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Research background. Tiger milk mushroom (Lignosus rhinocerus) is a medicinal mushroom that is geographically distributed in the region of South China, Thailand, Malaysia, Indonesia, Philippines and Papua New Guinea. Consumption of its sclerotium has been reported to treat various ailments. However, its anticancer potential towards oral cancer cell lines is yet to be determined considering the traditional method of its consumption by biting/chewing of the sclerotium. Experimental approach. Mushroom sclerotial powder of cultivar TM02® was extracted and fractionated in a chromatographic column prior to cytotoxicity testing against a panel of human oral cancer cell lines. The capability of the identified bioactive fraction in regulating several molecules associated with its tumour necrosis factor (TNF) pathway was investigated. Results and conclusions. 2,5-Diphenyl-2H-tetrazolium bromide (MTT) proliferation assay indicated that cell lines ORL-48 (derived from gingiva), ORL-188 (derived from the tongue) and ORL-204 (derived from buccal mucosa) were inhibited by cold water extract of L. rhinocerus sclerotia and its high-molecular-mass fraction (HMM) in varying degrees with ORL-204 being most affected. Hence, the treatment of ORL-204 with HMM mushroom extract was further investigated. HMM mushroom extract induced apoptosis and G0/G1 phase cell cycle arrest through caspase-3/7 cleavage. Activities of MIP2 and COX-2 were downregulated by 0.2- and 4.6-fold respectively in the HMM mushroom extract-treated ORL-204 cells. Novelty and scientific contribution. Using ORL-204, we showed that HMM mushroom extract may act via the TNF pathway at various network sites as a potential dietary compound for cancer prevention and natural adjunct therapeutic to conventional cancer treatment., Pozadina istraživanja. Medicinska gljiva Lignosus rhinocerus rasprostranjena je na području južne Kine, Tajlanda, Malezije, Indonezije, Filipina i Papua Nove Gvineje. Sklerocij gljive koristi se za liječenje različitih oboljenja. Međutim, dosad još nije ispitan antikacerogeni učinak sklerocija ove medicinske gljive, koja se tradicionalno konzumira tako da se grize odnosno žvače, na stanice karcinoma usne šupljine. Eksperimentalni pristup. Ekstrakt praha sklerocija kultivara gljive TM02® frakcioniran je pomoću kromatografske kolone, te je zatim ispitan njegov citokosični učinak na različite stanične linije humanih karcinoma usne šupljine. Ispitana je sposobnost bioaktivne frakcije da regulira molekule koje sudjeluju u sintezi faktora nekroze tumora (TNF). Rezultati i zaključci. Ispitivanjem proliferacije stanica pomoću testa redukcije 2,5-difenil-2H-tetrazolijeva bromida (MTT) utvrđeno je da ekstrakt sklerocija L. rhinocerus dobiven hladnom vodom, i to frakcija velike molekulske mase, u različitoj mjeri inhibira rast staničnih linija ORL-48 (izoliranih iz desni), ORL-188 (izoliranih ih jezika), a ponajviše onih linije ORL-204 (izoliranih iz sluznice obraza). Stoga smo dodatno istražili učinak ekstrakta gljive velike molekulske mase na staničnu liniju ORL-204. Ektrakt je potaknuo apoptozu i zaustavio stanični ciklus u fazi G0/G1 cijepanjem kaspaze 3/7. U stanicama ORL-204 tretiranim ekstraktom gljive velike molekulske mase smanjila se aktivnost enzima MIP2 za 0,2 puta, a enzima COX-2 za 4,6 puta. Novina i znanstveni doprinos. Pomoću stanične linije ORL-204 pokazali smo da ekstrakt medicinske gljive velike molekulske mase može djelovati na sintezu faktora nekroze tumora, te se upotrijebiti kao prirodni dodatak prehrani za prevenciju razvoja karcinoma ili kao dodatak konvencionalnom liječenju karcinoma.

Tiger milk mushroom (Mushroom sclerotial powder of cultivar TM02® was extracted and fractionated in a chromatographic column prior to cytotoxicity testing against a panel of human oral cancer cell lines. The capability of the identified bioactive fraction in regulating several molecules associated with its tumour necrosis factor (TNF) pathway was investigated.2,5-Diphenyl-2H-tetrazolium bromide (MTT) proliferation assay indicated that cell lines ORL-48 (derived from gingiva), ORL-188 (derived from the tongue) and ORL-204 (derived from buccal mucosa) were inhibited by cold water extract ofUsing ORL-204, we showed that HMM mushroom extract may act

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.

Background Lignosus tigris, a recently discovered species of the unique Lignosus family, has been traditionally used by the indigenous communities in Peninsular Malaysia to treat various ailments and as an alternative medicine for cancer treatment. The L. tigris cultivar sclerotia (Ligno TG-K) was found to contain numerous bioactive compounds with beneficial biomedicinal properties and the sclerotial extract exhibited potent antioxidant activity. However, the anticancer property of the Ligno TG-K including in vitro and in vivo antitumor effects as well as its anticancer active compounds and the mechanisms has yet to be investigated. Methods The cytotoxicity of the Ligno TG-K against human breast (MCF7), prostate (PC3) and lung (A549) adenocarcinoma cell lines was evaluated using MTT cytotoxicity assay. The cytotoxic mechanisms of the active high molecular weight proteins (HMWp) fraction were investigated through detection of caspases activity and apoptotic-related proteins expression by Western blotting. The in vivo antitumor activity of the isolated HMWp was examined using MCF7 mouse xenograft model. Shotgun LC-MS/MS analysis was performed to identify the proteins in the HMWp. Results and Discussion Cold water extract of the sclerotia inhibited proliferation of MCF7, A549 and PC3 cells with IC50 ranged from 28.9 to 95.0 µg/mL. Bioassay guided fractionation of the extract revealed that HMWp exhibited selective cytotoxicity against MCF7 cells via induction of cellular apoptosis by the activation of extrinsic and intrinsic signaling pathways. HMWp activated expression of caspase-8 and -9 enzymes, and pro-apoptotic Bax protein whilst inhibiting expression of tumor survivor protein, Bcl-2. HMWp induced tumor-cell apoptosis and suppressed growth of tumor in MCF-7 xenograft mice. Lectins, serine proteases, RNase Gf29 and a 230NA deoxyribonuclease are the major cytotoxic proteins that accounted for 55.93% of the HMWp. Conclusion The findings from this study provided scientific evidences to support the traditional use of the L. tigris sclerotia for treatment of breast cancer. Several cytotoxic proteins with high abundance have been identified in the HMWp of the sclerotial extract and these proteins have potential to be developed into new anticancer agents or as adjunct cancer therapy.

Background Lignosus tigris, a recently discovered species of the unique Lignosus family, has been traditionally used by the indigenous communities in Peninsular Malaysia to treat various ailments and as an alternative medicine for cancer treatment. The L. tigris cultivar sclerotia (Ligno TG-K) was found to contain numerous bioactive compounds with beneficial biomedicinal properties and the sclerotial extract exhibited potent antioxidant activity. However, the anticancer property of the Ligno TG-K including in vitro and in vivo antitumor effects as well as its anticancer active compounds and the mechanisms has yet to be investigated. Methods The cytotoxicity of the Ligno TG-K against human breast (MCF7), prostate (PC3) and lung (A549) adenocarcinoma cell lines was evaluated using MTT cytotoxicity assay. The cytotoxic mechanisms of the active high molecular weight proteins (HMWp) fraction were investigated through detection of caspases activity and apoptotic-related proteins expression by Western blotting. The in vivo antitumor activity of the isolated HMWp was examined using MCF7 mouse xenograft model. Shotgun LC-MS/MS analysis was performed to identify the proteins in the HMWp. Results and Discussion Cold water extract of the sclerotia inhibited proliferation of MCF7, A549 and PC3 cells with IC50 ranged from 28.9 to 95.0 µg/mL. Bioassay guided fractionation of the extract revealed that HMWp exhibited selective cytotoxicity against MCF7 cells via induction of cellular apoptosis by the activation of extrinsic and intrinsic signaling pathways. HMWp activated expression of caspase-8 and -9 enzymes, and pro-apoptotic Bax protein whilst inhibiting expression of tumor survivor protein, Bcl-2. HMWp induced tumor-cell apoptosis and suppressed growth of tumor in MCF-7 xenograft mice. Lectins, serine proteases, RNase Gf29 and a 230NA deoxyribonuclease are the major cytotoxic proteins that accounted for 55.93% of the HMWp. Conclusion The findings from this study provided scientific evidences to support the traditional use of the L. tigris sclerotia for treatment of breast cancer. Several cytotoxic proteins with high abundance have been identified in the HMWp of the sclerotial extract and these proteins have potential to be developed into new anticancer agents or as adjunct cancer therapy.

Background Genome mining facilitated by heterologous systems is an emerging approach to access the chemical diversity encoded in basidiomycete genomes. In this study, three sesquiterpene synthase genes, GME3634, GME3638, and GME9210, which were highly expressed in the sclerotium of the medicinal mushroom Lignosus rhinocerotis, were cloned and heterologously expressed in a yeast system. Results Metabolite profile analysis of the yeast culture extracts by GC–MS showed the production of several sesquiterpene alcohols (C15H26O), including cadinols and germacrene D-4-ol as major products. Other detected sesquiterpenes include selina-6-en-4-ol, β-elemene, β-cubebene, and cedrene. Two purified major compounds namely (+)-torreyol and α-cadinol synthesised by GME3638 and GME3634 respectively, are stereoisomers and their chemical structures were confirmed by 1H and 13C NMR. Phylogenetic analysis revealed that GME3638 and GME3634 are a pair of orthologues, and are grouped together with terpene synthases that synthesise cadinenes and related sesquiterpenes. (+)-Torreyol and α-cadinol were tested against a panel of human cancer cell lines and the latter was found to exhibit selective potent cytotoxicity in breast adenocarcinoma cells (MCF7) with IC50 value of 3.5 ± 0.58 μg/ml while α-cadinol is less active (IC50 = 18.0 ± 3.27 μg/ml). Conclusions This demonstrates that yeast-based genome mining, guided by transcriptomics, is a promising approach for uncovering bioactive compounds from medicinal mushrooms. Electronic supplementary material The online version of this article (doi:10.1186/s12934-017-0713-x) contains supplementary material, which is available to authorized users.

The emergence of Escherichia coli resistant to extended-spectrum cephalosporins (ESCs) is of concern as ESC is often used to treat infections by Gram-negative bacteria. One-hundred and ten E. coli strains isolated in 2009-2010 from children warded in a Malaysian tertiary hospital were analyzed for their antibiograms, carriage of extended-spectrum beta-lactamase (ESBL) and AmpC genes, possible inclusion of the beta-lactamase genes on an integron platform, and their genetic relatedness. All E. coli strains were sensitive to carbapenems. About 46% of strains were multidrug resistant (MDR; i.e., resistant to ≥3 antibiotic classes) and almost half (45%) were nonsusceptible to ESCs. Among the MDR strains, high resistance rates were observed for ampicillin (98%), tetracycline (75%), and trimethoprim/sulfamethoxazole (73%). Out of 110 strains, bla(TEM-1) (49.1%), bla(CTX-M) (11.8%), and bla(CMY-2) (6.4%) were detected. Twenty-one strains were ESBL producers. CTX-M-15 was the predominant CTX-M variant found and this is the first report of a CTX-M-27-producing E. coli strain from Malaysia. Majority (3.1%) of the strains harbored class 1 integron-encoded integrases with a predominance of aadA and dfr genes within the integron variable region. No gene cassette encoding ESBL genes was found and integrons were not significantly associated with ESBL or non-ESBL producers. Possible clonal expansion was observed for few CTX-M-15-positive strains but the O25-ST131 E. coli clone known to harbor CTX-M-15 was not detected while CMY-2-positive strains were genetically diverse.

Background Salmonella enterica serovar Typhi (S. Typhi) is strictly a human intracellular pathogen. It causes acute systemic (typhoid fever) and chronic infections that result in long-term asymptomatic human carriage. S. Typhi displays diverse disease manifestations in human infection and exhibits high clonality. The principal factors underlying the unique lifestyle of S. Typhi in its human host during acute and chronic infections remain largely unknown and are therefore the main objective of this study. Methodology/Principal Findings To obtain insight into the intracellular lifestyle of S. Typhi, a high-throughput phenotypic microarray was employed to characterise the catabolic capacity of 190 carbon sources in S. Typhi strains. The success of this study lies in the carefully selected library of S. Typhi strains, including strains from two geographically distinct areas oftyphoid endemicity, an asymptomatic human carrier, clinical stools and blood samples and sewage-contaminated rivers. An extremely low carbon catabolic capacity (27% of 190 carbon substrates) was observed among the strains. The carbon catabolic profiles appeared to suggest that S. Typhi strains survived well on carbon subtrates that are found abundantly in the human body but not in others. The strains could not utilise plant-associated carbon substrates. In addition, α-glycerolphosphate, glycerol, L-serine, pyruvate and lactate served as better carbon sources to monosaccharides in the S. Typhi strains tested. Conclusion The carbon catabolic profiles suggest that S. Typhi could survive and persist well in the nutrient depleted metabolic niches in the human host but not in the environment outside of the host. These findings serve as caveats for future studies to understand how carbon catabolism relates to the pathogenesis and transmission of this pathogen.

Acinetobacter baumannii, genomic species 3 and 13TU are being increasingly reported as the most important Acinetobacter species that cause infections in hospitalized patients. These Acinetobacter species are grouped in the Acinetobacter calcoaceticus- Acinetobacter baumannii (Acb) complex. Differentiation of the species in the Acb-complex is limited by phenotypic methods. Therefore, in this study, amplified ribosomal DNA restriction analysis (ARDRA) was applied to confirm the identity A. baumannii strains as well as to differentiate between the subspecies. One hundred and eighty-five strains from Intensive Care Unit, Universiti Malaya Medical Center (UMMC) were successfully identified as A. baumannii by ARDRA. Acinetobacter genomic species 13TU and 15TU were identified in 3 and 1 strains, respectively. ARDRA provides an accurate, rapid and definitive approach towards the identification of the species level in the genus Acinetobacter. This paper reports the first application ARDRA in genospecies identification of Acinetobacter in Malaysia.

Additional file 1: Table S1. Coding sequences of GME3634, GME3638, and GME9210. Table S2. PCR primers information. Table S3. 13C (150.903 MHz) and 1H (600.130 MHz) NMR spectral data for torreyol and α-cadinol (using CDCl3 as internal standard, 77.16 ppm). Figure S1. Mass spectrums of putative torreyol (1) and α-cadinol (2) to that of known compounds in NIST 05 library. Figure S2. Proposed general mechanism for the biosynthesis of torreyol and α-cadinol from farnesyl pyrophosphate.