Curvularia eragrostidis C52 that strongly degraded silica-based glasses coated with fluorine and hafnium, as expressed by the hyphal surface coverage of 46.16 ± 3.3% and reduced light transmission of 50.93 ± 1.45%. The genome of C. eragrostidis C52 is 36.9 Mb long with a GC content of 52.1% and contains 14,913 protein-coding genes, which is the largest genome ever recorded in the genus Curvularia. Phylogenomic analysis revealed C. eragrostidis C52 formed a distinct cluster with Curvularia sp. IFB-Z10 and was not evolved from compared genomes. Genome-wide comparison showed that strain C52 harbored significantly higher proportion of proteins involved in carbohydrate-active enzymes, peptidases, secreted proteins, and transcriptional factors, which may be potentially attributed to a lifestyle adaptation. Furthermore, 72 genes involved in the biosynthesis of 6 different organic acids were identified and expected to be crucial for the fungal survival in the glass environment. To form biofilm against stress, the fungal strain utilized 32 genes responsible for exopolysaccharide production. These findings will foster a better understanding of the biology of C. eragrostidis and the mechanisms behind fungal biodeterioration in the future.Glass biodeterioration by fungi has caused irreversible damage to valuable glass materials such as cultural heritages and optical devices. To date, knowledge about metabolic potential and genomic profile of biodeteriorative fungi is still scarce. Here, we report for the first time the whole genome sequence of  The phenomenon is known as the result of abiotic processes that cause alteration to a glass surface and biotic processes that leach elements from a glass surface through microbial activity2. With respect to biotic alteration, numerous investigations have been carried out to prove that the damage is also caused by metabolic activities of complex microbial communities including fungi, bacteria, archaea3. Once microbial community is typically shaped depending on water availability, temperature, and nutrients, biofilm is formed to protect microbial community and mineralize on the surface of glassy material2. Microbial cell death can attain physical stress on the glass surface and/or possibly detach, leading to the delamination of all or part of the glass alteration volume. As a result, etching, pitting, leaching, discoloration, and glass degradation are common features of biogenic attack, which are usually observed in medieval stained and optical glasses5.Glass, a non-crystalline material, is widely used for practical, technological, and decorative purposes however its originality has relatively been reduced by physicochemical damages or biodeterioration. Once used for long periods of time, the glass is subjected to weathering and drastic changes in appearance, color, and structureAspergillus sp. is the major fungus causing biodeterioration of historical stained-glass windows in the fifteenth century Cartuja de Miraflores monastery, Spain1; Cladosporium sp. and Phoma sp. are major fungi of Catalonian churches, Spain3; Penicillium sp. and Cladosporium sp. were the two predominant fungal genera isolated from historical glass windows, Portugal5. In our previous study, 40 fungal strains classified into 14 genera were recovered from optical observation instruments of museums in Vietnam where Aspergillus and Penicillium were the most abundant genera6. The biodeterioration ability of fungi on glass is identified by a combination of parameters, including organic acids, exopolysaccharides (EPSs), the interaction of carbohydrate-active enzymes, temperature, humidity, and so on. Organic residues present on the glass surface, as dead microbial material, metabolites of autotrophic bacteria, and dust deposits, accompanied by substrates from water vapor in the air initiate the life cycle of filamentous fungi3. As a result, biofilm composed of EPSs and specialized substrates is developed to absorb water and increase the chemical destruction of glasses. The surrounding environments are then acidified through the production of organic acids leading to pH changes, redox-reaction leaching and chelation of metal oxides such as Na2O, K2O, CaO, Al2O3, and B2O, present in glass3. As a consequence of bio-pitting corrosion and pitting7, cracks, Na, K, and Ca were proved to be the most leached elements, while an ionic alteration was recorded for Fe and Mn5. Another hypothesis is that the leached elements may react with other components yielding high solubility salts that support fungal growth. As for the medieval stained-window glass, moisture and rain remove the soluble salts, causing patinas and crusts, such as sulfates (gypsum and syngenite), calcite, Ca-oxalates8. Until now, most studies have only analyzed colonizing fungal communities on glass and the underlying mechanisms behind fungal biodeterioration remain largely unknown.Compared to other microorganisms, filamentous fungi play an important role in the biodeterioration of glass. For example, Curvularia is reported to be a species-rich genus consisting of 131 species listed in Index Fungorum (assessed on 4 January 2018)9. The genus Curvularia acts predominantly as endophytes and pathogens of plants, animals, and humans12. Of note, Curvularia spp. have been recovered from binocular eyepieces and air, indicating the widespread distribution of this genus in the natural environments13. Identification by morphology is not enough to delimit species within the genus, thus DNA barcodes and whole-genome sequencing are needed to allow accurate identification and comparison. However, there are only 7 genomes available on Genbank (NCBI), among which Curvularia genomes associated with plants occupied the majority. In the previous study, the genome of C. lunata, an important pathogen of maize, was sequenced to demonstrate the evolutionary relationship with the fungal pathogen Bipolaris maydis and provide a better understanding of pathogen development and infection mechanism14. In this study, we isolated and evaluated for the first time glass biodeterioration ability of C. eragrostidis C52 of which genome was then sequenced by Illumina platform. Genome-wide analysis and metabolic annotation shed light on lifestyle adaptation and the genetic basis involved in organic acid and EPS production of C. eragrostidis C52 under its interaction with the glass surface. The findings from this study will enrich the database of Curvularia genome and pave a new way for investigating glass biodeterioration and corrosion by fungi.16. Taken together, the isolate C52 severely damaged the surface layer of the experimental glasses in short-term incubation.A total of 12 fungal isolates were isolated from the surface of infected eyepieces of 3 binoculars. Most isolates showed strong growth and acidified the medium to a pH of around 3.0, except for C23, C33, and C34. Glass biodeterioration experiments further revealed that all strains were able to adhere to the glass surface by developing mycelium networks (9.5–46.2%), decrease light transmission through the glass (15.7–50.9%), and produce EPS (0.9–19.0 g/L) (Supplementary Table S1). Among them, fungal isolate C52 caused a remarkable decrease in pH of 2.6, produced the highest EPS concentration (19.0 ± 2.3 g/L), and grew on the glass surface with a hyphal coverage of 46.2 ± 3.3% recorded after 28 days of assessment. SEM analysis showed massive fingerprints on the glass surface by the isolate C52 after the cleaning procedure , C. eragrostidis CBS 189.48T (99.7% SI), and C. papendorifii MUCL 10191T (93.6% SI). In the neighbor-joining tree, strain C52 and reference taxa C. eragrostidis CBS 189.48 (HG778986) formed a clade with 100% bootstrap support . A reverse plate of strain C52 had olivaceous grey to olivaceous black pigmentation. The hyphae were branches, septate, and thin-walled with the size of 1.5–3 µm in diameter. Conidiophores are represented as singly or in groups, straight or flexuous. The size of cells did not decrease towards the apex and was sometimes branched. The cell walls were thicker than those of vegetative hyphae, mononematous, macronematous, reddish-brown to brown, paler towards apex, up to 700 μm long × 3–6 μm wide. The conidia were ellipsoidal to fusiform, pale brown, apical, and basal cells paler than the middle cells. The conidia varied in size ranging from 11 to 16 μm long × 16–34 μm wide. Furthermore, molecular identification using ITS gene sequencing analysis showed that strain C52 was most related to C. eragrostidis C52 was sequenced using Illumina platform, resulting in 11,419,284 reads containing 1,585,458,996 bases (Supplementary Table S3). The de novo assembly of the C. eragrostidis C52 genome yielded approximately 36.9 Mb consisting of 3594 contigs among which the total length of the 200 largest contigs occupied 83.2% of genome size  database under accession number: JAIRCK000000000.The genome of 17. In total, TEs were predicted to be approximately 1.1% of the assembled genome in which Class I TEs (retrotransposons) occupied most in the interspersed repeat (Supplementary Table S4). Long interspersed nuclear elements (LINEs) were the most abundant Class I TEs accounting for 0.3%. Tandem repeat sequences represented 0.8% of the assembled genome. C. eragrostidis C52 genome also harbored 194 rRNA genes.It was important to determine transposable elements (TEs) since they were reported to be involved in genome size expansion and evolutionC. eragrostidis C52 were assigned to 24 functional categories. Genes involved in “function unknown” accounted for the majority (3262 genes), followed by “carbohydrate transport and metabolism” (766 genes), “posttranslational modification, protein turnover, chaperones” (647 genes), and “amino acid transport and metabolism” (631 genes) . Genome-wide comparison across Curvularia genomes revealed that the 4 largest groups found in C. eragrostidis C52 were far more than that in C. kusanoi 30M1, C. lunata CX-3; 6, C. papendorfii UM 226; 7, Curvularia sp. IFB-Z10, and C. geniculata P1 (Supplementary Table S5). This could be due to the discrepancy in the isolation source and life cycle of these fungi. In support of COG analysis, KEGG analysis was conducted to show that “metabolism” was the most abundant group, among which the most representative pathways were “global and overview maps”, “carbohydrate metabolism”, “amino acid metabolism”, and “energy metabolism” .The genome was predicted to have 14,913 protein-coding genes with an average sequence length of 1318.5 bp. Among them, 8,622 (57.8%) were successfully assigned to GO terms, 11,883 (79.7%) were similar to the InterPro, 14,207 (95.2%) were mapped to COG, and 10,093 (67.7%) homologs were similar to sequences in Pfam, 7121 (47.8%) were annotated by KEGG pathway (Supplementary Table S4). Among them, the genes in clusters of COG families of Curvularia varied from 84.1 to 99.9%. The fungal strain C52 exhibited high ANI values ranging from 84.5 to 86.8% as compared to 7 selected genomes, in which the highest ANI value was observed in Curvularia sp. IFB-Z10. Phylogenetic analysis revealed the close genetic relatedness between C. eragrostidis C52 and Curvularia sp. IFB-Z10  which measures the nucleotide-level genomic similarity between two genomes was conducted. The ANI values between members of the genus C. eragrostidis C52 was performed through the dbCAN server. The genome of strain C52 revealed the existence of 596 CAZymes with high diversity of families that included 262 glycoside hydrolases (GH), 116 auxiliary activity (AA), 101 glycosyl transferases (GT), 77 carbohydrate esterases (CE), 22 polysaccharide lyases (PL), 18 carbohydrate-binding modules (CBM) , C. lunata CX-3 (500), C. kusanoi 30M1 (515), and C. geniculata W3 (502), this was the highest number of CAZymes reported. Given that GH enzymes were crucial for carbohydrate metabolism and organic acid production, the genome of strain C52 contained 75 GH families. GH3 (18 genes) was the most abundant GH family, followed by GH43 (17 genes), and GH16 (14 genes). Additionally, 29 out of 75 GH families comprised only one gene, which was comparable to other Curvularia strains. The fungal genome was also found to be rich in AA families that were divided into 8 families of ligninolytic enzymes and 3 families of lytic polysaccharide monooxygenases18. In this study, 30 AA3 , 25 AA9 (lytic polysaccharide monooxygenase), and 22 AA7 (glucooligosaccharide oxidase) were detected as the most abundant CAZyme families  Fig. A. As comC. eragrostidis C52 genome contained 36 GT family members which were involved in the biosynthesis of oligosaccharides, polysaccharides, and glycoconjugates based on catalytic activities on glycosidic linkages20. Out of 22 most abundant family members represented by heatmap, GT family occupied 5 members including GT83, GT9, GT2, GT1, and GT4, which was only lesser than GH family , followed by serine protease S9X (43 genes) , 14 metallo peptidases , and 5 other families  that were absent in 7 compared genomes.To decipher a lifestyle adaptation and glass biodeterioration ability of the strain C52, a BLAST search against MEROPS protease database was carried out for all selected fungi. A total of 470 peptidases were classified into 8 big families, including serine (198 genes), metallo peptidase 130 genes), cysteine peptidase (84 genes), theronine peptidase (26 genes), aspartic peptidase (18 genes), protease inhibitors (10 genes), mixed peptidase (2 genes), glutamic peptidase (1 gene), asparagine peptide lyase (1 gene), accounted for 119 sub-families , C. geniculata P1 (845 proteins), C. lunata W3 (807 proteins), C. geniculata W_3 (804 proteins) or human pathogenic fungi C. kusanoi 30M1 (859 proteins), C. papendorfii UM 226 (770 proteins). In addition, eukaryotic and prokaryotic pathways of protein export were found in the C52 genome, including Sec-dependent pathway, twin-arginine translocation system, and signal peptidase. Out of 39 genes, 10 genes encoding for SecE, SecG, YajC, SecM, Ffs, SRP9, TatE, RN7SL, SPCS2, and IMP1 were not predicted , followed by oxalic acid (0.15 ± 0.031 g/L) and citric acid (0.07 ± 0.016 g/L)  yielding lactic acid  contribute to oxaloacetate accumulation in the cytoplasm, which were catabolized by malate dehydrogenase  to malate and fumarate that are exported outside the membrane. At the end of this metabolic route, the presence of 11 succinate dehydrogenase sdh and one l-aspartate oxidase nadB might significantly improve succinic acid yield and productivity (Supplementary Table S6). Depending on induction condition, the rest of the pyruvate was converted into acetyl-coenzyme A (acetyl-CoA), which was the substrate of 8 citrate synthases . Due to citrate acceleration, citrate was transported to cytoplasm provoking strong oxalic acid production as indicated by phenotypic result above. Parallelly, citrate was commonly catabolized by aconitase (orf_5714) and aconitate hydratase  to produce cis-aconitate following the TCA cycle  and 2 UDP-glucose-1-phosphate uridylyltransferases  genes. One gene encoding β-1,3-glucan synthases (orf_6760) and β-glucan biosynthesis-associated proteins (orf_8141) was directly involved in β-1,3-glucan and β-1,6-glucan biosynthesis, respectively. The linear glucans were delivered to the outside membrane and then elongated by 2 copies of β-1,3-glucanosyltransferases  that belong to the GH72 family. In parallel, the short linear glucans were conjugated to another short 1,3-β-glucan by 5 glycoside hydrolases  through a linear β--linkage. Moreover, some genes associated directly and indirectly to β-1,3-glucan and β-1,6-glucan biosynthesis were also predicted  that functioned in the deacetylation of the synthesized GAG polymer was found. Searching on bacteria-type EPS also revealed the presence of 8 genes including 3 copies of epsA and 1 copy each of epsL, epsR, exoA, exoM, exgA, and waaL.To deeply understand biodeteriorative mechanisms of the fungal strains C52, polysaccharide biosynthesis was investigated at both genotypic and phenotypic levels. Increasing the incubation time led to a rise in EPS production. The highest EPS concentration (19 ± 0.2 g/L) was recorded after 7 days of incubation . After 10 days of incubation, a relative decrease was observed (16 ± 1.2 g/L). In support of this result, a total of 32 genes contributing to EPS biosynthesis were identified in the genome of C. eragrostidis C52 that produced clear damages on silica-based glasses coated with fluorine and hafnium elements, presented as fingerprints. The findings provide a better understanding of genomic features of C. eragrostidis C52 in deteriorating optical glass.Most of the current investigations have been focusing on the medical and agricultural applications of fungi, little is known about glass deterioration of fungi, especially genetic information. With the development of next generation sequencing techniques, genome research of fungi has recently started getting more attention due to the complexity of genomic and physiological characteristics. In this present study, we reported for the first time a genome sequence of C. eragrostidis C52 is the largest sequenced genome within the genus Curvularia. At the time of writing, there are only 7 Curvularia genomes sequenced and deposited onto GenBank (NCBI). The C. eragrostidis C52 genome size of 36.9 Mb is by far larger than other reported sizes ranging from 33 Mb (C. geniculata P1) to 35.5 Mb (C. lunata W3). At the species level, C. eragrostidis C52 is the first sequenced genome. The number of predicted protein-coding genes of C. eragrostidis C52  was remarkably higher than that of other species such as C. lunata W3 , C. kusanoi 30M1 , and Curvularia sp. IFB-Z10 . TEs are known as mobile genetic units, which cause mutations, gene expression and chromosomal rearrangement, thereby aiding the populations to adapt successfully to changes in the environment23. The genome size of plant pathogens such as Phytophthora infestans, and Blumeria grami f.sp. hordei is expanded due to the abundance of TE proliferation that accounted for approximately 29% of the genome25. In addition, TE repertoires vary not only among genus levels but also in the closely related fungal species. TE was estimated to be 1.1% of the C. eragrostidis C52 genome, which was not different from other Curvularia genomes. These results suggested that the expansion of protein-coding gene inventory might lead to the larger genome size of C. eragrostidis C52, which may be due to the lifestyle and ecological niche. Apart from that, TEs also function as novel promoters interfering in transcription processes that play an important role in fungal development and evolution26. Genomic analysis determined 372 genes related to transcription factors (TFs) accounting for approximately 0.03% of the total predicted genes, which is relatively similar to 7 compared genomes. Although the TF profile of C52 was compatible to others, lambda repressors containing helix-turn-helix domain, PAS fold, and bacterial regulatory HTH proteins were around fourfold, sixfold and 18-fold higher, respectively, than those of genes in other compared genomes . Hence, this finding could help explore the evolutionary relationships and lifestyle adaptation of Curvularia species upon different ecological niches that remain to be investigated in the future.The genome assembly revealed that C. eragrostidis C52 displayed a great expansion of certain gene families potentially involved in lifestyle adaptation and colonization. Given that airborne fungal spores have to germinate and develop into hyphae to colonize on glass surface6, C. eragrostidis C52 might successively undergo asexual and sexual stages. The sexual stage remains unknown, while the asexual stage of Curvularia species is reported to be crucial for causing disease to the hosts such as plant and human. As for the important maize pathogenic fungus C. lunata CX-3, the colonization process including the attachment to the plant surface, the germination on the plant surface and the formation of infectious structures, and the penetration and colonization of the host tissue attributed to a high proportion of CAZymes, proteases, and secreted proteins14. C. eragrostidis C52 was rich of CAZymes responsible for colonization cycle, nutrient acquisition and dispersal that are shaped by utilized substrate, lifestyle, and host preference27. A recent study proved that the repertoire of CE1 and CE10 genes was significantly reduced in biotrophic pathogens28. As it stands, plant-derived Curvularia strains  and human-derived strains  had an average CE10 of 16 genes. By contrast, a higher number of CE10 (26 genes) was shown in C. eragrostidis C52 recovered from glass. It could be found through genome-wide comparison that 23 CAZy family members were missing in 7 compared genomes, but not in C. eragrostidis C52. Furthermore, serine protease genes also contribute to adaptive natural selection and genome expansion as proved in the mycoparasitic and nematode-parasitic fungus Clonostachys rosea29. The ability to degrade lignocellulosic substrates such as dead plant material and cell wall components of Trichoderma is subjected to the expansion of proteases and CAZymes30. In this study, significantly expanded gene sets of S08A, S09C and S33 serine proteases were observed in C52 genome compared to others. Given that strain C52 is the first fungal genome sequence associated with glass biodeterioration, the remarked question is that whether these protease families are coupled to the glass biodeterioration lifestyle and evolutionary history within Curvularia genera.C. eragrostidis C52 also contributed to the biodegradation of glass. As described previously, EPSs such as β-1,3-glucan were required for cell walls and especially biofilm development, resulting in colonization of the fungi on the surface of glass31. In C. eragrostidis C52 genome, the presence of β-1,3-glucan synthase (Fks), β-glucan synthesis-associated protein (Bms), Rho-GTPase-activating protein , glycosyltransferase , and glycoside hydrolase  resulted in the production of β-1,3-glucan. By contrast, only one out of 4 genes attributed to exopolysaccharide GAG formation was predicted, and some genes related to bacterial EPSs also were also determined. These results suggested that C. eragrostidis C52 might produce different kinds of EPS to survive and thrive on the glass, which corresponds to the high concentration of EPS obtained from the supernatant. Since fungal biodeterioration mechanisms are poorly characterized to date, the involvement of EPSs in fungal biodeterioration will still be an interesting subject for future studies.The EPS production by C. eragrostidis C52 at the aspect of phenotypic and genotypic levels. It is believed that fungal colonization on the glass through biofilm formation is linked to EPS production, which subsequently produces various organic acids to dissolve metal oxides such as Na2O, K2O, CaO, Al2O3, B2O, and ZnO32. Another nutrient source is the lipid layer that comprises long-chain hydrocarbons, fatty acids, and some alkaline metals such as Li, Na, K, Ca, Ba, Al, Zn, and Pb. Under sufficient conditions, fungal growth results in glass corrosion. A. niger was reported to produce oxalic acid, formic acid, tartaric acid, malic acid, and citric acid at every pH value, while organic acid secretion produced by Penicillium ochrochloron and Penicillium oxalicum was inhibited under acidic pH34. It is clear that organic acid production is pH- and strain-dependent manners. In this study, when grown on the mineral medium containing 2.5 g/L glucose, C. eragrostidis C52 was able to produce 7 different organic acids at acidic pH, which was higher than filamentous fungi reported previously that were found to produce 3–6 different organic acids in rich liquid medium35. In line with this, low organic acid concentrations were produced by the strain C52, which could be due to low glucose concentration supplemented in the culture medium. The presence of organic acids was in agreement with metabolic pathways annotated in the genome of strain C52, except for gluconic acid. A recent study demonstrated that organic acids were excreted fully protonated or as anions accompanied by protons expelled via the plasma membrane H+-ATPase, which served important purposes such as charge balance, energy spilling, chelation of trace elements, and nutrient uptake. In some fungi like Schizosaccharomyces pombe, Hansenula anomala, Penicillium ochrochloron37, the reuptake of organic acids was observed. Since iron is required for virtually all biological systems, in A. niger, citric acid was demonstrated to function as iron siderophore to increase the bioavailability of iron represented as Fe(III) citrate38. Moreover, arbuscular mycorrhizal fungi such as Rhizophagus irregularis secretes a significant amount of organic acids  to sequester low-accessible phosphorus from Fe oxides39. Combining our results, it seems that the production of organic acids is such a critical mechanism to help fungi survive under metal-rich environments.Organic acids were shown to be secreted by C. eragrostidis C52 was sequenced to strengthen the Curvularia genome database and to provide a better understanding of factors involved in biodeterioration mechanisms. Although the genomic evolution within Curvularia genera upon different ecological niches has been mentioned14, it is necessary to sequence whole genomes of more species to illustrate this hypothesis more accurately.To date, a number of fungi related to glass decay have been isolated and identified but at the time of writing, the underlying mechanisms and genome sequence of such fungal strains have not been revealed. In the present study, the genome of biodeteriorative fungus 6. In brief, sterile cotton swabs were used to wipe on the surface of eyepieces, then transferred to a sterile tube containing 1 mL of 0.05% Tween 80, and homogenized by shaking at 200 rpm for 30 min. About 100 μL of aliquots were spread on a Czapek–Dox agar medium for 4–5 days at 30 °C. Hyphae tip of the grown fungal colony was cut by a syringe needle (29 gauge) under a stereo microscope and then transferred it onto new Czapek–Dox agar plates. The pure isolates were maintained on a Czapek–Dox agar medium at 4 °C or silicone beads at − 20 °C. Evaluation of the glass biodeterioration by fungal isolates was carried out following the guideline of the ISO 9022-11:2015 document . In brief, fungal spores yielded on potato dextrose agar (PDA) medium were resuspended in a mineral-salt medium containing 0.05% Tween806 to attain approximately 106 spores/mL. After that, the spore solution was spread equally on the surface of silica-based glasses (10 × 20 cm) that had fluorine and hafnium coatings. The control samples were prepared in the same way, in which fungal spores were not added to the glass. These experimental glasses were kept at 30 °C with relative humidity of 90% for 28 days. The hyphal surface coverage was analyzed using a digital camera via OPTIKA Vision Pro and ImageJ v.1.51 softwares8. The colonization of fungal strain on glass samples was visualized under a JEOL 5410 scanning electron microscope (SEM) (Japan). The light transmittance through glass samples was measured by a spectrophotometer UV-2550 at wavelengths of visible light range from 400 to 800 nm6. Elemental compositions on the glass surface of untreated and fungus-treated glasses were quantified by the Oxford instruments Energy dispersive X-ray spectroscopy (EDS) microanalysis system  carried out alongside scanning electron microscopy (SEM)  with default parameters at 15 kV accelerating voltage40. The weight and atomic percentages of elements were calculated as described previously41.Three binoculars highly contaminated by fungi (model 6nu5 8 × 30 M) were collected from Bien Hoa city, Vietnam during 2018–2019, which were then placed in sterile plastic bags and transported to the laboratory for isolation. Fungal strains were derived from the surface of eyepieces comprising multi-layer anti-reflective coatings made from essential elements such as fluorine and hafnium as described by Ngo et al.42. PCR was then performed to amplify the internal transcribed spacer (ITS) region sequences by using primer pairs ITS1F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′). Purification and sequencing of PCR product were done by FIRST BASE Laboratories Sdn. Bhd. . The resulting sequence was analyzed by using BioEdit v7.2.5 and compared with those available in GenBank via BLASTn search on GenBank (http://www.ncbi.nlm.nih.gov/). Phylogenetic analyses were conducted using the neighbor joining (NJ) methods in MEGA v7.0 and the bootstrap was 1000 replications to assess the reliability level of the nodes tree43. The type strain Bipolaris maydis CBS 136.29 (NR_138224) was used as the outgroup branch.The strain C52 was incubated on the Czapek–Dox agar medium at 30 °C for 4–5 days to identify the morphological characteristics as described previously. Genomic DNA of fungal strain C52 was extracted using the microwave method as described previously44. Strain C52 was grown on a Czapek–Dox broth agar medium for 72 h at 30 °C. The culture was used as inoculum in 100 mL of Czapek–Dox medium containing 5 g/L glucose. At intervals, a sample was taken, and EPS was then precipitated by the addition of an equal volume of chilled ethanol to the cell-free supernatant. After keeping at 4 °C for 24 h, the precipitated EPS was washed with chilled ethanol, freeze-dried, and weighted to determine the raw EPS concentration.EPS production was determined as g/L according to the procedure described by Jaroszuk-Ściseł et al. (2020)C. eragrostidis C52 was cultivated on a Czapek–Dox medium at 30 °C for 3 days as the seed culture. Assessment of the ability to produce organic acids of the strain C52 was carried out in 500 mL conical flasks containing 200 mL MT1 medium 2SO4 0.75 g, MgSO4·7H2O 1.0 g, NaCl 1.0 g, CaCl2·2H2O 0.1 g, 0.5 ml trace element , pH 7.2, in a liter) at 30 °C for 6 days with an inoculation rate of 2% (v/v). Cell-free supernatant was harvested by centrifugation and then passed through ultra-filtration using Vivaspin 5kD  tubes to eliminate proteins. Organic acids in the solution were determined using gas chromatography-mass spectrometry (GC–MS) with some modifications45. Briefly, 2.5 M NaOH was added to alkalize the solution, and 200 μL of the alkalized solution was then derivatized by the addition of methylchloroformate which was extracted with dichloromethane. About 2 μL of the extract were injected in the split injector port of Agilent 5973 N system, equipped with an Omegawax , 30 m × 250 μm i.d. × 0.25 μm thick films. Before injection, the oven temperature was programmed  and was isothermally held for 15 min. Compounds were determined after comparing with available data in the NIST library and using the standard organic acids proposed.C. eragrostidis C52 were harvested from liquid cultures grown in a Czapek–Dox medium at 30 °C for 3 days for DNA extraction. Mycelia were harvested and ground into a fine powder by using liquid nitrogen. The genomic DNA was extracted from the resulting powder using DNeasy Plant Minikit  according to the manufacturer’s instructions. DNA quantity and purity were assessed using a Nanodrop UV–Vis spectrophotometer  and a Qubit 2.0 fluorometer . The whole genome of C. eragrostidis C52 was de novo sequenced using Illumina MiSeq platform. Approximately, 11.4 million paired-end raw reads were generated and then quality was checked by FastQC. Low-quality reads (< Q15) and adapters were removed using Fastp v0.20.0. De novo assembly of Illumina reads data was performed using SPAdes v3.15.1 (https://github.com/ablab/spades) and MegaHit v1.2.9 (https://github.com/voutcn/megahit) with default parameters. The completeness of the fungal genome was analyzed by BUSCO v346.The mycelia of C. eragrostidis C52 was performed using Funannotate v1.8.1 (https://github.com/nextgenusfs/funannotate/tree/v1.8.1) which employs GeneMark-ES v4.48_3.60_lic47, Augustus v3.3.248, SNAP v2006-07-2849, and GlimmerHMM v3.0.450. Ribosomal RNA and tRNA genes were predicted using RNAmmer v1.251 and tRNAscan-SE v2.0.552. Noncoding RNAs (ncRNAs) in the genome of fungal strain C52 were predicted using Infernal v1.1.253. The repetitive elements in the C52 genome were identified by RepeatModeler v2.0.154. Pseudogene was determined using PseudoPipe55. All desirable open reading frames (ORFs) were blasted against the NCBI curated refseq_protein database (reference proteins) by Blastp56. Gene Ontology (GO) and Cluster of Orthologous Groups (COG) were annotated based on EggNOG-mapper v1.0.357. KEGG Automatic Annotation Server (KAAS) was performed by an assignment method of ‘bi-directional best hit’ against data of any species on the KEGG database to identify functional protein sequences following KEGG Orthology58. Default parameters were applied for all software tools.Genome annotation of Curvularia genomes available on GenBank (NCBI) divided into 3 groups including plant-derived strains , human-derived strains , and fish-derived strain (Curvularia sp. IFB-Z10) were used for comparative genome analysis. Orthologous protein domains between C. eragrostidis C52 and selected fungal genomes were determined using OrthoMCL (version 2.0.9)59. Furthermore, the matrix of ANI and AAI was carried out using CompareM v0.1.2 (https://github.com/dparks1134/CompareM) and PYANI v0.1.2 (https://github.com/widdowquinn/pyani/releases/tag/v0.1.2), respectively to identify the relationship within Curvularia species.Seven http://bcb.unl.edu/dbCAN2/) including the dbCAN CAZyme domain (HMMER search), short conserved motifs (Hotpep search), and CAZy databases (DIAMOND search)60. All positive hits were manually checked for final validation. The MEROPS peptidase database61 was applied to classify enzyme classes including aspartic, cysteine, glutamic, serine, metallo, threonine, asparagine, mixed peptidase, and protease inhibitor. Protein sequences with positive hits were checked again with a BLAST search  against the NCBI NR protein database. Apart from that, secreted proteins were identified based on the combination of SignalP v4.162, TargetP v2.0 server , TMHMM v2.0 , and Big-GPI (https://mendel.imp.ac.at/gpi/fungi_server.html) software. Transcriptional factors were predicted through analyzing InterPro IDs in the Fungal Transcriptional Factors63. Default parameters were applied for all software tools.Identification of genes related to Carbohydrate-Active enZYme (CAZyme) families including glycoside hydrolase (GH), carbohydrate-binding module (CBM), glycosyl transferase (GT), polysaccharide lyase (PL), carbohydrate esterase (CE), and auxiliary activity (AA) in the genome of C52 and 7 other selected genomes was carried out using the dbCAN meta server  against the genome of strain C52 using reference protein sequences.Genes encoding for proteins involved in organic acid and polysaccharide biosynthesis were predicted using the KEGG and BlastKOALASupplementary Information.


Aspergillus calidoustus, Trichoderma longibrachiatum, Rhizopus arrhizus and Paecilomyces variotii, as well as less likely pathogens: Aspergillus versicolor, Chaetomium cochliodes, Chaetomium globosum and Chaetomium rectangulare. Opportunistic pathogens such as Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus tubingensis and less likely pathogens such as Aspergillus westerdijkiae, Chaetomium globosum and Dichotomopilus finlandicus were isolated both from ordinary and from problematic buildings. Aspergillus was the dominant, most diverse genus found during screening for potentially pathogenic isolates in the indoor strain collection. Studies on Aspergillus niger and Aspergillus calidodoustus revealed that tolerance to cleaning chemicals may contribute to the adaptation of Aspergillus species to indoor environments.Viable airborne pathogenic fungi represent a potential health hazard when exposing vulnerable persons in quantities exceeding their resilience. In this study, 284 indoor fungal isolates from a strain collection of indoor fungi were screened for pathogenic potential through the ability to grow in neutral pH at 37 °C and 30 °C. The isolates were collected from 20 locations including 14 problematic and 6 non-problematic ordinary buildings. Out of the screened isolates, 170 isolates were unable to grow at 37 °C, whereas 67 isolates growing at pH 7.2 at 37 °C were considered as potential opportunistic pathogens. Forty-seven isolates growing at 30 °C but not at 37 °C were considered as less likely pathogens. Out of these categories, 33 and 33 strains, respectively, were identified to the species level. The problematic buildings included known opportunistic pathogens:  Invasive infections by opportunistic pathogenic fungi mostly affect immunocompromised persons. They represent a serious problem in buildings such as hospitals and nursing homes housing vulnerable persons ,2,3,4. OAspergillus, Rhizopus and Trichoderma are associated with renovations of mold-infested buildings tef1α, [CaM [rbp2  markers ergillus , Paeciloilomyces , RhizopuRhizopus , Trichodchoderma  and Chaeaetomium  species.A. calidoustus strain after dehydration due to long incubation were determined as follows: The tests were performed in malt extract broth  12 g in 1 L, pH 5.5, glucose 7.1 mmol L−1, in microtiter plates with 96 wells. The biocides dissolved in ethanol and the tenside dissolved in water were diluted by two-fold dilutions in 100 µL MEB. Fungal spore suspensions, 100 µL PBS containing 106 spores mL−1, were added to each well. The effects of the chemicals on spore germinantion, hyphal growth, resporulation of new conidia and occurrence of Hülle cells were inspected by a phase-contrast microscope  and an image recording software  after 3 days and 9 weeks of incubation at 24 °C. The biocides and the tenside were purchased from Sigma-Aldrich . The selected biocides were Borax , Triclosan  phenol), CAS: 3380-34-5) and the tenside and wetting agent Genapol X-080 (CAS: 9043-30-5).The effects of 2 biocides and a tenside on an Aspergillus niger strain A21 was performed by filling the wells of the microtiter plate with MEA , 200 µL supplemented with 50 µL of PBS Genapol dilutions, 300 µg mL−1, 5 µg mL−1, and 50 µL spore suspensions, 106 spores per ml of indoor isolates of A. niger, C. cochliodes and Paecilomyces sp. The plate was incubated for 20 d at room temperature, the lid was opened every third day and the plate was photographed. After the last day, the fungi growing in the wells were identified by microscopic inspection of conidiophores and ascomata.The competitiveness test on solid media used on the


Impaired cerebral glucose metabolism is an early event that contributes to the pathogenesis of Alzheimer’s disease (AD). Importantly, restoring glucose availability by pharmacological agents or genetic manipulation has been shown to protect against Aβ toxicity, ameliorate AD pathology, and increase lifespan. Lithium, a therapeutic agent widely used as a treatment for mood disorders, has been shown to attenuate AD pathology and promote glucose metabolism in skeletal muscle. However, despite its widespread use in neuropsychiatric disorders, lithium’s effects on the brain have been poorly characterized. Here we evaluated the effect of lithium on glucose metabolism in hippocampal neurons from wild-type (WT) and APPSwe/PS1ΔE9 (APP/PS1) mice. Our results showed that lithium significantly stimulates glucose uptake and replenishes ATP levels by preferential oxidation of glucose through glycolysis in neurons from WT mice. This increase was also accompanied by a strong increase in glucose transporter 3 (Glut3), the major carrier responsible for glucose uptake in neurons. Similarly, using hippocampal slices from APP-PS1 mice, we demonstrate that lithium increases glucose uptake, glycolytic rate, and the ATP:ADP ratio in a process that also involves the activation of AMPK. Together, our findings indicate that lithium stimulates glucose metabolism and can act as a potential therapeutic agent in AD. Alzheimer’s disease (AD) is the most prevalent age-related degenerative disease characterized by a dramatic loss of neurons and a disruption in synaptic activity ,2,3. TheFor over seven decades, lithium salts have been used in psychopharmacology, especially for treating bipolar disorder and depression ,14,15. M14C-glucose) in hippocampal neurons treated with either 1-, 10-, or 50-mM lithium over 180 s. Our initial analysis showed no differences in glucose uptake after 1 mM lithium treatment compared to control cells  decreased from 7.5 nmol/106 cells in control cells to 6.9 nmol/106 cells, respectively  A. To fur the PPP C. To constration D,E.Given the increase in glycolysis, next, we evaluated whether lithium affects ATP synthesis. We noticed a significant 2.6-fold increase in ATP levels in lithium-treated cells relative to the control A. As expNext, we analyzed the effect of lithium on the expression of several genes related to glucose metabolism in hippocampal neurons. Due to the increase in glycolysis, first we quantified the expression levels of hexokinase and phosphofructokinase-1 (PFK-1), which catalyze the conversion of fructose-6-phosphate and ATP into fructose 1,6-biphosphate and ADP. Our results showed no differences in the expression levels of either of these enzymes in lithium-treated neurons as compared to control A,B. FurtFinally, to determine whether lithium can restore the impaired glucose homeostasis in AD, we used hippocampal slices from APP/PS1 mice to follow changes in glucose metabolism. Consistent with our results, the administration of lithium significantly increased glucose uptake in hippocampal slices A. MoreovAmong the pathological hallmarks of AD is the progressive impairment of cerebral metabolism ,40,41. TConsistent with previous results, lithium caused a significant increase in the uptake of glucose, which once internalized, was preferentially oxidized via glycolysis, resulting in a higher ATP/ADP ratio in hippocampal cultures. A lithium-dependent rise in glucose uptake and glycolysis in the brains of rodents has been described previously ,50,51. S2+-ATP complex, an association that can act in a tissue-dependent manner  glucose , was added, with a final specific activity of 1–3 disintegrations/min/pmol (~1 mCi/mmol). Aliquots of 100 μL were then transferred to another tube, placed inside a capped scintillation vial containing 0.5 mL of water, and incubated at 45 °C for 48 h. After this vapor-phase equilibration step, the tube was removed from the vial, a scintillation mixture was added, and the 3H2O content was measured by counting over a 5-min period. Glycolytic rates were determined by measuring the rate of 3H2O production from D-[3-3H] glucose.Glycolytic rates were determined as previously described ,93. Hippg for 5 min at 4 °C. Lysates were then resuspended in isolation medium  at a 1:3 dilution, sonicated at 4 °C, and then centrifuged at 1500× g for 5 min at 4 °C. The HK activity of the supernatant was quantified. For the assay, the purified fraction was mixed with the reaction medium , and the mixture was incubated at 37 °C for 30 min. The reaction was stopped by the addition of 10% trichloroacetic acid , and the generation of NADPH was measured at 340 nm.Hippocampal cultures were treated with lithium (10 mM) or 2-deoxy-D-glucose  for 30 min, and cultures were washed with PBS, treated with trypsin/EDTA, and centrifuged at 500× + ratio was measured using a commercial colorimetric assay , according to the manufacturer’s instructions, as described previously  glucose  and [6-14C] glucose (decarboxylated only in the Krebs cycle) [2-saturated Krebs–Henseleit buffer and 500 μL of this suspension (~106 cells) were placed in Erlenmeyer flasks with another 0.5 mL of Krebs–Henseleit solution containing 0.5 μCi D-[1-14C] glucose or 2 μCi D-[6-14C] glucose and 5.5 mM D-glucose . The Erlenmeyer flasks were equipped with a central well containing an Eppendorf tube with 500 μL of benzethonium hydroxide. The flasks were flushed with O2 for 20 s, sealed with rubber caps, and incubated for 60 min in a 37 °C water bath with shaking. The incubations were stopped by the addition of 0.2 mL of 1.75 M HClO4 into the main well, and shaking was continued for another 20 min to facilitate the trapping of 14CO2 by benzethonium hydroxide. Radioactivity was quantified as previously described by liquid scintillation spectrometry [14C] glucose (#NEC043) and D-[1-16C] glucose (#NEC045) were purchased from Perkin-Elmer.Glucose oxidation via the PPP was measured as previously described based on the difference in s cycle) . Hippocatrometry ,101. Botw/v)), and pelleted. The cultures were then resuspended in isolation medium  at a 1:3 dilution, sonicated at 4 °C, and centrifuged for 5 min at 1500× g at 4 °C. Subsequently, the pellet was discarded, and the supernatant was further separated by centrifugation at 13,000× g for 30 min at 4 °C. Finally, the G6PDH activity of the supernatant was quantified in a reaction buffer containing 1 mM ATP and 10 mM glucose-6-phosphate (G6P) for 30 min at 37 °C. The reaction was stopped by the addition of 10% TCA. The generation of NADPH was measured at 340 nm, as described previously [Slices or neurons were washed with PBS, collected by trypsinization . Differences were considered significant when * p < 0.05, ** p < 0.01 and *** p < 0.001.All experiments were performed 3–5 times , with triplicates of each condition in each experimental run. The results are expressed as the mean ± SEM. Data were analyzed by either Student’s


The epidermal growth factor–epidermal growth factor receptor (EGF-EGFR) pathway has become the main focus of selective chemotherapeutic intervention. As a result, two classes of EGFR inhibitors have been clinically approved, namely monoclonal antibodies and small molecule kinase inhibitors. Despite an initial good response rate to these drugs, most patients develop drug resistance. Therefore, new treatment approaches are needed. In this work, we aimed to find a new EGFR-specific, short cyclic peptide, which could be used for targeted drug delivery. Phage display peptide technology and biopanning were applied to three EGFR expressing cells, including cells expressing the EGFRvIII mutation. DNA from the internalized phage was extracted and the peptide inserts were sequenced using next-generation sequencing (NGS). Eleven peptides were selected for further investigation using binding, internalization, and competition assays, and the results were confirmed by confocal microscopy and peptide docking. Among these eleven peptides, seven showed specific and selective binding and internalization into EGFR positive (EGFR+ve) cells, with two of them—P6 and P9—also demonstrating high specificity for non-small cell lung cancer (NSCLC) and glioblastoma cells, respectively. These peptides were chemically conjugated to camptothecin (CPT). The conjugates were more cytotoxic to EGFR+ve cells than free CPT. Our results describe a novel cyclic peptide, which can be used for targeted drug delivery to cells overexpressing the EGFR and EGFRvIII mutation. The genetic and cellular heterogeneities of almost all cancer types continue to confound the development of effective cancer chemotherapies. To help solve this issue, several studies have highlighted the potential of combined treatment with drugs differing in their mechanism of action ,2,3,4, bThe EGFR gene can undergo several mutations, particularly within exons 18–21, which encode a portion of the EGFR kinase domain that has been associated with some types of lung cancer . The mosOne strategy that could effectively overcome the hurdle of drug resistance is to employ drug delivery systems that target overexpressed survival and proliferation-related receptors ,20,21,22The aim of this study was to discover new cyclic peptide ligands that selectively bind to both EGFR and EGFRvIII and evaluate the targeting cytotoxicity of their PDCs bearing the topoisomerase I inhibitor camptothecin (CPT) for NSCLC and glioblastoma cell lines. 1H NMR and 13C NMR spectra were measured in CD3OD at 300 K on a Bruker AvanceIII HD (1H 400 MHz and 13C 100 MHz) spectrometer and a BBO probe equipped with a Z gradient coil. IR spectra of the different peptides were measured with a Jasco FTIR 4700 instrument. The spectral resolution was set to 2 cm−1 and 16 scans were taken per measurement. Absorption spectra were recorded on a Jasco V-730 UV–Vis spectrophotometer. Flow cytometry was performed on a Beckman CytoFLEX analyzer, and the data were evaluated with FlowJo software. Fluorescence images were recorded at a magnification of ×200 with a Zeiss LSM700 confocal laser microscope. PDCs were purified by preparative HPLC on an ECOM preparative system, with dual UV detection. A Phenomenex Gemini® 10 µm RP18 (250 × 21.2 mm) column was used. The column was kept at ambient temperature. Eluents A (0.1% TFA in water) and B (0.1% TFA in CH3CN) were used. A typical elution was a gradient from 100% A to 100% B over 35 min at a flow rate of 25 mL/min−1. Chemical reactions were monitored by TLC  and LC/MS.The LC/MS analysis was performed using an Agilent Technologies 1260 Infinity (LC) 6120 quadruple (MS), column Agilent SB-C18, 1.8 mm, 2.1 × 50 mm, column temperature 50 °C, eluent water–acetonitrile (ACN) + 0.1% formic acid. HRMS was performed in an ESI-positive mode by using an Agilent 6550 iFunnel Q-TOF LC/MS instrument. 2HPO4), potassium dihydrogen phosphate (KH2PO4), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside , isopropyl-β-D-thio galactopyranoside (IPTG), 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), glycine, sodium dodecyl sulfate (SDS), Triton X-100, NaCl, polyethylene glycol (PEG) 8000, agarose, and glycerol were purchased from Alfa Aesar, Israel or Sigma-Aldrich, Israel. Epidermal growth factor (EGF) was purchased from Abcam . The Ph.D.-C7C Phage Display Peptide Library kit containing Escherichia coli host strain ER2738, a robust F+ strain with rapid growth and M13 phage displaying 7 mer cyclic peptides, was from New England Biolabs, Ipswich, MA, USA. The Dulbecco’s Modified Eagle Medium (DMEM), RPMI-1640, trypsin, fetal bovine serum (FBS), XTT, penicillin, and streptomycin were obtained from Sartorius, Israel. Mouse antibody APC anti-human EGFR, APC Goat anti-mouse IgG, cell staining buffer, and DMSO were obtained from ENCO, Israel. Mouse monoclonal antibody anti-EGFRvIII mutation, clone L8A4, was obtained from Merck Millipore.All solutions were prepared with Milli-Q water . Tryptone, yeast extract, disodium phosphate  [DKMG cells were purchased from the DSMZ cell bank . Lung cancer , myeloid leukemia (K562), breast cancer (MDA-435), normal breast (MCF-10A), and embryonic (HEK-293) cell lines were obtained from the ATCC. All cell lines were cultured in RPMI 1640 or DMEM medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1% penicillin/streptomycin . Cells were maintained at 37 °C in humidified 5% CO of EGFR . DKMG ceR [vIII) . H1975 cR [vIII) .5 cells/mL in a 6-well plate. When the culture reached 80% confluence, one well was incubated with the phage library (1011 pfu/10 µL) for 1 h at 37 °C with gentle stirring. The medium containing the unbound phage was collected and transferred to a second well and incubated again. The procedure was repeated with the third well. Cells in the first and third wells were washed four times with 0.5% Tween-20 in PBS. Then, 0.5 mL of elution buffer  was added and incubated for 10 min at 4 °C; the pH was then neutralized by the addition of 75 µL of 1 M Tris–HCl buffer, pH 9.0. The supernatant containing the cell surface binding phage was collected. The cells were then lysed by incubation for 1 h at 4 °C with 2 mL of 30 mM Tris–HCl and 1 mM EDTA, pH 8.0, the medium was collected and centrifuged at 265× g for 5 min and the supernatant containing the cell-internalized phage was transferred to a new tube. The internalized and surface-bound phages were amplified according to the manufacturer’s instructions. An aliquot of the phages was retained for sequencing (see below) and the remainder was subjected to two additional rounds of biopanning. For K562 cells, the same scheme was used, except that the cells were washed by centrifugal pelleting for 5 min, RT, 265× g.The phage display kit was used for the in vitro biopanning experiments according to the manufacturer’s instructions. All cell lines were individually incubated with phages from the stock library. K562 cells, which do not express EGFR, were used for negative biopanning selection. Adherent cells  were each plated at a density of 10Following biopanning, DNA was extracted from first and third round phages as well as from the original phage pool, according to the manufacturer’s instructions. Libraries were prepared for NGS, and sequencing was performed by Hylabs Pty, Ltd. using MiSeq technology .For the analysis of DNA sequencing data, a specific Perl script was written. The workflow of the data processing included translation; generation of a report list sorted by the number of amino acid sequence repeats; and curation of the list by removal of sequences internalized by K562 cells or those that did not appear in the original Ph.D.-C7C Phage Display Peptide Library. From this workflow, 11 peptides were selected, from which, two sub-lists were generated: one list included those clones internalized by all 3 cell lines H1299, H1975, and DKMG; the second included a list of peptides internalized uniquely by each cell line.2 (10 eq.) in DMF/H2O (4:1) for 2 h [Eleven 7-mer S-S bridged cyclic peptides were synthesized by solid-phase peptide synthesis on 2-chlorotrityl chloride resin , as free peptides or conjugated to fluorescein isothiocyanate (FITC) or camptothecin (CPT) through γ-aminobutyric acid GABA linker using standard Fmoc protocols . The cyc for 2 h . The pur2O) during the 2 h. Then, the solvent was evaporated (N2) and the crude PDCs were purified by preparative HPLC on an ECOM preparative system, with dual UV detection. A Phenomenex Gemini® 10 µm RP18 (250 × 21.2 mm) column was used. The column was kept at 25 °C. Eluents A (0.1 % TFA in water) and B (0.1% TFA in CH3CN) were used. A typical elution was a gradient from 100% A to 100% B over 35 min at a flow rate of 25 mL/min. For the P6 FITC conjugate:  LC-MS: RT = 8.66 min; ESI-HRMS m/z (M+) calc. 1449.4940 (C66H77N15O17S3), found 1449.5023. For the P6 CPT conjugate: ; LC-MS: RT = 4.79 min; ESI-HRMS m/z (M+) calc. 1434.5612 (C67H83N15O17S2), found 1434.5562. For the P9 FITC conjugate: . LC-MS: RT = 7.94 min; ESI-HRMS m/z (M+) calc. 1489.4607 (C65H80N14O19S4), found 1489.4673. For the P9 CPT conjugate: . LC–MS: RT = 5.21 min; ESI-HRMS m/z (M+) calc.: 1474.5252 (C65H83N15O19S3) found: 1474.5281. The deprotected cyclic peptides on the resin were coupled to the Fmoc-GABA-OH linker (2 eq.) for 2 h using benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) (2 eq.) as a coupling reagent and diisopropylethylamine (DIPEA), (6 eq.), as a base in DMF during the 2 h. Further, the Fmoc protecting group was removed , the resin was washed with DMF 3 × 5 min, DCM 3 × 5 min, followed by conjugation to FITC: FITC (2 eq.) in DIPEA (6 eq.) in NMP for 2 h. For CPT: 4-Nitrophenyl camptothecin-10-yl carbonate (CPT–4NFCF)  (2 eq.) 20 . The samples were centrifuged at 1400 rpm for 15 min. Supernatants were collected, filtered, and analyzed by liquid chromatography–mass spectrometry (LC–MS). PDC breakdown rates were determined as described elsewhere . Brieflyg. K562 suspension cells were directly resuspended in FCSB. A population of 106 cells/mL was then incubated with mouse anti-human EGFRvIII primary followed by two washings with FCSB and incubation with APC anti-mouse Ig antibody at 4 °C for 20 min in the dark or directly stained with anti-human EGFR-APC antibodies at 4 °C for 1 h in the dark. After washing, cells were resuspended in 200 μL of FCSB and analyzed by flow cytometry. For each sample, 5 × 104 cells were examined. H1299 and DKMG cells were collected after treatment with trypsin, resuspended in Flow Cytometry Staining Buffer , and centrifuged for 5 min 265× Cell cultures of 80–90% confluence were incubated with 2.5 µM FITC-conjugated peptides in RPMI–5% FBS for 1 h at 37 °C. Cells were scrapped from the culture flask, washed twice with 2 mL FCSB, and analyzed by flow cytometry to measure internalization. The cells were trypsinized after removal of the unbound conjugate, washed twice with FCSB, resuspended in 200 μL of FCSB, and analyzed by flow cytometry. 4, 1.0 × 105, and 2.5 × 104 cells/well, respectively. The cells were cultured in a complete growth medium for 24 h. After incubation, the medium was removed, the cells were twice washed with PBS containing Ca2+Mg2+, and a fresh medium (0.5 mL) containing 25 µM of FITC labeled P6 or P9 peptide conjugate was added. The cells were incubated at 37 °C under 5% CO2 for 0 h and 3 h, washed twice with PBS and stained with Hoechst 33258  for 10 min. The cells were washed twice with PBS and fixed with 4% paraformaldehyde.H1299, DKMG, and HEK-293 cells were seeded in 12-well glass bottom black plates in concentrations of 5.0 × 102 to allow for peptide internalization. The cells were washed twice by centrifugation at 265× g for 5 min and analyzed by flow cytometry. H1299 and DKMG cell lines were cultured in a complete RPMI growth medium in a 6-well plate overnight, until 80% confluent. Pre-incubation was performed with a blocking buffer consisting of 5 mg/mL BSA in PBS, pH 7.4, at 4 °C for 30 min. Then, cells were washed twice in PBS and incubated with 2.5 µM of FITC-labeled peptides with or without 80 nM EGF at 4 °C for 1 h. The cells were then incubated for 15 min at 37 °C under 5% COThe three-dimensional structures of the peptides were predicted using the PEPstrMOD ,39 serve4 cells/well) and incubated overnight in a complete growth medium. They were then washed and re-cultured in 100 µL of fresh complete medium containing various concentrations of free drugs or PDCs, for 24, 48, and 72 h. The XTT solution was added to each well and the plate was further incubated for 2–3 h at 37 °C. The optical densities in the wells were measured at both 480 and 680 nm using a TECAN Infinite M200 ELISA reader. All tests were performed in triplicate, and each experiment was repeated three times. Cell growth in the presence of PDCs was measured by a commercial XTT assay kit . The cells were seeded in 96-well plates  . StatistWT and EGFRvIII were confirmed using flow cytometry. As expected, H1299, H1975, and DKMG cells expressed EGFRWT   A, whereactively) B. K562 cctively) A,B.To isolate peptides that specifically bind and internalize into EGFR+ve cells, the Ph.D.-C7C Phage Display Peptide Library was used. For each cell line, three rounds of biopanning were performed and both surface and internalized phages were recovered. DNA from phage pools was extracted from the first and the third rounds sequenced by NGS and translated.To select the target-specific peptides, we first validated that all of the peptide sequences appeared in the original phage display library. Then, peptides derived from the EGFR−ve K562 cells were excluded from the list. After this step, 617,503 sequences remained. Finally, two groups of peptides were constructed. The first included sequences that were present in all three EGFR+ve cell lines; the second was divided into three subgroups, one for each cell line . From th−1 [−1 and 1449 cm−1 [–1 [The 11 selected peptides were synthesized together with a FITC tag. The formation of the thiourea group upon the FITC-to-peptide binding was confirmed by FTIR. In the FITC spectrum, there is a characteristic signal of the N=C=S group at 2015 cm−1 , which d449 cm−1  cannot bcm−1 [–1 .The binding and internalization capabilities of the obtained conjugates were tested at 2.5 µM on K562, H1299, and DKMG cells. This concentration was chosen based on a preliminary dose-dependence study, which showed receptor saturation from 5 µM . The totTo validate whether the seven internalized peptides target the EGFR receptor, a competitive binding test with the natural ligand EGF was performed. WT expression on these cell lines, they were stained with an APC-labeled monoclonal anti-human EGFR antibody followed by a flow cytometry analysis. EGFRWT expression on HEK-293 and MCF-10A cells was negligible (less than 10%), while about 97% of MDA-MB-435 cells were EGFRWT-positive  . Notably−1 and 1610 cm−1 in the FTIR spectra [−1 [–1 upon the conjugation could not be detected because of the existence of the C-terminus carboxylic group of the peptides formed upon the cleavage from the Cl-Trt resin.For the CPT–peptide conjugates, the formed carbonate bond  of P6, P9, P6-CPT, or P9-CPT for 6 h, after which, the medium was replaced with fresh, drug-free, complete medium, and the cells were cultured for an additional 24 h (designated as 6h24h) or 48 h (6h48h). In the second protocol, cells were exposed continuously to free drugs or PDCs for 24, 48, and 72 h. For each case, during the culture period, cell cytotoxicity was determined as described above and ICOverall, CPT did not result in pronounced cytotoxicity with the first protocol; however, the second protocol (continuous exposure) resulted in lower IC50 values as exposure time increased.50 was higher than for CPT alone  compared to the free CPT (IC50 = 25 µM). After 48 and 72 h of incubation, P9-CPT demonstrated a similar cytotoxic effect as the free CPT. The cytotoxic effect of the peptide CPT conjugate was not tested on HEK normal cells, as the peptide did not bind or internalize into these EGFR-ve cells.For DKMG cells, a 24 h pre-incubation with P9 and P9-CPT did not affect cell viability. The cytotoxic effect of P9-CPT on DKMG was noted after 48 h while its ICTs alone . After cIn the present study, phage display technology and biopanning on viable cells were used to discover novel EGFR-specific cyclic peptides that could act as drug carriers in a targeted drug delivery system. For peptide selection, we utilized viable cells instead of purified target proteins. It might be suggested that the use of an isolated biomarker is a more direct approach to isolating a specific ligand from a library, as irrelevant molecules would not obscure the receptor of interest; however, it is difficult to isolate membrane proteins in a form that retains their natural configuration. Moreover, the use of whole cells allows for a more natural expression of cell surface receptors . ValidatPhage display biopanning was used previously to identify EGFR-binding peptides. For example, 17 peptides were selected for tumor imaging and targeted chemo- or photodynamic therapy in a murine model of colorectal cancer ,64. OtheWhen working with phage display technology, it is possible that a target receptor may bind a displayed peptide in associated with structures in the phage itself. Therefore, it is always important to synthesize the peptides displayed by selected candidate phage clones and then validate the target specificity of the free peptides. Indeed, we found that only 7 peptides of 11 originally selected candidates for target cell binding had the ability to be taken up by the cells and were therefore selected for further study. Four of these peptides showed high internalization into DKMG cells carrying the EGFRvIII mutation ,64.−1. P6 and P9 showed higher steric clashes with EGF than P11. These docking results are in good agreement with the competitive uptake experiment (We did not directly investigate the mechanism(s) of the internalization of the selected peptides. However, peptide in silico docking studies with three peptides, P6, P9, and P11, confirmed their target specificity and showed binding at the active site of the EGFR. These three peptides showed good binding energy that ranged from −13.0 to −11.3 kcal molperiment , where tSince the natural role of EGF is to stimulate intracellular signaling pathways important for cell proliferation , it is iThe cytotoxicity effects of PDCs depend on several factors, including biostability, the rate of penetration, efficiency of the drug release, and drug toxicity. The ability of the peptide–drug conjugates to remain stable and to specifically enter the target cells is an important factor in the delivery of highly toxic drugs ,69. Our 50 would require an increase in the PDC dose, this would be offset by the enhanced target specificity and consequent decrease in off-target toxicity. An additional approach would be to arm the peptide with additional copies of the drug, as we have described elsewhere [As found in previous studies, ,71, conjlsewhere .One of the features of glioblastoma (GBM) is the overexpression of EGFR  and its In this work, we discovered a series of cyclic nonapeptides specific to EGFR-overexpressed H1299 and its mutation EGFRvIII-expressed DKMG cell lines using a combined phage-display cell-based positive biopanning selection method, followed by NGS sequencing of the collected DNA phages.Apparently, two of these peptide ligands, P6 and P9. exhibited an effective binding and internalization to EGFR and EGFRvIII cell lines, as confirmed using FACS and fluorescent microscopy. Moreover, P6 and P9 conjugated to CPT enabled the nuclear accumulation of cytotoxic CPT. CPT intracellular accumulation resulted in DNA damage and induction of cell death. Hence, coupling of CPT to these peptides did not interfere with CPT cytotoxic effects, while contributing a specific targeting tool to EGFR and EGFRvIII overexpressed cancer cell lines. Docking simulations revealed that the peptides interact with the EGFR receptor in the vicinity of the EGF binding site, showing substantial overlap with EGF. We assume that P6 and P9 are promising peptide carriers for drug delivery to EGFR overexpressed cancers and their EGFRvIII mutation.Further investigations of the therapeutic efficacies of PDCs based on these peptide carriers in various preclinical models are undergoing.


The present investigation aims to highlight the role of salt priming in mitigating salt stress on faba bean. In the absence of priming, the results reflected an increase in H2O2 generation and lipid peroxidation in plants subjected to 200 mM salt shock for one week, accompanied by a decline in growth, photosynthetic pigments, and yield. As a defense, the shocked plants showed enhancements in ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), peroxidase (POX), and superoxide dismutase (SOD) activities. Additionally, the salt shock plants revealed a significant increase in phenolics and proline content, as well as an increase in the expression levels of glutathione (GSH) metabolism-related genes (the L-ascorbate peroxidase (L-APX) gene, the spermidine synthase (SPS) gene, the leucyl aminopeptidase (LAP) gene, the aminopeptidase N (AP-N) gene, and the ribonucleo-side-diphosphate reductase subunit M1 (RDS-M) gene). On the other hand, priming with increasing concentrations of NaCl (50–150 mM) exhibited little significant reduction in some growth- and yield-related traits. However, it maintained a permanent alert of plant defense that enhanced the expression of GSH-related genes, proline accumulation, and antioxidant enzymes, establishing a solid defensive front line ameliorating osmotic and oxidative consequences of salt shock and its injurious effect on growth and yield. Rhizobium bacteria, which enriches soil nitrogen  × 100.A total of 0.2 g of a freshly collected leaves were incubated in 10 mL of double-distilled water at 40 °C for 30 min, then the electrical conductivity (C formula : MSI (%)v/v) ethanol, according to [−1 DW, compared with the gallic acid standard curve.Polyphenols were extracted from dried leaf samples in 70% (rding to . Thereafrding to  method av/v) glycerol, and 4% (w/v) polyvinylpolypyrrolidone (PVP). The homogenate was centrifuged at 10,000× g for 10 min at 4 °C and supernatant was collected for enzymes assay.The activities of guaiacol peroxidase , catalase , superoxide dismutase , glutathione reductase , and ascorbate peroxidase  were measured according to the method outlined by Grace and Logan , with so2O2, and 100 µL of crude enzyme extract at 465 nm, based on a molar extinction coefficient (ε) of 26.6 mM−1 cm−1. The activity was expressed as µmol tetraguaiacol mg−1 protein min−1.POD activity was determined according to  by measu2O2 (ε = 43.6 M−1cm−1) decomposition at 240 nm in a mixture of 3 mLKP buffer  containing 25 mM of H2O2 mixed with 100 µL of crude enzyme extract [2O2 mg−1 protein min−1.CAT activity was assayed by monitoring the rate of H extract . CAT actSOD activity was estimated as described by , dependi2O2, and 0.1 mM of EDTA, based on ε of 2.8 mM−1 cm−1 for ascorbate.APX activity was measured according to  by monit−1 cm−1), and 1 mM of GSSG [GR activity was assessed by monitoring the rate of decrease in absorbance at 340 nm in a mixture of the crude enzyme extract and KP buffer  containing 3 mM of EDTA, 0.1 mM of NADPH  aqueous sulfosalicylic acid, as described in [w/v) ninhydrin reagent and quantified at 508 nm based on a proline standard curve, then expressed as mg proline g−1 DW.For proline determination, 10 mg of dry leaves material was homogenized in 3% , while first-strand cDNA was synthesized from 0.5 µg of total RNA using a RevetAid First Strand cDNA Synthesis Kit , following the manufacturer’s instructions.The faba bean leaves stored in liquid nitrogen were utilized for RNA extraction with a Qiagen RNeasyL-APX, SPS, LAP, AP-N, and RDS-M genes) were quantitatively amplified with the aid of StepOnePlus  Real-Time PCR system using specific primers . The transcription level of the Vicia faba Elongation Factor-alpha (VfEFα) gene was used as an endogenous control, to which the transcription level of the addressed genes were normalized using the 2−DDCt method. The amplification protocol consisted of 40 amplification cycles (95 °C for 15 s and 60 °C for 60 s) preceded by 95 °C for 10 min. For each gene, expression estimated in Ctrl was used as a quantification unit.The genes related to the glutathione metabolism pathways ( primers  and a SYp < 0.05) using a one-way analysis of variance (ANOVA). The mean values were compared through a Duncan multiple comparison test at the 5% level of significance.The results were expressed as the mean value of three replicates with a calculated standard error (±SE). The significance of the difference between mean values was determined at , proline accumulation, and antioxidant enzymes, which establish a solid defensive front line that counteracts the osmotic and oxidative consequences of salt stress and ameliorates its injurious effect on growth and yield. Thus, the current results open the door for wide application of salt priming to mitigate the injurious effects of salt stress and support food security in the adverse conditions created with climate change.


Background: In clinical studies, some patients who achieve deep molecular response (DMR) can successfully discontinue tyrosine kinase inhibitor (TKI). TKI dose reduction is also an important aspect of alleviating adverse effects and improving quality of life. This study aimed to explore the outcome after drug withdrawal in Chinese CML patients.Methods: We conducted a retrospective analysis of the outcome of 190 patients who stopped TKI. 27 patients experienced dose reduction before TKI discontinuation. The median duration of TKI treatment and MR4 before discontinuation was 82 months and 61 months.Results: With median follow-up after stopping TKI treatment of 17 months, the estimated TFR (Treatment Free Remission) were 76.9% , 68.8% , and 65.5%  at 6, 12 and 24 months. For full-dose and low-dose TKI groups, the TFR at 24 months was 66.7% and 55.8% . Most patients (56/57) quickly achieved MMR after restarting TKI treatment. Multivariable analysis showed that patients with TKI resistance had a higher risk of molecular relapse than patients without TKI resistance (p < 0.001).Conclusion: TFR rates were not impaired in patients experiencing dose reduction before TKI discontinuation compared to patients with full-dose TKI. Our data on Chinese population may provide a basis for the safety and feasibility of TKI discontinuation, including discontinuation after dose reduction, in clinical practice. The advent of tyrosine kinase inhibitor (TKI) has revolutionized the treatment of chronic myeloid leukemia (CML) and improved the prognosis of patients to near-normal levels . HoweverWe collected data from CML-CP patients who discontinued TKI between June 2013 and July 2022 from the three hospitals in China . All patients were diagnosed with CML-CP by bone marrow cell morphology, cytogenetics, and molecular biology examination. All patients who discontinued TKI were treated with TKI for at least 3 years, and MR4 lasted at least 2 years.BCR-ABL 1 level was expressed on the international scale (IS). The Molecular response was assessed according to ELN criteria . The criteria . In detaU test, and comparison of categorical variables was tested by χ2 or Fisher exact test. TFR was estimated using the Kaplan-Meier method, and comparison between groups was used log-rank tests. Univariate analysis of potential clinical factors associated with molecular relapse was performed using the Kaplan-Meier method. Multivariate analysis was performed using the Cox regression analysis. Proportionality hazards assumptions were checked for factors included in Cox regression analysis. All analyses were carried out using SPSS, version 22 statistical software, and GraphPad Prism 7.Comparison of continuous variables was tested by Student t-test or Mann-Whitney n = 48) before TKI discontinuation according to ELN criteria  , Table 1criteria .n = 17), desire to stop TKI (n = 8), and finical burden (n = 2)  . The med (n = 2) .p = 0.242, log-rank)  , 58 patients lost MMR, nine patients lost MR4, and 123 patients remained MR4 or deeper. The estimated overall TFR were 76.9% , 68.8% , and 65.5%  at 6, 12 and 24 months . For fulog-rank) . Moreoveog-rank) . Our datn = 39) or not (n = 9), patients without imatinib-resistant who switched to 2G-TKI therapy (n = 42) or not (n = 92) and patients treated with 2G-TKI in the first line (n = 8). For patients with imatinib resistance, those who switched to 2G-TKI therapy had remarkably higher TFR rates than those who did not    . Similarog-rank) . We alsoog-rank) . These rn = 27; imatinib 600 mg/d, n = 2; imatinib 200 mg/d, n = 4; dasatinib 100 mg, n = 2; nilotinib 300 mg/d, n = 2; nilotinib 600 mg/d, n = 9; Flumatinib 600 mg/d, n = 3), of which 49 patients achieved MMR and 41 patients achieved MR4. For the low-dose group, eight patients were restarted on imatinib treatment  and six of them obtained MR4. At last follow-up, one patient died of non-CML-related disease, one patient was not in MMR, 18 patients were in MMR, and 170 patients were in MR4 or deeper response.In the present study, 58 patients lost MMR at a median time of 4 months , with 42 (72.4%) patients losing MMR within 6 months, 54 (93.1%) patients lost MMR within 12 months. At the last follow-up, 56/57 patients who were restarted TKI regained MMR with a median time of 3 months , and 47 patients regained MR4 with a median time of 4 months . For the full-dose group, 49 patients were retreated with TKI  developed transient musculoskeletal pain within 12 months after TKI discontinuation, of which 10 patients were on imatinib and 4 patients were on nilotinib. 13 patients were in standard-dose group, only one patient was in the de-escalation group. Thirteen patients reported a grade 1–2 TKI withdrawal syndrome and one patient reported a grade 3 TKI withdrawal syndrome. Three patients lost MMR after the occurrence of musculoskeletal pain.Currently, long-term TKI treatment is no longer indicated for all patients with CML, as some patients who obtain DMR can safely discontinue TKI. For example, the DASFREE study showed the 1-year TFR rate was 48%, which was 58% in the ENESTop study . The EURn = 17), desire to stop TKI (n = 8), and finical burden (n = 2). Patients with low-dose TKI treatment had a TFR of 63.8% at 6 months and 55.8% at 12 months. Similarly, patients with full-dose displayed a TFR of 78.4% and 70.3% at 6 months and 12 months, respectively. In the study of At present, dose optimization is increasingly emphasized as an important part of individualized treatment for CML patients with different demands. In the present study, 27 patients experienced dose reduction before stopping TKI for adverse events (p = 0·017) compared with patients treated with imatinib (In the univariate analysis, the type of TKI, TKI resistance, line of therapy, and time to MR4, was the clinical variable significantly associated with molecular recurrence. Multivariate analysis showed patients with TKI resistance presented with significantly lower TFR rates than those without resistance. Similarly, imatinib resistance is an important risk factor for molecular relapse in the DADI trial . In ENESimatinib . In geneSeveral studies reported some patients developed or worsened musculoskeletal pain after discontinuing TKI, suggesting the presence of withdrawal syndrome. For example, the KID study reported that 30% of patients underwent withdrawal syndrome after TKI withdrawal . Berger In conclusion, this retrospective study showed TKI discontinuation in the real-world is as feasible and safe as in clinical studies. Indeed, dose optimization is an important consideration in individualized treatment for patients with different demands. Moreover, we found TFR rates were not impaired in patients experiencing dose reduction before TKI discontinuation compared to patients with full-dose TKI. Our data may provide a basis for the safety and feasibility of dose optimization before drug withdrawal. For patients with a need for discontinuation, second-generation TKI therapy may yield higher TFR rates than imatinib, especially for imatinib-resistant patients. However, our study still has limitations as a retrospective study. In addition, only 27 patients discontinued low-dose TKI in this study, and the data on discontinuation after dose reduction may be biased. Some patients with a long history of disease did not have BCR-ABL1 mutations test, so we did not analyze the effect of BCR-ABL1 mutations on TFR. As real-world data are still increasing, more studies are needed to accumulate data in daily practice to confirm the feasibility of dose reduction.