Identification and Characterization of the Determinants of Copper Resistance in the Acidophilic Fungus Acidomyces richmondensis MEY-1 Using the CRISPR/Cas9 System.

Copper (Cu) homeostasis has not been well documented in filamentous fungi, especially extremophiles. One of the main obstacles impeding their characterization is the lack of a powerful genome-editing tool. In this study, we applied a CRISPR/Cas9 system for efficient targeted gene disruption in the acidophilic fungus Acidomyces richmondensis MEY-1, formerly known as Bispora sp. strain MEY-1. Using this system, we investigated the basis of Cu tolerance in strain MEY-1. This strain has extremely high Cu tolerance among filamentous fungi, and the transcription factor ArAceA (A. richmondensis AceA) has been shown to be involved in this process. The ArAceA deletion mutant (ΔArAceA) exhibits specific growth defects at Cu concentrations of ≥10 mM and is transcriptionally more sensitive to Cu than the wild-type strain. In addition, the putative metallothionein ArCrdA was involved in Cu tolerance only under high Cu concentrations. MEY-1 has no Aspergillus nidulans CrpA homologs, which are targets of AceA-like transcription factors and play a role in Cu tolerance. Instead, we identified the Cu-transporting P-type ATPase ArYgA, homologous to A. nidulans YgA, which was involved in pigmentation rather than Cu tolerance. When the ΔArYgA mutant was grown on medium supplemented with Cu ions, the black color was completely restored. The lack of CrpA homologs in A. richmondensis MEY-1 and its high tolerance to Cu suggest that a novel Cu detoxification mechanism differing from the AceA-CrpA axis exists. IMPORTANCE Filamentous fungi are widely distributed worldwide and play an important ecological role as decomposers. However, the mechanisms of their adaptability to various environments are not fully understood. Various extremely acidophilic filamentous fungi have been isolated from acidic mine drainage (AMD) with extremely low pH and high heavy metal and sulfate concentrations, including A. richmondensis. The lack of genetic engineering tools, particularly genome-editing tools, hinders the study of these acidophilic and heavy metal-resistant fungi at the molecular level. Here, we first applied a CRISPR/Cas9-mediated gene-editing system to A. richmondensis MEY-1. Using this system, we identified and characterized the determinants of Cu resistance in A. richmondensis MEY-1. The conserved roles of the Cu-binding transcription factor ArAceA in Cu tolerance and the Cu-transporting P-type ATPase ArYgA in the Cu-dependent production of pigment were confirmed. Our findings provide insights into the molecular basis of Cu tolerance in the acidophilic fungus A. richmondensis MEY-1. Furthermore, the CRISPR/Cas9 system used here would be a powerful tool for studies of the mechanisms of adaptability of acidophilic fungi to extreme environments.

Casp8 acts through A20 to inhibit PD-L1 expression: The mechanism and its implication in immunotherapy.

Immunotherapy targeting the PD-L1/PD-1 pathway is a novel type of clinical cancer treatment, but only small subsets of patients can benefit from it because of multiple factors. PD-L1/PD-1 expression is a biomarker for predicting the efficacy of anti-PD-L1/PD-1 therapy, which highlights the importance of understanding the regulatory mechanisms of PD-L1 expression in cancer cells. Casp8 is an apical caspase protease involved in mediating cell apoptosis, but it also has multiple nonapoptotic functions. Casp8 mutations are associated with increased risks of cancer, and low expression of Casp8 is closely connected with poor prognosis in patients with cancer. In addition, mutations of Casp8 in lymphocytes also lead to human immunodeficiency, thereby causing dysfunction of the innate immune system, but the roles of Casp8 in antitumor immunity remain unclear. Here, we found that knocking down Casp8 in mouse melanoma cells promoted tumor progression in an immune system-dependent manner. Mechanistically, Casp8 induced PD-L1 degradation by upregulating TNFAIP3 (A20) expression, a ubiquitin-editing enzyme that results in PD-L1 ubiquitination. In addition, compared with Casp8fl/fl mice, mice with conditional deletion of Casp8 in natural killer (NK) cells (Ncr1iCre/+ Casp8fl/fl mice) showed a decreased frequency of IFN-γ+ and CD107a+ NK cells but an increased frequency of PD-1+ and CTLA-4+ NK cells. Melanoma cells with Casp8 knocked down exhibited sensitivity to anti-PD-1 or anti-CTLA-4 antibody treatments, particularly in Ncr1iCre/+Casp8fl/fl mice. Together, the results indicate that Casp8 induces PD-L1 degradation by upregulating A20 expression and that decreased Casp8 expression is a potential biomarker for predicting the sensitivity to anti-PD-L1/PD-1 immunotherapy.

Cisplatin/capecitabine with intensity-modulated radiation therapy in anal squamous cell carcinoma: a preliminary study.

OBJECTIVE: Mitomycin (MMC)/5-fluoroural (5-FU) with concurrent radiation is the standard treatment of anal squamous cell carcinoma (ASCC). The aim of this study is to evaluate the efficacy and safety of cisplatin/capecitabine (XP) as an alternative with intensity-modulated radiation therapy (IMRT) in ASCC setting. METHODS: We retrospectively screened all patients with stage I-IV ASCC from January 2010 to June 2019. The records of patients who received definitive chemoradiation with cisplatin/capecitabine (XP) and IMRT were collected and analyzed. RESULTS: The first patient was treated with XP in 2017, so totally 11 patients were included in our study from January 2017 to June 2019. All patients have experienced clinical complete response (cCR). After a median follow-up of 30 months (range, 18-39 months), no patient had local recurrence or distant metastasis. Two-year colostomy-free survival (CFS) and two-year disease-free survival (DFS) were both 100%. The median overall survival (OS) has not reached. Grade 3 acute toxicities included leukopenia (1, 9.1%), neutropenia (2, 18.2%) and thrombocytopenia (2, 18.2%). No grade 4 acute adverse events occurred. CONCLUSION: In our study, cisplatin/capecitabine combined with IMRT was safe in ASCC patients, with favorable efficacy as an alternative, and is expected to be explored in study with larger sample.

Cross-talk between TNF-α and IFN-γ signaling in induction of B7-H1 expression in hepatocellular carcinoma cells.

Clinical benefit from immunotherapy of B7-H1/PD-1 checkpoint blockade indicates that it is important to understand the regulatory mechanism of B7-H1 expression in cancer cells. As an adaptive response to the endogenous antitumor immunity, B7-H1 expression is up-regulated in HCC cells. B7-H1 expression is induced mainly by IFN-γ released from tumor-infiltrating T cells in HCC. In addition, HCC is a prototype of inflammation-related cancer and TNF-α is a critical component of inflammatory microenvironment of HCC. In the present study, we asked whether TNF-α can promote the expression of B7-H1 induced by IFN-γ in HCC cells. We found that JAK/STAT1/IRF1 was the primary pathway responsible for induction of B7-H1 expression by IFN-γ in human HCC cell lines. TNF-α and IFN-γ synergistically induced the expression of B7-H1 in the HCC cells. Moreover, the mechanism of the synergy was that TNF-α enhanced IFN-γ signaling by upregulating the expression of IFN-γ receptors. Furthermore, B7-H1 expression induced synergistically by TNF-α and IFN-γ in murine HCC cells facilitated tumor growth in vivo. Our findings suggest that TNF-α may enhance the adaptive immune resistance mediated by IFN-γ-induced B7-H1 in HCC cells.

The analysis of brain functional connectivity of post-stroke cognitive impairment patients: an fNIRS study.

Background: Post-stroke cognitive impairment (PSCI) is a considerable risk factor for developing dementia and reoccurrence of stroke. Understanding the neural mechanisms of cognitive impairment after stroke can facilitate early identification and intervention. Objectives: Using functional near-infrared spectroscopy (fNRIS), the present study aimed to examine whether resting-state functional connectivity (FC) of brain networks differs in patients with PSCI, patients with Non-PSCI (NPSCI), and healthy controls (HCs), and whether these features could be used for clinical diagnosis of PSCI. Methods: The present study recruited 16 HCs and 32 post-stroke patients. Based on the diagnostic criteria of PSCI, post-stroke patients were divided to the PSCI or NPSCI group. All participants underwent a 6-min resting-state fNRIS test to measure the hemodynamic responses from regions of interests (ROIs) that were primarily distributed in the prefrontal, somatosensory, and motor cortices. Results: The results showed that, when compared to the HC group, the PSCI group exhibited significantly decreased interhemispheric FC and intra-right hemispheric FC. ROI analyses showed significantly decreased FC among the regions of somatosensory cortex, dorsolateral prefrontal cortex, and medial prefrontal cortex for the PSCI group than for the HC group. However, no significant difference was found in the FC between the PSCI and the NPSCI groups. Conclusion: Our findings provide evidence for compromised interhemispheric and intra-right hemispheric functional connectivity in patients with PSCI, suggesting that fNIRS is a promising approach to investigate the effects of stroke on functional connectivity of brain networks.

Heterologous expression and characterization of novel GH12 ß-glucanase and AA10 lytic polysaccharide monooxygenase from Streptomyces megaspores and their synergistic action in cellulose saccharification.

BACKGROUND: The combination of cellulase and lytic polysaccharide monooxygenase (LPMO) is known to boost enzymatic saccharification of cellulose. Although the synergy between cellulases (GH5, 6 or 7) and LPMOs (AA9) has been extensively studied, the interplay between other glycoside hydrolase and LPMO families remains poorly understood. RESULTS: In this study, two cellulolytic enzyme-encoding genes SmBglu12A and SmLpmo10A from Streptomyces megaspores were identified and heterologously expressed in Escherichia coli. The recombinant SmBglu12A is a non-typical endo-ß-1,4-glucanase that preferentially hydrolyzed ß-1,3-1,4-glucans and slightly hydrolyzed ß-1,4-glucans and belongs to GH12 family. The recombinant SmLpmo10A belongs to a C1-oxidizing cellulose-active LPMO that catalyzed the oxidation of phosphoric acid swollen cellulose to produce celloaldonic acids. Moreover, individual SmBglu12A and SmLpmo10A were both active on barley ß-1,3-1,4-glucan, lichenan, sodium carboxymethyl cellulose, phosphoric acid swollen cellulose, as well as Avicel. Furthermore, the combination of SmBglu12A and SmLpmo10A enhanced enzymatic saccharification of phosphoric acid swollen cellulose by improving the native and oxidized cello-oligosaccharides yields. CONCLUSIONS: These results proved for the first time that the AA10 LPMO was able to boost the catalytic efficiency of GH12 glycoside hydrolases on cellulosic substrates, providing another novel combination of glycoside hydrolase and LPMO for cellulose enzymatic saccharification.

BAF60a Deficiency in Macrophage Promotes Diet-Induced Obesity and Metabolic Inflammation.

Adipose tissue macrophage (ATM) has been shown to play a key role in the pathogenesis of obesity-associated adipose tissue inflammation and metabolic diseases. However, the upstream factors that integrate the environmental signals to control ATM activation and adipose inflammation in obesity remain elusive. Here, we identify BAF60a, a subunit of the switch/sucrose-nonfermentable (SWI/SNF) chromatin remodeling complexes, as the central checkpoint regulator of obesity-induced ATM activation, adipose tissue inflammation, and systemic metabolic impairment. BAF60a expression was robustly downregulated in the adipose tissue stromal vascular fractions in type 2 diabetic mice. Myeloid-specific BAF60a knockout (BaMKO) promotes ATM proinflammatory activation, exacerbating diet-induced obesity, insulin resistance, and metabolic dysfunction. Conversely, myeloid-specific overexpression of BAF60a in mice attenuates macrophage proinflammatory activation. Mechanistically, transcriptome and chromatin landscape analyses demonstrate that BAF60a inactivation triggers the expression of proinflammatory gene program through chromatin remodeling. Moreover, motif analysis of ATAC-Seq and CUT&Tag-Seq data identifies the transcription factor Atf3 that physically interacts with BAF60a to suppress the proinflammatory gene expression, thereby controlling ATM activation and metabolic inflammation in obesity. Consistently, myeloid-specific Atf3 deficiency also promotes the proinflammatory activation of macrophage. This work uncovers BAF60a/Atf3 axis as the key regulator in obesity-associated ATM activation, adipose tissue inflammation, and metabolic diseases.

Deciphering the efficient cellulose degradation by the thermophilic fungus Myceliophthora thermophila focused on the synergistic action of glycoside hydrolases and lytic polysaccharide monooxygenases.

The thermophilic fungus Myceliophthora thermophila as an efficient decomposer secretes various glycoside hydrolases and auxiliary oxidation enzymes to deconstruct cellulose. However, the core enzymes critical for efficient cellulose degradation and their interactions with other cellulolytic enzymes remain unclear. Herein, the transcriptomic analysis of M. thermophila grown on Avicel exhibited that cellulases from GH5_5, GH6 and GH7, and lytic polysaccharide monooxygenases (LPMOs) from AA9 contributed to cellulose degradation. Moreover, the peptide mass fingerprinting analysis of major extracellular proteins and corresponding gene-knockout strains studies revealed that MtCel7A and MtCel5A were the core cellulolytic enzymes. Furthermore, synergistic experiments found that hydrolytic efficiencies of MtCel7A and MtCel5A were both improved by mixture C1/C4 oxidizing MtLPMO9H, but inhibited by C1 oxidizing MtLPMO9E and C4 oxidizing MtLPMO9J respectively. These results demonstrated the potential application of C1/C4 oxidizing LPMOs for future designing novel cellulolytic enzyme cocktails on the efficient conversion of cellulose into biofuels and biochemicals.

Efficient Degradation of Aflatoxin B1 and Zearalenone by Laccase-like Multicopper Oxidase from Streptomyces thermocarboxydus in the Presence of Mediators.

Multicopper oxidases (MCOs) are a diverse group of enzymes that could catalyze the oxidation of different xenobiotic compounds, with simultaneous reduction in oxygen to water. Aside from laccase, one member of the MCO superfamily has shown great potential in the biodegradation of mycotoxins; however, the mycotoxin degradation ability of other MCOs is uncertain. In this study, a novel MCO-encoding gene, StMCO, from Streptomyces thermocarboxydus, was identified, cloned, and heterologously expressed in Escherichia coli. The purified recombinant StMCO exhibited the characteristic blue color and bivalent copper ion-dependent enzyme activity. It was capable of oxidizing the model substrate ABTS, phenolic compound DMP, and azo dye RB5. Notably, StMCO could directly degrade aflatoxin B1 (AFB1) and zearalenone (ZEN) in the absence of mediators. Meanwhile, the presence of various lignin unit-derived natural mediators or ABTS could significantly accelerate the degradation of AFB1 and ZEN by StMCO. Furthermore, the biological toxicities of their corresponding degradation products, AFQ1 and 13-OH-ZEN-quinone, were remarkably decreased. Our findings suggested that efficient degradation of mycotoxins with mediators might be a common feature of the MCOs superfamily. In summary, the unique properties of MCOs make them good candidates for degrading multiple major mycotoxins in contaminated feed and food.

M1 Macrophages Induce PD-L1 Expression in Hepatocellular Carcinoma Cells Through IL-1ß Signaling.

Hepatocellular carcinoma (HCC) is a prototype of inflammation-related cancer, harboring M1-like and M2-like tumor-associated macrophages. M1 macrophages are thought to be tumoricidal, but some studies report its pro-tumor role. The programmed cell death-ligand (PD-L) 1 expressed in HCC cells is a critical checkpoint molecule to mediate immune escape of HCC. The PD-L1 expression in HCC cells is inducible. In the present study, we ask whether M1 macrophages induce the expression of PD-L1 in HCC cells. First, an association between M1 macrophage infiltration and PD-L1 expression in HCC tissues was determined by bioinformatics and immunohistochemistry experiments. The enrichment score of M1 macrophages was correlated to PD-L1 expression in 90 HCC samples from GEO database. Besides, infiltration of CD68+HLA-DR+ M1-like macrophages correlated with PD-L1 expression level in HCC cells. Moreover, M1-conditioned media was prepared from M1 macrophages derived from THP-1 cell, RAW264.7 cell or murine bone marrow. These supernatants induced expression of PD-L1 in HCC cells. Furthermore, inflammatory cytokine IL-1ß in the supernatants was identified to account for the inducible PD-L1 expression by siRNA assay and receptor blockade assay. Additionally, transcription factor p65 and IRF1 in the HCC cells were revealed by CHIP assay to mediate the inducible PD-L1 expression. All the results demonstrate that M1 macrophages induced expression of PD-L1 in HCC cells, supporting the pro-tumor role of M1 macrophages.

MYC inhibition increases PD-L1 expression induced by IFN-γ in hepatocellular carcinoma cells.

The effectiveness of immunotherapy targeting the immune checkpoint PD-L1/PD-1 pathway highlights importance of elucidating the regulatory mechanisms of PD-L1 expression in cancer cells. Previous studies demonstrate that oncogene MYC up-regulates PD-L1 expression in lymphomas. In the present study, we investigated the regulatory role of MYC in the PD-L1 expression induced by IFN-γ in HCC cells. Unexpectedly, knockdown of MYC expression using siRNA assay increased the inducible expression of PD-L1 both at mRNA and protein levels. Mechanistically, the inhibition of MYC elevated expression of STAT1, a critical component of IFN-γ signaling pathway, leading to the elevation of PD-L1 expression in HCC cells exposed to IFN-γ. These results suggest that MYC may down-regulate PD-L1 expression in the context of HCC. This study implicates that a combination therapy targeting MYC function and PD-L1/PD-1 pathway might be effective for treatment of HCC.