Metabolomics of airways disease in cystic fibrosis

While discovery metabolomic studies have identified many potential biomarkers of cystic fibrosis (CF) airways disease, relatively few have been validated. We review the recent literature to identify the most promising metabolomic findings as those repeatedly observed over multiple studies. Reproducible metabolomic findings include increased airway amino acids and small peptides in CF airways, as well as changes in phospholipids and sphingolipids. Other commonly altered pathways include adenosine metabolism, polyamine synthesis, and oxidative stress. These pathways represent potential biomarkers and therapeutic targets, though findings require reevaluation in the era of highly effective modulator therapies. Analysis of airway biomarkers in exhaled breath holds promise for non-invasive detection, though technical challenges will need to be overcome.     

Defining the role of CFTR channel blocker and ClC-2 activator in DNBS induced gastrointestinal inflammation

In the present study, we have investigated and/or compared the role of glibenclamide, G as cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor, and lubiprostone, L as chloride channel-2 (ClC-2) activator in the 2,4-dinitrobenzene sulfonic acid (DNBS)-induced gastrointestinal inflammation. GI inflammation was induced by intrarectal administration of DNBS. Rats were randomly allocated in 5 groups as sham control, distilled water + DNBS, sulfasalazine (S) + DNBS, G + DNBS, and L + DNBS. All the groups were pre-treated successively for five days before the induction of colitis. One day before and the first four days after DNBS administration various parameters were studied. Later, blood chemistry, colon’s gross structure, histology, and the antioxidant load was examined. Pre-treatment with G significantly protected the change induced by DNBS concerning the change in body weight, food intake, diarrhea, occult blood in the feces, wet weight of the colon, and spleen. G because of its anti-inflammatory property down-regulated the neutrophil and WBC count and up-regulated the lymphocyte number. Moreover, G efficiently ameliorates the oxidative stress in the colon and declines the level of myeloperoxidase and malondialdehyde and up-regulated the level of superoxide dismutase and glutathione. Lubiprostone has not shown any promising effects, in fact, it causes an increase in diarrheal frequency. Our findings from this study represent that G has good potential to ameliorate GI inflammation induced by DNBS by its multiple actions including CFTR blockage and reducing the release of inflammatory markers from the MCs, anti-inflammatory and free radical scavenging property.     

First small-molecule PROTACs for G protein-coupled receptors: inducing α1A-adrenergic receptor degradation

Proteolysis targeting chimeras (PROTACs) are dual-functional hybrid molecules that can selectively recruit an E3 ubiquitin ligase to a target protein to direct the protein into the ubiquitin-proteasome system (UPS), thereby selectively reducing the target protein level by the ubiquitin-proteasome pathway. Nowadays, small-molecule PROTACs are gaining popularity as tools to degrade pathogenic protein. Herein, we present the first small-molecule PROTACs that can induce the α_1A-adrenergic receptor (α_1A-AR) degradation, which is also the first small-molecule PROTACs for G protein-coupled receptors (GPCRs) to our knowledge. These degradation inducers were developed through conjugation of known α₁-adrenergic receptors (α₁-ARs) inhibitor prazosin and cereblon (CRBN) ligand pomalidomide through the different linkers. The representative compound 9c is proved to inhibit the proliferation of PC-3 cells and result in tumor growth regression, which highlighted the potential of our study as a new therapeutic strategy for prostate cancer.     

Molecular Dynamics Simulation to Uncover the Mechanisms of Protein Instability During Freezing

Freezing is a common process applied in the pharmaceutical industry to store and transport biotherapeutics. Herewith, multi-scale molecular dynamics simulations of Lactate dehydrogenase (LDH) protein in phosphate buffer with/without ice formation performed to uncover the still poorly understood mechanisms and molecular details of protein destabilization upon freezing. Both fast and slow ice growing conditions were simulated at 243 K from one or two-side of the simulation box, respectively. The rate of ice formation at all-atom simulations was crucial to LDH stability, as faster freezing rates resulted in enhanced structural stability maintained by a higher number of intramolecular hydrogen bonds, less flexible protein's residues, lower solvent accessibility and greater structural compactness. Further, protein aggregation investigated by coarse-grained simulations was verified to be initiated by extended protein structures and retained by electrostatic interactions of the salt bridges between charged residues and hydrogen bonds between polar residues of the protein. Lastly, the study of free energy of dissociation through steered molecular dynamics simulation revealed LDH was destabilized by the solvation of the hydrophobic core and the loss of hydrophobic interactions. For the first time, experimentally validated molecular simulations revealed the detailed mechanisms of LDH destabilization upon ice formation and cryoconcentration of solutes.     

Icariin regulates miR-23a-3p-mediated osteogenic differentiation of BMSCs via BMP-2/Smad5/Runx2 and WNT/β-catenin pathways in osteonecrosis of the femoral head

Icariin is commonly used for the clinical treatment of osteonecrosis of the femoral head (ONFH). miR-23a-3p plays a vital role in regulating the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). The present study aimed to investigate the roles of icariin and miR-23a-3p in the osteogenic differentiation of BMSCs and an ONFH model. BMSCs were isolated and cultured in vitro using icariin-containing serum at various concentrations, and BMSCs were also transfected with a miR-23a inhibitor. The alkaline phosphatase (ALP) activity and cell viability as well as BMP-2/Smad5/Runx2 and WNT/β-catenin pathway-related mRNA and protein expression were measured in BMSCs. Additionally, a dual-luciferase reporter assay and pathway inhibitors were used to verify the relationship of icariin treatment/miR-23a and the above pathways. An ONFH rat model was established in vivo, and a 28-day gavage treatment and lentivirus transfection of miR-23a-3p inhibitor were performed. Then, bone biochemical markers (ELISA kits) in serum, femoral head (HE staining and Digital Radiography, DR) and the above pathway-related proteins were detected. Our results revealed that icariin treatment/miR-23a knockdown promoted BMSC viability and osteogenic differentiation as well as increased the mRNA and protein expression of BMP-2, BMP-4, Runx2, p-Smad5, Wnt1 and β-catenin in BMSCs and ONFH model rats. In addition, icariin treatment/miR-23a knockdown increased bone biochemical markers (ACP-5, BAP, NTXI, CTXI and OC) and improved ONFH in ONFH model rats. In addition, a dual-luciferase reporter assay verified that Runx2 was a direct target of miR-23a-3p. These data indicated that icariin promotes BMSC viability and osteogenic differentiation as well as improves ONFH by decreasing miR-23a-3p levels and regulating the BMP-2/Smad5/Runx2 and WNT/β-catenin pathways.     

Bile Duct Obstruction Leads to Increased Intestinal Expression of Breast Cancer Resistance Protein With Reduced Gastrointestinal Absorption of Imatinib

Liver dysfunction reduces systemic clearance of drugs that are primarily eliminated by the liver. However, liver dysfunction can cause a reduction in the plasma concentration profiles of certain drugs, including several tyrosine kinase inhibitors, after oral administration. The aim of the present study was to clarify the reduction in oral absorption of a tyrosine kinase inhibitor, imatinib, and the mechanisms of action involved under conditions of hepatic dysfunction, focusing on intestinal transporters. The maximum plasma concentration of imatinib after oral administration in mice subjected to bile duct ligation (BDL) was lower than that in sham-operated mice, whereas the plasma concentration profile after intravenous administration was essentially unaffected by BDL. The change in maximum plasma concentration was compatible with a reduction in small intestinal permeability of imatinib observed in the in situ closed loop. Gene expression of abcg2 was increased in BDL mice compared with that in sham-operated mice. Expression of breast cancer resistance protein and P-glycoprotein in the small intestinal brush border membrane fraction from BDL mice was also increased compared with that in sham-operated mice. In summary, the intestinal absorption and permeability of imatinib was decreased in BDL mice, and this may be attributed to the up-regulation of the efflux transporter(s).     

Tubeimoside-1 induces TFEB-dependent lysosomal degradation of PD-L1 and promotes antitumor immunity by targeting mTOR

Programmed cell death ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) cascade is an effective therapeutic target for immune checkpoint blockade (ICB) therapy. Targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity. Using flow cytometry-based assay, we identify tubeimoside-1 (TBM-1) as a promising antitumor immune modulator that negatively regulates PD-L1 level. TBM-1 disrupts PD-1/PD-L1 interaction and enhances the cytotoxicity of T cells toward cancer cells through decreasing the abundance of PD-L1. Furthermore, TBM-1 exerts its antitumor effect in mice bearing Lewis lung carcinoma (LLC) and B16 melanoma tumor xenograft via activating tumor-infiltrating T-cell immunity. Mechanistically, TBM-1 triggers PD-L1 lysosomal degradation in a TFEB-dependent, autophagy-independent pathway. TBM-1 selectively binds to the mammalian target of rapamycin (mTOR) kinase and suppresses the activation of mTORC1, leading to the nuclear translocation of TFEB and lysosome biogenesis. Moreover, the combination of TBM-1 and anti-CTLA-4 effectively enhances antitumor T-cell immunity and reduces immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells. Our findings reveal a previously unrecognized antitumor mechanism of TBM-1 and represent an alternative ICB therapeutic strategy to enhance the efficacy of cancer immunotherapy.     

Identification of potential therapeutic target of naringenin in breast cancer stem cells inhibition by bioinformatics and in vitro studies

Cancer therapy is a strategic measure in inhibiting breast cancer stem cell (BCSC) pathways. Naringenin, a citrus flavonoid, was found to increase breast cancer cells’ sensitivity to chemotherapeutic agents. Bioinformatics study and 3D tumorsphere in vitro modeling in breast cancer (mammosphere) were used in this study, which aims to explore the potential therapeutic targets of naringenin (PTTNs) in inhibiting BCSCs. Bioinformatic analyses identified direct target proteins (DTPs), indirect target proteins (ITPs), naringenin-mediated proteins (NMPs), BCSC regulatory genes, and PTTNs. The PTTNs were further analyzed for gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein–protein interaction (PPI) networks, and hub protein selection. Mammospheres were cultured in serum-free media. The effects of naringenin were measured by MTT-based cytotoxicity, mammosphere forming potential (MFP), colony formation, scratch wound-healing assay, and flow cytometry-based cell cycle analyses and apoptosis assays. Gene expression analysis was performed using real-time quantitative polymerase chain reaction (q-RT PCR). Bioinformatics analysis revealed p53 and estrogen receptor alpha (ERα) as PTTNs, and KEGG pathway enrichment analysis revealed that TGF-ß and Wnt/ß-catenin pathways are regulated by PTTNs. Naringenin demonstrated cytotoxicity and inhibited mammosphere and colony formation, migration, and epithelial to mesenchymal transition in the mammosphere. The mRNA of tumor suppressors P53 and ERα were downregulated in the mammosphere, but were significantly upregulated upon naringenin treatment. By modulating the P53 and ERα mRNA, naringenin has the potential of inhibiting BCSCs. Further studies on the molecular mechanism and formulation of naringenin in BCSCs would be beneficial for its development as a BCSC-targeting drug.     

Gambogic acid alleviates inflammation and apoptosis and protects the blood-milk barrier in mastitis induced by LPS

Mastitis is one of the most common diseases among dairy cows. There is still much debate worldwide as to whether antibiotic therapy should be given to dairy cows, or if natural products should be taken as a substitute for antibacterial therapy. As the antibiotic treatment leads to the bacterial resistance and drug residue in milk, introducing natural products for mastitis is becoming a trend. This study investigates the mechanisms of the protective effects of the natural product gambogic acid (GA) in lipopolysaccharide (LPS)-induced mastitis. For in vitro treatments, it was found that GA reduced IL-6, TNF-α, and IL-1β levels by inhibiting the phosphorylation of proteins in the nuclear factor κB (NF-κB) and the mitogen-activated protein kinase (MAPK) pathway. GA also maintained a stable membrane mitochondrial potential and inhibited the overproduction of reactive oxygen species, which protected the cells from apoptosis. On the other hand, in vivo treatments with GA were found to reduce pathological symptoms markedly, and protected the blood-milk barrier from damage induced by LPS. The results demonstrate that GA plays a vital role in suppressing inflammation, alleviating the apoptosis effect, and protecting the blood-milk barrier in mastitis induced by LPS. Thus, these results suggest that the natural product GA plays a potential role in mastitis treatment.     

The role of nuclear factors as “Find-Me”/alarmin signals and immunostimulation in defective efferocytosis and related disorders

Efferocytosis as an apoptotic cell (AC) clearance mechanism facilitates the removal of dangerous and damaged cells, an important process in regulating normal homeostasis. Failure to correctly execute apoptosis and efferocytosis is associated with atherosclerosis, as well as chronic inflammatory and autoimmune disorders such as systemic lupus erythematosus (SLE). Effective and timely efferocytosis involves various molecules that act as “Find-Me” signals or as alarmins to quickly allow identification by phagocytic cells. In recent years, most of these molecules have been investigated, but less attention has been paid to the nuclear molecules associated with efferocytosis of ACs and necrotic cells (NCs). These molecules have several functions including acting as alarmin signals for faster recognition of ACs, facilitating the cleanup of ACs and for maintaining self-tolerance. The same group of molecules is also implicated in several inflammatory and autoimmune diseases. Previous studies have shown that these molecules also serve as targets for pharmacological agents such as necrostatins, recombinant Fcnb, anti-histone, neutralizing antibodies, calbiochem, aminophylline, activated protein C, CD24IgG recombinant fission protein, and recombinant thrombomodulin. Thus, greater understanding of these molecules/pathways will enable developments in the treatment and/or prevention of various disorders, especially autoimmune diseases. Here, we review current knowledge about the mechanisms by which nucleic acids, histones, nucleosomes and monosodium urate microcrystals (MSU) can act as alarmins/“Find-Me” signals, how they might be stimulated in defective efferocytosis and their function and importance as biomarkers for prognosis and treatment of atherosclerosis, inflammatory disorders and autoimmune diseases.     

Drug repurposing in skeletal muscle ion channelopathies

Skeletal muscle ion channelopathies are rare genetic diseases mainly characterized by myotonia (muscle stiffness) or periodic paralysis (muscle weakness). Here, we reviewed the available therapeutic options in non-dystrophic myotonias (NDM) and periodic paralyses (PP), which consists essentially in drug repositioning to address stiffness or weakness attacks. Empirical use followed by successful randomized clinical trials eventually led to the orphan drug designation and marketing authorization granting of mexiletine for NDM and dichlorphenamide for PP. Yet, these treatments neither consider the genetic cause of the diseases nor address the individual variability in drug response. Thus, ongoing research aims at the identification of repurposed drugs alternative to mexiletine and dichlorphenamide to allow personalization of treatment. This review highlights how drug repurposing may represent an efficient strategy in rare diseases, allowing reduction of drug development time and costs in a context in which the return on investment may be particularly challenging.     

Challenges and hopes for Alzheimer’s disease

Recent drug development efforts targeting Alzheimer’s disease (AD) have failed to produce effective disease-modifying agents for many reasons, including the substantial presymptomatic neuronal damage that is caused by the accumulation of the amyloid β (Aβ) peptide and tau protein abnormalities, deleterious adverse effects of drug candidates, and inadequate design of clinical trials. New molecular targets, biomarkers, and diagnostic techniques, as well as alternative nonpharmacological approaches, are sorely needed to detect and treat early pathological events. This article analyzes the successes and debacles of pharmaceutical endeavors to date, and highlights new technologies that may lead to the more effective diagnosis and treatment of the pathologies that underlie AD. The use of focused ultrasound, deep brain stimulation, stem cell therapy, and gene therapy, in parallel with pharmaceuticals and judicious lifestyle adjustments, holds promise for the deceleration, prevention, or cure of AD and other neurodegenerative disorders.     

Interference with megalin expression/endocytic function by montelukast mitigates gentamicin nephrotoxicity: Downregulation of ClC-5 expression

Megalin receptor-mediated endocytosis participates a crucial role in gentamicin (GM) uptake, accumulation, and toxicity. In this study, we investigated the potential effects of montelukast (MLK) on megalin expression/endocytic function against GM nephrotoxicity. Male Wistar rats were administered GM (120 mg/kg; i.p.) daily in divided doses along 4 hr; 30 mg/kg/hr; for 7 days. MLK (30 mg/kg/day) was orally administered 7 days before and then concurrently with GM. The protein expressions of megalin and chloride channel-5 (ClC-5); one of the essential regulators of megalin endocytic function; were determined by Western blotting. Besides, the endocytic function of megalin was evaluated by the uptake of bovine serum albumin labeled with fluorescein isothiocyanate (FITC-BSA) into proximal tubular epithelial cells. Moreover, kidney function biomarkers (Cr, BUN, GFR, KIM-1, cystatin-C) and apoptosis markers (p-AKT1, cleaved caspase-3) were estimated. Co-treatment with MLK downregulated ClC-5 expression leading to reduced recycling of megalin to the plasma membrane, reduced expression, and so impaired endocytic function that was evidenced by reduced uptake of FITC-BSA in proximal tubular epithelial cells. The protein expression of the apoptotic executioner cleaved caspase-3 was significantly reduced, while that of the antiapoptotic p-AKT1 was elevated. These results were confirmed by the improvement of kidney functions and histological findings. Our data suggest that MLK could interfere with megalin expression/endocytic function that could be attributed to downregulation of ClC-5 protein expression. That eventually reduces renal cell apoptosis and improves kidney functions after GM administration without affecting the antibacterial activity of GM. Therefore, reduced expression of ClC-5 and interference with megalin expression/endocytic function by MLK could be an effective strategy against GM nephrotoxicity.     

The carboxyl-terminal di-lysine motif is essential for catalytic activity of UDP-glucuronosyltransferase 1A9

UDP-Glucuronosyltransferase (UGT) is a type I membrane protein localized to the endoplasmic reticulum (ER). UGT has a di-lysine motif (KKXX/KXKXX) in its cytoplasmic domain, which is defined as an ER retention signal. However, our previous study has revealed that UGT2B7, one of the major UGT isoform in human, localizes to the ER in a manner that is independent of this motif. In this study, we focused on another UGT isoform, UGT1A9, and investigated the role of the di-lysine motif in its ER localization, glucuronidation activity, and homo-oligomer formation. Immunofluorescence microscopy indicated that the cytoplasmic domain of UGT1A9 functioned as an ER retention signal in a chimeric protein with CD4, but UGT1A9 itself could localize to the ER in a di-lysine motif-independent manner. In addition, UGT1A9 formed homo-oligomers in the absence of the motif. However, deletion of the di-lysine motif or substitution of lysines in the motif for alanines, severely impaired glucuronidation activity of UGT1A9. This is the first study that re-defines the cytoplasmic di-lysine motif of UGT as an essential peptide for retaining glucuronidation capacity.     

Evaluation of Encapsulated Eugenol by Chitosan Nanoparticles on the aggressive model of rheumatoid arthritis

Chitosan Nanoparticles Eugenol recognizes as a potent antioxidant that can use the first therapeutic chemical to treat rheumatoid arthritis (RA) instead of Methotrexate. The purpose of this study was to investigate the effects of Chitosan Nanoparticles Eugenol as a potent Nano-herbal agent in the healing process of experimental neonatal RA compared to Methotrexate.The neonatal Wistar rats induced rheumatoid arthritis in both genders were divided into sham, control, the treatment receiving Methotrexate, and the second treatment receiving encapsulated Eugenol by Chitosan Nanoparticles groups. Afterward, Malondialdehyde, for assessment of lipid peroxidation as an oxidative stress biomarker by assay kit, FOXO3 protein as an antioxidant up-regulating by western blotting and expression of the TGF-β and CCL2/MCP-1 genes by real-time PCR evaluation, supported by a cartilage histopathology analysis. Based on these results, Methotrexate and Eugenol encapsulated by Chitosan Nanoparticles, a significant decrease is observed in the serum level of MDA and FOXO3 protein expression in comparison to the control group. Additionally, Nanoparticle herbal agent and Methotrexate has a decreasing effect on the expression of TGF-β and MCP-1 genes and a significant positive correlation was observed between MCP-1 and TGF-β. Inflammation, synovial hyperplasia, and pannus formation were extreme in the Collagen Induced Arthritis rats. It can be concluded that Encapsulated Eugenol by Chitosan Nanoparticles and Methotrexate, probably by dint of their immunomodulatory, anti-inflammatory, and antioxidant potential has a protective effect against RA. Nano Eugenol is capable of delivering promising lines results to treat autoimmune diseases such as RA can also be suggested.     

Celiac disease: New therapies on the horizon

Celiac Disease (CeD) is a chronic intestinal disease which occurs in 0.7–1.4% of the global population. Since the discovery of gluten as its disease-inducing antigen, CeD patients are treated with a gluten-free diet which is effective but has limitations for certain groups of patients. Accordingly, over the past few years, there is a growing interest in alternative treatment options. This review summarizes emerging pharmacological approaches, including tolerance induction strategies, tissue transglutaminase inhibition, gluten degradation, and inhibition of interleukin (IL)-15.     

Conditional reprogramming: next generation cell culture

Long-term primary culture of mammalian cells has been always difficult due to unavoidable senescence. Conventional methods for generating immortalized cell lines usually require manipulation of genome which leads to change of important biological and genetic characteristics. Recently, conditional reprogramming (CR) emerges as a novel next generation tool for long-term culture of primary epithelium cells derived from almost all origins without alteration of genetic background of primary cells. CR co-cultures primary cells with inactivated mouse 3T3-J2 fibroblasts in the presence of RHO-related protein kinase (ROCK) inhibitor Y-27632, enabling primary cells to acquire stem-like characteristics while retain their ability to fully differentiate. With only a few years’ development, CR shows broad prospects in applications in varied areas including disease modeling, regenerative medicine, drug evaluation, drug discovery as well as precision medicine. This review is thus to comprehensively summarize and assess current progress in understanding mechanism of CR and its wide applications, highlighting the value of CR in both basic and translational researches and discussing the challenges faced with CR.