(5′S) 5′,8-cyclo-2′-deoxyadenosine Cannot Stop BER. Clustered DNA Lesion Studies

As a result of external and endocellular physical-chemical factors, every day approximately ~10⁵ DNA lesions might be formed in each human cell. During evolution, living organisms have developed numerous repair systems, of which Base Excision Repair (BER) is the most common. 5′,8-cyclo-2′-deoxyadenosine (cdA) is a tandem lesion that is removed by the Nucleotide Excision Repair (NER) mechanism. Previously, it was assumed that BER machinery was not able to remove (5′S)cdA from the genome. In this study; however, it has been demonstrated that, if (5′S)cdA is a part of a single-stranded clustered DNA lesion, it can be removed from ds-DNA by BER. The above is theoretically possible in two cases: (A) When, during repair, clustered lesions form Okazaki-like fragments; or (B) when the (5′S)cdA moiety is located in the oligonucleotide strand on the 3′-end side of the adjacent DNA damage site, but not when it appears at the opposite 5′-end side. To explain this phenomenon, pure enzymes involved in BER were used (polymerase β (Polβ), a Proliferating Cell Nuclear Antigen (PCNA), and the X-Ray Repair Cross-Complementing Protein 1 (XRCC1)), as well as the Nuclear Extract (NE) from xrs5 cells. It has been found that Polβ can effectively elongate the primer strand in the presence of XRCC1 or PCNA. Moreover, supplementation of the NE from xrs5 cells with Polβ (artificial Polβ overexpression) forced oligonucleotide repair via BER in all the discussed cases.     

cADPR Does Not Activate TRPM2

cADPR is a second messenger that releases Ca²⁺ from intracellular stores via the ryanodine receptor. Over more than 15 years, it has been controversially discussed whether cADPR also contributes to the activation of the nucleotide-gated cation channel TRPM2. While some groups have observed activation of TRPM2 by cADPR alone or in synergy with ADPR, sometimes only at 37 °C, others have argued that this is due to the contamination of cADPR by ADPR. The identification of a novel nucleotide-binding site in the N-terminus of TRPM2 that binds ADPR in a horseshoe-like conformation resembling cADPR as well as the cADPR antagonist 8-Br-cADPR, and another report that demonstrates activation of TRPM2 by binding of cADPR to the NUDT9H domain raised the question again and led us to revisit the topic. Here we show that (i) the N-terminal MHR1/2 domain and the C-terminal NUDT9H domain are required for activation of human TRPM2 by ADPR and 2′-deoxy-ADPR (2dADPR), (ii) that pure cADPR does not activate TRPM2 under a variety of conditions that have previously been shown to result in channel activation, (iii) the cADPR antagonist 8-Br-cADPR also inhibits activation of TRPM2 by ADPR, and (iv) cADPR does not bind to the MHR1/2 domain of TRPM2 while ADPR does.     

Synergistic Effects of A Combined Treatment of Glioblastoma U251 Cells with An Anti-miR-10b-5p Molecule and An AntiCancer Agent Based on 1-(3′,4′,5′-Trimethoxyphenyl)-2-Aryl-1H-Imidazole Scaffold

(1) Background: In the development of new and more effective anticancer approaches, combined treatments appear of great interest. Combination therapy could be of importance in the management of glioblastoma (GBM), a lethal malignancy that accounts for 42% of cancer of the central nervous system, with a median survival of 15 months. This study aimed to verify the activity on a glioblastoma cancer cell line of one of the most active compounds of a novel series of tubulin polymerization inhibitors based on the 1-(3′,4′,5′-trimethoxyphenyl)-2-aryl-1H-imidazole scaffold, used in combination with a miRNA inhibitor molecule targeting the oncomiRNA miR-10b-5p. This microRNA was selected in consideration of the role of miR-10b-5p on the onset and progression of glioblastoma. (2) Methods: Apoptosis was analyzed by Annexin-V and Caspase 3/7 assays, efficacy of the anti-miR-10b-5p was assessed by determining the miR-10b-5p content by RT-qPCR. (3) Results: The results obtained show that a “combination therapy” performed by combining the use of an anti-miR-10b-5p and a 1-(3′,4′,5′-trimethoxyphenyl)-2-aryl-1H-imidazole derivative is an encouraging strategy to boost the efficacy of anticancer therapies and at the same time to reduce side effects.     

Synthesis and Antimicrobial Evaluation of Some Novel Bis-α,β-Unsaturated Ketones,Nicotinonitrile, 1,2-Dihydropyridine-3-carbonitrile,Fused Thieno[2,3-b]pyridine and Pyrazolo[3,4-b]pyridine Derivatives

The title compounds were prepared by reaction of 1,1′-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)diethanone (1) with different aromatic aldehydes 2a–c, namely Furfural (2a), 4-chlorobenzaldehyde (2b) and 4-methoxybenzaldhyde (2c) to yield the corresponding α,β-unsaturated ketones 3a–c. Compound 3 was reacted with malononitrile, 2-cyanoacetamide or 2-cyanothioacetamide yielded the corresponding bis[2-amino-6-(aryl)nicotinonitrile] 4a–c, bis[6-(2-aryl)-2-oxo-1,2-dihydropyridine-3-carbonitrile] 5a–c or bis[6-(2-aryl)-2-thioxo-1,2-dihydropyridine-3-carbonitrile] 6a,b, respectively. The reaction of compound 6a with each of 2-chloro-N-(4-bromophenyl) acetamide (7a), chloroacetamide (7b) in ethanolic sodium ethoxide solution at room temperature to give the corresponding 4,4′-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis-6-(2-furyl)thieno[2,3-b]pyridine-2-carboxamide] derivatives 9a,b. While compound 6a reacted with hydrazine hydrate yielded the 4,4′-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis[6-(2-furyl)-1H-pyrazolo[3,4-b]pyridin-3-amine] 11. The structures of the products were elucidated based on their spectral properties, elemental analyses and, wherever possible, by alternate synthesis. Antimicrobial evaluation of the products was carried out.     

Design,Synthesis and Evaluation of Novel Derivatives of Curcuminoids with Cytotoxicity

Curcumin and curcuminoids have been discussed frequently due to their promising functional groups (such as scaffolds of α,β-unsaturated β-diketone, α,β-unsaturated ketone and β′-hydroxy-α,β-unsaturated ketone connected with aromatic rings on both sides) that play an important role in various bioactivities, including antioxidant, anti-inflammatory, anti-proliferation and anticancer activity. A series of novel curcuminoid derivatives (a total of 55 new compounds) and three reference compounds were synthesized with good yields using three-step organic synthesis. The anti-proliferative activities of curcumin derivatives were examined for six human cancer cell lines: HeLaS3, KBvin, MCF-7, HepG2, NCI-H460 and NCI-H460/MX20. Compared to the IC₅₀ values of all the synthesized derivatives, most α,β-unsaturated ketones displayed potent anti-proliferative effects against all six human cancer cell lines, whereas β′-hydroxy-α,β-unsaturated ketones and α,β-unsaturated β-diketones presented moderate anti-proliferative effects. Two potent curcuminoid derivatives were found among all the novel derivatives and reference compounds: (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a). These were selected for further analysis after the evaluation of their anti-proliferative effects against all human cancer cell lines. The results of apoptosis assays revealed that the number of dead cells was increased in early apoptosis and late apoptosis, while cell proliferation was also decreased after applying various concentrations of (E)-5-hydroxy-7-phenyl-1-(3,4,5-trimethoxyphenyl)hept-1-en-3-one (compound 3) and (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) to MCF-7 and HpeG2 cancer cells. Analysis of the gene expression arrays showed that three genes (GADD45B, SESN2 and BBC3) were correlated with the p53 pathway. From the quantitative PCR analysis, it was seen that (1E,4E)-1,7-bis(3,4,5-trimethoxyphenyl)hepta-1,4-dien-3-one (compound MD12a) effectively induced the up-regulated expression of GADD45B, leading to the suppression of MCF-7 cancer cell formation and cell death. Molecular docking analysis was used to predict and sketch the interactions of the GADD45B-α,β-unsaturated ketone complex for help in drug design.     

Investigation of the Anti-Methicillin-Resistant Staphylococcus aureus Activity of (+)-Tanikolide- and (+)-Malyngolide-Based Analogues Prepared by Asymmetric Synthesis

Herein, we report antibacterial and antifungal evaluation of a series of previously prepared (+)-tanikolide analogues. One analogue, (4S,6S)-4-methyltanikolide, displayed promising anti-methicillin-resistant Staphylococcus aureus activity with a MIC of 12.5 µg/mL. Based on the antimicrobial properties of the structurally related (−)-malyngolide, two further analogues (4S,6S)-4-methylmalyngolide and (4R,6S)-4-methylmalyngolide bearing a shortened n-nonyl alkyl side chain were prepared in the present study using a ZrCl₄-catalysed deprotection/cyclisation as the key step in their asymmetric synthesis. When these were tested for activity against anti-methicillin-resistant Staphylococcus aureus, the MIC increased to 50 µg/mL.     

Reciprocal Changes in miRNA Expression with Pigmentation and Decreased Proliferation Induced in Mouse B16F1 Melanoma Cells by l-Tyrosine and 5-Bromo-2′-Deoxyuridine

Background: Many microRNAs have been identified as critical mediators in the progression of melanoma through its regulation of genes involved in different cellular processes such as melanogenesis, cell cycle control, and senescence. However, microRNAs’ concurrent participation in syngeneic mouse B16F1 melanoma cells simultaneously induced decreased proliferation and differential pigmentation by exposure to 5-Brd-2′-dU (5’Bromo-2-deoxyuridine) and L-Tyr (L-Tyrosine) respectively, is poorly understood. Aim: To evaluate changes in the expression of microRNAs and identify which miRNAs in-network may contribute to the functional bases of phenotypes of differential pigmentation and reduction of proliferation in B16F1 melanoma cells exposed to 5-Brd-2′-dU and L-Tyr. Methods: Small RNAseq evaluation of the expression profiles of miRNAs in B16F1 melanoma cells exposed to 5-Brd-2′-dU (2.5 μg/mL) and L-Tyr (5 mM), as well as the expression by qRT-PCR of some molecular targets related to melanogenesis, cell cycle, and senescence. By bioinformatic analysis, we constructed network models of regulation and co-expression of microRNAs. Results: We confirmed that stimulation or repression of melanogenesis with L-Tyr or 5-Brd-2′-dU, respectively, generated changes in melanin concentration, reduction in proliferation, and changes in expression of microRNAs 470-3p, 470-5p, 30d-5p, 129-5p, 148b-3p, 27b-3p, and 211-5p, which presented patterns of coordinated and reciprocal co-expression, related to changes in melanogenesis through their putative targets Mitf, Tyr and Tyrp1, and control of cell cycle and senescence: Cyclin D1, Cdk2, Cdk4, p21, and p27. Conclusions: These findings provide insights into the molecular biology of melanoma of the way miRNAs are coordinated and reciprocal expression that may operate in a network as molecular bases for understanding changes in pigmentation and decreased proliferation induced in B16F1 melanoma cells exposed to L-Tyr and 5-Brd-2′-dU.     

Control of TRPM3 Ion Channels by Protein Kinase CK2-Mediated Phosphorylation in Pancreatic β-Cells of the Line INS-1

In pancreatic β-cells of the line INS-1, glucose uptake and metabolism induce the openings of Ca²⁺-permeable TRPM3 channels that contribute to the elevation of the intracellular Ca²⁺ concentration and the fusion of insulin granules with the plasma membrane. Conversely, glucose-induced Ca²⁺ signals and insulin release are reduced by the activity of the serine/threonine kinase CK2. Therefore, we hypothesized that TRPM3 channels might be regulated by CK2 phosphorylation. We used recombinant TRPM3α2 proteins, native TRPM3 proteins from INS-1 β-cells, and TRPM3-derived oligopeptides to analyze and localize CK2-dependent phosphorylation of TRPM3 channels. The functional consequences of CK2 phosphorylation upon TRPM3-mediated Ca²⁺ entry were investigated in Fura-2 Ca²⁺-imaging experiments. Recombinant TRPM3α2 channels expressed in HEK293 cells displayed enhanced Ca²⁺ entry in the presence of the CK2 inhibitor CX-4945 and their activity was strongly reduced after CK2 overexpression. TRPM3α2 channels were phosphorylated by CK2 in vitro at serine residue 1172. Accordingly, a TRPM3α2 S₁₁₇₂A mutant displayed enhanced Ca²⁺ entry. The TRPM3-mediated Ca²⁺ entry in INS-1 β-cells was also strongly increased in the presence of CX-4945 and reduced after overexpression of CK2. Our study shows that CK2-mediated phosphorylation controls TRPM3 channel activity in INS-1 β-cells.     

Similarities and Differences between the Orai1 Variants: Orai1α and Orai1β

Orai1, the first identified member of the Orai protein family, is ubiquitously expressed in the animal kingdom. Orai1 was initially characterized as the channel responsible for the store-operated calcium entry (SOCE), a major mechanism that allows cytosolic calcium concentration increments upon receptor-mediated IP₃ generation, which results in intracellular Ca²⁺ store depletion. Furthermore, current evidence supports that abnormal Orai1 expression or function underlies several disorders. Orai1 is, together with STIM1, the key element of SOCE, conducting the Ca²⁺ release-activated Ca²⁺ (CRAC) current and, in association with TRPC1, the store-operated Ca²⁺ (SOC) current. Additionally, Orai1 is involved in non-capacitative pathways, as the arachidonate-regulated or LTC4-regulated Ca²⁺ channel (ARC/LRC), store-independent Ca²⁺ influx activated by the secretory pathway Ca²⁺-ATPase (SPCA2) and the small conductance Ca²⁺-activated K⁺ channel 3 (SK3). Furthermore, Orai1 possesses two variants, Orai1α and Orai1β, the latter lacking 63 amino acids in the N-terminus as compared to the full-length Orai1α form, which confers distinct features to each variant. Here, we review the current knowledge about the differences between Orai1α and Orai1β, the implications of the Ca²⁺ signals triggered by each variant, and their downstream modulatory effect within the cell.     

Acute and Delayed Effects of Mechanical Injury on Calcium Homeostasis and Mitochondrial Potential of Primary Neuroglial Cell Culture: Potential Causal Contributions to Post-Traumatic Syndrome

In vitro models of traumatic brain injury (TBI) help to elucidate the pathological mechanisms responsible for cell dysfunction and death. To simulate in vitro the mechanical brain trauma, primary neuroglial cultures were scratched during different periods of network formation. Fluorescence microscopy was used to measure changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and mitochondrial potential (ΔΨm) a few minutes later and on days 3 and 7 after scratching. An increase in [Ca²⁺]_i and a decrease in ΔΨm were observed ~10 s after the injury in cells located no further than 150–200 µm from the scratch border. Ca²⁺ entry into cells during mechanical damage of the primary neuroglial culture occurred predominantly through the NMDA-type glutamate ionotropic channels. MK801, an inhibitor of this type of glutamate receptor, prevented an acute increase in [Ca²⁺]_i in 99% of neurons. Pathological changes in calcium homeostasis persisted in the primary neuroglial culture for one week after injury. Active cell migration in the scratch area occurred on day 11 after neurotrauma and was accompanied by a decrease in the ratio of live to dead cells in the areas adjacent to the injury. Immunohistochemical staining of glial fibrillary acidic protein and β-III tubulin showed that neuronal cells migrated to the injured area earlier than glial cells, but their repair potential was insufficient for survival. Mitochondrial Ca²⁺ overload and a drop in ΔΨm may cause delayed neuronal death and thus play a key role in the development of the post-traumatic syndrome. Preventing prolonged ΔΨm depolarization may be a promising therapeutic approach to improve neuronal survival after traumatic brain injury.     

Ditopic, flexible hydrazone-based building blocks with pendant 2,2′:6′,2′′-terpyridine metal-binding domains

The synthesis and characterization of three new bis(2,2′:6′,2′-terpyridine) (tpy) ligands containing different hydrazone spacers between the metal-binding domains are described. Treatment of 1,4-benzenedicarbaldehyde bis(2,2′:6′,2′′-terpyridin-4′-ylhydrazone) (1) with [(tpy)RuCl₃] in the presence of N-ethylmorpholine results in the formation of [(tpy)Ru(μ-1)Ru(tpy)]⁴⁺. Single crystal X-ray diffraction data for [(tpy)Ru(μ-1)Ru(tpy)][PF₆]₄·8MeCN confirm the ability of the hydrazone-based ligand to bridge two ruthenium(II) centres, providing proof-of-principle for the application of this class of flexible ligand in the design of coordination polymers.     

A Central Role for TRPM4 in Ca2+-Signal Amplification and Vasoconstriction

Transient receptor potential melastatin-4 (TRPM4) is activated by an increase in intracellular Ca²⁺ concentration and is expressed on smooth muscle cells (SMCs). It is implicated in the myogenic constriction of cerebral arteries. We hypothesized that TRPM4 has a general role in intracellular Ca²⁺ signal amplification in a wide range of blood vessels. TRPM4 function was tested with the TRPM4 antagonist 9-phenanthrol and the TRPM4 activator A23187 on the cardiovascular responses of the rat, in vivo and in isolated basilar, mesenteric, and skeletal muscle arteries. TRPM4 inhibition by 9-phenanthrol resulted in hypotension and a decreased heart rate in the rat. TRPM4 inhibition completely antagonized myogenic tone development and norepinephrine-evoked vasoconstriction, and depolarization (high extracellular KCl concentration) evoked vasoconstriction in a wide range of peripheral arteries. Vasorelaxation caused by TRPM4 inhibition was accompanied by a significant decrease in intracellular Ca²⁺ concentration, suggesting an inhibition of Ca²⁺ signal amplification. Immunohistochemistry confirmed TRPM4 expression in the smooth muscle cells of the peripheral arteries. Finally, TRPM4 activation by the Ca²⁺ ionophore A23187 was competitively inhibited by 9-phenanthrol. In summary, TRPM4 was identified as an essential Ca²⁺-amplifying channel in peripheral arteries, contributing to both myogenic tone and agonist responses. These results suggest an important role for TRPM4 in the circulation. The modulation of TRPM4 activity may be a therapeutic target for hypertension. Furthermore, the Ca²⁺ ionophore A23187 was identified as the first high-affinity (nanomolar) direct activator of TRPM4, acting on the 9-phenanthrol binding site.     

Electron-Induced Repair of 2′-Deoxyribose Sugar Radicals in DNA: A Density Functional Theory (DFT) Study

In this work, we used ωB97XD density functional and 6-31++G** basis set to study the structure, electron affinity, populations via Boltzmann distribution, and one-electron reduction potentials (E°) of 2′-deoxyribose sugar radicals in aqueous phase by considering 2′-deoxyguanosine and 2′-deoxythymidine as a model of DNA. The calculation predicted the relative stability of sugar radicals in the order C4′^• > C1′^• > C5′^• > C3′^• > C2′^•. The Boltzmann distribution populations based on the relative stability of the sugar radicals were not those found for ionizing radiation or OH-radical attack and are good evidence the kinetic mechanisms of the processes drive the products formed. The adiabatic electron affinities of these sugar radicals were in the range 2.6–3.3 eV which is higher than the canonical DNA bases. The sugar radicals reduction potentials (E°) without protonation (−1.8 to −1.2 V) were also significantly higher than the bases. Thus the sugar radicals will be far more readily reduced by solvated electrons than the DNA bases. In the aqueous phase, these one-electron reduced sugar radicals (anions) are protonated from solvent and thus are efficiently repaired via the “electron-induced proton transfer mechanism”. The calculation shows that, in comparison to efficient repair of sugar radicals by the electron-induced proton transfer mechanism, the repair of the cyclopurine lesion, 5′,8-cyclo-2′-dG, would involve a substantial barrier.     

Highly Selective and Potent Human β-Secretase 2 (BACE2) Inhibitors against Type 2 Diabetes: Design,Synthesis, X-ray Structure and Structure–Activity Relationship Studies

Herein we present the design, synthesis, and biological evaluation of potent and highly selective β-secretase 2 (memapsin 1, beta-site amyloid precursor protein cleaving enzyme 2, or BACE 2) inhibitors. BACE2 has been recognized as an exciting new target for type 2 diabetes. The X-ray structure of BACE1 bound to inhibitor 2 a {N³-[(1S,2R)-1-benzyl-2-hydroxy-3-[[(1S,2S)-2-hydroxy-1-(isobutylcarbamoyl)propyl]amino]propyl]-5-[methyl(methylsulfonyl)amino]-N¹-[(1R)-1-phenylpropyl]benzene-1,3-dicarboxamide} containing a hydroxyethylamine isostere was determined. Based on this structure, a computational docking study was performed which led to inhibitor 2 a -bound BACE2 models. These were used to optimize the potency and selectivity of inhibitors. A systematic structure–activity relationship study led to the identification of determinants of the inhibitors’ potency and selectivity toward the BACE2 enzyme. Inhibitors 2 d [N³-[(1S,2R)-1-benzyl-2-hydroxy-3-[[(1S,2S)-2-hydroxy-1-(isobutylcarbamoyl)pentyl]amino]propyl]-N¹-methyl-N¹-[(1R)-1-phenylpropyl]benzene-1,3-dicarboxamide; K_i=0.031 nm , selectivity over BACE1: ≈174 000-fold] and 3 l [N¹-((2S,3R)-3-hydroxy-1-phenyl-4-((3-(trifluoromethyl)benzyl)amino)butan-2-yl)-N³,5-dimethyl-N³-((R)-1-phenylethyl)isophthalamide; K_i=1.6 nm , selectivity over BACE1: >500-fold] displayed outstanding potency and selectivity. Inhibitor 3 l is nonpeptide in nature and may pave the way to the development of a new class of potent and selective BACE2 inhibitors with clinical potential.     

Chemoenzymatic Preparation of Enantiopure Homoadamantyl β‐Amino Acid and β‐Lactam Derivatives

Racemic cis‐10‐azatetracyclo[7.2.0.1^2,6.1^4,8]tridecan‐11‐one was prepared from homoadamant‐4‐ene by chlorosulfonyl isocyanate addition. The transformation of the β‐lactam to the corresponding β‐amino ester followed by Candida antarctica lipase A‐catalyzed enantioselective (E>>200) N‐acylation with 2,2,2‐trifluoroethyl butanoate afforded methyl (1R,4R,5S,8S)‐5‐aminotricyclo[4.3.1.1^3,8]undecane‐4‐carboxylate and the (1S,4S,5R,8R)‐butanamide with>99% ee at 50% conversion. Alternatively, transformation of the β‐lactam to the corresponding N‐hydroxymethyl‐β‐lactam and the following Pseudomonas cepacia (currently Burkholderia cepacia) lipase‐catalyzed enantioseletive O‐acylation provided the (1S,4S,6R,9R)‐alcohol (ee=87%) and the corresponding (1R,4R,6S,9S)‐butanoate (ee>99%). In the latter method, competition for the enzyme between the (1R,4R,6S,9S)‐butanoate, 2,2,2‐trifluoroethyl butanoate and the hydrolysis product, butanoic acid, tended to stop the reaction at about 45% conversion and finally gave racemization in the (1S,4S,6R,9R)‐alcohol with time.     

Molecular and Biochemical Analysis of Two Rice Flavonoid 3’-Hydroxylase to Evaluate Their Roles in Flavonoid Biosynthesis in Rice Grain

Anthocyanins and proanthocyanidins, the major flavonoids in black and red rice grains, respectively, are mainly derived from 3′,4′-dihydroxylated leucocyanidin. 3′-Hydroxylation of flavonoids in rice is catalyzed by flavonoid 3′-hydroxylase (F3′H: EC 1.14.13.21). We isolated cDNA clones of the two rice F3′H genes (CYP75B3 and CYP75B4) from Korean varieties of white, black, and red rice. Sequence analysis revealed allelic variants of each gene containing one or two amino acid substitutions. Heterologous expression in yeast demonstrated that CYP75B3 preferred kaempferol to other substrates, and had a low preference for dihydrokaempferol. CYP75B4 exhibited a higher preference for apigenin than for other substrates. CYP75B3 from black rice showed an approximately two-fold increase in catalytic efficiencies for naringenin and dihydrokaempferol compared to CYP75B3s from white and red rice. The F3′H activity of CYP75B3 was much higher than that of CYP75B4. Gene expression analysis showed that CYP75B3, CYP75B4, and most other flavonoid pathway genes were predominantly expressed in the developing seeds of black rice, but not in those of white and red rice, which is consistent with the pigmentation patterns of the seeds. The expression levels of CYP75B4 were relatively higher than those of CYP75B3 in the developing seeds, leaves, and roots of white rice.     

Novel N-Substituted 3-Aryl-4-(diethoxyphosphoryl)azetidin-2-ones as Antibiotic Enhancers and Antiviral Agents in Search for a Successful Treatment of Complex Infections

A novel series of N-substituted cis- and trans-3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones were synthesized by the Kinugasa reaction of N-methyl- or N-benzyl-(diethyoxyphosphoryl)nitrone and selected aryl alkynes. Stereochemistry of diastereoisomeric adducts was established based on vicinal H3–H4 coupling constants in azetidin-2-one ring. All the obtained azetidin-2-ones were evaluated for the antiviral activity against a broad range of DNA and RNA viruses. Azetidin-2-one trans-11f showed moderate inhibitory activity against human coronavirus (229E) with EC₅₀ = 45 µM. The other isomer cis-11f was active against influenza A virus H1N1 subtype (EC₅₀ = 12 µM by visual CPE score; EC₅₀ = 8.3 µM by TMS score; MCC > 100 µM, CC₅₀ = 39.9 µM). Several azetidin-2-ones 10 and 11 were tested for their cytostatic activity toward nine cancerous cell lines and several of them appeared slightly active for Capan-1, Hap1 and HCT-116 cells values of IC₅₀ in the range 14.5–97.9 µM. Compound trans-11f was identified as adjuvant of oxacillin with significant ability to enhance the efficacy of this antibiotic toward the highly resistant S. aureus strain HEMSA 5. Docking and molecular dynamics simulations showed that enantiomer (3R,4S)-11f can be responsible for the promising activity due to the potency in displacing oxacillin at β-lactamase, thus protecting the antibiotic from undesirable biotransformation.     

Human Milk Oligosaccharide 2′-Fucosyllactose Modulates Local Viral Immune Defense by Supporting the Regulatory Functions of Intestinal Epithelial and Immune Cells

Human milk contains bioactive components that provide protection against viral infections in early life. In particular, intestinal epithelial cells (IEC) have key regulatory roles in the prevention of enteric viral infections. Here we established an in vitro model to study the modulation of host responses against enteric viruses mimicked by poly I:C (pIC). The effects of 2′-fucosyllactose (2′FL), abundantly present in human milk, were studied on IEC and/or innate immune cells, and the subsequent functional response of the adaptive immune cells. IEC were pre-incubated with 2′FL and stimulated with naked or Lyovec™-complexed pIC (LV-pIC). Additionally, monocyte-derived dendritic cells (moDC) alone or in co-culture with IEC were stimulated with LV-pIC. Then, conditioned-moDC were co-cultured with naïve CD4⁺ T helper (Th)-cells. IEC stimulation with naked or LV-pIC promoted pro-inflammatory IL-8, CCL20, GROα and CXCL10 cytokine secretion. However, only exposure to LV-pIC additionally induced IFNβ, IFNλ1 and CCL5 secretion. Pre-incubation with 2′FL further increased pIC induced CCL20 secretion and LV-pIC induced CXCL10 secretion. LV-pIC-exposed IEC/moDC and moDC cultures showed increased secretion of IL-8, GROα, IFNλ1 and CXCL10, and in the presence of 2′FL galectin-4 and -9 were increased. The LV-pIC-exposed moDC showed a more pronounced secretion of CCL20, CXCL10 and CCL5. The moDC from IEC/moDC cultures did not drive T-cell development in moDC/T-cell cultures, while moDC directly exposed to LV-pIC secreted Th1 driving IL-12p70 and promoted IFNγ secretion by Th-cells. Hereby, a novel intestinal model was established to study mucosal host-defense upon a viral trigger. IEC may support intestinal homeostasis, regulating local viral defense which may be modulated by 2′FL. These results provide insights regarding the protective capacity of human milk components in early life.