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Background: Hepatic metastasis is the primary cause of colorectal cancer (CRC)-induced death. Our previous results showed the anti-metastatic effects of Coptidis rhizoma using in vitro model. Aim: The present study aimed to investigate whether berberine, the main active compound of C. rhizoma, inhibits colon–liver metastasis in an animal model, and to elucidate the underlying mechanisms. Methods: Murine colon carcinoma (CT26) tumor tissue was implanted into the cecum of balb/c mice with/without oral administration of berberine (100 mg/kg) for 21 days, after which liver metastasis was evaluated. In addition, the pharmacological actions of berberine were explored using 5-fluorouracil-resistant human colon cancer cells (HCT116/R). Result: The administration of berberine significantly decreased the number of tumor nodules in the liver, while significant activation of E-cadherin (an epithelial marker), and suppression of vimentin, Snail and TGF-β (mesenchymal markers) were observed in primary colon tumor tissues. Berberine treatment also notably lowered the levels of inflammatory cytokines (TGF-β, TNF- α, IL-6 and IL-1β) in the blood. In HCT116/R cells, berberine significantly inhibited migration and invasion and modulated the expression of TGF-β and representative molecules related to its pathway. The results obtained with a TGF-β inhibitor (SB431542) and a TGF-β siRNA, strongly suggest that the mechanism of action of berberine is linked to TGF-β signaling. Conclusion: In conclusion, berberine evidently possess an anti-colon–liver metastatic effect, and its underlying mechanisms involve the inhibition of epithelial–mesenchymal transition (EMT) through the TGF-β signaling pathway. Thus, we cautiously propose the pharmacological potential of berberine in drug research studies targeting hepatic metastasis from CRC. [ABSTRACT FROM AUTHOR]

Background: Osthole is extracted from Fructus Cnidii and is proved to be effective in the treatment of osteoporosis in rats. However, data are still scarce and the mechanism remains elusive. Objective: To investigate the effect of Osthole on proliferation and differentiation of osteoblast. Materials and Methods: Cells were divided into five groups: control group, β-estradiol group (10−8 M), and Osthole groups (10−6 M, 10−5 M, and 10−4 M). Osteoblast proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay. Alkaline phosphatase (ALP) activity was detected by ALP staining and enzymatic measurement. Mineralization was detected by alizarin-red staining. The level of osteocalcin was measured by enzyme-linked immunosorbent assay (ELISA). The expression of key proteins of Wnt/β-catenin signaling pathway and endoplasmic reticulum stress (ERS) was analyzed by Western blot. Results: Cell proliferation was retarded in moderate-dose and high-dose Osthole group at 1d, 2d and 3d and in low-dose Osthole group at 1d and 2d (P < 0.05). ALP activity was enhanced in high-dose Osthole group from 1d to 3d and in moderate-dose Osthole group at 2d (P < 0.05). Mineralization of bone matrix was promoted in high-dose Osthole group at 21d (P < 0.05). The secretion of osteocalcin was promoted in Osthole groups at 21d (P < 0.05). Expression of CHOP, GRP78, PDI, Wnt1, and β-catenin was upregulated in high-dose Osthole group at 2d, indicating that both of ERS and Wnt/β-catenin signaling pathway were activated. Conclusion: It can be concluded that the effect of Osthole on inhibition of proliferation is relevant with activation of ERS, and activation of Wnt/β-catenin signaling pathway is one of the mechanisms how Osthole promotes osteoblast differentiation. In summary, this study provided more evidence for Osthole as a potential anti-osteoporosis medicine. Abbreviations used: ALP: Alkaline phosphatase; ELISA: Enzyme-linked immunosorbent assay; CCK-8: Cell Counting Kit-8; ERS: endoplasmic reticulum stress; DMEM: Dulbecco's Modified Eagle Medium; α-MEM: α-minimum essential medium; EDTA: trypsin/ethylenediaminetetraacetic acid; DMF: N,N-dimethylformamide; TBS-T: Tris-buffered saline-Tween 20; RIPA: Radio Immuno Precipitation Assay; HRP: Horseradish peroxidase; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; BCA: Bicinchoninic acid; ANOVA: Analysis of variance. [ABSTRACT FROM AUTHOR]

Natural metabolites from beneficial fungi were recognized for their potential to inhibit multidrug-resistant human and plant fungal pathogens. The present study describes the isolation, metabolite profiling, antibacterial, and antifungal, antioxidant, and anticancer activities of soil fungi. Among the 17 isolates, the AK-7 isolate was selected based on the primary screening. Further, the identification of isolate AK-7 was performed by 18S rRNA sequencing and identified as Penicillium limosum (with 99.90% similarity). Additionally, the ethyl acetate extract of the Penicillium limosum strain AK-7 (AK-7 extract) was characterized by Fourier Transform Infrared Spectroscopy (FTIR) and a Gas Chromatography-Mass Spectroscopy (GC-MS) analysis, and the results showed different functional groups and bioactive metabolites. Consequently, a secondary screening of antibacterial activity by the agar well diffusion method showed significant antibacterial activity against Gram-negative and Gram-positive bacterial pathogens. The AK-7 extract exhibited notable antifungal activity by a food poisoning method and showed maximum inhibition of 77.84 ± 1.62%, 56.42 ± 1.27%, and 37.96 ± 1.84% against Cercospora canescens, Fusarium sambucinum and Sclerotium rolfsii phytopathogens. Consequently, the AK-7 extract showed significant antioxidant activity against DPPH and ABTS•+ free radicals with IC50 values of 59.084 μg/mL and 73.36 μg/mL. Further, the anticancer activity of the AK-7 extract against the human ovarian teratocarcinoma (PA-1) cell line was tested by MTT and Annexin V flow cytometry. The results showed a dose-dependent reduction in cell viability and exhibited apoptosis with an IC50 value of 82.04 μg/mL. The study highlights the potential of the Penicillium limosum strain AK-7 as a source of active metabolites and natural antibacterial, antifungal, antioxidant, and anticancer agent, and it could be an excellent alternative for pharmaceutical and agricultural sectors. [ABSTRACT FROM AUTHOR]

Background: The F11R/JAM-A cell adhesion protein was examined as the therapeutic target in triple negative breast cancer (TNBC) with the use of the peptide antagonist to F11R/JAM-A, that previously inhibited the early stages of breast cancer metastasis in vitro. Methods: The online in silico analysis was performed by TNMPlot, UALCAN, and KM plotter. The in vitro experiments were performed to verify the effect of peptide 4D (P4D) on human endothelial cell lines EA.hy926 and HMEC-1 as well as on human TNBC cell line MDA-MB-231. The cell morphology upon P4D treatment was verified by light microscopy, while the cell functions were assessed by colony forming assay, MTT cell viability assay, BrdU cell proliferation assay, and Transepithelial/Endothelial Electrical Resistance measurements. The in vivo experiments on 4T1 murine breast cancer model were followed by histopathological analysis and a series of quantitative analyses of murine tissues. Results: By in silico analysis we have found the elevated gene expression in breast cancer with particular emphasis on TNBC. The elevated F11R expression in TNBC was related with poorer survival prognosis. Peptide 4D has altered the morphology and increased the permeability of endothelial monolayers. The colony formation, viability, and proliferation of MDA-MB-231 cells were decreased. P4D inhibited the metastasis in 4T1 breast cancer murine model in a statistically significant manner that was demonstrated by the resampling bootstrap technique. Conclusions: The P4D peptide antagonist to F11R/JAM-A is able to hinder the metastasis in TNBC. This assumption needs to be confirmed by additional 4T1 mouse model study performed on larger group size, before making the decision on human clinical trials. [ABSTRACT FROM AUTHOR]

Wound is tissue damage that occurs in the skin. Helichrysum species (Altınotu) are rich in phenolic compounds used in traditional medicine for wound healing. The main component in their flower head (capitulum) is phenolic compounds. The present study investigates the proliferative, oxidative stress, and wound healing properties of the methanolic extract of Helichrysum plicatum subsp. pseudoplicatum capitulum on a human dermal fibroblast (HDF) cell line in this study. H plicatum subsp. pseudoplicatum capitulums were collected in Erzurum, Turkey (altitude 1950 m), dried, pulverized, and extracted with methanol. Firstly, total phenolic contents were determined and secondly, the proliferative effect, oxidative stress activities, and wound healing effects on HDF cells were evaluated by the cell proliferation kit (XTT) test, total antioxidant status (TAS), and total oxidant status (TOS) commercial kits, and the scratch experiment by taking microscopic images of the cells at 0, 12, 18, and 24 h, respectively. Total phenolic content was found to be 142.00 ± 0.73 mg gallic acid equivalent per gram (GAE/g) extract. The capitulum extract has a proliferative effect at 0.5 to 10 µg/mL concentrations according to the XTT test results. It was observed that TAS levels significantly increased in the plant extract at the concentration ranges 1 to 10 µg/mL (P <.01). About 1 to 5 µg/mL plant extract started to increase cell migration at the 12 h and significantly closed the wound area at the 24 h. At the doses between 1 to 5 μg/mL, it has the most substantial effect on both cell viability and antioxidant effect, and wound healing was found to be in this concentration range. These findings suggested that the H plicatum subsp. pseudoplicatum capitulum is a valuable source of phenolic content with important antioxidant activity at wound healing and it was concluded that the capitulum extract accelerates wound healing by increasing cell migration in low doses. [ABSTRACT FROM AUTHOR]

Background: Clinically, metastasis and recurrence occurred after routine radiochemotherapy in dozens of cases of gastric cancer, mainly attributed to the role of cancer stem cells (CSCs). Actually, radiochemotherapy could induce DNA damages, leading to activation of DNA repair which might be associated with acquisition of stem cell phenotype. Hitherto, the contribution made by active DNA repair to stemness induction has not been extensively explored. Methods: Cisplatin/doxorubicin treatment and X-ray exposure were conducted in gastric cancer cell lines and gastric cancer cells derived from solid tumors to model clinical therapy. Quantitative real-time PCR, Western blot, and tumorsphere/tumor formation assay were further used to characterize CSCs and assess activation of DNA repair. RNA-seq was performed to identify which DNA repair genes were crucial for CSC traits induction, followed by the investigation of underlying mechanism and functional significance via in vitro and in vivo experiments. Results: Here, we report a mechanism through which gastric cancer cells in response to radiochemotherapy were reprogrammed into gastric cancer stem cell-like cells. In this mechanism, radiochemotherapy triggers DNA damage response accompanied by elevated levels of EID3, a typical DNA repair gene, which interacts with NAMPT to promote stemness via upregulating Wnt signaling pathway, manifested by enhanced tumorsphere/tumor formation in gastric cancer. In addition to involvement of EID3 in stemness acquisition, it also shows impacts on proliferation, cell cycle, apoptosis and therapy resistance to maintain the characteristics of CSC populations. Conclusion: Our study indicates that gastric cancer cells can be endowed with stemness traits via EID3-NAMPT-Wnt/β-catenin axis in response to radiochemotherapy. Blocking this axis (i.e., targeting EID3) along with radiochemotherapy might represent a potential strategy to sensitize CSCs to radiochemotherapy and further reinforce the anti-tumor effects of conventional treatments. [ABSTRACT FROM AUTHOR]

Advances in skin tissue engineering have promoted the development of artificial skin substitutes to treat large burns and other major skin loss conditions. However, one of the main drawbacks to bioengineered skin is the need to obtain a large amount of viable epithelial cells in short periods of time, making the skin biofabrication process challenging and slow. Enhancing skin epithelial cell cultures by using mesenchymal stem cells secretome can favor the scalability of manufacturing processes for bioengineered skin. The effects of three different types of secretome derived from human mesenchymal stem cells, e.g. hADSC-s (adipose cells), hDPSC-s (dental pulp) and hWJSC-s (umbilical cord), were evaluated on cultured skin epithelial cells during 24, 48, 72 and 120 h to determine the potential of this product to enhance cell proliferation and improve biofabrication strategies for tissue engineering. Then, secretomes were applied in vivo in preliminary analyses carried out on Wistar rats. Results showed that the use of secretomes derived from mesenchymal stem cells enhanced currently available cell culture protocols. Secretome was associated with increased viability, proliferation and migration of human skin epithelial cells, with hDPSC-s and hWJSC-s yielding greater inductive effects than hADSC-s. Animals treated with hWJSC-s and especially, hDPSC-s tended to show enhanced wound healing in vivo with no detectable side effects. Mesenchymal stem cells derived secretomes could be considered as a promising approach to cell-free therapy able to improve skin wound healing and regeneration. [ABSTRACT FROM AUTHOR]

Context: Plantar ulcers occur in patients with leprosy not because of the disease but because of its neuropathic effects on the skin on the feet. Neuropathy increases the risk of trauma to patients' feet, leading to the development of ulcers. With the introduction of new therapeutic regimens, leprosy can now be cured. However, complications of the disease, such as sensory loss, muscle palsy, absorption of extremities, and recurrent ulcers, still lead to substantial morbidity. This short article reviews the current management of leprosy plantar ulceration in such (nonhealing chronic ulcers) patients. Aims: To evaluate the efficacy of autologous smashed follicular dermal graft and epidermal cell suspension in the treatment of chronic nonhealing trophic ulcers in Hansen's patients. Materials and Methods: A total of 23 chronic nonhealing trophic ulcers were treated with autologous smashed follicular dermal graft and epidermal cell suspension. Ulcers were assessed based on the rate of ulcer size reduction every week till 12 weeks and then once a month till the sixth month. Statistical Analysis: Analysis was done using SPSS 26 software. Independent t-test was used and a P value of <0.05 was considered statistically significant. Results: Amongst the Hansen's patients who were released from treatment, all 23 (100%) ulcers had healed within the study period of six months, and all 23 (100%) ulcers had healed within 8 weeks. Conclusion: With this modified technique of combining autologous smashed follicular dermal graft with epidermal cell suspension, trophic ulcers heal faster with good results of reepithelialization of the ulcer bed. [ABSTRACT FROM AUTHOR]

3D cell cultures are regarded as a better and more relevant approach for screening drugs and therapeutics, particularly due to their likeness with the in vivo conditions. Spheroids offer an intermediate platform between in vitro and in vivo models, for conducting tumor‐based investigations. In this study, a simple setup was developed for consistent generation of lung co‐culture spheroids, which were developed using the cancer cell lines A549, NCI H460, and fibroblast cells WI‐38. The potential of these spheroids for evaluating the toxicity of Eudragit® RL 100 nanoparticles (ENP) was explored. Monodisperse ENP, having the size range of 140–200 nm was prepared using the nanoprecipitation method. These were loaded with the poorly water‐soluble anticancer drug paclitaxel. The evaluation of toxicity and uptake of drug‐loaded ENP revealed that 2D monolayers were more sensitive to treatment than 3D spheroids. Within spheroids, co‐cultures were more resistant to the treatment than monocultures. Overall, our findings demonstrated that the lung co‐culture spheroids were a suitable model for accelerating the efficacy and toxicity‐related investigations of novel drug delivery systems. [ABSTRACT FROM AUTHOR]

Detection, quantification and monitoring of virus – host cell interactions are of great importance when evaluating the safety of pharmaceutical products. With the wide usage of viral based vector systems in combination with mammalian cell lines for the production of biopharmaceuticals, the presence of replication competent viral particles needs to be avoided and potential hazards carefully assessed. Consequently, regulatory agencies recommend viral clearance studies using plaque assays or TCID50 assays to evaluate the efficiency of the production process in removing viruses. While plaque assays provide reliable information on the presence of viral contaminations, they are still tedious to perform and can take up to two weeks to finish. To overcome some of these limitations, we have automated, miniaturized and integrated the dual cell culture bioassay into a common lab-on-a-chip platform containing embedded electrical sensor arrays to enrich and detect infectious viruses. Results of our microfluidic single step assay show that a significant reduction in assay time down to 3 to 4 days can be achieved using simultaneous cell-based viral amplification, release and detection of cytopathic effects in a target cell line. We further demonstrate the enhancing effect of continuous fluid flow on infection of PG-4 reporter cells by newly formed and highly active virions by M. dunni cells, thus pointing to the importance of physical relevant viral–cell interactions. [ABSTRACT FROM AUTHOR]

The world of biomedical research is in constant evolution, requiring more and more conditions and norms through pre-clinic and clinic studies. Nanodiamonds (NDs) with exceptional optical, thermal and mechanical properties emerged on the global scientific scene and recently gained more attention in biomedicine and bioanalysis fields. Many problematics have been deliberated to better understand their in vitro and in vivo efficiency and compatibility. Light was shed on their synthesis, modification and purification steps, as well as particle size and surface properties in order to find the most suitable operating conditions. In this review, we present the latest advances of NDs use in bioapplications. A large variety of subjects including anticancer and antimicrobial systems, wound healing and tissue engineering management tools, but also bioimaging and labeling probes are tackled. The key information resulting from these recent works were evidenced to make an overview of the potential features of NDs, with a special look on emerging therapeutic and diagnosis combinations. [ABSTRACT FROM AUTHOR]

Background: T helper 17 cells (Th17)/regulatory T cells (Treg), as subtypes of CD4+ T cells, play an important role in the inflammatory response of acute respiratory distress syndrome (ARDS). However, there is still a lack of effective methods to regulate the differentiation balance of Th17/Treg. It was proven that mesenchymal stem cells (MSCs) could regulate the differentiation of CD4+ T cells, but the mechanism is still unclear. TGFβ1, a paracrine cytokine of MSCs, could also regulate the differentiation of Th17/Treg but is lowly expressed in MSCs. Therefore, mouse MSCs (mMSCs) overexpressing TGFβ1 were constructed by lentivirus transduction and intratracheally transplanted into LPS-induced ARDS mice in our study. The aim of this study was to evaluate the therapeutic effects of mMSCs overexpressing TGFβ1 on inflammation and immunoregulation by impacting the Th17/Treg balance in LPS-induced ARDS mice. Methods: mMSCs overexpressing TGFβ1 were constructed using lentiviral vectors. Then, mouse bone-marrow-derived MSCs (mBM-MSC) and mBM-MSC-TGFβ1 (mBM-MSC overexpressing TGFβ1) were transplanted intratracheally into ARDS mice induced by lipopolysaccharide. At 3 and 7 days after transplantation, the mice were sacrificed, and the homing of the mMSCs was assayed by ex vivo optical imaging. The relative numbers of Th17 and Treg in the lungs and spleens of mice were detected by FCM. IL-17A and IL-10 levels in the lungs of mice were analysed by western blot. Permeability and inflammatory cytokines were evaluated by analysing the protein concentration of BALF using ELISA. Histopathology of the lungs was assessed by haematoxylin and eosin staining and lung injury scoring. Alveolar lung fibrosis was assessed by Masson's trichrome staining and Ashcroft scoring. The mortality of ARDS mice was followed until 7 days after transplantation. Results: The transduction efficiencies mediated by the lentiviral vectors ranged from 82.3 to 88.6%. Overexpressing TGFβ1 inhibited the proliferation of mMSCs during days 5–7 (p < 0.05) but had no effect on mMSC differentiation or migration (p > 0.05). Compared to that in the LPS + mBM-MSC-NC group mice, engraftment of mMSCs overexpressing TGFβ1 led to much more differentiation of T cells into Th17 or Treg (p < 0.05), improved permeability of injured lungs (p < 0.05) and ameliorative histopathology of lung tissue in ARDS mice (p < 0.05). Moreover, IL-17A content was also decreased while IL-10 content was increased in the LPS + mBM-MSC-TGFβ1 group compared with those in the LPS + mBM-MSC-NC group (p < 0.05). Finally, mMSCs overexpressing TGFβ1 did not aggravate lung fibrosis in ARDS mice (p > 0.05). Conclusion: MSCs overexpressing TGFβ1 could regulate lung inflammation and attenuate lung injuries by modulating the imbalance of Th17/Treg in the lungs of ARDS mice. [ABSTRACT FROM AUTHOR]

Background: Wood combustion emissions have been studied previously either by in vitro or in vivo models using collected particles, yet most studies have neglected gaseous compounds. Furthermore, a more accurate and holistic view of the toxicity of aerosols can be gained with parallel in vitro and in vivo studies using direct exposure methods. Moreover, modern exposure techniques such as air-liquid interface (ALI) exposures enable better assessment of the toxicity of the applied aerosols than, for example, the previous state-of-the-art submerged cell exposure techniques. Methods: We used three different ALI exposure systems in parallel to study the toxicological effects of spruce and pine combustion emissions in human alveolar epithelial (A549) and murine macrophage (RAW264.7) cell lines. A whole-body mouse inhalation system was also used to expose C57BL/6 J mice to aerosol emissions. Moreover, gaseous and particulate fractions were studied separately in one of the cell exposure systems. After exposure, the cells and animals were measured for various parameters of cytotoxicity, inflammation, genotoxicity, transcriptome and proteome. Results: We found that diluted (1:15) exposure pine combustion emissions (PM1 mass 7.7 ± 6.5 mg m− 3, 41 mg MJ− 1) contained, on average, more PM and polycyclic aromatic hydrocarbons (PAHs) than spruce (PM1 mass 4.3 ± 5.1 mg m− 3, 26 mg MJ− 1) emissions, which instead showed a higher concentration of inorganic metals in the emission aerosol. Both A549 cells and mice exposed to these emissions showed low levels of inflammation but significantly increased genotoxicity. Gaseous emission compounds produced similar genotoxicity and a higher inflammatory response than the corresponding complete combustion emission in A549 cells. Systems biology approaches supported the findings, but we detected differing responses between in vivo and in vitro experiments. Conclusions: Comprehensive in vitro and in vivo exposure studies with emission characterization and systems biology approaches revealed further information on the effects of combustion aerosol toxicity than could be achieved with either method alone. Interestingly, in vitro and in vivo exposures showed the opposite order of the highest DNA damage. In vitro measurements also indicated that the gaseous fraction of emission aerosols may be more important in causing adverse toxicological effects. Combustion aerosols of different wood species result in mild but aerosol specific in vitro and in vivo effects. [ABSTRACT FROM AUTHOR]

This study aimed at evaluating the effects of collagen scaffold with Bcl-2-modified adipose-derived stem cells (ADSCs) on wound repair in streptozotocin-induced diabetic mice. A round full thickness skin defect with a diameter of 7 mm was made in the mice model. The experimental mice were divided into 4 groups (n = 12 each): group A (control group), group B (scaffold group), group C (ADSCs-scaffold group), and group D (Bcl-2-ADSCs-scaffold group). On days 3, 7, 10, and 14 after surgery, characteristics of wound healing was observed, and wound tissues were sampled for histology characteristics via hematoxylin-eosin staining and immunohistochemical staining. Compared with other groups, the wound healing rate was significantly higher in group D a week after operation (P <.05). On the seventh day postoperation, group D exhibited higher blood vessel in the wounds granulation tissue than other groups according to results of hematoxylin-eosin staining and immunohistochemistry. In conclusion, these findings demonstrated that collagen scaffold with Bcl-2 modified ADSCs may effectively improve the wound healing process in diabetic mice. [ABSTRACT FROM AUTHOR]

Background: Various treatment modalities exist for vitiligo, yet none of them are curative. Vitiligo is still considered a challenging disease to manage. Surgical treatment offers an excellent option for patients with stable vitiligo, especially those who fail to respond to medical treatment. Cell suspension techniques carry the advantage of covering large surface areas. Objective: To compare noncultured extracted hair follicle outer root sheath cell suspension (NCORSHFS) and noncultured epidermal cell suspension (NCES) in producing repigmentation. Subjects and Methods: Twenty patients were randomly allocated into two groups. They were objectively evaluated for the extent of repigmentation (after 1, 2, and 3 months), complications, cosmetic outcome, and satisfaction. Results: In NCORSHFS group, 10% showed excellent pigmentation, 20% showed good pigmentation, 50% fair, and 20% poor pigmentation. In NCES group, 10% showed excellent pigmentation, 10% good pigmentation, 40% fair, and 40% poor pigmentation. This difference was not statistically significant. Excellent color match was observed in 80% of NCORSHFS and in 70% of NCES. Donor area complications were absent in NCORSHFS group, whereas in NCES, mild scarring in 20% and hyperpigmentation in 40% of patients was observed. The difference in patients' satisfaction between the two groups was not statistically significant. Conclusion: Both NCORSHFS and NCES are effective in producing good repigmentation with perfect color match and patients' satisfaction. NCORSHFS has no donor area complications because it is a scarless procedure. [ABSTRACT FROM AUTHOR]

Osteoporosis and vascular lesions induced by glucolipotoxicity are common complications of diabetes mellitus (DM). In order to deal with these complications, we designed a new therapeutic strategy, i.e. coculture system containing human amnion-derived mesenchymal stem cells (HAMSCs) and human bone marrow mesenchymal stem cells (HBMSCs). Two in vitro coculture models, transwell and mixed cocultures, were proposed for 7 days with variable HAMSCs: HBMSCs ratios. Then, supernatant from each coculture was used to reverse the deficiency of HBMSCs and human umbilical vein endothelial cells (HUVECs) impaired by high glucose and palmitic acid (GP). We found that glucolipotoxicity caused by GP remarkably inhibited cell proliferation, osteogenic differentiation and superoxide dismutase (SOD) activity, as well as induced the reactive oxygen species (ROS) level in HBMSCs. Meanwhile, glucolipotoxicity suppressed cell proliferation, tube formation capacity and angiogenic potential of HUVECs. Though, HAMSCs/HBMSCs coculture system reduced HBMSCs dysfunction by antioxidant properties and promoted angiogenesis in HUVECs. The mixed HAMSCs/HBMSCs coculture at the optimal ratio of 3/1 showed significantly greater cell proliferation, antioxidant properties, osteogenic and angiogenic differentiation than HBMSCs or HUVECs alone. In conclusion, the current coculture system of HAMSCs/HBMSCs can be a potential therapeutic material for advancing bone and vascular regeneration against DM-induced glucolipotoxicity. [ABSTRACT FROM AUTHOR]

Autologous skin transplantation is today's 'gold standard' treatment for full-thickness burns. However, when > 30% of total body surface area is damaged, there is an important shortage of autologous donor sites for skin grafting; then, treatment alternatives become crucial. Such alternatives can be based on polymeric scaffolds capable of functioning as protective covers and cells/factors carriers. Chitosan (CTS) is a natural-derived polymer with relevant biological-related properties but poor mechanical performance. Improved mechanical properties can be achieved through lactic acid grafting (LA-g); nevertheless, LA-g affects the biological response towards the CTS-based materials. In this work, CTS-LA scaffolds with different LA-g percentages were synthesized and evaluated to determine appropriate LA-g degrees for full-thickness burns treatment. In vitro results indicated that the higher the LA-g percentage, the lower the capability of the scaffolds to sustain fibroblasts culture. Scaffolds with LA-g around 28% (CTS-LA28) sustained cell culture and allowed normal cell functionality. Further evaluation of CTS-LA28 as acellular and cellular grafts in a full-thickness burn mouse model showed that at 28 days post-burn, macroscopic characteristic of the reparation tissue were closer to healthy skin when cellular grafts were used for treatment; histological evaluation also showed that dermis cellularity and collagenous fibers structure were similar to those in healthy skin when cellular grafts were used for burns treatment. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2875-2891, 2017. [ABSTRACT FROM AUTHOR]

Introduction: Biliary strictures after bile duct injury or duct-to-duct biliary reconstruction are serious complications that markedly reduce patients' quality of life because their treatment involves periodic stent replacements. This study aimed to create a scaffold-free tubular construct as an interposition graft to treat biliary complications., Methods: Scaffold-free tubular constructs of allogeneic pig fibroblasts, that is, fibroblast tubes, were created using a Bio-3D Printer and implanted into pigs as interposition grafts for duct-to-duct biliary reconstruction., Results: Although the fibroblast tube was weaker than the native bile duct, it was sufficiently strong to enable suturing. The pigs' serum hepatobiliary enzyme levels remained stable during the experimental period. Micro-computed tomography showed no biliary strictures, no biliary leakages, and no intrahepatic bile duct dilations. The tubular structure was retained in all resected specimens, and the fibroblasts persisted at the graft sites. Immunohistochemical analyses revealed angiogenesis in the fibroblast tube and absence of extensions of the biliary epithelium into the fibroblast tube's lumen., Conclusions: This study's findings demonstrated successful reconstruction of the extrahepatic bile duct with a scaffold-free tubular construct created from pig fibroblasts using a novel Bio-3D Printer. This construct could provide a novel regenerative treatment for patients with hepatobiliary diseases., Competing Interests: K. Nakayama is a cofounder and shareholder of Cyfuse Biomedical KK. The other authors declare no conflicts of interest., (© 2021 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography–tandem mass spectrometry (LC–MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS E ) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC–MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest. [ABSTRACT FROM AUTHOR]

Bisphenol-A-glycidyl methacrylate (Bis-GMA) released from dental resin materials causes various toxic effects on gingival epithelium. Thus the underlying mechanisms of its cytotoxicity should be elucidated for safety use. One potential cause of cell damage is the generation of reactive oxygen species (ROS) beyond the capacity of a balanced redox regulation. In this study, we found that exposure of human oral keratinocytes (HOKs) to Bis-GMA caused apoptosis and G1/S cell cycle arrest in parallel with an increased ROS level. Moreover, Bis-GMA induced a depletion of mitochondrial membrane potential, an increase in the Bax/Bcl-2 ratio, an activation of caspase-3 and altered expressions of cell cycle-related proteins (p21, PCNA, cyclinD1). Furthermore, the co-treatment of the ROS scavenger N-acetyl cysteine (NAC) obviously attenuated Bis-GMA-induced toxicity. Here we also evaluated the effects of Bis-GMA on the ROS-related PI3k/Akt pathway. We found that Bis-GMA inhibited the phosphorylation of Akt, whereas the amount of phosphorylated Akt was reverted to the control level in the presence of NAC. Our findings suggested that the toxic effects of Bis-GMA were related to ROS production and the antioxidant NAC effectively reduced Bis-GMA-mediated cytotoxicity. [ABSTRACT FROM AUTHOR]

Inkjet printing of drug nanosuspension on edible porous substrates was carried out for the first time with the objective of preparing personalized dosage forms of poorly soluble drugs. Amorphous drug–polysaccharide nanoparticle complex (or drug nanoplex in short) was used as the nanosuspension ink, instead of the conventional crystalline nanodrug. The amorphous drug nanoplex exhibited low propensity to Ostwald ripening growth, high colloidal stability, and supersaturation generation capability making it ideal for printing. Nanoplexes of ciprofloxacin – a BCS Class IV compound – prepared by complexation with dextran sulfate were used as the nanosuspension ink at two different sizes (i.e. ≈265 nm and 188 nm). Inkjet printing was performed on cellulose substrate at 0.25% (w/v) nanosuspension concentration and 5% (w/v) polyethylene glycol. For both nanoplex sizes, the results indicated that the printed dose could be increased by increasing the number of droplets dispensed. However, exact correlations between the achievable dose and the number of droplets dispensed were not evident, which was likely caused by the spatial non-homogeneity in the nanosuspension concentration. Compared to the larger nanoplex, printed nanodrugs of the smaller nanoplex consistently exhibited higher payload with better batch-to-batch reproducibility (<6%). The maximum achievable payload was equal to ≈2.5 μg/cm 2 , which was multifold higher than that achieved had inkjet printing of ciprofloxacin solution been performed. Nevertheless, print substrate with higher liquid uptake capacity is needed to increase the payload nearer to the therapeutic dose. Lastly, the drug release and non-cytotoxicity of the printed nanodrug were successfully established in vitro . [ABSTRACT FROM AUTHOR]

Multidrug resistance (MDR) is one of the major obstacles in drug delivery, and it is usually responsible for unsuccessful cancer treatment. MDR may be overcome by using MDR inhibitors. Among different classes of these inhibitors that block drug efflux mediated by permeability-glycoprotein (P-gp), less toxic amphiphilic diblock copolymers composed of methoxypolyethyleneglycol-block-polycaprolactone (MePEG-b-PCL) have been studied extensively. The purpose of this work is to evaluate how these copolymer molecules can reduce the efflux, thereby enhancing the accumulation of P-gp substrates (e.g., daunorubicin or DNR) in MDR cells. Using conventional methods, it was found that the low-molecular-weight diblock copolymer, MePEG-b-PCL (PCL5), enhanced drug accumulation in MDCKII-MDR1 cells, but the high-molecular-weight version, MePEG-b-PCL (PCL200), did not. However, when PCL200 was mixed with PCL5 (and DNR) in order to encapsulate them to facilitate drug delivery, there was no drug enhancement effect attributable to PCL5, and the reason for this negative result was unclear. Since drug accumulation measured on different cell batches originated from single cells, we employed the same-single-cell analysis in the accumulation mode ( SASCA- A) to find out the reason. A microfluidic biochip was used to select single MDR cells, and the accumulation of DNR was fluorescently measured in real time on these cells in the absence and presence of PCL5. The SASCA- A method allowed us to obtain drug accumulation information faster in comparison to conventional assays. The SASCA- A results, and subsequent curve-fitting analysis of the data, have confirmed that when PCL5 was encapsulated in PCL200 nanoparticles as soon as they were synthesized, the ability of PCL5 to enhance DNR accumulation was retained, thus suggesting PCL200 as a promising delivery system for encapsulating P-gp inhibitors, such as PCL5. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The present study involves molecular docking, molecular dynamics (MD) simulation studies, and Caco-2 cell monolayer permeability assay to investigate the effect of structural modifications on PepT1-mediated transport of thyrotropin releasing hormone (TRH) analogs. Molecular docking of four TRH analogs was performed using a homology model of human PepT1 followed by subsequent MD simulation studies. Caco-2 cell monolayer permeability studies of four TRH analogs were performed at apical to basolateral and basolateral to apical directions. Inhibition experiments were carried out using Gly-Sar, a typical PepT1 substrate, to confirm the PepT1-mediated transport mechanism of TRH analogs. Papp of the four analogs follows the order: NP-1894 < NP-2378 < NP-1896 < NP-1895. Higher absorptive transport was observed in the case of TRH analogs, indicating the possibility of a carrier-mediated transport mechanism. Further, the significant inhibition of the uptake of Gly-Sar by TRH analogs confirmed the PepT1-mediated transport mechanism. Glide docking scores of all the four analogues were in good agreement with their transport rates, suggesting the role of substrate binding affinity in the PepT1-mediated transport of TRH analogs. MD simulation studies revealed that the polar interactions with amino acid residues present in the active site are primarily responsible for substrate binding, and a downward trend was observed with the increase in bulkiness at the N-histidyl moiety of TRH analogs. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

We previously reported that insulin-like growth factor-II mRNA-binding protein-3 (IMP-3) depletion (IMP-3Δ) was shown to inhibit invadopodia formation and extracellular matrix degradation capacity in oral squamous cell carcinoma (OSCC) cells. In this study, we found that IMP-3Δ cells significantly downregulated the podoplanin (PDPN) level, which resulted in a loss of extracellular matrix degradation activity, although invadopodia was still thriving. From RNA in situ hybridization using a digoxigenin-labeled 3′UTR recognition probe of PDPN and reporter assay with 3′UTR of the PDPN gene cloned downstream from the luciferase reporter gene, we revealed that IMP-3 depletion was shown to be downregulated, which most probably lowered PDPN gene expression by reducing mRNA stabilization. In a xenograft model, PDPN depletion was the cause of a decrease in tumor volume and regional infiltration into nearby stroma. Taken together, transforming growth factor beta 1 increased PDPN expression, which potentiated cancer invasion through increased invadopodia formation and extracellular matrix degradation in the low invasive OSCC cell line. Reciprocally, interleukin-1 beta secreted by OSCC cells, stimulated transforming growth factor beta 1 secretion from stromal fibroblasts to induce PDPN expression in OSCC cells. In addition, a retrospective investigation of OSCC patients found that IMP-3 and PDPN expression significantly correlated with lymph node metastasis of OSCC patients. Moreover, co-expression of IMP-3 and PDPN were frequently detected both in primary and lymph nodes metastatic OSCC cells using immunohistochemical dual staining. Thus, the IMP-3-PDPN axis may be a sensitive target molecule in anti-invadopodia therapy for the treatment of metastatic cancers. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is characterized by progressive memory loss and cognitive impairment. The aggregation of amyloid β (Aβ) and hyperphosphorylated tau protein are two major pathological features of AD. Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, NOX) has been indicated in Aβ pathology; however, whether and how it affects tau pathology are not yet clear. Methods: The role of NOX2 in cognitive function, amyloid plaque formation, and tau hyperphosphorylation were examined in APP/PS1 transgenic mice mated with p47phox-deficient mice (with deletion of the gene of neutrophil cytosolic factor 1, Ncf1) and/or in p47phox-deficient mice receiving intracerebroventricular (ICV) injection of streptozotocin (STZ). The cognitive and non-cognitive functions in these mice were assessed by Morris water maze, Rotarod test, open field, and elevated plus maze. Aβ levels, amyloid plaques, p47phox expression, and astrocyte activation were evaluated using immunofluorescence staining, ELISA, and/or Western blotting. Cultured primary neuronal cells were treated with okadaic acid or conditioned media (CM) from high glucose-stimulated primary astrocytes. The alteration in tau pathology was determined using Western blotting and immunofluorescence staining. Results: Deletion of the gene coding for p47phox, the organizer subunit of NOX2, significantly attenuated cognitive impairment and tau pathology in these mice. p47phox deficiency decreased the activation of astrocytes but had no effect on Aβ levels and amyloid plaque formation in the brains of aged APP/PS1 mice, which displayed markedly increased expression of p47phox in neurons and astrocytes. Cell culture studies found that neuronal p47phox deletion attenuated okadaic acid-induced tau hyperphosphorylation at specific sites in primary cultures of neurons. CM from high glucose-treated WT astrocytes increased tau hyperphosphorylation in primary neurons, whereas this effect was absent from p47phox-deficient astrocytes. Conclusions: These results suggest that p47phox is associated with cognitive function and tau pathology in AD. p47phox expressed in neurons contributes to tau hyperphosphorylation directly, while p47phox in astrocytes affect tau hyperphosphorylation by activating astrocytes indirectly. Our results provide new insights into the role of NOX2 in AD and indicate that targeted inhibition of p47phox may be a new strategy for the treatment of AD. [ABSTRACT FROM AUTHOR]

A heparin-modified thermoresponsive surface bound with heparin-binding epidermal growth factor-like growth factor (HB-EGF) was designed to allow creation of transferrable and functional hepatocyte sheets. A heparin-modified thermoresponsive surface was prepared by covalently tethering heparin onto poly( N -isopropylacrylamide- co -2-carboxyisopropylacrylamide)-grafted tissue culture polystyrene surfaces (Heparin-IC). HB-EGFs were able to stably bind to heparin-IC via affinity interaction. The survival of primary rat hepatocytes was maintained through HB-EGF-bound heparin-IC (HB-EGF/heparin-IC). Moreover, cultured rat primary hepatocytes on HB-EGF/heparin-IC exhibited higher albumin-secretion than hepatocytes cultured on PIPAAm-grafted and collagen-coated surfaces with soluble HB-EGF in the culture medium, regardless of whether soluble EGF was added. These results suggested that HB-EGF/heparin-IC is able to effectively maintain hepatic function via continuous signaling of HB-EGF. After a 4-day cultivation, the cultured hepatocytes on HB-EGF/heparin-IC detached as a cell sheet with fibronectin and HB-EGF only after the temperature was lowered to 20 °C. In addition, higher expression of hepatocyte-specific genes (albumin, hepatocyte nuclear factor 4 alpha, coagulation factor VII, and coagulation factor IX) in hepatocyte sheets was detected on HB-EGF/heparin-IC than on a PIPAAm surface with soluble HB-EGF, indicating that HB-EGF/heparin-IC suppressed the dedifferentiation of cultured hepatocytes. Hence, heparin-modified thermoresponsive surfaces bound with HB-EGF facilitate the fabrication of transferrable hepatocyte sheets with intact hepatic functions and have the potential to provide an in vitro culture system using functional hepatocyte sheet tissues, which may serve as an effective hepatocyte-based tissue engineering platform for liver disease treatments.

Background: Bronchial carcinoids are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive, invasive and metastatic. Studies of tumor initiating cell (TIC) biology in bronchial carcinoids has been hindered by the lack of appropriate in-vitro and xenograft models representing the bronchial carcinoid phenotype and behavior.Methods: Bronchial carcinoid cell lines (H727, TC and H720, AC) were cultured in serum-free growth factor supplemented medium to form 3D spheroids and serially passaged up to the 3rd generation permitting expansion of the TIC population as verified by expression of stemness markers, clonogenicity in-vitro and tumorigenicity in both subcutaneous and orthotopic (lung) models. Acetazolamide (AZ), sulforaphane (SFN) and the AZ + SFN combination were evaluated for targeting TIC in bronchial carcinoids.Results: Data demonstrate that bronchial carcinoid cell line 3rd generation spheroid cells show increased drug resistance, clonogenicity, and tumorigenic potential compared with the parental cells, suggesting selection and expansion of a TIC fraction. Gene expression and immunolabeling studies demonstrated that the TIC expressed stemness factors Oct-4, Sox-2 and Nanog. In a lung orthotopic model bronchial carcinoid, cell line derived spheroids, and patient tumor derived 3rd generation spheroids when supported by a stroma, showed robust tumor formation. SFN and especially the AZ + SFN combination were effective in inhibiting tumor cell growth, spheroid formation and in reducing tumor formation in immunocompromised mice.Conclusions: Human bronchial carcinoid tumor cells serially passaged as spheroids contain a higher fraction of TIC exhibiting a stemness phenotype. This TIC population can be effectively targeted by the combination of AZ + SFN. Our work portends clinical relevance and supports the therapeutic use of the novel AZ+ SFN combination that may target the TIC population of bronchial carcinoids. [ABSTRACT FROM AUTHOR]