Programming Peptide Liquid Crystal Media to Acquire Independent Sets of Residual Dipolar Couplings and Enantiodiscrimination in Multiple Solvent Systems.

Multiple independent sets of residual dipolar couplings (RDCs) acquired by relying on different alignment media show the great potential for de novo structure determination of organic compounds. However, this methodology is severely compromised by the limited availability of multialignment media. In this work, an engineering strategy was developed to program the oligopeptide amphiphiles (OPAs) to create different peptide liquid crystal (LC) media for the acquisition of independent sets of RDCs. With no need for de novo design on peptide sequences, the molecular alignment can be simply modulated by varying the length of the hydrophobic tails within OPAs. Relying on these programmed peptide LC media, five independent sets of RDCs were extracted in a highly efficient and accurate manner. Because of the similar bulk composition of OPAs, this approach offers the significant advantage in circumventing the possible incompatibilities of analytes with one or several different alignment media, therefore avoiding the analysis complication. Notably, these peptide LC media show enantiodifferentiating properties, and the enantiodiscriminating capabilities could also be optimized through the programmed strategy. Furthermore, we show that these media are compatible with different polar solvents, allowing the possible de novo structure elucidation of organic compounds with varied polarities and solubilities.

Talaromyces marneffei suppresses macrophage inflammation by regulating host alternative splicing.

Talaromyces marneffei (T. marneffei) immune escape is essential in the pathogenesis of talaromycosis. It is currently known that T. marneffei achieves immune escape through various strategies. However, the role of cellular alternative splicing (AS) in immune escape remains unclear. Here, we depict the AS landscape in macrophages upon T. marneffei infection via high-throughput RNA sequencing and detect a truncated protein of NCOR2 / SMRT, named NCOR2-013, which is significantly upregulated after T. marneffei infection. Mechanistic analysis indicates that NCOR2-013 forms a co-repression complex with TBL1XR1 / TBLR1 and HDAC3, thereby inhibiting JunB-mediated transcriptional activation of pro-inflammatory cytokines via the inhibition of histone acetylation. Furthermore, we identify TUT1 as the AS regulator that regulates NCOR2-013 production and promotes T. marneffei immune evasion. Collectively, these findings indicate that T. marneffei escapes macrophage killing through TUT1-mediated alternative splicing of NCOR2 / SMRT, providing insight into the molecular mechanisms of T. marneffei immune evasion and potential targets for talaromycosis therapy.

Construction of a murine model of latent infection and reactivation induced by Talaromyces marneffei.

OBJECTIVE: To establish a murine model of Talaromyces marneffei (T. marneffei) latent infection and reactivation, providing a foundation for exploring the molecular mechanisms underlying disease relapse. METHODS: BALB/c mice were tail vein injected with T. marneffei at 0 days post-infection (dpi) and treated with cyclophosphamide (CTX) intraperitoneally every four days, starting from 21 dpi or 42 dpi. Mice were observed for body weight changes, liver and spleen indices, histological characteristics of liver and spleen, fungal load detection in liver and spleen, and Mp1p qualitation in liver and spleen to assess T. marneffei infection severity. RESULTS: T. marneffei-infected mice exhibited a trend of initial weight loss followed by recovery and a subsequent decrease in weight after CTX injection throughout the observation period. Liver and spleen indices, as well as tissue damage, significantly increased during infection but later returned to normal levels, with a gradual rise observed after immunosuppression. Fungal load analysis revealed positive T. marneffei cultures in the liver and spleen at 7 dpi and 14 dpi, followed by negative T. marneffei cultures from 21 dpi until day 21 post-immunosuppression (42 dpi or 63 dpi); however, the spleen remained T. marneffei-cultured negative, consistent with the trend observed in Mp1p detection results. CONCLUSION: A latent infection and reactivation model of T. marneffei in mice was successfully established, with the liver likely serving as a key site for latent T. marneffei.

Effect and Mechanism of Cotrimoxazole Against Talaromyces marneffei in vitro.

BACKGROUND: Talaromyces marneffei (formerly Penicillium marneffei) is an important thermally dimorphic fungus endemic which is characterized by one of the most frequent opportunistic infections in HIV/AIDS patients, mainly prevalent in Southeast Asia, southern China, and northeastern India. Cotrimoxazole(CTX) inhibits folic acid synthesis which is important for the survival of many bacteria, protozoa, and fungi, thereby commonly used to prevent several opportunistic infections among HIV/AIDS patients. In addition to preventing other HIV-associated opportunistic infections, CTX prophylaxis are considered to have the potential to prevent T. marneffei infection in HIV/AIDS patients receiving antiretroviral therapy (ART). However, the effect of cotrimoxazole towards T. marneffei fungus in vitro remains unclear. METHODS: Human THP-1 macrophages were used as cell model in vitro to explore the effect and mechanism of cotrimoxazole resistance towards T. marneffei. Cell viability assay and drug sensitivity colony forming units (CFU) experiments were conducted to determine the minimum inhibitory concentration (MIC) of cotrimoxazole inside and outside THP-1 macrophages respectively. Enzyme-linked immunosorbent assay (Elisa) was used to measure the concentration of Dihydropteroic acid synthetase (DHPS), Dihydrofolate synthetase (DHFS) and Dihydrofolate reductase (DHFR) between T. marneffei adding TMP/SMX and without adding TMP/SMX group respectively. Real-time fluorescence quantitative PCR(qPCR) was performed to detect the mRNA expression levels in Dectin-1 mediated signaling pathway and downstream inflammatory cytokines including IL-6, IL-10, IL-23A, CXCL8 and TNF-α released by T. marneffei-infected macrophages between adding TMP/SMX and without adding TMP/SMX group respectively. RESULTS: Cotrimoxazole can inhibit the proliferation of T. marneffei within safe concentration inside and outside THP-1 macrophages. Drug susceptibility results showed the minimal inhibit concentration(MIC) of 1:5 TMP/SMX was ranging from 14/70 to 68/340 µg/ml. The MIC of SMX was ranging from 100 to 360 µg/ml. The MIC of TMP was ranging from 240 to 400 µg/ml outside macrophages. The MIC of TMP/SMX was ranging from 36/180 to 68/340 µg/ml. The MIC of SMX was ranging from 340 to 360 µg/ml. The MIC of TMP was ranging from 320 to 400 µg/ml inside macrophages. The synergistic interaction of 1:5 TMP/SMX was more effective in inhibiting T. marneffei than separate SMX and TMP. DHPS, DHFS and DHFR can be inhibited by cotrimoxazole within safe and effective concentration. Dectin-1 expression is increased following T. marneffei infection, leading to the increase of IL-6, IL-10, IL-23A and the decrease of CXCL8 and TNF-α. Conversely, cotrimoxazole decrease the levels of Dectin-1, IL-6, IL-10, IL-23A and increase the levels of CXCL8 and TNF-α, thereby enhancing the intracellular killing-T. marneffei capacity of macrophages. CONCLUSIONS: Our findings indicated that cotrimoxazole directly inhibited T. marneffei growth by blocking DHPS, DHFS and DHFR and indirectly inhibited T. marneffei growth perhaps by regulating the Dectin-1 signaling pathway, which may effectively interfere with the defense ability of the host against T. marneffei infection.

A Pan-Cancer Analysis Revealing the Dual Roles of Lysine (K)-Specific Demethylase 6B in Tumorigenesis and Immunity.

Introduction: Epigenetic-targeted therapy has been increasingly applied in the treatment of cancers. Lysine (K)-specific demethylase 6B (KDM6B) is an epigenetic enzyme involved in the coordinated control between cellular intrinsic regulators and the tissue microenvironment whereas the pan-cancer analysis of KDM6B remains unavailable. Methods: The dual role of KDM6B in 33 cancers was investigated based on the GEO (Gene Expression Omnibus) and TCGA (The Cancer Genome Atlas) databases. TIMER2 and GEPIA2 were applied to investigate the KDM6B levels in different subtypes or stages of tumors. Besides, the Human Protein Atlas database allowed us to conduct a pan-cancer study of the KDM6B protein levels. GEPIA2 and Kaplan-Meier plotter were used for the prognosis analysis in different cancers. Characterization of genetic modifications of the KDM6B gene was analyzed by the cBioPortal. DNA methylation levels of different KDM6B probes in different TCGA tumors were analyzed by MEXPRESS. TIMER2 was applied to determine the association of the KDM6B expression and immune infiltration and DNA methyltransferases. Spearman correlation analysis was used to assess the association of the KDM6B expression with TMB (tumor mutation burden) and MSI (microsatellite instability). The KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis and GO (Gene ontology) enrichment analysis were used to further investigate the potential mechanism of KDM6B in tumor pathophysiology. Results: KDM6B was downregulated in 11 cancer types and upregulated across five types. In KIRC (kidney renal clear cell carcinoma) and OV (ovarian serous cystadenocarcinoma), the KDM6B level was significantly associated with the pathological stage. A high level of KDM6B was related to poor OS (overall survival) outcomes for THCA (thyroid carcinoma), while a low level was correlated with poor OS and DFS (disease-free survival) prognosis of KIRC. The KDM6B expression level was associated with TMB, MSI, and immune cell infiltration, particularly cancer-associated fibroblasts, across various cancer types with different correlations. Furthermore, the enrichment analysis revealed the relationship between H3K4 and H3K27 methylation and KDM6B function. Conclusion: Dysregulation of the DNA methyltransferase activity and methylation levels of H3K4 and H3K27 may involve in the dual role of KDM6B in tumorigenesis and development. Our study offered a relatively comprehensive understanding of KDM6B's dual role in cancer development and response to immunotherapy.

Talaromyces marneffei activates the AIM2-caspase-1/-4-GSDMD axis to induce pyroptosis in hepatocytes.

Talaromyces marneffei tends to induce systemic infection in immunocompromised individuals, which is one of the causes of the high mortality. The underlying molecular mechanisms of T.marneffei-induced abnormal liver function are still poorly understood. In this study, we found that T.marneffei-infected patients could develop abnormal liver function, evidenced by reduced albumin and increased levels of aspartate aminotransferase (AST) and AST/alanine aminotransferase (ALT). T. marneffei-infected mice exhibited similar characteristics. In vitro investigations showed that T.marneffei induced the death of AML-12 cells. Furthermore, we determined that T.marneffei infection induced pyroptosis in hepatocytes of C57BL/6J mice and AML-12 cells, demonstrated by the increase of AIM2, caspase-1/-4, Gasdermin D(GSDMD) and pyroptosis-related cytokines in T.marneffei-infected mice/cells. Importantly, cell death was markedly suppressed in the presence of VX765 (an inhibitor of caspase-1/-4). Furthermore, in the presence of VX765, T.marneffei-induced pyroptosis was blocked. Nevertheless, necroptosis and apoptosis were also detected in infected animal model at 14 days post-infection. In conclusion, T.marneffei induces pyroptosis in hepatocytes through activation of the AIM2-caspase-1/-4-GSDMD axis, which may be an important cause of liver damage, and other death pathways including necroptosis and apoptosis may also be involved in the later stage of infection.

Talaromyces marneffei promotes M2-like polarization of human macrophages by downregulating SOCS3 expression and activating the TLR9 pathway.

Little is known about how Talaromyces marneffei, a thermally dimorphic fungus that causes substantial morbidity and mortality in Southeast Asia, evades the human immune system. Polarization of macrophages into fungal-inhibiting M1-like and fungal-promoting M2-like types has been shown to play an important role in the innate immune response against fungal pathogens. This mechanism has not been defined for T. marneffei. Here, we demonstrated that T. marneffei promotes its survival in human macrophages by inducing them toward M2-like polarization. Our investigations of the mechanism revealed that T. marneffei infection led to SOCS3 protein degradation by inducing tyrosine phosphorylation, thereby relieving the inhibitory effect of SOCS3 on p-STAT6, a key factor for M2-like polarization. Our SOCS3-overexpression experiments showed that SOCS3 is a positive regulator of M1-like polarization and plays an important role in limiting M2-like polarization. Furthermore, we found that inhibition of the TLR9 pathway partially blocked T. marneffei-induced M2-like polarization and significantly enhanced the killing activity of macrophages against T. marneffei. Collectively, these results reveal a novel mechanism by which T. marneffei evades the immune response of human macrophages.

Identification and analysis of lncRNA, microRNA and mRNA expression profiles and construction of ceRNA network in Talaromyces marneffei-infected THP-1 macrophage.

BACKGROUND: Competitive endogenous RNA (ceRNA) reveals new mechanisms for interactions between RNAs, which have been considered to play a significant role in pathogen-host innate immune response. However, knowledge of ceRNA regulatory networks in Talaromyces marneffei (TM)-macrophages is still limited. METHODS: Next-generation sequencing technology (NGS) was used to obtain mRNA, miRNA and lncRNA expression profiles in TM-infected macrophages. The R package DESeq2 was used to identify differentially expressed lncRNA, miRNA and mRNA. The R package GOseq was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and the ceRNA network of lncRNA-miRNA-mRNA interaction was constructed in Cytoscape. Similarly, functional enrichment analysis on mRNA in the ceRNA network. Finally, two mRNAs and four lncRNAs in the ceRNA network were randomly selected to verify the expression using qRT-PCR. RESULTS: In total, 119 lncRNAs, 28 miRNAs and 208 mRNAs were identified as differentially expressed RNAs in TM-infected macrophages. The constructed ceRNA network contains 38 lncRNAs, 10 miRNAs and 45 mRNAs. GO and KEGG analysis of mRNA in the ceRNA network indicated that activated pathways in TM-infected macrophages were related to immunity, inflammation and metabolism. The quantitative validation of the expression of four randomly selected differentially expressed lncRNAs, AC006252.1, AC090197.1, IL6R-AS1, LINC02009 and two mRNAs, CSF1, NR4A3 showed that the expression levels were consistent with those in the RNA-sequencing. CONCLUSIONS: The ceRNA network related to immunity, inflammation and metabolism plays an important role in TM-macrophage interaction. This study may provide effective and novel insights for further understanding the underlying mechanism of TM infection.