Actomyosin-II protects axons from degeneration induced by mild mechanical stress.

Whether, to what extent, and how the axons in the central nervous system (CNS) can withstand sudden mechanical impacts remain unclear. By using a microfluidic device to apply controlled transverse mechanical stress to axons, we determined the stress levels that most axons can withstand and explored their instant responses at nanoscale resolution. We found mild stress triggers a highly reversible, rapid axon beading response, driven by actomyosin-II-dependent dynamic diameter modulations. This mechanism contributes to hindering the long-range spread of stress-induced Ca2+ elevations into non-stressed neuronal regions. Through pharmacological and molecular manipulations in vitro, we found that actomyosin-II inactivation diminishes the reversible beading process, fostering progressive Ca2+ spreading and thereby increasing acute axonal degeneration in stressed axons. Conversely, upregulating actomyosin-II activity prevents the progression of initial injury, protecting stressed axons from acute degeneration both in vitro and in vivo. Our study unveils the periodic actomyosin-II in axon shafts cortex as a novel protective mechanism, shielding neurons from detrimental effects caused by mechanical stress.

Neutrophil-to-lymphocyte ratio associated with renal function in type 2 diabetic patients.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a leading risk factor for the development and progression of chronic kidney disease (CKD). However, an accurate and convenient marker for early detection and appropriate management of CKD in individuals with T2DM is limited. Recent studies have demonstrated a strong correlation between the neutrophil-to-lymphocyte ratio (NLR) and CKD. Nonetheless, the predictive value of NLR for renal damage in type 2 diabetic patients remains understudied. AIM: To investigate the relationship between NLR and renal function in T2DM patients. METHODS: This study included 1040 adults aged 65 or older with T2DM from Shanghai's Community Health Service Center. The total number of neutrophils and lymphocytes was detected, and NLR levels were calculated. CKD was defined as an estimated glomerular filtration rate ≤ 60 mL/min/1.73 m². Participants were divided into four groups based on NLR levels. The clinical data and biochemical characteristics were compared among groups. A multivariate logistic regression model was used to analyze the association between NLR levels and CKD. RESULTS: Significant differences were found in terms of sex, serum creatinine, blood urea nitrogen, total cholesterol, and low-density lipoprotein cholesterol among patients with T2DM in different NLR groups (P < 0.0007). T2DM patients in the highest NLR quartile had a higher prevalence of CKD (P for trend = 0.0011). Multivariate logistic regression analysis indicated that a high NLR was an independent risk factor for CKD in T2DM patients even after adjustment for important clinical and pathological parameters (P = 0.0001, odds ratio = 1.41, 95% confidence intervals: 1.18-1.68). CONCLUSION: An elevated NLR in patients with T2DM is associated with higher prevalence of CKD, suggesting that it could be a marker for the detection and evaluation of diabetic kidney disease.

Targeting inhibition of TCTP could inhibit proliferation and induce apoptosis in AML cells.

Translationally controlled tumor protein (TCTP) is a highly conserved multifunctional protein, which participates in many important physiological processes. Recently, the roles of TCTP in cell proliferation and apoptosis, especially its close relationship with various tumors, have attracted widespread attention. In this study, we found that the protein level of TCTP was significantly reduced in acute promyelocytic leukemia cell line NB4 transfected with retinoic acid-induced gene G (RIG-G). The RIG-G was found in our previous work as a key mediator of anti-proliferative activity in retinoid/interferon-related pathways. Here, we tried to further explore the function of TCTP in the development of acute myeloid leukemia (AML) from different levels. Our results showed that inhibiting TCTP expression could attenuate AML cells proliferation and induce apoptosis both in AML cell lines and in xenograft of NOD-SCID mice. In addition, either compared with patients in complete remission or non-leukemia patients, we detected that the expression of TCTP was generally high in the fresh bone marrow of AML patients, suggesting that there was a certain correlation between TCTP and AML disease progression. Taken together, our study revealed the role of TCTP in AML development, and provided a potential target for AML treatment.

Hyperbaric oxygen improves cerebral ischemia-reperfusion injury in rats via inhibition of ferroptosis.

BACKGROUND: Our previous study found that hyperbaric oxygen (HBO) attenuated cognitive impairment in mice induced by cerebral ischemia-reperfusion injury (CIRI). However, its mechanism of action is not fully understood. In this study, we aimed to establish a rat model of cerebral ischemia-reperfusion, explore the possible role of ferroptosis in the pathogenesis of CIRI, and observe the effect of HBO on ferroptosis-mediated CIRI. METHODS: Sprague Dawley (SD) rats were randomly divided into control, model, Ferrostatin-1 (Fer-1), HBO and Fer-1+ HBO groups. Morris water maze, myelin basic protein (MBP) and ß-tubulin immunoreactivity were assessed to evaluate the neuroprotective effects of HBO on cerebral ischemia reperfusion injury. Ferroptosis were examined to investigate the mechanism underlying the effects of HBO. RESULTS: Our result showed that Fer-1 and HBO improved learning and memory ability in the navigation trail and probe trail of the Morris water maze and increased MBP and ß-tubulin immunoreactivity of the cortex in the model rats. The levels of ferritin, malondialdehyde (MDA) and glutathione (GSH) in the serum were also reversed by Fer-1 and HBO treatment. Mitochondrial cristae dissolution and vacuolization were observed in the model group by transmission electron microscopy and these conditions were improved in the Fer-1 and HBO groups. Furthermore, Fer-1 and HBO treatment reversed Prostaglandin-Endoperoxide Synthase 2 (PTGS2), Iron Responsive Element Binding Protein 2 (IREB2), acyl-CoA synthetase long chain family member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11) mRNA levels and Transferrin Receptor 1 (TFR1), ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), glutathione peroxidase 4 (GPX4), Nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (P-JNK) phosphorylated Extracellular signal-regulated protein kinase (P-ERK) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) protein levels. The above changes were more pronounced in Fer-1+ HBOGroup. DISCUSSION: The results of the present study indicated that HBO improves cerebral ischemia-reperfusion injury in rats, which may be related to inhibition of ferroptosis. This also means that ferroptosis may become a new target of HBO against CIRI.

Prevalence and associating variables with fear of progression in Chinese pediatric cancer patients: A cross-sectional study.

Fear of progression (FoP) is a prevalent psychological strain for cancer patients associated with poor quality of life and psychological morbidity. However, little evidence exists on FoP in children with cancer. Our study aimed to determine prevalence and correlates of FoP of cancer in children. From December 2018 to March 2019, cancer patients from Children's Hospital in Chongqing, Southwest China, were recruited. A Chinese version of Fear of Progression Questionnaire-Short Form (FoP-Q-SF) was adopted to assess children' FoP. Descriptive statistics (percentages, median, and interquartile range), non-parametric tests, and multiple regression analyses were performed on these data. Prevalence of high-level FoP was 43.75% among these 102 children. Multiple regression analysis showed that reproductive system tumors (beta = 0.315, t = 3.235 95% CI [3.171, 13.334]), and level of psychological care needs (beta = -0.370, t = -3.793 95% CI [-5.396, -1.680]) were independent predictors of FoP. Regression model explained 27.10% of all included variables (adjusted R square = 27.10%). As with adults with cancer, children with cancer also have FoP. More attention should be paid to FoP in children with reproductive tumors and in children who need psychological support. More access to psychological support should be offered to reduce FoP and to improve their quality of life.

Axons-on-a-chip for mimicking non-disruptive diffuse axonal injury underlying traumatic brain injury.

Diffuse axonal injury (DAI) is the most severe pathological feature of traumatic brain injury (TBI). However, how primary axonal injury is induced by transient mechanical impacts remains unknown, mainly due to the low temporal and spatial resolution of medical imaging approaches. Here we established an axon-on-a-chip (AoC) model for mimicking DAI and monitoring instant cellular responses. Integrating computational fluid dynamics and microfluidic techniques, DAI was induced by injecting a precisely controlled micro-flux in the transverse direction. The clear correlation between the flow speed of injecting flux and the severity of DAI was elucidated. We next used the AoC to investigate the instant intracellular responses underlying DAI and found that the dynamic formation of focal axonal swellings (FAS) accompanied by Ca2+ surge occurs during the flux. Surprisingly, periodic axonal cytoskeleton disruption also occurs rapidly after the flux. These instant injury responses are spatially restricted to the fluxed axon, not affecting the overall viability of the neuron in the acute stage. Compatible with high-resolution live microscopy, the AoC provides a versatile system to identify early mechanisms underlying DAI, offering a platform for screening effective treatments to alleviate TBI.

Hyperbaric oxygen protects HT22 cells and PC12 cells from damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of Nrf2/System Xc-/GPX4 axis-mediated ferroptosis.

This study was to investigate the protective effect of hyperbaric oxygen (HBO) on HT22 and PC12 cell damage caused by oxygen-glucose deprivation/reperfusion-induced ferroptosis. A 2-h oxygen-glucose deprivation and 24-h reperfusion model on HT22 and PC12 cells was used to simulate cerebral ischemia-reperfusion injury. Cell viabilities were detected by Cell Counting Kit-8 (CCK-8) method. The levels of reactive oxygen species (ROS) and lipid reactive oxygen species (Lipid ROS) were detected by fluorescent probes Dihydroethidium (DHE) and C11 BODIPY 581/591. Iron Colorimetric Assay Kit, malondialdehyde (MDA) and glutathione (GSH) activity assay kits were used to detect intracellular iron ion, MDA and GSHcontent. Cell ferroptosis-related ultrastructures were visualized using transmission electron microscopy (TEM). Furthermore, PCR and Western blot analyses were used to detect the expressions of ferroptosis-related genes and proteins. After receiving oxygen-glucose deprivation/reperfusion, the viabilities of HT22 and PC12 cells were significantly decreased; ROS, Lipid ROS, iron ions and MDA accumulation occurred in the cells; GSH contents decreased; TEM showed that cells were ruptured and blebbed, mitochondria atrophied and became smaller, mitochondrial ridges were reduced or even disappeared, and apoptotic bodies appeared. And the expressions of Nrf2, SLC7A11 and GPX4 genes were reduced; the expressions of p-Nrf2/Nrf2, xCT and GPX4 proteins were reduced. Notably, these parameters were significantly reversed by HBO, indicating that HBO can protect HT22 cells and PC12 cells from damage caused by oxygen-glucosedeprivation/reperfusion via the inhibition of Nrf2/System Xc-/GPX4 axis-mediated ferroptosis.

Simultaneous versus staged major hepatectomy (≥3 liver segments) for outcomes of synchronous colorectal liver metastases: A systematic review and meta-analysis.

BACKGROUND: Hepatectomy is an effective treatment for synchronous colorectal liver metastases (SCLM) patients. However, whether to choose simultaneous hepatectomy (SIH) or staged hepatectomy (STH) is still controversial, especially during major hepatectomy (≥3 liver segments). AIMS: Compare the difference between the SCLM patients underwent SIH and STH, especially during major hepatectomy (≥3 liver segments). METHODS AND RESULTS: A meta-analysis was conducted by analyzing the published data on the outcomes of SCLM patients underwent SIH or STH from January 2010 to December 2020 from the electronic databases. A random-effects model was used to derive pooled estimates of odds ratio (OR) with 95% confidence interval (CI) for the explored outcomes. Eventually, 18 studies, including 5101 patients, were included this study. The result of meta-analysis showed that SIH did not increase postoperative complications (pooled OR: 1.037; 95% CI: 0.897-1.200), perioperative mortality (pooled OR: 0.942; 95% CI: 0.552-1.607), 3-year mortality (pooled OR: 1.090; 95% CI: 0.903-1.316) or 5-year mortality (pooled OR: 1.077; 95% CI: 0.926-1.253), as compared with STH. Subgroup analysis showed that, simultaneous major hepatectomy (SIMH) also did not increase postoperative complications (pooled OR: 0.863; 95% CI: 0.627-1.188) or perioperative mortality (pooled OR: 0.689; 95% CI: 0.290-1.637) as compared with staged major hepatectomy (STMH). CONCLUSION: Postoperative complications, perioperative mortality and long-term prognosis had no significant difference between SIH and STH for SCLM patients. Besides, postoperative complications and perioperative mortality also had no significant difference between SIMH and STMH.

Identification of potential biomarkers for digestive system cancers from serum-derived extracellular vesicle RNA.

BACKGROUND: Poor prognosis of digestive system cancers is mainly owing to lack of accurate and timely diagnosis. The exploration of novel tumor biomarkers from extracellular vesicle (EV) might be helpful to clinical diagnosis for digestive system cancers. METHODS: Several public databases were first used for a preliminary screening of candidate genes. The RNA levels of these candidate genes were then detected in cancer cell lines and the patients serum-derived EVs by PCR Array or digital PCR, respectively. RESULTS: We found that 4 EV-RNAs, ANLN, ITGA6, KRT18, and MMP9, had a lower level in gastrointestinal cancer patients than in benign gastrointestinal diseases patients and healthy controls, while 3 EV-RNAs, ANLN, ITGA6, and KRT18, had a lower level in pancreatic cancer patients than in benign pancreatic diseases patients or healthy individuals. And EV-RNA of MMP9 had a relatively higher level in advanced pancreatic cancer patients than in early-stage patients. Moreover, ROC analysis demonstrated that the determination of the above EV-RNAs could increase the ability of traditional tumor biomarkers to distinguish benign and malignant diseases. CONCLUSIONS: The serum-derived EV-RNAs of ANLN, ITGA6, KRT18, and MMP9 could be served as novel, non-invasive biomarkers for digestive system cancers.

Factors influencing emotional and behavioral problems in preschool children with cancer based on self-regulation shift theory.

OBJECTIVE: The objective is to investigate the factors influencing emotional and behavioral problems in preschool children with cancer and to provide a scientific basis for developing predictive intervention strategies. METHODS: We recruited 375 preschool children with cancer, from March 2019 to February 2020, via convenience sampling. The self-regulation shift theory was implemented as a theoretical framework and a structural equation model was applied to construct and validate this framework and to analyze the relationships among various influencing factors. RESULTS: Children's effortful control, parent-child attachment, the family environment, family upbringing, parents' marital quality, parents' education level, and social support significantly affected children's emotions and behaviors. Additionally, children's effortful control and parent-child attachment mediated the effect of the family environment, family upbringing, and parents' marital quality on children's emotions and behaviors." CONCLUSION: This study identified several factors that influence children's emotions and behaviors, which should be considered while developing predictive intervention strategies to promote children's rehabilitation and improve parents' education, thus offering improved support for children with cancer.

The axonal radial contractility: Structural basis underlying a new form of neural plasticity.

Axons are the longest cellular structure reaching over a meter in the case of human motor axons. They have a relatively small diameter and contain several cytoskeletal elements that mediate both material and information exchange within neurons. Recently, a novel type of axonal plasticity, termed axonal radial contractility, has been unveiled. It is represented by dynamic and transient diameter changes of the axon shaft to accommodate the passages of large organelles. Mechanisms underpinning this plasticity are not fully understood. Here, we first summarised recent evidence of the functional relevance for axon radial contractility, then discussed the underlying structural basis, reviewing nanoscopic evidence of the subtle changes. Two models are proposed to explain how actomyosin rings are organised. Possible roles of non-muscle myosin II (NM-II) in axon degeneration are discussed. Finally, we discuss the concept of periodic functional nanodomains, which could sense extracellular cues and coordinate the axonal responses. Also see the video abstract here: https://youtu.be/ojCnrJ8RCRc.

GE11 Modified PLGA/TPGS Nanoparticles Targeting Delivery of Salinomycin to Breast Cancer Cells.

Salinomycin (Sal) is a potent inhibitor with effective anti-breast cancer properties in clinical therapy. The occurrence of various side effect of Sal greatly limits its application. The epidermal growth factor receptor (EGFR) family is a family of receptors highly expressed in most breast cancer cells. GE11 is a dodecapeptide which shows excellent EGFR affinity. A series of nanoparticles derivatives with GE11 peptide conjugated PLGA/TPGS were synthesized. Nanoprecipitation method was used to prepare the Sal loaded nanoparticles at the optimized concentration. The characterization, targeting efficacy, and antitumor activity were detected both in vitro and in vivo. Encapsulation of Sal in GE11 modified PLGA/TPGS nanoparticles shows an improved therapy efficacy and lower systemic side effect. This represents the delivery system a promising strategy to enhance the therapeutic effect against EGFR highly expressed breast cancer.

Machine learning based tissue analysis reveals Brachyury has a diagnosis value in breast cancer.

BACKGROUND: The aim of the present study was to confirm the role of Brachyury in breast cancer and to verify whether four types of machine learning models can use Brachyury expression to predict the survival of patients. METHODS: We conducted a retrospective review of the medical records to obtain patient information, and made the patient's paraffin tissue into tissue chips for staining analysis. We selected 303 patients for research and implemented four machine learning algorithms, including multivariate logistic regression model, decision tree, artificial neural network and random forest, and compared the results of these models with each other. Area under the receiver operating characteristic (ROC) curve (AUC) was used to compare the results. RESULTS: The chi-square test results of relevant data suggested that the expression of Brachyury protein in cancer tissues was significantly higher than that in paracancerous tissues (P=0.0335); patients with breast cancer with high Brachyury expression had a worse overall survival (OS) compared with patients with low Brachyury expression. We also found that Brachyury expression was associated with ER expression (P=0.0489). Subsequently, we used four machine learning models to verify the relationship between Brachyury expression and the survival of patients with breast cancer. The results showed that the decision tree model had the best performance (AUC = 0.781). CONCLUSIONS: Brachyury is highly expressed in breast cancer and indicates that patients had a poor prognosis. Compared with conventional statistical methods, decision tree model shows superior performance in predicting the survival status of patients with breast cancer.

A Protocol for Cancer-Related Mutation Detection on Exosomal DNA in Clinical Application.

BACKGROUND: Recently, some genomic mutations in exosomal DNA have been found to be related to disease progress and clinical outcomes of patients in several cancers. Unfortunately, the methods for exosome isolation and exosomal DNA analysis are still lack of relevant research to ensure their optimal performance and the comparability. Here we aim to establish a protocol for cancer-related mutation detection on exosomal DNA in clinical application. METHODS: Taking KRAS mutation in pancreatic cancer as an example, we tested whether the types of blood samples, the potential factors in the courses of exosome isolation and exosomal DNA preparation, as well as the detail in mutation detection by droplet digital PCR (ddPCR) could influence the exosomal DNA analysis. RESULTS: We found that the concentration of exosomal DNA from serum was higher than that from plasma, whereas the mutant allele fraction (MAF) of KRAS in serum-derived exosomal DNA was obviously lower. The membrane-based method for exosome isolation showed no evident difference in both exosomal DNA yield and KRAS MAF from the classical ultracentrifugation method. DNase I pretreatment on exosomes could remove the wild-type DNA outside of exosomes and increase the KRAS MAF. PBS might interfere with the effect of DNase I and should not be recommended as resuspension buffer for exosomes if the subsequent experiments would be done with exosomal DNA. Besides, the denaturation of exosomal DNA before droplet generation during ddPCR could effectively improve the total KRAS copy number and mutation-positive droplet number. CONCLUSION: This study provides some methodological evidences for the selection of the optimal experimental conditions in exosomal DNA analysis. We also suggest a protocol for mutation detection on exosomal DNA, which might be suitable for the clinical testing and could be helpful to the comparison of results from different laboratories.

Hyperbaric oxygen protects against myocardial ischemia­reperfusion injury through inhibiting mitochondria dysfunction and autophagy.

Our previous study demonstrated that hyperbaric oxygen (HBO) improves heart function predominantly through reducing oxygen stress, modulating energy metabolism and inhibiting cell apoptosis. The present study aimed to investigate the protective effects of HBO on mitochondrial function and autophagy using rats with a ligated left anterior descending artery. The cardioprotective effects of HBO were mainly evaluated using ELISA, fluorescent probes, transmission electron microscopy and reverse transcription­quantitative PCR (RT­qPCR). HBO pretreatment for 14 days (once a day) using a 0.25 MPa chamber improved mitochondrial morphology and decreased the number of autophagic vesicles, as observed using a transmission electron microscope. HBO pretreatment significantly increased the levels of ATP, ADP, energy charge and the opening of the mitochondrial permeability transition pore, but decreased the levels of AMP, cytochrome c and reactive oxygen species. Moreover, HBO pretreatment significantly increased the gene or protein expression levels of eIF4E­binding protein 1, mammalian target of rapamycin (mTOR), mitochondrial DNA, NADH dehydrogenase subunit 1, mitofusin 1 and mitofusin 2, whereas it decreased the gene or protein expression levels of autophagy­related 5 (Atg5), cytochrome c, dynamin­related protein 1 and p53, as determined using RT­qPCR or immunohistochemistry. In conclusion, HBO treatment was observed to protect cardiomyocytes during myocardial ischemia­reperfusion injury (MIRI) by preventing mitochondrial dysfunction and inhibiting autophagy. Thus, these results provide novel evidence to support the use of HBO as a potential agent for the mitigation of MIRI.

Proteomic Profiling of Serum Exosomes From Patients With Metastatic Gastric Cancer.

Background: Clinical management of metastatic gastric cancer (mGC) remains a major challenge due to a lack of specific biomarkers and effective therapeutic targets. Recently, accumulating evidence has suggested that exosomes play an essential role in cancer metastasis and can be an excellent reservoir of novel biomarkers and candidate therapeutic targets for cancer. Therefore, in this study, we aimed to reveal the proteomic profile of mGC-derived exosomes. Methods: Exosomes were isolated from pooled serum samples of 20 mGC patients and 40 healthy controls (HC) by ultracentrifugation. Next, quantitative proteomic analyses were applied to analyze the protein profiles of the exosomes, and bioinformatic analyses were conducted on the proteomic data. Finally, the expression of exosomal protein candidates was selectively validated in individual subjects by western blot analysis. Results: We isolated exosomes from serum samples. The size of the serum derived exosomes ranged from 30 to 150 nm in diameter. The exosomal markers CD9 and CD81 were observed in the serum exosomes. However, the exosomal negative marker calnexin, an endoplasmic reticulum protein, was not detected in exosomes. Overall, 443 exosomal proteins, including 110 differentially expressed proteins (DEPs) were identified by quantitative proteomics analyses. The bioinformatics analyses indicated that the upregulated proteins were enriched in the process of protein metabolic, whereas the downregulated proteins were largely involved in cell-cell adhesion organization. Surprisingly, 10 highly vital proteins (UBA52, PSMA1, PSMA5, PSMB6, PSMA7, PSMA4, PSMA3, PSMB1, PSMA6, and FGA) were filtered from DEPs, most of which are proteasome subunits. Moreover, the validation data confirmed that PSMA3 and PSMA6 were explicitly enriched in the serum derived exosomes from patients with mGC. Conclusion: The present study provided a comprehensive description of the serum exosome proteome of mGC patients, which could be an excellent resource for further studies of mGC.

Lipopolysaccharide induces the differentiation of hepatic progenitor cells into myofibroblasts constitutes the hepatocarcinogenesis-associated microenvironment.

Hepatocellular carcinoma (HCC) generally occurs in the presence of chronic liver injury, often as a sequela of liver fibrosis. Hepatic progenitor cells (HPCs) are known to be capable of forming both hepatocytes and cholangiocytes in chronic liver injury, which are also considered a source of myofibroblasts and tumor-initiating cells, under carcinogenic circumstances. However, the underlying mechanisms that activate HPCs to give rise to HCC are still unclear. In current study, the correlation between HPCs activation and liver fibrosis and carcinogenesis was investigated in rats and human specimens. We analyzed the role of HPCs in tumorigenesis, by transplanting exogenous HPCs in a diethylnitrosamine-induced rat HCC model. Our data indicated that HPC activation correlated with hepatic fibrosis and hepatocarcinogenesis. We further found that exogenous HPC infusion promoted liver fibrosis and hepatocarcinogenesis, while lipopolysaccharides (LPS) played an important role in this process. However, results of our study indicated that LPS did not induce HPCs to form tumor in nude mice directly. Rather, LPS induced myofibroblast-like morphology in HPCs, which enhanced the tumorigenic potential of HPCs. Further experiments showed that LPS/Toll-like receptor 4 (TLR4) signaling mediated the differentiation of HPCs into myofibroblasts and enhanced the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which led to the aberrant expression of Ras and p53 signaling pathways in HPCs, and finally, promoted the proliferation and malignant transformation of HPCs, by long non-coding RNA regulation. Besides, examination of HCC clinical samples demonstrated that IL-6 and TNF-α production correlated with HPC activation, hepatic fibrosis, and HCC recurrence. Our study indicates that both myofibroblasts and tumor cells are derived from HPCs. HPC-derived myofibroblasts create tumor microenvironment and contribute to the proliferation and malignant transformation of HPCs. Furthermore, LPS present in the chronic liver inflammation microenvironment might play an important role in hepatocarcinogenesis, by regulating the plastic potential of HPCs.

Hyperbaric oxygen alleviated cognitive impairments in mice induced by repeated cerebral ischemia-reperfusion injury via inhibition of autophagy.

AIMS: In this study, we investigate the effect and underlying mechanism of hyperbaric oxygen (HBO) treatment on a model of repeated cerebral ischemia-reperfusion injury (IR). MAIN METHODS: Eighty rats were randomly separated into sham, vehicle, hyperbaric air (HBA; 0.25 MPa, 60 min), and HBO (0.25 MPa, 60 min) groups. Repeated cerebral IR was induced by ligating the right and left bilateral common carotid arteries for 10 min and then allowing reperfusion for 10 min. This pattern was repeated three times. The neuroprotective effects of HBO were assessed by animal behavior, neuron morphology, inflammatory markers, intracellular calcium ion content, and autophagy-related protein and gene expression. KEY FINDINGS: Our result showed that HBO improved learning and memory in the navigation trail and probe trail of the Morris water maze, and these findings were supported by the observation data from 2,3,5-Triphenyltet-razolium chloride staining, Nissl staining, and electron microscopic. Importantly, we found that HBO reduced excessive autophagy in the prefrontal cortex, which was evidenced by activating of the mammalian target of the rapamycin (mTOR) and 4E-BP1, as well as suppression of LC3II and ATG5. Moreover, HBO significantly inhibited the cerebral IR-induced inflammatory reaction. Furthermore, HBO treatment modulated autophagy pathway-related factors, including producing a decrease in the intracellular calcium ion concentration and p53 level; meanwhile, the levels of BDNF and p-Akt were increased. SIGNIFICANCE: Our results indicated that HBO protected against IR-induced neuron injury by attenuating autophagy, inflammation, and calcium overload. These results provide a new mechanism and laboratory evidence for clinical treatment of VD.

KRAS Mutant Allele Fraction in Circulating Cell-Free DNA Correlates With Clinical Stage in Pancreatic Cancer Patients.

Background: The research on circulating tumor DNA (ctDNA) in pancreatic cancer (PC) has emerged recently. Although the detection rate of the KRAS mutation in ctDNA was relatively consistent with that in tumor tissue, whether the KRAS mutant allele fraction (MAF) differed was still not reported. So far, the clinical application of ctDNA detection in PC remains inconclusive. Methods: Plasma samples were collected from 110 PC and 52 pancreatic benign (PB) disease patients. The detection of KRAS mutation in ctDNA was performed using droplet digital PCR and compared with that in matched tumor tissue. We assessed the utility of KRAS MAFs in ctDNA and tissue for pancreatic malignancy assessment. Results: We found that KRAS MAF in ctDNA of PC patients was higher than that of PB patients, and was obviously associated with tumor staging and distant metastasis. However, KRAS MAF in ctDNA was significantly different from that in matched tissue. KRAS MAF in tumor tissue had no significant correlation with the clinical status. In addition, a ROC curve analysis revealed that mutant KRAS ctDNA combined with CA19-9 could increase the sensitivity rate of early-stage PC prediction, compared with CA19-9 test alone. Conclusion: The MAF of KRAS in ctDNA was related to the clinical stage of PC (p = 0.001). Mutant KRAS ctDNA could improve the sensitivity in early diagnosis of PC as a complement to CA19-9. Our study suggested that KRAS mutation in ctDNA could be a valuable circulating biomarker for the malignancy assessment in PC.

[Detection of Exosomal PML-RARA Fusion Gene Expression Level by Droplet Digital PCR].

OBJECTIVE: To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR). METHODS: By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique. RESULTS: The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected. CONCLUSION: A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.