Three-Component Electrochemical Aminoselenation of 1,3-Dienes.

Azoles and organoselenium compounds are pharmacologically important scaffolds in medicinal chemistry and natural products. We developed an efficient regioselective electrochemical aminoselenation reaction of 1,3-dienes, azoles, and diselenide derivatives to access selenium-containing allylazoles skeletons. This protocol is more economical and environmentally friendly and features a broad substrate scope; pyrazole, triazole, and tetrazolium were all tolerated under the standard conditions, which could be applied to the expedient synthesis of bioactive molecules and in the pharmaceutical industry.

Sulfur mustard analog 2-chloroethyl ethyl sulfide increases triglycerides by activating DGAT1-dependent biogenesis and inhibiting PGC1ɑ-dependent fat catabolism in immortalized human bronchial epithelial cells.

Using sulfur mustard analog 2-chloroethyl ethyl sulfide (CEES), we established an in vitro model by poisoning cultured immortalized human bronchial epithelial cells. Nile Red staining revealed lipids accumulated 24 h after a toxic dose of CEES (0.9 mM). Lipidomics analysis showed most of the increased lipids were triglycerides (TGs), and the increase in TGs was further confirmed using a Triglyceride-Glo™ Assay kit. Protein and mRNA levels of DGAT1, an important TG biogenesis enzyme, were increased following 0.4 mM CEES exposure. Under higher dose CEES (0.9 mM) exposure, protein and mRNA levels of PPARγ coactivator-1ɑ (PGC-1ɑ), a well-known transcription factor that regulates fatty acid oxidation, were decreased. Finally, application with DGAT1 inhibitor A 922500 or PGC1ɑ agonist ZLN005 was able to block the CEES-induced TGs increase. Overall, our dissection of CEES-induced TGs accumulation provides new insight into energy metabolism dysfunction upon vesicant exposure.HIGHLIGHTSIn CEES (0.9 mM)-injured cells:Triglycerides (TGs) were abundant in the accumulated lipids.Expression of DGAT1, not DGAT2, was increased.Expression of PGC1ɑ, not PGC1ß, was reduced.DGAT1 inhibitor or PGC1ɑ agonist blocked the CEES-mediated increase in TGs.

Mitochondrial ATP-sensitive potassium channel regulates mitochondrial dynamics to participate in neurodegeneration of Parkinson's disease.

Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. Mitochondrial dysfunction has been the focus of the pathogenesis of PD. The mitochondrial ATP-sensitive potassium channel (mitoKATP) plays a significant role in mitochondrial physiology and has been extensively shown to protect against ischemic and brain reperfusion injury. However, there have long been controversies regarding its role in Parkinson's disease. We investigated the role of mitoKATP channels in rotenone-induced PD model in vivo and vitro and the interactions of mitoKATP channels, mitochondrial dynamics and PD. The results indicated that the use of diazoxide to activate mitoKATP channels resulted in the aggravation of rotenone-induced dopamine neurodegeneration in PC12 cells and SD rats. In contrast, the use of 5-hydroxydecanoate (5-HD) to inhibit mitoKATP channels improved rotenone-induced dopamine neurodegeneration, which was not consistent with mitoKATP channels in ischemic and brain reperfusion injury. Further analysis determined that the mitoKATP channel was involved in PD mainly via the regulation of mitochondrial biogenesis and fission/fusion. And the pore subunits of Kir6.1, the major component of mitoKATP channels, was the key contributor in its interaction with mitochondrial dynamics in rotenone-induced dopamine neurodegeneration. Therefore, it can be concluded that mitoKATP channels regulate mitochondrial dynamics to participate in rotenone-induced PD mainly attributes to the pore subunits of Kir6.1. And additionally, though mitoKATP channels may represent a direction of one potential target for neuroprotection, it should be noted that the effects are different in the activation or inhibition of mitoKATP channels in different models.

miR-17-92 promotes leukemogenesis in chronic myeloid leukemia via targeting A20 and activation of NF-κB signaling.

miR-17-92 cluster are overexpressed in hematological malignancies including chronic myeloid leukemia (CML). However, their roles and mechanisms that regulate BCR-ABL induced leukemogenesis remain unclear. In this study, we demonstrated that genomic depletion of miR-17-92 inhibited the BCR-ABL induced leukemogenesis by using a mouse model of transplantation of BCR-ABL transduced hematopoietic stem cells. Furthermore, we identified that miR-19b targeted A20 (TNFAIP3). A20 overexpression results in inactivation of NF-κB activity including decrease of phosphorylation of P65 and IκBα, leads to induce apoptosis and inhibit proliferation and cycle in CML CD34 + cells. Thus we proved that miR-17-92 is a critical contributor to CML leukemogenesis via targeting A20 and activation of NF-κB signaling. These findings indicate that miR-17-92 will be important resources for developing novel treatment strategies of CML and better understanding long-term disease control.

Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells.

Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair.

Nitrogen mustard induces dynamic nuclear protein spectrum change and DNA-protein crosslinking, with p97 mediating repair.

Nitrogen mustard (NM) is a chemotherapeutic agent capable of alkylating nucleophilic proteins and DNA, causing severe cell damage. However, no reports have been on the dynamic changes in proteomics induced by NM. In this study, we established a model of acute exposure to NM for 1 h and a continuous cultured model for 24 h after NM removal (repair stage) using 16HBE cells. The nuclear protein spectrum and nuclear proteins crosslinked with DNA were analyzed, and the function of p97 during NM damage was examined. An hour of NM exposure resulted in severe changes in the nuclear protein spectrum and protein into the cell nucleus, which is mainly involved in nuclear acid-related issues. After 24 h, the return to normal process of the types and amounts of differentially expressed proteins was inhibited by si-p97. The main processes involved in si-p97 intervention were nucleocytoplasmic transport, processing in the endoplasmic reticulum, metabolic abnormalities, and DNA-response; however. An hour of exposure to NM increased DNA-protein crosslinking (DPC), total-H2AX, and p-H2AX. In contrast, si-p97 only further increased or maintained their levels at 24 h yet not at 1 h. The effect of the proteasome inhibitor, MG132, was similar to that of si-p97. The siRNA of DVC1, a partner of p97, also increased the DPC content. Both si-p97 and si-DVC1 increased the cytoplasmic levels of the proteasome (PSMD2). These results suggest acute NM exposure induces severe nuclear protein spectral changes, rapid protein influx into the nucleus, DPC formation, and DNA double-strand breaks. Furthermore, our data indicated that p97 is involved in normal protein spectrum maintenance and DPC removal after NM withdrawal, requiring the participation of DVC1 and the proteasome.

Electrochemical promoted three-component reaction to unsymmetric thiosulfonates.

Compounds comprising S-S bonds serve as significant pharmacological scaffolds in medicinal chemistry and natural products. We have devised an efficient electrochemical method for the construction of asymmetric disulfide bonds, leading to the synthesis of unsymmetric thiosulfonates. Compared with existing synthesis methods, our work not only avoids the use of metals and oxidants, but also realizes the operation of a one-pot three-component method, which makes this strategy extremely attractive.

Preparation and epitope mapping of monoclonal antibodies against African swine fever virus p22 protein.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is now widespread in many countries and severely affects the commercial rearing of swine. Rapid and early diagnosis is crucial for the prevention of ASF. ASFV mature virions comprise the inner envelope protein, p22, making it an excellent candidate for the serological diagnosis and surveillance of ASF. In this study, the prokaryotic-expressed p22 recombinant protein was prepared and purified for immunization in mice. Four monoclonal antibodies (mAbs) were identified using hybridoma cell fusion, clone purification, and immunological assays. The epitopes of mAbs 14G1 and 22D8 were further defined by alanine-scanning mutagenesis. Our results showed that amino acids C39, K40, V41, D42, C45, G48, E49, and C51 directly bound to 14G1, while the key amino acid epitope for 22D8 included K161, Y162, G163, D165, H166, I167, and I168. Homologous and structural analysis revealed that these sites were highly conserved across Asian and European ASFV strains, and the amino acids identified were located on the surface of p22. Thus, our study contributes to a better understanding of the antigenicity of the ASFV p22 protein, and the results could facilitate the prevention and control of ASF.

14-3-3 Proteins in the regulation of rotenone-induced neurotoxicity might be via its isoform 14-3-3epsilon's involvement in autophagy.

14-3-3 proteins have been confirmed to be involved in Parkinson's disease. It has been reported that an increase of 14-3-3 (theta, epsilon, and gamma) expression has neuroprotective effect in response to rotenone and MPP(+) in dopaminergic cell culture and transgenic C. elegans with alpha-synuclein overexpression. To further investigate the detail mechanism of 14-3-3 proteins in rotenone-induced dopamine neurotoxicity, we observed the expression of 14-3-3 isoforms, and the influence of 14-3-3epsilon knockdown on autophagic activity and cell function. The results showed that rotenone led to a decrease in expression of 14-3-3 protein and mRNA, and an increase in expression and aggregation of alpha-synuclein protein. Knockdown of 14-3-3epsilon expression in turn further aggravated PC12 cell damage, such as an enhancement of ROS formation, and a reduction of cell viability and ATP production. Further experiments confirmed that the autophagic activity was promoted with 14-3-3epsilon siRNA transfection, including an enhancement of autophagosome formation and the ratio of LC3-II/LC3-I. Therefore, we concluded that the regulation of 14-3-3 proteins in rotenone-induced neurotoxicity might be associated with its isoform 14-3-3epsilon's involvement in autophagy, which might be considered a mechanism in addition to the currently known function of 14-3-3 proteins in neurodegenerative disease pathogenesis.

Dopamine Release Suppression Dependent on an Increase of Intracellular Ca(2+) Contributed to Rotenone-induced Neurotoxicity in PC12 Cells.

Rotenone is an inhibitor of mitochondrial complex I that produces a model of Parkinson's disease (PD), in which neurons undergo dopamine release dysfunction and other features. In neurons, exocytosis is one of the processes associated with dopamine release and is dependent on Ca(2+) dynamic changes of the cell. In the present study, we have investigated the exocytosis of dopamine and the involvement of Ca(2+) in dopamine release in PC12 cells administrated with rotenone. Results demonstrated that rotenone led to an elevation of intracellular Ca(2+) through Ca(2+) influx by opening of the voltage-gated Ca(2+) channel and influenced the soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins expression (including syntaxin, vesicle-associated membrane protein 2 (VAMP2) and synaptosome-associated protein 25 (SNAP-25)); pretreatment with a blocker of L-type voltage-activated Ca(2+) channels (nifedipine) decreased the intracellular dopamine levels and ROS formation, increased the cell viability and enhanced the neurite outgrowth and exocytosis of synaptic vesicles. These results indicated that the involvement of intracellular Ca(2+) was one of the factors resulting in suppression of dopamine release suppression in PC12 cells intoxicated with rotenone, which was associated with the rotenone-induced dopamine neurotoxicity.

HSP90/CDC37 inactivation promotes degradation of LKB1 protein to suppress AMPK signaling in bronchial epithelial cells exposed to sulfur mustard analog, 2-chloroethyl ethyl sulfide.

To investigate the role of the liver kinase (LK) B1 protein, an activator of AMP-activated protein kinase (AMPK), in AMPK signaling suppression when exposed to vesicant, a kind of chemical warfare agent. Cultured human bronchial epithelial cells were inflicted with sulfur mustard (SM) analog, 2-chloroethyl ethyl sulfide (CEES) of 0.2-1.0 mM concentration, and cell proliferation, apoptosis, autophagy, and cellular ATP level were analyzed up to 24 h after the exposure. Focusing on LKB1, heat shock protein (HSP) 90, and cell division cycle (CDC) 37 proteins, the protein expression, phosphorylation, and interaction were examined with western blot, immunofluorescence staining, and/or immunoprecipitation. AMPK signaling was found to be inhibited 24 h after being exposed to either sub-cytotoxic (0.5 mM) or cytotoxic (1.0 mM) concentration of CEES based on MTS assay. Consistently, the degradation of the LKB1 protein and its less interaction with the HSP90/CDC37 complex was confirmed. It was found that 1.0, not 0.5 mM CEES also decreased the CDC37 protein, proteasome activity, and cellular ATP content that modulates HSP90 protein conformation. Inhibiting proteasome activity could alternatively activate autophagy. Finally, either 0.5 or 1.0 mM CEES activated HSP70 and autophagy, and the application of an HSP70 inhibitor blocked autophagy and autophagic degradation of the LKB1 protein. In conclusion, we reported here that AMPK signaling inactivation by CEES was a result of LKB1 protein loss via less protein complex formation and enhanced degradation.

The Mitochondrial-Derived Peptide (MOTS-c) Interacted with Nrf2 to Defend the Antioxidant System to Protect Dopaminergic Neurons Against Rotenone Exposure.

MOTS-c is a 16-amino acid mitochondrial-derived peptide reported to be involved in regulating energy metabolism. However, few studies have reported the role of MOTS-c on neuron degeneration. In this study, it was aimed to explore the action of MOTS-c in rotenone-induced dopaminergic neurotoxicity. In an in vitro study, it was observed that rotenone could influence the expression and localization of MOTS-c significantly in PC12 cells, with more MOTS-c translocating into the nucleus from mitochondria. Further study showed that the translocation of MOTS-c from the mitochondria into the nucleus could directly interact with Nrf2 to regulate HO-1 and NQO1 expression in PC12 cells exposed to rotenone, which had been suggested to be involved in the antioxidant defense system. In vivo and in vitro experiments demonstrated that exogenous MOTS-c pretreatment could protect PC12 cells and rats from mitochondrial dysfunction and oxidative stress induced by rotenone. Moreover, MOTS-c pretreatment significantly decreased the loss of TH, PSD95, and SYP protein expression in the striatum of rats exposed to rotenone. In addition, MOTS-c pretreatment could clearly alleviate the downregulated expression of Nrf2, HO-1, and NQO1, as well as the upregulated Keap1 protein expression in the striatum of rotenone-treated rats. Taken together, these findings suggested that MOTS-c could directly interact with Nrf2 to activate the Nrf2/HO-1/NQO1 signal pathway to defend the antioxidant system to prevent dopaminergic neurons from rotenone-induced oxidative stress and neurotoxicity in vitro and in vivo.

[Prediction of PAHs Content in Soil Around Gas Stations in Beijing Based on BP Neural Network].

With the rapid development of urbanization in China, the number of gas stations in cities is increasing. The composition of oil products in gas stations is complex and diverse, and a series of pollutants will be generated in the process of oil diffusion. Polycyclic aromatic hydrocarbons (PAHs) produced by gas stations can pollute the nearby soil and affect human health. In this study, soil samples (0-20 cm) near 117 gas stations in Beijing were collected, and the contents of seven PAHs were analyzed. Based on the BP neural network model, the contents of PAHs in soil of Beijing gas stations in 2025 and 2030 were predicted. The results showed that the total concentrations of the seven PAHs were 0.01-3.53 mg·kg-1. The concentrations of PAHs were lower than the soil environmental quality risk control standard for soil contamination of development land (Trial) GB 36600-2018. At the same time, the toxic equivalent concentrations (TEQ) of the above seven PAHs were lower than the standard value (1 mg·kg-1) of the World Health Organization (WHO), which they indicate a lower risk to human health. The prediction results showed that the rapid development of urbanization had a positive correlation with the increase in soil PAHs content. By 2030, the content of PAHs in Beijing gas station soil will continue to grow. The predicted concentrations of PAHs in the soil of Beijing gas stations in 2025 and 2030 were 0.085-4.077 mg·kg-1and 0.132-4.412 mg·kg-1, respectively. The contents of seven PAHs were lower than the soil pollution risk screening value of GB 36600-2018; however, the concentration of PAHs increased over time.The contents of PAHs in Chaoyang, Fengtai, and Haidian were relatively higher, which requires further attention.

Transcriptomic investigation of the effects of TDCPP on PC12 and GC2 cells with experimental validation.

TDCPP is a flame retardant which has nervous and reproductive toxicity. Although there is a close association between nervous and reproductive system, the exact toxic mechanism of TDCPP in these systems is still seldom, especially in a genome scale. In this study, we explored the transcriptomic landscape of TDCPP in PC12 and GC2 cells using RNAseq method. A total of 465 co-differential expressed genes were found. These genes were mainly enriched in extra-cellular matrix, cell adhesion, cell cycle arrest, oxidoreductase activity GO terms, and PI3K/AKT, focal adhesion, ECM-receptor interaction KEGG pathways. Hub genes (ANXA1, COL27A1, GAS6, GNB4 and THBS1) were extracted using STRING and confirmed by qPCR experiment. Vimentin, HSPA5 and Caspase3 were proved to be responsible to TDCPP in GC2 and PC12 cells. Knockdown assay in PC12 cells showed that these hub genes could also affect the protein expression of vimentin, HSPA5 and Caspase3. In summary, TDCPP might exert its toxic effect through disturbing focal adhesion, ECM-receptor interaction and PI3K/Akt pathways. One of the mechanisms could be influence on the cytoskeleton (vimentin), ER stress (HSPA5) and apoptosis (Caspase3). The sequence data in this study might be a useful resource for future TDCPP related researches.

Vitamin D3 protects against nitrogen mustard-induced apoptosis of the bronchial epithelial cells via activating the VDR/Nrf2/Sirt3 pathway.

Respiratory system injury is the main cause of mortality for nitrogen mustard (NM)-induced damage. Previous studies indicate that reactive oxygen species (ROS) participates in NM-mediated respiratory injuries, but the detailed mechanism is not quite clear. Human bronchial epithelial cell lines 16HBE and BEAS-2B were treated with HN2, a type of NM. In detail, it was shown that HN2 treatment induced impaired cell viability, excessive mitochondrial ROS production and enhanced cellular apoptosis in bronchial epithelial cells. Moreover, impaired Sirt3/SOD2 axis was observed upon HN2 treatment, with decreased Sirt3 and increased acetylated SOD2 expression levels. Sirt3 overexpression partially ameliorated HN2-induced cell injury. Meanwhile, vitamin D3 treatment partially attenuated HN2-induced apoptosis and improved the mitochondrial functions upon HN2 intervention. In addition, HN2 exposure decreased VDR expression, thus inhibiting the Nrf2 phosphorylation and Sirt3 activation. Inhibition of Nrf2 or Sirt3 could decrease the protective effects of vitamin D3 and enhance mitochondrial ROS production via modulating mitochondrial redox balance. In conclusion, impaired VDR/Nrf2/Sirt3 axis contributed to NM-induced apoptosis, while vitamin D3 supplementation provides protective effects via the activation of VDR and the improvement of mitochondrial functions. This study provides novel mechanism and strategy for NM exposure-induced pulmonary injuries.

First in-human trial and prospective case series of an articulated laparoscopic camera system in minimally invasive surgery in gynecology: an IDEAL stage 1 and 2a study.

Objectives: Precision Robotics' Sirius Robotic Flexible Endoscopic System is a new, fully integrated, compact three-dimensional laparoscopic camera system with a disposable single-use flexible tip that can change its viewing direction. This IDEAL Stage 1 and 2a study assessed its safety, reliability and potential efficacy particularly for single incision laparoscopic surgery and vaginal natural orifice transluminal endoscopic surgery. Design: Prospective single-institution, single-surgeon study. Setting: The study was conducted in a multispecialty hospital. Participants: Women aged 18-70 years scheduled for gynecological laparoscopic surgery were invited to participate. An information sheet and consent was available for the women and an informed consent was obtained. Thirteen participants completed this study. Interventions: The laparoscopic procedures were done in the usual manner. The only difference was the Sirius System was used in place of the conventional laparoscope. All other procedures and instruments remained the same. Main outcome measures: Primary outcome was the proportion of women who successfully completed the intended procedure using the Sirius System without conversion to another camera system, camera users and surgeon's view and experience, and iterations and modifications to the system. Secondary outcomes were the incidence of intraoperative and postoperative complications during the first 6 weeks following surgery, and duration of surgery. Results: 85% (11/13) of women had their procedure completed successfully using the Sirius System. Two women required immediate conversion to the conventional laparoscope due to technical issues. There were no intraoperative complications. Users agreed that the improved field of view was beneficial for laparoscopic surgery. Iterative improvements were made in the imaging quality, user interface and manufacturing quality. Conclusions: Sirius System has early indications for safety and efficacy for intermediate and major minimally invasive laparoscopic procedures in gynecology. Further studies are needed to confirm it can replace a conventional laparoscope in the surgical workflow. Trial registration number: NCT05048407.

Small-interfering RNA for c-Jun attenuates cell death by preventing JNK-dependent PARP1 cleavage and DNA fragmentation in nitrogen mustard-injured immortalized human bronchial epithelial cells.

Sulfur mustard (a type of vesicant) can directly damage lung bronchial epithelium via aerosol inhalation, and prevalent cell death is an early event that obstructs the respiratory tract. JNK/c-Jun is a stress response pathway, but its role in cell death of the injured cells is not clear. Here, we report that JNK/c-Jun was activated in immortalized human bronchial epithelial (HBE) cells exposed to a lethal dose (20 µM) of nitrogen mustard (NM, a sulfur mustard analog). c-Jun silencing using small-interfering RNA (siRNA) rendered the cells resistant to NM-mediated cell death by blocking poly(ADP-ribose) polymerase 1 (PARP1) cleavage and DNA fragmentation. In addition, the transduction of upstream extrinsic (Fasl-Fas-caspase-8) and intrinsic (loss of Bcl-2 and mitochondrial membrane potential, ΔΨm) apoptosis pathways, as well as phosphorylated (p)-H2AX (Ser139), an epigenetic marker contributing to DNA fragmentation and PARP1 activity, was partially suppressed. To mimic the detachment of cells by NM, HBE cells were trypsinized and seeded on culture plates that were pre-coated with poly-HEMA to prevent cell adhesion. The JNK/c-Jun pathway was found to be activated in the detached cells. In conclusion, our results indicate that JNK/c-Jun pathway activation is necessary for NM-caused HBE cell death and further suggest that c-Jun silencing may be a potential approach to protect HBE cells from vesicant damage.

PGC-1ɑ Mediated-EXOG, a Specific Repair Enzyme for Mitochondrial DNA, Plays an Essential Role in the Rotenone-Induced Neurotoxicity of PC12 Cells.

Mitochondria harbor small circular genomes (mtDNA) that encode 13 oxidative phosphorylation (OXPHOS) proteins, and types of damage to mtDNA may contribute to neuronal damage. Recent studies suggested that regulation of mtDNA repair proteins may be a potential strategy for treating neuronal damage. The mtDNA repair system contains its own repair enzymes and is independent from the nuclear DNA repair system. Endo/exonuclease G-like(EXOG) is a mitochondria-specific 5-exo/endonuclease required for repairing endogenous single-strand breaks (SSBs) in mtDNA. However, whether EXOG plays a key role in neuronal damage induced by rotenone remains unknown. Thus, in this study, we aimed to investigate the effect of EXOG on mtDNA repair and mitochondrial functional maintenance in rotenone-induced neurotoxicity. Our results indicated that rotenone influenced the expression and location of EXOG in PC12 cells. Meanwhile, after rotenone exposure, the expression was reduced for proteins responsible for mtDNA repair, including DNA polymerase γ (POLG), high-temperature requirement protease A2 (HtrA2), and the heat-shock factor 1-single-stranded DNA-binding protein 1 (HSF1-SSBP1) complex. Further analysis demonstrated that EXOG knockdown led to reduced mtDNA copy number and mtDNA transcript level and increased mtDNA deletion, which further aggravated the mtDNA damage and mitochondrial dysfunction under rotenone stress. In turn, EXOG overexpression protected PC12 cells from mtDNA damage and mitochondrial dysfunction induced by rotenone. As a result, EXOG knockdown reduced cell viability and tyrosine hydroxylase expression, while EXOG overexpression alleviated rotenone's effect on cell viability and tyrosine hydroxylase expression in PC12 cells. Further, we observed that EXOG influenced mtDNA repair by regulating protein expression of the HSF1-SSBP1 complex and POLG. Furthermore, our study showed that PGC-1α upregulation with ZLN005 led to increased protein levels of EXOG, POLG, HSF1, and SSBP1, all of which contribute to mtDNA homeostasis. Therefore, PGC-1α may be involved in mtDNA repair through interacting with multiple mtDNA repair proteins, especially with the help of EXOG. In summary, EXOG regulation by PGC-1α plays an essential role in rotenone-induced neurotoxicity in PC12 cells. EXOG represents a protective effect strategy in PC12 cells exposed to rotenone.

Targeting TRPV1-mediated autophagy attenuates nitrogen mustard-induced dermal toxicity.

Nitrogen mustard (NM) causes severe vesicating skin injury, which lacks effective targeted therapies. The major limitation is that the specific mechanism of NM-induced skin injury is not well understood. Recently, autophagy has been found to play important roles in physical and chemical exposure-caused cutaneous injuries. However, whether autophagy contributes to NM-induced dermal toxicity is unclear. Herein, we initially confirmed that NM dose-dependently caused cell death and induced autophagy in keratinocytes. Suppression of autophagy by 3-methyladenine, chloroquine, and bafilomycin A1 or ATG5 siRNA attenuated NM-induced keratinocyte cell death. Furthermore, NM increased transient receptor potential vanilloid 1 (TRPV1) expression, intracellular Ca2+ content, and the activities of Ca2+/calmodulin-dependent kinase kinase ß (CaMKKß), AMP-activated protein kinase (AMPK), unc-51-like kinase 1 (ULK1), and mammalian target of rapamycin (mTOR). NM-induced autophagy in keratinocytes was abolished by treatment with inhibitors of TRPV1 (capsazepine), CaMKKß (STO-609), AMPK (compound C), and ULK1 (SBI-0206965) as well as TRPV1, CaMKKß, and AMPK siRNA transfection. In addition, an mTOR inhibitor (rapamycin) had no significant effect on NM-stimulated autophagy or cell death of keratinocytes. Finally, the results of the in vivo experiment in NM-treated skin tissues were consistent with the findings of the in vitro experiment. In conclusion, NM-caused dermal toxicity by overactivating autophagy partially through the activation of TRPV1-Ca2+-CaMKKß-AMPK-ULK1 signaling pathway. These results suggest that blocking TRPV1-dependent autophagy could be a potential treatment strategy for NM-caused cutaneous injury.

Integrating real-time data analysis into automatic tracking of social insects.

Automatic video tracking has become a standard tool for investigating the social behaviour of insects. The recent integration of computer vision in tracking technologies will probably lead to fully automated behavioural pattern classification within the next few years. However, many current systems rely on offline data analysis and use computationally expensive techniques to track pre-recorded videos. To address this gap, we developed BACH (Behaviour Analysis maCHine), a software that performs video tracking of insect groups in real time. BACH uses object recognition via convolutional neural networks and identifies individually tagged insects via an existing matrix code recognition algorithm. We compared the tracking performances of BACH and a human observer (HO) across a series of short videos of ants moving in a two-dimensional arena. We found that BACH detected ant shapes only slightly worse than the HO. However, its matrix code-mediated identification of individual ants only attained human-comparable levels when ants moved relatively slowly, and fell when ants walked relatively fast. This happened because BACH had a relatively low efficiency in detecting matrix codes in blurry images of ants walking at high speeds. BACH needs to undergo hardware and software adjustments to overcome its present limits. Nevertheless, our study emphasizes the possibility of, and the need for, further integrating real-time data analysis into the study of animal behaviour. This will accelerate data generation, visualization and sharing, opening possibilities for conducting fully remote collaborative experiments.