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

Medicinska gljiva Lignosus rhinocerus svojim imunomodulacijskim učinkom i reguliranjem signalnog puta posredovanog faktorom nekroze tumora uzrokuje apoptozu i zaustavlja stanični ciklus stanica karcinoma usne šupljine ORL-204

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

The first whole genome and transcriptome of the cinereous vulture reveals adaptation in the gastric and immune defense systems and possible convergent evolution between the Old and New World vultures

Background: The cinereous vulture, Aegypius monachus, is the largest bird of prey and plays a key role in the ecosystem by removing carcasses, thus preventing the spread of diseases. Its feeding habits force it to cope with constant exposure to pathogens, making this species an interesting target for discovering functionally selected genetic variants. Furthermore, the presence of two independently evolved vulture groups, Old World and New World vultures, provides a natural experiment in which to investigate convergent evolution due to obligate scavenging. Results: We sequenced the genome of a cinereous vulture, and mapped it to the bald eagle reference genome, a close relative with a divergence time of 18 million years. By comparing the cinereous vulture to other avian genomes, we find positively selected genetic variations in this species associated with respiration, likely linked to their ability of immune defense responses and gastric acid secretion, consistent with their ability to digest carcasses. Comparisons between the Old World and New World vulture groups suggest convergent gene evolution. We assemble the cinereous vulture blood transcriptome from a second individual, and annotate genes. Finally, we infer the demographic history of the cinereous vulture which shows marked fluctuations in effective population size during the late Pleistocene. Conclusions: We present the first genome and transcriptome analyses of the cinereous vulture compared to other avian genomes and transcriptomes, revealing genetic signatures of dietary and environmental adaptations accompanied by possible convergent evolution between the Old World and New World vulturesopen

Scalable and extensible data Structures and methods for big data in phylogenomics and phylogenetics

Technology, data processing, and availability have led to large increases in data set sizes and computing resources. Traditional solutions can neither handle large data sets nor leverage more resources, prompting a need for advanced data structures, algorithms, and solutions. In particular, phylogenetics is a long-standing field with a rich history in the design of novel algorithms, heuristics, and visualization. It is a challenging field; no polynomial-time algorithms are known for many problems and many heuristics techniques have been developed and used in solving these problems. Scalable and extensible approaches are key requirements for modern solutions. We explore data structures and algorithms to generalize problems faced in genomics with examples in phylogenetics in a scalable and extensible manner. Scalable explicitly refers to the ability to process increasing inputs efficiently. In general, the solution should be unrestricted to small inputs or niche cases and be efficient regarding time complexity. Extensibility can be defined as the ability for the solution to be general, not restricted to special cases, and the solution can be extended for multiple purposes or applied to various applications. Numerous problem-specific approaches in phylogenetics, as in many fields, work extremely well but are limited to that problem. We present a step back in data structures for a common problem and show how this data structure can be both a scalable and extensible base for many algorithms as solutions to common phylogenetic problems. This successful approach to developing a common data structure shows promise as an alternative to inter-node distance matrix calculation as a precursor or prerequisite. Additionally, many algorithms can take advantage of this bipartition frequency hash to reduce complexity by at least an order of magnitude. Use cases showing the value of this data structure are presented, including Robinson-Foulds calculations, fast alternative tree inference, and tree refinement sampling alternatives demonstrated on multiple popular real-world data sets and simulations. The real-world data sets give us first-hand comparisons with other methods and show the advantages and disadvantages of the methods, while the simulations give us data points for time and space complexity for a scalability analysis. We also demonstrate a reference based alternative to a specific usage of Multiple Sequence Alignment. Lastly, we explore state-of-the-art Machine Learning platforms to learn features in a semi-supervised manner. Examples of data structures, use cases, and this platform are discussed

Airway Relaxation Effects of Water-Soluble Sclerotial Extract From Lignosus rhinocerotis

Lignosus rhinocerotis has a long history of use by the indigenous community within East Asia to treat a range of health conditions including asthma and chronic cough. To date, there is limited scientific evidence to support its therapeutic effects in relieving these airways conditions. In this study, we examined the effects of the different molecular weight fractions [high-molecular-weight (HMW), medium-molecular-weight (MMW), and low-molecular-weight (LMW)] obtained from the cold water sclerotial extract (CWE) of L. rhinocerotis on airways patency using airway segments isolated from Sprague Dawley rat in an organ bath set-up. It is demonstrated that the HMW and MMW fractions exhibited higher efficacy in relaxing the pre-contracted airways when compared to the CWE and LMW fraction. In addition, the HMW fraction markedly supressed carbachol-, 5-hydroxytrptamine-, and calcium-induced airway contractions. CWE demonstrated a lower efficacy than the HMW fraction but it also significantly attenuated carbachol- and calcium-induced airway contractions. Results showed that the bronchorelaxation effect of CWE and fractions is mediated via blockade of extracellular Ca 2+ influx. The composition analysis revealed the following parts of carbohydrate and proteins, respectively: HMW fraction: 71 and 4%; MMW fraction: 35 and 1%; and LMW fraction: 22 and 0.3%. Our results strongly suggest that the polysaccharide-protein complex or proteins found in the HMW and MMW fractions is likely to contribute to the bronchorelaxation effect of CWE

Whole genome survey of big cats (Genus: Panthera) identifies novel microsatellites of utility in conservation genetic study

Big cats (Genus: Panthera) are among the most threatened mammal groups of the world, owing to hunting, habitat loss, and illegal transnational trade. Conservation genetic studies and effective curbs on poaching are important for the conservation of these charismatic apex predators. A limited number of microsatellite markers exists for Panthera species and researchers often cross-amplify domestic cat microsatellites to study these species. We conducted data mining of seven Panthera genome sequences to discover microsatellites for conservation genetic studies of four threatened big cat species. A total of 32 polymorphic microsatellite loci were identified in silico and tested with 152 big cats, and were found polymorphic in most of the tested species. We propose a set of 12 novel microsatellite markers for use in conservation genetics and wildlife forensic investigations of big cat species. Cumulatively, these markers have a high discriminatory power of one in a million for unrelated individuals and one in a thousand for siblings. Similar PCR conditions of these markers increase the prospects of achieving efficient multiplex PCR assays. This study is a pioneering attempt to synthesise genome wide microsatellite markers for big cats