Cytidylic Acid Research Articles

Prebiotic studies on the interaction of zirconia nanoparticles and ribose nucleotides and their role in chemical evolution

AbstractStudies on the interaction of biomolecules with inorganic compounds, mainly mineral surfaces, are of great concern in identifying their role in chemical evolution and origins of life. Metal oxides are the major constituents of earth and earth-like planets. Hence, studies on the interaction of biomolecules with these minerals are the point of concern for the study of the emergence of life on different planets. Zirconium oxide is one of the metal oxides present in earth's crust as it is a part of several types of rocks found in sandy areas such as beaches and riverbeds, e.g. pebbles of baddeleyite. Different metal oxides have been studied for their role in chemical evolution but no studies have been reported about the role of zirconium oxide in chemical evolution and origins of life. Therefore, studies were carried out on the interaction of ribonucleic acid constituents, 5′-CMP (cytidine monophosphate), 5′-UMP (uridine monophosphate), 5′-GMP (guanosine monophosphate) and 5′-AMP (adenosine monophosphate), with zirconium oxide. Synthesized zirconium oxide particles were characterized by using vibrating sample magnetometer, X-Ray Diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy. Zirconia particles were in the nanometre range, from 14 to 27 nm. The interaction of zirconium oxide with ribonucleic acid constituents was performed in the concentration range of 5 × 10−5–300 × 10−5 M. Interaction studies were carried out in three mediums; acidic (pH 4.0), neutral (pH 7.0) and basic (pH 9.0). At neutral pH, maximum interaction was observed. The interaction of zirconium oxide with 5′-UMP was 49.45% and with 5′-CMP 67.98%, while with others it was in between. Interaction studies were Langmurian in nature. Xm and KL values were calculated. Infrared spectral studies of ribonucleotides, metal oxide and ribonucleotide–metal oxide adducts were carried out to find out the interactive sites. It was observed that the nitrogen base and phosphate moiety of ribonucleotides interact with the positive charge surface of metal oxide. SEM was also carried out to study the adsorption. The results of the present study favour the important role of zirconium oxide in concentrating the organic molecules from their dilute aqueous solutions in primeval seas.

Synthesis of a rod-based porous coordination polymer from a nucleotide as a sequential chiral inductor

We report the two-step synthesis of a novel chiral rod-based porous coordination polymer (PCP). The chemical approach consists of the use of a previously prepared bis(ethylenediamine) copper monomer [Cu(en)]2(NO3)2 [where en = ethylenediamine] reacting with the cytidine 5′-monophosphate (CMP) nucleotide. The bis(ethylenediamine) copper compound—stabilized by axial coordination of nitrate counter-anions—reacts in the presence of sodium salt of CMP to yield right-handed copper(II) chains of P helicity with formula [Cu2(en)2(CMP)2]·5H2O (1). The axial coordination of the CMP2- ligands through the N3 and O2 sites (free nitrogen and carbonyl groups) of the cytosine nucleobase and oxygen atoms of phosphate moieties, ensure stabilization of the neutral chiral polymer. The supramolecular organization involves strong hydrogen bonding interactions to build supramolecular chains of the same helicity. The resulting PCP constitutes one of the few examples of CMP compounds exhibiting coordination involving both nucleobase and phosphate moieties, where a highly stable metal complex is used as precursor for the rational construction of rod-based hydrogen metal-organic frameworks (HMOFs). Furthermore, it shows the intrinsic capability of biomolecules to act as chiral transfer systems.

Adaptive Chirality of an Achiral Cage: Chirality Transfer, Induction, and Circularly Polarized Luminescence through Aqueous Host–Guest Complexation

Chirality transfer, induction, and circularly polarized luminescence (CPL) using supramolecular hosts, such as macrocycles and cages, have been explored for wide-ranging applications in chiral reco...

Dietary guanosine‐monophosphate improves growth performance, feed utilization and intestinal morphology of whiteleg shrimp ( Litopenaeus vannamei ) maintained on soybean meal‐based diets

The present study evaluated the effects of dietary supplementation of individual or a combination of nucleotides in soya bean meal‐based diet on growth, survival, apparent digestibility and intestinal condition of whiteleg shrimp, Litopenaeus vannamei. The experimental protocol comprised a control set, and four test diets that were separately supplemented with 0.1% inosine monophosphate (IMP, D + I), 0.1% guanosine monophosphate (GMP, D + G), 0.1% mixture of IMP and GMP (D + IG) and 0.1% mixture of IMP, GMP, cytidine monophosphate (CMP) and uridine monophosphate (UMP) (D + IGCU). Triplicate groups of the shrimp (initial body weight = 0.99 ± 0.01 g) were fed four times daily for 8 weeks. Results demonstrated that D + G significantly enhanced body weight gain and specific growth rate in the shrimp compared with control and other dietary treatments with the various combinations of nucleotides (p 0.05). Shrimp fed D + G showed significantly higher feed intake and apparent net protein utilization compared with D + IGCU and, control and D + IG (p < 0.05) respectively. Nucleotide supplementation did not significantly improve the crude protein and lipid digestibility coefficients. This was evident when the data on these parameters were compared with those of the control. Shrimp fed D + G showed significantly improved intestinal villus height and body crude protein content (p < 0.05). In conclusion, GMP supplementation in soya bean meal‐based diet enhanced the growth, feed intake, protein utilization and villus height of L. vannamei.

Long term feeding effects of functional supplement ‘cytidine monophosphate’ on red sea bream, Pagrus major performances

There is no definitive evidence associated with administration duration of nucleotide on aquaculture organisms. This study evaluates the long-term feeding effects of cytidine monophosphate (CMP) nucleotide as functional supplement on growth and health conditions of red sea bream, Pagrus major. A formulated basal diet added with 0% (control), 0.2% (CMP-0.2) and 0.4% (CMP-0.4) individual CMP nucleotide to prepare three experimental feeds. Each experimental feed was fed to triplicate tanks of red sea bream (3.0 ± 0.01 g) for 150 days. At day 50th, growth performances were influenced significantly by dietary CMP inclusion and fish fed CMP-0.4 diet showed significantly higher value. In day 100 and 150 growth performances also non-significantly enhanced by CMP supplementation. Feed utilization performances also showed improved values in nucleotide included diets compared to the non-supplemented control diet (P > 0.05). No significant changes observed on measured hematological parameters by nucleotide administration duration. Lysozyme activity (LA) and total serum protein (TSP) significantly rose to their highest level on 50 days in CMP-0.4 diet group. In 100 and 150th day supplementation also improved TSP and LA values and it reaches maximum in CMP-0.2 diet group (P > 0.05). Oxidative radical production (nitro-blue-tetrazolium assay) and the activity of peroxidase and catalase were not affected significantly with feeding nucleotide in all sampling days. Supplementation of nucleotide improved oxidative stress tolerance capacity. Remarkably, CMP-0.2 diet group showed the least oxidative stress condition in 50th and 100th day. In 150th day fish showed acceptable but reduced oxidative stress resistances. On the basis of the overall findings, finally we concluded that, CMP nucleotide administration regime influences the optimum growth, health performance and immune response of Pagrus major and its mostly after feeding for 50 days, but not after 100 days.

In-situ construction of enzyme-copper nucleotide composite for efficient chemo-enzymatic cascade reaction

The integration of chemical catalyst and biocatalyst is of great interest due to the accompanying high designability of chemo-enzymatic cascade reactions for sustainable catalytic processes in chemical engineering. Here, a facile strategy is proposed by preparation of a copper ions coordinated cytidine monophosphate (CMP) encapsulated lipase composite via in-situ assembly in aqueous solution which followed by reduction to obtain an enzyme-copper hybrid catalyst. The as-synthesized enzyme-copper nucleotide composite exhibited both high enzymatic activity (6.05 U/mg) and copper-catalytic activity (with reaction rate constant of 8.85 min−1) in the chemo-enzymatic cascade reaction for hydrolysis of p-nitrophenyl butyrate (p-NPB) and generation of p-aminophenol (p-AP), which is a pharmaceutical intermediate for production of analgesics and antipyretics. This study thus sheds light on fabrication of hybrid catalyst under facile conditions, exhibiting potential in development of biosensors, industrial catalysis and biomanufacturing.

Cytidine Monophosphate N-Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection.

Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

Metabolic drug targets of the cytosine metabolism pathways in the dromedary camel (Camelus dromedarius) and blood parasite Trypanosoma evansi.

Trypanosomiasis is a major illness affecting camels in tropical and subtropical regions. Comparisons of camel and Trypanosoma evansi genomes can lead to the discovery of new drug targets for treating Trypanosoma infections. The synthesis pathways of cytosine, cytidine, cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP) deoxycytidine, deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP) were compared in the dromedary camel (Camelus dromedarius) and T. evansi. None of the enzymes involved in cytosine pathway were detected in camels and T. evansi. Notably, cytidine kinase (CK) and 5'-nucleotidase, which interconverts cytidine to CMP, were not detected in T. evansi but were present in camels. UMP/CMP kinase was not predicted in T. evansi. Therefore, the presence of enzymes involved in the CTP synthesis cascade was not predicted in T. evansi. CMP synthesis might also be encoded by other enzymes, e.g., purine nucleotides kinases. Both camel and T. evansi share an efficient enzyme system for converting CDP to CTP. In conclusion, CTP synthase is important for homeostasis of cytosine nucleotides in T. evansi and could be a potential drug target against the parasite. In addition, the inhibition of UMP synthesis might contribute to parasite death as it is a shared source for CTP synthesis.

Multi-Enzymatic Cascade One-Pot Biosynthesis of 3'-Sialyllactose Using Engineered Escherichia coli.

Among the human milk oligosaccharides (HMOs), one of the most abundant oligosaccharides and has great benefits for human health is 3′-sialyllactose (3′-SL). Given its important physiological functions and the lack of cost-effective production processes, we constructed an in vitro multi-enzymatic cofactor recycling system for the biosynthesis of 3′-SL from a low-cost substrate. First, we constructed the biosynthetic pathway and increased the solubility of cytidine monophosphate kinase (CMK) with chaperones. We subsequently identified that β-galactosidase (lacZ) affects the yield of 3′-SL, and hence with the lacZ gene knocked out, a 3.3-fold increase in the production of 3′-SL was observed. Further, temperature, pH, polyphosphate concentration, and concentration of divalent metal ions for 3′-SL production were optimized. Finally, an efficient biotransformation system was established under the optimized conditions. The maximum production of 3′-SL reached 38.7 mM, and a molar yield of 97.1% from N-acetylneuraminic acid (NeuAc, sialic acid, SA) was obtained. The results demonstrate that the multi-enzymatic cascade biosynthetic pathway with cofactor regeneration holds promise as an industrial strategy for producing 3′-SL.

Essential Functions of Glycans in Human Epithelia Dissected by a CRISPR-Cas9-Engineered Human Organotypic Skin Model.

SummaryThe glycome undergoes characteristic changes during histogenesis and organogenesis, but our understanding of the importance of select glycan structures for tissue formation and homeostasis is incomplete. Here, we present a human organotypic platform that allows genetic dissection of cellular glycosylation capacities and systematic interrogation of the roles of distinct glycan types in tissue formation. We used CRISPR-Cas9 gene targeting to generate a library of 3D organotypic skin tissues that selectively differ in their capacity to produce glycan structures on the main types of N- and O-linked glycoproteins and glycolipids. This tissue library revealed distinct changes in skin formation associated with a loss of features for all tested glycoconjugates. The organotypic skin model provides phenotypic cues for the distinct functions of glycoconjugates and serves as a unique resource for further genetic dissection and identification of the specific structural features involved. The strategy is also applicable to other organotypic tissue models.

Concomitant use of immobilized uridine-cytidine kinase and polyphosphate kinase for 5'-cytidine monophosphate production

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.

Molecular Interactions of the Polysialytransferase Domain (PSTD) in ST8Sia IV with CMP-Sialic Acid and Polysialic Acid Required for Polysialylation of the Neural Cell Adhesion Molecule Proteins: An NMR Study.

Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of α2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction, the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases.

Pigment epithelium‑derived factor facilitates NLRP3 inflammasome activation through downregulating cytidine monophosphate kinase 2: A potential treatment strategy for missed abortion.

A number of conditions may underlie the occurrence of missed abortion (MA), including inflammation. Pigment epithelium-derived factor (PEDF) is a novel mediator of the inflammation-related nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, which is associated with several human diseases. However, the association between MA and NLRP3 inflammasome, and whether PEDF is reduced in MA, remain unknown. In the present study, the decidua and chorion tissues of patients who had suffered a MA were examined, and a lipopolysaccharide (LPS)-induced human chorionic trophoblast HTR8/SVneo cell model was established to mimic MA in vitro. The results revealed that cytidine monophosphate kinase 2 (CMPK2) expression and NLRP3 inflammasome activation, downstream pro-IL-18 and pro-IL-1β expression, and IL-18 and IL-1β release, were all significantly increased in MA tissues or LPS-induced HTR8/SVneo cells. PEDF reversed the increase in CMPK2 expression and activation of the NLRP3 inflammasome axis and, thus, downregulated the production of mitochondrial reactive oxygen species and mitochondrial DNA release, resulting in reduced lactate dehydrogenase release, and a resultant decrease in cell viability. Recovery of CMPK2 expression abolished all the effects of PEDF, indicating that CMPK2 may be an effector downstream of PEDF. PEDF reduced CMPK2 protein levels but did not affect the mRNA levels, and treatment with the proteasomal inhibitor MG132 significantly reversed this reduction in CMPK2 protein levels. Furthermore, a ubiquitination assay of immunoprecipitation demonstrated that CMPK2 was polyubiquitinated in the presence of LPS, PEDF and MG132. These results indicated that the NLRP3 inflammasome is implicated in the pathogenesis of MA, and PEDF may reduce MA through ubiquitin-dependent proteasomal degradation of CMPK2 to inhibit NLRP3 activation, which may serve as a novel strategy for preventing or reducing the risk of MA.

Effects of dietary nucleotides on growth, survival and metabolic response in whiteleg shrimp, Litopenaeus vannamei against ammonia stress condition

This trial was conducted to evaluate the effects of nucleotides on growth of whiteleg shrimp, Litopenaeus vannamei, and the survival and metabolic responses to ammonia stress test. Experimental diets were as follows: low fish meal diet (LFMD), and four LFMD test diets, each supplemented with 0.1% guanosine monophosphate (GMP), 0.1% inosine monophosphate (IMP), 0.1% mixture of GMP and IMP and 0.1% mixture of GMP, IMP, uridine monophosphate (UMP) and cytidine monophosphate (CMP). The shrimp specimens (initial body weight: 0.99 ± 0.01 g) were randomly allocated into five groups and fed four times daily for 8-weeks. After the trial, final body weight was recorded and haemolymph was withdrawn for haematological analysis. The shrimp was then challenged with 70 mg/L ammonia (LC50) for 10 days. Survival and haemolymph of the shrimp were taken after exposure to ammonia. The highest growth performance was observed in the shrimp fed diet supplemented with GMP (p < .05), while survival was not influenced by the test diets in the feeding trial. In the ammonia challenge test, the highest survival was observed in the shrimp fed GMP supplemented diet compared to others. The plasma protein, glucose and cholesterol levels increased in all the treatments while triglycerides level decreased post challenge. Cortisol level recovered at day 10th after the challenge. Shrimps fed with nucleotides diets showed higher protein and glucose level compared to control groups post challenge. In general, nucleotides supplemented in the diet enhanced growth, improved stress resistance while modulating the haemolymph metabolites in L. vannamei under ammonia stress.

Prediction of VLCAD deficiency phenotype by a metabolic fingerprint in newborn screening bloodspots

PurposeNewborns who test positive for very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) in newborn screening may have a severe phenotype with early onset of life-threatening symptoms but may also have an attenuated phenotype and never become symptomatic. The objective of this study is to investigate whether metabolomic profiles in dried bloodspots (DBS) of newborns allow early phenotypic prediction, permitting tailored treatment and follow-up. MethodsA metabolic fingerprint was generated by direct infusion high resolution mass spectrometry in DBS of VLCADD patients (n = 15) and matched controls. Multivariate analysis of the metabolomic profiles was applied to differentiate subgroups. ResultsConcentration of six acylcarnitine species differed significantly between patients and controls. The concentration of C18:2- and C20:0-carnitine, 13,14-dihydroretinol and deoxycytidine monophosphate allowed separation between mild and severe patients. Two patients who could not be prognosticated on early clinical symptoms, were correctly fitted for severity in the score plot based on the untargeted metabolomics. ConclusionDistinctive metabolomic profiles in DBS of newborns with VLCADD may allow phenotypic prognostication. The full potential of this approach as well as the underlying biochemical mechanisms need further investigation.

Engineering substrate and energy metabolism for living cell production of cytidine-5'-diphosphocholine.

Cytidine-5'-diphosphocholine (CDP-choline) is a widely used neuroprotective drug for multiple indications. In industry, CDP-choline is synthesized by a two-step cell culture/permeabilized cell biotransformation method because substrates often do not enter cells in an efficient manner. This study develops a novel one-step living cell fermentation method for CDP-choline production. For this purpose, the feasibility of Pichia pastoris as a chassis was demonstrated by substrate feeding and CDP-choline production. Overexpression of choline phosphate cytidylyltransferase and choline kinase enhanced the choline transformation pathway and improved the biosynthesis of CDP-choline. Furthermore, co-overexpression of ScHnm1, which is a heterologous choline transporter, highly improved the utilization of choline substrates, despite its easy degradation in cells. This strategy increased CDP-choline titer by 55-folds comparing with the wild-type (WT). Overexpression of cytidine-5'-monophosphate (CMP) kinase and CDP kinase in the CMP transformation pathway showed no positive effects. An increase in the ATP production by citrate stimulation or metabolic pathway modification further improved CDP-choline biosynthesis by 120%. Finally, the orthogonal optimization of key substrates and pH was carried out, and the resulting CDP-choline titer (6.0 g/L) at optimum conditions increased 88 times the original titer in the WT. This study provides a new paradigm for CDP-choline bioproduction by living cells.

Separation of Cytidine 5′-Monophosphate and Guanosine 5′-Monophosphate Based on the Hyper-Cross-Linked Resin XDA-1 with pH Gradient Elution

Cytidine 5′-monophosphate (CMP) and guanosine 5′-monophosphate (GMP) were separated based on hyper-cross-linked resins. The adsorption performances of different resins to the two 5′-ribonucleotides were compared. The adsorption equilibria, kinetics, and dynamic separation process of CMP and GMP with pH gradient elution were studied experimentally and theoretically. The nonpolar resin XDA-1 was selected as the best adsorbent for separating CMP and GMP. A pH-dependent Langmuir isotherm model was developed. The model predicted the adsorption isotherms of CMP and GMP at different pH levels satisfactorily. The surface diffusion model can describe the adsorption kinetics curves of CMP and GMP better than the pseudo-first-order rate model and the pseudo-second-order rate model. CMP and GMP could be both separated with a high separation resolution when the loading amount is 0.031 or 0.093 bed volume (BV). A novel pH-dependent film-surface diffusion model was developed and the model predicted the dynamic separation process of CMP and GMP with pH gradient elution satisfactorily.

ADGRL4/ELTD1 Silencing in Endothelial Cells Induces ACLY and SLC25A1 and Alters the Cellular Metabolic Profile.

Adhesion G Protein-Coupled Receptor L4 (ADGRL4/ELTD1) is an endothelial cell adhesion G protein-coupled receptor (aGPCR) which regulates physiological and tumour angiogenesis, providing an attractive target for anti-cancer therapeutics. To date, ADGRL4/ELTD1′s full role and mechanism of function within endothelial biology remains unknown, as do its ligand(s). In this study, ADGRL4/ELTD1 silencing, using two independent small interfering RNAs (siRNAs), was performed in human umbilical vein endothelial cells (HUVECS) followed by transcriptional profiling, target gene validation, and metabolomics using liquid chromatography-mass spectrometry in order to better characterise ADGRL4/ELTD1′s role in endothelial cell biology. We show that ADGRL4/ELTD1 silencing induced expression of the cytoplasmic metabolic regulator ATP Citrate Lyase (ACLY) and the mitochondria-to-cytoplasm citrate transporter Solute Carrier Family 25 Member 1 (SLC25A1) but had no apparent effect on pathways downstream of ACLY (fatty acid and cholesterol synthesis or acetylation). Silencing induced KIT expression and affected the Notch signalling pathway, upregulating Delta Like Canonical Notch Ligand 4 (DLL4) and suppressing Jagged Canonical Notch Ligand 1 (JAG1) and Hes Family BHLH Transcription Factor 2 (HES2). The effect of ADGRL4/ELTD1 silencing on the cellular metabolic profile was modest but several metabolites were significantly affected. Cis-aconitic acid, uridine diphosphate (UDP)-glucoronate, fructose 2,6-diphosphate, uridine 5-diphosphate, and aspartic acid were all elevated as a result of silencing and phosphocreatine, N-acetylglutamic acid, taurine, deoxyadenosine triphosphate, and cytidine monophosphate were depleted. Metabolic pathway analysis implicated ADGRL4/ELTD1 in pyrimidine, amino acid, and sugar metabolism. In summary, this study shows that ADGRL4/ELTD1 impacts core components of endothelial metabolism and regulates genes involved in endothelial differentiation/homeostasis and Notch signalling.

Comparative Molecular Immunological Activity of Physiological Metal Oxide Nanoparticle and its Anticancer Peptide and RNA Complexes.

Currently, there is a great interest in nanoparticle-based vaccine delivery. Recent studies suggest that nanoparticles when introduced into the biological milieu are not simply passive carriers but may also contribute immunological activity themselves or of their own accord. For example there is considerable interest in the biomedical applications of one of the physiologically-based inorganic metal oxide nanoparticle, zinc oxide (ZnO). Indeed zinc oxide (ZnO) NP are now recognized as a nanoscale chemotherapeutic or anticancer nanoparticle (ANP) and several recent reports suggest ZnO NP and/or its complexes with drug and RNA induce a potent antitumor response in immuno-competent mouse models. A variety of cell culture studies have shown that ZnO NP can induce cytokines such as IFN-γ, TNF-α, IL-2, and IL-12 which are known to regulate the tumor microenvironment. Much less work has been done on magnesium oxide (MgO), cobalt oxide (Co3O4), or nickel oxide (NiO); however, despite the fact that these physiologically-based metal oxide NP are reported to functionally load and assemble RNA and protein onto their surface and may thus also be of potential interest as nanovaccine platform. Here we initially compared in vitro immunogenicity of ZnO and Co3O4 NP and their effects on cancer-associated or tolerogenic cytokines. Based on these data we moved ZnO NP forward to testing in the ex vivo splenocyte assay relative to MgO and NiO NP and these data showed significant difference for flow cytometry sorted population for ZnO-NP, relative to NiO and MgO. These data suggesting both molecular and cellular immunogenic activity, a double-stranded anticancer RNA (ACR), polyinosinic:poly cytidylic acid (poly I:C) known to bind ZnO NP; when ZnO-poly I:C was injected into B16F10-BALB/C tumor significantly induced, IL-2 and IL-12 as shown by Cohen’s d test. LL37 is an anticancer peptide (ACP) currently in clinical trials as an intratumoral immuno-therapeutic agent against metastatic melanoma. LL37 is known to bind poly I:C where it is thought to compete for receptor binding on the surface of some immune cells, metastatic melanoma and lung cells. Molecular dynamic simulations revealed association of LL37 onto ZnO NP confirmed by gel shift assay. Thus using the well-characterized model human lung cancer model cell line (BEAS-2B), poly I:C RNA, LL37 peptide, or LL37-poly I:C complexes were loaded onto ZnO NP and delivered to BEAS-2B lung cells, and the effect on the main cancer regulating cytokine, IL-6 determined by ELISA. Surprisingly ZnO-LL37, but not ZnO-poly I:C or the more novel tricomplex (ZnO-LL37-poly I:C) significantly suppressed IL-6 by >98–99%. These data support the further evaluation of physiological metal oxide compositions, so-called physiometacomposite (PMC) materials and their formulation with anticancer peptide (ACP) and/or anticancer RNA (ACR) as a potential new class of immuno-therapeutic against melanoma and potentially lung carcinoma or other cancers.

Temporal release of a three-component protein subunit vaccine from polymer multilayers

Sustained antigen and adjuvant availability have been shown to improve antiviral immune responses following vaccination. Transcutaneous delivery of vaccines using microneedles has also shown promise and may be particularly relevant for mosquito-borne viruses. We aim to combine these traits to create a three-component Protein Subunit vaccine on Microneedle Arrays (PSMNs) for transcutaneous delivery using layer-by-layer (LbL) assembly. Polymer multilayer thin films were generated to co-deliver a model combination of three chemically distinct vaccine components, a dengue virus Envelope protein Domain III (EDIII) subunit antigen and two adjuvants, a double-stranded RNA (Poly (inosinic:cytidylic acid) (PolyI:C)) and an amphiphilic hexapeptide, Pam3CSK4. Following application of PSMNs to the skin, implanted thin films facilitated sustained and temporal release of individual vaccine components from polymer multilayers. By modulating LbL composition and architecture, component release profiles in the skin could be independently tuned to allow release of adjuvants and antigen from days up to two weeks. Uptake of antigen and adjuvant from implanted vaccine films by antigen-presenting cells was demonstrated using in vivo mouse and ex vivo human skin models. Overall, we believe that such modular vaccine strategies offer design principles for enhancing the immunogenicity of protein subunit vaccines.