Diversity of Secondary Metabolites from Fungi of the Ascomycete Genus Tolypocladium

Abstract

Fungi in the genus of Tolypocladium are often discovered as endophytes, soil inhabitants, and insect pathogens. Many biologically active secondary metabolites have been discovered from them in the past half-century, including medicinally important oligopeptide cyclosporine A, and other compounds like terpenes, polyketides, polyketide-non-ribosomal peptides. This review has summarized the chemistry and pharmacology of natural products discovered from Tolypocladium genus fungi, to provide a full record of all Tolypocladium secondary metabolites.

Keywords

cyclosporin meroterpenes bioactivity cyclic oligopeptides nonribosomal pepetides

Introduction

The genus Tolypocladium was first established in 1970s, when 3 species of fungi were isolated from soil samples by Grams,¹ and was synonymized with Chaunopycnis and Elaphocordyceps by genetic phylogenetic analyses.² Distribution of this genus is wide, despite the original discovery from soil, most members in this genus are discovered as endophytic fungi, and they were possibly favored by plants for their abilities of producing insecticidal substances.

Fungi of this genus are medicinally and biologically important for their secondary metabolites (SMs), many of which are pharmacologically potent. Cyclosporin A, an oligopeptide discovered from T. inflatum, has been developed as a good immunosuppressant. Thus, metabolites in Tolypocladium sp. are worth exploring for their structural diversity and biological significance. Terpenes, polyketides (PKs) and PK-non-ribosomal peptides (PK-NRPs) and peptides including cyclosporines, have been discovered from fungus of Tolypocladium sp. Currently, there are review articles on taxonomy of Tolypocladium and research on cyclosporin A,^3–5 but chemical and pharmacological properties of Tolypocladium SMs have never been summarized in full details. Thus, in this review, we have retrieved all papers on the chemistry and bioactivities of SMs isolated from fungi of Tolypocladium genus, and summarized structures and pharmacology of all the compounds, in order to show what we already discovered from this genus, and what to expect from it.

Secondary Metabolites

In the past half century, approximately 100 new Tolypocladium SMs have been found, they are mostly meroterpenoids, oligopeptides, PK-NRP and so forth. In this section, we summarized all the compounds isolated from Tolypocladium spp., and they are listed according to their structure types.

Terpenes and Meroterpenoids

Most commonly found terpenes in Tolypocladium sp. are diterpenes and terpenoid indole alkaloids. In 2011, Lin et al⁶ first obtained 4 diterpenes from T inflatum and named them as aphidicolins (1-4, Figure 1, Table 1). Compounds 1 and 2 are to some degree toxic to 8 human cancer cell lines (see Table S1), IC₅₀ of 2 to Hep3B-2 was 7.3 μM. Nogawa et al⁷ discovered a compound named metarhizin C (5) which showed IC₅₀ up to 0.79 μg/mL against MG-63 human osteosarcoma cells in a glucose-free medium and > 30 μg/mL in normal medium. Xu et al^6,8 discovered 12 new terpenoid indole alkaloids, tolypocladin A-L (6-17) and a known compound terpendole L (18). These compounds are suspected to be originated from tryptophan. Tolypocladin A (6) showed significant antifungal effect against 6 strains of plant pathogens (Alternaria fragariae, Corynespora cassiicola, Alternaria alternata, Botrytis cinereal Pers., Cercospora personata, Verticillium dahlia Kleb, and Sclerotinia sclerotiorum), all MICs were below 25 µg/mL, also it was toxic to multiple cancer cell lines and patient-derived tumor cells (Table S1). Tolypocladin H (13) showed MICs below 25 µg/mL against all tested bacteria (Bacillus cereus, methicillin-resistant Staphylococcus aureus, Micrococcus lysodeikticus, B paratyphosum, B subtilis, Enterobacter aerogenes, Salmonella typhi, and Proteus vulgaris). Terpendole L (18) inhibited Micrococcus luteus with MIC value of 6.25 µg/mL. Lin et al⁹ reported isolation of inflatin G (19), a diterpene structurally similar to aphidicolins. Terpendole E (20) was discovered by Motoyama et al¹⁰ from Chaunopcnis alba and it was a kinesin Eg5 inhibitor. On the basis of gene knock-out experiments, they proposed that terpendole E was a biosynthetic intermediate of indole-diterpene.

Figure 1.

Terpenes and meroterpenes isolated from fungi of the Tolypocladium genus.

Table 1.

Terpenes and Meroterpenoids and Their Origins.

No.	Name	Origins	References
1	Inflatin A	T inflatum	⁶
2	Inflatin B	T inflatum	⁶
3	Aphidicolin	T inflatum	⁶
4	Aphidicolin-17-monoacetate	T inflatum	⁶
5	Metarhizin C	T album RK17-F0007	⁷
6	Tolypocladin A	T. sp. XL115	⁶
7	Tolypocladin B	T. sp. XL115	⁶
8	Tolypocladin C	T. sp. XL115	⁶
9	Tolypocladin D	T. sp. XL115	⁶
10	Tolypocladin E	T. sp. XL115	⁶
11	Tolypocladin F	T. sp. XL115	⁶
12	Tolypocladin G	T. sp. XL115	⁶
13	Tolypocladin H	T. sp. XL115	⁶
14	Tolypocladin I	T. sp. XL115	⁶
15	Tolypocladin J	T. sp. XL115	⁶
16	Tolypocladin K	T. sp. XL115	⁸
17	Tolypocladin L	T. sp. XL115	⁸
18	Terpendole L	T. sp. XL115	⁸
19	Inflatin G	T inflatum	⁹
20	Terpendole E	Chaunopycnis alba	¹⁰

Polyketides

A quite diversified collection of PKs can be produced by species of Tolypocladium, including pyrones, benzophenones, coumarins, etc, though mostly not active in bio-assays (Table 2 and Table S1). Structures and tested activities are illustrated in Figure 2 (Table 2 and Table S1).

Figure 2.

Polyketides isolated from fungi of the Tolypocladium genus.

Table 2.

Polyketides Discovered from Fungi in Tolypocladium Genus.

No.	Name	Origins	References
21	Tolypocladin	T inflatum DSM 915	¹¹
22	Inflatin C	T inflatum	⁶
23	Inflatin D	T inflatum	⁶
24	Inflatin E	T inflatum	⁶
25	Inflatin F	T inflatum	⁶
26	Isochlamydosporol	T inflatum	⁶
27	Cylindromicin	T. sp. SCSIO 40433	¹²
28	Epi-citrinin H1	T. sp. SCSIO 40433	¹²
29	Dicitrinin A	T. sp. SCSIO 40433	¹²
30	Citreorosein	T. sp. SCSIO 40433	¹²
31	Phenol A	T. sp. SCSIO 40433	¹²
32	Phenol A acid	T. sp. SCSIO 40433	¹²
33	tolypocladone A	T. sp. (4259a)	¹³
34	Tolypocladone B	T. sp. (4259a)	¹³
35	Glycine, n-(2,3-dihydroxybenzoyl)-methyl ester	T. sp. (4259a)	¹³
36	2h-pyran-2-one, 4-methyoxy-6-(1,3-pentadienyl)	T. sp. (4259a)	¹³
37	Gulypyrone A	T inflatum	⁹
38	3,8-dihydroxy-4-(4-hydroxyphenyl)-6-methylcoumarin	T cylindrosporum	¹⁴
39	Malettinin A	T. sp. Sup5-1 (T2)	¹⁵
40	Malettinin B	T. sp. Sup5-1 (T2)	¹⁵
41	Malettinin F	T. sp. Sup5-1 (T2)	¹⁵
42	Malettinin G	T. sp. Sup5-1 (T2)	¹⁵
43	Malettinin H	T. sp. Sup5-1 (T2)	¹⁵

Aza-anthraquinones are not commonly discovered as natural products. Tolypocladin (21), an aza-anthraquinone was discovered in 1990 from T inflatum.¹¹ The authors suspected that it was a hydrogen acceptor for maintaining normal physiological functions of T inflatum during oxygen limitation. Lin et al⁶ discovered 5 chlamydosporol derivatives from T inflatum, namely inflatins C-F (22-25) and isochlamydosporol (26). Inflatin C was a chlamydosporol monomer while the latter 3 were dimers, all of which were inactive in either antimicrobial or cytotoxic assays. In the research of Khan et al,¹² 2 phenols, 1 pyrone, 2 benzophenones and anthraquinones (27-32) were isolated from T cylindrosporum, whereas Hu et al¹³ also obtained 2 new pyrones (33 and 34) and 2 known compounds (35 and 36) from Tolypocladium sp. (4259a). The discovery of Du et al¹⁵ indicated that malettinins (39-43), a family of antibacterial PKs originally isolated from Cladosporium sp.,¹⁶ were also produced by Tolypocladium.

Polyketide-Non-Ribosomal Peptides

Tolypocladium-originated PK-NRPs are mostly tyrosine or alanine-derived pyridones and pyrrolidones, the latter is often referred to as tetramic acids. Tolypoalbin (44) discovered from Tolypocladium Album TAMA 479 can induce cellular differentiation of ST-31 preadipocytes to mature adipycytes which produced adiponectin.¹⁷ The authors suspected that the effect could be related to PPARγ activation, and a reporter gene assay was conducted to show that 44 can activate PPARγ. A similar effect was not shown by the other derivative, F-14329 (45). Iqalisetins A and B (46 and 47) were 2 tetramic acids isolated by Grunwald et al¹⁸ from a Tolypocladium sp., an arctic marine sediment-originated fungal species. Interestingly, judging from the structures, only iqalisetins A and B were derived from alanine-lovastatin whereas most PK-NRPs isolated from Tolypocladium spp. were biosynthetically originated from tyrosine and methylated fatty acids.

Li et al¹⁴ discovered 6 PK-NRPs (48-53, Figure 3 and Table 3) from an endolichen fungus strain T cylindrosporum which inhabits Lethariella zahlbruckneri. Pyridoxatin (52) was significantly toxic to 4 human cancer cell lines (MDA-MB-231, A549, A2780, and K562, Table S1). Furthermore, 52 can cause cell cycle arrest at G0/G1 phase in A549 cells.

Figure 3.

Polyketide-non-ribosomal peptides isolated from fungi of the Tolypocladium genus.

Table 3.

Polyketide-Non-Ribosomal Peptides Discovered from Fungi in Tolypocladium Genus.

No.	Name	Origins	Reference
44	Tolypoalbin	T album TAMA 479	¹⁷
45	F-14329	T album TAMA 479	¹⁷
46	Iqalisetin A	T. sp. (RKAG373)	¹⁸
47	Iqalisetin B	T. sp. (RKAG373)	¹⁸
48	Tolypocladenol A₁	T cylindrosporum	¹⁴
49	Tolypocladenol A₂	T cylindrosporum	¹⁴
50	Tolypocladenol B	T cylindrosporum	¹⁴
51	Tolypyridone A	T cylindrosporum	¹⁴
52	Pyridoxatin	T cylindrosporum	¹⁴
53	Tolypyridone B	T cylindrosporum	¹⁴
54	Tolypyridone C	T. sp. 49Y*	¹⁹
55	Tolypyridone D	T. sp. 49Y*	¹⁹
56	Tolypyridone E	T. sp. 49Y	¹⁹
57	Tolypyridone F	T. sp. 49Y	¹⁹
58	Tolypyridone G	T. sp. 49Y	¹⁹
59	Tolypyridone H	T. sp. 49Y	¹⁹
60	Chaunolidine A	Chaunopycnis sp. (CMB-MF028)	²⁰
61	Chaunolidine B	Chaunopycnis sp. (CMB-MF028)	²⁰
62	Chaunolidine C	Chaunopycnis sp. (CMB-MF028)	²⁰
63	Chaunolidone A	Chaunopycnis sp. (CMB-MF028)	²⁰
64	Tolypyridone I	T album dws120	²¹
65	Tolypyridone J	T album dws120	²¹
66	Tolypyridone K	T album dws120	²¹
67	Tolypyridone L	T album dws120	²¹
68	Tolypyridone M	T album dws120	²¹

*Obtained from heterologous expression of gene clusters of T. sp. 49Y in Aspergillus oryzae NSAR1.

Zhang et al¹⁹ heterologously expressed a pyridone biosynthetic gene cluster from Tolypocladium sp. 49Y in Aspergillus oryzae NSAR1 and obtained 2 new compounds, tolypyridones C and D (54, 55), the former inhibited growth of Cryptococcus neoformans (MIC 6.25 μM, 2.22 μg/mL). Besides, 4 new pyridones tolypyridones E-H (56-59) were isolated from the rice ferments of the host strain of this gene cluster. This research also confirmed that tolypyridones originated from a tenellin-like biosynthetic pathway, the tolypyridone synthase gene (tolA) shared high homology with tenellin synthase gene (tenS), the construction of biosynthetic gene cluster was also same to that of tenellin's (ten gene cluster).²² Biosynthesis of these compounds involves cyclization by condensation between the amino acid and PK chain²² and free-radical-mediated ring expansion from pyrrolidone to pyridone.²³

From a Chaunopycnis sp., Shang et al²⁰ discovered 4 new PK-NRPs (60-63). Chaunolidine C (62) was mildly antibacterial, whereas chaunolidone A (63) was potently and selectively inhibitory to human non-small cell lung carcinoma cells (NCI-H460). They also mentioned these pyrrolidones can form metal chelates.

Recently, 5 new pyridone derivatives (tolypyridones I-M, 64-68) were isolated from rice medium ferments of T album dws120 by Wu et al,²¹ and they revealed tolypyridone I (64) can protect liver cells against ethanol-induced injury.

NonRibosomal Peptides

Nonribosomal peptides (NRPs) are the most structurally diversified among all Tolypocladium SMs, over 50 cyclic or linear oligopeptides have been discovered till now. NRPs discovered in this genus are usually linear, cyclic, or branched oligopeptides consisted of at most 22 amino acid units which are sometimes acylated, glycosylated or N-methylated. This family of compounds is medicinally important. Cyclosporine A (amino acid sequence shown in Table 4, and structure in Figure 4), a cyclopeptide consists of 11 amino acid moieties, is now a first-line immunosuppressant (Sandimmun, Novartis) that is commonly used in transplantation and autoimmune diseases (eg psoriasis, rheumatoid arthritis, etc).²⁴

Figure 4.

Structure of cyclosporin A.

Table 4.

Amino Acid Sequences of Cyclic Peptides in Tolypocladium sp. (Cyclosporines B-Z were from Traber²⁵).

Name	1	2	3	4	5	6	7	8	9	10	11
A²⁶	MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
B	MeBmt	Ala	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
C	MeBmt	Thr	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
D	MeBmt	Val	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
E	MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	Val
F	Desoxy-MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
G	MeBmt	Nva	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
H	MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	D-MeVal
I	MeBmt	Val	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	Leu	MeVal
K	Desoxy-MeBmt	Val	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
L	Bmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
M	MeBmt	Nva	Sar	MeLeu	Nva	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
N	MeBmt	Nva	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	Leu	MeVal
O	MeLeu	Nva	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
P	Bmt	Thr	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
Q	MeBmt	Abu	Sar	Val	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
R	MeBmt	Abu	Sar	MeLeu	Val	Leu	Ala	D-Ala	MeLeu	Leu	MeVal
S	MeBmt	Thr	Sar	Val	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
T	MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	Leu	MeVal
U	MeBmt	Abu	Sar	MeLeu	Val	Leu	Ala	D-Ala	MeLeu	MeLeu	MeVal
V	MeBmt	Abu	Sar	MeLeu	Val	MeLeu	Abu	D-Ala	MeLeu	MeLeu	MeVal
W	MeBmt	Thr	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	Val
X	MeBmt	Nva	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	Leu	MeLeu	MeVal
Y	MeBmt	Nva	Sar	MeLeu	Val	Leu	Ala	D-Ala	MeLeu	MeLeu	MeVal
Z	Methylamino-octanoic acid	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
MeIle²⁷	MeBmt	Abu	Sar	MeIle	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
4-Leu A²⁸	MeBmt	Abu	Sar	Leu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
J²⁸	MeLeu	Abu	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
D hydro-peroxide²⁹	Xaa	Val	Sar	MeLeu	Val	MeLeu	Ala	D-Ala	MeLeu	MeLeu	MeVal
FR190293³⁰	DiOHOrn-R	Thr	OHPro	DiOHTyr	OHGln	OHMePro

Abbreviations: Bmt, (2S, 3R, 4R, 6E)-2-amino-3-hydroxy-4-methyl-6-octenoic acid; MeBmt, N-methyl Bmt; Xaa, 3-hydroxy-7-hydroperoxy-4-methyl-2-methyl-amino-5E-octenoic acid; R, 10,12-dimethyl myristic acid; Nva, norvaline.

Cyclic Oligopeptides

Cyclosporin was first isolated from Trichoderma polysporum (Link ex Pers) (later redesignated as T inflatum Grams)³¹ and identified in 1970s by Petcher et al.²⁶ It was a sarcosine-containing cyclo-undecapeptide (Figure 4). Then Traber et al^25,32–34 discovered over 20 cyclosporins from T niveum, namely cyclosporins B-Z (Table 4). Structures ofcyclosporins were featured with unnatural amino acid moieties, (2S, 3R, 4R, 6E)-2-amino-3-hydroxy-4-methyl-6-octenoic acid (Bmt) and Bmt derivatives, and it was believed that this moiety was critical for immunosuppression, alternation of Bmt (deoxidation, replacement by other amino acids) caused loss of activity. They also found anti-HIV cyclosporine (MeIle) from T niveum fermentations supplemented with D-threonine, this compound can inhibit HIV-1 (strain IIIB)-induced cytopathic effect in MT4 cells.²⁷ With 60 cyclosporin derivatives, Loor et al³⁵ investigated the structure-activity relationship (SAR) of cyclosporin inhibiting human MDR-1 P-glycoprotein (P-gp) ABC transporter, the results indicated that hydrophobic residues favored P-gp inhibition, while N-desmethylation mostly abolished the activity.

Biosynthesis of Cyclosporin A

As cyclosporin A is chemically unique and clinically important, it is worth to give a brief summary on the biosynthesis of cyclosporin. Currently, it is commonly recognized that 3 major steps have been involved in the biosynthesis of cyclosporin A: isomerization of l-alanine, formation of Bmt and assembly of undecapeptide chain (Figure 6).

Figure 5.

Diketopiperazines isolated from fungi of the Tolypocladium genus.

Figure 6.

Biosynthesis of cyclosporin A involves 3 steps: (1) l-Ala is converted into d-Ala by a racemase; (2) the backbone of Bmt is synthesized by a polyketide synthase; (3) CsSyn is both multidomain and multifunctional, each amino acid unit is activated by adenylation, transferred to the thiolation domain (PCP) or linked to the amino group of adjacent moiety with the help of condensation domain; methylation occurs when there is a methyltransferase domain. Abbreviations: PCP, peptidyl carrier protein; CsSyn, cyclosporin A synthase; C, condensation domain; A, adenylation domain; T, thiolation domain; MT, methyltransferase domain.

The unnatural amino acid d-alanine was yielded by alanine racemase acting upon l-alanine. Hoffman et al³⁶ isolated this alanine racemase from T niveum, and revealed that it can catalyze the reversible racemase of l- and d- alanines. The Bmt building block was a threonine derivative synthesized by oxidation and transamination of a PK.³⁷ Isotope labelling^38,39 indicated that T inflatum can incorporate ¹³C in glucoses into the Bmt moiety, and Offenzeller et al³⁷ showed that in T niveum NRRL 8044 a carbon chain was synthesized by a PK synthase and then was transformed and released as amino acid coenzyme A thioester.

Assembly of the undecapeptide cycle was the most sophisticate step and it was catalyzed by a NRP synthase,⁴⁰ cyclosporin synthase (CySyn). CySyn, encoded by a 45.8 kb open reading frame (assigned as simA) in T niveum NRRL 8044, was a giant multimodule enzyme that consisted 11 sets of condensation-adenylation-thiolation domains (the C-A-T domains). Adenylation domain activated amino acids by adding adenyl group onto the amino group, thiolation domain with a free thiol group acted as a carrier of activated amino acids and peptidyls, and condensation domain catalyzed the formation of peptide bonds.^41,42 Seven of the 11 modules contained a methyltransferase (MT) domain, and their function was catalyzation of N-methylation on the corresponding amino acid units (Bmt-1, Sar-3, Leu-4, Leu-6, Leu-9, Leu-10, and Ala-11).⁴⁰

Linear Oligopeptides

Efrapeptins are linear oligopeptides that are featured by their characteristic constituents α-amino-isobutyric acid (Aib) and pipecolic acid (Pip) (Table 5). Efrapeptins A-G were isolated from T niveum.^43–45 Efrapeptins D and F were insecticidal (LC₅₀ were 18.9 and 8.4 ppm) against Colorado potato beetle.

Table 5.

Sequences of Linear Peptides Discovered from Tolypocladium Species.

Name	Sequences	Origins	References
LP237-F8	Lol-Ala-Gln-EtNor-Aib-Gln-Gln-Aib-Phe-Pro-Aib-Oc	T geodes	⁴⁶
LP237-F5	Lol-Ala-Gln-EtNor-Aib-Gln-Gln-Aib-Tyr-Pro-Aib-Oc	T geodes	⁴⁷
LP237-F7	Lol-Ala-Gln-Aib-Aib-Gln-Gln-Aib-Phe-Pro-Aib-Dec	T geodes	⁴⁷
Efrapeptin C	AcPip-Aib-Pip-Aib-Aib-Leu-bAla-Gly-Aib-Aib-Pip-Aib-Gly-Leu-Aib-HPP	T inflatum	⁴⁸
Efrapeptin D	AcPip-Aib-Pip-Aib-Aib-Leu-bAla-Gly-Aib-Aib-Pip-Aib-Gly-Leu-Iva-HPP	T inflatum	⁴⁸
Efrapeptin E	AcPip-Aib-Pip-Iva-Aib-Leu-bAla-Gly-Aib-Aib-Pip-Aib-Gly-Leu-Iva-HPP	T inflatum	⁴⁸
Efrapeptin F	AcPip-Aib-Pip-Aib-Aib-Leu-bAla-Gly-Aib-Aib-Pip-Aib-Ala-Leu-Iva-HPP	T inflatum	⁴⁸
Efrapeptin G	AcPip-Aib-Pip-Iva-Aib-Leu-bAla-Gly-Aib-Aib-Pip-Aib-Ala-Leu-Iva-HPP	T inflatum	⁴⁸
Efrapeptin J	AcPip-Aib-Pip-Aib-Leu-β-Ala-Gly-Aib-Aib-Pip-Aib-Pip-Aib-Ala-Leu-Aib-HPP	Tolypocladium sp.	⁴⁹
Efrapeptin K	AcPip-Aib-Pip-Aib-Leu-β-Ala-Gly-Aib-Aib-Pip-Aib-Pip-Aib-Ala-Leu-Iva-HPP	Tolypocladium sp.	⁴⁹
Efrapeptin L	AcPip-Aib-Pip-Aib-Leu-β-Ala-Gly-Aib-Aib-Pip-Aib-Pip-Aib-Ala-Leu-Iva-HPP	Tolypocladium sp.	⁴⁹
Gichigamin A	AcAib-Pro-Aib-Pro-Phe-Iva-Pro-Ala-Aib-bAla-Ala-Iva-bAla-Leu-Aib-bAla-Leu-Aib-Aib-Leu-bAla-GlyOH	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin B	AcAib-Pro-Aib-Pro-Phe-Iva-Pro-Ala-Aib-bAla-Ala-Iva-bAla-Leu-Aib-bAla-Leu-Iva-Aib-Leu-bAla-GlyOH	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin C	AcAib-Pro-Aib-Pro-Phe-Iva-Pro-Ala-Aib-bAla-Ala-Iva-bAla-Leu-Aib-bAla-Leu-Aib-Aib-Leu-bAla	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin D	AcAib-Pro-Aib-Pro-Phe-Iva-Pro-Ala-Aib-bAla-Ala-Iva-bAla-Leu-Aib-bAla-Leu-Iva-Aib-Leu-bAla	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin E	AcAib-Pro-Aib-Pro-Phe-Iva-Pro-Ala-Aib-bAla-Ala-Aib-bAla-Leu-Aib-bAla-Leu-Aib-Aib-Leu-bAla-GlyOH	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin F	AcAib-Pro-Aib-Pro-Phe-Aib-Pro-Ala-Aib-bAla-Ala-Iva-bAla-Leu-Aib-bAla-Leu-Aib-Aib-Leu-bAla-GlyOH	T. sp. Sup5-1 (T2)	¹⁵
Gichigamin G	AcAib-Pro-Aib-Pro-Phe-Aib-Pro-Ala-Aib-bAla-Ala-Aib-bAla-Leu-Aib-bAla-Leu-Aib-Aib-Leu-bAla-GlyOH	T. sp. Sup5-1 (T2)	¹⁵
Dakwaabakain A	Dec-Aib-Pro-Leu-Aib-Gln-Gln-Aib-Aib-Gln-Ala-Lol	T. sp. Sup5-1 (T2)	¹⁵
Dakwaabakain B	Dec-Aib-Pro-Tyr-Aib-Gln-Gln-Aib-Aib-Gln-Ala- Lol	T. sp. Sup5-1 (T2)	¹⁵
Dakwaabakain C	Dec-Aib-Pro-Tyr-Aib-Gln-Gln-Aib-Iva-Gln-Ala- Lol	T. sp. Sup5-1 (T2)	¹⁵
Dakwaabakain D	Dec-Aib-Pro-Phe-Aib-Gln-Gln-Aib-Iva-Gln-Ala- Lol	T. sp. Sup5-1 (T2)	¹⁵
Dakwaabakain E	Non-Aib-Pro-Leu-Aib-Gln-Gln-Aib-Aib-Gln-Ala- Lol	T. sp. Sup5-1 (T2)	¹⁵
Tolypocladamide A	Dec-Aib-Pro-Bmt-Iva-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide B	Dec-Iva-Pro-Bmt-Iva-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide C	Dec-Aib-Pro-Bmt-Iva-Gln-Gln-Aib-Iva-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide D	Oct-Aib-Pro-Bmt-Iva-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide E	Dec-Aib-Pro-Bmt-Aib-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide F	Oct-Aib-Pro-Leu-Iva-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide G	Dec-Iva-Pro-Leu-Iva-Gln-Gln-Aib-Aib-Gln-Leu-Lol	T inflatum	⁵⁰
Tolypocladamide H	Dec-Aib-Pro-Bmt-Aib-Gln-Gln-Aib-Aib-Gln-Val-Lol	T inflatum	⁵¹
Tolypocaibol A	Dec-Aib-Pro-Phe-Aib-Gln-Gln-Aib-Aib-Gln-Leu-Valol	Tolypocladium sp.	⁵²
Tolypocaibol B	Dec-Aib-mPro-Phe-Aib-Gln-Gln-Aib-Aib-Gln-Leu-Valol	Tolypocladium sp.	⁵²

Abbreviations: AcAib, acetyl-2-aminoisobytyric acid; GlyOH, 2-hydroxyethylamine; Dec, decanoic acid; Non, nonanoic acid; Oc, octanoic acid; Lol, D-leucinol; Valol, valinol; mPro, 4-methyl proline, bAla, D-alanine.

Gichigamins are a family of 21 and 22-residue peptides, Du et al¹⁵ comprehensively investigated the structures of gichigamins A-G by NMR, mass spectroscopy, electronic circular dichroism (ECD) or single-crystal x-ray diffraction. Gichigamin A was cytotoxic in vitro (GI₅₀ 0.55 ± 0.04 μM) and it showed antitumor activity in tumor xenograft mice as well. Gichigamin A can disrupt mitochondrial functions. In their semisynthetic structural modification research, by linking a coumarin to its C-terminus, antitumor potency increased to 60-fold of the prototype. Along with these peptides, the author also isolated 6 new 11-residue lipopeptaibols named dakwaabakains A-E and LP237-F7 along with 5 PKs.

In 2022, an array of new 11-residue peptaibols named tolypocladamides were discovered from T inflatum by 2 groups of researchers separately.^50,51 Tolypocladamides A-G were tested for their Ras/Raf inhibitory activities, and tolypocladaminde C showed the lowest IC₅₀ at 0.56 μM.

Recently in 2023, an endophytic Tolypocladium sp. from marine alga Spongomorpha arcta was investigated by Morehouse et al⁵² Isolation led to the discovery of 2 antibacterial peptides named tolypocaibols A and B and an NRPS-PK-shikimate metabolite mixture maxinmiscin. Both peptides showed MIC values at 4 to 16 μM against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and other pathogenic bacteria.

Besides, diketopiperazines and other small-molecule peptides have been discovered alongside (69-73, Fig. 5 and Table 6).^53–56 Bioactivities of the compounds mentioned above were listed in the Supplemental Materials (Table S2).

Table 6.

Diketopiperazines and Other Small-Molecule Peptides.

No.	Name	Origins	References
64	Sch54794	Tolypocladium sp.	⁵³
65	Sch54796	Tolypocladium sp.	⁵³
66	Sch56396	Tolypocladium sp.	⁵⁴
67	1-Alaninechlamydocin	Tolypocladium sp.	⁵⁵
68	Tolyprolinol	T. sp. FKI-7981	⁵⁶

Discussion and Conclusions

Over the past half century since the genus Tolypocladium has been described and established, much has been done to understand its chemistry and discover pharmacologically active natural products from species in this genus. Gratifyingly, a potent immunosuppressant, cyclosporin A, is found and developed into a useful medicine.

Though species of this genus can be found from different sources including plants and insects, small molecules like PK-NPRs are rather similar (eg tolypyridones vs chaunolidones) among strains. This indicated that they are biosynthetically similar, and secondary metabolic gene clusters of small molecules are conserved between strains.

We believe that Tolypocladium genus is still a good resource of new natural products, especially oligopeptides. With the help of bio-informative methods and gene manipulation techniques, more cryptic biosynthetic gene clusters can hopefully be disclosed in the future. Noteworthily, pharmacology of many of the compounds, especially the oligopeptides, is somehow inadequately investigated, thus it is necessary to revisit these compounds for more useful bioactivities.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231225538 - Supplemental material for Diversity of Secondary Metabolites from Fungi of the Ascomycete Genus Tolypocladium

Supplemental material, sj-docx-1-npx-10.1177_1934578X231225538 for Diversity of Secondary Metabolites from Fungi of the Ascomycete Genus Tolypocladium by Ting-Ting Niu, Wei-Qiong Chen, Wen-Xing Li, Li Zhang, Ying-Qiao Shan and Li-Wei Ren in Natural Product Communications

Footnotes

Author Contributions

Investigation and Project administration were done by T.-T. N., L.-W. R.. Supervision was done by L. Z., L.-W. R. Data collection was done by T.-T. N., Y.-Q. S. Writing—original draft was done by T.-T. N, W.-Q. C. Writing—review & editing was done by W.-X. L, L.-W. R.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Science, Technology and Innovation Commission of Shenzhen Municipality, Shenzhen Key Medical Discipline Construction Fund (grant numbers JCYJ20230807112402005, SZXK059).

ORCID iD

Ting-Ting Niu

Supplemental Material

Supplemental material for this article is available online.

References

Gams

. Tolypocladium, eine Hyphomycetengattung mit geschwollenen Phialiden. Persoonia—Mol Phylogeny Evol Fungi. 1971;6(2):185-191.

Quandt

Kepler

Gams

, et al. Phylogenetic-based nomenclatural proposals for Ophiocordycipitaceae (Hypocreales) with new combinations in Tolypocladium. IMA Fungus. Jun 2014;5(1):121-134. doi:10.5598/imafungus.2014.05.01.12

F-M

Thilini Chethana

Wei

D-P

, et al. Comprehensive review of Tolypocladium and description of a novel lineage from southwest China. Pathogens. 2021;10(11):1389. doi:10.3390/pathogens10111389

Patocka

Nepovimova

Kuca

. Cyclosporine A: chemistry and toxicity - A review. Curr Med Chem. 2021;28(20):3925-3934. doi:10.2174/0929867327666201006153202

Lawen

. Biosynthesis of cyclosporins and other natural peptidyl prolyl cis/trans isomerase inhibitors. Biochimica Et Biophysica Acta-General Subjects. Oct 2015;1850(10):2111-2120. doi:10.1016/j.bbagen.2014.12.009

Lin

Chen

Cai

, et al. Isolation and characterization of aphidicolin and chlamydosporol derivatives from Tolypocladium inflatum. J Nat Prod. Aug 26 2011;74(8):1798-1804. doi:10.1021/np200431k

Nogawa

Kawatani

Okano

, et al. Structure and biological activity of Metarhizin C, a stereoisomer of BR-050 from Tolypocladium album RK17-F0007. J Antibiot Dec 2019;72(12):996-1000. doi:10.1038/s41429-019-0229-1

L-L

Pang

X-J

Shi

Xian

P-J

Tao

Y-D

Yang

X-L

. Two new prenylated indole diterpenoids from Tolypocladium sp. and their antimicrobial activities. Chem Biodiversity Jun 2019;16(6):e1900116. doi:10.1002/cbdv.201900116

Lin

Niu

Ding

, et al. Isolation and characterization of aphidicolin derivatives from Tolypocladium inflatum. Molecules. Jul 2017;22(7):1168. doi:10.3390/molecules22071168

10.

Motoyama

Hayashi

Hirota

Ueki

Osada

. Terpendole E, a kinesin Eg5 inhibitor, is a key biosynthetic intermediate of indole-diterpenes in the producing fungus Chaunopycnis alba. Chem Biol . Dec 21 2012;19(12):1611-1619. doi:10.1016/j.chembiol.2012.10.010

11.

Gräfe

Ihn

Tresselt

, et al. Tolypocladin—a new metal-chelating 2-aza-anthraquinone from Tolypocladium inflatum. Biol Met. 1990;3(1):39-44. doi:10.1007/BF01141176

12.

Khan

Peng

Fang

, et al. Cylindromicin from arctic-derived fungus Tolypocladium sp. SCSIO 40433. Molecules. Feb 2021;26(4):1080. doi:10.3390/molecules26041080

13.

C-H

Zhou

Y-H

Xie

Y-L

Zhao

Z-T

Lou

H-X

. Two new—pyrone derivatives from an endolichenic fungus Tolypocladium sp. J Asian Nat Prod Res. 2017 2017;19(8):786-792. doi:10.1080/10286020.2017.1283311

14.

X-B

Zhu

R-X

, et al. Tetramic acids and pyridone alkaloids from the endolichenic fungus Tolypocladium cylindrosporum. J Nat Prod Sep 2015;78(9):2155-2160. doi:10.1021/np501018w

15.

Risinger

Mitchell

, et al. Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides. Proc Natl Acad Sci USA. Oct 24 2017;114(43):E8957-E8966. doi:10.1073/pnas.1707565114

16.

Silber

Ohlendorf

Labes

Wenzel-Storjohann

Näther

Imhoff

. Malettinin E, an antibacterial and antifungal tropolone produced by a marine Cladosporium strain. Front Mar Sci. 2014;1(35): 1–6 doi:10.3389/fmars.2014.00035.

17.

Fukuda

Sudoh

Tsuchiya

, et al. Tolypoalbin, a new tetramic acid from Tolypocladium album TAMA 479. J Antibiot. Jun 2015;68(6):399-402. doi:10.1038/ja.2014.165

18.

Grunwald

Berrue

Overy

Kerr

. Isolation of iqalisetins A and B from a Tolypocladium sp isolated from marine sediment from frobisher bay in Canada's arctic. Can J Chem. Apr 2016;94(4):444-448. doi:10.1139/cjc-2015-0439

19.

Zhang

W-Y

Zhong

, et al. 4-Hydroxy pyridones from heterologous expression and cultivation of the native host. J Nat Prod. 2020;83(11):3338-3346. doi:10.1021/acs.jnatprod.0c00675

20.

Shang

Esposito

, et al. New PKS-NRPS tetramic acids and pyridinone from an Australian marine-derived fungus, Chaunopycnis sp. Org Biomol Chem. 2015;13(28):7795-7802. doi:10.1039/c5ob01058f

21.

X-Q

Zhou

, et al. Liver-cell protective pyridones from the fungi Tolypocladium album dws120. Phytochemistry. Aug 2023;212:113730. doi:10.1016/j.phytochem.2023.113730

22.

Halo

Marshall

Yakasai

, et al. Authentic heterologous expression of the tenellin iterative polyketide synthase nonribosomal peptide synthetase requires coexpression with an enoyl reductase. Chembiochem. Mar 2008;9(4):585-594. doi:10.1002/cbic.200700390

23.

Halo

Heneghan

Yakasai

, et al. Late-stage oxidations during the biosynthesis of the 2-pyridone tenellin in the entomopathogenic fungus Beauveria bassiana. J Am Chem Soc. Dec 2008;130(52):17988-17996. doi:10.1021/ja807052c

24.

Yang

Feng

Yin

Bushley

Spatafora

Wang

. Cyclosporine biosynthesis in Tolypocladium inflatum benefits fungal adaptation to the environment. Mbio. Sep-Oct 2018;9(5):e01211-18, 1–14. doi:10.1128/mBio.01211-18

25.

Traber

Hofmann

Loosli

Ponelle

Vonwartburg

. Novel cyclosporins from Tolypocladium-inflatum - the cyclosporins-K-Z. Helv Chim Acta. 1987;70(1):13-36. doi:10.1002/hlca.19870700103

26.

Petcher

Weber

Rüegger

. Crystal and molecular structure of an iodo-derivative of the cyclic undecapeptide cyclosporin A. Helv Chim Acta. Jun 14 1976;59(5):1480-1489. doi:10.1002/hlca.19760590509

27.

Traber

Kobel

Loosli

Senn

Rosenwirth

Lawen

. Meile(4) cyclosporin, a novel natural cyclosporine with anti-hiv activity - structural elucidation, biosynthesis. Antiviral Chem Chemother. Sep 1994;5(5):331-339. doi:10.1177/095632029400500507

28.

Jegorov

Matha

Sedmera

, et al. Cyclosporines from Tolypocladium-terricola. Phytochemistry. Jan 1995;38(2):403-407. doi:10.1016/0031-9422(94)00711-2

29.

Sedmera

Havlicek

Jegorov

Segre

. Cyclosporine-D hydroperoxide, a new metabolite of Tolypocladium-terricola. Tetrahedron Lett. Sep 18 1995;36(38):6953-6956. doi:10.1016/0040-4039(95)01378-u

30.

Kanasaki

Kobayashi

Fujine

, et al. FR227673 And FR190293, novel antifungal lipopeptides from Chalara sp no. 22210 and Tolypociadium parasiticum no. 16616. J Antibiot. Mar 2006;59(3):158-167. doi:10.1038/ja.2006.23

31.

Rüegger

Kuhn

Lichti

, et al. Cyclosporin A, ein immunsuppressiv wirksamer Peptidmetabolit aus Trichoderma polysporum (LINK ex PERS.) Rifai. Helv Chim Acta. 1976;59(4):1075-1092. doi:10.1002/hlca.19760590412

32.

Traber

Kuhn

Ruegger

Lichti

Loosli

Wartburg

. Structure of cyclosporin-C. Helv Chim Acta. 1977;60(4):1247-1255. doi:10.1002/hlca.19770600414

33.

Traber

Loosli

Pache

Wartburg

. New cyclopeptides from Trichoderma-polysporum (link ex pers) rifai - cyclosporins B, D and E. Helv Chim Acta. 1977;60(5):1568-1578. doi:10.1002/hlca.19770600513

34.

Traber

Loosli

Hofmann

Kuhn

Vonwartburg

. Isolation and structure determination of the new cyclosporins E, F, G, H and I. Helv Chim Acta. 1982;65(5):1655-1677. doi:10.1002/hlca.19820650538

35.

Loor

Tiberghien

Wenandy

Didier

Traber

. Cyclosporins: structure−activity relationships for the inhibition of the human MDR1 P-glycoprotein ABC transporter. J Med Chem. 2002;45(21):4598-4612. doi:10.1021/jm0109863

36.

Hoffmann

Schneiderscherzer

Kleinkauf

Zocher

. Purification and characterization of eukaryotic alanine racemase acting as key enzyme in cyclosporine biosynthesis. Article. Journal of Biological Chemistry. Apr 29 1994;269(17):12710-12714. doi:10.1016/S0021-9258(18)99934-5

37.

Offenzeller

Santer

Totschnig

, et al. Biosynthesis of the unusual amino acid (4R)-4- (E)-2-butenyl −4-methyl-L-threonine of cyclosporin A: Enzymatic analysis of the reaction sequence including identification of the methylation precursor in a polyketide pathway. Biochemistry. Jun 25 1996;35(25):8401-8412. doi:10.1021/bi960224n

38.

Kobel

Loosli

Voges

. Contribution to knowledge of the biosynthesis of cyclosporin-a. Note. Experientia. 1983;39(8):873-876. doi:10.1007/bf01990413

39.

Senn

Weber

Kobel

Traber

. Selective C-13-labeling of cyclosporine-A. Eur J Biochem. Aug 1 1991;199(3):653-658. doi:10.1111/j.1432-1033.1991.tb16167.x

40.

Weber

Schorgendorfer

Schneiderscherzer

Leitner

. The peptide synthetase catalyzing cyclosporine production in Tolypocladium-niveum is encoded by a giant 45.8-kilobase open reading frame. Curr Genet. Aug 1994;26(2):120-125. doi:10.1007/bf00313798

41.

Bloudoff

Schmeing

. Structural and functional aspects of the nonribosomal peptide synthetase condensation domain superfamily: Discovery, dissection and diversity. Biochimica Et Biophysica Acta-Proteins and Proteomics. Nov 2017;1865(11):1587-1604. doi:10.1016/j.bbapap.2017.05.010

42.

D-Y

F-C

Zhang

S-W

Zhan

J-X

. Decoding and reprogramming fungal iterative nonribosomal peptide synthetases. Nat Comm. May 2017:815349, 1–11. doi:10.1038/ncomms15349

43.

Jackson

Linnett

Beechey

Henderson

PJF

. Purification and preliminary structural analysis of the efrapeptins, a group of antibiotics that inhibit the mitochondrial adenosine triphosphatase. Biochem Soc Trans. Feb 1979;7:224-226. doi:10.1042/bst0070224

44.

Krasnoff

Gupta

. Identification and directed biosynthesis of efrapeptins in the fungus Tolypocladium-geodes gams (Deuteromycotina, Hyphomycetes). J Chem Ecol. Oct 1991;17(10):1953-1962. doi:10.1007/bf00992580

45.

Krasnoff

Gupta

Stleger

Renwick

JAA

Roberts

. Antifungal and insecticidal properties of the efrapeptins - metabolites of the fungus Tolypocladium-niveum. J Invertebr Pathol. Sep 1991;58(2):180-188. doi:10.1016/0022-2011(91)90062-u

46.

Tsantrizos

Pischos

Sauriol

. Structural assignment of the peptide antibiotic LP237-F8, a metabolite of Tolypocladium geodes. J Org Chem. Mar 22 1996;61(6):2118-2121. doi:10.1021/jo951837t

47.

Tsantrizos

Pischos

Sauriol

Widden

. Peptaibol metabolites of Tolypocladium geodes. Canad J Chem-Revue Canadienne De Chimie. Feb 1996;74(2):165-172. doi:10.1139/v96-020

48.

Gupta

Krasnoff

Roberts

Renwick

JAA

Brinen

Clardy

. Structure of efrapeptins from the fungus Tolypocladium niveum: peptide inhibitors of mitochondrial ATPase. J Org Chem. 1992;57(8):2306-2313. doi:10.1021/jo00034a022

49.

Hayakawa

Hattori

Kawasaki

, et al. Efrapeptin J, a new down-regulator of the molecular chaperone GRP78 from a marine Tolypocladium sp. J Antibiot. Jun. 2008;61(6):365-371. doi:10.1038/ja.2008.51

50.

Senadeera

SPD

Wang

Kim

C-K

, et al. Tolypocladamides A–G: cytotoxic peptaibols from Tolypocladium inflatum. J Nat Prod. 2022;85(6):1603-1616. doi:10.1021/acs.jnatprod.2c00240

51.

Tehan

Blount

Goold

, et al. Tolypocladamide H and the proposed tolypocladamide NRPS in Tolypocladium species. J Nat Prod. 2022;85(5):1363-1373. doi:10.1021/acs.jnatprod.2c00153

52.

Morehouse

NJJ

Flewelling

AJJ

Liu

DYY

, et al. Tolypocaibols: antibacterial lipopeptaibols from a Tolypocladium sp. endophyte of the marine macroalga Spongomorpha arcta. J Nat Prod. 2023. doi:10.1021/acs.jnatprod.3c00233

53.

Chu

Mierzwa

Truumees

, et al. Two novel diketopiperazines isolated from the fungus Tolypocladium sp. Tetrahedron Lett. Nov 19 1993;34(47):7537-7540. doi:10.1016/s0040-4039(00)60393-3

54.

Chu

Truumees

Mierzwa

Patel

Puar

. Sch 56396: a new c-fos proto-oncogene inhibitor produced by the fungus Tolypocladium sp. J Antibiot. Dec 1997;50(12):1061-1063. doi:10.7164/antibiotics.50.1061

55.

Risinger

King

Powell

Cichewicz

. A potent HDAC inhibitor, 1-alaninechlamydocin, from a Tolypocladium sp induces G2/M cell cycle arrest and apoptosis in MIA PaCa-2 cells. J Nat Prod. Jul 2014;77(7):1753-1757. doi:10.1021/np500387h

56.

Fukasawa

Mori

Iwatsuki

, et al. Tolyprolinol, a new dipeptide from Tolypocladium sp FKI-7981. J Antibiot. Jul 2018;71(7):682-684. doi:10.1038/s41429-018-0041-3

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