Conyza blinii H. Lév is a medicinal plant that has a variety of pharmacological activities, but its study is at a standstill due to the shortage of resources.2-Al(NO)3 colorimetry and further identified by the ITS sequence. Additionally, we used five antioxidant assays  to systematically evaluate the antioxidant capacity of total flavonoids , and we also determined their antibacterial activity.This study utilized the surface sterilization method to isolate endophytic fungi, and they were preliminarily identified by morphology. Flavonoid-producing strains were screened by NaNOConyza blinii H. Lév for the first time. There were six flavonoid-producing strains, especially CBL11, whose total flavonoid content reached 50.78 ± 2.4 mg/L. CBL12, CBL12-2 and CBL1-1 all exhibited excellent antioxidant activity. The effect of CBL12 was similar to that of ascorbic acid at low concentrations, and its radical scavenging rates for DPPH and ABTS were 94.56 ± 0.29 % and 99.88 ± 0.27%, respectively, while its IC50 values were only 0.11 ± 0.01 mg/mL and 0.2 ± 0.01 mg/mL. Through LC-MS, we found that CBL12 could produce many high-value flavonoids, such as 3-methoxyflavone, nobiletin, formononetin, scopoletin, and daidzein. Additionally, CBL9 had good antibacterial activity against both gram-positive and gram-negative bacteria. Notably, we obtained the high-yield strains CBL12 and CBL9, which not only had high yields  but also had excellent biological activity. Hence, the results of this study provide new ideas for endophytic fungi that can be exploited as a source of flavonoids and other medicinal components from Conyza blinii H. Lév. Moreover, this study can serve as a reference for the development of rare medicinal materials.In this study, 21 endophytic fungi were isolated from wild  Taxomyces andreanae) that produced Taxol from Taxus brevifolia , Pseudomonas aeruginosa (ATCC9027), Bacillus subtilis (ATCC6633), and Staphylococcus aureus (ATCC6538), were used to test the antibacterial activities of the extraction.Four bacteria, including Conyza blinii H. Lév, two control groups were set up. In one group, the disinfected tissue was directly inoculated on the medium without cutting, and in another group, the last rinse solution was spread on the medium. If both control groups grew without any microorganisms, we could be sure that the fungi we obtained were the endophytic fungi of Conyza blinii H. Lév.The leaves and stems were washed with running water and immersed in distilled water until the samples were saturated and restored. First, the tissue masses were immersed in 75% ethanol solution for 1 min, followed by 4% sodium hypochlorite solution for 3 min. Then, they were rinsed with sterile distilled water 3–4 times. Next, the surface-sterilized parts were cut into 1 cm × 1 cm tissue masses with a sterile scalpel and inoculated onto potato dextrose agar (PDA) medium with 50 mg/L ampicillin (AMP). Finally, these plates were incubated in a 28 °C constant temperature incubator for 7–9 days. Meanwhile, to ensure the fungi isolated from this experiment were the endophytic fungi of When hyphae grew out at the edge of the tissue wound, different hyphae were inoculated separately into fresh PDA culture medium (AMP) and placed in a 28 °C constant temperature incubator for 5–7 days until the hyphae were separated and stored at 4 °C.We observed and recorded the growth rate of hyphae, the morphology of colonies and pigment production. Microscopic identification was carried out by the cover slip culture method, which could determine the characteristics of the different hyphae. A sterilized cover slip was obliquely inserted into the growth edge of the colony. When the hyphae were attached to the cover slip, the cover slip was removed from the medium. Then, conidial structure and a hyphal septum were observed under a 10 × 40 field of vision (microscope model: OLYMPUS CX23) that was recorded by a camera. Finally, these fungi were classified through a fungal identification manual.Twenty-one endophytic fungi were inoculated into 100 mL of fresh PDA liquid medium  and then cultured on a shaker for 7 days (160 rpm/min and 28 °C).After fermentation, the liquid was filtered out and extracted twice with 25 mL of ethyl acetate. After that, the extracted liquid was collected and concentrated by vacuum rotary evaporation until the product became dry. Then, methanol was added up to 5 mL, and the solution was filtered with a 0.22 µm syringe filter.2-Al(NO)3 colorimetry, which was used for generating a rutin standard curve. First, a 20 mg rutin standard was dissolved with 100 mL of 60% ethanol to obtain the mother liquor, and 2.5 mL of the solution was pipetted into 2.5 mL of 60% ethanol. Eventually, the standard solution concentration was 0.1 mg/mL and diluted to a gradient of 6 concentrations. In each test tube, 60 µL of one of six different concentrations of the standard solution was added, followed by 20 µL of 5% NaNO2, standing for 6 min, the addition of 20 µL of 10% Al(NO3)3, standing for 6 min, and the addition of 1 mol/L NaOH. Finally, a solution of 60% ethanol was utilized to reach a volume of 200 µL, followed by standing for 15 min, and the absorbance of the mixture was measured at 510 nm .The total flavonoid content of ethyl acetate was evaluated by NaNOThe fermentation broth concentration was determined according to the standard system. All the steps were the same as those above, and each group had 3 parallel controls. The flavonoid content of each sample was calculated with reference to the rutin standard curve.Total flavonoid content = (mass concentration × dilution ratio × methanol solution volume)/total volume of fungal fermentation broth × 100%.The fungal genome DNA was extracted by the SDS method, and the universal primers ITS1 and ITS4 were used to amplify the internal transcribed spacer (ITS) region (5.8S rRNA gene) of these fungi by PCR. The reaction product was detected by agarose gel electrophoresis, and when the fragment sizes were all between 500 bp and 750 bp (DNA Marker: Solarbio D2000), the results showed that ITS sequence amplification was successful. Then, the PCR products were recovered, and the purified DNA samples were sequenced by Invitrogen/Thermo Fisher Scientific (Shanghai).After successful sequencing, the DNA sequences were compared by BLAST, similar sequences were downloaded, and a phylogenetic tree alignment was performed with MEGA 7.0 software by the neighbor-joining method.PDA liquid medium was used for fermenting the 6 flavonoid-producing endophytic fungi for 7 days under the same conditions , and the fermentation was extracted twice with ethyl acetate according to the method in 2.4. The extraction was performed through vacuum rotary evaporation until the product became dry; 5 mL of absolute ethanol was added to dissolve the sample and then filtered with a 0.22 µm syringe filter.3+ reducing power, and total antioxidant capacity assays) and stored in a freezer at −20 °C. SPSS 23.0 was used for statistical analyses.The prepared solution was freeze-dried and adjusted to 1 mg/mL. The abovementioned 6 kinds of fermentation broth and the positive control (ascorbic acid) were adjusted to five concentrations  for five antioxidant activity assays  free radical scavenging, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, hydroxyl radical scavenging, FeThe DPPH radical has a single unpaired electron that results in strong absorption at 517 nm, while its alcohol solution is purple. When a free radical scavenger is present, its absorption gradually disappears due to the pairing of its single electron, which could be widely used in the analysis of antioxidant capacity .DPPH powder (25 mg) was accurately weighed and dissolved in 50 mL of anhydrous ethanol. Then, it was transferred to a 100 mL volumetric flask and diluted to a constant volume. Finally, a 0.25 mg/mL DPPH-ethanol solution was obtained and stored in a 4 °C refrigerator.Y is the DPPH clearance rate, A0 is the absorbance of 100 µL of DPPH solution plus 100 µL of anhydrous ethanol, A1 is the absorbance of 100 µL of DPPH solution mixed with 100 µL of gradient sample solution, and A2 is the absorbance of 100 µL of gradient sample solution plus 100 µL of anhydrous ethanol.Then, 100 µL of DPPH solution and 100 µL of gradient sample solution were added to an enzyme label plate and mixed on the basis of the concentration gradient, and the sample solution was incubated in the dark for 30 min at room temperature (24 °C). The OD value was measured at 517 nm  with anhydrous ethanol as the blank control, and each experimental group had 3 parallel controls. ABTS is oxidized to green ABTS ⋅ + with appropriate oxidants, and the production of ABTS ⋅ + is inhibited in the presence of antioxidants .ABTS powder (78 mg) was accurately weighed and added to 13.2 mg of potassium persulfate into 20 mL of ultrapure water at 4 °C, which was stored in the dark for 16 h. After stabilization, the solution was stored as an ABTS stock solution. Before the ABTS radical scavenging experiment, the ABTS stock solution was diluted with anhydrous ethanol to an absorbance of 0.7  ± 0.02. Then, 2 mL of ABTS solution was mixed with 100 µL of gradient sample solution and incubated for 6 min. Finally, 200 µL of supernatant was aspirated and added to an ELISA plate. The absorbance was measured at 734 nm using anhydrous ethanol as a blank control, and each experimental group had 3 parallel controls. Y is the clearance rate of ABTS, A0 is the absorbance of ABTS solution mixed with anhydrous ethanol, A1 is the absorbance of ABTS solution mixed with the sample, and A2 is the absorbance of the sample mixed with anhydrous ethanol.Hydroxyl radicals have a strong ability to acquire electrons, and hydroxyl radical scavenging activity is a vital indicator of antioxidant activity. We used the Fenton reaction to assay this activity .2O2 (0.1%) solutions and a sample concentration gradient of 0∼1.0 mg/mL were prepared for this experiment. The reaction system was as follows: 75 µL of 1,10-phenanthroline monohydrate solution, 200 µL of PBS buffer solution, and 500 µL of each gradient sample in sequence. The sample was mixed, and then 50 µL of ferrous sulfate solution and 100 µL of H2O2 solution were added and mixed; the sample was incubated in a 37 °C water bath for 30 min. The absorbance was measured at 510 nm with 200 µL samples added to the ELISA plate, and each experimental group had 3 parallel controls. First, 1,10-phenanthroline monohydrate (5 mmol/L), PBS  buffer , ferrous sulfate (7.5 mmol/L), and HY is the clearance rate of the hydroxyl group, A1 is the absorbance of a reaction solution containing the sample solution and H2O2, A0 is the absorbance of a blank solution with the sample solution replaced with anhydrous ethanol, and A2 is the absorbance of a solution with distilled water instead of H2O2.3+-reducing antioxidant power (FRAP) assays measure the antioxidant capacity of samples using ferrous ions and TPTZ (tripyridyltriazine) to form a blue-violet complex at low pH. They are widely used in the analysis of antioxidant capacity  and 250 µL of 1% potassium ferricyanide solution were added to an Eppendorf tube, vortexed, and then placed in a 50 °C water bath for 20 min. At this time, the solution was removed and cooled to room temperature (24 °C), and 250 µL of 10% trichloroacetic acid (TCA) solution was added; the mixture should not be exposed to light during this step. Thereafter, the sample was centrifuged at 3,000 rpm for 10 min, and then 300 µL of supernatant was aspirated and added to a solution containing 300 µL of distilled water and 60 µL of 0.1% FeCl1 is the absorbance of the supernatant mixed with distilled water and FeCl3 solution, and A2 is the absorbance of supernatant mixed with distilled water.The absorbance indicates the reducing power, N is the reducing power of Fe, AThe following reaction system  was cons2O was set as the blank control. The absorbance of each tube was measured at 520 nm with a light beam diameter of 1 cm. A total antioxidant capacity unit was defined as a milliliter of sample increasing the absorbance (OD) value by 0.01 per minute at 37 °C. Each experimental group had 3 parallel controls. Y represents the antioxidant capacity of total flavonoids, A0 is the absorbance of the test tube used as a control, and A1 isthe absorbance of the test tube.Samples were fully mixed by a vortex generator, placed into a 37 °C water bath for 30 min, and then removed and cooled with cold water immediately. Then, another system  was addeSix flavonoid endophytic fungi were inoculated in 100 mL of fresh PDA liquid medium and cultured at 28 °C and 160 rpm for 7 days. The fermentation broth was then removed, and the filtrate was extracted with 25 mL of ethyl acetate. Ethyl acetate was evaporated to dryness with vacuum rotary evaporation, and the sample was dissolved by adding 5 mL of methanol. Then, the solution was stored in a freezer at −20 °C.Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus) were activated by 10 mL of LB medium and cultured at 37 °C, then shaken at 180 rpm for 12 h. Next, the sterilized LB liquid was used to dilute the bacterial solution 1,000 fold.Four bacteria  for the non-targeting LC-MS metabolomic test.Conyza blinii H. Lév, comprising 12 strains from the leaves and 9 strains from the stems, and the corresponding endophytic fungi were numbered in order. The endophytic fungi isolated from Conyza blinii H. Lév are shown in In our study, 21 different endophytic fungi were isolated from the leaves and stems of Different endophytic fungi were cultured in fresh PDA medium, and then the morphological characteristics of each colony were recorded and observed to determine whether any pigment was produced. Pictures of each fungus colony are shown in Conyza blinii H. Lév mainly belong to basidiomycetes, deuteromycota, ascomycota and rhizopus, which indicated that there were diverse endophytic fungi isolated from Conyza blinii H. Lév . Then, on this basis, the total flavonoid contents from 21 different fermentation broths were determined by NaNO2-Al(NO3)3 colorimetry, and the results suggested that six strains had the potential to produce flavonoids . The results showed that all fragments were between 500 bp and 750 bp in length, which proved that ITS sequence amplification was successful, and the DNA marker was D2000 .Chaetomium globosum and a species of Xylariaceae. After that, the sequence information of 10 strains with the best aligned sequences was downloaded, and MEGA7.0 software was used for phylogenetic tree analysis to further analyze the relationships of these fungi. Combining phylogenetic tree and previous morphological identification, we can further determine the species of the flavonoid-producing endophytic fungi from Conyza blinii H. Lév ; subsequently, a homologous sequence alignment of ITS sequences was uploaded to NCBI BLAST for comparison to obtain relevant homologous strain and evolution information. The results are shown in i H. Lév .50 values were 0.11 ± 0.01 mg/mL (CBL12), 0.14 ± 0.01 mg/mL (CBL1), 0.15 ± 0.03 mg/mL (CBL9), and 0.10 mg/mL ascorbic acid .All samples within the concentration range of 0∼1.0 mg/mL scavenged DPPH free radicals, and this activity was positively correlated with the sample concentration. The DPPH radical scavenging rates of each sample at the highest concentration (1.0 mg/mL) followed the descending order of CBL1-1 (97.89 ± 0.38%), ascorbic acid (96.86 ± 0.20%), CBL12 (94.56 ± 0.29%), CBL1 (94.55 ± 0.47%), CBL9 (94.24 ± 0.30%), CBL12-2 (84.86 ± 1.01%) and CBL11 (62.36 ± 1.88%). Moreover, at the lowest concentration (0.2 mg/mL), scavenging abilities of CBL12, CBL1, and CBL9 for DPPH free radicals were almost the same as that of the positive control (ascorbic acid) . Their I50values of these samples were 0.2 ± 0.01 mg/mL (CBL12), 0.13 ± 0.06 mg/mL (CBL12-2), and 0.11 mg/mL (ascorbic acid) .The results also showed that the ABTS radical scavenging rates of each sample at the highest concentration (1.0 mg/mL) followed the descending order of CBL12 (99.88 ± 0.27%), CBL12-2 (99.63 ± 0.34%), ascorbic acid (99.40 ± 0.29%), CBL11 (95.30 ± 2.27%), CBL1 (93.69 ± 2.79%), CBL1-1 (53.08 ± 1.60%), and CBL9 (42.63 ± 3.48%). At 0.4 mg/mL, the ABTS radical scavenging rate of CBL12 was basically equal to that of ascorbic acid . The IC550values of the active samples were 0.19 ± 0.02 mg/mL (CBL12), 0.19 ± 0.03 mg/mL (CBL1-1), and 0.96 ± 0.06 mg/mL (ascorbic acid) . These results fully demonstrate that the flavonoids from CBL12 and CBL1-1 exhibit excellent antioxidant activity.Hydroxyl free radical scavenging activity was observed in all samples in the concentration range of 0∼1.0 mg/mL, and the hydroxyl radical scavenging rates of each sample at the highest concentration (1.0 mg/mL) followed the descending order of CBL12 (81.89 ± 6.53%), CBL1-1 (74.28 ± 4.95%), ascorbic acid (58.60 ± 1.53%), CBL9 (57.36 ± 3.56%), and CBL11 (54.13%). The effects of CBL12 and CBL1-1 were obviously better than that of ascorbic acid . AdditioThe statistical analysis of each IC50 value is shown in 3+-reducing power of other samples increased with increasing sample concentration. The absorbance values of each sample at the highest concentration (1.0 mg/mL) followed the descending order of CBL12 (2.346 ± 0.023), ascorbic acid (2.288 ± 0.010), CBL12-2 (1.313 ± 0.049), and CBL1 (1.093 ± 0.081)   .The T-AOC of each sample followed the descending order of ascorbic acid, CBL12, CBL1, CBL1-1, CBL12-2, CBL11, and CBL9, while the T-AOC of CBL12 was almost as same as that of ascorbic acid .Through these five antioxidant experiments, we can easily summarize that the flavonoid-producing endophytic fungi have extremely high antioxidant activity associated with their flavonoids and have excellent prospects. The flavonoids from strain CBL12, whose activity was similar to that of ascorbic acid, have good medical implications and deserve further research.Escherichia coli, with an inhibition zone diameter of up to 24.67 ± 1.15 mm. Other inhibition zone diameters of CBL9 were as follows: S. aureus (19.67 ± 2.52 mm), Bacillus subtilis (23 ± 2 mm), and Pseudomonas aeruginosa (17.33 ± 2.08 mm) (P < 0.05). . . In addiAmpicillin and methanol were set as positive and negative controls, respectively, and each experimental group had 3 parallel controls.Because of the excellent antioxidant activity of CBL12, we used LC-MS to preliminarily identify the flavonoids from CBL12. The results showed that there were various flavonoids produced by the endophytic fungus CBL12, including multiple high-value flavonoids, such as 3-methoxyflavone, nobiletin, formononetin, scopoletin, daidzein, etc. The representative compounds and specific information are shown in Conyza blinii H. Lév for the first time. Endophytic fungi from medicinal plants produce a large number of biologically active secondary metabolites due to the interactions between endophytic fungi and the host plant, so we isolated endophytic fungi with rich secondary metabolites that have excellent biological activity from Conyza blinii H. Lév. Because endophytic fungi have host specificity and tissue specificity and are relevant to the plant environment  to set a good standard for evaluating the antioxidant capacity of samples  with an IC50 of only 0.11 ± 0.01 mg/mL, which was similar to ascorbic acid at an equivalent concentration. Furthermore, according to the LC-MS results, CBL12 had the potential to produce abundant high-value flavonoids, so there was no doubt that it will have broad prospects as a resource for natural antioxidant agents and promote more medicinal research of Xylariales. For instance, based on our study, these flavonoids can be produced by microbial fermentation, allowing economical production of these compounds on an industrial scale  but also excellent biological activity. This meant that the two flavonoid-producing endophytic fungi have better application prospects for medicine and will promote research on endothytic fungi from rare medicinal plants.Meanwhile, the results of a recent study suggested that flavonoids also have significant antibacterial activity due to their function of inhibiting several bacterial virulence factors . In our Plant accumulation of secondary metabolites has spatiotemporal specificity , and theSalvia miltiorrhiza endophytic fungus (Chaetomium globosum D38) significantly enhances the accumulation of tanshinone and salvianolic acid by the host. Moreover, this study combined rare medicinal materials with endophytic fungi to give excellent guidance for the modernization of Traditional Chinese Medicine, providing a new way to explore the high-value compounds from these insufficient sources and promote related research. This is the first study on the endophytic fungi of Conyza blinii H. Lév, which increases the knowledge in this field, and we find that these endophytic fungi can produce high-value flavonoids. We also hope that we can find additional novel compounds from these sources in the future and will continue to explore whether the fungi can accelerate Conyza blinii H. Lév growth or expedite the accumulation of secondary metabolites.In addition, as endophytic fungi have a close relationship with their host plant, some endophytes can produce the same compound as the host plant , impact Conyza blinii H. Lév for the first time, and via morphology identification, the species were preliminarily determined. We obtained six flavonoid-producing strains, and among them, strain CBL11 had the highest yield, reaching up to 50.78 ± 2.4 mg/L, which was significantly higher than other strains previously reported. ITS sequence analysis indicated that the six strains mainly belong to Chaetomium globosum and Xylariaceae. Then, we used five experiments  to systematically evaluate the antioxidant capacity of total flavonoids in fermentation broth. The results showed that CBL12, CBL12-2 and CBL1-1 all had excellent antioxidant activity; remarkably, the effect of CBL12 was similar to that of ascorbic acid at low concentrations (0.2 mg/mL), and its scavenging rates of DPPH, ABTS, and hydroxyl radicals were 94.56 ± 0.29%, 99.88 ± 0.27%, and 81.89 ± 6.53%, respectively. The IC50 values were only 0.11 ± 0.01 mg/mL, 0.2 ± 0.01 mg/mL, and 0.13 ± 0.06 mg/mL, respectively. Notably, LC-MS results suggested that there were various flavonoids produced by the endophytic fungus CBL12, including multiple high-value flavonoids, such as 3-methoxyflavone, nobiletin, formononetin, scopoletin, and daidzein. In addition, CBL9 showed good antimicrobial activity; specifically, it was found to inhibit both gram-positive and gram-negative bacterial growth, with inhibition zone diameters reaching 24.67 ± 1.15 mm for Escherichia coli and 23 ± 2 mm for Bacillus subtilis.In this study, 21 strains of endophytic fungi were isolated from the rare medicinal plant Conyza blinii H. Lév and screen the high-flavonoid strain CBL11; the high-total flavonoid strain CBL12, with excellent antioxidant activity; and the strain CBL9, with antibacterial activity. Furthermore, this work compensates for the absence of research on Conyza blinii H. Lév, provides a new idea for the development of rare medicinal materials, and lays the foundation for the drug exploitation of Conyza blinii H. Lév.This study was the first to isolate endophytic fungi from wild 10.7717/peerj.8978/supp-1Table S1Click here for additional data file.10.7717/peerj.8978/supp-2Data S1Click here for additional data file.10.7717/peerj.8978/supp-3Data S2Click here for additional data file.


Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens, which are encoded by a multigene family called var genes, are exported and inserted onto the surface of the infected erythrocytes. PfEMP1 plays a key role in the pathogenesis of severe malaria and are major targets of naturally acquired immunity. Studying the expression pattern of var genes in P. falciparum clinical isolates is crucial for understanding disease mechanism and immunity to malaria. However, var genes are highly variable, which makes it difficult to study their expression in clinical isolates obtained directly from malaria patients. In this chapter, we describe an approach for analysis of var gene expression that targets a region referred to as DBLα tag, which is relatively conserved in all var genes.The  There are about 60 var genes in the haploid genome of each isolate [var genes are made up of two exons. Exon 1 is highly variable and encodes for the part of the PfEMP1 that is exposed on the infected erythrocyte surface. This part of PfEMP1 is composed of a combination of multiple domains of Duffy binding-like (DBL) and cysteine-rich interspersed region (CIDR) domains and the N-terminal segment (NTS) [nt (NTS) . Even thnt (NTS) , 4. Exonvar genes in severe malaria [var genes, makes it difficult to study var gene transcription in clinical isolates. However, a number of studies have shown that var genes have a semiconserved head structure [var genes and formed part of the semiconserved head structure [var sequence [Several studies have demonstrated the importance of  malaria –12. Sero malaria –17. Clintructure  designedsequence .var genes containing DBLα-tag sequence with a reduced number of cysteine residues have been shown to predominantly fall under group A and to be preferentially transcribed by isolates from children with severe malaria and low host immunity [var gene transcription using the DBLα tag. We describe the use of DBLα sequencing from clinical isolates and counting of the var gene sub-groups as a proportion of the var, as well as the use of var expression homogeneity (VEH) [immunity , 20–22. ty (VEH) .22.1Incomplete medium “yellow RPMI”: Roswell Park Memorial Institute (RPMI) 1640, 1 mM L-glutamine, 25 μg/mL gentamicin sulphate, 200 mg D-glucose/mL.Lymphoprep or any density gradient medium.Plasmion.Pasteur pipettes.Sterile heparinized vacutainers.15 mL centrifuge tubes.2.2TRIzol.2.0 mL Apex tubes.2.3Chloroform.98% Isoamyl alcohol (IAA).75% Ethanol made with RNase free water. Store at –20 °C.GlycoBlue.RNA Secure (RNA resuspension reagent).RNase-free water (DEPC water).RNase Zap.1.5 mL Safe T-seal conical tubes and caps.2 mL Safe T-seal tubes and caps sterile.RNase-free filter tips.RNA pipettes and rack specific for RNA work only.2.4Superscript III reverse transcriptase and kit.Ambion DNAse enzyme.Ambion DNase inactivation reagent.PCR strip tubes.2.5Forward primer: DBLαAF (GCACGMAGTTTYGC).Reverse primer: DBLαBR (GCCCATTCSTCGAACCA).High fidelity DNA polymerase kit with proof reading ability.2.PCR reaction mixture: 0.2 μM for each primer, 0.2 mM dNTP mix, 0.2 U Amplitaq polymerase and 1.5 mM MgClAgarose powder.1× TAE Buffer containing 40 mM Tris Base, 40 mM Acetate and 1 mM EDTA, pH 8.5.DNA stain.DNA gel loading dye.2.61× TE buffer:10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA.Sephacryl S-400 high resolution.Microspin columns.2.7Topo pCR2.1 TA vector and Kit.E. coli cells.One Shot chemically competent Top 10 LB agar: 20 g/L Agar, 10 g/L NaCl, 10 g/L Tryptone, 5 g/L Yeast extract.SOC medium: 5% yeast extract, 2% tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4. Add 20 mM glucose after autoclaving.20 mg/mL 5-bromo-4-chloro-3-indolyl-β-D-galactopyrano-side .100 mg/mL ampicillin antibiotic.2.8For capillary sequencing, you will require a sequencing kit, which includes sequencing buffer and Big Dye 3.1, and the primers targeting the plasmid used for cloning such as M13 forward (5-′-GTAAAACGACGGCCAG-3′) and M13 reverse (5-′-CAGGAAACAGCTATGAC-3′).33.1Collect 2–5 mL venous blood sample into sterile heparinized vacutainers and store at 4 °C.Transfer the blood to a sterile 15 mL centrifuge tube under a laminar flow hood.g for 5 min and remove supernatant (plasma).To separate plasma from the cellular components, centrifuge the blood at 440 × Resuspend the remaining cells in 5 mL buffered incomplete RPMI 1640 medium.g for 20 min to separate PBMCs  from the freezer and let them thaw in the fume hood.Transfer the thawed TRIzol sample directly into prelabeled RNA tubes.Using P1000 filtered tips, add 200 μL of chloroform, shake vigorously for 15 s and let the sample stand for 2–3 min.g at 4 °C for 35 min.Spin at 1660 × Label fresh RNA tubes and add 2 μL GlycoBlue into them.Gently remove the samples from the centrifuge and let them rest for 2 min.Harvest the clear aqueous phase into the GlycoBlue-containing RNA tubes and add 500 μL isoamyl alcohol.Mix thoroughly by inverting and shaking the tubes at least ten times.Incubate for at least 2 h at 4 °C for the RNA to precipitate.g for 30 min at room temperature.Spin at 15,900 × Gently wash the pellet in ice-cold 500 μL of cold 75% ethanol by inverting and pour off the supernatant. Do a short spin to bring down the remaining ethanol and aspirate off the supernatant using a P20 pipette and filtered tip.Invert the tubes on tissue paper and let them dry for no longer than 5 min. Make sure not to disrupt the RNA pellet.Add 20 μL RNA secure and heat at 60 °C for 10 min.g for 10 s on the microfuge (seeGently mix the RNA suspension by pipetting up and down using a P20 pipette then spin at 376 × fuge see.Store at –80 °C awaiting complementary DNA (cDNA) synthesis.RNA can be extracted using commercial kits; here we describe a method traditionally used for RNA extraction .Turn on3.4To remove any contaminating DNA, digest RNA using 1 μL of Ambion DNAse enzyme for 20 min at 37 °C.To remove the DNase, add 3 μL of Ambion DNase inactivation reagent to the reaction, mix and stand for 2 min at room temperature.g for 1.5 min and transfer two aliquots of 8 μL of supernatant containing RNA into clean PCR strip tubes.Centrifuge at 9408 × seeNext, reverse transcribe all the RNA to make the first strand using the SuperScript III kit in the presence of random hexamers and dNTPs according to the manufacturer’s instructions see.Two microliters of extracted RNA is used to make (cDNA) using the Superscript III kit according to the manufacturer’s protocol.3.5var genes being expressed by each P. falciparum isolate, degenerate primers DBLαAF (GCACGMAGTTTYGC) and DBLαBR (GCCCATTCSTCGAACCA) targeting the semiconserved DBLα-tag sequence are used for amplification [Prepare a 25 μL PCR reaction mixture including a 2 μL template from the cDNA reaction.Run the PCR reaction using the following conditions: denaturation 95 °C, annealing 42 °C, extension 65 °C, and a final extension of 65 °C for 30 cycles on thermocycler.Prepare a 2% agarose gel by mixing 2 g agarose powder with 100 mL 1 × TAE buffer in a microwavable flask and microwave for 30 s or until the powder melts completely.After cooling to about 50 °C, add a preferred gel stain see  to a finUsing a suitable loading dye, load and fractionate 5 μL of the amplified PCR product on the agarose gel for 90 min at 90 kV.View the stained gel under ultraviolet light in a transilluminator for the expected product size of 350–450 bp.To capture the majority of the fication .Prepare3.6To prepare the column, place empty microspin columns in eppendorf tubes/collection tubes.g for 1 min at room temperature.Add 700 μL of the Sephacryl S-400 into the empty microspin columns and centrifuge at 738 × Empty the collection tube and add 200 μL TE buffer into the microspin column.steps 2 and 3 above centrifuging columns at 738 × g for 1 min at room temperature.Repeat Transfer microspin column with Sephacryl into a clean and labeled collection tube.g for 1 min at room temperature.Add 20 μL of the PCR product  into the microspin column and centrifuge at 738 × Collect and store the flow-through as cleaned PCR product and discard microspin column.For capillary sequencing, aliquot 2 μL for the ligation reaction described below (Subheading 3.7) while for next generation, process the product for sequencing directly using appropriate library preparation kit.3.7Prepare a ligation mixture using 1 μL salt solution, 2.5 μL water, 0.5 μL TOPOpCR2.1 vector.Add 2 μL or the cleaned PCR product on the bench and incubate at room temperature for 5 min.E. coli cells from liquid nitrogen and thaw on ice.Retrieve frozen One Shot chemically competent Top 10 Prewarm SOC medium at 37 °C.E. coli cells and allow to stand for a few minutes.Gently mix 2 μL of the ligation reaction into a vial of Transfer the mixture into a water bath at 42 °C for 30 s before putting back on ice.Add 1 mL of the prewarmed SOC medium into each transformation reaction and incubate at 37 °C for 1 h.Inoculate the transformation culture on plates containing LB agar, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside  substrate, and ampicillin antibiotic for selection of recombinant clones.Incubate the plates overnight at 37 °C.seePick and sequence white single colonies see using M1We have used both capillary sequencing and next-generation 454 sequencing for the DBLα tag. Here, we describe the cloning and sequencing approach we use for capillary sequencing.3.8Following base-calling using Phred software, remove/clip low quality ends and assemble the forward and reverse reads.Translate to obtain an open reading frame and capture DBLα tags by use of semiconserved features including DIGDI, VW, WW, and PQYLR motifs as described in Bull et al. .var1 sequences from the analysis.Exclude any sequences that encode peptides shorter than 100 amino acids  and remove the constitutively expressed Classify the tags obtained into Cys2 for those containing two cysteines, Cys4 for those with four and CysX for those containing one, three, five, or six cysteines.Further classification can be done based on the presence or absence of the MFK/REY motifs into Cys/PoLV groups 1–6  as folloAn alternative approach is the use of a network of recombining sequences to define blocks of sequences that tend to recombine with each other. This algorithm uses block sharing groups (BS groups) made up of polymorphic sequence blocks together with the number of cysteines in the DBLα tag .var genes  belong to group A-like enes see.P. falciparum isolate falling into each of these groups and express as a proportion of the total number of reads obtained for the isolate.Count all the reads per var expression homogeneity (VEH) index can also be calculated. VEH is defined as the extent to which a small number of var gene sequences dominate an isolates expression profile [var profile. Thus, the lower VEH the more heterogeneous an isolate’s var expression profile.Following DBLα classification,  profile . VEH is We use two main approaches to classify the DBLα tags. These approaches and algorithms were developed and published by Bull et al. , 24. In


Trichoderma reesei is one of the best producers of cellulase and has been widely studied for the production of cellulosic ethanol and bio-based products. We previously reported that Mn2+ and N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via Ca2+ bursts and calcium signalling in T. reesei under cellulase-inducing conditions. To further understand the regulatory networks involved in cellulase overexpression in T. reesei, we characterised the Mn2+/DMF-induced calcium signalling pathway involved in the stimulation of cellulase overexpression.The filamentous fungus 2+/DMF stimulation significantly increased the intracellular levels of cAMP in an adenylate cyclase (ACY1)-dependent manner. Deletion of acy1 confirmed that cAMP is crucial for the Mn2+/DMF-stimulated cellulase overexpression in T. reesei. We further revealed that cAMP elevation induces a cytosolic Ca2+ burst, thereby initiating the Ca2+ signal transduction pathway in T. reesei, and that cAMP signalling causes the Ca2+ signalling pathway to regulate cellulase production in T. reesei. Furthermore, using a phospholipase C encoding gene plc-e deletion strain, we showed that the plc-e gene is vital for cellulase overexpression in response to stimulation by both Mn2+ and DMF, and that cAMP induces a Ca2+ burst through PLC-E.We found that Mn2+/DMF stimulation produces cAMP. Increase in the levels of cAMP activates the calcium signalling pathway via phospholipase C to regulate cellulase overexpression under cellulase-inducing conditions. These findings provide insights into the molecular mechanism of the cAMP–PLC–calcium signalling pathway underlying cellulase expression in T. reesei and highlight the potential applications of signal transduction in the regulation of gene expression in fungi.The findings of this study reveal the presence of a signal transduction pathway in which MnThe online version contains supplementary material available at 10.1186/s13068-021-01914-0. Trichoderma reesei is one of the best-studied model organisms for the production of hydrolytic enzymes [T. reesei harbours a variety of cellulase- and hemicellulase-encoding genes. Their expression is controlled by a complex regulatory network [T. reesei at the molecular level will lead to new metabolic engineering approaches to construct strains capable of more efficient production of cellulase than is presently possible [Lignocellulosic biomass is one of the most abundant, renewable, and relatively inexpensive raw materials for biorefineries. This biomass is used for the production of value-added chemicals and fuels –3. Filam enzymes –9. T. re network , 10. A bpossible , 8.2+ stimulated hyphal growth, growth-independent cellulase production, and total protein secretion of T. reesei Rut-C30 involving the Ca2+/calmodulin–calcineurin–CRZ1 signal transduction pathway. Other authors have reported that high levels of Ca2+ and Mn2+ concentrations generate a prolonged nuclear accumulation of CrzA in Aspergillus nidulans [2+ acts synergistically with CRZ1 to modulate cellulase gene expression in T. reesei. We previously reported that Mn2+ and the organic solvent N,N-dimethylformamide (DMF) can stimulate cellulase overexpression via a calcium signal transduction pathway in T. reesei [2+, which is a ubiquitous second messenger, is an important intracellular signalling molecule involved in the regulation of gene expression in fungi stimulated by environmental signals. However, the details of the signal transduction pathway from environmental stimulation to calcium signalling in regulating cellulase gene expression remain unknown in filamentous fungi.Cellulase production is regulated by a complex signalling cascade and regulatory network . The prenidulans , 17. Marnidulans  demonstr. reesei , 20. AboT. reesei [T. reesei adenylate cyclase (ACY1) has a consistently positive effect on cellulase gene expression [Extensive progress has been made in understanding the importance of the other ubiquitous second messenger, cyclic AMP (cAMP), in filamentous fungi. Exogenous cAMP leads to increased endoglucanase synthesis in . reesei . A posit. reesei , 22. Adepression . As a sepression  and carbpression . However2+/DMF stimulation to calcium signalling and cellulase production in T. reesei under cellulase-inducing conditions. Our results demonstrate that phospholipase C is an important link between cAMP and calcium signalling in cellulase production that occurs in response to Mn2+/DMF stimulation. These findings provide evidence for the mechanism of the environmental regulation of cellulase expression in filamentous fungi.In this study, we assessed the signal transduction pathway from Mn2+ or DMF markedly stimulates cellulase overexpression in T. reesei [2+/DMF stimulation had any effect on cAMP concentration in T. reesei, precultured mycelia of T. reesei QM6a was inoculated into fresh liquid MM containing 1% Avicel (cellulase-inducing conditions) as the sole carbon source with no further supplementation, or with the addition of 10 mM Mn2+ or 1% DMF, as previously described [We previously demonstrated that a biologically relevant level of extracellular Mn. reesei , 20. As escribed .T. reesei QM6a after the addition of Mn2+ and DMF are presented in Fig. 2+ produced a 76.5% increase in the intracellular cAMP concentration compared with that of the control (no addition) under the same growth conditions. Similarly, stimulation with 1% DMF resulted in a 74% increase in intracellular cAMP concentration compared with the control. These results showed that Mn2+/DMF stimulation could induce cAMP accumulation in T. reesei. Supplementation with forskolin, a direct adenylate cyclase activator [2+/DMF stimulation can result in cAMP accumulation.The intracellular cAMP concentrations of ctivator , also in2+/DMF stimulation, we constructed an adenylate cyclase gene acy1 deletion strain (Δacy1) to detect its function during cAMP accumulation following Mn2+/DMF stimulation. The growth rate of the Δacy1 strains was clearly slower than that of the wild-type strain QM6a  in wild-type QM6a, but did not result in increased cellulase production in the Δacy1 strains. The pNPCase activity in the acy1 re-complementation strain Racy1 was similar to that in QM6a  in both Δacy1 strains and wild-type QM6a.RT-qPCR was performed to determine the transcription levels of the main cellulase genes 2+/DMF-stimulated cellulase overexpression in T. reesei.These results indicate that cAMP signalling mediates Mn2+/DMF-stimulated cellulase overexpression in T. reesei [2+/DMF-stimulated cellulase overexpression  in wild-type QM6a compared to untreated cells. However, the expression levels of cam, cna1, and crz1 remained stable in acy1 deletion strains, irrespective of whether Mn2+, DMF, and forskolin were added to the cells. The addition of dbcAMP resulted in an increase in the expression levels of cam, cna1, and crz1 in both wild-type QM6a and Δacy1 strains compared to untreated cells. These data suggest that an increase in cAMP levels induces a cytosolic Ca2+ burst that activates the Ca2+ signal transduction pathway in T. reesei.The effect of cAMP on Ca2+ signalling, we measured cellulase production in response to exogenous dbcAMP and dbcAMP in combination with LaCl3. LaCl3 is a plasma membrane Ca2+ channel blocker that inhibits the cytoplasmic Ca2+ burst. As shown in Additional File pNPCase activities) in wild-type QM6a. However, cellulase activities decreased significantly when LaCl3 supplements were added compared to the no-LaCl3 control. These results suggest that cAMP signalling causes Ca2+ signalling to regulate cellulase production in T. reesei.To further investigate the link between cAMP and Ca2+/DMF stimulates cellulase overexpression, we compared the transcriptomes of three T. reesei QM6a cultures, with no addition, with the addition of 10 mM Mn2+, or with the addition of 1% DMF (liquid MM containing 1% Avicel as the sole carbon source) incubated at 28 °C and 200 rpm for 36 h [2+ addition compared to the control (no addition). Of these 846 genes, 580 were up-regulated and 266 were down-regulated , three endoglucanase-encoding genes , including two main endoglucanase genes (egl1 and egl2), one beta-glucosidase-encoding gene , a xylanase-encoding gene , and transcription of accessory protein-encoding genes, including those of swollenin gene swo1  and cip2  [2+ and DMF addition. The transcriptome data agreed with our previous cellulase activity and qPCR results [Of the genes that were consistently differentially expressed by both Mn results , 20.2+ and DMF, the transcriptional levels of plc-e, which encodes a phospholipase C protein, were significantly upregulated in response to 10 mM Mn2+ and DMF addition , which is correlated with calcium signalling , 27. Preon Figs. , 4 and [ion Fig. . Thus, w2+ bursts induced by cAMP, the Ca2+ concentration was compared between the QM6a and Δplc-e strains treated with Mn2+, DMF, forskolin, and dbcAMP. The cytosolic Ca2+ levels remained almost stable or were only slightly enhanced in the Δplc-e strains in the presence of Mn2+/DMF stimulation, forskolin supplementation, or exogenous dbcAMP addition. A significant Ca2+ burst was observed in wild-type QM6a under all these conditions compared with that of the untreated control  in wild-type QM6a induced by cAMP were significantly weakened in the Δplc-e strains , and two GprK-type GPCR-encoding genes (Tr37525 and Tr81383)  was significantly upregulated in the presence of 1% DMF , act as sensors to connect to the environment , 29. GPCynthesis . In Cryp pathway . In T. rtrations . These d2+ concentration to activate appropriate downstream responses [2+ signals under Mn2+/DMF−stimulated cellulase overexpression  was used throughout the study. E. coli and A. tumefaciens were cultured in Luria broth (LB) medium. All strains of T. reesei were maintained on potato dextrose agar (PDA) plates at 28 °C. Conidia were collected from the PDA plates. All strains were cultured in the dark.ormation . T. rees4)2SO4 5 g/L; KH2PO4 15 g/L; MgSO4 0.6 g/L; CaCl2 0.6 g/L; FeSO4·7H2O 5 mg/L; ZnSO4·7H2O 1.4 mg/L; CoCl2·6H2O 2 mg/L; pH 5.5) with 2% glucose or 1% Avicel was used to assess hyphal growth and cellulase production. Conidia (2 × 106) were cultivated at 28 °C (200 rpm) in 50 mL of MM (2% glucose as the sole carbon source) for 36–48 h. The mycelia were inoculated into 100 mL of freshly prepared MM containing 1% Avicel as the sole carbon source with no further addition or the addition of Mn2+ , DMF , forskolin , or dibutyryl cAMP  as described previously [Minimal medium  as the substrate in 50 mM sodium acetate buffer at pH 5.0 and 50 °C for 30 min. One unit of pNPCase activity was defined as 1 μmol of p-nitrophenol released per min. CMCase activity was determined using 1% carboxymethylcellulose  as the substrate in 50 mM sodium acetate buffer at pH 5.0, and 50 °C for 30 min. One unit of CMCase activity was defined as the amount of enzyme producing 1 μmol of reducing sugar per min. Biomass concentration was indirectly measured by calculating the amount of total intracellular proteins, as described by Chen et al. [pNPCase activities were used to represent cellulase activity.Cellulase activity was measured as previously described . In brien et al. . CMCase cbh1 (encoding cellobiohydrolase I) and egl1 (encoding endoglucanase I) using the 2−ΔΔCt method. The sequences of the primers used in RT-qPCR are described in Additional File sar1 were measured for data normalisation [RNA extraction and RT-qPCR were performed as described by Chen et al. . In brielisation .Cultures of liquid mycelia were harvested and frozen in liquid nitrogen. Intracellular cAMP extraction and determination were performed as previously described  with som2+-specific probe to assess cytoplasmic Ca2+ concentrations according to the manufacturer’s protocol [Fluo-4 AM  was used as a Caprotocol . The mycacy1 deletion mutant, the 799 bp upstream and 718 bp downstream fragments of acy1 were generated from the genome of T. reesei QM6a using KOD-Plus-Neo . The primers used are listed in Additional File PacI and XbaI linearized LML2.1 [acy1. Subsequently, the downstream fragment was inserted into SwaI-linearised pFacy1 to form the binary vector pDacy1  generated by double crossover were verified by diagnostic PCR using the primers acy1-CF and acy1-CR and acy1-OF and acy1-OR  were generated using Agrobacterium-mediated transformation  were constructed similarly.The re-complementation cassettes of T. reesei QM6a cultured alone or with 10 mM Mn2+ or 1% DMF [6) were cultivated at 28 °C (200 rpm) in 50 mL of MM (2% glucose as the sole carbon source) for 36–48 h. The mycelia were inoculated in 100 mL of freshly prepared MM containing 1% Avicel as the sole carbon source, with either no further addition of components or with the addition of 10 mM Mn2+ or 1% DMF grown for 36 h. Mycelia were then harvested from the cultures. All the mycelia of the WT , Mn , and DMF  were pooled, resulting in six samples. The samples were sent to a company  for preparation and RNA sequencing using a HiSeq X Ten apparatus . Two biological replicates of each condition were submitted for RNA sequencing. Differential expression analysis was performed using DESeq [P values were lower than 0.01, and whose log2-fold change values were lower than -1 or higher than 1 were selected as differentially expressed genes (DEGs).The transcriptomes of r 1% DMF  were comng DESeq . Genes wT. reesei reference genome (genome.jgi.doe.gov/Trire2/Trire2.home.html) with 93.87 to 94.80% coverage  between the two biological replicates of each condition used in the transcriptional analysis  under accession number PRJNA510366.The raw whole transcriptome shotgun sequencing data and the related protocols are available at the NCBI SRA web site  from the mean of triplicate determinations. Student’s Additional file 1: Figure S1. Construction and verification of Δacy1 strains. a. Schematic representation of the acy1 locus from the QM6a and Δacy1 strains. The region from +1 to +6649 bp relative to the translation start site of acy1 (grey box) was replaced with the hygromycin cassette (black box). The binding sites of primers on the genome of QM6a and Δacy1 are indicated by red arrows. The binding sites of primers on the hygromycin cassette are indicated by blue arrows. The expected sizes of the PCR verification products in the Δacy1 strains are indicated as numbers. Primer pairs (acy1-T1/ acy1-T2 and acy1-T3/acy1-T4) shown in purple were used to identify the copy number of integrated genes. b. Schematic representation of the Pacy1-acy1-Tacy1 cassette in Racy1 strains. The acy1 complementation cassette was constructed by ligating the whole gene sequence  into LML2.1. The primer pairs indicated were used in the verification of the expression cassette. c. PCR verification of Δacy1 strains. Lane M, DNA molecular mass maker Lane F, PCR amplification results using the acy1-CF/D70-4 pair. Lane R, PCR amplification results using the HG3.6/acy1-CR pair. Lane O, PCR amplification results using the acy1-OF/acy1-OR pair. Lane N, PCR amplification results using acy1-OF/ acy1-OR pair with water as a negative control. Δacy1-1, Δacy1-2, and Δacy1-3 represent three Δacy1 strains. QM6a as controls. d. PCR verification of Racy1 strains. Lane M, DNA molecular mass maker Lanes, PCR amplification results using the acy1-OF/acy1-OR pair with three Racy1 strains and Δacy1 strains as templates. e. Verification of copy numbers for Δacy1 transformants by qPCR. The genome of QM6a was used as a reference with a single copy. f. Growth rates of wild-type QM6a, Δacy1, and Racy1 strains in MM plates.Additional file 2: Figure S2. Cellulase activities of T. reesei complementary strains Racy1 and Rplc-e under different addition conditions. pNPCase activity/mg biomass of T. reesei QM6a, Racy1 and Rplc-e strains supplemented with 10 mM Mn2+ or 1% DMF. T. reesei QM6a was used as the control. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences from the control .Additional file 3: Figure S3. Effect of Mn2+/DMF/cAMP-induced cellulase overexpression. a and b. pNPCase activity/mg biomass (a) and CMCase activity/mg biomass (b) of T. reesei QM6a and Δacy1 strains supplemented with 5 mM dbcAMP, 10 mM Mn2+ or (and) 1% DMF. Values are the means ± SD of the results from three independent experiments. Asterisks indicate significant differences from the control .Additional file 4: Figure S4. Relative expression levels of cam (c), cna1 (d), and crz1 (e) in T. reesei QM6a and Δacy1 strains under different conditions. The QM6a and Δacy1 strains were cultured in MM with 2% glucose as the carbon source and then inoculated in fresh MM supplemented with 10 mM Mn2+, 1% DMF, 0.02 mΜ Forskolin, or 5 mM dbcAMP, with 1% Avicel as the carbon source. Cultures that were not supplemented with Mn2+, DMF, Forskolin, or dbcAMP were used as controls. QM6a cells cultured without the addition of Mn2+, DMF, Forskolin, or dbcAMP were used as the reference sample. Values are expressed as the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences from the control .Additional file 5: Figure S5. Effect of cAMP/LaCl3-induced cellulase overexpression. a and b. pNPCase activity/mg biomass (a) and CMCase activity/mg biomass (b) of T. reesei QM6a strains supplemented with 5 mM dbcAMP and 5 mM LaCl3. Values are the means ± SD of the results from three independent experiments. Asterisks indicate significant differences from the control .Additional file 6: Figure S6. Volcano plot analysis of up- and downregulated genes of strains treated with or without 10 mM Mn2+ treatment. Volcano plot for differences in gene expression with no addition or 10 mM Mn2+ addition. Red dots indicate significantly upregulated genes, green dots indicate significantly downregulated genes, and grey dots indicate non-significantly different gene expression. The x-axis represents the logarithm of the differential multiple of a gene expression in two samples. The y-axis represents the negative log of a statistically significant change in gene expression. Control: parental strain QM6a without 10 mM Mn2+ treatment; Sample 1: parental strain QM6a with 10 mM Mn2+ treatment.Additional file 7: Table S1. Genes that are significantly up- or down-regulated in T. reesei QM6a with 10 mM Mn2+ addition when compared with no addition.Additional file 8: Table S2. Differential transcription of genes in response to Mn2+ and DMF as measured by RNA-sequencing.Additional file 9: Table S3. The log2 fold changes of the major cellulose degradation-related genes under Mn2+/DMF addition conditions.Additional file 10: Table S4. Whole transcriptome shotgun sequencing data and RT-qPCR verification of the plc-e gene under Mn2+ addition conditions.Additional file 11: Figure S7. The growth rate related cellulase activity and transcription levels of cbh1 and egl1 after Mn2+ stimulation in wild-type QM6a and Δplc-e mutant.Additional file 12: Figure S8. Relative expression levels of cam (c), cna1 (d), and crz1 (e) in T. reesei QM6a and Δplc-e strains. The QM6a and Δplc-e strains were cultured in MM with 2% glucose as the carbon source and then inoculated in fresh MM supplemented with 10 mM Mn2+, 1% DMF, 0.02 mΜ Forskolin, or 5 mM dbcAMP, with 1% Avicel as the carbon source. Cultures with no addition were used as controls. QM6a cells cultured with no addition were used as the reference sample. Values are expressed as the mean ± SD of the results from three independent experiments. Asterisks indicate significant differences from the control .Additional file 13: Table S5. The log2 fold changes of the putative GPCRs genes under Mn2+/DMF addition conditions.Additional file 14: Table S6. Primers used in this study.Additional file 15: Table S7. Sequencing statistics for whole transcriptome shotgun sequencing results from this study.Additional file 16: Figure S9. Biological replicates used for whole-transcriptome shotgun sequencing analysis. Graphs representing the Pearson correlation between biological replicates of each sample. A high Pearson correlation was obtained, demonstrating the reliability of whole transcriptome shotgun sequencing analysis (r2 ≥ 0.898). The x-axis and y-axis correspond to the gene expression in different treatments  after conversion by log2(FPKM+1).


Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen. O-glycosylation, which is generated after lysyl hydroxylation, is involved in cross-link formations, and the amount depends on the type of tissue, the rate of triple helix formation, and the presence or absence of ER chaperones   occur before triple helix formation , and theractions , 20. Wheractions , 23, 24.ractions , 26. Cypwith LH1 . CypB wawith LH1 , and as with LH1 , 29, 30.with LH1 . LH2 is with LH1 , while awith LH1 , 31, 32.ctively) , 35, 36.ctively) , 37. Fewctively) . Additioctively) . The fun for LH1 . Collect for LH1 . HoweverIn this study, we aimed to investigate the precise role of LH1 for PTMs in collagen triple helices and to clarify how tightly P3H3 affects the activity of LH1 in the rER. To achieve these aims, we delved deeply into the levels of overall lysyl hydroxylation and PTM occupancy at individual sites between collagens extracted from LH1 and P3H3 null mice. We analyzed different collagens from different tissues to identify if LH1 is a global or specific enzyme, as described above , 35, 36.A and with more detailed information in the methods section. Figure 1B and Fig. S1A display the results of PCR genotyping showing that the P3H3 null allele product is smaller than wild type (WT) due to the deletion of Exon 1. To confirm that the expression of P3H3 protein was abolished, western blotting was performed using a whole kidney lysate, and the protein signal corresponding to the molecular weight of P3H3 (79 kDa) was absent in the P3H3 null lysate . Our P3H3 null mice were viable, and we did not observe any apparent growth or skeletal phenotypes by growth curve analysis and X-ray imaging (D and E respectively).LH1 null mice were generated and characterized previously . P3H3 nuFigure 1O-glycosylated and can be isolated to high purity from the skin (A). A difference in collagen migration in SDS-PAGE gels is one of the classic indications of altered levels of PTMs, particularly in the case of OI (B). Both mouse lines are in a C57BL/6 background, but slight differences between their backgrounds exist as the mouse lines originated from different laboratories . However, the calculated p values are higher than or close to 0.05. Also, the ratios of lysine and hydroxylysine do not show any statistically significant difference between the WT mice ; however, this difference is not statistically significant , the level of glucosylgalactosyl hydroxylysine (GGHL) was decreased at both sites in P3H3 null mice, although the decrease at α1(V) K84 did not quite reach statistical significance , at least α2(V) K87.Next, we determined the occupancy of PTMs at individual lysyl hydroxylation sites in type V collagen from WT, P3H3, and LH1 null mice by liquid chromatography–mass spectrometry (LC-MS) after trypsin digestion. Since lysine residues at the Yaa position of collagenous Gly-Xaa-Yaa triplets are extensively glycosylated in type V collagen , site-spA). AAA was carried out to quantify the total number of PTMs in the purified type I collagen. Neither P3H3 nor LH1 null mice had consistent changes in proline hydroxylations (prolyl 3- and 4-hydroxylation) , and unmodified hydroxylysine showed a difference with statistical significance between the genotypes in both tissues . Electron microscopy showed that the average diameter of collagen fibrils is similar between P3H3 null (84.9 ± 35.6 nm) and WT (85.1 ± 25.9 nm) mice (D). However, the distribution of fibril diameters was broader in P3H3 null mice (E), and this was also reported in LH1 null skin  mice D. Howeveull mice E, and thull skin . In summTo clarify how LH1 functionally cooperates with the LH1-associated protein P3H3, we conducted quantitative analyses and directly compared the level of PTMs between WT, P3H3 null, and LH1 null mouse tissues. As a result, we discovered novel collagen type-dependent and site-differential effects of LH1 and P3H3 for precise lysyl hydroxylation in the collagen triple helix in the rER. We showed that type I collagen (without telopeptide regions) from the tendon and skin from LH1 and P3H3 null mice was differentially modified between the null mice and tissues . We alsoC). Rather than the presence of specific binding or enhancer sequences, we envision that a three-dimensionally and temporally precise molecular interplay between LH1, P3H3, and CypB is required, particularly around the cross-link formation sites according to We also looked for specific binding or enhancer sequence motifs in type I and V collagen close to the lysyl hydroxylation sites based on our results . As suggFig. 3B). Similar observations have been made before: the results from the lung and kidney of LH3 mutant mice demonstrated that the amount of hydroxylysine was not changed in type I collagen-rich fractions but was reduced by 30% in type IV and type V collagen-rich fractions . Tissue isolation from the LH1 null and corresponding WT mice was performed under an animal experiment license number ESAVI/8179/04.10.07/2017 approved by the Animal Experiment Board of Finland, following the regulations of the EU Directive 86/609/EEC, the European Convention ETS123, and the national legislation of Finland. The LH1 null mice were housed in the University of Oulu Laboratory Animal Center, and the recommendations given by the Federation of European Laboratory Animal Science Associations and the Finnish and EU legislations concerning laboratory animal experiments and handling were followed.Leprel2  gene (UniProt entry number: Q8CG70), encoding P3H3, was deleted. The procedure is briefly described as follows. The PGK-neo selection cassette flanked by FRT sites was generated by PCR from C57BL/6J genomic DNA and inserted downstream of the exon 1, which was flanked by loxP sites. The targeted locus was eliminated using an FLP recombinase and a Cre recombinase. ES cell clones were confirmed by southern hybridization. P3H3 inactivation was verified in mice by DNA preparation from tissues and PCR with primer sets (Fwd: 5’-CTTACCCACACTAGACCCATGTGTC-3’ and Rev: 5’-GTTGCATTCATTAGCCTAGACCCGCTA-3’). The sizes of the PCR products for the WT and null alleles were approximately 1000 and 200 pb, respectively (P3H3 null mice were purchased from Ozgene. Directed knockouts were created in which exon 1 of the mouse ectively .Plod1) knockout mice using in-frame insertion of the lacZ-neo cassette into exon 2 has been described earlier  and rabbit polyclonal antibody against GAPDH were purchased from Proteintech (16023-1-AP) and Sigma-Aldrich (PLA0125), respectively. Alkaline phosphatase-conjugated anti-rabbit IgG (A9919: Sigma-Aldrich) was used as a secondary antibody. Western blotting was performed three times using three independently prepared kidneys.A kidney was extracted from WT, heterozygous, and null P3H3 mouse and homogenized using T-PER (Thermo Fisher Scientific) containing protease inhibitors at 4 °C. After centrifugation, soluble proteins in the extract were mixed with NuPAGE LDS sample buffer with reducing agents. The protein solutions were separated by Bolt 4 to 12% Bis-Tris Plus (Thermo Fisher Scientific) gel electrophoresis and electrotransferred onto PVDF membranes. The membranes were blocked in PBS solution containing 5% (w/v) skim milk for antibody staining. All proteins were detected by alkaline phosphatase developed with 5-bromo-4-chloro-3-indolyl phosphate and Nitro blue tetrazolium. Rabbit polyclonal antibody against P3H3 . Voltages and exposure times were optimized for best-resolution images.Tendon, skin, and bone were taken from adult mice . All proCD spectra were recorded on an AVIV 202 spectropolarimeter  using a Peltier thermostat controlled cell holder and a 1-mm path length rectangular quartz cell (Starna Cells Inc). The temperature scanning curves were monitored at 221 nm with 10 °C/h scan rate. All curves shown were the average of at least three independent measurements using independently prepared type V and I collagen samples from tissuesAcid hydrolysis was performed in 6 x 50-mm Pyrex culture tubes placed in Pico Tag reaction vessels fitted with a sealable cap . Samples were placed in culture tubes, dried in a SpeedVac , and then placed into a reaction vessel that contained 250 ml of 6 M HCl (Pierce) containing 2% phenol (Sigma-Aldrich). The vessel was then purged with argon gas and evacuated using the automated evacuation workstation Eldex hydrolysis/derivatization workstation . Closing the valve on the Pico Tag cap maintained the vacuum during hydrolysis at 105 °C for 24 h. The hydrolyzed samples were then dried in a Savant SpeedVac. The dried samples were dissolved in 100 ml of 0.02 M HCl containing an internal standard . The analysis was performed by ion-exchange chromatography with postcolumn ninhydrin derivatization and visible detection (440 nm/570 nm) with a Hitachi L-8800A amino acid analyzer  running the EZChrom Elite software . Three technical replicates were performed in each analysis.2) after adding stable isotope-labeled collagen as an internal standard. The alkaline hydrolysates were neutralized with 30% acetic acid and then desalted using a mixed-mode cation-exchange sorbent . Unmodified and glycosylated hydroxylysines were quantitated by LC-MS in multiple reaction monitoring mode using a 3200 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (AB Sciex) coupled to an Agilent 1200 Series HPLC system (Agilent Technologies) with a ZIC-HILIC column . Chromatographic separation was performed at a flow rate of 300 μl/min with a binary gradient as follows: 90% solvent B (100% acetonitrile) for 3 min, linear gradient of 10 to 95% solvent A (0.1% acetic acid/5 mM ammonium acetate) for 10 min, 95% solvent A for 4 min, and 90% solvent B for 3 min.Glycosylation of hydroxylysine was estimated by LC-MS after alkaline hydrolysis, as described previously . In brieGrimontia hollisae (Nippi) and pepsin (Sigma-Aldrich) after heat denaturation at 60 °C for 30 min. On the other hand, type V collagen was digested with trypsin not only in solution but also in SDS-PAGE gel. The samples were separated by SDS-PAGE, and the regions containing α1(V) and α2(V) were subjected to in-gel digestion with trypsin. The protease digests of type I and type V collagen were analyzed by LC-MS on a maXis II quadrupole time-of-flight mass spectrometer  coupled to a Shimadzu Prominence UFLC-XR system (Shimadzu). Peptides were separated on an Ascentis Express C18 HPLC Column  at a flow rate of 500 μl/min with a binary gradient as follows: 100% solvent A (0.1% formic acid) for 2.5 min, linear gradient of 2 to 50% solvent B (100% acetonitrile) for 12.5 min, 90% solvent B for 2.5 min, and 100% solvent A for 2.5 min. Peptides were ionized and detected in positive ion mode with data acquisition using otofControl version 4.0 . The MS scan (type I and type V collagen samples) and MS/MS acquisition (type V collagen samples) were performed over the m/z ranges of 50 to 2500 with a frequency of 2 Hz (peptide quantification) or 5 Hz (peptide identification). The acquired MS/MS data were converted to.mgf files for database search against the UniProtKB/Swiss-Prot database (release 2018_05) for Mus musculus species  using ProteinPilot software 4.0 (AB Sciex) with the Paragon algorithm  KAll procedures for sample preparation were performed at 4 °C. Skin was taken from adult mice  and protOHSU histology core facility performed sectioning and staining of the skin samples. Ventral skin tissues (n = 3) were fixed in 4% paraformaldehyde for 24 h at 4 °C. The fixed tissues were dehydrated using an ethanol gradient, cleared in xylene, and embedded in paraffin wax, after which 5 μm sections were cut using a microtome. The tissue sections were stained with Masson’s trichrome for collagen fiber analysis. Regions of interest (n = 2–4) were selected per image (n = 5), and the thickness of dermis and hypodermis was measured. The thickness was normalized by each scale bar.4, then dehydrated in a graded series of ethanol to 100%, rinsed in propylene oxide, and infiltrated in Spurr’s epoxy. Samples were polymerized at 70 °C for 18 h. Ultrathin sections (∼80 nm) were cut on a Leica EM UC7 ultramicrotome and mounted on formvar-coated, copper palladium 1 x 2 mm slot grids. Sections were stained in saturated uranyl acetate followed by lead citrate and photographed using an AMT 2K x 2K side entry camera (AMT) mounted on a FEI G2 transmission electron microscope operated at 120 kV, and at least four images were taken per sample.The three independently prepared skin samples from P3H3 null and WT mice were fixed in 1.5% glutaraldehyde/1.5% paraformaldehyde (Electron Microscopy Sciences) in Dulbecco’s serum-free media (SFM) containing 0.05% tannic acid. The samples were rinsed in SFM, postfixed in 1% OsO2. Spatial calibration for diameter measurements was applied against each scale bar. The numbers were counted for 1845 and 3616 fibrils of P3H3 WT and null samples, respectively.The number and cross-sectional area of fibrils were measured using the Fiji software (ImageJ). Regions of interest were selected from the images of WT (n = 13) and P3H3 null (n = 17) skin, and fibril areas were measured by counting the pixels per fibril, which accounts for μmp value of less than 0.05 was considered statistically significant.For comparisons between two groups, one-way ANOVA was performed to determine whether differences between groups are significant using ORIGIN Pro ver. 9.1 (OriginLab Corp). The via the jPOST partner repository with the data set identifier PXD019748 (https://repository.jpostdb.org/entry/JPST000744) (The MS data sets for identification of lysine PTM sites in type V collagen have been deposited to the ProteomeXchange consortium T000744) , 61. IdeT000744) , 29. AllThis article contains The authors declare that they have no competing interests related to this work.


The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication. Coronaviridae, whose members share a distinct pattern in their genomic organization (Lai The current worldwide COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely impacted nearly every area of human life. In the absence of reliable therapeutic options or vaccination, research efforts have been increased in order to understand molecular characteristics of the functional components of the virus. SARS-CoV-2 belongs to the family of tion Lai . In contThe RNA genome of SARS-CoV-2 encodes at least 14 polypeptides, some of which are cleaved by viral proteases into their functional forms. The non-structural protein 10 (nsp10) is generated from the first open reading frame of the virus (ORF1) through cleavage by the main protease nsp5 to yield a 139 amino acid protein of 14.8 kDa.Previous work has been reported on the homologous proteins of nsp10 from SARS-CoV  vector (Genscript), carrying an additional sequence at the N-terminus (MGSDKIHHHHHH) including a hexa-histidine (His6)-tag for purification.The amino acid sequence of SARS-CoV-2 nsp10 was obtained from the NCBI reference genome entry NC_045512.2, identical to GenBank entry MN908947.3  and supplemented with 50 µM ZnCl2. The final preparative expression was performed at 20 °C, 120 rpm for 12 h. Cells from a 2 L culture were harvested at 4000 g for 15 min using a Beckmann centrifuge with a JLA 8.1000 rotor. The pellet was resuspended in 100 mL of buffer A  and supplemented with 50 µM ZnCl2, and two protease-inhibitor tablets . Cells were mechanically lysed using Microfluidics M-110P at 15,000 PSI (pounds per square inch) under continuous ice cooling, followed by centrifugation at 4 °C and 38,400×g for 45 min using a Beckmann centrifuge with a JA 20 rotor.The pET21b(+) plasmid containing the nsp10 sequence was transformed into T7-Express GE Healthcare, USA) connected to an FPLC system . Elution of the bound protein was achieved with a linear gradient of 500 mM imidazole containing buffer A. Protein-containing fractions were combined, concentrated using centrifugal concentrator devices  and further purified with a 320 mL HiLoad 26/600 Superdex 75 pg gel filtration column  using 50 mM sodium phosphate buffer (pH 7.5), 50 mM NaCl, 5 mM dithiothreitol (DTT). Purity of the produced protein was verified by SDS-PAGE analysis and confirmed using mass spectrometry (MALDI). Finally, the protein was concentrated using centrifugal concentrator devices .The supernatant was further purified using immobilized metal ion affinity chromatography (IMAC) followed by size exclusion chromatography (SEC). After centrifugation, the supernatant was loaded onto a 5 mL HisTrap HP column  were performed at 600 MHz and 298 K using a 0.15 mM and a 1.6 mM sample. For determining the longitudinal T115N relaxation time, a series of spectra with the following relaxation delays was used: 20, 60, 100, 200, 400, 600, 800, 1200, 1400, 1600, 1800 ms. The transverse 15N relaxation time T2 was determined from spectra with the following relaxation delays: 16.96, 33.92, 67.84, 101.76, 135.68, 169.60, 203.52, 271.36 ms. The {1H}-15N hetNOEs were calculated from the signal intensity ratio (Ion/Ioff) obtained from spectra recorded with and without saturation of amide protons with a recovery (d1) and saturation delay of 10 s.Heteronuclear TRACT experiments . When excluding the first twelve N-terminal residues including the His6-tag, the backbone assignment comprises 92% of the 1H,15N pairs and 95.5% of the total resonances . Residues M1, G2, F28, C29, T59 H60 and the N-terminal His6-tag were not assigned. Assignment of the residues K5 and W135 is tentative.The backbone chemical shift assignment of nsp10 protein was conducted manually by using standard double- and triple-resonance NMR experiments on uniformly labeled samples at 298 K. With the heteronuclear 3D experiments listed above we could assign 89% of the The number of signals observed in BEST-TROSY experiments is approximately 5% higher than expected for the nsp10 protein. Lower intensities for a subset of the signals are indicative of the presence of a second minor conformation in slow exchange on the NMR time scale. The resonances in question were assigned to the last seven C-terminal amino acids  and analyzing intramolecular hydrogen bonds using temperature factors  is 10.7 ns, which is agreement with the monomer value predicted by HydroNMR (10.8 ns) , which is directly involved in interactions with the complex partner nsp16 in the crystal structure (PDB 6WQ3). A second difference can be observed for some of the C-terminal amino acids, which adopt a zinc finger fold, but are classified as ß-strands in the TALOS prediction. However, all of the assigned cysteine residues show Cβ chemical shifts that are indicative of a Zn4–60, whiorm Fig. .Fig. 513


Composites were manufactured by leveraging the in situ reaction of the components during printing at 150–300 W laser power, 500–1000 mm·s−1 laser scanning speed, and 100–134 J·mm−3 volumetric energy density. Microcomputed tomography scans indicated a negligible induced porosity throughout the specimens. The fully dense Mo2-based composites, with hardness exceeding 545 HV1 and low roughness for both the top  and side  surfaces, demonstrated that laser-based additive manufacturing can be exploited to create unique structures containing hexagonal Mo(Si0.67Al0.33)2.The laser power bed fusion approach has been successfully employed to manufacture Mo The device is equipped with an Ytterbium laser with a maximum power of 400 W and a wavelength of 1.07µm. Cylindrical samples with dimensions of ∅7 mm × 7 mm were built. The meander scan strategy was used, whereby scan patterns rotate by 67° after each printed layer. The whole manufacturing process was carried out inside a chamber containing a precisely controlled atmosphere of argon at an oxygen level below 500 ppm. The hatching distance (h) and laser spot size were set as 85 µm and 90 µm, respectively. The laser power (P) was in the range of 150–300 W and the scanning speed (ν) was in the range of in 500–1000 mm·s−1 range. The layer thickness (d) was chosen as 35 µm. The process parameters for preparation of the three samples are listed in Consolidation of the MoSiνhd, respectively.The laser volumetric energy density (LED) and build rate were calculated according to E = P/νhd and BR = The density of printed parts was measured by Archimedes’ principle  and by calculating the dimensions  and weight  of the samples. The size, morphology, and alignment of the pores in the SLM-fabricated samples were inspected via X-ray computed tomography (CT) . The surface roughness analysis was performed using a Keyence VK-X250 profile analyzing confocal laser microscope . On each studied surface, 4 areas with dimensions of 500 μm × 700 μm were scanned using a 20× lens. The maximum height (Sz) and arithmetical mean height (Sa) were averaged for 4 measurements.Samples were ground and polished by conventional metallographic methods to a 0.5 μm diamond polish and to a finer 0.1 μm finish using a nylon disc and polishing suspension. The morphology and microstructure of the powders and printed bulks were examined by TESCAN VEGA3  and HR-SEM Zeiss Merlin scanning electron microscope  equipped with an EDS detector . Phase compositions were analyzed with the help of an X-ray diffractometer (Siemens/Bruker D5000 X-ray Powder Diffraction (XRD) System, Billerica, MA, USA) with CuKα radiation in the 2θ range of 20° to 80°. The concentrations of compounds and elements were estimated via the Rietveld refinement method. The Vickers hardness values were measured on the polished surfaces of the printed specimens using a tester  at a load of 9.8 N, applied over 10 s for 10 indents.2 consists of agglomerates of fine (1–5 µm) particles , but at different scanning speeds and laser powers. The sample S3 possesses the lowest number of pores  and the maximum height (Sz) of the top and side surfaces of the printed samples are listed in Spectrum 2 c shows tThe SEM images of the polished top surfaces of samples S1, S2, and S3 are depicted in −1  phase). A high scanning speed decreases the interaction time between a laser and a material, thus decreasing the solidification time and inducing a high temperature gradient [The cores consisted of fine submicron- to micron-sized grains d–f, whil−1 c, a highgradient ; therefoSignals of Mo were recorded everywhere expect the darkest regions, which correspond to the Al-rich phase a,b repreThe EDS maps corresponding to the SEM image in 2, face-centered cubic Al, the substituted face-centered Si, the coexistence of the in-situ-formed hexagonal Mo3(Al2Si4)/MoAl0.6Si1.4 phases 2, x = 0.3–0.33) composition), and the Si-saturated Al-rich Al0.85Si0.15 phase.The diffractograms of sample S3 confirm the presence of tetragonal MoSi2 to C40 Mo3(Al2Si4), with weak peaks of unreacted C11b, negligible MoAl0.6SiSi1.4, and with Al0.85Si0.15 being formed. For the applied high scanning speed, apparently the unreacted MoSi2 and face-centered Al were found in the final product. In samples S1 and S2, no pure Al was detected, but the Si-saturated Al-rich Al0.85Si0.15 phase was detected. Under the chosen conditions (100.8–134.4 J·mm−3 ED) and with the addition of 30 wt.% AlSi10Mg to the MoSi2, the C11b tetragonal lattice of MoSi2 expanded until achieving a hexagonal Mo(Si0.67Al0.33)2 (x = 0.33 mol) structure, while no further C40 lattice expansion was detected in forming the Al-rich orthorhombic C54 structure. In contrast, the formation of C54 orthorhombic structures with Mo10Si7Al13, MoAl1.3Si0.7, and Al4Mo3Si2 is claimed in [8Mo3 phase is reported in [The patterns for samples S1 and S2 evidenced the complete transformation of MoSiaimed in ,10,11; torted in .2–AlSi10Mg interaction, the thermogravimetric analysis for MoSi2-30 wt.% AlSi10Mg was performed. 2-30 wt.% AlSi10Mg mixture heated up to 1450 °C. The DSC curves exhibit the endothermic peak of the reaction of the aluminum melting and the Al-Si eutectic phase formation, where the heat absorption recorded was 0.9575W·g−1. The weak exothermic reaction was observed starting from 944 °C and the maximum heat flow was 0.2393 W·g−1 at 1019 °C.To study the mechanism of the MoSi3(Al2Si4), 0.7% MoSi2, 12.4% Si, 3.3% MoAl0.6Si1.4, and 21.2% Al; however, the Al0.85Si0.15 phase was not detected.No further chemical reactions were detected. According to the XRD patterns in 2 addition and the development of new phases during solidification.The measured hardness of all SLM-processed materials was HV1 ~ 545 ± 48, which was around four times higher than for the printed AlSi10Mg alloy . The higNowadays, there is huge interest in the development of new alloys for AM processes. The in situ SLM of mixed powders allows the production of hard materials with tailored properties, taking into consideration specific needs.1-x,Alx)2-based composites with hardness exceeding 500 HV1 were successfully SLM-processed at laser volumetric energy density values of 100.8 and 134.4 J·mm−3 and with scanning speeds of either 500 or 1000 mm s−1 from a MoSi2-30 wt.% AlSi10Mg powder mixture. The bulks demonstrated relatively low roughness for both the top  and side  surfaces, indicating the good quality of the produced parts.Fully dense Mo2 (x = 0.33 mol) structure, while no further C40 lattice expansion was detected in forming the Al-rich orthorhombic C54 structure.Under the chosen conditions, with the addition of 30 wt.% AlSi10Mg to the MoSi


Kluyveromyces marxianus (K. marxianus) is an increasingly popular industrially relevant yeast. It is known to possess a highly efficient non-homologous end joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus otherwise wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the gross chromosomal rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in eukaryotes, which could potentially provide insights for cancer research. Kluyveromyces marxianus is a thermotolerant yeast [Saccharomyces cerevisiae, are lacking. As a result, K. marxianus is becoming a potentially valuable industrial yeast. Therefore, many molecular tools [Kluyveromyces lactis [K. marxianus also embraces a robust non-homologous end joining (NHEJ) pathway in the absence of homology. It can be transformed with non-homologous DNA fragments by illegitimate recombination (IR) that involves the NHEJ pathway [nt yeast  and is tnt yeast . It has ar tools ,4,5,6,7 s lactis , K. marx pathway . In the  pathway ,11.Kluyveromyces lactis that were strongly biased toward intergenic regions. If they induced ectopic double strand breaks (DSBs) using restriction cleavage, then there was no bias for genic versus intergenic sequence, suggesting that genome-wide IR occurred at spontaneous mitotic DSBs that are preferentially distributed in intergenic sites. Nonklang et al. [Saccharomyces cerevisiae URA3 (ScURA3) gene to transform a K. marxianus DMKU3-1042 ura3∆ mutant. They analyzed 9 Ura+ transformants by Southern blot hybridization. The ScURA3 insertions were in different genome locations and one transformant had multiple insertions. Abdel-Banat et al. [ScURA3 DNA into the genome of K. marxianus DMKU3-1042 (2×106 transformants/µg DNA) by the NHEJ pathway. They suggested that a high-density ScURA3 insertion map analogous to that obtained by global transposon mutagenesis [K. marxianus, it is desired to have the precise sequence at the junctions of insert sites in the genome. This is not possible to be achieved by Southern blot hybridization due to its low resolution, not to mention its laboriousness.Kegel et al.  reportedg et al.  used a Pagenesis ,14 couldK. marxianus transformants by transforming a 1123-bp PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) and took advantage of Illumina MiSeq and whole-genome Oxford Nanopore sequencing to produce a detailed analysis of 24 Ura+ transformant clones. We found a surprising variety of insertion events. In addition to three tandem dimer and two trimer ScURA3 concatemer insertions, two insertions produced inversions, one produced a large deletion, and another three Ura+ transformants produced chromosomal translocations in which each end of ScURA3 were inserted in a different chromosome. Sequencing analysis, especially the long reads obtained with Nanopore sequencing, was instrumental in detecting and analyzing GCRs that are a hallmark of cancers. Therefore, our results and method may provide insights to understanding the basic mechanisms of DNA repair and cancer biology.We generated our own Ura+ ScURA3 head or tail junctions , insertions were accompanied by small deletions of the genome sequence, ranging from 2 to 30 bp. Several of these also involved alterations of the terminal head or tail sequences at the junctions. In clone 19, the ScURA3 tail had lost 24 terminal bases followed by 4 base substitutions . As a result of the disruption of the ADE1 gene, K. marxianus colonies are pink on low adenine medium. This is consistent with Ade mutants in other yeasts. It appeared to be precise except for an inserted 65 bp DNA sequence between the ScURA3 tail junction and the genome sequence. This DNA did not convincingly match any of the genome sequence. Its source is not known. It seems unlikely, because of its length, to have been inserted by an NHEJ polymerase not requiring a template. One result of this insertion was the inability to identify a tail junction sequence for clone 1 in the MiSeq data. The head junction sequence was readily identified. Only by Oxford Nanopore sequencing was the insertion detected and the point of insertion of ScURA3 DNA into the genome identified.Clone 1 has an insertion in the ade1p gene at position 397,946 on chromosome 1, which is in the open reading frame of  electrophoresis gel analysis of this clone was identical to the wild type . Strain G13 (ura3∆) was constructed as in KmURA3 ORF in K. marxianus NRRL Y-6860 was removed by homologous recombination (HR)-mediated 5-FOA counter selection. Primers used for strain construction and confirmation are listed in ScURA3 cassette contained in the plasmid pRS316  was PCR-amplified using the two primers 5′-tgagagtgcaccacgcttttcaattc and 5’-cagggtaataactgatataattaaattg. The 5´ OH PCR product  was then mixed with 50 µL of the K. marxianus cell suspension (containing approximately 109 cells) and incubated at 42 °C for 30 min. In total, 100 µL of CAA-U medium  were added and the cells were spread on a CAA-U/2% agar plate and incubated at 30 °C for 2 days. Then, 24 isolated colonies were picked and patched onto a YPD plate. After incubation for a day at 30 °C, the 24 patches were re-patched on a CAA-U plate and grown another day. Twenty-four 50-mL tubes containing 10 mL of CAA-U medium were inoculated from the patches and grown at 30 °C for 24h on a shaker. Cells were harvested from each of the cultures by centrifugation and resuspended in 200 µL of P1 cocktail , 5 µL of 14M β-mercaptoethanol, and 125 µL of Zymolyase 20mg/mL) in 1.5-mL Eppendorf tubes. The cells were incubated at 37 °C for 30 min followed by addition of 20 µL of 3M sodium acetate, and extraction with an equal volume of phenol. After centrifugation, the supernatants were harvested and precipitated with 2 volumes of ethanol. The precipitates were washed with ethanol and dissolved in 200 µL of TE buffer . The 24 transformant DNAs ranged in concentration from approximately 50 ng/µL to 200 ng/µL.Transformation buffer (TFB) consists of 9 parts PEG/Li acetate solution , 1.5 mL of 4M lithium acetate, and 5.5 mL of sterile water) and 1 part of fresh 1M dithiothreitol. For transformation, In addition to the individual transformant DNAs, cells were pooled from a plate of 24 patches and extracted as above to yield 24-clone pool DNA at approximately 200 ng/µL.K. marxianus genome contains approximately 37,500 Sau3AI restriction sites (5′GATC) occurring on average every 280 bp along the genome. There is a single site in the 1123-bp ScURA3 sequence , 2 µL Sau3AI (10 ug/µL), and 38 µL water. Incubation was at 37 °C for 2h followed by inactivation of Sau3AI enzyme at 65 °C for 30 min. A 5-µL aliquot of the fragments was mixed with 20 µL of 10× T4 ligase buffer (NEB), 5 µL T4 ligase , and 170 µL water. Incubation was at 23 °C for 17 h followed by 72 °C for 10 min to inactivate the ligase. Two PCR reactions were performed. The first contained 5 µL of the ligated DNA, 16 µL of water, 25 µL of 2× Q5 master mix , and 2 µL of each of the primers ura3 + 720c and ura3 R1-1-DN at 25 µM. The second PCR was the same except that the primers were ura3 + 61c and ura3 TestF  following the manufacturer’s manual. The plugs were inserted into wells of 1% 0.5X TBE agarose gel and sealed with 1% 0.5× TBE agarose.® III Pulsed Field Electrophoresis Systems  was used for running PFGE with the following settings: initial switch time: 26.3 s, final switch time: 228 s, gradient: 6 V/cm, angle: 120°, temperature: 14 °C, and total time: 36 h. Gel was stained with 0.5 µg/mL ethidium bromide solution in water for 30 min and de-stained in distilled water for 1 h. Genomic DNA was visualized using Typhoon 9410 Variable Mode Imager.CHEF-DR