Triple-negative breast cancer (TNBC) is a distinct subtype characterized by the absence of estrogen, progesterone, and human epidermal growth factor receptor-2 expression. Representing 15–20% of newly diagnosed breast cancer cases, TNBC carries a 40% mortality rate within the first 5 years post diagnosis. This underscores the urgency in enhancing TNBC chemoresistance, one trustworthy option being the use of natural compounds as chemosensitizers. The main objective of this study was to improve the sensitivity of TNBC cells to docetaxel by combining it with cytotoxic natural compounds and to elucidate the precise molecular mechanisms of action using a cellular and metabolomic approach. In pursuit of this, we conducted cytotoxicity profiling for docetaxel, genistein, leontopodic acid, and kaempferol individually and in dual combinations on a panel of TNBC cell lines (HS578T, MDA-MB-231, MDA-MB-462), using the MTT assay. The results indicated that the genistein–docetaxel combination exhibited the most potent cytotoxic effects across all cell lines. For this combination, we investigated the biochemical mechanisms involved using a gas chromatography–mass spectrometry (GC-MS) metabolomics approach. The analysis identified 69 intracellular metabolites, with 17 quantified relative to internal standards. These metabolites belong to different chemical classes, including phosphorylated amino acids, carbohydrates, fatty acids, or nucleotides, and are key intermediates in different metabolic pathways. Quantitative enrichment analysis revealed distinct pathway alterations for each cell line, encompassing beta-alanine metabolism, nicotinamide and nicotinate metabolism, purine and pyrimidine base metabolism, as well as lysine degradation and biotin metabolism. In conclusion, the combination of genistein and docetaxel demonstrates a potent chemosensitizing effect in TNBC cells, inducing cytotoxicity through diverse metabolic pathways. This underscores the considerable potential of this combination in addressing the TNBC subtype. Acknowledgments: This work was supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS— UEFISCDI, project number PN-III-P1-1.1-PD-2021-0093, within PNCDI III, awarded to Alina Uifălean.

Abstract

Introduction: Protease-activated receptor 2 (PAR2) plays a pivotal role in activating pain and inflammation pathways in rheumatoid arthritis (RA), contributing to cartilage destruction, synovial inflammation, and heightened joint pain. This study aims at identifying mast cell stabilizers with potential functionality in inhibiting PAR2 activation, focusing on FDA-approved candidates for their safety and efficacy.

Methods: Twelve mast cell stabilizers were selected based on toxicity screening and clearance time from PubChem. Molecular docking simulations were performed, assessing their interactions with the PAR2 molecule. Docking scores were meticulously analyzed to identify potential candidates with superior functionality compared to existing RA treatments, namely, Bicalutamide and Methotrexate. Lipinski’s rules of drug validity were applied to ensure drug-like properties.

Results: Among the mast cell stabilizers, S-Azelastine and Cromoglicic acid exhibited superior functionality, surpassing Bicalutamide and Methotrexate. The evaluation considered patient safety aspects and the efficacy of inhibiting PAR2 activation. Notably, the selected mast cell stabilizers demonstrated compliance with Lipinski’s rules, indicating their potential suitability for drug development.

Conclusions: This study highlights PAR2 as a promising target for drug repurposing in RA treatment. Mast cell stabilizers, particularly S-Azelastine and Cromoglicic acid, show potential for safer pain management in RA patients. Their superior functionality, especially in PAR2 inhibition and patient safety, suggests a viable avenue for drug repurposing strategies. Further exploration and development of these mast cell stabilizers could lead to novel therapeutic options for RA, addressing the critical need for more effective and safe pain management strategies in this patient population.

Background: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer among the breast cancer subtypes. Race plays a crucial role in TNBC, with a African American (AA) women having higher incidence rates compared to European Americans (EAs). In the present study, we aim to analyze differentially expressed genes (DEGs) among African Ghanaian (AG), African Ethiopian (AE), and African American populations using the bioinformatics approach.

Material and Methods: Gene expression profiles for Ghanaian, Ethiopian, and African American populations were downloaded from the GEO database. DEG analysis was performed using GEO2R. Tumor-infiltrating immune cells were analyzed for the DEGs using TIMER. Upstream regulatory networks from signatures of DEGs were identified using X2kweb.

Results: Our results showed that 863 genes were differentially expressed between AA versus AE, 160 genes were differentially expressed between AE versus AG, and 35 genes were differentially expressed between AA versus AE. Among the identified DEGs, genes CSF3, CXCL2, CXCL3, PRKCB, and PTGER4 were found to be common among all the population groups. Further, gene enrichment analysis on the DEGs showed that these genes were found to be significantly enriched among the B-cell receptor signaling, B-cell differentiation, and B-cell activation pathways.

Conclusion: These findings indicate that these genes could be potential immune biomarkers for identifying TNBC in the African-descent population.

Breast cancer is the most common type of cancer in women. Breast cancer affects 200,000 women in the United States each year. Some of its treatments include chemotherapy, radiation, and surgery. In the present study, we aim to analyze the race-specific differences in gene expression profiles of breast cancer subtypes among the African American and White/Caucasian populations. We used The Cancer Genome Atlas (TCGA) to study invasive breast carcinoma tumors and the correlated genes, TACSTD2, PIK3CA, and NTRK1. Cbioportal was used to find the expression levels of the three genes (NTRK1, PIK3CA, TACSTD2) and further understand their potential roles as DNA biomarkers for breast cancer. The Gene Cards database helped determine the genes with a direct correlation to breast cancer and identify which of them are its promoters or enhancers. Genes PIK3CA and TACSTD2 were shown to be directly correlated. UALCAN was used to analyze the different gene expression levels between the two races, White/Caucasian and African American. Additionally, the KEGG database found gene pathways related to breast cancer. Gene Ontology identified the phenotype of the PIK3CA gene concerning breast cancer. Reference sequencing analysis was conducted on the genes to find mRNA expressions relating to breast cancer. The findings showed genes NTRK1 and TACSTD2 were negatively correlated and gene PIK3CA was positively correlated. The purpose of this study was to measure the correlation of the genetic biomarkers of the African American and White/Caucasian ethnicities with breast cancer subtypes. The genes identified in our study could be potential targets to address different probable causes of and treatments for breast cancer in the African American and White/Caucasian ethnicities.

Introduction

Pulegone is a natural biomolecule that may be found in essential oils from many medicinal and aromatic plants, especially those from the Lamiaceae family. It is known for its distinguished minty aroma and taste, and it has been studied for its potential use in various therapies due to its antibacterial, anti-inflammatory, and cytotoxic effects. Even with its recognized toxicity, pulegone is still widely used as a versatile and valuable compound in many industrial fields.

Methodology

To identify trends in the application of pulegone, we prospecte patents from specialized patent databases. A search was carried out on the titles, abstracts, and claims of the patents. The patents were then sorted by applicant names, and then the applicant patent portfolio was studied to identify the targeted trends.

Results

The patents reveal that the leading jurisdiction is the United States, with about 44% of the filed patents. The study of the top 10 applicants allows us to distinguish three major areas of innovation. The field of daily-use cleaning, health, and hygiene encompasses 121 patents that take advantage of the aromatic characteristics of pulegone to provide hygiene and cosmetic products. The plant protection field stands out with 58 patents. In this field, pulegone is used for its pesticide and pest-repellent action to develop crop protection products. The third field is the use of pulegone with cannabinoids for pharmacological innovations as well as for recreational uses, as represented by 42 patents.

Conclusion

The pulegone-related patents were based on preparations for medical, dental, or toiletry purposes. Pulegone is valued for its refreshing minty scent; it is utilized in perfumes and body care, as well as being an odor neutralizer and odorizing agent for toilet and cleaning compositions. Furthermore, pulegone is used as a natural pesticide and pest repellent, reducing the need for chemicals.

Introduction: Pharmaceutical availability testing is one of the quality control methods used to assess the ability of a medicinal substance to diffuse from a substrate, through a membrane, into a suitable acceptor fluid. The most commonly used membranes for this type of testing are those made of polymers (cellulose, cellophane, or nylon membranes) or natural membranes (mouse/rat/pig skin, human cadaver skin). An alternative to the above membranes is the Strat-M® membrane, designed to mimic human and animal skin. Our study aimed to compare the pharmaceutical availability of insulin from a hydrogel matrix based on Sepineo™ P600 and Sepineo™ PHD100 (Seppic, Paris, France) using a Strat-M® membrane (Merck Millipore, Burlington, USA) and cellulose dialysis membrane Spectra/Por®2 (MWCO of 12-14 kDa; Spectrum Laboratories, Inc., California, United States). Methods: Insulin hydrogels (Insulatard Penfil) based on Sepineo™ P600 and Sepineo™ PHD100 were prepared. The insulin dose was set at 1mg/g. Pharmaceutical availability studies of insulin from hydrogel formulations were conducted in vitro in an ERWEKA DT600 paddle apparatus (Husenstamm, Germany), using Enhancer CellTM (Erweka, Husenstamm, Germany). The volume of PBS acceptor fluid was 50 mL. Testing was carried out at 32±1°C (human skin surface temperature). The stirrer speed was 100 rpm. The amount of insulin released was determined by spectrophotometry at λ = 271 nm. The Strat-M® membrane and cellulose dialysis membrane Spectra/Por® 2 were used in the study. Results: In a pharmaceutical availability study using the Strat-M® membrane, 53.36% and 47.4% of the API were released from the Sepineo™ P600 and Sepineo™ PHD100 hydrogel after 6.5 hours, respectively. In contrast, 69.6% and 64.3% of insulin were released through the cellulose dialysis membrane Spectra/Por®2 after 5 hours. The release of hormones from the hydrogel matrices followed the Peppas/Sahlin model. Conclusions: The amount of INS released by the Strat-M® membrane (high correlation with human skin) was lower than the cellulose dialysis membrane Spectra/Por®2.

Background: Breast cancer is one of the most common cancer types and the second most frequent one among women in the United States. Race plays a key role in the prevalence and prognosis of breast cancer, specifically in triple-negative breast cancer, with African American women being highly prevalent compared to white women. Material and Methods: MDA-MB-453 and 231 cell lines were cultured in RPMI-1640 media with 10%FBS. Upon 70-80% confluency, cells were treated with doxorubicin (2uM for MDA-MB-453 and 1uM for MDA-MB-231). After 24 hrs, sensitized cells were treated with inhibitors (Olaparib, O; Niraparib, N; Atezolizumab, A; durvalumab, D; and Pembrolizumab, P) alone as well as with different combinations at a concentration of 100 uM. The drug combinations include: O+N, O+A, O+D, O+P, N+A, N+D, N+P, D+A, A+P, and P+D. Statistical analysis was performed between the treatment groups using the one-way ANOVA. Results: Our results showed that MDA-MB-453 cells treated with the combinations O+N, O+A, O+D, N+A, N+D, N+P, and A+P showed a significant change in viability compared to the doxorubicin-treated group. Results showed that MDA-MB-231 cells, treated with the combinations O+N, O+D, N+A, N+D, N+P, A+P, and D+A, showed a significant change in viability compared to the doxorubicin-treated group. Conclusion: These findings indicate that the inclusion of these inhibitors, along with the chemotherapeutic drug, not only significantly affects the cell viability of breast cancer cells but also may be helpful in the better therapeutic regime and patient prognosis.

Background and Aims

Severe traumatic brain injury (TBI) is a complex disease and understanding the injury-induced cellular pathobiology is vital to predicting outcome and providing effective treatment and precision healthcare. This can be achieved through studying brain tissue obtained at biopsy soon after injury from severe TBI patients, which has been proven a safe procedure. Distinguishing the various cellular markers within the biopsy tissue can provide us with a better understanding of the complex cellular interactions.

Methods

Using multilayer immunohistochemistry, 12 immunostaining markers were applied on brain tissue obtained at biopsy (n=2). One was from a patient who recovered, one from a patient who died. The cryostat-sectioned tissue was stained with two immunostaining markers per staining procedure and imaged using a fluorescent microscope. Thereafter, the immunostaining was removed using a stripping buffer and successful removal of immunostaining was confirmed by absence of the previous immunostains. The staining and stripping process was then repeated to achieve additional 5 sets of double immunostaining.

Results

The multilayer immunohistochemistry using markers for neurones (NeuN, N52, SMI31, SMI32, MAP2, somatostatin), oligodendrocytes (CNPase), astrocytes (GFAP), microglia (Iba1, P2Y12), and vasculature (claudin5, vWF), successfully stained the same brain tissue section. Merging the immunostaining images together allows the visualisation of the complex cellular interactions, and the increase or decrease expression levels of immunostaining markers between a patient with good functional outcome and another patient with poor functional outcome.

Conclusion

Multilayer immunohistochemistry enabled the identification and simultaneous visualisation of 12 immunostaining markers on a single brain biopsy. This is the first study to conduct a cheap multilayer immunohistochemistry technique on brain biopsy from patients with severe TBI.

Elevated cholesterol levels, or hypercholesterolemia, have been recognized as the underlying cause of various diseases, most notably cardiovascular diseases. Unfortunately, most cholesterol-lowering (or anti-hypercholesterolemic) drugs are associated with several adverse effects, emphasizing the need to identify new cholesterol-lowering strategies. Natural products, particularly bioactive phytochemicals, have gained significant attention for their safer profile, fewer side effects, and potential health benefits, including cholesterol-lowering properties. The citrus fruit bergamot (Citrus bergamia) is renowned for its diverse array of bioactive phytochemicals. In this study, an in silico approach was utilized to assess the cholesterol-lowering potential of phytochemicals derived from C. bergamia. Molecular docking using AutoDock Vina of the selected phytochemicals was performed against the HMG-CoA reductase (HMGR), an enzyme targeted for hypercholesterolemia. Results indicated that among the selected 20 phytochemicals, 8, namely Eriocitrin, Narirutin, Scolymoside, Neodiosmin, Brutieridin, Neohesperidin, Rhoifolin, and Naringin, exhibited better binding affinities than the conventional HMG-CoA reductase inhibitor, Atorvastatin (-9.2 kcal/mol). Notably, among these top eight phytochemicals, Eriocitrin displayed the most favorable binding affinity of -10.0 kcal/mol. These findings strongly imply that C. bergamia possesses potential HMGR inhibitory activity and anti-hypercholesterolemic activity, primarily due to the high binding affinities exhibited by its phytochemical constituents. Therefore, further studies must be considered to comprehensively explore the cholesterol-lowering properties of C. bergamia phytochemicals.

Introduction. Amperometry is arguably the most widely used technique for studying the exocytosis of biological amines, allowing the study of the dynamics and kinetics of this cellular mechanism in real time. In recent decades, multielectrode array (MEA) devices have been developed and increase the efficiency and speed of amperometric measurements.

Platelets are the most accessible cells to study the exocytosis of amines as they have90% of blood serotonin included in their granules, which is avidly uptaken from the blood and released by exocytosis. Furthermore, platelets are not able to synthesize this amine.

Methods. We have optimized boron-doped diamond (BDD) MEA systems that allow the detection of amperometric recordings from the quantum release of serotonin from human platelets. With this technique, exocytotic release phenomena are recorded as a succession of discrete signals in the form of peaks that result from electric current intensities measured during the oxidation of the released serotonin.

Our initial results were carried out with 16 microelectrode devices on silicone matrices (BDD-on-silicon MEA)¹. In this conference, we present transparent MEA devices with 16 microelectrodes that allow microscopy observation too: BDD-on-quartz MEA.

Results. BDD-on-quartz MEA devices exhibit the same excellent electrochemical properties as BDD-on-silicon MEA¹: We present a comparative study of quantum and kinetics data obtained with both MEA chips from unloaded platelets and after loading the platelets with 10 µM serotonin for 2 h. And finally, we show examples of the different types of peaks detected.

Conclusions. We demonstrate the effectiveness of BDD-on-quartz MEA devices foramperometrical studies of serotonin exocytosis from human platelets.

References. ¹ González Brito, R; Montenegro, P; Méndez, A; Carabelli, V; Tomagra, G; Shabgahi, R.E.; Pasquarelli, A.; Borges, R. Multielectrode Arrays as a Means to Study Exocytosis in Human Platelets. Biosensors 2023, 13, 86. https://doi.org/10.3390/bios13010086.