Analysis of cases with co-infection of COVID-19 and pulmonary aspergillosis.

The objective of this study was to investigate the risk factors and diagnosis measure of COVID-19-associated pulmonary aspergillosis (CAPA). This study included 201 COVID-19 patients from December 1, 2022, to January 31, 2023; 7 (3.5%) were diagnosed with CAPA. The main risk factors were age, MV, ICU admission and COPD, and the presence of comorbidities such as ARDS and hypoproteinemia in COVID-19 patients, more susceptible to Aspergillus infection. In addition to specimen culture in the lower respiratory tract, the 1,3-ß-D-glucan antigen test can serve as an important screening indicator for early CAPA diagnosis in non-granulocytopenia patients.

NMDARs activation regulates endothelial ferroptosis via the PP2A-AMPK-HMGB1 axis.

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated, voltage-dependent channels of the ionotropic glutamate receptor family. The present study explored whether NMDAR activation induced ferroptosis in vascular endothelial cells and its complicated mechanisms in vivo and in vitro. Various detection approaches were used to determine the ferroptosis-related cellular iron content, lipid reactive oxygen species (LOS), siRNA molecules, RNA-sequence, MDA, GSH, and western blotting. The AMPK activator Acadesine (AICAR), HMGB1 inhibitor glycyrrhizin (GLY), PP2A inhibitor LB-100, and NMDAR inhibitor MK801 were used to investigate the involved in vivo and in vitro pathways. The activation of NMDAR with L-glutamic acid (GLU) or NMDA significantly promoted cellular ferroptosis, iron content, MDA, and the PTGS2 expression, while decreasing GPX4 expression and GSH concentration in human umbilical vein endothelial cells (HUVECs), which was reversed by ferroptosis inhibitors Ferrostatin-1(Fer-1), Liproxstatin-1 (Lip-1), or Deferoxamine (DFO). RNA-seq revealed that ferroptosis and SLC7A11 participate in NMDA or GLU-mediated NMDAR activation. The PP2A-AMPK-HMGB1 pathway was majorly associated with NMDAR activation-induced ferroptosis, validated using the PP2A inhibitor LB-100, AMPK activator AICAR, or HMGB1 siRNA. The role of NMDAR in ferroptosis was validated in HUVECs induced with the ferroptosis activator errasin or RSL3 and counteracted by the NMDAR inhibitor MK-801. The in vivo results showed that NMDA- or GLU-induced ferroptosis and LOS production was reversed by MK-801, LB-100, AICAR, MK-801, and GLY, confirming that the PP2A-AMPK-HMGB1 pathway is involved in NMDAR activation-induced vascular endothelium ferroptosis. In conclusion, the present study demonstrated a novel role of NMDAR in endothelial cell injury by regulating ferroptosis via the PP2A-AMPK-HMGB1 pathway.

Long-term MNNG exposure promotes gastric carcinogenesis by activating METTL3/m6A/miR1184 axis-mediated epithelial-mesenchymal transition.

As the representative item of environmental chemical carcinogen, MNNG was closely associated with the onset of Gastric cancer (GC), while the underlying mechanisms remain largely unknown. Here, we comprehensively analyzed the potential clinical significance of METTL3 in multiple GC patient cohorts. Additionally, we demonstrated that long-term exposure to MNNG elevated METTL3 and EMT marker expression by in vitro and in vivo models. Furthermore, the depletion of METTL3 impacted the proliferation, migration, invasion, and tumorigenesis of MNNG malignant transformation cells and GC cells. By me-RIP sequencing, we identified a panel of vital miRNAs potentially regulated by METTL3 that aberrantly expressed in MNNG-induced GC cells. Mechanistically, we showed that METTL3 meditated miR-1184/TRPM2 axis by regulating the process of miRNA-118. Our results provide novel insights into critical epigenetic molecular events vital to MNNG-induced gastric carcinogenesis. These findings suggest the potential therapeutic targets of METTL3 for GC treatment.

Histone methyltransferase Ezh2 coordinates mammalian axon regeneration via regulation of key regenerative pathways.

Current treatments for neurodegenerative diseases and neural injuries face major challenges, primarily due to the diminished regenerative capacity of neurons in the mammalian CNS as they mature. Here, we investigated the role of Ezh2, a histone methyltransferase, in regulating mammalian axon regeneration. We found that Ezh2 declined in the mouse nervous system during maturation but was upregulated in adult dorsal root ganglion neurons following peripheral nerve injury to facilitate spontaneous axon regeneration. In addition, overexpression of Ezh2 in retinal ganglion cells in the CNS promoted optic nerve regeneration via both histone methylation-dependent and -independent mechanisms. Further investigation revealed that Ezh2 fostered axon regeneration by orchestrating the transcriptional silencing of genes governing synaptic function and those inhibiting axon regeneration, while concurrently activating various factors that support axon regeneration. Notably, we demonstrated that GABA transporter 2, encoded by Slc6a13, acted downstream of Ezh2 to control axon regeneration. Overall, our study underscores the potential of modulating chromatin accessibility as a promising strategy for promoting CNS axon regeneration.

[Research and application of model fruit fly in prevention and treatment of neuropsychiatric disorders by traditional Chinese medicine and natural compounds: a review].

As an important model animal, fruit fly is characterized by outstanding genetic characteristics, relatively perfect nervous system, rapid reproduction, and low cost. Thus, it has been applied in the research on neuropsychiatric disorders in recent years, showing great potential in life science. The incidence of neuropsychiatric disorders has been on the rise, and the disorders have high disability rate and low case fatality rate. The global drug demand for such diseases is second only to cardiovascular and cerebrovascular diseases. At the moment, the demand of the drugs for the diseases have been rising, and it is an urgent task to develop related drugs. However, the research and development of the drugs are time-intensive and have a high failure rate. A suitable animal model can help shorten the time for drug screening and development, thereby reducing the cost and failure rate. This study reviews the application of fruit flies in several common neuropsychiatric disorders, which is expected to provide new ideas for the research and application of the model animals in traditional Chinese medicine.

Cerebrospinal fluid cytokine and chemokine patterns correlate with prognosis of HIV-uninfected cryptococcal meningitis: A prospective observational study.

The cerebrospinal fluid (CSF) immune responses in HIV-uninfected cryptococcal meningitis (CM) have not been well studied. In this study, we aimed to explore the phenotype of CSF immune response during the course of disease and to examine relationships between phenotypes and disease severity. We profiled the CSF immune response in 128 HIV-uninfected CM and 30 pulmonary cryptococcosis patients using a 27-plex Luminex cytokine kit. Principal component analyses (PCA) and logistic regression model were performed. Concentrations of 23 out of 27 cytokines and chemokines in baseline CSF were significantly elevated in CM patients compared with pulmonary cryptococcosis cases. In CM patients with Cryptococcus neoformans infection, IL-1ra, IL-9, and VEGF were significantly elevated in immunocompetent cases. Cytokine levels usually reached peaks within the first 2 weeks of antifungal treatment and gradually decreased over time. PCA demonstrated a co-correlated CSF cytokine and chemokine response consisting of Th1, Th2, and Th17 type cytokines. Prognostic analysis showed that higher scores for the PCs loading pro-inflammatory cytokines, IFN-γ, TNF-α, and IL-12; and anti-inflammatory cytokine, IL-4; and chemokines, Eotaxin, FGF-basis, and PDGF-bb; as well as lower scores for the PCs loading RANTES were associated with disease severity, as defined by a Glasgow Coma Scale of <15 or death. In conclusion, combined inflammatory responses in CSF involving both pro- and anti-inflammatory cytokines and chemokines are upregulated in HIV-uninfected CM, and associated with disease severity.

Synthesis and Structure Elucidation of Two Essential Metal Complexes: In-Vitro Studies of Their BSA/HSA-Binding Properties, Docking Simulations, and Anticancer Activities.

Imidazole and tetrazole derivatives are widely used as clinical drugs since they possess a variety of pharmaceutical function. Zinc and iron are essential trace elements of the human body, with less toxicity and good biocompatibility. In this paper, two new essential metal mononuclear complexes [M(H2tmidc)2(H2O)2]·2H2O (M = Zn (1), Fe (2)) were synthesized through the reaction of 2-((1H-tetrazol-1-yl)methylene)-1H-imidazole-4,5-dicarboxylic acid (H3tmidc) and ZnSO4·7H2O or FeSO4·7H2O. The crystal structures were determined by means of the X-ray single crystal diffraction technique. Results from fluorescence investigations show that both complexes could interact with BSA as well as HSA through the static quenching mechanism. van der Waals forces and hydrogen bonds play important roles in the interaction of complexes and BSA/HSA since both ΔH and ΔS values are negative. The results of molecular docking are consistent with those in experimental studies. Furthermore, the anticancer activity of H3tmidc and both complexes against Eca-109 were preliminarily evaluated and the results show that both complexes have better anticancer activity than the corresponding ligand H3tmidc.

Immune reconstitution inflammatory syndrome in non-HIV cryptococcal meningitis: Cross-talk between pathogen and host.

BACKGROUND: Cryptococcal meningitis (CM)-associated immune reconstitution inflammatory syndrome (IRIS) is associated with high mortality, the epidemiology and pathophysiology of which is poorly understood, especially in non-HIV populations. OBJECTIVES: We aim to explore the incidence, clinical risk factors, immunological profiles and potential influence of leukotriene A4 hydroxylase (LTA4H) on non-HIV CM IRIS populations. METHODS: In this observational cohort study, 101 previously untreated non-HIV CM patients were included. We obtained data for clinical variables, 27 cerebrospinal fluid (CSF) cytokines levels and LTA4H genotype frequencies. Changes of CSF cytokines levels before and at IRIS occurrence were compared. RESULTS: Immune reconstitution inflammatory syndrome was identified in 11 immunocompetent males, generating an incidence of 10.9% in non-HIV CM patients. Patients with higher CrAg titres (> 1:160) were more likely to develop IRIS, and titre of 1:1280 is the optimum level to predict IRIS occurrence. Baseline CSF cytokines were significantly higher in IRIS group, which indicated a severe host immune inflammation response. Four LTA4H SNPs (rs17525488, rs6538697, rs17525495 and rs1978331) exhibited significant genetic susceptibility to IRIS in overall non-HIV CM, while five cytokines were found to be associated with rs1978331, and baseline monocyte chemotactic protein 1 (MCP-1) became the only cytokine correlated with both IRIS and LTA4H SNPs. CONCLUSIONS: Our study suggested that non-HIV CM patients with high fungal burden and severe immune inflammation response were more likely to developed IRIS. LTA4H polymorphisms may affect the pathogenesis of IRIS by regulating the level of baseline CSF MCP-1.

Assessing Empathy in Anesthesia Residents with the Chinese Version of the Consultation and Relational Empathy: A Pilot Standardized Patient Program.

Objectives To validate the reliability of the Chinese version of the Consultation and Relational Empathy (CARE) in physician-standardized patient (SP) encounter. We also tried to examine the agreement between video-based ratings and in-room ratings, as well as the agreement between the faculty ratings and SP ratings. Methods The CARE was translated into Chinese. Forty-eight anesthesia residents were recruited to make preoperative interview in SP-counter. Performance of each resident was graded by in-room raters, video raters and SP raters. Consistency between different raters was examined. Results The Chinese-CARE measure demonstrated high scale reliability with a Cronbach's alpha value of 0.95 and high consistency in the in-room ratings in intraclass correlation (coefficient=0.888,P<0.001). Despite a good consistency in intraclass correlation, video ratings were significantly higher than in-room ratings (39.6±7.1vs. 24.0±10.0,P<0.001), and Wilcoxon signed-rank test indicated that the pass/fail rate was significantly higher based on video ratings than based on in-room ratings (45/48vs. 22/48, P<0.001). SP ratings had a moderate consistency with in-room faculty ratings (coefficient=0.568,P<0.001), and there was no significant difference between the pass/fail rates based on the in-room ratings and SP ratings (22/48vs. 28/48, P=0.12). Conclusions The Chinese-CARE measure is reliable in the assessment of empathy during preoperative anesthesia interview. In-room and video ratings are not equivalent, while SP may provide a feasible alternative for the faculty rater in the assessment of communication skills with an appropriate measure.

Adiponectin antagonises LPS-regulated secretion of inflammatory factors in airway epithelial cells, and its expression is regulated by many factors.

Many studies have shown that adiponectin is closely related to chronic obstructive pulmonary disease (COPD), but the specific role of adiponectin in COPD is still not well understood. Adiponectin and IL-6 expression in patients with acute exacerbation of COPD (AECOPD) was detected by ELISA. Human bronchial epithelial cells (HBECs) were stimulated with TNF-α, IL-6, apoptotic cells or LPS. Then, the expression of adiponectin was detected by qRT-PCR and western blotting, and pro- and anti-inflammatory factors were detected by ELISA. Adiponectin expression in AECOPD patients increased after treatment. TNF-α and apoptotic cells promoted adiponectin expression in HBECs in a dose-dependent manner, and apoptotic cells significantly promoted adiponectin secretion. IL-6 also promoted adiponectin expression, but it inhibited adiponectin expression at high doses and with long treatment times. LPS inhibited adiponectin expression, but when HBECs were pretreated with anti-TNF-α and then treated with LPS, the expression and secretion of adiponectin increased significantly with increasing anti-TNF-α concentrations. Adiponectin stimulated the secretion of pro-inflammatory factors in HBECs, but this effect was not concentration dependent. Adiponectin promoted the secretion of anti-inflammatory factors in a dose-dependent manner. Although LPS also stimulated HBECs to secrete pro-inflammatory and anti-inflammatory factors, adiponectin inhibited LPS-induced pro-inflammatory factor secretion and enhanced anti-inflammatory factor secretion. Many factors regulate the expression and secretion of adiponectin, and adiponectin regulates the balance of the inflammatory response and inhibits further expansion of inflammation. SIGNIFICANCE OF THE STUDY: Many studies have shown that adiponectin is closely related to chronic obstructive pulmonary disease (COPD), but the specific role of adiponectin in COPD is still not well understood. Adiponectin expression in AECOPD patients increased after treatment. TNF-α, IL-6 and apoptotic cells promoted adiponectin expression in HBECs. Adiponectin stimulated the secretion of pro-inflammatory factors in HBECs, but this effect was not concentration dependent. Adiponectin promoted the secretion of anti-inflammatory factors in a dose-dependent manner. Adiponectin inhibited LPS-induced pro-inflammatory factor secretion and enhanced anti-inflammatory factor secretion. Therefore, many factors regulate the expression and secretion of adiponectin, and adiponectin regulates the balance of the inflammatory response and inhibits further expansion of inflammation.

Selenium Supplementation Protects Against Lipopolysaccharide-Induced Heart Injury via Sting Pathway in Mice.

Sepsis-induced myocardial dysfunctions are associated with high morbidity and mortality. Selenium, an essential trace element, has been reported to exert anti-inflammation, anti-oxidative stress, and anti-apoptosis. However, the protective effects of selenium on LPS-induced heart injury are still poorly illustrated. Therefore, in the present study, we sought to explore the effects of selenium pretreatment on LPS-induced myocardial injury in mice. We firstly found that selenium pretreatment significantly improved markers of myocardial injury and alleviated LPS-induced myocardial dysfunctions. Moreover, selenium supplementation reduced pro-inflammatory cytokines expression, decreased oxidative stress, and inhibited myocardial apoptosis. In addition, selenium supplementation inactivated the Sting pathway. In conclusion, our study suggests that selenium exerts protective effects on LPS-induced myocardial injury, and the underlying molecular mechanism may be related to the inactivation of Sting pathway, implying a potential therapy for sepsis-induced myocardial dysfunctions.

Calenduloside E Ameliorates Myocardial Ischemia-Reperfusion Injury through Regulation of AMPK and Mitochondrial OPA1.

Calenduloside E (CE) is a natural triterpenoid saponin isolated from Aralia elata (Miq.) Seem., a well-known traditional Chinese medicine. Our previous studies have shown that CE exerts cardiovascular protective effects both in vivo and in vitro. However, its role in myocardial ischemia/reperfusion injury (MIRI) and the mechanism involved are currently unknown. Mitochondrial dynamics play a key role in MIRI. This study investigated the effects of CE on mitochondrial dynamics and the signaling pathways involved in myocardial ischemia/reperfusion (MI/R). The MI/R rat model and the hypoxia/reoxygenation (H/R) cardiomyocyte model were established in this study. CE exerted significant cardioprotective effects in vivo and in vitro by improving cardiac function, decreasing myocardial infarct size, increasing cardiomyocyte viability, and inhibiting cardiomyocyte apoptosis associated with MI/R. Mechanistically, CE restored mitochondrial homeostasis against MI/R injury through improved mitochondrial ultrastructure, enhanced ATP content and mitochondrial membrane potential, and reduced mitochondrial permeability transition pore (MPTP) opening, while promoting mitochondrial fusion and preventing mitochondrial fission. However, genetic silencing of OPA1 by siRNA abolished the beneficial effects of CE on cardiomyocyte survival and mitochondrial dynamics. Moreover, we demonstrated that CE activated AMP-activated protein kinase (AMPK) and treatment with the AMPK inhibitor, compound C, abolished the protective effects of CE on OPA1 expression and mitochondrial function. Overall, this study demonstrates that CE is effective in mitigating MIRI by modulating AMPK activation-mediated OPA1-related mitochondrial fusion.

Evaluation of low cryptococcal antigen titer as determined by the lateral flow assay in serum and cerebrospinal fluid among HIV-negative patients: a retrospective diagnostic accuracy study.

Cryptococcosis is one of the most common opportunistic infections in both immunocompetent and immunocompromised hosts. Although the cryptococcal antigen (CrAg) lateral flow assay (LFA) has been widely used in clinical settings due to its high sensitivity and specificity, the diagnostic value of a low CrAg LFA titers remains unclear. In this study, we performed a retrospective analysis of 149 HIV-negative patients with low CrAg LFA titers (≤1:10) in a Chinese tertiary hospital from January 2013 to December 2017, to evaluate the diagnostic value of low CrAg LFA titers in serum and cerebrospinal fluid (CSF) at different thresholds. Sensitivity and specificity of low CrAg LFA titers in patients with definitive diagnoses of cryptococcosis were 39.6% (95% CI, 29.7-50.1%) and 100% (95% CI, 69.2-100%), respectively, at a threshold of 1:10 in serum. A sensitivity of 72.9% (95% CI, 62.9-81.5%) and a decreased specificity of 70.0% (95% CI, 34.8-93.3%) were observed at a threshold of 1:5 in serum. No false-positive cases were identified in patients with low CrAg titers in CSF and all positive predictive values (PPVs) were 100%. Among the cases with low serum CrAg titers, lumbar puncture was performed in 97 patients and positive CSF CrAg titers were reported in 6 patients. In conclusion, the results of this study imply that low CrAg LFA titer, either in serum or CSF, is crucial for early diagnosis of cryptococcosis in HIV-negative patients, and lumbar puncture is recommended to be performed routinely for CSF testing when a positive low serum titer is reported. Cryptococcal meningitis should be considered seriously when the CSF CrAg titer is positive.

Diallyl Trisulfide Suppresses Angiotensin II-Induced Vascular Remodeling Via Inhibition of Mitochondrial Fission.

OBJECTIVE: We have shown previously that diallyl trisulfide (DATS) ameliorates mitochondrial fission and oxidative stress in a hyperglycemia-induced endothelial apoptosis and diabetic mouse model. The aim of this study was to investigate whether DATS mitigates Ang II-induced vascular smooth muscle cell (VSMC) phenotypic switching and vascular remodeling, and if so, to determine the underlying molecular events. METHODS: Male C57BL/6 mice were used to establish a vascular remodeling model by continuous 2-week Ang II infusion using a subcutaneous osmotic pump. Animals were intraperitoneally injected with DATS or vehicle. Physiological parameters, vascular morphology, and molecular markers were assessed. For in vitro studies, VSMCs were pretreated with or without DATS for 1 h, then were stimulated with Ang II, and mitochondrial morphology and phenotypic switching of VSMCs were also measured. RESULTS: In primary mouse VSMCs, we found that Drp1-dependent mitochondrial fission regulated mitochondrial reactive oxygen species (mtROS) generation, which eventually promoted Ang II-induced VSMC proliferation, migration, and phenotypic switching. Moreover, Ang II was found to up-regulate the Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which regulated mitochondrial fission and VSMC phenotypic switching by phosphorylating Drp1. However, the biological effect of Ang II was abrogated by DATS. Consistent with the effects in VSMCs, we found that DATS markedly alleviated mitochondrial fission, VSMC differentiation, and vessel wall thickening in an animal model of Ang II-induced vascular remodeling, which was regulated by the ROCK1/Drp1 signal. CONCLUSIONS: Our findings showed that DATS mitigated Ang II-induced vascular remodeling by suppressing Drp1-mediated mitochondrial fission in an ROCK1-dependent manner.

Genetic polymorphisms of transient receptor potential melastatin 1 correlate with voriconazole-related visual adverse events.

BACKGROUND: Causes of voriconazole-related visual adverse events (VVAE) remained controversial. OBJECTIVES: We aimed to explore the relationship between voriconazole serum concentrations and VVAE as well as the potential influence of transient receptor potential melastatin 1 (TRPM1) on VVAE. PATIENTS/METHODS: This prospective observational cohort study was done in two stages. Patients who received voriconazole for invasive fungal diseases were consecutively enrolled. Correlations between voriconazole trough levels and VVAE were explored in 76 patients. Genotyping was further conducted for 17 tag SNPs of TRPM1 in a larger population of 137 patients. Genotype distributions were compared between patients with and without VVAE. RESULT: Of the 76 patients, a total of 229 steady-state voriconazole trough levels were evaluated, 69.9% of which were within the target range (1-5.5 mg/L). No correlations were found between voriconazole trough levels and VVAE. Of the total 137 patients, VVAE occurred in 37 (27.0%) patients, including visual hallucination (13.9%, 19/137) and visual disturbances (19.0%, 26/137). Significant difference in TRPM1 genotype distribution was only observed in patients with visual hallucination but not with visual disturbances. We found that rs890160 G/T genotype was under-presented (OR, 0.11; 95% CI, 0.01-0.84; P = .011) and rs1378847 C/C genotype was more frequently detected (OR, 8.89; 95% CI, 1.14-69.02; P = .013) in patients with visual hallucination when compared with those without. CONCLUSION: Transient receptor potential melastatin 1 was genetically associated with voriconazole-related visual hallucination. The correlation was failed to found between voriconazole trough levels and VVAE.

Hydroxysafflor yellow A inhibits hypoxia/reoxygenation-induced cardiomyocyte injury via regulating the AMPK/NLRP3 inflammasome pathway.

Hydroxysafflor yellow A (HSYA) is an effective therapeutic agent that alleviates myocardial ischaemia/reperfusion injury (MIRI), but the exact mechanisms remain elusive. The aim of this study was to investigate the potential protective effect of HSYA against MIRI through mechanisms related to NLRP3 inflammasome regulation. In this study, hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocytes were treated with HSYA or the AMPK inhibitor, compound C (CC). Our results showed that HSYA pretreatment improved cardiomyocyte viability, maintained mitochondrial membrane potential, reduced apoptotic cardiomyocytes, decreased caspase-3 activity, and inhibited NOD-like receptor 3 (NLRP3) inflammasome activation during H/R injury. Moreover, the inhibition of AMPK activation by the CC inhibitor partially abolished the effects of HSYA treatment, including suppressing the upregulation of NLRP3 inflammasome components (NLRP3, caspase-1 and interleukin-1ß) and promoting autophagy (LC3-II/LC3-I and p62). In conclusion, the protective mechanism of HSYA in H/R-induced cardiomyocyte injury is associated with inhibiting NLRP3 inflammasome activation through the AMPK signalling pathway.

CYP2E1 changes the biological function of gastric cancer cells via the PI3K/Akt/mTOR signaling pathway.

The present study investigated the role of cytochrome P450 family 2 subfamily E polypeptide 1 (CYP2E1) in the development and progression of gastric cancer (GC). The expression levels of CYP2E1 in MGC­803 GC cells and normal GES­1 cells were investigated via western blotting, and it was identified that the expression of CYP2E1 was different between GES­1 and MGC­803 cells. CYP2E1 was overexpressed in MGC­803 cells using a lentiviral vector GV358. Cell Counting Kit­8, flow cytometry, cell migration and Matrigel invasion assays suggested that overexpression of CYP2E1 promoted the proliferation and invasion, and inhibited the apoptosis of GC cells. The relationship between CYP2E1 expression and key signaling molecules in the PI3K/Akt/mTOR signaling pathway was assessed. Reverse transcription­quantitative PCR analysis showed that mTOR mRNA expression was significantly increased after overexpression of CYP2E1 (P<0.05). Western blotting results showed that overexpression of CYP2E1 upregulated the expression of phosphorylated (p)­Akt, p­mTOR and p­p70 ribosomal protein S6 kinase (P70S6K; Ser371) proteins (P<0.05). To further investigate the relationship between CYP2E1 and the PI3K/Akt/mTOR signaling pathway in GC cells, MGC­803 cells were treated with the PI3K inhibitor LY294002, and changes in the expression levels of PI3K, AKT, mTOR, P70S6K and CYP2E1 were observed. The present results showed that LY294002 downregulated the expression of PI3K, CYP2E1, AKT, mTOR and P70S6K (P<0.05). Therefore, changes in the biological function of GC cells induced by CYP2E1 overexpression may be via the PI3K/Akt/mTOR signaling pathway.

Time course analysis of sensory axon regeneration in vivo by directly tracing regenerating axons.

Most current studies quantify axon regeneration by immunostaining regeneration-associated proteins, representing indirect measurement of axon lengths from both sensory neurons in the dorsal root ganglia and motor neurons in the spinal cord. Our recently developed method of in vivo electroporation of plasmid DNA encoding for enhanced green fluorescent protein into adult sensory neurons in the dorsal root ganglia provides a way to directly and specifically measure regenerating sensory axon lengths in whole-mount nerves. A mouse model of sciatic nerve compression was established by squeezing the sciatic nerve with tweezers. Plasmid DNA carrying enhanced green fluorescent protein was transfected by ipsilateral dorsal root ganglion electroporation 2 or 3 days before injury. Fluorescence distribution of dorsal root or sciatic nerve was observed by confocal microscopy. At 12 and 18 hours, and 1, 2, 3, 4, 5, and 6 days of injury, lengths of regenerated axons after sciatic nerve compression were measured using green fluorescence images. Apoptosis-related protein caspase-3 expression in dorsal root ganglia was determined by western blot assay. We found that in vivo electroporation did not affect caspase-3 expression in dorsal root ganglia. Dorsal root ganglia and sciatic nerves were successfully removed and subjected to a rapid tissue clearing technique. Neuronal soma in dorsal root ganglia expressing enhanced green fluorescent protein or fluorescent dye-labeled microRNAs were imaged after tissue clearing. The results facilitate direct time course analysis of peripheral nerve axon regeneration. This study was approved by the Institutional Animal Care and Use Committee of Guilin Medical University, China (approval No. GLMC201503010) on March 7, 2014.

Protective effects of Myrica rubra flavonoids against hypoxia/reoxygenation-induced cardiomyocyte injury via the regulation of the PI3K/Akt/GSK3ß pathway.

Myrica rubra is well known for its delicious taste and high nutritional value. The present study investigated the potential protective effects and mechanisms of M. rubra flavonoids (MRF) extract on isoproterenol (ISO)­induced myocardial injury in rats and hypoxia/reoxygenation (H/R) injury in H9c2 cardiomyocytes. An in vivo study revealed that MRF decreased serum cardiac enzyme levels, ameliorated pathological heart alterations and increased the antioxidant potential. The in vitro investigation demonstrated that MRF inhibited cell death, reactive oxygen species (ROS) accumulation, mitochondrial membrane depolarization, apoptosis rate and caspase­3 activation and enhanced the Bcl­2/Bax ratio during H/R injury. These effects were accompanied by the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase (GSK)­3ß. Further mechanism studies demonstrated that LY294002, a specific inhibitor of phosphoinositide 3­kinase (PI3K), abolished the MRF­mediated cardioprotection against H/R­induced apoptosis and ROS overproduction. Collectively, these results suggested that MRF exerts cardioprotective effects by attenuating oxidative damage and cardiomyocyte apoptosis most likely via a PI3K/Akt/GSK3ß­dependent mechanism.