-
Organic Letters Jun 2024Protein adenosine diphosphate (ADP)-ribosylation is crucial for a proper immune response. Accordingly, viruses have evolved ADP-ribosyl hydrolases to remove these...
Protein adenosine diphosphate (ADP)-ribosylation is crucial for a proper immune response. Accordingly, viruses have evolved ADP-ribosyl hydrolases to remove these modifications, a prominent example being the SARS-CoV-2 NSP3 macrodomain, "Mac1". Consequently, inhibitors are developed by testing large libraries of small molecule candidates, with considerable success. However, a relatively underexplored angle in design pertains to the synthesis of structural substrate mimics. Here, we present the synthesis and biophysical activity of novel adenosine diphosphate ribose (ADPr) analogues as SARS-CoV-2 NSP3 Mac1 inhibitors.
PubMed: 38935522
DOI: 10.1021/acs.orglett.4c01792 -
Arteriosclerosis, Thrombosis, and... Jun 2024Despite being in an oxygen-rich environment, endothelial cells (ECs) use anaerobic glycolysis (Warburg effect) as the primary metabolic pathway for cellular energy...
BACKGROUND
Despite being in an oxygen-rich environment, endothelial cells (ECs) use anaerobic glycolysis (Warburg effect) as the primary metabolic pathway for cellular energy needs. PFKFB (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase)-3 regulates a critical enzymatic checkpoint in glycolysis and has been shown to induce angiogenesis. This study builds on our efforts to determine the metabolic regulation of ischemic angiogenesis and perfusion recovery in the ischemic muscle.
METHODS
Hypoxia serum starvation (HSS) was used as an in vitro peripheral artery disease (PAD) model, and hind limb ischemia by femoral artery ligation and resection was used as a preclinical PAD model.
RESULTS
Despite increasing PFKFB3-dependent glycolysis, HSS significantly decreased the angiogenic capacity of ischemic ECs. Interestingly, inhibiting PFKFB3 significantly induced the angiogenic capacity of HSS-ECs. Since ischemia induced a significant in PFKFB3 levels in hind limb ischemia muscle versus nonischemic, we wanted to determine whether glucose bioavailability (rather than PFKFB3 expression) in the ischemic muscle is a limiting factor behind impaired angiogenesis. However, treating the ischemic muscle with intramuscular delivery of D-glucose or L-glucose (osmolar control) showed no significant differences in the perfusion recovery, indicating that glucose bioavailability is not a limiting factor to induce ischemic angiogenesis in experimental PAD. Unexpectedly, we found that shRNA-mediated PFKFB3 inhibition in the ischemic muscle resulted in a numerical increase in perfusion recovery and significantly higher vascular density compared with control shRNA (consistent with the increased angiogenic capacity of PFKFB3 silenced HSS-ECs). Based on these data, we hypothesized that inhibiting HSS-induced PFKFB3 in ischemic ECs activates alternative metabolic pathways that revascularize the ischemic muscle in experimental PAD. A comprehensive glucose metabolic gene qPCR arrays in PFKFB3 silenced HSS-ECs, and PFKFB3-inhibited ischemic muscle versus respective controls identified UGP2 (uridine diphosphate-glucose pyrophosphorylase 2), a regulator of protein glycosylation and glycogen synthesis, is induced upon PFKFB3 inhibition in vitro and in vivo. Antibody-mediated inhibition of UGP2 in the ischemic muscle significantly impaired perfusion recovery versus IgG control. Mechanistically, supplementing uridine diphosphate-glucose, a metabolite of UGP2 activity, significantly induced HSS-EC angiogenic capacity in vitro and enhanced perfusion recovery in vivo by increasing protein glycosylation (but not glycogen synthesis).
CONCLUSIONS
Our data present that inhibition of maladaptive PFKFB3-driven glycolysis in HSS-ECs is necessary to promote the UGP2-uridine diphosphate-glucose axis that enhances ischemic angiogenesis and perfusion recovery in experimental PAD.
PubMed: 38934117
DOI: 10.1161/ATVBAHA.124.320665 -
Microorganisms May 2024Stevioside is a secondary metabolite of diterpenoid glycoside production in plants. It has been used as a natural sweetener in various foods because of its high...
Stevioside is a secondary metabolite of diterpenoid glycoside production in plants. It has been used as a natural sweetener in various foods because of its high sweetness and low-calorie content. In this study, we constructed a strain for the complete synthesis of stevioside using a metabolic engineering strategy. Firstly, the synthesis pathway of steviol was modularly constructed in BY4742, and the precursor pathway was strengthened. The yield of steviol was used as an indicator to investigate the expression effect of different sources of diterpene synthases under different combinations, and the strains with further improved steviol yield were screened. Secondly, glycosyltransferases were heterologously expressed in this strain to produce stevioside, the sequence of glycosyltransferase expression was optimized, and the uridine diphosphate-glucose (UDP-Glc) supply was enhanced. Finally, the results showed that the strain SST-302III-ST2 produced 164.89 mg/L of stevioside in a shake flask experiment, and the yield of stevioside reached 1104.49 mg/L in an experiment employing a 10 L bioreactor with batch feeding, which was the highest yield reported. We constructed strains with a high production of stevioside, thus laying the foundation for the production of other classes of steviol glycosides and holding good prospects for application and promotion.
PubMed: 38930507
DOI: 10.3390/microorganisms12061125 -
International Journal of Molecular... Jun 2024Pseudoxanthoma elasticum (PXE) is a rare inherited systemic disease responsible for a juvenile peripheral arterial calcification disease. The clinical diagnosis of PXE...
Pseudoxanthoma elasticum (PXE) is a rare inherited systemic disease responsible for a juvenile peripheral arterial calcification disease. The clinical diagnosis of PXE is only based on a complex multi-organ phenotypic score and/or genetical analysis. Reduced plasma inorganic pyrophosphate concentration [PPi]p has been linked to PXE. In this study, we used a novel and accurate method to measure [PPi]p in one of the largest cohorts of PXE patients, and we reported the valuable contribution of a cutoff value to PXE diagnosis. Plasma samples and clinical records from two French reference centers for PXE (PXE Consultation Center, Angers, and FAVA-MULTI South Competent Center, Nice) were assessed. Plasma PPi were measured in 153 PXE and 46 non-PXE patients. The PPi concentrations in the plasma samples were determined by a new method combining enzymatic and ion chromatography approaches. The best match between the sensitivity and specificity (Youden index) for diagnosing PXE was determined by ROC analysis. [PPi]p were lower in PXE patients (0.92 ± 0.30 µmol/L) than in non-PXE patients (1.61 ± 0.33 µmol/L, < 0.0001), corresponding to a mean reduction of 43 ± 19% (SD). The PPi cutoff value for diagnosing PXE in all patients was 1.2 µmol/L, with a sensitivity of 83.3% and a specificity of 91.1% (AUC = 0.93), without sex differences. In patients aged <50 years (i.e., the age period for PXE diagnosis), the cutoff PPi was 1.2 µmol/L (sensitivity, specificity, and AUC of 93%, 96%, and 0.97, respectively). The [PPi]p shows high accuracy for diagnosing PXE; thus, quantifying plasma PPi represents the first blood assay for diagnosing PXE.
Topics: Humans; Pseudoxanthoma Elasticum; Female; Male; Middle Aged; Adult; Diphosphates; Aged; ROC Curve; Young Adult; Sensitivity and Specificity; Biomarkers; Adolescent
PubMed: 38928212
DOI: 10.3390/ijms25126502 -
Biomolecules May 2024Platelets play essential roles in the formation of blood clots by clumping with coagulation factors at the site of vascular injury to stop bleeding; therefore, a...
Platelets play essential roles in the formation of blood clots by clumping with coagulation factors at the site of vascular injury to stop bleeding; therefore, a reduction in the platelet number or disorder in their function causes bleeding risk. In our research, we developed a method to assess platelet aggregation using an optical approach within a microfluidic chip's channel by evaluating the size of laser speckles. These speckles, associated with slowed blood flow in the microfluidic channel, had a baseline size of 28.54 ± 0.72 µm in whole blood. Removing platelets from the sample led to a notable decrease in speckle size to 27.04 ± 1.23 µm. Moreover, the addition of an ADP-containing agonist, which activates platelets, resulted in an increased speckle size of 32.89 ± 1.69 µm. This finding may provide a simple optical method via microfluidics that could be utilized to assess platelet functionality in diagnosing bleeding disorders and potentially in monitoring therapies that target platelets.
Topics: Blood Platelets; Humans; Platelet Aggregation; Platelet Function Tests; Lab-On-A-Chip Devices; Microfluidic Analytical Techniques; Microfluidics; Adenosine Diphosphate
PubMed: 38927016
DOI: 10.3390/biom14060612 -
Genes To Cells : Devoted To Molecular &... Jun 2024The second messenger guanosine 3',5'-bis(diphosphate)/guanosine tetraphosphate (ppGpp) and guanosine 3'-diphosphate 5'-triphosphate/guanosine pentaphosphate (pppGpp)...
The second messenger guanosine 3',5'-bis(diphosphate)/guanosine tetraphosphate (ppGpp) and guanosine 3'-diphosphate 5'-triphosphate/guanosine pentaphosphate (pppGpp) ((p)ppGpp) has been shown to be crucial for the survival of mycobacteria under hostile conditions. Unexpectedly, deletion of primary (p)ppGpp synthetase-Rel did not completely diminish (p)ppGpp levels leading to the discovery of novel bifunctional enzyme-RelZ, which displayed guanosine 5'-monophosphate,3'-diphosphate (pGpp), ppGpp, and pppGpp ((pp)pGpp) synthesis and RNAseHII activity. What conditions does it express itself under, and does it work in concert with Rel? The regulation of its transcription and whether the Rel enzyme plays a role in such regulation remain unclear. In this article, we have studied relZ promoter and compared its activity with rel promoter in different growth conditions. We observed that the promoter activity of relZ was constitutive; it is weaker than rel promoter, lies within 200 bp upstream of translation-start site, and it increased under carbon starvation. Furthermore, the promoter activity of relZ was compromised in the rel-knockout strain in the stationary phase. Our study unveils the dynamic regulation of relZ promoter activity by SigA and SigB sigma factors in different growth phases in mycobacteria. Importantly, elucidating the regulatory network of RelZ would enable the development of the targeted interventions for treating mycobacterial infections.
PubMed: 38923083
DOI: 10.1111/gtc.13135 -
Medicinal Research Reviews Jun 2024Breast cancer (BC) is a highly heterogeneous disease, and the presence of germline breast cancer gene mutation (gBRCAm) is associated with a poor prognosis.... (Review)
Review
Breast cancer (BC) is a highly heterogeneous disease, and the presence of germline breast cancer gene mutation (gBRCAm) is associated with a poor prognosis. Triple-negative breast cancer (TNBC) is a BC subtype, characterized by the absence of hormone and growth factor receptor expression, making therapeutic decisions difficult. Defects in the DNA damage response pathway due to mutation in breast cancer genes (BRCA 1/2) lead to homologous recombination deficiency (HRD). However, in HRD conditions, poly (adenosine diphosphate-ribose) polymerase (PARP) proteins repair DNA damage and lead to tumor cell survival. Biological understanding of HRD leads to the development of PARP inhibitors (PARPi), which trap PARP proteins and cause genomic instability and tumor cell lysis. HRD assessment can be an important biomarker in identifying gBRCAm patients with BC who could benefit from PARPi therapy. HRD can be identified by homologous recombination repair (HRR) gene-based assays, genomic-scarring assays and mutational signatures, transcription and protein expression profiles, and functional assays. However, gold standard methodologies that are robust and reliable to assess HRD are not available currently. Hence, there is a pressing need to develop accurate biomarkers identifying HRD tumors to guide targeted therapies such as PARPi in patients with BC. HRD assessment has shown fruitful outcomes in chemotherapy studies and preliminary evidence on PARPi intervention as monotherapy and combination therapy in HRD-stratified patients. Furthermore, ongoing trials are exploring the potential of PARPi in BC and clinically complex TNBC settings, where HRD testing is used as an adjunct to stratify patients based on BRCA mutations.
PubMed: 38922930
DOI: 10.1002/med.22058 -
Marine Drugs May 2024The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in...
The formation of phytoene by condensing two geranylgeranyl diphosphate molecules catalyzed by phytoene synthase (PSY) is the first committed and rate-limiting step in carotenoid biosynthesis, which has been extensively investigated in bacteria, land plants and microalgae. However, this step in macroalgae remains unknown. In the present study, a gene encoding putative phytoene synthase was cloned from the economic red alga -a species that has long been used in food and pharmaceuticals. The conservative motifs/domains and the tertiary structure predicted using bioinformatic tools suggested that the cloned should encode a phytoene synthase; this was empirically confirmed by pigment complementation in . This phytoene synthase was encoded by a single copy gene, whose expression was presumably regulated by many factors. The phylogenetic relationship of PSYs from different organisms suggested that red algae are probably the progeny of primary endosymbiosis and plastid donors of secondary endosymbiosis.
Topics: Rhodophyta; Phylogeny; Geranylgeranyl-Diphosphate Geranylgeranyltransferase; Carotenoids; Escherichia coli; Cloning, Molecular; Edible Seaweeds; Porphyra
PubMed: 38921568
DOI: 10.3390/md22060257 -
Current Gene Therapy Jun 2024The Ribonucleoside-diphosphate Reductase subunit M2 (RRM2) is known to be overexpressed in various cancers, though its specific functional implications remain unclear....
INTRODUCTION
The Ribonucleoside-diphosphate Reductase subunit M2 (RRM2) is known to be overexpressed in various cancers, though its specific functional implications remain unclear. This aims to elucidate the role of RRM2 in the progression of Lung Adenocarcinoma (LUAD) by exploring its involvement and potential impact.
METHODS
RRM2 data were sourced from multiple databases to assess its diagnostic and prognostic significance in LUAD. We evaluated the association between RRM2 expression and immune cell infiltration, analyzed its function, and explored the effects of modulating RRM2 expression on LUAD cell characteristics through laboratory experiments.
RESULTS
RRM2 was significantly upregulated in LUAD tissues and cells compared to normal counterparts (p<0.05), with rare genetic alterations noted (approximately 2%). This overexpression clearly distinguished LUAD from normal tissue (area under the curve (AUC): 0.963, 95% confidence intervals (CI): 0.946-0.981). Elevated RRM2 expression was significantly associated with adverse clinicopathological characteristics and poor prognosis in LUAD patients. Furthermore, a positive association was observed between RRM2 expression and immune cell infiltration. Pathway analysis revealed a critical connection between RRM2 and the cell cycle signaling pathway within LUAD. Targeting RRM2 inhibition effectively suppressed LUAD cell proliferation, migration, and invasion while promoting apoptosis. This intervention also modified the expression of several crucial proteins, including the downregulation of CDC25A, CDC25C, RAD1, Bcl-2, and PPM1D and the upregulation of TP53 and Bax (p < 0.05).
CONCLUSION
Our findings highlight the potential utility of RRM2 expression as a biomarker for diagnosing and predicting prognosis in LUAD, shedding new light on the role of RRM2 in this malignancy.
PubMed: 38920074
DOI: 10.2174/0115665232286359240611051307 -
Genetic Testing and Molecular Biomarkers Jun 2024Obesity is a major nutritional problem with an increasing prevalence among children and adolescents. The () gene encodes the UDP-glucuronosyl transferase enzyme,...
Obesity is a major nutritional problem with an increasing prevalence among children and adolescents. The () gene encodes the UDP-glucuronosyl transferase enzyme, converting the toxic form of bilirubin to a soluble, nontoxic form. There are yet to be studies on the evaluation of the variant types detected by next-generation sequencing (NGS) and their effects on bilirubin levels in nonsyndromic obese children. Forty-five children with body mass index (BMI) >95 percentile (p) constituted the obesity group and fourteen healthy children with BMI <85p constituted the control group. Anthropometric, clinical features, and biochemical parameters were evaluated. Furthermore, the gene was sequenced by NGS. The obese patients had lower total, direct, and indirect bilirubin levels ( = 0.422, 0.026, and 0.568, respectively). In addition, obese patients had more genetic variations in the gene compared with the control group (62.2% and 50%, respectively). We found that children with variations had higher total direct and indirect bilirubin levels compared with those without variation ( = 0.016, 0.028, and 0.015, respectively). Children diagnosed with obesity in the first two years of their life had fewer genetic variations and lower total bilirubin levels ( = 0.000 and 0.013, respectively). It is assumed that bilirubin can be protective against many chronic diseases. Although bilirubin levels are found to be lower in obese children compared with the control group, some variations in the gene may be supported by raising bilirubin. We suggest that high bilirubin levels caused by those variations may be protective against obesity and its many negative effects.
PubMed: 38916116
DOI: 10.1089/gtmb.2023.0365