Dynamically Mapping the Topography and Stiffness of the Leading Edge of Migrating Cells Using AFM in Fast-QI Mode.

Cell migration profoundly influences cellular function, often resulting in adverse effects in various pathologies including cancer metastasis. Directly assessing and quantifying the nanoscale dynamics of living cell structure and mechanics has remained a challenge. At the forefront of cell movement, the flat actin modules─the lamellipodium and the lamellum─interact to propel cell migration. The lamellipodium extends from the lamellum and undergoes rapid changes within seconds, making measurement of its stiffness a persistent hurdle. In this study, we introduce the fast-quantitative imaging (fast-QI) mode, demonstrating its capability to simultaneously map both the lamellipodium and the lamellum with enhanced spatiotemporal resolution compared with the classic quantitative imaging (QI) mode. Specifically, our findings reveal nanoscale stiffness gradients in the lamellipodium at the leading edge, where it appears to be slightly thinner and significantly softer than the lamellum. Additionally, we illustrate the fast-QI mode's accuracy in generating maps of height and effective stiffness through a streamlined and efficient processing of force-distance curves. These results underscore the potential of the fast-QI mode for investigating the role of motile cell structures in mechanosensing.

miR-301a Deficiency Attenuates the Macrophage Migration and Phagocytosis through YY1/CXCR4 Pathway.

(1) Background: the miR-301a is well known involving the proliferation and migration of tumor cells. However, the role of miR-301a in the migration and phagocytosis of macrophages is still unclear. (2) Methods: sciatic nerve injury, liver injury models, as well as primary macrophage cultures were prepared from the miR-301a knockout (KO) and wild type (WT) mice to assess the macrophage's migration and phagocytosis capabilities. Targetscan database analysis, Western blotting, siRNA transfection, and CXCR4 inhibition or activation were performed to reveal miR301a's potential mechanism. (3) Results: the macrophage's migration and phagocytosis were significantly attenuated by the miR-301a KO both in vivo and in vitro. MiR-301a can target Yin-Yang 1 (YY1), and miR-301a KO resulted in YY1 up-regulation and CXCR4 (YY1's down-stream molecule) down-regulation. siYY1 increased the expression of CXCR4 and enhanced migration and phagocytosis in KO macrophages. Meanwhile, a CXCR4 inhibitor or agonist could attenuate or accelerate, respectively, the macrophage migration and phagocytosis. (4) Conclusions: current findings indicated that miR-301a plays important roles in a macrophage's capabilities of migration and phagocytosis through the YY1/CXCR4 pathway. Hence, miR-301a might be a promising therapeutic candidate for inflammatory diseases by adjusting macrophage bio-functions.

Effect of Solanum lyratum Polysaccharide on Malignant Behaviors of Lung Cancer Cells by Regulating the Circ_UHRF1/miR-513b-5p Axis.

This study aimed to investigate the effect of Solanum lyratum polysaccharide on the malignant behavior of lung cancer cells and its possible mechanism. For this purpose, lung cancer A549 cells were cultured in vitro and treated with different doses (0.4, 0.8, 1.2 mg/mL) of Solanum lyratum polysaccharide for 24 h. Then cell proliferation was detected by the CCK-8 method and clone formation test. Transwell test was used to detect cell migration and invasion, and flow cytometry was used to detect cell apoptosis. The protein expressions of Bax and Bcl-2 in cells were detected by Western blotting, and the protein expressions of circ_UHRF1 and miR-513b-5p were detected by the qRT-PCR method. Pearson correlation was used to analyze the correlation between circ_UHRF1 and miR-513b-5p expressions in lung cancer tissues. Results showed that compared with the control group, the proliferation inhibition rate and apoptosis rate of A549 cells that intervened with the Solanum lyratum polysaccharide and expression of Bax protein in the cells were all increased (P<0.05), but the number of clones, migration and invasion and the protein expression of Bcl-2 were all decreased (P<0.05), and were dose-dependent. The expression of circ_UHRF1 in A549 cells that intervened with the S. lyratum polysaccharide was decreased (P<0.05), but the expression of miR-513b-5p was increased (P<0.05). The expression of circ_UHRF1 in lung cancer tissues was higher than that of adjacent tissues (P<0.05), and the expression of miR-513b-5p was lower than that of adjacent tissues (P<0.05). The expressions of circ_UHRF1 and miR-513b-5p in lung cancer tissues were negatively correlated (r=-0.861, P<0.05). Circ_UHRF1 could target miR-513b-5p, and the expression of miR-513b-5p in A549 cells knocking down circ_UHRF1 was increased. After knocking down circ_UHRF1, the proliferation inhibition rate and apoptosis rate of A549 cells and protein expression of Bax in the cells were all increased (P<0.05), but the number of clones, migration and invasion and the protein expression of Bcl-2 were all decreased (P<0.05). Up-regulation of circ_UHRF1 reduced the effects of S. lyratum polysaccharide on the proliferation, migration, invasion and apoptosis of A549 cells. In general, S. lyratum polysaccharide could inhibit the proliferation, migration and invasion of lung cancer A549 cells, and induce cell apoptosis. Its mechanism may be related to the regulation of the circ_UHRF1/miR-513b-5p axis.

[Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts].

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.

Assembly and Use of a Microfluidic Device to Study Nuclear Mechanobiology During Confined Migration.

Cancer metastasis, that is, the spreading of tumor cells from the primary tumor to distant sites, requires cancer cells to travel through pores substantially smaller than their cross section . This "confined migration" requires substantial deformation by the relatively large and rigid nucleus, which can impact nuclear compartmentalization, trigger cellular mechanotransduction pathways, and increase genomic instability. To improve our understanding of how cells perform and respond to confined migration, we developed polydimethylsiloxane (PDMS) microfluidic devices in which cells migrate through a precisely controlled "field of pillars" that closely mimic the intermittent confinement of tumor microenvironments and interstitial spaces. The devices can be designed with various densities of pillars, ranging from a very low density that does not require nuclear deformation to high densities that present microenvironment conditions with severe confinement. The devices enable assessment of cellular fitness for confined migration based on the distance traveled through the constriction area over several days. In this protocol, we present two complementary techniques to generate silicon master molds for the device fabrication: (1) SU-8 soft lithography for rapid prototyping and for devices with relatively large features; and (2) reactive ion etching (RIE) to achieve finer features and more durable molds. In addition, we describe the production, use, and validation of the devices, along with the analysis pipeline for experiments using the devices with fluorescently labeled cells. Collectively, this protocol enables the study of confined migration and is readily amendable to investigate other aspects of confined migration mechanobiology, such as nuclear pore complex function in response to nuclear deformation.

Mathematical modeling of therapeutic neural stem cell migration in mouse brain with and without brain tumors.

Neural stem cells (NSCs) offer a potential solution to treating brain tumors. This is because NSCs can circumvent the blood-brain barrier and migrate to areas of damage in the central nervous system, including tumors, stroke, and wound injuries. However, for successful clinical application of NSC treatment, a sufficient number of viable cells must reach the diseased or damaged area(s) in the brain, and evidence suggests that it may be affected by the paths the NSCs take through the brain, as well as the locations of tumors. To study the NSC migration in brain, we develop a mathematical model of therapeutic NSC migration towards brain tumor, that provides a low cost platform to investigate NSC treatment efficacy. Our model is an extension of the model developed in Rockne et al. (PLoS ONE 13, e0199967, 2018) that considers NSC migration in non-tumor bearing naive mouse brain. Here we modify the model in Rockne et al. in three ways: (i) we consider three-dimensional mouse brain geometry, (ii) we add chemotaxis to model the tumor-tropic nature of NSCs into tumor sites, and (iii) we model stochasticity of migration speed and chemosensitivity. The proposed model is used to study migration patterns of NSCs to sites of tumors for different injection strategies, in particular, intranasal and intracerebral delivery. We observe that intracerebral injection results in more NSCs arriving at the tumor site(s), but the relative fraction of NSCs depends on the location of injection relative to the target site(s). On the other hand, intranasal injection results in fewer NSCs at the tumor site, but yields a more even distribution of NSCs within and around the target tumor site(s).

FoxO1 Regulates Neuropeptide Y and Pro-opiomelanocortin in the Hypothalamus of Rat Offspring Small for Gestational Age.

Adulthood obesity, diabetes, and metabolic diseases are associated with small for gestational age (SGA) newborns. This association could be related to abnormal appetite signaling pathways in the hypothalamus. This study investigated the appetite regulation by the hypothalamus of SGA newborns by establishing an SGA rat model and culturing SGA neural progenitor cells (NPCs) in vitro. Models of SGA were established by maternal food restriction embryonic day 10 (E10). At E18, postpartum day 1 (P1), and P5, hypothalamic neural precursor cells (NPCs) of offspring were cultured in vitro. Immunofluorescence, Western blot (WB), and qRT-PCR were used to assess NPY, POMC, and FoxO1 expression levels. The effects on mRNA expression of the FoxO1-specific inhibitor AS1842856 were examined. The results indicated that compared with controls, NPY was higher, and POMC was lower at embryonic day 18 (E18), postpartum day 1 (P1), and P5. The proliferation and migration of NPCs in the third ventricle of SGA hypothalami were lower than in controls. After treatment with the FoxO1 inhibitor AS1842856, the differences in the mRNA expression of NPY and POMC between the two groups disappeared. NPY and POMC mRNA levels in the SGA group treated with AS1842856 were not significantly different compared with the control group without AS1842856 treatment. In conclusion, SGA pups showed an increase in appetite-promoting NPY and a decrease in appetite-reducing POMC, probably contributing to adulthood weight gain, obesity, and endocrine disorders.

Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts / 中华烧伤杂志

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.

Curcumol inhibits EBV-positive Nasopharyngeal carcinoma migration and invasion by targeting nucleolin.

Metastasis is a major cause of recurrence and death in patients with EBV-positive Nasopharyngeal carcinoma (NPC). Previous reports documented that curcumol has both anti-cancer and anti-viral effects, but there is little literature systematically addressing the mechanism of curcumol in EBV-positive tumors. Previously we found that nucelolin (NCL) is a target protein of curcumol in CNE2 cells, an EBV-negative NPC, and in this experiment, we reported a critical role for NCL in promoting migration and invasion of C666-1 cells, an EBV-positive NPC, and found that the expression of NCL determined the level of curcumol's efficacy. Mechanistically, NCL interacted with Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) to activate VEGFA/VEGFR1/PI3K/AKT signaling pathway, which in turn promoted NPC cell invasion and metastasis. Moreover, further study showed that the differential expression of NCL and curcumol intervention only had a regulatory effect on the nuclear accumulation of VEGFR1, which strengthened the anti-cancer effect of curcumol mediated through NCL. Our findings indicated that curcumol exerted anti EBV-positive NPC invasion and metastasis by downregulating EBNA1 and inhibiting VEGFA/VEGFR1/PI3K/AKT signaling by targeting NCL, which provides a novel pharmacological basis for curcumol's clinical use in treating patients with EBV-positive NPC.

Ruyiping extract reduces lung metastasis in triple negative breast cancer by regulating macrophage polarization.

Lung metastasis of Triple-negative breast cancer (TNBC) causes severe breath-related events and poor prognosis. Ruyiping (RYP), a traditional Chinese medicine prescription, is used to treat breast cancer lung metastasis in clinical practice. This study was to explore the anti-lung-metastatic activities and mechanism of RYP extract by regulating macrophage polarization. The results showed that RYP can inhibit the viability and induce the apoptosis of TNBC cells. In in vitro experiments, RYP significantly inhibited the invasion and migration ability of TNBC cells promoted by M2, the subtype of macrophage which increased TNBC metastasis related genes. In in vivo experiments, RYP reduced the TNBC progression and lung metastasis. M2/M1 ration in the lung and M2 in the tumor was reduced by RYP, as well as M2 master regulator Stat6. Therefore, RYP extract may exhibit anti-lung metastasis function by reducing M2 in both tumor and lung through reducing Stat6.

Kaempferia parviflora extract inhibits TNF-α-induced release of MCP-1 in ovarian cancer cells through the suppression of NF-κB signaling.

Ovarian clear cell carcinoma (OCCC) is an uncommon subtype of epithelial cell ovarian cancers (EOCs) that has poor response to conventional platinum-based therapy. Therefore, finding new potential therapeutic agents is required. Since inflammatory cytokine, tumor necrosis factor alpha (TNF-α), is strongly expressed in EOCs and associated with the level of tumor grade, disruption of this inflammation pathway may provide another potential target for OCCC treatment. We previously reported that Kaempferia parviflora (KP) extract decreased cell proliferation and induced apoptosis. However, the effects of KP on OCCC, especially the aspects related to inflammatory cytokines, have not been elucidated. Our current study demonstrated the effects of KP extract on cytokine production in TNF-α-induced OCCC TOV-21G cell line. This study showed that KP extract inhibited interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) production at both transcription and translation levels via the suppression of nuclear factor-kappa B (NF-κB) signal transduction. In contrast, KP extract increased the expression of inhibitor kappa B (IκB) protein which may delay NF-κB translocation into the nucleus upon TNF-α activation. Moreover, the suppression of cytokines released from KP treated-TOV-21G reduced the migration of monocyte cell (THP-1). KP extract also exhibited the inhibition of IL-6 and MCP-1 production from THP-1 activated by lipopolysaccharides (LPS). Cells treated with KP extract exhibited a decrease in extracellular signal-regulated kinases (ERK1/2) and protein kinase B (AKT) phosphorylation and induced myeloid leukemia cell differentiation protein Mcl-1 (MCL-1) expression. Suppression of inflammatory cytokine and chemokine production and inhibition of tumor-associated macrophage (TAM) migration support the possibility of using KP for OCCC treatment.

Therapeutic candidates for keloid scars identified by qualitative review of scratch assay research for wound healing.

The scratch assay is an in vitro technique used to analyze cell migration, proliferation, and cell-to-cell interaction. In the assay, cells are grown to confluence and then 'scratched' with a sterile instrument. For the cells in the leading edge, the resulting polarity induces migration and proliferation in attempt to 'heal' the modeled wound. Keloid scars are known to have an accelerated wound closure phenotype in the scratch assay, representing an overactivation of wound healing. We performed a qualitative review of the recent literature searching for inhibitors of scratch assay activity that were already available in topical formulations under the hypothesis that such compounds may offer therapeutic potential in keloid treatment. Although several shortcomings in the scratch assay literature were identified, caffeine and allicin successfully inhibited the scratch assay closure and inflammatory abnormalities in the commercially available keloid fibroblast cell line. Caffeine and allicin also impacted ATP production in keloid cells, most notably with inhibition of non-mitochondrial oxygen consumption. The traditional Chinese medicine, shikonin, was also successful in inhibiting scratch closure but displayed less dramatic impacts on metabolism. Together, our results partially summarize the strengths and limitations of current scratch assay literature and suggest clinical assessment of the therapeutic potential for these identified compounds against keloid scars may be warranted.

Quail-chick grafting experiments corroborate that Tbr1-positive eminential prethalamic neurons migrate along three streams into hypothalamus, subpallium and septocommissural areas.

The prethalamic eminence (PThE), a diencephalic caudal neighbor of the telencephalon and alar hypothalamus, is frequently described in mammals and birds as a transient embryonic structure, undetectable in the adult brain. Based on descriptive developmental analysis of Tbr1 gene brain expression in chick embryos, we previously reported that three migratory cellular streams exit the PThE rostralward, targeting multiple sites in the hypothalamus, subpallium and septocommissural area, where eminential cells form distinct nuclei or disperse populations. These conclusions needed experimental corroboration. In this work, we used the homotopic quail-chick chimeric grafting procedure at stages HH10/HH11 to demonstrate by fate-mapping the three predicted tangential migration streams. Some chimeric brains were processed for Tbr1 in situ hybridization, for correlation with our previous approach. Evidence supporting all three postulated migration streams is presented. The results suggested a slight heterochrony among the juxtapeduncular (first), the peripeduncular (next), and the eminentio-septal (last) streams, each of which followed differential routes. A possible effect of such heterochrony on the differential selection of medial to lateral habenular hodologic targets by the migrated neurons is discussed.

Marine Seagrass Extract of Thalassia testudinum Suppresses Colorectal Tumor Growth, Motility and Angiogenesis by Autophagic Stress and Immunogenic Cell Death Pathways.

Marine plants have become an inexhaustible reservoir of new phytopharmaceuticals for cancer treatment. We demonstrate in vitro/in vivo antitumor efficacy of a standardized polyphenol extract from the marine angiosperm Thalassia testudinum (TTE) in colon tumor cell lines (RKO, SW480, and CT26) and a syngeneic allograft murine colorectal cancer model. MTT assays revealed a dose-dependent decrease of cell viability of RKO, CT26, and SW480 cells upon TTE treatment with IC50 values of, respectively, 175, 115, and 60 µg/mL. Furthermore, TTE significantly prevented basal and bFGF-induced angiogenesis in the chicken chorioallantoic membrane angiogenesis assay. In addition, TTE suppressed bFGF-induced migration of endothelial cells in a wound closure assay. Finally, TTE treatment abrogated CT26 colorectal cancer growth and increased overall organism survival in a syngeneic murine allograft model. Corresponding transcriptome profiling and pathway analysis allowed for the identification of the mechanism of action for the antitumor effects of TTE. In line with our in vitro/in vivo results, TTE treatment triggers ATF4-P53-NFκB specific gene expression and autophagy stress pathways. This results in suppression of colon cancer cell growth, cell motility, and angiogenesis pathways in vitro and in addition promotes antitumor immunogenic cell death in vivo.

Sim1-expressing cells illuminate the origin and course of migration of the nucleus of the lateral olfactory tract in the mouse amygdala.

We focus this report on the nucleus of the lateral olfactory tract (NLOT), a superficial amygdalar nucleus receiving olfactory input. Mixed with its Tbr1-expressing layer 2 pyramidal cell population (NLOT2), there are Sim1-expressing cells whose embryonic origin and mode of arrival remain unclear. We examined this population with Sim1-ISH and a Sim1-tauLacZ mouse line. An alar hypothalamic origin is apparent at the paraventricular area, which expresses Sim1 precociously. This progenitor area shows at E10.5 a Sim1-expressing dorsal prolongation that crosses the telencephalic stalk and follows the terminal sulcus, reaching the caudomedial end of the pallial amygdala. We conceive this Sim1-expressing hypothalamo-amygdalar corridor (HyA) as an evaginated part of the hypothalamic paraventricular area, which participates in the production of Sim1-expressing cells. From E13.5 onwards, Sim1-expressing cells migrated via the HyA penetrate the posterior pallial amygdalar radial unit and associate therein to the incipient Tbr1-expressing migration stream which swings medially past the amygdalar anterior basolateral nucleus (E15.5), crosses the pallio-subpallial boundary (E16.5), and forms the NLOT2 within the anterior amygdala by E17.5. We conclude that the Tbr1-expressing NLOT2 cells arise strictly within the posterior pallial amygdalar unit, involving a variety of required gene functions we discuss. Our results are consistent with the experimental data on NLOT2 origin reported by Remedios et al. (Nat Neurosci 10:1141-1150, 2007), but we disagree on their implication in this process of the dorsal pallium, observed to be distant from the amygdala.

Berberine inhibits intestinal epithelial barrier dysfunction in colon caused by peritoneal dialysis fluid by improving cell migration.

ETHNOPHARMACOLOGICAL RELEVANCE: Berberine is generally extracted from Rhizoma Coptidis (Coptis chinensis Franch), a traditional Chinese medicine, which can be used in the treatment of intestinal diseases, respiratory infections and cardiovascular diseases. Berberine is especially effective for the treatment of gastrointestinal disorders such as diarrhea because of the effect of heat-clearing and detoxifying in traditional Chinese medicine theory. AIM OF THE STUDY: This study aimed to examine the protective effect of berberine (BBR) on the damaged colonic epithelial barrier caused by peritoneal dialysis fluid (PDF). METHODS: The damage to intestinal epithelial barrier was examined by intraperitoneally injecting 4.25% dextrose-containing PDF in mice and establishing a long-term PD model in rats with renal failure. Then, the therapeutic potential of berberine on PD-related colonic injuries was examined. T84 colonic epithelial cells were used to test the effect of PDF and berberine in vitro. The damaging effect of PDF and the protective effect of berberine were evaluated by histology staining, histofluorescence and transmission electron microscopy. The migration of colonic epithelial cell and actin-related protein 2 (Arp2) were tested by wound healing assay and Western blot to determine the possible mechanism in vitro. RESULTS: PD administration induced intestinal epithelial barrier dysfunction in the colon, and berberine alleviated the injury by increasing the tight junction and adhesion junction protein, both in vivo and in vitro. Berberine could also improve the morphology of microvillus. In the wound healing assay, berberine exhibited the ability to promote cell migration, indicating that berberine could probably recover the function of intestinal epithelial cells when the intestinal epithelial barrier was damaged by the PDF. CONCLUSIONS: The present study demonstrates that berberine can ameliorate intestinal epithelial barrier dysfunction in the colon caused by long-term PDF through improving cell migration.

In vitro antibacterial activity of extracts from Samoan medicinal plants and their effect on proliferation and migration of human fibroblasts.

ETHNOPHARMACOLOGICAL RELEVANCE: The prevalence of different types of chronic wounds, due to the ageing population and increase incidence of diseases, is becoming a worldwide problem. Various medicinal plants used in folk medicine have demonstrated wound healing and antimicrobial properties, and some of these species are currently used in commercial preparations. Despite the well-documented and rich tradition of the use of local herbs for the treatment of skin injuries in Samoan folk medicine, their wound healing potential has not yet been systematically studied. AIM OF THE STUDY: Investigation into the in vitro antibacterial activity of ethanol extracts from 14 medicinal plants used in Samoan traditional medicine for the healing of wounds, burns and sores, and their effects on the proliferation and migration of human fibroblasts. MATERIALS AND METHODS: The antibacterial activity of these extracts was tested against pathogens associated with infected skin injuries, using the broth microdilution method. The effect on migration, proliferation and viability of human dermal fibroblasts was evaluated using wound healing scratch assay, cell proliferation assay, and thiazolyl blue tetrazolium bromide cytotoxicity test. RESULTS: The extracts from Cerbera manghas, Commelina diffusa, Kleinhovia hospita, Mikania micrantha, Omalanthus nutans, Peperomia pellucida, Phymatosorus scolopendria, Piper graeffei, Psychotria insularum, and Schizostachyum glaucifolium inhibited the growth of Staphylococcus aureus at the minimum inhibitory concentration (MIC) of ≥4 µg/mL, whereas C. manghas and P. pellucida produced the same MIC against both Escherichia coli and Pseudomonas aeruginosa. Among the antibacterially active species, C. diffusa, K. hospita, P. scolopendria, P. insularum, and S. glaucifolium did not produce toxicity towards the standard line of normal adult human dermal fibroblasts (IC80 > 128 µg/mL). In addition, extracts from Barringtonia asiatica, C. manghas, M. micrantha, O. nutans, P. insularum, and Piper graeffei stimulated significant migration of dermal fibroblasts, while M. micrantha, O. nutans, and P. insularum did not affect cell proliferation at a concentration of 32 µg/mL. CONCLUSIONS: The results suggest that the above-mentioned species of Samoan medicinal plants can be used for the development of new wound healing agents. However, further phytochemical and pharmacological research is needed regarding the isolation and identification of their active constituents.

Kirenol, darutoside and hesperidin contribute to the anti-inflammatory and analgesic activities of Siegesbeckia pubescens makino by inhibiting COX-2 expression and inflammatory cell infiltration.

ETHNOPHARMACOLOGICAL RELEVANCE: The Chinese traditional medicine of Siegesbeckia pubescens Makino (SM), which has the effect of healing rheumatism and promoting joint health, is often used to treat rheumatoid arthritis and ischemic stroke. AIM OF THE STUDY: To clarify the mechanisms underlying the anti-inflammatory and analgesic influence of active components in the ethanol extract of Siegesbeckia pubescens Makino (ESM). MATERIALS AND METHODS: The active ingredients in the ESM were identified practicing high-performance liquid chromatography-diode array detection (HPLC-DAD). Four models including xylene-induced ear oedema, complete Freund's adjuvant (CFA)-induced hind paw oedema, acetic acid-induced pain writhing and lipopolysaccharide (LPS)-induced RAW264.7 cell migration, were used to clarify the anti-inflammatory and analgesic mechanisms of the active ingredients in the ESM. RESULTS: (1) Three active ingredients of kirenol, darutoside and hesperidin were identified in the ESM, with relative proportion of 0.6%, 0.2% and 0.01%, respectively; hesperidin was reported for the first time in the ESM. (2) Both the ESM and its active ingredients could effectively alleviate the degree of swelling of the auricle and toes, increase the threshold of heat pain, decrease the overexpression of inflammatory protein cyclooxygenase-2 (COX-2) in the skin tissue of the tested parts of the toes, and reduce the number of writhes induced by acetic acid in mice. (3) ESM and its active ingredients also dose-dependently inhibited the migration of RAW264.7 cells. CONCLUSIONS: ESM and its active ingredients can effectively attenuate the expression of inflammatory factors induced by chemical inflammation, prevent the infiltration of inflammatory cells, and exert good anti-inflammatory and antinociceptive activities.

Sodium Danshensu Inhibits Oral Cancer Cell Migration and Invasion by Modulating p38 Signaling Pathway.

Background: Oral squamous cell carcinoma (OSCC) that comprises about 90% of all oral cancer cases is associated with poor prognosis due to its highly metastatic nature. The majority of OSCC treatment options are related detrimental side-effects. Hypothesis/Purpose: The present study aimed at deciphering the effects of a bioactive phytochemical, sodium danshensu, on human oral cancer cell metastasis. Methods and Results: The treatment of FaDu and Ca9-22 cells with different doses of sodium danshensu (25, 50, and 100 µM) caused a significant reduction in cellular motility, migration, and invasion, as compared to the untreated cells. This effect was associated with a reduced expression of MMP-2, vimentin and N-cadherin, together with an enhanced expression of E-cadherin and ZO-1. Further investigation on the molecular mechanism revealed that treatment with sodium danshensu caused significant reduction in p38 phosphorylation; however, phosphorylation of ERK1/2 significantly decreased only in FaDu cells, whereas p-JNK1/2 did not show any alteration. A combination of p38 and JNK1/2 inhibitors with sodium danshensu also reduced the migration in the FaDu and Ca9-22 cell lines. Conclusion: Collectively, the present study findings reveal that sodium danshensu execute anti-metastatic effect by suppressing p38 phosphorylation in human oral cancer. The study identifies sodium danshensu as a potential natural anticancer agent that can be used therapeutically to manage highly metastatic OSCC.

Increased antitumor efficacy of ginsenoside Rh2 via mixed micelles: in vivo and in vitro evaluation.

The aim of this work is to apply Solutol® HS15 and TPGS to prepare self-assembled micelles loading with ginsenoside Rh2 to increase the solubility of ginsenoside Rh2, hence, improving the antitumor efficacy. Ginsenoside Rh2-mixed micelles (Rh2-M) were prepared by thin film dispersion method. The optimal Rh2-M was characterized by particle size, morphology, and drug encapsulation efficiency. The enhancement of in vivo anti-tumor efficacy of Rh2-M was evaluated by nude mice bearing tumor model. The solubility of Rh2 in self-assembled micelles was increased approximately 150-folds compared to free Rh2. In vitro results demonstrated that the particle size of Rh2-M is 74.72 ± 2.63 nm(PDI = 0.147 ± 0.15), and the morphology of Rh2-M is spherical or spheroid, and the EE% and LE% are 95.27 ± 1.26% and 7.68 ± 1.34%, respectively. The results of in vitro cell uptake and in vivo imaging showed that Rh2-M could not only increase the cell uptake of drugs, but also transport drug to tumor sites, prolonging the retention time. In vitro cytotoxicity and in vivo antitumor results showed that the anti-tumor effect of Rh2 can be effectively improved by Rh2-M. Therefore, Solutol® HS15 and TPGS could be used to entrapping Rh2 into micelles, enhancing solubility and antitumor efficacy.