Biolmpacts
Volume:14 Issue: 3, May 2024

 A new analytical method based on the coupling of microextraction and microfluidics was developed and investigated for the pre-concentration, separation, and electrochemical detection of fenitrothion (FT) and parathion (PA) at the sub-ppm concentrations.

 In the first step, the microchip capillary electrophoresis technique was used to serve as a separation and detection system. Analytes were injected in the 40 mm long microchannel with 10 mm sidearms. Then, they were separated by applying a direct electrical field (+1800 V) between the buffer and detection reservoirs. 2-(n-morpholino)ethanesulfonic acid (MES) buffer (20 mM, pH 5) was used as a running buffer. The electrochemical detection was performed using three Pt microelectrodes with the width of working, counter, and reference electrodes (50, 250, and 250 µm, respectively) in the out-channel approach.

 The system was devised to have the optimum detection potential equal to -1.2 V vs. pseudo-reference electrode. The dimensions of the SU-8 channel have 20 µm depth and 50 µm width. In the second step, an air-assisted liquid-liquid microextraction technique was used to extract and preconcentration of analytes from human blood plasma. Then, 1, 2 di-bromoethan was used as extractant solvent, the analytes were preconcentrated, and the sedimented solvent (50 µL) was evaporated in a 60 ˚C water bath followed by substitution of running buffer containing 10% ethanol. The optimal extraction cycles were found to be 8 with adding 1% NaCl to the aqueous phase. Analyzing time of the mentioned analytes was less than 100s, the precision range was 3.3 – 8.2 with a linear range of 0.8–100 ppm and 1.2–100 ppm for FT and PA, respectively. The extraction recoveries were about 91% and 87% for FT and PA, respectively. The detection limits for FT and PA were 240 and 360 ppb, respectively. Finally, the reliability of the method was investigated by GC-FID.

 The proposed method and device were validated and can be used as in situ and portable detection systems for detecting fenitrothion and parathion insecticides.

 Understanding the key role of the tumor microenvironment in specifying molecular markers of breast cancer subtypes is of a high importance in diagnosis and treatment. Therefore, the possibility of interconversion of luminal states and their specific markers alteration under the control of tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs) deserves to be further investigated.

 To activate normal human fibroblasts, liquid overlay technique or nemosis was used and α-SMA protein expression, CAFs marker, in fibroblastic spheroids was measured by blotting. The luminal A, MCF-7, and luminal B, MDA-MB 361, cell lines were treated with normal and spheroidal/activated fibroblast conditioned medium for 48 hours. The morphological changes of both luminal A and B cells were evaluated by invert light microscopy and analyzed through the shape factor formula. Moreover, chemo-sensitivity, proliferation, and changes in ER-related and proliferative genes expression levels were assessed respectively via MTT assay, Ki67 expression Immunofluorescence assay, real time PCR and Annexin V-FITC techniques.

 Activated (spheroidal) fibroblasts, expressed αSMA marker two folds more than monolayer cultured fibroblasts. Our study indicated a significant increase in IC50 of both luminal A and B cell lines after being treated with conditioned medium particularly in treated group with spheroidal conditioned medium. Studying Morphological changes using shape factor formula demonstrated more aggressiveness with gaining mesenchymal features in both luminal A and B subtypes by increasing exposure time. Changes in the expression of Ki67 were observed following treatment with fibroblastic and spheroidal paracrine secretome. Driven Data from Ki67 assay supports the luminal A and B interconversion by elevated Ki67 expression in luminal A and lowered Ki67 expression in luminal B. Gene expression analysis revealed that anti-apoptotic Bcl2 gene expression in both luminal types treated with condition medium has been increased though there has seen no interchange in expression of ER-related and proliferative genes between luminal A (MCF7) and luminal B (MDA-MB361) subtypes, the results of Annexin V-FITC flow cytometry test indicated a decrease in the population of both early and late apoptotic cells in groups treated with both fibroblastic and spheroidal condition medium compared to of control group.

 Under the paracrine influence of fibroblast cells, both luminal A (MCF7) and luminal B (MDA-MB) subtypes of breast cancer gained invasive, anti-apoptotic, and chemoresistance features which are mostly increased by activated(spheroidal) fibroblasts conditioned medium mimicking CAFs. There was no strong proof for interconversion of luminal A and luminal B which share more similarities among breast cancer molecular subtypes.

 Neuroglioma, a classification encompassing tumors arising from glial cells, exhibits variable aggressiveness and depends on tumor grade and stage. Unraveling the EGFR gene alterations, including amplifications (unaltered), deletions, and missense mutations (altered), is emerging in glioma. However, the precise understanding of emerging EGFR mutations and their role in neuroglioma remains limited. This study aims to identify specific EGFR mutations prevalent in neuroglioma patients and investigate their potential as therapeutic targets using FDA- approved drugs for repurposing approach.

 Neuroglioma patient’s data were analyzed to identify the various mutations and survival rates. High throughput virtual screening (HTVS) of FDA-approved (1615) drugs using molecular docking and simulation was executed to determine the potential hits.

 Neuroglioma patient samples (n=4251) analysis reveals 19% EGFR alterations with most missense mutations at V774M in exon 19. The Kaplan-Meier plots show that the overall survival rate was higher in the unaltered group than in the altered group. Docking studies resulted the best hits based on each target's higher docking score, minimum free energy (MMGBSA), minimum kd, ki, and IC50 values. MD simulations and their trajectories show that compounds ZINC000011679756 target unaltered EGFR and ZINC000003978005 targets altered EGFR, whereas ZINC000012503187 (Conivaptan, Benzazepine) and ZINC000068153186 (Dabrafenib, aminopyrimidine) target both the EGFRs. The shortlisted compounds demonstrate favorable residual interactions with their respective targets, forming highly stable complexes. Moreover, these shortlisted compounds have drug- like properties as assessed by ADMET profiling.

 Therefore, compounds (ZINC000012503187 and ZINC000068153186) can effectively target both the unaltered/altered EGFRs as multi-target therapeutic repurposing drugs towards neuroglioma.

Introduction: 

Urinary extracellular vesicles (uEVs) can be considered biomarkers of kidney diseases. EVs derived from podocytes may reflect podocyte damage in different glomerular diseases. IgA nephropathy (IgAN) is one of the most common forms of glomerulonephritis (GN) characterized by proteinuria and hematuria. This study aimed to analyze the uEVs of IgAN patients to understand the pathophysiological processes of the disease at the protein level.

Methods:

 Patients with GN [biopsy-proven IgAN (n = 16) and membranous glomerulonephritis (MGN, n = 16)], and healthy controls (n = 16) were included in this study. The uEVs were extracted, characterized, and analyzed to evaluate the protein levels of candidate markers of IgAN, including vasorin precursor, aminopeptidase N, and ceruloplasmin by western-blot analysis.

Results:

 Higher levels of both podocytes and EVs-related proteins were observed in the pooled urine samples of GN patients compared to the healthy controls. In IgAN patients, uEV-protein levels of vasorin were statistically lower while levels of ceruloplasmin were significantly higher compared to MGN (P = 0.002, P = 0.06) and healthy controls, respectively (P = 0.020, P= 0.001).

Conclusion:

 Different levels of the studied proteins in uEVs may indicate podocyte injury and represent a direct association with the pathology of IgAN and MGN.

 The endothelial cells derived from the human vein cord (HUVECs) are used as in-vitro models for studying cellular and molecular pathophysiology, drug and hormones transport mechanisms, or pathways. In these studies, the proliferation and quantity of cells are important features that should be monitored and assessed regularly. So rapid, easy, noninvasive, and inexpensive methods are favorable for this purpose.

 In this work, a novel method based on fast Fourier transform square-wave voltammetry (FFTSWV) combined with a 3D printed electrochemical cell including two inserted platinum electrodes was developed for non-invasive and probeless rapid in-vitro monitoring and quantification of human umbilical vein endothelial cells (HUVECs). The electrochemical cell configuration, along with inverted microscope images, provided the capability of easy use, online in-vitro monitoring, and quantification of the cells during proliferation.

 HUVECs were cultured and proliferated at defined experimental conditions, and standard cell counts in the initial range of 12 500 to 175 000 were prepared and calibrated by using a hemocytometer (Neubauer chamber) counting for electrochemical measurements. The optimum condition, for FFTSWV at a frequency of 100 Hz and 5 mV amplitude, were found to be a safe electrochemical measurement in the cell culture medium. In each run, the impedance or admittance measurement was measured in a 5 seconds time window. The total measurements were fulfilled at 5, 24, and 48 hours after the seeding of the cells, respectively. The recorded microscopic images before every electrochemical assay showed the conformity of morphology and objective counts of cells in every plate well. The proposed electrochemical method showed dynamic linearity in the range of 12 500-265 000 HUVECs 48 hours after the seeding of cells.

 The proposed electrochemical method can be used as a simple, fast, and noninvasive technique for tracing and monitoring of HUVECs population in in-vitro studies. This method is highly cheap in comparison with other traditional tools. The introduced configuration has the versatility to develop electrodes for the study of various cells and the application of other electrochemical designations.

 As the most common aggressive primary brain tumor, glioblastoma is inevitably a recurrent malignancy whose patients’ prognosis is poor. miR-143 and miR-145, as tumor suppressor miRNAs, are downregulated through tumorigenesis of multiple human cancers, including glioblastoma. These two miRNAs regulate numerous cellular processes, such as proliferation and migration. This research was intended to explore the simultaneous replacement effect of miR-143, and miR-145 on in vitro tumorgenicity of U87 glioblastoma cells.

 U87 cells were cultured, and transfected with miR-143-5p and miR-145-5p. Afterward, the changes in cell viability, and apoptosis induction were determined by MTT assay and Annexin V/PI staining. The accumulation of cells at the cell cycle phases was assessed using the flow cytometry. Wound healing and colony formation assays were performed to study cell migration. qRT-PCR and western blot techniques were utilized to quantify gene expression levels.

 Our results showed that miR-143-5p and 145-5p exogenous upregulation cooperatively diminished cell viability, and enhanced U-87 cell apoptosis by modulating Caspase-3/8/9, Bax, and Bcl-2 protein expression. The combination therapy increased accumulation of cells at the sub-G1 phase by modulating CDK1, Cyclin D1, and P53 protein expression. miR-143/145-5p significantly decreased cell migration, and reduced colony formation ability by the downregulation of c-Myc and CD44 gene expression. Furthermore, the results showed the combination therapy of these miRNAs could remarkably downregulate phosphorylated-AKT expression levels.

 In conclusion, miR-143 and miR-145 were indicated to show cooperative anti- cancer effects on glioblastoma cells via modulating AKT signaling as a new therapeutic approach.

This study aimed to assess the potential of poly (acrylic acid)/tricalcium phosphate nanoparticles (PAA/triCaPNPs) scaffold in terms of biocompatibility and osteoconductivity properties the in-vivo evaluation as well as to investigate the performance of PAA/triCaPNPs scaffold (with or without exosomes derived from UC-MSCs) for bone regeneration of rat critical-sized defect.

PAA/triCaPNPs scaffold was made from acrylic acid (AA) monomer, N,N’-methylenebisacrylamide (MBAA), sodium bicarbonate (SBC), and ammonium persulfate (APS) through freeze-drying method. For in vivo evaluation, we randomly divided 24 rats into three groups. The rat calvarial bone defects were treated as follows: (1) Control group: defects without any treatment, (2) scaffold group: defects treated with scaffold only, (3) scaffold+exo group: defects treated with scaffold enriched with exosomes (1 μg/ μL, 150 μg per rat). Eight- and 12-weeks post-surgery, half of the animals were sacrificed and bone regeneration was examined through micro-computerized tomography (μ-CT), histological staining, and immunohistochemistry (IHC).

Quantitative analysis based on μ-CT scan images at 8 and 12 weeks post-implantation clearly indicated that healing rate for defects that were filled with scaffold enriched with exosome was significantly higher than defects filled with scaffold without exosome. The H&E and Masson staining results revealed that more new bone-like form developed in the scaffold+exo group than that in control and scaffold groups. Further, IHC staining for osteocalcin and CD31 confirmed that more bone healing in the scaffold+exo group at 12 weeks could be associated with osteogenesis and angiogenesis concurrently.

In the present study, we aimed to investigate the therapeutic potential of PAA/ triCaPNPs scaffold as a carrier of human UC-MSC-derived exosome to achieve the exosomecontrolled release on calvarial bone defect. The in vivo results indicated that the exosome-enriched scaffold could effectively minify the defect area and improve the bone healing in rat model, and as such it could be an option for exosome-based therapy.

Cell culture-based technologies are widely utilized in various domains such as drug evaluation, toxicity assessment, vaccine and biopharmaceutical development, reproductive technology, and regenerative medicine. It has been demonstrated that pre-adsorption of extracellular matrix (ECM) proteins including collagen, laminin and fibronectin provide more degrees of support for cell adhesion. The purpose of cell imprinting is to imitate the natural topography of cell membranes by gels or polymers to create a reliable environment for the regulation of cell function. The results of recent studies show that cell imprinting is a tool to guide the behavior of cultured cells by controlling their adhesive interactions with surfaces. Therefore, in this review we aim to compare different cell cultures with the imprinting method and discuss different cell imprinting applications in regenerative medicine, personalized medicine, disease modeling, and cell therapy.