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The vagus nerve (VN) extensively innervates the gastric enteric nervous system (ENS), but its influence on gastric ENS functionality and motility in vivo remains unclear due to technical challenges. Here we describe a method for stable, long-term observation of gastric ENS activity and muscle dynamics at cellular resolution, which can also be extended to intestinal applications. This method involves ENS-specific labeling and the implantation of an abdominal wall window for optical recording in male mice. In vivo calcium imaging reveals a linear relationship between vagal stimulation frequency and myenteric neuron activation in gastric antrum. Furthermore, the motility of gastric antrum is significantly enhanced and shows a positive correlation with the intensity and number of activated myenteric neurons. While vagal stimulation also activates proximal colonic myenteric neurons, this activation is not frequency-dependent and does not induce proximal colonic motility. The method and results provide important insights into VN-ENS interactions in vivo, advancing our understanding of gastrointestinal motility regulation. In vivo optical recording of the real-time effects of extrinsic afferents on the activity of the enteric nervous system has been challenging. Here, the authors show a method that allows long-term imaging of the mouse gastric antrum and stable recording of the gastric myenteric plexus in response to vagal activation.

Background Accumulating evidence suggests that cancer stem cells (CSCs) play a critical role in tumor initiation, progression and therapy, and recent studies have indicated that Forkhead box C1 (FOXC1) is strongly associated with CSCs. This study investigates the regulatory effects of FOXC1 on CSC-like properties in non-small cell lung cancer (NSCLC). Methods We analyzed FOXC1 expression in NSCLC using the Cancer Genome Atlas (TCGA) database on UALCANC and performed survival analyses of NSCLC patients on Human Protein Atlas. CSC-like properties were analyzed based on CSC marker-positive cell population, self-renewal ability, stemness-related gene expression, tumorigenicity and drug resistance. The percentage of CD133 + cells was analyzed by flow cytometric analysis. Self-renewal ability was detected by sphere-formation analysis. Real-time PCR, western blotting and immunohistochemical staining were employed to detect mRNA and protein levels. Tumorigenicity was determined based on a xenograft formation assay, and effects of FOXC1 on drug resistance were assessed by cell viability and apoptosis assays. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to investigate the binding of FOXC1 to beta-catenin promoter. Results FOXC1 expression was found to be elevated in NSCLC tissues and negatively correlated with patient survival. FOXC1 knockdown reduced CD133 + cell percentage, suppressed self-renewal ability, decreased expression of stemness-related genes (Oct4, NANOG, SOX2 and ABCG2) and inhibited NSCLC cell tumorigenicity in vivo. Moreover, FOXC1 knockdown increased cisplatin and docetaxel sensitivity and reduced gefitinib resistance, whereas FOXC1 overexpression enhanced CSC-like properties. Luciferase reporter and ChIP assays showed beta-catenin to be a direct transcriptional target of FOXC1. Furthermore, overexpression of beta-catenin reversed the CSC-like property inhibition induced by FOXC1 knockdown, and knockdown of beta-catenin attenuated the CSC-like properties induced by FOXC1 overexpression. Conclusions This study demonstrates that FOXC1 induces CSC-like properties in NSCLC by promoting beta-catenin expression. The findings indicate that FOXC1 is a potential molecular target for anti-CSC-based therapies in NSCLC.

Gefitinib (Gef) provides clinical benefits to non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. However, acquired resistance (AR) is a major obstacle to effective Gef therapy. This study demonstrated that resveratrol (Res) could synergize with Gef to inhibit the proliferation of Gef-resistant NSCLC cells. The underlying mechanisms of synergism were investigated and the results showed that cotreatment with Gef and Res could inhibit EGFR phosphorylation by increasing intracellular Gef accumulation through the impairment of Gef elimination from PC9/G cells. Consistently, CYP1A1 and ABCG2 expression were inhibited. Meanwhile, the cotreatment significantly induced cell apoptosis, autophagy, cell cycle arrest and senescence accompanied by increased expression of cleaved caspase-3, LC3B-II, p53 and p21. Further studies revealed that autophagy inhibition enhanced apoptosis and abrogated senescence while apoptosis inhibition had no notable effect on cell autophagy and senescence during cotreatment with Gef and Res. These results indicated that in addition to apoptosis, senescence promoted by autophagy contributes to the antiproliferation effect of combined Gef and Res on PC9/G cells. In conclusion, combined treatment with Gef and Res may represent a rational strategy to overcome AR in NSCLC cells.

Background Estrogen receptors (ERs) are thought to play an important role in non-small cell lung cancer (NSCLC). However, the effect of ERs in NSCLC is still controversial and needs further investigation. A new consideration is that ERs may affect NSCLC progression through complicated molecular signaling networks rather than individual targets. Therefore, this study aims to explore the effect of ERs in NSCLC from the perspective of cancer systems biology. Methods The gene expression profile of NSCLC samples in TCGA dataset was analyzed by bioinformatics method. Variations of cell behaviors and protein expression were detected in vitro. The kinetic process of molecular signaling network was illustrated by a systemic computational model. At last, immunohistochemical (IHC) and survival analysis was applied to evaluate the clinical relevance and prognostic effect of key receptors in NSCLC. Results Bioinformatics analysis revealed that ERs might affect many cancer-related molecular events and pathways in NSCLC, particularly membrane receptor activation and signal transduction, which might ultimately lead to changes in cell behaviors. Experimental results confirmed that ERs could regulate cell behaviors including cell proliferation, apoptosis, invasion and migration; ERs also regulated the expression or activation of key members in membrane receptor signaling pathways such as epidermal growth factor receptor (EGFR), Notch1 and Glycogen synthase kinase-3β/β-Catenin (GSK3β/β-Catenin) pathways. Modeling results illustrated that the promotive effect of ERs in NSCLC was implemented by modulating the signaling network composed of EGFR, Notch1 and GSK3β/β-Catenin pathways; ERs maintained and enhanced the output of oncogenic signals by adding redundant and positive-feedback paths into the network. IHC results echoed that high expression of ERs, EGFR and Notch1 had a synergistic effect on poor prognosis of advanced NSCLC. Conclusions This study indicated that ERs were likely to promote NSCLC progression by modulating the integrated membrane receptor signaling network composed of EGFR, Notch1 and GSK3β/β-Catenin pathways and then affecting tumor cell behaviors. It also complemented the molecular mechanisms underlying the progression of NSCLC and provided new opportunities for optimizing therapeutic scheme of NSCLC.

Tumor heterogeneity and tumor evolution contribute to cancer treatment failure. To understand how selective pressures drive heterogeneous tumor evolution, it would be useful to image multiple important components and tumor subclones in vivo. We propose a supercontinuum-tailoring two-photon microscope (SCT-TPM) and realize simultaneous observation of nine fluorophores with a single light beam, breaking through the ‘color barrier’ of intravital two-photon fluorescence imaging. It achieves excitation multiplexing only by modulating the phase of fiber supercontinuum (SC), allowing to capture rapid events of multiple targets with maintaining precise spatial alignment. We employ SCT-TPM to visualize the spatiotemporal dynamics of heterogeneous tumor evolution under host immune surveillance, particularly the behaviors and interactions of six tumor subclones, immune cells and vascular network, and thus infer the trajectories of tumor progression and clonal competition. SCT-TPM opens up the possibility of tumor lineage tracking and mechanism exploration in living biological systems. Studying tumour heterogeneity and tumour evolution remains a challenging task. Here, the authors propose a supercontinuum-tailoring two-photon microscope to enable the imaging of multiple fluorophores with a single light beam. They use this tool to monitor the dynamics of heterogeneous tumour evolution.

Introduction ERBB3, one of the four members of the ErbB family of receptor tyrosine kinases, plays an important role in breast cancer etiology and progression. In the present study, we aimed to identify novel miRNAs that can potentially target ERBB3 and their biological functions. Method The expression levels of miR-143/145 and target mRNA were examined by relative quantification RT-PCR, and the expression levels of target protein were detected by Western blot. We used bioinformatic analyses to search for miRNAs that can potentially target ERBB3. Luciferase reporter plasmids were constructed to confirm direct targeting. Furthermore, the biological consequences of the targeting of ERBB3 by miR-143/145 were examined by cell proliferation and invasion assays in vitro and by the mouse xenograft tumor model in vivo. Results We identified an inverse correlation between miR-143/145 levels and ERBB3 protein levels, but not between miR-143/145 levels and ERBB3 mRNA levels, in breast cancer tissue samples. We identified specific targeting sites for miR-143 and miR-145 (miR-143/145) in the 3’-untranslated region (3’-UTR) of the ERBB3 gene and regulate ERBB3 expression. We demonstrated that the repression of ERBB3 by miR-143/145 suppressed the proliferation and invasion of breast cancer cells, and that miR-143/145 showed an anti-tumor effect by negatively regulating ERBB3 in the xenograft mouse model. Interestingly, miR-143 and miR-145 showed a cooperative repression of ERBB3 expression and cell proliferation and invasion in breast cancer cells, such that the effects of the two miRNAs were greater than with either miR-143 or miR-145 alone. Conclusion Taken together, our findings provide the first clues regarding the role of the miR-143/145 cluster as a tumor suppressor in breast cancer through the inhibition of ERBB3 translation. These results also support the idea that different miRNAs in a cluster can synergistically repress a given target mRNA.

Adaptive optics (AO) is a powerful tool for optical microscopy to counteract the effects of optical aberrations and improve the imaging performance in biological tissues. The diversity of sample characteristics entails the use of different AO schemes to measure the underlying aberrations. Here, we present an indirect wavefront sensing method leveraging a virtual imaging scheme and a structural-similarity-based shift measurement algorithm to enable aberration measurement using intrinsic structures even with temporally varying signals. We achieved high-resolution two-photon imaging in a variety of biological samples, including fixed biological tissues and living animals, after aberration correction. We present AO-incorporated subtractive imaging to show that our method can be readily integrated with resolution enhancement techniques to obtain higher resolution in biological tissues. The robustness of our method to signal variation is demonstrated by both simulations and aberration measurement on neurons exhibiting spontaneous activity in a living larval zebrafish.

Insulin resistance (IR) is a pivotal pathological characteristic that affects the occurrence and development of type 2 diabetes mellitus (T2DM). Thus, the effective control of IR is of great significance for diabetes prevention and treatment. Traditional Chinese medicine (TCM) represents a valuable tool handed down to the world by the Chinese nation and has a long history of use for diabetes clinical therapy. In this study, we focused on a self-drafted TCM-patented formula, Sanghuang Tongxie Formula (SHTXF), which exhibits clinical efficacy in the treatment of diabetes. To explore the effect and molecular mechanism of SHTXF on IR in vivo , Drosophila melanogaster was used and a ( Collagen ) Cg > InR K1409A diabetic IR fly model was established. SHTXF water extract was found to contribute toward carbohydrate clearance from the circulating system by converting it into triglycerides (TAG), not glycogen, for nutrient storage. In addition, SHTXF activated phosphatidylinositol-3-kinase (PI3K) activity and improved protein kinase B (PKB, also termed Akt) phosphorylation. Finally, SHTXF promoted Drosophila Forkhead Box O (dFoxO) cytoplasmic localization and inhibited its transcriptional activity. Taken together, these findings not only highlight the positive role of SHTXF in ameliorating IR via the PI3K/Akt pathway but also provide potential drug targets and key insights for use in T2DM clinical treatment strategies.

Background Many patients complain of pain following laparoscopic surgery. Clinicians have used ultrasound-guided posterior transversus abdominis plane block (TAPB) and rectus sheath block (RSB) for multimodal analgesia after surgery. We investigated the analgesic effects of US-guided posterior TAPB with RSB on postoperative pain following laparoscopy-assisted radical resection of early-stage rectal cancer. Methods Seventy-eight adults scheduled for laparoscopy-assisted radical resection of rectal cancer were enrolled in this double-blind placebo-controlled trial. Patients were randomized into 3 groups: the TR Group underwent US-guided bilateral posterior TAPB (40 mL 0.33% ropivacaine) with RSB (20 mL 0.33% ropivacaine); the T Group underwent US-guided bilateral posterior TAPB alone; and the Control Group received saline alone. All patients also had access to patient-controlled intravenous analgesia (PCIA) with sufentanil. The primary outcome was postoperative sufentanil consumption at 0–24, 24–48, and 48–72 h. The secondary outcomes were postoperative pain intensity and functional activity score at rest and while coughing for the same three time periods, intraoperative medication dosage, use of rescue analgesia, recovery parameters, and adverse effects. Results The three groups had no significant differences in baseline demographic and perioperative data, use of intraoperative medications, recovery parameters, and adverse effects. The TR group had significantly lower postoperative use of PCIA and rescue analgesic than in the other two groups ( P  < 0.05), but the Control Group and T Group had no significant differences in these outcomes. Conclusions Postoperative US-guided posterior TAPB with RSB reduced postoperative opioid use in patients following laparoscopy-assisted radical resection of rectal cancer. Trial registration The trial was registered with chictr.org (ChiCTR2000029326) on January 25, 2020.

Background: The anesthetic characteristics of ultrasound-guided bi-level erector spinae plane block (ESPB) plus dexmedetomidine (Dex) remain unclear. We compared the efficacy and safety of ultrasound-guided bi-level ESPB plus different doses of Dex in patients undergoing video-assisted thoracic surgery (VATS). Methods: One-hundred eight patients undergoing VATS were randomized into three groups: R group ( n = 38, 15 ml of 0.375% ropivacaine with 0.1 mg/kg dexamethasone), RD1 group ( n = 38, 15 ml of 0.375% ropivacaine plus 0.5 μg/kg DEX with 0.1 mg/kg dexamethasone) and RD2 group ( n = 38, 15 ml of 0.375% ropivacaine plus 1.0 μg/kg DEX with 0.1 mg/kg dexamethasone). The primary outcome was the pain 12 h after surgery. Secondary outcomes included the Prince Henry Hospital Pain Score; hemodynamics; consumption of sufentanil; anesthetized dermatomal distribution; recovery time; rescue analgesia; satisfaction scores of patients and surgeon; quick recovery index; adverse effects; the prevalence of chronic pain and quality of recovery. Results: The visual analog scale (VAS) and the Prince Henry pain score were significantly lower in both the RD1 and RD2 groups during the first 24 h after surgery ( P < 0.05). Both VAS with coughing and the Prince Henry pain score were significantly lower in the RD2 group than in the RD1 group 8–24 h after surgery ( P < 0.05). Both heart rate and mean arterial pressure were significantly different from T2 to T6 in the RD1 and RD2 groups ( P < 0.05). The receipt of remifentanil, propofol, Dex, and recovery time was significantly reduced in the RD2 group ( P < 0.05). The requirement for sufentanil during the 8–72 h after surgery, less rescue medication, and total press times were significantly lower in the RD2 group ( P < 0.05). The time to the first dose of rescue ketorolac was significantly longer in the RD2 group ( P < 0.05). Further, anal exhaust, removal of chest tubes, and ambulation were significantly shorter in the RD2 group ( P < 0.05). The incidence of tachycardia, post-operative nausea and vomiting, and chronic pain was significantly reduced in the RD2 group, while the QoR-40 score was significantly higher in the RD2 group ( P < 0.05). Conclusions: Pre-operative bi-level, single-injection ESPB plus 1 μg/kg DEX provided superior pain relief and long-term post-operative recovery for patients undergoing VATS. Clinical Trial Registration: http://www.chictr.org.cn/searchproj.aspx .