Induction of in vitro flowering in the orchid Dendrobium Sonia 17.

In this study, Dendrobium Sonia 17 plantlets were used to induce in vitro flowering. Infloresce nces were induced and rooting was inhibited in the half-strength Murashige and Skoog medium containing 20μ MN6-benzyladenine (BA). The medium with high P and low N contents was effective to in duce inflorescences while the medium with low P and high N contents was only effective to promote forming of shoots. In addition, the induced in vitro inflorescences were able to multiply and maintain without exhibiting a distinctive vegetative phase. Different morphologies of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed

Production of the virus-like particles of nipah virus matrix protein in Pichia pastoris as diagnostic reagents

The matrix (M) protein of Nipah virus (NiV) is a peripheral protein that plays a vital role in the envelopment of nucleocapsid protein and acts as a bridge between the viral surface and the nucleocapsid proteins. The M protein is also proven to play an important role in production of virus-like particles (VLPs) and is essential for assembly and budding of NiV particles. The recombinant M protein produced in Escherichia coli assembled into VLPs in the absence of the viral surface proteins. However, the E. coli produced VLPs are smaller than the native virus particles. Therefore, the aims of this study were to produce NiV M protein in Pichia pastoris, to examine the structure of the VLPs formed, and to assess the potential of the VLPs as a diagnostic reagent. The M protein was successfully expressed in P. pastoris and was detected with anti-myc antibody using Western blotting. The VLPs formed by the recombinant M protein were purified with sucrose density gradient ultracentrifugation, high-performance liquid chromatography (HPLC), and Immobilized Metal Affinity Chromatography (IMAC). Immunogold staining and transmission electron microscopy confirmed that the M protein assembled into VLPs as large as 200 nm. ELISA revealed that the NiV M protein produced in P. pastoris reacted strongly with positive NiV sera demonstrating its potential as a diagnostic reagent

Selection of co-transformed Dendrobium Sonia 17 using hygromycin and green fluorescent protein

Dendrobium Sonia 17 calluses were used for co-transformation study using particle bombardment. The bombarded transformed callus tissues were selected using half-strength MS medium containing 25 mg dm−3 hygromycin. Expression of green fluorescent protein (GFP) was observed in the callus and protocorm-like body (PLBs) tissues survived on the selection medium. The presence of green fluorescence protein (sgfp), hygromycin-B-phosphotransferase (hptII) and β-glucuronidase (uidA) genes in the transformed tissues were verified using PCR, Southern blot and dot blot analyses. Based on the results from PCR and expression of sgfp and uidA genes in the calluses and PLBs survived from hygromycin selection, we reported the co-transformation of sgfp, hptII and uidA genes into Dendrobium Sonia 17. GFP-expressing tissues were also observed in the regenerated transformed plantlets

Immunogenic properties of recombinant ectodomain of Newcastle disease virus hemagglutinin-neuraminidase protein expressed in Escherichia coli.

Hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a vital role in the viral infectivity, host immunity, and disease diagnosis. A portion of the HN gene encoding the ectodomain (nt 142-1739) was cloned and expressed in Escherichia coli yielding an insoluble HN protein and a soluble NusA-HN protein containing N-utilization substance A (NusA) fusion component. Both recombinant proteins were purified and used for immunization of chickens. The recombinant HN protein induced higher antibody titers as compared to the recombinant NusA-HN protein. These antibodies were able to react in immunoblot analysis with the corresponding recombinant proteins as well as with the HN protein of NDV

The genome of the Tiger Milk mushroom, Lignosus rhinocerotis, provides insights into the genetic basis of its medicinal properties

BACKGROUND The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden or Tiger milk mushroom (Polyporales, Basidiomycota) is a valuable folk medicine for indigenous peoples in Southeast Asia. Despite the increasing interest in this ethnobotanical mushroom, very little is known about the molecular and genetic basis of its medicinal and nutraceutical properties. RESULTS The de novo assembled 34.3 Mb L. rhinocerotis genome encodes 10,742 putative genes with 84.30% of them having detectable sequence similarities to others available in public databases. Phylogenetic analysis revealed a close evolutionary relationship of L. rhinocerotis to Ganoderma lucidum, Dichomitus squalens, and Trametes versicolor in the core polyporoid clade. The L. rhinocerotis genome encodes a repertoire of enzymes engaged in carbohydrate and glycoconjugate metabolism, along with cytochrome P450s, putative bioactive proteins (lectins and fungal immunomodulatory proteins) and laccases. Other genes annotated include those encoding key enzymes for secondary metabolite biosynthesis, including those from polyketide, nonribosomal peptide, and triterpenoid pathways. Among them, the L. rhinocerotis genome is particularly enriched with sesquiterpenoid biosynthesis genes. CONCLUSIONS The genome content of L. rhinocerotis provides insights into the genetic basis of its reported medicinal properties as well as serving as a platform to further characterize putative bioactive proteins and secondary metabolite pathway enzymes and as a reference for comparative genomics of polyporoid fungi.This research is supported by High Impact Research Grant UM.C/625/1/HIR/ MoE/E20040-20001 from the University of Malaya/Ministry of Education, Malaysia. H-YYY is supported by the postgraduate research grant (PPP) PV024/ 2012A from University of Malaya, Malaysia. Y-HC is a recipient of Australian Research Council Discovery Early Career Researcher Award (ARC DECRA)

Transient expression of an immunogenic envelope attachment glycoprotein of nipah virus in Nicotiana benthamiana

The Nipah virus is highly virulent to swine and humans. The envelope attachment glycoprotein (G) of Nipah virus plays a key role in viral entry and induction of neutralizing antibody in mammalian hosts, thus is considered a good candidate for vaccine development. Plant transient expression systems are gaining recognition as a viable alternative for the production of vaccine antigens. In this study, we expressed the Nipah virus G protein heterologously in Nicotiana benthamiana using an agroinfiltration approach. The highest expression of recombinant G protein in N. benthamiana at RNA and protein levels was detected on day 9 post-infiltration. Western blot analysis demonstrated that the purified G protein reacted specifically with rabbit anti-Nipah Virus serum, indicating its potential for vaccine use

The genome of the Tiger Milk mushroom, Lignosus rhinocerotis, provides insights into the genetic basis of its medicinal properties

BACKGROUND: The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden or Tiger milk mushroom (Polyporales, Basidiomycota) is a valuable folk medicine for indigenous peoples in Southeast Asia. Despite the increasing interest in this ethnobotanical mushroom, very little is known about the molecular and genetic basis of its medicinal and nutraceutical properties. RESULTS: The de novo assembled 34.3 Mb L. rhinocerotis genome encodes 10,742 putative genes with 84.30% of them having detectable sequence similarities to others available in public databases. Phylogenetic analysis revealed a close evolutionary relationship of L. rhinocerotis to Ganoderma lucidum, Dichomitus squalens, and Trametes versicolor in the core polyporoid clade. The L. rhinocerotis genome encodes a repertoire of enzymes engaged in carbohydrate and glycoconjugate metabolism, along with cytochrome P450s, putative bioactive proteins (lectins and fungal immunomodulatory proteins) and laccases. Other genes annotated include those encoding key enzymes for secondary metabolite biosynthesis, including those from polyketide, nonribosomal peptide, and triterpenoid pathways. Among them, the L. rhinocerotis genome is particularly enriched with sesquiterpenoid biosynthesis genes. CONCLUSIONS: The genome content of L. rhinocerotis provides insights into the genetic basis of its reported medicinal properties as well as serving as a platform to further characterize putative bioactive proteins and secondary metabolite pathway enzymes and as a reference for comparative genomics of polyporoid fungi. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-635) contains supplementary material, which is available to authorized users

Quantitation of green fluorescent protein using a gel-based imaging method.

Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately

Production of long helical capsid of Nipah virus by Pichia pastoris.

The nucleocapsid (N) protein of Nipah virus (NiV) produced in a recombinant host can replace the use of inactivated virus as a diagnostic reagent because it is safer and affordable. The aim of this study was to express the N protein in Pichia pastoris. The N gene of NiV was cloned into the yeast expression vector, pPICZ B and expressed in P. pastoris. The recombinant N protein of NiV was purified using sucrose density gradient ultracentrifugation and was confirmed with Western blotting using rabbit anti-N antibody. The P. pastoris expressed N protein self-assembled into helical structures as large as 1.5 μm as shown in an electron micrograph. ELISA analysis performed with the swine sera obtained during the viral outbreak proved that the recombinant N protein to be highly antigenic. The NiV N protein produced in P. pastoris serves as an alternative to the recombinant N protein produced in Escherichia coli

Identifikacija bioaktivnih proteina gljive Ophiocordyceps sinensis i određivanje njihovog antioksidacijskog i citotoksičnog učinka pomoću shotgun analize proteoma

Research background. Ophiocordyceps sinensis, a highly valued medicinal fungus, is close to extinction due to overexploitation. Successful cultivation of O. sinensis fruiting body (OCS02®) shows that the cultivar has a promising nutritional value and numerous bioactive compounds. Antioxidant and antiproliferative properties and biologically active proteins of the OCS02® are investigated for possible development into nutraceuticals. Experimental approach. The chemical composition of the OCS02® cold water extract was determined, and the antioxidant activities were examined using ferric reducing, DPPH• and O2•- scavenging assays. Tetrazolium dye (MTT) cytotoxic assay was performed to assess the antiproliferative activity of the extract. Bioactive proteins in the active fraction of the extract were identified using liquid chromatography (LC) and tandem-mass spectrometry (MS/MS). Results and conclusions. The OCS02® extract exhibited strong O2•- scavenging (expressed as Trolox equivalents (18.4±1.1) mol/g) and potent cytotoxic activities against adenocarcinomic human alveolar basal epithelial (A549) cells (IC50=(58.2±6.8) µg/mL). High molecular mass polysaccharides, proteins and protein-polysaccharide complexes could have contributed to the antioxidant and cytotoxic selectivity of the OCS02®. LC-MS/MS analysis identified several potential cytotoxic proteases and an oxalate decarboxylase protein which may exhibit protection effects on kidneys. Novelty and scientific contribution. The findings demonstrate the potential of OCS02® to be developed into functional food due to its promising superoxide anion radical scavenging capacity, cytotoxic effect and presence of biopharmaceutically active proteins.Pozadina istraživanja. Vrlo cijenjena medicinska gljiva Ophiocordyceps sinensis je na rubu izumiranja zbog njezine prekomjerne eksploatacije. Uspješnim uzgojem plodišta gljive O. sinensis (OCS02®) potvrđeno je da taj kultivar ima obećavajuća hranjiva svojstva te sadržava brojne bioaktivne spojeve. Ispitana su njegova antioksidacijska i antiproliferacijska svojstva te sastav biološki aktivnih proteina, s ciljem mogućeg razvoja nutraceutika. Eksperimentalni pristup. Utvrđen je kemijski sastav ekstrakta gljive u hladnoj vodi, a antioksidacijska je aktivnost ispitana pomoću FRAP metode te metodama uklanjanja DPPH˙ i O2 radikala. Citotoksičnost odnosno antiproliferacijska aktivnost ekstrakta ispitana je testom na osnovi tetrazolija (MTT test). Bioaktivni proteini su identificirani u aktivnoj frakciji ekstrakta pomoću tekućinske kromatografije i tandemske spektrometrije masa. Rezultati i zaključci. Ekstrakt gljive OCS02® imao je izrazito jako svojstvo uklanjanja superoksid radikala (izraženo u ekvivalentima Troloxa (18,4±1,1) mol/g) i snažan citotoksični učinak (IC50=(58,2±6,8) µg/mL) na humane epitelne stanice adenokarcinoma pluća (A549). Moguće je da polisaharidi, proteini i kompleksi proteina s polisaharidima velike molekularne mase pridonose antioksidacijskoj i citotoksičnoj selektivnosti gljive OCS02®. Tekućinskom kromatografijom i tandemskom spektrometrijom masa identificirano je nekoliko potencijalno citotoksičnih proteaza te protein oksalat dekarboksilaza koji bi mogli imati zaštitni učinak na bubrege. Novina i znanstveni doprinos. Dobiveni rezultati pokazuju da se gljiva OCS02® može upotrijebiti u proizvodnji funkcionalne hrane zbog njezine obećavajuće sposobnosti uklanjanja superoksidnih aniona, citotoksičnog učinka te prisutnosti biofarmaceutski aktivnih proteina