Genomic insights into Dactylosporangium sp. AC04546, a rubber degrader with three latex clearing proteins.

With more than 100 rubber-degrading strains being reported, only 9 Lcp proteins isolated from Nocardia, Gordonia, Streptomyces, Rhodococcus, Actinoplanes, and Solimonas have been purified and biochemically characterized. A new strain, Dactylosporangium sp. AC04546 (strain JCM34239), isolated from soil samples collected in Sarawak Forest, was able to grow and utilize natural or synthetic rubber as the sole carbon source. Complete genome of Strain AC04546 was obtained from the hybrid assembly of PacBio Sequel II and Illumina MiSeq. Strain AC04546 has a large circular genome of 13.08 Mb with a G+C content of 72.1%. The genome contains 11,865 protein-coding sequences with 3 latex clearing protein (lcp) genes located on its chromosome. The genetic organization of the lcp gene cluster is similar to two other reported rubber-degrading strains-Actinoplanes sp. OR16 and Streptomyces sp. CFMR 7. All 3 Lcp from strain AC04546 were expressed in Escherichia coli and exhibited degrading activity against natural rubber. The distinctiveness of strain AC04546, along with other characterized rubber-degrading strains, is reported here.

Broad anti-pathogen potential of DEAD box RNA helicase eIF4A-targeting rocaglates.

Inhibition of eukaryotic initiation factor 4A has been proposed as a strategy to fight pathogens. Rocaglates exhibit the highest specificities among eIF4A inhibitors, but their anti-pathogenic potential has not been comprehensively assessed across eukaryotes. In silico analysis of the substitution patterns of six eIF4A1 aa residues critical to rocaglate binding, uncovered 35 variants. Molecular docking of eIF4A:RNA:rocaglate complexes, and in vitro thermal shift assays with select recombinantly expressed eIF4A variants, revealed that sensitivity correlated with low inferred binding energies and high melting temperature shifts. In vitro testing with silvestrol validated predicted resistance in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. Our analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens with rocaglates. Finally, our findings might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens.

Streptomyces sp. AC04842: Genomic Insights and Functional Expression of Its Latex Clearing Protein Genes (lcp1 and lcp2) When Cultivated With Natural and Vulcanized Rubber as the Sole Carbon Source.

Rubber-degrading Actinobacteria have been discovered and investigated since 1985. Only recently, through the advancement of genomic sequencing and molecular techniques, genes and pathways involved in rubber degradation are being revealed; however, the complete degradation pathway remains unknown. Streptomyces sp. AC04842 (JCM 34241) was discovered by screening at a Culture Collection Centre in Sarawak for Actinomycetes forming a clear zone on natural rubber latex agar. Streptomyces is a dominant and well-studied soil bacterium playing an important role in soil ecology including carbon recycling and biodegradation. Streptomyces sp. AC04842 draft genome revealed the presence of 2 putative latex clearing protein (lcp) genes on its chromosome and is closely related to Streptomyces cellulosae. Under the Streptomyces genus, there are a total of 64 putative lcp genes deposited in the GenBank and UniProt database. Only 1 lcp gene from Streptomyces sp. K30 has been characterized. Unlike Streptomyces sp. K30 which contained 1 lcp gene on its chromosome, Streptomyces sp. AC04842 contained 2 lcp genes on its chromosome. Streptomyces sp. AC04842 lcp1 and lcp2 amino acid sequences showed 46.13 and 69.11%, respectively, similarity to lcp sequences of Streptomyces sp. K30. Most rubber degrading strains were known to harbor only 1 lcp gene, and only recently, 2-3 lcp homologs have been reported. Several studies have shown that lcp-homolog expression increased in the presence of rubber. To study the expression of lcp1 and lcp2 genes for Streptomyces sp. AC04842, the strain was incubated in different types of rubber as the sole carbon source. In general, the lcp1 gene was highly expressed, while the lcp2 gene expression was upregulated in the presence of vulcanized rubber. Mixtures of natural and vulcanized rubber did not further increase the expression of both lcp genes compared with the presence of a specific rubber type. In this study, we paved the way to the exploration of lcp homologs and their function in degrading different types of rubber.

Rubber Degrading Strains: Microtetraspora and Dactylosporangium.

Rubber composed of highly unsaturated hydrocarbons, modified through addition of chemicals and vulcanization are widely used to date. However, the usage of rubber, faces many obstacles. These elastomeric materials are difficult to be re-used and recovered, leading to high post-consumer waste and vast environmental problems. Tyres, the major rubber waste source can take up to 80 years to naturally degrade. Experiments show that the latex clearing proteins (Lcp) found in Actinobacteria were reportedly critical for the initial oxidative cleavage of poly(cis-1,4-isoprene), the major polymeric unit of rubber. Although, more than 100 rubber degrading strains have been reported, only 8 Lcp proteins isolated from Nocardia (3), Gordonia (2), Streptomyces (1), Rhodococcus (1), and Solimonas (1) have been purified and biochemically characterized. Previous studies on rubber degrading strains and Lcp enzymes, implied that they are distinct. Following this, we aim to discover additional rubber degrading strains by randomly screening 940 Actinobacterial strains isolated from various locations in Sarawak on natural rubber (NR) latex agar. A total of 18 strains from 5 genera produced clearing zones on NR latex agar, and genes encoding Lcp were identified. We report here lcp genes from Microtetraspora sp. AC03309 (lcp1 and lcp2) and Dactylosporangium sp. AC04546 (lcp1, lcp2, lcp3), together with the predicted genes related to rubber degradation. In silico analysis suggested that Microtetraspora sp. AC03309 is a distinct species closely related to Microtetraspora glauca while Dactylosporangium sp. AC04546 is a species closely related to Dactylosporangium sucinum. Genome-based characterization allowed the establishment of the strains taxonomic position and provided insights into their metabolic potential especially in biodegradation of rubber. Morphological changes and the spectrophotometric detection of aldehyde and keto groups indicated the degradation of the original material in rubber samples incubated with the strains. This confirms the strains' ability to utilize different rubber materials (fresh latex, NR product and vulcanized rubber) as the sole carbon source. Both strains exhibited different levels of biodegradation ability. Findings on tyre utilization capability by Dactylosporangium sp. AC04546 is of interest. The final aim is to find sustainable rubber treatment methods to treat rubber wastes.

Inhibition Effect of Extract of Psychotria viridiflora Stem on α-Amylase and α-Glucosidase and Its Application in Lowering the Digestibility of Noodles.

A collection of tropical medicinal plants from East Malaysia's rainforests are used by indigenous tribes for their curative properties. Despite their purported healing properties, these forest plant species are largely unexplored and hence remain virtually unknown to the outside world. In this study, antidiabetic properties of Psychotria viridiflora, a plant used to treat diabetes by a local community in Sarawak, Malaysia were investigated. Ethyl acetate (EA) extract of P. viridiflora stem was found to exhibit high starch hydrolase inhibition activity with an IC50 value of 15.4 ± 2.1 µg/ml against porcine α-amylase and an IC50 value of 32.4 ± 3.7 µg/ml against rat intestinal α-glucosidase. A complex mixture of A-type oligomeric proanthocyanidins containing (epi)fisetinidol, (epi)afzelechin, (epi)guibourtinidol, and (epi)catechin were found. These compounds may be responsible for the starch hydrolase inhibition activity. Ethyl acetate (EA) extract of P. viridiflora stem was incorporated into wheat and rice flour to reformulate noodles with slow digestibility and was assessed under in vitro simulated gastrointestinal conditions. A dose-dependent effect on digestibility was observed for both noodles upon incorporation of 1-6% (w/w) of EA extract, with noodles containing 6% (w/w) extract exhibiting the greatest reduction in digestibility. As compared to rice noodles containing 6% extract (31.16% inhibition), wheat noodles with the same extract concentration had a smaller decline in digestibility (27.25% inhibition) after 180 min. Overall, our findings highlight the potential of P. viridiflora in the prevention of postprandial hyperglycaemia.

Transcriptional alterations in Caenorhabditis elegans following exposure to an anthelmintic fraction of the plant Picria fel-terrae Lour.

BACKGROUND: Natural compounds from plants are known to provide a source of anthelmintic molecules. In previous studies, we have shown that plant extracts from the plant Picria fel-terrae Lour. and particular fractions thereof have activity against the free-living nematode Caenorhabditis elegans, causing quite pronounced stress responses in this nematode. We have also shown that a fraction, designated Pf-fraction 5, derived from this plant has a substantial adverse effect on this worm; however, nothing is known about the molecular processes affected in the worm. In the present study, we explored this aspect. RESULTS: Key biological processes linked to upregulated genes (n = 214) included 'response to endoplasmic reticulum stress' and 'lipid metabolism', and processes representing downregulated genes (n = 357) included 'DNA-conformation change' and 'cellular lipid metabolism'. CONCLUSIONS: Exposure of C. elegans to Pf-fraction 5 induces significant changes in the transcriptome. Gene ontology analysis suggests that Pf-fraction 5 induces endoplasmic reticulum and mitochondrial stress, and the changes in gene expression are either a direct or indirect consequence of this. Further work is required to assess specific responses to sub-fractions of Pf-fraction 5 in time-course experiments in C. elegans, to define the chemical(s) with potent anthelmintic properties, to attempt to unravel their mode(s) of action and to assess their selectivity against nematodes.

Metabolic profiling and in vitro assessment of anthelmintic fractions of Picria fel-terrae Lour.

Anthelmintic resistance is widespread in gastrointestinal nematode populations, such that there is a consistent need to search for new anthelmintics. However, the cost of screening for new compounds is high and has a very low success rate. Using the knowledge of traditional healers from Borneo Rainforests (Sarawak, Malaysia), we have previously shown that some traditional medicinal plants are a rich source of potential new anthelmintic drug candidates. In this study, Picria fel-terrae Lour. plant extract, which has previously shown promising anthelmintic activities, was fractionated via the use of a solid phase extraction cartridge and each isolated fraction was then tested on free-living nematode Caenorhabditis elegans and the parasitic nematode Haemonchus contortus. We found that a single fraction was enriched for nematocidal activity, killing ≥90% of C. elegans adults and inhibiting the motility of exsheathed L3 of H. contortus, while having minimal cytotoxic activity in mammalian cell culture. Metabolic profiling and chemometric analysis of the effective fraction indicated medium chained fatty acids and phenolic acids were highly represented.

Inhibition of nasopharyngeal carcinoma cell proliferation and synergism of cisplatin with silvestrol and episilvestrol isolated from Aglaia stellatopilosa.

Nasopharyngeal carcinoma (NPC) is a type of tumour that arises from the epithelial cells that line the surface of the nasopharynx. NPC is treated with radiotherapy and cytotoxic chemotherapeutic drugs such as cisplatin and 5-fluorouracil. However, current strategies are often associated with potential toxicities. This has prompted efforts to identify alternative methods of treatment. The present study aimed to investigate silvestrol and episilvestrol-mediated inhibition of cell proliferation in human NPC cells. The growth kinetics of NPC cells treated with silvestrol or episilvestrol were monitored dynamically using a real-time, impedance-based cell analyzer, and dose-response profiles were generated using a colorimetric cell viability assay. Furthermore, apoptosis was evaluated using flow cytometry and high content analysis. In addition, flow cytometry was performed to determine cell cycle distribution. Finally, the effects of combining silvestrol or episilvestrol with cisplatin on NPC cells was examined. Apoptosis was not observed in silvestrol and episilvestrol-treated NPC cells, although cell cycle perturbation was evident. Treatment with both compounds induced a significant increase in the percentage of cells in the G2/M phase, as compared with the control. In vitro cultures combining silvestrol or episilvestrol with cisplatin showed synergistic effects against NPC cells. The results of the present study suggested that silvestrol and episilvestrol had an anti-tumour activity in NPC cells. Silvestrol and episilvestrol, particularly in combination with cisplatin, merit further investigation, so as to determine the cellular mechanisms underlying their action(s) as anti-NPC agents.

Anthelmintic activity of selected ethno-medicinal plant extracts on parasitic stages of Haemonchus contortus.

BACKGROUND: Parasitic roundworms (nematodes) cause substantial morbidity and mortality in livestock animals globally, and considerable productivity losses to farmers. The control of these nematodes has relied largely on the use of a limited number of anthelmintics. However, resistance to many of these these anthelmintics is now widespread, and, therefore, there is a need to find new drugs to ensure sustained and effective treatment and control into the future. METHODS: Recently, we developed a screening assay to test natural, plant extracts with known inhibitory effects against the free-living worm Caenorhabditis elegans. Using this assay, we assessed here the effects of the extracts on motility and development of parasitic larval stages of Haemonchus contortus, one of the most important nematodes of small ruminants worldwide. RESULTS: The study showed that two of five extracts from Picria fel-terrae Lour. have a significant inhibitory effect (at concentrations of 3-5 mg/ml) on the motility and development of H. contortus larvae. Although the two extracts originated from the same plant, they displayed different levels of inhibition on motility and development, which might relate to the presence of various active constituents in these extracts, or the same constituents at different concentrations in distinct parts of the plant. CONCLUSIONS: These results suggest that extracts from P. fel-terrae Lour. have promising anthelmintic activity and that more broadly, plant extracts are a potential rich source of anthelmintics to combat helminthic diseases.

Flemingin-Type Prenylated Chalcones from the Sarawak Rainforest Plant Desmodium congestum.

In an ongoing program to identify new anti-infective leads, an extract derived from whole plant material of Desmodium congestum collected in the Sarawak rainforest was found to have anti-MRSA activity. Bioassay-guided isolation led to the isolation of two new prenylated chalcones, 5'-O-methyl-3-hydroxyflemingin A (1) and 5'-O-methylflemingin C (2), which were closely related to the flemingins previously isolated from various Flemingia species. Chalcones 1 and 2, which were determined to be 4:6 enantiomeric mixtures by chiral HPLC, exhibited moderate activity against a panel of Gram-positive bacteria and were also cytotoxic to the HEK293 human embryonic kidney cell line.

Luteipulveratus halotolerans sp. nov., an actinobacterium (Dermacoccaceae) from forest soil.

The taxonomic position of an actinobacterium strain, C296001T, isolated from a soil sample collected in Sarawak, Malaysia, was established using a polyphasic approach. Phylogenetically, strain C296001T was closely associated with the genus Luteipulveratus and formed a distinct monophyletic clade with the only described species, Luteipulveratus mongoliensis NBRC 105296T. The 16S rRNA gene sequence similarity between strain C296001T and L. mongoliensis was 98.7 %. DNA-DNA hybridization results showed that the relatedness of strain C296001T to L. mongoliensis was only 21.5 %. The DNA G+C content of strain C296001T was 71.7 mol%. Using a PacBio RS II system, whole genome sequences for strains C296001T and NBRC 105296T were obtained. The genome sizes of 4.5 Mbp and 5.4 Mbp determined were similar to those of other members of the family Dermacoccaceae. The cell-wall peptidoglycan contained lysine, alanine, aspartic acid, glutamic acid and serine, representing the peptidoglycan type A4α l-Lys-l-Ser-d-Asp. The major menaquinones were MK-8(H4), MK-8 and MK-8(H2). Phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol and phosphoglycolipid were the polar lipids, while the whole-cell sugars were glucose, fucose and lesser amounts of ribose and galactose. The major fatty acids were iso-C16 : 0, anteiso-C17 : 0, iso-C16 : 1 H, anteiso-C17 : 1ω9c, iso-C18 : 0 and 10-methyl C17 : 0. Chemotaxonomic analyses showed that C296001T had typical characteristics of members of the genus Luteipulveratus, with the main differences occurring in phenotypic characteristics. On the basis of the phenotypic and chemotaxonomic evidence, it is proposed that strain C296001T be classified as a representative of a novel species in the genus Luteipulveratus, for which the name Luteipulveratus halotolerans sp. nov. is recommended. The type strain is C296001T ( = ATCC TSD-4T = JCM 30660T).

A nucleotide signature for identification of Aglaia stellatopilosa Pannell.

Members of the genus Aglaia have been reported to contain bioactive phytochemicals. The genus, belonging to the Meliaceae family, is represented by at least 120 known species of woody trees or shrubs in the tropical rain forest. As some of these species are very similar in their morphology, taxonomic identification can be difficult. A reliable and definitive molecular method which can identify Aglaia to the level of the species will hence be useful in comparing the content of specific bioactive compounds between the species of this genus. Here, we report the analysis of DNA sequences in the internal transcribed spacer (ITS) of the nuclear ribosomal DNA and the observation of a unique nucleotide signature in the ITS that can be used for the identification of Aglaia stellatopilosa. The nucleotide signature consists of nine bases over the length of the ITS sequence (654 bp). This uniqueness was validated in 37 samples identified as Aglaia stellatopilosa by an expert taxonomist, whereas the nucleotide signature was lacking in a selection of other Aglaia species and non-Aglaia genera. This finding suggests that molecular typing could be utilized in the identification of Aglaia stellatopilosa.

Building a discovery partnership with Sarawak Biodiversity Centre: a gateway to access natural products from the rainforests.

The Sarawak Biodiversity Centre (SBC) is a state government agency which regulates research and promotes the sustainable use of biodiversity. It has a program on documentation of traditional knowledge (TK) and is well-equipped with facilities for natural product research. SBC maintains a Natural Product Library (NPL) consisting of local plant and microbial extracts for bioprospecting. The NPL is a core discovery platform for screening of bioactive compounds by researchers through a formal agreement with clear benefit sharing obligations. SBC aims to develop partnerships with leading institutions and the industries to explore the benefits of biodiversity.

Photodynamic activity of plant extracts from Sarawak, Borneo.

Photodynamic therapy (PDT) is a medical treatment that involves the irradiation of an administered photosensitizing drug with light of a particular wavelength to activate the photosensitizer to kill abnormal cells. To date, only a small number of photosensitizers have been clinically approved for PDT, and researchers continue to look for new molecules that have more desirable properties for clinical applications. Natural products have long been important sources of pharmaceuticals, and there is a great potential for discovery of novel chemotypes from under-explored biodiversities in the world. The objective of this study is to mine the terrestrial plants in Sarawak, Borneo Island, for new photosensitizers for PDT. In a screening program from 2004 to 2008, we prepared and studied 2,400 extracts from 888 plants for their photosensitizing activities. This report details the bioprospecting process, preparation and testing of extracts, analysis of the active samples, fractionation of four samples, and isolation and characterization of photosensitizers.