Characterization of tryptanthrin as an antibacterial reagent inhibiting Vibrio splendidus.

Isolates of Vibrio splendidus are ubiquitously presented in various marine environments, and they can infect diverse marine culture animals, leading to high mortality and economic loss. Therefore, a control strategy of the infection caused by V. splendidus is urgently recommended. Tryptanthrin is a naturally extracted bioactive chemical with antimicrobial activity to other bacteria. In this study, the effects of tryptanthrin on the bacterial growth and virulence-related factors of one pathogenic strain V. splendidus AJ01 were determined. Tryptanthrin (10 µg/mL) could completely inhibit the growth of V. splendidus AJ01. The virulence-related factors of V. splendidus AJ01 were affected in the presence of tryptanthrin. Tryptanthrin resulted an increase in biofilm formation, but lead to reduction in the motility and hemolytic activity of V. splendidus cells. In the cells treated with tryptanthrin, two distinctly differentially expressed extracellular proteins, proteases and flagellum, were identified using SDS-PAGE combined with LC-MS. Real-time reverse transcriptase PCR confirmed that the genes involved in the flagellar formation and hemolysin decreased, whereas specific extracellular proteases and the genes involved in the biofilm formation were upregulated. Two previously annotated luxOVs genes were cloned, and their expression levels were analyzed at different cell densities. Molecular docking was performed to predict the interaction between LuxOVs and ATP/tryptanthrin. The two sigma-54-dependent transcriptional regulators showed similar ATP or tryptanthrin binding capacity but with different sites, and the direct competitive binding between ATP and tryptanthrin was present only in their binding to LuxO1. These results indicated that tryptanthrin can be used as a bactericide of V. splendidus by inhibiting the growth, bacterial flagella, and extracellular proteases, but increasing the biofilm. Sigma-54-dependent transcriptional regulator, especially the quorum sensing regulatory protein LuxO1, was determined to be the potential target of tryptanthrin. KEY POINTS: • Tryptanthrin inhibited the growth of V. splendidus in a dose-dependent manner. • The effect of tryptanthrin on the virulence factors of V. splendidus was characterized. • LuxO was the potential target for tryptanthrin based on molecular docking.

Phosphorylated CPI-17 and MLC2 as Biomarkers of Coronary Artery Spasm-Induced Sudden Cardiac Death.

Coronary artery spasm (CAS) plays an important role in the pathogeneses of various ischemic heart diseases and has gradually become a common cause of life-threatening arrhythmia. The specific molecular mechanism of CAS has not been fully elucidated, nor are there any specific diagnostic markers for the condition. Therefore, this study aimed to examine the specific molecular mechanism underlying CAS, and screen for potential diagnostic markers. To this end, we successfully constructed a rat CAS model and achieved in vitro culture of a human coronary-artery smooth-muscle cell (hCASMC) contraction model. Possible molecular mechanisms by which protein kinase C (PKC) regulated CAS through the C kinase-potentiated protein phosphatase 1 inhibitor of 17 kDa (CPI-17)/myosin II regulatory light chain (MLC2) pathway were studied in vivo and in vitro to screen for potential molecular markers of CAS. We performed hematoxylin and eosin staining, myocardial zymogram, and transmission electron microscopy to determine myocardial and coronary artery injury in CAS rats. Then, using immunohistochemical staining, immunofluorescence staining, and Western blotting, we further demonstrated a potential molecular mechanism by which PKC regulated CAS via the CPI-17/MLC2 pathway. The results showed that membrane translocation of PKCα occurred in the coronary arteries of CAS rats. CPI-17/MLC2 signaling was observably activated in coronary arteries undergoing CAS. In addition, in vitro treatment of hCASMCs with angiotensin II (Ang II) increased PKCα membrane translocation while consistently activating CPI-17/MLC2 signaling. Conversely, GF-109203X and calphostin C, specific inhibitors of PKC, inactivated CPI-17/MLC2 signaling. We also collected the coronary artery tissues from deceased subjects suspected to have died of CAS and measured their levels of phosphorylated CPI-17 (p-CPI-17) and MLC2 (p-MLC2). Immunohistochemical staining was positive for p-CPI-17 and p-MLC2 in the tissues of these subjects. These findings suggest that PKCα induced CAS through the CPI-17/MLC2 pathway; therefore, p-CPI-17 and p-MLC2 could be used as potential markers for CAS. Our data provide novel evidence that therapeutic strategies against PKC or CPI-17/MLC2 signaling might be promising in the treatment of CAS.

The effects of restraint stress on ceramide metabolism disorders in the rat liver: the role of CerS6 in hepatocyte injury.

BACKGROUND: Stress is implicated in various pathological conditions leading to liver injury. Existing evidence suggests that excessive stress can induce mitochondrial damage in hepatocytes, yet the underlying mechanism remains unclear. Ceramide synthase 6 (CerS6)-derived C16:0 ceramide is recognised as a lipotoxic substance capable of causing mitochondrial damage. However, the role of CerS6 in stress has received insufficient attention. This study aimed to explore the involvement of CerS6 in stress-induced hepatic damage and its associated mechanisms. METHODS: The rat restraint stress model and a corticosterone (CORT)-induced hepatocyte stress model were employed for in vivo and in vitro experimental analyses, respectively. Changes in mitochondrial damage and ceramide metabolism in hepatocytes induced by stress were evaluated. The impact of CORT on mitochondrial damage and ceramide metabolism in hepatocytes was assessed following CerS6 knockdown. Mitochondria were isolated using a commercial kit, and ceramides in liver tissue and hepatocytes were detected by LC-MS/MS. RESULTS: In comparison to the control group, rats subjected to one week of restraint exhibited elevated serum CORT levels. The liver displayed significant signs of mitochondrial damage, accompanied by increased CerS6 and mitochondrial C16:0 ceramide, along with activation of the AMPK/p38 MAPK pathway. In vitro studies demonstrated that CORT treatment of hepatocytes resulted in mitochondrial damage, concomitant with elevated CerS6 and mitochondrial C16:0 ceramide. Furthermore, CORT induced sequential phosphorylation of AMPK and p38 MAPK proteins, and inhibition of the p38 MAPK pathway using SB203580 mitigated the CORT-induced elevation in CerS6 protein. Knocking down CerS6 in hepatocytes inhibited both the increase in C16:0 ceramide and the release of mitochondrial cytochrome c induced by CORT. CONCLUSIONS: CerS6-associated C16:0 ceramide plays a mediating role in stress-induced mitochondrial damage in hepatocytes. The molecular mechanism is linked to CORT-induced activation of the AMPK/p38 MAPK pathway, leading to upregulated CerS6.

Vibrio splendidus AJ01 Promotes Pathogenicity via L-Glutamic Acid.

Vibrio splendidus is a pathogen that infects a wide range of hosts, especially the sea cucumber species Apostichopus japonicus. Previous studies showed that the level of L-glutamic acid (L-Glu) significantly increased under heat stress, and it was found to be one of the best carbon sources used by V. splendidus AJ01. In this study, the effects of exogenous L-Glu on the coelomocyte viability, tissue status, and individual mortality of sea cucumbers were analyzed. The results showed that 10 mM of L-Glu decreased coelomocyte viability and increased individual mortality, with tissue rupture and pyknosis, while 0.1 mM of L-Glu slightly affected the survival of sea cucumbers without obvious damage at the cellular and tissue levels. Transcriptomic analysis showed that exogenous L-Glu upregulated 343 and downregulated 206 genes. Gene Ontology (GO) analysis showed that differentially expressed genes (DEGs) were mainly enriched in signaling and membrane formation, while a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that DEGs were significantly enriched in the upregulated endocytosis and downregulated lysosomal pathways. The coelomocyte viability further decreased by 20% in the simultaneous presence of exogenous L-Glu and V. splendidus AJ01 compared with that in the presence of V. splendidus AJ01 infection alone. Consequently, a higher sea cucumber mortality was also observed in the presence of exogenous L-Glu challenged by V. splendidus AJ01. Real-time reverse transcriptase PCR showed that L-Glu specifically upregulated the expression of the fliC gene coding the subunit protein of the flagellar filament, promoting the swimming motility activity of V. splendidus. Our results indicate that L-Glu should be kept in a state of equilibrium, and excess L-Glu at the host-pathogen interface prompts the virulence of V. splendidus via the increase of bacterial motility.

Interleukin 6 (IL-6) Regulates GABAA Receptors in the Dorsomedial Hypothalamus Nucleus (DMH) through Activation of the JAK/STAT Pathway to Affect Heart Rate Variability in Stressed Rats.

The dorsomedial hypothalamus nucleus (DMH) is an important component of the autonomic nervous system and plays a critical role in regulating the sympathetic outputs of the heart. Stress alters the neuronal activity of the DMH, affecting sympathetic outputs and triggering heart rate variability. However, the specific molecular mechanisms behind stress leading to abnormal DMH neuronal activity have still not been fully elucidated. Therefore, in the present study, we successfully constructed a stressed rat model and used it to investigate the potential molecular mechanisms by which IL-6 regulates GABAA receptors in the DMH through activation of the JAK/STAT pathway and thus affects heart rate variability in rats. By detecting the c-Fos expression of neurons in the DMH and electrocardiogram (ECG) changes in rats, we clarified the relationship between abnormal DMH neuronal activity and heart rate variability in stressed rats. Then, using ELISA, immunohistochemical staining, Western blotting, RT-qPCR, and RNAscope, we further explored the correlation between the IL-6/JAK/STAT signaling pathway and GABAA receptors. The data showed that an increase in IL-6 induced by stress inhibited GABAA receptors in DMH neurons by activating the JAK/STAT signaling pathway, while specific inhibition of the JAK/STAT signaling pathway using AG490 obviously reduced DMH neuronal activity and improved heart rate variability in rats. These findings suggest that IL-6 regulates the expression of GABAA receptors via the activation of the JAK/STAT pathway in the DMH, which may be an important cause of heart rate variability in stressed rats.

Endoplasmic reticulum stress induced by turbulence of mitochondrial fusion and fission was involved in stressed cardiomyocyte injury.

Mitochondria are sensitive organelles that sense intrinsic and extrinsic stressors and maintain cellular physiological functions through the dynamic homeostasis of mitochondrial fusion and fission. Numerous pathological processes are associated with mitochondrial fusion and fission disorders. However, the molecular mechanism by which stress induces cardiac pathophysiological changes through destabilising mitochondrial fusion and fission is unclear. Therefore, this study aimed to investigate whether the endoplasmic reticulum stress signalling pathway initiated by the turbulence of mitochondrial fusion and fission under stressful circumstances is involved in cardiomyocyte damage. Based on the successful establishment of the classical stress rat model of restraint plus ice water swimming, we measured the content of serum lactate dehydrogenase. We used haematoxylin-eosin staining, special histochemical staining, RT-qPCR and western blotting to clarify the cardiac pathology, ultrastructural changes and expression patterns of mitochondrial fusion and fission marker proteins and endoplasmic reticulum stress signalling pathway proteins. The results indicated that mitochondrial fusion and fission markers and proteins of the endoplasmic reticulum stress JNK signalling pathway showed significant abnormal dynamic changes with the prolongation of stress, and stabilisation of mitochondrial fusion and fission using Mdivi-1 could effectively improve these abnormal expressions and ameliorate cardiomyocyte injury. These findings suggest that stress could contribute to pathological cardiac injury, closely linked to the endoplasmic reticulum stress JNK signalling pathway induced by mitochondrial fusion and fission turbulence.

Ajpacifastin-like is involved in the immune response of Apostichopus japonicus challenged by Vibrio splendidus.

Pacifastin proteins are previously found to regulate the phenoloxidase system in invertebrates and arthropods. In this study, the immune response that was regulated by Ajpacifastin-like in the sea cucumber Apostichopus japonicus was determined. RNA interference was used to knock down the expression of the Ajpacifastin-like gene in A. japonicus, followed by challenge with Vibrio splendidus, and the colony count showed that the survival of V. splendidus in the si-Ajpacifastin group increased 4.64-fold compared to that of the control group. The purified recombinant Ajpacifastin-like showed an inhibitory effect on the extracellular protease activity of the supernatant collected from the V. splendidus culture. Consequently, a comparative transcriptome analysis of the coelomocytes from the control group and the si-Ajpacifastin group was performed to explore the global regulatory effect of the Ajpacifastin-like. A total of 1486 differentially expressed genes (DEGs) were identified, including 745 upregulated genes and 741 downregulated genes. GO enrichment showed that the DEGs were mainly enriched in translation, cytosolic ribosomal subunit and structural constituent of ribosome. KEGG analysis showed that the DEGs were significantly enriched in the retinoic acid-inducible gene I (RIG-I)-like receptor signaling pathway, antigen processing and presentation, toll-like receptor signaling pathway, mitogen-activated protein kinase signaling pathway, nuclear factor-kappa B signaling pathway and other immune-related pathways. Furthermore, real-time reverse transcriptase PCR was used to determine the RNA levels of six DEGs, i.e., cathepsinB, CYLD, caspase8, TRAF6, hsp90 and FADD, to verify the RNA-seq results. Overall, our results specified the immune response and pathways of A. japonicus in which Ajpacifastin-like was involved in.

Bioinformatics Analysis of Molecular Interactions between Endoplasmic Reticulum Stress and Ferroptosis under Stress Exposure.

Stress has become a universal biological phenomenon in the body, which leads to pathophysiological changes. However, the molecular network interactions between endoplasmic reticulum (ER) stress and ferroptosis under stressful conditions are not clear. For this purpose, we screened the gene expression profile of GSE173795 for intersection with ferroptosis genes and screened 68 differentially expressed genes (DEGs) (63 up-regulated, 5 down-regulated), mainly related to lipid and atherosclerosis, autophagy-animal, mitophagy-animal, focal adhesion, DNA replication, proteasome, oocyte meiosis, toll-like receptor signaling pathway, cell cycle, etc. Immune infiltration analysis revealed that stress resulted in decreased B cells memory, T cells CD8 and T cells CD4 memory resting, monocytes, macrophages M2, and increased B cells naive, T cells follicular helper, and macrophages M1. 19 core-DEGs (ASNS, TRIB3, ATF4, EIF2S1, CEBPG, RELA, HSPA5, DDIT3, STAT3, MAP3K5, HIF1A, HNF4A, MAPK14, HMOX1, CDKN1A, KRAS, SP1, SIRT1, EGFR) were screened, all of which were up-regulated DEGs. These biological processes and pathways were mainly involved in responding to ER stress, lipid and atherosclerosis, cellular response to stress, cellular response to chemical stress, and regulation of DNA-templated transcription in response to stress, etc. Spearman analysis did not find MAPK14 to be significantly associated with immune cells. Other core-DEGs were associated with immune cells, including B cells naive, T cells follicular helper, and monocytes. Based on core-DEGs, 283 miRNAs were predicted. Among the 22 miRNAs with highly cross-linked DEGs, 11 had upstream lncRNA, mainly targeting STAT3, SP1, CDKN1A, and SIRT1, and a total of 39 lncRNA were obtained. 85 potential drugs targeting 11 core-DEGs were identified and were expected to be potential immunotherapeutic agents for stress injury. Our experiments also confirmed that Liproxstatin-1 alleviates common cross-linked proteins between ER stress and ferroptosis. In conclusion, our study explored the molecular mechanisms and network interactions among stress-ER stress-ferroptosis from a novel perspective, which provides new research ideas for studying stressful injury.

Exogenous c-di-GMP inhibited the biofilm formation of Vibrio splendidus.

Vibrio splendidus, a gram-negative bacterium that is ubiquitously present in marine environments, has been increasingly deemed an important opportunistic pathogen of marine animals. In this study, the biofilm formation of V. splendidus was quantitatively determined and morphologically characterized. Three stages of biofilm formation, including adhesion, aggregation and maturation were observed in the biofilm formed by V. splendidus. The inhibitory effect of exogenous bis (3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) on the biofilm formation from the scratch and preformed established biofilms of V. splendidus was determined. When 200 µmol/L c-di-GMP was added, the quantity of biofilm decreased by 88.1% or 66.7% under the two conditions. To explore the preliminary mechanism of exogenous c-di-GMP on the biofilm formed by V. splendidus, proteomic analysis was performed. GO enrichment analysis showed that exogenous c-di-GMP upregulated biological processes, including the tricarboxylic acid cycle, oxidation‒reduction reactions and organonitrogen compound catabolism and significantly downregulated tRNA threonylcarbamoyladenosine modification, protein dephosphorylation, and lactate transmembrane transporter activity. Sequence-specific DNA binding activity was the most markedly downregulated molecular function. KEGG analysis showed that the valine, leucine and isoleucine degradation pathway was the most enriched pathway, followed by nitrogen metabolism, among the 20 upregulated pathways. Among the downregulated pathways, a nonribosomal peptide structure pathway and the streptomycine, polyketide sugar unit, acarbose and validamycin biosynthesis pathways were significantly enriched. Our present study provides basic data for the biofilm formation of V. splendidus and the preliminary inhibitory mechanism of exogenous c-di-GMP on the biofilm formation of V. splendidus.

Case report: Myocarditis following COVID-19 protein subunit vaccination.

We report findings in a 34-year-old female patient who presented with fulminant myocarditis 8 days after receiving the first dose of the ZF2001 RBD-subunit vaccine against coronavirus disease 2019 (COVID-19). Autopsy showed severe interstitial myocarditis, including multiple patchy infiltrations of lymphocytes and monocytes in the myocardium of the left and right ventricular walls associated with myocyte degeneration and necrosis. This report highlights the details of clinical presentations and autopsy findings of myocarditis after ZF2001 (RBD-subunit vaccine) vaccination. The correlation between vaccination and death due to myocarditis is discussed.

A polypropylene (PP) supported solid polymer electrolyte enables high-stability organic lithium batteries at low temperature.

We innovatively used a polypropylene (PP) separator as a substrate and PEO-LiTFSI-SN as a paste to coat on both of the PP surfaces, and formed a sandwich-like solid polymer electrolyte (SPE). The SPE shows a conductivity of 4.22 × 10-3 S cm-1 at room temperature and 7.75 × 10-5 S cm-1 at 0 °C. The pyrene-4,5,9,10-tetraone (PTO)||SPE||Li battery shows a maximum discharge specific capacity of 187.8 mA h g-1 at a current density of 20 mA g-1 under 0 °C. After 100 cycles, the capacity could still be obtained at 88.4 mA h g-1, and the coulombic efficiency stayed stable at 98%. This work paved a new way for the development of solid-state organic batteries (SSOBs) at low temperatures.

A fatal case of accidental oral formaldehyde poisoning and its pathomorphological characteristics.

Formaldehyde is a colourless irritating gas at room temperature, which, therefore, is usually stored in liquid form. This compound is often used as an antiseptic, disinfectant and fumigant in biology and medicine. Formaldehyde, as a potential carcinogen confirmed by the World Health Organization (WHO), is seriously harmful to human systems, such as the respiratory system, immune system and reproductive system. This article reports a case of a 50-year-old woman who died after accidentally drinking 25% formaldehyde solution in a transparent plastic bottle. Anatomical examination revealed fixed tissue morphology of the stomach and adjacent organs. The toxicity test results showed that the concentrations of formaldehyde in the blood and gastric tissue were 36.56 mg/kg and 274.48 mg/kg, respectively, which was consistent with death from formaldehyde poisoning. Due to the particular smell of formaldehyde, poisoning by accidentally drinking formaldehyde solution is rare. Of late, the mechanism of death from formaldehyde poisoning is that it rapidly causes coagulation of tissue cell protein, which may lose its normal function. Based on the pathological characteristics of the case, we put forward a new viewpoint on the mechanism of death from formaldehyde poisoning in which formaldehyde causes rapid fixation of blood in the tissue, thus leading to acute circulatory disturbance.

An Experimental Study on Combustion and Cycle-by-Cycle Variations of an N-Butanol Engine with Hydrogen Direct Injection under Lean Burn Conditions.

This study experimentally investigated the effects of hydrogen direct injection on combustion and the cycle-by-cycle variations in a spark ignition n-butanol engine under lean burn conditions. For this purpose, a spark ignition engine installed with a hydrogen and n-butanol dual fuel injection system was specially developed. Experiments were conducted at four excess air ratios, four hydrogen fractions(φ(𝐻2)) and pure n-butanol. Engine speed and intake manifold absolute pressure (MAP) were kept at 1500 r/min and 43 kPa, respectively. The results indicate that the θ0-10 and θ10-90 decreased gradually with the increase in hydrogen fraction. Additionally, the indicated mean effective pressure (IMEP), the peak cylinder pressure (Pmax) and the maximum rate of pressure rise ((dP/dφ)max) increased gradually, while their cycle-by-cycle variations decreased with the increase in hydrogen fraction. In addition, the correlation between the (dP/dφ)max and its corresponding crank angle became weak with the increase in the excess air coefficient (λ), which tends to be strongly correlated with the increase in hydrogen fraction. The coefficient of variation of the Pmax and the IMEP increased with the increase in λ, while they decreased obviously after blending in the hydrogen under lean burn conditions. Furthermore, when λ was 1.0, a 5% hydrogen fraction improved the cycle-by-cycle variations most significantly. While a larger hydrogen fraction is needed to achieve the excellent combustion characteristics under lean burn conditions, hydrogen direct injection can promote combustion process and is beneficial for enhancing stable combustion and reducing the cycle-by-cycle variations.

11,11,12,12-tetracyano-9,10-anthraquinonedimethane as a high potential and sustainable cathode for organic potassium-ion batteries.

We fabricated a potassium-ion battery by using 11,11,12,12-tetracyano-9,10-anthraquinonedimethane (TCAQ) as the cathode for the first time. Owing to the unique molecular structure and configuration of ionic liquid electrolytes, TCAQ shows a high redox potential of 2.6 V vs. K+/K while delivering a capacity of 88 mAh g-1 at a current density of 17 mA g-1 and a capacity retention of 61% after 50 cycles. The mechanism of the reaction of TCAQ with K was investigated. The results prove that TCAQ holds great promise for broad applications in potassium-ion batteries while revealing new scientific insights into K+-organic cathode batteries.

Bibliometric analysis of post-traumatic stress disorder in forensic medicine: Research trends, hot spots, and prospects.

Background: Post-traumatic stress disorder (PTSD) has various risk factors, complex pathogenesis, and diverse symptoms, and is often comorbid with other injuries and diseases, making forensic diagnosis difficult. Methods: To explore the current research status and trends of PTSD, we used the Web of Science Core Collection databases to screen PTSD-related literature published between 2010 and 2021 and CiteSpace to perform bibliometric analysis. Results: In recent years, PTSD-related research has grown steadily. The countries and institutions with the most research results were the United States and England, and King's College London and Boston University, respectively. Publications were identified from 2,821 different journals, including 13 forensic-related journals, but the journal distribution was relatively scattered and there was a lack of professional core journals. Keyword co-occurrence and clustering identified many hot topics; "rat model," "mental health," and "satisfaction" were the topics most likely to have a clear effect on future research. Analysis extracted nine turning points from the literature that suggested that neural network centers, the hypothalamic-pituitary-adrenal axis, and biomarkers were new research directions. It was found that COVID-19 can cause severe psychological stress and induce PTSD, but the relationship needs further study. The literature on stress response areas and biomarkers has gradually increased over time, but specific systemic neural brain circuits and biomarkers remain to be determined. Conclusion: There is a need to expand the collection of different types of biological tissue samples from patients with different backgrounds, screen PTSD biomarkers and molecular targets using multi-omics and molecular biology techniques, and establish PTSD-related molecular networks. This may promote a systematic understanding of the abnormal activation of neural circuits in patients with PTSD and help to establish a personalized, accurate, and objective forensic diagnostic standard.

Mechanism of Chronic Stress-Induced Glutamatergic Neuronal Damage in the Basolateral Amygdaloid Nucleus.

Stress is a ubiquitous part of our life, while appropriate stress levels can help improve the body's adaptability to the environment. However, sustained and excessive levels of stress can lead to the occurrence of multiple devastating diseases. As an emotional center, the amygdala plays a key role in the regulation of stress-induced psycho-behavioral disorders. The structural changes in the amygdala have been shown to affect its functional characteristics. The amygdala-related neurotransmitter imbalance is closely related to psychobehavioral abnormalities. However, the mechanism of structural and functional changes of glutamatergic neurons in the amygdala induced by stress has not been fully elucidated. Here, we identified that chronic stress could lead to the degeneration and death of glutamatergic neurons in the lateral amygdaloid nucleus, resulting in neuroendocrine and psychobehavioral disorders. Therefore, our studies further suggest that the Protein Kinase R-like ER Kinase (PERK) pathway may be therapeutically targeted as one of the key mechanisms of stress-induced glutamatergic neuronal degeneration and death in the amygdala.

Endoplasmic Reticulum Stress Is Associated with the Mesencephalic Dopaminergic Neuron Injury in Stressed Rats.

An increasing number of people are in a state of stress due to social and psychological pressures, which may result in mental disorders. Previous studies indicated that mesencephalic dopaminergic neurons are associated with not only reward-related behaviors but also with stress-induced mental disorders. To explore the effect of stress on dopaminergic neuron and potential mechanism, we established stressed rat models of different time durations and observed pathological changes in dopaminergic neurons of the ventral tegmental area (VTA) through HE and thionine staining. Immunohistochemistry coupled with microscopy-based multicolor tissue cytometry (MMTC) was employed to investigate the number changes of dopaminergic neurons. Double immunofluorescence labelling was used to investigate expression changes of endoplasmic reticulum stress (ERS) protein GRP78 and CHOP in dopaminergic neurons. Our results showed that prolonged stress led to pathological alteration in dopaminergic neurons of VTA, such as missing of Nissl bodies and pyknosis in dopaminergic neurons. Immunohistochemistry with MMTC indicated that chronic stress exposure resulted in a significant decrease in dopaminergic neurons. Double immunofluorescence labelling showed that the endoplasmic reticulum stress protein took part in the injury of dopaminergic neurons. Taken together, these results indicated the involvement of ERS in mesencephalic dopaminergic neuron injury induced by stress exposure.

Transcriptome analysis reveals roles of polian vesicle in sea cucumber Apostichopus japonicus response to Vibrio splendidus infection.

Polian vesicle is originally regarded as a hematopoietic and inflammatory response organ in sea cucumber by the operations of cell depletion and heterogeneous cells injection, respectively. In the present study, to reveal the role and immune mechanisms of polian vesicle in response to pathogen, Vibrio splendidus, we first performed a comparative transcriptome analysis for the cells from polian vesicle wall in V. splendidus-challenged Apostichopus japonicus through RNA high-throughput sequencing technology. Briefly, 465,356,848 clean reads were obtained after cleaning up low-quality reads in total. Approximately 73% of the sequenced reads could be aligned to the reference genome of A. japonicus. The DEGs of CG (control group) vs TG 24 h (24 h post-infection group), CG vs TG 72 h (72 h post-infection group) and TG 24 h vs TG 72 h were 3762, 1391 and 3258, respectively. Gene Ontology (GO) annotation assay revealed that those genes associated with the processes such as cell process, cell, binding and catalytic activity were significantly induced in all three groups post V. splendidus infection. KEGG enrichment analysis suggested the DEGs in TG 24 h were enriched in Toll-like receptor (TLR) signaling pathway, complement and coagulation cascades, antigen processing and presentation and IL-17 signaling pathway compared with that in CG, while the pathways including ribosome biogenesis in eukaryotes, DNA replication, and cell cycle related with cell proliferation were mainly enriched in TG 72 h than that of CG. Furthermore, six important DEGs were chosen and showed the consistent expression patterns with the results of RNA-seq by qPCR. Overall, our analysis towards the current data demonstrates that polian vesicle may play an essential role in the regulation of immune response in A. japonicus and provide new insights into hematopoietic function of polian vesicle in response to pathogen infection.

The Pathology of Morphine-Inhibited Nerve Repair and Morphine-Induced Nerve Damage Is Mediated via Endoplasmic Reticulum Stress.

OBJECTIVE: The aim of the present study was to observe the pathological damage in the cerebral cortex of rats under acute morphine exposure (AME) and different durations of morphine dependence (MD), explore whether endoplasmic reticulum stress (ERS) is involved in the damage process, and assess the effect of morphine exposure on the proliferation and differentiation of newborn neurons. METHODS: Rat models of AME and different durations of MD were established. Pathological changes in cortical neurons were assessed by hematoxylin and eosin (H&E) and thionine staining. The expression of nuclear receptor-related factor 1 (NURR1) and that of the ERS-related proteins glucose-regulated protein 78 (GRP78), p-eIF2α, activating transcription factor 6 (ATF6), and CHOP in cortical neurons was assessed by immunohistochemistry. Double immunofluorescence labeling was used to observe the expression of Ki-67. RESULTS: H&E and thionine staining revealed that AME resulted in pyknotic changes in cortical neurons. With prolonged morphine exposure, the number of pyknotic neurons was significantly increased, the protein expression of Ki-67 and NURR1 was significantly decreased, and the protein levels of GRP78, p-eIF2α, ATF6, and CHOP showed marked dynamic changes. CONCLUSION: AME and different durations of MD caused varying degrees of pathological changes in the cortex. Furthermore, the dynamic changes observed in ERS-related protein expression suggested that ERS may be associated with cortical injury. Different durations of MD inhibited the proliferation, differentiation, and migration of newborn neurons, which may affect the nerve repair process after injury.

Endoplasmic Reticulum Stress Is Involved in Glucocorticoid-Induced Apoptosis in PC12 Cells.

OBJECTIVE: The present study selected PC12 cells to construct a neuronal injury model induced by glucocorticoids (GC) in vitro, aiming to explore whether the endoplasmic reticulum stress (ERS) PKR-like endoplasmic reticulum kinase (PERK)-activating transcription factor 4 (ATF4)-C/EBP-homologous protein (CHOP) and inositol requirement 1 (IRE1)-apoptosis signal regulating kinase 1 (ASK1)-C-Jun amino-terminal kinase (JNK) signaling pathways are associated with the neuronal injury process induced by GC and provide morphological evidence. METHODS: Cell models with different doses and different durations of GC exposure were established. The viability of PC12 cells was detected by the CCK-8 assay, and the apoptosis rate of PC12 cells was detected by the flow cytometry assay. The expression of microtubule-associated protein 2 (Map2); glucocorticoids receptor (GR); cellular oncogene fos (C-fos); and ERS-related proteins, glucose-regulated protein 78 (GRP78), p-PERK, p-IRE1, ATF4, ASK1, JNK, and CHOP, was observed by immunofluorescence staining. RESULTS: The results of immunofluorescence staining showed that PC12 cells abundantly expressed Map2 and GR. The CCK-8 assay revealed that high-concentration GC exposure significantly inhibited the cell viability of PC12 cells. The flow cytometry assay indicated that high-concentration GC exposure significantly increased the apoptosis rate of PC12 cells. Immunofluorescence staining showed that GC exposure significantly increased the expression of C-fos, GRP78, p-PERK, p-IRE1, ATF4, ASK1, JNK, and CHOP. Treatment with ERS inhibitor 4-phenylbutyric acid (4-PBA) and GR inhibitor RU38486 attenuated related damage and downregulated the expression of the abovementioned proteins. CONCLUSION: High-concentration GC exposure can significantly inhibit the viability of PC12 cells and induce apoptosis. PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways are involved in the above damage process.