Characterization and antimicrobial activity of endophytic fungi from medicinal plant Agave americana.

Endophytic microorganisms associated with medicinal plants are of particular interest as they are a potential source of new bioactive chemicals effective against novel emerging and drug-resistant pathogens. Agave americana is a tropical medicinal plant with antibacterial, antifungal, and anticancer properties. We studied the biodiversity of fungal endophytes of A. americana and their antimicrobial production potential. Isolated endophytic fungi were classified into 32 morphotypes (15 from stem and 17 from leaf) based on their cultural and morphological characteristics. Among the fungal crude extracts tested, 82% of isolates from the leaves and 80% of the isolates from the stem showed antibacterial activity against the bacterial strains (Escherichia coli ATTC 25902, Staphylococcus aureus ATTC 14775, and Bacillus subtilis NRRL 5109) tested. Extracts from four fungal isolates from leaves showed antifungal activity against at least one of the fungal strains (Candida albicans ATTC 10231 and Aspergillus fumigatus NRRL 5109) tested. Crude extracts of seven fungal isolates showed a zone of inhibition of more than 11 mm at 10 mgml-1 against both Gram-positive and Gram-negative bacteria tested. Penicillium, Colletotrichum, Curvularia, Pleosporales, Dothideomycetes, and Pleurotus are the main endophytes responsible for bioactive potential. These results indicate that A. americana harbors endophytes capable of producing antimicrobial metabolites.

The impact of ultrasound treatment on glycolytic enzymes when applied to crude extracts from early post-mortem bovine muscle.

The rate of pH decline post - mortem and its interaction with temperature influences the final tenderness of meat, and therefore, the manipulation of the rate of pH decline is a strategy of interest in order to obtain consistent high quality meat. Ultrasound is a potential early post - mortem carcass intervention, which may alter the rate of glycolysis based on its ability to alter enzyme activity. In this study, homogenates (prepared from early post-mortem Longissimus thoracis et lumborum muscle) were subjected to different ultrasound intensities (0 %/60 %/100 % amp) and treatment durations (15/ 30 min). The effect of these treatments on the inherent activity of the glycolytic enzymes was investigated using an in vitro glycolytic buffer model system. It was found that ultrasound treatment intensity and duration had a significant interactive effect on the rate of pH decline, and on reducing sugars and lactic acid concentrations, specifically following the 100 % amp ultrasound for 30 min treatment and between 30 and 240 min incubation. No significant differences in pH or metabolites content were observed between treatments after 1440 min of incubation. No effect of ultrasound intensity or treatment duration was observed on the degradation of glycogen. Under the reported conditions of this trial, it can be concluded that the application of ultrasound has limited potential to have an impact on the glycolytic pathways in bovine muscle.

Comparative Proteomics Reveals Prolonged Corneal Preservation Impaired Ocular Surface Immunity Accompanied by Fibrosis in Human Stroma.

Cornea transplantation is one of the most commonly performed allotransplantations worldwide. Prolonged storage of donor corneas leads to decreased endothelial cell viability, severe stromal edema, and opacification, significantly compromising the success rate of corneal transplantation. Corneal stroma, which constitutes the majority of the cornea, plays a crucial role in maintaining its shape and transparency. In this study, we conducted proteomic analysis of corneal stroma preserved in Optisol-GS medium at 4 °C for 7 or 14 days to investigate molecular changes during storage. Among 1923 identified proteins, 1634 were quantifiable and 387 were significantly regulated with longer preservation. Compared to stroma preserved for 7 days, proteins involved in ocular surface immunomodulation were largely downregulated while proteins associated with extracellular matrix reorganization and fibrosis were upregulated in those preserved for 14 days. The increase in extracellular matrix structural proteins together with upregulation of growth factor signaling implies the occurrence of stromal fibrosis, which may compromise tissue clarity and cause vision impairments. This study is the first to provide insights into how storage duration affects corneal stroma from a proteomic perspective. Our findings may contribute to future research efforts aimed at developing long-term preservation techniques and improving the quality of preserved corneas, thus maximizing their clinical application.

New Cytotoxic Monoalkyl Glycerol Ether from the Red Sea Soft Coral Nephthea mollis.

A new monoalkyl glycerol ether, 3-(n-henicosyloxy)propane-1,2-diol (1), was isolated from the CH2 Cl2 /MeOH crude extract of the Red Sea soft coral Nephthea mollis. Additionally, three known related analogs were identified: chimyl alcohol (2), batyl alcohol (3), and 3-(icosyloxy)propane-1,2-diol (4). The chemical structure of 3-(n-henicosyloxy)propane-1,2-diol was determined using advanced spectroscopic analyses, including 1D, 2D Nuclear Magnetic Resonance (NMR), Electron Ionization mass spectra (EI-MS), and High-Resolution Electron Spray Ionization mass spectra (HR-ESI-MS) analyses. Furthermore, the identification of chimyl alcohol, batyl alcohol and 3-(icosyloxy)propane-1,2-diol was achieved by studying their EI mass fragmentation analyses and comparing their mass data with those previously reported in the literature. The cytotoxic activity of the Nephthea mollis crude extract and 3-(n-henicosyloxy)propane-1,2-diol was evaluated against five human cancer cell lines: HepG2 (hepatocellular carcinoma), MCF-7 (breast carcinoma), NCI-1299 (lung carcinoma), HeLa (cervical cancer cell), and HT-29 (colon adenocarcinoma). Moreover, 3-(n-henicosyloxy)propane-1,2-diol revealed moderate cytotoxicity against the HeLa cell lines with an IC50 value of 24.1 µM, while showing inactivity against the remaining cell lines (IC50 >100 µM).

Fungal Secondary Metabolites/Dicationic Pyridinium Iodide Combinations in Combat against Multi-Drug Resistant Microorganisms.

The spread of antibiotic-resistant opportunistic microbes is a huge socioeconomic burden and a growing concern for global public health. In the current study, two endophytic fungal strains were isolated from Mangifera Indica roots and identified as Aspergillus niger MT597434.1 and Trichoderma lixii KU324798.1. Secondary metabolites produced by A. niger and T. lixii were extracted and tested for their antimicrobial activity. The highest activity was noticed against Staphylococcus aureus and E. coli treated with A. niger and T. lixii secondary metabolites, respectively. A. niger crude extract was mainly composed of Pentadecanoic acid, 14-methyl-, methyl ester and 9-Octadecenoic acid (Z)-, methyl ester (26.66 and 18.01%, respectively), while T. lixii crude extract's major components were 2,4-Decadienal, (E,E) and 9-Octadecenoic acid (Z)-, and methyl ester (10.69 and 10.32%, respectively). Moreover, a comparative study between the fungal extracts and dicationic pyridinium iodide showed that the combination of A. niger and T. lixii secondary metabolites with dicationic pyridinium iodide compound showed a synergistic effect against Klebsiella pneumoniae. The combined formulae inhibited the bacterial growth after 4 to 6 h through cell wall breakage and cells deformation, with intracellular components leakage and increased ROS production.

Investigating ethanol production using the Zymomonas mobilis crude extract.

Cell-free systems have become valuable investigating tools for metabolic engineering research due to their easy access to metabolism without the interference of the membrane. Therefore, we applied Zymomonas mobilis cell-free system to investigate whether ethanol production is controlled by the genes of the metabolic pathway or is limited by cofactors. Initially, different glucose concentrations were added to the extract to determine the crude extract's capability to produce ethanol. Then, we investigated the genes of the metabolic pathway to find the limiting step in the ethanol production pathway. Next, to identify the bottleneck gene, a systemic approach was applied based on the integration of gene expression data on a cell-free metabolic model. ZMO1696 was determined as the bottleneck gene and an activator for its enzyme was added to the extract to experimentally assess its effect on ethanol production. Then the effect of NAD+ addition at the high concentration of glucose (1 M) was evaluated, which indicates no improvement in efficiency. Finally, the imbalance ratio of ADP/ATP was found as the controlling factor by measuring ATP levels in the extract. Furthermore, sodium gluconate as a carbon source was utilized to investigate the expansion of substrate consumption by the extract. 100% of the maximum theoretical yield was obtained at 0.01 M of sodium gluconate while it cannot be consumed by Z. mobilis. This research demonstrated the challenges and advantages of using Z. mobilis crude extract for overproduction.

A comprehensive method for modeling and simulating ion exchange chromatography of complex mixtures.

In recent years, many biological-based products have been developed, representing a significant fraction of income in the pharmaceutical market. Ion exchange chromatography is an important downstream step for the purification of target recombinant proteins present in clarified cell extracts, together with many other unknown impurities. This work develops a robust approach to model and simulate the purification of untagged heterologous proteins, so that the improved conditions to carry out an ion exchange chromatography are identified in a rational basis prior to the real purification run itself. Purification of the pneumococcal surface protein A (PspA4Pro) was used as a case study. This protein is produced by recombinant Escherichia coli and is a candidate for the manufacture of improved pneumococcal vaccines. The developed method combined experimental and computational procedures. Different anion exchange operating conditions were mapped in order to gather a broad range of representative experimental data. The equilibrium dispersive and the steric mass action equations were used to model and simulate the process. A training strategy to fit the model and separately describe the elution profiles of PspA4Pro and other proteins of the cell extract was applied. Based on the simulation results, a reduced ionic strength was applied for PspA4Pro elution, leading to increases of 14.9% and 11.5% for PspA4Pro recovery and purity, respectively, compared to the original elution profile. These results showed the potential of this method, which could be further applied to improve the performance of ion exchange chromatography in the purification of other target proteins under real process conditions.

Advanced DNA Zipper Probes for Detecting Cell Membrane Lipid Domains.

The cell membrane is a complex mixture of lipids, proteins, and other components. By forming dynamic lipid domains, different membrane molecules can selectively interact with each other to control cell signaling. Herein, we report several new types of lipid-DNA conjugates, termed as "DNA zippers", which can be used to measure cell membrane dynamic interactions and the formation of lipid domains. Dependent on the choice of lipid moieties, cholesterol- and sphingomyelin-conjugated DNA zippers specifically locate in and detect membrane lipid-ordered domains, while in contrast, a tocopherol-DNA zipper can be applied for the selective imaging of lipid-disordered phases. These versatile and programmable probes can be further engineered into membrane competition assays to simultaneously detect multiple types of membrane dynamic interactions. These DNA zipper probes can be broadly used to study the correlation between lipid domains and various cellular processes, such as the epithelial-mesenchymal transition.

Two-Dimensional Thin Layer Chromatography of Melatonin and Related Compounds.

Two-dimensional thin layer chromatography has been used by workers in the field to separate radiolabeled serotonin derivatives from complex mixtures of culture media and homogenates of glands. The compounds resolved include N-acetylserotonin, melatonin, hydroxytryptophol, methoxytryptophol, hydroxyindole acetic acid, and methoxyindole acetic acid. The method requires either radiolabeled tryptophan or serotonin, if an investigator wants to study conversion. It is also useful in the chemical synthesis of serotonin metabolites because it is relatively fast. It pointed to the enzyme that converts serotonin to N-acetylserotonin as being key in controlling the nocturnal increase in vertebrate melatonin production. This enzyme, arylalkylamine N-acetyltransferase (E.C. 2.3.1.87), has been the focus of hundreds of papers which probed its biology, biochemistry, molecular biology, structural biology, neural regulation, development, evolution, and genetics.

Liver dysfunction and energy storage mobilization in traíra, Hoplias malabaricus (Teleostei, Erythrinidae) induced by subchronic exposure to toxic cyanobacterial crude extract.

Microcystins (MC) are hepatotoxic for organisms. Liver MC accumulation and structural change are intensely studied, but the functional hepatic enzymes and energy metabolism have received little attention. This study investigated the liver and hepatocyte structures and the activity of key hepatic functional enzymes with emphasis on energetic metabolism changes after subchronic fish exposure to cyanobacterial crude extract (CE) containing MC. The Neotropical erythrinid fish, Hoplias malabaricus, were exposed intraperitoneally to CE containing 100 µg MC-LR eq kg-1 for 30 days and, thereafter, the plasma, liver, and white muscle was sampled for analyses. Liver tissue lost cellular structure organization showing round hepatocytes, hyperemia, and biliary duct obstruction. At the ultrastructural level, the mitochondria and the endoplasmic reticulum exhibited disorganization. Direct and total bilirubin increased in plasma. In the liver, the activity of acid phosphatase (ACP) increased, and the aspartate aminotransferase (AST) decreased; AST increased in plasma. Alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were unchanged in the liver, muscle, and plasma. Glycogen stores and the energetic metabolites as glucose, lactate, and pyruvate decrease in the liver; pyruvate decreased in plasma and lactate decreased in muscle. Ammonia levels increased and protein concentration decreased in plasma. CE alters liver morphology by causing hepatocyte intracellular disorder, obstructive cholestasis, and dysfunction in the activity of key liver enzymes. The increasing energy demand implies glucose mobilization and metabolic adjustments maintaining protein preservation and lipid recruitment to supply the needs for detoxification allowing fish survival.

A swamp forest Streptomyces sp. strain KF15 with broad spectrum antifungal activity against chilli pathogens exhibits anticancer activity on HeLa cells.

During the investigation, soil actinomycetes were isolated from Kathlekanu swamp forest and the crude ethyl acetate extract from the potent isolate KF15 was analysed with GC-MS and HPTLC to identify bioactive metabolites. The crude extract was examined for in-vitro antifungal activity on pathogens of chilli; MTT cytotoxicity assay was performed against HeLa cell line to determine the anticancer potential. The isolate Streptomyces sp. strain KF15 exhibited antagonistic activity against fungal pathogens by inhibiting growth and altering growth pattern with increased antimicrobial activity in dose-dependent manner. GC-MS revealed many bioactive compounds and HPTLC depicted metabolite fingerprint. The IC50 of 99.85 µg/ml indicated the high potential of KF15 extract to prevent proliferation of HeLa cells. Therefore, the findings of this study indicate that the crude extract from Streptomyces sp. strain KF15 contains antifungal and anticancer metabolites; further study on purification could help in controlling many fungal diseases as well as cervical cancer in humans.

Elucidation of Chemical Interactions between Crude Drugs Using Quantitative Thin-Layer Chromatography Analysis.

To elucidate the interactions between crude drugs in Kampo medicines (traditional Japanese medicines), it is important to determine the content of the constituents in a cost-effective and simple manner. In this study, we quantified the constituents in crude drug extracts using thin-layer chromatography (TLC), an inexpensive and simple analytical method, to elucidate the chemical interactions between crude drugs. We focused on five crude drugs, for which quantitative high-performance liquid chromatography (HPLC) methods are stipulated in the Japanese Pharmacopoeia XVIII (JP XVIII) and compared the analytical data of HPLC and TLC, confirming that the TLC results corresponded with the HPLC data and satisfied the criteria of JP XVIII. (Z)-ligustilide, a major constituent in Japanese Angelica Root, for which a method of quantification has not been stipulated in JP XVIII, was also quantitatively analyzed using HPLC and TLC. Furthermore, Japanese Angelica Root was combined with 26 crude drugs to observe the variation in the (Z)-ligustilide content from each combination by TLC. The results revealed that combinations with Phellodendron Bark, Citrus Unshiu Peel, Scutellaria Root, Coptis Rhizome, Gardenia Fruit, and Peony Root increased the (Z)-ligustilide content. Quantifying the constituents in crude drug extracts using the inexpensive and simple TLC method can contribute to elucidating interactions between crude drugs in Kampo medicines, as proposed by the herbal-pair theory.

Study the Effect of P. minor Seaweed Crude Extract as a Biostimulant on Soybean.

<b>Background and Objective:</b> Seaweed biostimulants are often used in agriculture because of their benefits in increasing growth, production and quality of plants and are safe for the environment. <i>Padina minor</i> is one of the potential seaweeds that contains high macro and micronutrients and has also been shown to increase the vegetative growth of several plants. This study aims to determine the effect of <i>P. minor</i> seaweed extract in various concentrations and frequencies as a biostimulant on the growth and production of soybean plants. <b>Materials and Methods:</b> <i>Padina minor</i> extract was applied to soybean plants with several concentrations (0, 10, 20, 30 and 40%) at three different application times. Where 1 application (2 weeks after planting), 2 applications (2 and 4 weeks after planting) and 3 applications (2, 4 and 5 weeks after planting). <b>Results:</b> <i>Padina minor</i> extract with a concentration of 40% with 1 application was able to increase plant height and shorten soybean harvest life. While the <i>P. minor</i> extract with a concentration of 40% with two and three applications was able to increase the gross and dry weight of plants, the number of pods, gross and dry mass of whole seeds. <b>Conclusion:</b> <i>Padina minor</i> seaweed extract with a concentration of 40% was able to increase the growth and production of soybean plants.

A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation.

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout cells and recombinant TBK1. We observed agonist-, TBK1-, "ER-to-Golgi" traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-ß-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.

Chemical Variation among Castes, Female Life Stages and Populations of the Facultative Eusocial Sweat Bee Halictus rubicundus (Hymenoptera: Halictidae).

In eusocial insects, chemical communication is crucial for mediating many aspects of social activities, especially the regulation of reproduction. Though queen signals are known to decrease ovarian activation of workers in highly eusocial species, little is known about their evolution. In contrast, some primitively eusocial species are thought to control worker reproduction through physical aggression by the queen rather than via pheromones, suggesting the evolutionary establishment of chemical signals with more derived sociality. However, studies supporting this hypothesis are largely missing. Socially polymorphic halictid bees, such as Halictus rubicundus, with social and solitary populations in both Europe and North America, offer excellent opportunities to illuminate the evolution of caste-specific signals. Here we compared the chemical profiles of social and solitary populations from both continents and tested whether (i) population or social level affect chemical dissimilarity and whether (ii) caste-specific patterns reflect a conserved queen signal. Our results demonstrate unique odor profiles of European and North American populations, mainly due to different isomers of n-alkenes and macrocyclic lactones; chemical differences may be indicative of phylogeographic drift in odor profiles. We also found common compounds overproduced in queens compared to workers in both populations, indicating a potential conserved queen signal. However, North American populations have a lower caste-specific chemical dissimilarity than European populations which raises the question if both use different mechanisms of regulating reproductive division of labor. Therefore, our study gives new insights into the evolution of eusocial behavior and the role of chemical communication in the inhibition of reproduction.

Effect of the Crude Extract of Coprophilous Fungi on Some Bacterial Species Isolated from Cases of Mastitis.

Coprophilous fungi are a large group of fungi mostly found in herbivore dung and have an exclusive life cycle. This group of fungi produces many important metabolites which can be consumed in medicine or agriculture. The present study aimed to investigate the antibacterial effects of these fungi on bacterial mastitis. A total of 50 dung samples were collected from four herbivores (cows, buffalos, sheep, and camels) from different areas of Basra. The moist chamber method was used for each sample to establish a fungal fruiting body and detect the type of the fungi. The coprophilous fungi included Aspergillus sp. (A. niger, A. fumigatus, A. flavus, A. terrus), Chaetomium sp., Sordaria sp., and Podospora sp. which belong to the Ascomycetes class. PCR test was performed using the ITS region for confirmatory detection of species. The highest and the lowest number of isolated species was associated with cow dung and camel dung, respectively. The antimicrobial property of three different partitioned extracts (petroleum ether [F1], ethanol [F2], and water [F3]) prepared from some fungal mycelia was evaluated in vitro. All fractions were tested to detect antimicrobial activity using the disc diffusion assay against five pathogenic bacteria Staphylococcus aureus, Streptococcus Enterobacter, Proteus mirabilis, and E. coli. which is isolated from bovine mastitis. Data revealed that all fractions could inhibit the tested bacteria. However, inhibitory activity was found to be dependent on (6i) the used fungal strains; (ii) the extracted solvent; and (iii) the tested bacteria. In general, the petroleum ether extracts (F1) derived from all fungi displayed the highest inhibitory activity against the testing bacteria. In conclusion, the present study concluded that the extracts prepared from the fungal mycelia contain bioactive compounds with antibacterial properties. This study was first conducted in Iraq and further studies are required to develop new treatments.

Effects of photo-stimulation with laser or LED on the composition of Xanthan gum produced in media containing distilled water or dialyzed or not produced water by means of Raman spectroscopy.

Oil is expected to continue to be one of the most important sources of energy in the world and world's energy matrix for the foreseeable future. However, high demand for energy and the decline of the production of oil fields makes oil recovery a challenge. Most techniques used for the recovery process are expensive, non-sustainable and technically difficult to implement. In this context, microbial enhanced oil recovery (MEOR) represents an attractive alternative. It employs products derived from the metabolism of microorganisms that produce biopolymers. Certain bacteria species (e.g., Xanthomonas campestris) produce polysaccharides (exopolysaccharides - EPS) such as the well-known Xanthan gum (XG). We hypothesized that the use of produced water (PW) water in combination photo-stimulation with laser/LED could influence the production and composition of XG. Raman spectroscopy has been used for qualitative and quantitative evaluation of the biochemical composition of XG biopolymer under light stimulation. X. campestris cultures in either distilled water or dialysis-produced water were studied under the absence or presence of laser irradiation (λ = 660 nm, CW, spot size 0.040 cm2, 40 mW, 444 s, 8.0 J/cm2) or LED (λ = 630 nm ± 2 nm, CW, spot size 0.50 cm2, 140 mW, 500 s, 12 J/cm2). XG produced by these cultures was analyzed by Raman spectroscopy at 1064 nm excitation and subjected to principal component analysis (PCA). Results of the exploratory analysis and ANOVA general linear model (GLM) suggested that the extent of XG and pyruvate (pyruvyl mannose) production was affected differentially in X. campestris when cultured in distilled water plus LED photo-stimulation versus dialysis-produced water plus LED photo-stimulation. XG production increased in the distilled water culture. In contrast, both pyruvate acetyl mannose content went up in the dialysis-water culture. These results open a wide field of opportunities in the use of metal-enriched cultures in combination with photo-biomodulation to direct and optimize bacterial production of compounds (i.e., XG) that may be of great benefit in the implementation of sustainable practices for oil extraction.

Arsenite suppresses the transcriptional activity of EVI1 through the binding to CCHC-type Zn finger domain.

Transcription factor EVI1 is essential for normal hematopoiesis in embryos but is aberrantly elevated in bone marrow cells of myelodysplastic syndrome (MDS) patients. EVI1 and its downstream GATA-2 appear to be a possible therapeutic target of MDS. Here we found that treatment of EVI1-expressing K562 cells with arsenite (As(III)) reduced the mRNA and protein levels of EVI1 and GATA-2. A gel shift assay using the nuclear extract of K562 cells showed that As(III) suppressed the DNA-binding activity of EVI1. The DNA-binding activity of the recombinant EVI1 protein was also suppressed by As(III) but was recovered by excess amounts of dithiothreitol, suggesting the involvement of cysteine residues of EVI1. Since the 7th Zn finger domain of EVI1, having a motif of CCHC, is known to be involved in DNA-binding, the synthetic peptide of 7th Zn finger domain was reacted with As(III) and subjected to MALDI-TOF-MS analysis. The results showed that As(III) binds to this peptide via three cysteine residues. As(III)-induced reduction of the DNA-binding activity of the recombinant EVI1 was abolished by the mutations of each of three cysteine residues to alanine in the 7th Zn finger domain. These results demonstrate that As(III) causes the down-regulation of EVI1 and GATA-2 by inhibiting the transcriptional activity of EVI1 through the binding to the cysteine residues of CCHC-type Zn finger domain.

Development and validation of a GC-FID method for the analysis of short chain fatty acids in rat and human faeces and in fermentation fluids.

Short-chain fatty acids (SCFAs) are gut microbiota metabolites recognized for their beneficial effects on the host organism. In this study, a simple and rapid sample preparation method combined to SCFAs analysis by direct injection and gas chromatography coupled with flame ionization detection (GC-FID), for the determination and quantification of eight SCFAs (acetic, propionic, i-butyric, butyric, i-valeric, valeric, i-caproic and caproic acids) in rat, mice and human faeces and in fermentation fluids samples, has been developed and validated. The method consists of extraction of the SCFAs by ethyl ether after acidification of the samples. The effect of the number of extractions has been assessed in order to optimize the procedure and to obtain a satisfactory yield for all the analyzed SCFAs. The increase of the extracted analytes quantity was significant passing from 1 to 2 and from 2 to 3 extractions (P < 0.05), while no significant differences were found performing 3, 4 or 5 extractions (P > 0.05). The SCFAs extracted are directly analyzed by GC-FID without derivatization and separated on a polyethylene glycol nitroterephthalic acid modified coated capillary column, with a chromatographic run time of 13 min. The proposed method showed good sensitivity, with limits of quantifications in the range 0.14-0.48 µM for SCFAs from propionic to caproic acids and 2.12 µM for acetic acid; recovery was between 80.8 and 108.8% and intraday and interday repeatability in the range 0.6-5.0% of precision (RSD, %) The optimized method is suitable for the quantitative analysis of SCFAs in real samples of rat, mouse and human faeces and in fermentation fluids, and it can be applied also to very small amount of faecal sample (20 mg).

Screening for tyrosinase inhibitors from actinomycetes; identification of trichostatin derivatives from Streptomyces sp. CA-129531 and scale up production in bioreactor.

In the course of a primary screening of 614 microbial actinomycete extracts for the discovery of tyrosinase inhibitors, the EtOAc extract of the fermentation broth of the strain Streptomyces sp. CA-129531 isolated from a Martinique sample, exhibited in cell free and cell-based assays the most promising activity (IC50 value of 63 µg/mL). Scaled-up production in a bioreactor led to the isolation of one new trichostatic acid analogue, namely trichostatic acid B (1), along with six known trichostatin derivatives (2-7), four diketopiperazines (8-11), two butyrolactones (12-13) and one hydroxamic acid siderophore (14). Among them, trichostatin A (4) showed a Ki value of 6.1 µM and six times stronger anti-tyrosinase activity (IC50 2.18 µΜ) than kojic acid (IC50 14.07 µΜ) used as a positive control. Deoxytrichostatin A (6) displayed also strong inhibitory activity against tyrosinase (IC50 19.18 µΜ). Trichostatin A production in bioreactor started together with the exponential phase of growth (day 4) and the maximum concentration was reached at day 9 (2.67 ± 0.13 µg/mL). Despite the cytotoxicity of some individual components, the EtOAc extract showed no cytotoxic effect on HepG2, A2058, A549, MCF-7 and MIA PaCa-2 cell lines, (IC50 >2.84 mg/mL) and against BG fibroblasts at the concentrations where the whitening effect was exerted, reassuring its safety and great tyrosinase inhibitory potential.