The role of FERMT2 in the tumor microenvironment and immunotherapy in pan-cancer using comprehensive single-cell and bulk sequencing.

FERMT2 has been identified as a participant in integrin-linked kinase signaling pathways, influencing epithelial-mesenchymal transition and thereby affecting tumor initiation, progression, and invasion. While the character of FERMT2 in the tumor microenvironment (TME) as well as its implications for immunotherapy remain unclear. Thus, we conducted a comprehensive analysis to assess the prognostic significance of FERMT2 using Kaplan-Meier analysis. In addition, we employed enrichment analysis to uncover potential underlying molecular mechanisms. Using "Immunedeconv" package, we evaluated the immune characteristics of FERMT2 within TME. Furthermore, we determined the expression levels of FERMT2 in various cell types within TME, based on single-cell sequencing data. To confirm the co-expression of FERMT2 and markers of cancer-associated fibroblasts (CAFs), we performed multiplex immunofluorescence staining on tissue paraffin sections across various cancer types. Our analysis disclosed a significant correlation between elevated FERMT2 expression and unfavorable prognosis in specific cancer types. Furthermore, we identified a strong correlation between FERMT2 expression and diverse immune-related factors, including immune checkpoint molecules, immune cell infiltration, microsatellite instability (MSI), and tumor mutational burden (TMB). Additionally, there was a significant correlation between FERMT2 expression and immune-related pathways, particularly those associated with activating, migrating, and promoting the growth of fibroblasts in diverse cancer types. Interestingly, we observed consistent co-expression of FERMT2 in both malignant tumor cells and stromal cells, particularly within CAFs. Notably, our findings also indicated that FERMT2, in particular, exhibited elevated expression levels within tumor tissues and co-expressed with α-SMA in CAFs based on the multiplex immunofluorescence staining results.

Na,K-ATPase activity promotes macropinocytosis in colon cancer via Wnt signaling.

Recent research has shown that membrane trafficking plays an important role in canonical Wnt signaling through sequestration of the ß-catenin destruction complex inside multivesicular bodies (MVBs) and lysosomes. In this study, we introduce Ouabain, an inhibitor of the Na,K-ATPase pump that establishes electric potentials across membranes, as a potent inhibitor of Wnt signaling. We find that Na,K-ATPase levels are elevated in advanced colon carcinoma, that this enzyme is elevated in cancer cells with constitutively activated Wnt pathway and is activated by GSK3 inhibitors that increase macropinocytosis. Ouabain blocks macropinocytosis, which is an essential step in Wnt signaling, probably explaining the strong effects of Ouabain on this pathway. In Xenopus embryos, brief Ouabain treatment at the 32-cell stage, critical for the earliest Wnt signal in development-inhibited brains, could be reversed by treatment with Lithium chloride, a Wnt mimic. Inhibiting membrane trafficking may provide a way of targeting Wnt-driven cancers.

Salvia guidongensis sp. nov.: unraveling a critical evolutionary link in East Asian Salvia from Central China integrating morphology, phylogeny, and plastid genomics.

Introduction: Salvia L., representing the largest genus within the mint family, is noted for its global distribution of approximately 1000 species, with East Asia, and particularly China, recognized as a critical center of diversity for the genus. Methods: Our research was conducted through extensive fieldwork in Guidong County, Hunan Province, China, where we identified a previously undescribed species of Salvia. The identification process involved detailed morphological observations, phylogenetic analyses, and plastid genomics. Results: The newly discovered species, Salvia guidongensis, exhibits unique characteristics not commonly observed in the East Asian lineage of Salvia, including dual floral colors within natural populations-either pale purple or pale yellow. Morphologically, while it shares similarities with members of sect. Glutinaria, S. guidongensis is distinct in its floral morphology, stature, and specific foliar traits. Phylogenetic analysis places S. guidongensis in a unique clade within the East Asian lineage of Salvia, suggesting it may serve as an important evolutionary link. Additionally, we explored the plastome features of S. guidongensis, comparing them with those of closely related species. Discussion: The discovery of S. guidongensis not only entriches the taxonomic tapestry of Salvia but also provides critical insights into the biogeography and evolutionary pathways of the genus in East Asia. By integrating morphological and molecular data, we validate the novel status of S. guidongensis and highlight its significance in bridging taxonomic and evolutionary gaps within Sect. Glutinaria of Salvia.

Calreticulin P-domain-derived "Eat-me" peptides for enhancing liposomal uptake in dendritic cells.

Discovering new ligands for enhanced drug uptake and delivery has been the core interest of the drug delivery field. This study capitalizes on the natural "eat-me" signal of calreticulin (CRT), proposing a novel strategy for functionalizing liposomes to improve cellular uptake. CRT is presented on the surfaces of apoptotic cells, and it plays a crucial role in immunogenic cell death (ICD). This is because it is essential for antigen uptake via low-density lipoprotein (LDL) receptor-mediated phagocytosis. Inspired by this mechanism, we interrogated CRT's "eat-me" feature using CRT-derived peptides to functionalize liposomes. We studied liposomal formulation stability, properties, cellular uptake, toxicity, and intracellular trafficking in dendritic cells. We identified key peptide fragments of CRT, specifically from the hydrophilic P-domain, that are compatible with liposomal formulations. Contrary to the more hydrophobic N-domain peptides, the P-domain peptides induced significantly higher liposomal uptake in DC2.4 dendritic cells than cationic DOTAP and anionic DPPG liposomes without inducing toxicity. The P-domain-derived peptides led to enhanced liposomal uptake into DC2.4 dendritic cells compared to the standard DPPC liposomes. The uptake can be partially blocked by the receptor-associated protein (RAP). Upon internalization, P-domain-peptide-decorated liposomes showed higher co-localization with lysosomes compared to the standard DPPC liposomes. Our findings illuminate CRT's operational role and identify P-domain peptides as promising agents for developing biomimetic drug delivery systems that can potentially replicate CRT's "eat-me" function.

Synergistic Combination of Irinotecan and Rapamycin Orally Delivered by Nanoemulsion for Enhancing Therapeutic Efficacy of Pancreatic Cancer.

In recent years, combining different types of therapy has emerged as an advanced strategy for cancer treatment. In these combination therapies, oral delivery of anticancer drugs is more convenient and compliant. This study developed an irinotecan/rapamycin-loaded oral lecithin-based self-nanoemulsifying nanoemulsion preconcentrate (LBSNENPir/ra) and evaluated its synergistic combination effects on pancreatic cancer. LBSNENP loaded with irinotecan and rapamycin at a ratio of 1:1 (LBSNENPir10/ra10) had a better drug release profile and smaller particle size (<200 nm) than the drug powder. Moreover, LBSNENPir10/ra10 exhibited a strong synergistic effect (combination index [CI] < 1.0) in cell viability and combination effect studies. In the tumor inhibition study, the antitumor activity of LBSNENPir10/ra10/sily20 against MIA PaCa-2 (a human pancreatic cancer cell line) was significantly increased compared with the other groups. When administered with rapamycin and silymarin, the area under the curve and the maximum concentration of irinotecan significantly improved compared with the control. We successfully developed an irinotecan/rapamycin-loaded oral self-nanoemulsifying nanoemulsion system to achieve treatment efficacy for pancreatic cancer.

Advances of development and application amino acid biosensors / 生物工程学报

Amino acids are the basic building blocks of protein that are very important to the nutrition and health of humans and animals, and widely used in feed, food, medicine and daily chemicals. At present, amino acids are mainly produced from renewable raw materials by microbial fermentation, forming one of the important pillar industries of biomanufacturing in China. Amino acid-producing strains are mostly developed through random mutagenesis- and metabolic engineering-enabled strain breeding combined with strain screening. One of the key limitations to further improvement of production level is the lack of efficient, rapid, and accurate strain screening methods. Therefore, the development of high-throughput screening methods for amino acid strains is very important for the mining of key functional elements and the creation and screening of hyper-producing strains. This paper reviews the design of amino acid biosensors and their applications in the high-throughput evolution and screening of functional elements and hyper-producing strains, and the dynamic regulation of metabolic pathways. The challenges of existing amino acid biosensors and strategies for biosensor optimization are discussed. Finally, the importance of developing biosensors for amino acid derivatives is prospected.

Dietary metal intake and the prevalence of erectile dysfunction in US men: Results from National Health and Nutrition Examination Survey 2001-2004.

Background: Erectile dysfunction (ED) mainly affects men over 40 years of age and is a common clinical condition. In addition to hypertension and diabetes, environment, and lifestyle are also significantly associated with erectile dysfunction. The relationship between dietary trace metal intake and ED has not been studied. Materials and methods: Data on participants were obtained from the National Health and Nutrition Examination Survey for this study, and those with incomplete information on clinical variables were excluded. Dose-response curve analysis was used to investigate the relationship between dietary trace metal intake and ED prevalence. Multivariate logistic regression analysis was used to adjust for confounders to further investigate the relationship between dietary trace metal intake and ED prevalence. 1:1 propensity score matching (PSM) was performed to adjust for differences between clinical variables for data reanalysis to confirm the reliability of the results. Results: A total of 3,745 individuals were included in the study, including 1096 ED patients and 2,649 participants without ED. Dietary intake of trace metals (Mg, Zn, Cu, and Se) was significantly higher in participants without ED than in ED patients (all P < 0.001). Dose-response curve analysis showed a significant negative association between these dietary metal intakes and ED prevalence (all P < 0.001). Multivariate logistic regression analysis adjusted for confounders (age, education, BMI, annual household income, hypertension, diabetes, marital status, race, and current health status) revealed that increased dietary metal intake reduced the odds ratio of ED. 1:1 PSM reanalysis further confirmed the validity of the results. Conclusion: Increasing dietary intake of trace metals (magnesium, zinc, copper, and selenium) within the upper limit is beneficial in reducing the prevalence of ED.

Clinical Study on Efficiency of Using Traditional Direct Bonding or OrthGuide Computer-Aided Indirect Bonding in Orthodontic Patients.

Objective: This study aims to clinically investigate and compare the therapeutic effects and treatment cycle between traditional direct bonding and OrthGuide computer-aided indirect bonding in orthodontic treatment. Methods: Forty patients treated at the Department of Orthodontics, Beijing Rytime Dental Hospital between July 1, 2016, and December 31, 2019, were included. The patients were divided into a control group (n = 20, traditional direct bonding) and a test group (n = 20, OrthGuide computer-aided indirect bonding). The American Board of Orthodontics (ABO) measurement was performed on patients using Uceph cephalometric analysis software to compare intragroup and intergroup differences, and the treatment cycles of all patients were recorded. Results: After treatment, U1-NA (mm), ∠U1-SN (°), LL-EP (mm), and UL-EP (mm) in the control group were significantly lower than before treatment, and there was no significant difference in other ABO measurement indexes, while the test group showed no marked difference in all ABO measurements between pre- and posttreatment. Further, intergroup comparison showed no significant difference in ABO measurements in pre- and posttreatment between the two groups. The test group had a shorter treatment cycle than the control group, with an average treatment cycle of 21.20 ± 7.14 months in the control group and 17.17 ± 4.16 months in the test group. Conclusion: There was no significant difference in the therapeutic effects between the direct and indirect bonding techniques. However, OrthGuide computer-assisted indirect bonding demonstrated a significantly shorter treatment cycle and might be more efficient than traditional direct bonding.

In situ subcutaneously injectable thermosensitive PEG-PLGA diblock and PLGA-PEG-PLGA triblock copolymer composite as sustained delivery of bispecific anti-CD3 scFv T-cell/anti-EGFR Fab Engager (BiTEE).

In this study, PEGylated poly (lactide-co-glycolide) (PLGA) thermosensitive composite hydrogels (DTgels) loaded with bispecific anti-cluster of differentiation 3 (CD3) scFv T-cell/anti-epidermal growth factor receptor (EGFR) Fab engager (BiTEE) were subcutaneously (s.c.) injected for the in situ formation of a drug deposit to resolve limitations of the clinical application of the BiTEE of a short half-life and potential side effects. Three kinds of DTgels prepared with different ratios of methoxy poly (ethylene glycol) (mPEG)-PLGA (diblock copolymer, DP) and PLGA-PEG-PLGA (triblock copolymer, TP) were designated DTgel-1, DTgel-2, and DTgel-2S. All three DTgel formulations showed thermosensitive properties with a sol-gel transition temperature at 28-34 °C, which is suitable for an injection. An in vitro release study showed that all DTgel formulations loaded with stabilized BiTEE extended the release of the BiTEE for up to 7 days. In an animal pharmacokinetics study, an s.c. injection of BiTEE/DTgel-1, BiTEE/DTgel-2, or BiTEE/DTgel-2S respectively prolonged the half-life of the BiTEE by 3.5-, 2.0-, and 2.2-fold compared to an intravenous injection of the BiTEE solution. Simultaneously, BiTEE/DTgel formulations showed almost no proinflammatory cytokine release in mice injected with T cells after s.c. administration. Results of an animal antitumor (MDA-MB-231) study indicated that an s.c. injection of the BiTEE/DTgel formulations significantly improved the antitumor efficacy compared to an intravenous (i.v.) or s.c. injection of the BiTEE solution. Moreover, BiTEE/DTgel formulations led to enhanced T-cell recruitment to solid-tumor sites. In conclusion, the in situ formation of injectable PEGylated PLGA thermosensitive hydrogels loaded with the BiTEE was successfully carried out to increase its half-life, maintain a constant blood level within therapeutic windows, and enhance T-cell recruitment to solid-tumor sites resulting in exceptional treatment efficacy.

The Role of Androgen Receptor Splicing Variant 7 in Predicting the Prognosis of Metastatic Castration-Resistant Prostate Cancer: Systematic Review and Meta-Analysis.

OBJECTIVE: The purpose of this meta-analysis was to study the prognostic effects of androgen receptor splicing variant 7 (AR-V7) on metastatic castration-resistant prostate cancer (mCRPC) under different treatment options (chemotherapy, hormone therapy). METHODS: We conducted a systematic search of PubMed, EMBASE and Cochrane databases for clinical studies up to June 4, 2021, and used prostate-specific antigen (PSA) progression free-survival (PSA-PFS), radiologic PFS (r-PFS), overall survival (OS) and PSA response rate (PSA RR) as the main endpoints. Subgroup analyses were conducted based on the source of the specimens. STATA v.15 software was used for data analysis. RESULTS: Twenty-one studies were included in this meta-analysis, with a total of 1578 samples. In the abiraterone (AA)/enzalutamide (E) treatment group, AR-V7 positive patients had worse PSA-PFS (hazard ratio [HR] = 3.40; 95% confidence interval [95%CI] 2.56-4.51; P < 0.05) and worse r-PFS (HR = 2.69; 95%CI 1.70-4.24; P < 0.05) and OS (HR = 3.02; 95%CI 1.73-5.30; P < 0.05). Multivariate Cox regression results showed that AR-V7 positive status was an independent risk factor for OS in the AA/E treatment group. In the taxane treatment group, AR-V7-positive and negative patients had similar PSA-PFS (HR = 0.87; 95%CI 0.46-1.63; P = 0.657), r-PFS (HR = 1.01; 95%CI 0.53-1.96; P = 0.965) and OS (HR = 1.50; 95%CI 0.89-2.52; P = 0.127). For AR-V7-positive patients, the difference in OS between taxane and AA/E treatment was not statistically significant (HR = 1.03; 95%CI 0.52-2.06; P = 0.930). However, multivariate Cox regression results suggested that for AR-V7-positive patients, taxane therapy was a protective factor for OS (HR = 0.35; 95%CI 0.20-0.60; P < 0.05). CONCLUSION: The expression of AR-V7 indicates a poor prognosis and is an independent risk factor for OS in AA/E-treated mCRPC patients. However, AR-V7 positive status does not play the same role in taxane-treated patients. In addition, compared to AA/E, taxane treatment is a protective factor for OS in AR-V7-positive patients. AR-V7 may thus be an effective biomarker for treatment prognosis in patients with mCRPC.

Exposure to environmental bisphenol A inhibits HTR-8/SVneo cell migration and invasion.

Environmental pollutants, such as bisphenol A (BPA) have recently been implicated in the development of adverse birth outcomes. However, the underlying teratogenic mechanisms remain unclear. We investigated the effects of BPA on the migration and invasion of human primary extravillous trophoblast HTR-8/SVneo cells. Our results indicated that BPA reduced cell migration and invasion. Moreover, it altered the ratio of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) by downregulating MMP-2 and MMP-9, and upregulating TIMP-1 and TIMP-2. Furthermore, BPA suppressed integrin ß1, integrin α5, and vimentin. Interestingly, BPA-induced invasion was partially restored by G15, a membrane G-protein-coupled estrogen receptor 30 antagonist. We further revealed that 42 proteins were differentially expressed by mass spectrometry analysis, which could be divided into three categories based on gene ontology including biological process, cellular component, and molecular function. These results suggest that BPA reduces HTR-8/SVneo cell migration and invasion by downregulating MMP-2 and MMP-9, up-regulating TIMP-1 and TIMP-2, and suppressing adhesion molecules.

A Novel Composite Hydrogel Composed of Formic Acid-Decellularized Pepsin-Soluble Extracellular Matrix Hydrogel and Sacchachitin Hydrogel as Wound Dressing to Synergistically Accelerate Diabetic Wound Healing.

Extracellular matrix (ECM) hydrogel can create a favorable regenerative microenvironment and act as a promising dressing for accelerating the healing of diabetic wound. In this study, a simple and effective decellularization technique was developed and optimized to obtain acellular extracellular matrix (aECM) from porcine skin. It was found that decellularization at 30% formic acid for 72 h effectively decellularized porcine skin while retaining >75% collagen and ~37% GAG in the aECM with no presence of nuclei of cellular remnants. aECM hydrogel was fabricated by digesting aECM with pepsin in various acidic solutions (0.1 N HCl, glycolic acid (GA) and 2-pyrrolidone-5-carboxylic acid (PCA)) and then treated with a pH-controlled neutralization and temperature-controlled gelation procedure. Based on physical characterizations, including SDS-PAGE, rheological analysis and SEM analysis, aECMHCl hydrogels fabricated at 25 mg/mL in 0.1 N HCl were selected. Four polymeric ECM-mimic hydrogels, including sacchachitin (SC), hyaluronic acid (HA) and chitosan (CS) and three composite hydrogels of combining SC either with aECMHCl,25 (aECMHCl/SC), HA (HA/SC) or CS (SC/CS) were prepared and evaluated for WS-1 cell viability and wound-healing effectiveness. Cell viability study confirmed that no hydrogel dressings possessed any toxicity at all concentrations examined and ECMHCl, HA and ECMHCl/SC at higher concentrations (>0.05%) induced statistically significant proliferation. Diabetic wound healing study and histological examinations revealed that ECMHCl/SC hydrogel was observed to synergistically accelerate wound healing and ultimately stimulated the growth of hair follicles and sweat glands in the healing wound indicating the wound had healed as functional tissues. The results support the great potential of this newly produced ECMHCl/SC composite hydrogel for healing and regeneration of diabetic wounds.

Ginsenoside Rb1 improving autophagic flux against myocardial ischemia reperfusion injury in isolated-heart of rat / 解剖学报

Objective To explore the protective effect of ginsenoside Rb1 on the myocardial ischemia / reperfusion (I / R) injury in rats in vitro. Methods Totally 60 adult male SD rats were randomly divided into 6 groups:sham group,I / R group,ginsenosde Rb1 pretreatment groups(at the doses of 1 μmol / L,5 μmol / L,10 μmol / L and 20 μmol / L,respectively), 10 in each group. The Langendorff perfusion system was used to establish I / R model. The Lab Chart electrophysiological system was used to monitor real-time heart function by monitoring heart rate (HR), left ventricular development pressure (LVDP) and left ventricular development pressure (± dp / dtmax). TTC staining method was used to measure myocardial infarct size. The Western blotting were used to assay Beclin 1, LC3, p62 and Lamp 2 expression, respectively. The immunohistochemistry were used to assay Beclin 1 expression. Results Ginsenoside Rbl of all the four different concent rations improved the decrease of LVDP and ± dp / dtmax arising from myocardial I / R injury. Meanwhile, ginsenoside Rbl significantly decreased the area of cardial infarction. Ginsenoside Rb1 (10 μmol / L) precondition group protected the heart most significantly (P<0. 05). The expression of Beclin 1 with I / R increased significantly in the cytoplasm of cardiomyocytes. Moreover, Beclin 1 expression decreased after addition pretreatment with ginsenoside Rb1 (10 μmol / L) (P < 0. 05). Compared with sham group, we found that the autophagic flux was impaired in I / R group which the expression of Beclin 1, LC3 and p62 increased significantly, as well as the expression of Lamp 2 decreased significantly. On the other hand, pretreatment with ginsenoside Rb1 (10 μmol / L) could reverse impaired autophagic flux (P < 0. 05). Conclusion Ginsenoside Rbl demonstrates pharmacological preconditioning effect and protects against myocardial I / R injury by improving damaged-autophagy flux, the dose of 10 μmol / L precondition protectes the heart most significantly.

Effect of Shaoyaotang on Expressions of TLR4, NF-κB p65 and IL-6 in Rats with Damp-heat Ulcerative Colitis / 中国实验方剂学杂志

Objective:To explore the mechanism of Shaoyaotang in the treatment of ulcerative colitis (UC) based on toll-like receptor 4 (TLR4)/nuclear factor kappaB (NF-κB) signaling pathway. Method:A total of 50 Wistar rats were selected, including half male and half female. The damp-heat UC rat model was replicated by the methods of the combination of diseases and syndromes and the combination of 2, 4, 6-nitrobenzene sulfonic acid (TNBS) and ethanol. After the successful modeling, the model rats were randomly divided into model group, salazulesulfonate group, and low, medium and high-dose Shaoyaotang groups, and 10 rats (half male and half female) were selected as the blank control group. Low, medium and high-dose Shaoyaotang groups were given 6, 12, 24 g·kg-1 by gavage, and salazonyl arsenic group was given 1 g·kg-1 by gavage. Blank control group was given the equal volume of normal saline for 21 consecutive days. Colon samples were collected after the last administration, and the expressions of TLR4, NF-κB p65 and IL-6 mRNA in colon tissues were detected by fluorescent quantitative polymerase chain reaction (Real-time PCR）, and the expressions of TLR4, NF-κB p65 and IL-6 protein in colon tissues were detected by Western blot. Result:Compared with the blank control group, the relative expressions of TLR4, NF-κB p65, IL-6 mRNA and protein in the model group were significantly increased (P<0.05). Compared with the model group, the expression levels of TLR4, NF-κB p65 and IL-6 mRNA and protein in the salazopyridine group and Shaoyaotang groups were significantly decreased (P<0.05). Conclusion:Shaoyaotang can inhibit the development of UC by regulating the expressions of TLR4, NF-κB p65 and IL-6 mRNA and proteins in the TLR4/NF-κB pathway.

Hybrid driving variable-focus optofluidic lens.

Conventional optofluidic lens usually has only one interface, which means that the zoom range is small, and the ability to correct aberrations is poor. In this paper, we propose a hybrid driving variable-focus optofluidic lens. It has one water-oil interface shifted by an applied voltage and one tunable Polydimethylsiloxane (PDMS) lens deformed by pumping liquid in or out of the cavity. The proposed lens combines the advantages of electrowetting lens and mechanical lens. Therefore, it can provide a large focal length tuning range with good image quality. The shortest positive and negative focal length are ∼6.02 mm and ∼-11.15 mm, respectively. The maximum resolution of our liquid lens can be reached 18 lp/mm. We also designed and fabricated a zoom system using the hybrid driving variable-focus optofluidic lens. In the experiment, the zoom range of the system is 14 mm∼30 mm and the zoom ratio is ∼2.14× without any mechanical moving parts. Its applications for zoom telescope system and zoom microscope and so on are foreseeable.

Analysis and Pinning Control for Output Synchronization and H∞ Output Synchronization of Multiweighted Complex Networks.

The output synchronization and H∞ output synchronization problems for multiweighted complex network are discussed in this paper. First, we analyze the output synchronization of multiweighted complex network by exploiting Lyapunov functional and Barbalat's lemma. In addition, some nodes- and edges-based pinning control strategies are developed to ensure the output synchronization of multiweighted complex network. Similarly, the H∞ output synchronization problem of multiweighted complex network is also discussed. Finally, two numerical examples are presented to verify the correctness of the obtained results.

Effect of Shaoyaotang on Expressions of AP-1 and TNF-α in Colon of Rats with Hygrothermal Ulcerative Colitis / 中国实验方剂学杂志

Objective: To observe the effect of Shaoyaotang on mRNA and protein expressions of colon tissue activated protein-1 (AP-1) and tumor necrosis factor-α (TNF-α) of hot and humid-type intrinsic ulcerative colitis (UC) model in rats, in order to explore the mechanism of action of herbaceous peony decoction in the treatment of UC. Method: Totally 60 Wistar rats were randomly divided into blank group, model group, SASP group, and low, medium and high-dose Shaoyaotang groups. The damp-heat intrinsic UC rat model was replicated based on integrated disease and syndrome, namely, high-fat and high-sugar spicy food and immune complex method combined with 2,4,6-trinitrobenzene sulfolnic acid (TNBS) and ethanol complex method. After the successful modeling, low, medium and high-dose Shaoyaotang (6, 12, 24 g·kg-1) was given by gavage, and 1 g·kg-1 dose of salazol sulfadiazine was given to by gavage. The blank group was given constant volume normal saline for 21 d. Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) was used to detect mRNA expressions of AP-1 and TNF-α in colon tissues, and Western blot was used to detect protein expressions of AP-1 and TNF-α in colon tissues. Result: Compared with the blank group, relative mRNA and protein expressions of AP-1, TNF-α in the model group were significantly increased (Pα in the treatment groups were significantly decreased (PConclusion: Shaoyaotang can inhibit the expression of TNF-α and stimulate AP-1 protein expression in rats with damp-heat UC.

Expression changes of cell cycle related molecules in palatal tissue of fetal mice with cleft palate induced by TCDD / 中华整形外科杂志

Objective@#The purpose of this study is to investigate the expression change of cell cycle-related molecules in platal tissue of fetal mice with cleft palate, induced by 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin (TCDD), and to explore the mechanism of cell cycle-related molecules in cleft palate.@*Methods@#In vivo, 48 pregnant mice were randomly divided into TCDD treatment group and control group with Random number table, 24 mice in each group. On the embryonic day 10.5 (E10.5), pregnant mice were orally administrated with TCDD 28 μg/kg (containing 5 μg/ml TCDD of corn oil) in TCDD treatment group. The same volume of corn oil was given to the mice in control group. The pregnant mice in each group were sacrificed on E13.5, E14.5 and E15.5, to collect the fetal palates for analysis. Fetal palates were used to extract total RNA and total protein, so as to detect the expression levels of cell cycle-related molecules, using RT-PCR and western blotting respectively. In vitro, human kidney embryo 293t (HEK293t) cells were treated with different concentrations of TCDD (0.01, 0.1, 0.5 and 1 nmol/L), and cells proliferation activity was detected using MTT assay. Statistical analysis was performed with IBM SPSS 24.0. Kolmogorov-Smimov test was used for normal distribution check, and the distribution was normal. Independent t-test was carried out among two groups. P<0.05 was considered statistically significant.@*Results@#At E13.5, E14.5 and E15.5, the expression level of interferon regulatory factor 6 (Irf6) protein were higher in the control group (1.26 ± 0.13, 1.67 ± 0.14 and 1.42 ± 0.15, respectively) compared to that in the TCDD group (0.81 ± 0.08, 1.04± 0.02 and 0.86 ± 0.12, respectively), on each time point (t value were 2.836, 3.662 and 2.867, respectively; P values were 0.0471, 0.0146 and 0.0241, respectively). The expression level of cyclin-dependent kinase inhibitor 1A (P21) protein on E13.5 and E14.5 of the control group (2.26 ± 0.21, 1.99 ± 0.21)were higher than that in the TCDD group on each time point(1.43 ± 0.12、0.93 ± 0.22), (t value were 3.398 and 3.378; P value were 0.8726 and 0.0273). The expression level of cyclin D1 in the control group (1.00±0.02, 0.94±0.03 and 1.11±0.09, respectively)were higher than that of the TCDD group (0.28±0.01, 0.33±0.06 and 0.88±0.01, respectively) on each time point (t value are were 22.53, 22.35 and 14.27, respectively, P value <0.001, <0.001 and<0.001, respectively). The expression of cyclin E1, cyclin A2, cyclin B1, CDK6, CDK2 and CDK1 in TCDD groups were higher than that of the controls (P<0.05). However, there was no significant difference of cyclin B1 on E13.5 and Cdk2 on E15.5. As treatment with TCDD (0.1 nmol/L) at 1, 2 and 3 days (0.70 ± 0.05, 1.05 ± 0.03 and 1.39 ± 0.04, respectively), the proliferation of HEK293t cells increased compared with the control group (0.49 ± 0.04, 0.98 ± 0.03 and 1.55 ± 0.02, respectively). The differences were statistically significant (t value were 2.829, 1.395 and 2.692, respectively; P value were 0.0198, 0.1320 and 0.0247, respectively).@*Conclusions@#TCDD down-regulates Irf6 and P21, and interferes with the normal expression of cell cycle-associated molecules, which in turn interferes with medial edge epithelia (MEE) cells cycle arrest and proliferation. These indicate that the disorder of spatiotemporal expression of cell cycle-related molecules during palatal development may be involved with the mechanism of TCDD-induced cleft palate..

Clustered Regularly Interspaced Short Palindromic Repeats-Based Genome Surgery for the Treatment of Autosomal Dominant Retinitis Pigmentosa.

PURPOSE: To develop a universal gene therapy to overcome the genetic heterogeneity in retinitis pigmentosa (RP) resulting from mutations in rhodopsin (RHO). DESIGN: Experimental study for a combination gene therapy that uses both gene ablation and gene replacement. PARTICIPANTS: This study included 2 kinds of human RHO mutation knock-in mouse models: RhoP23H and RhoD190N. In total, 23 RhoP23H/P23H, 43 RhoP23H/+, and 31 RhoD190N/+ mice were used for analysis. METHODS: This study involved gene therapy using dual adeno-associated viruses (AAVs) that (1) destroy expression of the endogenous Rho gene in a mutation-independent manner via an improved clustered regularly interspaced short palindromic repeats-based gene deletion and (2) enable expression of wild-type protein via exogenous cDNA. MAIN OUTCOME MEASURES: Electroretinographic and histologic analysis. RESULTS: The thickness of the outer nuclear layer (ONL) after the subretinal injection of combination ablate-and-replace gene therapy was approximately 17% to 36% more than the ONL thickness resulting from gene replacement-only therapy at 3 months after AAV injection. Furthermore, electroretinography results demonstrated that the a and b waves of both RhoP23H and RhoD190N disease models were preserved more significantly using ablate-and-replace gene therapy (P < 0.001), but not by gene replacement monotherapy. CONCLUSIONS: As a proof of concept, our results suggest that the ablate-and-replace strategy can ameliorate disease progression as measured by photoreceptor structure and function for both of the human mutation knock-in models. These results demonstrate the potency of the ablate-and-replace strategy to treat RP caused by different Rho mutations. Furthermore, because ablate-and-replace treatment is mutation independent, this strategy may be used to treat a wide array of dominant diseases in ophthalmology and other fields. Clinical trials using ablate-and-replace gene therapy would allow researchers to determine if this strategy provides any benefits for patients with diseases of interest.

Mitochondrial A3243G mutation results in corneal endothelial polymegathism.

PURPOSE: The mitochondrial DNA point mutation A3243G leads to a spectrum of syndromes ranging from MIDD to MELAS. Ocular manifestations include pattern macular dystrophy and concentric perifoveal atrophy. Given the high metabolic demand of corneal endothelial cells, we performed specular biomicroscopy analysis in patients harboring the mitochondrial DNA point mutation A3243G to assess for the associated presence of corneal endothelial abnormalities. METHODS: We present a case series with participants from two institutions. Patients diagnosed with macular dystrophy associated with MIDD or MELAS, and the mitochondrial DNA point mutation A3243G were recruited. Exclusion criteria included a prior diagnosis, or a positive family history, of endothelial corneal dystrophy. Slit-lamp corneal examination and specular biomicroscopy were performed. Corneal endothelial cell count, cell size and polymegathism, and central corneal thickness were assessed. Patients diagnosed with MIDD or MELAS based on clinical history and examination were genetically tested for the mitochondrial DNA point mutation A3243G using pyrosequencing. RESULTS: Five patients (two male and three female participants) from five different families, and with different ethnic backgrounds, met the inclusion criteria. Their ages ranged from 41 to 60 years. Corneal endothelial changes observed using slit-lamp examination were primarily mild to rare guttata. Specular biomicroscopy displayed mainly polymegathism associated with guttata. The average endothelial cell count was 2358 ± 456 cells per mm2, the average endothelial cell size was 442 ± 103 µm2 and the average central corneal thickness (CCT) was 551 ± 33 µm. These values were similar to that of the average population. The average coefficient of variation (COV), an index of heterogeneity in cell size, was 42.0 ± 4.1%. When compared to the average population, the average COV was significantly higher than predicted for the patients' age. None of the patients had signs of corneal edema. One patient had a pre-Descemet's opacity. CONCLUSIONS: In patients with the mitochondrial DNA point mutation A3243G, corneal endothelial polymegathism is present. This is mainly associated with mild guttata. The findings of corneal endothelial cell polymegathism may be a biomarker of mitochondrial disease, specifically in patients with the mitochondrial DNA A3243G mutation.