Long noncoding RNA Regulating ImMune Escape regulates mixed lineage leukaemia protein-1-H3K4me3-mediated immune escape in oesophageal squamous cell carcinoma.

BACKGROUND: Predictive biomarkers for oesophageal squamous cell carcinoma (ESCC) immunotherapy are lacking, and immunotherapy resistance remains to be addressed. The role of long noncoding RNA (lncRNA) in ESCC immune escape and immunotherapy resistance remains to be elucidated. METHODS: The tumour-associated macrophage-upregulated lncRNAs and the exosomal lncRNAs highly expressed in ESCC immunotherapy nonresponders were identified by lncRNA sequencing and polymerase chain reaction assays. CRISPR-Cas9 was used to explore the functional roles of the lncRNA. RNA pull-down, MS2-tagged RNA affinity purification (MS2-TRAP) and RNA-binding protein immunoprecipitation (RIP) were performed to identify lncRNA-associated proteins and related mechanisms. In vivo, the humanized PBMC (hu-PBMC) mouse model was established to assess the therapeutic responses of specific lncRNA inhibitors and their combination with programmed cell death protein 1 (PD-1) monoclonal antibody (mAb). Single-cell sequencing, flow cytometry, and multiplex fluorescent immunohistochemistry were used to analyze immune cells infiltrating the tumour microenvironment. RESULTS: We identified a lncRNA that is involved in tumour immune evasion and immunotherapy resistance. High LINC02096 (RIME) expression in plasma exosomes correlates with a reduced response to PD-1 mAb treatment and poor prognosis. Mechanistically, RIME binds to mixed lineage leukaemia protein-1 (MLL1) and prevents ankyrin repeat and SOCS box containing 2 (ASB2)-mediated MLL1 ubiquitination, improving the stability of MLL1. RIME-MLL1 increases H3K4me3 levels in the promoter regions of programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1), constitutively increasing the expression of PD-L1/IDO-1 in tumour cells and inhibiting CD8+ T cells infiltration and activation. RIME depletion in huPBMC-NOG mice significantly represses tumour development and improves the effectiveness of PD-1 mAb treatment by activating T-cell-mediated antitumour immunity. CONCLUSIONS: This study reveals that the RIME-MLL1-H3K4me3 axis plays a critical role in tumour immunosuppression. Moreover, RIME appears to be a potential prognostic biomarker for immunotherapy and developing drugs that target RIME may be a new therapeutic strategy that overcomes immunotherapy resistance and benefits patients with ESCC.

Which is the best combination of surgery for hepatocellular carcinoma with hepatic/portal vein thrombosis in China: a network meta-analysis of randomized controlled trials.

PURPOSE: To assess the efficacy and safety of different perioperative regimens using network meta-analysis for hepatocellular carcinoma (HCC) with hepatic/portal vein thrombosis. The interested modalities included neoadjuvant three-dimensional conformal radiotherapy (3D-CRT), post-operative intensity modulated radiation therapy (IMRT). post-operative transarterial chemoembolization (TACE) and surgery alone. METHODS: PubMed and Cochrane Library electronic databases were systematically searched for eligible studies published up to November 2020. Data related to treatment efficacy including overall survival (OS), and disease-free survival (DFS) were extracted and compared using a Bayesian approach. Adverse events (AEs) were assessed and compared. RESULTS: Four studies published between 2005 and 2020 involving a total of 422 patients were enrolled in this network meta-analysis. The comparison showed that surgery with IMRT ranked relatively higher in prolonging OS in advanced HCC patients, followed by neoadjuvant 3DCRT and surgery plus TACE. Postoperative IMRT appeared better choice in terms of DFS. The rate of AEs did not significantly differ. CONCLUSION: Adjuvant IMRT showed more favorable treatment responses compared with other regimens in HCC patients with hepatic/portal vein thrombosis.

Decreased Serum Brain-Derived Neurotrophic Factor May Indicate the Development of Poststroke Depression in Patients with Acute Ischemic Stroke: A Meta-Analysis.

BACKGROUND: Depression is a common complication after stroke and has been associated with poor outcome. Thus, it is of great importance to identify potential biomarkers that can aid in predicting and detecting patients with stroke at high risk of poststroke depression (PSD) development. Previous studies showed that brain-derived neurotrophic factor (BDNF) had potential use as a biomarker for discriminating patients with stroke at high risk of PSD. However, the results were inconsistent. METHODS: A meta-analysis was performed to evaluate the correlation between the peripheral BDNF levels and the development of PSD in the acute stage of stroke. RESULTS: Data were obtained from 4 studies including 499 patients with stroke. Among them, 171 patients were diagnosed with PSD at follow-ups. Our results showed that patients with stroke who were predisposed to developing PSD had significantly lower serum BDNF concentrations at the early stage of stroke. CONCLUSIONS: This study suggests a potential association between circulating BDNF concentrations at admission and subsequent PSD development, and provides additional support for the involvement of BDNF in the PSD development.

[Preparation and evaluation of risperidone-loaded microsphere/sucrose acetate isobutyrate in situ forming complex depot with double diffusion barriers].

In the present study, a risperidone loaded microsphere/sucrose acetate isobutyrate (SAIB) in situ forming complex depot was designed to reduce the burst release of SAIB in situ forming depot and to continuously release risperidone for a long-term period without lagime. The model drug risperidone (Ris) was first encapsulated into microspheres and then the Ris-microspheres were embedded into SAIB depot to reduce the amount of dissolved drug in the depot. The effects of different types of microsphere matrix, including chitosan and poly(lactide-coglycolide) (PLGA), matrix/Ris ratios in microspheres and morphology of microspheres on the drug release behavior of complex depot were investigated. In comparison with the Ris-loaded SAIB depot (Ris-SAIB), the complex depot containing chitosan microspheres (in which chitosan/Ris = 1 : 1, w/w) (Ris-Cm-SAIB) decreased the burst release from 12.16% to 5.80%. However, increased drug release rate after 4 days was observed in Ris-Cm-SAIB, which was caused by the high penetration of the medium to Ris-Cm-SAIB due to the hydrophilie of chitosan. By encapsulation of risperidone in PLGA microspheres, most drugs can be prevented from dissolving in the depot and meanwhile the hydrophobic PLGA can reduce the media penetration effect on the depot. The complex depot containing PLGA microspheres (in which PLGA/ drug=4 : 2, w/w) (Ris-Pm-SAIB) showed a significant effectiveness on reducing the burst release both in vitro and in vivo whereby only 0.64% drug was released on the first day in vitro and a low AUC0-4d value [(105.2± 24.4) ng.mL-1.d] was detected over the first 4 days in vivo. In addition, drug release from Ris-Pm-SAIB can be modified by varying the morphology of microspheres. The porous PLGA microspheres could be prepared by adding medium chain triglyceride (MCT) in the organic phase which served as pore agents during the preparation of PLGA microspheres. The complex depot containing porous PLGA microspheres (which were prepared by co-encapsulation of 20% MCT) (Ris-PPm-SAIB) exhibited a slightly increased AUC0-4d of (194.6±15.8) ng.mL-1d and high plasma concentration levels from 4 to 78 days [Cs(4-78d)=(7.8±1.2) ng.mL-1]. The plasma concentration on 78 day C78d was (9.0 2.5) ng.mL-1 which was higher than that of Ris-Pm-SAIB [C78d= (1.6 ± 0.6) ng.mL-1]. In comparison with Ris-Pm-SAIB, the AUC4-78d of Ris-PPm-SAIB increased from (379.0±114.3) ng.mL-1.d to (465.0 ±149.2) ng.mL-1.d, indicating sufficient drug release from the Ris-PPm-SAIB. These results demonstrate that the risperidone loaded porous PLGA microsphere/SAIB in situ forming complex depot could not only efficiently reduce the burst release of SAIB depot both in vitro and in vivo, but also release the drug sufficiently in vivo, and be capable to continuously release the drug for 78 days.

The Relationship between Gentle Tactile Stimulation on the Fetus and Its Temperament 3 Months after Birth.

OBJECTIVE: The aim of this study was to evaluate the effect of gentle tactile stimulation on the fetus in its temperament 3 months after birth. METHOD: A total of 302 mother-3-month-infant dyads enrolled the retrospective cohort study. 76 mothers had regular gentle tactile stimulation on the fetus in their pregnancy; 62 mothers had irregular tactile stimulation on the fetus, and the rest of 164 mothers who had no tactile stimulation served as nonexposure group. Temperament was assessed using the EITS (a nine-dimensional scale of temperament). RESULTS: Significant difference in temperament type was found among infants in 3 groups at 3 months of age. In the regular practice group, the babies with easy type temperament accounted for 73.7%, which was higher than that in irregular practice group (53.2%, P = 0.012) and that in the control group (42.1%, P < 0.001). Compared to infants in no practice group, the infants who had received regular gentle tactile stimulation before birth were lower in negative mood (P = 0.047) while higher in adaptability (P < 0.001), approach (P = 0.001), and persistence (P = 0.001), respectively. CONCLUSION: Regular gentle tactile stimulation on fetus may promote the formation of easy type infant temperament.

[Effect of local injection of rhTGF-α1 on osteoclasts during orthodontic tooth movement in rats].

PURPOSE: To investigate the effect of exogenous TGF-ß1 on improving periodontal tissues remodeling during orthodontic tooth movement in rats. METHODS: Eighty male SD rats were randomly divided into 2 groups. Rats in the experimental group were injected respectively with rhTGF-ß1 in dosages of 0.1 mL (5 ng/mL) in the buccal submucosal area of the molar every other day, while rats in the control group received equivalent volumes of PBS. Each group (n=40) was subdivided equally into 5 subgroups. An orthodontic appliance, consisting of a 5 mm nickel titanium closed coil spring, was ligated between the maxillary left incisor and first molar of each rat to deliver an initial force of 0.49N. Eight rats in each subgroup were sacrificed at one of the five time points (1, 4, 7, 10 and 14 days) after appliance placement. The distance of the tooth movement was measured by using stereomicroscope. Tissue sections around the first maxillary left molar were stained with tartrate-resistant acid phosphatase (TRAP) histochemistry to analyze the changes of the amount and distribution of osteoclasts on the compression side of tooth. Statistical analysis was performed using SPSS 17.0 software package. RESULTS: Molars treated with rhTGF-ß1 moved mesially more rapidly than the control group. The distance of tooth movement of the experimental group showed a significant increase compared with the control group at day 7 (P<0.05) and significant increase compared with the control group at day 10 and 14 (P<0.01). The number of TRAP positive cells appearing in the periodontal ligament space on the pressure side of the alveolar bone were increased markedly in experimental group. There were statistically significant differences in the amount of osteoclasts between experimental and control groups (P<0.01). CONCLUSIONS: Local injection of rhTGF-ß1 in the periodontal tissues can accelerate orthodontic tooth movement via enhancing the numbers of osteoclasts. At the histologic level, increased numbers of mononuclear osteoclasts are recruited and activated, resulting in greater amounts of alveolar bone resorption on the pressure side of the periodontal ligament.

Discovery of loperamide as an antagonist of angiopoietin1 and angiopoietin2 by virtual screening.

The angiopoietin-Tie2 binding and related signal transduction pathways are crucial for vascular angiogenesis, blood vessel integrity and maturation. In this study, we preformed a virtual screening of small molecules targeting to Tie2. The binding site was selected at the extracellular ligand binding region of Tie2, rather than its conventional endocellular ATP binding region. It was found that loperamide, a widely-used antidiarrhea drug, was among the top hits. The binding between loperamide and Tie2 was confirmed by surface plasmon resonance (SPR) assay. Loperamide competitively inhibited the binding of both angiopoietin1 and angiopoietin2. These results indicate that loperamide is an antagonist of angiopoietin1 and angiopoietin2.

Loperamide, an antidiarrhea drug, has antitumor activity by inducing cell apoptosis.

Loperamide, an antidiarrhea drug, is a peripheral opiate agonist. Some other opiate agonists have been shown to promote cell apoptosis. In this research, we studied the apoptosis-inducing and cytotoxic activities of loperamide. MTT assay was used to determine its cytotoxicity on nine established human tumor cell lines. Cell apoptosis was detected by flow cytometry. Hypodiploid cells and cell cycles were analyzed by propidium iodide (PI) staining, while early apoptotic cells were detected by annexin V-FITC/PI staining. It was found that loperamide could inhibit the proliferation of the tested tumor cell lines. The IC50 values for SMMC7721, MCF7, SPC-A1, SKOV3-DDP, H460, HepG2, SGC7901, U2OS, and ACHN cells were 24.2±2.1 µM, 23.6±2.5 µM, 25.9±3.1 µM, 27.1±2.5 µM, 41.4±2.1 µM, 23.7±1.3 µM, 35.4±3.5 µM, 11.8±2.8 µM, and 28.5±3.4 µM, respectively. Loperamide was more effective to the human osteosarcoma U2OS cells with an IC50 value of 11.8±2.8 µM. Meanwhile, it could induce cell apoptosis and cause G2/M-phase cell cycle arrest. The apoptotic cells could be found when treating with loperamide for 6h and most of them belonged to early apoptosis. In loperamide-treated cells, activation of caspase-3 was found, namely that caspase-3 was involved in the loperamide-induced apoptosis. The results of these studies indicate that loperamide is a potential antitumor agent. To our knowledge, this is the first report on antitumor activity of loperamide.

High performance liquid chromatography assay with ultraviolet detection for moxifloxacin: validation and application to a pharmacokinetic study in Chinese volunteers.

A specific, sensitive and widely applicable high performance liquid chromatography with ultraviolet detection (HPLC-UV) method for the determination of moxifloxacin in human plasma was developed and validated in this study. The method involved a single step of liquid-liquid extraction with dichloromethane and the extraction yields more than 80% were achieved. The separation was performed on a common Kromasil C(8) column with an isocratic mobile phase. The total time was within 10 min per run. The calibration curve for moxifloxacin was linear in the concentration range of 0.05-5.0 µg/ml with correlation coefficient of 0.9997. The developed method was validated with excellent specificity, sensitivity, accuracy, precision and stability. Using this developed method, the pharmacokinetics of moxifloxacin in healthy Chinese volunteers was studied.

Non-cytolytic antigen clearance in DNA-vaccinated mice with electroporation.

AIM: To explore the potential of electroporation (EP)-mediated hepatitis B virus (HBV) DNA vaccination for the treatment of chronic HBV infection. METHODS: BALB/c mice were vaccinated with HBV DNA vaccine encoding for the HBV preS(2)-S antigen, combined with or without EP. HBV surface antigen expression plasmid was administered into mice liver via a hydrodynamic injection to mimic HBV infection. The clearance of antigen in the serum and liver was detected by ELISA assay and immunohistochemical staining. The histopathology of the liver tissues was examined by HE staining and serum alanine aminotransferase assay. RESULTS: The immunogenicity of HBV DNA vaccine encoding for the HBV preS(2)- S antigen can be improved by EP-mediated vaccine delivery. The elicited immune responses can indeed reduce the expression of HBV surface antigen (HBsAg) in hepatocytes of the mouse model that was transfected to express HBsAg using the hydrodynamic injection method. The antigen clearance process did not cause significant toxicity to liver tissue, suggesting a non-cytolytic mechanism. CONCLUSION: The EP-aided DNA vaccination may have potential in mediating viral clearance in chronic hepatitis B patients.

Application of EGFP-EGF fusions to explore mechanism of endocytosis of epidermal growth factor.

AIM: To develop a simple method for monitoring protein localization of epidermal growth factor (EGF) in living cells. METHODS: Enhanced green fluorescent protein (EGFP) was used as an autofluorescent tag to label EGF ligands. SDS-PAGE and Western blot analysis were used to detect the expression of the EGFP-tagged EGF (EGFP-EGF) protein. The cell-binding and internalization activity of EGFP-EGF were analyzed by fluorescence-activated cell sorting (FACS) and confocal microscopy. RESULTS: EGFP-EGF protein was expressed in Escherichia coli and purified. A cell-binding assay demonstrated that the EGFP-EGF protein could bind efficiently to the cells expressing EGFR. The binding and internalization of EGFP-EGF can be visualized even at a very low concentration under confocal microscopy. The FACS-based assay for internalization activity indicated the accumulation of internalized EGFP-EGF over time. Furthermore, the results of the competition assay indicated its EGFR binding specificity. Using such a method, it does not need to label EGF with chemicals and avoid light in the experimental process. CONCLUSION: The fusion protein EGFP-EGF has several characters including high sensitivity, stability and convenience for manipulation, and is a powerful tool for the study of EGF endocytosis.

Inhibitory effect of Ca2+ on in vivo gene transfer by electroporation.

AIM: To investigate the specific effects of Ca2+ on transgene expression during electroporation-mediated gene transfer in mice. METHODS: Skeletal muscle and skin were subjected to in vivo electroporation with a luciferase reporter plasmid, with or without Ca2+ and various other ions. RESULTS: For in vivo electroporation, the presence of just 10 mmol/L Ca2+ in the DNA solution drastically reduced the resulting transgene expression, to less than 5% of control values. Only Ca2+, not other ions, caused inhibition, and the effect was not tissue specific. More surprisingly, even when Ca2+ ions were delivered by electroporation before or after DNA administration, similar effects were still observed. CONCLUSION: The inhibitory effect of Ca2+ on in vivo gene transfer by electroporation is specific, ie, the inhibitory effect may be related to the cell membrane properties after electroporation and the subsequent resealing event.

Multivesicular liposome formulations for the sustained delivery of interferon alpha-2b.

AIM: To develop and optimize a sustained release multivesicular liposome (MVL) formulation of interferon (IFN) alpha-2b. METHODS: IFN alpha-2b MVL were prepared using a typical double-emulsion procedure. The sustained release effects of IFN alpha-2b MVL were investigated by monitoring the blood IFN alpha-2b concentration using an enzyme-linked immunosorbent assay test after subcutaneous administration to healthy mice. RESULTS: IFN alpha-2b was successfully encapsulated in MVL with high efficiency, and the integrity of encapsulated protein was maintained. After subcutaneous injection, the MVL slowly released IFN alpha-2b into systemic circulation in a sustained manner. The estimated serum half-life of IFN alpha-2b was approximately 30 h. In addition, varying the size of the MVL preparations could further modify the in vivo release profile. CONCLUSION: IFN alpha-2b MVL may be a useful sustained release formulation in the clinical treatment of viral diseases.

Conjugate of bovine hemoglobin and human serum albumin as a candidate for blood substitute: characteristics and effects on rats.

Conjugate of bovine hemoglobin (bHb) and human serum albumin (HSA) was prepared. The product was simply composed of 89.7% one-to-one Hb-HSA conjugate, 6.0% oligomer of Hb and HSA, 3.5% unconjugated HSA and 0.8% unconjugated Hb, with an average molecular weight of 157 kD. The physicochemical characteristics were determined. Effects of single replacement on blood pressure and long-term survival of rats with 30% and 60% acute blood loss were studied, in comparison with Ringer-lactate solution, stroma-free hemoglobin (SFHb), 5% HSA in Ringer-lactate, whole blood and no resuscitation fluid. Results showed that Hb-HSA conjugate maintained the mean arterial pressure of rats to initial level with no pressor effect. Long-term effects of the replacement fluids on 30% bleeding rats showed that, for the group infused with Hb-HSA conjugate, histology of five major organs, heart, kidney, liver, spleen and lung, were essentially normal, similar to that of whole blood, while obviously renal side-effects appeared in other groups. The efficacy of the conjugate was further demonstrated by the resuscitation of lethal hemorrhagic shock rats (60% acute blood loss) with 100% survival rate (followed for 14 days), the same result as whole blood. The Hb-HSA conjugate can thus be another candidate for blood substitute in emergency.

Modulation of the interactions of isolated ryanodine receptors of rabbit skeletal muscle by Na+ and K+.

Ryanodine receptors (RyRs) of skeletal muscle, as calcium release channels, have been found to form semicrystalline arrays in the membrane of sarcoplasmic reticulum. Recently, both experimental observations and theoretical simulations suggested cooperative coupling within interlocking RyRs. To better understand the interactions between RyRs and their modulation, the aggregation and dissociation of isolated RyRs in aqueous medium containing various Na(+) and K(+) concentrations were investigated using photon correlation spectroscopy (PCS) and atomic force microscopy (AFM). RyRs aggregated readily at low salt concentrations. However, a different behavior was observed in the presence of Na(+) or K(+). Detectable aggregates were formed in 5 microg/mL RyR sample when the concentration of Na(+) and K(+) was reduced from 1 M to below 0.28 and 0.23 M, respectively. The dissociation of RyR aggregates was also examined when raising the salt concentration. While aggregates formed in 0.15 M NaCl medium could reverse almost completely, those formed in 0.15 M KCl medium only dissolved partly. When keeping the total salt concentration at 0.15 M, the aggregation and dissociation of RyRs were seen to evidently depend on the relative concentration of Na(+) and K(+). The interaction between RyRs was strengthened with increasing Na(+)/K(+) ratios in the mixed medium. Accompanying this, a decrease of [(3)H]ryanodine binding occurred. The results obtained with PCS and AFM provide further evidence for the interaction between RyRs and suggest the importance of Na(+), K(+), and their relative composition in modulating the interaction and cooperation between RyRs in vivo.