use in patients with NUDT15 R139C in view of its adverse effects on HSCs in the form of DNA damage.
Topics: Animals; Mice; DNA Damage; Hematopoietic Stem Cells; Immunosuppressive Agents; Leukopenia; Mercaptopurine; Pyrophosphatases
PubMed: 37488179
DOI: 10.1038/s41598-023-38952-7 -
Frontiers in Microbiology 2023[This corrects the article DOI: 10.3389/fmicb.2021.788500.].
[This corrects the article DOI: 10.3389/fmicb.2021.788500.].
PubMed: 37469423
DOI: 10.3389/fmicb.2023.1229396 -
BMC Gastroenterology Jul 2023Thiopurines continue to play an important role in the treatment of inflammatory bowel disease (IBD). It is well known that thiopurines can cause several adverse...
BACKGROUND
Thiopurines continue to play an important role in the treatment of inflammatory bowel disease (IBD). It is well known that thiopurines can cause several adverse reactions. Especially, hematopoietic toxicity may lead to severe agranulocytosis. In a previous prospective study, we investigated the relationship between inosine triphosphate pyrophosphatase (ITPA) c.94c > a polymorphism, 6-thioguanine nucleotide (6-TGN) concentration and toxicity.
METHODS
To clarify the cause of thiopurine toxicity, we analysed nucleoside disphosphate-linked moiety X-type motif 15 (NUDT15) gene polymorphisms, i.e., R139C, V18I, and V19_V19insGV, and measured 6-mercaptopurines and 6-methylmercaptopurines (6-MMP) using the archived blood samples collected from 49 IBD patients for our previous study.
RESULTS
The ITPA c.94c > a polymorphism was detected in 19 patients (38.7%, all heterozygous). The R139C polymorphism was found in 10 patients (20.4%, 1 homozygous, 9 heterozygous), V18_V19insGV in 7 patients (14.3%, all heterozygous), and V18I in 2 patients (4.08%, all heterozygous). Although R139C was more strongly associated with leukopenia than c.94c > a, there were no significant correlations with 6-TGN and 6-MMP levels, as for c.94c > a. The leukopenia incidence rates for each gene polymorphism were 0% in those with all wild-type genes, 21.4% for c.94c > a only, 42.9% for NUDT15 polymorphism (s) only, and 80.0% for both polymorphisms.
CONCLUSIONS
All cases of leukopenia were associated with ITPA c.94c > a and/or polymorphism of NUDT15 and the risk of developing leukopenia was synergistically increased by ITPA and NUDT15 gene polymorphism. However, there was no association between the level of azathioprine metabolites and these polymorphisms.
Topics: Humans; Azathioprine; East Asian People; Inflammatory Bowel Diseases; Leukopenia; Mercaptopurine; Pyrophosphatases
PubMed: 37454061
DOI: 10.1186/s12876-023-02881-6 -
Analytical Chemistry Jul 2023Recent discoveries of noncanonical RNA caps, such as nicotinamide adenine dinucleotide (NAD) and 3'-dephospho-coenzyme A (dpCoA), have expanded our knowledge of RNA...
Recent discoveries of noncanonical RNA caps, such as nicotinamide adenine dinucleotide (NAD) and 3'-dephospho-coenzyme A (dpCoA), have expanded our knowledge of RNA caps. Although dpCoA has been known to cap RNAs in various species, the identities of its capped RNAs (dpCoA-RNAs) remained unknown. To fill this gap, we developed a method called dpCoA tagSeq, which utilized a thiol-reactive maleimide group to label dpCoA cap with a tag RNA serving as the 5' barcode. The barcoded RNAs were isolated using a complementary DNA strand of the tag RNA prior to direct sequencing by nanopore technology. Our validation experiments with model RNAs showed that dpCoA-RNA was efficiently tagged and captured using this protocol. To confirm that the tagged RNAs are capped by dpCoA and no other thiol-containing molecules, we used a pyrophosphatase NudC to degrade the dpCoA cap to adenosine monophosphate (AMP) moiety before performing the tagSeq protocol. We identified 44 genes that transcribe dpCoA-RNAs in mouse liver, demonstrating the method's effectiveness in identifying and characterizing the capped RNAs. This strategy provides a viable approach to identifying dpCoA-RNAs that allows for further functional investigations of the cap.
Topics: Animals; Mice; Nanopore Sequencing; Nanopores; RNA Caps; Coenzyme A; Maleimides
PubMed: 37439785
DOI: 10.1021/acs.analchem.3c02063 -
Archives of Razi Institute Apr 2023The chromogenic in situ hybridization (CISH) test is the gold standard for detecting Epstein-Barr virus (EBV)-associated gastric carcinoma (GC). Real-time (RT) PCR...
The chromogenic in situ hybridization (CISH) test is the gold standard for detecting Epstein-Barr virus (EBV)-associated gastric carcinoma (GC). Real-time (RT) PCR method is also a sensitive test that can detect the viral load in samples. As such, three EBV oncogenes were investigated in this study. RNA extraction and cDNA synthesis were performed on GC tissues of nine patients, who were previously confirmed to have EBVGC subtype. In addition, 44 patients that had positive RT-PCR but negative CISH results were also included as the control group. TaqMan RT-PCR analysis was performed to determine the expression of EBV-encoded microRNAs, and the expression of EBV-encoded , as well as , was analyzed by SYBR Green RT-PCR. EBV-encoded microRNAs and were identified in 2 out of 9 (22%) EBVGC subtypes. In addition, EBV-encoded was detected in 4 out of 9 (44.5%) EBVGC subtypes. EBV-encoded was also expressed in a sample of the control group. The expression of , EBV-encoded microRNAs, and EBV-encoded viral oncogenes in patients with high EBV viral loads indicates that these expressions correlate with viral loads. Our findings indicate that the EBV-encoded gene may have a role in EBVGC patients' non-response to treatment and might be considered a Biomarker-targeted therapy.
Topics: Humans; MicroRNAs; Herpesvirus 4, Human; Epstein-Barr Virus Infections; Viral Load; Stomach Neoplasms; Oncogenes; Carcinoma
PubMed: 37396720
DOI: 10.22092/ARI.2022.359408.2415 -
Frontiers in Microbiology 2023Nudix hydrolases comprise a large and ubiquitous protein superfamily that catalyzes the hydrolysis of a nucleoside diphosphate linked to another moiety X (Nudix)....
Nudix hydrolases comprise a large and ubiquitous protein superfamily that catalyzes the hydrolysis of a nucleoside diphosphate linked to another moiety X (Nudix). possesses four Nudix domain-containing proteins (SACI_RS00730/Saci_0153, SACI_RS02625/Saci_0550, SACI_RS00060/Saci_0013/Saci_NudT5, and SACI_RS00575/Saci_0121). Deletion strains were generated for the four individual Nudix genes and for both Nudix genes annotated to encode ADP-ribose pyrophosphatases () and did not reveal a distinct phenotype compared to the wild-type strain under standard growth conditions, nutrient stress or heat stress conditions. We employed RNA-seq to establish the transcriptome profiles of the Nudix deletion strains, revealing a large number of differentially regulated genes, most notably in the Δ double knock-out strain and the Δ single deletion strain. The absence of Nudix hydrolases is suggested to impact transcription differentially regulated transcriptional regulators. We observed downregulation of the lysine biosynthesis and the archaellum formation iModulons in stationary phase cells, as well as upregulation of two genes involved in the NAD biosynthesis pathway. Furthermore, the deletion strains exhibited upregulation of two thermosome subunits (α, β) and the toxin-antitoxin system VapBC, which are implicated in the archaeal heat shock response. These results uncover a defined set of pathways that involve archaeal Nudix protein activities and assist in their functional characterization.
PubMed: 37396357
DOI: 10.3389/fmicb.2023.1197877