Antibacterial Mechanism, Control Efficiency, and Nontarget Toxicity Evaluation of Actinomycin X2 against Xanthomonas citri Subsp. citri.

The present study investigated the antibacterial mechanism, control efficiency, and nontarget toxicity of actinomycin X2 (Act-X2) against Xanthomonas citri subsp. citri (Xcc) for the first time. Act-X2 almost completely inhibited the proliferation of Xcc in the growth curve assay at a concentration of 0.25 MIC (minimum inhibitory concentration, MIC = 31.25 µg/mL). This inhibitory effect was achieved by increasing the production of reactive oxygen species (ROS), blocking the formation of biofilms, obstructing the synthesis of intracellular proteins, and decreasing the enzymatic activities of malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) of Xcc. Molecular docking and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis results indicated that Act-X2 steadily bonded to the RNA polymerase, ribosome, malate dehydrogenase, and succinate dehydrogenase to inhibit their activities, thus drastically reducing the expression levels of related genes. Act-X2 showed far more effectiveness than the commercially available pesticide Cu2(OH)3Cl in the prevention and therapy of citrus canker disease. Furthermore, the nontarget toxicity evaluation demonstrated that Act-X2 was not phytotoxic to citrus trees and exhibited minimal toxicity to earthworms in both contact and soil toxic assays. This study suggests that Act-X2 has the potential as an effective and environmentally friendly antibacterial agent.

Control of citrus blue and green molds by Actinomycin X2 and its possible antifungal mechanism.

Citrus blue and green molds caused by Penicillium digitatum, P. italicum, and P. polonicum, are the major postharvest diseases of citrus fruit. In the present study, Actinomycin X2 (Act-X2), a naturally occurring antibiotic produced by Streptomyces species, was found to show excellent antifungal effect against these three pathogens with a minimum inhibitory concentration (MIC) value of 62.5 µg/mL for them all, which was better than the positive control thiophanate-methyl. Act-X2 significantly reduced the percentage of spore germination, and highly inhibited the mycelial growth of P. italicum, P. digitatum, and P. polonicum with EC50 values being 34.34, 13.76, and 37.48 µg/mL, respectively. In addition, Act-X2 greatly decreased the intracellular protein content while increasing the reactive oxygen species (ROS) level and superoxide anion (O2-) content in the mycelia of pathogens. In vivo test indicated that Act-X2 strongly inhibited the infection of navel oranges by these three Penicillium species, with an inhibition percentage of >50% for them all at the concentration of 10 MIC. Transcriptome analysis suggested that Act-X2 might highly influence the ribosomal functions of P. polonicum, which was supported as well by the molecular docking analysis of Act-X2 with some key functional proteins and RNAs of the ribosome. Furthermore, Act-X2 significantly reduced the decay percentage and improved the firmness, color, and sugar-acid ratio of navel oranges spray-inoculated with P. polonicum during the postharvest storage at 4 °C for 60 d.

Discovery of actinomycin L, a new member of the actinomycin family of antibiotics.

Streptomycetes are major producers of bioactive natural products, including the majority of the naturally produced antibiotics. While much of the low-hanging fruit has been discovered, it is predicted that less than 5% of the chemical space of natural products has been mined. Here, we describe the discovery of the novel actinomycins L1 and L2 produced by Streptomyces sp. MBT27, via application of metabolic analysis and molecular networking. Actinomycins L1 and L2 are diastereomers, and the structure of actinomycin L2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X2, prior to the condensation of the actinomycin halves. Such a structural feature has not previously been identified in naturally occurring actinomycins. Adding anthranilamide to cultures of the actinomycin X2 producer Streptomyces antibioticus, which has the same biosynthetic gene cluster as Streptomyces sp. MBT27, resulted in the production of actinomycin L. This supports a biosynthetic pathway whereby actinomycin L is produced from two distinct metabolic routes, namely those for actinomycin X2 and for anthranilamide. Actinomycins L1 and L2 showed significant antimicrobial activity against Gram-positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families.

Separation and characterization of two series of unknown degradation impurities caused by light irradiation and autoclaving in xinfujunsu injection using liquid chromatography tandem mass spectrometry.

RATIONALE: A series of photodegradation impurities and a series of degradation impurities produced in autoclaving in xinfujunsu injection were discovered, and these unknown impurities were separated and characterized thoroughly using liquid chromatography tandem quadrupole time-of-flight mass spectrometry. METHODS: The column was a Platisil 5 µm ODS (4.6 × 250 mm, 5 µm). For the analysis of degradation impurities caused by light irradiation and autoclaving, the mobile phase was composed of 0.01 M ammonium formate aqueous solution and acetonitrile/isopropanol (90:10, V/V). Full scan LC-MS and LC-MS2 was carried out to obtain as much structural information as possible. The fragmentation behavior of actinomycin D, actinomycin S3 , and its impurities was studied and used to obtain information about the structures of these impurities. RESULTS: Based on MS2 spectral data and exact mass measurements, the chemical structures of two series of degradation impurities were characterized, among which five unknown impurities were photodegradation impurities and seven unknown impurities were degradation impurities produced in autoclaving of xinfujunsu injection. CONCLUSIONS: Based on characterization of impurities, this study also revealed the cause of impurity production and provided guidance for enterprises to improve the process and drug packaging material to reduce impurity content. Furthermore, this study also provided scientific basis for further improvement of official monographs in pharmacopoeias.

Actinomycin V Induces Apoptosis Associated with Mitochondrial and PI3K/AKT Pathways in Human CRC Cells.

Actinomycin (Act) V, an analogue of Act D, presented stronger antitumor activity and less hepatorenal toxicity than Act D in our previous studies, which is worthy of further investigation. We hereby report that Act V induces apoptosis via mitochondrial and PI3K/AKT pathways in colorectal cancer (CRC) cells. Act V-induced apoptosis was characterized by mitochondrial dysfunction, with loss of mitochondria membrane potential (MMP) and cytochrome c release, which then activated cleaved caspase-9, cleaved caspase-3, and cleaved PARP, revealing that it was related to the mitochondrial pathway, and the apoptotic trendency can be reversed by caspase inhibitor Z-VAD-FMK. Furthermore, we proved that Act V significantly inhibited PI3K/AKT signalling in HCT-116 cells using cell experiments in vitro, and it also presented a potential targeted PI3Kα inhibition using computer docking models. Further elucidation revealed that it exhibited a 28-fold greater potency than the PI3K inhibitor LY294002 on PI3K inhibition efficacy. Taken together, Act V, as a superior potential replacement of Act D, is a potential candidate for inhibiting the PI3K/AKT pathway and is worthy of more pre-clinical studies in the therapy of CRC.

Synergistic Interactions of Cannabidiol with Chemotherapeutic Drugs in MCF7 Cells: Mode of Interaction and Proteomics Analysis of Mechanisms.

Cannabidiol (CBD), a nonpsychoactive phytocannabinoid, has recently emerged as a potential cytotoxic agent in addition to its ameliorative activity in chemotherapy-associated side effects. In this work, the potential interactions of CBD with docetaxel (DOC), doxorubicin (DOX), paclitaxel (PTX), vinorelbine (VIN), and 7-ethyl-10-hydroxycamptothecin (SN-38) were explored in MCF7 breast adenocarcinoma cells using different synergy quantification models. The apoptotic profiles of MCF7 cells after the treatments were assessed via flow cytometry. The molecular mechanisms of CBD and the most promising combinations were investigated via label-free quantification proteomics. A strong synergy was observed across all synergy models at different molar ratios of CBD in combination with SN-38 and VIN. Intriguingly, synergy was observed for CBD with all chemotherapeutic drugs at a molar ratio of 636:1 in almost all synergy models. However, discording synergy trends warranted the validation of the selected combinations against different models. Enhanced apoptosis was observed for all synergistic CBD combinations compared to monotherapies or negative controls. A shotgun proteomics study highlighted 121 dysregulated proteins in CBD-treated MCF7 cells compared to the negative controls. We reported the inhibition of topoisomerase II ß and α, cullin 1, V-type proton ATPase, and CDK-6 in CBD-treated MCF7 cells for the first time as additional cytotoxic mechanisms of CBD, alongside sabotaged energy production and reduced mitochondrial translation. We observed 91 significantly dysregulated proteins in MCF7 cells treated with the synergistic combination of CBD with SN-38 (CSN-38), compared to the monotherapies. Regulation of telomerase, cell cycle, topoisomerase I, EGFR1, protein metabolism, TP53 regulation of DNA repair, death receptor signalling, and RHO GTPase signalling pathways contributed to the proteome-wide synergistic molecular mechanisms of CSN-38. In conclusion, we identified significant synergistic interactions between CBD and the five important chemotherapeutic drugs and the key molecular pathways of CBD and its synergistic combination with SN-38 in MCF7 cells. Further in vivo and clinical studies are warranted to evaluate the implementation of CBD-based synergistic adjuvant therapies for breast cancer.

Integrated pan-cancer of AURKA expression and drug sensitivity analysis reveals increased expression of AURKA is responsible for drug resistance.

INTRODUCTION: The AURKA gene encodes a protein kinase involved in cell cycle regulation and plays an oncogenic role in many cancers. The main objective of this study is to analyze AURKA expression in 13 common cancers and its role in prognostic and drug resistance. METHOD: Using the cancer genome atlas (TCGA) as well as CCLE and GDSC data, the level of AURKA gene expression and its role in prognosis and its association with drug resistance were evaluated, respectively. In addition, the expression level of AURKA was assessed in colorectal cancer (CRC) and gastric cancer (GC) samples. Besides, using Gene Expression Omnibus (GEO) data, drugs that could affect the expression level of this gene were also identified. RESULTS: The results indicated that the expression level of AURKA gene in 13 common cancers increased significantly compared to normal samples or it survived poorly (HR >1, p < 0.01) in lung, prostate, kidney, bladder, and uterine cancers. Also, the gene expression data showed increased expression in CRC and GC samples compared to normal ones. The level of AURKA was significantly associated with the resistance to SB 505124, NU-7441, and irinotecan drugs (p < 0.01). Eventually, GEO data showed that JQ1, actinomycin D1, and camptothecin could reduce the expression of AURKA gene in different cancer cell lines (logFC < 1, p < 0.01). CONCLUSION: Increased expression of AURKA is observed in prevalent cancers and associated with poor prognostic and the development of drug resistance. In addition, some chemotherapy drugs can reduce the expression of this gene.

Mutasynthesis of Antibacterial Halogenated Actinomycin Analogues.

Through precursor-directed biosynthesis, feeding halogenated (F-, Cl-, Br-, I-) or methoxy-substituted 4-methyl-3-hydroxyanthranilic acid (4-MHA) analogues to the acnGHLM-deleted mutant strain of Streptomyces costaricanus SCSIO ZS0073 led to the production of ten new actinomycin analogues (4-13). Several of the actinomycin congeners displayed impressive antimicrobial activities, with MIC values spanning 0.06-64 µg/mL to clinically derived antibiotic resistant pathogens, including Staphylococcus aureus, Enterococcus faecium, and Candida albicans, with low cytotoxicity.

Actinomycin X2, an Antimicrobial Depsipeptide from Marine-Derived Streptomyces cyaneofuscatus Applied as a Good Natural Dye for Silk Fabric.

Actinomycins as clinical medicine have been extensively studied, while few investigations were conducted to discover the feasibility of actinomycins as antimicrobial natural dye contributing to the medical value of the functional fabrics. This study was focused on the application of actinomycin X2 (Ac.X2), a peptide pigment cultured from marine-derived Streptomyces cyaneofuscatus, in the dyeing and finishing of silk fabric. The dyeing potential of Ac.X2 with silk vs. cotton fabrics was assessed. As a result, the silk fabric exhibited greater uptake and color fastness with Ac.X2. Through Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and X-ray diffraction (XRD) analyses, some changes of chemical property for the dyed fabric and Ac.X2 were studied. The silk fabric dyed with Ac.X2 exhibited good UV protection ability. The antibacterial properties of dyed and finished silk were also evaluated, which exhibited over 90% antibacterial activity even after 20 washing cycles. In addition, the brine shrimp assay was conducted to evaluate the general toxicity of the tested fabric, and the results indicated that the dyed silk fabrics had a good biological safety property.

Exploring the Thermodynamics of 7-Amino Actinomycin D-Induced Single-Stranded DNA Hairpin by Spectroscopic Techniques and Computational Simulations.

NMR studies have indicated that the anti-tumor therapeutic agent actinomycin D (ACTD) can induce seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3GTGG-3' to form a hairpin structure with tandem GT mismatches at the stem region next to a loop of three stacked thymine bases. In an effort to uncover the preference of binding sequence and to elucidate the thermodynamics properties of the binding, a combination of spectroscopic techniques and computational simulation studies was performed with d(CCGTTnGTGG) and d(CCGAAnGAGG) (denoted as GTTn and GAAn, respectively; n = 3, 5, and 7) sequences. In the presence of 7-amino actinomycin D (7AACTD), all the six oligomers formed stable hairpin structures. The GTT5-7AACTD/GAA5-7AACTD hairpin structure was more stable than the corresponding GTTn-7AACTD and GAAn-7AACTD (n = 3, 7). No significant ΔG difference was observed between GTTn-7AACTD and GAAn-7AACTD complexes with the same loop length. In agreement with the 7AACTD-induced hairpin stability results, the binding affinity of GTTn and GAAn with 7AACTD increased from n = 3 to n = 5 and then decreased when n is 7. Moreover, GTTn and GAAn with the same loop length showed comparable binding affinities to 7AACTD. Furthermore, molecular dynamics simulations found that van der Waals interactions between GTTn/GAAn and 7AACTD were the primary attractive forces for 7AACTD binding, and the electrostatic interactions between the carbonyl groups of 7AACTD and bases in the hairpin were the major unfavorable forces. These findings furthered our understanding that 7AACTD is sensitive to the loop size and sequence as well as tandem GT/GA mismatches of their deoxyribonucleic acid (DNA) targets. A deep understanding of the thermodynamics and the molecular recognition mechanism of 7AACTD with ssDNAs would further the development of ACTD-like antitumor agents.

Involvement of Reactive Oxygen Species in the Hepatorenal Toxicity of Actinomycin V In Vitro and In Vivo.

The high toxicity of actinomycin D (Act D) severely limits its use as a first-line chemotherapeutic agent in the clinic. Actinomycin V (Act V), an analog of Act D, exhibited strong anticancer activity in our previous studies. Here, we provide evidence that Act V has less hepatorenal toxicity than Act D in vitro and in vivo, associated with the reactive oxygen species (ROS) pathway. Compared to Act D, Act V exhibited considerably stronger sensitivity for cancer cells and less toxicity to human normal liver LO-2 and human embryonic kidney 293T cells using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay. Notably, Act V caused less damage to both the liver and kidney than Act D in vivo, indicated by organ to body weight ratios, as well as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and serum creatinine (Scr) levels. Further experiments showed that the ROS pathway is involved in Act V-induced hepatorenal toxicity. Act V generates ROS and accumulates malondialdehyde (MDA), reducing levels of superoxide dismutase (SOD) and glutathione (GSH) in LO-2 and 293T cells. These findings indicate that Act V induces less hepatorenal toxicity than Act D in vitro and in vivo and merits further development as a potential therapeutic agent for the treatment of cancer.

Insights into Streptomyces spp. isolated from the rhizospheric soil of Panax notoginseng: isolation, antimicrobial activity and biosynthetic potential for polyketides and non-ribosomal peptides.

BACKGROUND: Streptomycetes from the rhizospheric soils are a rich resource of novel secondary metabolites with various biological activities. However, there is still little information related to the isolation, antimicrobial activity and biosynthetic potential for polyketide and non-ribosomal peptide discovery associated with the rhizospheric streptomycetes of Panax notoginseng. Thus, the aims of the present study are to (i) identify culturable streptomycetes from the rhizospheric soil of P. notoginseng by 16S rRNA gene, (ii) evaluate the antimicrobial activities of isolates and analyze the biosynthetic gene encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) of isolates, (iii) detect the bioactive secondary metabolites from selected streptomycetes, (iv) study the influence of the selected isolate on the growth of P. notoginseng in the continuous cropping field. This study would provide a preliminary basis for the further discovery of the secondary metabolites from streptomycetes isolated from the rhizospheric soil of P. notoginseng and their further utilization for biocontrol of plants. RESULTS: A total of 42 strains representing 42 species of the genus Streptomyces were isolated from 12 rhizospheric soil samples in the cultivation field of P. notoginseng and were analyzed by 16S rRNA gene sequencing. Overall, 40 crude cell extracts out of 42 under two culture conditions showed antibacterial and antifungal activities. Also, the presence of biosynthesis genes encoding type I and II polyketide synthase (PKS I and PKS II) and nonribosomal peptide synthetases (NRPSs) in 42 strains were established. Based on characteristic chemical profiles screening by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD), the secondary metabolite profiles of strain SYP-A7257 were evaluated by High Performance Liquid Chromatography-High Resolution Mass Spectrometry (HPLC-HRMS). Finally, four compounds actinomycin X2 (F1), fungichromin (F2), thailandin B (F7) and antifungalmycin (F8) were isolated from strain SYP-A7257 by using chromatography techniques, UV, HR-ESI-MS and NMR, and their antimicrobial activities against the test bacteria and fungus were also evaluated. In the farm experiments, Streptomyces sp. SYP-A7257 showed healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field. CONCLUSIONS: We demonstrated the P. notoginseng rhizospheric soil-derived Streptomyces spp. distribution and diversity with respect to their metabolic potential for polyketides and non-ribosomal peptides, as well as the presence of biosynthesis genes PKS I, PKS II and NRPSs. Our results showed that cultivatable Streptomyces isolates from the rhizospheric soils of P. notoginseng have the ability to produce bioactive secondary metabolites. The farm experiments suggested that the rhizospheric soil Streptomyces sp. SYP-A7257 may be a potential biological control agent for healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field.

Sensitive monitoring of RNA transcription by optical amplification of cationic conjugated polymers.

We reported a new strategy for sensitive monitoring in vitro RNA synthesis in real time based on fluorescence resonance energy transfer (FRET) from water-soluble conjugated polymer poly (9, 9-bis (6'-N, N, N,-trimethylammonium) hexyl) fluorene-co-alt-1,4-phenylene) bromide (PFP) to fluorogenic RNA aptamer/fluorophore (Spanich2/DFHBI and Broccoli/DFHBI) system. In this strategy, RNA of interest was transcribed accompanied by the Spanich2 or Broccoli. Then the 3, 5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) bound to the RNA aptamer sequence and thereby induced a fluorescence signal. PFP was used as the fluorescence energy donor, and Spanich2/DFHBI was the fluorescence energy acceptor. The fluorescence signal of Spanich2/DFHBI was amplified by light-harvesting and fluorescence amplification ability of PFP via FRET. And the limit of detection (LOD) (0.29 nM) was near 10-fold lower than that of RNA aptamer/DFHBI (LOD is 2.8 nM) alone by measuring the FRET ratio, which greatly reduced the variation of background signals. Most importantly, the addition of PFP did not interfere with RNA transcription in vitro, so this method was successfully applied to sensitively monitor RNA transcription and effect of T7 RNA polymerase inhibitor in real time, supplying a sensitive and simple method to study the modulation and inhibitor of RNA polymerase in vitro.

Purification and characterization of actinomycins from Streptomyces strain M7 active against methicillin resistant Staphylococcus aureus and vancomycin resistant Enterococcus.

BACKGROUND: The increased rate of resistance among two highly concerned pathogens i.e. methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) necessitates the discovery of novel anti-MRSA and anti-VRE compounds. In microbial drug discovery, Streptomyces are well known source of two-thirds of natural antibiotics used clinically. Hence, screening of new strains of streptomycetes is the key step to get novel bioactive compounds with antimicrobial activity against drug resistant bacteria. RESULTS: In the present study, Streptomyces antibioticus strain M7, possessing potent antibacterial activity against different pathogenic bacteria, was isolated from rhizospheric soil of Stevia rebudiana. 16S rRNA sequence of M7 (1418 bp) showed 96.47-100% similarity with different Streptomyces spp. and the maximum similarity (100%) was observed with Streptomyces antibioticus NBRC 12838T (AB184184). Phylogenetic analysis using neighbor joining method further validated its similarity with Streptomyces antibioticus NBRC 12838 T (AB184184) as it formed clade with the latter and showed high boot strap value (99%). Antibacterial metabolites isolated from the fermentation broth were characterized using NMR, FT-IR and LC-MS as actinomycins V, X2 and D. The purified actinomycins exhibited potent antibacterial activities against test bacteria viz. B. subtilis, K. pneumoniae sub sp. pneumoniae, S. aureus, S. epidermidis, S. typhi, E. coli, MRSA and VRE. Among these actinomycins, actinomycin X2 was more effective as compared to actinomycins D and V. The minimum inhibitory concentration values of purified compounds against a set of test bacterial organisms viz. VRE, MRSA, E. coli (S1-LF), K. pneumoniae sub sp. pneumoniae and B. subtilis ranged between 1.95 and 31.25 µg/ml. CONCLUSIONS: This study demonstrates that actinomycins V, X2 and D produced by S. antibioticus strain M7 hold the potential to be used against multidrug resistant bacteria, particularly VRE and MRSA.

Antimicrobial potential of haloalkaliphilic Nocardiopsis sp. AJ1 isolated from solar salterns in India.

Antagonistic haloalkaliphilic Nocardiopsis sp. AJ1 (GenBank JX575136.1), isolated and identified from the saline soil of Kovalam solar salterns was able to produce antimicrobial secondary metabolites and effectively suppressed several bacterial and fungal pathogens. The metabolite extracted from ethyl acetate precipitation suppressed the bacterial and fungal pathogens to the range between 2.14 and 20.14 mm and also controlled the shrimp killer virus WSSV by 83% than the control and significantly (p < 0.05) differed. GC-MS analysis revealed that, the ethyl acetate precipitation contains pyrrolo (1,2-A(pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-) and actinomycin C2. Non ribosomal peptide synthetase (NRPS) was amplified by PCR with the amplicon size of 750-800 bp length and further predicted the secondary structure by Iterative Threading Assembly Refinement (I-TASSER) bioinformatics approach. I-TASSER prediction helped to find out the secondary, 3-D structure, and ligand binding sites. The top ten modelling concluded that, the NRPS gene is closely similar to surfactin synthesizing gene, surfactin A synthetase C (SRFA-C). The findings revealed that, the active compounds from the secondary metabolites effectively suppressed the pathogenic bacteria, fungi, and virus and useful to develop antimicrobials.

A new actinomycin Z analogue with an additional oxygen bridge between chromophore and ß-depsipentapeptide from Streptomyces sp. KIB-H714.

Actinomycin Z6 (1), a new member of the actinomycin family, along with three congeners of the Z-type (Z1, Z3, Z5) actinomycins, are produced from Streptomyces sp. KIB-H714. Their structures were authenticated by comprehensive spectroscopic data interpretation. Different from all the reported Z-type actinomycins, the ß-ring of the new compound actinomycin Z6 includes an additional ring linked between the actinoyl chromophore and ß-peptidolactone. In Z3 and Z5, the L-threonine in ß-depsipeptide is replaced by the unusual 4-chlorothreonine, an amino acid rarely found in actinomycin family. All isolates were evaluated for cytotoxicity against five human tumor cell lines and for inhibitory activity against Candida albicans and Staphylococcus aureus.

Actinomycin-Producing Endophytic Streptomyces parvulus Associated with Root of Aloe vera and Optimization of Conditions for Antibiotic Production.

Endophytic actinomycetes are a rich source of novel antimicrobial compounds. The aim of this study was to evaluate the production of antimicrobial compound by endophytic Streptomyces sp. Av-R5 associated with root of Aloe vera against multidrug-resistant human pathogens. The 16S rRNA sequence of the isolate Av-R5 has been identified as Streptomyces parvulus NBRC 13193T (AB184326) and the sequence was submitted to the National Center for Biotechnology Information (NCBI) GenBank database (accession number KY771080). Streptomyces parvulus Av-R5 grown under submerged fermentation condition optimized by central composite design (glucose 11.16 g/L, soybean meal 10.25 g/L, sodium chloride 11.18 g/L, calcium carbonate 1.32 g/L at pH 7.19 at 31.42 °C with 6.04% seed inoculum for 10 days of incubation) exhibited the highest activity against multidrug-resistant Staphylococcus aureus JNMC-3, Staphylococcus epidermidis JNMC-4, Klebsiella pneumoniae MTCC-3384, Klebsiella pneumoniae JNMC-6, Pseudomonas aeruginosa MTCC-741, Proteus vulgaris JNMC-7, Candida albicans MTCC-183, and Aspergillus niger MTCC-872. The structures of the active compounds were elucidated by UV-Vis spectroscopy, 1H and 13C NMR, FT-IR, and ESIMS. Actinomycin D and actinomycin X0ß were detected in crude extracts and major components were eluted by HPLC at 10.96 and 6.81 min, respectively. In this case, a high yield of actinomycin D and actinomycin X0ß (400 mg/L) was achieved with Streptomyces parvulus Av-R5, fermented in glucose soybean meal broth media, which can be used in industrial fermentation process to obtain high yields.

Improved flow cytometry crossmatching in kidney transplantation.

Flow cytometry crossmatching (FC-XM) assay is the most sensitive cell-based method for detecting donor-specific antibodies (DSAs). However, the use of FC-XM remains limited by methodological and clinical variations. This basic assay cannot discriminate between complement-fixing and noncomplement-fixing antibodies. FC-XM also detects patient all antibodies bound to donor cells and not only DSAs against to HLA molecules. Pretest factors associated with a donor's medical care can affect test results by changing the number, viability and target on lymphocytes (such as rituximab on CD20+ B-cells). Assay adjustment can be performed to improve the sensitivity and specificity of FC-XM. Pronase treatment (0.5-1 mg/mL) prevents false-positive B-cell FC-XM due to nonspecific immunoglobulin binding by Fc receptors and binding of surface immunoglobulins onto the surface of B-cells. Pronase treatment (2 mg/mL) or a serum incubation step with an anti-rituximab monoclonal antibody (Ab) prevents the interference induced by rituximab therapy. The use of 7 aminoactinomycin-D (7-AAD) or fluorochrome-conjugated C4d Ab, after complement incubation, allows complement-fixing antibodies to be distinguished from noncomplement-fixing antibodies. The use of donor endothelial precursor cells as target cells allows the detection of nonmajor histocompatibility complex Ab-binding endothelial cells. However, lymphocyte crossmatches still had some limits in specificity and sensitivity. This implies that this assay must be interpreted with the virtual crossmatch.

Current practices for viability testing of cryopreserved cord blood products: an international survey by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative.

BACKGROUND: Viability testing is a common practice in laboratories. The goal of this study was to ascertain current laboratory practices internationally for performing viability testing for cryopreserved cord blood (CB) products and glean information about how to standardize the method to improve interlaboratory reproducibility. STUDY DESIGN AND METHODS: A survey to evaluate current laboratory practices for viability testing was designed and distributed internationally. The question topics included sampling and testing methods, responses to unexpected results, and the rating of the reliability of the CB quality tests, together with expectations for standardization. RESULTS: There were 32 respondents to the survey, of whom 28 responded to the more detailed questionnaire about viability methods. Overall, responses indicated that various stains were used among the laboratories, and when multiple sites used the same viability stain the methods differed. The majority of the respondents were in favor of standardizing the viability testing methods. A wide variety of preferences were communicated about how to standardize the method, but a majority did advocate the use of 7-aminoactinomycin D (7-AAD) with flow cytometry. CONCLUSIONS: The survey results revealed a variety of tests and inconsistent interlaboratory practices for performing the viability assay. Flow cytometry with a 7-AAD dye was suggested as a first step toward standardization.

Flow cytometry in formamide treated cells.

The use of formamide for the study in flow cytometry of cell cycle phases, by DNA content measurement in human cancer cell lines, was recently published. In this manuscript, we verify the possibility of extending the procedure to simultaneous analysis of other parameters. The results obtained, here reported, show that the treatment of samples by formamide is compatible with the simultaneous detection of DNA content and surface phenotypes, with quantification of replicating DNA and with measurement of cells with fractional content of DNA. For each of these three applications, we have adapted the procedure to gain simple, reproducible and above all advantageous protocols. Regarding the simultaneous analysis of DNA content and phenotyping the use of formamide achieves optimal DNA stoichiometric staining (C.V. < 3; G2/G1 ratio = 2 ± 0.05) and sufficient maintenance of physical parameters and membrane fluorescence. In the study of duplicating DNA labeled with click chemistry, our procedure eliminates paraformaldehyde (PFA) fixation improving the DNA stoichiometric staining and allows the use of 7-aminoactinomycin D (7-AAD) preserving the Alexa Fluor 488 quantum efficiency. Concerning the detection of cells with fractional content of DNA, permeabilization and fixation by formamide gives the advantage of resolve on linear scale sub-G1 cells from debris and to allow optimal sample recovery (>90%) which is essential in the study of cell necrobiology. Cells treatment by formamide, suitably modified for different applications, can be used to prepare cell samples for flow cytometry analyses that go far beyond stoichiometric staining of DNA.