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Viruses Mar 2024Glycosylation, a dynamic modification prevalent in viruses and higher eukaryotes, is principally regulated by uridine diphosphate (UDP)-glycosyltransferases (UGTs) in...
Glycosylation, a dynamic modification prevalent in viruses and higher eukaryotes, is principally regulated by uridine diphosphate (UDP)-glycosyltransferases (UGTs) in plants. Although UGTs are involved in plant defense responses, their responses to most pathogens, especially plant viruses, remain unclear. Here, we aimed to identify UGTs in the whole genome of () and to analyze their function in Chinese wheat mosaic virus (CWMV) infection. A total of 147 were identified in . To conduct a phylogenetic analysis, the UGT protein sequences of and were aligned. The gene structure and conserved motifs of the UGTs were also analyzed. Additionally, the physicochemical properties and predictable subcellular localization were examined in detail. Analysis of cis-acting elements in the putative promoter revealed that were involved in temperature, defense, and hormone responses. The expression levels of 20 containing defense-related cis-acting elements were assessed in CWMV-infected , revealing a significant upregulation of 8 . Subcellular localization analysis of three NbUGTs (NbUGT12, NbUGT16 and NbUGT17) revealed their predominant localization in the cytoplasm of leaves, and NbUGT12 was also distributed in the chloroplasts. CWMV infection did not alter the subcellular localization of NbUGT12, NbUGT16, and NbUGT17. Transient overexpression of , , and enhanced CWMV infection, whereas the knockdown of , and inhibited CWMV infection in . These could serve as potential susceptibility genes to facilitate CWMV infection. Overall, the findings throw light on the evolution and function of .
Topics: Nicotiana; Plant Diseases; Glycosyltransferases; Phylogeny; Disease Resistance; Gene Expression Regulation, Plant; Plant Proteins; Genome, Plant; Uridine Diphosphate; Potyvirus; Genome-Wide Association Study
PubMed: 38675832
DOI: 10.3390/v16040489 -
Journal of Pediatric Gastroenterology... Jun 2024The safety and efficacy of sofosbuvir-velpatasvir in children aged 3-17 years with chronic hepatitis C virus (HCV) infection of any genotype were evaluated.
BACKGROUND
The safety and efficacy of sofosbuvir-velpatasvir in children aged 3-17 years with chronic hepatitis C virus (HCV) infection of any genotype were evaluated.
METHODS
In this Phase 2, multicenter, open-label study, patients received once daily for 12 weeks either sofosbuvir-velpatasvir 400/100 mg tablet (12-17 years), 200/50 mg low dose tablet or oral granules (3-11 years and ≥17 kg), or 150/37.5 mg oral granules (3-5 years and <17 kg). The efficacy endpoint was sustained virologic response 12 weeks after therapy (SVR12). Dose appropriateness was confirmed by intensive pharmacokinetics in each age group.
FINDINGS
Among 216 patients treated, 76% had HCV genotype 1% and 12% had genotype 3. Rates of SVR12 were 83% (34/41) among 3-5-year-olds, 93% (68/73) among 6-11-year-olds, and 95% (97/102) among 12-17-year-olds. Only two patients experienced virologic failure. The most common adverse events were headache, fatigue, and nausea in 12-17-year-olds; vomiting, cough, and headache in 6-11-year-olds; and vomiting in 3-5-year-olds. Three patients discontinued treatment because of adverse events. Four patients had serious adverse events; all except auditory hallucination (n = 1) were considered unrelated to study drug. Exposures of sofosbuvir, its metabolite GS-331007, and velpatasvir were comparable to those in adults in prior Phase 2/3 studies. Population pharmacokinetic simulations supported weight-based dosing for children in this age range.
INTERPRETATION
The pangenotypic regimen of sofosbuvir-velpatasvir is highly effective and safe in treating children 3-17 years with chronic HCV infection.
Topics: Humans; Sofosbuvir; Heterocyclic Compounds, 4 or More Rings; Child; Carbamates; Male; Child, Preschool; Female; Antiviral Agents; Adolescent; Hepatitis C, Chronic; Drug Combinations; Treatment Outcome; Hepacivirus; Sustained Virologic Response; Genotype; Benzimidazoles; Benzopyrans
PubMed: 38644678
DOI: 10.1002/jpn3.12045 -
Microbial Cell Factories Apr 2024Isoquercitrin (quercetin-3-O-β-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents....
BACKGROUND
Isoquercitrin (quercetin-3-O-β-D-glucopyranoside) has exhibited promising therapeutic potentials as cardioprotective, anti-diabetic, anti-cancer, and anti-viral agents. However, its structural complexity and limited natural abundance make both bulk chemical synthesis and extraction from medical plants difficult. Microbial biotransformation through heterologous expression of glycosyltransferases offers a safe and sustainable route for its production. Despite several attempts reported in microbial hosts, the current production levels of isoquercitrin still lag behind industrial standards.
RESULTS
Herein, the heterologous expression of glycosyltransferase UGT78D2 gene in Bacillus subtilis 168 and reconstruction of UDP-glucose (UDP-Glc) synthesis pathway led to the synthesis of isoquercitrin from quercetin with titers of 0.37 g/L and 0.42 g/L, respectively. Subsequently, the quercetin catabolism blocked by disruption of a quercetin dioxygenase, three ring-cleavage dioxygenases, and seven oxidoreductases increased the isoquercitrin titer to 1.64 g/L. And the hydrolysis of isoquercitrin was eliminated by three β-glucosidase genes disruption, thereby affording 3.58 g/L isoquercitrin. Furthermore, UDP-Glc pool boosted by pgi (encoding glucose-6-phosphate isomerase) disruption increased the isoquercitrin titer to 10.6 g/L with the yield on quercetin of 72% and to 35.6 g/L with the yield on quercetin of 77.2% in a 1.3-L fermentor.
CONCLUSION
The engineered B. subtilis strain developed here holds great potential for initiating the sustainable and large-scale industrial production of isoquercitrin. The strategies proposed in this study provides a reference to improve the production of other flavonoid glycosides by engineered B. subtilis cell factories.
Topics: Quercetin; Metabolic Engineering; Bacillus subtilis; Uridine Diphosphate
PubMed: 38641799
DOI: 10.1186/s12934-024-02390-5 -
Experimental Eye Research Jun 2024Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by injury to the ocular surface due to exposure to ultraviolet (UV) radiation. UV-induced damage in...
Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by injury to the ocular surface due to exposure to ultraviolet (UV) radiation. UV-induced damage in the cells leads to the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts that are repaired by the NER (Nucleotide Excision Repair) pathway. Mutations in the genes coding for NER proteins, as reported in XP patients, would lead to sub-optimal damage repair resulting in clinical signs varying from photo-keratitis to cancerous lesions on the ocular surface. Here, we aimed to provide evidence for the accumulation of DNA damage and activation of DNA repair pathway proteins in the corneal cells of patients with XP. Corneal buttons of patients who underwent penetrating keratoplasty were stained to quantify DNA damage and the presence of activated DNA damage response proteins (DDR) using specific antibodies. Positive staining for pH2A.X and thymidine dimers confirmed the presence of DNA damage in the corneal cells. Positive cells were found in both control corneas and XP samples however, unlike normal tissues, positive cells were found in all cell layers of XP samples indicating that these cells were sensitive to very low levels of UV. pH2A.X-positive cells were significantly more in XP corneas (p < 0.05) indicating the presence of double strand breaks in these tissues. A positive expression of phosphorylated-forms of DDR proteins was noted in XP corneas (unlike controls) such as ataxia telangiectasia mutated/Rad-3 related proteins (ATM/ATR), breast cancer-1 and checkpoint kinases-1 and -2. Nuclear localization of XPA was noted in XP samples which co-localized (calculated using Pearson's correlation) with pATM (0.9 ± 0.007) and pATR (0.6 ± 0.053). The increased presence of these in the nucleus confirms that unresolved DNA damage was accumulating in these cells thereby leading to prolonged activation of the damage response proteins. An increase in pp53 and TUNEL positive cells in the XP corneas indicated cell death likely driven by the p53 pathway. For comparison, cultured normal corneal epithelial cells were exposed to UV-radiation and stained for DDR proteins at 3, 6 and 24 h after irradiation to quantify the time taken by cells with intact DDR pathway to repair damage. These cells, when exposed to UV showed nuclear translocation of DDR proteins at 3 and 6 h which reduced significantly by 24 h confirming that the damaged DNA was being actively repaired leading to cell survival. The persistent presence of the DDR proteins in XP corneas indicates that damage is being actively recognized and DNA replication is stalled, thereby causing accumulation of damaged DNA leading to cell death, which would explain the cancer incidence and cell loss reported in these patients.
Topics: Humans; Ultraviolet Rays; DNA Damage; Xeroderma Pigmentosum; DNA Repair; Pyrimidine Dimers; Keratoplasty, Penetrating; Cornea; Female; Adult; Histones; Male; Middle Aged; Ataxia Telangiectasia Mutated Proteins; Adolescent; Young Adult
PubMed: 38641197
DOI: 10.1016/j.exer.2024.109901 -
Biochemical and Biophysical Research... Jun 2024Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify...
Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify that RNA-like polymers can be produced by hot wet-dry cycling of nucleotides. However, it has not been shown that these RNA-like polymers are similar to biological RNA with 3'-5' phosphodiester bonds. In the results described here, RNA-like polymers were generated from 5'-monophosphate nucleosides AMP and UMP. To confirm that the polymers resemble biological RNA, ribonuclease A should catalyze hydrolysis of the 3'-5' phosphodiester bonds between pyrimidine nucleotides to each other or to purine nucleotides, but not purine-purine nucleotide bonds. Here we show AFM images of specific polymers produced by hot wet-dry cycling of AMP, UMP and AMP/UMP (1:1) solutions on mica surfaces, before and after exposure to ribonuclease A. AMP polymers were unaffected by ribonuclease A but UMP polymers disappeared. This indicates that a major fraction of the bonds in the UMP polymers is indeed 3'-5' phosphodiester bonds. Some of the polymers generated from the AMP/UMP mixture also showed clear signs of cleavage. Because ribonuclease A recognizes the ester bonds in the polymers, we show for the first time that these prebiotically produced polymers are in fact similar to biological RNA but are likely to be linked by a mixture of 3'-5' and 2'-5' phosphodiester bonds.
Topics: RNA; Ribonuclease, Pancreatic; Uridine Monophosphate; Microscopy, Atomic Force; Hot Temperature; Polymers; Adenosine Monophosphate; Hydrolysis; Polymerization
PubMed: 38640739
DOI: 10.1016/j.bbrc.2024.149938 -
Luminescence : the Journal of... Apr 2024A simple, rapid, and low-cost technique was developed to allow reliable analysis of the anti-hepatitis C drug sofosbuvir in bulk, tablet form, and spiked human plasma....
A simple, rapid, and low-cost technique was developed to allow reliable analysis of the anti-hepatitis C drug sofosbuvir in bulk, tablet form, and spiked human plasma. This method depends on the ability of sofosbuvir to quench the fluorescence of the newly synthesized 2-amino-3-cyano-4,6-dimethylpyridine (reagent 3). Elemental analysis and spectral data were used to validate the structure of the synthesized reagent. The newly synthesized reagent exhibited a satisfactory level of fluorescence emission at 365 nm after excitation at 247 nm. All experimental variables that might affect the quenching process were analyzed and optimized. Linearity, range, accuracy, precision, limit of detection (LOD), and limit of quantitation (LOQ) were all validated in accordance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines. The concentration range was shown to be linear between 0.1 and 1.5 μg/mL. The technique was effectively utilized for sofosbuvir analysis in both its tablet dosage form and spiked human plasma, with mean percentage recoveries of 100.13 ± 0.35 and 94.26 ± 1.69, respectively.
Topics: Humans; Sofosbuvir; Fluorescent Dyes; Spectrometry, Fluorescence; Tablets
PubMed: 38637644
DOI: 10.1002/bio.4742 -
Nucleic Acids Research May 2024It has been proposed that coronavirus nsp15 mediates evasion of host cell double-stranded (ds) RNA sensors via its uracil-specific endoribonuclease activity. However,...
It has been proposed that coronavirus nsp15 mediates evasion of host cell double-stranded (ds) RNA sensors via its uracil-specific endoribonuclease activity. However, how nsp15 processes viral dsRNA, commonly considered as a genome replication intermediate, remains elusive. Previous research has mainly focused on short single-stranded RNA as substrates, and whether nsp15 prefers single-stranded or double-stranded RNA for cleavage is controversial. In the present work, we prepared numerous RNA substrates, including both long substrates mimicking the viral genome and short defined RNA, to clarify the substrate preference and cleavage pattern of SARS-CoV-2 nsp15. We demonstrated that SARS-CoV-2 nsp15 preferentially cleaved pyrimidine nucleotides located in less thermodynamically stable areas in dsRNA, such as AU-rich areas and mismatch-containing areas, in a nicking manner. Because coronavirus genomes generally have a high AU content, our work supported the mechanism that coronaviruses evade the antiviral response mediated by host cell dsRNA sensors by using nsp15 dsRNA nickase to directly cleave dsRNA intermediates formed during genome replication and transcription.
Topics: RNA, Double-Stranded; SARS-CoV-2; RNA, Viral; Viral Nonstructural Proteins; Humans; Endoribonucleases; Virus Replication; Substrate Specificity; Genome, Viral; COVID-19
PubMed: 38634805
DOI: 10.1093/nar/gkae290 -
International Journal of Biological... May 2024Current management of HCV infection is based on Direct-Acting Antiviral Drugs (DAAs). However, resistance-associated mutations, especially in the NS3 and NS5B regions...
Current management of HCV infection is based on Direct-Acting Antiviral Drugs (DAAs). However, resistance-associated mutations, especially in the NS3 and NS5B regions are gradually decreasing the efficacy of DAAs. Among the most effective HCV NS3/4A protease drugs, Sofosbuvir also develops resistance due to mutations in the NS3 and NS5B regions. Four mutations at positions A156Y, L36P, Q41H, and Q80K are classified as high-level resistance mutations. The resistance mechanism of HCV NS3/4A protease toward Sofosbuvir caused by these mutations is still unclear, as there is less information available regarding the structural and functional effects of the mutations against Sofosbuvir. In this work, we combined molecular dynamics simulation, molecular mechanics/Generalized-Born surface area calculation, principal component analysis, and free energy landscape analysis to explore the resistance mechanism of HCV NS3/4A protease due to these mutations, as well as compare interaction changes in wild-type. Subsequently, we identified that the mutant form of HCV NS3/4A protease affects the activity of Sofosbuvir. In this study, the resistance mechanism of Sofosbuvir at the atomic level is proposed. The proposed drug-resistance mechanism will provide valuable guidance for the design of HCV drugs.
Topics: Antiviral Agents; DEAD-box RNA Helicases; Drug Resistance, Viral; Hepacivirus; Molecular Dynamics Simulation; Mutation; Nucleoside-Triphosphatase; Serine Endopeptidases; Serine Proteases; Sofosbuvir; Viral Nonstructural Proteins; Viral Proteases
PubMed: 38631585
DOI: 10.1016/j.ijbiomac.2024.131629 -
Zhongguo Zhong Yao Za Zhi = Zhongguo... Feb 2024Uridine diphosphate glycosyltransferase(UGT) is involved in the glycosylation of a variety of secondary metabolites in plants and plays an important role in plant growth...
Uridine diphosphate glycosyltransferase(UGT) is involved in the glycosylation of a variety of secondary metabolites in plants and plays an important role in plant growth and development and regulation of secondary metabolism. Based on the genome of a diploid Chrysanthemum indicum, the UGT gene family from Ch. indicum was identified by bioinformatics methods, and the physical and chemical properties, subcellular localization prediction, conserved motif, phylogeny, chromosome location, gene structure, and gene replication events of UGT protein were analyzed. Transcriptome and real-time fluorescence quantitative polymerase chain reaction(PCR) were used to analyze the expression pattern of the UGT gene in flowers and leaves of Ch. indicum. Quasi-targeted metabolomics was used to analyze the differential metabolites in flowers and leaves. The results showed that a total of 279 UGT genes were identified in the Ch. indicum genome. Phylogenetic analysis showed that these UGT genes were divided into 8 subfamilies. Members of the same subfamily were distributed in clusters on the chromosomes. Tandem duplications were the main driver of the expansion of the UGT gene family from Ch. indicum. Structural domain analysis showed that 262 UGT genes had complete plant secondary metabolism signal sequences(PSPG box). The analysis of cis-acting elements indicated that light-responsive elements were the most ubiquitous elements in the promoter regions of UGT gene family members. Quasi-targeted metabolome analysis of floral and leaf tissue revealed that most of the flavonoid metabolites, including luteolin-7-O-glucoside and kaempferol-7-O-glucoside, had higher accumulation in flowers. Comparative transcriptome analysis of flower and leaf tissue showed that there were 72 differentially expressed UGT genes, of which 29 genes were up-regulated in flowers, and 43 genes were up-regulated in leaves. Correlation network and phylogenetic analysis showed that CindChr9G00614970.1, CindChr2G00092510.1, and CindChr2G00092490.1 may be involved in the synthesis of 7-O-flavonoid glycosides in Ch. indicum, and real-time fluorescence quantitative PCR analysis further confirmed the reliability of transcriptome data. The results of this study are helpful to understand the function of the UGT gene family from Ch. indicum and provide data reference and theoretical basis for further study on the molecular regulation mechanism of flavonoid glycosides synthesis in Ch. indicum.
Topics: Glycosyltransferases; Chrysanthemum; Uridine Diphosphate; Phylogeny; Reproducibility of Results; Plants; Flavonoids; Glycosides; Gene Expression Regulation, Plant
PubMed: 38621874
DOI: 10.19540/j.cnki.cjcmm.20231115.102 -
Neuron Jun 2024Microglial calcium signaling is rare in a baseline state but strongly engaged during early epilepsy development. The mechanism(s) governing microglial calcium signaling...
Microglial calcium signaling is rare in a baseline state but strongly engaged during early epilepsy development. The mechanism(s) governing microglial calcium signaling are not known. By developing an in vivo uridine diphosphate (UDP) fluorescent sensor, GRAB, we discovered that UDP release is a conserved response to seizures and excitotoxicity across brain regions. UDP can signal through the microglial-enriched P2Y receptor to increase calcium activity during epileptogenesis. P2Y calcium activity is associated with lysosome biogenesis and enhanced production of NF-κB-related cytokines. In the hippocampus, knockout of the P2Y receptor prevents microglia from fully engulfing neurons. Attenuating microglial calcium signaling through calcium extruder ("CalEx") expression recapitulates multiple features of P2Y knockout, including reduced lysosome biogenesis and phagocytic interactions. Ultimately, P2Y knockout mice retain more CA3 neurons and better cognitive task performance during epileptogenesis. Our results demonstrate that P2Y signaling impacts multiple aspects of myeloid cell immune function during epileptogenesis.
Topics: Animals; Microglia; Mice; Phagocytosis; Receptors, Purinergic P2; Mice, Knockout; Calcium Signaling; Epilepsy; Uridine Diphosphate; Lysosomes; Neurons; Mice, Inbred C57BL; Male; Hippocampus; Neuroimmunomodulation
PubMed: 38614103
DOI: 10.1016/j.neuron.2024.03.017