Effects of adding Bacillus subtilis natto NTU-18 in paste feed on growth, intestinal morphology, gastrointestinal microbiota diversity, immunity, and disease resistance of Anguilla japonica glass eels.

Japanese eel, Anguilla japonica, holds significant importance in Taiwanese aquaculture. With the intensification of eel farming, the impact of Edwardsiella tarda has become increasingly severe. Consequently, the abusive use of antibiotics has risen. Bacillus subtilis natto NTU-18, a strain of Bacillus with a high survival rate in feed processing, plays a crucial role in promoting intestinal health through competitive rejection, enhancing immune responses against bacterial pathogens, and improving intestinal health by modulating gastrointestinal microbiota to produce beneficial metabolites of mice and grass carp, Ctenopharyngodon idella. This study investigated the effects of different proportions (control, 0.25 %, 0.5 %, 1 %, and 2 %) of B. subtilis natto NTU-18 added to paste feed on the growth performance, intestinal morphology, and microbiota, expression of immune-related genes, and resistance to E. tarda in Japanese glass eel. The results indicated that the growth performance of all groups with B. subtilis natto NTU-18 added was significantly higher than that of the control group and did not impact the villi morphology. The expression of immune-related genes in the kidney, specifically HSP70 and SOD, was significantly higher from 0.5 % and above than the control; however, no significant differences were observed in CAT, POD, and HSP90. In the liver, significant differences were found in HSP70 and IgM above 0.25 % compared to the control group, with no significant differences in SOD, CAT, POD, and HSP90 among all groups. Additionally, intestinal microbiota analysis revealed that the 2 % additional group had significantly lower diversity than other groups, with Cetobacterium as the dominant species. The challenge test observed that the survival rates of the 0.5 % and 1 % groups were significantly higher. This research suggests that adding 0.5 % and 1 % of B. subtilis natto NTU-18 to the diet is beneficial for Japanese glass eel's immunity, growth performance, and disease resistance.

Probiotic Bacillus Affects Enterococcus faecalis Antibiotic Resistance Transfer by Interfering with Pheromone Signaling Cascades.

Enterococcus faecalis, a member of the commensal flora in the human gastrointestinal tract, has become a threatening nosocomial pathogen because it has developed resistance to many known antibiotics. More concerningly, resistance gene-carrying E. faecalis cells may transfer antibiotic resistance to resistance-free E. faecalis cells through their unique quorum sensing-mediated plasmid transfer system. Therefore, we investigated the role of probiotic bacteria in the transfer frequency of the antibiotic resistance plasmid pCF10 in E. faecalis populations to mitigate the spread of antibiotic resistance. Bacillus subtilis subsp. natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited pCF10 transfer by suppressing peptide pheromone activity from chromosomally encoded CF10 (cCF10) without inhibiting E. faecalis growth. The inhibitory effect was attributed to at least one 30- to 50-kDa extracellular protease present in B. subtilis subsp. natto. Nattokinase of B. subtilis subsp. natto was involved in the inhibition of pCF10 transfer and cleaved cCF10 (LVTLVFV) into LVTL plus VFV fragments. Moreover, the cleavage product LVTL (L peptide) interfered with the conjugative transfer of pCF10. In addition to cCF10, faecalis-cAM373 and gordonii-cAM373, which are mating inducers of vancomycin-resistant E. faecalis, were also cleaved by nattokinase, indicating that B. subtilis subsp. natto can likely interfere with vancomycin resistance transfer in E. faecalis. Our work shows the feasibility of applying fermentation products of B. subtilis subsp. natto and L peptide to mitigate E. faecalis antibiotic resistance transfer. IMPORTANCE Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, antibiotic resistance genes can undergo efficient intra- and interspecies transfer via E. faecalis peptide pheromone-mediated plasmid transfer systems. Therefore, this study provided the first experimental demonstration that probiotics are a feasible approach for interfering with conjugative plasmid transfer between E. faecalis strains to stop the transfer of antibiotic resistance. We found that the extracellular protease(s) of Bacillus subtilis subsp. natto cleaved peptide pheromones without affecting the growth of E. faecalis, thereby reducing the frequency of conjugative plasmid transfer. In addition, a specific cleaved pheromone fragment interfered with conjugative plasmid transfer. These findings provide a potential probiotic-based method for interfering with the transfer of antibiotic resistance between E. faecalis strains.

Production of 1-Dodecanol, 1-Tetradecanol, and 1,12-Dodecanediol through Whole-Cell Biotransformation in Escherichia coli.

Medium- and long-chain 1-alkanol and α,ω-alkanediols are used in personal care products, in industrial lubricants, and as precursors for polymers synthesized for medical applications. The industrial production of α,ω-alkanediols by alkane hydroxylation primarily occurs at high temperature and pressure using heavy metal catalysts. However, bioproduction has recently emerged as a more economical and environmentally friendly alternative. Among alkane monooxygenases, CYP153A from Marinobacter aquaeolei VT8 (CYP153A M.aq ; the strain is also known as Marinobacter hydrocarbonoclasticus VT8) possesses low overoxidation activity and high regioselectivity and thus has great potential for use in terminal hydroxylation. However, the application of CYP153A M.aq is limited because it is encoded by a dysfunctional operon. In this study, we demonstrated that the operon regulator AlkR M.aq is functional, can be induced by alkanes of various lengths, and does not suffer from product inhibition. Additionally, we identified a transposon insertion in the CYP153A M.aq operon. When the transposon was removed, the expression of the operon genes could be induced by alkanes, and the alkanes could then be oxyfunctionalized by the resulting proteins. To increase the accessibility of medium- and long-chain alkanes, we coexpressed a tunable alkane facilitator (AlkL) from Pseudomonas putida GPo1. Using a recombinant Escherichia coli strain, we produced 1.5 g/liter 1-dodecanol in 20 h and 2 g/liter 1-tetradecanol in 50 h by adding dodecane and tetradecane, respectively. Furthermore, in 68 h, we generated 3.76 g/liter of 1,12-dodecanediol by adding a dodecane-1-dodecanol substrate mixture. This study reports a very efficient method of producing C12/C14 alkanols and C12 1,12-alkanediol by whole-cell biotransformation.IMPORTANCE To produce terminally hydroxylated medium- to long-chain alkane compounds by whole-cell biotransformation, substrate permeability, enzymatic activity, and the control of overoxidability should be considered. Due to difficulties in production, small amounts of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol are typically produced. In this study, we identified an alkane-inducible monooxygenase operon that can efficiently catalyze the conversion of alkane to 1-alkanol with no detection of the overoxidation product. By coexpressing an alkane membrane facilitator, high levels of 1-dodecanol, 1-tetradecanol, and 1,12-dodecanediol could be generated. This study is significant for the bioproduction of medium- and long-chain 1-alkanol and α,ω-alkanediols.

Chemical Profiles of Incense Smoke Ingredients from Agarwood by Headspace Gas Chromatography-Tandem Mass Spectrometry.

Agarwood, the resinous wood in the heartwood of Aquilaria trees, has been used as incense in traditional Chinese medicine for its sedative, aphrodisiac, carminative, and anti-emetic effects. Grading of agarwood is usually based on its physical properties. Therefore, it is important to develop analytic methods for judgment and grading of agarwood. Here, we created a headspace (HS) preheating system that is combined with gas chromatography-mass spectrometry (HS GC-MS) to analyze the chemical constituents in the incense smoke produced by agarwood. Incense smoke generated in the HS preheating system was injected directly to GC-MS for analysis. A total of 40 compounds were identified in the incense smoke produced by Kynam agarwood, the best agarwood in the world. About half of the compounds are aromatics and sesquiterpenes. By analyzing chemical constituents in the incense smoke produced by Vietnamese, Lao, and Cambodian varieties of agarwood, we found that butyl hexadecanoate, butyl octadecanoate, bis(2-ethylhexyl) 1,2-benzenedicarboxylate, and squalene were common in the aforementioned four varieties of agarwoods. 2-(2-Phenylethyl) chromone derivatives were identified only in the incense smoke produced by Kynam agarwood, and were the major ingredient (27.23%) in the same. In conclusion, this is the first study that analyzes chemical profiles of incense smoke produced by agarwood using HS GC-MS. Our data showed that 2-(2-phenylethyl) chromone derivatives could be used to assess quality of agarwoods. Moreover, HS GC/MS may be a useful tool for grading quality of agarwood.

Transcriptomic analysis reveals that reactive oxygen species and genes encoding lipid transfer protein are associated with tobacco hairy root growth and branch development.

The hairy root, a specialized plant tissue that emerges from a cell transformed with transfer DNA (T-DNA) from Agrobacterium rhizogenes, can be used to study root biology and utilized in biotechnological applications. The rol genes are known to participate in the generation of hairy roots; however, the means by which the rol genes contribute to the initiation and the maintenance of hairy roots remains largely unknown. We demonstrated that tobacco hairy roots lacking either rolB or rolC exhibited fewer branch roots and lost their growth ability in long-term subculture. Additionally, a microarray analysis revealed that the expression of several genes encoding lipid transfer proteins (LTP) and reactive oxygen species (ROS)-related genes was significantly suppressed in rolB- or rolC-deficient hairy roots. We found that hairy root clones that exhibited greater branching expressed higher levels of RolB or RolC and the genes encoding LTP identified from the microarray. When hairy roots were compared with intact roots, the expression levels of LTP-encoding genes were dramatically different. In addition, ROS were present at lower levels in rolB- and rolC-deficient hairy roots. We therefore suggest that upregulating LTP and increasing the level of ROS is important for hairy root growth.

Bioconversion of pinoresinol into matairesinol by use of recombinant Escherichia coli.

Lignans, a class of dimeric phenylpropanoid derivative found in plants, such as whole grains and sesame and flax seeds, have anticancer activity and can act as phytoestrogens. The lignans secoisolariciresinol and matairesinol can be converted in the mammalian proximal colon into enterolactone and enterodiol, respectively, which reduce the risk of breast and colon cancer. To establish an efficient bioconversion system to generate matairesinol from pinoresinol, the genes encoding pinoresinol-lariciresinol reductase (PLR) and secoisolariciresinol dehydrogenase (SDH) were cloned from Podophyllum pleianthum Hance, an endangered herb in Taiwan, and the recombinant proteins, rPLR and rSDH, were expressed in Escherichia coli and purified. The two genes, termed plr-PpH and sdh-PpH, were also linked to form two bifunctional fusion genes, plr-sdh and sdh-plr, which were also expressed in E. coli and purified. Bioconversion in vitro at 22°C for 60 min showed that the conversion efficiency of fusion protein PLR-SDH was higher than that of the mixture of rPLR and rSDH. The percent conversion of (+)-pinoresinol to matairesinol was 49.8% using PLR-SDH and only 17.7% using a mixture of rPLR and rSDH. However, conversion of (+)-pinoresinol by fusion protein SDH-PLR stopped at the intermediate product, secoisolariciresinol. In vivo, (+)-pinoresinol was completely converted to matairesinol by living recombinant E. coli expressing PLR-SDH without addition of cofactors.

Nattokinase attenuates endothelial inflammation through the activation of SRF and THBS1.

Natto contains a potent fibrinolytic enzyme called nattokinase (NK), which has thrombolytic, antihypertensive, antiatherosclerotic and lipid-lowering effects. Although NK has been recognized for its beneficial effect on humans with atherosclerotic cardiovascular disease (ASCVD), the underlying mechanisms involved in vascular inflammation-atherosclerosis development remain largely unknown. The current study aimed to explore the effects of NK on gene regulation, autophagy, necroptosis and inflammasome in vascular inflammation. The transcriptional profiles of NK in endothelial cells (ECs) by RNA sequencing (RNA-seq) revealed that NK affected THBS1, SRF and SREBF1 mRNA expression. In Q-PCR analysis, SRF and THBS1 were upregulated but SREBF1 was unaffected in ECs treated with NK. NK treatment induced autophagy and inhibited NLRP3 inflammasome and necroptosis in ECs. Furthermore, the inhibition of SRF or THBS1 by siRNA suppressed autophagy and enhanced the NLRP3 inflammasome and necroptosis. In a mouse model, NK reduced vascular inflammation by activating autophagy and inhibiting NLRP3 inflammasome and necroptosis. Our findings provide the first evidence that NK upregulates SRF and THBS1 genes, subsequently increasing autophagy and decreasing necroptosis and NLRP3 inflammasome formation to reduce vascular inflammation. Therefore, NK could serve as nutraceuticals or adjuvant therapies to reduce vascular inflammation and possible atherosclerosis progression.

A comparison of strategies for multiple-gene co-transformation via hairy root induction.

Hairy root is a transformed root tissue in which transfer DNA (T-DNA) is inserted in the genome by Agrobacterium rhizogenes. To establish a system for multiple-gene co-transformation in hairy roots, we evaluated four different strategies using A. rhizogenes. The genes gusA and mgfp5 were located in separate plasmids, which were transformed into two different batches of A. rhizogenes (strategy 2AR) or a single batch (strategy 2BV). The two reporter genes were also inserted in one T-DNA (strategy 1TD) or two different T-DNAs (strategy 2TD) in a binary vector. Over 90 % of infected Nicotiana tabacum leaf discs formed hairy roots in all four groups, which was not significantly different from the infection efficiency of wild-type A. rhizogenes. Proportions of co-transformed hairy roots with strategies 2AR, 2BV, 1TD, and 2TD were 65.4, 40.0, 78.6, and 82.1 %, respectively, which indicated that all of the strategies were suitable for co-transformation of multiple genes. High variation in growth rate and heterologous protein expression indicated that further screening is required to identify the clone with the highest productivity. Our results indicated that strategies 1TD and 2TD achieved the highest co-transformation efficiency. Combination with strategy 2AR or 2BV provides additional options for co-transformation of multiple transgenes.

A process for high-efficiency isoflavone deglycosylation using Bacillus subtilis natto NTU-18.

In order to produce isoflavone aglycosides effectively, a process of isoflavone hydrolysis by Bacillus subtilis natto NTU-18 (BCRC 80390) was established. This process integrates the three stages for the production of isoflavone aglycosides in one single fermenter, including the growth of B. subtilis natto, production of ß-glucosidase, deglycosylation of fed isoflavone glycosides. After 8 h of batch culture of B. subtilis natto NTU-18 in 2 L of soy medium, a total of 3 L of soy isoflavone glucoside solution containing 3.0 mg/mL of daidzin and 1.0 mg/mL of genistin was fed continuously over 34 h. The percentage deglycosylation of daidzin and genistin was 97.7% and 94.6%, respectively. The concentration of daidzein and genistein in the broth reached 1,066.8 µg/mL (4.2 mM) and 351 µg/mL (1.3 mM), respectively, and no residual daidzin or genistin was detected. The productivity of the bioconversion of daidzein and genistein over the 42 h of culture was 25.6 mg/L/h and 8.5 mg/L/h, respectively. This showed that this is an efficient bioconversion process for selective estrogen receptor modulator production.

Anti-inflammatory sesquiterpene and triterpene acids from Mesona procumbens Hemsley.

Mesona procumbens Hemsley is a plant conventionally processed to provide popular food materials and herbal medicines in Asia. In this study, six triterpene acids, including five new ones (mesonaic acids D-H, 1-5), and one proximadiol-type sesquiterpene (7) were isolated from the methanolic extract of the air-dried M. procumbens. Chemical structures of 1‒7 were established by spectroscopic methods, especially 2D NMR techniques (1H-1H COSY, HSQC, HMBC, and NOESY) and HRESIMS. Concerning their biological activities, compounds 1, 2, 6, and 7 were examined manifesting high inhibition toward the pro-inflammatory NO production with EC50 values ranging from 12.88 to 21.21 µM, outrunning the positive control quercetin (24.12 µM). The mesoeudesmol B (7) identified from M. procumbens is the very first example, which exhibited high anti-inflammatory activity diminishing the level of the lipopolysaccharide-induced NO in RAW264.7 macrophage cells, thereby suppressing the secretion of pro-inflammatory cytokines TNF-α and IL-6 and the level of two critical downstream inflammatory mediators iNOS and COX-2.

Involvement of the azorhizobial chromosome partition gene (parA) in the onset of bacteroid differentiation during Sesbania rostrata stem nodule development.

A parA gene in-frame deletion mutant of Azorhizobium caulinodans ORS571 (ORS571-ΔparA) was constructed to evaluate the roles of the chromosome-partitioning gene on various bacterial traits and on the development of stem-positioned nodules. The ΔparA mutant showed a pleiomorphic cell shape phenotype and was polyploid, with differences in nucleoid sizes due to dramatic defects in chromosome partitioning. Upon inoculation of the ΔparA mutant onto the stem of Sesbania rostrata, three types of immature nodule-like structures with impaired nitrogen-fixing activity were generated. Most showed signs of bacteroid early senescence. Moreover, the ΔparA cells within the nodule-like structures exhibited multiple developmental-stage phenotypes. Since the bacA gene has been considered an indicator for bacteroid formation, we applied the expression pattern of bacA as a nodule maturity index in this study. Our data indicate that the bacA gene expression is parA dependent in symbiosis. The presence of the parA gene transcript was inversely correlated with the maturity of nodule; the transcript was switched off in fully mature bacteroids. In summary, our experimental evidence demonstrates that the parA gene not only plays crucial roles in cellular development when the microbe is free-living but also negatively regulates bacteroid formation in S. rostrata stem nodules.

Mesonosides A-H, primeverose derivatives from Mesona procumbens suppress adipogenesis by downregulating PPARγ and C/EBPα in 3T3-L1 cells.

Obesity is becoming a worldwide epidemic, especially in industrialized countries. We hereby report a methanolic extract of Mesona procumbens (known as Hsian-tsao in Taiwan) significantly inhibits lipid accumulation in 3T3-L1 adipocytes, and eight new primeverose derivatives, mesonosides A-H (1-8), were isolated from the methanolic extract of M. procumbens. Structural elucidation of 1-8 was established by spectroscopic methods, especially 2D NMR techniques (1H-1H COSY, HSQC, HMBC, and NOESY) and HRESIMS. Anti-obesity evaluation revealed that isolates 1-5, 7, and 8 showed inhibitory effects on lipid accumulation and protein levels of adipogenic transcription factor, PPARγ and C/EBPα in 3T3-L1 cells. Our study suggests that M. procumbens extract including new primeverose isolates may be potentially used as a natural source to ameliorate fat accumulation and even obesity.

Novel serotonin-boosting effect of incense smoke from Kynam agarwood in mice: The involvement of multiple neuroactive pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Stress is a state of feeling that inhibits one from responding properly in the face of a threat. Agarwood smoke has been used in traditional medicine as a sedative anti-anxious, and anti-restless therapy. Its scent emitted from heat induces people to enter a stable state; however, the underlying molecular effect is still unclear. AIM OF THE STUDY: This study analyzed novel biological events and gene expression signatures induced by agarwood incense smoke in mice. MATERIALS AND METHODS: Incense smoke was produced by heating at 150 °C for 30 min in a headspace autosampler oven. We treated mice with exposure to incense smoke from Kynam agarwood for 45 min/day for 7 consecutive days. After a 7-day inhalation period, the potent agarwood smoke affected-indicators in serum were measured, and the RNA profiles of the mouse brains were analyzed by microarray to elucidate the biological events induced by agarwood incense smoke. RESULTS: Chemical profile analysis showed that the major component in the incense smoke of Kynam was 2-(2-phenylethyl) chromone (26.82%). Incense smoke from Kynam induced mice to enter a stable state and increased the levels of serotonin in sera. The emotion-related pathways, including dopaminergic synapse, serotonergic synapse, GABAergic synapse, long-term depression and neuroactive ligand-receptor interaction, were significantly affected by incense smoke. Moreover, the expression of Crhr2 and Chrnd genes, involved with neuroactive ligand-receptor interaction pathway, was upregulated by incense smoke. CONCLUSIONS: By a newly-established incense smoke exposure system, we first identified that anti-anxious and anti-depressant effects of agarwood incense smoke were likely associated with the increase of serotonin levels and multiple neuroactive pathways in mice.

Triterpene Acids from Mesona procumbens Exert Anti-inflammatory Effects on LPS-Stimulated Murine Macrophages by Regulating the MAPK Signaling Pathway.

Five new triterpene acids, mesonaic acids A-C (1-3), 2α,3α,19α-trihydroxy-24-nor-4(23),12-oleanadien-28-oic acid (4), and 3α,19α,22α-trihydroxy-2-oxo-12-ursen-28-oic acid (5), and 10 known triterpene acid compounds (6-15) were isolated from a methanolic extract of Mesona procumbens. Triterpenes 1-3 possess unusual hexacyclic skeletons with a 13α,27-cyclopropane ring. Regarding their anti-inflammatory activity, compounds 1-3, 6, and 7 inhibited NO production with EC50 values lower than 15 µM, which were better than that of the positive control quercetin. Compounds 1-3, 6, and 7 markedly decreased levels of the inducible iNOS and COX-2 proteins in macrophages by inhibiting the activation of NF-κB through interference with the MAPK signaling pathway. Based on these data, compounds 1-3, 6, and 7 have great potential as NO inhibitors.

Bacillus subtilis natto Derivatives Inhibit Enterococcal Biofilm Formation via Restructuring of the Cell Envelope.

Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, the presence of E. faecalis biofilms can make infections with E. faecalis more difficult to eradicate with current antibiotic therapies. Thus, our aim in this study was to investigate the effects of probiotic derivatives against E. faecalis biofilm formation. Bacillus subtilis natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited adherence to Caco-2 cell monolayers, aggregation, and biofilm production without inhibiting the growth of E. faecalis. An apparent decrease in the thickness of E. faecalis biofilms was observed through confocal laser scanning microscopy. In addition, exopolysaccharide synthesis in E. faecalis biofilms was reduced by B. subtilis natto culture fluid treatment. Carbohydrate composition analysis also showed that carbohydrates in the E. faecalis cell envelope were restructured. Furthermore, transcriptome sequencing revealed that the culture fluid of B. subtilis natto downregulated the transcription of genes involved in the WalK/WalR two-component system, peptidoglycan biosynthesis and membrane glycolipid biosynthesis, which are all crucial for E. faecalis cell envelope synthesis and biofilm formation. Collectively, our work shows that some derivatives present in the culture fluid of B. subtilis natto may be useful for controlling E. faecalis biofilms.

Corrigendum: A Systematic Study of the Stability, Safety, and Efficacy of the de novo Designed Antimicrobial Peptide PepD2 and Its Modified Derivatives Against Acinetobacter baumannii.

[This corrects the article DOI: 10.3389/fmicb.2021.678330.].

De Novo Design of Antimicrobial Peptides With a Special Charge Pattern and Their Application in Combating Plant Pathogens.

Plant diseases are important issues in agriculture, and the development of effective and environment-friendly means of disease control is crucial and highly desired. Antimicrobial peptides (AMPs) are known as potential alternatives to chemical pesticides because of their potent broad-spectrum antimicrobial activity and because they have no risk, or have only a low risk, of developing chemical-resistant pathogens. In this study, we designed a series of amphipathic helical peptides with different spatial distributions of positive charges and found that the peptides that had a special sequence pattern "BBHBBHHBBH" ("B" for basic residue and "H" for hydrophobic residue) displayed excellent bactericidal and fungicidal activities in a wide range of economically important plant pathogens. The peptides with higher helical propensity had lower antimicrobial activity. When we modified the peptides with a long acyl chain at their N-terminus, their plant protection effect improved. Our application of the fatty acyl-modified peptides on the leaves of tomato and Arabidopsis plants lessened the infection caused by Pectobacterium carotovorum subsp. carotovorum and Botrytis cinerea. Our study provides important insights on the development of more potent novel AMPs for plant protection.

New Diterpenoids from Mesona procumbens with Antiproliferative Activities Modulate Cell Cycle Arrest and Apoptosis in Human Leukemia Cancer Cells.

Mesona procumbens is a popular material used in foods and herbal medicines in Asia for clearing heat and resolving toxins. However, phytochemical research on this plant is very rare. In this study, eleven new diterpenoids, mesonols A-K (1-11), comprising seven ent-kauranes, three ent-atisanes, and one sarcopetalane, were isolated from its methanolic extract. Structural elucidation of compounds 1-11 was performed by spectroscopic methods, especially 2D NMR, HRESIMS, and X-ray crystallographic analysis. All isolates were assessed for their antiproliferative activity, and compounds 1-4 showed potential antiproliferative activities against A549, Hep-3B, PC-3, HT29, and U937 cancer cells, with IC50 values ranging from 1.97 to 19.86 µM. The most active compounds, 1 and 2, were selected for further investigation of their effects on cell cycle progression, apoptosis, and ROS generation in U937 human leukemia cancer cells. Interestingly, it was found that compounds 1 and 2 induced antiproliferative effects in U937 cells through different mechanisms. Compound 1 caused cell cycle arrest at the G2/M phase and subsequent cell death in a dose- and time-dependent manner. However, 2-mediated antiproliferation of U937 cells triggered ROS-mediated mitochondrial-dependent apoptosis. These results provide insight into the molecular mechanism involved in the antiproliferative activities of compounds 1 and 2 in U937 cells. Altogether, the study showed that new diterpenoid compounds 1 and 2 from M. procumbens are potent and promising anticancer agents.

A Systematic Study of the Stability, Safety, and Efficacy of the de novo Designed Antimicrobial Peptide PepD2 and Its Modified Derivatives Against Acinetobacter baumannii.

Searching for new antimicrobials is a pressing issue to conquer the emergence of multidrug-resistant (MDR) bacteria and fungi. Antimicrobial peptides (AMPs) usually have antimicrobial mechanisms different from those of traditional antibiotics and bring new hope in the discovery of new antimicrobials. In addition to antimicrobial activity, stability and target selectivity are important concerns to decide whether an antimicrobial peptide can be applied in vivo. Here, we used a simple de novo designed peptide, pepD2, which contains only three kinds of amino acid residues (W, K, L), as an example to evaluate how the residues and modifications affect the antimicrobial activity against Acinetobacter baumannii, stability in plasma, and toxicity to human HEK293 cells. We found that pepI2 with a Leu→Ile substitution can decrease the minimum bactericidal concentrations (MBC) against A. baumannii by one half (4 µg/mL). A D-form peptide, pepdD2, in which the D-enantiomers replaced the L-enantiomers of the Lys(K) and Leu(L) residues, extended the peptide half-life in plasma by more than 12-fold. PepD3 is 3-residue shorter than pepD2. Decreasing peptide length did not affect antimicrobial activity but increased the IC50 to HEK293 cells, thus increased the selectivity index (SI) between A. baumannii and HEK293 cells from 4.7 to 8.5. The chain length increase of the N-terminal acyl group and the Lys→Arg substitution greatly enhanced the hemolytic activity, hence those modifications are not good for clinical application. Unlike colistin, the action mechanism of our peptides relies on negatively charged lipids rather than lipopolysaccharides. Therefore, not only gram-negative bacteria but also gram-positive bacteria can be killed by our peptides.

Cloning, expression, and characterization of two beta-glucosidases from isoflavone glycoside-hydrolyzing Bacillus subtilis natto.

On the basis of the genomic sequence of Bacillus subtilis 168, two beta-glucosidase genes (bglH and yckE) from B. subtilis natto, which has been reported to have high isoflavone glucoside-hydrolyzing activity, were cloned and overexpressed in E. coli M15. The temperature for the optimal p-nitrophenyl-beta-D-glucoside hydrolyzing activity of both enzymes was between 37 and 45 degrees C, but BglH had a higher thermal stability than YckE. Both showed high activity at pH 6.0, but YckE was stable over a wider pH range than BglH. Recombinant BglH was inhibited 73%, 63%, and 43% by 1.0 mM Cd(2+), Fe(2+), or Cu(2+), respectively, while other divalent metal ions resulted in 0-23% inhibition, whereas YckE was inhibited by less than 20% by any of the divalent metal ions we tested. Among the substrate we used, BglH showed the highest affinity for genistin and YckE showed the highest affinity for p-nitrophenyl-beta-D-fructopyranoside. Both BglH and YckE hydrolyzed genistin and daidzin into their isoflavone aglycones, genistein and daidzein, but BglH was more efficient than YckE in isoflavone glucoside hydrolysis (20-fold higher kcat). Our results suggest that recombinant BglH may be applicable in the process of isoflavones deglycosylation.