Single-cell calcium monitoring of Caco-2 cell co-cultured with intestinal microbiome through carbon fiber based potentiometric microelectrode.

The Caco-2 cells were used as intestinal epithelial cell model to illustrate the hyperuricemia (HUA) mechanism under the co-culture of the imbalanced intestinal microbiome in this work. The uric acid (UA) concentration in the HUA process was monitored, and could be up to 425 µmol/L at 8 h co-cultured with the imbalanced intestinal microbiome. Single-cell potentiometry based on ion-selective microelectrode was used to study extracellular calcium change, which is hypothesized to play an important role in the UA excretion. The potential signal of the calcium in the extremely limited microenvironment around single Caco-2 cell was recorded through the single-cell analysis platform. The potential signal of sharp decrease and slow increase followed within a few seconds indicates the sudden uptake and gradually excretion process of calcium through the cell membrane. Moreover, the value of the potential decrease increases with the increase of the time co-cultured with the imbalanced intestinal microbiome ranging from 0 to 8 h. The Ca2+ concentration around the cell membrane could decrease from 1.3 mM to 0.4 mM according to the potential decrease of 27.0 mV at the co-culture time of 8 h. The apoptosis ratio of the Caco-2 cells also exhibits time dependent with the co-culture of the imbalanced intestinal microbiome, and was 39.1 ± 3.6 % at the co-culture time of 8 h, which is much higher than the Caco-2 cells without any treatment (3.9 ± 2.9 %). These results firstly provide the links between the UA excretion with the apoptosis of the intestinal epithelial cell under the interaction of the imbalanced intestinal microbiome. Moreover, the apoptosis could be triggered by the calcium signaling.

Predictive Effect of Cervical Sagittal Parameters and Corresponding Segmental Paravertebral Muscle Degeneration on the Occurrence of Cervical Kyphosis Following Cervical Laminoplasty.

BACKGROUND: Cervical sagittal parameters and paravertebral muscle degeneration are important factors for the occurrence of cervical spondylotic myelopathy. However, the relationship between the 2 risk factors and cervical kyphosis following cervical laminoplasty remains unknown. METHODS: A total of 130 patients undergoing cervical laminoplasty were enrolled from July 2018 to July 2020 and were followed up for at least 24 months. Clinical recovery was recorded, including the Japanese Orthopedic Association, neck disability index and visual analog scale scores. Radiographic sagittal parameters were measured on cervical lateral radiographs: T1 slope (T1S), C2-C7 Cobb lordotic angle (CLA), C2-C7 sagittal vertical axis, O-C2 angle, and T1S-CLA. The magnetic resonance imaging (MRI) parameters of the paraspinal muscles were also measured, including cross-sectional area and fat infiltration (FI). The patients were divided into a kyphosis group and a lordosis group based on the last follow-up results of CLA. Multivariate logistic analysis was performed to analyze risk factors for kyphosis following laminoplasty. RESULTS: Thirty-two patients were assigned to the kyphosis group and 98 were assigned to the lordosis group. Patient baseline and surgical information in the 2 groups showed no statistically significant difference. In the comparison of clinical recovery, patients with kyphosis showed a lower Japanese Orthopedic Association recovery rate than the lordosis group. For the radiographic parameters and muscle condition comparison, CLA, T1S-CLA, and FI were the most significant parameters. The logistic regression revealed that T1S-CLA and FI were the most important variables that predicted kyphosis. CONCLUSIONS: We concluded that FI remarkably differed in the paraspinal muscles in the 2 groups. Multivariate logistic regression demonstrated that T1S-CLA and FI significantly influenced the process of kyphosis after cervical laminoplasty.

Aquaporin-1 facilitates proliferation and invasion of gastric cancer cells via GRB7-mediated ERK and Ras activation.

Gastric cancer, one of the most common malignant tumors of the digestive tract, is devoid of effective treatment owing to its highly invasive ability. Aquaporins (AQPs), transmembrane water channel proteins, has been shown to be involved in the malignancy of gastric cancer. This study aims to investigate the pathophysiological roles of AQP-1 in gastric cancer. We first demonstrated quantitative real-time polymerase chain reaction analysis and found up-regulation of AQP-1 in gastric cancer cell lines. Additionally, silence of AQP-1 inhibited cell proliferation via decrease of proliferating cell nuclear antigen (PCNA) and minichromosome maintenance complex component 2 (MCM2). Moreover, migration and invasion of gastric cancer cells were also suppressed by the interference of AQP-1. However, the tumorigenic mechanism of AQP-1 on gastric cancer is yet to be found. We demonstrated western blot analysis and found that knockdown of AQP-1 decreased protein expression of phospho (p)-GRB7 (growth factor receptor-bound protein 7) and led to a remarkable reduction of p-extracellular signal-regulated kinase (ERK) via inactivation of RAS. In general, our findings indicated that AQP-1 facilitates proliferation and invasion of gastric cancer cells via GRB7-mediated ERK and Ras activation, illuminating a novel AQP-1-RAS/ERK molecular axis as regulator in gastric cancer progression and suggesting potential implications in the treatment of gastric cancer.

The Effect and Mechanism of Negative Pressure Wound Therapy on Lymphatic Leakage in Rabbits.

BACKGROUND: Lymphatic leakage is one of the severe complications after lymphadenectomy. However, efficient treatment it still unclear. MATERIALS AND METHODS: We employed inguinal lymphadenectomy and saphenous lymphatic vessel excision to establish a inguinal lymphatic leakage rabbit model. Rabbits with bilateral lymphatic leakage were divided in two groups, which were subject to negative pressure wound therapy (NPWT) on right sides and dressing change on left sides, respectively. Following 7-11 d of treatment, skin thickness and drainage volume were measured. Western blot and RT-PCR were used for analyzing the VEGF-C level. Tissues of wound were dissected and subject to anti-LYVE-1 immunohistochemical for lymphatic average positive staining area percentage and the ratio of lymphatic lumen area evaluation. RESULTS: Our lymphatic leakage model showed significant lymph stasis, delayed wound healing, and skin swelling and was confirmed by methylene blue instillation. Using this rabbit model, we found that NPWT could largely promote wound healing and resolution of skin edema. Compared with the dressing change group, the thickness of the dermis layer in the NPWT group was significantly reduced. Western blot and RT-PCR analysis showed a decrease of VEGF-C in the NPWT group. The immunohistochemical result of the NPWT group did not show a significant change in lymphatic average positive staining area percentage, whereas the ratio of lymphatic lumen area was significantly decreased, suggesting that NPWT treatment can significantly compress the dilated lymphatic vessels. CONCLUSIONS: We successfully established the first clinically relevant lymphatic leakage model in rabbits. NPWT can be an effective treatment for lymphatic leakage via reducing edema and lymphatic stasis by compressing dilated lymph vessels and promoting lymphatic drainage.

Nitrostyrene-Modified 2-(2-Hydroxyphenyl)benzothiazole: Enol-Emission Solvatochromism by ESICT-ESIPT and Aggregation-Induced Emission Enhancement.

Excited-state intramolecular charge transfer (ESICT) and excited-state intramolecular proton transfer (ESIPT) are two competitive reactions that occur in the excited states of organic dyes that contain intramolecular hydrogen-bonds and electron acceptors and donors. Determining the mechanisms of these processes is key to understanding their multiple emission features, as the manner in which these processes interact can be modulated by modifying the dye structure. In addition, donor-π-acceptor (D-π-A) molecules often suffer from aggregation-induced quenching. Herein, we report the synthesis of three nitrophenyl-modified 2-(2-hydroxyphenyl)benzothiazole (HBT) derivatives, HBT-s-NO2 , HBT-d-NO2 , and HBT-t-NO2 , which have C-C, C=C, and C≡C bonds between their HBT and nitrophenyl moieties, respectively. Compared with the enol emissions from HBT-s-NO2 and HBT-t-NO2 , that from HBT-d-NO2 exhibits outstanding solvatochromism owing to consecutive ESICT-ESIPT. In addition, X-ray diffraction reveals that despite the highly planar and polar nature of HBT-d-NO2 , which is strongly H-aggregated, it exhibits highly efficient fluorescence. Hence, this study provides new insight into the design of ESICT/ESIPT-coupled systems and for engendering planar dipolar molecules with excellent emission properties in the solid state.

Cationic poly-l-lysine-encapsulated melanin nanoparticles as efficient photoacoustic agents targeting to glycosaminoglycans for the early diagnosis of articular cartilage degeneration in osteoarthritis.

Cartilage degeneration is the hallmark of osteoarthritis (OA) and its early diagnosis is essential for effective cartilage repair. However, until now, there was still a lack of imaging modalities that can accurately detect and evaluate cartilage degeneration in its early stage. Herein, we introduce endogenous melanin nanoparticles (MNPs) encapsulated by poly-l-lysine (PLL) as positively charged contrast agents for the accurate photoacoustic (PA) imaging of cartilage degeneration through its strong electrostatic interaction with anionic glycosaminoglycans (GAGs) in the cartilage. PLL-MNPs presented high PA intensity, photostability and biocompatibility. In vitro PAI studies showed that PLL-MNPs with a zeta potential of +32.5 ± 9.3 mV had more cartilage uptake and longer retention time than anionic MNPs, and generated a positive relationship with the GAG content in the cartilage. After administration via intra-articular injection in living mouse models, PLL-MNPs exhibited about a two-fold stronger PA signal in a normal joint (with high GAG content) than an OA joint (with low GAG content). Furthermore, the obtained PAI results provided accurate information of the GAG content distribution in the OA knee joint. Consequently, by detecting and analyzing the changes of the GAG content in OA cartilage using PAI, we can clearly distinguish early OA from late OA and monitor the therapeutic efficacy in OA after drug treatment. All PAI results were examined histologically.

Acid-Induced Shift of Intramolecular Hydrogen Bonding Responsible for Excited-State Intramolecular Proton Transfer.

The significant progress recently achieved in designing smart acid-responsive materials based on intramolecular charge transfer inspired us to utilize excited-state intramolecular proton transfer (ESIPT) for developing a turn-on acid-responsive fluorescent system with an exceedingly large Stokes shift. Two ESIPT-active fluorophores, 2-(2-hydroxyphenyl)pyridine (HPP) and 2-(2-hydroxyphenyl)benzothiazole (HBT), were fused into a novel dye (HBT-HPP) fluorescent only in the protonated state. Moreover, we also synthesized three structurally relevant control compounds to compare their steady-state fluorescence spectra and optimized geometric structures in neutral and acidic media. The results suggest that the fluorescence turn-on was caused by the acid-induced shift of the ESIPT-responsible intramolecular hydrogen bond from the HPP to HBT moiety. This work presents a systematic comparison of the emission efficiencies and basicity of HBT and HPP for the first time, thereby utilizing their differences to construct an acid-responsive smart organic fluorescent material. As a practical application, red fluorescent letters can be written using the acid as an ink on polymer film.

Negative pressure wound therapy: Regulating blood flow perfusion and microvessel maturation through microvascular pericytes.

Negative pressure wound therapy (NPWT) has been demonstrated to accelerate wound healing by promoting angiogenesis. However, whether blood flow perfusion is regulated by microvessel maturation and pericytes following NPWT remains unclear, as well as the exact association between pericytes and collagen type IV. The aim of this study was to investigate the relevant association between blood flow perfusion and microvessel maturation and pericytes following NPWT, and to further explore the underlying molecular mechanisms. We also aimed to investigate the association between pericytes and collagen type IV. For this purpose, we created a rat model of diabetic wounds and microvascular blood flow perfusion was detected using a laser Doppler blood perfusion imager. The expression levels of angiogenin-1, tyrosine phosphorylation of tyrosine kinase receptor-2 (Tie-2), α-smooth muscle actin (α-SMA) and collagen type IV were detected and analyzed through immunohistochemistry, immunofluorescence, RT-qPCR and western blot analysis. The results revealed that NPWT promoted the overexpression of angiogenin-1, Tie-2, α-SMA and collagen type IV, and significantly increased blood flow perfusion coupled with microvessel maturation in the NPWT group at the later stages (7-10 days) of wound healing. Our results suggested that NPWT can preferentially enhance vessel maturation and increase the number of pericytes, thus regulating blood flow perfusion. On the other hand, pericytes and collagen type IV had a mutual interaction, promoting microvessel maturation.

Hierarchical Microspheres Constructed from Chitin Nanofibers Penetrated Hydroxyapatite Crystals for Bone Regeneration.

Chitin exists abundantly in crab and shrimp shells as the template of the minerals, which inspired us to mineralize it for fabricating bone grafting materials. In the present work, chitin nanofibrous microspheres were used as the matrix for in situ synthesis of hydroxyapatite (HA) crystals including microflakes, submicron-needles, and submicron-spheres, which were penetrated by long chitin nanofibers, leading to the hierarchical structure. The shape and size of the HA crystals could be controlled by changing the HA synthesis process. The tight interface adhesion between chitin and HA through the noncovanlent bonds occurred in the composite microspheres, and HAs were homogeneously dispersed and bounded to the chitin nanofibers. In our findings, the inherent biocompatibilities of the both chitin and HA contributed the bone cell adhesion and osteoconduction. Moreover, the chitin microsphere with submicron-needle and submicron-sphere HA crystals remarkably promoted in vitro cell adhesion and in vivo bone healing. It was demonstrated that rabbits with 1.5 cm radius defect were almost cured completely within three months in a growth factor- and cell-free state, as a result of the unique surface microstructure and biocompatibilities of the composite microspheres. The microsphere scaffold displayed excellent biofunctions and an appropriate biodegradability. This work opened up a new avenue to construct natural polymer-based organic-inorganic hybrid microspheres for bone regeneration.

Enhancement of Bone-Marrow-Derived Mesenchymal Stem Cell Angiogenic Capacity by NPWT for a Combinatorial Therapy to Promote Wound Healing with Large Defect.

Poor viability of engrafted bone marrow mesenchymal stem cells (BMSCs) often hinders their application for wound healing, and the strategy of how to take full advantage of their angiogenic capacity within wounds still remains unclear. Negative pressure wound therapy (NPWT) has been demonstrated to be effective for enhancing wound healing, especially for the promotion of angiogenesis within wounds. Here we utilized combinatory strategy using the transplantation of BMSCs and NPWT to investigate whether this combinatory therapy could accelerate angiogenesis in wounds. In vitro, after 9-day culture, BMSCs proliferation significantly increased in NPWT group. Furthermore, NPWT induced their differentiation into the angiogenic related cells, which are indispensable for wound angiogenesis. In vivo, rat full-thickness cutaneous wounds treated with BMSCs combined with NPWT exhibited better viability of the cells and enhanced angiogenesis and maturation of functional blood vessels than did local BMSC injection or NPWT alone. Expression of angiogenesis markers (NG2, VEGF, CD31, and α-SMA) was upregulated in wounds treated with combined BMSCs with NPWT. Our data suggest that NPWT may act as an inductive role to enhance BMSCs angiogenic capacity and this combinatorial therapy may serve as a simple but efficient clinical solution for complex wounds with large defects.

Excited State Intramolecular Proton Transfer in Ethynyl-Extended Regioisomers of 2-(2'-Hydroxyphenyl)benzothiazole: Effects of the Position and Electronic Nature of Substituent Groups.

Although the organic dyes based on excited state intramolecular proton transfer (ESIPT) mechanism have attracted significant attention, the structure-property relationship of ESIPT dyes needs to be further exploited. In this paper, three series of ethynyl-extended regioisomers of 2-(2'-hydroxyphenyl)benzothiazole (HBT), at the 3'-, 4'- and 6-positions, respectively, have been synthesized. Changes in the absorption and emission spectra were correlated with the position and electronic nature of the substituent groups. Although 4'- and 6-substituted HBT derivatives exhibited absorption bands at longer wavelengths, the keto-emission of 3'-substituted HBT derivatives was found at a substantially longer wavelength. The gradual red-shifted fluorescence emission was found for 3'-substituted HBT derivatives where the electron-donating nature of substituent group increased, which was opposite to what was observed for 4'- and 6-substituted HBT derivatives. The results derived from the theoretical calculations were in conformity with the experimental observations. Our study could potentially provide experimental and theoretical basis for designing novel ESIPT dyes that possess unique fluorescent properties.

The iron chelator Dp44mT suppresses osteosarcoma's proliferation, invasion and migration: in vitro and in vivo.

Di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), the novel iron chelator, has been reported to inhibit the tumorigenesis and progression of various cancer cells, including neuroblastoma, neuroepithelioma and prostate cancer. However, whether Dp44mT has anticancer effects in osteosarcoma is still unknown. Here, we investigated the antitumor action of Dp44mT in osteosarcoma and its underlying mechanisms. A human osteosarcoma 143B cell line in vitro and 143B xenograft in nude mice in vivo were utilized, the anticancer effects of Dp44mT were examined through methods of MTT assay, transwell, wound healing assay, flow cytometry, western blot, immunohistochemistry and H&E staining. We showed that Dp44mT inhibits cell proliferation, invasion and migration in vitro. In addition, flow cytometry further illustrated that Dp44mT suppression of 143B cell proliferation, invasion and migration were partially due to induction of cell apoptosis, cell cycle arrest in S phase and ROS production. Also in vitro and in vivo, the expression levels of Bcl2, Bax, Caspase3, Caspase9, LC3-II, ß-catenin and its downstream targets such as C-myc and Cyclin D1 demonstrated that cell apoptosis and autophagy, as well as Wnt/ß-catenin pathway were involved in Dp44mT induced osteosarcoma suppression. The Dp44mT inhibition of osteosarcoma was further verified via animal models. The findings indicated that in vivo Dp44mT showed a significant reduction in the 143B xenograft tumor growth and metastasis. In conclusion, our data demonstrated that Dp44mT has effective anticancer capability in osteosarcoma and that may represent a promising treatment strategy for osteosarcoma.

[Study on tissue culture and plant regeneration of the stem-tips and buds of Periploca forrestii].

OBJECTIVE: To establish the rapid propagation systems of the stem-tips and buds of Periploca forrestii. METHODS: Inserted the stem-tips and buds of Periploca forrestii into MS medium with different concentrations of 6-BA, NAA and 2.4-D and induced them growing into complete plants. RESULTS AND CONCLUSION: The optimal culture medium for bud induction is MS + 6-BA 1.0 mg/L + NAA 0.3 mg/L and the bud induction rate can reach 86.29%. The optimal culture medium for stem-tips induction is MS +6-BA 2.0 mg/L + NAA 0.5 mg/L and the bud induction rate can reach 86.29%. The optimal culture medium for bud multiplication is MS + 6-BA 2.0 mg/L + NAA 0.1 mg/L and the multiplication coefficient can reach 2.10. The best rooting medium is 1/2 MS + IBA 0.5 mg/L and the rooting rate is 53.33%.