A Multifunctional Aptamer Decorated Lipid Nanoparticles for the Delivery of EpCAM-targeted CRISPR/Cas9 Plasmid for Efficacious In Vivo Tumor Regression.

Epithelial cell adhesion molecule (EpCAM) gene encodes a type-I trans-membrane glycoprotein that is overexpressed in many cancerous epithelial cells and promotes tumor progression by regulating the expression of several oncogenes like c-myc and other cyclins. Because of this tumorigenic association, the EpCAM gene has been a potential target for anti-cancer therapy in recent days. Herein, it is attempted to knockout the proto-oncogenic EpCAM expression by efficiently delivering an all-in-one Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) plasmid via a lipid nanoparticle system made out of synthetic stimuli-sensitive lipids. The plasmid possesses the necessary information in the form of a guide RNA targeted to the EpCAM gene. The aptamer decorated system selectively targets EpCAM overexpressed cells and efficiently inhibits the genetic expression. It has explored the pH-responsive property of the developed lipid nanoparticles and monitored their efficacy in various cancer cell lines of different origins with elevated EpCAM levels. The phenomenon has further been validated in vivo in non-immunocompromised mouse tumor models. Overall, the newly developed aptamer decorated lipid nanoparticle system has been proven to be efficacious for the delivery of EpCAM-targeted CRISPR/Cas9 plasmid.

miR-29a-3p orchestrates key signaling pathways for enhanced migration of human mesenchymal stem cells.

BACKGROUND: The homing of human mesenchymal stem cells (hMSCs) is crucial for their therapeutic efficacy and is characterized by the orchestrated regulation of multiple signaling modules. However, the principal upstream regulators that synchronize these signaling pathways and their mechanisms during cellular migration remain largely unexplored. METHODS: miR-29a-3p was exogenously expressed in either wild-type or DiGeorge syndrome critical region 8 (DGCR8) knockdown hMSCs. Multiple pathway components were analyzed using Western blotting, immunohistochemistry, and real-time quantitative PCR. hMSC migration was assessed both in vitro and in vivo through wound healing, Transwell, contraction, and in vivo migration assays. Extensive bioinformatic analyses using gene set enrichment analysis and Ingenuity pathway analysis identified enriched pathways, upstream regulators, and downstream targets. RESULTS: The global depletion of microRNAs (miRNAs) due to DGCR8 gene silencing, a critical component of miRNA biogenesis, significantly impaired hMSC migration. The bioinformatics analysis identified miR-29a-3p as a pivotal upstream regulator. Its overexpression in DGCR8-knockdown hMSCs markedly improved their migration capabilities. Our data demonstrate that miR-29a-3p enhances cell migration by directly inhibiting two key phosphatases: protein tyrosine phosphatase receptor type kappa (PTPRK) and phosphatase and tensin homolog (PTEN). The ectopic expression of miR-29a-3p stabilized the polarization of the Golgi apparatus and actin cytoskeleton during wound healing. It also altered actomyosin contractility and cellular traction forces by changing the distribution and phosphorylation of myosin light chain 2. Additionally, it regulated focal adhesions by modulating the levels of PTPRK and paxillin. In immunocompromised mice, the migration of hMSCs overexpressing miR-29a-3p toward a chemoattractant significantly increased. CONCLUSIONS: Our findings identify miR-29a-3p as a key upstream regulator that governs hMSC migration. Specifically, it was found to modulate principal signaling pathways, including polarization, actin cytoskeleton, contractility, and adhesion, both in vitro and in vivo, thereby reinforcing migration regulatory circuits.

Systemic cellular migration: The forces driving the directed locomotion movement of cells.

Directional motility is an essential property of cells. Despite its enormous relevance in many fundamental physiological and pathological processes, how cells control their locomotion movements remains an unresolved question. Here, we have addressed the systemic processes driving the directed locomotion of cells. Specifically, we have performed an exhaustive study analyzing the trajectories of 700 individual cells belonging to three different species (Amoeba proteus, Metamoeba leningradensis, and Amoeba borokensis) in four different scenarios: in absence of stimuli, under an electric field (galvanotaxis), in a chemotactic gradient (chemotaxis), and under simultaneous galvanotactic and chemotactic stimuli. All movements were analyzed using advanced quantitative tools. The results show that the trajectories are mainly characterized by coherent integrative responses that operate at the global cellular scale. These systemic migratory movements depend on the cooperative nonlinear interaction of most, if not all, molecular components of cells.

Altered neuroepithelial morphogenesis and migration defects in iPSC-derived cerebral organoids and 2D neural stem cells in familial bipolar disorder.

Bipolar disorder (BD) is a severe mental illness that can result from neurodevelopmental aberrations, particularly in familial BD, which may include causative genetic variants. In the present study, we derived cortical organoids from BD patients and healthy (control) individuals from a clinically dense family in the Indian population. Our data reveal that the patient organoids show neurodevelopmental anomalies, including organisational, proliferation and migration defects. The BD organoids show a reduction in both the number of neuroepithelial buds/cortical rosettes and the ventricular zone size. Additionally, patient organoids show a lower number of SOX2-positive and EdU-positive cycling progenitors, suggesting a progenitor proliferation defect. Further, the patient neurons show abnormal positioning in the ventricular/intermediate zone of the neuroepithelial bud. Transcriptomic analysis of control and patient organoids supports our cellular topology data and reveals dysregulation of genes crucial for progenitor proliferation and neuronal migration. Lastly, time-lapse imaging of neural stem cells in 2D in vitro cultures reveals abnormal cellular migration in BD samples. Overall, our study pinpoints a cellular and molecular deficit in BD patient-derived organoids and neural stem cell cultures.

Mechanistic insights into the antitumoral potential and in vivo antiproliferative efficacy of a silver-based core@shell nanosystem.

This study delves into the biomolecular mechanisms underlying the antitumoral efficacy of a hybrid nanosystem, comprised of a silver core@shell (Ag@MSNs) functionalized with transferrin (Tf). Employing a SILAC proteomics strategy, we identified over 150 de-regulated proteins following exposure to the nanosystem. These proteins play pivotal roles in diverse cellular processes, including mitochondrial fission, calcium homeostasis, endoplasmic reticulum (ER) stress, oxidative stress response, migration, invasion, protein synthesis, RNA maturation, chemoresistance, and cellular proliferation. Rigorous validation of key findings substantiates that the nanosystem elicits its antitumoral effects by activating mitochondrial fission, leading to disruptions in calcium homeostasis, as corroborated by RT-qPCR and flow cytometry analyses. Additionally, induction of ER stress was validated through western blotting of ER stress markers. The cytotoxic action of the nanosystem was further affirmed through the generation of cytosolic and mitochondrial reactive oxygen species (ROS). Finally, in vivo experiments using a chicken embryo model not only confirmed the antitumoral capacity of the nanosystem, but also demonstrated its efficacy in reducing cellular proliferation. These comprehensive findings endorse the potential of the designed Ag@MSNs-Tf nanosystem as a groundbreaking chemotherapeutic agent, shedding light on its multifaceted mechanisms and in vivo applicability.

Selenium Compounds Affect Differently the Cytoplasmic Thiol/Disulfide State in Dermic Fibroblasts and Improve Cell Migration by Interacting with the Extracellular Matrix.

Deficient wound healing is frequently observed in patients diagnosed with diabetes, a clinical complication that compromises mobility and leads to limb amputation, decreasing patient autonomy and family lifestyle. Fibroblasts are crucial for secreting the extracellular matrix (ECM) to pave the wound site for endothelial and keratinocyte regeneration. The biosynthetic pathways involved in collagen production and crosslinking are intimately related to fibroblast redox homeostasis. In this study, two sets of human dermic fibroblasts were cultured in normal (5 mM) and high (25 mM)-glucose conditions in the presence of 1 µM selenium, as sodium selenite (inorganic) and the two selenium amino acids (organic), Se-cysteine and Se-methionine, for ten days. We investigated the ultrastructural changes in the secreted ECM induced by these conditions using scanning electron microscopy (SEM). In addition, we evaluated the redox impact of these three compounds by measuring the basal state and real-time responses of the thiol-based HyPer biosensor expressed in the cytoplasm of these fibroblasts. Our results indicate that selenium compound supplementation pushed the redox equilibrium towards a more oxidative tone in both sets of fibroblasts, and this effect was independent of the type of selenium. The kinetic analysis of biosensor responses allowed us to identify Se-cysteine as the only compound that simultaneously improved the sensitivity to oxidative stimuli and augmented the disulfide bond reduction rate in high-glucose-cultured fibroblasts. The redox response profiles showed no clear association with the ultrastructural changes observed in matrix fibers secreted by selenium-treated fibroblasts. However, we found that selenium supplementation improved the ECM secreted by high-glucose-cultured fibroblasts according to endothelial migration assessed with a wound healing assay. Direct application of sodium selenite and Se-cysteine on purified collagen fibers subjected to glycation also improved cellular migration, suggesting that these selenium compounds avoid the undesired effect of glycation.

Sphingosine 1-phosphate signaling axis mediates neuropeptide S-induced invasive phenotype of endometriotic cells.

Endometriosis is a chronic gynecological syndrome characterized by endometrial cell invasion of the extra-uterine milieu, pelvic pain and infertility. Treatment relies on either symptomatic drugs or hormonal therapies, even though the mechanism involved in the onset of endometriosis is yet to be elucidated. The signaling of sphingolipid sphingosine 1-phosphate (S1P) is profoundly dysregulated in endometriosis. Indeed, sphingosine kinase (SK)1, one of the two isoenzymes responsible for S1P biosynthesis, and S1P1, S1P3 and S1P5, three of its five specific receptors, are more highly expressed in endometriotic lesions compared to healthy endometrium. Recently, missense coding variants of the gene encoding the receptor 1 for neuropeptide S (NPS) have been robustly associated with endometriosis in humans. This study aimed to characterize the biological effect of NPS in endometriotic epithelial cells and the possible involvement of the S1P signaling axis in its action. NPS was found to potently induce cell invasion and actin cytoskeletal remodeling. Of note, the NPS-induced invasive phenotype was dependent on SK1 and SK2 as well as on S1P1 and S1P3, given that the biological action of the neuropeptide was fully prevented when one of the two biosynthetic enzymes or one of the two selective receptors was inhibited or silenced. Furthermore, the RhoA/Rho kinase pathway, downstream to S1P receptor signaling, was found to be critically implicated in invasion and cytoskeletal remodeling elicited by NPS. These findings provide new information to the understanding of the molecular mechanisms implicated in endometriosis pathogenesis, establishing the rationale for non-hormonal therapeutic targets for its treatment.

Efeito da ingestão do suco de uva integral sobre a migração de leucócitos em mulheres com sobrepeso. Participação da delfinidina-3-glicosídeo como potencial modulador do perfil inflamatório e da migração celular / Effect of whole grape juice intake on leukocyte migration in overweight women. Participation of delphinidin-3-glycoside as a potential modulator of inflammatory profile and cell migration

Concentrações aumentadas de tecido adiposo corporal observadas no sobrepeso e obesidade, podem gerar a produção de diversos mediadores inflamatórios com ação direta ou indireta em influenciar a capacidade de proliferação e diferenciação das células hematopoéticas e, consequentemente, a complexa regulação que envolve os processos de migração celular. Sabe-se que o recrutamento contínuo de leucócitos durante vários estágios do processo inflamatório apresenta importante papel na gênese desse processo, participando intensamente na perpetuação da inflamação. Na literatura, vários estudos demostraram acapacidade anti-inflamatória das antocianinas sobre vários órgãos; contudo poucos estudos avaliam a influência das antocianinas sobre a migração celular. As antocianinas pertencem à ampla classe dos flavonoides que estão presentes em uma ampla variedade de frutas, vegetais e bebidas e são os compostos responsáveis pelas cores azul, violeta e vermelha desses alimentos. Sendo assim, pretende-se nesse estudo avaliar o efeito da delfinidina-3-glicosídeo, uma antocianina presente em abundância em diversas frutas com destaque para o suco de uva integral, sobre os processos de migração leucocitária. Para tanto esse trabalho foi dividido em duas etapas: (i) uma realizada em pacientes com sobrepeso que consumiram suco de uva integral e (ii) uma segunda etapa, in vitro, onde foi avaliado o efeito da delfinidina-3-glicosídeo sobre mecanismos envolvidos na modulação dos processos de migração leucocitária. Na etapa inicial com estudos in vivo, foram avaliados parâmetros bioquímicos, hematológicos, bem como a expressão de moléculas de adesão de células polimorfonucleares do sangue periférico e a quantificação de citocinas inflamatórias e alguns genes envolvidos nos processos de inflamação e migração celular. Na primeira etapa, a ingestão de suco de uva não alterou o perfil lipídico/inflamatório ou a contagem de leucócitos, entretanto, reduziu os valores circulantes de sICAM e sVCAM. Na segunda etapa, os resultados in vitro mostraram que a delfinidina reduziu a taxa de migração e a expressão de células CD11/CD18 positivas, reduziu a expressão gênica de ICAM-1 e a fosforilação e expressão gênica de NFkB, reduzindo também a produção de IL-6, IL-8 e CCL2

Increased concentrations of body adipose tissue observed in overweight and obesity may generate the production of several inflammatory mediators that can act directly or indirectly on the hematopoietic cells capacity of proliferation and differentiation and, consequently, the complex regulation that involves the processes of cell migration. It is known that the continuous recruitment of leukocytes during various stages of the inflammatory process plays an important role in the genesis of this process, intensely participating in the perpetuation of inflammation. In the literature, several studies have demonstrated the anti-inflammatory capacity of anthocyanins on various organs; however, few studies have evaluated the influence of anthocyanins on cell migration. Anthocyanins belong to the broad class of flavonoids that are present in a wide variety of fruits, vegetables and beverages and are the compounds responsible for the blue, violet and red colors of these foods. Thus, this study intends to evaluate the effect of delphinidin-3-glycoside, an anthocyanin present in abundance in several fruits, especially whole grape juice, on leukocyte migration processes. Therefore, this study was divided into two stages: (i) one performed in overweight patients who consumed whole grape juice and (ii) a second stage, in vitro, where the effect of delphinidin-3-glycoside on mechanisms involved in the modulation of leukocyte migration processes was evaluated. In the initial stage with in vivo studies, biochemical and hematological parameters were evaluated, as well as the expression of adhesion molecules of polymorphonuclear cells of peripheral blood and the quantification of inflammatory cytokines and some genes involved in the processes of inflammation and cell migration. In the first stage, the intake of grape juice did not alter the lipid/inflammatory profile or the leukocyte count, however, it reduced the circulating values of sICAM and sVCAM. In the second step, in vitro results showed that delphinidine reduced the migration rate and expression of CD11/CD18 positive cells, reduced ICAM-1 gene expression and NFkB phosphorylation and gene expression, and also reduced IL-6, IL-8 and CCL2 production

Green synthesis of ginger-encapsulated zinc oxide nanoparticles: Unveiling their characterization and selective cytotoxicity on MDA-MB 231 breast cancer cells.

Zinc oxide nanoparticles (ZnO-NPs) were synthesized using ginger (Zingiber officinale) extracts in a green synthesis approach and evaluated their in vitro cytotoxicity effect on the MDA-MB 231 breast cancer cell line. The bottom-up approach was employed to develop the green-synthesized ginger-encapsulated ZnO-NPs (GZnO-NPs) without using hazardous substances. The most substantial Fourier-transform infrared absorption peak of the ginger root extract was seen at 1634.24 cm-1. The peak also confirmed the presence of ginger root extract-encapsulated ZnO-NPs at 1556.79, 1471.54, and 1019.83 cm-1. It indicates that the biomolecules found in plant extracts behave as capping agents, aiding in the formation of nanoparticles. The mean particle sizes (PSs) of optimized GZnO-NPs of the ratios 1:2 were found to be 104.01 ± 7.12 nm with a zeta potential of -11.5 ± 1.31 mV. The X-ray diffraction and scanning electron microscope analysis confirmed that the prepared nanoparticles were spherical and crystalline, with PS ranging from 100 to 150 nm. The GZnO-NPs were subjected to MTT assay and cellular migration potential, and it was found that the inhibitory concentration on the MDA-MB 231 (breast) cancer cell line and scratch area showed a dose-dependent efficacy. The successfully green-synthesized GZnO-NPs effectively induced cell death in the MDA-MB 231 cancer cell line. The scratch assay results confirmed that prepared GZnO-NPs inhibited the proliferation and migration of cancerous cells.

miRNAs from Inflamed Gingiva Link Gene Signaling to Increased MET Expression.

Several array-based microRNA (miRNA) expression studies independently showed increased expression of miRNAs hsa-miR-130a-3p, -142-3p, -144-3p, -144-5p, -223-3p, -17-5p, and -30e-5p in gingiva affected by periodontal inflammation. We aimed to determine direct target genes and signaling pathways regulated by these miRNAs to identify processes relevant to gingival inflammatory responses and tissue homeostasis. We transfected miRNA mimics (mirVana) for each of the 7 miRNAs separately into human primary gingival fibroblasts cultured from 3 different donors. Following RNA sequencing, differential gene expression and second-generation gene set enrichment analyses were performed. miRNA inhibition and upregulation was validated at the transcript and protein levels using quantitative reverse transcriptase polymerase chain reaction, Western blotting, and reporter gene assays. All 7 miRNAs significantly increased expression of the gene MET proto-oncogene, receptor tyrosine kinase (MET). Expression of known periodontitis risk genes CPEB1, ABCA1, and ATP6V1C1 was significantly repressed by hsa-miR-130a-3p, -144-3p, and -144-5p, respectively. The genes WASL, ENPP5, ARL6IP1, and IDH1 showed the most significant and strongest downregulation after hsa-miR-142-3p, -17-5p, -223-3p, and -30e-5p transfection, respectively. The most significantly regulated gene set of each miRNA related to cell cycle (hsa-miRNA-144-3p and -5p [Padj = 4 × 10-40 and Padj = 4 × 10-6], -miR-17-5p [Padj = 9.5 × 10-23], -miR-30e-5p [Padj = 8.2 × 10-18], -miR-130a-3p [Padj = 5 × 10-15]), integrin cell surface interaction (-miR-223-3p [Padj = 2.4 × 10-7]), and interferon signaling (-miR-142-3p [Padj = 5 × 10-11]). At the end of acute inflammation, gingival miRNAs bring together complex regulatory networks that lead to increased expression of the gene MET. This underscores the importance of mesenchymal cell migration and invasion during gingival tissue remodeling and proliferation in restoring periodontal tissue homeostasis after active inflammation. MET, a receptor of the mitogenic hepatocyte growth factor fibroblast secreted, is a core gene of this process.

Epithelial-to-mesenchymal transition status correlated with ultrastructural features, and TP53 mutation in patient-derived oral cancer cell lines.

BACKGROUND: Oral Squamous Cell Carcinoma (OSCC) is a highly prevalent cancer in the Indian subcontinent. The major cause of mortality in OSCC patients is metastasis. Epithelial-to-mesenchymal transition (EMT) marks an important step in the metastatic process. Additionally, TP53, an important tumor suppressor gene, is also a significant determinant of the treatment outcome, and also plays a role in EMT. Therefore, understanding the interconnections between ultrastructural features, EMT status and TP53 mutational status is of vital importance. METHODS AND RESULTS: The ultrastructure of five OSCC cell lines was visualized by transmission electron microscopy. Trans-well invasion and migration assays as well as scratch-wound assay, and the expression of various EMT-related genes were utilized to assess the EMT status of the cell lines. The TP53 exons were amplified for the ACOSC3, ACOSC4 and ACOSC16 cell lines and sequenced and the mutations in the gene were identified by sequence alignment. The TP53 mutation in the UPCI:SCC029B cell line has been previously reported, while UPCI:SCC040 has been reported to harbor a wild type TP53. The ACOSC4 cell line which showed the shortest intercellular gaps, also had the least invasive and migratory potential. Interestingly, ACOSC4 showed the highest expression of E-cadherin and the lowest expression of Vimentin, TWIST1, ZEB1, and MMPs. Additionally, TP53 gene of ACOSC4 was unmutated, whereas the ACOSC3 and ACOSC16 harbored TP53 mutations. The mutation in ACOSC3 (R196*) was also found in 7 TCGA samples. Similarly, the UPCI:SCC040 cell line that harbors a wild type TP53 showed shorter intracellular gaps. CONCLUSIONS: Cellular migratory properties are associated with cellular ultrastructure, epithelial-to-mesenchymal transition status and the status of TP53 mutation in the genome.

Starvation-inactivated MTOR triggers cell migration via a ULK1-SH3PXD2A/TKS5-MMP14 pathway in ovarian carcinoma.

ABBREVIATIONS: AMPK: AMP-activated protein kinase; CHX: cycloheximide; RAD001: everolimus; HBSS: Hanks' balanced salt solution; LC-MS/MS: liquid chromatography-mass spectrometry/mass spectrometry; MMP14: matrix metallopeptidase 14; MTOR: mechanistic target of rapamycin kinase; MAPK: mitogen-activated protein kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; PtdIns3P: phosphatidylinositol-3-phosphate; PX: phox homology; SH3: Src homology 3; SH3PXD2A/TKS5: SH3 and PX domains 2A; SH3PXD2A-[6A]: S112A S142A S146A S147A S175A S348A mutant; ULK1: unc-51 like autophagy activating kinase 1.

Starch-based films doped with porphyrinoid photosensitizers for active skin wound healing.

Starch is a biodegradable and biocompatible carbohydrate that, when combined with bioactive molecules, can be processed as biomimetic platforms with enhanced performance, allowing its use as active wound dressing materials. Porphyrinoid photosensitizers can tune the physicochemical/functional profile of biomacromolecules, allowing their use in anti-infective strategies. In this work, the feasibility of using the cationic 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetraiodide (TMPyP) to enhance the physicochemical, mechanical, antimicrobial performance, and wound healing ability of casted starch-based films was studied. TMPyP conferred a reddish coloration to the films, maintaining their pristine transparency. It increased by 87 % the films hydrophobicity and, depending on the TMPyP used, conferred mobility to the starch polymeric chains. Starch/TMPyP-based films effectively photoinactivated Escherichia coli (>99.99 %) and favored the wound healing process, even in the absence of light. Therefore, the incorporation of TMPyP into starch-based formulations revealed to be a promising strategy to tune the films compaction degree while giving rise to water tolerant and photosensitive biomaterials that can act as multitarget antimicrobial medical dressings and glycocarriers of active compounds relevant for effective skin wound healing.

Inhibitory effect of hydroalcoholic extract of Cunila spicata Benth. on phlogistic agents-induced cellular migration in the airways of mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cunila spicata Benth. Is a creeping and aromatic plant that has an ethnopharmacological indication in the southern region of Brazil, in the Araucaria Forest biome. It's used as an alternative therapy for respiratory diseases for men and animals since the "tropeirista" movement in colonial Brazil. AIM OF THE STUDY: Investigate the influence of oral administration of hydroalcoholic extract of C. spicata (HECS) on cellular inflammatory processes in the airways of mice. MATERIAL AND METHODS: The HECS was obtained by the ultrasound-assisted technique. Phytochemical analysis of the volatile compounds was performed using gas-chromatography with mass spectrometry. To investigate the biological activity of HECS, the animals were pre-treated with HECS orally 1 h before and the bronchoalveolar lavage (BAL) model was used, in which the animals were subjected to inhalation of different pro-inflammatory agents such as carrageenan (CAR), histamine (HIST), capsaicin (CAP), bradykinin (BK), and 48/80 compound (C48/80). After sacrifice, BAL sample was collected from the bronchi and was analyzed for total and differential white blood cell counts, compared with control groups. RESULTS: Fourteen volatile phytocompounds were identified in the HECS, the main ones being 2-bornanone, menthofuran and camphene. Among the HECS treated animals, all showed significant maximal inhibition (MI) when challenged with pro-inflammatory agents by nebulization. In the group of animals that received CAR, the MI was 72.77 ± 3.88%, compared to the negative control (NC). There was a predominance of lymphocytes (59.18 ± 4.07%). For the HIST group, MI was 64.03 ± 4.33%, in relation to the NC, with predominance in macrophages number (76.53 ± 2.78%). In the CAP group, the MI obtained was 38.90 ± 11.31%, with greater macrophage migration occurring (80.98 ± 3.44%) in the higher dose. For animals subjected to BK inhalation, the MI was 48.63 ± 3.95% with macrophages predominance (64.33 ± 10.04%). In the group that received C48/80 inhalation, the MI was 40.25 ± 5.30% (100 mg kg-1), with higher occurrence of macrophages (72.97 ± 4.23%). CONCLUSION: Our results suggest that HECS had a non-specific inhibitory effect on cellular migration induced by different pro-inflammatory agents, reducing inflammation in airways of mice. These effects coincide and support its ethnopharmacological use as an alternative medicine for respiratory diseases in regions where the plant is prevalent.

Suppressed Migration and Enhanced Cisplatin Chemosensitivity in Human Cancer Cell Lines by Tuning the Molecular Mobility of Supramolecular Biomaterials.

Cancer cells recognize physical cues transmitted from the surrounding microenvironment, and accordingly alter the migration and chemosensitivity. Cell adhesive biomaterials with tunable physical properties can contribute to the understanding of cancer cell responses, and development of new cancer therapies. Previously, it was reported that polyrotaxane-based surfaces with molecular mobility effectively modulate cellular functions via the yes-associated protein (YAP)-related signaling pathway. In the present study, the impact of molecular mobility of polyrotaxane surfaces on the migration and chemosensitivity of lung (A549), pancreatic (BxPC-3), and breast cancer (MDA-MB-231) cell lines is investigated, and it is found that the cellular spreading of adherent A549 and BxPC-3 cells and nuclear YAP translocation are promoted on low-mobility surfaces, suggesting that cancer cells alter their subcellular YAP localization in response to molecular mobility. Furthermore, low-mobility surfaces suppress cellular migration more than high-mobility surfaces. Additionally, low-mobility surfaces promote the cisplatin chemosensitivity of each cancer cell line to a greater extent than high-mobility surfaces. These results suggest that the molecular mobility of polyrotaxane surfaces suppresses cellular migration and enhances chemosensitivity via the subcellular translocation of YAP in cancer cells. Biointerfaces based on polyrotaxanes can thus be a new platform for elucidating cancer cell migration and chemoresistance mechanisms.

Discovery and characterization of a functional scFv for CCR2 inhibition via an extracellular loop.

The chemokine receptor CCR2 plays a key role in cellular migration and inflammatory processes. While tremendous progress has been made in elucidating CCR2 function and inhibition, the majority of approaches target its N-terminal domain and less is known about the function of the remaining extracellular loops and their potential as targets. Here, we used phage display to identify an antibody-derived scFv (single chain variable fragment) clone that specifically targets the second extracellular epitope of CCR2 (ECL2) for inhibition. Using in silico molecular docking, we identified six potential primary binding conformations of the novel scFv to the specified CCR2 epitope. In silico molecular dynamic analysis was used to determine conformational stability and identify protein-protein interactions. Umbrella sampling of a range of configurations with incrementally increasing separation of scFv and target generated by force pulling simulations was used to calculate binding energies. Downstream characterization by ELISA showed high binding affinity of the ECL2-scFv to CCR2. Furthermore, we showed that blocking the second extracellular loop inhibits macrophage migration and polarized macrophages towards M1 inflammatory cytokine production as potently as lipopolysaccharide (LPS). These studies highlight the applicability of epitope-specific targeting, emphasize the importance of in silico predictive modeling, and warrant further investigation into the role of the remaining epitopes of CCR2.

Magnetic Field Intervention Enhances Cellular Migration Rates in Biological Scaffolds.

The impact of magnetic fields on cellular function is diverse but can be described at least in part by the radical pair mechanism (RPM), where magnetic field intervention alters reactive oxygen species (ROS) populations and downstream cellular signaling. Here, cellular migration within three-dimensional scaffolds was monitored in an applied oscillating 1.4 MHz radiofrequency (RF) magnetic field with an amplitude of 10 µT and a static 50 µT magnetic field. Given that cellular bioenergetics can be altered based on applied RF magnetic fields, this study focused on a magnetic field configuration that increased cellular respiration. Results suggest that RF accelerated cell clustering and elongation after 1 day, with increased levels of clustering and cellular linkage after 7 days. Cell distribution analysis within the scaffolds revealed that the clustering rate during the first day was increased nearly five times in the RF environment. Electron microscopy provided additional topological information and verified the development of fibrous networks, with a cell-derived matrix (CDM) visualized after 7 days in samples maintained in RF. This work demonstrates time-dependent cellular migration that may be influenced by quantum biology (QB) processes and downstream oxidative signaling, enhancing cellular migration behavior.

Primary TSC2-/meth Cells Induce Follicular Neogenesis in an Innovative TSC Mouse Model.

Cutaneous lesions are one of the hallmarks of tuberous sclerosis complex (TSC), a genetic disease in which mTOR is hyperactivated due to the lack of hamartin or tuberin. To date, novel pharmacological treatments for TSC cutaneous lesions that are benign but still have an impact on a patient's life are needed, because neither surgery nor rapamycin administration prevents their recurrence. Here, we demonstrated that primary TSC2-/meth cells that do not express tuberin for an epigenetic event caused cutaneous lesions and follicular neogenesis when they were subcutaneously injected in nude mice. Tuberin-null cells localized in the hair bulbs and alongside mature hairs, where high phosphorylation of S6 and Erk indicated mTOR hyperactivation. Interestingly, 5-azacytidine treatment reduced hair follicles, indicating that chromatin remodeling agents might be effective on TSC lesions in which cells lack tuberin for an epigenetic event. Moreover, we demonstrated that the primary TSC2-/meth cells had metastatic capability: when subcutaneously injected, they reached the bloodstream and lymphatics and invaded the lungs, causing the enlargement of the alveolar walls. The capability of TSC2-/meth cells to survive and migrate in vivo makes our mouse model ideal to follow the progression of the disease and test potential pharmacological treatments in a time-dependent manner.

Characterizing cell recruitment into isotropic and anisotropic biomaterials by quantification of spatial density gradients in vitro.

The success of cell-free in situ tissue engineering approaches depends on an appropriate recruitment of autologous cells from neighboring tissues. This identifies cellular migration as a critical parameter for the pre-clinical characterization of biomaterials. Here, we present a new method to quantify both the extent and the spatial anisotropy of cell migration in vitro. For this purpose, a cell spheroid is used as a cell source to provide a high number of cells for cellular invasion and, at the same time, to guarantee a controlled and spatially localized contact to the material. Therefore, current limitations of assays based on 2D cell sources can be overcome. We tested the method on three biomaterials that are in clinical use for soft tissue augmentation in maxilla-facial surgery and a substrate used for 3D in vitro cell culture. The selected biomaterials were all collagen-derived, but differed in their internal architecture. The analysis of cellular isodensity profiles within the biomaterials allowed the identification of the extent and the preferential directions of migration, as well as their relation to the biomaterials and their specific pore morphologies. The higher cell density within the biomaterials resulting from the here-introduced cell spheroid assay compared to established 2D cell layer assays suggests a better representation of the in vivo situation. Consequently, the presented method is proposed to advance the pre-clinical evaluation of cell recruitment into biomaterials, possibly leading to an improved prediction of the regeneration outcome.

Optical Cellular Micromotion: A New Paradigm to Measure Tumor Cells Invasion within Gels Mimicking the 3D Tumor Environments.

Measuring tumor cell invasiveness through 3D tissues, particularly at the single-cell level, can provide important mechanistic understanding and assist in identifying therapeutic targets of tumor invasion. However, current experimental approaches, including standard in vitro invasion assays, have limited physiological relevance and offer insufficient insight into the vast heterogeneity in tumor cell migration through tissues. To address these issues, here the concept of optical cellular micromotion is reported on, where digital holographic microscopy is used to map the optical nano- to submicrometer thickness fluctuations within single-cells. These fluctuations are driven by the dynamic movement of subcellular structures including the cytoskeleton and inherently associated with the biological processes involved in cell invasion within tissues. It is experimentally demonstrated that the optical cellular micromotion correlates with tumor cells motility and invasiveness both at the population and single-cell levels. In addition, the optical cellular micromotion significantly reduced upon treatment with migrastatic drugs that inhibit tumor cell invasion. These results demonstrate that micromotion measurements can rapidly and non-invasively determine the invasive behavior of single tumor cells within tissues, yielding a new and powerful tool to assess the efficacy of approaches targeting tumor cell invasiveness.