Dissecting climate change risk and financial market instability: Implications for ecological risk management.

This research investigates the impact of climate challenges on financial markets by introducing an innovative approach to measure climate risk, specifically the aggregate climate change concern (ACCC) index. The study aims to assess and quantify the potential influence of climate change and risk-related factors on the performance and dynamics of financial markets. In this paper, concern is defined as the attention paid to the risk of climate change and the associated negative consequences. The findings demonstrate that the aggregate index exhibits robust predictability of market risk premiums, both within the sample and out-of-sample. By comparison, the index contains additional information beyond 14 economic predictors and 12 risk/uncertainty indexes in forecasting stock market return. In addition, the index proves valuable for mean-variance investors in asset allocation, leading to significant economic gains. The study identifies the index's ability to capture the reversal of temporary price crashes caused by overreactions to climate change risk. Furthermore, it exhibits stronger return forecasting capability for green stocks, non-state-owned enterprise (non-SOE) stocks, and stocks in regions with low air pollution. Particularly during periods of low air pollution and relaxed regulation, the index displays an enhanced ability to forecast returns. The study's findings provide valuable insights for policymakers and financial institutions as they address 21st-century environmental challenges. Moreover, these findings can inform the design of adaptive measures and interventions aimed at mitigating ecological risks and promoting sustainable economic growth.

Combined Therapies of Modified Taiyi Miraculous Moxa Roll and Cupping for Patients with Lumbar Intervertebral Disc Herniation.

Lumbar intervertebral disc herniation is a kind of syndrome caused by stimulation or pressure of nerve root and cauda equina due to intervertebral disc disorder, fibrous ring rupture, and pulpiform nucleus protrusion. Application of traditional Chinese medicine (TCM) including acupuncture therapy and cupping therapy is unique and effective treatment for lumbar intervertebral disc herniation in China. Hence, we try to investigate the combined clinical efficacy of modified Taiyi miraculous moxa roll and cupping therapy on patients with lumbar intervertebral disc herniation. Seventy patients were randomly assigned into combined treatment group (n = 35) and control group (n = 35). The treatment group received combined therapy of modified Taiyi miraculous moxa roll and cupping therapy, while control group received acupuncture therapy alone. Diagnostic criteria of TCM syndrome, Japanese Orthopedic Association (JOA) score, and simplified McGill pain questionnaire (MPQ) were used to evaluate the therapy. 11 and 13 out of 35 subjects in the combined treatment group had improvement > 75% and between 50% and 75%, respectively. The corresponding number was 2 and 22 of 35 subjects in the acupuncture group. There was significant difference in the clinical efficacy between the treatment group and control group (P = 0.036). The scores of JOA and MPQ detected in the patients of the two groups (P < 0.05) also showed statistically significant differences. Moreover, no serious adverse events occurred in the patients, who received cupping therapy or acupuncture. The combined or alone therapies can effectively improve the treatment efficacy in the patients with lumbar intervertebral disc herniation, while the combined therapies show more comparative effectiveness. Furthermore, the combined therapies are potentially safe and cost-effective and also benefit the improvement of short-term pain. Therefore, the combined therapies of the two ancient TCM deserve further clinical applications.

The Antitumor Effects of Vaccine-Activated CD8+ T Cells Associate with Weak TCR Signaling and Induction of Stem-Like Memory T Cells.

To understand why vaccine-activated tumor-specific T cells often fail to generate antitumor effects, we studied two α-fetoprotein-specific CD8+ T cells (Tet499 and Tet212) that had different antitumor effects. We found that Tet499 required high antigen doses for reactivation, but could survive persistent antigen stimulation and maintain their effector functions. In contrast, Tet212 had a low threshold of reactivation, but underwent exhaustion and apoptosis in the presence of persistent antigen. In vivo, Tet499 cells expanded more than Tet212 upon reencountering antigen and generated stronger antitumor effects. The different antigen responsiveness and antitumor effects of Tet212 and Tet499 cells correlated with their activation and differentiation states. Compared with Tet212, the population of Tet499 cells was less activated and contained more stem-like memory T cells (Tscm) that could undergo expansion in vivo The TCR signaling strength on Tet499 was weaker than Tet212, correlating with more severe Tet499 TCR downregulation. Weak TCR signaling may halt T-cell differentiation at the Tscm stage during immune priming and also explains why Tet499 reactivation requires a high antigen dose. Weak TCR signaling of Tet499 cells in the effector stage will also protect them from exhaustion and apoptosis when they reencounter persistent antigen in tumor lesion, which generates antitumor effects. Further investigation of TCR downregulation and manipulation of TCR signaling strength may help design cancer vaccines to elicit a mix of tumor-specific CD8+ T cells, including Tscm, capable of surviving antigen restimulation to generate antitumor effects. Cancer Immunol Res; 5(10); 908-19. ©2017 AACR.

A comprehensive proteogenomic study of the human Brucella vaccine strain 104 M.

BACKGROUND: Brucella spp. are Gram-negative, facultative intracellular pathogens that cause brucellosis in both humans and animals. The B. abortus vaccine strain 104 M is the only vaccine available in China for the prevention of brucellosis in humans. Although the B. abortus 104 M genome has been fully sequenced, the current genome annotations are not yet complete. In addition, the main mechanisms underpinning its residual toxicity and vaccine-induced immune protection have yet to be elucidated. Mapping the proteome of B. abortus 104 M will help to improve genome annotation quality, thereby facilitating a greater understanding of its biology. RESULTS: In this study, we utilized a proteogenomic approach that combined subcellular fractionation and peptide fractionation to perform a whole-proteome analysis and genome reannotation of B. abortus 104 M using high-resolution mass spectrometry. In total, 1,729 proteins (56.3% of 3,072) including 218 hypothetical proteins were identified using the culture conditions that were employed this study. The annotations of the B. abortus 104 M genome were also refined following identification and validation by reverse transcription-PCR. In addition, 14 pivotal virulence factors and 17 known protective antigens known to be involved in residual toxicity and immune protection were confirmed at the protein level following induction by the 104 M vaccine. Moreover, a further insight into the cell biology of multichromosomal bacteria was obtained following the elucidation of differences in protein expression levels between the small and large chromosomes. CONCLUSIONS: The work presented in this report used a proteogenomic approach to perform whole-proteome analysis and genome reannotation in B. abortus 104 M; this work helped to improve genome annotation quality. Our analysis of virulence factors, protective antigens and other protein effectors provided the basis for further research to elucidate the mechanisms of residual toxicity and immune protection induced by the 104 M vaccine. Finally, the potential link between replication dynamics, gene function, and protein expression levels in this multichromosomal bacterium was detailed.

Recombinant HSA-CMG2 Is a Promising Anthrax Toxin Inhibitor.

Anthrax toxin is the major virulence factor produced by Bacillus anthracis. Protective antigen (PA) is the key component of the toxin and has been confirmed as the main target for the development of toxin inhibitors. The inhibition of the binding of PA to its receptor, capillary morphogenesis protein-2 (CMG2), can effectively block anthrax intoxication. The recombinant, soluble von Willebrand factor type A (vWA) domain of CMG2 (sCMG2) has demonstrated potency against anthrax toxin. However, the short half-life of sCMG2 in vivo is a disadvantage for its development as a new anthrax drug. In the present study, we report that HSA-CMG2, a protein combining human serum albumin (HSA) and sCMG2, produced in the Pichia pastoris expression system prolonged the half-life of sCMG2 while maintaining PA binding ability. The IC50 of HSA-CMG2 is similar to those of sCMG2 and CMG2-Fc in in vitro toxin neutralization assays, and HSA-CMG2 completely protects rats from lethal doses of anthrax toxin challenge; these same challenge doses exceed sCMG2 at a sub-equivalent dose ratio and overwhelm CMG2-Fc. Our results suggest that HSA-CMG2 is a promising inhibitor of anthrax toxin and may contribute to the development of novel anthrax drugs.

Targeting the WASF3-CYFIP1 Complex Using Stapled Peptides Suppresses Cancer Cell Invasion.

Activation of the WASF3 protein by extracellular stimuli promotes actin cytoskeleton reorganization and facilitates cancer cell invasion, whereas WASF3 depletion suppresses invasion and metastasis. In quiescent cells, the interaction between WASF3 and a complex of proteins, including CYFIP1, acts as a conformational restraint to prevent WASF3 activation. Therefore, we took advantage of this endogenous regulatory mechanism to investigate potential sites that disrupt WASF3 function. Here, we show that genetic knockdown of CYFIP1 in cancer cells led to the destabilization of the WASF3 complex, loss of WASF3 function, and suppressed invasion. Based on existing crystallographic data, we developed stapled peptides, referred to as WASF Helix Mimics (WAHM), that target an α-helical interface between WASF3 and CYFIP1. Treatment of highly invasive breast and prostate cancer cells with WAHM inhibitor peptides significantly reduced motility and invasion in vitro. Mechanistic investigations revealed that these inhibitors suppressed the interaction between Rac and the WASF3 complex, which has been shown to promote cell migration. Furthermore, peptide-mediated inhibition of WASF3 also resulted in the dysregulation of known downstream targets such as MMP-9 and KISS1. Finally, we demonstrate that this invasive phenotype is specific to WASF3 as depletion of WASF1 and WASF2, which can also bind to CYFIP1, did not affect invasion. Collectively, our findings suggest that targeting WASF3 function with WAHM peptides could represent a promising therapeutic strategy for preventing tumor invasion and metastasis.

Deletion modification enhances anthrax specific immunity and protective efficacy of a hepatitis B core particle-based anthrax epitope vaccine.

Protective antigen (PA) is one of the major virulence factors of anthrax and is also the major constituent of the current anthrax vaccine. Previously, we found that the 2ß2-2ß3 loop of PA contains a dominant neutralizing epitope, the SFFD. We successfully inserted the 2ß2-2ß3 loop of PA into the major immunodominant region (MIR) of hepatitis B virus core (HBc) protein. The resulting fusion protein, termed HBc-N144-PA-loop2 (HBcL2), can effectively produce anthrax specific protective antibodies in an animal model. However, the protective immunity caused by HBcL2 could still be improved. In this research, we removed amino acids 79-81 from the HBc MIR of the HBcL2. This region was previously reported to be the major B cell epitope of HBc, and in keeping with this finding, we observed that the short deletion in the MIR not only diminished the intrinsic immunogenicity of HBc but also stimulated a higher titer of anthrax specific immunity. Most importantly, this deletion led to the full protection of the immunized mice against a lethal dose anthrax toxin challenge. We supposed that the conformational changes which occurred after the short deletion and foreign insertion in the MIR of HBc were the most likely reasons for the improvement in the immunogenicity of the HBc-based anthrax epitope vaccine.

[ESX-1 secreted protein ESAT-6 of Mycobacterium tuberculosis enhances the phagocytosis of RAW264.7 macrophages].

OBJECTIVE: To investigate the effects of Mycobacterium tuberculosis ESX-1 protein early secreted antigenic target of 6 kDa (ESAT-6) in modulating phagocytosis of RAW264.7 cells. METHODS: RAW264.7 cells were transfected with recombinant plasmids pFLAG-ESAT-6 and pFLAG-EGFP by liposome. After screening with a high level of G418, the macrophage cell lines stably expressing flag-ESAT-6 or flag-EGFP proteins were obtained. The cell lines were further identified by PCR, RT-PCR and western blot. The phagocytosis of those cell lines was analyzed for ingested fluorescent beads by flow cytometry and for phagocytized Escherichia coli (E. coli) by colony count and confocalmicroscopy. RESULTS: We established successfully RAW-E6 cell line stably expressing flag-ESAT-6 and RAW-EGFP cell line stably expressing flag-EGFP. Flow cytometric analysis shows that the percentage of phagocytosis of RAW-E6 was higher than that of RAW264.7 and RAW-EGFP. Colony count and confocal microscopy test also show that RAW-E6 had higher phagocytosis ability than RAW264.7 and RAW-EGFP. CONCLUSION: The secreted protein ESAT-6 can enhance the phagocytosis of macrophages, which provides new evidence to understand the pathogenesis of Mycobacterium tuberculosis.

Intracellular delivery of artificial transcription factors fused to the protein transduction domain of HIV-1 Tat.

Protein transduction domains (PTDs), such as the TAT peptide derived from HIV Tat protein, may transduce macromolecules into cells. In the present study, the TAT peptide-fused artificial transcription factors (ATFs) were generated by fusion of the N-terminal TAT peptide with SV40 promoter-targeted three-fingered C2H2 zinc finger proteins and the KRAB transcriptional repression domain. The fusion proteins were then expressed in an E .coli system and purified by Ni-NTA affinity chromatography. The purified fusion proteins were tested on mammalian cell lines CHO DG44 and L929. TAT-ATF-S, which contains the zinc fingers that bind to the SV40 promoter with high specificity, exhibited the desired transcriptional repression activity to the reported genes, indicating the successful cellular delivery and desired conformation of TAT-ATF-S. Our study has provided a new strategy for intracellular ATF delivery.

Human IgG subclasses against enterovirus Type 71: neutralization versus antibody dependent enhancement of infection.

The emerging human enterovirus 71 (EV71) represents a growing threat to public health, and no vaccine or specific antiviral is currently available. Human intravenous immunoglobulin (IVIG) is clinical used in treating severe EV71 infections. However, the discovery of antibody dependent enhancement (ADE) of EV71 infection illustrates the complex roles of antibody in controlling EV71 infection. In this study, to identify the distinct role of each IgG subclass on neutralization and enhancement of EV71 infection, different lots of pharmaceutical IVIG preparations manufactured from Chinese donors were used for IgG subclass fractionation by pH gradient elution with the protein A-conjugated affinity column. The neutralization and ADE capacities on EV71 infection of each purified IgG subclass were then assayed, respectively. The neutralizing activity of human IVIG is mainly mediated by IgG1 subclass and to less extent by IgG2 subclass. Interestingly, IgG3 fraction did not have neutralizing activity but enhanced EV71 infection in vitro. These results revealed the different roles of human IgG subclasses on EV71 infection, which is of critical importance for the rational design of immunotherapy and vaccines against severe EV71 diseases.

Secretory expression and efficient purification of recombinant anthrax toxin lethal factor with full biological activity in E. coli.

Lethal factor (LF), a virulence factor of Bacillus anthracis, plays key roles in anthrax pathogenesis and host-pathogen interactions. The detailed mechanisms by which LF contributes to infection are still under investigation. While these studies require pure, homogeneous and reliable LF preparations, most methods reported for production of recombinant LF (rLF) in B. anthracis or Escherichia coli either are complicated or add extra residues to the protein. In this work, we modified our previous method by codon optimization and chromatograph workflow refinement and developed an improved strategy for efficient production of rLF from the periplasm of E. coli. We were able to obtain fully functional rLF with a purity above 95% and with a considerable yield of 5 mg/L. The preparation was characterized by SDS-PAGE, Western blot, and N-terminal sequencing, and the activity was validated by intoxication of macrophages and Fischer 344 rats. Our final product is suitable for most research involving drug development and mechanism analysis of anthrax pathogenesis.

[Stable expression of Mycobacterium tuberculosis protein EspB in RAW264.7].

OBJECTIVE: We established a cell line stably expressing Mycobacterium tuberculosis secretory protein EspB in order to provide evidences for studying EspB in modulating the functions of macrophage. METHODS: The recombinant plasmid pEGFP-C1-EspB was first constructed, then RAW264.7 cell was transfected with pEGFP-C1-EspB and pEGFP-C1 by liposome respectively. After screening with a high level of G418, the macrophage cell lines that stably expressed EGFP-EspB fusion protein or EGFP were established. The gene and protein expression levels were further analyzed by RT-PCR, fluorescence microscopy and western blot. RESULTS: The EGFP-EspB fusion gene was integrated into the chromosome and the protein was stably expressed in the selected macrophage cell line. The macrophage cell lines that stably expressed EGFP-EspB fusion protein or EGFP were established. CONCLUSION: These results gave us a tool for the future study in the effects of EspB protein in modulating the functions of macrophage and its interaction with other molecules of macrophage.

[Establishment of RAW264.7 cell line stably expressing Mycobacterium tuberculosis protein ESAT-6].

For studying the effects of Mycobacterium tuberculosis secretory protein ESAT-6 on the related functions of macrophages, RAW264.7 cells were transfected with pEGFP-C1-ESAT-6 and pEGFP-C1 by liposome respectively. After screening with a high level of G418, the macrophage cell lines that stably expressed EGFP-ESAT-6 fusion protein or EGFP were established. The gene and protein expression levels were further analyzed by RT-PCR, fluorescence microscopy and Western blotting. The results indicated that the EGFP-ESAT6 fusion gene was integrated into the chromosome and the protein could be stably expressed in the selected macrophage cell line. These results gave us a tool for the future study in the mechanisms of ESAT-6 protein in modulating the macrophage cells.

Antiviral potential of exogenous human omega interferon to inhibit pandemic 2009 A (H1N1) influenza virus.

The pandemic 2009 H1N1 influenza virus broke out in North America and spread rapidly throughout the world. The type I interferon (IFN) response represents one of the first lines of defense against influenza virus infections. In this study, the protective potential of human exogenous IFN-ω against pandemic 2009 A (H1N1) influenza virus was assessed both in vitro and in guinea pigs. The viral loads of pandemic 2009 A (H1N1) influenza virus strains A/California/04/2009 and A/Beijing/501/2009 were reduced by up to 5000-fold in Caco-2 cells by the addition of human IFN-ω. With daily intranasal treatment with human IFN-ω the viral load of pandemic 2009 A (H1N1) influenza virus strain A/California/04/2009 decreased by 1000-fold in lung tissues of guinea pigs. These results provide strong support for the application of human IFN-ω pretreatment to human influenza control.

High-level expression of Mycobacterium tuberculosis protein EspB in E. coli and preparation of novel anti-EspB monoclonal antibodies.

The ESX-1 secretion system plays a critical role in the virulence of Mycobacterium tuberculosis. The ESX-1 secreted protein EspB is cleaved close to its C-terminus during secretion and is necessary for inhibiting phagosome maturation. In this study, the EspB gene of M. tuberculosis H37Rv was cloned into the expression vector of pET21a(+) and was effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as a soluble form in cell lysate and was first purified by a column packed with Ni-NTA resin and then a column packed with DEAE-Sepharose Fast Flow matrix. Using the purified protein to immunize BALB/c mice, five monoclonal antibodies were produced. As shown by ELISA and immunoblotting, the five respective antibodies could recognize the EspB protein. These monoclonal antibodies will provide powerful reagents for further investigation of EspB protein functions.

Hepatitis B virus core particles displaying Mycobacterium tuberculosis antigen ESAT-6 enhance ESAT-6-specific immune responses.

Early secreted antigenic target-6 (ESAT-6), an important Mycobacterium tuberculosis T-cell antigen, is an attractive candidate antigen for tuberculosis subunit vaccine development. Because ESAT-6 has a low inherent immunogenicity, we used Hepatitis B virus core (HBc) protein as an immune carrier to enhance ESAT-6 immunogenicity. The ESAT-6 gene was inserted into the major immunodominant region of the HBc molecule by fusion PCR. The recombinant protein, HBc-ESAT-6 (HE6), was expressed in Escherichia coli, and electron microscopy confirmed the formation of virus-like particles. The immunogenicity of the chimeric particles was assessed in mice. Serological assays and in vitro Th1-biased cytokine assays found that immunization with HE6 particles elicited significantly higher ESAT-6-specific antibodies and CD4⁺/CD8⁺ T cell responses in mice compared to immunization with recombinant ESAT-6 protein. These data demonstrate the feasibility of HBc particles serving as an efficient immune carrier for ESAT-6 and suggest that HE6 has potential for use in a tuberculosis subunit vaccine.

Protection against anthrax and plague by a combined vaccine in mice and rabbits.

The protective antigen (PA) of Bacillus anthracis and the Fraction 1 Capsular Antigen (F1 antigen), V antigen of Yersinia pestis have been demonstrated to be potential immunogens and candidate vaccine sub-units against anthrax and plague respectively. In this study, the authors have investigated the antibody responses and the protective efficacy when the antigens were administered separately or in combination intramuscularly formulation adsorbed to an aluminum hydroxide adjuvant. Results show that immunized rF1 + rV and rPA antigen together was as effective as separately for induction of serological antibody response, and these titers were maintained for over 1 year in mice. An isotype analysis of the serum indicates that the co-administration of these antigens did not influence the antigen-specific IgG1/IgG2a ratio which was consistent with a Th2 bias. Furthermore, the combined vaccine comprising the protein antigens rF1 + rV + rPA has been demonstrated to protect mice from subcutaneous challenge with 10(7) colony-forming units (CFU) virulent Y. pestis strain, and to fully protect rabbit against subcutaneous challenge with 1.2x10(5) colony-forming units (CFU) virulent B. anthracis spores. These data show that the protective efficacy was unaffected when the antigens were administered in combination.

Fusion protein of Delta 27LFn and EFn has the potential as a novel anthrax toxin inhibitor.

PA-binding domain of LF (LFn) or PA-binding domain of EF (EFn) is the anthrax protective antigen (PA) binding domain of anthrax lethal factor (LF) or edema factor (EF). Here we show the development of a novel anthrax toxin inhibitor, fusion protein of N-terminal 27 amino acids deletion of LFn (Delta27LFn) and EFn. In a cell model of intoxication, fusion protein of Delta27LFn and EFn (Delta27LFn-EFn) was a 62-fold more potent toxin inhibitor than LFn or EFn, and this increased activity corresponded to a 39-fold higher PA-binding affinity by Biacore analysis. More importantly, Delta27LFn-EFn could protect the highly susceptible Fischer 344 rats from anthrax lethal toxin challenge. This work suggested that Delta27LFn-EFn has the potential as a candidate therapeutic agent against anthrax.

Chimeric hepatitis B virus core particles carrying an epitope of anthrax protective antigen induce protective immunity against Bacillus anthracis.

The major aim of present study is to develop and evaluate chimeric virus-like particles (VLPs) displaying a neutralizing epitope of anthrax protective antigen (PA) as a potential vaccine against anthrax. The truncated hepatitis B virus core (HBc) protein (aa 1-144) was used as a carrier, and the 2beta2-2beta3 loop of the PA domain 2 (aa 302-325) which has been shown contains a dominant neutralizing epitope was inserted into the major immunodominant region (MIR) of the HBc. The recombinant protein HBc-N144-PA-loop2 was expressed in Escherichia coli, and was able to form HBc-like particles confirmed by electron microscopy. The immunogenicity of these chimeric particles was evaluated in mice and guinea pigs. In mice the HBc-N144-PA-loop2 was able to induce PA-epitope specific antibodies; in guinea pigs it was able to induce PA-epitope specific antibodies and anthrax toxin-neutralizing antibodies regardless of whether alum adjuvant was used or not, and was able to partially protect the immunized guinea pigs against virulent anthrax spores challenge. This study suggests chimeric HBc particles carrying a neutralizing epitope of PA can induce protective immunity against Bacillus anthracis.

The 2beta2-2beta3 loop of anthrax protective antigen contains a dominant neutralizing epitope.

Anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. PA is the major component in the current anthrax vaccine, but the antigenic epitopes on it are not well-defined. We generated a pool of toxin-neutralizing anti-PA monoclonal antibodies (MAbs) to analyze the neutralizing epitopes of PA. Nine toxin-neutralizing MAbs obtained were found bound to three different domains of PA respectively, among which three MAbs with the strongest toxin-neutralizing activity recognized the same epitope within domain 2. This epitope was fine mapped to the chymotrypsin-sensitive site, (312)SFFD(315), in the 2beta(2)-2beta(3) loop of PA, using phage-displayed random peptide libraries and mutation analysis. The result demonstrated for the first time that the 2beta(2)-2beta(3) loop, which is involved in the transition of PA oligomers from prepore to pore, contains a dominant neutralizing epitope. This work contributes to the immunological and functional analysis of PA and offers perspective for the development of a new epitope vaccine against anthrax.