Comparative Metabolome and Transcriptome Analysis Revealed the Accumulative Mechanism of Rubusoside in Chinese Sweet Tea.

Terpenoids are important secondary metabolites in Rubus. Rubusoside is a relatively specific diterpenoid bioactive component in the leaves of Chinese Sweet Tea (Rubus suavissimus). However, the terpenoid anabolic pathway of Rubus and the molecular mechanism underlying the specific accumulation of rubusoside in R. suavissimus remain unclear. Here, metabolomics and transcriptomics analyses were performed on differences in terpenoid metabolism levels between R. suavissimus (sweet leaves) and Rubus chingii (bitter leaves). Steviol glycosides and goshonosides primarily accumulated in R. suavissimus and R. chingii, respectively. Three pairs of highly homologous glycosyltransferase genes (UGT85A57, UGT75L20, and UGT75T4) associated with rubusoside biosynthesis in the two Rubus species were identified. The three pairs of UGT proteins in both species could glycosylate steviol. Thus, the transcriptional regulation of UGTs in R. suavissimus appears to play a pivotal role in rubusoside accumulation. Our findings provide insights into the differences in terpenoid metabolism between R. suavissimus and R. chingii and reveal the molecular mechanism of rubusoside accumulation in R. suavissimus.

Uridine triphosphate hybrid catalyst for carbon­carbon bond formation reactions with enhanced enantioselectivity by mercury(II) ions.

DNA hybrid catalysts are constructed by embedding active metal species into the chiral scaffolds of DNA, which have been successfully applied to some important aqueous-phase enantioselective transformations. Owing to simple components and inherent chirality, nucleotide hybrid catalysts are emerging in response to soving the unclear locations of catalytic centers and the plausible catalytic mechanisms in DNA-based asymmetric catalysis. However, the tertiary structure of nucleotides lacks tunability, severely impeding further design of nucleotide hybrid catalysts for potential applications. To this end, a design strategy for tunable nucleotide hybrid catalysts is put forward by introducing metal-mediated base pairs. Herein, we found that the formation of uracil­mercury(II)-uracil (U-Hg2+-U) base pairs could enhance the enantioselectivity in uracil-containing nucleotide-based asymmetric reactions. Compared with uracil triphosphate (UTP) complexing with Cu2+ ions (UTP∙Cu2+), the presence of Hg2+ ions gave rise to an increased enantiomeric excess (ee) of 38 % in Diels-Alder reactions and 22 % ee in Michael reactions. The Hg2+-tuning behaviors of UTP hybrid catalyst have been demonstrated to largely depend on nucleotides, Hg2+ concentrations, metal cofactors, additives and reaction types. Based on ultraviolet-visible, circular dichroism and nuclear magnetic resonance spectroscopic techniques, the chiral enhancement of Hg2+-containing UTP hybrid catalyst is proved to largely depend on the formation of U-Hg2+-U base pairs and the plausible cross-linked structure of UTP-Hg2+-UTP/Cu2+ assembly. This work provides a tunable strategy based on the concept of metal-mediated base pairs, allowing further design of potent oligonucleotide-based catalysts for other enantioselective reactions.

[Production of neohesperidin from hesperetin by an engineered strain of Escherichia coli].

Neohesperidin is a flavonoid glycoside widely used in the food and pharmaceutical industries. The current production of neohesperidin mainly relies on extraction from plants. Microbial fermentation demonstrates a promising prospect as an environmentally friendly, efficient, and economical method. In this study, we designed and constructed the biosynthetic pathway of neohesperidin in an Escherichia coli strain by introducing the glycosyltransferase UGT73B2 from Arabidopsis thaliana, rhamnose synthase VvRHM-NRS from Vitis vinifera, and rhamnose transferase Cm1,2RhaT from Citrus maxima. After optimization of the module and the uridine diphosphate (UDP)-glucose synthetic pathway, the engineered strain produced 4.64 g/L neohesperidin in a 5 L bioreactor, and the molar conversion rate of hesperetin was 45.8%. This has been the highest titer reported to date for the biosynthesis of neohesperidin in microorganisms. This study lays a foundation for the construction and application of strains with high yields of neohesperidin and provides a potential choice for the microbial production of other flavonoid glycosides.

[Efficient synthesis of salidroside from tyrosol based on UDPG recycling system].

Salidroside is a functional ingredient with wide applications in food and pharmaceutical fields. It is conventionally produced by extraction from plants, the application of which is limited by the scarcity of raw materials and cumbersome process. This study achieved the efficient production of salidroside by biosynthesis with tyrosol as the substrate. While utilizing glycosyltransferases for tyrosol glycosylation, we introduced sucrose synthase to construct the uridine diphosphate glucose (UDPG) recycling system. The glycosyltransferase UGT33 and sucrose synthase AtSUS were screened out by comparison, and the recombinant strain Escherichia coli BL21/pETDuet-AtSUS-UGT33 was constructed. The copy number of the gene was optimized and the optimal copy number ratio of glycosyltransferase to sucrose synthase was determined to be 3:1. The whole-cell transformation conditions (temperature, pH, inoculum amount, substrate concentration, and concentrations of metal ions) of the recombinant strain were optimized, and the highest yield of salidroside reached 8.17 g/L after fermentation under the optimal conditions in a 5 L fermenter for 24 h. This study provides a reference for the efficient production of salidroside by microorganisms.

A review on rebaudioside M: The next generation steviol glycoside and noncaloric sweetener.

So far, the use of artificial low-calorie sweeteners, like sucralose, saccharin, and so on, to replace the conventional-based sugars has not succeeded due to the long-term adverse health effects, for example, hypertension, and not well-known safety stand. In this review, we discussed the next generation SvGl (rebaudioside M [Reb M]), their biosynthetic pathway in plant, high-yield production via microbial fermentation and enzyme engineering, physicochemical properties, taste modification, kinetic metabolism, application in food and beverages, safety and toxicological evaluation, regulation and dosage recommendation, and health benefits. In stevia, the biosynthesis of stevia glycosides, especially Reb M, is derived from the bifurcation of the pathway leading to gibberellin, followed by subsequent enzymatic modification of rubusoside. Reb M is more economically produced via microbial fermentation of modified yeast Yarrowia lipolytica and enzymatic bioconversion of rebaudioside A (Reb A) or Reb E. Reb M can serve as a suitable alternative to the conventional-based sugars.

Unveiling genetic variants: Tetra-primer ARMS-PCR diagnosis and structural insights into BLAD, BC, and DUMPS in Pakistani cattle herds.

BACKGROUND: Bovine leukocyte adhesion deficiency (BLAD), bovine citrullinemia (BC), and deficiency of Uridine monophosphate synthetase (DUMPS) are the common autosomal recessive disorders affecting the global dairy industry. BLAD leads to poor wound healing and recurrent infections. In BC, ammonia builds up leading to neurological disorders and death. DUMPS results in developmental abnormalities. METHODOLOGY: In this study, tetra-primer amplification refractory mutation system polymerase chain reaction (ARMS PCR) based diagnostic tests were optimized for BLAD, BC, and DUMPS. A total of 250 animals (58 indigenous and 192 Holstein Friesian (HF)) were screened from all across Pakistan. In addition to validation of ARMS-PCR results through Sanger sequencing, the protein modeling provided structural insights of the disease-associated reported SNPs. Pathway analysis illustrated gene functions under normal and mutated conditions. Furthermore, haplotype and phylogenetic analysis of ASS1 (Argininosuccinate synthetase) gene were performed on study samples and NCBI retrieved sequences. RESULTS: The study's focus was to screen the herds for prevalence of carriers of genetic disorders, as they are the main source of disease dissemination. One animal was found carrier for BC, whereas no carriers were found for BLAD and DUMPS. The protein models corroborated the reported amino acid change in BLAD, and protein truncation in both BC and DUMPS proteins. SNPs found in NCBI retrieved sequences were either silent or missense and had no effect on protein structure. DNA network presented graphical illustration of haplotype interactions and phylogenetic analysis conferred evolutionary landscape of ASS1 gene. The combination of these approaches produced an in-depth genetic picture of BC in Pakistani cattle. CONCLUSION: The development of diagnostic tests and identification of the heterozygous BC sample underscores the significance of constant monitoring to avoid the unwanted dissemination of mutant alleles among Pakistani cattle, thereby promoting the general well-being and sustainability of the dairy sector.

Biphasic effects of single-dose intravenous injection of uridine adenosine tetraphosphate on blood pressure in mice.

PURPOSE: To explore the effects of a single dose of uridine adenosine tetraphosphate (Up4A) administered through the tail vein, on the blood pressure of mice. METHODS: The mice were separated into three groups: the Up4A group, the norepinephrine (NA) group, and the α, ß-methylene adenosine triphosphate (α, ß-meATP) group. Each group of mice were injected drugs through the tail vein at 1, 3, 10, and 30 nmol/kg doses in an ascending order. Additionally, six mice were injected Up4A through the tail vein at 20, 40, 60, and 80 nmol/kg doses in an ascending order. The administration intervals for each dose were 20 min. RESULTS: Mice in these groups experienced a rapid increase in blood pressure, reaching its peak within 10 s after drug administration. It took approximately 120 s for the blood pressure to return to baseline levels after the administration of the drugs in both the NA and α, ß-meATP groups. After higher doses of Up4A were administered to the mice, their blood pressure exhibited biphasic changes. Initially, blood pressure of the mice rapidly dropped to a minimum within 10 s, then rose rapidly to a peak within 30 s. Subsequently, it gradually declined, taking around 10 min to return to the levels before the drug administration. CONCLUSION: Compared to NA and α, ß-meATP, Up4A, which contains purine and pyrimidine components, displayed a weaker blood pressure-elevating potency. Through its corresponding structure, Up4A exerted vasodilatory and vasoconstrictive effects throughout the entire experiment resulting in biphasic changes in blood pressure.

Development of Biolayer Interferometry (BLI)-Based Double-Stranded RNA Detection Method with Application in mRNA-Based Therapeutics and Vaccines.

Background: In vitro-transcribed (IVT) mRNA has been established as a promising platform for therapeutics and vaccine development. Double-stranded RNA (dsRNA) is a major impurity of IVT mRNA and can trigger unfavored immune responses, potentially causing adverse events in patients. Existing dsRNA detection and quantitation methods, such as gel electrophoresis, ELISA, or homogeneous time-resolved fluorescence (HTRF), have low sensitivity or are time-consuming. A recently published lateral flow immunoassay (LFSA) was shown to be fast, but it lacks the sensitivity for dsRNA with uridine modifications. Methods: In this study, we provided a possible explanation for the reduced sensitivity of existing quantitation methods for dsRNA with modified uridines by characterizing the binding affinities of commonly used anti-dsRNA antibodies. Then, a rapid and sensitive biolayer interferometry (BLI) dsRNA detection assay utilizing Flock House Virus (FHV) B2 protein was developed to overcome the challenges in dsRNA detection and the reduced sensitivity. Results: This assay allows the detection of dsRNA with different uridine modifications (ψ, m1ψ, 5 moU) with similar sensitivity as dsRNA without modification. Furthermore, we demonstrated this method can be used to quantify both short and long dsRNA, as well as hairpin-structured dsRNA, providing a more comprehensive detection for dsRNA impurities. Moreover, we applied this assay to monitor dsRNA removal through a purification process. Conclusions: Taken together, this BLI method could enable real-time monitoring of impurities in IVT mRNA production to prevent immunogenicity stemming from dsRNA.

Short-term Uridine Treatment Alleviates Endoplasmic Reticulum Stress via Regulating Inflammation and Oxidative Stress in Lithium-Pilocarpine Model of Status Epilepticus.

BACKGROUND: Status Epilepticus (SE) leads to the development of epilepsy with the contribution of Endoplasmic Reticulum (ER) stress. Uridine, a pyrimidine nucleoside, has been shown to have neuroprotective and antiepileptogenic effects in animal models. This study aimed to determine whether uridine ameliorates ER stress and apoptosis following epileptogenic insult. Secondly, this study aimed to establish the effect of uridine on inflammatory and oxidative stress parameters that contribute to ER stress. METHODS: Status epilepticus was induced using lithium-pilocarpine in adult male Sprague-Dawley rats. Following SE termination, rats were treated with uridine, 4-phenylbutyric acid (4-PBA), or saline twice daily for 48 h. Expressions of hippocampal glucose-regulated protein 78 (GRP78), Inositol- Requiring Protein 1 (IRE1α), Protein kinase RNA-like Endoplasmic Reticulum Kinase (PERK), and C/EBP Homologous Protein (CHOP) were determined by western blotting 48 h after SE. Uridine's effects on apoptosis, inflammation or oxidation were evaluated by analyses of cleaved caspase-3 and poly(ADP-ribose) polymerase 1 (PARP1) protein expressions or pro-inflammatory cytokine levels or levels of oxidative stress markers, respectively. RESULTS: Expressions of all ER stress-related proteins significantly increased 48 h after SE. Uridine treatment markedly decreased GRP78, IRE1α, and CHOP levels. A decrease in the PERK level was observed following the administration of 4-PBA; however, uridine had no effect. Cleaved caspase-3 and PARP1 levels were increased in the SHAM group, while uridine and 4-PBA treatment effectively decreased their expressions. Treatment with uridine significantly reduced Myeloperoxidase (MPO) and Malondialdehyde (MDA) levels while tending to increase Catalase (CAT) and Glutathione Peroxidase (GPx) levels. Uridine treatment also significantly attenuated levels of TNF-α and IL-1ß, the pro-inflammatory cytokines, which increased 48 h post-SE. CONCLUSION: Our data indicate that uridine alleviates ER stress after SE. This effect may be attributed to the regulation of inflammation and oxidative stress. Uridine shows promise as a potential preventive agent for epilepsy.

Thermodynamic Studies of Complexes in Cu(II)/Uridine-5'-Diphosphoglucuronic Acid System.

A binary system of uridine-5'-diphosphoglucuronic acid with copper (II) ions was studied. Potentiometric studies in aqueous solutions using computer data analysis were carried out. The pH of dominance, the overall stability constants (logß), and the equilibrium constants of the formation reaction (logKe) were determined for each complex compound formed in the studied system. Spectroscopic studies were carried out to determine the mode of coordination in the compounds studied. Cytotoxicity and metabolic activity tests of the compounds obtained showed an increase in the biological activity of the complexes tested against the free ligand. The current research may contribute to the knowledge of complex compounds of biomolecules found in the human body and may also contribute to the characterization of a group of complex compounds with potential anticancer properties.

Pharmacological targets of SGLT2 inhibition on prostate cancer mediated by circulating metabolites: a drug-target Mendelian randomization study.

Background: The relationship between sodium-glucose cotransporter 2 (SGLT2) inhibitors and prostate cancer is still unknown. Although these inhibitors can influence tumor glycolysis, the underlying mechanism requires further exploration. Methods: A two-sample two-step MR was used to determine 1) causal effects of SGLT2 inhibition on prostate cancer; 2) causal effects of 1,400 circulating metabolites or metabolite ratios on prostate cancer; and 3) mediation effects of these circulating metabolites. Genetic proxies for SGLT2 inhibition were identified as variants in the SLC5A2 gene and glycated hemoglobin level (HbA1c). Additionally, positive control analysis on type 2 diabetes mellitus (T2DM) was conducted to test the selection of genetic proxies. Phenome Wide Association Study (PheWAS) and MR-PheWAS analysis were used to explore potential treatable diseases and adverse outcomes of SGLT2 inhibitors. Results: Genetically predicted SGLT2 inhibition (per 1 SD decrement in HbA1c) was associated with reduced risk of T2DM [odds ratio (OR) = 0.66 (95% CI 0.53, 0.82), P = 1.57 × 10-4]; prostate cancer [0.34 (0.23, 0.49), P = 2.21 × 10-8] and prostate-specific antigen [0.26 (0.08, 0.81), P = 2.07 × 10-2]. The effect of SGLT2 inhibition on prostate cancer was mediated by uridine level, with a mediated proportion of 9.34% of the total effect. In MR-PheWAS, 65 traits were found to be associated with SLGT2 inhibitors (P < 1.78 × 10-5), and among them, 13 were related to diabetes. Conclusion: Our study suggested that SGLT2 inhibition could lower prostate cancer risk through uridine mediation. More mechanistic and clinical research is necessary to explore how uridine mediates the link between SGLT2 inhibition and prostate cancer.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are associated with insecticide resistance in the major malaria vectors Anopheles gambiae s.l. and Anopheles funestus.

Malaria remains one of the highest causes of morbidity and mortality, with 249 million cases and over 608,000 deaths in 2022. Insecticides, which target the Anopheles mosquito vector, are the primary method to control malaria. The widespread nature of resistance to the most important insecticide class, the pyrethroids, threatens the control of this disease. To reverse the stall in malaria control there is urgent need for new vector control tools, which necessitates understanding the molecular basis of pyrethroid resistance. In this study we utilised multi-omics data to identify uridine-diphosphate (UDP)-glycosyltransferases (UGTs) potentially involved in resistance across multiple Anopheles species. Phylogenetic analysis identifies sequence similarities between Anopheline UGTs and those involved in agricultural pesticide resistance to pyrethroids, pyrroles and spinosyns. Expression of five UGTs was characterised in An. gambiae and An. coluzzii to determine constitutive over-expression, induction, and tissue specificity. Furthermore, a UGT inhibitor, sulfinpyrazone, restored susceptibility to pyrethroids and DDT in An. gambiae, An. coluzzii, An. arabiensis and An. funestus, the major African malaria vectors. Taken together, this study provides clear association of UGTs with pyrethroid resistance as well as highlighting the potential use of sulfinpyrazone as a novel synergist for vector control.

Study of the RNA splicing kinetics via in vivo 5-EU labeling.

Splicing is an important step of gene expression in all eukaryotes. Splice sites might be used with different efficiency, giving rise to alternative splicing products. At the same time, splice sites might be used at a variable rate. We used 5-ethynyl uridine labeling to sequence a nascent transcriptome of HeLa cells and deduced the rate of splicing for each donor and acceptor splice site. The following correlation analysis showed a correspondence of primary transcript features with the rate of splicing. Some dependencies we revealed were anticipated, such as a splicing rate decrease with a decreased complementarity of the donor splice site to U1 and acceptor sites to U2 snRNAs. Other dependencies were more surprising, like a negative influence of a distance to the 5' end on the rate of the acceptor splicing site utilization, or the differences in splicing rate between long, short, and RBM17-dependent introns. We also observed a deceleration of last intron splicing with an increase of the distance to the poly(A) site, which might be explained by the cooperativity of the splicing and polyadenylation. Additional analysis of splicing kinetics of SF3B4 knockdown cells suggested the impairment of a U2 snRNA recognition step. As a result, we deconvoluted the effects of several examined features on the splicing rate into a single regression model. The data obtained here are useful for further studies in the field, as they provide general splicing rate dependencies as well as help to justify the existence of slowly removed splice sites.

Lipidomic and transcriptomic profiles provide new insights into the triacylglycerol and glucose handling capacities of the Arctic fox.

The Arctic fox (Vulpes lagopus) is a species indigenous to the Arctic and has developed unique lipid metabolism, but the mechanisms remain unclear. Here, the significantly increased body weight of Arctic foxes was consistent with the significantly increased serum very-low-density lipoprotein (VLDL), and the 40% crude fat diet further increased the Arctic fox body weight. The enhanced body weight gain stems primarily from increased subcutaneous adipose tissue accumulation. The adipose triacylglycerol and phosphatidylethanolamine were significantly greater in Arctic foxes. The adipose fatty-acid synthase content was significantly lower in Arctic foxes, highlighting the main role of exogenous fatty-acids in fat accumulation. Considering the same diet, liver-derived fat dominates adipose expansion in Arctic foxes. Liver transcriptome analysis revealed greater fat and VLDL synthesis in Arctic foxes, consistent with the greater VLDL. Glucose homeostasis wasn't impacted in Arctic foxes. And the free fatty-acids in adipose, which promote insulin resistance, also did not differ between groups. However, the hepatic glycogen was greater in Arctic foxes and transcriptome analysis revealed upregulated glycogen synthesis, improving glucose homeostasis. These results suggest that the superior fat accumulation capacity and distinct characteristics of hepatic and adipose lipid and glucose metabolism facilitate glucose homeostasis and massive fat accumulation in Arctic foxes.

5'-UMP inhibited muscle atrophy due to detraining: a randomized, double-blinded, placebo-controlled, parallel-group comparative study.

Purpose: A short period of disuse of 1-2 weeks due to factors such as illness or injury can lead to muscle atrophy, affecting both athletic performance and health. Recent research has shown that uridine 5'-monophosphate (5'-UMP) can counteract disuse-induced muscle atrophy by increasing PGC-1α expression and inhibiting atrogin-1 expression. However, the effect of 5'-UMP on disuse muscle atrophy in humans remains unknown. Therefore, the aimed of this study was to explore the effects of 5'-UMP supplementation during detraining on short-term disuse muscle atrophy in healthy men. Methods: Following a 6-week resistance training program on upper arm, healthy men were randomized to either a UMP group (n = 11) or a placebo group (n = 10), taking their respective supplements during the 2-week detraining period. Muscle thickness, an indicator of muscle hypertrophy and atrophy, was measured at 3 positions (MT50, MT60, and MT70) at baseline, 1 week, and 2 weeks after detraining. Results: Both groups showed a significant decrease in muscle thickness at MT70. The relative decrease was greater in the placebo group (2.4 ± 2.8%) than in the UMP group (0.0 ± 2.0%), significantly (p = 0.034) at 1 week. However, no significant difference was observed at MT50 and MT60. Conclusion: After the hypertrophy, 5'-UMP may prevent muscle atrophy due to the detraining within the first week.

Abnormal metabolism in melanocytes participates in the activation of dendritic cell in halo nevus.

A comprehensive analysis of spatial transcriptomics was carried out to better understand the progress of halo nevus. We found that halo nevus was characterized by overactive immune responses, triggered by chemokines and dendritic cells (DCs), T cells, and macrophages. Consequently, we observed abnormal cell death, such as apoptosis and disulfidptosis in halo nevus, some were closely related to immunity. Interestingly, we identified aberrant metabolites such as uridine diphosphate glucose (UDP-G) within the halo nevus. UDP-G, accompanied by the infiltration of DCs and T cells, exhibited correlations with certain forms of cell death. Subsequent experiments confirmed that UDP-G was increased in vitiligo serum and could activate DCs. We also confirmed that oxidative response is an inducer of UDP-G. In summary, the immune response in halo nevus, including DC activation, was accompanied by abnormal cell death and metabolites. Especially, melanocyte-derived UDP-G may play a crucial role in DC activation.

The Protective Effect of Uridine in a Rotenone-Induced Model of Parkinson's Disease: The Role of the Mitochondrial ATP-Dependent Potassium Channel.

The effect of the modulators of the mitochondrial ATP-dependent potassium channel (mitoKATP) on the structural and biochemical alterations in the substantia nigra and brain tissues was studied in a rat model of Parkinson's disease induced by rotenone. It was found that, in experimental parkinsonism accompanied by characteristic motor deficits, both neurons and the myelin sheath of nerve fibers in the substantia nigra were affected. Changes in energy and ion exchange in brain mitochondria were also revealed. The nucleoside uridine, which is a source for the synthesis of the mitoKATP channel opener uridine diphosphate, was able to dose-dependently decrease behavioral disorders and prevent the death of animals, which occurred for about 50% of animals in the model. Uridine prevented disturbances in redox, energy, and ion exchanges in brain mitochondria, and eliminated alterations in their structure and the myelin sheath in the substantia nigra. Cytochemical examination showed that uridine restored the indicators of oxidative phosphorylation and glycolysis in peripheral blood lymphocytes. The specific blocker of the mitoKATP channel, 5-hydroxydecanoate, eliminated the positive effects of uridine, suggesting that this channel is involved in neuroprotection. Taken together, these findings indicate the promise of using the natural metabolite uridine as a new drug to prevent and, possibly, stop the progression of Parkinson's disease.

Glycolytic PFKFB3 and Glycogenic UGP2 Axis Regulates Perfusion Recovery in Experimental Hind Limb Ischemia.

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 an increased perfusion recovery and 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 expression/levels 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-knock-down 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.

Nanoarchitectonics and Biological Properties of Nanocomposite Thermosensitive Chitosan Hydrogels Obtained with the Use of Uridine 5'-Monophosphate Disodium Salt.

Currently, an important group of biomaterials used in the research in the field of tissue engineering is thermosensitive chitosan hydrogels. Their main advantage is the possibility of introducing their precursors (sols) into the implantation site using a minimally invasive method-by injection. In this publication, the results of studies on the new chitosan structures in the form of thermosensitive hydrogels containing graphene oxide as a nanofiller are presented. These systems were prepared from chitosan lactate and chitosan chloride solutions with the use of a salt of pyrimidine nucleotide-uridine 5'-monophosphate disodium salt-as the cross-linking agent. In order to perform the characterization of the developed hydrogels, the sol-gel transition temperature of the colloidal systems was first determined based on rheological measurements. The hydrogels were also analyzed using FTIR spectroscopy and SEM. Biological studies assessed the cytotoxicity (resazurin assay) and genotoxicity (alkaline version of the comet assay) of the nanocomposite chitosan hydrogels against normal human BJ fibroblasts. The conducted research allowed us to conclude that the developed hydrogels containing graphene oxide are an attractive material for potential use as scaffolds for the regeneration of damaged tissues.

Preliminary X-ray diffraction and ligand-binding analyses of the N-terminal domain of hypothetical protein Rv1421 from Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis can reside and persist in deep tissues; latent tuberculosis can evade immune detection and has a unique mechanism to convert it into active disease through reactivation. M. tuberculosis Rv1421 (MtRv1421) is a hypothetical protein that has been proposed to be involved in nucleotide binding-related metabolism in cell-growth and cell-division processes. However, due to a lack of structural information, the detailed function of MtRv1421 remains unclear. In this study, a truncated N-terminal domain (NTD) of MtRv1421, which contains a Walker A/B-like motif, was purified and crystallized using PEG 400 as a precipitant. The crystal of MtRv1421-NTD diffracted to a resolution of 1.7 Šand was considered to belong to either the C-centered monoclinic space group C2 or the I-centered orthorhombic space group I222, with unit-cell parameters a = 124.01, b = 58.55, c = 84.87 Å, ß = 133.12° or a = 58.53, b = 84.86, c = 90.52 Å, respectively. The asymmetric units of the C2 or I222 crystals contained two or one monomers, respectively. In terms of the binding ability of MtRv1421-NTD to various ligands, uridine diphosphate (UDP) and UDP-N-acetylglucosamine significantly increased the melting temperature of MtRv1421-NTD, which indicates structural stabilization through the binding of these ligands. Altogether, the results reveal that a UDP moiety may be required for the interaction of MtRv1421-NTD as a nucleotide-binding protein with its ligand.