Isolation, Culture and Bioactive Substances Elicitation of Sanghuangporus Baumii

Sanghuangporus baumii is a forest pathogenic fungus and also a medicine mushroom. In the wild, S. baumii parasitize on living host plants and there have been no reports of cultivating fruiting bodies under artificial conditions. In this study, we identified and isolated a S. baumii strain, and successfully cultivated the fruiting bodies on sawdust medium by optimizing culture conditions. The optimum medium, culture temperature and pH for mycelial growth of S. baumii were WBA and YPA, at pH 5.5–7.5 and 28℃, respectively. The contents of total flavonoids, total polysaccharides and total triterpenoids in S. baumii were compared with those in other two medicinal sanghuang, S. vaninii and S. sanghuang. The results showed that both the fruiting bodies and mycelia of S. baumii were rich in bioactive substances. The content of total flavonoids was higher in the fruiting bodies (34.91 mg/g), while the contents of total polysaccharides and total triterpenoids were higher in the mycelia (45.41 mg/g, 14.06 mg/g, respectively). In addition, the mycelia of S. baumii could be elicited to produce more bioactive substances. The use of sealing film in culture increased the polysaccharides content in mycelia by 51.8%, while the light increased the flavonoids content by 151.0%. The cultivation of fruiting bodies and the elicitation of bioactive substances from mycelia provide biological materials for the study and utilization of S. baumii.

In vitro culture of Dendrobium lineale Rolfe orchid for plant breeding and propagation

D.lineale is included in the CITES appendix II due to D. lineale has great potential as a cut flower and the parent of hybrid orchids. In vitro culture can be used for mass propagation of orchids for commercial breeding as well as conservation. Basic culture medium with the addition of organic substances can increase the growth of plantlets. The objective of this research was to determine the best condition for in vitro culture of D.lineale orchids for producing a mass number of plantlets. Methods used include planting orchid seedlings in various variations of culture media, plantlet subcultures, observation of the anatomy of leaves and roots, as well as the measurement of chlorophyll content. This research uses ‘pisang raja’ as organic material in the culture medium. The highest chlorophyll content in the leaves was 1.58 mg/g, while the highest chlorophyll content in the roots was 0.34 mg/g. Both were found in medium NP+100 g/L bananas in light conditions (1317 lux). The anatomical structure of the leaves shows that the mesophyll is thicker in plants placed under lighting 1317 lux because it shows good effectiveness in photosynthesis. The anatomy of the orchid roots showed differences in diameter in each treatment because the addition of bananas to the medium stimulated rapid and abundant root formation, but the size was smaller. Based on these results, it can be concluded that the addition of 100 gr/L bananas as organic substances in in vitro medium culture increases the growth rate of D. lineale orchids, which will be beneficial for conservation efforts.

Accumulation of 4-Deoxy-7-Hydroxytrichothecenes, but Not 4,7-Dihydroxytrichothecenes, in Axenic Culture of a Transgenic Nivalenol Chemotype Expressing the NX-type FgTri1 Gene

Fusarium graminearum species complex produces type B trichothecenes oxygenated at C-7. In axenic liquid culture, F. graminearum mainly accumulates one of the three types of trichothecenes, namely 3-acetyldeoxyinvalenol, 15-acetyldeoxyinvalenol, or mixtures of 4,15-diacetylnivalenol/4-acetylnivalenol, depending on each strain’s genetic background. The acetyl groups of these trichothecenes are slowly deacetylated to give deoxynivalenol (DON) or nivalenol (NIV) on solid medium culture. Due to the evolution of F. graminearum FgTri1, encoding a cytochrome P450 monooxygenase responsible for hydroxylation at both C-7 and C-8, new derivatives of DON, designated as NX-type trichothecenes, have recently emerged. To assess the risks of emergence of new NX-type trichothecenes, we examined the effects of replacing FgTri1 in the three chemotypes with FgTri1_NX chemotype, which encodes a cytochrome P450 monooxygenase that can only hydroxylate C-7 of trichothecenes. Similar to the transgenic DON chemotypes, the transgenic NIV chemotype strain accumulated NX-type 4-deoxytrichothecenes in axenic liquid culture. C-4 oxygenated trichothecenes were marginal, despite the presence of a functional FgTri13 encoding a C-4 hydroxylase. At present, outcrossing of the currently occurring NX chemotype with NIV chemotype strains of F. graminearum in the natural environment likely will not yield a new strain that produces a C-4 oxygenated NX-type trichothecene.

Fecal screening of Dientamoeba fragilis: relative efficacy of permanent smear and culture

Dientamoeba fragilis (D. fragilis) is an enteric trichmonad protozoan parasite that remains obscure and neglected. The aim of this study is to detect D. fragilis as a neglected pathogen in children aged 6-12 years old complaining of gastrointestinal illness by stool culture and light microscopy with comparison between the results of both techniques. A total of 100 fresh stool samples were included in this current study. All specimens were subjected to microscopic examination using iron- hematoxylin stained stool smears and stool culture using a Loeffler’s culture medium. Culture detected 2 positive stool samples (2%) while microscopy detected (1%). Sensitivities of culture and microscopy were 100% and 50% respectively. Specificity of culture and microscopy were 100% and 95% respectively. There is a moderate agreement between culture and microscopy (K = 0.4). In conclusion, culture had a high performance compared to microscopy in the diagnosis of D. fragilis infection. Culture can be applied for routine diagnosis for the detection of D. fragilis in clinical samples.

Early Detection of Mycobacterium avium subsp. paratuberculosis Infected Cattle: Use of Experimental Johnins and Innovative Interferon-Gamma Test Interpretative Criteria

Paratuberculosis (PTB), also known as Johne's disease, is a chronic proliferative enteritis of ruminants caused by Mycobacterium avium subsp.paratuberculosis (MAP). To date, PTB diagnosis, based on serology, fecal culture, and real-time polymerase chain reaction, has identified animals in advanced stages of infection. To detect MAP infection in animals earlier, the interferon-gamma (IFN-γ) test may be applied. This assay detects cytokines produced by T-lymphocytes of infected subjects after stimulation with purified protein derivatives (PPDs), extracted from Mycobacterium bovis (MB) and from M. avium (MA). The study involved three bovine herds: one PTB-infected herd, one PTB-free herd, and one with an outbreak of bovine tuberculosis. The IFN-γ test was performed on 235 animals, using bovine PPD (PPDB), avian PPD (PPDA), and three experimental PPD Johnins (PPDJs) extracted from a synthetic liquid medium culture of MAP (PPDJ A, B, and C), to assess early MAP detection and avoid false reactions to MB. Furthermore, IFN-γ results were evaluated using 12 interpretative criteria (ICs), based on the differences and ratio between PPD optical density (OD) and IFN-γ basal OD values after lymphocytic stimulation. IC accuracy was expressed as area under the receiver operating characteristic curve. Through a longitudinal study, PPDJs proved to be specific and sensitive in the detection of MAP-infected animals. Among the evaluated ICs, six showed the best performance in terms of accuracy (p < 0.0001), highlighting PTB subclinical infections. In particular, the two best criteria reached sensitivity values of 100% [confidence interval (CI) 95%, 94.1–100%] with a specificity of 91.8% (CI 95%, 81.9–97.3%) and sensitivity levels of 80.6% (CI 95%, 69.1–89.2%) with a specificity of 100% (CI 95%, 94.1–100%). Thus, the IFN-γ assay proved to be a useful diagnostic tool to identify early subclinical MAP-infected animals, in order to manage infected cattle or those exposed to MAP and to monitor younger calves within a herd. Furthermore, the IFN-γ test can be considered an additional test to avoid the introduction of MAP-infected animals, especially in herds where disease has already been eradicated and preservation of the health status is required to maintain the PTB certification level.

Benefits of understanding the enzymatic activities in saline Lake Letea in the Danube Delta

The purpose of this paper was to isolate halophiles from Letea saline lake and to performed a screening for industrially relevant extracellular enzymes. The investigations were conducted from October 2016 until May 2018. After a random selection of colonies that grew on the medium culture, 82 isolates were investigated. Based on their salt requirements and tolerance, it was remarked the presence of isolates belonging to halotolerant and moderate halophilic bacteria. Morphological and biochemical tests were used to characterize them. The ability of the tested isolates to produce extracellular enzymes was evaluated on the culture medium with salinity varying between 0-4M and supplemented with a specific substrate. The highest hydrolytic activities were recorded for casein at 0M NaCl, 1M NaCl, 2M NaCl, and Tween 80 and inulin at 0M NaCl, 1M NaCl, 2M NaCl, and 3M NaCl.

Mst1 Knockout Alleviates Mitochondrial Fission and Mitigates Left Ventricular Remodeling in the Development of Diabetic Cardiomyopathy

The disruption of mitochondrial dynamics is responsible for the development of diabetic cardiomyopathy (DCM). However, the mechanisms that regulate the balance of mitochondrial fission and fusion are not well-understood. Wild-type, Mst1 transgenic and Mst1 knockout mice were induced with experimental diabetes by streptozotocin injection. In addition, primary neonatal cardiomyocytes were isolated and cultured to simulate diabetes to explore the mechanisms. Echocardiograms and hemodynamic measurements revealed that Mst1 knockout alleviated left ventricular remodeling and cardiac dysfunction in diabetic mice. Mst1 knockdown significantly decreased the number of TUNEL-positive cardiomyocytes subjected to high-glucose (HG) medium culture. Immunofluorescence study indicated that Mst1 overexpression enhanced, while Mst1 knockdown mitigated mitochondrial fission in DCM. Mst1 participated in the regulation of mitochondrial fission by upregulating the expression of Drp1, activating Drp1S616 phosphorylation and Drp1S637 dephosphorylation, as well as promoting Drp1 recruitment to the mitochondria. Furthermore, Drp1 knockdown abolished the effects of Mst1 on mitochondrial fission, mitochondrial membrane potential and mitochondrial dysfunction in cardiomyocytes subjected to HG treatment. These results indicated that Mst1 knockout inhibits mitochondrial fission and alleviates left ventricular remodeling thus prevents the development of DCM.

Screening of essential oils activity against a gram negative psychrophilic bacterium isolated from aquatic environment (Water & Biofilm)

Among the applications of medicinal plants, it is their use as antimicrobial agents. The objective of this study was to investigate the effect of some essential oils against an etiological pathogen Flavobacterium spp. responsible for several lost in rainbow trout, (Oncorhynchus mykiss) hatcheries, the strains used in this study were isolated from rearing tanks water and biofilm, identified as Flavobacterium spp. based on phenotypic, biochemical and enzymatic characterizations. A collection of eight essential oils were extracted, analyzed and tested for an inhibitory activity against the isolated strains, the effect on this bacterium has been demonstrated by the aromatogram method based on a screening of bacterial growth in a solid medium culture with disks containing essential oils. Our study’s results show that the chemical composition of the extracted essential oils play a crucial role in their antibacterial activity, which varies from 6 mm up to 34 mm as maximal inhibitory diameter.

Improvement of a recombinant β-glucosidase activity of a sequence derived from metagenome database of Binh Chau hotspring

Metabolic products obtaining from microorganisms of geothermal ecologies often show special characteristics which help their cells to survive, grow and develop under extreme conditions. Exploiting the microbial gene resource of those environments demands a new approach via uncultured methods. Thanks to the development of metagenomics and bioinformatic softwares, we can exploit novel genes from environment directly. Based on Binh Chau hotspring’s DNA metagenome sequencing, ORF [denovogenes]_32768 encoding for β-glucosidase is selected for expression into pET17b vector because it shown a low similartity of amino acid sequence as compared to others in Genbank, a high alkali and Tm predicted values. To improve the expression efficiency of β-glucosidase, some factors (host strains, medium culture, IPTG concentration, aeration…) are investigated. The results showed that the recombinant E. coli C43(DE3) reached the highest dried biomass at 8.26 g/L and the maximum enzymatic activity at 0.34 U/mL in shaking condition (TB medium plus 0.25 mM IPTG with the ratio of cultured /flask volume is 20%, 42-48 hours, 30°C). This study demonstrates the capacity of mining a novel gene encoded for enzyme from DNA metagenome of Vietnam hot spring as well as produces recombinant enzyme for biomass conversion.