A CAR-T response prediction model for r/r B-NHL patients based on a T cell subset nomogram.

BACKGROUND: Chimeric antigen receptor (CAR) T cells for refractory or relapsed (r/r) B cell no-Hodgkin lymphoma (NHL) patients have shown promising clinical effectiveness. However, the factors impacting the clinical response of CAR-T therapy have not been fully elucidated. We here investigate the independent influencing factors of the efficacy of CD19 CAR-T cell infusion in the treatment of r/r B-NHL and to establish an early prediction model. METHODS: A total of 43 r/r B-NHL patients were enrolled in this retrospective study. The patients' general data were recorded, and the primary endpoint is the patients' treatment response. The independent factors of complete remission (CR) and partial remission (PR) were investigated by univariate and binary logistic regression analysis, and the prediction model of the probability of CR was constructed according to the determined independent factors. Receiver operating characteristic (ROC) and calibration plot were used to assess the discrimination and calibration of the established model. Furthermore, we collected 15 participators to validate the model. RESULTS: Univariate analysis and binary logistic regression analysis of 43 patients showed that the ratio of central memory T cell (Tcm) and naïve T cell (Tn) in cytotoxic T cells (Tc) was an independent risk factor for response to CD19 CAR-T cell therapy in r/r B-NHL. On this basis, the area under the curve (AUC) of Tcm in the Tc and Tn in the Tc nomogram model was 0.914 (95%CI 0.832-0.996), the sensitivity was 83%, and the specificity was 74.2%, which had excellent predictive value. We did not found the difference of the progression-free survival (PFS). CONCLUSIONS: The ratio of Tcm and Tn in Tc was found to be able to predict the treatment response of CD19 CAR-T cells in r/r B-NHL. We have established a nomogram model for the assessment of the CD19 CAR-T therapy response presented high specificity and sensitivity.

Rogersonins C-F, 9H-Imidazo[2,1-i]purine-Incorporating Adenine-Polyketide Hybrids from an Ophiocordyceps-Associated Clonostachys rogersoniana.

Rogersonins C-F (1-4), four unprecedented adenine-polyketide hybrids featuring a rare 9H-imidazo[2,1-i]purine (1,N6-ethenoadenine) moiety, were isolated from an Ophiocordyceps-associated fungus, Clonostachys rogersoniana. Their structures were elucidated primarily by NMR experiments. The absolute configurations of 1-4 were assigned by a combination of the modified Mosher method, chemical degradation, electronic circular dichroism (ECD) calculations, and X-ray crystallography using Cu Kα radiation. Compound 3 downregulated the expression of PD-L1 protein in MDA-MB-231 and A549 cells, but did not show detectable effect on mRNA transcription of the PD-L1-encoding gene CD274.

DhFIG_2, a gene of nematode-trapping fungus Dactylellina haptotyla that encodes a component of the low-affinity calcium uptake system, is required for conidiation and knob-trap formation.

Calcium ion (Ca2+) is a universal second messenger involved in regulating diverse processes in animals, plants, and fungi. The low-affinity calcium uptake system (LACS) participates in acquiring Ca2+ from extracellular environments under high extracellular Ca2+ concentration. Unlike most fungi, which encode only one protein (FIG1) for LACS, nematode-trapping fungi (NTF) encode two related proteins. AoFIG_2, the NTF-specific LACS component encoded by adhesive network-trap forming Arthrobotrys oligospora, was shown to be required for conidiation and trap formation. We characterized the role of DhFIG_2, an AoFIG_2 ortholog encoded by knob-trap forming Dactylellina haptotyla, in growth and development to expand our understanding of the role of LACS in NTF. Because repeated attempts to disrupt DhFIG_2 failed, knocking down the expression of DhFIG_2 via RNA interference (RNAi) was used to study its function. RNAi of DhFIG_2 significantly decreased its expression, severely reduced conidiation and trap formation, and affected vegetative growth and stress responses, suggesting that this component of LACS is crucial for trap formation and conidiation in NTF. Our study demonstrated the utility of RNAi assisted by ATMT for studying gene function in D. haptotyla.

Identification of new bisabosqual-type meroterpenoids reveals non-enzymatic conversion of bisabosquals into seco-bisabosquals.

Bisabosqual-type meroterpenoids are fungi-derived polyketide-terpenoid hybrids bearing a 2,3,3a,3a1,9,9a-hexahydro-1H-benzofuro[4,3,2-cde]chromene skeleton (6/6/6/5 ring system) or its seco-C-ring structure, and exhibit diverse bioactivities. Their unique structural architecture and impressive biological activities have led to considerable interest in discovering new analogues. However, to date, only nine analogues have been identified. Herein, we reported the isolation and identification of six new bisabosqual-type meroterpenoids stachybisbins C-H (1-6), together with one known compound bisabosqual C (7), from Stachybotrys bisbyi PYH05-7. Intriguingly, we found that 7, which contains the intact tetracyclic skeleton, can be non-enzymatically converted into its seco derivative stachybisbin I (8), unveiling the biosynthetic relationship between bisabosquals and seco-bisabosquals. Moreover, based on CRISPR/Cas9-mediated gene disruption, we revealed that the three-gene cluster responsible for the formation of LL-Z1272ß is associated with the biosynthesis of bisabosqual-type meroterpenoids, and then proposed a plausible route to 1-8.

Altersteroids A-D, 9,11-Secosteroid-Derived γ-Lactones from an Alternaria sp.

Altersteroids A-D (1-4), four new 9,11-secosteroid-derived γ-lactones, were isolated from cultures of the ascomycete fungus Alternaria sp. Their structures were elucidated primarily by NMR experiments. The absolute configuration of 1 was established by X-ray crystallographic analysis of its di-p-nitrobenzenesulfonate 1a using Cu Kα radiation, whereas those for 2-4 were assigned by quantum-chemical calculations. Compounds 1-4 incorporate a γ-lactone moiety fused to the steroid D ring at C-13/C-14. Compound 3 showed moderate cytotoxicity toward four tumor cell lines and induced an apoptotic process in A549 cells. Notably, compound 3 showed equipotent activity against the cisplatin-sensitive MB49 and -resistant MB49 CisR cells, with an IC50 value of 12.7 µM.

Cladoxanthones A and B, Xanthone-Derived Metabolites with a Spiro[cyclopentane-1,2'-[3,9a]ethanoxanthene]-2,4',9',11'-tetraone Skeleton from a Cladosporium sp.

Cladoxanthones A (1) and B (2), two xanthone-derived metabolites featuring a new spiro[cyclopentane-1,2'-[3,9a]ethanoxanthene]-2,4',9',11'(4a'H)-tetraone skeleton, were isolated from cultures of the ascomycete fungus Cladosporium sp., together with the known mangrovamide J (3). Their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2 were assigned by X-ray crystallography using Cu Kα radiation. Compound 1 could be generated from the hypothetical precursors related to α-methylene ketone and dihydro-xanthone via a Diels-Alder reaction, while 2 could be an oxidative coupling product resulting from 1 and 3. Compounds 1 and 2 showed weakly cytotoxic effects.

Bacterial Volatile-Mediated Suppression of Root-Knot Nematode (Meloidogyne incognita).

Root-knot nematodes (Meloidogyne spp.) are obligate plant parasites that cause severe economic losses to agricultural crops worldwide. Because of serious health and environmental concerns related to the use of chemical nematicides, the development of efficient alternatives is of great importance. Biological control through exploiting the potential of rhizosphere microorganisms is currently accepted as an important approach for pest management in sustainable agriculture. In our research, during screening of rhizosphere bacteria against the root-knot nematodes Meloidogyne incognita, Ochrobactrum pseudogrignonense strain NC1 from the rhizosphere of healthy tomatoes showed strong nematode inhibition. A volatile nematicidal assay showed that the cell-free fermentation filtrate in the first-row wells of 12-well tissue culture plates caused M. incognita juvenile mortality in the second-row wells. Gas chromatography-mass spectrometry analysis revealed that dimethyl disulfide (DMDS) and benzaldehyde were the main volatile compounds produced by strain NC1. The nematicidal activity of these compounds indicated that the lethal concentration 50 against the M. incognita juveniles in the second-row wells and the fourth-row wells were 23.4 µmol/ml and 30.7 µmol/ml for DMDS and 4.7 µmol/ml and 15.2 µmol/ml for benzaldehyde, respectively. A greenhouse trial using O. pseudogrignonense strain NC1 provided management efficiencies of root-knot nematodes of 88 to 100% compared with the untreated control. This study demonstrated that nematode-induced root-gall suppression mediated by the bacterial volatiles DMDS and benzaldehyde presents a new opportunity for root-knot nematode management.

Plant Defense Responses to a Novel Plant Elicitor Candidate LY5-24-2.

Plant elicitors enhance plant defense against pathogen attacks by inducing systemic acquired resistance (SAR) with no or low direct fungicidal activity. Here we report the synthesis of a novel plant elicitor candidate LY5-24-2 [3,4-dichloro-N-(3-chloro-5-(trifluoromethyl)pyridin-2-yl)isothiazole-5-carboxamide] and evaluation of its SAR inducing activity. Bioassays indicated that LY5-24-2 did not show significant anti-fungal activity but provided long-lasting resistance in Arabidopsis thaliana (A. thaliana) through promoting the accumulation of lignin, cellulose and pectin by 60.1%, 82.4% and 305.6%, respectively, at a concentration of 100 µM. LY5-24-2 also facilitated the closure of leaf stomata and increased the intracellular free Ca2+ by 47.8%, induced reactive oxygen species (ROS) accumulation, and inhibited the activity of ascorbate peroxidase (APX, EC 1.11.1.11) and catalase (CAT, EC 1.11.1.6) by 38.9% and 34.0%, respectively, as compared with the control at a concentration of 100 µM. LY5-24-2 induced SAR in plants and was dependent on the NPR1-mediated SA pathway by up-regulating expression of 2273 genes in A. thaliana. Meanwhile, LY5-24-2 also improved cucumber (Cucumis sativus L.) defense against Pseudoperonospora cubensis (P. cubensis) through promoting ROS accumulation and inhibiting activity of APX and CAT by 30.7% and 23.1%, respectively. Its expression of SA signaling genes CsNPR1, CsPR4 and CsPR5 was enhanced by 10.8, 5.8 and 6.6 times, respectively. These results demonstrated that LY5-24-2 is a novel elicitor candidate for plant protection via inducing SAR.

DdaSTE12 is involved in trap formation, ring inflation, conidiation, and vegetative growth in the nematode-trapping fungus Drechslerella dactyloides.

Ste12 transcription factors, downstream of mitogen-activated protein kinase (MAPK) signalling pathways, are exclusively found in the fungal kingdom and regulate fungal mating, development, and pathogenicity. The nematode-trapping fungus Drechslerella dactyloides can capture free-living nematodes using constricting rings by cell inflation within 1 s when stimulated by nematodes entering the rings. The MAPK signalling pathways are involved in the trap formation of nematode-trapping fungi, but their downstream regulation is not clearly understood. In this study, disruption of the DdaSTE12 gene in D. dactyloides disabled cell inflation of constricting rings and led to an inability to capture nematodes. The number of septa of constricting rings and the ring cell vacuoles were changed in ΔDdaSTE12. Compared with the wild type, ΔDdaSTE12 reduced trap formation, conidiation, and vegetative growth by 79.3%, 80.3%, and 21.5%, respectively. The transcriptomes of ΔDdaSTE12-3, compared with those of the wild type, indicated that the expression of genes participating in trap formation processes, including signal transduction (Gpa2 and a 7-transmembrane receptor), vesicular transport and cell fusion (MARVEL domain-containing proteins), and nematode infection (PEX11 and CFEM domain-containing proteins), is regulated by DdaSTE12. The results suggest that DdaSTE12 is involved in trap formation and ring cell inflation, as well as conidiation and vegetative growth, by regulating a wide range of downstream functions. Our findings expanded the roles of Ste12 homologous transcription factors in the development of constricting rings and provided new insights into the downstream regulation of the MAPK signalling pathway involved in nematode predation. KEY POINTS: • DdaSTE12 was the first gene disrupted in D. dactyloides. • DdaSTE12 is related to ring cell inflation, vegetative growth, and conidiation. • DdaSTE12 deletion resulted in defects in trap formation and ring development.

Two transcription factors cooperatively regulate DHN melanin biosynthesis and development in Pestalotiopsis fici.

Fungal 1,8-dihydroxynaphthalene (DHN) melanin plays important roles in UV protection, oxidative stress and pathogenesis. However, knowledge of the regulatory mechanisms of its biosynthesis is limited. Previous studies showed two transcription factors, PfmaF and PfmaH, located in the DHN melanin biosynthetic gene cluster (Pfma) in Pestalotiopsis fici. In this study, deletion of PfmaH resulted in loss of melanin and affected conidia cell wall integrity. Specifically, PfmaH directly regulates the expression of scytalone dehydratase, which catalyzes the transition of scytalone to T3 HN. However, PfmaF disruption using CRISPR/Cas9 system affected neither DHN melanin distribution nor conidia cell wall integrity in P. fici. Unexpectedly, overexpression of PfmaF leads to heavy pigment accumulation in P. fici hyphae. Transcriptome and qRT-PCR analyses provide insight into the roles of PfmaF and PfmaH in DHN melanin regulation. PfmaH, as a pathway specific regulator, mainly regulates melanin biosynthesis that contributes to cell wall development. Furthermore, PfmaF functions as a broad regulator to stimulate PfmaH expression in melanin production, secondary metabolism as well as fungal development.

Disulfide Cleavage in a Dimeric Epipolythiodioxopiperazine Natural Product Diminishes Its Apoptosis-Inducing Effect but Enhances Autophagy in Tumor Cells.

Gliocladicillin C (3) is a cytotoxic epipolythiodioxopiperazine (ETP) isolated from the Ophiocordyceps-associated fungus Clonostachys rogersoniana. Although the disulfides/polysulfides in ETPs are believed to account for their cytotoxicity, and 11'-deoxyverticillin A was demonstrated to induce apoptosis and autophagy, how they mediate apoptosis and autophagy remained unknown. Here, we revealed that 3 activated caspase-dependent apoptosis and autophagy in human tumor cells, while the prepared disulfide-cleavage product failed to induce reactive oxygen species production and PARP cleavage, but further enhanced the autophagic flux compared to 3. Gliocladicillin C and its derivative also increased the phosphorylation of AMP-activated protein kinase and stimulated autophagy by affecting the glycolytic pathway. These results demonstrated that the disulfides played an essential role in inducing apoptosis, but not autophagy.

A new regulator RsdA mediating fungal secondary metabolism has a detrimental impact on asexual development in Pestalotiopsis fici.

Secondary metabolite (SM) production and development are correlated processes in fungi that are often coordinated by pleiotropic regulators. The eukaryotic regulators are critical players in mediating SM production related to fungal development, yet little data are available to support this hypothesis. In this study, a global regulator, RsdA (regulation of secondary metabolism and development), was identified through genome-wide analysis and deletion of the regulator gene in the endophytic fungus Pestalotiopsis fici. Here, we established that RsdA regulation of SMs is accompanied by the repression of asexual development. Deletion of rsdA significantly reduces not only asexual development, resulting in low sporulation and abnormal conidia, but also the major SM production, while remarkably increasing the melanin production. Overproduction of melanin leads to the formation of unusual, heavily pigmented hyphae. Transcriptome analysis data provide the evidence that RsdA globally regulates genes involved in secondary metabolism and asexual development. Double deletion of rsdA and the melanin polyketide synthase gene PfmaE confirm that RsdA regulation of asexual development is independent of the melanin biosynthetic pathway. Finally, our results demonstrate that RsdA can be used for the discovery of secondary metabolites in filamentous fungi.

Identification of the gene cluster for bistropolone-humulene meroterpenoid biosynthesis in Phoma sp.

Eupenifeldin, a bistropolone meroterpenoid, was first discovered as an antitumor agent from the fungus Eupenicillium brefeldianum. We also isolated this compound and a new congener from a strain of Phoma sp. (CGMCC 10481), and evaluated their antitumor effects. Eupenifeldin showed potent in vitro anti-glioma activity. This tropolone-humulene-tropolone meroterpenoid could be originated from two units of tropolone orthoquinone methides and a 10-hydroxyhumulene moiety via hetero-Diels-Alder reactions. To explore the biosynthesis of this class of tropolonic sesquiterpenes, the genome of a eupenifeldin-producing Phoma sp. was sequenced and analyzed. The biosynthetic gene cluster of eupenifeldin (eup) was identified and partially validated by genomic analysis, gene disruption, and product analysis. A nonreducing polyketide synthase EupA, a FAD-dependent monooxygenase EupB, and a non-heme Fe (II)-dependent dioxygenase EupC, were identified as the enzymes responsible for tropolone formation. While the terpene cyclase EupE of an unknown family was characterized to catalyze humulene formation, and a cytochrome P450 enzyme EupD was responsible for hydroxylation of humulene. This study sheds light on the biosynthesis of eupenifeldin, and paves the way to further decipher its biosynthetic pathway.

A breakthrough in the artificial cultivation of Chinese cordyceps on a large-scale and its impact on science, the economy, and industry.

Chinese cordyceps, an entity of the Chinese caterpillar fungus (Ophiocordyceps sinensis, syn. Cordyceps sinensis) that parasitizes ghost moth larvae, is one of the best known traditional Chinese medicines and is found exclusively on the Tibetan Plateau with limited natural resources. Although the fungus O. sinensis can grow on artificial substrates and the ghost moth has been successfully reared, the large-scale artificial cultivation of Chinese cordyceps has only recently been accomplished after several decades of efforts and attempts. In this article, research progress related to this breakthrough from living habitats, the life history of the fungus, its host insect, fungal isolation and culture, host larvae rearing, infection cycle of the fungus to the host, primordium induction, and fruiting body development have been reviewed. An understanding of the basic biology of O. sinensis, its host insect and the simulation of the Tibetan alpine environment resulted in the success of artificial cultivation on a large scale. Practical workshop production has reached annual yields of 2.5, 5, and 10 tons in 2014, 2015, and 2016, respectively. There was no difference in the chemical components detected between the cultivated and natural Chinese cordyceps. However, the artificial cultivation system can be controlled to avoid heavy metal contamination and results in high-quality products. Although omics studies, including genomic, transcriptomic, proteomic, and metabolomic studies, have helped to understand the biology of the fungus, the success of the artificial cultivation of the Chinese cordyceps is clearly a milestone and provides the possibility for research on the in-depth mechanisms of the interaction between the fungus and host insects and their adaptation to the harsh habitats. This cultivation will not only result in a large industry to alleviate the pressure of human demand but also protect the limited natural resources for sustainable utilization.

The disruption of verM activates the production of gliocladiosin A and B in Clonostachys rogersoniana.

Gliocladiosin A (1) and B (2), two dipeptides conjugated with macrolides, were identified from a verM disruption mutant of the Cordycep-colonizing fungus Clonostachys rogersoniana. The structures and absolute configurations of 1 and 2 were determined on the basis of spectroscopic data analysis, including MS, NMR, CD and X-ray diffraction. A biogenetic pathway for 1 and 2 was proposed. These two compounds showed moderate antibacterial effects.

Phomanolides C-F from a Phoma sp.: Meroterpenoids Generated via Hetero-Diels-Alder Reactions.

Phomanolides C-F (1-4), four new meroterpenoids, were isolated from a Phoma sp., together with the known phomanolides A (5) and B (6); their structures were elucidated primarily by NMR experiments. The absolute configurations of 1-3 were assigned by electronic circular dichroism calculations, and that of 4 was established by X-ray diffraction analysis using Cu Kα radiation. Compounds 1-3 incorporate an unprecedented trioxa[4.4.3]propellane subunit in their skeletons. Compounds 2 and 4 were weakly cytotoxic.

Rogersonins A and B, Imidazolone N-Oxide-Incorporating Indole Alkaloids from a verG Disruption Mutant of Clonostachys rogersoniana.

Rogersonins A (1) and B (2), two new indole alkaloid-polyketide hybrids, have been isolated from cultures of a verG disruption mutant of the Cordyceps-colonizing fungus Clonostachys rogersoniana; their structures were elucidated primarily by NMR experiments. The absolute configurations of 1 and 2 were assigned using the modified Mosher method and via electronic circular dichroism and NMR calculations. Compounds 1 and 2 incorporate an imidazolone N-oxide moiety that has not been reported in any natural products.

Volatile Compounds Emitted by Diverse Verticillium Species Enhance Plant Growth by Manipulating Auxin Signaling.

Some volatile compounds (VC) play critical roles in intra- and interspecies interactions. To investigate roles of VC in fungal ecology, we characterized how VC produced by Verticillium spp., a group of broad-host-range soilborne fungal pathogens, affect plant growth and development. VC produced by 19 strains corresponding to 10 species significantly enhanced the growth of Arabidopsis thaliana and Nicotiana benthamiana. Analysis of VC produced by four species revealed the presence of diverse compounds, including those previously shown to affect plant growth. Using A. thaliana, we investigated the mechanism underpinning plant growth enhancement by Verticillium dahliae VC. Allometric analysis indicated that VC caused preferential resource allocation for root growth over shoot growth. Growth responses of A. thaliana mutants defective in auxin or ethylene signaling suggested the involvement of several components of auxin signaling, with TIR3 playing a key role. AUX1, TIR1, and AXR1 were also implicated but appeared to play lesser roles. Inhibition of auxin efflux using 1-naphthylphthalamic acid blocked VC-mediated growth enhancement. Spatial and temporal expression patterns of the auxin-responsive reporter DR5::GUS indicated that the activation of auxin signaling occurred before enhanced plant growth became visible. Results from this study suggest critical yet overlooked roles of VC in Verticillium ecology and pathology.

A cryptic pigment biosynthetic pathway uncovered by heterologous expression is essential for conidial development in Pestalotiopsis fici.

Spore pigmentation is very common in the fungal kingdom. The best studied pigment in fungi is melanin which coats the surface of single cell spores. What and how pigments function in a fungal species with multiple cell conidia is poorly understood. Here, we identified and deleted a polyketide synthase (PKS) gene PfmaE and showed that it is essential for multicellular conidial pigmentation and development in a plant endophytic fungus, Pestalotiopsis fici. To further characterize the melanin pathway, we utilized an advanced Aspergillus nidulans heterologous system for the expression of the PKS PfmaE and the Pfma gene cluster. By structural elucidation of the pathway metabolite scytalone in A. nidulans, we provided chemical evidence that the Pfma cluster synthesizes DHN melanin. Combining genetic deletion and combinatorial gene expression of Pfma cluster genes, we determined that the putative reductase PfmaG and the PKS are sufficient for the synthesis of scytalone. Feeding scytalone back to the P. fici ΔPfmaE mutant restored pigmentation and multicellular adherence of the conidia. These results cement a growing understanding that pigments are essential not simply for protection of spores from biotic and abiotic stresses but also for spore structural development.

Genomics-driven discovery of a novel self-resistance mechanism in the echinocandin-producing fungus Pezicula radicicola.

The echinocandins are antifungal lipopeptides targeting fungi via noncompetitive inhibition of the ß-1,3-d-glucan synthase FKS1 subunit. A novel echinocandin resistance mechanism involving an auxiliary copy of FKS1 in echinocandin-producing fungus Pezicula radicicola NRRL 12192 was discovered. We sequenced the genome of NRRL 12192 and predicted two FKS1-encoding genes (prfks1n and prfks1a), rather than a single FKS1 gene typical of filamentous ascomycetes. The prfks1a gene sits immediately adjacent to an echinocandin (sporiofungin) gene cluster, which was confirmed by disruption of prnrps4 and abolishment of sporiofungin production. Disruption of prfks1a dramatically increased the strain's sensitivity to exogenous echinocandins. In the absence of echinocandins, transcription levels of prfks1a relative to ß-tubulin in the wild type and in Δprnrps4 stains were similar. Moreover, prfks1a is consistently transcribed at low levels and is upregulated in the presence of exogenous echinocandin, but not during growth conditions promoting endogenous production of sporiofungin. Therefore, we conclude that prfks1a is primarily responsible for protecting the fungus against extracellular echinocandin toxicity. The presence of unclustered auxiliary copies of FKS1 with high similarity to prfks1a in two other echinocandin-producing strains suggests that this previously unrecognized resistance mechanism may be common in echinocandin-producing fungi of the family Dermataceae of the class Leotiomycetes.