Cajanus cajan is cultivated in different regions of the world as an economical source of carbohydrates, fiber, proteins, and fodder for animals. In the current study, 39 K+ transport genes were identified in C. cajan, including 25 K+ transporters (17 carrier-like K+ transporters (KUP/HAK/KTs), 2 high-affinity potassium transporters (HKTs), and 6 K+ efflux transporters (KEAs) and 14 K+ channels (9 shakers and 5 tandem-pore K+ channels (TPKs). Chromosomal mapping indicated that these genes were randomly distributed among 10 chromosomes. A comparative phylogenetic analysis including protein sequences from Glycine max, Arabidopsis thaliana, Oryza sativa, Medicago truncatula Cicer arietinum, and C. cajan suggested vital conservation of K+ transport genes. Gene structure analysis showed that the intron/exon organization of K+ transporter and channel genes is highly conserved in a family-specific manner. In the promoter region, many cis-regulatory elements were identified related to abiotic stress, suggesting their role in abiotic stress response. Abiotic stresses  adversely affect chlorophyll, carotenoids contents, and total soluble proteins. Furthermore, the activities of catalase, superoxide, and peroxidase were altered in C. cajan leaves under applied stresses. Expression analysis  revealed that several K+ transport genes were expressed in abiotic stress-responsive manners. The present study provides an in-depth understanding of K+ transport system genes in C. cajan and serves as a basis for further characterization of these genes.Potassium is the most important and abundant inorganic cation in plants and it can comprise up to 10% of a plant’s dry weight. Plants possess complex systems of transporters and channels for the transport of K The variants of all predicted genes were checked manually and only the largest open reading frames (ORF) were selected for further analysis. Subsequently, genes were further analyzed by different databases, i.e., Pfam database (http://pfam.janelia.org/ (accessed on 14 August 2021)), NCBI conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 14 August 2021)), and SMART database (http://smart.embl-heidelberg.de/ (accessed on 14 August 2021)). Highly conserved domains containing genes were selected for further analysis. All the genomic information, protein sequences, chromosomal locations, exon numbers, and protein lengths of K+ transporters and channels were taken from NCBI.The protein sequences of already-identified KC. cajan ,16,17. Ahttp://meme.sdsc.edu/meme/meme.html (accessed on 14 August 2021)) was used to find conserved motifs in protein sequences and default parameters were used for motif analysis. Weblogo (https://weblogo.berkeley.edu/logo.cgi (accessed on 14 August 2021)) was used to represent the conserved motifs. NCBI was used to retrieve the genomic and coding sequence of all identified genes. Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/ (accessed on 14 August 2021)) was used to schematically represent the exon–intron structure of genes. Analysis of gene structure was performed using selected sequences.MEME (https://itol.embl.de (accessed on 14 August 2021)) was used to visualize and modify the phylogenetic tree. Cis-regulatory elements (CREs) were searched from upstream regions (1000–1100 bp) of all characterized genes. The cis-regulatory elements in the promoter region were analyzed by the Plant CARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 14 August 2021)).ClustalW was used for multiple sequence alignment. Phylogenetic analysis was performed using MEGAX Neighbor-Joining with 1000 bootstrap methods to construct phylogenetic trees. iTOL  ).Gene duplication events were determined by DNAsp and the Ka/Ks ratio was calculated to determine duplication events. Divergence time was also calculated using the Ks and Ka values to predict the evolutionary events. Genomic Loci of putative KC. cajan were obtained from a local market of Faisalabad, Pakistan. Plants were grown in plastic pots filled with peat moss in a growth chamber. Pots were kept under the following growth conditions: 22 °C night/25 °C day temperature, 8 h dark and 16 h light period, and 68% humidity. Plants were grown in four different treatments groups: control, heat stress, drought stress, and salt stress, each with three replicates. After 12 days of germination, plants were challenged with heat, drought, and salt stresses. Hoagland solution (10 mL/day) was applied to provide necessary nutrients for plant growth. Drought stress was imposed by limiting the water supply for 7 days, while control was supplied with ample water. Salt stress was imposed by supplying 100 mM NaCl solution for 8 days from the 13th day of germination [Seeds of domesticated mination . Plants a, b, total chlorophyll, and chlorophyll a/b ratios:Fresh leaves (0.1 g) were taken, ground, and homogenized in 80% methanol for estimation of chlorophyll content. These samples were kept at 4 °C overnight. Subsequently, a spectrophotometer  was used to take the absorbance at various wavelengths, i.e., 645 nm and 663 nm. Then, 80% methanol was used as blank to normalize the absorbance value of the solvent. The following formulas were used to measure chlorophyll V = volume of the extract (mL)W = weight of fresh leaf tissue (g)For estimation of carotenoid contents, fresh leaves (0.1 g) were ground and homogenized in 80% methanol. Samples were centrifuged for 15 min and the supernatant was used to measure the carotenoid contents at 480 nm. The following formula was used to measure total carotenoid content:A = absorbanceCa = alpha-caroteneCb = beta-caroteneThe activity of biochemical entities  was determined by following protocols reported in different studies. Superoxide dismutase activity was measured by following . Peroxid+ transport genes was analyzed in two different cultivars of C. cajan . For this purpose, RNA-seq data  were downloaded from NCBI-SRA (https://www.ncbi.nlm.nih.gov/sra (accessed on 14 August 2021)). Moreover, to analyze the expression pattern of potassium-transporting genes in C. cajan under heat stress at different time points  and salt stress in ICP7: salt-tolerant and ICP1071: salt susceptible, data  were downloaded from the NCBI-SRA database. These paired-end clean reads were mapped to the C. cajan genome. C. cajan genome was downloaded from NCBI and indexes were built using bowtie2. The cufflinks program was used to calculate the expression level of annotated genes in the reference genome using the GFF format of the genome. The normalized FPKM value was used to construct a heatmap using TBtools. Furthermore, these identified potassium transporters and channels were subjected to STRING v11.0 (https://string-db.org/ (accessed on 14 August 2021)) to analyze network of protein–protein interactions (PPIs). The minimum required interaction score was 0.400, corresponding to medium confidence.The expression pattern of KC. cajan by Trizol method and quantified by Thermo Nanodrop 2000 . One mg of RNA was used for the synthesis of cDNA using the First-Strand Synthesis kit. cDNA was stored at −20 °C for further analysis. qRT-PCR was performed for expression analysis of transcripts level using a qRT-PCR detection system  with iTaq Universal SYBR Green SuperMix. The “Oligo Calculator”, an online tool ), was used to design gene-specific primers, which were further verified by the NCBI-primer BLAST program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 14 August 2021)). The expression analysis was triplicated for each of the genes and the GAPDH gene was considered as the housekeeping gene [Total RNA was extracted from leaf samples of ing gene .+ is one of the major nutrients (NPK), yet it has attracted very little attention from farmers and researchers. C. cajan is cultivated on soils that have low nutrients. Various studies have reported on growth and development by K+ transport systems in plants. Therefore, a comprehensive investigation into K+ homeostasis mechanisms is required in C. cajan to understand their influence on plant development, growth, and stress response. The present research focused on revealing K+-transporting genes in C. cajan. In this study, 39 genes of the K+ transport system were identified, comprising K+ transporters (25 genes) and K+ channels (14 genes), in C. cajan on the basis of their sequence and structure similarity with A. thaliana. Evolutionary conservation of K+ transport system genes in monocots/dicots and legumes/non-legumes was also observed by gene structure and phylogenetic analysis. Gene promoter analysis provided significant information about the importance of K+ transport-related genes in response to abiotic stress in C. cajan. Physio-chemical analysis provided insights about the contrary effects of abiotic stresses on C. cajans under different physiological parameters such as chlorophyll, carotenoids, and total soluble protein and antioxidant activities, i.e., SOD, POD, and CAT under heat, salt, and drought. The antioxidant activity of these compounds was increased under applied stress conditions; catalase activity was significantly decreased under all three stresses. This study provides initial insight into the C. cajan K+ transport system, which is helpful for further studies hoping to explore all-genes function in different biological processes and under different stress conditions.In plants, K


Aspergillus fumigatus. Here we present the largest analysis of PQS in fungi and identify significant heterogeneity of these sequences throughout phyla, genera and species. PQS were genetically conserved in Aspergillus spp. and frequently pathogenic species appeared to contain fewer PQS than their lesser/non-pathogenic counterparts. GO-term analysis identified that PQS-containing genes were involved in processes linked with virulence such as zinc ion binding, the biosynthesis of secondary metabolites and regulation of transcription in A. fumigatus. Although the genome frequency of PQS was lower in A. fumigatus, PQS could be found enriched in genes involved in virulence, and genes upregulated during germination and hypoxia. Moreover, PQS were found in genes involved in drug resistance. Quadruplexes could have important roles within fungal biology and virulence, but their roles require further elucidation.Fungal infections cause >1 million deaths annually and the emergence of antifungal resistance has prompted the exploration for novel antifungal targets. Quadruplexes are four-stranded nucleic acid secondary structures, which can regulate processes such as transcription, translation, replication and recombination. They are also found in genes linked to virulence in microbes, and ligands that bind to quadruplexes can eliminate drug-resistant pathogens. Using a computational approach, we quantified putative quadruplex-forming sequences (PQS) in 1359 genomes across the fungal kingdom and explored their presence in genes related to virulence, drug resistance and biological processes associated with pathogenicity in  The genomes used were obtained from public repositories  base pairs (G-quadruplexes (G4s) and i-motifs (iMs) are intriguing four-stranded (quadruplex) secondary structures in nucleic acids that are enriched in regulatory regions, particularly the promoters and telomeric regions of prokaryotic and eukaryotic genomes . G4s canse pairs  21]. St. St+ , and eukaryotic pathogens  [S. aureus and vancomycin-resistant Enterococcus spp.; potentially providing a novel target to overcome drug resistance [There is a growing number of pathogens in which G4s have been shown to contribute to virulence phenotypes, including viruses , prokaryotic bacterial pathogens (ciparum) . Notablysistance . In contsistance . AlthougA. fumigatus, and their possible contribution to the virulence of this microorganism.Practically all organisms possess quadruplexes and due to the advances in the development of drugs that bind and stabilize these structures, the ability to target specific quadruplexes is starting to become a reality . A thorohttps://doi.org/10.5281/zenodo.3783970). These genomes encompassed the Saccharomycotina (332 genomes from 12 major clades), Pezizomycotina (761 genomes representing 9 of 16 classes) and Taphrinomycotina (14 genomes representing 4 of 5 classes). The completeness of these genomes has been assessed previously [The 1107 genome sequences from fungi in the Ascomycota that were analysed in this study were obtained from a publicly available repository  and GenBank via FTP access number (ftp://ftp.ncbi.nlm.nih.gov/genomes/). The completeness of these genomes was not assessed, and thus the analysis of these genomes can be found in the Supplementary Material for reference. Information for all the genomes used in this study can be found in Table S1 (available in the online version of this article).A further 252 published genomes from the Basidiomycota, Mucoromycota, Zoopagomycota, Chytridiomycota, Microsporidia and Cryptomycota were also obtained from the Joint Genome Institute MycoCosm portal  were extracted. The algorithm used specifically recognizes PQS, therefore all sequences obtained from the G4Hunter application can be considered PQS .To identify the location of PQS within annotated genomic features, the file containing the annotations for known genomic features within the genome of the selected fungi were downloaded from the NCBI database. The presence of PQS within a pre-defined genomic feature  or within ±100 bp of these genomic features were analysed. Discrepancies in the frequencies of PQS between mRNA and genes are due to how the feature tables downloaded from the NCBI database present their annotations. mRNA length is slightly shorter than the gene length due to the absence of intronic sequences and alternative splicing .http://bioinformatics.ibp.cz:8080/#/) [https://pypi.org/project/dna-analyser-ibp/. The protein classes of genes containing PQS were determined via PANTHER Protein Class v.15.0 [https://elbe.hki-jena.de/fungifun/) and significance was determined via Fisher’s exact test [http://bioinformatics.ramapo.edu/QGRS/analyze.php) [Analysis of the genomes for PQS was conducted using the G4Hunter DNA analyser web application (8080/#/) . The locs v.15.0 . Gene onact test . The PQSyze.php) . Sequenchttps://3dproteinimaging.com/protein-imager/) [ChemDraw Ultra v12.0 was used to draw structures and The Protein Imager (imager/)  was usedhttps://fungidb.org/) [A. fumigatus biofilms [Q=1.0 %) and normality was tested with Shapiro–Wilk, prior to analysis via one-sample T test. A P-value<0.05 was considered statistically significant.Upregulated genes in germinating conidia and hyphae , during db.org/) . Upregulbiofilms  were obtT test, one-sample T test or Pearson’s correlation coefficient; or non-parametric Kruskal–Wallis tests with post-hoc Dunn’s test and Bonferroni corrections. Outliers were removed when necessary using the ROUT method (Q=1.0 %) and normality was tested using Shapiro–Wilk. All analyses were performed using GraphPad Prism software v6.01. P<0.05 were considered statistically significant.Statistical analyses were conducted via parametric one-way ANOVA with Tukey’s multiple comparisons, Student’s A thorough analysis of putative quadruplex-forming sequences in fungi has not been conducted to date. Using G4Hunter, the number of PQS were quantified in 1359 genomes across the fungal kingdom. Due to the high variability in genome size and chromosome number between fungal species, to normalize the data, the total number of PQS in addition to the frequency of PQS/kbp and number of PQS relative to the GC content (PQS/GC%) were noted.Across the divisions, the Chytridiomycota had the largest average number of PQS (24 734 PQS) and highest PQS frequencies , the Fumigati (0.204–0.224), the Candidi (0.747–0.816), the Circumdati (0.395–0.467), the Flavi (0.225–0.289), the Ochraceoros (0.421–0.422), the Usti (0.385–0.396), the Versicolores (0.282–0.308) and the Nigri (0.495–0.782). The Nigri displayed the largest variation in PQS, however the lesser related species A. carbonarius and A. aculeatus skewed this range and if only the closest related species were noted, this range would be from 0.495 to 0.611  pathogenic species had a significantly lower frequency of PQS/kbp (0.364 vs. 0.523) and PQS/GC% (249.9 vs. 380.5) within their genomes on average, compared to non-pathogenic species (Cryptococcus spp. (containing the important basidiomycete pathogen Cryptococcus neoformans) but not for Candida spp. . Furthermore, the most pathogenic species, A. fumigatus, had a lower than average frequency of PQS  serve as hotspots for genome rearrangement, and mRNA splicing events in around 30 % of genes (~3000 genes) contribute to significant functional diversity, which likely contributes to the rapid adaptation to new environments [A. fumigatus to provide some insight into their potential functional roles.Genomic rearrangements have been suggested to contribute to the success of fungal pathogens, their ability to colonize and invade, and their adaptation to host niches. Repetitive genomic regions in ronments . Quadrupronments . TherefoA. fumigatus, the annotation information for the strain Af293 was obtained from the NCBI database and the presence of PQS within defined genomic features, or within 100 bp before or after these features were analysed. Both the total number of PQS per feature and the frequency of PQS/kbp of the described features were noted.To evaluate the position of PQS within C. albicans and C. neoformans . However, this was not the same when considering the frequency of PQS/kbp of the genomic features. In A. fumigatus, the greatest frequency of PQS could be found in the repeat regions and mRNAs, whilst the lowest frequency could be found within the tRNAs (A. fumigatus telomeres was high (14.95 PQS/kbp). However, PQS were also abundant in non-telomeric repeat regions  of these genes contained at least one PQS in their tandem repeat regions (Table S3). These observations were suggestive of the potential involvement of quadruplexes in the promotion of genetic diversity and adaptation in A. fumigatus.When only total PQS were considered, the largest number of PQS could be found within the genes and mRNA, with few PQS found in other genomic features . This wahe tRNAs . It is w et al.,  highlighA. fumigatus, 35.1 % of genes contained at least one PQS likely to form quadruplex structures. In C. neoformans, this number was almost double, with 59.9 % of genes containing PQS. Conversely, PQS were only found in 5.6 % of genes in C. albicans. Despite the discrepancies in the number of genes where PQS can be found between the organisms, in all cases, PQS were primarily located in genes that encoded proteins involved in metabolism, nucleic acid binding, cell transport and protein modification  were investigated for the presence and frequency of PQS. Notably, PQS could be found in 77.5 % of the genes investigated  and echinocandin resistance (fks1). Genes implicated in virulence, which were enriched in PQS compared to the average frequency for the entire genome (PQS/kbp in brackets), included mkk2 (4.78), zafA (4.04), laeA (2.36), rasB (4.97), medA (4.25), glfA (2.56), pacC (3.60), sidG (7.06), hapX (2.71), calA (7.44), zrfC (5.23), aspf13 (8.65), cpcA (2.46), gel2 (2.35) and mscA (2.63). Furthermore, numerous genes also contained high-scoring sequences likely to form quadruplexes, which were potentially composed of three or more G-tetrads; including, zafA, pes1, laeA, pkac1, hasA, pacC, lysF, kre2, zrfC, hscA, mcsA, atrR, cyp51A, cyp51B, abcC, atrF and hmg1  and were shown to have QGRS scores of around 21, 42 or 63 . All the PQS-containing virulence genes contained at least one sequence scoring higher than 20, with many sequences scoring between 29 and 42 thus, very likely to form quadruplex structures, at least in vitro .The highest scoring potential quadruplex-forming sequences for each of these genes were then re-analysed in an alternative PQS predictive algorithm called QGRS Mapper. The scores of sequences known to form quadruplexes were compared to scores of the PQS identified in A. fischeri is one of the closest genetic relatives of A. fumigatus and around ~90 % of the proteome is shared between the two species [A. fumigatus is regularly isolated from patients, only a few known cases of human infection with A. fischeri have ever been reported. Indeed, A. fischeri has been shown to be far less virulent compared to A. fumigatus in several murine models, but interestingly, A. fischeri appears to intrinsically display reduced susceptibility to azoles [A. fumigatus genes could be found in A. fischeri. Therefore, the PQS frequencies in orthologous genes in A. fischeri were quantified. Only quadruplex frequencies in the coding regions were compared between species, as other genetic regions of the A. fischeri orthologues are poorly understood. species . Howevero azoles . FurthersidG, aspf13, cpcA, gliZ, gliG, gliK, gliT and gliH were higher in A. fumigatus compared to A. fischeri. Conversely, the PQS frequencies for laeA, sidA, medA, glfA, sidD, cat1, lysF, rodA, kre2, rasA, atrF, hmg1 and gliI were higher in A. fischeri compared to A. fumigatus . Moreover, A. fischeri orthologues of hmg1, pkaC1, hasA, glfA, kre2 and hscA also do not share the high-scoring PQS that were found in A. fumigatus. The implication of these differences to the functions of these organisms, and whether the altered PQS frequencies are associated with virulence, are currently unknown. However, future studies investigating these differences might unveil how these species differ in their pathogenicity.Considering the genetic similarity between the two species and high sequence similarities in orthologues of the virulence genes under investigation (average similarities of 96.4 % and average sequence coverage of 99 %), the distribution of the PQS between the two appeared quite different . The PQSA. fumigatus, their potential involvement in key biological processes, and discussed their presence in genes associated with virulence and drug resistance.In this study, we computationally predicted PQS within the genomes of fungi. We made several important observations. This was the first study to investigate the frequency of PQS amongst numerous genetically distinct fungal species. Moreover, we highlighted the genomic location of PQS in Escherichia coli contain around 0.028 PQS/kbp [Saccharomyces spp.) and as suggested for bacteria, it is possible that G4s may also have been deselected through evolution in ascomycete yeasts [Cryptococcus spp. and Malassezia spp. Cryptococcal species such as C. wingfieldii, C. amylolentus and C. floricola possessed some of the highest PQS frequencies in the study. Alternatively, all the Malassezia species investigated had much lower PQS frequencies than expected, which was surprising given their genomes were GC rich compared to many other fungi (>55 % GC content). We also identified many PQS within genes, and mRNA of A. fumigatus, but a higher frequency of PQS within the repeat regions and mRNA. It has previously been observed that S. cerevisiae promoters and open reading frames are enriched with sequences capable of potentially forming intramolecular G4s [et al., also demonstrated that inverted repeats were enriched in the centromeres and rDNA/rRNA regions, and G4s were enriched in the telomeres and tRNAs of S. cerevisiae [The frequency of PQS throughout genomes is highly variable; for example, human genomes were shown to contain around 0.228 PQS/kbp, whereas the genomes of  PQS/kbp . In thise yeasts . Howeverular G4s . A recenrevisiae .Candida species or within the Saccharomycotina. Interestingly, loss of PQS has recently also been observed in pathogenic Coronaviridae [Aspergillus and Cryptococcus species, this loss of PQS might also be linked with evasion of the immune response. Furthermore, the low PQS frequency observed within Candida and Malassezia species might be associated with their ability to form commensal relationships and avoid stimulating a host response. The importance of quadruplex stabilization may therefore be situational and could be detrimental or beneficial to the fungus depending on the environment. It is known that fungal pathogens such as A. fumigatus and C. neoformans can survive the acidic conditions within intracellular organelles to propagate infection [Notably, the genomes of pathogenic aspergilli and cryptococci contained significantly lower PQS frequencies relative to their non-pathogenic/infrequently pathogenic counterparts. Conversely, there was no correlation between PQS frequency and pathogenicity between aviridae . The exaaviridae . Therefoaviridae . In thisnfection . TherefoA. fumigatus was one of the lowest amongst the aspergilli, the average PQS frequencies in genes involved in virulence and gliotoxin synthesis, or those upregulated in response to conditions such as hypoxia, iron limitation and oxidative stress, appeared to be much higher than the average genome frequency. GO-term enrichment analysis implicated potential roles for PQS-containing genes in functions associated with virulence in pathogenic fungi such as zinc-ion binding [Although the overall genome PQS frequency in  binding , ribofla binding , melanin binding  and seco binding , metallo binding , and the binding . MoreoveC. neoformans and melanization [S. cerevisiae, humans and bacteria were also primarily associated with metabolism [A. fumigatus and implicates an important role for quadruplexes in metabolism throughout different organisms. Thus, the enrichment of PQS within these genes is suggestive of the potential importance of quadruplexes in the regulation of key biological functions and stress responses, and it would be interesting to explore the effects quadruplex-targeting ligands could have on these processes.It is widely acknowledged that metabolism in fungi is central to its virulence, pathogenicity and survival . The abinization . Moreovenization . Not onlnization . Furthertabolism . These dhsdS, recD and pmrA genes of S. pneumoniae, and var genes of P. falciparum can modulate host–pathogen interactions [espK, espB and cyp51 from Mycobacterium tuberculosis with the G4/iM-binding ligand TMPyP4 could inhibit transcription of these essential virulence genes [S. aureus and vancomycin-resistant Enterococcus spp. [C. albicans and C. neoformans [There are now an ever-increasing number of G4s identified within genes linked to microbial pathogenicity. G4-forming motifs located in the ractions . Moreovece genes . Bindingce genes . Interesus spp. , and anooformans .3+L1-12) were also found in important genes implicated in azole resistance  and virulence . Many of which were not found to be shared with the close non-pathogenic relative, A. fischeri. Mutations in cyp51A are a major contributing factor to azole resistance, but there is growing evidence that mutations in cyp51B and the involvement other members of the ergosterol synthesis pathway contribute to this resistance phenotype [laeA (encoding a master regulator of secondary metabolite biosynthesis) and pacC (encoding a pH-responsive regulator), regulate key virulent processes, such as gliotoxin production and host-cell adaption, respectively [in vivo and if they are implicated in regulating their transcription, they could pose an interesting target to modulate fungal pathogenic processes, as observed for other pathogenic microorganisms, such as viruses.Notably, sequences highly likely to form quadruplexes (Ghenotype . Moreoveectively . Thus, iin vivo and are involved in regulating transcription and biological processes in fungi, then they could have the potential to represent novel antifungal targets. Understanding whether quadruplexes could form under pathophysiological conditions, whether fungi themselves could release quadruplex-binding ligands to modulate host responses, or identifying whether there are fungal specific quadruplex-forming sequences could also be important factors to consider. Thus, targeting quadruplexes in fungi could represent a novel antifungal target, although experimental validation is necessary.Here, we provide a representative overview of potential quadruplex-forming sequences across the fungal kingdom and discuss their potential implications in fungal virulence and drug resistance. If these structures form Click here for additional data file.Click here for additional data file.


Pholiota carbonaria, and a moss, Polytrichum commune, were visually examined under laboratory conditions. Fungal appressoria were visualized on germinating spores and living protonemata within two weeks of inoculation in most growth chambers. Appressoria were pigmented, reddish gold to braun, and with a penetration peg. Pigmented, reddish gold to braun fungal hyphae were associated with living tissue, and numerous mature rhizoids contained fungal hyphae at six months. Inter-rhizoidal hyphae were pigmented and reddish gold to braun, but no structures were visualized on mature gametophyte leaf or stem tissues. Based on our visual evidence and previous work, we provide additional support for P. carbonaria having multiple strategies in how it obtains nutrients from the environment, and provide the first visual documentation of these structures in vitro.Wildfires play a critical role in maintaining biodiversity and shaping ecosystem structure in fire-prone regions, and successional patterns involving numerous plant and fungal species in post-fire events have been elucidated. Evidence is growing to support the idea that some post-fire fungi can form endophytic/endolichenic relationships with plants and lichens. However, no direct observations of fire-associated fungal–moss interactions have been visualized to date. Therefore, physical interactions between a post-fire fungus,  Wildfires play a critical role in shaping ecosystems, maintaining biodiversity, and influencing ecological processes in fire-prone regions . Early pAnthracobia spp., Ascobolus spp., Morchella exuberans Clowez, Hugh Sm. and S. Sm., Peziza echinospora P. Karst., Plicaria spp., Psathyrella pennata (Fr.) A. Pearson and Dennis, Pholiota carbonaria (Fr.) Singer, Pyronema spp., and Rhizina undulata Fr. are some species that require fire to fruit. Fungal species with enhanced fruit body production after a fire include Hygrocybe conica (Schaeff.) P. Kumm., Mycena galericulata (Scop.) Gray, Sphaerosporella spp., and Thelephora terrestris Ehrh. [Post-fire fungi are categorized into two main categories based on their response to fruiting in burnt habitats : (1) fruis Ehrh. .Bryum argenteum Hedw., B. sauteri Bruch and Schimp., Campylopus introflexus (Hedw.) Brid., Ceratodon purpureus (Hedw.) Brid., Funaria hygrometrica Hedw., Polytrichum juniperinum Hedw., and the post-fire liverwort, Marchantia polymorpha L. [B. argenteum, C. purpureus, and F. hygrometrica typically appear around six weeks after a fire, whereas M. polymorpha typically appears after several months [P. carbonaria, basidiomata of this species start to appear about five months after a fire event and continue fruiting for at least 18 months [Similar to post-fire fungi, there are species of mosses and liverworts that rapidly colonize burnt areas after a forest fire. They are often the first species to colonize burnt substrates, especially after intense fires, and their role at the beginning of succession should not be underestimated. These include, but are not limited to, orpha L. ,7. Juvenl months . There i8 months , placingAnthracobia, Geopyxis, and Pyronema, suggesting an important relationship between bryophytes and some post-fire fungi taxa.In times between fire events, studies have indicated that some post-fire fungi remain in the soil as active saprobes  or dormaPholiota carbonaria (syn. P. highlandensis) is a member of the family Strophariaceae, order Agaricales, and is a well-known post-fire fungus [Pinus and Taxus), bryophytes, and lichens [Pholiota carbonaria has been previously isolated into pure culture  from sudium sp. , and as dium sp. . Due to P. carbonaria, and the moss, P. commune, and discuss the environmental relevance of this interaction.Therefore, although there is currently ample evidence to support that some pyrophilous fungi can associate with bryophytes, there is a lack of visual documentation as to the extent (structures and location) of these specific fungal–moss interactions. Consequently, this study aimed to visually document the physical interactions (structures produced) in vitro between a post-fire fungus, P. carbonaria cultures from surface-sterilized moss tissues and from basidiomata collected from the Great Smoky Mountains National Park  mixed per manufacturer instructions and one mL L-1 of this orchid fertilizer solution was added to the BCD medium before autoclaving. Ferrous sulfate was not added due to its inclusion within the orchid fertilizer.Modified BCD medium  was heatPolytrichum moss spores were obtained from Carolina Biological Supply (item # 156661). Each growth chamber was inoculated with moss spores under a laminar flow hood. The outer surface of moss spore vials were wiped down with 100% ethanol before opening, Polytrichum spores were allowed to fall onto the medium surface in a fine layer, recapped, and set by a windowsill exposed to both partial direct and partial indirect light at 22 °C. After one week, growth chambers containing Polytrichum spores were visually inspected for bacterial and fungal contamination. Four growth chambers were randomly selected, and one corner was inoculated with one 4 × 4 mm agar plug containing actively growing P. carbonaria mycelia. This was repeated for 4 weeks to allow four different stages of Polytrichum development to be exposed to P. carbonaria. Negative controls consisted of growth chambers without Polytrichum spores and uninoculated Polytrichum spores. Polytrichum, species level identification, was determined at the end of the experiment to be P. commune, the most widely distributed Polytrichum species in the eastern United States [d States , based oPolytrichum spores was excised and examined for the presence of fungal–host interactions. Staining procedures using lactophenol cotton blue, trypan blue, and acidified trypan were used in an attempt to visualize the resulting structures [Light microscopic evaluation was conducted using an Olympus BX51 microscope with differential interference contrast (DIC) equipped with an Olympus DP22 camera. Images were captured and measured using Olympus cellSens standard software version 1.18 . Each week a small piece of medium with germinated ructures  better. ructures .Polytrichum gametophytes from inoculated and uninoculated control growth chambers were surface sterilized following a modified version of [Six-month-old rsion of . In shorg for 2 min, and 5 μL of the resulting supernatant was added to 495 μL 100 mM Tris-HCl buffered with NaOH to pH 8.5–8.9 , 1 μL of each 10 μM primer ITSIF and ITS4 [® SV Gel and PCR Clean-Up System (Promega). A BigDye® Terminator 3.1 cycle sequencing kit  was used to sequence the ITS in one direction using the ITS5 primer [P. carbonaria (GenBank number MT307288) [Fungal DNA was extracted by grinding fresh mycelium in 200 μL 0.5 M NaOH. The extract was centrifuged at 16,873× solution ). PCR waand ITS4 ,33, 3 μL5 primer  on an ApP. commune germinating spores showed numerous hyphae of P. carbonaria in close proximity to germinating spores and protonemata. Hyphae of P. carbonaria had prominent clamp connections. No noticeable difference with inoculation time was perceived. Fungal appressoria were visualized on germinating spores and living protonemata  original inoculants. No fungi were observed within negative controls and P. carbonaria was not isolated from uninoculated spore controls. In two months, several fruit body initials and mature P. carbonaria basidiomata were produced with viable basidiospores capable of generating new cultures  and was found in a large number  of surface-sterilized non-necrotic moss and lichen tissues [ tissues . Althoug tissues  suggest The historical understanding of fungal–moss associations appears to vary within the literature. Pressel et al.  reportedGalerina paludosa (Fr.) Kühner and Sphagnum capillifolium (Ehrh.) Hedw.  Schrank). Moreover, it was concluded that they form a parasitic relationship. Redhead [Species within the Basidiomycota have also demonstrated the ability to form associations with bryophytes, including parasitic/pathogenic , and sap Redhead  visualiz Redhead . Further Redhead . They foG. paludosa infects only the initial filaments from germinating spores, this relationship may be a pioneering life-history strategy or that G. paludosa attacks immature gametophytes before the host can mount a resistance. Based on the current visual evidence and previous work, P. carbonaria has plasticity in its nutritional relationship [P. commune, which is not uncommon among fungal species [G. paludosa but with unapparent and asymptomatic infection. Furthermore, P. carbonaria is also likely saprobic within the dead tissue layers through the invasion of dead rhizoids over time and is capable of growing independently in vitro on malt extract and potato dextrose [P. carbonaria was previously isolated from surface-sterilized non-necrotic leaf and stem tissues from several moss species.Our study visualized what appeared to be appressoria with penetration pegs b–d occurtionship  with P.  species . The timdextrose ,18. AlthPholiota. Chen et al. [Pholiota from eastern North Carolina from culture-dependent studies of the moss Dicranum scopiarum Hedw. ITS sequence data of these Pholiota isolates indicated that they were P. castanea, another post-fire Pholiota [P. spumosa, a non-post-fire species of Pholiota. Raudabaugh et al. [P. carbonaria from surface-sterilized moss samples collected from recently burned sites in the Great Smoky Mountains National Park. The moss species from which these isolates were obtained included one fire-response moss, Funaria hygrometrica Hedw., and several common moss species such as Ceratodon purpureus (Hedw.) Brid., Ditrichum pallidum (Hedw.) Hampe, and P. commune. Another species, Pholiota castanea, was also identified from Atrichum angustatum (Brid.) Bruch and Schimp. [Pholiota peleae E. Horak and Desjardin was described from Hawaii, and although not a post-fire Pholiota species, this species was initially described from bryophyte covered bark of living or dead Metrosideros polymorpha Gaudich [Cibotium glaucum (Sm.) Hook. and Arn. (a tree fern). Pholiota henningsii (Bres.) P.D. Orton is considered sphagnicolous, inhabiting or growing on Sphagnum, and is found rarely in several European countries [Pholiota species likely break down lignin-like polymers and is not considered to parasitize Sphagnum species [Pholiota–bryophyte interaction, several Pholiota species were found in biological soil crusts  in glacier forelands in Southern Norway [Pholiota species have many life-history strategies that allow them to occupy a wide variety of poor nutrient habitats.Fungal–moss associations may be common within the genus n et al.  detectedPholiota  and P. sh et al.  isolated Schimp. . Pholiot Gaudich  in additountries . It is b species . Finallyn Norway , suggestPholiota associate with mosses in nature. Notably, the broad range of moss species that P. carbonaria and other fire-associated fungi have been identified with in nature, along with the current in vitro observations of this association, suggest that the fungi–moss interaction may provide a more favorable environment  than the surrounding soil. Depending on the fire intensity, burnt soil is low in some nutrients and has altered soil physical properties [These previous studies and our current visual confirmation provide additional evidence that some fire-associated and non-post-fire species of operties , includioperties  demonstrP. carbonaria can form appressoria with penetration pegs, and invade mature rhizoid in vitro on P. commune and suggest that this post-fire species has multiple strategies in how it obtains nutrients from the environment. Future analyses of stable carbon and nitrogen signatures would be of value in fully understanding the nutritional relationship, electron microscopy visualization would be beneficial in further refining the interactions between P. carbonaria and P. commune, and a long-term study is needed to determine the temporal stability of this association. Although additional research is needed to understand this relationship fully, this study suggests that early post-fire colonizers may have more interconnected life histories than previously thought, which is essential when considering the contribution of post-fire fungi and bryophytes to post-forest fire system recovery.In summary, based on visual evidence and previous work, we show that


Staphylococcus aureus, as a proof of principle in examining the promise and limitations of statins in recalcitrant infection.An emergent approach to bacterial infection is the use of host rather than bacterial-directed strategies. This approach has the potential to improve efficacy in especially challenging infection settings, including chronic, recurrent infection due to intracellular pathogens. For nearly two decades, the pleiotropic effects of statin drugs have been examined for therapeutic usefulness beyond the treatment of hypercholesterolemia. Interest originated after retrospective studies reported decreases in the risk of death due to bacteremia or sepsis for those on a statin regimen. Although subsequent clinical trials have yielded mixed results and earlier findings have been questioned for biased study design, in vitro and in vivo studies have provided clear evidence of protective mechanisms that include immunomodulatory effects and the inhibition of host cell invasion. Ultimately, the benefits of statins in an infection setting appear to require attention to the underlying host response and to the timing of the dosage. From this examination of statin efficacy, additional novel host-directed strategies may produce adjunctive therapeutic approaches for the treatment of infection where traditional antimicrobial therapy continues to yield poor outcomes. This review focuses on the opportunistic pathogen,  Staphylococcus aureus colonizes approximately 30% of the human population, yet colonized individuals typically remain asymptomatic [S. aureus is also an opportunistic pathogen and is the causative agent in life-threatening infections associated with high morbidity and mortality. Wisplinghoff et al. reported S. aureus as the second leading cause of bacteremia in hospitals in the United States, exceeded only by coagulase-negative Staphylococcus species [S. aureus as the leading cause of infective endocarditis worldwide [S. aureus infections. Noskin et al. reported a 5-fold increase in the risk of in-hospital death for S. aureus infection compared to non-S. aureus infection [S. aureus bloodstream infections are associated with mortality [S. aureus pneumonia [S. aureus (MSSA) bloodstream infections [ptomatic ,2. Howevorldwide . High moneumonia . High mofections . Thus, tS. aureus strains are an important cause of chronic, relapsing infection, especially notable in cystic fibrosis [S. aureus is an initial isolate identified in the colonization of the respiratory tract of cystic fibrosis patients, as indicated by Armstrong et al., where 66.6% of infants less than 6 months old with cystic fibrosis had lower respiratory infections caused by S. aureus [S. aureus infection persists includes findings from Schwerdt et al. showing 61% of cystic fibrosis patients chronically infected with S. aureus for more than 50% of a 22-year observation period [S. aureus strain for 12–28 months [S. aureus per year from 56.2% in 2002 to 70.2% in 2019 [S. aureus (MRSA) infection (24.6%). Thus, in addition to acute pathogenesis, both MSSA and MRSA represent an important cause of severe, chronic infections associated with high mortality.Invasive fibrosis ,9. S. au. aureus . Evidencn period . Persist in 2019 . Of thesS. aureus had been characterized as an extracellular pathogen; yet more recent evidence indicates a clear role for host cell invasion in pathogenesis. Multiple factors determine the fate of invasive S. aureus and the contribution to pathogenesis. The response appears dependent on factors of both the host cell and of the invasive strain. One potential fate is the host cell killing of invading bacteria via the host endocytic pathway [Conventionally,  pathway . During S. aureus survives inside macrophages by preventing the formation of the host lysosome [S. aureus can reside within the host cell, protected from antimicrobial therapy, as indicated in the report by Lehar et al., where intracellular S. aureus was found to establish infection in a mouse model despite intravenous treatment with the antibiotic vancomycin [S. aureus [S. aureus persisting for weeks within host cells in vivo and in vitro, suggesting the insufficiency of the host immune response in clearing intracellular infection. Thus, microbial factors can drive persistence through alteration of the host response.However, invasive staphylococcal strains have been identified that exhibit the capacity to evade lysosomal destruction. Tranchemontagne et al. found that lysosome . By evadncomycin . Krut et. aureus . Invasio. aureus . TuchschS. aureus can persist within the host cell as latent bacterial populations [S. aureus invasion into human peritoneal mesothelial cells induces cell death initially via apoptosis followed by necrosis [S. aureus invasion into bovine mammary epithelial cells induces apoptosis as noted by DNA fragmentation and morphological changes associated with apoptosis [S. aureus can lead to the release of viable bacteria into the extracellular space. These newly released bacteria can then begin a chronic cycle of relapsing infection by infecting neighboring host cells [S. aureus invasion into circulating immune cells provides protection and transport via the bloodstream to initiate infection at secondary sites [S. aureus contributes to pathogenesis through apoptotic and necrotic host cell death, resultant tissue damage, and the unleashing of intracellular bacterial populations to spread infection into previously sterile sites.Invasive ulations ,19,20. Dulations ,22. Haslpoptosis . Ultimatst cells . S. aurery sites ,24,25. TS. aureus have been identified: wildtype and a phenotype that develops from and can revert to the wildtype phenotype: small colony variants (SCVs) [S. aureus, SCVs of S. aureus exhibit a slow growth rate and diminished susceptibility to first-line antibiotics oxacillin or vancomycin [S. aureus induces the expression of CCL5 and CXCL10, genes involved in the immune response [S. aureus infection. SCVs of S. aureus do, however, exhibit an increase in the expression of bacterial adhesion molecules used to invade host cells [S. aureus of fibronectin-binding protein A (FnBPA), an adhesin important for staphylococcal attachment to the host cell [S. aureus exhibit the increased expression of bacterial FNBPs and bacterial clumping factor A (CflA) and an associated increase in adhesion to host fibrinogen and host fibronectin, which are important for invasion [Clinically, two distinct phenotypes of s (SCVs) ,19,26,27ncomycin ,29. Infest cells ,30. Tuchost cell . VaudauxS. aureus has been recovered from epithelial cells two weeks after infection [S. aureus can persist in their new cellular niche for extended periods, and upon release, these variant bacteria, expressing high levels of adhesion molecules, rapidly infect neighboring cells [Both phenotypes can persist intracellularly. Viable wildtype nfection , from kenfection , and frong cells . Taken tS. aureus virulence factors involved in adhesion and invasion [S. aureus surface proteins that mediate binding to host extracellular matrix (ECM) components, including fibrinogen, collagen, and fibronectin. Fibronectin-binding proteins (FnBPs) are members of the MSCRAMM family that bind, with high affinity, to host fibronectin. The major mechanism of S. aureus invasion into host cells utilizes FnBPs [S. aureus mutants that do not express FnBPs lose their invasive ability [Staphylococcus carnosus and Lactococcus lactis, confers invasiveness [The microbial surface component recognizing adhesive matrix molecule (MSCRAMM) family of proteins is the largest class of invasion . The MSCes FnBPs ,32,33. SS. aureus bind to host fibronectin, which then binds to the host cell integrin receptor α5β1  [3, including the binding of PIP3 to α-actinin, dislodging α-actinin from actin and enabling the reordering of actin subunits [During membrane   . Downstrsubunits .S. aureus, indicating a role for PI3K catalytic activity in host cell entry [S. aureus [The expression of kinase-dead PI3Kp110α diminishes host cell invasion by ll entry . Supportll entry . Moreove. aureus . Based oS. aureus at reduced concentrations relative to lipophobic antibiotics [S. aureus by three days post-infection compared to the persistence of viable intracellular bacteria at six days post-infection in clindamycin-, linezolid-, and azithromycin-treated samples [Efficacy in killing intracellular infection is limited for first-line, hydrophilic antibiotics that exhibit limited permeability across the host cell membrane . Rifampiibiotics . Krut et samples . Thus, rRifampin prevents bacterial protein synthesis by binding to and inhibiting bacterial RNA polymerase (RNAP) . BacteriC. difficile isolates were resistant to rifampin [Mycobacterium tuberculosis isolates from the Qaem Hospital were rifampin-resistant [S. aureus infections, including evidence reported by Zhou et al., where 31.1% of S. aureus isolates, over a twelve-month period, were rifampin-resistant [Although rifampin can penetrate host cells and kill intracellular bacteria, treatment is associated with rapid resistance development and cross-resistance to other antibiotics ,57,58,59rifampin . Tajbakhesistant .rpoB gene, which encodes the β subunit of bacterial RNAP, confer resistance to rifampin [rpoB gene have all been implicated in resistance development [rpoB gene produce an altered β subunit that is still able to function in the core enzyme of RNAP but has a decreased affinity for rifampin [rpoB gene also confer cross-resistance to vancomycin and daptomycin, two last-line antibiotics for treating S. aureus infections [rpoB mutations are associated with a thickening of the cell wall, although the mechanism of thickening remains to be fully characterized [2+ and these calcium–daptomycin complexes insert into the plasma membrane of the bacteria [rpoB mutations, may serve as an obstacle for the penetration of the large daptomycin–calcium oligomers to the cell membrane [Mutations in the rifampin ,56. Singelopment ,54,61. Mrifampin . Interesfections . rpoB mucterized ,63,64. Icterized . This bicterized . The redbacteria . The insmembrane . Cross-rRifampin treatment is associated with severe adverse side effects ,68,69. HS. aureus strains in vitro [S. aureus strains in vitro [S. aureus relative to monotherapy with any of the three antibiotics [Due to the development of rifampin resistance and adverse side effects, rifampin is commonly administered with other antibiotics . Rifampiin vitro . Additioin vitro . Contrarin vitro ,74. Greiibiotics . DworkinS. aureus, to develop antibiotic resistance, an emerging approach is to target the host rather than promote resistance by targeting the bacterium [S. aureus bloodstream infections than non-statin users [Given the propensity of bacteria, especially acterium ,41. One acterium ,80,81,82in users . Almog ein users . Björkhein users . The proHowever, in a series of randomized clinical trials, statin’s efficacy in treating sepsis or acute respiratory distress syndromes was not supported ,84,85, cStatins inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in cholesterol biosynthesis  89,90,9,990,91. In addition to cholesterol lowering, statins yield cholesterol-independent effects, also known as pleiotropic effects. Statin inhibition of HMG-CoA reductase diminishes synthesis not only of cholesterol, but also of hydrophobic isoprenoid intermediates Fpp and geranylgeranyl pyrophosphate (GGpp) that form from Fpp. Statin pleiotropic effects in part are due to decreased synthesis of these intermediates. These long hydrophobic molecules are modified and attached covalently through the process of post-translational prenylation of proteins containing the conserved CaaX domain . In thisStatins interrupt specific stages of host cell invasion through non-cholesterol-dependent pleiotropic effects. The centrality of isoprenoid depletion in these effects is evidenced by the restoration of invasion during simvastatin treatment if Fpp or GGpp are replenished. Invasion was not restored by replenishing cholesterol  with thi3. Evidence of this disruption is that simvastatin treatment limits actin stress fiber disassembly, the endocytic process dependent on PIP3 binding α-actinin.Although CDC42 remains sequestered within the cytosol, simvastatin stimulates GTP-loading within the CDC42 activation site . In the S. aureus invasion into host cells through decreased synthesis of isoprenoid intermediates, sequestration of RAC, RHO, and CDC42 in the cytosol, decreased membrane localization of these small-GTPases coupled to PI3Kp85α, reduced actin stress fiber depolymerization, decreased host cell binding to fibronectin, and decreased internalization and recycling of β1-integrin receptor complexes to the host cell surface.Simvastatin reduces host cell binding to fibronectin . MoreoveS. aureus [S. aureus and decreases lethality. Simvastatin blunts pulmonary pathogenesis and the inflammatory response to infection, in addition to lowering markers of the inflammatory response both within lung tissue and systemically. Thus, in vivo evidence supports the potential efficacy of statin use for limiting pulmonary infection.In vivo, simvastatin treatment aids in clearing pulmonary infection by invasive . aureus . Similar. aureus ,98, simvAlthough pleiotropic effects of statins include inhibition of infection, the use of statins in the critically ill has been challenged due to altered pharmacokinetics within this patient population that renders statins more toxic ,102,103.S. aureus [S. aureus invasion [Alternative small molecule inhibitors have been examined that might limit host cell invasion yet circumvent adverse effects and limitations associated with statins. In characterizing the underlying mechanism of simvastatin, RAC, RHO, and CDC42 had emerged as potential molecular targets central to host cell invasion by . aureus . Earlierinvasion  and CDC4invasion . We therinvasion . To examinvasion .-/- mouse embryonic fibroblasts (MEF) [ML141 differs from simvastatin in its target and mechanism of action. While simvastatin demonstrates specificity for HMG-CoA reductase at the early, rate limiting step of cholesterol/isoprenoid biosynthesis and thereby indirectly decreases the prenylation of CDC42, RAC, and RHO ,106,107,ts (MEF) ,109. Thets (MEF) ,40,43,46Streptococcus pyogenes, a Gram-positive invasive bacterium that shares the fibronectin/integrin invasion mechanism used by S. aureus [ML141 decreases host cell invasion by MSSA and MRSA strains [. aureus . SimilarGiven that simvastatin and ML141 decrease the number of bacteria invading host cells and rifampin can kill intracellular bacteria, it was hypothesized the use of rifampin with simvastatin or with ML141 could decrease the number of intracellular bacteria with greater efficacy than rifampin alone . TherefoTo test this hypothesis in vitro, host cells were cotreated with simvastatin and rifampin or with ML141 and rifampin. Interestingly, the response to simvastatin diverged from the response to ML141 . As antiIt is plausible that differential host cell responses are contributing factors in the divergence. Host cell membrane integrity remained intact in response to ML141 yet decreased in response to simvastatin . CotreatS. aureus is an important cause of severe, life-threatening infections and chronic, recurrent infections associated with high morbidity and mortality. Infections are challenging to treat due to multiple factors, including the propensity of S. aureus to develop antibiotic resistance and the ability of invasive strains to reside within host cells, protecting the invading bacteria from host immune defenses and antibiotic therapy. Rifampin, although able to penetrate host cells and kill intracellular bacteria, is associated with the development of rapid resistance and with adverse side effects. Emerging evidence indicates the usefulness of alternative strategies that enhance clearance of intracellular infection through host rather than bacterial-directed mechanisms. Statin pleiotropic effects include host-directed protection during non-systemic infection, especially when an ongoing statin regimen can be achieved. However, the novel host-directed small molecule inhibitor ML141 may provide an alternative strategy that circumvents statin limitations by selectively targeting CDC42, a key host cell-signaling molecule implicated in invasive infection. Cotreatment strategies combining host-directed therapeutics with antimicrobials may aid in reducing intracellular bacterial infection and the likelihood of developing antibiotic resistance. Evidence of unintended host responses to both host-directed and antimicrobial therapeutics indicates the complexity of adjunctive therapeutic development. The ongoing challenge of treatment failures in infectious disease necessitates continued expansion of such alternative strategies.


Childhood-onset hypertrophic cardiomyopathy (HCM) is far less common than adult-onset disease, thus natural history is not well characterized. We aim to describe the characteristics and outcomes of childhood-onset HCM.adj 2.39 (1.36–4.20), P = 0.003] and 67% increased risk of the overall composite outcome . When compared with adult-onset HCM, childhood-onset was 36% more likely to develop life-threatening ventricular arrhythmias [HRadj 1.36 (1.03–1.80)] and twice as likely to require transplant or ventricular assist device [HRadj 1.99 (1.23–3.23)].We performed an observational cohort study of 7677 HCM patients from the Sarcomeric Human Cardiomyopathy Registry (SHaRe). Hypertrophic cardiomyopathy patients were stratified by age at diagnosis  and assessed for composite endpoints reflecting heart failure (HF), life-threatening ventricular arrhythmias, atrial fibrillation (AF), and an overall composite that also included stroke and death. Stratifying by age of diagnosis, 184 (2.4%) patients were diagnosed in infancy; 1128 (14.7%) in childhood; and 6365 (82.9%) in adulthood. Childhood-onset HCM patients had an ∼2%/year event rate for the overall composite endpoint, with ventricular arrhythmias representing the most common event in the 1st decade following baseline visit, but HF and AF becoming more common by the end of the 2nd decade. Sarcomeric variants were more common in childhood-onset HCM (63%) and carried a worse prognosis than non-sarcomeric disease, including a greater than two-fold increased risk of HF [HRPatients with childhood-onset HCM are more likely to have sarcomeric disease, carry a higher risk of life-threatening ventricular arrythmias, and have greater need for advanced HF therapies. These findings provide insight into the natural history of disease and can help inform clinical risk stratification.  Patients with HCM may experience atrial and ventricular arrhythmias and heart failure (HF); however, disease expression and severity are highly variable. Furthermore, there is marked diversity in the age of diagnosis. The estimated prevalence of HCM is 1:500 in the general adult population.Prevailing beliefs are that HCM typically develops during adolescence and that childhood-onset disease is associated with an increased risk of ventricular arrhythmias, but lower risk of HF and atrial fibrillation (AF). However, data to support these beliefs are limited as prior studies have been small, included a large proportion of infant-onset cardiomyopathy (diagnosed in the 1st year of life), and often included syndromic causes of cardiac hypertrophy, such as Friedreich’s ataxia, Noonan’s syndrome/RASopathies, and inborn errors of metabolism—all of which are distinct disorders from primary HCM.In this study, we aimed to describe the characteristics and outcomes of childhood-onset primary HCM in a large multicentre cohort. This study focuses on patients diagnosed between 1 and 18 years of age, describing adverse cardiac outcomes and risk predictors, determining the impact of pathogenic variation in sarcomeric genes, and characterizing how natural history differs in patients diagnosed with HCM during childhood vs. adulthood.The Sarcomeric Human Cardiomyopathy Registry (SHaRe) is an ongoing longitudinal registry study drawing from HCM referral centres across the USA, Brazil, Europe, and Australia. Data captured include historical events prior to SHaRe entry, baseline demographic and clinical data at first visit to a SHaRe site, genetic testing results, serial cardiac imaging results, and longitudinal cardiovascular outcomes. Each site provides updated data on a quarterly basis. A detailed description of SHaRe has previously been published.Patients included in this study carried a site-designated diagnosis of HCM. This was defined as maximal left ventricular wall thickness of at least 13 mm , myosin heavy chain (MYH7), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), α-tropomyosin (TPM1), myosin essential and regulatory light chains , and actin (ACTC). Sarcomeric variants with ambiguous classifications underwent additional systematic review by a subgroup of four investigators  to adjudicate and standardize classification., LAMP2) and α-galactosidase (GLA)] as these identify genocopies resulting in LVH and different disease states.Genetic testing was performed by sites as part of research protocols or clinical care. As previously described,The endpoints of interest included an HF composite, life-threatening ventricular arrhythmia composite, AF, and an overall cardiac event composite. The HF composite included left ventricular ejection fraction <35%, New York Heart Association (NYHA) functional class III/IV symptoms, cardiac transplantation, or implantation of a left ventricular assist device. The life-threatening ventricular arrhythmia composite included sudden cardiac death, resuscitated cardiac arrest, or appropriate implantable cardioverter-defibrillator therapy. The overall composite included components of the HF composite (excluding left ventricular ejection fraction <35%), components of the ventricular arrhythmia composite, and additionally AF, stroke, and all-cause death.2 test. Kaplan–Meier analyses were performed using the first occurrence of any component of the composite outcome of interest. Cox-proportional hazards regression model was used to generate hazard ratios (HRs) and 95% confidence intervals (CIs). A multivariable model including risk predictors selected based on biological importance in disease was performed on patients who had complete data available for all relevant variables. All reported P-values are two-sided. All analyses were performed using R version 3.5.4 .This analysis included patient data obtained through the 3rd quarter of 2019. All analyses were stratified by age at diagnosis, and time to first event was evaluated from initial visit at a SHaRe site forward. Analyses comparing sarcomeric to non-sarcomeric patients were limited to patients who underwent genetic testing. Continuous variables within each age group are presented as median and interquartile ranges and compared using Kruskal–Wallis test. Categorical variables are compared using a χFigure  shows the distribution of age at HCM diagnosis. Across the lifespan, there appear to be three predominant peaks when HCM is diagnosed: the first is within the 1st year of life (infancy), the second is in the teenage to early adult years, and the third is in mid-adulthood. To ensure the peak during the teenage years was not related to more frequent performance of cascade family screening, a proband-only sensitivity analysis was performed, and the pattern remained unchanged.There were 7677 HCM patients included in this cohort. Figure  highlights the association between age of diagnosis and genotype in the 4746 patients who underwent genetic testing. Although infants are less likely to have sarcomeric disease, patients with sarcomeric HCM are diagnosed an average of 9 years earlier than those with non-sarcomeric HCM (mean of 38 vs. 47 years of age). Sarcomeric HCM is more prevalent than non-sarcomeric until approximately age 50 years, when non-sarcomeric HCM becomes more prevalent. Focusing on sarcomeric HCM, MYH7 variants are the most common in infant- and childhood-onset HCM. The proportion of MYBPC3 variants increases with age at diagnosis, such that it is the most common by adulthood . A listing of all pathogenic and likely pathogenic variants is presented in Table 1. Patients with infant-onset HCM were the least likely to have a family history of HCM (P < 0.001) or have pathogenic/likely pathogenic sarcomere gene variants (P < 0.001). Conversely, patients with childhood-onset HCM were the most likely to have a family history  and have sarcomeric disease .Based on defined age of diagnosis strata, 184 patients (2.4%) were diagnosed in infancy (<1 year old), 1128 (14.7%) in childhood (ages 1–18 years), and 6365 (82.9%) in adulthood (>18 years old). Baseline characteristics for these three ages of diagnosis are displayed in Of the 184 patients diagnosed during infancy, morbidity and mortality were initially greater than in those diagnosed during childhood Figure S1Table 1). Of these, 64% were male, 49% had a family history, and 85% were probands. In the first decade after initial SHaRe visit, nearly 20% of patients had a cardiac event as reflected by the overall composite endpoint encompassing HF, ventricular arrhythmias, AF, stroke, and all-cause death . The mean age of diagnosis was similar in sarcomeric and non-sarcomeric HCM  and both groups were male-predominant . Patients with childhood-onset sarcomeric HCM had a 63% increased risk for the overall cardiac composite outcome compared with non-sarcomeric childhood-onset HCM  . The presence of a sarcomeric variant was associated with a 67% increased risk of overall cardiac composite outcomes  and greater than two-fold increased risk of HF . Age at diagnosis (within childhood) was the only significant predictor of AF and had only modest effect. Sex . Analysing patients from the time of first visit, those diagnosed in childhood were 36% more likely to develop life-threatening ventricular arrhythmias [HRadj 1.36 (1.03–1.80)] and twice as likely to require transplant or ventricular assist device [HRadj 1.99 (1.23–3.23)] than patients with adult-onset HCM. Given the broad age range encompassed in adult-onset HCM, we performed sensitivity analyses, dividing the adult population into early (age of diagnosis 19–40 years) and later adult-onset (age of diagnosis >40 years) cohorts. Findings remained consistent when comparing childhood-onset to both early and later adult-onset HCM . And finally, the natural history of sarcomeric HCM may differ depending on age of diagnosis with HCM.The goal of this study was to better characterize the natural history of childhood-onset HCM, particularly examining the impact of genetic background (sarcomeric vs. non-sarcomeric disease) and contrasting outcomes to adult-onset disease. We identified four main findings. First, there appear to be three distinct peaks when HCM is diagnosed over the lifespan: infancy, adolescence, and middle adulthood, with childhood-onset HCM being predominantly sarcomeric disease. Second, childhood-onset HCM is clinically different from both infant-onset and adult-onset HCM. Third, patients with childhood-onset HCM are at the highest risk for life-threatening ventricular arrhythmias and requiring advanced HF therapies , we found that the majority (63%) of childhood-onset HCM is due to sarcomeric disease. At 63%, this is a higher prevalence of sarcomeric HCM than previously identified in smaller cohorts or cohorts with lower utilization of genetic testing., , , , The association between age of HCM onset, genotype, and outcomes has clinical relevance. Overall, patients with sarcomeric HCM presented an average of 9 years earlier than patients with non-sarcomeric HCM and constituted the majority of patients diagnosed during the teenage years. Sarcomeric HCM appeared to be more common throughout young adulthood but continued to be relatively prevalent until the latter part of the 5th decade, before declining. Conversely, non-sarcomeric HCM was most commonly diagnosed after age 50, with peak age at diagnosis during the 6th decade of life, comprising the bulk of the 3rd peak in age of HCM diagnosis during middle adulthood. Sarcomeric variants have been associated with worse outcomes in HCM.This study has limitations inherent to observational studies using partially retrospective data, including missing data, and survival bias. Childhood-onset HCM is relatively rare, limiting power to detect differences between subgroups and identify predictors of outcomes. However, our study is the largest study of childhood-onset HCM to date,Patients with childhood-onset HCM have important differences in clinical characteristics, genotype, and cardiovascular outcomes. They are more likely to have sarcomeric disease, have a higher risk of ventricular arrhythmias, and a greater frequency of advanced HF than patients diagnosed during adulthood. These findings help improve patient stratification and underscore the need to better understand disease biology.European Heart Journal online.The SHaRe Registry data is not universally available but we encourage parties interested in collaborating to contact the corresponding author directly.Conflict of interest: N.A.M. reports involvement in clinical trials with Amgen, Pfizer, Novartis, and AstraZeneca without personal fees, payments, or increase in salary. L.H., E.A.A., A.C.P., C.S., S.D.C., and S.G.W. have nothing to disclose. I.O. reports grants and personal fees from MyoKardia, Genzyme, Shire, and Amicus; personal fees from Cytokinetics; grants from Bayer and Menarini International, outside the submitted work. S.M.D. reports grants from MyoKardia, during the conduct of the study. M.M. reports personal fees from MyoKardia, outside the submitted work. J.I. reports grants from MyoKardia, Inc. during the conduct of the study. D.J. reports grants from MyoKardia, during the conduct of the study; grants and personal fees from MyoKardia, personal fees from Cytokinetics, outside the submitted work. J.W.R. reports personal fees from Amgen, Abiomed, Novartis, Cytokinetics, MyoKardia, and Bayer, outside the submitted work. J.S.W. reports grants and non-financial support from the Wellcome Trust [107469/Z/15/Z], Medical Research Council (UK), British Heart Foundation, NIHR Royal Brompton Cardiovascular Biomedical Research Unit, and the NIHR Imperial College Biomedical Research Centre, during the conduct of the study; grants and personal fees from MyoKardia, outside the submitted work. S.S. reports personal fees from MyoKardia, Inc., from null, outside the submitted work. A.S.H. reports grants from MyoKardia, during the conduct of the study; personal fees from Tenaya Therapeutics, outside the submitted work. C.Y.H. reports grants and personal fees from MyoKardia, personal fees from Novartis and Tenaya, during the conduct of the study.ehab148_Supplementary_AppendixClick here for additional data file.


Neuroblastoma is a common childhood cancer possessing a significant risk of death. This solid tumor manifests variable clinical behaviors ranging from spontaneous regression to widespread metastatic disease. The lack of promising treatments calls for new research approaches which can enhance the understanding of the molecular background of neuroblastoma. The high proliferation of malignant neuroblastoma cells requires efficient energy metabolism. Thus, we focus our attention on energy pathways and their role in neuroblastoma tumorigenesis. Recent studies suggest that neuroblastoma-driven extracellular vesicles stimulate tumorigenesis inside the recipient cells. Furthermore, proteomic studies have demonstrated extracellular vesicles (EVs) to cargo metabolic enzymes needed to build up a fully operative energy metabolism network. The majority of EV-derived enzymes comes from glycolysis, while other metabolic enzymes have a fatty acid β-oxidation and tricarboxylic acid cycle origin. The previously mentioned glycolysis has been shown to play a primary role in neuroblastoma energy metabolism. Therefore, another way to modify the energy metabolism in neuroblastoma is linked with genetic alterations resulting in the decreased activity of some tricarboxylic acid cycle enzymes and enhanced glycolysis. This metabolic shift enables malignant cells to cope with increasing metabolic stress, nutrition breakdown and an upregulated proliferation ratio. MYCN, mutations of the ALK gene or germline mutations in SDHA and SDHB genes .,48.47,48activity . Proteomactivity ,7,35,39.activity ,7,35,39.activity .MYCN oncogene correlates with a poor diagnosis in onco-pediatric NB patients .,73.SDHA, in SDHB . Apart f in SDHB . Both mu in SDHB . The acc in SDHB ,74. Succ in SDHB ,74. Cyto in SDHB ,74. Cons in SDHB ,74.FH gene is known to promote renal cell tumorigenesis and also neuro-encephalopathy and early death in childhood (FH deficiency syndrome) . Ma. Ma69]. yndrome) ,81,82,83tropenia ,81,82,83utations . The FH tectable . A lowermutation  [5,69,74In this review, we discussed different molecular tools by which neuroblastoma controls energy metabolism in the microenvironment. We argued that NB derived exosomes support the transformation of healthy cells into malignant cells. We opened this review with the role of neuroblastoma-derived exosomes and closed it with known genetic mutations affecting the tricarboxylic acid cycle leading to a metabolic shift in cancer cells. A growing body of evidence has emerged indicating that energy metabolism determines the overall neuroblastoma progression.


Oxidative stress plays a dominant role in inflammatory skin diseases. Emerging evidence has shown that the close interaction occurred between oxidative stress and the gut microbiome. Overall, in this review, we have summarized the impact of oxidative stress and gut microbiome during the progression and treatment for inflammatory skin diseases, the interactions between gut dysbiosis and redox imbalance, and discussed the potential possible role of oxidative stress in the gut-skin axis. In addition, we have also elucidated the promising gut microbiome/redox-targeted therapeutic strategies for inflammatory skin diseases. Endogenous H2S is produced by l-cysteine catalyzed by cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS), while produced by β-mercaptopyruvate pyruvic acid in the presence of 3-mercaptopyruvate sulfotransferase (3-MST) in mitochondria might result in oxidative stress reactions. Additionally, these active substances are called reactive species, mainly including reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS), which have made stimulating effects on the progress of cell metabolism . In detamed skin . RSS hasochondria. At presochondria. Meanwhiochondria. The shoochondria.et al. and Xanthine oxidase (XO). Increased NADPH oxidase activity of neutrophils and macrophages subsequently produce large amounts of O2⋅−, which may contribute to the cytotoxic reactions during inflammatory skin diseases  and involucrin (IVN) in cultured human keratinocytes , while the average concentration of disulfide, SS/SH, SS/total SH, SH/total SH were significantly higher than those of healthy infants (p < 0.05), which suggested that the higher antioxidant defense level of AD infants . A high level of ROS was observed in skin biopsies from AD patients . Meanwhi infants . The env infants . Staphylduce ROS . Furtherduce ROS  and aggrduce ROS .et al. also showed that mtROS-dependent IL-1β is involved in exacerbating psoriatic inflammation, and total antioxidant capacity (TAC) in serum of psoriasis patients. Furthermore, Sorokin et al. found that assaying oxidation-modification of lipids revealed a significant association with oxidized LDL and oxHDL in psoriasis patients  cells (a type of DCs), which are the major cell types in the skin lesions of psoriasis, are the sites for the expression of inducible NOS . Moreoveet al. showed that inhibition of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC1-α) and enhanced carbonylation in melanocytes of vitiligo could lead to dysregulation of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase Sirtuin 3 , which led to mitochondrial severe dysfunction and apoptosis of melanocyte  and total antioxidant capacity (TAC), while increases in the levels of MDA and 8-OHdG. Moreover, these abnormal indexes about oxidative stress were correlated with activity and disease severity . The ROSlanocyte .2+ interference and translocation of calreticulin (CRT) may be the main pathways leading to melanocyte death. Other studies have shown that the UPR is activated during keratinocyte differentiation  , and Lachnospiraceae were associated with decreased risks of eczema   . Lee et < 0.001) . Recent < 0.001) . Meanwhi7, 1.00) . Marrs erollment . Furtherh cohort . A studyh cohort . Combiniet al. and carbohydrates (Prevotella)  . Based ovotella) . Dao et ted diet . Moreoveted diet  and multted diet  via gut E. coli, and finally increased levels of circulating trimethylamine N-oxide, which is a potentially harmful metabolite produced by the gut microbiome  can impair the gut barrier , change crobiome . In retuifficile . Moreovee health . A recene health . More onet al. found that high doses of iron can lead to oxidative stress in the body   levels, down-regulation of tight junction-related genes in the mucosa, and disturbance of cecal microbiota, indicating that particular diet increased not only oxidative stress levels in the body but also caused mucosal barrier damage and gut dysbiosis )  is closethe body . It has the body . A studyiomarker , is highve tract . Zhang e < 0.05) . Ge et aysbiosis .in vivo and in vitro studies have developed reliable dietary recommendations based on the fundamental mechanisms, such as abnormal activation of autoimmune, oxidative stress, and gut dysbiosis. In vitiligo, for example, a disorder of redox balance (an increase in hydrogen peroxide) enhances the process by which phenolic chemicals compete with tyrosine to produces reactive quinones. The reactive quinone acts as a covalent binding center of incomplete antigens to tyrosinase and produce new antigens. The micromole (non-cytotoxic) O-quinone also gains immunogenicity to induce an immune response (P. copri (significantly decreased in AD children) had been attributed mainly to the high consumption of complex carbohydrates. Furthermore, AD children with high sugar content in their diet had a significantly lower P. copri , suggested the protection role of diet-related gut microbes expression and the death of intestinal epithelial cells . As mentet al. have found that intestinal symbiotic bacteria genus Lactobacillus can induce intestinal phagocytes to produce ROS, dependent on NADPH oxidase 1(NOX1)  . Lee et al cells . Local RWe defined the gut microbiome as a ROS-stressed gut microbiome, which exists in oxidative stress. As previously described, local excess of ROS in the gut can be caused by diet ingestion or gut-specific bacteria. There are preliminary findings in the microbiome and inflammatory skin diseases. For example, the detection of intestinal bacterial DNA in the blood of patients with psoriasis may indicate the presence of intestinal barrier dysfunction and bacterial translocation. Further study requires whether patients with other inflammatory skin diseases have similar intestinal characteristics.p = 0.002) , and celiac disease (CD) are complications . A 10-ye= 0.002) . CD is a= 0.002) . In a st= 0.002) , a case-= 0.002)  atopic d= 0.002)  in patieClostridioides difficile infection , and live biological agents (LBPs), et al. . Many minfection .Bifidobacterium longum HK003. 89% of the patients in the treatment group showed significant improvement or complete recovery of their skin lesions , macromolecular organic compounds . However, the treatment of some inorganic compounds needs specific drug delivery conditions to ensure the stability of drugs and the therapeutic effect.With the development of oxidative stress-related research in inflammatory skin diseases, antioxidant therapy is widely studied. Traditional antioxidant therapy studies include oral or topical treatment of plant/animal biologically active components  production , organic isothiocyanates, et al. , a neuropeptide, reduced the proliferation of dermal fibroblasts and keratinocytes in a dose-dependent manner. Moreover, the OXTR knock induced ROS levels and decreases in glutathione  transduced mesenchymal stem cells (MSCs) to treat AD mice. Moreover, they found this approach effectively inhibits the inflammatory response subcutaneously . Phospho oxidase . Recentloduction . Polymeroduction showed thIn inflammatory skin diseases, various redox-related signaling pathways mediated by reactive species are involved in cells inducing inflammation and cell death. Research of gut microbiome and skin diseases is still at an early exploration stage. Meanwhile, there is no conclusive evidence for the specific mechanisms between oxidative stress and gut microbiome in inflammatory skin diseases. Our review suggests that the complex microbial-host cross-talk, which occurs through the gut-skin axis, may affect the local/systemic redox status through reactive species, activating local/systemic inflammation, eventually leading to inflammatory skin diseases . The gut