Genetically predicted gut microbiome and risk of oral cancer.

PURPOSE: Mounting evidence suggests a possible link between gut microbiome and oral cancer, pointing to some potential modifiable targets for disease prevention. In the present study, Mendelian randomization (MR) was used to explore whether there was a causal link between gut microbiome and oral cancer. METHODS: The single nucleotide polymorphisms (SNPs) significantly associated with gut microbiome were served as instrumental variables. MR analyses were performed using genetic approaches such as inverse variance weighting (IVW), MR Egger and weighted median, with IVW as the primary approach, supplemented by MR Egger and weighted median. Mendelian randomization pleiotropy residual sum and outlier (MR-PRESSO) and MR-Egger regression were used to detect the presence of horizontal pleiotropy and identify outlier SNPs. RESULTS: Causal effect estimates indicated that genetically predicted abundance of Prevotellaceae was associated with higher risk of oral cancer (odds ratio (OR) 1.80, 95% confidence interval (CI) 1.16-2.81, p = 0.009). There was no evidence of notable heterogeneity and horizontal pleiotropy. CONCLUSION: Genetically derived estimates suggest that Prevotellaceae may be associated with the risk of oral cancer. Such robust evidence should be given priority in future studies and explore the underlying mechanisms.

Association of lifestyle behaviors and oral health care needs: Mediating effects of inflammatory markers.

OBJECTIVE: Mounting evidence indicates that modifiable risk factors such as lifestyle behaviors may be involved in the occurrence of oral diseases. However, existing research doesn't come to a unanimous consent. This study aims to evaluate the association between lifestyle behaviors and oral health care needs. METHODS: This study used the nationally representative dataset from the National Health and Nutrition Examination Survey (NHANES) from March 2017 to 2020 pre-pandemic. Binary logistic regression analysis was used to evaluate lifestyle behavioral factors that influence oral health care needs. Mediation analysis was performed to explore the roles of inflammatory markers in the relationship between physical activities and oral problems. RESULTS: After adjusting for covariates, multivariate analysis indicated that flossing (OR = 0.590, 95% CI, 0.510-0.682, P < 0.001), moderate alcohol consumption (per week: OR = 0.717, 95% CI, 0.588-0.873, P < 0.001; per month/year: OR = 0.794, 95% CI, 0.669-0.942, P = 0.008) and participation in recreational activities (vigorous recreational activities: OR = 0.548, 95% CI, 0.462-0.648, P < 0.001; moderate recreational activities: OR = 0.629, 95% CI, 0.549-0.721, P < 0.001) significantly reduced oral health care needs. In addition, sleep duration of 7-9 h was associated with lower oral health care needs compared to less or more sleep duration (<7 h or > 9 h) (OR = 0.851, 95% CI, 0.741-0.976, P = 0.021). Mediation analysis suggested that white blood cell (WBC) counts and high-sensitivity C-reactive protein (hs-CRP) concentrations acted significant mediating roles in the association between recreational activities and oral problems. CONCLUSIONS: The possible beneficial effects of healthy lifestyle behaviors on oral health will guide individuals to develop good habits, thereby reducing the burden of oral diseases.

PoxCbh, a novel CENPB-type HTH domain protein, regulates cellulase and xylanase gene expression in Penicillium oxalicum.

The essential transcription factor PoxCxrA is required for cellulase and xylanase gene expression in the filamentous fungus Penicillium oxalicum that is potentially applied in biotechnological industry as a result of the existence of the integrated cellulolytic and xylolytic system. However, the regulatory mechanism of cellulase and xylanase gene expression specifically associated with PoxCxrA regulation in fungi is poorly understood. In this study, the novel regulator PoxCbh (POX06865), containing a centromere protein B-type helix-turn-helix domain, was identified through screening for the PoxCxrA regulon under Avicel induction and genetic analysis. The mutant ∆PoxCbh showed significant reduction in cellulase and xylanase production, ranging from 28.4% to 59.8%. Furthermore, PoxCbh was found to directly regulate the expression of important cellulase and xylanase genes, as well as the known regulatory genes PoxNsdD and POX02484, and its expression was directly controlled by PoxCxrA. The PoxCbh-binding DNA sequence in the promoter region of the cellobiohydrolase 1 gene cbh1 was identified. These results expand our understanding of the diverse roles of centromere protein B-like protein, the regulatory network of cellulase and xylanase gene expression, and regulatory mechanisms in fungi.

Regulatory function of the novel transcription factor CxrC in Penicillium oxalicum.

Numerous transcription factors (TFs) in ascomycete fungi play crucial roles in cellular processes; however, how most of them function is poorly understood. Here, we identified and characterized a novel TF, CxrC (POX01387), acting downstream of the key TF CxrA, which is essential for plant-biomass-degrading-enzyme (PBDE) production in Penicillium oxalicum. Deletion of cxrC in P. oxalicum significantly affected the production of PBDEs, as well as mycelial growth and conidiospore production. CxrA directly repressed the expression of cxrC after about 12 hr following switch to Avicel culture. CxrC bound the promoters of major PBDE genes and genes involved in conidiospore development. CxrC was found to bind the TSSGTYR core sequence (S: C and G; Y: T and C; R: G and A) of the important cellulase genes cbh1 and eg1. Both N- and C-terminal peptides of CxrC and the CxrC phosphorylation were found to mediate its homodimerization. The conserved motif LPSVRSLLTP (65-74) in CxrC was found to be required for regulating cellulase production. This study reveals novel mechanisms of TF-mediated regulation of the expression of PBDE genes and genes involved in cellular processes in an ascomycete fungus.

G protein γ subunit modulates expression of plant-biomass-degrading enzyme genes and mycelial-development-related genes in Penicillium oxalicum.

Heterotrimeric-G-protein-mediated signaling pathways modulate the expression of the essential genes in many fundamental cellular processes in fungi at the transcription level. However, these processes remain unclear in Penicillium oxalicum. In this study, we generated knockout and knockout-complemented strains of gng-1 (POX07071) encoding the Gγ protein and found that GNG-1 modulated the expression of genes encoding plant-biomass-degrading enzymes (PBDEs) and sporulation-related activators. Interestingly, GNG-1 affected expression of the cxrB that encodes a known transcription factor required for the expression of major cellulase and xylanase genes. Constitutive overexpression of cxrB in ∆gng-1 circumvented the dependence of PBDE production on GNG-1. Further evidence indicated that CxrB indirectly regulated the transcription levels of key amylase genes by controlling the expression of the regulatory gene amyR. These data extended the diversity of Gγ protein functions and provided new insight into the signal transduction and regulation of PBDE gene expression in filamentous fungi. KEY POINTS: • GNG-1 modulates the expression of PBDE genes and sporulation-related genes. • GNG-1 controls expression of the key regulatory gene cxrB. • Overexpression of cxrB circumvents dependence of PBDE production on GNG-1.

A mitogen-activated protein kinase PoxMK1 mediates regulation of the production of plant-biomass-degrading enzymes, vegetative growth, and pigment biosynthesis in Penicillium oxalicum.

Mitogen-activated protein kinase (MAPK) cascades are broadly conserved and play essential roles in multiple cellular processes, including fungal development, pathogenicity, and secondary metabolism. Their function, however, also exhibits species and strain specificity. Penicillium oxalicum secretes plant-biomass-degrading enzymes (PBDEs) that contribute to the carbon cycle in the natural environment and to utilization of lignocellulose in industrial processes. However, knowledge of the MAPK pathway in P. oxalicum has been relatively limited. In this study, comparative transcriptomic analysis of P. oxalicum, cultured on different carbon sources, found ten putative kinase genes with significantly modified transcriptional levels. Six of these putative kinase genes were knocked out in the parental strain ∆PoxKu70, and deletion of the gene, Fus3/Kss1-like PoxMK1 (POX00158), resulted in the largest reduction (91.1%) in filter paper cellulase production. Further tests revealed that the mutant ∆PoxMK1 lost 37.1 to 92.2% of PBDE production, under both submerged- and solid-state fermentation conditions, compared with ∆PoxKu70. In addition, the mutant ∆PoxMK1 had reduced vegetative growth and increased pigment biosynthesis. Comparative transcriptomic analysis showed that PoxMK1 deletion from P. oxalicum downregulated the expression of major PBDE genes and known regulatory genes such as PoxClrB and PoxCxrB, whereas the transcription of pigment biosynthesis-related genes was upregulated. Comparative phosphoproteomic analysis revealed that PoxMK1 deletion considerably modified phosphorylation of key transcription- and signal transduction-associated proteins, including transcription factors Mcm1 and Atf1, RNA polymerase II subunits Rpb1 and Rpb9, MAPK-associated Hog1 and Ste7, and cyclin-dependent kinase Kin28. These findings provide novel insights into understanding signal transduction and regulation of PBDE gene expression in fungi.Key points• PoxMK1 is involved in expression of PBDE- and pigment synthesis-related genes.• PoxMK1 is required for vegetative growth of P. oxalicum.• PoxMK1 is involved in phosphorylation of key TFs, kinases, and RNA polymerase II.

Involvement of phospholipase PLA2 in production of cellulase and xylanase by Penicillium oxalicum.

Phospholipases play vital roles in immune and inflammatory responses in mammals and plants; however, knowledge of phospholipase functions in fungi is limited. In this study, we investigated the effects of deleting predicted phospholipase genes on cellulase and xylanase production, and morphological phenotype, in Penicillium oxalicum. Individual deletion of nine of the ten predicted phospholipase genes resulted in alteration of cellulase and xylanase production, and the morphological phenotypes, to various degrees. The mutant ∆POX07277 lost 22.5 to 82.8% of cellulase (i.e., filter paper cellulase, carboxymethylcellulase, and p-nitrophenyl-ß-cellobiosidase) and xylanase production, whereas p-nitrophenyl-ß-glucopyranosidase production increased by 5.8-127.8 fold. POX07277 (P. oxalicum gene No. 07277) was predicted to encode phospholipase A2 and was found to negatively affect the sporulation of P. oxalicum. Comparative transcriptomic and quantitative reverse transcription-PCR analysis indicated that POX07277 dynamically affected the expression of cellulase and xylanase genes and the regulatory genes for fungal sporulation, under micro-crystalline cellulose induction. POX07277 was required for the expression of the known regulatory gene PoxCxrB (cellulolytic and xylanolytic regulator B in P. oxalicum), which is involved in cellulase and xylanase gene expression in P. oxalicum. Conversely, POX07277 expression was regulated by PoxCxrB. These findings will aid the understanding of phospholipase functions and provide novel insights into the mechanism of fungal cellulase and xylanase gene expression. KEY POINTS : • The roles of phospholipases were investigated in Penicillium oxalicum. • POX07277 (PLA2) is required for the expression of cellulase and xylanase genes. • PoxCxrB dynamically regulated POX07277 expression.

Identification of a unique 1,4-ß-D-glucan glucohydrolase of glycoside hydrolase family 9 from Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-ß-D-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl ß-D-cellobioside (ρ-NPC) and ρ-nitrophenyl ß-D-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The kcat/Km values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its kcat/Km for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth. KEY POINTS: • CHU_0961 and its homologues formed a novel group (group C) of GH9 enzymes. • CHU_0961 was identified as a 1,4-ß-d-glucan glucohydrolase with unique properties. • CHU_0961 may play an important role in the early stage of C. hutchinsonii growth.

Transcription Factor Atf1 Regulates Expression of Cellulase and Xylanase Genes during Solid-State Fermentation of Ascomycetes.

Transcriptional regulation of cellulolytic and xylolytic genes in ascomycete fungi is controlled by specific carbon sources in different external environments. Here, comparative transcriptomic analyses of Penicillium oxalicum grown on wheat bran (WB), WB plus rice straw (WR), or WB plus Avicel (WA) as the sole carbon source under solid-state fermentation (SSF) revealed that most of the differentially expressed genes (DEGs) were involved in metabolism, specifically, carbohydrate metabolism. Of the DEGs, the basic core carbohydrate-active enzyme-encoding genes which responded to the plant biomass resources were identified in P. oxalicum, and their transcriptional levels changed to various extents depending on the different carbon sources. Moreover, this study found that three deletion mutants of genes encoding putative transcription factors showed significant alterations in filter paper cellulase production compared with that of a parental P. oxalicum strain with a deletion of Ku70 (ΔPoxKu70 strain) when grown on WR under SSF. Importantly, the ΔPoxAtf1 mutant (with a deletion of P. oxalicumAtf1, also called POX03016) displayed 46.1 to 183.2% more cellulase and xylanase production than a ΔPoxKu70 mutant after 2 days of growth on WR. RNA sequencing and quantitative reverse transcription-PCR revealed that PoxAtf1 dynamically regulated the expression of major cellulase and xylanase genes under SSF. PoxAtf1 bound to the promoter regions of the key cellulase and xylanase genes in vitro This study provides novel insights into the regulatory mechanism of fungal cellulase and xylanase gene expression under SSF.IMPORTANCE The transition to a more environmentally friendly economy encourages studies involving the high-value-added utilization of lignocellulosic biomass. Solid-state fermentation (SSF), that simulates the natural habitat of soil microorganisms, is used for a variety of applications such as biomass biorefinery. Prior to the current study, our understanding of genome-wide gene expression and of the regulation of gene expression of lignocellulose-degrading enzymes in ascomycete fungi during SSF was limited. Here, we employed RNA sequencing and genetic analyses to investigate transcriptomes of Penicillium oxalicum strain EU2101 cultured on medium containing different carbon sources and to identify and characterize transcription factors for regulating the expression of cellulase and xylanase genes during SSF. The results generated will provide novel insights into genetic engineering of filamentous fungi to further increase enzyme production.

Purification and characterization of an endo-xylanase from Trichoderma sp., with xylobiose as the main product from xylan hydrolysis.

Fungal endo-ß-1,4-xylanases (endo-xylanases) can hydrolyze xylan into xylooligosaccharides (XOS), and have potential biotechnological applications for the exploitation of natural renewable polysaccharides. In the current study, we aimed to screen and characterize an efficient fungal endo-xylanase from 100 natural humus-rich soil samples collected in Guizhou Province, China, using extracted sugarcane bagasse xylan (SBX) as the sole carbon source. Initially, 182 fungal isolates producing xylanases were selected, among which Trichoderma sp. strain TP3-36 was identified as showing the highest xylanase activity of 295 U/mL with xylobiose (X2) as the main product when beechwood xylan was used as substrate. Subsequently, a glycoside hydrolase family 11 endo-xylanase, TXyn11A, was purified from strain TP3-36, and its optimal pH and temperature for activity against beechwood xylan were identified to be 5.0 and 55 °C, respectively. TXyn11A was stable across a broad pH range (3.0-10.0), and exhibited strict substrate specificity, including xylan from beechwood, wheat, rye, and sugarcane bagasse, with Km and Vmax values of 5 mg/mL and 1250 µmol/mg min, respectively, toward beechwood xylan. Intriguingly, the main product obtained from hydrolysis of beechwood xylan by TXyn11A was xylobiose, whereas SBX hydrolysis resulted in both X2 and xylotriose. Overall, these characteristics of the endo-xylanase TXyn11A indicate several potential industrial applications.

Transcription Factor NsdD Regulates the Expression of Genes Involved in Plant Biomass-Degrading Enzymes, Conidiation, and Pigment Biosynthesis in Penicillium oxalicum.

Soil fungi produce a wide range of chemical compounds and enzymes with potential for applications in medicine and biotechnology. Cellular processes in soil fungi are highly dependent on the regulation under environmentally induced stress, but most of the underlying mechanisms remain unclear. Previous work identified a key GATA-type transcription factor, Penicillium oxalicum NsdD (PoxNsdD; also called POX08415), that regulates the expression of cellulase and xylanase genes in P. oxalicum PoxNsdD shares 57 to 64% identity with the key activator NsdD, involved in asexual development in Aspergillus In the present study, the regulatory roles of PoxNsdD in P. oxalicum were further explored. Comparative transcriptomic profiling revealed that PoxNsdD regulates major genes involved in starch, cellulose, and hemicellulose degradation, as well as conidiation and pigment biosynthesis. Subsequent experiments confirmed that a ΔPoxNsdD strain lost 43.9 to 78.8% of starch-digesting enzyme activity when grown on soluble corn starch, and it produced 54.9 to 146.0% more conidia than the ΔPoxKu70 parental strain. During cultivation, ΔPoxNsdD cultures changed color, from pale orange to brick red, while the ΔPoxKu70 cultures remained bluish white. Real-time quantitative reverse transcription-PCR showed that PoxNsdD dynamically regulated the expression of a glucoamylase gene (POX01356/Amy15A), an α-amylase gene (POX09352/Amy13A), and a regulatory gene (POX03890/amyR), as well as a polyketide synthase gene (POX01430/alb1/wA) for yellow pigment biosynthesis and a conidiation-regulated gene (POX06534/brlA). Moreover, in vitro binding experiments showed that PoxNsdD bound the promoter regions of the above-described genes. This work provides novel insights into the regulatory mechanisms of fungal cellular processes and may assist in genetic engineering of Poxalicum for potential industrial and medical applications.IMPORTANCE Most filamentous fungi produce a vast number of extracellular enzymes that are used commercially for biorefineries of plant biomass to produce biofuels and value-added chemicals, which might promote the transition to a more environmentally friendly economy. The expression of these extracellular enzyme genes is tightly controlled at the transcriptional level, which limits their yields. Hitherto our understanding of the regulation of expression of plant biomass-degrading enzyme genes in filamentous fungi has been rather limited. In the present study, regulatory roles of a key regulator, PoxNsdD, were further explored in the soil fungus Penicillium oxalicum, contributing to the understanding of gene regulation in filamentous fungi and revealing the biotechnological potential of Poxalicum via genetic engineering.

Secretory overproduction of a raw starch-degrading glucoamylase in Penicillium oxalicum using strong promoter and signal peptide.

Raw starch-degrading enzymes (RSDEs) are capable of directly degrading raw starch granules below the gelatinization temperature of starch, which may significantly reduce the cost of starch-based biorefining. However, low yields of natural RSDEs from filamentous fungi limit their industrial application. In this study, transcriptomic and secretomic profiling was employed to screen strongest promoters and signal peptides for use in overexpression of a RSDE gene in Penicillium oxalicum. Top five strong promoters and three signal peptides were detected. Using a green fluorescent protein (GFP) as the reporter, the inducible promoter pPoxEgCel5B of an endoglucanase gene PoxEgCel5B and the signal peptide spPoxGA15A of a raw starch-degrading glucoamylase PoxGA15A were respectively identified as driving the highest GFP production in P. oxalicum. PoxGA15A-overexpressed P. oxalicum strain OXPoxGA15A, which was constructed based on both pPoxEgCel5B and spPoxGA15A, produced significantly higher amounts of recombinant PoxGA15A than the parental strain ∆PoxKu70. Furthermore, crude enzyme from the OXPoxGA15A strain exhibited high activities towards raw starch from cassava, potato, and uncooked soluble starch. Specifically, raw cassava starch-degrading enzyme activity reached 241.6 U/mL in the OXPoxGA15A, which was 3.4-fold higher than that of the ∆PoxKu70. This work provides a feasible method for hyperproduction of RSDEs in P. oxalicum.

Characterization of novel roles of a HMG-box protein PoxHmbB in biomass-degrading enzyme production by Penicillium oxalicum.

High-mobility group (HMG)-box proteins are involved in chromatin organization in eukaryotes, especially in sex determination and regulation of mitochondrial DNA compaction. Although a novel HMG-box protein, PoxHmbB, had been initially identified to be required for filter paper cellulase activity by Penicillium oxalicum, the biological roles of HMG-box proteins in biomass-degrading enzyme production have not been systematically explored. The P. oxalicum mutant ∆PoxHmbB lost 34.7-86.5% of cellulase (endoglucanase, p-nitrophenyl-ß-cellobiosidase, and p-nitrophenyl-ß-glucopyranosidase) activities and 60.3% of xylanase activity following Avicel induction, whereas it exhibited about onefold increase in amylase activity following soluble corn starch induction. Furthermore, ∆PoxHmbB presented delayed conidiation and hyphae growth. Transcriptomic profiling and real-time quantitative reverse transcription-PCR revealed that PoxHmbB regulated the expression of major genes encoding plant biomass-degrading enzymes such as PoxCel7A-2, PoxCel5B, PoxBgl3A, PoxXyn11B, and PoxGA15A, as well as those involved in conidiation such as PoxBrlA. In vitro binding experiments further confirmed that PoxHmbB directly binds to the promoter regions of these major genes. These results further indicate the diversity of the biological functions of HMG-box proteins and provide a novel and promising engineering target for improving plant biomass-degrading enzyme production in filamentous fungi.

Deletion of TpKu70 facilitates gene targeting in Talaromyces pinophilus and identification of TpAmyR involvement in amylase production.

Talaromyces pinophilus is a promising filamentous fungus for industrial production of biomass-degrading enzymes used in biorefining, and its genome was recently sequenced and reported. However, functional analysis of genes in T. pinophilus is rather limited owing to lack of genetic tools. In this study, a putative TpKu70 encoding the Ku70 homolog involved in the classic non-homologous end-joining pathway was deleted in T. pinophilus 1-95. ΔTpKu70 displayed no apparent defect in vegetative growth and enzyme production, and presented similar sensitivity to benomyl, bleomycin, and UV, when compared with the wild-type T. pinophilus strain 1-95. Seven genes that encode putative transcription factors, including TpAmyR, were successfully knocked out in ΔTpKu70 at 61.5-100% of homologous recombination frequency, which is significantly higher than that noted in the wild-type. Interestingly, ΔTpAmyR produced approximately 20% of amylase secreted by the parent strain ΔTpKu70 in medium containing soluble starch from corn as the sole carbon source. Real-time quantitative reverse transcription PCR showed that TpAmyR positively regulated the expression of genes encoding α-amylase and glucoamylase. Thus, this study provides a useful tool for genetic analysis of T. pinophilus, and identification of a key role for the transcription factor TpAmyR in amylase production in T. pinophilus.

IL-10-dependent Effect of Chinese Medicine Abelmoschus manihot on Alleviating Intestinal Inflammation and Modulating Gut Microbiota.

Inflammatory bowel disease (IBD) is a recurrent disease associated with a potential risk of colorectal cancer. Abelmoschus manihot (AM), a Chinese herbal medicine, is known to alleviate IBD. However, its mechanism of action requires further clarification. Here, we focused on the role of IL-10 and the gut microbiota in the mechanism of action of AM. The effects of AM on intestinal inflammation, mucus production, and gut microbes were evaluated in dextran sodium sulfate (DSS)-induced acute and chronic IBD models and in IL-10-deficient mice (IL-10[Formula: see text]). AM exhibited protective effects on acute and chronic models of IBD in wild-type mice by restoring body weight and colon length, promoting IL-10 secretion, and decreasing TNF-[Formula: see text] levels. Moreover, AM alleviated inflammatory infiltration, increased mucin 2 transcription, and increased the number of goblet cells in the colon. On the contrary, these effects were diminished in IL-10[Formula: see text] mice, which implied that the effect of AM on intestinal inflammation is IL-10-dependent. A gut microbial sequencing analysis showed that gut microbial dysbiosis was modulated by AM intervention. The regulatory effects of AM on Eggerthellaceae, Sutterellaceae, Erysipelotrichaceae, Burkholderiaceae, Desulfovibrionaceae, and Enterococcaceae were dependent on IL-10. These results revealed that AM ameliorated IBD and modulated gut microbes by promoting IL-10 secretion, indicating that AM has the potential to improve IBD and that AM is IL-10-dependent.

Improvement of triterpenoid production in mycelia of Antrodia camphorata through mutagenesis breeding and amelioration of CCl4-induced liver injury in mice.

Due to the scarcity of wild fruiting bodies, submerged fermentation of the medicinal fungus Antrodia camphorata is attracting much attention, but the production of bioactive triterpenoids is low. Therefore, there is an urgent need to improve the triterpenoid yield of submerged fermentation. Here, the A. camphorata mutant E3-64 was generated from strain AC16101 through random mutagenesis breeding, producing 172.8 mg triterpenoid per gram of dry mycelia. Further optimization of culture parameters resulted in a yield of 255.5 mg/g dry mycelia (i.e., an additional >1.4-fold increase), which is the highest reported yield thus far. Notably, mutant E3-64 produced 94% and 178% more of the triterpenoid components antcin A and antcamphin A, respectively, while it produced 52% and 15% less antcin B and G, respectively. Mutant E3-64 showed increased expression of key genes involved in triterpenoid biosynthesis, as well as different genome-wide single-nucleotide polymorphisms as compared with AC16101. Triterpenoids of the E3-64 mycelia exhibited remarkably protective activity against acute CCl4-induced liver injury in mice. This study shows the potential of A. camphorata for scientific research and commercial application.

Three-Dimensional Genome Map of the Filamentous Fungus Penicillium oxalicum.

Higher-order spatial organization of the chromatin in the nucleus plays crucial roles in the maintenance of cell functions and the regulation of gene expression. Three-dimensional (3D) genome sequencing has been used to great effect in mammal and plants, but the availability of 3D genomes of filamentous fungi is severely limited. Here, we performed a chromosome-level genome assembly of Penicillium oxalicum through single-molecule real-time sequencing (Pacific Biosciences) and chromatin interaction mapping (Hi-C), with a scaffold N50 of 4.07 Mb and a contig N50 of 3.81 Mb, and further elucidated the 3D genome architecture of P. oxalicum. High-frequency interchromosomal contacts occurred within the centromeres and telomeres, as well as within individual chromosomes. There were 12,203 cis-interactions and 7,884 trans-interactions detected at a resolution of 1 kb. Moreover, a total of 1,099 topologically associated domains (or globules) were found, ranging in size from 2.0 to 76.0 kb. Interestingly, transcription factor-bound motifs were enriched in the globule boundaries. All the cellulase and xylanase genes were discretely distributed in the 3D model of the P. oxalicum genome as a result of few cis- and trans-interactions. Our results from this study provide a global view of chromatin interactions in the P. oxalicum genome and will act as a resource for studying spatial regulation of gene expression in filamentous fungi. IMPORTANCE The spatial structure of chromatin plays important roles in normal cell functions and the regulation of gene expression. The three-dimensional (3D) architectures of the genomes of many mammals and plants have been elucidated, but corresponding studies on filamentous fungi, which play vital roles as decomposers of organic matter in the soil, are very limited. Penicillium oxalicum is one of the predominant cellulolytic aerobic fungi in subtropical and tropical forest soils and can secrete integrative cellulase and xylanase under integrated regulatory control, degrading plant biomass highly efficiently. In the present study, we employed Hi-C technology to construct the 3D genome model of P. oxalicum strain HP7-1 and to further investigate cellulase and xylanase as well as transcription factor genes in 3D genome. These results provide a resource to achieve a deeper understanding of cell function and the regulation of gene expression in filamentous fungi.

[Discussion on the essential thought of Ma's warm moxibustion technique for "unblocking fu-organs"].

"Unblocking fu organs" is one of the essential principles of Ma's warm moxibustion technique, characterized as "dredging" and "harmonizing" for either deficiency or excess condition. Under the guidance of this therapeutic thought, the acupoints for moxibustion are mainly selected from the middle and lower parts of the body. Regarding the therapeutic approach, the acupoint prescription for moxibustion should be formed in line with warming and promoting circulation of fu organs; the moxibustion degree should be specially considered, in which, the mild moxibustion is recommended to induce promoting action; and the systematic moxibustion technique should be the root for dredging fu organs and regulating zang organs. Ma's mild moxibustion technique stresses on removing the obstruction of fu organs and emphasizes promoting the qi activity of sanjiao (triple energizer) and regulating the balance of five zang organs.

Improvement of cellulase and xylanase production in Penicillium oxalicum under solid-state fermentation by flippase recombination enzyme/ recognition target-mediated genetic engineering of transcription repressors.

Penicillium oxalicum has received increasing attention as a potential cellulase-producer. In this study, a copper-controlled flippase recombination enzyme/recognition target (FLP/FRT)-mediated recombination system was constructed in P. oxalicum, to overcome limited availability of antibiotic resistance markers. Using this system, two crucial transcription repressor genes atf1 and cxrC for the production of cellulase and xylanase under solid-state fermentation (SSF) were simultaneously deleted, thereby leading to 2.4- to 29.1-fold higher cellulase and 78.9% to 130.8% higher xylanase production than the parental strain under SSF, respectively. Glucose and xylose released from hydrolysis of pretreated sugarcane bagasse achieved 10.6%-13.5% improvement by using the crude enzymes from the engineered strain Δatf1ΔcxrC::flp under SSF in comparison with that of the parental strain. Consequently, these results provide a feasible strategy for improved cellulase and xylanase production by filamentous fungi.

A transcriptomic analysis of sugarcane response to Leifsonia xyli subsp. xyli infection.

Sugarcane ratoon stunting disease (RSD) caused by Leifsonia xyli subsp. xyli (Lxx) is a common destructive disease that occurs around the world. Lxx is an obligate pathogen of sugarcane, and previous studies have reported some physiological responses of RSD-affected sugarcane. However, the molecular understanding of sugarcane response to Lxx infection remains unclear. In the present study, transcriptomes of healthy and Lxx-infected sugarcane stalks and leaves were studied to gain more insights into the gene activity in sugarcane in response to Lxx infection. RNA-Seq analysis of healthy and diseased plants transcriptomes identified 107,750 unigenes. Analysis of these unigenes showed a large number of differentially expressed genes (DEGs) occurring mostly in leaves of infected plants. Sugarcane responds to Lxx infection mainly via alteration of metabolic pathways such as photosynthesis, phytohormone biosynthesis, phytohormone action-mediated regulation, and plant-pathogen interactions. It was also found that cell wall defense pathways and protein phosphorylation/dephosphorylation pathways may play important roles in Lxx pathogeneis. In Lxx-infected plants, significant inhibition in photosynthetic processes through large number of differentially expressed genes involved in energy capture, energy metabolism and chloroplast structure. Also, Lxx infection caused down-regulation of gibberellin response through an increased activity of DELLA and down-regulation of GID1 proteins. This alteration in gibberellic acid response combined with the inhibition of photosynthetic processes may account for the majority of growth retardation occurring in RSD-affected plants. A number of genes associated with plant-pathogen interactions were also differentially expressed in Lxx-infected plants. These include those involved in secondary metabolite biosynthesis, protein phosphorylation/dephosphorylation, cell wall biosynthesis, and phagosomes, implicating an active defense response to Lxx infection. Considering the fact that RSD occurs worldwide and a significant cause of sugarcane productivity, a better understanding of Lxx resistance-related processes may help develop tools and technologies for producing RSD-resistant sugarcane varieties through conventional and/or molecular breeding.