This page has only limited features, please log in for full access.
Deoxynivalenol (DON) is synthesized by Fusarium species that frequently infect crops during storage, and it’s harm risk to human is reflected in the consumption of infected food crops or indirectly through foods of animal origin. In this study, Hela and Chang liver cells were used to research the cellular apoptosis induced by deoxynivalenol. Cells were treated by DON toxin with a series of concentration and incubated for different time. MTT, fluorescence microscope, flow cytometer and Western blot methods were used to analyze the effect of DON on the cell apoptosis in vitro and in vivo systematically. The results showed that DON was toxic to the cells tested. After being treated by DON, the morphology of Chang livers and Hela cells changed significantly. The DON promoted apoptosis in a dose- and time-dependent manner. The activity of Caspase 3 was significantly increased in DON-induced apoptosis. Moreover, endogenous Glutathione (GSH) level in these cell lines was gradually decreased. In the early apoptosis progress, oxidative stress was induced by DON. When DON reached 10 µg/mL, a markedly increased content of Malondialdehyde (MDA) was detected in both Hela and Chang liver cells. Furthermore, an in vivo test indicated that DON had toxicity to mice by causing weight loss and swollen spleen, and significantly increased expression of AST and ALT. In conclusion, the DON was toxic to mice and could induce the apoptosis of tested cells undergoing a Caspase-3 related pathway.
Qing’Ai Chen; Ying Cui; Jiaru Zhao; Wanlin Zeng; Ni Jin; Lan Yang; Jun Yuan. Cellular Apoptosis Induced by Deoxynivalenol. Indian Journal of Microbiology 2021, 1 -9.
AMA StyleQing’Ai Chen, Ying Cui, Jiaru Zhao, Wanlin Zeng, Ni Jin, Lan Yang, Jun Yuan. Cellular Apoptosis Induced by Deoxynivalenol. Indian Journal of Microbiology. 2021; ():1-9.
Chicago/Turabian StyleQing’Ai Chen; Ying Cui; Jiaru Zhao; Wanlin Zeng; Ni Jin; Lan Yang; Jun Yuan. 2021. "Cellular Apoptosis Induced by Deoxynivalenol." Indian Journal of Microbiology , no. : 1-9.
The RNA polymerase II (Pol II) transcription process is coordinated by the reversible phosphorylation of its largest subunit-carboxy terminal domain (CTD). Ssu72 is identified as a CTD phosphatase with specificity for phosphorylation of Ser5 and Ser7 and plays critical roles in regulation of transcription cycle in eukaryotes. However, the biofunction of Ssu72 is still unknown in Aspergillus flavus, which is a plant pathogenic fungus and produces one of the most toxic mycotoxins-aflatoxin. Here, we identified a putative phosphatase Ssu72 and investigated the function of Ssu72 in A. flavus. Deletion of ssu72 resulted in severe defects in vegetative growth, conidiation and sclerotia formation. Additionally, we found that phosphatase Ssu72 positively regulates aflatoxin production through regulating expression of aflatoxin biosynthesis cluster genes. Notably, seeds infection assays indicated that phosphatase Ssu72 is crucial for pathogenicity of A. flavus. Furthermore, the Δssu72 mutant exhibited more sensitivity to osmotic and oxidative stresses. Taken together, our study suggests that the putative phosphatase Ssu72 is involved in fungal development, aflatoxin production and pathogenicity in A. flavus, and may provide a novel strategy to prevent the contamination of this pathogenic fungus.
Guang Yang; Xiaohong Cao; Ling Qin; Lijuan Yan; Rongsheng Hong; Jun Yuan; Shihua Wang. Ssu72 Regulates Fungal Development, Aflatoxin Biosynthesis and Pathogenicity in Aspergillus flavus. Toxins 2020, 12, 717 .
AMA StyleGuang Yang, Xiaohong Cao, Ling Qin, Lijuan Yan, Rongsheng Hong, Jun Yuan, Shihua Wang. Ssu72 Regulates Fungal Development, Aflatoxin Biosynthesis and Pathogenicity in Aspergillus flavus. Toxins. 2020; 12 (11):717.
Chicago/Turabian StyleGuang Yang; Xiaohong Cao; Ling Qin; Lijuan Yan; Rongsheng Hong; Jun Yuan; Shihua Wang. 2020. "Ssu72 Regulates Fungal Development, Aflatoxin Biosynthesis and Pathogenicity in Aspergillus flavus." Toxins 12, no. 11: 717.
As a pathogenic fungus, Aspergillus flavus can produce carcinogenic aflatoxins (AFs), which poses a great threat to crops and animals. Msb2, the signalling mucin protein, is a part of mitogen‐activated protein kinase (MAPK) pathway which contributes to a range of physiological processes. In this study, the roles of membrane mucin Msb2 were explored in A. flavus by the application of gene disruption. The deletion of msb2 gene (Δmsb2) caused defects in vegetative growth, sporulation and sclerotia formation when compared to WT and complement strain (Δmsb2C) in A. flavus. Using thin‐layer chromatography (TLC) and high‐performance liquid chromatography (HPLC) analysis, it was found that deletion of msb2 down‐regulated aflatoxin B1 (AFB1) synthesis and decreased the infection capacity of A. flavus. Consistently, Msb2 responds to cell wall stress and osmotic stress by positively regulating the phosphorylation of MAP kinase. Notably, Δmsb2 mutant exhibited cell wall defect, and it was more sensitive to inhibitor caspofungin when compared to WT and Δmsb2C. Taking together, these results revealed that Msb2 plays key roles in morphological development process, stresses adaptation, secondary metabolism and pathogenicity in fungus A. flavus.
Ling Qin; Ding Li; Jiaru Zhao; Guang Yang; Yinchun Wang; Kunlong Yang; Elisabeth Tumukunde; Shihua Wang; Jun Yuan. The membrane mucin Msb2 regulates aflatoxin biosynthesis and pathogenicity in fungus Aspergillus flavus. Microbial Biotechnology 2020, 14, 628 -642.
AMA StyleLing Qin, Ding Li, Jiaru Zhao, Guang Yang, Yinchun Wang, Kunlong Yang, Elisabeth Tumukunde, Shihua Wang, Jun Yuan. The membrane mucin Msb2 regulates aflatoxin biosynthesis and pathogenicity in fungus Aspergillus flavus. Microbial Biotechnology. 2020; 14 (2):628-642.
Chicago/Turabian StyleLing Qin; Ding Li; Jiaru Zhao; Guang Yang; Yinchun Wang; Kunlong Yang; Elisabeth Tumukunde; Shihua Wang; Jun Yuan. 2020. "The membrane mucin Msb2 regulates aflatoxin biosynthesis and pathogenicity in fungus Aspergillus flavus." Microbial Biotechnology 14, no. 2: 628-642.
Various signaling pathways in filamentous fungi help cells receive and respond to environmental information. Previous studies have shown that the mitogen-activated protein kinase (MAPK) pathway is phosphorylation-dependent and activated by different kinase proteins. Serine/threonine kinase plays a very important role in the MAPK pathway. In this study, we selected the serine/threonine kinase AflSte20 in Aspergillus flavus for functional study. By constructing Aflste20 knockout mutants and complemented strains, it was proven that the Aflste20 knockout mutant (ΔAflste20) showed a significant decrease in growth, sporogenesis, sclerotinogenesis, virulence, and infection compared to the WT (wild type) and complemented strain (ΔAflste20C). Further research indicated that ΔAflste20 has more sensitivity characteristics than WT and ΔAflste20C under various stimuli such as osmotic stress and other types of environmental stresses. Above all, our study showed that the mitogen-activated kinase AflSte20 plays an important role in the growth, conidia production, stress response and sclerotia formation, as well as aflatoxin biosynthesis, in A. flavus.
Ding Li; Ling Qin; Yinchun Wang; Qingchen Xie; Na Li; Shihua Wang; Jun Yuan. AflSte20 Regulates Morphogenesis, Stress Response, and Aflatoxin Biosynthesis of Aspergillus flavus. Toxins 2019, 11, 730 .
AMA StyleDing Li, Ling Qin, Yinchun Wang, Qingchen Xie, Na Li, Shihua Wang, Jun Yuan. AflSte20 Regulates Morphogenesis, Stress Response, and Aflatoxin Biosynthesis of Aspergillus flavus. Toxins. 2019; 11 (12):730.
Chicago/Turabian StyleDing Li; Ling Qin; Yinchun Wang; Qingchen Xie; Na Li; Shihua Wang; Jun Yuan. 2019. "AflSte20 Regulates Morphogenesis, Stress Response, and Aflatoxin Biosynthesis of Aspergillus flavus." Toxins 11, no. 12: 730.
Woronin bodies are fungal-specific organelles whose formation is derived from peroxisomes. The former are believed to be involved in the regulation of mycotoxins biosynthesis, but not in their damage repair function. The hexagonal peroxisome protein (HexA or Hex1) encoded by hexA gene in Aspergillus is the main and the essential component of the Woronin body. However, little is known about HexA in Aspergillus flavus. In this study, hexA knock-out mutant (ΔhexA) and complementation strain (ΔhexAC) were produced using homologous recombination. The results showed that, ΔhexA and ΔhexAC were successfully constructed. And the data analysis indicated that the colony diameter, stress sensitivity and the sclerotia formation of A. flavus were nearly not affected by the absence of HexA. Yet, the deletion of hexA gene reduced the production of asexual spores and lessened virulence on peanuts and maize seeds markedly. In addition, it was also found that there was a significant decrease of Aflatoxin B1 production in deletion mutant, when compared to wild type. Therefore, it suggested that the hexA gene has an essential function in conidia production and secondary metabolism in A. flavus. The gene is also believed to be playing an important role in the invasion of A. flavus to the host.
Jun Yuan; Ding Li; Ling Qin; Jiaojiao Shen; Xiaodong Guo; Elisabeth Tumukunde; Mingzhu Li; Shihua Wang. HexA is required for growth, aflatoxin biosynthesis and virulence in Aspergillus flavus. BMC Molecular Biology 2019, 20, 1 -13.
AMA StyleJun Yuan, Ding Li, Ling Qin, Jiaojiao Shen, Xiaodong Guo, Elisabeth Tumukunde, Mingzhu Li, Shihua Wang. HexA is required for growth, aflatoxin biosynthesis and virulence in Aspergillus flavus. BMC Molecular Biology. 2019; 20 (1):1-13.
Chicago/Turabian StyleJun Yuan; Ding Li; Ling Qin; Jiaojiao Shen; Xiaodong Guo; Elisabeth Tumukunde; Mingzhu Li; Shihua Wang. 2019. "HexA is required for growth, aflatoxin biosynthesis and virulence in Aspergillus flavus." BMC Molecular Biology 20, no. 1: 1-13.
Aspergillus flavus is one of the fungi from the big family of Aspergillus genus and it is capable of colonizing a large number of seed/crops and living organisms such as animals and human beings. SakA (also called hogA/hog1) is an integral part of the mitogen activated protein kinase signal of the high osmolarity glycerol pathway. In this study, the AfsakA gene was deleted (∆AfsakA) then complemented (∆AfsakA::AfsakA) using homologous recombination and the osmotic stress was induced by 1.2 mol/L D-sorbital and 1.2 mol/L sodium chloride. The result showed that ∆AfsakA mutant caused a significant influence on conidial formation compared to wild-type and ∆AfsakA::AfsakA strains. It was also found that AfsakA responds to both the osmotic stress and the cell wall stress. In the absence of osmotic stress, ∆AfsakA mutant produced more sclerotia in contrast to other strains, whereas all strains failed to generate sclerotia under osmotic stress. Furthermore, the deletion of AfsakA resulted in the increase of Aflatoxin B1 production compared to other strains. The virulence assay on both maize kernel and peanut seeds showed that ∆AfsakA strain drastically produced more conidia and Aflatoxin B1 than wild-type and complementary strains. AfSakA-mCherry was located to the cytoplasm in the absence of osmotic stress, while it translocated to the nucleus upon exposure to the osmotic stimuli. This study provides new insights on the development and evaluation of aflatoxin biosynthesis and also provides better understanding on how to prevent Aspergillus infections which would be considered the first step towards the prevention of the seeds damages caused by A. flavus.
Elisabeth Tumukunde; Ding Li; Ling Qin; Yu Li; Jiaojiao Shen; Shihua Wang; Jun Yuan. Osmotic-Adaptation Response of sakA/hogA Gene to Aflatoxin Biosynthesis, Morphology Development and Pathogenicity in Aspergillus flavus. Toxins 2019, 11, 41 .
AMA StyleElisabeth Tumukunde, Ding Li, Ling Qin, Yu Li, Jiaojiao Shen, Shihua Wang, Jun Yuan. Osmotic-Adaptation Response of sakA/hogA Gene to Aflatoxin Biosynthesis, Morphology Development and Pathogenicity in Aspergillus flavus. Toxins. 2019; 11 (1):41.
Chicago/Turabian StyleElisabeth Tumukunde; Ding Li; Ling Qin; Yu Li; Jiaojiao Shen; Shihua Wang; Jun Yuan. 2019. "Osmotic-Adaptation Response of sakA/hogA Gene to Aflatoxin Biosynthesis, Morphology Development and Pathogenicity in Aspergillus flavus." Toxins 11, no. 1: 41.
(3S)-Acetoin and (2S,3S)-2,3-butanediol are important platform chemicals widely applied in the asymmetric synthesis of valuable chiral chemicals. However, their production by fermentative methods is difficult to perform. This study aimed to develop a whole-cell biocatalysis strategy for the production of (3S)-acetoin and (2S,3S)-2,3-butanediol from meso-2,3-butanediol. First, E. coli co-expressing (2R,3R)-2,3-butanediol dehydrogenase, NADH oxidase and Vitreoscilla hemoglobin was developed for (3S)-acetoin production from meso-2,3-butanediol. Maximum (3S)-acetoin concentration of 72.38 g/L with the stereoisomeric purity of 94.65% was achieved at 24 h under optimal conditions. Subsequently, we developed another biocatalyst co-expressing (2S,3S)-2,3-butanediol dehydrogenase and formate dehydrogenase for (2S,3S)-2,3-butanediol production from (3S)-acetoin. Synchronous catalysis together with two biocatalysts afforded 38.41 g/L of (2S,3S)-butanediol with stereoisomeric purity of 98.03% from 40 g/L meso-2,3-butanediol. These results exhibited the potential for (3S)-acetoin and (2S,3S)-butanediol production from meso-2,3-butanediol as a substrate via whole-cell biocatalysis.
Yuanzhi He; Feixue Chen; Meijing Sun; Huifang Gao; Zewang Guo; Hui Lin; Jiebo Chen; Wensong Jin; Yunlong Yang; Liaoyuan Zhang; Jun Yuan. Efficient (3S)-Acetoin and (2S,3S)-2,3-Butanediol Production from meso-2,3-Butanediol Using Whole-Cell Biocatalysis. Molecules 2018, 23, 691 .
AMA StyleYuanzhi He, Feixue Chen, Meijing Sun, Huifang Gao, Zewang Guo, Hui Lin, Jiebo Chen, Wensong Jin, Yunlong Yang, Liaoyuan Zhang, Jun Yuan. Efficient (3S)-Acetoin and (2S,3S)-2,3-Butanediol Production from meso-2,3-Butanediol Using Whole-Cell Biocatalysis. Molecules. 2018; 23 (3):691.
Chicago/Turabian StyleYuanzhi He; Feixue Chen; Meijing Sun; Huifang Gao; Zewang Guo; Hui Lin; Jiebo Chen; Wensong Jin; Yunlong Yang; Liaoyuan Zhang; Jun Yuan. 2018. "Efficient (3S)-Acetoin and (2S,3S)-2,3-Butanediol Production from meso-2,3-Butanediol Using Whole-Cell Biocatalysis." Molecules 23, no. 3: 691.
Domoic acid (DA) is a potent toxin, marine biotoxin, and primarily produced by Pseudo-nitzschia. The DA hapten was coupled with bovine serum albumin (BSA), and ovalbumin (OVA) as carrier proteins. DA-BSA conjugate was used as immunogen and DA-OVA as coating antigen. Cell fusion between spleen cells and sp2/0 myeloma cells developed 1C3 hybridoma clone producing 1C3 monoclonal antibody (mAb). Hybridoma was injected into the mice to produce ascites, and further purified by caprylic acid/ammonium sulfate method. The mAb was of IgG3 subclass, and was specific to DA with high affinity (2.5 × 108 L/mol). Moreover, western blot exhibited significant specificity to the DA antigens. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) showed DA working range of 0.006–0.2 ng/mL. The IC50 was 0.03 ng/mL with low limit of detection (LOD) of 0.006 ng/mL. Average DA recovery from spiked shellfish extract was 100.56% ± 2.8% with the coefficient variation of 0.01–0.1%. Hence, mAb producing 1C3 hybridoma was successfully developed and could be used to detect DA in contaminated samples.
Abdullah F. U. H. Saeed; Sumei Ling; Jun Yuan; Shihua Wang. The Preparation and Identification of a Monoclonal Antibody against Domoic Acid and Establishment of Detection by Indirect Competitive ELISA. Toxins 2017, 9, 250 .
AMA StyleAbdullah F. U. H. Saeed, Sumei Ling, Jun Yuan, Shihua Wang. The Preparation and Identification of a Monoclonal Antibody against Domoic Acid and Establishment of Detection by Indirect Competitive ELISA. Toxins. 2017; 9 (8):250.
Chicago/Turabian StyleAbdullah F. U. H. Saeed; Sumei Ling; Jun Yuan; Shihua Wang. 2017. "The Preparation and Identification of a Monoclonal Antibody against Domoic Acid and Establishment of Detection by Indirect Competitive ELISA." Toxins 9, no. 8: 250.
Citrinin (CTN) is a hepato-nephrotoxic mycotoxin produced by fungi genera of Aspergillus, Monauscus, and Penicillium. CTN contaminates grains, fruits, juices and vegetables, and causes various toxic effects to humans and animals. It has small molecular weight, which is non-immunogenic to animals. Thus, CTN was conjugated to bovine serum albumin (BSA) and ovalbumin (OVA), respectively, by amide bonds using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Mice were immunized with CTN-BSA conjugates, and spleen cells of the immunized mice were fused with Sp2/0 myeloma cells to obtain 21H27 hybriodoma cell. Ascitic fluid of hybridoma cell was produced in mice abdomen, and purified using caprylic/ammonium sulfate precipitation method. The 21H27 anti-CTN mcAb was the IgG2a antibody subclass, and cross-reactivity results indicated that anti-CTN mcAb is specific to CTN with high affinity (2.0 × 108 L/mol). Indirect competitive ELISA (ic-ELISA) results showed that the linear range of detection was 0.01–5.96 ng/mL and the IC50 was 0.28 ng/mL with a lower detection limit (LOD) of 0.01 ng/mL. The average recovery was 93.8% ± 1.6% with a coefficient variation of 1.0%–4.3%. Hence, anti-CTN mcAb secreted by 21H27 hybridoma cell was successfully produced and can be used to detect CTN contaminated feed and foodstuffs.
Shimuye Kalayu Yirga; Sumei Ling; Yanling Yang; Jun Yuan; Shihua Wang. The Preparation and Identification of a Monoclonal Antibody against Citrinin and the Development of Detection via Indirect Competitive ELISA. Toxins 2017, 9, 110 .
AMA StyleShimuye Kalayu Yirga, Sumei Ling, Yanling Yang, Jun Yuan, Shihua Wang. The Preparation and Identification of a Monoclonal Antibody against Citrinin and the Development of Detection via Indirect Competitive ELISA. Toxins. 2017; 9 (3):110.
Chicago/Turabian StyleShimuye Kalayu Yirga; Sumei Ling; Yanling Yang; Jun Yuan; Shihua Wang. 2017. "The Preparation and Identification of a Monoclonal Antibody against Citrinin and the Development of Detection via Indirect Competitive ELISA." Toxins 9, no. 3: 110.
The bacteria involved in organic pollutant degradation in pelagic deep-sea environments are largely unknown. In this report, the diversity of polycyclic aromatic hydrocarbon ( PAH)-degrading bacteria was analyzed in deep-sea water on the Southwest Indian Ridge (SWIR). After enrichment with a PAH mixture (phenanthrene, anthracene, fluoranthene and pyrene), 9 nine bacterial consortia were obtained from depths of 3946 m to 4746 m. PAH degradation occurred to all components of the mixture, but when using a single PAH as the sole carbon and energy source, only phenanthrene can be degraded obviously. This indicates the cometabolism of anthracene, fluoranthene and pyrene with phenanthreneWhile the consortia degraded all four PAHs when supplied in a mixture, when PAHs were tested individually, only phenanthrene supported growth. Thus, degradation of the PAH mixture reflected a cometabolism of anthracene, fluoranthene and pyrene with phenanthrene. Further, both culture-dependent and independent methods revealed many new bacteria involved in PAH degradation. Specifically, the alpha and gamma subclasses of Proteobacteria were confirmed as the major groups within the communities. Additionally, Actinobacteria, the CFB group and Firmicutes were detected. Denaturing Gradient Gel Electrophoresis (DGGE) analysis showed that bacteria closely affiliated with Alcanivorax, Novosphingobium and Rhodovulum occurred most frequently in different PAH-degrading consortia. More than half of the isolates (34 of 51 isolates) were confirmed to have the ability to grow with the PAH mixture By using general heterotrophic media, 51 bacteria were isolated from the consortia and of these 34 grew with the PAH mixture as a sole carbon source. Of these, isolates most closely related to Alterierythrobacter, Citricella, Erythrobacter, Idiomarina, Lutibacterium, Maricaulis, Marinobacter, Martelella, Pseudidiomarina, Rhodobacter, Roseovarius, Salipiger, Sphingopyxis and Stappia were found to be PAH degraders.
Jun Yuan; Zongze Shao; Qiliang Lai; TianLing Zheng. The diversity of PAH-degrading bacteria in a deep-sea water column above the Southwest Indian Ridge. Frontiers in Microbiology 2015, 6, 853 .
AMA StyleJun Yuan, Zongze Shao, Qiliang Lai, TianLing Zheng. The diversity of PAH-degrading bacteria in a deep-sea water column above the Southwest Indian Ridge. Frontiers in Microbiology. 2015; 6 ():853.
Chicago/Turabian StyleJun Yuan; Zongze Shao; Qiliang Lai; TianLing Zheng. 2015. "The diversity of PAH-degrading bacteria in a deep-sea water column above the Southwest Indian Ridge." Frontiers in Microbiology 6, no. : 853.
Today, thrombosis is one of the most widely occurring diseases in modern life. Drugs with thrombolytic functions are the most effective methods in the treatment of thrombosis. Among them, Douchi fibrinolytic enzyme (DFE) is a promising agent. DFE was isolated from Douchi, a typical and popular soybean-fermented food in China, and it can dissolve fibrin directly and efficiently. A strain, Bacillus subtilis LD-8547 produced DFE with high fibrinolytic activity has been isolated in our lab previously. In the study, thrombolytic effect of DFE from Bacillus subtilis LD-8547 was studied in vitro and in vivo systematically. The results showed that DFE played a significant role in thrombolysis and anticoagulation in vitro. And the thrombolytic effects correlated with DFE in a dose-dependent manner. In vivo, the acute toxicity assay showed that DFE had no obvious acute toxicity to mice. Test of carrageenan-induced thrombosis in mice indicated that the DFE significantly prevented tail thrombosis, and arterial thrombosis model test indicated that Douchi fibrinolytic enzyme DFE had thrombolytic effect on carotid thrombosis of rabbits in vivo. Other results in vivo indicated that DFE could increase bleeding and clotting time obviously. The DFE isolated from Bacillus subtilis LD-8547 has obvious thrombolytic effects in vitro and in vivo. This function demonstrates that this enzyme can be a useful tool for preventing and treating clinical thrombus.
Jun Yuan; Jing Yang; Zhenhong Zhuang; Yanling Yang; Ling Lin; Shihua Wang. Thrombolytic effects of Douchi Fibrinolytic enzyme from Bacillus subtilis LD-8547 in vitro and in vivo. BMC Biotechnology 2012, 12, 36 -36.
AMA StyleJun Yuan, Jing Yang, Zhenhong Zhuang, Yanling Yang, Ling Lin, Shihua Wang. Thrombolytic effects of Douchi Fibrinolytic enzyme from Bacillus subtilis LD-8547 in vitro and in vivo. BMC Biotechnology. 2012; 12 (1):36-36.
Chicago/Turabian StyleJun Yuan; Jing Yang; Zhenhong Zhuang; Yanling Yang; Ling Lin; Shihua Wang. 2012. "Thrombolytic effects of Douchi Fibrinolytic enzyme from Bacillus subtilis LD-8547 in vitro and in vivo." BMC Biotechnology 12, no. 1: 36-36.