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.     

Synthesis of Reactive Sulfur Species in Cultured Vascular Endothelial Cells after Exposure to TGF-β1: Induction of Cystathionine γ-Lyase and Cystathionine β-Synthase Expression Mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 Pathways

Transforming growth factor-β₁ (TGF-β₁) occurs at high levels at damage sites of vascular endothelial cell layers and regulates the functions of vascular endothelial cells. Reactive sulfur species (RSS), such as cysteine persulfide, glutathione persulfide, and hydrogen persulfide, are cytoprotective factors against electrophiles such as reactive oxygen species and heavy metals. Previously, we reported that sodium trisulfide, a sulfane sulfur donor, promotes vascular endothelial cell proliferation. The objective of the present study was to clarify the regulation and significance of RSS synthesis in vascular endothelial cells after exposure to TGF-β₁. Bovine aortic endothelial cells in a culture system were treated with TGF-β₁ to assess the expression of intracellular RSS, the effect of RSS on cell proliferation in the presence of TGF-β₁, induction of RSS-producing enzymes by TGF-β₁, and intracellular signal pathways that mediate this induction. The results suggest that TGF-β₁ increased intracellular RSS levels to modulate its inhibitory effect on proliferation. The increased production of RSS, probably high-molecular-mass RSS, was due to the induction of cystathionine γ-lyase and cystathionine β-synthase, which are RSS-producing enzymes, and the induction was mediated by the ALK5-Smad2/3/4 and ALK5-Smad2/3-ATF4 pathways in vascular endothelial cells. TGF-β₁ regulates vascular endothelial cell functions such as proliferation and fibrinolytic activity; intracellular high-molecular-mass RSS, which are increased by TGF-β₁, may modulate the regulation activity in vascular endothelial cells.     

Preparation and Characterization of a Chloroperoxidase-like Catalytic Antibody

The small molecule, meso-tetra(α,α,α,α-o-phenylacetamidophenyl) porphyrin (Mr1147.0) was used as complete antigen to elicit MAb through the immunization and cell fusion techniques. The MAb 1F2 obtained was demonstrated to be very pure by MALDI/TOFMS. The subtype of MAb 1F2 is IgG2a, which has a relative molecular weight of 156,678.8 Da.No significant change in the intensity of absorption peaks in UV and CD spectra was observed over a pH range between 6 and 12. The high stability of the abzyme and the tight binding between Fe porphyrin and antibody were also demonstrated. V_max, K_m, κcat, κcat/K_m for abzyme are 5.18 × 10⁻⁸ Ms⁻¹, 1.50 × 10⁻⁸ M, 0.518 s⁻¹, 3.45 × 10⁷ M⁻¹s⁻¹, respectively. The data obtained indicate that catalytic antibody has high catalytic activity. The chloroperoxidase activity of MAb 1F2-Fe porphyrin complex is stable from 10 °C to 60 °C.     

Design and Evaluation of a Polypeptide That Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity

Fibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sought to target and interfere with interactions between EDA-FN and its integrin receptors to effectively inhibit profibrotic activity and myofibroblast formation. Molecular docking was used to assist in the design of a blocking polypeptide (antifibrotic 38-amino-acid polypeptide, AF38Pep) for specific inhibition of EDA-FN associations with the fibroblast-expressed integrins α₄β₁ and α₄β₇. Blocking peptides were designed and evaluated in silico before synthesis, confirmation of binding specificity, and evaluation in vitro. We identified the high-affinity EDA-FN C-C′ loop binding cleft within integrins α₄β₁ and α₄β₇. The polypeptide with the highest predicted binding affinity, AF38Pep, was synthesized and could achieve specific binding to myofibroblast fibronectin-rich ECM and EDA-FN C-C′ loop peptides. AF38Pep demonstrated potent myofibroblast inhibitory activity at 10 µg/mL and was not cytotoxic. Treatment with AF38Pep prevented integrin α₄β₁-mediated focal adhesion kinase (FAK) activation and early signaling through extracellular-signal-regulated kinases 1 and 2 (ERK1/2), attenuated the expression of pro-matrix metalloproteinase 9 (MMP9) and pro-MMP2, and inhibited collagen synthesis and deposition. Immunocytochemistry staining revealed an inhibition of α-smooth muscle actin (α-SMA) incorporation into actin stress fibers and attenuated cell contraction. Increases in the expression of mRNA associated with fibrosis and downstream from integrin signaling were inhibited by treatment with AF38Pep. Our study suggested that AF38Pep could successfully interfere with EDA-FN C-C′ loop-specific integrin interactions and could act as an effective inhibitor of fibroblast of myofibroblast differentiation.     

Synthesis of Oxidized 3β,3′β-Disteryl Ethers and Search after High-Temperature Treatment of Sterol-Rich Samples

It was proven that sterols subjected to high-temperature treatment can be concatenated, which results in polymeric structures, e.g., 3β,3′β-disteryl ethers. However, it was also proven that due to increased temperature in oxygen-containing conditions, sterols can undergo various oxidation reactions. This study aimed to prove the existence and perform quantitative analysis of oxidized 3β,3′β-disteryl ethers, which could form during high-temperature treatment of sterol-rich samples. Samples were heated at 180, 200 and 220 °C for 0.5 to 4 h. Quantitative analyses of the oxidized 3β,3′β-disteryl ethers were performed with liquid extraction, solid-phase extraction and liquid chromatography coupled with mass spectrometry. Additionally, to perform this analysis, the appropriate standards of all oxidized 3β,3′β-disteryl ethers were prepared. Eighteen various oxidized 3β,3′β-disteryl ethers (derivatives of 3β,3′β-dicholesteryl ether, 3β,3′β-disitosteryl ether and 3β,3′β-distigmasteryl ether) were prepared. Additionally, the influence of metal compounds on the mechanism of ether formation at high temperatures was investigated.     

Turn-Directed α-β Conformational Transition of α-syn12 Peptide at Different pH Revealed by Unbiased Molecular Dynamics Simulations

The transition from α-helical to β-hairpin conformations of α-syn12 peptide is characterized here using long timescale, unbiased molecular dynamics (MD) simulations in explicit solvent models at physiological and acidic pH values. Four independent normal MD trajectories, each 2500 ns, are performed at 300 K using the GROMOS 43A1 force field and SPC water model. The most clustered structures at both pH values are β-hairpin but with different turns and hydrogen bonds. Turn_9-6 and four hydrogen bonds (HB_9-6, HB_6-9, HB_11-4 and HB_4-11) are formed at physiological pH; turn_8-5 and five hydrogen bonds (HB_8-5, HB_5-8, HB_10-3, HB_3-10 and HB_12-1) are formed at acidic pH. A common folding mechanism is observed: the formation of the turn is always before the formation of the hydrogen bonds, which means the turn is always found to be the major determinant in initiating the transition process. Furthermore, two transition paths are observed at physiological pH. One of the transition paths tends to form the most-clustered turn and improper hydrogen bonds at the beginning, and then form the most-clustered hydrogen bonds. Another transition path tends to form the most-clustered turn, and turn_5-2 firstly, followed by the formation of part hydrogen bonds, then turn_5-2 is extended and more hydrogen bonds are formed. The transition path at acidic pH is as the same as the first path described at physiological pH.     

Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry

HCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their phenotype. To map the various anti-HCMV cellular responses, we used a combination of three HLA_peptide tetramer complexes (HLA-E_VMAPRTLIL, HLA-E_VMAPRSLLL, and HLA-A2_NLVPMVATV) and antibodies for 18 surface markers (CD3, CD4, CD8, CD16, CD19, CD45RA, CD56, CD57, CD158, NKG2A, NKG2C, CCR7, TCRγδ, TCRγδ2, CX3CR1, KLRG1, 2B4, and PD-1) in a 20-color spectral flow cytometry analysis. This immunostaining protocol was applied to PBMCs isolated from HCMV⁻ and HCMV⁺ individuals. Our workflow allows the efficient determination of events featuring HCMV infection such as CD4/CD8 ratio, CD8 inflation and differentiation, HCMV peptide-specific HLA-E_UL40 and HLA-A2_pp65CD8 T cells, and expansion of γδT and NK subsets including δ2⁻γT and memory-like NKG2C⁺CD57⁺ NK cells. Each subset can be further characterized by the expression of 2B4, PD-1, KLRG1, CD45RA, CCR7, CD158, and NKG2A to achieve a fine-tuned mapping of HCMV immune responses. This assay should be useful for the analysis and monitoring of T-and NK cell responses to HCMV infection or vaccines.     

Alicyclobacillus mali FL18 as a Novel Source of Glycosyl Hydrolases: Characterization of a New Thermophilic β-Xylosidase Tolerant to Monosaccharides

A thermo-acidophilic bacterium, Alicyclobacillus mali FL18, was isolated from a hot spring of Pisciarelli, near Naples, Italy; following genome analysis, a novel putative β-xylosidase, AmβXyl, belonging to the glycosyl hydrolase (GH) family 3 was identified. A synthetic gene was produced, cloned in pET-30a(+), and expressed in Escherichia coli BL21 (DE3) RIL. The purified recombinant protein, which showed a dimeric structure, had optimal catalytic activity at 80 °C and pH 5.6, exhibiting 60% of its activity after 2 h at 50 °C and displaying high stability (more than 80%) at pH 5.0–8.0 after 16 h. AmβXyl is mainly active on both para-nitrophenyl-β-D-xylopyranoside (K_M 0.52 mM, k_cat 1606 s⁻¹, and k_cat/K_M 3088.46 mM⁻¹·s⁻¹) and para-nitrophenyl-α-L-arabinofuranoside (K_M 10.56 mM, k_cat 2395.8 s⁻¹, and k_cat/K_M 226.87 mM⁻¹·s⁻¹). Thin-layer chromatography showed its ability to convert xylooligomers (xylobiose and xylotriose) into xylose, confirming that AmβXyl is a true β-xylosidase. Furthermore, no inhibitory effect on enzymatic activity by metal ions, detergents, or EDTA was observed except for 5 mM Cu²⁺. AmβXyl showed an excellent tolerance to organic solvents; in particular, the enzyme increased its activity at high concentrations (30%) of organic solvents such as ethanol, methanol, and DMSO. Lastly, the enzyme showed not only a good tolerance to inhibition by xylose, arabinose, and glucose, but was activated by 0.75 M xylose and up to 1.5 M by both arabinose and glucose. The high tolerance to organic solvents and monosaccharides together with other characteristics reported above suggests that AmβXyl may have several applications in many industrial fields.     

The α2δ Calcium Channel Subunit Accessorily and Independently Affects the Biological Function of Ditylenchus destructor

The α₂δ subunit is a high-voltage activated (HVA) calcium channel (Ca_v1 and Ca_v2) auxiliary subunit that increases the density and function of HVA calcium channels in the plasma membrane of mammals. However, its function in plant parasitic nematodes remains unknown. In this study, we cloned the full-length cDNA sequence of the voltage-gated calcium channel (VGCC) α₂δ subunit (named DdCa_vα₂δ) in Ditylenchus destructor. We found that DdCa_vα₂δ tends to be expressed in the egg stage, followed by the J3 stage. RNA-DIG in situ hybridization experiments showed that the DdCa_vα₂δ subunit was expressed in the body wall, esophageal gland, uterus, post uterine, and spicules of D. destructor. The in vitro application of RNA interference (RNAi) affected the motility, reproduction, chemotaxis, stylet thrusting, and protein secretion of D. destructor to different degrees by targeting DdCα1D, DdCα1A, and DdCa_vα₂δ in J3 stages, respectively. Based on the results of RNAi experiments, it was hypothesized that L-type VGCC may affect the motility, chemotaxis, and stylet thrusting of D. destructor. Non-L-type VGCC may affect the protein secretion and reproduction of D. destructor. The DdCa_vα₂δ subunit gene also affected the motility, chemotaxis, and reproduction of D. destructor. These findings reveal the independent function of the VGCC α₂δ subunit in D. destructor as well as give a theoretical foundation for future research on plant parasitic nematode VGCC.     

α2δ-4 and Cachd1 Proteins Are Regulators of Presynaptic Functions

The α₂δ auxiliary subunits of voltage-gated calcium channels (VGCC) were traditionally regarded as modulators of biophysical channel properties. In recent years, channel-independent functions of these subunits, such as involvement in synapse formation, have been identified. In the central nervous system, α₂δ isoforms 1, 2, and 3 are strongly expressed, regulating glutamatergic synapse formation by a presynaptic mechanism. Although the α₂δ-4 isoform is predominantly found in the retina with very little expression in the brain, it was recently linked to brain functions. In contrast, Cachd1, a novel α₂δ-like protein, shows strong expression in brain, but its function in neurons is not yet known. Therefore, we aimed to investigate the presynaptic functions of α₂δ-4 and Cachd1 by expressing individual proteins in cultured hippocampal neurons. Both α₂δ-4 and Cachd1 are expressed in the presynaptic membrane and could rescue a severe synaptic defect present in triple knockout/knockdown neurons that lacked the α₂δ-1-3 isoforms (α₂δ TKO/KD). This observation suggests that presynaptic localization and the regulation of synapse formation in glutamatergic neurons is a general feature of α₂δ proteins. In contrast to this redundant presynaptic function, α₂δ-4 and Cachd1 differentially regulate the abundance of presynaptic calcium channels and the amplitude of presynaptic calcium transients. These functional differences may be caused by subtle isoform-specific differences in α₁-α₂δ protein–protein interactions, as revealed by structural homology modelling. Taken together, our study identifies both α₂δ-4 and Cachd1 as presynaptic regulators of synapse formation, differentiation, and calcium channel functions that can at least partially compensate for the loss of α₂δ-1-3. Moreover, we show that regulating glutamatergic synapse formation and differentiation is a critical and surprisingly redundant function of α₂δ and Cachd1.     

Korean Red Ginseng Improves Astrocytic Mitochondrial Function by Upregulating HO-1-Mediated AMPKα–PGC-1α–ERRα Circuit after Traumatic Brain Injury

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the peroxisome proliferator-activating receptor-γ coactivator-1α (PGC-1α)–estrogen-related receptor α (ERRα) pathway in astrocytes. However, the role of Korean red ginseng extract (KRGE) in HO-1-mediated mitochondrial function in traumatic brain injury (TBI) is not well-elucidated. We found that HO-1 was upregulated in astrocytes located in peri-injured brain regions after a TBI, following exposure to KRGE. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HO-1 levels highly correlated with increased AMP-activated protein kinase α (AMPKα) activation, which led to the PGC-1α-ERRα axis-induced increases in mitochondrial functions (detected based on expression of cytochrome c oxidase subunit 2 (MTCO2) and cytochrome c as well as O₂ consumption and ATP production). Knockdown of ERRα significantly reduced the p-AMPKα/AMPKα ratio and PGC-1α expression, leading to AMPKα–PGC-1α–ERRα circuit formation. Inactivation of HO by injecting the HO inhibitor Sn(IV) protoporphyrin IX dichloride diminished the expression of p-AMPKα, PGC-1α, ERRα, MTCO2, and cytochrome c in the KRGE-administered peri-injured region of a brain subjected to TBI. These data suggest that KRGE enhanced astrocytic mitochondrial function via a HO-1-mediated AMPKα–PGC-1α–ERRα circuit and consequent oxidative phosphorylation, O₂ consumption, and ATP production. This circuit may play an important role in repairing neurovascular function after TBI in the peri-injured region by stimulating astrocytic mitochondrial biogenesis.     

Overexpression of Glucocorticoid Receptor β Enhances Myogenesis and Reduces Catabolic Gene Expression

Unlike the glucocorticoid receptor α (GRα), GR β (GRβ) has a truncated ligand-binding domain that prevents glucocorticoid binding, implicating GRα as the mediator of glucocorticoid-induced skeletal muscle loss. Because GRβ causes glucocorticoid resistance, targeting GRβ may be beneficial in impairing muscle loss as a result of GRα activity. The purpose of this study was to determine how the overexpression of GRβ affects myotube formation and dexamethasone (Dex) responsiveness. We measured GR isoform expression in C₂C₁₂ muscle cells in response to Dex and insulin, and through four days of myotube formation. Next, lentiviral-mediated overexpression of GRβ in C₂C₁₂ was performed, and these cells were characterized for cell fusion and myotube formation, as well as sensitivity to Dex via the expression of ubiquitin ligases. GRβ overexpression increased mRNA levels of muscle regulatory factors and enhanced proliferation in myoblasts. GRβ overexpressing myotubes had an increased fusion index. Myotubes overexpressing GRβ had lower forkhead box O3 (Foxo3a) mRNA levels and a blunted muscle atrophy F-box/Atrogen-1 (MAFbx) and muscle ring finger 1 (MuRF1) response to Dex. We showed that GRβ may serve as a pharmacological target for skeletal muscle growth and protection from glucocorticoid-induced catabolic signaling. Increasing GRβ levels in skeletal muscle may cause a state of glucocorticoid resistance, stabilizing muscle mass during exposure to high doses of glucocorticoids.     

Activation of β-Adrenoceptors Promotes Lipid Droplet Accumulation in MCF-7 Breast Cancer Cells via cAMP/PKA/EPAC Pathways

Physiologically, β-adrenoceptors are major regulators of lipid metabolism, which may be reflected in alterations in lipid droplet dynamics. β-adrenoceptors have also been shown to participate in breast cancer carcinogenesis. Since lipid droplets may be seen as a hallmark of cancer, the present study aimed to investigate the role of β-adrenoceptors in the regulation of lipid droplet dynamics in MCF-7 breast cancer cells. Cells were treated for up to 72 h with adrenaline (an endogenous adrenoceptor agonist), isoprenaline (a non-selective β-adrenoceptor agonist) and salbutamol (a selective β₂-selective agonist), and their effects on lipid droplets were evaluated using Nile Red staining. Adrenaline or isoprenaline, but not salbutamol, caused a lipid-accumulating phenotype in the MCF-7 cells. These effects were significantly reduced by selective β₁- and β₃-antagonists (10 nM atenolol and 100 nM L-748,337, respectively), indicating a dependence on both β₁- and β₃-adrenoceptors. These effects were dependent on the cAMP signalling pathway, involving both protein kinase A (PKA) and cAMP-dependent guanine-nucleotide-exchange (EPAC) proteins: treatment with cAMP-elevating agents (forskolin or 8-Br-cAMP) induced lipid droplet accumulation, whereas either 1 µM H-89 or 1 µM ESI-09 (PKA or EPAC inhibitors, respectively) abrogated this effect. Taken together, the present results demonstrate the existence of a β-adrenoceptor-mediated regulation of lipid droplet dynamics in breast cancer cells, likely involving β₁- and β₃-adrenoceptors, revealing a new mechanism by which adrenergic stimulation may influence cancer cell metabolism.     

Ionic Environment Affects Biomolecular Interactions of Amyloid-β: SPR Biosensor Study

In early stages of Alzheimer’s disease (AD), amyloid beta (Aβ) accumulates in the mitochondrial matrix and interacts with mitochondrial proteins, such as cyclophilin D (cypD) and 17β-hydroxysteroid dehydrogenase 10 (17β-HSD10). Multiple processes associated with AD such as increased production or oligomerization of Aβ affect these interactions and disbalance the equilibrium between the biomolecules, which contributes to mitochondrial dysfunction. Here, we investigate the effect of the ionic environment on the interactions of Aβ (Aβ_1–40, Aβ_1–42) with cypD and 17β-HSD10 using a surface plasmon resonance (SPR) biosensor. We show that changes in concentrations of K⁺ and Mg²⁺ significantly affect the interactions and may increase the binding efficiency between the biomolecules by up to 35% and 65% for the interactions with Aβ_1–40 and Aβ_1–42, respectively, in comparison with the physiological state. We also demonstrate that while the binding of Aβ_1–40 to cypD and 17β-HSD10 takes place preferentially around the physiological concentrations of ions, decreased concentrations of K⁺ and increased concentrations of Mg²⁺ promote the interaction of both mitochondrial proteins with Aβ_1–42. These results suggest that the ionic environment represents an important factor that should be considered in the investigation of biomolecular interactions taking place in the mitochondrial matrix under physiological as well as AD-associated conditions.     

The Function of Transthyretin Complexes with Metallothionein in Alzheimer’s Disease

Alzheimer’s disease (AD) is one of the most frequently diagnosed types of dementia in the elderly. An important pathological feature in AD is the aggregation and deposition of the β-amyloid (Aβ) in extracellular plaques. Transthyretin (TTR) can cleave Aβ, resulting in the formation of short peptides with less activity of amyloid plaques formation, as well as being able to degrade Aβ peptides that have already been aggregated. In the presence of TTR, Aβ aggregation decreases and toxicity of Aβ is abolished. This may prevent amyloidosis but the malfunction of this process leads to the development of AD. In the context of Aβplaque formation in AD, we discuss metallothionein (MT) interaction with TTR, the effects of which depend on the type of MT isoform. In the brains of patients with AD, the loss of MT-3 occurs. On the contrary, MT-1/2 level has been consistently reported to be increased. Through interaction with TTR, MT-2 reduces the ability of TTR to bind to Aβ, while MT-3 causes the opposite effect. It increases TTR-Aβ binding, providing inhibition of Aβ aggregation. The protective effect, assigned to MT-3 against the deposition of Aβ, relies also on this mechanism. Additionally, both Zn₇MT-2 and Zn₇MT-3, decrease Aβ neurotoxicity in cultured cortical neurons probably because of a metal swap between Zn₇MT and Cu(II)Aβ. Understanding the molecular mechanism of metals transfer between MT and other proteins as well as cognition of the significance of TTR interaction with different MT isoforms can help in AD treatment and prevention.     

Chondrocytes from Osteoarthritis Patients Adopt Distinct Phenotypes in Response to Central TH1/TH2/TH17 Cytokines

Chronic low-grade inflammation plays a central role in the pathogenesis of osteoarthritis (OA), and several pro- and anti-inflammatory cytokines have been implicated to mediate and regulate this process. Out of these cytokines, particularly IFNγ, IL-1β, IL-4 and IL-17 are associated with different phenotypes of T helper (T_H) cells and macrophages, both examples of cells known for great phenotypic and functional heterogeneity. Chondrocytes also display various phenotypic changes during the course of arthritis. We set out to study the hypothesis of whether chondrocytes might adopt polarized phenotypes analogous to T_H cells and macrophages. We studied the effects of IFNγ, IL-1β, IL-4 and IL-17 on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were harvested from the cartilage of OA patients undergoing knee replacement surgery and then cultured with or without the cytokines for 24 h. Total RNA was isolated and sequenced, and GO (Gene Ontology) functional analysis was performed. We also separately investigated genes linked to OA in recent genome wide expression analysis (GWEA) studies. The expression of more than 2800 genes was significantly altered in chondrocytes treated with IL-1β [in the C(IL-1β) phenotype] with a fold change (FC) > 2.5 in either direction. These included a large number of genes associated with inflammation, cartilage degradation and attenuation of metabolic signaling. The profile of genes differentially affected by IFNγ (the C(IFNγ) phenotype) was relatively distinct from that of the C(IL-1β) phenotype and included several genes associated with antigen processing and presentation. The IL-17-induced C(IL-17) phenotype was characterized by the induction of a more limited set of proinflammatory factors compared to C(IL-1β) cells. The C(IL-4) phenotype induced by IL-4 displayed a differential expression of a rather small set of genes compared with control, primarily those associated with TGFβ signaling and the regulation of inflammation. In conclusion, our results show that OA chondrocytes can adopt diverse phenotypes partly analogously to T_H cells and macrophages. This phenotypic plasticity may play a role in the pathogenesis of arthritis and open new therapeutic avenues for the development of disease-modifying treatments for (osteo)arthritis.     

Characterization of a Mouse Model of Alzheimer’s Disease Expressing Aβ4-42 and Human Mutant Tau

The relationship between the two most prominent neuropathological hallmarks of Alzheimer’s Disease (AD), extracellular amyloid-β (Aβ) deposits and intracellular accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFT), remains at present not fully understood. A large body of evidence places Aβ upstream in the cascade of pathological events, triggering NFTs formation and the subsequent neuron loss. Extracellular Aβ deposits were indeed causative of an increased tau phosphorylation and accumulation in several transgenic models but the contribution of soluble Aβ peptides is still controversial. Among the different Aβ variants, the N-terminally truncated peptide Aβ_4–42 is among the most abundant. To understand whether soluble Aβ_4–42 peptides impact the onset or extent of tau pathology, we have crossed the homozygous Tg4–42 mouse model of AD, exclusively expressing Aβ_4–42 peptides, with the PS19 (P301S) tau transgenic model. Behavioral assessment showed that the resulting double-transgenic line presented a partial worsening of motor performance and spatial memory deficits in the aged group. While an increased loss of distal CA1 pyramidal neurons was detected in young mice, no significant alterations in hippocampal tau phosphorylation were observed in immunohistochemical analyses.     

Inhibition of Inducible Nitric Oxide Synthase Prevents IL-1β-Induced Mitochondrial Dysfunction in Human Chondrocytes

Interleukin (IL)-1β is an important pro-inflammatory cytokine in the progression of osteoarthritis (OA), which impairs mitochondrial function and induces the production of nitric oxide (NO) in chondrocytes. The aim was to investigate if blockade of NO production prevents IL-1β-induced mitochondrial dysfunction in chondrocytes and whether cAMP and AMP-activated protein kinase (AMPK) affects NO production and mitochondrial function. Isolated human OA chondrocytes were stimulated with IL-1β in combination with/without forskolin, L-NIL, AMPK activator or inhibitor. The release of NO, IL-6, PGE₂, MMP3, and the expression of iNOS were measured by ELISA or Western blot. Parameters of mitochondrial respiration were measured using a seahorse analyzer. IL-1β significantly induced NO release and mitochondrial dysfunction. Inhibition of iNOS by L-NIL prevented IL-1β-induced NO release and mitochondrial dysfunction but not IL-1β-induced release of IL-6, PGE₂, and MMP3. Enhancement of cAMP by forskolin reduced IL-1β-induced NO release and prevented IL-1β-induced mitochondrial impairment. Activation of AMPK increased IL-1β-induced NO production and the negative impact of IL-1β on mitochondrial respiration, whereas inhibition of AMPK had the opposite effects. NO is critically involved in the IL-1β-induced impairment of mitochondrial respiration in human OA chondrocytes. Increased intracellular cAMP or inhibition of AMPK prevented both IL-1β-induced NO release and mitochondrial dysfunction.     

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.