Targeting the chromatin structural changes of antitumor immunity.

Epigenomic imbalance drives abnormal transcriptional processes, promoting the onset and progression of cancer. Although defective gene regulation generally affects carcinogenesis and tumor suppression networks, tumor immunogenicity and immune cells involved in antitumor responses may also be affected by epigenomic changes, which may have significant implications for the development and application of epigenetic therapy, cancer immunotherapy, and their combinations. Herein, we focus on the impact of epigenetic regulation on tumor immune cell function and the role of key abnormal epigenetic processes, DNA methylation, histone post-translational modification, and chromatin structure in tumor immunogenicity, and introduce these epigenetic research methods. We emphasize the value of small-molecule inhibitors of epigenetic modulators in enhancing antitumor immune responses and discuss the challenges of developing treatment plans that combine epigenetic therapy and immunotherapy through the complex interaction between cancer epigenetics and cancer immunology.

Indirubin-3'-monoxime exhibits potent antiviral and anti-inflammatory effects against human adenoviruses in vitro and in vivo.

Human adenovirus (HAdV) infection is a major cause of respiratory disease, yet no antiviral drugs have been approved for its treatment. Herein, we evaluated the antiviral and anti-inflammatory effects of cyclin-dependent protein kinase (CDK) inhibitor indirubin-3'-monoxime (IM) against HAdV infection in cells and a transgenic mouse model. After evaluating its cytotoxicity, cytopathic effect reduction, antiviral replication kinetics, and viral yield reduction assays were performed to assess the anti-HAdV activity of IM. Quantitative real-time polymerase chain reaction (qPCR), quantitative reverse transcription PCR (qRT-PCR), and western blotting were used to assess the effects of IM on HAdV DNA replication, transcription, and protein expression, respectively. IM significantly inhibited HAdV DNA replication as well as E1A and Hexon transcription, in addition to significantly suppressing the phosphorylation of the RNA polymerase II C-terminal domain (CTD). IM mitigated body weight loss, reduced viral burden, and lung injury, decreasing cytokine and chemokine secretion to a greater extent than cidofovir. Altogether, IM inhibits HAdV replication by downregulating CTD phosphorylation to suppress viral infection and corresponding innate immune reactions as a promising therapeutic agent.

A novel humanized tri-receptor transgenic mouse model of HAdV infection and pathogenesis.

Human adenovirus (HAdV) is a highly virulent respiratory pathogen that poses clinical challenges in terms of diagnostics and treatment. Currently, no effective therapeutic drugs or prophylactic vaccines are available for HAdV infections. One factor contributing to this deficiency is that existing animal models, including wild-type and single-receptor transgenic mice, are unsuitable for HAdV proliferation and pathology testing. In this study, a tri-receptor transgenic mouse model expressing the three best-characterized human cellular receptors for HAdV (hCAR, hCD46, and hDSG2) was generated and validated via analysis of transgene insertion, receptor mRNA expression, and protein abundance distribution. Following HAdV-7 infection, the tri-receptor mice exhibited high transcription levels at the early and late stages of the HAdV gene, as well as viral protein expression. Furthermore, the tri-receptor mice infected with HAdV exhibited dysregulated cytokine responses and multiple tissue lesions. This transgenic mouse model represents human HAdV infection and pathogenesis with more accuracy than any other reported animal model. As such, this model facilitates the comprehensive investigation of HAdV pathogenesis as well as the evaluation of potential vaccines and therapeutic modalities for HAdV.

An outbreak of acute respiratory disease caused by HAdV-55 in Beijing, China, 2020.

Human adenoviruses (HAdVs) can cause acute respiratory diseases (ARDs) worldwide, and HAdV-55 is a reemergent pathogen in recent years. In the study, we investigated an outbreak of ARD at a school due to HAdV-55 in Beijing, China, during the early outbreak of coronavirus disease 2019 (COVID-19). The epidemic prevention team was dispatched to the school to collect epidemiologic data and nasopharyngeal samples. Then, real-time reverse transcription polymerase chain reaction (PCR) and multiplex PCR assays were used to detect severe acute respiratory syndrome coronavirus 2 and other respiratory pathogens, respectively. One representative HAdV-55 isolate was selected and submitted for whole-genome sequencing using a MiSeq system and the whole-genome phylogenetic tree was conducted based on the maximum likelihood method. The outbreak lasted from January 27 to February 6, 2020, and 108 students developed fever, among whom 60 (55.56%) cases were diagnosed with HAdV-55 infection in the laboratory using real-time PCR and 56 cases were hospitalized. All the confirmed cases had a fever and 11 cases (18.33%) presented with a fever above 39°C. Other main clinical symptoms included sore throat (43.33%) and headache (43.33%). We obtained and assembled the full genome of one isolate, BJ-446, with 34 761 nucleotides in length. HAdV-55 isolate BJ-446 was 99.85% identical to strain QS-DLL, which was the first HAdV-55 strain in China isolated from an ARD outbreak in Shanxi in 2006. One and four amino acid mutations were observed in the hexon gene and the coding region of L2 pV 40.1 kDa protein, respectively. We identified the first HAdV-55 infection associated with the ARD outbreak in Beijing since the emergence of COVID-19. The study suggests that improved surveillance of HAdV is needed, although COVID-19 is still prevalent in the world.

Structural Modifications and Biological Activities of Natural α- and ß-Cembrenediol: A Comprehensive Review.

As one of the most characteristic ingredients of glandular trichome secretions from Nicotiana tabacum L. (tobacco), natural cembrenediols, namely, (1S,2E,4S,6R,7E,11E)-2,7,11-cembratriene-4,6-diol (α-cembrenediol/α-CBD) and its C-4 epimer (ß-cembrenediol/ß-CBD), have attracted considerable attention for their potent antitumor, neuroprotective, antimicrobial, and other activities. Many researchers are committed to exploring the possibility of utilizing these two cembrenediols and their derivatives both in human medicine and in agricultural fungicides. To the best of our knowledge, this review is the first to provide a comprehensive summary of the chemical modifications and bioactivities of α- and ß-CBD from their discovery to the present day; the review highlights their potential medicinal value for humans. The extensive references from 1962 to 2022 provided herein were systematically gathered from the SciFinder, Web of Science, and Google Scholar databases. We expect this review to assist in providing practical ideas for future drug development based on α- and ß-CBD and in further facilitating the utilization of the tobacco cembrenediols.

Pneumonia Patients Caused by Co-infection With SARS-CoV-2 and Human Adenovirus in China.

Objectives: To data, no patients with obvious epidemiological relationship co-infected with SARS-CoV-2 and other pathogens have been reported. Here, we investigated 10 patients caused by co-infection with SARS-CoV-2 and human adenovirus (HAdV), resulting in third-generation transmission. Materials and Methods: From Jan 15, 2020, we enrolled 10 patients with pneumonia in Hunan Province, China. Epidemiological, clinical, and laboratory investigation results from these patients were analyzed. An epidemiological investigation was performed to assess whether patient infections were linked using conventional methods and metagenomic sequencing. Results: The presence of co-infection with SARS-CoV-2 and HAdV was determined via RT-PCR and metagenomic sequencing. Phylogenetic analysis revealed that SARS-CoV-2 and HAdV genomes clustered together, with similar genetic relationships. The first patient likely became co-infected during meetings or travel in Wuhan. The patient transmitted the virus via dinners and meetings, which resulted in four second-generation cases. Then, a second-generation case transmitted the virus to her family members or relatives via presymptomatic transmission. Conclusions: This study described an example of co-infection with SARS-CoV-2 and HAdV in pneumonia patients, which caused third-generation cases and inter-regional transmission via meetings, household interactions, and dinner parties. We also observed the persistent and presymptomatic transmission of co-infection, which has the potential to make the continued control of the COVID-19 pandemic challenging. Continuous surveillance is needed to monitor the prevalence, infectivity, transmissibility, and pathogenicity of SARS-CoV-2 co-infection with other pathogens to evaluate its real risk.

Circulation of HAdV-41 with diverse genome types and recombination in acute gastroenteritis among children in Shanghai.

Human adenovirus F (HAdV-F) is one of the major causative species detected in acute gastroenteritis in children worldwide. HAdV-F is composed of serotypes 40 and 41. Most studies have reported the prevalence of HAdV-41 and focused on its epidemiologic characteristics. In this study, seventeen samples were identified as HAdV-41 out of 273 fecal specimens from children with acute diarrhea in Shanghai. Five isolates were isolated and subjected to whole genome sequencing and analysis to characterize the genetic variation and evolution. Full genome analysis revealed low genetic variation (99.07-99.92% identity) among the isolates, and InDels are observed in the E2A gene and the hexon gene compared to the reference strain NIVD103. Phylogenetic analysis showed that all isolates mainly formed two genome-type clusters but with incongruence in the trees of whole genomes and individual genes. The recombination breakpoints of the five isolates were inferred by the Recombination Detection Program (RDP) and varied in the number and location of the recombination events, indicating different evolution origins. Overall, our study highlights the genetic diversity of HAdV-41 isolates circulating in Shanghai, which may have evolved from inter-strain recombination.

Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9.

The presence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the permanent integration of HBV DNA into the host genome confers the risk of viral reactivation and hepatocellular carcinoma. Nucleoside/nucleotide analogs alone have little or no capacity to eliminate replicative HBV templates consisting of cccDNA or integrated HBV DNA. Recently, CRISPR/Cas9 technology has been widely applied as a promising genome-editing tool, and HBV-specific CRISPR-Cas9 systems were shown to effectively mediate HBV cccDNA disruption. However, the integrated HBV DNA fragments are considered as important pro-oncogenic properties and it serves as an important template for viral replication and expression in stable HBV cell line. In this study, we completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV cccDNA in a stable HBV cell line. In HBV-excised cell line, the HBV cccDNA inside cells, supernatant HBV DNA, HBsAg, and HBeAg remained below the negative critical values for more than 10 months. Besides, by whole genome sequencing, we analyzed off-target effects and excluded cell contamination. It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line. These findings demonstrate that the CRISPR-Cas9 system is a potentially powerful tool capable of promoting a radical or "sterile" HBV cure.

An outbreak of acute respiratory disease caused by a virus associated RNA II gene mutation strain of human adenovirus 7 in China, 2015.

Human adenovirus 7 (HAdV-7) strains are a major cause of acute respiratory disease (ARD) among adults and children, associated with fatal pneumonia. An ARD outbreak caused by HAdV-7 that involved 739 college students was reported in this article. To better understand the underlying cause of this large-scale epidemic, virus strains were isolated from infected patients and sequence variations of the whole genome sequence were detected. Evolutionary trees and alignment results indicated that the major capsid protein genes hexon and fibre were strongly conserved among serotype 7 strains in China at that time. Instead, the HAdV-7 strains presented three thymine deletions in the virus associated RNA (VA RNA) II terminal region. We also found that the mutation might lead to increased mRNA expression of an adjacent gene, L1 52/55K, and thus promoted faster growth. These findings suggest that sequence variation of VA RNA II gene was a potential cause of such a severe HAdV-7 infection and this gene should be a new-emerging factor to be monitored for better understanding of HAdV-7 infection.

Inhibition of TLR4 signaling by Brucella TIR-containing protein TcpB-derived decoy peptides.

Brucella spp. avoid host immune recognition and thus, weaken the immune response to infection. The Toll/interleukin-1 receptor (TIR) domain-containing protein (TcpB/Btp1) of Brucella spp. is thought to be involved in blocking host innate immune responses by binding to adaptors downstream of Toll-like receptors. In this study, based on the observation that TcpB binds to the host target proteins, MAL, through the TIR domain, we examined decoy peptides from TcpB TIR domains and found that TB-8 and TB-9 substantially inhibit lipopolysaccharide (LPS)-induced signaling in vitro and in vivo. Both these peptides share a common loop, the DD loop, indicating a novel structural region mediating TIR interactions. The inhibition of LPS signaling by TB-8 and TB-9 shows no preference to MyD88-dependent cytokines, such as TNF-α and IL-1ß or TRIF-dependent cytokines including IFN-ß and IL-6. Furthermore, these two peptides rescue the virulence of Brucella ΔtcpB mutants at the cellular level, indicating key roles of the DD loop in Brucella pathogenesis. In conclusion, identification of inhibitors from the bacterial TIR domains is helpful not only for illustrating interacting mechanisms between TIR domains and bacterial pathogenesis, but also for developing novel signaling inhibitors and therapeutics for human inflammatory diseases.

Large-scale identification of small noncoding RNA with strand-specific deep sequencing and characterization of a novel virulence-related sRNA in Brucella melitensis.

Brucella is the causative agent of brucellosis, a worldwide epidemic zoonosis. Small noncoding RNAs (sRNAs) are important modulators of gene expression and involved in pathogenesis and stress adaptation of Brucella. In this study, using a strand-specific RNA deep-sequencing approach, we identified a global set of sRNAs expressed by B. melitensis 16M. In total, 1321 sRNAs were identified, ranging from 100 to 600 nucleotides. These sRNAs differ in their expression levels and strand and chromosomal distributions. The role of BSR0441, one of these sRNAs, in the virulence of B. melitensis 16M was further characterized. BSR0441 was highly induced during the infection of macrophages and mice. The deletion mutant of BSR0441 showed significantly reduced spleen colonization in the middle and late phases of infection. The expression of the BSR0441 target mRNA genes was also altered in the BSR0441 mutant strain during macrophage and mice infection, which is consistent with its reduced intracellular survival capacity. In summary, Brucella encodes a large number of sRNAs, which may be involved in the stress adaptation and virulence of Brucella. Further investigation of these regulators will extend our understanding of the Brucella pathogenesis mechanism and the interactions between Brucella and its hosts.

Impact of Hfq on global gene expression and intracellular survival in Brucella melitensis.

Brucella melitensis is a facultative intracellular bacterium that replicates within macrophages. The ability of brucellae to survive and multiply in the hostile environment of host macrophages is essential to its virulence. The RNA-binding protein Hfq is a global regulator that is involved in stress resistance and pathogenicity. Here we demonstrate that Hfq is essential for stress adaptation and intracellular survival in B. melitensis. A B. melitensis hfq deletion mutant exhibits reduced survival under environmental stresses and is attenuated in cultured macrophages and mice. Microarray-based transcriptome analyses revealed that 359 genes involved in numerous cellular processes were dysregulated in the hfq mutant. From these same samples the proteins were also prepared for proteomic analysis to directly identify Hfq-regulated proteins. Fifty-five proteins with significantly affected expression were identified in the hfq mutant. Our results demonstrate that Hfq regulates many genes and/or proteins involved in metabolism, virulence, and stress responses, including those potentially involved in the adaptation of Brucella to the oxidative, acid, heat stress, and antibacterial peptides encountered within the host. The dysregulation of such genes and/or proteins could contribute to the attenuated hfq mutant phenotype. These findings highlight the involvement of Hfq as a key regulator of Brucella gene expression and facilitate our understanding of the role of Hfq in environmental stress adaptation and intracellular survival of B. melitensis.

The 16MΔvjbR as an ideal live attenuated vaccine candidate for differentiation between Brucella vaccination and infection.

Brucellosis brings great economic burdens for developing countries. Live attenuated vaccines are the most efficient means for prevention and control of animal Brucellosis. However, the difficulties of differentiating of infection from vaccine immunization, which is essential for eradication programs, limit their applications. Therefore, the development of a vaccine that could differentiate infection from immunization will overcome the limitations and get extensive application. VjbR is a quorum sensing regulator involving in Brucella's intracellular survival. The vjbR∷Tn5 mutants have been proven effective against wild type strain challenge, implying its possibility of use in vaccine candidate development. To further evaluate this candidate gene, in the present study, the antigenicity of purified recombinant VjbR protein was analyzed. Antibodies to Brucella melitensis VjbR could be detected in sera from patients and animals with brucellosis but not in control ones, implying the potential use of this protein as a diagnostic antigen. Then a vjbR mutant of B. melitensis 16M was constructed by replacing the vjbR with kanamycin gene. The mutant showed reduced survival in macrophage and mice. Vaccination of BALB/c mice with 16MΔvjbR conferred significant protective immunity against B. melitensis strain 16M challenges, being equivalent to which induced by the license vaccine Rev.1. The vjbR deletion mutant elicited an anti-Brucella-specific immunoglobulin G response and induced the secretion of gamma interferon and interleukin-10. The most importance is that, the use of vjbR mutants as vaccines in association with diagnostic tests based on the VjbR antigen would allow the serological differentiation between infected and vaccinated animals. These results suggest that 16MΔvjbR is an ideal live attenuated vaccine candidate against B. melitensis and deserves further evaluation for vaccine development.

Cytotoxicity of Brucella smooth strains for macrophages is mediated by increased secretion of the type IV secretion system.

Some Brucella rough mutants cause cytotoxicity that resembles oncosis and necrosis in macrophages. This cytotoxicity requires the type IV secretion system (T4SS). In rough mutants, the cell-surface O antigen is shortened and the T4SS structure is thus exposed on the surface. Cytotoxicity effector proteins can therefore be more easily secreted. This enhanced secretion of effector proteins might cause the increased levels of cytotoxicity observed. However, whether this cytotoxicity is unique to the rough mutant and is mediated by overexpression of the T4SS has not been definitively determined. To test this, in the present study, a virB inactivation mutant (BMDeltavirB) and an overexpression strain (BM-VIR) of a smooth Brucella melitensis strain (BM) were constructed and their cytotoxicity for macrophages and intracellular survival capability were analysed and compared. Cytotoxicity was detected in macrophages infected with higher concentrations of strains BM or BM-VIR, but not in those infected with BMDeltavirB. The quorum sensing signal molecule N-dodecanoyl-dl-homoserine lactone (C(12)-HSL), a molecule that can inhibit expression of virB, inhibited the cytotoxicity of BM and BM-VIR, but not of BMDeltavirB. These results indicated that overexpression of virB is responsible for Brucella cytotoxicity in macrophages. Transcription analysis showed that virB is regulated in a cell-density-dependent manner both in in vitro culture and during macrophage infection. When compared with BM, BM-VIR showed a reduced survival capacity in macrophages and mice, but both strains demonstrated similar resistance to in vitro stress conditions designed to simulate intracellular environments. Taken together, the cytotoxicity of Brucella for macrophages is probably mediated by increased secretion of effector proteins that results from overexpression of virB or an increase in the number of bacterial cells. The observation that both inactivation and overexpression of virB are detrimental for Brucella intracellular survival also indicated that the expression of virB is tightly regulated in a cell-density-dependent manner.

[Construction of BP26 tagged vaccine strain and development of discriminating PCR for Brucella].

OBJECTIVE: The wide application of live attenuated vaccine strains is limited because of drawbacks of residual virulence, similar antigenenicity to virulent strain and the difficulty to differentiate vaccination and natural infection. In this study, we modified the vaccine strain to prevent the drawbacks. METHODS: By using homologous recombination, we replaced the BP26 gene with the kanamycin gene in a live attenuated vaccine strain M5. The new tagged vaccine strain, M5DeltaBP26, was generated. The wild type strain and M5DeltaBP26 were used to infect macrophage and mice to compare their intracellular survival capability. According to the conservative sequence of dnaK and the deleted region of BP26, primers were designed to develop a duplex PCR for discriminating the wild type strain and M5DeltaBP26. RESULTS: A new tagged strain, M5DeltaBP26, was successfully constructed. The tagged strain could survive in both macrophage and mice, indicating the feasibility as live attenuated vaccine strain. Results from mice infection showed that, at 2 weeks p.i., 10(2.9) CFU of Brucella were isolated from M5 infected mice, whereas only 10(1.1) CFU of Brucella were isolated from M5DeltaBP26 infected mice (P < 0.01). At 3 weeks p. i., 10(2.2) CFU of Brucella whereas no M5DeltaBP26 were isolated. These results indicated that infection capability of M5DeltaBP26 was decreased. Based on the sequence differences between M5DeltaBP26 and M5, a new discriminating duplex PCR was developed. With the duplex PCR, only one product was amplified from M5DeltaBP26, by which it can be differentiated from wild type and virulent strains. CONCLUSION: The construction of tagged strain and the development of discriminating PCR provide a new candidate for further vaccine development.

Comparative proteomics analyses reveal the virB of B. melitensis affects expression of intracellular survival related proteins.

BACKGROUND: Brucella melitensis is a facultative, intracellular, pathogenic bacterium that replicates within macrophages. The type IV secretion system encoded by the virB operon (virB) is involved in Brucella intracellular survival. However, the underlying molecular mechanisms, especially the target proteins affected by the virB, remain largely unclear. METHODOLOGY/PRINCIPAL FINDINGS: In order to define the proteins affected by virB, the proteomes of wild-type and the virB mutant were compared under in vitro conditions where virB was highly activated. The differentially expressed proteins were identified by MALDI-TOF-MS. Forty-four down-regulated and eighteen up-regulated proteins which exhibited a 2-fold or greater change were identified. These proteins included those involved in amino acid transport and metabolism, lipid metabolism, energy production, cell membrane biogenesis, translation, post-translational modifications and protein turnover, as well as unknown proteins. Interestingly, several important virulence related proteins involved in intracellular survival, including VjbR, DnaK, HtrA, Omp25, and GntR, were down-regulated in the virB mutant. Transcription analysis of virB and vjbR at different growth phase showed that virB positively affect transcription of vjbR in a growth phase dependent manner. Quantitative RT-PCR showed that transcription of these genes was also affected by virB during macrophage cell infection, consistent with the observed decreased survival of the virB mutant in macrophage. CONCLUSIONS/SIGNIFICANCE: These data indicated that the virB operon may control the intracellular survival of Brucella by affecting the expression of relevant proteins.