Development of novel humanized CD19/BAFFR bicistronic chimeric antigen receptor T cells with potent antitumor activity against B-cell lineage neoplasms.

Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy in treating advanced B-cell malignancies by targeting CD19, but antigen-negative relapses and immune responses triggered by murine-derived antibodies remain significant challenges, necessitating the development of novel humanized multitarget CAR-T therapies. Here, we engineered a second-generation 4-1BB-CD3ζ-based CAR construct incorporating humanized CD19 single-chain variable fragments (scFvs) and BAFFR single-variable domains on heavy chains (VHHs), also known as nanobodies. The resultant CAR-T cells, with different constructs, were functionally compared both in vitro and in vivo. We found that the optimal tandem and bicistronic (BI) structures retained respective antigen-binding abilities, and both demonstrated specific activation when stimulated with target cells. At the same time, BI CAR-T cells (BI CARs) exhibited stronger tumour-killing ability and better secretion of interleukin-2 and tumour necrosis factor-alpha than single-target CAR-T cells. Additionally, BI CARs showed less exhaustion phenotype upon repeated antigen stimulation and demonstrated more potent and persistent antitumor effects in mouse xenograft models. Overall, we developed a novel humanized CD19/BAFFR bicistronic CAR (BI CAR) based on a combination of scFv and VHH, which showed potent and sustained antitumor ability both in vitro and in vivo, including against tumours with CD19 or BAFFR deficiencies.

Epidemiological characteristics of human metapneumovirus among children in Nanjing, China.

PURPOSE: The objective of this study was to examine the molecular epidemiology and clinical characteristics of HMPV infection among children with ARIs in Nanjing. METHODS: The respiratory samples were collected from 2078 children (≤ 14 years) with acute respiratory infections and were tested for HMPV using real-time RT-PCR. Amplification and sequencing of the HMPV G gene were followed by phylogenetic analysis using MEGA 7.0. RESULT: The detection rate of HMPV among children was 4.7% (97/2078), with a concentration in those under 5 years of age. Notably, the peak season for HMPV prevalence was observed in winter. Among the 97 HMPV-positive samples, 51.5% (50/97) were available for characterization of the HMPV G protein gene. Phylogenetic analysis indicated that the sequenced HMPV strains were classified into three sublineages: A2c111nt - dup (84.0%), B1 (2.0%), and B2 (14.0%). CONCLUSION: There was an incidence of HMPV among hospitalized children during 2021-2022 in Nanjing with A2c111nt - dup being the dominant strain. This study demonstrated the molecular epidemiological characteristics of HMPV among children with respiratory infections in Nanjing, China.

A Bispecific Antibody Targeting RBD and S2 Potently Neutralizes SARS-CoV-2 Omicron and Other Variants of Concern.

Emerging severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) variants, especially the Omicron variant, have impaired the efficacy of existing vaccines and most therapeutic antibodies, highlighting the need for additional antibody-based tools that can efficiently neutralize emerging SARS-CoV-2 variants. The use of a "single" agent to simultaneously target multiple distinct epitopes on the spike is desirable in overcoming the neutralizing escape of SARS-CoV-2 variants. Herein, we generated a human-derived IgG-like bispecific antibody (bsAb), Bi-Nab35B5-47D10, which successfully retained parental specificity and simultaneously bound to the two distinct epitopes on receptor-binding domain (RBD) and S2. Bi-Nab35B5-47D10 showed improved spike binding breadth among wild-type (WT) SARS-CoV-2, variants of concern (VOCs), and variants being monitored (VBMs) compared with its parental monoclonal antibodies (MAbs). Furthermore, pseudotyped virus neutralization demonstrated that Bi-Nab35B5-47D10 can efficiently neutralize VBMs, including Alpha (B.1.1.7), Beta (B.1.351), and Kappa (B.1.617.1), as well as VOCs, including Delta (B.1.617.2), Omicron BA.1, and Omicron BA.2. Crucially, Bi-Nab35B5-47D10 substantially improved neutralizing activity against Omicron BA.1 (IC50 = 0.15 nM) and Omicron BA.2 (IC50 = 0.67 nM) compared with its parental MAbs. Therefore, Bi-Nab35B5-47D10 represents a potential effective countermeasure against SARS-CoV-2 Omicron and other variants of concern. IMPORTANCE The new, highly contagious SARS-CoV-2 Omicron variant caused substantial breakthrough infections and has become the dominant strain in countries across the world. Omicron variants usually bear high mutations in the spike protein and exhibit considerable escape of most potent neutralization monoclonal antibodies and reduced efficacy of current COVID-19 vaccines. The development of neutralizing antibodies with potent efficacy against the Omicron variant is still an urgent priority. Here, we generated a bsAb, Bi-Nab35B5-47D10, which simultaneously targets SARS-CoV-2 RBD and S2 and improves the neutralizing potency and breadth against SARS-CoV-2 WT and the tested variants compared with their parental antibodies. Notably, Bi-Nab35B5-47D10 has more potent neutralizing activity against the VOC Omicron pseudotyped virus. Therefore, Bi-Nab35B5-47D10 is a feasible and potentially effective strategy by which to treat and prevent COVID-19.

Kinetic and isotherm of competitive adsorption cadmium and lead onto Saccharomyces cerevisiae autoclaved cells.

Heavy metal pollution has been regarded as a significant public health hazard during the industrialization, which also have exhibited various types of toxicological manifestations. Moreover, due to the high cost and toxic by-products, some conventional remediation methods were limited to heavy metals pollution control. In this work, autoclaved Saccharomyces cerevisiae was used as a biosorbent for the removal of Cd2+ and Pb2+ from single and binary ions aqueous solution system. The kinetics and isotherm of Cd2+ and Pb2+ were studied in different ion systems. The results showed that the competitive adsorption ability of S. cerevisiae to Pb2+ was stronger than that to Cd2+ in binary ions solution. To all the single ion solution of Cd2+ or Pb2+ and binary ions solution of Cd2+-Pb2+, there always existed that the adsorption of metal ions on S. cerevisiae fitted well with pseudo-second-order kinetic model and Langmuir isotherms model. The adsorption quantity qt in different solutions followed the sequence as qt (Cd2+-Pb2+) > qt (Pb2+-single) > qt (Pb2+-binary) > qt (Cd2+-single) > qt (Cd2+-binary). The autoclaved S. cerevisiae used in this research was one kind of rapid and favourable biosorbent for Pb2+ and Cd2+. In Pb2+ and Cd2+-containing solutions, sites competition and jointed toxicity of Pb2+ and Cd2+ on S. cerevisiae cells were the key to the total adsorption effect, and further researches were necessary in the next work. Thus, the current research presented that the autoclaved S. cerevisiae could be applied as an effective biosorbent for heavy metal adsorption from water environment and the design of eco-friendly technologies for the treatment of waste liquor.

Bortezomib activation of mTORC1 pathway mediated by NOX2-drived reactive oxygen species results in apoptosis in primary dorsal root ganglion neurons.

Bortezomib (Bort), a chemotherapeutic agent, is widely used for the clinical treatment of cancers. However, Bort-induced peripheral neurotoxicity (BIPN) significantly restricts its clinical application, which is difficult to deal with since the underlying mechanisms of BIPN are unclear. Here, we showed that Bort activates mTORC1 pathway leading to dorsal root ganglion (DRG) neuronal apoptosis. Inhibition of mTORC1 with rapamycin or knockdown of raptor, regulatory-associated protein of mTORC1, with shRNA dramatically rescued the cells from Bort-caused apoptosis. In addition, we found that Bort-activated mTORC1 pathway was attributed to Bort elevation of reactive oxygen species (ROS). This is supported by the evidence that using ROS scavenger N-acetyl cysteine (NAC) significantly alleviated Bort-activated mTORC1 pathway. Furthermore, we revealed that upregulation of NOX2 contributed to Bort-elicited ROS overproduction, leading to mTORC1 pathway-dependent apoptosis in DRG neurons. Inhibition of NOX2 with apocynin remarkably diminished Bort-induced overgeneration of ROS, activation of mTORC1 pathway and apoptosis in the cells. Taken together, these results indicate that Bort activation of mTORC1 pathway mediated by NOX2-drived ROS leads to apoptotic death in DRG neurons. Our findings highlight that manipulation of intracellular ROS level or NOX2 or mTORC1 activity may be exploited for prevention of BIPN.

Solamargine induces autophagy-mediated apoptosis and enhances bortezomib activity in multiple myeloma.

Multiple myeloma (MM) is an incurable plasma cell malignancy with a poor survival rate. Conventional chemotherapeutic agent-induced adverse events, including toxicity, neuropathy or drug resistance, significantly decrease the patients' quality of life and can even lead to interruption of treatment. Therefore, novel therapeutic drugs and strategies are urgently needed to improve MM therapy and patient outcomes. Here, we show that solamargine (SM), a steroidal alkaloid glycoside isolated from a Chinese herb Solanum nigrum L., exhibits promising anti-MM activity. In particular, SM suppressed the viability of MM cell lines (ARP-1 and NCI-H929) in a concentration- and time-dependent manner, inducing apoptosis in these cells. RNA-seq analysis showed that treatment with SM led to the upregulation of genes associated with cell death and autophagy in H929 cells. Further, we found that treatment with SM activated autophagy in the MM cells, as incubation with 3-Methyladenine, an inhibitor of autophagy, significantly alleviated SM-triggered apoptosis and inhibition of viability in MM cells. Interestingly, we also observed a synergistic effect between SM and bortezomib (BTZ), a common chemotherapeutic agent for MM, in both MM cells and human bone marrow CD138+ primary myeloma cells. We also confirmed the single-agent efficacy of SM and the synergistic effects between SM and BTZ in an MM xenograft mouse model. Collectively, these findings indicate that SM exerts an anti-MM effect, at least in part, by activating cell autophagy and reveal that SM alone or in combination with BTZ is a potential therapeutic strategy for treating MM.

Overcoming the blood-brain barrier by using a multistage exosome delivery system to inhibit central nervous system lymphoma.

Due to the presence of blood-brain barrier (BBB), various chemotherapy drugs against B-cell lymphoma cannot be effectively transmitted into the brain, leading to poor prognosis of primary central nervous system lymphoma (PCNSL). Exosomes can cross the BBB as a bio- and immune-compatible drug carrier. In this study, we developed a novel drug delivery system, in which the exosomes (Exo) are conjugated with anti-CD22 monoclonal antibody fragments (CD22-F(ab')2) and encapsulate doxorubicin (DOX) to form CD22-F(ab')2-Exo-DOX. We showed that CD22-F(ab')2-Exo-DOX can cross BBB and deliver DOX precisely to tumor cells. The average apoptosis rate of lymphoma cells was 84.60% ±â€¯10.69%. The tumor-bearing mice treated with CD22-F(ab')2-Exo-DOX have significantly prolonged life expectancy and the enhanced anti-tumor activity. CD22-F(ab')2-Exo-DOX might be ingested by brain microvascular endothelial cells through endocytosis to cross the BBB. Therefore, targeted chemotherapy mediated by CD22-F(ab')2-Exo-DOX is a promising option for the treatment of PCNSL.

Cadmium Impairs Autophagy Leading to Apoptosis by Ca2+-Dependent Activation of JNK Signaling Pathway in Neuronal Cells.

Autophagy, a process for self-degradation of intracellular components and dysfunctional organelles, is closely related with neurodegenerative diseases. It has been shown that cadmium (Cd) induces neurotoxicity partly by impairing autophagy. However, the underlying mechanism is not fully elucidated. In this study, we show that Cd induced expansion of autophagosomes with a concomitant abnormal expression of autophagy-related (Atg) proteins in PC12 cells and primary murine neurons. 3-MA, a classical inhibitor of autophagy, attenuated Cd-induced expansion of autophagosomes and apoptosis in the cells. Further investigation demonstrated that Cd activated JNK pathway contributing to autophagosome expansion-dependent neuronal apoptosis. This is supported by the findings that pharmacological inhibition of JNK with SP600125 or expression of dominant negative c-Jun markedly attenuated Cd-induced expansion of autophagosomes and abnormal expression of Atg proteins, as well as apoptosis in PC12 cells and/or primary neurons. Furthermore, we noticed that chelating intracellular free Ca2+ ([Ca2+]i) with BAPTA/AM profoundly blocked Cd-elicited activation of JNK pathway and consequential expansion of autophagosomes, abnormal expression of Atg proteins, and apoptosis in the neuronal cells. Similar events were also seen following prevention of [Ca2+]i elevation with EGTA or 2-APB, implying a Ca2+-dependent mechanism involved. Taken together, the results indicate that Cd impairs autophagy leading to apoptosis by Ca2+-dependent activation of JNK signaling pathway in neuronal cells. Our findings highlight that manipulation of intracellular Ca2+ level and/or JNK activity to ameliorate autophagy may be a promising intervention against Cd-induced neurotoxicity and neurodegeneration.

Resveratrol inhibits Erk1/2-mediated adhesion of cancer cells via activating PP2A-PTEN signaling network.

Resveratrol, a natural polyphenol compound, has been shown to possess anticancer activity. However, how resveratrol inhibits cancer cell adhesion has not been fully elucidated. Here, we show that resveratrol suppressed the basal or type I insulin-like growth factor (IGF)-1-stimulated adhesion of cancer cells (Rh1, Rh30, HT29, and HeLa cells) by inhibiting the extracellular signal-regulated kinase 1/2 (Erk1/2) pathway. Inhibition of Erk1/2 with U0126, knockdown of Erk1/2, or overexpression of dominant-negative mitogen-activated protein kinase kinase 1 (MKK1) strengthened resveratrol's inhibition of the basal or IGF-1-stimulated of Erk1/2 phosphorylation and cell adhesion, whereas ectopic expression of constitutively active MKK1 attenuated the inhibitory effects of resveratrol. Further research revealed that both protein phosphatase 2A (PP2A) and phosphatase and tensin homolog (PTEN)-Akt were implicated in resveratrol-inactivated Erk1/2-dependent cell adhesion. Inhibition of PP2A with okadaic acid or overexpression of dominant-negative PP2A rendered resistance to resveratrol's suppression of the basal or IGF-1-stimulated phospho-Erk1/2 and cell adhesion, whereas expression of wild-type PP2A enhanced resveratrol's inhibitory effects. Overexpression of wild-type PTEN or dominant-negative Akt or inhibition of Akt with Akt inhibitor X strengthened resveratrol's inhibition of the basal or IGF-1-stimulated Erk1/2 phosphorylation and cell adhesion. Furthermore, inhibition of mechanistic/mammalian target of rapamycin (mTOR) with rapamycin or silencing mTOR enhanced resveratrol's inhibitory effects on the basal and IGF-1-induced inhibition of PP2A-PTEN, activation of Akt-Erk1/2, and cell adhesion. The results indicate that resveratrol inhibits Erk1/2-mediated adhesion of cancer cells via activating PP2A-PTEN signaling network. Our data highlight that resveratrol has a great potential in the prevention of cancer cell adhesion.

Maduramicin induces cardiac muscle cell death by the ROS-dependent PTEN/Akt-Erk1/2 signaling pathway.

Maduramicin (Mad), a polyether ionophore antibiotic, has been reported to be toxic to animals and humans because of being used at high doses or for long time, resulting in heart failure. However, the toxic mechanism of Mad in cardiac muscle cells is not well understood. Here, we show that Mad induced cell viability reduction and apoptosis in cardiac-derived H9c2, HL-1 cells, primary cardiomyocytes, and murine cardiac muscles, which was because of the inhibition of extracellular-signal-regulated kinase 1/2 (Erk1/2). Expression of constitutively active mitogen-activated protein kinase kinase 1 (MKK1) attenuated Mad-induced cell death in H9c2 cells, whereas silencing Erk1/2 or ectopic expression of dominant negative MKK1 strengthened Mad-induced cell death. Moreover, we found that both phosphatase and tensin homolog on chromosome 10 (PTEN) and protein kinase B (Akt) were implicated in the regulation of Erk1/2 inactivation and apoptosis in the cells and tissues exposed to Mad. Overexpression of dominant negative PTEN and/or constitutively active Akt, or constitutively active Akt and/or constitutively active MKK1 rescued the cells from Mad-induced dephosphorylated-Erk1/2 and cell death. Furthermore, Mad-induced reactive oxygen species (ROS) activated PTEN and inactivated Akt-Erk1/2 contributing to cell death, as N-acetyl- L-cysteine ameliorated the event. Taken together, the results disclose that Mad inhibits Erk1/2 via ROS-dependent activation of PTEN and inactivation of Akt, leading to cell death in cardiac muscle cells. Our findings suggest that manipulation of the ROS-PTEN-Akt-Erk1/2 pathway may be a potential approach to prevent Mad-induced cardiotoxicity.

Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations.

Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. The TGs identified to date do not have a typical endoplasmic reticulum (ER)-signal peptide, and the molecular mechanisms of their secretion under physiologic conditions are unclear. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. We also found that TG-A, but not TG-B, was modified concomitantly by N-myristoylation and S-palmitoylation, and N-myristoylation was a pre-requisite for S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca2+ ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. Ultracentrifugation, electron microscopy analyses, and treatments with inhibitors of multivesicular body formation revealed that TG-A was secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes.

Rapamycin attenuates BAFF-extended proliferation and survival via disruption of mTORC1/2 signaling in normal and neoplastic B-lymphoid cells.

B cell activating factor from the TNF family (BAFF) stimulates B-cell proliferation and survival, but excessive BAFF promotes the development of aggressive B cells leading to malignant and autoimmune diseases. Recently, we have reported that rapamycin, a macrocyclic lactone, attenuates human soluble BAFF (hsBAFF)-stimulated B-cell proliferation/survival by suppressing mTOR-mediated PP2A-Erk1/2 signaling pathway. Here, we show that the inhibitory effect of rapamycin on hsBAFF-promoted B cell proliferation/survival is also related to blocking hsBAFF-stimulated phosphorylation of Akt, S6K1, and 4E-BP1, as well as expression of survivin in normal and B-lymphoid (Raji and Daudi) cells. It appeared that both mTORC1 and mTORC2 were involved in the inhibitory activity of rapamycin, as silencing raptor or rictor enhanced rapamycin's suppression of hsBAFF-induced survivin expression and proliferation/viability in B cells. Also, PP242, an mTORC1/2 kinase inhibitor, repressed survivin expression, and cell proliferation/viability more potently than rapamycin (mTORC1 inhibitor) in B cells in response to hsBAFF. Of interest, ectopic expression of constitutively active Akt (myr-Akt) or constitutively active S6K1 (S6K1-ca), or downregulation of 4E-BP1 conferred resistance to rapamycin's attenuation of hsBAFF-induced survivin expression and B-cell proliferation/viability, whereas overexpression of dominant negative Akt (dn-Akt) or constitutively hypophosphorylated 4E-BP1 (4EBP1-5A), or downregulation of S6K1, or co-treatment with Akt inhibitor potentiated the inhibitory effects of rapamycin. The findings indicate that rapamycin attenuates excessive hsBAFF-induced cell proliferation/survival via blocking mTORC1/2 signaling in normal and neoplastic B-lymphoid cells. Our data underscore that rapamycin may be a potential agent for preventing excessive BAFF-evoked aggressive B-cell malignancies and autoimmune diseases.

Non-thermal plasma reduces periodontitis-induced alveolar bone loss in rats.

Periodontitis, a chronic infectious disease induced by microbial biofilm, is one of the most common diseases worldwide. Scaling and root planning (SRP) has always been recognized as the typical treatment. However, the therapeutic efficiency is often limited due to the intraoperative bleeding and the limitations of instruments. Non-thermal atmospheric plasma (NTP) appears to be a potential tool for periodontitis due to its promising biofilm degradation and decontamination effects. In this study, we investigated the role of NTP, as an adjuvant approach for the treatment of ligature-induced periodontitis in rats. Herein we showed that SRP or SRP-NTP application attenuated the periodontitis-induced alveolar bone loss, reflected by the increased BV/TV value and the decreased CEJ-AB distance, which might be related to the lower detection rate of periodontal pathogen in SRP and SRP-NTP groups. Besides, SRP-NTP rats showed less bone loss and lower CEJ-AB distance than that of SRP group at 30d post treatment, indicating a more comprehensive and long-lasting effect of SRP-NTP. A remarkable decrease of osteoclast number and lower expression of RANKL was also detected in SRP-NTP rats. In addition, expression of inflammatory-related cytokines such as TNF-α and IL-1ß decreased significantly in SRP-NTP group, while IL-10 level increased substantially. These results together illustrated that a combination of SRP and NTP treatment was an effective way to prevent periodontitis progress, which reduced alveolar bone loss and promoted periodontium restoration in ligature-induced periodontitis rats. In conclusion, non-thermal plasma treatment may be considered as a feasible and effective supplementary approach to control periodontitis.

Celastrol Attenuates Cadmium-Induced Neuronal Apoptosis via Inhibiting Ca2+ -CaMKII-Dependent Akt/mTOR Pathway.

Cadmium (Cd), an environmental and industrial pollutant, affects the nervous system and consequential neurodegenerative disorders. Recently, we have shown that celastrol prevents Cd-induced neuronal cell death partially by suppressing Akt/mTOR pathway. However, the underlying mechanism remains to be elucidated. Here, we show that celastrol attenuated Cd-elevated intracellular-free calcium ([Ca2+ ]i ) level and apoptosis in neuronal cells. Celastrol prevented Cd-induced neuronal apoptosis by inhibiting Akt-mediated mTOR pathway, as inhibition of Akt with Akt inhibitor X or ectopic expression of dominant negative Akt reinforced celastrol's prevention of Cd-induced phosphorylation of S6K1/4E-BP1 and cell apoptosis. Furthermore, chelating intracellular Ca2+ with BAPTA/AM or preventing [Ca2+ ]i elevation using EGTA potentiated celastrol's repression of Cd-induced [Ca2+ ]i elevation and consequential activation of Akt/mTOR pathway and cell apoptosis. Moreover, celastrol blocked Cd-elicited phosphorylation of CaMKII, and pretreatment with BAPTA/AM or EGTA enhanced celastrol's suppression of Cd-increased phosphorylation of CaMKII in neuronal cells, implying that celastrol hinders [Ca2+ ]i -mediated CaMKII phosphorylation. Inhibiting CaMKII with KN93 or silencing CaMKII attenuated Cd activation of Akt/mTOR pathway and cell apoptosis, and this was strengthened by celastrol. Taken together, these data demonstrate that celastrol attenuates Cd-induced neuronal apoptosis via inhibiting Ca2+ -CaMKII-dependent Akt/mTOR pathway. Our findings underscore that celastrol may act as a neuroprotective agent for the prevention of Cd-induced neurodegenerative disorders. J. Cell. Physiol. 232: 2145-2157, 2017. © 2016 Wiley Periodicals, Inc.

Celastrol ameliorates Cd-induced neuronal apoptosis by targeting NOX2-derived ROS-dependent PP5-JNK signaling pathway.

Celastrol, a plant-derived triterpene, has neuroprotective benefit in the models of neurodegenerative disorders that are characterized by overproduction of reactive oxygen species (ROS). Recently, we have reported that cadmium (Cd) activates c-Jun N-terminal kinase (JNK) pathway leading to neuronal cell death by inducing ROS inactivation of protein phosphatase 5 (PP5), and celastrol prevents Cd-activated JNK pathway against neuronal apoptosis. Therefore, we hypothesized that celastrol could hinder Cd induction of ROS-dependent PP5-JNK signaling pathway from apoptosis in neuronal cells. Here, we show that celastrol attenuated Cd-induced expression of NADPH oxidase 2 (NOX2) and its regulatory proteins (p22phox , p40phox , p47phox , p67phox , and Rac1), as well as the generation of ROS in PC12 cells and primary neurons. Also, N-acetyl-l-cysteine, a ROS scavenger, potentiated celastrol's inhibition of the events in the cells triggered by Cd, implying neuroprotection by celastrol via blocking Cd-evoked NOX2-derived ROS. Further research revealed that celastrol was involved in the regulation of PP5 inactivation and JNK/c-Jun activation induced by Cd, as celastrol prevented Cd from reducing PP5 expression, and over-expression of wild-type PP5 or dominant negative c-Jun strengthened celastrol's inhibition of Cd-induced phosphorylation of JNK and/or c-Jun, as well as apoptosis in neuronal cells. Of importance, inhibiting NOX2 with apocynin or silencing NOX2 by RNA interference enhanced the inhibitory effects of celastrol on Cd-induced inactivation of PP5, activation of JNK/c-Jun, ROS, and apoptosis in the cells. Furthermore, we noticed that expression of wild-type PP5 or dominant negative c-Jun, or pretreatment with JNK inhibitor SP600125 reinforced celastrol's suppression of Cd-induced NOX2 and its regulatory proteins, and consequential ROS in neuronal cells. These findings indicate that celastrol ameliorates Cd-induced neuronal apoptosis via targeting NOX2-derived ROS-dependent PP5-JNK signaling pathway. Our data highlight a beneficial role of celastrol in the prevention of Cd-induced oxidative stress and neurodegenerative diseases.

[Role of NF-κB in the protective effects of L-carnitine against oxidative injury in hepatocytes].

OBJECTIVE: To investigate whether the protective effects of L-carnitine( LC) against hydrogen peroxide( H_2O_2)-induced injury in hepatocytes were related to nuclear factor-kappa B( NF-κB). METHODS: CCK-8 and lactate dehydrogenase( LDH)methods were used to detect the influences of NF-κB inhibitors on the cell damage induced by H_2O_2. The effects of LC on the NF-κB expressions in H_2O_2-treated HL7702 cells were determined by Western blot. The translocation of NF-κB was observed by immunofluorescence staining. Electrophoretic mobility shift assay( EMSA) was used to evaluate NF-κB-DNA binding activities. RESULTS: Compared with H_2O_2 group, NF-κB inhibitor groups showed increased cell activities and decreased LDH release( P < 0. 05, P < 0. 01). Western blot and immunofluorescence staining both demonstrated that the nucleus NF-κB expressions elevated in H_2O_2 group and LC had inhibitory effect on them( P < 0. 01). LC also inhibited H_2O_2-induced increase of NF-κB-DNA binding activity inHL7702 cells. CONCLUSION: LC protects against H_2O_2-induced injury in HL7702 cells by inhibiting NF-κB activities.

Effects of L-carnitine against H2O2-induced oxidative stress in grass carp ovary cells (Ctenopharyngodon idellus).

This study was designed in vitro to investigate the effects of L-carnitine against H2O2-induced oxidative stress in a grass carp (Ctenopharyngodon idellus) ovary cell line (GCO). GCO cells were pre-treated with different concentrations of L-carnitine, followed by incubation with 2.5 mM H2O2 for 1 h to induce oxidative damage. The results indicated that adding L-carnitine at concentrations of 0.01-1 mM into the medium for 12 h significantly increased cell viability. Pre-treatment with L-carnitine at concentrations of 0.1-5 mM for 12 h significantly inhibited 2.5 mM H2O2-induced cell viability loss. The significant decreases in the level of reactive oxygen species and cell apoptosis were observed in 0.5 mM L-carnitine group compared to the H2O2 group. Malondialdehyde values of all of the L-carnitine groups were significantly lower than those of the H2O2 group, while total glutathione levels of all of the L-carnitine groups were significantly higher than of the H2O2 group. The activity of antioxidant enzymes, such as total superoxide dismutase (0.1 and 0.5 mM L-carnitine), catalase (0.5 mM L-carnitine) and γ-glutamyl cysteine synthetase (0.5 and 1 mM L-carnitine), was significantly increased. In addition, pre-treatment of L-carnitine in GCO cells exposed to 2.5 mM H2O2 significantly increased the mRNA expression of copper, zinc superoxide dismutase, catalase (0.5 mM L-carnitine), glutamate cysteine ligase catalytic subunit (0.1-1 mM) and glutathione peroxidase (0.1 mM L-carnitine). In conclusion, L-carnitine promotes GCO cell growth and improves antioxidant function, it plays a protective role against oxidative stress induced by H2O2 in GCO cells, and the appropriate supplemental amount of L-carnitine is 0.1-1 mM.

Effects of antimicrobial peptides (AMPs) on blood biochemical parameters, antioxidase activity, and immune function in the common carp (Cyprinus carpio).

Antibiotic use in livestock feed additives has resulted in harmful residue accumulation and spread of drug-resistance. We examined the use of antimicrobial peptides (AMPs) as a safer alternative to antibiotics in feeding the common carp. AMPs were added to common carp basal diets (Control) as additives at four concentrations: 100 mg kg(-1) (B1), 200 mg kg(-1) (B2), 400 mg kg(-1) (B3), 600 mg kg(-1) (B4) by dry weight of basal diet. After a 60-day feeding experiment, the final weight, DG and SGR of carps on B1, B2 and B3 diet were significantly higher than the control (p < 0.05). The FCR of carps on B1, B2 and B3 diet were significantly lower than the control (p < 0.05). Carps on B2, B3, and B4 diets showed significantly lower (p < 0.05) levels of triglyceride than the control. B4-fed carps showed significantly lower (p < 0.05) levels of total protein, albumin, and total cholesterol than the control. However there was no remarkable difference (p > 0.05) in levels of uric ammonia, globulin, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, lactic dehydrogenase and blood glucose in all groups. The serum superoxide dismutase and catalase activity of B1-fed carps was significantly higher (p < 0.05) than the control and B4-fed carps. The serum alkaline phosphate activity of carps on B1 diets was significantly higher (p < 0.05) than B4-fed carps. The serum acid phosphatase activity of B1-fed carps was significantly higher (p < 0.05) than the control and other antimicrobial peptide-fed groups. The serum lysozyme activity of carps on B1, B2, and B3 diets was significantly higher (p < 0.05) than the control- and B4-fed carps. Regarding immune factors in serum, the levels of immunoglobulin (Ig) and interleukin (IL)-1ß in B1-fed carps were significantly higher (p < 0.05) than the control and other groups, while IL-1α levels in B1-fed carps was significantly higher (p < 0.05) than the control-, B2-, and B3-fed carps. Furthermore, there were no significant differences in the content of MHC among the five groups. In conclusion, antimicrobial peptide can reduce triglyceride levels in serum, enrich oxidation resistance, and improve immunity of the common carp. It showed that appropriate concentration of antibacterial peptide as supplements in diets for common carp increased the final weight, DG, SGR and decreased the FCR.

[Protective effects of inhaled hydrogen gas on cognitive function in mice with sepsis-associated encephalopathy].

OBJECTIVE: To evaluate protective effects of inhaled hydrogen gas (H2) on cognitive function in a murine model of sepsis-associated encephalopathy (SAE). METHODS: A total of 84 male ICR mice, weighing 20-25 g, aged 6-8 weeks, were randomly divided into 4 groups of sham, sham+H2, sepsis and sepsis+H2. Sepsis was established by cecal ligation and puncture (CLP). Mice in sham+H2 and sepsis+H2 groups received 2% H2 inhalation for 1 h at 1 h and 6 h after sham operation or CLP operation respectively. The changes of neurological function and neuronal damage in hippocampal CA1 region were observed at 24 h post-operation. The activities of superoxide dismutase (SOD) and catalase (CAT) and the levels of malondialdehyde (MDA) and 8-iso-prostaglandin F2α (8-iso-PGF2α) in sera and hippocampus were detected at 24 h post-operation. The changes of cognitive function were observed by Y-maze test and fear conditional test at days 3 to 14 post-operation. RESULTS: Compared with sham group, the neurological function significantly declined and neurons in hippocampal CA1 region were significantly damaged; the activities of SOD and CAT markedly decreased while the levels of MDA and 8-iso-PGF2α markedly increased in sera and hippocampus; the time in new zone and the percentage of freezing time dramatically decreased at days 3 to 14 post-operation in sepsis group (P < 0.05) . Compared with sepsis group, neurological function significantly improved and damaged neurons in hippocampal CA1 region significantly reduced; the activities of SOD and CAT markedly increased and the levels of MDA and 8-iso-PGF2α markedly decreased in sera and hippocampus; the time in new zone and the percentage of freezing time dramatically increased at days 3 to 14 post-operations in sepsis+H2 group (P < 0.05). CONCLUSION: H2 inhalation can significantly alleviate neuronal damage and improve cognitive dysfunction in CLP-induced SAE mice. And it is probably associated with the increased activities of antioxidant enzymes and the reduced levels of oxidative products.

Scenario-based assessment of emergency management of urban infectious disease outbreaks.

Infectious diseases pose a severe threat to human health and are accompanied by significant economic losses. Studies of urban outbreaks of infectious diseases are diverse. However, previous studies have neglected the identification of critical events and the evaluation of scenario-based modeling of urban infectious disease outbreak emergency management mechanisms. In this paper, we aim to conduct an empirical analysis and scenario extrapolation using a questionnaire survey of 18 experts, based on the CIA-ISM method and scenario theory, to identify the key factors influencing urban infectious disease outbreaks. Subsequently, we evaluate the effectiveness of urban infectious disease outbreak emergency management mechanisms. Finally, we compare and verify the actual situation of COVID-19 in China, drawing the following conclusions and recommendations. (1) The scenario-based urban infectious disease emergency management model can effectively replicate the development of urban infectious diseases. (2) The establishment of an emergency command center and the isolation and observation of individuals exposed to infectious diseases are crucial factors in the emergency management of urban outbreaks of infectious disease.