Development of a new assay for quantification of parasite load of intracellular Leishmania sp. in macrophages using flow cytometry.

Finding novel methodologies that enhance the precision, agility, and standardization of drug discovery is crucial for studying leishmaniasis. The slide count is the technique most used to assess the leishmanicidal effect of a given drug in vitro. Despite being consolidated in the scientific environment, it presents several difficulties in its execution, assessment, and results. In addition to being laborious, this technique takes time, both for the preparation of the material for analysis and for the counting itself. Our research group suggests a fresh approach to address this requirement, which involves utilizing nuclear labeling with propidium iodide and flow cytometry to determine the quantity of Leishmania sp. parasites present in macrophages in vitro. Our results show that the fluorescence of infected samples increases as the infection rate increases. Using Pearson's Correlation analysis, it was possible to establish a correlation coefficient (Pearson r = 0.9473) that was strongly positive, linear, and directly proportional to the fluorescence and infection rate variables. Thus, it is possible to infer a mathematical equation through linear regression to estimate the number of parasites in each sample using the Relative Fluorescence Units (RFU) values. This new methodology opens space for the possibility of using this methodological resource in the in vitro quantification of Leishmania in macrophages.

Spectrofluorometric determination of ascorbic acid in the plasma matrix: Exploring correlation with autism spectrum disorder.

Ascorbic acid (Vitamin C) is crucial for bodily functions, including collagen synthesis, immune system support and antioxidant defense. Despite autism spectrum disorder's multifactorial nature involving genetic, environmental and neurological factors, robust evidence exploring the association between ascorbic acid and this disorder is notably lacking. This study introduces an innovative spectrofluorometric method to quantify ascorbic acid in the plasma of healthy children and those with autism spectrum disorder. The method relies on the interaction of ascorbic acid with the fluorescent dye propidium iodide. In acidic conditions, propidium iodide undergoes protonation and selectively binds to the negatively charged ascorbic acid forming an ion-pair complex. This complex alters the molecular structure of propidium iodide inducing chemical fluorescence quenching, that can be utilized for ascorbic acid quantification. The developed method undergoes rigorous validation following ICH guidelines, demonstrating a linear relationship within a concentration range of 4-40 µg/mL, with high precision and accuracy metrics. Analysis of real plasma samples from autistic and healthy children reveals clinically and statistically elevated levels of ascorbic acid in those with autism spectrum disorder.

Staining Properties of Selected Commercial Fluorescent Dyes Toward B- and Z-DNA.

The properties of six commonly used, commercially available, fluorescent dyes were compared in staining right-handed B-DNA and left-handed Z-DNA. All showed different degree of fluorescence turn-on in the presence of B-DNA, but very little in the presence of Z-DNA. The optimal range of dye-DNA ratios of DNA was determined. While these dyes do not provide a turn-on type probe for Z-DNA, staining between B- and Z-DNA using dyes such as SYBR Green I was shown to be useful in tracking the kinetics of conformational changes between these two forms of DNA. Finally, SYBR Green I showed unique circular dichroism patterns in 4 M NaCl that change in the presence of double stranded DNA, both in the visible and UV range.

Practical Characterization Strategies for Comparison, Qualification, and Selection of Cell Viability Detection Methods for Cellular Therapeutic Product Development and Manufacturing.

Cellular therapy development and manufacturing has focused on providing novel therapeutic cell-based products for various diseases. The International Organization for Standardization (ISO) has provided guidance on critical quality attributes (CQAs) that shall be considered when testing and releasing cellular therapeutic products. Cell count and viability measurements are two of the CQAs that are determined during development, manufacturing, testing, and product release. The ISO Cell Counting Standard Part 1 and 2 addressed the needs for improving the quality of cell counting results. However, there is currently no guidance on the qualification and selection of a fit-for-purpose cell viability detection method. In this work, we present strategies for the characterization and comparison of AO/PI and AO/DAPI staining methods using the heat-killed (HK) and low temperature/nutrient-deprived (LT/ND) cell death models to evaluate the comparability of cell viability measurements and identify potential causes of differences. We compared the AO/PI and AO/DAPI staining methods using HK and LT/ND-generated dead cells, investigated the staining time effects on cell viability measurements, and determined their viability linearity with different mixtures of live and dead cells. Furthermore, we validated AO/PI and AO/DAPI cell viability measurement with a long-term cell proliferation assay. Finally, we demonstrate a practical example of cell viability measurement comparison using AO/PI and AO/DAPI on antibiotic-selected transduced Jurkat and THP-1 cells to select a fit-for-purpose method for functional genomics screening. The proposed strategies may potentially enable scientists to properly characterize, compare, and select cell viability detection methods that are critical for cellular therapeutic product development and manufacturing.

Multiscale reconstruction of bronchus and cancer cells in human lung adenocarcinoma.

BACKGROUND: While previous studies primarily focused on the structure of the normal whole mouse lung, the whole bronchus and cytoarchitectural details of the mouse intact lung lobe have been discovered at single-cell resolution. Revealing the sophisticated lung adenocarcinoma structure at three-dimensional (3D) and single-cell level remains a fundamental and critical challenge for the pathological mechanism research of lung adenocarcinoma (LA). METHODS: Fluorescence micro-optical Sectioning Tomography (fMOST) combined with PI staining were used to obtain the 3D imaging of the human LA tissue at single-cell resolution. RESULTS: With a spatial resolution of 0.32 × 0.32 × 1.0 µm3, the dataset of human LA with single-cell precision consists of two channels, each of which contains information about the bronchi and the cytoarchitecture. The bronchial wall is thicker and the lumen is smaller in the cancer tissue, in which its original normal structure is vanished. More solid components, more clustered cancer cells with larger nucleoli, and more significant atypia are found in cancer tissue. In paracancerous tissue, the bronchial wall cells have a monolayer or bilayer structure, cluster along the wall, and are relatively dispersed. Few fibrous structures and occasional dissemination of spread through air spaces (STAS) are observed. CONCLUSIONS: Based on the human LA tissue dataset obtained by fMOST and PI staining, the bronchi and cells were reconstructed and visualized. This work provides a technical roadmap for studying the bronchus and cytoarchitectural structure and their spatial relationship in LA tissue, which may help with the understanding of the main histological structure of LA among pathologists.

Clostridium perfringens Beta2 toxin forms highly cation-selective channels in lipid bilayers.

Clostridium perfringens is a potent producer of a variety of toxins. Well studied from these are five toxins (alpha, Beta (CPB), epsilon, iota and CPE) that are produced by seven toxinotype strains (A-G) of C. perfringens. Besides these toxins, C. perfringens produces also another toxin that causes necrotizing enterocolitis in piglets. This toxin termed consensus Beta2 toxin (cCPB2) has a molecular mass of 27,620 Da and shows only little homology to CPB and no one to the other toxins of C. perfringens. Its primary action on cells remained unknown to date. cCPB2 was heterogeneously expressed as fusion protein with GST in Escherichia coli and purified to homogeneity. Although cCPB2 does not exhibit the typical structure of beta-stranded pore-forming proteins and contains no indication for the presence of amphipathic alpha-helices we could demonstrate that cCPB2 is a pore-forming component with an extremely high activity in lipid bilayers. The channels have a single-channel conductance of about 700 pS in 1 M KCl and are highly cation-selective as judged from selectivity measurements in the presence of salt gradients. The high cation selectivity is caused by the presence of net negative charges in or near the channel that allowed an estimate of the channel size being about 1.4 nm wide. Our measurements suggest that the primary effect of cCPB2 is the formation of cation-selective channels followed by necrotic enteritis in humans and animals. We searched in databases for homologs of cCPB2 and constructed a cladogram representing the phylogenetic relationship to the next relatives of cCPB2.

Lysinibacillus macroides-mediated control of cellulose-producing morphotype of Salmonella.

BACKGROUND: Soil-dwelling human pathogens like Salmonella are transmitted by fresh produce such as tomato, spinach, onion and cabbage. With >2600 serovars, it is difficult to classify the good plant colonizers from the non-colonizers. Generally, soil microbiota are classified as autochthonous or zymogenous organisms, based on their ability to survive in soil. However, such information for soil-dwelling human pathogens is not available Thus there is a need to classify these organisms for designing a strategy to prevent their outbreak. Moreover, soil harbours a plethora of microbes, which can be screened for competitive organisms to control such human pathogens. RESULTS: In this study, we examined whether the morphotype based on the attachment factors (e.g., cellulose and curli fimbri) of Salmonella was important for its colonization of roots. Secondly, we tracked the location of the bacteria in the plant cell. Interestingly, most of the epidermal cells occupied by Salmonella showed propidium iodide-positive nuclei. As an extension of the study, a screening of competitive rhizospheric bacteria was performed. One isolate, identified as Lysinibacillus macroides, was able to inhibit the biofilm of Salmonella and subsequently reduced its colonization on roots. CONCLUSION: Based on this study, we classified the Rdar (red, dry and rough) morphotypes as good plant colonists. The ability to colonize and subsequent kill the live plant cell throws light on the zymogenous life cycle of soil-dwelling Salmonella. Additionally, Lysinibacillus macroides served as a biocontrol agent by reducing the burden of Salmonella in various vegetables. Such organisms can further be explored to prevent contamination of the food chain. © 2022 Society of Chemical Industry.

Evaluation of anti-methicillin-resistant Staphylococcus aureus property of zerumbone.

CONTEXT AND OBJECTIVE: Zerumbone has been reported to exert anti-microbial effects, but the mechanism by which the compound exerts its action is not known. Thus, this study aimed to investigate the mechanism of action of zerumbone against methicillin-resistance Staphylococcus aureus (MRSA), using the atomic force microscopy (AFM), scanning electron microscopy (SEM), and flow cytometry techniques. METHODS: MRSA (NCTC 13277) cell viability was determined using the microplate AlamarBlue assay. AFM and SEM were used to determine the morphology of zerumbone-treated MRSA cells. Flow cytometric analysis was used to determine the effect of zerumbone on bacterial membrane permeability and membrane potential, using the propidium iodide (PI) staining method, membrane potential-sensitive fluorescence probe, and DiBAC4(3) dye. DCFDA dye was used to determine the generation of reactive oxygen species (ROS) by MRSA. RESULTS: Zerumbone significantly inhibited MRSA growth with a minimum inhibitory concentration (MIC) of 125 µg/ml. The AFM analysis showed that zerumbone caused leakage of cytoplasmic content from the bacterial cells. Ultrastructure analysis showed small colonies of the bacteria with pores on the membrane surface. There were increases in zerumbone-treated MRSA PI and DiBAC4(3) fluorescence, indicating an increase in cell membrane permeability and a decrease in membrane potential that culminated in the loss of membrane structural integrity and bacterial death. Based on DCFDA dye analysis, zerumbone also reduced ROS production by MRSA. CONCLUSIONS: Zerumbone exerts anti-MRSA effects by causing membrane depolarization, increasing membrane permeability, and finally disrupting cell membrane and bacterial killing.

A Combined Immunofluorescence and Fluorescent Viability Cocktail Staining Procedure for Rapid Microscopic Detection and Enumeration of Live Legionella pneumophila.

This report describes a combined immunofluorescence and fluorescence viability stain applied as one staining solution for rapid detection of live Legionella pneumophila in mixed bacterial populations. Instead of sequential viability staining with the Invitrogen BacLight LIVE/DEAD staining kit followed by antibody-Alexa Fluor (AF) 647 conjugate staining to identify live L. pneumophila, a combined single cocktail solution staining protocol was developed to simplify and accelerate the time to detection of viable L. pneumophila serogroup-1 (SG-1) in mixed species populations on a filter membrane. The stain cocktail will aid in accelerating fluorescence microscopic analysis of cooling tower, air conditioner and water fountain or other liquid samples for the presence of L. pneumophila and its viability status. Visibly red stained cells were identified as dead non-L. pneumophila SG-1 cells, while green fluorescing cells represented viable non-L. pneumophila SG-1 cells. Due to also staining red with antibody-AF 647, L. pneumophila SG-1 cells were pseudocolorized as blue to distinguish them from other dead cells. Fluorescence color emission mixing from the viability dyes (SYTO 9 and propidium iodide) with antibody-AF 647 stained L. pneumophila led to other fluorescent colors. For example, green plus pseudocolorized blue AF 647-antibody- labeled cells were identified as live cyan-colored L. pneumophila SG-1 cells. Magenta-colored cells resulted from dead L. pneumophila cells that combined red propidium iodide with blue pseudocolorized AF 647-antibody emissions. Analysis of measured RGB (red, green, blue) color values in microscopic images of mixed bacterial populations suggests the possibility of facile automated discrimination of subpopulations of live and dead L. pneumophila and non-L. pneumophila species by computers in 3-dimensional RGB color space after staining in the combined cocktail which will save time for more rapid microscopic detection of potential sources of Legionnaire's disease.

In vitro efficacy of anthelmintics on Angiostrongylus cantonensis L3 larvae.

Angiostrongylus cantonensis is the leading cause of eosinophilic meningitis worldwide, with life-threatening complications if not managed correctly. Previous in vitro studies have utilized change in motility patterns of adult female worms to assess the efficacy of anthelmintics qualitatively. However, it is the third stage larvae (L3) that are infectious to humans. With differential staining using propidium iodide penetration as the indicator of death, we can distinguish between dead and live larvae. This assay has enabled us to quantify the in vitro efficacy of nine clinically established anthelmintics on A. cantonensis L3. All drugs were tested at a 1 mm concentration. Piperazine and niclosamide were ineffective in inducing larval death; however, albendazole sulfoxide, pyrantel pamoate, diethylcarbamazine, levamisole and praziquantel were effective as compared to unexposed controls (P < 0.05). Ivermectin and moxidectin did not induce significant levels of mortality, but they considerably reduced larval motility almost immediately. This study indicates the need for further in vivo studies to determine the optimal dose and time frame for post-infection treatment with anthelmintics that demonstrated efficacy.

Measurement of membrane integrity in canine spermatozoa using a fluorescent computer-assisted spermatozoal quantification method after SYBR-14/PI staining compared with manual counting after CFDA/PI staining.

The aim of this study was to compare measurements of spermatozoal membrane status in dogs using computer-assisted spermatozoal quantification (CASQ) after staining with SYBR-14 and propidium iodide (PI) with manual counting after CFDA/PI staining. CASQ was performed on fresh (n = 11) and thawed cryopreserved canine semen (n = 91) using (1) a red long-pass (LP) filter on an untreated sample (membrane-disrupted spermatozoa, MDS count) and in a sample with all cellular membranes disrupted (total spermatozoal count, TC), (2) green LP filter for a TC and the red filter for an MDS count and (3) a green short-pass filter to obtain a membrane-intact spermatozoa (MIS) count and the red filter to obtain the MDS count, which were added to give a TC (red-green filter CASQ, n = 50). Spermatozoa were also stained with CFDA/PI, manually examined and classified as MIS or MDS. All measurements were performed in duplicate. The percentage of membrane-intact spermatozoa (MIS) was calculated. The percentage of progressively motile spermatozoa (PMS) was determined subjectively. The data were analysed to measure the agreement between the CASQ and CFDA/PI methods, repeatability of the methods and correlation between the MIS and PMS percentage. Compared with the CFDA/PI method, the agreement of MIS percentage with red filter CASQ was -12% to 34%, green LP filter CASQ -42% to 47% and red-green filter CASQ -23% to 29%. The repeatability of the CFDA/PI and red-green filter CASQ methods were the highest. The MIS and PMS percentages were always correlated (p < .05). Measurement of MIS percentage using red and red-green filter CASQ appeared to be the most reliable automated methods.

Inactivation Mechanism of Aspergillus flavus Conidia by High Hydrostatic Pressure.

This study investigated the inactivation mechanism of Aspergillus flavus conidia by high hydrostatic pressure (HHP). Activity counts, scanning electron microscopic (SEM) analysis, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to study the effects of the HHP treatment on the morphology and protein composition of A. flavus spores. The results showed that that a 3-min-lasting 600 MPa treatment could completely abolish 107 colony-forming units/mL of live fungi. Furthermore, we also observed that lower spore viability corresponded to a higher Propidium Iodide absorption rate. The SEM images revealed that HHP disrupted the spore morphology and resulted in pore formation that led to the release of intracellular molecules, such as nucleic acids and proteins. The nucleic acid and protein concentration in the spore suspension increased in parallel with the increasing treatment pressure. The SDS-PAGE analysis showed that there were differences in the protein bands between the HHP-treated and untreated A. flavus spores, as the HHP treatment caused partial protein degradation and extracellular release. Therefore, the results of this study proved that high pressure could induce a morphological disruption in the internal and external cellular structures and degrade intracellular and extracellular proteins leading to an inactive state in A. flavus.

Bacteriocin isolated from the natural inhabitant of Allium cepa against Staphylococcus aureus.

Extensive usage of antibiotics has led to the emergence of drug-resistant strains of pathogens and hence, there is an urgent need for alternative antimicrobial agents. Antimicrobial Peptides (AMPs) of bacterial origin have shown the potential to replace some conventional antibiotics. In the present study, an AMP was isolated from Bacillus subtilis subsp. spizizenii strain Ba49 present on the Allium cepa, the common onion and named as peptide-Ba49. The isolated AMP was purified and characterized. The purified peptide-Ba49, having a molecular weight of ~ 3.3 kDa as determined using mass spectroscopy, was stable up to 121 °C and in the pH range of 5-10. Its interaction with protein degrading enzymes confirmed the peptide nature of the molecule. The peptide exhibited low minimum inhibitory concentration (MIC) against Staphylococcus aureus and its (Methicillin-resistant Staphylococcus aureus) MRSA strains (MIC, 2-16 µM/mL). Further, time kill kinetic assay was performed and analysis of the results of membrane depolarization and permeabilization assays (TEM, DiBAC4 (3) and PI) suggested peptide-Ba49 to be acting through the change in membrane potential leading to disruption of S. aureus membrane. Additionally, cytotoxicity studies of peptide-Ba49, carried out using three mammalian cell lines viz. HEK 293T, RAW 264.7, and L929, showed limited cytotoxicity on these cell lines at a concentration much higher than its MIC values. All these studies suggested that the AMP isolated from strain Ba49 (peptide-Ba49) has the potential to be an alternative to antibiotics in terms of eradicating the pathogenic as well as drug-resistant microorganisms.

Induction of cell death and modulation of Annexin A1 by phytoestrogens in human leukemic cell lines.

BACKGROUND: Phytoestrogens are polyphenolic plant compounds which are structurally similar to the endogenous mammalian estrogen, 17ß-estradiol. Annexin A1 (ANXA1) is an endogenous protein which inhibits cyclo-oxygenase 2 (COX-2) and phospholipase A2, signal transduction, DNA replication, cell transformation, and mediation of apoptosis. OBJECTIVE: This study aimed to determine the effects of selected phytoestrogens on annexin A1 (ANXA1) expression, mode of cell death and cell cycle arrest in different human leukemic cell lines. METHODS: Cells viability were examined by MTT assay and ANXA1 quantification via Enzyme-linked Immunosorbent Assay. Cell cycle and apoptosis were examined by flow cytometer and phagocytosis effect was evaluated using haematoxylin-eosin staining. RESULTS: Coumestrol significantly (p < 0.05) reduced the total level of ANXA1 in both K562 and U937 cells and genistein significantly (p < 0.05) reduced it in K562, Jurkat and U937 cells, meanwhile estradiol and daidzein induced similar reduction in U937 and Jurkat cells. Coumestrol and daidzein induced apoptosis in K562 and Jurkat cells, while genistein and estradiol induced apoptosis in all tested cells. Coumestrol and estradiol induced cell cycle arrest at G2/M phase in K562 and Jurkat cells with an addition of U937 cells for estradiol. Genistein induced cell cycle arrest at S phase for both K562 and Jurkat cells. However, daidzein induced cell cycle arrest at G0/G1 phase in K562, and G2/M phase of Jurkat cells. Coumestrol, genistein and estradiol induced phagocytosis in all tested cells but daidzein induced significant (p < 0.05) phagocytosis in K562 and Jurkat cells only. CONCLUSION: The selected phytoestrogens induced cell cycle arrest, apoptosis and phagocytosis and at the same time they reduced ANXA1 level in the tested cells. The IC50 value of phytoestrogens was undetectable at the concentrations tested, their ability to induce leukemic cells death may be related with their ability to reduce the levels of ANXA1. These findings can be used as a new approach in cancer treatment particularly in leukemia.

Nanoparticles of cisplatin augment drug accumulations and inhibit multidrug resistance transporters in human glioblastoma cells.

BACKGROUND: Cisplatin (CSP) is a potent anticancer drug widely used in treating glioblastoma multiforme (GBM). However, CSP's clinical efficacy in GBM contrasted with low therapeutic ratio, toxicity, and multidrug resistance (MDR). Therefore, we have developed a system for the active targeting of cisplatin in GBM via cisplatin loaded polymeric nanoplatforms (CSP-NPs). METHODS: CSP-NPs were prepared by modified double emulsion and nanoprecipitation techniques. The physiochemical characterizations of CSP-NPs were performed using zeta sizer, scanning electron microscopy (SEM), drug release kinetics, and drug content analysis. Cytotoxicity, induction of apoptosis, and cell cycle-specific activity of CSP-NPs in human GBM cell lines were evaluated by MTT assay, fluorescent microscopy, and flow cytometry. Intracellular drug uptake was gauged by fluorescent imaging and flow cytometry. The potential of CSP-NPs to inhibit MDR transporters were assessed by flow cytometry-based drug efflux assays. RESULTS: CSP-NPs have smooth surface properties with discrete particle size with required zeta potential, polydispersity index, drug entrapment efficiency, and drug content. CSP-NPs has demonstrated an 'initial burst effect' followed by sustained drug release properties. CSP-NPs imparted dose and time-dependent cytotoxicity and triggered apoptosis in human GBM cells. Interestingly, CSP-NPs significantly increased uptake, internalization, and accumulations of anticancer drugs. Moreover, CSP-NPs significantly reversed the MDR transporters (ABCB1 and ABCG2) in human GBM cells. CONCLUSION: The nanoparticulate system of cisplatin seems to has a promising potential for active targeting of cisplatin as an effective and specific therapeutic for human GBM, thus eliminating current chemotherapy's limitations.

Characterisation of early responses in lead accumulation and localization of Salix babylonica L. roots.

BACKGROUND: Lead (Pb) is a harmful pollutant that disrupts normal functions from the cell to organ levels. Salix babylonica is characterized by high biomass productivity, high transpiration rates, and species specific Pb. Better understanding the accumulating and transporting Pb capability in shoots and roots of S. babylonica, the toxic effects of Pb and the subcellular distribution of Pb is very important. RESULTS: Pb exerted inhibitory effects on the roots and shoots growth at all Pb concentrations. According to the results utilizing inductively coupled plasma atomic emission spectrometry (ICP-AES), S. babylonica can be considered as a plant with great phytoextraction potentials as translocation factor (TF) value > 1 is observed in all treatment groups throughout the experiment. The Leadmium™ Green AM dye test results indicated that Pb ions initially entered elongation zone cells and accumulated in this area. Then, ions were gradually accumulated in the meristem zone. After 24 h of Pb exposure, Pb accumulated in the meristem zone. The scanning electron microscopy (SEM) and energy-dispersive X-ray analyses (EDXA) results confirmed the fluorescent probe observations and indicated that Pb was localized to the cell wall and cytoplasm. In transverse sections of the mature zone, Pb levels in the cell wall and cytoplasm of epidermal cells was the lowest compared to cortical and vessel cells, and an increasing trend in Pb content was detected in cortical cells from the epidermis to vascular cylinder. Similar results were shown in the Pb content in the cell wall and cytoplasm of the transverse sections of the meristem. Cell damage in the roots exposed to Pb was detected by propidium iodide (PI) staining, which was in agreement with the findings of Pb absorption in different zones of S. babylonica roots under Pb stress. CONCLUSION: S. babylonica L. is observed as a plant with great potential of Pb-accumulation and Pb-tolerance. The information obtained here of Pb accumulation and localization in S. babylonica roots can furthers our understanding of Pb-induced toxicity and its tolerance mechanisms, which will provide valuable and scientific information to phytoremediation investigations of other woody plants under Pb stress.

MAP30 protein from Momordica charantia is therapeutic and has synergic activity with cisplatin against ovarian cancer in vivo by altering metabolism and inducing ferroptosis.

Increasing evidence shows that Traditional Chinese Medicine (TCM) has an obvious appeal for cancer treatment, but there is still a lack of scientific investigation of its underlying molecular mechanisms. Bitter melon or bitter gourd (Momordica charantia) is an edible fruit that is commonly consumed, and it is used to cure different diseases in various ancient folk medical practices. We report that a bioactive protein, MAP30, isolated from bitter melon seeds exhibited potent anticancer and anti-chemoresistant effects on ovarian cancer cells. Functional studies revealed that MAP30 inhibited cancer cell migration, cell invasion, and cell proliferation in various ovarian cancer cells but not normal immortalized ovarian epithelial cells. When administered with cisplatin, MAP30 produced a synergistic effect on cisplatin-induced cell cytotoxicity in ovarian cancer cells. When low doses of cisplatin and MAP30 were co-injected intraperitoneally, a remarkable reduction of tumor dissemination and tumor growth was observed in an ovarian cancer ascites mouse model. Notably, blood tests confirmed that MAP30 did not cause any adverse effects on liver and kidney functions in the treated mice. MAP30 activated AMP-activated protein kinase (AMPK) signaling via CaMKKß and induced cell cycle arrest in the S-phase. MAP30 modulated cell metabolism of ovarian cancer cells via suppression of GLUT-1/-3-mediated glucose uptake, adipogenesis, and lipid droplet formation in tumor development and progression. MAP30 also induced an increase in intracellular Ca2+ ion concentration, which triggered ROS-mediated cancer cell death via apoptosis and ferroptosis. Collectively, these findings suggest that natural MAP30 is a non-toxic supplement that may enhance chemotherapeutic outcomes and benefit ovarian cancer patients with peritoneal metastases.

Influence of ClearT and ClearT2 Agitation Conditions in the Fluorescence Imaging of 3D Spheroids.

3D tumor spheroids have arisen in the last years as potent tools for the in vitro screening of novel anticancer therapeutics. Nevertheless, to increase the reproducibility and predictability of the data originated from the spheroids it is still necessary to develop or optimize the techniques used for spheroids' physical and biomolecular characterization. Fluorescence microscopy, such as confocal laser scanning microscopy (CLSM), is a tool commonly used by researchers to characterize spheroids structure and the antitumoral effect of novel therapeutics. However, its application in spheroids' analysis is hindered by the limited light penetration in thick samples. For this purpose, optical clearing solutions have been explored to increase the spheroids' transparency by reducing the light scattering. In this study, the influence of agitation conditions (i.e., static, horizontal agitation, and rotatory agitation) on the ClearT and ClearT2 methods' clearing efficacy and tumor spheroids' imaging by CLSM was characterized. The obtained results demonstrate that the ClearT method results in the improved imaging of the spheroids interior, whereas the ClearT2 resulted in an increased propidium iodide mean fluorescence intensity as well as a higher signal depth in the Z-axis. Additionally, for both methods, the best clearing results were obtained for the spheroids treated under the rotatory agitation. In general, this work provides new insights on the ClearT and ClearT2 clearing methodologies and their utilization for improving the reproducibility of the data obtained through the CLSM, such as the analysis of the cell death in response to therapeutics administration.

Phytochemical constituents and anticancer activities of Tarchonanthus camphoratus essential oils grown in Saudi Arabia.

Tarchonanthus Camphoratus L. is traditionally known for its various medicinal purposes. In this study, the T. camphoratus essential oil (TCEO) was isolated via steam distillation, and its chemical constituents were determined using GC-MS. The in vitro antiproliferative effects of TCEO on A549, HepG2, MCF-7 cancer cells, and HUVEC non-tumor cells was investigated using an MTT assay. Flow cytometry analysis was conducted to evaluate cell cycle distribution using propidium iodide staining, and cell death mode using Annexin V-FITC/PI assays. The expression of some apoptosis related genes was investigated using qRT-PCR. Major constituents of TCEO included fenchol, borneol, 3-cyclohexene-1-methanol and 3-ethyl-3-methyl. Cell viability test showed that TCEO is highly effective against MCF-7 cells with IC50 12.5 µg/mL. Cell cycle arrest at the G1/S phase, and apoptosis mediation were evident in the presence of TCEO. Gene expression analysis of several pro-apoptotic and anti-apoptotic genes revealed the initiation of apoptosis in TCEO-MCF-7 cells. In conclusion, our study confirms the antiproliferative activity of the T. camphoratus essential oil.

Codium fragile F2 sensitize colorectal cancer cells to TRAIL-induced apoptosis via c-FLIP ubiquitination.

This study demonstrates that combined treatment with subtoxic doses of Codium extracts (CE), a flavonoid found in many fruits and vegetables, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), induces apoptosis in TRAIL-resistant colorectal cancer (CRC) cells. Effective induction of apoptosis by combined treatment with CE and TRAIL was not blocked by Bcl-xL overexpression, which is known to confer resistance to various chemotherapeutic agents. While TRAIL-mediated proteolytic processing of procaspase-3 was partially blocked in various CRC cells treated with TRAIL alone, co-treatment with CE efficiently recovered TRAIL-induced caspase activation. We observed that CE treatment of CRC cells did not change the expression of anti-apoptotic proteins and pro-apoptotic proteins, including death receptors (DR4 and DR5). However, CE treatment markedly reduced the protein level of the short form of the cellular FLICE-inhibitory protein (c-FLIPS), an inhibitor of caspase-8, via proteasome-mediated degradation. Collectively, these observations show that CE recovers TRAIL sensitivity in various CRC cells via down-regulation of c-FLIPS.