Three functionalized thienopyrazines (TPs), TP-MN (1), TP-CA (2), and TPT-MN (3) were designed and synthesized as self-assembled monolayers (SAMs) deposited on the NiOx film for tin-perovskite solar cells (TPSCs). Thermal, optical, electrochemical, morphological, crystallinity, hole mobility, and charge recombination properties, as well as DFT-derived energy levels with electrostatic surface potential mapping of these SAMs, have been thoroughly investigated and discussed. The structure of the TP-MN (1) single crystal was successfully grown and analyzed to support the uniform SAM produced on the ITO/NiOx substrate. When we used NiOx as HTM in TPSC, the device showed poor performance. To improve the efficiency of TPSC, we utilized a combination of new organic SAMs with NiOx as HTM, the TPSC device exhibited the highest PCE of 7.7 % for TP-MN (1). Hence, the designed NiOx/TP-MN (1) acts as a new model system for the development of efficient SAM-based TPSC. To the best of our knowledge, the combination of organic SAMs with anchoring CN/CN or CN/COOH groups and NiOx as HTM for TPSC has never been reported elsewhere. The TPSC device based on the NiOx/TP-MN bilayer exhibits great enduring stability for performance, retaining ~80 % of its original value for shelf storage over 4000 h.
Mut L homolog-1 (MLH1) is a key DNA mismatch repair protein which participates in the sensitivity to DNA damaging agents. However, its role in the radiosensitivity of tumor cells is less well characterized. In this study, we investigated the role of MLH1 in cellular responses to ionizing radiation (IR) and explored the signaling molecules involved. The isogenic pair of MLH1 proficient (MLH1 + ) and deficient (MLH1 – ) human colorectal cancer HCT116 cells was exposed to IR for 24 h at the dose of 3 cGy. The clonogenic survival was examined by the colony formation assay. Cell cycle distribution was analyzed with flow cytometry. Changes in the protein level of MLH1, DNA damage marker γH2AX, and protein kinase A catalytic subunit (PRKAC), a common target for anti-tumor drugs, were examined with Western blotting. The results showed that the HCT116 (MLH1 + ) cells demonstrated increased radio-resistance with increased S population, decreased G2 population, a low level of γH2AX, a reduced ratio of phosphorylated PRKACαβ to total PRKAC, and an elevated level of total PRKAC and phosphorylated PRKACβII following IR compared with the HCT116 (MLH1 – ) cells. Importantly, silencing PRKAC in HCT116 (MLH1 + ) cells increased the cellular radiosensitivity. In conclusion, MLH1 may increase cellular resistance to IR by activating PRKAC. Our finding is the first to demonstrate the important role of PRKAC in MLH1-mediated radiosensitivity, suggesting that PRKAC has potential as a biomarker and a therapeutic target for increasing radio-sensitization.
The aim of this study was to analyze the dosimetric and radiobiologic differences of the left-sided whole breast and regional nodes in intensity-modulated radiotherapy (IMRT), volume-modulated arc therapy (VMAT), and helical tomotherapy (HT). The IMRT, VMAT, and HT plans in this study were generated for thirty-five left-sided breast cancer patients after breast-conserving surgery (BCS). The planning target volume (PTV) included the whole breast and supraclavicular nodes. PTV coverage, homogeneity index (HI), conformity index (CI), dose to organs at risk (OARs), secondary cancer complication probability (SCCP), and excess absolute risk (EAR) were used to evaluate the plans. Compared to IMRT, the VMAT and HT plans resulted in higher PTV coverage and homogeneity. The VMAT and HT plans also delivered a lower mean dose to the ipsilateral lung (9.19 ± 1.36 Gy, 9.48 ± 1.17 Gy vs. 11.31 ± 1.42 Gy) and heart (3.99 ± 0.86 Gy, 4.48 ± 0.62 Gy vs. 5.53 ± 1.02 Gy) and reduced the V5Gy, V10Gy, V20Gy, V30Gy, and V40Gy of the ipsilateral lung and heart. The SCCP and EAR for the ipsilateral lung were reduced by 3.67%, 3.09% in VMAT, and 22.18%, 19.21% in HT, respectively. While were increased for the contralateral lung and breast. This study showed that VMAT plans provide a more homogeneous dose distribution to the PTV, minimizing exposure to ipsilateral structures and significantly reducing SCCP and EAR, and slightly increasing dose to contralateral structures. Overall, the VMAT plan can be considered a beneficial technique for BCS patients whose PTV includes the whole breast and regional nodes.
Variability in plan quality of radiotherapy is commonly attributed to the planner's skill rather than technological parameters. While experienced planners can set reasonable parameters before optimization, less experienced planners face challenges. This study aimed to assess the quality of volumetric-modulated arc therapy (VMAT) in patients with left-sided breast cancer following breast-conserving surgery. Twenty-eight patients requiring whole-breast irradiation were randomly selected for inclusion. Each patient underwent two VMAT treatment plans: one optimized by an experienced planner (VMAT-EXP group) and the other designed by a less experienced planner using feasibility dose-volume histogram (FDVH) parameters from PlanIQ (VMAT-FDVH group). Both plans aimed to deliver a prescription dose of 50 Gy in 25 fractions to the planning target volume (PTV). Dosimetry parameters for the PTV and organs at risk (OARs) were compared between the two groups. Both the VMAT-EXP and VMAT-FDVH groups met the clinical plan goals for PTV and OARs. VMAT-FDVH demonstrated a PTV coverage and homogeneity comparable to those of VMAT-EXP. Compared to VMAT-EXP plans, VMAT-FDVH plans resulted in a significant reduction in the mean ipsilateral lung dose, with an average decrease of 0.9 Gy (8.5 Gy vs. 7.6 Gy, P < 0.001). The V5Gy and V20Gy of the ipsilateral lung were also reduced by 3.2% and 1.8%, respectively. Minor differences were observed in the heart, contralateral lung, breast, and liver. Personalized objectives derived from the feasibility DVH tool facilitated the generation of acceptable VMAT plans. Less experienced planners achieved lower doses to the ipsilateral lung while maintaining adequate target coverage and homogeneity. These findings suggest the potential for the effective use of VMAT in in patients with left-sided breast cancer following breast-conserving surgery, especially when guided by feasibility DVH parameters.
Fungal endophytes live asymptomatically within plants and are widespread inhabitants of leaves and other organs (Wilson, 1995). Likewise, endolichenic fungi live asymptomatically within lichens, occurring in healthy lichen thalli worldwide (Arnold et al., 2009). Endophytes and endolichenic fungi are ecologically similar, living in symbiosis with either a plant or the photobionts of lichens (Arnold et al., 2009), and both functional groups represent the same major lineages of fungi (U'Ren et al., 2012). As a whole, these fungi include diverse species whose life cycles often include pathogenic or saprobic phases (Porras-Alfaro & Bayman, 2011; Selosse, 2018; Terhonen et al., 2019). Endophytes and endolichenic fungi occur from polar regions to the tropics (Arnold et al., 2009), with most species transmitted horizontally (see Rodriguez et al., 2009). Although most endophyte–host interactions have not been examined, some have positive impacts on the physiology, growth or stress tolerance of their hosts (e.g. Arnold et al., 2003; Rodriguez et al., 2009; Porras-Alfaro & Bayman, 2011). Endophytic and endolichenic fungi are especially common and diverse among the largest nonlichenized lineages of the subphylum Pezizomycotina (Ascomycota), with variation among host lineages and biomes in the relative abundance of the five most common classes in which endophytism is known (Sordariomycetes, Dothideomycetes, Leotiomycetes, Eurotiomycetes and Pezizomycetes; Arnold et al., 2009). Among these, fungi in the class Pezizomycetes are of special interest because the biology of many taxa is incompletely known. This class consists of one order (Pezizales), 23 families and an estimated 2000 species (Pfister & Healy, 2021). Species of Pezizomycetes include well-documented plant pathogens (Marek et al., 2009), ectomycorrhizal (ECM) fungi (Tedersoo et al., 2006) and saprobes (Hobbie et al., 2001; Hansen & Pfister, 2006). Some species colonize specific substrates, acting as parasites of bryophytes (Döbbeler, 1997) or as specialized saprobes of dung (Pfister, 2015; Richardson, 2019) or postfire materials (Egger, 1986). Over the past decade, studies have shown that some species of Pezizomycetes are common as endophytes within bryophytes and occur frequently in lichen thalli (U'Ren et al., 2010, 2019). The life cycles and trophic ecology of some Pezizomycete species are unclear or controversial, and the endophytic habit has not been considered an important ecological strategy across the class (Pfister, 2015). Yet evidence suggests a more complex story. For example, stable isotope analyses indicate that members of the genus Morchella (morels) are able to access dead organic matter (Hobbie et al., 2001, 2016), but they also can live endophytically in roots (Baynes et al., 2012) and in conifer needles (Baroni et al., 2018). Likewise, the esteemed black truffles Tuber melanosporum and T. aestivum are ECM fungi, but in an interesting twist to truffle ecology, they have been shown to live as endophytes in roots of non-ECM plants (Schneider-Maunoury et al., 2020). While studying Pezizomycetes from an evolutionary and functional perspective, we noted that rDNA sequences from many Pezizomycete endophytes were available in GenBank, but that their phylogenetic affinities were not defined. Many sequences were generated from living cultures isolated from diverse plants and lichens sampled across the globe by Arnold and collaborators at the University of Arizona (UA) and maintained there as part of the Robert L. Gilbertson Mycological Herbarium (Myco-ARIZ; e.g. Hoffman & Arnold, 2010; U'Ren et al., 2010, 2012, 2014, 2019; Lau et al., 2013; Sandberg et al., 2014; Massimo et al., 2015; Huang et al., 2016, 2018a; U'Ren & Arnold, 2016; Bowman & Arnold, 2018; Oita et al., 2021a,b). The many DNA barcode matches among endophytes in GenBank and our unpublished sequences from fruit bodies suggested that endophytism and endolichenism might be more common, phylogenetically dispersed and ecologically important among Pezizomycetes than documented previously. Therefore, we assembled the available ecological and phylogenetic data on endophytic and endolichenic Pezizomycete species to ask: of the estimated 2000 Pezizomycete species known to date, how many occur as endophytes or endolichenic fungi?; in which lineages does endophytism or endolichenism occur across the Pezizomycetes?; and what are the main nutritional modes of endophytic or endolichenic Pezizomycete species when they are outside their hosts? We generated comprehensive phylogenies of Pezizomycetes based on 3315 sequences from the internal transcribed spacer region of nuclear ribosomal DNA (ITS1-5.8s-ITS2 nrDNA; hereafter ITS) and 1102 sequences of the large subunit nrDNA (28S) from fruit bodies, endophytes, and environmental sequences. We used maximum-likelihood (ML) phylogenetic analysis to determine operational taxonomic units (OTUs) (Table 1), but also compared our phylogeny-based OTUs with those recovered from a clustering approach based on 97% sequence similarity (Supporting Information Table S1). We included representatives of 3784 ITS sequences from the UA endophyte collection as well as new and reference sequences from Pezizomycete fruit bodies (Tables 1, S2). We preferentially incorporated available sequences from type specimens and used representative sequences in combination with Blast to obtain additional sequences of endophytic and endolichenic species (see flow chart in Fig. S1 for graphic of the methods). In order to examine placement of endophytes in Pezizomycetes, we first assembled a single 28S rDNA alignment with representatives of Pezizomycete lineages and ecological modes, including 266 endophytic and endolichenic sequences, and performed ML analyses via RAxML with 1000 bootstraps. The 50% majority rule tree (Figs 1, S2) was used in conjunction with Hansen & Pfister (2006) and Pfister (2015) as a guide for placement of endophytic and endolichenic taxa (Table 1). The ITS locus often is useful for species-level identification (Schoch et al., 2012; Kõljalg et al., 2013) and many endophyte studies generate only ITS sequences. Accordingly, we compiled 35 separate ITS alignments for individual families or lineages within families of Pezizomycetes, including 1046 endophytic or endolichenic sequences (Figs S3–S37). Details of methods, sequence alignments and accession numbers are provided in Notes S1, Fig. S1 and Tables S1 & S2. We detected endophytic and endolichenic species in 50 Pezizomycete genera and in 14 lineages that could not be assigned confidently to a genus. Together these represented ≥ 160 OTUs distributed across ≥ 16 families (Table 1; Figs 1, S2–S37). Some families had endophytism or endolichenism represented in only one or two genera (e.g. Desmazierella in Chorioactidaceae, Pseudombrophila in Pseudombrophilaceae), whereas others had species with these lifestyles in many genera, including Pezizaceae (27 OTUs in nine genera) and Pyronemataceae (45 OTUs in 17 genera; see also Tedersoo et al., 2013). Our conservative OTU delimitations based on ITS phylogenies yielded 160 OTUs whereas a clustering-based approach yielded 216 OTUs. However, the two methods yielded similar inferences regarding the ecology and phylogenetic distribution of Pezizomycete endophytes and endolichenic fungi (Table S1). The results presented here are based on the phylogenetic approach. Notably, we detected endophytism or endolichenism in two lineages for which this mode was previously undocumented: the ECM genus Otidea (Otideaceae) (Fig. S10) and dung saprobe genus Coprotus (Coprotaceae) (Fig. S6). In both genera we detected endophytic and endolichenic isolates from multiple hosts, suggesting that endophytism is a regular feature of their biology. Although a few Pezizomycete lineages with endophytic or endolichenic members were detected in only a limited range of hosts (e.g. Ascodesmidaceae was only isolated from vascular plants), most Pezizomycetes have a broad endophytic host range (as reported in detail by U'Ren et al., 2019). Endophytic and endolichenic Pezizomycetes are from lineages with a variety of trophic strategies, including saprobes, plant pathogens and mycorrhizal fungi (Tables S2, S4), but also include taxa for which the trophic strategies remain unresolved or unknown (Table S5). One particularly striking finding was an OTU that could not be assigned to a known family. This OTU is represented by five isolates from angiosperm leaves and lichens in an Alaskan boreal forest (USA) (U'Ren et al., 2019). It represents a unique lineage nested between Tuberaceae and Geomoriaceae, both of which consist exclusively of ECM species (Fig. 2; Notes S1; Tables S1–S3). The vast majority of Pezizomycete isolates in the culture-based UA dataset came from lichens (78%). Only one family (Ascodesmidaceae) was never detected in lichens, suggesting that lichens are important hosts for Pezizomycete species (Table S4). However, despite the importance of lichens as hosts, no Pezizomycete families were restricted to lichens alone and OTUs from most Pezizomycete families were detected in many photosynthetic hosts. For example, the most frequently isolated species were from Sarcosomataceae (Table S4), a family of largely saprobic taxa. At the genus level Pseudoplectania (Sarcoscomataceae) was especially well represented, comprising nearly 40% of Pezizomycete isolates in the UA dataset (Table S5). More generally, one conspicuous and recurrent phylogenetic pattern is that both endophytes and endolichenic fungi are common in clades containing well-characterized saprobes from wood, dung or postfire substrates, but infrequent in closely related ECM or plant pathogenic clades, as noted for Pyronemataceae by Tedersoo et al. (2013). For example, endophytism and endolichenism were commonly detected for Geopyxis, a genus of Tarzettaceae with putatively biotrophic, weakly parasitic and/or pyrophilous species (Egger, 1986; Vrålstad et al., 1998), but not detected for the ECM sister genus Tarzetta (Fig. S35). Likewise, endophytism and endolichenism was commonly detected for species of saprobic Pezizaceae (e.g. Peziza s.s., Plicaria and Geoscypha) (Figs S13, S17, S18) but rarely or never detected for ECM species such as those in Legaliana or Ruhlandiella (Fig. S11). In the Discinaceae, endophytism and endolichenism was common for species of Gyromitra, a genus of putative saprobes, but absent in species of the ECM sister genus Hydnotrya (Hobbie et al., 2001) (Fig. S7). Although endophytes were documented only rarely among ECM and pathogenic clades of Pezizomycetes, we detected endophytism and endolichenism in four lineages known previously only for ECM lifestyles (Otidea and several ECM Pezizaceae), and endophytism was also reported in species of the ECM genera Sphaerosporella (Hughes et al., 2020) and Tuber (Schneider-Maunoury et al., 2018). Although many ECM fungi are difficult or impossible to culture (Tedersoo et al., 2010), these endophytic or endolichenic isolates grow well in pure culture and thus enable future research in genomics, experimental manipulation and secondary metabolites. There is evidence from Sphaerosporella that endophytes of needles and colonization of ECM roots may rarely occur in the same individual host, but that the mode of infection for these two organs is different (Hughes et al., 2020). Additional studies are needed, perhaps employing culturing, resynthesis, inoculation and isotopic methods, to substantiate whether Pezizomycete endophytes with identical ITS sequences to those on their ECM host roots are actually the same genotype and are playing similar ecological roles. New approaches that allow visualization of the extent and morphology of fungal colonization, such as fluorescence in situ hybridization (Schneider-Maunoury et al., 2020), will be especially helpful in future work. By contrast, other ECM Pezizomycetes (e.g. Otidea, some ECM Pezizaceae) were not detected as endophytes in their ECM hosts but were instead found in bryophytes and lichens, suggesting possible compartmentalization of different trophic modes on different hosts. A similar pattern was also found among the two lineages of plant pathogens where endophytism was detected. The endophytic state of Pithya cupressina is putatively a dormant pathogen because this fungus is considered the cause of twig die-back on Juniperus, but it was also found as an endophyte in healthy tissue of the same Juniperus species. By contrast, Rhizina undulata, a root pathogen of Pinaceae, was endophytic only in lycopods and ferns. Interestingly, no bryophyte parasites (such as Octospora or Lamprospora) were detected as endophytic or endolichenic in any sampled host, including mosses. These patterns suggest that host preferences, compartmentalization and the ability to colonize different hosts or host organs may be species- or lineage-specific. Notably, endophytic and endolichenic species were particularly common among clades of fire-adapted and pyrophilous Pezizomycetes, including 24 OTUs from 19 genera (Table S6). Raudabaugh et al. (2020) found that pyrophilous taxa such as Anthracobia melaloma, Ascorhizoctonia praecox, Pyronema omphalodes and R. undulata are common as endophytes but appear to be rare to absent in soil. After wildfires, however, these fungi fruit prolifically on soil and burnt plant debris (e.g. Petersen, 1970; Reazin et al., 2016; Bruns et al., 2020). Likewise, in a study of endophytes of Bromus tectorum (cheatgrass), Baynes et al. (2012) identified several pyrophilous Pezizomycetes (e.g. Peziza ostracoderma, Pyronema domesticum, Morchella eximia and M. snyderi). Subsequent experiments showed that endophytic Morchella species increased B. tectorum growth and enhanced seed survival following fire, highlighting a previously unstudied benefit of this symbiosis. U'Ren et al. (2012) and Huang et al. (2016) reported the dominance of Pezizomycete endophytic and endolichenic species in Arizona forests where fire is common, indicating that this may be a widespread phenomenon. Our analysis revealed additional pyrophilous fungi that can be endophytic or endolichenic, including Geoscypha tenacella and Pyropyxis rubra. Available evidence suggests hidden roles of some pyrophilous Pezizomycetes as plant symbionts, setting the stage for studies of ecological effects of endophytism on host plants and endolichenism on host lichens. These and other hypotheses will be testable in the future due to living fungal libraries such as the UA culture collection. Metagenomics and other culture-free tools have been, and will continue to be, critical for elucidating plant–fungi interactions, especially because these methods typically detect a far greater diversity of fungi than culture-based methods alone (U'Ren et al., 2019). However, studies such as ours highlight the importance of maintaining living endophytic and endolichenic fungus cultures and generating ITS and 28S DNA to identify them (see U'Ren et al., 2019 for benefits and drawbacks of molecular vs culture-based detection). Fungal cultures can be used for diverse purposes: to test nutritional requirements, characterize novel metabolites, sequence genomes and transcriptomes, and inoculate plants to study the effects on plant and fungal fitness (e.g. Wijeratne et al., 2012; Sarmiento et al., 2017; Torres-Cruz et al., 2017; Huang et al., 2018b; Harrington et al., 2019). In this study, we identified endophytic and endolichenic species from an impressive 16 of 23 recognized Pezizomycete families, as well as a lineage that likely represents a new family (undetermined lineage in Fig. 2). Our results suggest that endophytism and endolichenism may indeed be the rule rather than the exception across families of Pezizomycetes. Our conservative phylogenetic approach to OTU delimitation detected a minimum of 160 OTUs of endophytic and endolichenic Pezizomycetes. This is equivalent to c. 8% of the estimated 2000 species in this class, but these are spread across c. 70% of the families. Given that only a small fraction of potential hosts and geographical areas have been sampled for endophytes or endolichenic fungi, the number of OTUs is probably a marked underestimate. Notably, relatively few studies have broadly sampled lichens wherein Pezizomycete species are dominant (but see U'Ren et al., 2012, 2019). It seems probable that more sampling will detect additional Pezizomycete species as cryptic residents in plants and lichens from biomes ranging from tropical forests to polar deserts, as illustrated by culture-based and culture-free studies (e.g. Higgins et al., 2007; U'Ren et al., 2019; Oita et al., 2021a). The ecologies of many rare or understudied species of Pezizomycetes in genera such as Carbomyces, Eremiomyces, Glaziella, Hydnocystis, Kalaharituber, Pseudotricharina and Sowerbyella remain mysterious and currently unclear (Læssøe & Hansen, 2007; Tedersoo et al., 2010; Tedersoo & Smith, 2013). Although the trophic nature for most endophytic Pezizomycetes outside their hosts is putatively saprobic, endophytism and endolichenism also appear to be a normal part of the life history in many ECM and pathogenic species. Functional roles during their endophytic or endolichenic phase are unknown for any of these trophic groups and require further investigation. Our results suggest that future studies to elucidate the lifestyles of these poorly known Pezizomycetes should look first to the nearest plants and lichens to see what fungi might be living inside. Funding for this project was provided by: NSF grant DEB-1946445 (to MES, RH and GB); NIFA-USDA award FLA-PLP-005289 (to MES); NSF DEB-1541496 (to AEA); NIFA-USDA ARZT-1361340-H25-242 to AEA and colleagues; diverse awards to AEA and colleagues that supported endophyte collections; the College of Agriculture and Life Sciences at UA; and the University of Florida's Institute for Food and Agricultural Sciences (IFAS). We thank Katherine Lobuglio from Harvard University for providing an ITS sequence of Pseudopithyella. We thank N. Reynolds at the Florida Museum of Natural History (FLAS) for helping to accession voucher specimens of Pezizomycetes. We thank J.M. U'Ren, F. Lutzoni, J. Miadlikowska, N. Massimo, E.A. Bowman, S. Oita, P. Cerda, N. Garber, J. Moy, M. Lau, M. Hoffman, F. Quintana, M. Lee, N. Colón-Carrión, and numerous students and collaborators for contributing to the isolation and barcoding of endophytes that were included in public depositories referenced in this study. We thank Autumn Anglin, Michael Beug, Marcos Caiafa Sepúlveda, Django Grootmyers, Arthur Grupe, Roger Heidt, Jason Karakehian, Ron Petersen, Jim Trappe, Nicolas Van Vooren, Else Vellinga and other collectors for their contributions of Pezizales fruit bodies and anamorphs that we used in our molecular analyses. Finally, we thank three anonymous reviewers and Editor M-A. Selosse for insightful comments and useful feedback that helped to improve this work. MES and RAH conceived the study; MES, AEA, DHP, RAH and GB obtained funding and contributed resources to the work; MES, RAH, BL, DHP and GB collected specimens and generated DNA sequences from museum specimens; AEA and Y-LH isolated endophytes, maintained fungal cultures and generated DNA sequences from endophyte cultures, in collaboration with authors who submitted cultures to the UA collection (see References and Acknowledgements); MES, RAH and BL compiled data, completed analyses, generated figures and deposited specimens and sequences; DHP provided advice and reviewed all taxonomy and nomenclature; and MES and RAH wrote the manuscript with input from all authors (MES, RH, DHP, BL, GB, AEA and Y-LH). Data in this manuscript are publicly available on GenBank and via OSF (see the Supporting Information). Fig. S1 Flow chart outlining the basic steps for assembling data on Pezizomycetes endophytes and endolichenics. Fig. S2 Phylogeny of endophytic fungi in Pezizomycetes based on 28S sequences analyzed with ML. Fig. S3 Phylogeny based on ITS sequences of Ascobolaceae and related endophytic or endolichenic fungi analyzed with ML. Fig. S4 Phylogeny based on ITS sequences of Ascodesmidaceae and related endophytic or endolichenic fungi analyzed with ML. Fig. S5 Phylogeny for Coprotaceae and related endophytic or endolichenic fungi analyzed with ML. Fig. S6 Phylogeny based on ITS sequences of Chorioactidaceae and related endophytic or endolichenic fungi analyzed with ML. Fig. S7 Phylogeny based on ITS sequences of Discinaceae endophytic or endolichenic fungi analyzed with ML. Figs S8 and S9 Phylogeny based on ITS sequences of Morchellaceae endophytic or endolichenic fungi analyzed with MLood. Fig. S10 Phylogeny based on ITS sequences of Otideaceae and related endophytic or endolichenic fungi analyzed with ML. Fig. S11 Phylogeny based on ITS sequences of Pezizaceae (pro parte) for placement of endophytes and endolichenic fungi analyzed with ML. Fig. S12 Phylogeny based on ITS sequences of Pezizaceae (Daleomyces, Malvipezia) and related endophytic or endolichenic fungi analyzed with ML. Fig. S13 Phylogeny based on ITS sequences of Pezizaceae (Geoscypha) and related endophytic or endolichenic fungi analyzed with ML. Fig. S14 Phylogeny based on ITS sequences of Pezizaceae (Iodophanus) and related endophytic or endolichenic fungi analyzed with ML. Fig. S15 Phylogeny based on ITS sequences of Pezizaceae (Lepidotia) and related endophyte analyzed with ML. Fig. S16 Phylogeny based on ITS sequences of Pezizaceae (Mattirolomyes, Elderia) and related endophytes analyzed with ML. Fig. S17 Phylogeny based on ITS sequences of Pezizaceae (Peziza sensu stricto) and related endophytic and endolichenic fungi analyzed with ML. Fig. S18 Phylogeny based on ITS sequences of Pezizaceae (Plicaria) and related endophytic and endolichenic fungi analyzed with ML. Fig. S19 Phylogeny based on ITS sequences of Pseudombrophilaceae and related endophytic and endolichenic fungi analyzed with ML. Fig. S20 Phylogeny based on ITS sequences of Pulvinulaceae and related endophytic and endolichenic fungi analyzed with ML. Fig. S21 Phylogeny based on ITS of Pyronemataceae (pro parte) for placement of endophytes and endolichenic fungi analyzed with ML. Fig. S22 Phylogeny based on ITS of Pyronemataceae (pro parte) for placement of endophytes and endolichenic fungi analyzed with ML. Fig. S23 Phylogeny based on ITS of Pyronemataceae (pro parte) for placement of endophytes and endolichenic fungi analyzed with ML. Fig. S24 Phylogeny based on ITS of Pyronemataceae (Lasiobolidium) for placement of endophytes and endolichenic fungi analyzed with ML. Fig. S25 Phylogeny based on ITS of Pyronemataceae (Perilachnea) for placement of endophytes and endolichenic fungi analyzed with ML. Fig. S26 Phylogeny based on ITS of Pyronemataceae (Jafnea, Pyropyxis, Smardaea) for placement of endophytes and endolichenic fungi analyzed with ML. Fig. S27 Phylogeny based on ITS of Pyronemataceae (pro parte) for placement of endophytes and endolichenic fungi analyzed with ML. Fig. S28 Maximum likelihood analysis of ITS of Pyronemataceae (Trichophaea, Wilcoxina) for placement of endophytes and endolichenic fungi analyzed with ML. Fig. S29 Phylogeny based on ITS sequences of Rhizinaceae and related endophytic and endolichenic fungi analyzed with ML. Fig. S30 Phylogeny based on ITS sequences of Sarcoscyphaceae and related endophytic and endolichenic fungi analyzed with ML. Fig. S31 Phylogeny based on ITS sequences of Sarcosomataceae (Donadinia) and related endophytic and endolichenic fungi analyzed with ML. Fig. S32 Phylogeny based on ITS sequences of Sarcosomataceae (Galiella, Plectania) and related endophytic and endolichenic fungi analyzed with ML. Fig. S33 Phylogeny based on ITS sequences of Sarcosomataceae (Pseudoplectania, Sarcosoma) and related endophytic and endolichenic fungi analyzed with ML. Fig. S34 Phylogeny based on ITS sequences of Sarcosomataceae (Urnula) and related endophytic and endolichenic fungi analyzed with ML. Fig. S35 Phylogeny based on ITS sequences of Tarzettaceae and related endophytic and endolichenic fungi analyzed with ML. Fig. S36 Phylogeny based on ITS sequences of Tuberaceae analyzed with ML. Fig. S37 Phylogeny based on ITS sequences of Geomoriaceae and an undetermined lineage of related endophytic and endolichenic fungi analyzed with ML. Notes S1 Additional method details for culture work, molecular work, and phylogenetic analyses. Table S1 Number of sequences, characters and endophyte OTUs included in the rDNA and multilocus analyses. Table S2 GenBank numbers for newly accessioned sequences and their sequence sources, herbaria of deposit, and geographical localities. Table S3 GenBank numbers for newly accessioned sequences and their sequence sources, herbaria of deposit, and geographical localities. Table S4 Synopsis of the 3784 records of endophytic and endolichenic Pezizomycete isolates in the UA database showing both the phylogenetic placement to the family level and the recorded host associations. Table S5 Synopsis of the most frequently isolated endophytic or endolichenic Pezizomycetes in the UA database, enumerated by genus in a particular type of host. Table S6 Endophytic or endolichenic species of Pezizomycetes that are obligately pyrophilous or commonly fruit after burns or volcanic eruptions. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. 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Biodiversity collections contain a wealth of information encapsulated both in specimens and in their metadata, providing the foundation for diverse studies in fields such as ecology. Yet biodiversity repositories can present a challenge for ecological inferences because collections rarely are structured with ecological questions in mind: collections may be opportunistic in space or time, may focus on particular taxonomic groups, may reflect different collection strategies in different places or times, or may not be exhaustive in terms of retaining every specimen or having similar metadata for each record. In addition to its primary holdings, the Robert L. Gilbertson Mycological Herbarium at the University of Arizona holds a collection of living specimens of fungi isolated from the interior of healthy plants and lichens (i.e., endophytic and endolichenic fungi). Over the past decade, more than 7000 isolates from the southwestern United States were accessioned, including strains from diverse hosts in more than 50 localities across the biotically rich state of Arizona. This collection is distinctive in that metadata and barcode sequences are available for each specimen, many localities have been sampled with consistent methods, and all isolates obtained in surveys have been retained. Here, we use this herbarium collection to examine endophyte community structure in an ecological and evolutionary context. We then artificially restructure the collection to resemble collections more typical of biodiversity repositories, providing a case study for ecological insights that can be gleaned from collections that were not structured explicitly to address ecological questions. Overall, our analyses highlight the relevance of biogeography, climate, hosts, and geographic separation in endophyte community composition. This study showcases the importance of extensive metadata in collections and highlights the utility of biodiversity collections that can yield emergent insights from many surveys to answer ecological questions in mycology, ultimately providing information for understanding and conserving fungal biodiversity.
Abstract Background: To compare the dosimetric normal tissue complication probability (NTCP), secondary cancer complication probabilities (SCCP), and excess absolute risk (EAR) differences of volumetric modulated arc therapy (VMAT) and intensity-modulated radiation therapy (IMRT) for left-sided breast cancer after mastectomy. Methods and materials: Thirty patients with left-sided breast cancer treated with post-mastectomy radiation therapy (PMRT) were randomly enrolled in this study. Both IMRT and VMAT treatment plans were created for each patient. Planning target volume (PTV) doses for the chest wall and internal mammary nodes, PTV1, and PTV of the supraclavicular nodes, PTV2, of 50 Gy were prescribed in 25 fractions. The plans were evaluated based on PTV1 and PTV2 coverage, homogeneity index (HI), conformity index, conformity number (CN), dose to organs at risk, NTCP, SCCP, EAR, number of monitors units, and beam delivery time. Results: VMAT resulted in more homogeneous chest wall coverage than did IMRT. The percent volume of PTV1 that received the prescribed dose of VMRT and IMRT was 95.9 ± 1.2% and 94.5 ± 1.6%, respectively ( p < 0.001). The HI was 0.11 ± 0.01 for VMAT and 0.12 ± 0.02 for IMRT, respectively ( p = 0.001). The VMAT plan had better conformity (CN: 0.84 ± 0.02 vs. 0.78 ± 0.04, p < 0.001) in PTV compared with IMRT. As opposed to IMRT plans, VMAT delivered a lower mean dose to the ipsilateral lung (11.5 Gy vs 12.6 Gy) and heart (5.2 Gy vs 6.0 Gy) and significantly reduced the V 5 , V 10 , V 20, V 30, and V 40 of the ipsilateral lung and heart; only the differences in V 5 of the ipsilateral lung did not reach statistical significance ( p = 0.409). Although the volume of the ipsilateral lung and heart encompassed by the 2.5 Gy isodose line (V 2.5 ) was increased by 6.7% and 7.7% ( p < 0.001, p = 0.002), the NTCP was decreased by 0.8% and 0.6%, and SCCP and EAR were decreased by 1.9% and 0.1% for the ipsilateral lung. No significant differences were observed in the contralateral lung/breast V 2.5 , V 5, V 10 , V 20 , mean dose, SCCP, and EAR. Finally, VMAT reduced the number of monitor units by 31.5% and the treatment time by 71.4%, as compared with IMRT. Conclusions: Compared with IMRT, VMAT is the optimal technique for PMRT patients with left-sided breast cancer due to better target coverage, a lower dose delivered, NTCP, SCCP, and EAR to the ipsilateral lung and heart, similar doses delivered to the contralateral lung and breast, fewer monitor units and a shorter delivery time.
Fungal endophytes are diverse and widespread symbionts that occur in the living tissues of all lineages of plants without causing evidence of disease. Culture-based and culture-free studies indicate that they often are abundant in the leaves of woody angiosperms, but only a few studies have visualized endophytic fungi in leaf tissues, and the process through which most endophytes colonize leaves has not been studied thoroughly. We inoculated leaf discs and the living leaves of a model woody angiosperm, Populus trichocarpa, which has endophytes that represent three distantly-related genera (Cladosporium, Penicillium, and Trichoderma). We used scanning electron microscopy and light microscopy to evaluate the timeline and processes by which they colonize leaf tissue. Under laboratory conditions with high humidity, conidia germinated on leaf discs to yield hyphae that grew epiphytically and incidentally entered stomata, but did not grow in a directed fashion toward stomatal openings. No cuticular penetration was observed. The endophytes readily colonized the interiors of leaf discs that were detached from living leaves, and could be visualized within discs with light microscopy. Although they were difficult to visualize within the interior of living leaves following in vivo inoculations, standard methods for isolating foliar endophytes confirmed their presence.
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