Ultrasonic microencapsulation of oil-soluble vitamins by hen egg white and green tea for fortification of food.

We report the microencapsulation of oil soluble vitamins (A, D and E) using a one pot ultrasonic process and raw egg white proteins as a shell material. Green tea catechin/iron complex coating method was further developed to impart UV filtering property to the microcapsules in order to protect the encapsulated nutrients from photodegradation. The microcapsules showed antibacterial properties and long shelf-life. The encapsulated vitamins were protected from degradation upon heating, UV irradiation, simulated storage/transit and cooking processes. The in-vitro digestion study showed that functional vitamin D can be potentially released in the gastrointestinal tract improving vitamin D availability by more than 2-fold compared to the free vitamin. The vitamin D microcapsules were highly stable and maintained their microstructures once incorporated into staple food products. The low-cost egg white shell encapsulated vitamins can improve the nutritional value of staple food products to combat maternal and child malnutrition.

Asperorydines N-P, three new cyclopiazonic acid alkaloids from the marine-derived fungus Aspergillus flavus SCSIO F025.

Three new tricyclic cyclopiazonic acid (CPA) related alkaloids asperorydines N-P (1-3), together with six known compounds (4-9) were isolated and characterized from the fungus Aspergillus flavus SCSIO F025 derived from the deep-sea sediments of South China Sea. The structures including absolute configurations of 1-3 were deduced from spectroscopic data, X-ray diffraction analysis, and electronic circular dichroism (ECD). All compounds were evaluated for the antioxidative activities against DPPH, cytotoxic activities against four tumor cell lines (SF-268, HepG-2, MCF-7, and A549), and antimicrobial activities. Compound 9 showed significant radical scavenging activities against DPPH with an IC50 value of 62.23 µM and broad-spectrum cytotoxicities against four tumor cell lines with IC50 values ranging from 24.38 to 48.28 µM. Furthermore, compounds 4-9 exhibited weak antimicrobial activities against E scherichia coli, and compound 9 also showed antibacterial activity against Bacillus thuringiensis, Micrococcus lutea, Staphylococcus aureus, Bacillus subtilis, Methicillin resistant Staphylococcus aureus.

Half of 'Micrococcus spp.' cases identified by conventional methods are revealed as other life-threatening bacteria with different drug susceptibility patterns by 16S ribosomal RNA gene sequencing.

Bacterial infection during chemotherapy is a fatal complication, therefore precise identification of the pathogenic microorganism is required for treatment. We report that 2 of 4 pediatric patients with malignancy who were diagnosed with Micrococcus spp. infection by conventional methods were finally revealed to have Kytococcus schroeteri and Kocuria marina infection by 16S ribosomal RNA gene sequence analysis (16S rRNA analysis). Although K. schroeteri is morphologically similar to Micrococcus spp., its drug susceptibility profile is quite different from that of Micrococcus spp. K. schroeteri is resistant to penicillin and cephalosporin, which are effective for Micrococcus spp. In fact, penicillin-resistant lethal pneumonia caused by K. schroeteri has been reported in compromised hosts. Based on our results, Micrococcus spp. determined by conventional methods could contain other life-threatening bacteria with different drug susceptibility patterns from Micrococcus spp. To develop an effective empirical treatment for immunocompromised hosts, accumulation of pathogen data by 16S rRNA analysis is required.

Effects of biochar-immobilized bacteria on phytoremediation of cadmium-polluted soil.

This work is the first report of the ability of biochar-immobilized cadmium-resistant bacteria (CRB) on promoting the efficiency of cadmium phytoextraction by Chlorophytum laxum R.Br. The survival of CRB immobilized on biochar in cadmium-contaminated soil at a concentration of 75.45 mg kg-1 was studied. The results found that both CRB, namely Arthrobacter sp. TM6 and Micrococcus sp. MU1, can survive and grow in cadmium-contaminated soil. To study phytoextraction in the pot experiments, 2-month-old C. laxum was individually planted in cadmium-contaminated soil and divided into four treatments, including (i) untreated control, (ii) biochar, (iii) biochar-immobilized (BC) Arthrobacter sp., and (iv) BC-Micrococcus sp. The results found that biochar-immobilized CRB did not cause any effect to the root lengths and shoot heights of plants compared to the untreated control. Interestingly, inoculation of biochar-immobilized CRB significantly increased cadmium accumulation in the shoots and roots compared to the untreated control. In addition, the highest cadmium content in a whole plant, best phytoextraction performance, and greatest bioaccumulation factor was found in plant inoculated with BC-Micrococcus sp., followed by BC-Arthrobacter sp. In conclusion, inoculation of biochar-immobilized CRB enhanced cadmium accumulation and translocation of cadmium from the roots to shoots, suggesting further applying biochar-immobilized CRB in cadmium-polluted soil for promoting cadmium phytoextraction efficiency of ornamental plants. Graphical abstract.

Ageritin, a Ribotoxin from Poplar Mushroom ( Agrocybe aegerita) with Defensive and Antiproliferative Activities.

Ribotoxins make up a group of extracellular rRNA endoribonucleases produced by ascomycetes that display cytotoxicity toward animal cells, having been proposed as insecticidal agents. Recently, the ribotoxin Ageritin has been isolated from the basidiomycetes Agrocybe aegerita (poplar mushroom), suggesting that ribotoxins are widely distributed among fungi. To gain insights into the protective properties of Ageritin against pathogens and its putative biotechnological applications, we have tested several biological activities of Ageritin, comparing them with those of the well-known ribotoxin α-sarcin, and we found that Ageritin displayed, in addition to the already reported activities, (i) antibacterial activity against Micrococcus lysodeikticus, (ii) activity against the tobacco mosaic virus RNA, (iii) endonuclease activity against a supercoiled plasmid, (iv) nuclease activity against genomic DNA, (v) cytotoxicity to COLO 320, HeLa, and Raji cells by promoting apoptosis, and (vi) antifungal activity against the green mold Penicillium digitatum. Therefore, Ageritin and α-sarcin can induce resistance not only to insects but also to viruses, bacteria, and fungi. The multiple biological activities of Ageritin could be exploited to improve resistance to different pathogens by engineering transgenic plants. Furthermore, the induction of cell death by different mechanisms turns these ribotoxins into useful tools for cancer therapy.

Absolute Configurations of 14,15-Hydroxylated Prenylxanthones from a Marine-Derived Aspergillus sp. Fungus by Chiroptical Methods.

Determination of the absolute configrations for natural products is one of the most important and challenging tasks, especially when the molecules display high conformational flexibility. In this paper, eight new prenylxanthones, aspergixanthones A-H (1-8), and one known analogue (9), were isolated from the marine-derived fungus Aspergillus sp. ZA-01. The absolute configurations of C-14 and C-15 in 1-8 were difficult to be assigned due to the high conformational flexibility of the chains. To solve this problem, the experimental ECD, ORD, and VCD spectra of 1 were combined for analysis with the corresponding theoretical predictions for its different diastereomers. This study suggested that a concerted application of more than one chiroptical methods could be used as a preferable approach for the stereochemical characterizations of flexible molecules. Compounds 1-9 were evaluated for their cytotoxic and antibacterial activities. Among them, 6 showed cytotoxicity against the A-549 cell line with the IC50 value of 1.1 µM, and 7 exhibited antibacterial activity against Micrococcus lysodeikticus with the MIC value of 0.78 µg/mL.

Phytoremediation of cadmium-polluted soil by Chlorophytum laxum combined with chitosan-immobilized cadmium-resistant bacteria.

This study examined the performance of the chitosan-immobilized cadmium-resistant bacteria Arthrobacter sp. and Micrococcus sp. on cadmium phytoremediation by Chlorophytum laxum in cadmium-polluted soil. These immobilized cadmium-resistant bacteria can survive in cadmium-contaminated soil and significantly increased soil cadmium solubility, but the ability of chitosan-immobilized cells to increase cadmium solubility was lower than that of free cells. A pot experiment demonstrated that chitosan-immobilized Micrococcus sp. promoted the growth of C. laxum planted in cadmium-contaminated soil. A significant increase in the cadmium concentration in the roots and aboveground parts of C. laxum was found in plants inoculated with free and chitosan-immobilized cells of these bacteria. The performance of Arthrobacter sp. free cells to augment cadmium accumulation in C. laxum was a little bit better than that of chitosan-immobilized Arthrobacter sp., except at 9 weeks after planting. The phytoextraction coefficient, bioaccumulation factor, and translocation factor of C. laxum inoculated with free and chitosan-immobilized cells of cadmium-resistant bacteria were higher than those of the uninoculated control and increased with time. Our findings suggest that chitosan-immobilized cells can be exploited to enhance the efficiency of cadmium phytoremediation by C. laxum.

Improvement of cadmium phytoremediation after soil inoculation with a cadmium-resistant Micrococcus sp.

Cadmium-resistant Micrococcus sp. TISTR2221, a plant growth-promoting bacterium, has stimulatory effects on the root lengths of Zea mays L. seedlings under toxic cadmium conditions compared to uninoculated seedlings. The performance of Micrococcus sp. TISTR2221 on promoting growth and cadmium accumulation in Z. mays L. was investigated in a pot experiment. The results indicated that Micrococcus sp. TISTR2221significantly promoted the root length, shoot length, and dry biomass of Z. mays L. transplanted in both uncontaminated and cadmium-contaminated soils. Micrococcus sp. TISTR2221 significantly increased cadmium accumulation in the roots and shoots of Z. mays L. compared to uninoculated plants. At the beginning of the planting period, cadmium accumulated mainly in the shoots. With a prolonged duration of cultivation, cadmium content increased in the roots. As expected, little cadmium was found in maize grains. Soil cadmium was significantly reduced with time, and the highest percentage of cadmium removal was found in the bacterial-inoculated Z. mays L. after transplantation for 6 weeks. We conclude that Micrococcus sp. TISTR2221 is a potent bioaugmenting agent, facilitating cadmium phytoextraction in Z. mays L.

Antibacterial activity of ruthenium(II) polypyridyl complex manipulated by membrane permeability and cell morphology.

This study investigates the antibacterial effects of the ruthenium(II) complex RuBP and the mechanism of RuBP action on bacteria. Results show that RuBP can inhibit the growth of Gram-positive bacteria, such as Staphylococcus aureus and Micrococcus tetragenus. Cellular uptake and laser confocal microscopic studies reveal the efficient uptake of RuBP by M. tetragenus cells. Scanning electron microscopic observations of the morphologies of M. tetragenus and S. aureus treated with RuBP further confirm that direct contact of both bacteria with RuBP can damage the cell membrane and membrane integrity, which may eventually induce growth inhibition and bacterial death. After RuBP treatment, the electrical conductivity of the bacterial suspensions increases. Spectroscopic studies and agarose gel electrophoresis indicate that intact DNA and RNA decrease or disappear in RuBP-treated bacterial cells, thus demonstrating that RuBP performs its antibacterial function by increasing the permeability of cell membranes. This study provides new insights for understanding the antibacterial actions of RuBP and designing metal complex antibiotics for other biomedical applications.

Covalent immobilization of lysozyme on ethylene vinyl alcohol films for nonmigrating antimicrobial packaging applications.

The objective of this study was to develop a new antimicrobial film, in which lysozyme was covalently attached onto two different ethylene vinyl alcohol copolymers (EVOH 29 and EVOH 44). The EVOH surface was modified with UV irradiation treatment to generate carboxylic acid groups, and lysozyme was covalently attached to the functionalized polymer surface. Surface characterization of control and modified films was performed using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and dye assay. The value of protein loading after attachment on the surface was 8.49 µg protein/cm(2) and 5.74 µg protein/cm(2) for EVOH 29 and EVOH 44, respectively, after 10 min UV irradiation and bioconjugation. The efficacy of the EVOH-lysozyme films was assessed using Micrococcus lysodeikticus. The antimicrobial activity of the films was tested against Listeria monocytogenes and was similar to an equivalent amount of free enzyme. The reduction was 1.08 log for EVOH 29-lysozyme, 0.95 log for EVOH 44-lysozyme, and 1.34 log for free lysozyme. This work confirmed the successful use of lysozyme immobilization on the EVOH surface for antimicrobial packaging.

Mass spectrometry-guided optimization and characterization of a biologically active transferrin-lysozyme model drug conjugate.

Transferrin is a promising drug carrier that has the potential to deliver metals, small organic molecules and therapeutic proteins to cancer cells and/or across physiological barriers (such as the blood-brain barrier). Despite this promise, very few transferrin-based therapeutics have been developed and reached clinical trials. This modest success record can be explained by the complexity and heterogeneity of protein conjugation products, which also pose great challenges to their analytical characterization. In this work, we use lysozyme conjugated to transferrin as a model therapeutic that targets the central nervous system (where its bacteriostatic properties may be exploited to control infection) and develop analytical protocols based on electrospray ionization mass spectrometry to characterize its structure and interactions with therapeutic targets and physiological partners critical for its successful delivery. Mass spectrometry has already become an indispensable tool facilitating all stages of the protein drug development process, and this work demonstrates the enormous potential of this technique in facilitating the development of a range of therapeutically effective protein-drug conjugates.

JBIR-78 and JBIR-95: phenylacetylated peptides isolated from Kibdelosporangium sp. AK-AA56.

The search for metabolites of Kibdelosporangium sp. AK-AA56 resulted in the discovery of novel N-phenylacetylated peptides, JBIR-78 (1) and JBIR-95 (2). Compounds 1 and 2 were established to be N-phenylacetylated heptapeptides by extensive NMR and HRESIMS analyses. The absolute configuration of the standard amino acids including a cysteic acid moiety was determined using Marfey's method on the acid hydrolysates of 1 and 2. The relative and absolute configurations of a nonstandard amino acid, ß-hydroxyleucine, were elucidated using the J-based and modified Mosher's methods, respectively. In an antimicrobial test, 1 showed antibacterial activity against Micrococcus luteus.

Prenylated acylphloroglucinols, chipericumins A-D, from Hypericum chinense.

Two new tetracyclic prenylated acylphloroglucinols, chipericumins A (1) and B (2), were isolated from the roots of Hypericum chinense, together with two new tricyclic prenylated acylphloroglucinols, chipericumins C (3) and D (4). Their structures were elucidated by spectroscopic data. Chipericumins A-D (1-4) are prenylated acylphloroglucinols having a spiro skeleton with an acyl group, a methyl group, a C(5) unit, and a monoterpene moiety in common.

Scalarane sesterterpenes from the sponge Hyatella sp.

Five new sesterterpenes (7-11) along with six known compounds (1-6) were isolated from the sponge Hyatella sp., collected off the coast of Soheuksan-do, Korea. Spectroscopic analyses revealed these compounds as scalarane sesterterpenes with oxidized furan moieties (7-10) and a corresponding lactam (11). The compounds exhibited moderate cytotoxicity, antibacterial activity, and weak inhibitory activity against isocitrate lyase.

Potent antifouling resorcylic acid lactones from the gorgonian-derived fungus Cochliobolus lunatus.

Three new 14-membered resorcylic acid lactones, two with a rare natural acetonide group and one with a 5-chloro-substituted lactone, named cochliomycins A-C (1-3), together with four known analogues, zeaenol (4), LL-Z1640-1 (5), LL-Z1640-2 (6), and paecilomycin F (7), were isolated from the culture broth of Cochliobolus lunatus, a fungus obtained from the gorgonian Dichotella gemmacea collected in the South China Sea. Their structures and the relative configurations of 1-3 were elucidated using comprehensive spectroscopic methods including NOESY spectra and chemical conversions. A transetherification reaction was also observed in which cochliomycin B (2) in a solution of CDCl(3) slowly rearranged to give cochliomycin A (1) at room temperature. These resorcylic acid lactones were evaluated against the larval settlement of barnacle Balanus amphitrite, and antifouling activity was detected for the first time for this class of metabolites. The antibacterial and cytotoxic activities of these compounds were also examined.

Simple purification of human antimicrobial peptide dermcidin (MDCD-1L) by intein-mediated expression in E.coli.

Among human antimicrobial peptides (hAMPs), DCD-1L has a broad spectrum of antimicrobial activity over a wide pH range and in high salt concentrations. It offers a promising alternative to conventional antibiotics. The 458-bp-long dermcidin cDNA was amplified by PCR using a human fetal cDNA library as a template. The 147-bp fragment of the MDCD-1L gene encoding an additional methionine residue was subcloned into the pTYB11 vector. Recombinant MDCD-1L was expressed as an intein fusion protein in E. coli, and then purified by affinity chromatography using chitin beads. A small peptide with a molecular mass of about 5 kDa was detected by tricine gel electrophoresis. The recombinant MDCD-1L peptide was purified from the gel and its amino acid sequence was determined by nanoLC-ESI-MS/MS analysis. The initiating amino acid, methionine, remained attached to the N-terminal region of recombinant MDCD-1L. Purified MDCD-1L showed antimicrobial activity against a Micrococcus luteus test strain.

Cytotoxic alkaloids and antibiotic nordammarane triterpenoids from the marine-derived fungus Aspergillus sydowi.

Three new diketopiperazine alkaloids, 6-methoxyspirotryprostatin B (1), 18-oxotryprostatin A (2), and 14-hydroxyterezine D (3), with an oxaspiro[4.4]lactam moiety, 14-norpseurotin A (4), and the 29-nordammarane triterpenoid 6beta,16beta-diacetoxy-25-hydroxy-3,7-dioxy-29-nordammara-1,17(20)-dien-21-oic acid (5), as well as 12 known compounds (6- 17), were isolated from the ethyl acetate extract of a marine-derived fungal strain, Aspergillus sydowi PFW1-13. The structures of compounds 1- 5 were elucidated by comprehensive spectroscopic analysis. Compounds 1- 3 exhibit weak cytotoxicity against A-549 cells, with IC 50 values of 8.29, 1.28, and 7.31 microM, respectively. Compound 1 also shows slight cytotoxicity against HL-60 cells, with an IC 50 value of 9.71 microM. Compounds 4 and 5 display significant antimicrobial activities against Escherichia coli, Bacillus subtilis, and Micrococcus lysoleikticus with MICs of 3.74, 14.97, and 7.49 microM and 10.65, 5.33, and 10.65 microM, respectively.

NMR structure of mussel mytilin, and antiviral-antibacterial activities of derived synthetic peptides.

Mytilin is a 34-residue antibacterial peptide from the mussel Mytilus galloprovincialis, which in addition possesses in vitro antiviral activity. The three-dimensional solution structure of the synthetic mytilin was established by using 1H NMR and consists of the common cysteine-stabilized alphabeta motif close to the one observed in the mussel defensin MGD-1. Mytilin is characterized by 8 cysteines engaged in four disulfide bonds (2-27, 6-29, 10-31, and 15-34) only involving the beta-strand II. Hydrophilic and hydrophobic areas of mytilin account for 63% and 37%, respectively, a ratio very close to that of MGD-1 (64% and 36%). One linear and three cyclic fragments were designed from the interstrand loop sequence known to retain the biological activities in MGD-1. Only the fragment of 10 amino acids (C10C) constrained by two disulfide bonds in a stable beta-hairpin structure was able to inhibit the mortality of Palaemon serratus shrimp injected with white spot syndrome virus (WSSV). Fifty percent inhibition was obtained by in vitro pre-incubation of WSSV with 45 microM of C10C compared with 7 microM for mytilin. Interaction between the fragment and the virus occurred very rapidly as 40% survival was recorded after only 1 min of pre-incubation. In addition, C10C was capable of inhibiting in vitro growth of Vibrio splendidus LGP32 (MIC 125 microM), Vibrio anguillarum (MIC 2mM), Micrococcus lysodeikticus and Escherichia coli (MIC 1mM). Destroying the cysteine-stabilized alphabeta structure or shortening the C10C fragment to the C6C fragment with only one disulfide bond resulted in loss of both antiviral and antibacterial activities. Increasing the positive net charge did not enforce the antibacterial activity and completely suppressed the antiviral one. The C10C-designed peptide from mytilin appeared comparable in composition and structure with protegrin, tachyplesin and polyphemusin.

In vitro and in vivo protein delivery from in situ forming poly(ethylene glycol)-poly(lactide) hydrogels.

Previous studies have shown that stereocomplexed hydrogels are rapidly formed in situ by mixing aqueous solutions of eight-arm poly(ethylene glycol)-poly(L-lactide) and poly(ethylene glycol)-poly(D-lactide) star block copolymers (denoted as PEG-(PLLA)(8) and PEG-(PDLA)(8), respectively). In this study, in vitro and in vivo protein release from stereocomplexed hydrogels was investigated. These hydrogels were fully degradable under physiological conditions. Proteins could be easily loaded into the stereocomplexed hydrogels by mixing protein containing aqueous solutions of PEG-(PLLA)(8) and PEG-(PDLA)(8) copolymers. The release of the relatively small protein lysozyme (d(h)=4.1 nm) followed first order kinetics and approximately 90% was released in 10 days. Bacteria lysis experiments showed that the released lysozyme had retained its activity. The relatively large protein IgG (d(h)=10.7 nm) could be released from stereocomplexed hydrogels with nearly zero order kinetics, wherein up to 50% was released in 16 days. The in vitro release of the therapeutic protein rhIL-2 from stereocomplexed hydrogels also showed nearly zero order kinetics, wherein up to 45% was released in 7 days. The therapeutic efficacy of stereocomplexed hydrogels loaded with 1x10(6) IU of rhIL-2 was studied using SL2-lymphoma bearing DBA/2 mice. The PEG-(PLLA)(8)/PEG-(PDLA)(8)/rhIL-2 mixture could be easily injected intratumorally. The released rhIL-2 was therapeutically effective as the tumor size was reduced and the cure rate was 30%, whereas no therapeutic effect was achieved when no rhIL-2 was given. However, the cure rate of rhIL-2 loaded stereocomplexed hydrogels was lower, though not statistically significant, compared to that of a single injection with 1x10(6) IU of free rhIL-2 at the start of the therapy (cure rate=70%). The therapeutic effect of rhIL-2 loaded stereocomplexed hydrogels was retarded for approximately 1-2 weeks compared to free rhIL-2, most likely due to a slow, constant release of rhIL-2 from the hydrogels.