Omic Studies on In Vitro Cystinosis Model: siRNA-Mediated CTNS Gene Silencing in HK-2 Cells.

Cystinosis is an autosomal recessive disease caused by mutations in the CTNS gene encoding a protein called cystinosine, which is a lysosomal cystine transporter. Disease-causing mutations lead to accumulation of cystine crystals in the lysosomes, thereby causing dysfunction of vital organs. Determination of the increased leukocyte cystine level is one of the most used methods for diagnosis. However, this method is expensive, difficult to perform, and may yield different results in different laboratories. In this study, a disease model was created with CTNS gene-silenced HK2 cells, which can mimic cystinosis in cell culture, and multiomics methods (ie, proteomics, metabolomics, and fluxomics) were implemented at this cell culture to investigate new biomarkers for the diagnosis. CTNS-silenced cell line exhibited distinct metabolic profiles compared with the control cell line. Pathway analysis highlighted significant alterations in various metabolic pathways, including alanine, aspartate, and glutamate metabolism; glutathione metabolism; aminoacyl-tRNA biosynthesis; arginine and proline metabolism; beta-alanine metabolism; ascorbate and aldarate metabolism; and histidine metabolism upon CTNS silencing. Fluxomics analysis revealed increased cycle rates of Krebs cycle intermediates such as fumarate, malate, and citrate, accompanied by enhanced activation of inorganic phosphate and ATP production. Furthermore, proteomic analysis unveiled differential expression levels of key proteins involved in crucial cellular processes. Notably, peptidyl-prolyl cis-trans isomerase A, translation elongation factor 1-beta (EF-1beta), and 60S acidic ribosomal protein decreased in CTNS-silenced cells. Additionally, levels of P0 and tubulin α-1A chain were reduced, whereas levels of 40S ribosomal protein S8 and Midasin increased. Overall, our study, through the utilization of an in vitro cystinosis model and comprehensive multiomics approach, led to the way toward the identification of potential new biomarkers while offering valuable insights into the pathogenesis of cystinosis.

Manipulating macrophage polarization with nanoparticles to control metastatic behavior in heterotypic breast cancer micro-tissues via exosome signaling.

This study aimed to investigate the effects of nanoparticles on macrophage polarization and their subsequent influence on post-tumorigenic behavior. Initially, seven different nanoparticles were applied to macrophages, and Zn-Ni-FeO (100 nm) and palladium nanoparticles (PdNPs, ∼25 nm) were found to induce M1-polarization in macrophages. A co-culture experiment was then conducted to examine the effects of macrophages on MCF-7 breast cancer micro-tissues. The M2-macrophages promoted tumor proliferation, while M1- and PdNPs-induced macrophages showed anti-tumor effects by suppressing cell proliferation. To reveal the mechanisms of effect, exosomes isolated from M1 (M1-Exo), M0 (M0-Exo), M2 (M2-Exo), and PdNPs-induced (PdNPs-Exo) macrophages were applied to the heterotypic tumor micro-tissues including MCF-7, human umbilical vein endothelial cells (HUVECs), and primary human dermal fibroblasts (phDFs). M2-Exo was seen to promote the migration of cancer cells and induce epithelial-mesenchymal transition (EMT), while M1-Exo suppressed these behaviors. PdNPs-Exo was effective in suppressing the aggressive nature of breast cancer cells similar to M1-Exo, moreover, the efficacy of 5-fluorouracil (5-FU) was increased in combination with PdNPs-Exo in both MCF-7 and heterotypic micro-tissues. In conclusion, PdNPs-Exo has potential anti-tumor effects, can be used as a combination therapy to enhance the efficacy of anti-cancer drugs, as well as innovative implants for breast cancer treatment.

Bioactive and chemically defined hydrogels with tunable stiffness guide cerebral organoid formation and modulate multi-omics plasticity in cerebral organoids.

Organoids are an emerging technology with great potential in human disease modelling, drug development, diagnosis, tissue engineering, and regenerative medicine. Organoids as 3D-tissue culture systems have gained special attention in the past decades due to their ability to faithfully recapitulate the complexity of organ-specific tissues. Despite considerable successes in culturing physiologically relevant organoids, their real-life applications are currently limited by challenges such as scarcity of an appropriate biomimetic matrix. Peptide amphiphiles (PAs) due to their well-defined chemistry, tunable bioactivity, and extracellular matrix (ECM)-like nanofibrous architecture represent an attractive material scaffold for organoids development. Using cerebral organoids (COs) as exemplar, we demonstrate the possibility to create bio-instructive hydrogels with tunable stiffness ranging from 0.69 kPa to 2.24 kPa to culture and induce COs growth. We used orthogonal chemistry involving oxidative coupling and supramolecular interactions to create two-component hydrogels integrating the bio-instructive activity and ECM-like nanofibrous architecture of a laminin-mimetic PAs (IKVAV-PA) and tunable crosslinking density of hyaluronic acid functionalized with tyramine (HA-Try). Multi-omics technology including transcriptomics, proteomics, and metabolomics reveals the induction and growth of COs in soft HA-Tyr hydrogels containing PA-IKVAV such that the COs display morphology and biomolecular signatures similar to those grown in Matrigel scaffolds. Our materials hold great promise as a safe synthetic ECM for COs induction and growth. Our approach represents a well-defined alternative to animal-derived matrices for the culture of COs and might expand the applicability of organoids in basic and clinical research. STATEMENT OF SIGNIFICANCE: Synthetic bio-instructive materials which display tissue-specific functionality and nanoscale architecture of the native extracellular matrix are attractive matrices for organoids development. These synthetic matrices are chemically defined and animal-free compared to current gold standard matrices such as Matrigel. Here, we developed hydrogel matrices with tunable stiffness, which incorporate laminin-mimetic peptide amphiphiles to grow and expand cerebral organoids. Using multi-omics tools, the present study provides exciting data on the effects of neuro-inductive cues on the biomolecular profiles of brain organoids.

Molecular docking, synthesis, anticancer activity, and metabolomics study of boronic acid ester-containing fingolimod derivatives.

In recent years, drugs that contain boronic acid groups, such as ixazomib (Ninlaro™) and bortezomib (Velcade™), have been used in the treatment of bone marrow cancer. The activity of compounds has been found to increase with the addition of boron atoms to the structure. In addition to these compounds, studies have found that fingolimod (FTY720) is more effective against breast cancer than cisplatin. Therefore, in this study, the first examples of boron-containing derivatives of fingolimod were designed and synthesized; in addition, their structures were confirmed by spectroscopic techniques. The synthesized boron-containing drug candidates were found to significantly inhibit cell proliferation and induce apoptosis-mediated cell death in HT-29 (colorectal cells), SaOs-2 (osteosarcoma cells), and U87-MG (glioblastoma cells). Moreover, we revealed that the anticancer effects of boron-containing fingolimod compounds were found to be significantly enhanced over boron-free control groups and, strikingly, over the widely used anticancer drug 5-fluorouracil. The metabolomic analysis confirmed that administration of the boron-containing drug candidates induces significant changes in the metabolite profiles in HT-29, SaOs-2, and U87-MG cells. Altogether, our results showed that boron-containing fingolimod compounds can be further examined to reveal their potential as anticancer drug candidates.

The effect of mitochondria-targeted slow hydrogen sulfide releasing donor AP39-treatment on airway inflammation.

Mitochondrial dysfunction has been shown to contribute to the pathophysiology of airway diseases. Therefore, mitochondria are targeted in the development of new therapeutic approaches. Hydrogen sulfide (H2S) has been shown to be involved in the pathophysiological processes of airway inflammation. We aimed to evaluate the effect of mitochondria-targeted slow H2S releasing donor AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl)triphenylphosphoniumbromide)] on lipopolysaccharide (LPS)-induced airway inflammation in mice. LPS was applied to female Balb/c mice by intranasal (i.n.) route to induce airway inflammation and the subgroups of mice were treated with i.n. AP39 (250-1000 nmol/kg). 48 h after LPS administration airway reactivity was evaluated in vivo, then bronchoalveolar lavage (BAL) fluid and lungs were collected. LPS application led to bronchial hyperreactivity and neutrophil infiltration into the lung tissues along with increased TNF-α, IL-1ß and IL-6 levels in BAL fluid. LPS also induced an increase in the rate of glycolysis, glycogenolysis and Krebs-cycle. AP39 treatment prevented the LPS-induced bronchial hyperreactivity and reversed the increase in TNF-α and IL-6 levels in BAL fluid. The increase in neutrophil numbers in BAL fluid was also prevented by AP39 treatment at the highest dose. Our results indicate that AP39 can prevent bronchial hyperreactivity and decrease airway inflammation. Targeting H2S to the mitochondria may be a new therapeutic approach in airway inflammation.

Metabolomic Analyses to Identify Candidate Biomarkers of Cystinosis.

Cystinosis is a rare, devastating hereditary disease secondary to recessive CTNS gene mutations. The most commonly used diagnostic method is confirmation of an elevated leukocyte cystine level; however, this method is expensive and difficult to perform. This study aimed to identify candidate biomarkers for the diagnosis and follow-up of cystinosis based on multiomics studies. The study included three groups: newly-diagnosed cystinosis patients (patient group, n = 14); cystinosis patients under treatment (treatment group, n = 19); and healthy controls (control group, n = 30). Plasma metabolomics analysis identified 10 metabolites as candidate biomarkers that differed between the patient and control groups [L-serine, taurine, lyxose, 4-trimethylammoniobutanoic acid, orotic acid, glutathione, PE(O-18:1(9Z)/0:0), 2-hydroxyphenyl acetic acid, acetyl-N-formil-5-metoxikinuramine, 3-indoxyl sulphate]. As compared to the healthy control group, in the treatment group, hypotaurine, phosphatidylethanolamine, N-acetyl-d-mannosamine, 3-indolacetic acid, p-cresol, phenylethylamine, 5-aminovaleric acid, glycine, creatinine, and saccharic acid levels were significantly higher, and the metabolites quinic acid, capric acid, lenticin, xanthotoxin, glucose-6-phosphate, taurine, uric acid, glyceric acid, alpha-D-glucosamine phosphate, and serine levels were significantly lower. Urinary metabolomic analysis clearly differentiated the patient group from the control group by means of higher allo-inositol, talose, glucose, 2-hydroxybutiric acid, cystine, pyruvic acid, valine, and phenylalanine levels, and lower metabolite (N-acetyl-L-glutamic acid, 3-aminopropionitrile, ribitol, hydroquinone, glucuronic acid, 3-phosphoglycerate, xanthine, creatinine, and 5-aminovaleric acid) levels in the patient group. Urine metabolites were also found to be significantly different in the treatment group than in the control group. Thus, this study identified candidate biomarkers that could be used for the diagnosis and follow-up of cystinosis.

Tuning the Cell-Adhesive Properties of Two-Component Hybrid Hydrogels to Modulate Cancer Cell Behavior, Metastasis, and Death Pathways.

This work presents a polysaccharide and protein-based two-component hybrid hydrogel integrating the cell-adhesive gelatin-tyramine (G-Tyr) and nonadhesive hyaluronic acid-tyramine (HA-Tyr) through enzyme-mediated oxidative coupling reaction. The resulting HA-Tyr/G-Tyr hydrogel reflects the precise chemical and mechanical features of the cancer extracellular matrix and is able to tune cancer cell adhesion upon switching the component ratio. The cells form quasi-spheroids on HA-Tyr rich hydrogels, while they tend to form an invasive monolayer culture on G-Tyr rich hydrogels. The metastatic genotype of colorectal adenocarcinoma cells (HT-29) increases on G-Tyr rich hydrogels which is driven by the material's adhesive property, and additionally confirmed by the suppressed gene expressions of apoptosis and autophagy. On the other hand, HA-Tyr rich hydrogels lead the cells to necrotic death via oxidative stress in quasi-spheroids. This work demonstrates the ideality of HA-Tyr/G-Tyr to modulate cancer cell adhesion, which also has potential in preventing primary metastasis after onco-surgery, biomaterials-based cancer research, and drug testing.

Liquid biopsy markers for early diagnosis of brain metastasis patients with breast cancer by metabolomics.

Introduction: Breast cancer is the most common cancer in women and is the second most common cause of cancer related mortality. Metabolomics, the identification of small metabolites, is a technique for determining the amount of these metabolites. Objectives: This study aimed to identify markers for the early diagnosis of brain metastasis by metabolomic methods in breast cancer patients. Methods: A total of 88 breast cancer patients with distant metastases were included in the study. The patients were divided into two groups according to their metastasis status: patients with brain metastases and distant metastases without any brain metastases. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qTOF-MS) and gas chromatography-mass spectrometry (GC-MS) analysis methods were used for metabolomic analyses. Results: 33 of them, 88 patients had brain metastasis, and 55 patients had distant metastases without brain metastasis. A total of 72 and 35 metabolites were identified by the GC-MS and LC-qTOF-MS analysis, respectively. 47 of them were found to be significantly different in patients with brain metastasis. The pathway analysis, performed with significantly altered metabolites, showed that aminoacyl tRNA biosynthesis, valine, leucine and isoleucine biosynthesis, alanine, aspartate, and glutamate metabolism, arginine biosynthesis, glycine, serine, and threonine metabolism pathways significantly altered in patients with brain metastasis. Predictive accuracies for have identifying the brain metastasis were performed with receiver operating characteristic (ROC) analysis, and the model with fifteen metabolites has 96.9% accuracy. Conclusions: While these results should be supported by prospective studies, these data are promising for early detection of brain metastasis with markers in liquid biopsy samples.

Xenogenic Neural Stem Cell-Derived Extracellular Nanovesicles Modulate Human Mesenchymal Stem Cell Fate and Reconstruct Metabolomic Structure.

Extracellular nanovesicles, particularly exosomes, can deliver their diverse bioactive biomolecular content, including miRNAs, proteins, and lipids, thus providing a context for investigating the capability of exosomes to induce stem cells toward lineage-specific cells and tissue regeneration. In this study, it is demonstrated that rat subventricular zone neural stem cell-derived exosomes (rSVZ-NSCExo) can control neural-lineage specification of human mesenchymal stem cells (hMSCs). Microarray analysis shows that the miRNA content of rSVZ-NSCExo is a faithful representation of rSVZ tissue. Through immunocytochemistry, gene expression, and multi-omics analyses, the capability to use rSVZ-NSCExo to induce hMSCs into a neuroglial or neural stem cell phenotype and genotype in a temporal and dose-dependent manner via multiple signaling pathways is demonstrated. The current study presents a new and innovative strategy to modulate hMSCs fate by harnessing the molecular content of exosomes, thus suggesting future opportunities for rSVZ-NSCExo in nerve tissue regeneration.

Mechanically robust hybrid hydrogels of photo-crosslinkable gelatin and laminin-mimetic peptide amphiphiles for neural induction.

Self-assembling bio-instructive materials that can provide a biomimetic tissue microenvironment with the capability to regulate cellular behaviors represent an attractive platform in regenerative medicine. Herein, we develop a hybrid neuro-instructive hydrogel that combines the properties of a photo-crosslinkable gelatin methacrylate (GelMA) and self-assembling peptide amphiphiles (PAs) bearing a laminin-derived neuro-inductive epitope (PA-GSR). Electrostatic interaction and ultraviolet light crosslinking mechanisms were combined to create dual-crosslinked hybrid hydrogels with tunable stiffness. Spectroscopic, microscopic and theoretical techniques show that the cationic PA-GSR(+) electrostatically co-assembles with the negatively charged GelMA to create weak hydrogels with hierarchically ordered microstructures, which were further photo-crosslinked to create mechanically robust hydrogels. Dynamic oscillatory rheology and micromechanical testing show that photo-crosslinking of the co-assembled GelMA and PA-GSR(+) hydrogel results in robust hydrogels displaying improved stiffness. Gene expression analysis was used to show that GelMA/PA-GSR(+) hydrogels can induce human mesenchymal stem cells (hMSCs) into neural-lineage cells and supports neural-lineage specification of neuroblast-like cells (SH-SY5Y) in a growth-factor-free manner. Also, metabolomics analysis suggests that the hydrogel alters the metabolite profiles in the cells by affecting multiple molecular pathways. This work highlights a new approach for the design of PA-based hybrid hydrogels with robust mechanical properties and biological functionalities for nerve tissue regeneration.

Decellularized spinal cord meninges extracellular matrix hydrogel that supports neurogenic differentiation and vascular structure formation.

Decellularization of extracellular matrices offers an alternative source of regenerative biomaterials that preserve biochemical structure and matrix components of native tissues. In this study, decellularized bovine spinal cord meninges (dSCM)-derived extracellular matrix hydrogel (MeninGEL) is fabricated by employing a protocol that involves physical, chemical, and enzymatic processing of spinal meninges tissue and preserves the biochemical structure of meninges. The success of decellularization is characterized by measuring the contents of residual DNA, glycosaminoglycans, and hydroxyproline, while a proteomics analysis is applied to reveal the composition of MeninGEL. Frequency and temperature sweep rheometry show that dSCM forms self-supporting hydrogel at physiological temperature. The MeninGEL possesses excellent cytocompatibility. Moreover, it is evidenced with immuno/histochemistry and gene expression studies that the hydrogel induces growth-factor free differentiation of human mesenchymal stem cells into neural-lineage cells. Furthermore, MeninGEL instructs human umbilical vein endothelial cells to form vascular branching. With its innate bioactivity and low batch-to-batch variation property, the MeninGEL has the potential to be an off-the-shelf product in nerve tissue regeneration and restoration.

Gas chromatography-mass spectrometry based 18O stable isotope labeling of Krebs cycle intermediates.

New technologies permit determining metabolomic profiles of human diseases by fingerprinting metabolites levels. However, to fully understand metabolomic phenotypes, metabolite levels and turnover rates are necessary to know. Krebs cycle is the major hub of energy metabolism and cell signaling. Traditionally, 13C stable isotope labeled substrates were used to track the carbon turnover rates in Krebs cycle metabolites. In this study, for the first time we introduce H2[18O] based stable isotope marker that permit tracking oxygen exchange rates in separate segments of Krebs cycle. The chromatographic and non-chromatographic parameters were systematically tested on the effect of labeling ratio of Krebs cycle mediators to increase selectivity and sensitivity of the method. We have developed a rapid, precise, and robust GC-MS method for determining the percentage of 18O incorporation to Krebs cycle metabolites. The developed method was applied to track the cancer-induced shift in the Krebs cycle dynamics of Caco-2 cells as compared to the control FHC cells revealing Warburg effects in Caco-2 cells. We demonstrate that unique information could be obtained using this newly developed 18O-labeling analytical technology by following the oxygen exchange rates of Krebs cycle metabolites. Thus, 18O-labeling of Krebs cycle metabolites expands the arsenal of techniques for monitoring the dynamics of cellular metabolism. Moreover, the developed method will allow to apply the 18O-labeling technique to numerous other metabolic pathways where oxygen exchange with water takes place.

Neuroactivity of the naturally occurring aporphine alkaloid, roemerine.

Roemerine is a naturally occurring aporphine alkaloid. In this study, we screened a conformer library of Food and Drug Administration (FDA)-approved drugs to identify similar drugs that can assist in identifying the biological targets of roemerine. To assess the neuroactivity in vitro, we measured the levels of cell metabolites, Brain-Derived Neurotrophic Factor (BDNF) and serotonin (5-HT) in SH-SY5Y cell line. By means of structure-based virtual screening, we identified five drugs that are similar to roemerine; mirtazapine, atomoxetine, epinastine, diphenhydramine and orphenadrine. GC-MS metabolomics study revealed that roemerine has a high impact on alanine-aspartate-glutamate pathway in cell lysate and cultured medium. Additionally, roemerine increased intercellular 5-HT level and intracellular BDNF protein expression at 10 µM. In conclusion, roemerine - a major alkaloid in antidepressant-like effect possessing plants (P. lacerum and P. syriacum) - has a neuronal activity through increasing BDNF protein expression and affecting serotonergic and glutamatergic systems in SH-SY5Y cell line.

Untargeted multi-omic analysis of colorectal cancer-specific exosomes reveals joint pathways of colorectal cancer in both clinical samples and cell culture.

Exosomes are naturally secreted nano-vesicles consisting of biochemical molecules including RNAs, metabolites, lipids, and proteins, that emerge as diagnostic tools and disease-specific reporters. Here we offer a systematic and integrative approach for the simultaneous analysis of altered molecules namely metabolites, lipids, and proteins. These components tend to augment the discovery of low abundance signature components, and assist in explanation of molecular basis of colorectal cancer (CRC). In order to investigate CRC-derived exosomes, we selected mi-R19a, miR-21, miR-92a, and miR-1246 positive exosomes for downstream experiments. The overall multi-omic changes were investigated comparatively in cell culture and serum samples. Following a systematic multi-omic study, 37 (cell culture) and 31 (serum) metabolites; 130 (cell culture) and 56 (serum) lipids; 9 (cell culture) and 13 (serum) proteins were seen to be differentially expressed (p < 0.05), enabling discrimination between CRC and control. By using these enriched components, we demonstrated that the joint pathways mainly involving fatty acid and amino acid metabolism related pathways changed in CRC significantly. We conclude that this study increases our understanding of molecular basis of CRC, and provides potential exosomal biomarkers for the non-invasive detection, and discrimination of CRC.