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Pharmaceuticals (Basel, Switzerland) Jun 2024Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In...
Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In inflammatory conditions, the synovial membrane (SM) is subjected to transient hypoxia, especially during movement. CPPD formation is supported by an increase in extracellular inorganic pyrophosphate (ePPi) levels, which are mainly controlled by the transporter Ank and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We demonstrated previously that transforming growth factor (TGF)-β1 increased ePPi production by inducing Ank and Enpp1 expression in chondrocytes. As the TGF-β1 level raises in synovial fluid under hypoxic conditions, we investigated whether hypoxia may transform SM as a major source of ePPi production. Synovial fibroblasts and SM explants were exposed to 10 ng/mL of TGF-β1 in normoxic or hypoxic (5% O) culture conditions. Ank and Enpp1 expression were assessed by quantitative PCR, Western blot and immunohistochemistry. ePPi was quantified in culture supernatants. RNA silencing was used to define the respective roles of and in TGF-β1-induced ePPi generation. The molecular mechanisms involved in hypoxia were investigated using an promoter reporter plasmid for transactivation studies, as well as gene overexpression and RNA silencing, the respective role of hypoxia-induced factor (HIF)-1 and HIF-2. Our results showed that TGF-β1 increased Ank, Enpp1, and therefore ePPi production in synovial fibroblasts and SM explants. Ank was the major contributor in ePPi production compared to ENPP1. Hypoxia increased ePPi levels on its own and enhanced the stimulating effect of TGF-β1. Hypoxic conditions enhanced promoter transactivation in an HIF-1-dependent/HIF-2-independent fashion. We demonstrated that under hypoxia, SM is an important contributor to ePPi production in the joint through the induction of and . These findings are of interest as a rationale for the beneficial effect of anti-inflammatory drugs on SM in crystal depositions.
PubMed: 38931405
DOI: 10.3390/ph17060738 -
BMC Infectious Diseases Jun 2024Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from...
BACKGROUND
Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes.
METHODS
Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm).
RESULTS
We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M.S. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and HO. Since schistosomes are damaged by HO, we determined if SmNPP5 could impede HO production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M.S.
CONCLUSIONS
The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.
Topics: Riboflavin; Flavin Mononucleotide; Animals; Flavin-Adenine Dinucleotide; Schistosoma mansoni; Mice; Humans; Schistosomiasis mansoni
PubMed: 38918706
DOI: 10.1186/s12879-024-09538-z -
Journal of Cancer 2024DNA damage-inducible transcript 3 (DDIT3) is a transcription factor central to apoptosis, differentiation, and stress response. DDIT3 has been extensively studied in...
DNA damage-inducible transcript 3 (DDIT3) is a transcription factor central to apoptosis, differentiation, and stress response. DDIT3 has been extensively studied in cancer biology. However, its precise implications in breast cancer progression and its interaction with the immune microenvironment are unclear. In this study, we utilized a novel multi-omics integration strategy, combining bulk RNA sequencing, single-cell sequencing, spatial transcriptomics and immunohistochemistry, to explore the role of DDIT3 in breast cancer and establish the correlation between DDIT3 and poor prognosis in breast cancer patients. We identified a robust prognostic signature, including six genes (unc-93 homolog B1, TLR signaling regulator, anti-Mullerian hormone, DCTP pyrophosphatase 1, mitochondrial ribosomal protein L36, nuclear factor erythroid 2, and Rho GTPase activating protein 39), associated with DDIT3. This signature stratified the high-risk patient groups, characterized by increased infiltration of the regulatory T cells and M2-like macrophages and fibroblast growth factor (FGF)/FGF receptor signaling activation. Notably, the high-risk patient group demonstrated enhanced sensitivity to immunotherapy, presenting novel therapeutic opportunities. Integrating multi-omics data helped determine the spatial expression pattern of DDIT3 in the tumor microenvironment and its correlation with immune cell infiltration. This multi-dimensional analysis provided a comprehensive understanding of the intricate interplay between DDIT3 and the immune microenvironment in breast cancer. Overall, our study not only facilitates understanding the role of DDIT3 in breast cancer but also offers innovative insights for developing prognostic models and therapeutic strategies. Identifying the DDIT3-related prognostic signature and its association with the immune microenvironment provided a promising avenue for personalized breast cancer treatment.
PubMed: 38911383
DOI: 10.7150/jca.96491 -
MBio Jun 2024Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising...
Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes , , and ) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP levels derepress the regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP toxicity is alleviated by deletion of , the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating () allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of hyper-repressed expression in phosphate-replete cells; suppressed the derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious lncRNA termination); and delayed induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the and promoters, but not for the promoters that drive the synthesis of the -repressive lncRNAs. Transcription profiling of ∆ and () cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast genes , , and by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic alleles derepress the genes via the action of IP as an agonist of precocious lncRNA 3'-processing/termination. IP toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP levels on termination. In this study, a forward genetic screen revealed that IP toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of -activating transcription factor Pho7 to its binding sites in the mRNA promoters.
PubMed: 38899862
DOI: 10.1128/mbio.01252-24 -
International Journal of Molecular... May 2024Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of...
Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.
Topics: Cystathionine beta-Synthase; Mutation; Protein Binding; Mutagenesis, Site-Directed; Adenine Nucleotides; Protein Domains; Pyrophosphatases; Adenosine Diphosphate; Adenosine Triphosphate; Bacterial Proteins; Inorganic Pyrophosphatase; Models, Molecular; Binding Sites
PubMed: 38891956
DOI: 10.3390/ijms25115768 -
Bioorganic & Medicinal Chemistry Letters Jun 2024The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide...
The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as its dominant negative regulator. Since activation of the innate immune system is a promising strategy for the treatment of various infectious diseases and cancers, ENPP1 inhibitors have attracted great attention as candidate drugs. We have previously identified small-molecule ENPP1 inhibitors having a [1,2,4]triazolo[1,5-a]pyrimidine scaffold by means of chemical screening using a fluorescence probe, TG-mAMP. In this study, we evaluated the structure-activity relationships of the hit and lead compounds in detail, and succeeded in developing compounds that strongly and selectively inhibit ENPP1 not only in vitro, but also in cellular systems.
PubMed: 38851358
DOI: 10.1016/j.bmcl.2024.129820 -
Scientific Reports Jun 2024The enzyme dUTPase has an essential role in maintaining genomic integrity. In mouse, nuclear and mitochondrial isoforms of the enzyme have been described. Here we...
The enzyme dUTPase has an essential role in maintaining genomic integrity. In mouse, nuclear and mitochondrial isoforms of the enzyme have been described. Here we present the isoform-specific mRNA expression levels in different murine organs during development using RT-qPCR. In this study, we analyzed organs of 14.5-day embryos and of postnatal 2-, 4-, 10-week- and 13-month-old mice. We demonstrate organ-, sex- and developmental stage-specific differences in the mRNA expression levels of both isoforms. We found high mRNA expression level of the nuclear isoform in the embryo brain, and the expression level remained relatively high in the adult brain as well. This was surprising, since dUTPase is known to play an important role in proliferating cells, and mass production of neural cells is completed by adulthood. Thus, we investigated the pattern of the dUTPase protein expression specifically in the adult brain with immunostaining and found that dUTPase is present in the germinative zones, the subventricular and the subgranular zones, where neurogenesis occurs and in the rostral migratory stream where neuroblasts migrate to the olfactory bulb. These novel findings suggest that dUTPase may have a role in cell differentiation and indicate that accurate dTTP biosynthesis can be vital, especially in neurogenesis.
Topics: Animals; Neurogenesis; Pyrophosphatases; Mice; Female; Male; Brain; Gene Expression Regulation, Developmental; RNA, Messenger
PubMed: 38849394
DOI: 10.1038/s41598-024-63405-0 -
BMC Oral Health Jun 2024Peri-implantitis (PI) is a frequent inflammatory disorder characterised by progressive loss of the supporting bone. Not all patients with recognised risk factors develop...
BACKGROUND
Peri-implantitis (PI) is a frequent inflammatory disorder characterised by progressive loss of the supporting bone. Not all patients with recognised risk factors develop PI. The aim of this study is to evaluate the presence of single nucleotide polymorphisms (SNP) of inflammatory and bone metabolism related proteins in a population treated with dental implants from the Basque Country (Spain).
METHODS
We included 80 patients with diagnosis of PI and 81 patients without PI, 91 women and 70 men, with a mean age of 60.90 years. SNPs of BMP-4, BRINP3, CD14, FGF-3, FGF-10, GBP-1, IL-1α, IL-1β, IL-10, LTF, OPG and RANKL proteins were selected. We performed a univariate and bivariate analysis using IBM SPSS® v.28 statistical software.
RESULTS
Presence of SNPs GBP1 rs7911 (p = 0.041) and BRINP3 rs1935881 (p = 0.012) was significantly more common in patients with PI. Patients with PI who smoked (> 10 cig/day) showed a higher presence of OPG rs2073617 SNP (p = 0.034). Also, BMP-4 rs17563 (p = 0.018) and FGF-3 rs1893047 (p = 0.014) SNPs were more frequent in patients with PI and Type II diabetes mellitus.
CONCLUSIONS
Our findings suggest that PI could be favoured by an alteration in the osseointegration of dental implants, based on an abnormal immunological response to peri-implant infection in patients from the Basque Country (Spain).
Topics: Humans; Male; Female; Case-Control Studies; Middle Aged; Spain; Polymorphism, Single Nucleotide; Peri-Implantitis; Dental Implants; Osteoprotegerin; Aged; Bone Morphogenetic Protein 4; GTP-Binding Proteins; RANK Ligand; Interleukin-1alpha; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 38840172
DOI: 10.1186/s12903-024-04319-1 -
BMC Medical Genomics May 2024Therapy with anti-cancer drugs remain the cornerstone of treating cancer. The effectiveness and safety of anti-cancer drugs vary significantly among individuals due to...
BACKGROUND
Therapy with anti-cancer drugs remain the cornerstone of treating cancer. The effectiveness and safety of anti-cancer drugs vary significantly among individuals due to genetic factors influencing the drug response and metabolism. Data on the pharmacogenomic variations in Sri Lankans related to anti-cancer therapy is sparse. As current treatment guidelines in Sri Lanka often do not consider local pharmacogenomic variants, this study aimed to explore the diversity of pharmacogenomic variants in the Sri Lankan population to pave the way for personalized treatment approaches and improve patient outcomes.
METHODS
Pharmacogenomic data regarding variant-drug pairs of genes CYP2D6, DPYD, NUDT15, EPAS1, and XRCC1 with clinical annotations labelled as evidence levels 1A-2B were obtained from the Pharmacogenomics Knowledgebase database. Their frequencies in Sri Lankans were obtained from an anonymized database that was derived from 541 Sri Lankans who underwent exome sequencing at the Human Genetics Unit, Faculty of Medicine, University of Colombo. Variations in DPYD, NUDT15, and EPAS1 genes are related to increased toxicity to fluoropyrimidines, mercaptopurines, and sorafenib respectively. Variations in CYP2D6 and XRCC1 genes are related to changes in efficacy of tamoxifen and platinum compounds, respectively. Minor allele frequencies of these variants were calculated and compared with other populations.
RESULTS
MAFs of rs1065852 c.100 C > T (CYP2D6), rs3918290 c.1905 + 1G > A (DPYD), rs56038477 c.1236G > A (DPYD), rs7557402 c.1035-7 C > G (EPAS1), rs116855232 c.415 C > T (NUDT15*3), and rs25487 c.1196 A > G (XRCC1) were: 12.9% [95%CI:10.9-14.9], 1.5% [95%CI:0.8-2.2], 1.2% [95%CI:0.5-1.8], 37.7% [95%CI:34.8-40.6], 8.3% [95%CI:6.7-10.0], and 64.0% [95%CI:61.1-66.8], respectively. Frequencies of rs1065852 c.100 C > T (CYP2D6), rs7557402 c.1035-7 C > G (EPAS1), and rs25487 (XRCC1) were significantly lower in Sri Lankans, while frequencies of rs116855232 c.415 C > T (NUDT15*3) and rs56038477 c.1236G > A (DPYD) were significantly higher in Sri Lankans when compared to some Western and Asian populations.
CONCLUSION
Sri Lankans are likely to show lower toxicity risk with sorafenib (rs7557402 c.84,131 C > G) and, higher toxicity risk with fluoropyrimidines (rs56038477 c.1236G > A) and mercaptopurine (rs116855232 c.415 C > T), and reduced effectiveness with tamoxifen (rs1065852 c.100 C > T) and platinum compounds (rs25487). These findings highlight the potential contribution of these genetic variations to the individual variability in anti-cancer dosage requirements among Sri Lankans.
Topics: Humans; Sri Lanka; Antineoplastic Agents; Pharmacogenomic Variants; X-ray Repair Cross Complementing Protein 1; Pyrophosphatases; Basic Helix-Loop-Helix Transcription Factors; Cytochrome P-450 CYP2D6; Neoplasms; Asian People; Pharmacogenetics; Gene Frequency; Nudix Hydrolases
PubMed: 38789983
DOI: 10.1186/s12920-024-01919-2 -
JBMR Plus Jun 2024Arterial media calcification or pathological deposition of calcium-phosphate crystals in the vessel wall contributes significantly to the high mortality rate observed in...
Arterial media calcification or pathological deposition of calcium-phosphate crystals in the vessel wall contributes significantly to the high mortality rate observed in patients with CKD. Extracellular nucleotides (ie, ATP or UTP) regulate the arterial calcification process by interacting with (1) purinergic receptors and (2) breakdown via ecto-nucleotidases, such as ectonucleotide pyrophosphatase/phosphodiesterase NPP1 or NPP3, affecting the local levels of calcification inhibitor, pyrophosphate, and stimulator inorganic phosphate (PP/P ratio). Also, it has been shown that ATP analogs (ie, β,γ-methylene-ATP [β,γ-meATP]) inhibit vascular smooth muscle cell calcification in vitro. In the first experiment, daily dosing of β,γ-meATP (2 mg/kg) was investigated in rats fed a warfarin diet to trigger the development of non-CKD-related arterial medial calcifications. This study showed that β,γ-meATP significantly lowered the calcium scores in the aorta and peripheral vessels in warfarin-exposed rats. In a second experiment, daily dosing of 4 mg/kg β,γ-meATP and its metabolite medronic acid (MDP) was analyzed in rats fed an adenine diet to promote the development of CKD-related arterial medial calcification. Administration of β,γ-meATP and MDP did not significantly decrease aortic calcification scores in this model. Moreover, both compounds induced deleterious effects on physiological bone mineralization, causing an imminent risk for worsening the already compromised bone status in CKD. Due to this, it was not possible to raise the dosage of both compounds to tackle CKD-related arterial calcification. Again, this points out the difficult task of targeting solely ectopic calcifications without negatively affecting physiological bone mineralization. On the other hand, aortic mRNA expression of and was significantly and positively associated with aortic calcification scores, suggesting that normalizing the aortic NPP1/3 activity to control values might be a possible target to treat (CKD-induced) arterial media calcifications.
PubMed: 38764790
DOI: 10.1093/jbmrpl/ziae057