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.     

αN-Acetyl β-Endorphin Is an Endogenous Ligand of σ1Rs That Regulates Mu-Opioid Receptor Signaling by Exchanging G Proteins for σ2Rs in σ1R Oligomers

The opioid peptide β-endorphin coexists in the pituitary and brain in its ^αN-acetylated form, which does not bind to opioid receptors. We now report that these neuropeptides exhibited opposite effects in in vivo paradigms, in which ligands of the sigma type 1 receptor (σ1R) displayed positive effects. Thus, ^αN-acetyl β-Endorphin reduced vascular infarct caused by permanent unilateral middle cerebral artery occlusion and diminished the incidence of N-methyl-D-aspartate acid-promoted convulsive syndrome and mechanical allodynia caused by unilateral chronic constriction of the sciatic nerve. Moreover, ^αN-acetyl β-Endorphin reduced the analgesia of morphine, β-Endorphin and clonidine but enhanced that of DAMGO. All these effects were counteracted by β-Endorphin and absent in σ1R^−/− mice. We observed that σ1Rs negatively regulate mu-opioid receptor (MOR)-mediated morphine analgesia by binding and sequestering G proteins. In this scenario, β-Endorphin promoted the exchange of σ2Rs by G proteins at σ1R oligomers and increased the regulation of G proteins by MORs. The opposite was observed for the ^αN-acetyl derivative, as σ1R oligomerization decreased and σ2R binding was favored, which displaced G proteins; thus, MOR-regulated transduction was reduced. Our findings suggest that the pharmacological β-Endorphin-specific epsilon receptor is a σ1R-regulated MOR and that β-Endorphin and ^αN-acetyl β-Endorphin are endogenous ligands of σ1R.     

The Dual Role of the β2-Adrenoreceptor in the Modulation of IL-17 and IFN-γ Production by T Cells in Multiple Sclerosis

Norepinephrine is a neurotransmitter that also has an immunomodulatory effect and is involved in multiple sclerosis (MS) pathogenesis. This study aimed to clarify the role of the β₂-adrenoreceptor in the norepinephrine-mediated modulation of interleukin-17 (IL-17) and interferon-γ (IFN-γ) production, which play a critical pathogenetic role in MS. CD4⁺ T cells obtained from twenty-five relapsing-remitting MS patients and sixteen healthy subjects were cultured ex vivo with norepinephrine and/or β₂-adrenoreceptor antagonist or agonist, followed by a cytokine production analysis using ELISA. Norepinephrine suppressed IL-17 and IFN-γ production by the anti-CD3/anti-CD28-microbead-stimulated CD4⁺ T cells in both groups. Blockade of the β₂-adrenoreceptor with the specific antagonist ICI 118.551 enhanced norepinephrine-mediated IL-17 suppression but decreased its inhibitory effect on IFN-γ production in MS patients. In contrast, the β₂-adrenoreceptor agonist formoterol did not influence norepinephrine’s inhibitory effect on cytokine production in both groups. The blockade of the β₂-adrenoreceptor, even in the absence of exogenous norepinephrine, suppressed IL-17 production but did not influence IFN-γ production in both groups. Conversely, β₂-adrenoreceptor activation by formoterol decreased IFN-γ production and did not affect IL-17 production in both groups. These data illustrate the inhibitory effect of norepinephrine on IL-17 and IFN-γ production by CD4⁺ T cells in MS. The inhibitory effect of norepinephrine on IFN-γ production by CD4⁺ T cells in MS could be mediated via β₂-adrenoreceptor activation.     

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.     

GSK3β Inhibition by Phosphorylation at Ser389 Controls Neuroinflammation

The inhibition of Glycogen Synthase Kinase 3 β (GSK3β) by Ser⁹ phosphorylation affects many physiological processes, including the immune response. However, the consequences of GSK3β inhibition by alternative Ser³⁸⁹ phosphorylation remain poorly characterized. Here we have examined neuroinflammation in GSK3β Ser³⁸⁹ knock-in (KI) mice, in which the phosphorylation of Ser³⁸⁹ GSK3β is impaired. The number of activated microglia/infiltrated macrophages, astrocytes, and infiltrated neutrophils was significantly higher in these animals compared to C57BL/6J wild-type (WT) counterparts, which suggests that the failure to inactivate GSK3β by Ser³⁸⁹ phosphorylation results in sustained low-grade neuroinflammation. Moreover, glial cell activation and brain infiltration of immune cells in response to lipopolysaccharide (LPS) failed in GSK3β Ser³⁸⁹ KI mice. Such effects were brain-specific, as peripheral immunity was not similarly affected. Additionally, phosphorylation of the IkB kinase complex (IKK) in response to LPS failed in GSK3β Ser³⁸⁹ KI mice, while STAT3 phosphorylation was fully conserved, suggesting that the NF-κB signaling pathway is specifically affected by this GSK3β regulatory pathway. Overall, our findings indicate that GSK3β inactivation by Ser³⁸⁹ phosphorylation controls the brain inflammatory response, raising the need to evaluate its role in the progression of neuroinflammatory pathologies.     

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes

Intracellular free zinc ([Zn²⁺]_i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn²⁺]_i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn²⁺ have been identified, proteins that are involved in Zn²⁺ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn²⁺ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn²⁺]_i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn²⁺]_i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca²⁺ oscillations. Assays with fluorescent Zn²⁺ indicators revealed that the basal [Zn²⁺]_i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn²⁺]_i level in the presence of 1 μM Zn²⁺ in inflammatory FLSs. Among the 17 out of 24 known Zn²⁺ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn²⁺]_i level. Taken together, Zn²⁺ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn²⁺]_i in inflammatory FLSs.     

Caffeic Acid Phenethyl Ester Suppresses Proliferation and Survival of TW2.6 Human Oral Cancer Cells via Inhibition of Akt Signaling

Caffeic acid phenethyl ester (CAPE) is a bioactive component extracted from honeybee hive propolis. Our observations indicated that CAPE treatment suppressed cell proliferation and colony formation of TW2.6 human oral squamous cell carcinoma (OSCC) cells dose-dependently. CAPE treatment decreased G1 phase cell population, increased G2/M phase cell population, and induced apoptosis in TW2.6 cells. Treatment with CAPE decreased protein abundance of Akt, Akt1, Akt2, Akt3, phospho-Akt Ser473, phospho-Akt Thr 308, GSK3β, FOXO1, FOXO3a, phospho-FOXO1 Thr24, phospho-FoxO3a Thr32, NF-κB, phospho-NF-κB Ser536, Rb, phospho-Rb Ser807/811, Skp2, and cyclin D1, but increased cell cycle inhibitor p27^Kip. Overexpression of Akt1 or Akt2 in TW2.6 cells rescued growth inhibition caused by CAPE treatment. Co-treating TW2.6 cells with CAPE and 5-fluorouracil, a commonly used chemotherapeutic drug for oral cancers, exhibited additive cell proliferation inhibition. Our study suggested that administration of CAPE is a potential adjuvant therapy for patients with OSCC oral cancer.     

Novel Epoxides of Soloxolone Methyl: An Effect of the Formation of Oxirane Ring and Stereoisomerism on Cytotoxic Profile,Anti-Metastatic and Anti-Inflammatory Activities In Vitro and In Vivo

It is known that epoxide-bearing compounds display pronounced pharmacological activities, and the epoxidation of natural metabolites can be a promising strategy to improve their bioactivity. Here, we report the design, synthesis and evaluation of biological properties of αO-SM and βO-SM, novel epoxides of soloxolone methyl (SM), a cyanoenone-bearing derivative of 18βH-glycyrrhetinic acid. We demonstrated that the replacement of a double-bound within the cyanoenone pharmacophore group of SM with α- and β-epoxide moieties did not abrogate the high antitumor and anti-inflammatory potentials of the triterpenoid. It was found that novel SM epoxides induced the death of tumor cells at low micromolar concentrations (IC₅₀^(24h) = 0.7–4.1 µM) via the induction of mitochondrial-mediated apoptosis, reinforced intracellular accumulation of doxorubicin in B16 melanoma cells, probably by direct interaction with key drug efflux pumps (P-glycoprotein, MRP1, MXR1), and the suppressed pro-metastatic phenotype of B16 cells, effectively inhibiting their metastasis in a murine model. Moreover, αO-SM and βO-SM hampered macrophage functionality in vitro (motility, NO production) and significantly suppressed carrageenan-induced peritonitis in vivo. Furthermore, the effect of the stereoisomerism of SM epoxides on the mentioned bioactivities and toxic profiles of these compounds in vivo were evaluated. Considering the comparable antitumor and anti-inflammatory effects of SM epoxides with SM and reference drugs (dacarbazine, dexamethasone), αO-SM and βO-SM can be considered novel promising antitumor and anti-inflammatory drug candidates.     

PLG-007 and Its Active Component Galactomannan-α Competitively Inhibit Enzymes That Hydrolyze Glucose Polymers

PLG-007 is a developmental therapeutic compound that has been clinically shown to reduce the magnitude of postprandial glucose excursions and has the potential to be an adjunct treatment for diabetes and inflammatory-related diseases. The present investigation is aimed at understanding the molecular mechanism of action of PLG-007 and its galactomannan (GM) components GMα and GMβ (in a 1:4 mass ratio, respectively) on enzyme (i.e., α-amylase, maltase, and lactase) hydrolysis of glucose polymers using colorimetric assays and ¹³C HSQC NMR spectroscopy. The starch–iodine colorimetric assay indicated that GMα strongly inhibits α-amylase activity (~16-fold more potent than GMβ) and thus is the primary active component in PLG-007. ¹³C HSQC experiments, used to follow the α-amylase-mediated hydrolysis of starch and amylopectin, further demonstrate the α-amylase inhibitory effect of GMα via α-amylase-mediated hydrolysis of starch and amylopectin. Maltohexaose (MT6) was used to circumvent the relative kinetic complexity of starch/amylopectin degradation in Michaelis–Menten analyses. The V_max, K_M, and K_i parameters were determined using peak volume integrals from ¹³C HSQC NMR spectra. In the presence of PLG-007 with α-amylase and MT6, the increase in K_M from 7.5 ± 0.6 × 10⁻³ M (control) to 21 ± 1.4 × 10⁻³ M, with no significant change in V_max, indicates that PLG-007 is a competitive inhibitor of α-amylase. Using K_M values, K_i was estimated to be 2.1 ± 0.9 × 10⁻⁶ M; however, the microscopic K_i value of GMα is expected to be larger as the binding stoichiometry is likely to be greater than 1:1. Colorimetric assays also demonstrated that GMα is a competitive inhibitor of the enzymes maltase and lactase. Overall, this study provides insight as to how PLG-007 (GMα) is likely to function in vivo.     

The PI-3-Kinase P110α Catalytic Subunit of T Lymphocytes Modulates Collagen-Induced Arthritis

The phosphatidylinositol 3-kinase (PI3K) family of enzymes plays a determinant role in inflammation and autoimmune responses. However, the implication of the different isoforms of catalytic subunits in these processes is not clear. Rheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory disease that entails innate and adaptive immune response elements in which PI3K is a potential hub for immune modulation. In a mouse transgenic model with T-cell-specific deletion of p110α catalytic chain (p110α^−/−ΔT), we show the modulation of collagen-induced arthritis (CIA) by this isoform of PI3K. In established arthritis, p110α^−/−ΔT mice show decreased prevalence of illness than their control siblings, higher IgG1 titers and lower levels of IL-6 in serum, together with decreased ex vivo Collagen II (CII)-induced proliferation, IL-17A secretion and proportion of naive T cells in the lymph nodes. In a pre-arthritis phase, at 13 days post-Ag, T-cell-specific deletion of p110α chain induced an increased, less pathogenic IgG1/IgG2a antibodies ratio; changes in the fraction of naive and effector CD4⁺ subpopulations; and an increased number of CXCR5⁺ T cells in the draining lymph nodes of the p110α^−/−ΔT mice. Strikingly, T-cell blasts in vitro obtained from non-immunized p110α^−/−ΔT mice showed an increased expression of CXCR5, CD44 and ICOS surface markers and defective ICOS-induced signaling towards Akt phosphorylation. These results, plus the accumulation of cells in the lymph nodes in the early phase of the process, could explain the diminished illness incidence and prevalence in the p110α^−/−ΔT mice and suggests a modulation of CIA by the p110α catalytic chain of PI3K, opening new avenues of intervention in T-cell-directed therapies to autoimmune diseases.     

The C-Terminal Domain of LRRK2 with the G2019S Substitution Increases Mutant A53T α-Synuclein Toxicity in Dopaminergic Neurons In Vivo

Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) play crucial roles in Parkinson’s disease (PD). They may functionally interact to induce the degeneration of dopaminergic (DA) neurons via mechanisms that are not yet fully understood. We previously showed that the C-terminal portion of LRRK2 (ΔLRRK2) with the G2019S mutation (ΔLRRK2^G2019S) was sufficient to induce neurodegeneration of DA neurons in vivo, suggesting that mutated LRRK2 induces neurotoxicity through mechanisms that are (i) independent of the N-terminal domains and (ii) “cell-autonomous”. Here, we explored whether ΔLRRK2^G2019S could modify α-syn toxicity through these two mechanisms. We used a co-transduction approach in rats with AAV vectors encoding ΔLRRK2^G2019S or its “dead” kinase form, ΔLRRK2^DK, and human α-syn with the A53T mutation (AAV-α-syn^A53T). Behavioral and histological evaluations were performed at 6- and 15-weeks post-injection. Results showed that neither form of ΔLRRK2 alone induced the degeneration of neurons at these post-injection time points. By contrast, injection of AAV-α-syn^A53T alone resulted in motor signs and degeneration of DA neurons. Co-injection of AAV-α-syn^A53T with AAV-ΔLRRK2^G2019S induced DA neuron degeneration that was significantly higher than that induced by AAV-α-syn^A53T alone or with AAV-ΔLRRK2^DK. Thus, mutated α-syn neurotoxicity can be enhanced by the C-terminal domain of LRRK2^G2019 alone^, through cell-autonomous mechanisms.     

The Neosartorya fischeri Antifungal Protein 2 (NFAP2): A New Potential Weapon against Multidrug-Resistant Candida auris Biofilms

Candida auris is a potential multidrug-resistant pathogen able to persist on indwelling devices as a biofilm, which serve as a source of catheter-associated infections. Neosartorya fischeri antifungal protein 2 (NFAP2) is a cysteine-rich, cationic protein with potent anti-Candida activity. We studied the in vitro activity of NFAP2 alone and in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against C. auris biofilms. The nature of interactions was assessed utilizing the fractional inhibitory concentration index (FICI), a Bliss independence model, and LIVE/DEAD viability assay. NFAP2 exerted synergy with all tested antifungals with FICIs ranging between 0.312–0.5, 0.155–0.5, 0.037–0.375, 0.064–0.375, and 0.064–0.375 for fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. These results were confirmed using a Bliss model, where NFAP2 produced 17.54 μM²%, 2.16 μM²%, 33.31 μM²%, 10.72 μM²%, and 111.19 μM²% cumulative synergy log volume in combination with fluconazole, amphotericin B, anidulafungin, caspofungin, and micafungin, respectively. In addition, biofilms exposed to echinocandins (32 mg/L) showed significant cell death in the presence of NFAP2 (128 mg/L). Our study shows that NFAP2 displays strong potential as a novel antifungal compound in alternative therapies to combat C. auris biofilms.     

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.     

Human β-Defensin 3 Inhibits Porphyromonas Gingivalis Lipopolysaccharide-Induced Oxidative and Inflammatory Responses of Microglia by Suppression of Cathepsins B and L

Recently, the effects of antibacterial peptides are suggested to have therapeutic potential in Alzheimer’s disease. Furthermore, systemic treatment of Porphyromonas gingivalis (Pg) lipopolysaccharide (LPS) induced Alzheimer’s disease-like neuropathological changes in middle-aged mice. Then, we examined whether human β-defensins (hBDs), antimicrobial peptides produced by the oral mucosa and salivary glands, can suppress Pg LPS-induced oxidative and inflammatory responses by microglia. hBD3 (1 μM) significantly suppressed Pg LPS-induced production of nitric oxide and interleukin-6 (IL-6) by MG6 cells, a mouse microglial cell line. hBD3 (1 μM) also significantly inhibited Pg LPS-induced expression of IL-6 by HMC3 cells, a human microglial cell line. In contrast, neither hBD1, hBD2 nor hBD4 failed to inhibit their productions. Furthermore, hBD3 suppressed Pg LPS-induced p65 nuclear translocation through the IκBα degradation. Pg LPS-induced expression of IL-6 was significantly suppressed by E64d, a cysteine protease inhibitor, and CA-074Me, a known specific inhibitor for cathepsin B, but not by pepstatin A, an aspartic protease inhibitor. Interestingly, hBD3 significantly inhibited enzymatic activities of recombinant human cathepsins B and L, lysosomal cysteine proteases, and their intracellular activities in MG6 cells. Therefore, hBD3 suppressed oxidative and inflammatory responses of microglia through the inhibition of cathepsins B and L, which enzymatic activities are necessary for the NF-κB activation.     

Association of IgG1 Antibody Clearance with FcγRIIA Polymorphism and Platelet Count in Infliximab-Treated Patients

The FcγRIIA/CD32A is mainly expressed on platelets, myeloid and several endothelial cells. Its affinity is considered insufficient for allowing significant binding of monomeric IgG, while its H131R polymorphism (histidine > arginine at position 131) influences affinity for multimeric IgG2. Platelet FcγRIIA has been reported to contribute to IgG-containing immune-complexe clearance. Given our finding that platelet FcγRIIA actually binds monomeric IgG, we investigated the role of platelets and FcγRIIA in IgG antibody elimination. We used pharmacokinetics analysis of infliximab (IgG1) in individuals with controlled Crohn’s disease. The influence of platelet count and FcγRIIA polymorphism was quantified by multivariate linear modelling. The infliximab half-life increased with R allele number (13.2, 14.4 and 15.6 days for HH, HR and RR patients, respectively). It decreased with increasing platelet count in R carriers: from ≈20 days (RR) and ≈17 days (HR) at 150 × 10⁹/L, respectively, to ≈13 days (both HR and RR) at 350 × 10⁹/L. Moreover, a flow cytometry assay showed that infliximab and monomeric IgG1 bound efficiently to platelet FcγRIIA H and R allotypes, whereas panitumumab and IgG2 bound poorly to the latter. We propose that infliximab (and presumably any IgG1 antibody) elimination is partly due to an unappreciated mechanism dependent on binding to platelet FcγRIIA, which is probably tuned by its affinity for IgG2.     

Fluorine Atoms on C6H5-Corrole Affect the Interaction with Mpro and PLpro Proteases of SARS-CoV-2: Molecular Docking and 2D-QSAR Approaches

The chymotrypsin-like cysteine protease (3CL^pro, also known as main protease—M^pro) and papain-like protease (PL^pro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been used as the main targets for screening potential synthetic inhibitors for posterior in vitro evaluation of the most promising compounds. In this sense, the present work reports for the first time the evaluation of the interaction between M^pro/PL^pro with a series of 17 porphyrin analogues-corrole (C1), meso-aryl-corrole (C2), and 15 fluorinated-meso-aryl-corrole derivatives (C3–C17) via molecular docking calculations. The impact of fluorine atoms on meso-aryl-corrole structure was also evaluated in terms of binding affinity and physical-chemical properties by two-dimensional quantitative structure–activity relationship (2D-QSAR). The presence of phenyl moieties increased the binding capacity of corrole for both proteases and depending on the position of fluorine atoms might impact positively or negatively the binding capacity. For M^pro the para-fluorine atoms might decrease drastically the binding capacity, while for PL^pro there was a certain increase in the binding affinity of fluorinated-corroles with the increase of fluorine atoms into meso-aryl-corrole structure mainly from tri-fluorinated insertions. The 2D-QSAR models indicated two separated regions of higher and lower affinity for M^pro:C1–C17 based on dual electronic parameters (σ_I and σ_R), as well as one model was obtained with a correlation between the docking score value of M^pro:C2–C17 and the corresponding ¹³C nuclear magnetic resonance (NMR) chemical shifts of the sp² carbon atoms (δ_C-1 and δ_C-2) of C2–C17. Overall, the fluorinated-meso-aryl-corrole derivatives showed favorable in silico parameters as potential synthetic compounds for future in vitro assays on the inhibition of SARS-CoV-2 replication.     

Loss of Heterozygosity for KrasG12D Promotes Malignant Phenotype of Pancreatic Ductal Adenocarcinoma by Activating HIF-2α-c-Myc-Regulated Glutamine Metabolism

Loss of heterozygosity (LOH) for KRAS, in which a wild-type KRAS allele is progressively lost, promotes invasive and migratory abilities of pancreatic ductal adenocarcinoma (PDAC) cells and tissues. Moreover, the occurrence of Kras^G12D-LOH activates nonclassical glutamine metabolism, which is related to the malignant behavior of PDAC cells. Herein, we aim to demonstrate the regulatory link between hypoxia-inducible factor-2α (HIF-2α) and glutamine metabolism that mediates malignant phenotypes in Kras^G12D-LOH PDAC cells. HIF-2α-shRNA knockdown lentivirus transfection and metabolite analysis were performed in Kras^G12D-LOH and Kras^G12D cell lines, respectively. Cell proliferation, migration, and invasion were examined using Cell Counting Kit-8, colony formation, and Transwell assays. Cell cycle phase and apoptosis were determined using flow cytometry. Western blotting and real-time quantitative PCR were also performed. Additionally, a subcutaneous xenograft mouse model was established. LOH stimulated HIF-2α activity and transactivated c-Myc, which has a central regulatory effect on glutamine metabolism independent of hypoxia. Meanwhile, HIF-2α silencing repressed Kras^G12D-LOH PDAC cell proliferation, invasion, and migration. HIF-2α knockdown inhibited glutamine uptake and GOT1 expression via a c-Myc-dependent pathway. Collectively, Kras^G12D-LOH can activate HIF-2α to regulate c-Myc-mediated glutamine metabolism and promote malignant phenotypes. Moreover, targeting HIF-2α-c-Myc regulated nonclassical glutamine metabolism, providing a new therapeutic perspective for Kras^G12D-LOH PDAC.