Culture Production Research Articles

In vitro study to assess modulation of Candida biofilm by Escherichia coli from vaginal strains

BackgroundVulvovaginal candidiasis (VVC) is caused by biofilm formation and epithelial invasion. In addition, Escherichia coli (EC) can establish a vaginal intracellular reservoir modulating Candida spp. biofilm production. We aimed to analyze the behavior of Candida albicans (CA) and EC biofilm both in single cultures and in co-cultures. MethodsWe prospectively collected CA and EC isolates from vaginal swabs over 6 months. We selected positive cultures with both CA and EC (cases) and a comparator group with either CA or EC (controls). We analyzed overall biomass production and metabolic activity in single cultures and in co-cultures based on staining assays, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) to assess biofilm occupation. We also analyzed clinical manifestations. ResultsWe cultured 455 samples, 16 (3.5%) of which had CA and EC (cases); only CA or EC (controls) was detected, respectively, in 72 (15.8%) and 98 (21.5%). Biomass production and metabolic activity were significantly more pronounced in co-cultures in both groups. CLSM and SEM, on the other hand, showed the biofilm of each species to be significantly reduced when they were cultured together, with higher values in CA (percentage biofilm reduction: CA, 95.8% vs. EC, 36.2%, p < 0.001). There were no clinically significant differences between co-infected patients and patients infected only by C. albicans. ConclusionOurs is the first study assessing co-cultures of CA and EC in a large collection of samples. We observed that coinfection of CA and EC was unusual (3.5%) and promoted high biomass, whereas microscopy enabled us to detect a reduction in biofilm production when microorganisms were co-cultured. No differences in symptoms were observed.

Fluxapyroxad Resistance Mechanisms in Sclerotinia sclerotiorum.

The necrotrophic pathogen Sclerotinia sclerotiorum has a global distribution and a wide host range making it one of the most damaging and economically important of all plant pathogens. The current study found that fluxapyroxad, a typical succinate dehydrogenase inhibitor (SDHI) fungicide, had a strong inhibitory effect against S. sclerotiorum, with EC50 values ranging from 0.021 to 0.095 μg/mL. Further investigation of five highly resistant S. sclerotiorum mutants, with EC50 values of 12.37-31.36 μg/mL, found that fluxapyroxad resistance was accompanied by a certain cost to fitness. All of the mutants were found to have significantly (p < 0.05) reduced mycelial growth and altered sclerotia production in artificial culture, as well as reduced pathogenicity compared to wild-type isolates, with one mutant completely losing the capacity to infect detached soybean leaves. Sequence analysis found that four of the mutants had point mutation leading to amino acid changes in the SsSdhB subunit of the fungicide target protein succinate dehydrogenase. In addition, two of the mutants were also found to have amino acid changes in the predicted sequence of their SsSdhD subunit, whilst the fifth mutant had no changes in any of its SsSdh sequences, indicating that an alternative mechanism might be responsible for the observed resistance in this mutant. No cross-resistance was found between fluxapyroxad and any of the other fungicides tested, including tebuconazole, prochloraz, dimethachlone, carbendazim, procymidone, pyraclostrobin, boscalid, fluazinam, fludioxonil, and cyprodinil, which indicates that fluxapyroxad has great potential as an alternative method of control for the SSR caused by S. sclerotiorum, and which could provide ongoing protection to the soybean fields of China.

Contribution of the decomposition of a macroalgal bloom to methane production in sea cucumber culture

CH4 is one of the most important greenhouse gases responsible for climate change. The effect of algal decomposition on CH4 emission fluxes in aquaculture environments is unknown. This study determined the relative abundance of methanogens in aquaculture in northern China. We determined various parameters in the water and sediment after the decomposition of Chaetomorpha valida. The results showed that the relative abundance of methanogens increased significantly during the decomposition of C. valida. The average CH4 emission flux was 50.64 mg m-2 h-1 when the density of C. valida was 25 mg cm-3, which was 11 times higher than that at a density of 0 mg cm-3. The concentrations of dissolved oxygen (DO), nitrite, nitrate and sulfate in the aquaculture pond significantly decreased during the decomposition of C. valida. The formation of a strong anaerobic reduction environment promoted the formation and release of CH4. Our results confirmed that the decomposition of C. valida enhanced the CH4 emission flux. The average CH4 emission fluxes ranged from 4.62 to 50.64 mg m-2 h-1. It is particularly important to remove decomposed algae in a timely manner and control greenhouse gas emissions from aquaculture ponds to mitigate climate change.

Developing an in vitro elicitation strategy for specialized secondary metabolites production in adventitious root cultures of Primula veris subsp. veris

The demand for new sources of bioactive plant secondary metabolites (SMs) with importance as pharmaceutical and food additives, cosmetics and flavors, have emerged as rising scientific topic in parallel to the protection of natural habitats due to overexploitation and decreasing of biodiversity. Cowslip (Primula veris L. subsp. veris) is a perennial medicinal plant in which pharmaceutically important SMs such as phenolic acids glycosides (primulaverin, primeverin) and triterpene saponins (primulic acid I and II) are produced in the roots and aerial parts. These metabolites serve as quality markers for the evaluation of raw materials used for the production of expectorant and diuretic drugs. Alternatively to traditional soil-cultivation of whole plants, in vitro adventitious root production systems have the potential for elevated and stable production of plant SMs through elicitation processes. Additionally, these systems have an advantageous perspective from ecological and economical aspect comparing with conventional method for mass production of valuable compounds. In the present study eight abiotic elicitors [salicylic acid (SA), cazeine hydrolysate (CH), L-proline, silver nitrate (AgNO3), cobalt chloride (CoCl2), sucrose, jasmonic acid (JA) and putrescine], under a 60-day culture period into 250 mL flasks were tested. Afterwards, the following three elicitors applied in single (100 mg/L CH, 100 mg/L L-proline, 25 μM JA) or combinations (JA + CH, JA + L-proline), under two exposure times (30 and 60 days). The results showed that primeverin [35.7 mg/g dry weight (DW)], and cumulative phenolic acids glycosides (52.9 mg/g) content, yield index (YI) of primeverin (97.4 mg of pure dry extract), cumulative phenolic acids glycosides (144.4 mg), primulic acid I (37.2 mg), primulic acid II (9.1 mg) and cumulative saponins (45.4 mg), as well as root fresh biomass growth rate (× 10.2-fold), and root DW (2.7 g) were promoted after elicitation with 100 mg/L L-proline for 60 days. Primulaverin (23.6 mg/g), primeverin (14.4 mg/g) and the cumulative phenolic acids glycosides (38.1 mg/g) content was considerably maximized after treatment with 25 μM JA for 30 days. Combined elicitation of roots with 25 μM JA + 100 mg/L L-proline for 30 days, indicated the highest content in primulic acid II (5.9 mg/g), cumulative saponins (22 mg/g), while primulic acid I represented the highest content under 100 μM JA for 60 days (27 mg/g). Among the eight elicitors tested, L-proline, JA, and L-proline + JA proved to be the most beneficial treatments for SMs production. Following that, root fresh biomass growth rate (× 8.1), content, and YI of primulaverin (14.3 mg/g, 149.1 mg), primeverin (0.3 mg/g, 3.2 mg), cumulative phenolic acids glycosides (14.6 mg/g, 152.3 mg), primulic acid I (10.3 mg/g, 93.3 mg), primulic acid II (2.5 mg/g, 19.2 mg), and cumulative saponins (12.8 mg/g, 112.5 mg) were considerably higher after 90 days of culture in a 2 L balloon bubble type bioreactor system than after 60 days. Elicitor type, elicitor concentration, and elicitation exposure duration are factors that play a key role in the production of SMs. In the present study, the importance of abiotic elicitation strategies, and bioreactor systems which are novel in the scale-up process, on adventitious root in vitro cultures of P. veris subsp. veris have been discussed in detail and are highlighted for the first time. The production systems applied herein lay the possibilities for the large-scale manufacturing of specialized plant-derived SMs including triterpene saponins and phenolic acid glycosides, presenting commercial applications due to higher root biomass and bioactive compounds productivity obtained.

The Eff ect of Precursors and Elicitors on the Production of Oleanolic Acid in Suspension Culture of Lantana camara

Oleanolic acid (OA) is an important pentacyclic triterpenoid. Traditionally used by Asian medicine, OA for anti-infl ammatory, anti-hyperlipidemic, and hepatoprotective. This study used triterpene precursors at 1 to 5 mg/L and elicitors in diff erent concentrations at 2, 5, 10 mg/L to enhance OA production in Lantana camara suspension cultures. Treated cells were evaluated for their OA contents by using HPLC and compared with the nontreated cells as a control. The results showed that FPP at 2 mg/L infl uenced the biomass and OA production more over the phytohormone combination, other precursors, and elicitors in Suspension cultures of L. camara.

Effects of branched-chain amino acids on Shiraia perylenequinone production in mycelium cultures

BackgroundPerylenequinones from Shiraia fruiting bodies are excellent photosensitizers and widely used for anti-cancer photodynamic therapy (PDT). The lower yield of Shiraia perylenequinones becomes a significant bottleneck for their medical application. Branched-chain amino acids (BCAAs) not only serve as important precursors for protein synthesis, but also are involved in signaling pathway in cell growth and development. However, there are few reports concerning their regulation of fungal secondary metabolism. In present study, the eliciting effects of BCAAs including l-isoleucine (l-Ile), l-leucine (l-Leu) and l-valine (l-Val) on Shiraia perylenequinone production were investigated.ResultsBased on the analysis of the transcriptome and amino acid contents of Shiraia in the production medium, we revealed the involvement of BCAAs in perylenequinone biosynthesis. The fungal conidiation was promoted by l-Val treatment at 1.5 g/L, but inhibited by l-Leu. The spore germination was promoted by both. The production of fungal perylenequinones including hypocrellins A (HA), HC and elsinochromes A-C (EA–EC) was stimulated significantly by l-Val at 1.5 g/L, but sharply suppressed by l-Leu. After l-Val treatment (1.5 g/L) in Shiraia mycelium cultures, HA, one of the main bioactive perylenequinones reached highest production 237.92 mg/L, about 2.12-fold than that of the control. Simultaneously, we found that the expression levels of key genes involved in the central carbon metabolism and in the late steps for perylenequinone biosynthesis were up-regulated significantly by l-Val, but most of them were down-regulated by l-Leu.ConclusionsOur transcriptome analysis demonstrated that BCAA metabolism was involved in Shiraia perylenequinone biosynthesis. Exogenous BCAAs exhibit contrasting effects on Shiraia growth and perylenequinones production. l-Val could promote perylenequinone biosynthesis via not only enhancing the central carbon metabolism for more precursors, but also eliciting perylenequinone biosynthetic gene expressions. This is the first report on the regulation of BCAAs on fungal perylenequinone production. These findings provided a basis for understanding physiological roles of BCAAs and a new avenue for increasing perylenequinone production in Shiraia mycelium cultures.Graphical

Stimulating Transcription in Antibiotic-Tolerant Escherichia coli Sensitizes It to Fluoroquinolone and Nonfluoroquinolone Topoisomerase Inhibitors.

Antibiotic tolerant bacteria and persistent cells that remain alive after a course of antibiotic treatment can foster the chronicity of infections and the development of antibiotic resistance. Elucidating how bacteria overcome antibiotic action and devising strategies to bolster a new drug's activity can allow us to preserve our antibiotic arsenal. Here, we investigate strategies to potentiate the activities of topoisomerase inhibitors against nongrowing Escherichia coli that are often recalcitrant to existing antibiotics. We focus on sensitizing bacteria to the fluoroquinolone (FQ) levofloxacin (Levo) and to the spiropyrimidinetrione zoliflodacin (Zoli)-the first antibiotic in its class of compounds in clinical development. We found that metabolic stimulation either alone or in combination with inhibiting the AcrAB-TolC efflux pump sensitized stationary-phase E. coli to Levo and Zoli. We demonstrate that the added metabolites increased proton motive force generation and ATP production in stationary-phase cultures without restarting growth. Instead, the stimulated bacteria increased transcription and translation, which rendered the populations more susceptible to topoisomerase inhibitors. Our findings illuminate potential vulnerabilities of antibiotic-tolerant bacteria that can be leveraged to sensitize them to new and existing classes of topoisomerase inhibitors. These approaches enable us to stay one step ahead of adaptive bacteria and safeguard the efficacy of our existing antibiotics.

Metal-based nanoadditives for increasing biomass and biohydrogen production in microalgal cultures: A review

Microalgae have emerged as an interesting alternative for the generation of biofuels, including bioethanol, biodiesel, biomethane, biohydrogen and syngas. They are also used for the production of biofertilizers and other products of interest, while acting as a CO2 sink and favoring water purification by absorbing nutrients. However, the economic viability of large-scale microalgae cultivation for any of these purposes is not yet a reality. One of the main factors affecting this viability refers to the microalgae growing phase. In this scenario, nanoparticles, e.g. metallic, are emerging as possible alternatives to improve the cultivation and harvesting of microalgae. The addition of certain nanoadditives in microalgal cultures can improve light conversion in the photobioreactor, as well as CO2 absorption performance. However, the way in which these nanoparticles act or the concentration at which they maximize their positive effects on the culture are aspects that remain unclear. Thus, this paper critically reviews recent advances in nanoparticulate additives when they are used as a supplement in microalgae cultures, with the specific objective of improving biohydrogen production, but also analyzing their effect on algal growth, lipid accumulation and CO2 capture. The effects of nanoparticles on the selection of metabolic pathways of microalgae and their potential toxicity are also reviewed. The reported studies reveal that the toxicity of nanoadditives depends on their concentration, typology (chemical nature, particle size, crystalline structure) and species of microalgae. Furthermore, the supplementation of certain nanoparticles clearly increases the accumulation of intracellular compounds of interest and the growth of the algae. Additional studies should focus on aspects such as optimizing the dose of nanoparticles, designing strategies to minimize toxicity risks, or working with several additives simultaneously to create synergistic effects.

Effect of incremental amounts of Asparagopsis taxiformis on ruminal fermentation and methane production in continuous culture with orchardgrass herbage

Enteric methane (CH4) emissions from ruminants are a significant contributor to total greenhouse gas emissions from the livestock sector. Previous research has demonstrated that the red seaweed Asparagopsis taxiformis strongly inhibits ruminal methanogenesis, but its effects on CH4 production, nutrient digestibility, and ruminal fermentation when added to a cool-season herbage diet have not been assessed. A 4-unit, single-flow continuous culture fermentor system fed orchardgrass (Dactylis glomerata L.) herbage was used to assess the effects of incremental addition of dietary A. taxiformis (0, 5, 10, or 15 g/kg DM) on nutrient digestibility, volatile fatty acid (VFA) concentration, pH, CH4 production, and N metabolism. Treatments were randomly assigned to fermentors in a randomized block design with 7 d of treatment adaptation and 3 d of sample collection. Fermentors were fed a total of 76 g of DM per day, split equally into 4 feedings (07:30, 10:30, 14:00, and 19:00 h). In the last 3 d of each experimental period, daily samples of total effluent were taken for analyses of ammonia N and VFA. One liter of effluent was collected daily (3 L total) for a composite sample that was blended and lyophilized for analysis of DM, ash, neutral and acid detergent fiber, crude protein, and total purines. Headspace CH4 concentration in each fermentor was measured every 15 min (192 readings/d) using a Fourier transform infrared gas analyzer, and pH was recorded every 2 min. Data were analyzed using the GLM procedure of SAS with orthogonal polynomial contrasts. Methane production in fermentors receiving all 3 levels of A. taxiformis was reduced by 99.9% within the first 24 h of each experimental period and remained predominantly below the limit of detection for the remaining 9 d. Apparent digestibility of neutral and acid detergent fiber, as well as apparent and true DM digestibilities, linearly decreased with A. taxiformis inclusion. Total VFA concentration decreased quadratically, and molar proportion of acetate decreased in a cubic manner. Overall, A. taxiformis acutely inhibited CH4 production in continuous cultures fed orchardgrass herbage, but also negatively affected fiber digestibility and total VFA concentration, especially at higher feeding rates. In vitro results indicated that A. taxiformis could be effective in reducing enteric CH4 production in grazing ruminants, but in vivo research is needed to determine how A. taxiformis may impact ruminal fermentation and milk production or average daily gain in grazing systems.

The effect of plant growth regulators on the antioxidant enzyme activity and secondary metabolite production in the cell suspension cultures of Melia azedarach L.

ABSTRACT In the present study, we investigated the effect of plant growth regulators and explant type on callus induction, cell suspension culture growth, antioxidant enzyme activity, and accumulation of rutin, quercetin, and kaempferol in M. azedarach L. cell cultures. Our studies showed that the inflorescence and petiole explants had a high percentage of callus induction compared to the leaf explants, whereas the highest callus growth was obtained from the leaf explants cultured on the MS medium containing 5 mg l−1 NAA and 5 mg l−1 BAP and 5 mg l−1 NAA and 3 mg l−1 kinetin Kin. Furthermore, the highest cell growth was obtained from the leaf and inflorescence calli transferred to liquid MS medium supplemented with 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D)+1 mg l−1 Kin/BAP. The highest activity of antioxidant enzymes and the lowest protein content was related to the leaf cell suspension cultures in liquid MS medium containing 3 mg l−1 NAA and 3 mg l−1 BAP. The inflorescence cell suspension cultures in the MS medium containing 3 mg l−1 NAA+1 mg l−1 BAP exhibited the highest rutin accumulation (47.536 mg g−1 FW). However, the highest amount of quercetin (8.570 mg g−1 FW) and kaempferol (5.420 mg g−1 FW) accumulation were obtained from the petiole cell suspension cultures in the MS medium with 1 mg l−1 2,4-D +1 mg l−1 Kin.

Composition and Bioactivity of a Placental Tissue Particulate (PTP-001) Indicate Greater Potential than Platelet-Rich Plasma for the Treatment of Osteoarthritis.

This study was conducted to compare therapeutically relevant properties of platelet-rich plasma (PRP), a commonly used autologous intra-articular treatment for osteoarthritis (OA), with those of a novel placental tissue particulate, PTP-001, which is in development as a regulated biologic treatment for knee OA. Quantitative immunoassays were performed to determine the content of key growth/regulatory biofactors in PTP-001, and in leukocyte-rich (LR)-PRP or leukocyte-poor (LP)-PRP. An anti-inflammatory bioassay was used to evaluate the effects of each treatment on pro-inflammatory cytokine (tumor necrosis factor (TNF)-α) production in a macrophage cell culture system. Gene expression experiments were conducted using a co-culture system of human synoviocytes (pre-stimulated with interleukin (IL)-1β) and articular chondrocytes, with quantitative polymerase chain reaction analyses of the separate cellular compartments. The concentrations of several biofactors (e.g., basic fibroblast growth factor, tissue inhibitor of metalloproteases-3, interleukin-1 receptor antagonist) representative of diverse disease-relevant mechanisms of action were significantly higher for PTP-001 relative to LR-PRP or LP-PRP. PTP-001 and PRP preparations were able to reduce TNF-α production in macrophage cell cultures; however, greater variability was observed for PRP in comparison with PTP-001. In the chondrocyte/synoviocyte co-culture experiments, PTP-001 and LR-PRP (but not LP-PRP) significantly reduced chondrocyte MMP13 expression in cultures containing IL-1-pretreated synoviocytes. In addition, ADAMTS5 expression was reduced in the chondrocyte compartment following treatment with PTP-001 relative to PRP. These findings support evidence of a potent, multifactorial mechanism of action for a consistently manufactured biologic (PTP-001), which may be of greater therapeutic benefit in comparison with more heterogeneous preparations of PRP which may be generated at the time of treatment.

Census of potential predators and competitors of sandfish, Holothuria scabra, juveniles during floating hapa ocean nursery culture

AbstractPredation and competition are among the most important factors affecting sandfish, Holothuria scabra, growth and survival in ocean‐based production systems. In this study, the potential predators and competitors of sandfish juveniles reared at two sites in the Philippines using the floating hapa ocean nursery system were assessed. A total of 26 species of potential predators and competitors from 16 different families of fishes, molluscs and crustaceans were recorded. These taxa were dominated by the opisthobranch Stylocheilus striatus at Bolinao (north–west Luzon) and the isopod Cymodoce sp. at Maliwaliw (eastern Visayas). Highest number of potential predators and competitors that recruited and/or migrated to the inside of floating hapa nets was recorded on day 23 at Bolinao and day 30 at Maliwaliw, then decreased over the 69‐day duration of the study. Mean length, absolute growth rates (AGRs) and survival of sandfish juveniles reared in floating hapas at the two sites varied during the study. AGRs were highest at 1.17 ± 0.06 mm day−1 at Bolinao, and 1 ± 0.08 mm day−1 at Maliwaliw, by days 23 and 60, respectively. Mean lengths of sandfish juveniles reared at Bolinao and Maliwaliw by the end of the study were 46.7 ± 0.15 and 58.8 ± 2.4 mm, respectively, and survival at both sites was high at 96% by the end of the study. Negative correlations were observed between the number of individual predators and competitors, and survival and growth rates of sandfish juveniles reared at Bolinao and Maliwaliw, respectively. Our results highlight the importance of predator and competitor management measures and appropriate site selection to optimize growth performance and survival of sandfish juveniles reared in ocean‐based culture production systems.

Nitric Oxide Production in mixed cultures of infected rats with Neospora caninum and treated with Jataí Honey (Tetragonisca angustula)

Neosporosis is a disease caused by Neospora caninum sp (JP Dubey), an intracellular parasite that affects many animal species, fostering abortions and neurological disorders. Recent studies on Neospora caninum have shown that glial cells have been considered a model of in vitro infections of these protozoa. Honey has been used since ancient times for its anti-inflammatory and antimicrobial properties. The present study aimed to evaluate the in vitro reactivity of glial cells (astrocytes and microglia) infected with N. caninum treated with Jataí honey at 1% (Tetragonisca angustula). After 72 hours from infection, mitochondrial metabolism, dehydrogenase lactate activity (LDH), nitric oxide production (NO) and the number of parasites were checked. Cultures treated with honey at 1% have shown mitochondrial metabolism and cell permeability increase. There was an increase in nitrite production, possible indicating a microglial activation. Nitric Oxide values found in this study may indicate an immune response against Neospora caninum tachyzoites, since such increase may contribute to reduce the number of tachyzoites. So, our results suggest that honey had a protective effect over astrocytes and microglia cultures infected with Neospora caninum.

Pumpkin Rind and Seeds Extracts Inhibit α - Glucosidase and Nitric Oxide Production and Promote Chlorella ellipsoidea Growth in Culture

The objective of this work was to investigate the potential of pumpkin rind and seed on antidiabetic and anti-inflammatory activities and the underlying mechanism. Therefore, this work was carried out to determine the antidiabetic activity using inhibitory activities of pumpkin rind and seeds extracts on α - glucosidase and the anti-inflammatory activity via inhibitory activity on nitric oxide production. And also, the potential of the pumpkin rind and seeds on culture of Chlorella ellipsoidea was determined. Determination of inhibitory activity on α - glucosidase was performed using α - glucosidase assay, while the Griess assay was employed for the inhibition on Nitric oxide (NO) -production. The pumpkin seed extract inhibited α - glucosidase more potent than the pumpkin rind extract (IC50 of 321.36 vs. 326.18 µg/mL). However, the activities of the extracts were less potent than that of Acarbose (IC50 of 317.26 µg/mL). Application of the extracts at the highest concentration, 500 µg/mL, the pumpkin seed extract displayed the inhibition of NO production higher than the pumpkin rind extract did (18.01 ± 1.57 % vs. 14.99 ± 1.94 %). Study on the effects of pumpkin rind and seeds on C. ellipsoidea culture revealed that the optimum media was the 7th medium (NPK+ mixture of pumpkin seed water and Blue-Green Medium (BG-11), 1:4) which generated the growth of C. ellipsoidea for 28 days with the Optical Density (OD) value of 1.43 ± 0.01 followed by BG-11 medium OD value of 1.41 ± 0.02) and the 3rd medium (NPK+ mixture of pumpkin rind water and BG-11, 1:1, OD value of 1.32 ± 0.01), respectively. The chemical contents of C. ellipsoidea cultured in 7th medium contained 54.25 ± 0.06 % protein, 15.95 ± 0.87 % carbohydrates, 9.35 ± 0.05 % fat, and 20.30 ± 0.33 % ash, meanwhile 53.37 ± 0.77 % protein, 12.83 ± 0.62 % carbohydrates, 17.80 ± 0.23 % fat, and 23.30 ± 1.33 % ash in the 3rd medium. The results obtained from this investigation indicate that pumpkin rind and seeds not only can be exploited for the antidiabetic and anti-inflammatory components but also can be applied instead of the conventional medium for the culture of C. ellipdoidea. Therefore, bio-waste from pumpkin could be potentially utilized as the source of natural antidiabetic inhibitors, anti-inflammatory drugs and the effective components of C. ellipdoidea culture media. HIGHLIGHTS The pumpkin rind and seed extracts possess antidiabetic and anti-inflammatory properties The extracts from pumpkin rind and seeds exhibit antidiabetic activity via α- glucosidase inhibition The extracts from pumpkin rind and seeds display anti-inflammatory activity by inhibiting on nitric oxide (NO) production The extracts from pumpkin rind and seeds could be potentially utilized as a source of natural antidiabetic inhibitors and anti-inflammatory drugs The bio-waste, pumpkin rind and seeds, could be used as the effective components of ellipdoidea culture medium GRAPHICAL ABSTRACT

Towards high-throughput screening (HTS) of polyhydroxyalkanoate (PHA) production via Fourier transform infrared (FTIR) spectroscopy of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99.

High-throughput screening (HTS) methods for characterization of microbial production of polyhydroxyalkanoates (PHA) are currently under investigated, despite the advent of such systems in related fields. In this study, phenotypic microarray by Biolog PM1 screening of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99 identified 49 and 54 carbon substrates to be metabolized by these bacteria, respectively. Growth on 15 (Halomonas sp. R5-57) and 14 (Pseudomonas sp. MR4-99) carbon substrates was subsequently characterized in 96-well plates using medium with low nitrogen concentration. Bacterial cells were then harvested and analyzed for putative PHA production using two different Fourier transform infrared spectroscopy (FTIR) systems. The FTIR spectra obtained from both strains contained carbonyl-ester peaks indicative of PHA production. Strain specific differences in the carbonyl-ester peak wavenumber indicated that the PHA side chain configuration differed between the two strains. Confirmation of short chain length PHA (scl-PHA) accumulation in Halomonas sp. R5-57 and medium chain length PHA (mcl-PHA) in Pseudomonas sp. MR4-99 was done using Gas Chromatography-Flame Ionization Detector (GC-FID) analysis after upscaling to 50 mL cultures supplemented with glycerol and gluconate. The strain specific PHA side chain configurations were also found in FTIR spectra of the 50 mL cultures. This supports the hypothesis that PHA was also produced in the cells cultivated in 96-well plates, and that the HTS approach is suitable for analysis of PHA production in bacteria. However, the carbonyl-ester peaks detected by FTIR are only indicative of PHA production in the small-scale cultures, and appropriate calibration and prediction models based on combining FTIR and GC-FID data needs to be developed and optimized by performing more extensive screenings and multivariate analyses.

Improving fed-batch culture efficiency of Rhodiola sachalinensis cells and optimizing flash extraction process of polysaccharides from the cultured cells by BBD–RSM

Fed-batch culture of plant cells can enhance biomass and bioactive compound accumulation compared with batch culture, in which the initial culture medium is one of the controllable factors in the fed-batch culture system. Rhodiola sachalinensis is a valuable medicinal herb and its cell culture is an alternative approach to obtaining plant material. R. sachalinensis cells has been previously cultured in a fed-batch culture system and the initial culture medium has been found to critically affect production yield. Therefore, this study investigated the effect of nitrogen concentration, ammonia and nitrate ratio (NH4+/NO3-), and phosphorus and calcium concentrations in initial culture medium on cell biomass, as well as salidroside and polysaccharide accumulation to improve the fed-batch culture efficiency. Then, the cultured cells were extracted by flash extraction and the extraction conditions were optimized by Box-Behnken Design Response Surface Method (BBD–RSM). Finally, the antibacterial and antibiofilm effects of the extract were examined to further utilize R. sachalinensis cell cultures. The results showed that the fed-batch culture efficiency was obviously improved when the initial culture medium was modified as 30 mM of nitrogen with 5/25 of NH4+/NO3-, 0.94 mM of phosphorus, and 2.3 mM of calcium, at which 277.3 mg/L of salidroside and 4.5 g/L of polysaccharides were obtained In flash extraction, polysaccharide yield was as the response value to optimize extraction process by controlling extraction time, extraction temperature, and liquid–solid ratio in the BBD–RSM experiments. The optimal flash extraction conditions were 55.7 s of extraction time, 56.5 °C of extraction temperature, and 37.0 mL/g of liquid–solid ratio, which the polysaccharide yield reached 12.76% under the optimized conditions. In antibacterial experiment, the cell extract (RCE) inhibited the growth of Bacillus subtilis, Pseudomonas aeruginosa, and Salmonella choleraesuis; the biofilm of B. subtilis and S. choleraesuis was scavenged in the groups of 8 mg/mL and 16 mg/mL RCE. The findings provided a basis for further industrial production of cell cultures and a theoretical reference in using R. sachalinensis cells in the production of antibacterial-related products.

The Clownfish (Amphiprion spp.) Larviculture Technique with Recirculating Aquaculture System (RAS) in Buleleng, Bali

Clownfish is one of the most demanding-marine ornamental fish. Due to the increased demand of the clownfish, wild clownfish catch has also been increasing, until reaching the overexploitation condition. One way to preserve the existence of this fish species and to sustainably fulfil the market demand is by conducting a culture technique with advanced application using a recirculating-aquaculture system, specifically larviculture. Thereby, this paper reported the procedure of the clownfish larviculture and determined the growth performance of the larvae during the culture activity. This paper reviewed the direct observational operation for 40 days at Rizky Mitra Karya Hatchery, Buleleng, Bali. All observational data were analyzed descriptively. During the culture, the process was composed of rearing media preparation, clownfish egg-clutch collecting and stocking, and larval rearing. This process also included the feeding management in each rearing part (broodstock and larval rearing), water quality control by setting the RAS standard and and harvest for juvenile rearing. However, the hatching rate was lower than the standard given. Egg collection with scrapping method produced a lower hatching rate due to more human error discovered. To gain a better improvement, rotifer as a live feed for clownfish larvae and fluctuated weather condition due to the seasonal transition, which impacted on the availability of live feed for clownfish larvae should be handled intensively. Therefore, proper live feed culture with indoor system, thorough live feed intake observation on larvae, and proper larvae stocking by directly taking from the broodstock tank are necessary to stabilize this culture production with high sustainability level.

Made in Italy

Climate change and resultant environmental catastrophes are a growing factor in the increased number of migrants crossing the Mediterranean. Some end up in Italian prisons where rehabilitation programs emphasize Italian culture production as a path toward inclusion in Italian society. These programs model how to address climate-induced eco-migration and new templates for tackling climate change through prison reform.

The Regeneration and Value Realization of Urban Cultural Resources in the Capital of Culture in East Asian Cities: The Examples from China

The development of a city is not only a long-term process of material environment construction, but also a long-term process of cultural development and civilization accumulation. The essence of a city is that the city can provide people with a high-quality lifestyle: “valuable, meaningful, tasteful, dignified and dreamy”. When a city's material civilization is built to a certain extent, the excessive materialization needs cultural harmony to be more perfect. The Capital of Culture in East Asia has rich cultural resources, and also excellent tourist cities. The core task of building the Capital of Culture in East Asia is to protect and utilize cultural resources fully, and promote the regeneration of urban cultural resources and cultural development. This study used research data derived from the relevant statistics officially released by the cities, and mainly using the case study with the Capital of Culture in East Asia in China as the example. Previous studies have shown that the realization of a city's cultural value is first to promote the regional development of the urban culture, so as to allow the residents to establish “Asian awareness” and “Asian vision”, promote the development of urban culture in life, and increase opportunities for host and guest participation. On this basis, the spatial carrier of urban culture production, trade and consumption is built through the construction of urban culture space and the production of space. Finally, through the implementation of a brand strategy for the Capital of Culture in East Asia, the city's cultural space, cultural taste and consumption level will be improved, the city brand based on local culture and regional culture will be built, and its competitiveness will be achieved on both regional and global levels.

Health benefits of butyrate and its producing bacterium, Clostridium butyricum, on aquatic animals

Aquaculture plays an important role in contributing to global food security and nutrition; thus, the intensification and diversification of aquaculture are increasingly considered. However, paralleling the development of the industrial scale in aquaculture, the occurrence of diseases is always an important issue that causes great losses in economics. The finding of approaches that not only improve culture production but also reduce the impact of diseases in cultured animals is crucially essential. Previously, several studies have addressed the potential application of feed additives, such as prebiotics, probiotics, synbiotics, and microbial-derived metabolites (including short-chain fatty acids-SCFAs), in aquaculture. In this review, we provide an update focusing on the health benefits of dietary supplementation with a type of SCFAs, butyrate, and its producer, Clostridium butyricum, including their effects on growth, feed utilization, body composition, intestinal structure and function, antioxidant activity, immune response, and tolerance against stress and infection in aquatic animals. The outcomes of this study may indicate more benefits of the use of C. butyricum than that of butyrate (and its forms). This review provides general knowledge of the efficacy of butyrate and C. butyricum in aquaculture.