Macromolecules with predominant ß-pleated sheet proteins in extracellular vesicles released from Raphanus sativus L. var. caudatus Alef microgreens induce DNA damage-mediated apoptosis in HCT116 colon cancer cells.

Plant-derived bioactive macromolecules (i.e., proteins, lipids, and nucleic acids) were prepared as extracellular vesicles (EVs). Plant-derived EVs are gaining pharmaceutical research interest because of their bioactive components and delivery properties. The spherical nanosized EVs derived from Raphanus sativus L. var. caudatus Alef microgreens previously showed antiproliferative activity in HCT116 colon cancer cells from macromolecular compositions (predominantly proteins). To understand the mechanism of action, the biological activity studies, i.e., antiproliferation, cellular biochemical changes, DNA conformational changes, DNA damage, apoptotic nuclear morphological changes, apoptosis induction, and apoptotic pathways, were determined by neutral red uptake assay, synchrotron radiation-based Fourier transform infrared microspectroscopy, circular dichroism spectroscopy, comet assay, 4',6-diamidino-2-phenylindole (DAPI) staining, flow cytometry, and caspase activity assay, respectively. EVs inhibited HCT116 cell growth in concentration- and time-dependent manners, with a half-maximal inhibitory concentration of 675.4 ± 33.8 µg/ml at 48 h and a selectivity index of 1.5 ± 0.076. HCT116 treated with EVs mainly changed the cellular biochemical compositions in the nucleic acids and carbohydrates region. The DNA damage caused no changes in DNA conformation. The apoptotic nuclear morphological changes were associated with the increased apoptotic cell population. The apoptotic cell death was induced by both extrinsic and intrinsic pathways. EVs have potential as antiproliferative bioparticles.

Development of a machine learning model for systematics of Aspergillus section Nigri using synchrotron radiation-based fourier transform infrared spectroscopy.

Aspergillus section Nigri (black aspergilli) fungi are economically important food spoilage agents. Some species in this section also produce harmful mycotoxins in food. However, it is remarkably difficult to identify this fungal group at the species level using morphological and chemical characteristics. The molecular approach for classification is preferable; however, it is time-consuming, making it inappropriate for rapid testing of large numbers of samples. To address this, we explored synchrotron radiation-based Fourier transform infrared microspectroscopy (SR-FTIR) as a rapid method for obtaining data suitable for species classification. SR-FTIR data were obtained from the mycelia/conidia of 22 black aspergilli species. The Convolutional Neural Network (CNN) approach, a supervised deep learning algorithm, was used with SR-FTIR data to classify black aspergilli at the species level. A subset of the data was used to train the CNN model, and the model classification performance was evaluated using the validation data subsets. The model demonstrated a 95.97% accuracy in species classification on the testing (blind) data subset. The technique presented herein could be an alternative method for identifying problematic black aspergilli in the food industry.

Effect of ultrasound and thermal pretreatments on antioxidant activity of egg white hydrolysate.

The use of ultrasonic (US) treatment of egg white prior to enzymatic hydrolysis to produce hydrolysate with antioxidant activity was investigated. The state of egg white (raw vs. cooked form) along with two levels of Alcalase (1% and 10% (w/w) protein) was applied. Hydrolysis and antioxidant activity of hydrolysate increased by US pretreatment at intensity of 41.53 W/cm2 . The hydrolysate prepared from US treatment on raw egg white hydrolyzed by 1% Alcalase (US-R1%) showed the lowest degree of hydrolysis (DH); however, its 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging and ferric reducing antioxidant power activities were the highest. In contrast, the highest cytoprotective effect and intracellular reactive oxygen species scavenging activity were more notable in the hydrolysate prepared from US treatment of boiled egg white hydrolyzed by 10% Alcalase (US-B10%), which also exhibited the highest DH and metal chelation ability. The hydrolysate possessing cellular antioxidant activity (CAA) showed the highest proportion of small molecular weight peptides (<200 Da). Fourier-transform infrared spectroscopy revealed an increase of N- and C-terminal ends at 1500 and 1400 cm-1 , respectively, in concomitant with a decrease of amide I. Principal component analysis showed clear differentiation of spectra from different levels of enzyme according to their DH, C-terminal ends, and antioxidant activity. Our findings suggested that cooked egg white followed by US pretreatment was beneficial to produce hydrolysate containing high CAA.

Applying synchrotron radiation-based attenuated total reflection-fourier transform infrared to chemically characterise organic functional groups in terrestrial soils of King George Island, Antarctica.

Anthropogenic activities, especially associated with fossil fuel combustion, are raising concerns worldwide, but remote areas with extreme climate conditions, such as Antarctica, are isolated from the adverse influence of human civilisation. Antarctica is considered as the most untouched place on Earth. Such pristine areas, which have extremely low chemical pollutant concentrations owing to restricted anthropogenic impacts, exemplify plausible model environments to test the reliability and sensitivity of advanced analytical techniques employed to chemically characterise and evaluate the spatial distribution of chemical pollutants. Here, synchrotron radiation-based attenuated total reflection-Fourier transform infrared (SR-ATR-FTIR) spectroscopy was employed to evaluate the variations in the organic functional groups (OFGs) of terrestrial soils of King George Island, Antarctica. Second-derivative SR-ATR-FTIR spectroscopy coupled with several multivariate statistical techniques highlighted the influence of anthropogenic activities on the alterations of OFGs in terrestrial soils collected near airports. Moreover, the daily activities of penguins could also have caused fluctuations in some OFGs of the samples the close to the Tombolo area and Ardley Island. The findings proved the effectiveness of SR-ATR-FTIR in evaluating the potential sources of variations in the chemical constituents, especially OFGs, in Antarctic terrestrial soils.

Isolation, identification, and mode of action of antibacterial peptides derived from egg yolk hydrolysate.

Egg yolk is a coproduct of egg white processing. The protein hydrolysis of egg yolks to exhibit antimicrobial activity is a strategy for its valorization. The objective of this study is to fractionate antibacterial peptides from pepsin-hydrolyzed egg yolks using flash chromatography. In addition, the mode of actions of the fractionated peptides were elucidated and plausible antibacterial peptides were reported. The fraction 6 (F6) obtained from a C18-flash column exhibited antibacterial activity against Staphylococcus aureus ATCC 29213 and Salmonella typhimurium TISTR 292 at minimal inhibitory concentration (MIC) values of 0.5 to 1 mmol/L (Leucine equivalent). The fractionated peptides induced DNA leakage as monitored by 260 nm. Propidium iodide and SYTO9 staining observed under a confocal microscope suggested the disintegration of cell membranes. Synchrotron-based Fourier-transform infrared spectroscopy analysis revealed that the egg yolk peptides at 1 × MIC induced an alteration of phospholipids at cell membranes and modified conformation of intracellular proteins and nucleic acids. Scanning electron microscopy revealed obvious cell ruptures when S. aureus was treated at 1 × MIC for 4 h, whereas damage of cell membranes and leakage of intracellular components were also observed for the transmission electron microscopy. Egg yolk peptides showed no hemolytic activity in human erythrocytes at concentrations up to 4 mmol/L. Peptide identification by LC-MS/MS revealed 3 cationic and 10 anionic peptides with 100% sequence similarity to apolipoprotein-B of Gallus gallus with hydrophobicity ranging from 27 to 75%. The identified peptide KGGDLGLFEPTL exhibited the highest antibacterial activity toward S. aureus at MIC of 2 mmol/L. Peptides derived from egg yolk hydrolysate present significant potential as antistaphylococcal agents for food and/or pharmaceutical application.

Investigating the Effect of Various Sous-Vide Cooking Conditions on Protein Structure and Texture Characteristics of Tilapia Fillet Using Synchrotron Radiation-Based FTIR.

The effects of various sous-vide (SV) cooking conditions (50-60℃, 30-60 min) on physicochemical properties related to the texture characteristics, protein structure/degradation, and sensory acceptability of tilapia fillet (Oreochromis niloticus) were investigated. With an increasing temperature and processing time of SV cooking, protein degradation (of both myofibrils and connective tissue) was more pronounced, as evaluated by the decrease in water- and salt-soluble proteins, total collagen, as well as the changes in the ratio of secondary protein structures (α-helix, ß-sheet, ß-turn, etc.), which were determined by synchrotron-FTIR (SR-FTIR). These degradations were associated with the improvement of meat tenderness, as estimated by shear force and texture profile analyzer (TPA) results. Among all SV conditions, using 60 ℃ for 45 min seems to be the optimal condition for tilapia meat, since it delivered the best results for texture characteristics and acceptability (p < 0.05). Moreover, principal component analysis (PCA) results clearly demonstrated that the highest texture-liking score of this condition was well associated with the intensity of ß-sheets, which seem to be the crucial component that affected the texture of SV-cooked tilapia more so than other parameters. The findings demonstrated the potential of SR-FTIR to decipher the biomolecular structure, particularly the secondary protein structure, of SV-cooked tilapia. This technique provided essential information for a better understanding of the changes in biomolecules related to the textural characteristics of this product.

Performance Evaluation of Focal Plane Array (FPA)-FTIR and Synchrotron Radiation (SR)-FTIR Microspectroscopy to Classify Rice Components.

The development of biochemical analysis techniques to study heterogeneous biological samples is increasing. These techniques include synchrotron radiation Fourier transform infrared (SR-FTIR) microspectroscopy. This method has been applied to analyze biological tissue with multivariate statistical analysis to classify the components revealed by the spectral data. This study aims to compare the efficiencies of SR-FTIR microspectroscopy and focal plane array (FPA)-FTIR microspectroscopy when classifying rice tissue components. Spectral data were acquired for mapping the same sample areas from both techniques. Principal component analysis and cluster imaging were used to investigate the biochemical variations of the tissue types. The classification was based on the functional groups of pectin, protein, and polysaccharide. Four layers from SR-FTIR microspectroscopy including pericarp, aleurone layer, sub-aleurone layer, and endosperm were classified using cluster imaging, while FPA-FTIR microspectroscopy could classify only three layers of pericarp, aleurone layer, and endosperm. Moreover, SR-FTIR microspectroscopy increased the image contrast of the biochemical distribution in rice tissue more efficiently than FPA-FTIR microspectroscopy. We have demonstrated the capability of the high-resolution synchrotron technique and its ability to clarify small structures in rice tissue. The use of this technique might increase in future studies of tissue characterization.

Extracellular vesicles derived from microgreens of Raphanus sativus L. var. caudatus Alef contain bioactive macromolecules and inhibit HCT116 cells proliferation.

Extracellular vesicles (EVs) are phospholipid bilayer vesicles released from cells, containing natural cargos. Microgreens of Raphanus sativus L. var. caudatus Alef were used in this study as the source of EVs. EVs were isolated by differential centrifugation. The physical properties were determined by dynamic light scattering (DLS) and electron microscopy. The biological and chemical composition were studied by Fourier-transform infrared (FTIR) microspectroscopy and high-performance liquid chromatography analysis, respectively. EVs had a median size of 227.17 and 234.90 ± 23.30 nm determined by electron microscopy and DLS, respectively with a polydispersity index of 0.293 ± 0.019. Electron microscopy indicated the intact morphology and confirmed the size. The FTIR spectra revealed that EVs are composed of proteins as the most abundant macromolecules. Using a curve-fitting analysis, ß-pleated sheets were the predominant secondary structure. Notably, the micromolecular biomarkers were not detected. EVs exerted anti-cancer activity on HCT116 colon cancer over Vero normal cells with an IC50 of 448.98 µg/ml and a selectivity index of > 2.23. To conclude, EVs could be successfully prepared with a simple and effective isolation method to contain nano-sized macromolecules possessing anti-cancer activity.

Metabolic sensor O-GlcNAcylation regulates erythroid differentiation and globin production via BCL11A.

BACKGROUND: Human erythropoiesis is a tightly regulated, multistep process encompassing the differentiation of hematopoietic stem cells (HSCs) toward mature erythrocytes. Cellular metabolism is an important regulator of cell fate determination during the differentiation of HSCs. However, how O-GlcNAcylation, a posttranslational modification of proteins that is an ideal metabolic sensor, contributes to the commitment of HSCs to the erythroid lineage and to the terminal erythroid differentiation has not been addressed. METHODS: Cellular O-GlcNAcylation was manipulated using small molecule inhibition or CRISPR/Cas9 manipulation of catalyzing enzyme O-GlcNAc transferase (OGT) and removing enzyme O-GlcNAcase (OGA) in two cell models of erythroid differentiation, starting from: (i) human umbilical cord blood-derived CD34+ hematopoietic stem/progenitor cells (HSPCs) to investigate the erythroid lineage specification and differentiation; and (ii) human-derived erythroblastic leukemia K562 cells to investigate the terminal differentiation. The functional and regulatory roles of O-GlcNAcylation in erythroid differentiation, maturation, and globin production were investigated, and downstream signaling was delineated. RESULTS: First, we observed that two-step inhibition of OGT and OGA, which were established from the observed dynamics of O-GlcNAc level along the course of differentiation, promotes HSPCs toward erythroid differentiation and enucleation, in agreement with an upregulation of a multitude of erythroid-associated genes. Further studies in the efficient K562 model of erythroid differentiation confirmed that OGA inhibition and subsequent hyper-O-GlcNAcylation enhance terminal erythroid differentiation and affect globin production. Mechanistically, we found that BCL11A is a key mediator of O-GlcNAc-driven erythroid differentiation and ß- and α-globin production herein. Additionally, analysis of biochemical contents using synchrotron-based Fourier transform infrared (FTIR) spectroscopy showed unique metabolic fingerprints upon OGA inhibition during erythroid differentiation, supporting that metabolic reprogramming plays a part in this process. CONCLUSIONS: The evidence presented here demonstrated the novel regulatory role of O-GlcNAc/BCL11A axis in erythroid differentiation, maturation, and globin production that could be important in understanding erythropoiesis and hematologic disorders whose etiology is related to impaired erythroid differentiation and hemoglobinopathies. Our findings may lay the groundwork for future clinical applications toward an ex vivo production of functional human reticulocytes for transfusion from renewable cell sources, i.e., HSPCs and pluripotent stem cells.

The significant influence of residual feed intake on flavor precursors and biomolecules in slow-growing Korat chicken meat.

OBJECTIVE: This study investigated the association between feed efficiency, physicochemical properties, flavor precursors and biomolecules in the thigh meat of Korat (KR) chickens. METHODS: The feed intake and body weight of individual male KR chickens were recorded from 1 to 10 weeks old to calculate the individual residual feed intake (RFI) of 75 birds. At 10 weeks of age, chickens with the 10 highest (HRFI) and lowest RFI (LRFI) were slaughtered to provide thigh meat samples. The physicochemical properties (ultimate pH, water holding capacity [WHC], drip loss) and flavor precursors (guanosine monophosphate, inosine monophosphate (IMP), adenosine monophosphate and inosine) were analyzed conventionally, and Fourier transform infrared spectroscopy was used to identify the composition of biomolecules (lipids, ester lipids, amide I, amide II, amide III, and carbohydrates) and the secondary structure of the proteins. A group t-test was used to determine significant differences between mean values and principal component analysis to classify thigh meat samples into LRFI and HRFI KR chickens. RESULTS: The physicochemical properties of thigh meat samples from LRFI and HRFI KR chickens were not significantly different but the IMP content, ratios of lipid, lipid ester, protein (amide I, amide II) were significantly different (p<0.05). The correlation loading results showed that the LRFI group was correlated with high ratios of lipids, lipid esters, collagen content (amide III) and beta sheet protein (rg loading >0.5) while the HRFI group was positively correlated with protein (amide I, amide II), alpha helix protein, IMP content, carbohydrate, ultimate pH and WHC (rg loading >0.5). CONCLUSION: The thigh meat from chickens with different RFI differed in physiochemical properties affecting meat texture, and in the contents of flavor precursors and biomolecules affecting the nutritional value of meat. This information can help animal breeders to make genetic improvements by taking more account of traits related to RFI.

Gymnema inodorum (Lour.) Decne. Extract Alleviates Oxidative Stress and Inflammatory Mediators Produced by RAW264.7 Macrophages.

Gymnema inodorum (Lour.) Decne. (G. inodorum) is widely used in Northern Thai cuisine as local vegetables and commercial herb tea products. In the present study, G. inodorum extract (GIE) was evaluated for its antioxidant and anti-inflammatory effects in LPS plus IFN-γ-induced RAW264.7 cells. Major compounds in GIE were evaluated using GC-MS and found 16 volatile compounds presenting in the extract. GIE exhibited antioxidant activity by scavenging the intracellular reactive oxygen species (ROS) production and increasing superoxide dismutase 2 (SOD2) mRNA expression in LPS plus IFN-γ-induced RAW264.7 cells. GIE showed anti-inflammatory activity through suppressing nitric oxide (NO), proinflammatory cytokine production interleukin 6 (IL-6) and also downregulation of the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and IL-6 mRNA levels in LPS plus IFN-γ-induced RAW264.7 cells. Mechanism studies showed that GIE suppressed the NF-κB p65 nuclear translocation and slightly decreased the phosphorylation of NF-κB p65 (p-NF-κB p65) protein. Our studies applied the synchrotron radiation-based FTIR microspectroscopy (SR-FTIR), supported by multivariate analysis, to identify the FTIR spectral changes based on macromolecule alterations occurring in RAW264.7 cells. SR-FTIR results demonstrated that the presence of LPS plus IFN-γ in RAW264.7 cells associated with the increase of amide I/amide II ratio (contributing to the alteration of secondary protein structure) and lipid content, whereas glycogen and other carbohydrate content were decreased. These findings lead us to believe that GIE may prevent oxidative damage by scavenging intracellular ROS production and activating the antioxidant gene, SOD2, expression. Therefore, it is possible that the antioxidant properties of GIE could modulate the inflammation process by regulating the ROS levels, which lead to the suppression of proinflammatory cytokines and genes. Therefore, GIE could be developed into a novel antioxidant and anti-inflammatory agent to treat and prevent diseases related to oxidative stress and inflammation.

Assessment of Antimicrobial Activity, Mode of Action and Volatile Compounds of Etlingera pavieana Essential Oil.

Etlingera pavieana (Pierre ex Gagnep.) R.M.S. is a rhizomatous plant in the Zingiberaceae family which could be freshly eaten, used as a condiment or as a traditional remedy. Our work investigated the chemical composition and antimicrobial activity of the E. pavieana essential oils extracted from the rhizome (EOEP). We extracted the EOEP from the rhizome by hydrodistillation and analyzed the chemical composition by headspace solid-phase microextraction coupled with gas chromatography/mass spectrometry (HS-SPME-GC/MS). A total of 22 volatile compounds were identified where trans-anethole (78.54%) and estragole (19.36%) were the major components in the EOEP. The antimicrobial activity of EOEP was evaluated based on the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) values using the broth dilution method and enumerating cell death overtime. Our work shows that the EOEP exhibits potent antibacterial activity against foodborne pathogenic Gram-positive bacteria, namely Bacillus cereus, Staphylococcus aureus and Listeria monocytogenes in the range of 0.1-0.3% (v/v). We further investigated the mechanism of EOEP inhibition using Synchrotron Fourier transformation infrared (FTIR) microspectroscopy. Here, we show significant differences in DNA/nucleic acid, proteins and cell membrane composition in the bacterial cell. To conclude, EOEP exhibited antimicrobial activity against foodborne pathogens, especially the Gram-positive bacteria associated with ready-to-eat (RTE) food and, thus, has the potential to serve as a natural preservative agent in RTE products.

Pupae protein extracts exert anticancer effects by downregulating the expression of IL-6, IL-1ß and TNF-α through biomolecular changes in human breast cancer cells.

The Pupae of Bombyx mori and Samia ricini are a source of high-quality proteins and essential nutrient elements for human. Recent studies revealed that protein extracted from pupae possessed therapeutic benefits for the treatment of many diseases. However, the anticancer activity of protein extracts from the pupae of B. mori and S. ricini has been rarely reported. Our objective was to study the effect of protein extracts from the pupae of B. mori and S. ricini on cytotoxicity and expression of pro-inflammatory cytokines; IL-6, IL-1ß and TNF-α, in breast cancer cells (MCF-7). Additionally, anticancer action of protein extracted from the pupae was further investigated through biomolecular changes in MCF-7 cells using Fourier transform infrared (FTIR) spectroscopy. Pupae protein extracts of B. mori exhibited cytotoxic effects with an IC50 value of 15.23 + 0.4 µg/mL with higher selectivity than doxorubicin on MCF-7 cells. Fourier transform infrared (FTIR) spectroscopy revealed that lipid contents in MCF-7 cells treated with pupae protein extracts of B. mori were higher than untreated cells. Treatment with protein extracts from pupae of B. mori or S. ricini caused significantly reduced protein and nucleic acid contents of MCF-7 cells. The expression of IL-6, IL-1ß and TNF-α in MCF-7 treated cells was investigated using RT-qPCR and ELISA. Our results revealed that protein extracts from the pupae of B. mori or S. ricini significantly decreased IL-6, IL-1ß and TNF-α in MCF-7 cells both at mRNA and protein levels. Expression of IL-6 and IL-1ß in MCF-7 treated cells, especially IL-6, was strongly reduced compared to untreated cells, while TNF-α expression was slightly decreased. These findings suggest that pupae protein extracted from B. mori or S. ricini may play a role in breast cancer through a down-regulatory action on the expression of IL-6, IL-1ß and TNF-α, and may also exert anticancer effects by causing biochemical changes of lipids, proteins and nucleic acids. These findings indicate that pupae protein extracted from B. mori or S. ricini may provide a potential novel therapeutic target for breast cancer.

Evaluation of Melanoma (SK-MEL-2) Cell Growth between Three-Dimensional (3D) and Two-Dimensional (2D) Cell Cultures with Fourier Transform Infrared (FTIR) Microspectroscopy.

Fourier transform infrared (FTIR) microspectroscopy was used to evaluate the growth of human melanoma cells (SK-MEL-2) in two-dimensional (2D) versus three-dimensional (3D) spheroid culture systems. FTIR microspectroscopy, coupled with multivariate analysis, could be used to monitor the variability of spheroid morphologies prepared from different cell densities. The characteristic shift in absorbance bands of the 2D cells were different from the spectra of cells from 3D spheroids. FTIR microspectroscopy can also be used to monitor cell death similar to fluorescence cell staining in 3D spheroids. A change in the secondary structure of protein was observed in cells from the 3D spheroid versus the 2D culture system. FTIR microspectroscopy can detect specific alterations in the biological components inside the spheroid, which cannot be detected using fluorescence cell death staining. In the cells from 3D spheroids, the respective lipid, DNA, and RNA region content represent specific markers directly proportional to the spheroid size and central area of necrotic cell death, which can be confirmed using unsupervised PCA and hierarchical cluster analysis. FTIR microspectroscopy could be used as an alternative tool for spheroid cell culture discrimination, and validation of the usual biochemical technique.

FTIR spectra signatures reveal different cellular effects of EGFR inhibitors on nonsmall cell lung cancer cells.

ATP-analogue inhibitors, Gefitinib (Iressa) and Erlotinib (Tarceva) had been approved for advanced and metastatic nonsmall cell lung cancer (NSCLC) cells against tyrosine kinase domain of epidermal growth factor receptor (EGFR). Many techniques have been developed to better understand the drug mechanism which is multistep, time-consuming and expensive. Herein, we performed Fourier-transform infrared (FTIR) microscopy for evaluating the biochemical change on NSCLC (A549) cells after treatment. At levels that produced equivalent effects, Gefitinib dramatically induced cell apoptosis via impaired mitochondrial transmembrane potential. Whereas, Erlotinib had a slight effect on A549. Principal component analysis was performed to distinguish the effect of EGFR inhibitors on A549. FTIR spectra regions were divided into three regions: lipids (3000-2800 cm-1 ), proteins (1700-1500 cm-1 ) and carbohydrates and nuclei acids (1200-1000 cm-1 ). Biochemical changes can be evaluated by these spectral regions. This work may be a novel concept for utilizing FTIR spectroscopy for high-throughput discriminative effects of a drug or compound and its derivatives on cells.

YAP as a key regulator of adipo-osteogenic differentiation in human MSCs.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent stem cells that are able to differentiate into several cell types, including cartilage, fat, and bone. As a common progenitor, MSC differentiation has to be tightly regulated to maintain the balance of their differentiation commitment. It has been reported that the decision process of MSCs into fat and bone cells is competing and reciprocal. Several factors have been suggested as critical factors that affect adipo-osteogenic decision, including melatonin and smad4. Yes-associated protein (YAP) is an important effector protein in the Hippo signaling pathway that acts as a transcriptional regulator by activating the transcription of the genes involved in cell proliferation and anti-apoptosis. The non-canonical role of YAP in regulating bone homeostasis by promoting osteogenesis and suppressing adipogenesis was recently demonstrated in a mouse model. However, it is unclear whether YAP is also crucial for modulating human MSC differentiation to fat and bone. METHODS: The expression level of YAP during MSC differentiation was modulated using pharmaceutical molecule and genetic experiments through gain- and loss-of-function approaches. RESULTS: We demonstrated for the first time that YAP has a non-canonical role in regulating the balance of adipo-osteogenic differentiation of human MSCs. The result from synchrotron radiation-based Fourier transform infrared (FTIR) microspectroscopy showed unique metabolic fingerprints generated from YAP-targeted differentiated cells that were clearly distinguished from non-manipulated control. CONCLUSIONS: These results, thus, identify YAP as an important effector protein that regulates human MSC differentiation to fat and bone and suggests the use of FTIR microspectroscopy as a promising technique in stem cell research.

Alyssin and Iberin in Cruciferous Vegetables Exert Anticancer Activity in HepG2 by Increasing Intracellular Reactive Oxygen Species and Tubulin Depolymerization.

To determine the chemopreventive potential of alyssin and iberin, the in vitro anticancer activities and molecular targets of isothiocyanates (ITCs) were measured and compared to sulforaphane in hepatocellular carcinoma cell HepG2. The SR-FTIR spectra observed a similar pattern vis-à-vis the biomolecular alteration amongst the ITCs-treated cells suggesting a similar mode of action. All of the ITCs in this study cause cancer cell death through both apoptosis and necrosis in concentration dependent manner (20-80 µM). We found no interactions of any of the ITCs studied with DNA. Notwithstanding, all of the ITCs studied increased intracellular reactive oxygen species (ROS) and suppressed tubulin polymerization, which led to cell-cycle arrest in the S and G2/M phase. Alyssin possessed the most potent anticancer ability; possibly due to its ability to increase intracellular ROS rather than tubulin depolymerization. Nevertheless, the structural influence of alkyl chain length on anticancer capabilities of ITCs remains inconclusive. The results of this study indicate an optional, potent ITC (viz., alyssin) because of its underlying mechanisms against hepatic cancer. As a consequence, further selection and development of effective chemotherapeutic ITCs is recommended.

Deciphering the Elevated Lipid via CD36 in Mantle Cell Lymphoma with Bortezomib Resistance Using Synchrotron-Based Fourier Transform Infrared Spectroscopy of Single Cells.

Despite overall progress in improving cancer treatments, the complete response of mantle cell lymphoma (MCL) is still limited due to the inevitable development of drug resistance. More than half of patients did not attain response to bortezomib (BTZ), the approved treatment for relapsed or refractory MCL. Understanding how MCL cells acquire BTZ resistance at the molecular level may be a key to the long-term management of MCL patients and new therapeutic strategies. We established a series of de novo BTZ-resistant human MCL-derived cells with approximately 15- to 60-fold less sensitivity than those of parental cells. Using gene expression profiling, we discovered that putative cancer-related genes involved in drug resistance and cell survival tested were mostly downregulated, likely due to global DNA hypermethylation. Significant information on dysregulated lipid metabolism was obtained from synchrotron-based Fourier transform infrared (FTIR) spectroscopy of single cells. We demonstrated for the first time an upregulation of CD36 in highly BTZ-resistant cells in accordance with an increase in their lipid accumulation. Ectopic expression of CD36 causes an increase in lipid droplets and renders BTZ resistance to various human MCL cells. By contrast, inhibition of CD36 by neutralizing antibody strongly enhances BTZ sensitivity, particularly in CD36-overexpressing cells and de novo BTZ-resistant cells. Together, our findings highlight the potential application of CD36 inhibition for BTZ sensitization and suggest the use of FTIR spectroscopy as a promising technique in cancer research.

Structures of isothiocyanates attributed to reactive oxygen species generation and microtubule depolymerization in HepG2 cells.

The structure of the isothiocyanates (ITCs)-erucin, sulforaphane, erysolin, sulforaphene, and phenethyl isothiocyanate-were assessed as well as their respective in vitro anticancer activity on the hepatocellular carcinoma cell line HepG2. All of these ITCs induced both apoptotic and necrotic cell death. FTIR analysis indicated that the ITCs caused changes in cellular components comparable to vinblastine. Despite no observable effect on DNA, the ITCs all induced generation of intracellular reactive oxygen species (ROS) and suppressed microtubule polymerization. The variation in sulfur oxidation states and the presence of an aromatic ring on the ITC side chain affected microtubule depolymerization and intracellular ROS generation, leading to apoptotic and necrotic cancer cell death. Knowing the influences of structural variations of the ITC side chain would be useful for selecting the more potent ITCs (i.e., erysolin) for the design and development of effective chemopreventive agents.

Partial least squares regression and fourier transform infrared (FTIR) microspectroscopy for prediction of resistance in hepatocellular carcinoma HepG2 cells.

We evaluated the feasibility of FTIR microspectroscopy combined with partial least squares regression (PLS-R) for determination of resistance in HepG2 cells. Cell viability testing was performed using neutral red assay for the concentration of cisplatin resulting in 50% antiproliferation (IC50). The resistance index (RI) is the ratio of the IC50 in resistant HepG2 cells vs. parental HepG2 cells. Principal component and unsupervised hierarchical cluster analyses were applied and a differentiation of samples of cells (parental, 1.8RI, 2.3RI, 3.0RI, and 3.5RI) was demonstrated (3000-2800cm-1 in the lipid and 1700-1500cm-1 in the protein regions. The FTIR spectra were preprocessed with several treatments to test the algorithm. PLS-R models were built using the 1170 spectra of the HepG2 cells. Cross-validation was used to evaluate prediction of the RI value using this model. PLS-R models-preprocessed with the second derivative FTIR spectra-yielded the best model (R2=0.99, RMSEE=0.095 and RPD=7.98). Most RI values were predicted with high accuracy (91-100%) such that the linear correlation between the actual and predicted RI values was nearly perfect (slope~1). FTIR microspectroscopy combined with chemometric analysis using PLS-R offers quick, accurate, and reliable quantitative analysis of HepG2 cell resistance.