Important role of DNA methylation hints at significant potential in tuberculosis.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (Mtb) infection, has persisted as a major global public health threat for millennia. Until now, TB continues to challenge efforts aimed at controlling it, with drug resistance and latent infections being the two main factors hindering treatment efficacy. The scientific community is still striving to understand the underlying mechanisms behind Mtb's drug resistance and latent infection. DNA methylation, a critical epigenetic modification occurring throughout an individual's growth and development, has gained attention following advances in high-throughput sequencing technologies. Researchers have observed abnormal DNA methylation patterns in the host genome during Mtb infection. Given the escalating issue of drug-resistant Mtb, delving into the role of DNA methylation in TB's development is crucial. This review article explores DNA methylation's significance in human growth, development and disease, and its role in regulating Mtb's evolution and infection processes. Additionally, it discusses potential applications of DNA methylation research in tuberculosis.

Statistical and network analyses reveal mechanisms for the enhancement of macrophage immunity by manganese in Mycobacterium tuberculosis infection.

Tuberculosis is a significant infectious disease that poses a serious risk to human health. Our previous research has indicated that manganese ions reduce the bacterial load of Mycobacterium tuberculosis in macrophages, but the exact immune defense mechanism remains unknown. Several critical proteins and pathways involved in the host's immune response during this process are still unidentified. Our research aims to identify these proteins and pathways and provide a rationale for the use of manganese ions in the adjuvant treatment of tuberculosis. We downloaded GSE211666 data from the GEO database and selected the RM (Post-infection manganese ion treatment group) and Ra (single-infection group) groups for comparison and analysis to identify differential genes. These differential genes were then enriched and analyzed using STRING, Cytoscape, and NDEx tools to identify the two most relevant pathways of the "Host Response Signature Network." After conducting an in-depth analysis of these two pathways, we found that manganese ions mainly mediate (1) the interferon -gamma (IFN-γ) and its receptor IFNGR and the downstream JAK-STAT pathway and (2) the NFκB pathway to enhance macrophage response to interferon, autophagy, polarization, and cytokine release. Using qPCR experiments, we verified the increased expression of CXCL10, MHCII, IFNγ, CSF2, and IL12, all of which are cytokines that play a key role in resistance to Mycobacterium tuberculosis infection, suggesting that macrophages enter a state of pro-inflammatory and activation after the addition of manganese ions, which enhances their immunosuppressive effect against Mycobacterium tuberculosis. We conclude that our study provides evidence of manganese ion's ability to treat tuberculosis adjuvantly.

Manganese enhances macrophage defense against Mycobacterium tuberculosis via the STING-TNF signaling pathway.

The host cell antiviral response pathway depends heavily on manganese (Mn), but its role in defense against Mycobacterium tuberculosis (M. tuberculosis) infection is rarely reported. In this study, we found that, in H37Ra-infected macrophages, Mn2+ increases the phosphorylation of stimulator of interferon genes (STING) and P65, as well as triggers the phosphorylation cascade of tumor necrosis factor (TNF) signaling pathway proteins, signal-regulated kinase (ERK), P38, and c-Jun N-terminal kinase (JNK). The activation of the TNF signaling pathway stimulated the expression of downstream inflammatory factors TNF-α, C-X-C Motif Chemokine Ligand 10(CXCL10), CC Motif Chemokine Ligand 20(CCL20), Colony Stimulating Factor 1(CSF1), Colony Stimulating Factor 2(CSF2), and Jagged Canonical Notch Ligand 1(JAG1), thereby triggering a strong inflammatory response in the cells. The excessive accumulation of TNF-α in macrophages induces necroptosis and inhibits the survival of M. tuberculosis in macrophages. When we treated macrophages with the STING inhibitor H-151, the phosphorylation of P38 was reduced, and the secretion of downstream inflammatory factors TNF-α and CXCL10, CCL20, CSF1, and CSF2 were also inhibited. Overall, this study reveals that Mn2+ plays a crucial role in host cell defense against M. tuberculosis infection, contributes to a deeper understanding of pathogen-host interactions, and offers theoretical support for the use of Mn2+ as a drug cofactor for the treatment of tuberculosis and the development of a new generation of drugs and vaccine adjuvants.

Sulforaphane kills Mycobacterium tuberculosis H37Ra and Mycobacterium smegmatis mc2155 through a reactive oxygen species dependent mechanism.

Mycobacterium tuberculosis (M. tuberculosis) is a highly pathogenic intracellular pathogen that causes tuberculosis (TB), the leading cause of mortality from single infections. Redox homeostasis plays a very important role in the resistance of M. tuberculosis to antibiotic damage and various environmental stresses. The antioxidant sulforaphane (SFN) has been reported to exhibit anticancer activity and inhibit the growth of a variety of bacteria and fungi. Nonetheless, it remains unclear whether SFN exhibits anti-mycobacterial activity. Our results showed that the SFN against M. tuberculosis H37Ra exhibited bactericidal activity in a time and dose-dependent manner. The anti-tubercular activity of SFN was significantly correlated with bacterial reactive oxygen species (ROS) levels. In addition, SFN promoted the bactericidal effect of macrophages on intracellular bacteria in a dose-dependent manner, mediated by increasing intracellular mitochondrial ROS levels and decreasing cytoplasmic ROS levels. Taken together, our data revealed the previously unrecognized antimicrobial functions of SFN. Future studies focusing on the mechanism of SFN in macrophages against M. tuberculosis are essential for developing new host-directed therapeutic approaches against TB.

Class â histone deacetylase inhibitor regulate of Mycobacteria-Driven guanylate-binding protein 1 gene expression.

Guanylate-binding proteins (GBPs) are a class of interferon (IFN)-stimulated genes with well-established activity against viruses, intracellular bacteria, and parasites. The effect of epigenetic modification on GBP activity upon Mycobacterium tuberculosis (Mtb) infection is poorly understood. In this study, we found that Mtb infection can significantly increase the expression of GBPs. Class Ⅰ histone deacetylase inhibitor (HDACi) MS-275 can selectively inhibit GBP1 expression, ultimately affecting the release of inflammatory cytokines IL-1ß and suppressing Mtb intracellular survival. Moreover, interfering with GBP1 expression could reduce the production of IL-1ß and the level of cleaved-caspase-3 in response to Mtb infection. GBP1 silencing did not affect Mtb survival. Besides, using the bisulfite sequencing PCR, we showed that the CpG site of the GBP1 promoter was hypermethylated, and the methylation status of the GBP1 promoter did not change significantly upon Mtb infection. Overall, this study sheds light on the role of GBP in Mtb infection and provides a link between epigenetics and GBP1 activity.

The role of neoantigens in tumor immunotherapy.

Tumor neoantigens are aberrant polypeptides produced by tumor cells as a result of genomic mutations. They are also tumor-specific antigens (TSA). Neoantigens are more immunogenic than tumor-related antigens and do not induce autoimmunity. Based on the rapid development of bioinformatics and the continuous update of sequencing technology, cancer immunotherapy with tumor neoantigens has made promising breakthroughs and progress. In this review, the generation, prediction, and identification of novel antigens, as well as the individualized treatments of neoantigens, were first introduced. Secondly, the mechanism of Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) therapy and immune checkpoint blockade therapy in the treatment of tumors were outlined, and the three treatment methods were compared. Thirdly, the application of neoantigens in CAR-T therapy and PD-1/PD-L1 blockade therapy was briefly described. The benefits of the neoantigen vaccines over common vaccines were summarized as well. Finally, the prospect of neoantigen therapy was presented.

Pneumonia caused by Mycobacterium tuberculosis.

Tuberculosis (TB) is one of the top ten leading causes of death worldwide. The causative agent of TB is Mycobacterium tuberculosis. Acute tuberculous pneumonia (TP) is an acute form of pulmonary TB. However, acute TP and non-tuberculous community-acquired pneumonia can be easily confused, resulting in deterioration of TP due to delayed treatment. Therefore, rapid and accurate diagnosis of acute TP is crucial in order to stop the transmission of TB. Moreover, development of new diagnostic tools (technologies and approaches), and flexible application of different therapy schemes will help to reduce the incidence of TP and promote the goal of ending the TB epidemic.

Housecleaning of pyrimidine nucleotide pool coordinates metabolic adaptation of nongrowing Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis (Mtb) to adopt a slowly growing or nongrowing state within the host plays a critical role for the bacilli to persist in the face of a prolonged multidrug therapy, establish latency and sustain chronic infection. In our previous study, we revealed that genome maintenance via MazG-mediated elimination of oxidized dCTP contributes to the antibiotic tolerance of nongrowing Mtb. Here, we provide evidence that housecleaning of pyrimidine nucleotide pool via MazG coordinates metabolic adaptation of Mtb to nongrowing state. We found that the ΔmazG mutant fails to maintain a nongrowing and metabolic quiescence state under dormancy models in vitro. To investigate bacterial metabolic changes during infection, we employed RNA-seq to compare the global transcriptional response of wild-type Mtb and the ΔmazG mutant after infection of macrophages. Pathway enrichment analyses of the differentially regulated genes indicate that the deletion of mazG in Mtb not only results in DNA instability, but also perturbs pyrimidine metabolism, iron and carbon source uptake, catabolism of propionate and TCA cycle. Moreover, these transcriptional signatures reflect anticipatory metabolism and regulatory activities observed during cell cycle re-entry in the ΔmazG mutant. Taken together, these results provide evidence that pyrimidine metabolism is a metabolic checkpoint during mycobacterial adaptation to nongrowing state.

Oxidation of dCTP contributes to antibiotic lethality in stationary-phase mycobacteria.

Growing evidence shows that generation of reactive oxygen species (ROS) derived from antibiotic-induced metabolic perturbation contribute to antibiotic lethality. However, our knowledge of the mechanisms by which antibiotic-induced oxidative stress actually kills cells remains elusive. Here, we show that oxidation of dCTP underlies ROS-mediated antibiotic lethality via induction of DNA double-strand breaks (DSBs). Deletion of mazG-encoded 5-OH-dCTP-specific pyrophosphohydrolase potentiates antibiotic killing of stationary-phase mycobacteria, but did not affect antibiotic efficacy in exponentially growing cultures. Critically, the effect of mazG deletion on potentiating antibiotic killing is associated with antibiotic-induced ROS and accumulation of 5-OH-dCTP. Independent lines of evidence presented here indicate that the increased level of DSBs observed in the ΔmazG mutant is a dead-end event accounting for enhanced antibiotic killing. Moreover, we provided genetic evidence that 5-OH-dCTP is incorporated into genomic DNA via error-prone DNA polymerase DnaE2 and repair of 5-OH-dC lesions via the endonuclease Nth leads to the generation of lethal DSBs. This work provides a mechanistic view of ROS-mediated antibiotic lethality in stationary phase and may have broad implications not only with respect to antibiotic lethality but also to the mechanism of stress-induced mutagenesis in bacteria.

Differences in weathering pattern, stress resistance and community structure of culturable rock-weathering bacteria between altered rocks and soils.

In this study, we isolated and characterized rock-weathering bacteria from the surfaces of less and more altered tuffs, along with the adjacent soils, with respect to their rock weathering pattern, stress resistance, community structure, and the changes in these rocks and soils. Using a moderate-nutrition medium, we obtained 150 isolates from the rocks and soils. The rock-weathering patterns of the isolates were characterized using batch cultures that measure the quantity of Si, Al, and Fe released from tuff under aerobic conditions. Based on the potential of the bacterial influence on the element releases, the isolates could be grouped into highly, moderately, and least effective element solubilizers, respectively. Significantly more highly effective Al and Fe solubilizers were observed in the altered rocks, while the soils had more highly effective Si solubilizers. Furthermore, more isolates from the altered rocks significantly acidified the culture medium in the rock weathering process. Dynamic changes in the element release showed the distinct element releasing patterns of three selected isolates. More isolates from the altered rocks could grow at 4 °C or at 55 °C or at pH 4. Some isolates from the altered rocks could grow at pH 10 and with 10-15% (w/v) NaCl. The altered rocks and the soils existed in diverse and different highly weathering-specific culturable rock-weathering community structures. The changes in the culturable weathering communities between the altered rocks and the soils were attributable not only to major bacterial groups but also to a change in the minor population structure.

Antimicrobial Activity of Quinazolin Derivatives of 1,2-Di(quinazolin-4-yl)diselane against Mycobacteria.

Mycobacterium tuberculosis (M. tuberculosis) is one of the leading causes of morbidity and mortality. Currently, the emergence of drug resistance has an urgent need for new drugs. In previous study, we found that 1,2-di(quinazolin-4-yl)diselane (DQYD), a quinazoline derivative, has anticancer activities against many cancers. However, whether DQYD has the activity of antimycobacterium is still little known. Here our results show that DQYD has a similar value of the minimum inhibitory concentration with clinical drugs against mycobacteria and also has the ability of bacteriostatic activity with dose-dependent and time-dependent manner. Furthermore, the activities of DQYD against M. tuberculosis are associated with intracellular ATP homeostasis. Meanwhile, mycobacterium DNA damage level was increased after DQYD treatment. But there was no correlation between survival of mycobacteria in the presence of DQYD and intercellular reactive oxygen species. This study enlightens the possible benefits of quinazoline derivatives as potential antimycobacterium compounds and furtherly suggests a new strategy to develop new methods for searching antituberculosis drugs.

Cloning identification and functional analysis of human IL-17A promoter.

OBJECTIVE: To conduct the cloning identification and characterization of the sequence of human IL-17A promoter so as to analyze the regulatory mechanism of the gene expression of IL-17. METHODS: First of all, the potential promoter region of IL-17A was found by means of the bioinformatics methods. Then, it was cloned into the reporter vector with PCR technique. Finally, the activity of the test promoter was determined by dual luciferase reporter system. RESULTS: Two transcriptional start points of the upper region, 600 bp and 1000 bp, of IL-17A were obtained by PCR clone and proved to have certain activities by dual luciferase reporter system. Also, they could be activated by IL-17A activator STAT3, which could start the expression of the reported gene. CONCLUSIONS: Clone established the regulatory region of human IL-17A promoter, which provided bases to the subsequent function research.

NLRP3 Activation Was Regulated by DNA Methylation Modification during Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (Mtb) infection activates the NLRP3 inflammasome in macrophages and dendritic cells. Much attention has been paid to the mechanisms for regulation of NLRP3 against Mtb. However, whether epigenetic mechanisms participated in NLRP3 activation is still little known. Here we showed that NLRP3 activation was regulated by DNA methylation modification. Mtb infection promoted NLRP3 activation and inflammatory cytokines expression. NLRP3 promoter was cloned and subsequently identified by Dual-Luciferase Reporter System. The results showed that NLRP3 promoter activity was decreased after methylation by DNA methylase Sss I in vitro. Meanwhile, DNA methyltransferases inhibitor DAC could upregulate the expression of NLRP3. Furthermore, promoter region of NLRP3 gene was demethylated after Mtb H37Rv strain infection. These data revealed that DNA methylation was involved in NLRP3 inflammasome activation during Mtb infection and provided a new insight into the relationship between host and pathogens.

XAGE-1b expression is associated with the diagnosis and early recurrence of hepatocellular carcinoma.

XAGE-1b is a 470 bp transcript of the XAGE-1 gene, which belongs to the cancer-testis antigens that exhibit a restricted pattern of expression in normal tissues. Recently, the expression of XAGE-1b has been shown to be frequent in patients with hepatocellular carcinoma (HCC). However, the underlying mechanism is not fully understood. To investigate the role of XAGE-1b in HCC diagnosis and postoperative evaluation, the expression level of XAGE-1b was first examined in the tissue and peripheral blood of HCC patients and controls by using quantitative polymerase chain reaction. Subsequently, the associations between XAGE-1b and the clinical variables were assessed using χ2 or Kaplan-Meier tests. The data showed that HCC tissues had increased XAGE-1b expression when compared to paired non-tumorous tissues. The blood samples from the HCC patients showed upregulated XAGE-1b mRNA, as compared to non-HCC patients. The patients with portal vein tumor thrombus or higher tumor-node metastasis stages (II~IV) were more likely to have increased levels of XAGE-1b mRNA. Furthermore, the 1-year recurrence rate of the patients with a high level of XAGE-1b mRNA was significantly greater compared to the patients with a low level. All these findings indicate that XAGE-1b is associated with the aggressive biological behavior of HCC cells and it may be a potential biomarker for HCC diagnosis and prognosis.

Mycobacterial MazG safeguards genetic stability via housecleaning of 5-OH-dCTP.

Generation of reactive oxygen species and reactive nitrogen species in phagocytes is an important innate immune response mechanism to eliminate microbial pathogens. It is known that deoxynucleotides (dNTPs), the precursor nucleotides to DNA synthesis, are one group of the significant targets for these oxidants and incorporation of oxidized dNTPs into genomic DNA may cause mutations and even cell death. Here we show that the mycobacterial dNTP pyrophosphohydrolase MazG safeguards the bacilli genome by degrading 5-OH-dCTP, thereby, preventing it from incorporation into DNA. Deletion of the (d)NTP pyrophosphohydrolase-encoding mazG in mycobacteria leads to a mutator phenotype both under oxidative stress and in the stationary phase of growth, resulting in increased CG to TA mutations. Biochemical analyses demonstrate that mycobacterial MazG can efficiently hydrolyze 5-OH-dCTP, an oxidized nucleotide that induces CG to TA mutation upon incorporation by polymerase. Moreover, chemical genetic analyses show that direct incorporation of 5-OH-dCTP into mazG-null mutant strain of Mycobacterium smegmatis (Msm) leads to a dose-dependent mutagenesis phenotype, indicating that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mycobacterium tuberculosis (Mtb) leads to reduced survival in activated macrophages and in the spleen of infected mice. This study not only characterizes the mycobacterial MazG as a novel pyrimidine-specific housecleaning enzyme that prevents CG to TA mutation by degrading 5-OH-dCTP but also reveals a genome-safeguarding mechanism for survival of Mtb in vivo.

[Histone acetylation modulates influenza virus replicative intermediate dsRNA-induced interleukin-6 expression in A549 cells].

OBJECTIVE: To investigate the role of histone acetylation in regulating influenza virus replicative intermediate double-stranded RNA (dsRNA)-induced interleukin-6 (IL-6) expression in A549 cells. METHODS: A549 cells were treated with influenza virus replicative intermediate dsRNA, histone deacetylase (HDAC) inhibitor trichostatin A (TSA), or HADC small interfering RNA (siRNA). The changes in the cellular IL-6 promoter activities were detected by dual-luciferase assay, and IL-6 mRNA and protein expressions in the cells were determined using real-time RT-PCR and ELISA, respectively. RESULTS: Influenza virus replicative intermediate dsRNA obviously up-regulated IL-6 expression in the cells. HDAC inhibitor TSA significantly enhanced the activity of IL-6 promoter and increased IL-6 mRNA expression in A549 cells, and HDAC3 may play an important role in this process. HDAC inhibitor TSA and DNMT inhibitor DAC showed no synergic effect in regulating IL-6 expression. CONCLUSIONS: Influenza virus replicative intermediate dsRNA-induced IL-6 expression in A549 cells is regulated by histone acetylation.

[Sequence homology analysis on lactate dehydrogenase (LDH) of Plasmodium vivax Anhui isolates].

DNA in dried blood spots of 39 vivax malaria patients (2009-2010) from Anhui (Bengbu urban district and counties of Wuhe, Huaiyuan, Mengcheng and Lixin) was extracted. The Plasmodium vivax LDH (PvLDH) gene was amplified, cloned and sequenced. The sequences were subjected to NCBI Blast program. The results showed that the targeted DNA fragment size was 951 bp without difference among the 39 samples (accession No. GU078391), and was more than 99% homologous to the PvLDH sequences in other strains from GenBank. There was only one different amino acid in the protein sequences between the isolates from Anhui and EJEU60134 or MIA061251 strains.

Interleukin-6 expression was regulated by epigenetic mechanisms in response to influenza virus infection or dsRNA treatment.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays critical roles in a wide range of biologic activities. However, it remains unknown whether epigenetic mechanisms are involved in these processes. The possible epigenetic mechanisms involved in IL-6 expression during influenza virus (IV) infection were investigated in this study. Our results showed that both IV and its replicative intermediate dsRNA activated the IL-6 promoter, increased its transcription and enhanced cytokine secretion. IL-6 was up-regulated by DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) treatment, while its promoter activity was decreased when promoter DNA was methylated in vitro. Further study found that the IL-6 proximal promoter region was demethylated after both IV infection and dsRNA treatment, subsequently impairing its binding by transcription factors. Moreover, the DNA methyltransferase (DNMT) activity was inhibited in dsRNA treated nuclei, and DNMT1 but not DNMT3a or DNMT3b was responsible for IL-6 expression in this process. These results implied that IL-6 expression was regulated by promoter demethylation induced by down-regulation of DNMT activity. Our work revealed that epigenetic mechanisms regulate host genes expression in IV infection, and provided a new insight into understanding the mechanisms of viral infection.

NLS-dependent and insufficient nuclear localization of XAGE-1 splice variants.

XAGE-1 is a member of the cancer/testis antigen family and it was first identified by searching for the PAGE/ GAGE-related genes. Four transcript variants XAGE-1a, -1b, -1c and XAGE-1d have been discovered and have a broad expression in cancer. As a prominent transcript, XAGE-1b is encoded by 81 amino acids with a molecular weight of 9 kDa. We determined the cellular localization of all four splice variants by confocal microscopy analysis. Among these, XAGE-1a, -1b and -1c showed distinct speckled nuclear localization, while XAGE-1d was distributed evenly both in the cytoplasm and nucleus. By deletion mutagenesis and site directed mutagenesis, we identified the bipartite nuclear localization signal and found that it contributes to the nuclear localization of XAGE-1 variants; but the nuclear localization signal (NLS) only cannot form the characterized distribution of XAGE-1b; amino acids 25-42 also play a role in the formation of nuclear speckles. XAGE-1b, the main transcript of XAGE-1, may act as a partner protein or a member of a protein complex, which plays a role in tumor development and progression.

[Effects of veratryl alcohol and tween 80 on ligninase production and its roles in decolorization of azo dyes by white-rot basidiomycete PM2].

Basidiomycete PM2, a lignin-degrading white rot fungus, produces lgnin peroxidase (Lip) and manganese peroxidase (Mnp) in nutrient nitrogen limited liquid cultures. This fungus was selected for its ability to decolorize azo group of dyes. In order to improve production of the peroxidases and rapid dye decolorizing activity by basidiomycete PM2, the addition of veratryl alcohol or Tween 80 to nutrient nitrogen limited liquid cultures were tested. It was found to have a large stimulatory effect on Mnp activities and decolorization rate of azo dyes. A maximum Mnp activities of 254.2 u/L with veratryl alcohol and 192.2 u/L with Tween 80 were achieved respectively. These values were about 3.4-fold and 2.5-fold higher than that obtained in the control cultures (without alcohol or Tween 80), whereas the levels of Lip activity detected were very low (about 12 u/L)in all the cultures. In further experiments using three kinds of azo dyes of congo red, orange G and orange IV, enzyme activities and dye decolorization were investigated in the above-mentioned cultures. The results showed that Mnp activities and decolorization were notably higher than those obtained in the control cultures in the presence of azo dyes. Cultures supplemented with Tween 80 were more adequate for dye decolorization. The rates of the decolorization with Tween 80 of congo red (95.4%), orange G (98.5%) and orange IV (54.4%) after 24 hours of dye incubation were higher than that supplemented with veratryl alcohol. According to the results, Mnp activities secreted by basidiomycete PM2 play an essential role in the process of dye decolorization. Tween 80 was the main factor affecting the decolorization. The analysis of structure of the three kinds of azo dyes indicats that the extent of decolorization is affected by the dye molecular structure. The types and quantity of the substituted groups on the aromatic ring of azo dyes have effect on the percentage of biological decolorization.