Sterilization protocols and the effect of plant growth regulators on callus induction and secondary metabolites production in in vitro cultures Melia azedarach L.

Melia azedarach L. is a valuable source of antioxidants and secondary metabolites. This study is a first extensive report about the effect of different serialization protocols and plant growth regulators (PGRs) on explant disinfection efficiency, callus induction and secondary metabolites production and accumulation in callus cultures of M. azedarach L. In this regard, the effect of plant growth regulators on callus induction and secondary metabolites production were examined. In addition, different sterilization agents were evaluated for disinfection of chinaberry leaf explants. The results showed that the lowest percentage of explant contamination and browning with the highest percentage of callus induction and callus growth obtained with explants pretreated with benomyl (2 g/L) for 2 h and sterilized with 7% H2O2 for 10 min and NaOCl 2% (without pH adjustment) for 12 min. Although adjusting the pH of NaOCl to pH = 7 and 10 significantly reduced the microbial contamination and increased the percentage of contamination-free cultures of M. azedarach L., adversely influenced the explant viability and callus induction and growth. The highest percentage of callus induction obtained on the MS medium containing 3 mg/L NAA/2,4-D and 1 or 3 mg/L Kin/BAP, and the highest callus yield (1804.833 mg/explant) belonged to the MS medium supplemented with 5 mg/L 2,4-D and 5 mg/L Kin. The callus cultures grown on the MS medium supplemented with 3 mg/L NAA and 1 mg/L Kin produced the highest amount of Quercetin (2.06 mg/g fresh weight), Rutin (5.56 mg/g fresh weight) and Kaempferol (1.84 mg/g fresh weight).

Chromogenic Escherichia coli reporter strain for screening DNA damaging agents

The presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

φYeO3-12 phage tail fiber Gp17 as a promising high specific tool for recognition of Yersinia enterocolitica pathogenic serotype O:3

Yersiniosis is an infectious zoonotic disease caused by two enteropathogenic species of Gram-negative genus Yersinia: Yersinia enterocolitica and Yersinia pseudotuberculosis. Pigs and other wild and domestic animals are reservoirs for these bacteria. Infection is usually spread to humans by ingestion of contaminated food. Yersiniosis is considered a rare disease, but recent studies indicate that it is overlooked in the diagnostic process therefore the infections with this bacterium are not often identified. Reliable diagnosis of Yersiniosis by culturing is difficult due to the slow growth of the bacteria easily overgrown by other more rapidly growing microbes unless selec-tive growth media is used. Phage adhesins recognizing bacteria in a specific manner can be an excellent diagnostic tool, es-pecially in the diagnosis of pathogens difficult for culturing. In this study, it was shown that Gp17, the tail fiber protein (TFP) of phage φYeO3-12, specifically recognizes only the pathogenic Yersinia enterocolitica serotype O:3 (YeO:3) bacteria. The ELISA test used in this work confirmed the specific interaction of this protein with YeO:3 and demonstrated a promising tool for developing the pathogen recognition method based on phage adhesins.

Yeasts from Chinese strong flavour Daqu samples: isolation and evaluation of their potential for fortified Daqu production

In total, 16 yeast were isolated from Chinese strong flavour Daqu samples and underwent RAPD analysis and identification. Totally, 11 different species were identified among these isolates including Saccharomyces cerevisiae, Hanseniaspora vineae, Pichia kluyveri, Trichosporon asahii, Wickerhamomyces anomalus, Kluyveromyces lactis, Yarrowia lipolytica, Wickerhamomyces mori, Galactomyces geotrichum, Dabaryomyces hansenii, and Saccharomyces kudriavzevii. To understand the impact of these yeast strains on the quality and flavour of Daqu, we then assessed volatile compounds associated with Daqu samples fermented with corresponding strains. These analyses revealed strain YE006 exhibited the most robust ability to produce ethanol via fermentation but yielded relatively low quantities of volatile compounds, whereas strain YE010 exhibited relatively poor fermentation efficiency but produced the greatest quantity of volatile compounds. These two yeast strains were then utilized in a mixed culture to produce fortified Daqu, with the optimal inoculum size being assessed experimentally. These analyses revealed that maximal fermentation, saccharifying, liquefying, and esterifying power as well as high levels of volatile compounds were achieved when using a 2% inoculum composed of YE006/YE010 at a 1:2 (v/v) ratio. When the liquor prepared using this optimized fortified Daqu was compared to unfortified control Daqu, the former was found to exhibit significantly higher levels of flavour compounds and better sensory scores. Overall, our findings may provide a reliable approach to ensuring Daqu quality and improving the consistency and flavour of Chinese strong-flavour liquor through bioaugmentation.

ADP-ribosyl transferase activity and gamma radiation cytotoxicity of Pseudomonas aeruginosa exotoxin A

This work explores the ADP-ribosyltransferase activity of Pseudomonas (P.) aeruginosa exotoxin A using the guanyl hydrazone derivative, nitrobenzylidine aminoguanidine (NBAG) and the impact of gamma radiation on its efficacy. Unlike the conventional detection methods, NBAG was used as the acceptor of ADP ribose moiety instead of wheat germ extract elongation factor 2. Exotoxin A was extracted from P. aeruginosa clinical isolates and screened for toxA gene using standard PCR. NBAG was synthesized using aminoguanidine bicarbonate and 4-nitrobenzaldehyde and its identity has been confirmed by UV, FTIR, Mass and 13C-NMR spectroscopy. The ADP-ribosyl transferase activity of exotoxin A on NBAG in the presence of Nicotinamide adenine dinucleotide (NAD+) was recorded using UV spectroscopy and HPLC. In vitro ADP-ribosyl transferase activity of exotoxin A protein extract was also explored by monitoring its cytotoxicity on Hep-2 cells using sulforhodamine B cytotoxicity assay. Bacterial broths were irradiated at 5, 10, 15, 24 Gy and exotoxin A protein extract activity were assessed post exposure. Exotoxin A extract exerted an ADP-ribosyltransferase ability which was depicted by the appearance of a new ʎmax after the addition of exotoxin A to NBAG/NAD+ mixture, fragmentation of NAD+ and development of new peaks in HPLC chromatograms. Intracellular enzyme activity was confirmed by the prominent cytotoxic effects of exotoxin A extract on cultured cells. In conclusion, the activity of Exotoxin A can be monitored via its ADP-ribosyltransferase activity and low doses of gamma radiation reduced its activity. Therefore, coupling radiotherapy with exotoxin A in cancer therapy should be carefully monitored.

Anti-tumor effect of polysaccharide from Pleurotus ostreatus on H22 mouse Hepatoma ascites in-vivo and hepatocellular carcinoma in-vitro model

Hepatocellular carcinoma is one of the leading causes of cancer-associated death across the globe. Malignant ascites are the major clinical attributes in cancer patients. Despite the advancements in HCC treatments such as chemotherapy, radiotherapy, surgery, and hormonal therapy, researchers are pursuing novel natural edible compounds for the treatment of cancer to eliminate dreadful side effects. Pleurotus ostreatus is one of the most edible cuisines in Asia as well as all over the world. It has been a source of nutritious diet since it was classified as an edible mushroom with no or negligible side effects. The present study focused on the natural anti-cancerous and anti-ascites capabilities of polysaccharides extracted from Pleurotus ostreatus in-vivo as well as in-vitro. Administration of polysaccharide Pleurotus ostreatus showed a significant decrease in tumor cell metastasis while the increase in the survival period among mice models of H22 malignant ascites. Downregulation of regenerative genes Foxp3 and Stat3 and secretion of immunological factors such as IL-2, TNF α, and INF γ were observed after treating with the partially pure extracted polysaccharide. Twining with the hypothesis of tumor suppression in-vivo model polysaccharide showed a decrease in invasion and migration abilities and henceforth responsible for the gene regulation such Cytochrome C which supposedly induced the chain of gene regulation process resulting in apoptosis in HCC cell lines observed in-vitro experiments. Collective research findings manifested that polysaccharide extracted from Pleurotus ostreatus bears anti-proliferative activity and thus influence tumor suppression in-vivo and in-vitro against hepatocellular carcinoma and can be used for therapeutic purposes as a potential anti-cancerous source in the future.

Thermococcus sp. KS-1 PPIase as a fusion partner improving soluble production of aromatic amino acid decarboxylase

Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS−1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS−1 (PPIase KS−1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS−1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS−1. The recombinant E. coli cells expressing the PPIase KS−1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS−1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS−1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.

Bacterial communities in paddy soil and ditch sediment under rice-crab co-culture system

As an important form of sustainable agriculture, rice-crab (Eriocheir sinensis) co-culture is rapid developing worldwide. However, the knowledge on the bacterial communities of the different components of the system is limited. In this study, we investigated the bacterial community structure in paddy soil and ditch sediment by using high-throughput sequencing technology. The results showed that compared with the ditch sediment, the content of NH4+-N in paddy soil decreased by 62.31%, and the content of AP (available phosphorus) increased by 172.02% (P < 0.05). The most abundant phyla in paddy soil and ditch sediment were Proteobacteria, Bacteroidetes and Chloroflexi, whose relative abundance was above 65%. Among the dominant genera, the relative abundance of an uncultured bacterium genus of Saprospiraceae and an uncultured bacterium genus of Lentimicrobiaceae in paddy soil was significantly lower than ditch sediment (P < 0.05). Alpha diversity indicated that the bacterial diversity of paddy soil and ditch sediment was similar. The bacterial community structure was affected by the relative abundance of bacteria, not the species of bacteria. Redundancy analysis (RDA) showed that the bacterial communities in paddy soil and ditch sediment were correlated with physicochemical properties. Our findings showed that the bacterial community structure was distinct in paddy soil and ditch sediment under rice-crab co-culture probably due to their different management patterns. These results can provide theoretical support for improving rice-crab co-culture technology.

Response of soil biological properties and bacterial diversity to different levels of nitrogen application in sugarcane fields

To select an eco-friendly nitrogen (N) application level for sugarcane production, soil fertility and soil bacterial diversity under different nitrogen application levels were analyzed. Four levels of urea applications were high Nitrogen (H, 964 kg ha−1), medium Nitrogen (M, 482 kg ha−1), low Nitrogen (L, 96 kg ha−1) and no Nitrogen (CK, 0 kg ha−1) treatments, respectively. The results showed that the soil microbial biomass carbon and phosphorus were altered significantly by CK and L treatments. Moreover, the indexes of soil bacterial richness and diversity in the sugarcane field could be significantly improved by L. At the genus level, SC-I-84, Mycobacterium, Micropepsaceae, Saccharimonadales, Subgroup_2 and Acetobacteraceae were the unique dominant bacteria in the soil with the H treatment. JG30-KF-CM45 and Jatrophihabitans were the unique dominant genera in the M treatment. Subgroup_6, HSB_OF53-F07, Streptomyces, 67–14, SBR1031 and KD4-96 were the unique dominant genera in the L treatment. In contrast, FCPS473, Actinospica, 1921–2, Sinomonas, and Ktedonobacteraceae were the unique dominant genera in the CK treatment. The findings suggest that soil fertility all could be changed by different N application levels, but the most increasing integral effect only could be found in L. Moreover, even though soil bacterial richness could be significantly promoted by the M and H treatments, but soil bacterial diversity could not be significantly improved. On the contrary, soil bacterial diversity and richness all could be improved by L treatment. In addition, higher abundance of unique soil dominant bacteria could be only found in L treatment which compared to the CK, M and H treatments. These findings suggest that the rate of 96 kg ha−1 N application is ecofriendly for sugarcane production in Guangxi.

Derivation of pb(II)-sensing Escherichia coli cell-based biosensors from arsenic responsive genetic systems

Heavy metal-responsive operons were used for the generation of Escherichia coli cell-based biosensors. The selectivity and specificity of the biosensors were determined based on the interaction between heavy metals and regulatory proteins; thereby, the modulating target selectivity of biosensors could be achieved by changing target sensing properties of regulatory proteins. The results of this study demonstrated that Pb(II)-sensing biosensors could be generated from an arsenic-responsive genetic system, which was originally used for arsenic-sensing biosensors. The amino acids around to As(III)-binding sites of ArsR were mutated and cysteine residues were relocated to modulate the metal selectivity. In addition, genes encoding metal ion-translocating P-type ATPases, such as copA and zntA, were deleted to enhance the specificity by increasing the intercellular levels of divalent metal ions. Based on the results, channel protein deleted E. coli cells harboring a pair of recombinant genes, engineered ArsR and arsAp::egfp, showed enhanced responses upon Pb exposure and could be used to quantify the amount of Pb(II) in artificially contaminated water and plants grown in media containing Pb(II). Although we focused on generating Pb(II)-specific biosensors in this study, the proposed strategy has a great potential for the generation of diverse heavy metal-sensing biosensors and risk assessment of heavy metals in environmental samples as well as in plants.