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Journal of Functional Biomaterials Mar 2024Utilizing the immune system as a strategy for disease prevention and treatment is promising, especially with dendritic cells (DCs) playing a central role in adaptive...
Utilizing the immune system as a strategy for disease prevention and treatment is promising, especially with dendritic cells (DCs) playing a central role in adaptive immune responses. The unique properties of DCs drive interest in developing materials for cell-based therapy and immune modulation. Injectable systems require syringe-compatible scaffolds, while hydrogels, like alginate, known for their programmability and biocompatibility, offer a versatile platform for immune medicine enhancement through easy preparation and room-temperature cross-linking. In this study, we synthesized alginate balls loaded with DCs or cytosine-phosphorothioate-guanine deoxyribonucleotide (CpG DNA) microparticles, aiming for long-term immune cell culture with potential immune stimulation effects. Encapsulated DCs exhibited proliferation within the alginate balls for up to 7 days, and CpG MPs were uniformly dispersed, which can facilitate uptake by DCs. This was supported by the result that DCs effectively phagocytosed CpG microparticles in a 2D environment. After the uptake of CpG MPs, the alginate balls with CpG-MP-uptaken DCs were synthesized successfully. The injectable properties of the alginate balls were easily modulated by adjusting the syringe needle gauges. This innovative strategy holds substantial promise for advancing medical treatments, offering effective and comfortable solutions for controlled immune modulation.
PubMed: 38535252
DOI: 10.3390/jfb15030059 -
Journal of the American Chemical Society Jul 2023Bacterial glycomes are rich in prokaryote-specific or "rare" sugars that are absent in mammals. Like common sugars found across organisms, rare sugars are typically...
Bacterial glycomes are rich in prokaryote-specific or "rare" sugars that are absent in mammals. Like common sugars found across organisms, rare sugars are typically activated as nucleoside diphosphate sugars (NDP-sugars) by nucleotidyltransferases. In bacteria, the nucleotidyltransferase RmlA initiates the production of several rare NDP-sugars, which in turn regulate downstream glycan assembly through feedback inhibition of RmlA via binding to an allosteric site. , RmlA activates a range of common sugar-1-phosphates to produce NDP-sugars for biochemical and synthetic applications. However, our ability to probe bacterial glycan biosynthesis is hindered by limited chemoenzymatic access to rare NDP-sugars. We postulate that natural feedback mechanisms impact nucleotidyltransferase utility. Here, we use synthetic rare NDP-sugars to identify structural features required for regulation of RmlA from diverse bacterial species. We find that mutation of RmlA to eliminate allosteric binding of an abundant rare NDP-sugar facilitates the activation of noncanonical rare sugar-1-phosphate substrates, as products no longer affect turnover. In addition to promoting an understanding of nucleotidyltransferase regulation by metabolites, this work provides new routes to access rare sugar substrates for the study of important bacteria-specific glycan pathways.
Topics: Animals; Nucleotides; Nucleotidyltransferases; Sugars; Feedback; Bacteria; Nucleoside Diphosphate Sugars; Mammals
PubMed: 37283497
DOI: 10.1021/jacs.3c02319 -
Nucleic Acids Research Jun 2024Cancer cells produce vast quantities of reactive oxygen species, leading to the accumulation of toxic nucleotides as 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate...
Cancer cells produce vast quantities of reactive oxygen species, leading to the accumulation of toxic nucleotides as 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP). The human MTH1 protein catalyzes the hydrolysis of 8-oxo-dGTP, and cancer cells are dependent on MTH1 for their survival. MTH1 inhibitors are possible candidates for a class of anticancer drugs; however, a reliable screening system using live cells has not been developed. Here we report a visualization method for 8-oxo-dGTP and its related nucleotides in living cells. Escherichia coli MutT, a functional homologue of MTH1, is divided into the N-terminal (1-95) and C-terminal (96-129) parts (Mu95 and 96tT, respectively). Mu95 and 96tT were fused to Ash (assembly helper tag) and hAG (Azami Green), respectively, to visualize the nucleotides as fluorescent foci formed upon the Ash-hAG association. The foci were highly increased when human cells expressing Ash-Mu95 and hAG-96tT were treated with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-dGTP. The foci formation by 8-oxo-dG(TP) was strikingly enhanced by the MTH1 knockdown. Moreover, known MTH1 inhibitors and oxidizing reagents also increased foci. This is the first system that visualizes damaged nucleotides in living cells, provides an excellent detection method for the oxidized nucleotides and oxidative stress, and enables high throughput screening for MTH1 inhibitors.
Topics: Humans; Deoxyguanine Nucleotides; DNA Repair Enzymes; Escherichia coli; Escherichia coli Proteins; Guanine Nucleotides; Oxidation-Reduction; Phosphoric Monoester Hydrolases; Pyrophosphatases
PubMed: 38738661
DOI: 10.1093/nar/gkae371 -
Pain Practice : the Official Journal of... Jul 2023Spinal cord stimulation (SCS) has been proven to be an effective treatment for patients suffering from intractable chronic neuropathic pain. Recent advances in the field...
BACKGROUND
Spinal cord stimulation (SCS) has been proven to be an effective treatment for patients suffering from intractable chronic neuropathic pain. Recent advances in the field include the utilization of programs that multiplex various signals to target different neural structures in the dorsal spinal cord associated with the painful area. Preclinical studies have been fundamental in understanding the mechanism by which this differential target multiplexed programming (DTMP) SCS approach works. Transcriptomic- and proteomic-based studies demonstrated that DTMP can modulate expression levels of genes and proteins involved in pain-related processes that have been affected by a neuropathic pain model. This work studied the effect of the intensity of DTMP signals on mechanical hypersensitivity and cell-specific transcriptomes.
METHODS
The spared nerve injury model (SNI) of neuropathic pain was induced in 20 animals which were 1:1 randomized into two SCS groups in which the intensity of the DTMP was adjusted to either 70% or 40% of the motor threshold (MT). SCS was applied continuously for 48 h via a quadripolar lead implanted in the dorsal epidural space of animals. Controls, which included a group of implanted SNI animals that received no SCS and a group of animals naive to the SNI, were assessed in parallel to the SCS groups. Mechanical hypersensitivity was assessed before SNI, before SCS, and at 48 h of SCS. At the end of SCS, the stimulated segment of the dorsal spinal cord was dissected and subjected to RNA sequencing to quantify expression levels in all experimental groups. Differential effects were assessed via fold-change comparisons of SCS and naive groups versus the no-SCS group for transcriptomes specific to neurons and glial cells. Standard statistical analyses were employed to assess significance of the comparisons (p < 0.05).
RESULTS
SCS treatments provided significant improvement in mechanical sensitivity relative to no SCS treatment. However, the change in the intensity did not provide a significant difference in the improvement of mechanical sensitivity. DTMP regulated expression levels back toward those found in the naive group in the cell-specific transcriptomes analyzed. There were no significant differences related to the intensity of the stimulation in terms of the percentage of genes in each transcriptome in which expression levels were reversed toward the naive state.
CONCLUSIONS
DTMP when applied at either 40% MT or 70% MT provided similar reduction of pain-like behavior in rats and similar effects in neuron- and glia-specific transcriptomes.
Topics: Rats; Animals; Pain Threshold; Pain Measurement; Proteomics; Thymidine Monophosphate; Disease Models, Animal; Neuralgia; Spinal Cord; Spinal Cord Stimulation
PubMed: 37067033
DOI: 10.1111/papr.13235 -
BMC Pediatrics Apr 2024Dilated cardiomyopathy (DCMP) is characterized by the enlargement and weakening of the heart and is a major cause of heart failure in children. Infection and nutritional...
BACKGROUND
Dilated cardiomyopathy (DCMP) is characterized by the enlargement and weakening of the heart and is a major cause of heart failure in children. Infection and nutritional deficiencies are culprits for DCMP. Zinc is an important nutrient for human health due to its anti-oxidant effect that protects cell against oxidative damage. This case-control study aimed to investigate the relationship between dietary intake of zinc and selenium and the risk of DCMP in pediatric patients.
METHODS
A total of 36 DCMP patients and 72 matched controls were recruited, and their dietary intakes were assessed via a validated food frequency questionnaire. We used chi-square and sample T-test for qualitative and quantitative variables, respectively. Logistic regression analysis was applied to assess the relationship between selenium and zinc intake with the risk of DCMP.
RESULTS
After fully adjusting for confounding factors, analyses showed that selenium (OR = 0.19, CI = 0.057-0.069, P trend < 0.011) and zinc (OR = 0.12, CI = 0.035-0.046, P trend < 0.002) intake were strongly associated with 81% and 88% lower risk of pediatric DCMP, respectively.
CONCLUSIONS
This study highlights the protective role of adequate dietary intake of selenium and zinc in decreasing the risk of DCMP in children. Malnutrition may exacerbate the condition and addressing these micronutrient deficiencies may improve the cardiac function. Further studies are recommended to detect the underlying mechanisms and dietary recommendations for DCMP prevention.
Topics: Humans; Child; Selenium; Case-Control Studies; Cardiomyopathy, Dilated; Deoxycytidine Monophosphate; Zinc; Malnutrition
PubMed: 38605385
DOI: 10.1186/s12887-024-04706-1 -
Nature Communications Jun 2024C2'-halogenation has been recognized as an essential modification to enhance the drug-like properties of nucleotide analogs. The direct C2'-halogenation of the...
C2'-halogenation has been recognized as an essential modification to enhance the drug-like properties of nucleotide analogs. The direct C2'-halogenation of the nucleotide 2'-deoxyadenosine-5'-monophosphate (dAMP) has recently been achieved using the Fe(II)/α-ketoglutarate-dependent nucleotide halogenase AdaV. However, the limited substrate scope of this enzyme hampers its broader applications. In this study, we report two halogenases capable of halogenating 2'-deoxyguanosine monophosphate (dGMP), thereby expanding the family of nucleotide halogenases. Computational studies reveal that nucleotide specificity is regulated by the binding pose of the phosphate group. Based on these findings, we successfully engineered the substrate specificity of these halogenases by mutating second-sphere residues. This work expands the toolbox of nucleotide halogenases and provides insights into the regulation mechanism of nucleotide specificity.
Topics: Substrate Specificity; Protein Engineering; Halogenation; Nucleotides; Deoxyguanine Nucleotides; Escherichia coli
PubMed: 38898020
DOI: 10.1038/s41467-024-49147-7 -
Frontiers in Cellular and Infection... 2023Maintenance of dNTPs pools in is dependent on both biosynthetic and degradation pathways that together ensure correct cellular homeostasis throughout the cell cycle...
Maintenance of dNTPs pools in is dependent on both biosynthetic and degradation pathways that together ensure correct cellular homeostasis throughout the cell cycle which is essential for the preservation of genomic stability. Both the salvage and pathways participate in the provision of pyrimidine dNTPs while purine dNTPs are made available solely through salvage. In order to identify enzymes involved in degradation here we have characterized the role of a trypanosomal SAMHD1 orthologue denominated TbHD82. Our results show that TbHD82 is a nuclear enzyme in both procyclic and bloodstream forms of . Knockout forms exhibit a hypermutator phenotype, cell cycle perturbations and an activation of the DNA repair response. Furthermore, dNTP quantification of null mutant cells revealed perturbations in nucleotide metabolism with a substantial accumulation of dATP, dCTP and dTTP. We propose that this HD domain-containing protein present in kinetoplastids plays an essential role acting as a sentinel of genomic fidelity by modulating the unnecessary and detrimental accumulation of dNTPs.
Topics: Deoxyribonucleotides; Trypanosoma brucei brucei; SAM Domain and HD Domain-Containing Protein 1; Genomic Instability; Genome, Protozoan; DNA Damage; Cell Cycle
PubMed: 37674581
DOI: 10.3389/fcimb.2023.1241305 -
Cellular and Molecular Biology... Sep 2023This research examined the effects of varying amounts of fibrinogen (the fibrin precursor) and HA-MA on mechanical strength, BMSC proliferation, and chondrogenesis...
Bone marrow concentration in combination with hyaluronan and fibrin for the treatment of primary and non-primary in orthopaedic cases of osteochondral defects of the ankle.
This research examined the effects of varying amounts of fibrinogen (the fibrin precursor) and HA-MA on mechanical strength, BMSC proliferation, and chondrogenesis potential in vitro. In order to start a culture, fibrinogen, aprotinin, doxycycline, and HA-MA were well mixed in 200 L of AMEM containing 1% (w/v) photoinitiator. In order to produce a fibrin gel, thrombin was used at 1, 3,5, and 7 days after implantation, live/dead staining and a metabolic-activity test were used to examine the viability and proliferation of BMSCs inside fibrin/HA-MA. The cell lysis solution from the Real Time ready Cell Lysis Kit was added to each gel. Both primer and probe mixes, as well as DNA polymerase, deoxyribonucleotide triphosphates, an activator, and an enhancer, were mixed before the lysis process began to asses mRNA expression. The mechanical strength of fibrin hydrogels was shown to be proportional to the quantity of HA-MA utilised in the reinforcement. Quantitative polymerase chain reaction demonstrated a reduction in the expression of collagen type 1 alpha 1 mRNA in BMSCs after they were treated in a fibrin/HA-MA hydrogel. Using fibrin/HA-MA hydrogel as a delivery technique for bone marrow stem cells may induce BMSC differentiation into chondrocytes and perhaps aid in articular cartilage repair for OA therapy (BMSCs). We concluded that the utilisation of bone marrow concentration in conjunction with a combination of fibrin and Hyaluronan treatment is safe for patients suffering from OCD of the ankle and is well tolerated by these patients as both a primary treatment and a non-primary treatment option.
Topics: Humans; Hyaluronic Acid; Ankle; Bone Marrow; Fibrin; Orthopedics; Hydrogels; RNA, Messenger; Fibrinogen; Chondrogenesis; Bone Marrow Cells
PubMed: 37807338
DOI: 10.14715/cmb/2023.69.9.4 -
Insect Science Apr 2024Spermatogenesis is critical for insect reproduction and the process is regulated by multiple genes. Glycosyltransferases have been shown to participate in the...
Spermatogenesis is critical for insect reproduction and the process is regulated by multiple genes. Glycosyltransferases have been shown to participate in the development of Drosophila melanogaster; however, their role in spermatogenesis is still unclear. In this study, we found that α1,4-galactosyltransferase 1 (α4GT1) was expressed at a significantly higher level in the testis than in the ovary of Drosophila. Importantly, the hatching rate was significantly decreased when α4GT1 RNA interference (RNAi) males were crossed with w females, with only a few mature sperm being present in the seminal vesicle of α4GT1 RNAi flies. Immunofluorescence staining further revealed that the individualization complex (IC) in the testes from α4GT1 RNAi flies was scattered and did not move synchronically, compared with the clustered IC observed in the control flies. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay showed that apoptosis signals in the sperm bundles of α4GT1 RNAi flies were significantly increased. Moreover, the expression of several individualization-related genes, such as Shrub, Obp44a and Hanabi, was significantly decreased, whereas the expression of several apoptosis-related genes, including Dronc and Drice, was significantly increased in the testes of α4GT1 RNAi flies. Together, these results suggest that α4GT1 may play dual roles in Drosophila spermatogenesis by regulating the sperm individualization process and maintaining the survival of sperm bundles.
PubMed: 38643371
DOI: 10.1111/1744-7917.13369 -
Molecular Biology and Evolution Dec 2023The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine...
Functional Prokaryotic-Like Deoxycytidine Triphosphate Deaminases and Thymidylate Synthase in Eukaryotic Social Amoebae: Vertical, Endosymbiotic, or Horizontal Gene Transfer?
The de novo synthesis of deoxythymidine triphosphate uses several pathways: gram-negative bacteria use deoxycytidine triphosphate deaminase to convert deoxycytidine triphosphate into deoxyuridine triphosphate, whereas eukaryotes and gram-positive bacteria instead use deoxycytidine monophosphate deaminase to transform deoxycytidine monophosphate to deoxyuridine monophosphate. It is then unusual that in addition to deoxycytidine monophosphate deaminases, the eukaryote Dictyostelium discoideum has 2 deoxycytidine triphosphate deaminases (Dcd1Dicty and Dcd2Dicty). Expression of either DcdDicty can fully rescue the slow growth of an Escherichia coli dcd knockout. Both DcdDicty mitigate the hydroxyurea sensitivity of a Schizosaccharomyces pombe deoxycytidine monophosphate deaminase knockout. Phylogenies show that Dcd1Dicty homologs may have entered the common ancestor of the eukaryotic groups of Amoebozoa, Obazoa, Metamonada, and Discoba through an ancient horizontal gene transfer from a prokaryote or an ancient endosymbiotic gene transfer from a mitochondrion, followed by horizontal gene transfer from Amoebozoa to several other unrelated groups of eukaryotes. In contrast, the Dcd2Dicty homologs were a separate horizontal gene transfer from a prokaryote or a virus into either Amoebozoa or Rhizaria, followed by a horizontal gene transfer between them. ThyXDicty, the D. discoideum thymidylate synthase, another enzyme of the deoxythymidine triphosphate biosynthesis pathway, was suggested previously to be acquired from the ancestral mitochondria or by horizontal gene transfer from alpha-proteobacteria. ThyXDicty can fully rescue the E. coli thymidylate synthase knockout, and we establish that it was obtained by the common ancestor of social amoebae not from mitochondria but from a bacterium. We propose horizontal gene transfer and endosymbiotic gene transfer contributed to the enzyme diversity of the deoxythymidine triphosphate synthesis pathway in most social amoebae, many Amoebozoa, and other eukaryotes.
Topics: DCMP Deaminase; Gene Transfer, Horizontal; Escherichia coli; Amoeba; Thymidylate Synthase; Dictyostelium; Deoxycytidine Monophosphate
PubMed: 38064674
DOI: 10.1093/molbev/msad268