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Additional file 1: Table S1. Scripts used in this study. Table S2. mRNA folding energy based Top 10 and Bottom 10 variants. Table S3. Plasmids used in this study. Table S4. Synthetic double strand DNA fragment used in this study. Table S5. Primers used in this study. Figure S1. Expression of TGm1 in different forms. E. coli transformed with A pET-22b/pro-TGm1, B pET-22b/proH-TGm1, and C pET-22b-trxA-proH-TGm1. 1, 3, and 5: insoluble fractions; 2, 4, and 6: soluble fractions. E. coli was cultivated at 20 ℃ for 32 h after induction by adding IPTG at a final concentration of 0.1 mM when OD600 reached 1.0. Red arrow: A pro-TGm1, B proH-TGm1, and C TrxA-proH-TGm1. Figure S2. SDS-PAGE analysis of smTG variants purified by Nickel affinity chromatography. 1: FRAP-TGm1; 2: FRAPD-TGm1. Figure S3. Edman sequencing the N-terminus of recombinant TGm1 variants activated by dispase in vitro. pro-TGm1 and proH-TGm1 were intracellularly expressed in E. coli using pET-22b/pro-TGm1 and pET-22b/proH-TGm1, recombinantly expressed TGm1 variants were in vitro activated and purified by affinity chromatography using HisTrap (GE Healthcare, USA) column. Figure S4. E. coli carrying pETDuet/pelB-TrxA-proH-TGm1/TAMEP and pKJE7 was cultivated under 20 ℃ after induction by IPTG. Figure S5. MD simulation of FRAP-TGm1 and FRAPD-TGm1. A Hydrogen bonds within the N-terminus (FRAPDPDD) of FRAP-TGm1. B Hydrogen bonds within the N-terminus (FRAPDPDD) of FRAPD-TGm1. MD simulation was conducted for 100 ns using Gromacs-2020. C The modeled structure of FRAPD-TGm1. The N-terminal loop of FRAPD-TGm1 was colored in cyan. Figure S6. Sequence analysis of pro-TGm1 (GenBank ID: MZ516369). The number of nucleotide sequences (left side); Single underline: pro-region; Double underline: mature region of TGm1; Box, His-tag. Figure S7. Sequence analysis of TAMEP (GenBank ID: MZ516816). A start codon (ATG) was placed after the predicted signal peptide. Single underline: signal peptide [2]. Numbers (left side): the number of nucleotide sequences. Box, His-tag. For purification purpose, a His-tag was placed as shown in box while constructing the plasmid pETDuet/TAMEP; for co-expressing TAMEP with TGm1, the His-tag (as shown in box) was removed.

The chemical compositions, bioactive ingredients, and in vitro antitumor activity were analyzed using water extracts from the dried fermented mycelia of Cephalosporium sinensis (C1), Paecilomyces hepiali (C2), Cordyceps cicadae (C3), as well as the dried fruiting bodies of C. militaris (C4). Total amino acid content was highest in C3, compared with C1, C2, C4, and natural C. militaris (S1). The contents of Fe, Zn, Mn, and Ca as well as the Zn/Cu ratio in C1-C3 were obviously different than those of C4 and S1, whereas Na contents and K/Na ratios in C3, C4, and S1 were obviously different from those in C1 and C2. Except for C4, cordycepin was not detected in C1-C3 or S1. The crude polysaccharide contents in C4 and total triterpenoid content in C4 were relatively lower than that in C1-C3. Detailed analysis of the crude polysaccharide from C4 did not detect fucose, but it contained the highest amount of glucose, compared with C1-C3. Additionally, the antitumor activities of the respective water extracts were ranked in the order of C3 > C4 > C1 > C2. These results can help us better understand products of Cordyceps strains.

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect 2.5 × 103 CFU/ml of pLR colonies spiked in 106 CFU/ml of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

Aims Piliated Lactobacillus rhamnosus (pLR) strains have attracted much attention owing to their excellent mucus adhering capacity and immunomodulatory effects. Here, we aimed to develop a rapid, sensitive method for isolating pLR strains in complex ecosystems using immunomagnetic separation (IMS) with colony immunoblotting (CIB). Methods and Results Magnetic nanobeads (diameter: 180 nm) conjugated with anti-pLR SpaA pilin antibodies (anti-SpaA) were prepared and used to preconcentrate pLR strains in samples, followed by confirmation with anti-SpaA-based CIB analysis. Under optimised experimental conditions, IMS-CIB selectively recovered pLR strains from 107 CFU ml−1 of faecal microbiota samples spiked with 2.9 × 101 CFU ml−1 to 2.4 × 106 CFU ml−1 of pLR strains. No positive colonies were detected in samples without addition of pLR strains. The detection limit of IMS-CIB was 29 CFU pLR ml−1 of faecal microbiota, which is much lower than that of CIB without IMS preconcentration (2.0 × 104 CFU ml−1). Conclusions IMS-CIB allowed selective preconcentration of pLR strains in highly heterogeneous bacterial suspensions and direct detection of pLR colonies, which remained readily available for subsequent isolation. Significance and Impact of the Study Our findings established an effective method for selective enrichment and detection of pLR strains. This article is protected by copyright. All rights reserved.