Search

Andrias davidianus (Chinese giant salamander, CGS) is the largest and oldest extant amphibian species in the world and is a source of prospective functional food in China. However, the progress of functional peptides mining was slow due to lack of reference genome and protein sequence data. In this study, we illustrated full-length transcriptome sequencing to interpret the proteome of CGS meat and obtain 10703 coding DNA sequences. By functional annotation and amino acid composition analysis, we have discovered various genes related to signal transduction, and 16 genes related to longevity. We have also found vast variety of functional peptides through protein coding sequence (CDS) analysis by comparing the data obtained with the functional peptide database. Val-Pro-Ile predicted by the CDS analysis was released from the CGS meat through enzymatic hydrolysis, suggesting that our approach is reliable. This study suggested that transcriptomic analysis can be used as a reference to guide polypeptide mining in CGS meat, thereby providing a powerful mining strategy for the bioresources with unknown genomic and proteomic sequences.

CD19 is a surface antigen on B cells that regulates B cell activation and proliferation, participating in B cell signaling. It is expressed in all B cell lineage tumor diseases, making CD19 a significant marker for detecting B cell tumor diseases and an important target for related immunotherapies. In recent years, with the deepening research on canine and feline diseases and the establishment of animal models, the demand for cat CD19 monoclonal antibodies (mAbs) has been steadily increasing. We successfully prepared cat CD19-specific monoclonal antibodies using a KLH-conjugated cat CD19 peptide as an antigen and optimized the antibody production method. The obtained monoclonal antibodies' molecular and cellular affinities were identified using CD19 peptides, eukaryotic overexpressed proteins, and peripheral blood mononuclear cells (PBMCs). The results indicate that the CD19-3H9 and CD19-8A7 monoclonal antibodies prepared in this study specifically bind to the CD19 molecule, demonstrating their suitability for use in ELISA, Western blot, and cell assays. This study successfully produced cat CD19 monoclonal antibodies with specificity and optimized the antibody preparation method, laying the foundation for the diagnosis and targeted drug combination therapy of B cell tumor diseases in both humans and pets.

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 amygdala is a core region in the limbic system that is highly sensitive to stress. Astrocytes are key players in stress disorders such as anxiety and depression. However, the effects of stress on the morphology and function of amygdala astrocytes and its potential mechanisms remain largely unknown. Hence, we performed in vivo and in vitro experiments using a restraint stress (RS) rat model and stress-induced astrocyte culture, respectively. Our data show that norepinephrine (NE) content increased, cytotoxic edema occurred, and aquaporin-4 (AQP4) expression was up-regulated in the basolateral amygdala (BLA) obtained from RS rats. Additionally, the p38 mitogen-activated protein kinase (MAPK) pathway was also observed to be significantly activated in the BLA of rats subjected to RS. The administration of NE to in vitro astrocytes increased the AQP4 level and induced cell edema. Furthermore, p38 MAPK signaling was activated. The NE inhibitor alpha-methyl-p-tyrosine (AMPT) alleviated cytotoxic edema in astrocytes, inhibited AQP4 expression, and inactivated the p38 MAPK pathway in RS rats. Meanwhile, in the in vitro experiment, the p38 MAPK signaling inhibitor SB203580 reversed NE-induced cytotoxic edema and down-regulated the expression of AQP4 in astrocytes. Briefly, NE-induced activation of the p38 MAPK pathway mediated cytotoxic edema in BLA astrocytes from RS rats. Thus, our data provide novel evidence that NE-induced p38 MAPK pathway activation may be one of the mechanisms leading to cytotoxic edema in BLA under stress conditions, which also could enable the development of an effective therapeutic strategy against cytotoxic edema in BLA under stress and provide new ideas for the treatment of neuropsychiatric diseases.

ABSTRACT Changes in diet and environment can lead to acute diarrhea in companion animals, but the composition and interactions of the gut microbiome during acute diarrhea remain unclear. In this multicenter case-control study, we investigated the relationship between intestinal flora and acute diarrhea in two breeds of cats. Acutely diarrheic American Shorthair (MD, n = 12) and British Shorthair (BD, n = 12) and healthy American Shorthair (MH, n = 12) and British Shorthair (BH, n = 12) cats were recruited. Gut microbial 16S rRNA sequencing, metagenomic sequencing, and untargeted metabolomic analysis were performed. We observed significant differences in beta-diversity (Adonis, P

Background: Methamphetamine (METH)-induced cardiovascular toxicity has been attributed to its destructive effect on mitochondrial function at least to some extent. Previous studies highlighted the benefits of dapagliflozin (DAPA) on the cardiovascular system, but the response of METH-induced cardiomyopathy to DAPA is never addressed before. The present study aimed to investigate the potential ability of DAPA in preventing METH-induced cardiomyopathy.Materials and Methods: C57BL/6 mice were randomly divided into control group (n = 24), METH group (n = 24), and METH + DAPA group (n = 24). The METH-induced cardiomyopathy group received intraperitoneal METH injections at gradually increasing doses thrice weekly for 14 weeks. Mice in the METH + DAPA group were simultaneously treated with DAPA 1 mg/kg/day by intragastric administration. Echocardiography was performed to assess cardiac function. Reactive oxygen species (ROS), JC-1, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays were performed to evaluate oxidative stress, mitochondrial damage, and apoptosis, respectively. Mitochondrial and apoptosis-related protein expression was measured by western blotting.Results: Mice exposed to METH exhibited reduced cardiac function (left ventricular ejection fraction [LVEF]: 56.51 ± 6.49 vs. 73.62 ± 1.42, p < 0.01), fibrotic remodeling, and mitochondrial dysfunction, leading to apoptosis (apoptotic cells%: 7.4 ± 1.3 vs. 1.3 ± 0.5, p < 0.01). DAPA significantly reduced mitochondrial dynamics and function, ROS, apoptosis (apoptotic cells%: 2.4 ± 0.8 vs. 7.4 ± 1.3, p < 0.01), cardiac function decline (LVEF: 70.99 ± 4.936 vs. 56.51 ± 6.49, p < 0.01), and fibrotic remodeling. These results indicated that DAPA could be considered as an effective therapeutic agent in the protection against METH-associated cardiomyopathy.Conclusion: DAPA protects against METH-induced cardiomyopathy in mice by decreasing mitochondrial damage and apoptosis.

Three medicinal fungi were used to carry out solid-state fermentation (SSF) of wheat bran. The results showed that the use of these fungi for SSF significantly improved wheat bran’s nutritional properties including the extraction yield of soluble dietary fiber (SDF), total phenolic content (TPC), total flavonoid content (TFC), physical properties containing swelling capacity (SC) and oil absorption capacity (OAC), as well as antioxidant activities. Electronic nose and GC–MS analyses showed that fermented wheat bran had different volatiles profiles compared to unfermented wheat bran. The results suggest that SSF by medicinal fungi is a promising way for the high-value utilization of wheat bran.

ObjectiveThe 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.MethodsRat 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.ResultsH&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.ConclusionAME 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.

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.

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.

The amygdala is an important center of fear learning and memory and plays a critical role in regulating stress disorders. Previous studies have shown that changes in the amygdala caused by stress are an important cause of mental disorders including anxiety, but the specific mechanism remains unclear. Therefore, the purpose of this study was to investigate whether mental disorders induced by stress are related to γ-aminobutyric acid (GABA)ergic neuron damage in the basolateral amygdala (BLA) and whether endoplasmic reticulum stress (ERS) is involved in the injury process. Rat models of different durations of stress were established by restraint and forced ice-water swimming. Behavioral tests and high-performance liquid chromatography (HPLC) were used to detect anxiety in rats and changes in neurotransmitter levels in the BLA. Morphological approaches and microscopy-based multicolor tissue cytometry (MMTC) were used to detect the damage-induced changes in GABAergic neurons in the BLA. Immunofluorescence double labeling was used to detect the expression of ERS-related proteins before and after the inhibition of protein kinase R-like endoplasmic reticulum kinase (PERK) pathway. Stress resulted in damage to GABAergic neurons in the BLA, decreased GABA and increased glutamic acid (GLU) levels, perturbation of the excitation/inhibition (E/I) ratio in the BLA, and obvious anxiety disorders in rats. Moreover, ERS-mediated GABAergic neuron injury was an important cause of neurotransmitter level changes in the BLA. These results suggested that ERS-mediated GABAergic neuron injury in the BLA may be an important cause of stress-induced mental disorders.

Aims: To investigate the effect of RU486 (mifepristone) on emotional disorders in chronic restraint stress-induced rats and to explore the mechanisms of that phenomenon. Methods: For this purpose, 80 healthy male Sprague Dawley rats were randomly divided into four groups: the normal group (Con group, The Con group members received no treatment, eating and drinking freely), the chronic restraint stress group (CRS group, normal Sprague Dawley rats treated with chronic restraint stress, 6 h/day for 21days), the propylene glycol group (CRS+propylene glycol) and the RU486 group (CRS+RU486). RU486 or propylene glycol was administered 30 mins before each CRS procedure. Twenty-four hours after CRS exposure, we investigated the effects of CRS on the anxiety-like behavior using an elevated plus-maze (EPM). To explore the mechanisms of RU486 on anxiety, we measured the expression of glial fibrillary acid protein (GFAP) and β-subunit of S100 (S100β) via immunohistochemistry and western blot analysis. Apoptosis was demonstrated by flow cytometry. In addition, endoplasmic reticulum (ER) stress markers, glucose regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and Cysteine aspartic acid specific protease-12 (Caspase-12), were detected by western blot analysis. Results: Compared to the control group, rats in the CRS and propylene glycol group showed decreased exploratory behavior on the open arms during EPM testing, and these reductions were accompanied by significantly reduced GFAP and S100β expression, increased apoptosis and GRP78, CHOP, and caspase-12 expression in the amygdala. However, RU486 increases the exploratory behavior and reverses the changes of GFAP, S100β, GRP78, CHOP, and caspase-12 and protects cells against apoptosis. Conclusions: Taken together, these data suggest that exposure to chronic restraint stress decreases the number of astrocytes and induces apoptosis and ER stress in the amygdala, which are possible causes for psychiatric disorders. RU486 can significantly ameliorate abnormal behaviors in CRS-induced anxiety model rats. The protective effects of RU486 could be attributed to its anti-ER stress, anti-apoptosis and astrocyte increasing effects.

The hypothalamus, which is the initial part of the hypothalamic-pituitary-adrenal (HPA) axis, plays a critical role in regulating stress in the central nervous system. The present study aimed to determine whether endoplasmic reticulum stress in hypothalamic neurons is differentially stimulated by varying durations of stress exposure, which ultimately leads to pathological changes in neurons by affecting HPA axis function. There is a need for better morphological evidence of the mechanisms involved in stress-induced neuron injury. A stress model was established in rats by restraining for 8 h and forced ice-water swimming for 5 min each day. The stress-inducing process lasted for 1, 3, 7, 14, and 21 days. Enzyme-linked immunosorbent assay (ELISA) was used to assay serum glucocorticoid levels. Thionine staining was used to observe morphological changes in hypothalamic neurons. Immunohistochemistry and microscopy-based multicolor tissue cytometry (MMTC) was used to detect changes in expression of endoplasmic reticulum stress protein GRP78, ATF4, and CHOP. Serum glucocorticoid levels significantly increased after 3 days of stress exposure and the levels peaked by 7 days. By 21 days, however, the levels were significantly decreased. Thionine staining revealed that prolonged stress exposure resulted in hypothalamic neurons with edema, a lack of Nissl bodies, and pyknotic neurons. Immunohistochemistry and MMTC showed that increasing stress periods significantly decreased GRP78 expression, although ATF4 and CHOP protein expression significantly increased. Stress resulted in pathological changes and significant dynamic changes because of endoplasmic reticulum stress in rat hypothalamic neurons. These results suggested that the endoplasmic reticulum stress PERK-ATF4-CHOP pathway may be associated with hypothalamic neuronal injury.

Copper, as the third most abundant transition metal ions of human, plays an essential role in the redox reaction, signal transduction, hematopoiesis, and other physiological processes. Abnormal content of copper ions in the body will cause some diseases such as anemia, coronary heart disease, Menkes' syndrome. In this article, a new fluorescence probe L for Cu2+ was designed and synthetized by using 4-bromo-1,8 naphthalene anhydride and 2-thiophene formaldehyde as raw materials. Fluorescent probe L itself exhibited strong fluorescence, upon the addition of Cu2+ ions, the fluorescence was quenched. The fluorescent detection limit for Cu2+ ions was determined to be 1.8 μM based on a 3δ/S method. UV-vis absorption and fluorescence spectra indicated that probe L showed good selectivity and sensitivity for Cu2+, and this selectivity was not interfered by other metal ions and anions. Further cell fluorescence imaging experiments indicated that the probe L had potential to be used to examine copper ions in vivo.

The ventral tegmental area (VTA), a critical portion of the mesencephalic dopamine system, is thought to be involved in the development and maintenance of addiction. It has been proposed that the dopaminergic regulatory factors TH, Nurr1, and Pitx3 are crucial for determining the survival and maintenance of dopaminergic neurons. Thus, the present study investigated whether abnormalities in these dopaminergic regulatory factors in the VTA were associated with neuronal injury induced by chronic morphine dependence. Rat models with different durations of morphine dependence were established. Thionine staining was used to observe morphological changes in the VTA neurons. Immunohistochemistry and western blot were used to observe changes in the expression of the dopaminergic regulatory proteins TH, Nurr1, and Pitx3. Thionine staining revealed that prolonged morphine dependence resulted in dopaminergic neurons with edema, a lack of Nissl bodies, and pyknosis. Immunohistochemistry showed that the number of TH+, Nurr1+, and Pitx3+ cells, and the number of TH+ cells expressing Nurr1 or Pitx3, significantly decreased in the VTA after a long period of morphine dependence. Western blot results were consistent with the immunohistochemistry findings. Chronic morphine exposure resulted in abnormalities in dopaminergic regulatory factors and pathological changes in dopaminergic neurons in the VTA. These results suggest that dysregulation of dopaminergic regulatory factors in the VTA are associated with neuronal injury induced by chronic morphine dependence.