Growth and autolysis of the kefir yeast Kluyveromyces marxianus in lactate culture.

Kluyveromyces marxianus is a yeast that could be identified from kefir and can use a broad range of substrates, such as glucose and lactate, as carbon sources. The lactate produced in kefir culture can be a substrate for K. marxianus. However, the complexity of the kefir microbiota makes the traits of K. marxianus difficult to study. In this research, we focused on K. marxianus cultured with lactate as the sole carbon source. The optimal growth and released protein in lactate culture were determined under different pH conditions, and the LC-MS/MS-identified proteins were associated with the tricarboxylic acid cycle, glycolysis pathway, and cellular stress responses in cells, indicating that autolysis of K. marxianus had occurred under the culture conditions. The abundant glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) was cocrystallized with other proteins in the cell-free fraction, and the low transcription level of the GAP1 gene indicated that the protein abundance under autolysis conditions was dependent on protein stability. These results suggest that lactate induces the growth and autolysis of K. marxianus, releasing proteins and peptides. These findings can be fundamental for K. marxianus probiotic and kefir studies in the future.

Sustainable and eco-friendly strategies for shrimp shell valorization.

Among the seafood used globally, shellfish consumption is in great demand. The utilization of these shellfish such as prawn/shrimp has opened a new market for the utilization of the shellfish wastes. Considering the trends on the production of wealth from wastes, shrimp shell wastes seem an important resource for the generation of high value products when processed on the principles of a biorefinery. In recent years, various chemical strategies have been tried to valorize the shrimp shell wastes, which required harsh chemicals such as HCl and NaOH for demineralization (DM) and deproteination (DP) of the shrimp wastes. Disposal of chemicals by the chitin and chitosan industries into the aquatic bodies pose harm to the aquatic flora and fauna. Thus, there has been intensive efforts to develop safe and sustainable technologies for the management of shrimp shell wastes. This review provides an insight about environmentally-friendly methods along with biological methods to valorize the shrimp waste compared to the strategies employing concentrated chemicals. The main objective of this review article is to explain the utilization shrimp shell wastes in a productive manner such that it would be offer environment and economic sustainability. The application of valorized by-products developed from the shrimp shell wastes and physical methods to improve the pretreatment process of shellfish wastes for valorization are also highlighted in this paper.

Anti-EMT properties of CoQ0 attributed to PI3K/AKT/NFKB/MMP-9 signaling pathway through ROS-mediated apoptosis.

BACKGROUND: Breast cancer is the most prevalent cancer among women. In triple-negative breast cancer (TNBC) cells, a novel quinone derivative, coenzyme Q0 (CoQ0), promotes apoptosis and cell-cycle arrest. This study explored the anti-epithelial-mesenchymal transition (EMT) and antimetastatic attributes of CoQ0 in TNBC (MDA-MB-231). METHODS: Invasion, as well as MTT assays were conducted. Lipofectamine RNAiMAX was used to transfect cells with ß-catenin siRNA. Through Western blotting and RT-PCR, the major signaling pathways' protein expressions were examined, and the biopsied tumor tissues underwent immunohistochemical and hematoxylin and eosin staining as well as Western blotting. RESULTS: CoQ0 (0.5-2 µM) hindered tumor migration, invasion, and progression. Additionally, it caused MMP-2/- 9, uPA, uPAR, and VEGF downregulation. Furthermore, in highly metastatic MDA-MB-231 cells, TIMP-1/2 expression was subsequently upregulated and MMP-9 expression was downregulated. In addition, CoQ0 inhibited metastasis and EMT in TGF-ß/TNF-α-stimulated non-tumorigenic MCF-10A cells. Bioluminescence imaging of MDA-MB-231 luciferase-injected live mice demonstrated that CoQ0 significantly inhibited metastasis of the breast cancer to the lungs and inhibited the development of tumors in MDA-MB-231 xenografted nude mice. Silencing of ß-catenin with siRNA stimulated CoQ0-inhibited EMT. Western blotting as well as histological analysis established that CoQ0 reduced xenografted tumor development because apoptosis induction, cell-cycle inhibition, E-cadherin upregulation, ß-catenin downregulation, and metastasis and EMT regulatory protein modulation were observed. CONCLUSIONS: CoQ0 inhibited the progression of metastasis as well as EMT (in vitro and in vivo). The described approach has potential in treating human breast cancer metastasis.

The in vitro and in vivo depigmenting activity of Coenzyme Q10 through the down-regulation of α-MSH signaling pathways and induction of Nrf2/ARE-mediated antioxidant genes in UVA-irradiated skin keratinocytes.

Coenzyme CoQ10 (CoQ10), a ubiquinone compound, has been reported to inhibit tyrosinase activity and melanin production in melanoma B16F10 cells. However, the molecular mechanism underlying this inhibitory effect is poorly understood. In this paper we aimed to investigate the molecular mechanisms involved in the anti-melanogenic activity of CoQ10 (1-2 µM) in UVA (5 J/cm2)-irradiated keratinocyte HaCaT cells and α-MSH stimulated B16-F10 cells. It was observed that CoQ10 suppressed p53/POMC, α-MSH production as well as inhibited ROS generation in UVA-irradiated keratinocyte HaCaT cells. CoQ10 down-regulated the melanin synthesis in α-MSH-stimulated B16-F10 cells by suppressing the MITF expression by down regulating the cAMP mediated CREB signaling cascades. Furthermore, in vivo evidence demonstrated the inhibitory effect of CoQ10 on endogenous pigmentation in zebrafish. Increased nuclear Nrf2 translocation accompanied by the induction of HO-1 and γ-GCLC genes were observed in CoQ10 treated keratinocyte HaCaT cells. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished CoQ10-mediated anti-melanogenic activity, as evidenced by impaired antioxidant HO-1 gene, uncontrolled ROS generation, and α-MSH production following UVA irradiation. To conclude, CoQ10 is an effective de-pigmention or skin-whitening agent and could be used in cosmetics for topical application.

Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death.

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0-15 µg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.

Antrodia camphorata inhibits epithelial-to-mesenchymal transition by targeting multiple pathways in triple-negative breast cancers.

Antrodia camphorata (AC) exhibits potential for engendering cell-cycle arrest as well as prompting apoptosis and metastasis inhibition in triple-negative breast cancer (TNBC) cells. We performed the current study to explore the anti-epithelial-to-mesenchymal transition (EMT) properties of fermented AC broth in TNBC cells. Our results illustrated that noncytotoxic concentrations of AC (20-60 µg/ml) reversed the morphological changes (fibroblastic-to-epithelial phenotype) as well as the EMT by upregulating the observed E-cadherin expression. Furthermore, we discovered treatment with AC substantially inhibit the Twist expression in human TNBC (MDA-MB-231) cells as well as in those that were transfected with Twist. In addition, we determined AC to decrease the observed Wnt/ß-catenin nuclear translocation through a pathway determined to be dependent on GSK3ß. Notably, AC treatment consistently inhibited the EMT by downregulating mesenchymal marker proteins like N-cadherin, vimentin, Snail, ZEB-1, and fibronectin; at that same time upregulating epithelial marker proteins like occludin and ZO-1. Bioluminescence imaging that was executed in vivo demonstrated AC substantially suppressed breast cancer metastasis to the lungs. Notably, we found that western blot analysis confirmed that AC decreased lung metastasis as demonstrated by upregulation of E-cadherin expression in biopsied lung tissue. Together with our results support the anti-EMT activity of AC, indicating AC as having the potential for acting as an anticancer agent for the treatment of human TNBC treatment.

Antrodia salmonea-induced oxidative stress abrogates HER-2 signaling cascade and enhanced apoptosis in ovarian carcinoma cells.

Antrodia salmonea is well known in Taiwan as a traditional Chinese medicinal fungus and has demonstrated antioxidant, anti-inflammatory, and anticancer effects. However, the anticancer activity of A. salmonea against human ovarian cancer is still elusive. Therefore, we investigated the antiovarian tumor activity of a fermented culture broth of A. salmonea and exhibits its underlying molecular mechanism. A. salmonea shows a significant effect on cell viability in human ovarian carcinoma (SKOV-3 or A2780) cell lines with an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Increased terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells and annexin V-propidium iodide stained cells indicate that A. salmonea induces late apoptosis in SKOV-3 cells. Notably, treatment with A. salmonea induced the following events: Apoptosis; caspase-3, -8, -9 and poly(ADP-ribose) polymerase activation; first apoptosis signal (Fas) and Fas ligand activation; Bid cleavage; and Bax2-B-cell lymphoma 2 dysregulation. The results show that A. salmonea-induced apoptosis was mediated by both mitochondrial and death receptor pathways. An increase in intracellular reactive oxygen species (ROS) was also observed in A. salmonea-treated cells, whereas the antioxidant N-acetylcysteine (NAC) prevented A. salmonea-induced cell death and DNA fragmentation, indicating that A. salmonea-induced apoptosis was mediated by ROS generation. Interestingly, A. salmonea-induced apoptosis is associated with the suppression of human epidermal growth factor receptor-2 (HER-2/neu) and phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) expression in HER-2/neu overexpressing SKOV-3 cells. NAC significantly prevented A. salmonea-induced HER-2/neu depletion and PI3K/AKT inactivation, indicating that A. salmonea-triggered apoptosis is mediated by ROS-inhibited HER-2/neu signaling cascades. To our knowledge, this is the first report describing the anticancer activity of this potentially beneficial mushroom against human ovarian carcinoma.

Ganoderma tsugae induced ROS-independent apoptosis and cytoprotective autophagy in human chronic myeloid leukemia cells.

The medicinal fungus Ganoderma, known in Chinese as Lingzhi or Reishi, traditionally has various medicinal uses and has been employed in cancer treatment in Asia for centuries. This study used ethanol-extracted Ganoderma tsugae (GT) and examined its antitumor activities on human chronic myeloid leukemia cells as well as its molecular mechanism of action. Treatment with GT (200-400 µg/mL) significantly reduced cell viability and caused G2/M arrest in K562 cells. In addition, GT induced mitochondrial and death receptor mediated apoptosis, correlated with DNA fragmentation, followed by cytochrome c release, caspase-3/8/9 activation, PARP cleavage, Fas activation, Bid cleavage, and Bax/Bcl-2 dysregulation. Cytoprotective autophagy was found to be induced by GT, as was revealed by increased LC3-II accumulation, Beclin-1/Bcl-2 dysregulation, acidic vesicular organelle formation, and p62/SQSTM1 activation. Notably, pretreatment of cells with the autophagy inhibitors 3-MA and CQ enhanced GT-induced apoptosis. Interestingly, reactive oxygen species production in cells was not triggered by GT administration; equally, the antioxidant N-acetylcysteine was found to be incapable of preventing apoptosis and autophagy induced by GT treatment. Finally, this study discovered that cytoprotective autophagy induced by GT was associated with EGFR and PI3K/AKT/mTOR signaling cascade suppression. In summary, GT demonstrated antitumor activity against human chronic myeloid leukemia.

Antrodia salmonea suppresses invasion and metastasis in triple-negative breast cancer cells by reversing EMT through the NF-κB and Wnt/ß-catenin signaling pathway.

Antrodia salonea (AS), a fungus that is indigenous to Taiwan has been well known for its anti-cancer properties. We investigated the anti-metastatic and anti-epithelial-mesenchymal transition (EMT) properties of AS in TNBC cells. To determine their EMT and metastasis levels, in vitro wound healing, wound invasion, Western blotting, RT-PCR, luciferase activity and immunofluorescence assays were performed, while the in vivo anti-metastatic efficacy of AS was evaluated in BALB/c-nu mice through bioluminescence imaging, HE staining, and immunohistochemical staining. MDA-MB-231 cells, when treated with AS concentrations (25-100 µg/mL) resulted in significant reduction of invasion and migration as well as the downregulation of VEGF, uPAR, uPA and MMP-9 (inhibition of PI3K/AKT/NFκB pathways). AS treatment prevented morphological changes and reversed EMT through the upregulation of E-cadherin and the downregulation of N-cadherin, Slug, Twist, and Vimentin. Inhibition of Smad3 signaling pathway, downregulation of ß-catenin pathway and upregulation of GSK3ß expression were also observed while, suppression of metastasis and EMT in TGF-ß1-stimulated non-tumorigenic MCF-10A cells was observed when treated with AS. Histological analysis confirmed that AS reduced tumor metastasis and upregulated E-cadherin expression in biopsied lung tissues. Our results indicated that AS exhibits anti-EMT and anti-metastatic activity, that could contribute to develop anticancer drugs against TNBC.

Chalcone flavokawain A attenuates TGF-ß1-induced fibrotic pathology via inhibition of ROS/Smad3 signaling pathways and induction of Nrf2/ARE-mediated antioxidant genes in vascular smooth muscle cells.

TGF-ß1 plays a crucial role in the pathogenesis of vascular fibrotic diseases. Chalcones are reportedly cancer chemo-preventive food components that are rich in fruits and vegetables. In this study, flavokawain A (FKA, 2-30 µM), a naturally occurring chalcone in kava extracts, was evaluated for its anti-fibrotic and antioxidant properties in TGF-ß1-stimulated vascular smooth muscle (A7r5) cells, as well as its underlying molecular mechanism of action. Immunofluorescence data showed down-regulated F-actin expression with FKA treatment in TGF-ß1-stimulated A7r5 cells. Western blotting demonstrated that FKA treatment suppressed the expression of α-SMA and fibronectin proteins under TGF-ß1 stimulation. Findings from wound-healing and invasion experiments showed that FKA inhibits TGF-ß1-mediated migration and invasion. Western blotting demonstrated that treatment with FKA down-regulated MMP-9 and MMP-2 and up-regulated TIMP-1 expression. Further evidence showed that FKA decreased TGF-ß1-mediated phosphorylation and the transcriptional activity of Smad3. TGF-ß1-induced excessive ROS production was remarkably reversed by FKA treatment in A7r5 cells, and inhibition by FKA or N-acetylcysteine (NAC) substantially diminished TGF-ß1-induced p-Smad3 activation and wound-healing migration. Interestingly, FKA-mediated antioxidant properties were associated with increased nuclear translocation of Nrf2 and elevated antioxidant response element (ARE) luciferase activity. Activation of Nrf2/ARE signaling was accompanied by the induction of HO-1, NQO-1 and γ-GCLC genes in FKA-treated A7r5 cells. Notably, silencing of Nrf2 (siRNA transfection) significantly diminished the FKA-mediated antioxidant effects, indicating that FKA may inhibit TGF-ß1-induced fibrosis through suppressing ROS generation in A7r5 cells. Our results suggested that anti-fibrotic and antioxidant activities of the chalcone flavokawain A may contribute to the development of food-based chemo-preventive drugs for fibrotic diseases.

Induction of autophagic cell death in human ovarian carcinoma cells by Antrodia salmonea through increased reactive oxygen species generation.

We reported in our previously executed studies that the fermented culture broth of Antrodia salmonea (AS), a mushroom used in Taiwanese folk medicine induced reactive oxygen species (ROS)-mediated apoptosis in human ovarian carcinoma cells. In this study, we studied the anticancer efficacies of AS (0-240 µg/ml) by examining the key molecular events implicated in cell death associated with autophagy in SKOV-3 and A2780 human ovarian carcinoma cells and clarified the fundamental molecular mechanisms. Treatment of ovarian carcinoma cells with AS-induced autophagic cell death mediated by increased microtubule-associated protein LC3-II, GFP-LC3 puncta, and acidic vesicular organelle (AVO) formation. These events are linked with the activation of p62/SQSTM1, the inhibition of ATG4B, the expression of ATG7, and the dysregulation of Beclin-1/Bcl-2 (i.e., B-cell lymphoma 2). N-acetylcysteine inhibited AS-induced ROS generation, which in turn constricted AS-induced LC3 conversion, AVO formation, and ATG4B inhibition, indicating ROS-mediated autophagy cell death. In addition, the 3-methyladenine (3-MA) or chloroquine (CQ)-induced autophagy inhibition decreased AS-induced apoptosis. Additionally, apoptosis inhibition by Z-VAD-FMK, a pan-caspase inhibitor, substantially suppressed AS-induced autophagy. Furthermore, AS-inhibited HER-2/ neu and PI3K/AKT signaling pathways which were reversed by autophagy inhibitors 3-MA and CQ. Thus, A. salmonea is a potential chemopreventive agent that is capable of activating ROS-mediated autophagic cell death in ovarian carcinoma cells.

Genomic sequence analysis of a plant-associated Photobacterium halotolerans MELD1: from marine to terrestrial environment?

Mercury impacts the function and development of the central nervous system in both humans and wildlife by being a potent neurotoxin. Microbial bioremediation is an important means of remediation of mercury-contaminated soil. The rhizospheric Photobacterium halotolerans strain MELD1 was isolated from mercury and dioxin contaminated site from Tainan, Taiwan. It has been shown to reduce Hg(2+) to Hg(0). The 4,758,027 bp genome of P. halotolerans MELD1 has a G + C content of 50.88 % and contains 4198 protein-coding and 106 RNA genes. Genomic analysis revealed the presence of a number of interesting gene cluster that maybe involved in heavy metal resistance, rhizosphere competence and colonization of the host plant.

Genome Sequence of Photobacterium halotolerans MELD1, with Mercury Reductase (merA), Isolated from Phragmites australis.

Here, we present the whole-genome sequence of Photobacterium halotolerans strain, MELD1, isolated from the roots of a terrestrial plant Phragmites australis grown in soil heavily contaminated with mercury and dioxin. The genome provides further insight into the adaptation of bacteria to the toxic environment from where it was isolated.

A rhizosphere-associated symbiont, Photobacterium spp. strain MELD1, and its targeted synergistic activity for phytoprotection against mercury.

Though heavy metal such as mercury is toxic to plants and microorganisms, the synergistic activity between them may offer benefit for surviving. In this study, a mercury-reducing bacterium, Photobacterium spp. strain MELD1, with an MIC of 33 mg x kg(-1) mercury was isolated from a severely mercury and dioxin contaminated rhizosphere soil of reed (Phragmites australis). While the whole genome sequencing of MELD1 confirmed the presence of a mer operon, the mercury reductase MerA gene showed 99% sequence identity to Vibrio shilloni AK1 and implicates its route resulted from the event of horizontal gene transfer. The efficiency of MELD1 to vaporize mercury (25 mg x kg(-1), 24 h) and its tolerance to toxic metals and xenobiotics such as lead, cadmium, pentachlorophenol, pentachloroethylene, 3-chlorobenzoic acid, 2,3,7,8-tetrachlorodibenzo-p-dioxin and 1,2,3,7,8,9-hexachlorodibenzo-p-dioxin is promising. Combination of a long yard bean (Vigna unguiculata ssp. Sesquipedalis) and strain MELD1 proved beneficial in the phytoprotection of mercury in vivo. The effect of mercury (Hg) on growth, distribution and tolerance was examined in root, shoot, leaves and pod of yard long bean with and without the inoculation of strain MELD1. The model plant inoculated with MELD1 had significant increases in biomass, root length, seed number, and increased mercury uptake limited to roots. Biolog plate assay were used to assess the sole-carbon source utilization pattern of the isolate and Indole-3-acetic acid (IAA) productivity was analyzed to examine if the strain could contribute to plant growth. The results of this study suggest that, as a rhizosphere-associated symbiont, the synergistic activity between the plant and MELD1 can improve the efficiency for phytoprotection, phytostabilization and phytoremediation of mercury.

Synergistic collaboration of gut symbionts in Odontotermes formosanus for lignocellulosic degradation and bio-hydrogen production.

In this work, gut microbes from the macrotermitine termite Odontotermes formosanus the cellulolytic Bacillus and fermentative Clostridium were studied in batch experiments using different carbon substrates to bio-mimic the termite gut for hydrogen production. Their fungus comb aging and the in vitro lignocellulosic degradation of the mango tree substrates by the synergistic interaction of Bacillus, Clostridium and Termitomyces were detected by Solid-state NMR. From the results, Bacillus species acted as a mutualist, by initiating an anaerobic environment for the growth of Clostridium, for bio-hydrogen production and the presence of Termitomyces enhanced the lignocellulosic degradation of substrates in vitro and in vivo. Thus, the synergistic collaboration of these three microbes can be used for termite-derived bio-fuel processing technology.

Selection and application of endophytic bacterium Achromobacter xylosoxidans strain F3B for improving phytoremediation of phenolic pollutants.

While phytoremediation has been considered as an in situ bioprocess to remediate environmental contaminants, the application of functional endophytic bacteria within plants remains a potential strategy that could enhance the plants' efficiency in phytoremediation. In this study, 219 endophytes were isolated from plants that are predominantly located in a constructed wetland, including reed (Phragmites australis) and water spinach (Ipomoea aquatica). Twenty-five strains of the isolated endophytes utilize aromatic compounds as sole carbon source; Achromobacter xylosoxidans strain F3B was chosen for the in planta studies using the model plant Arabidopsis thaliana. Phylogenetic analysis indicated that those endophytic isolates of A. xylosoxidans formed a cluster within its species, and a specific real-time PCR detection method was developed for confirming the stability of the isolates in plants. In the presence of either catechol or phenol, inoculation of A. thaliana with F3B could extend into the root lengths and fresh weights to promote pollutants removal rates. These results demonstrate the potential of the endophytic F3B strain for helping plants to tolerate stress from aromatic compounds and to improve phytoremediation of phenolic pollutants.

Microbial community analysis in the termite gut and fungus comb of Odontotermes formosanus: the implication of Bacillus as mutualists.

The microbial communities harbored in the gut and fungus comb of the fungus-growing termite Odontotermes formosanus were analyzed by both culture-dependent and culture-independent methods to better understand the community structure of their microflora. The microorganisms detected by denaturing gradient gel electrophoresis (DGGE), clonal selection, and culture-dependent methods were hypothesized to contribute to cellulose-hemicellulose hydrolysis, gut fermentation, nutrient production, the breakdown of the fungus comb and the initiation of the growth of the symbiotic fungus Termitomyces. The predominant bacterial cultivars isolated by the cultural approach belonged to the genus Bacillus (Phylum Firmicutes). Apart from their function in lignocellulosic degradation, the Bacillus isolates suppressed the growth of the microfungus Trichoderma harzianum (genus Hypocrea), which grew voraciously on the fungus comb in the absence of termites but grew in harmony with the symbiotic fungus Termitomyces. The in vitro studies suggested that the Bacillus sp. may function as mutualists in the termite-gut-fungus-comb microbial ecosystem.