Metabolomics can provide diagnostic, prognostic, and therapeutic biomarker profiles of individual patients because a large number of metabolites can be simultaneously measured in biological samples in an unbiased manner. Minor stimuli can result in substantial alterations, making it a valuable target for analysis. Due to the complexity and sensitivity of the metabolome, studies must be devised to maintain consistency, minimize subject-to-subject variation, and maximize information recovery. This effort has been aided by technological advances in experimental design, rodent models, and instrumentation. Proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy of biofluids, such as plasma, urine, and faeces provide the opportunity to identify biomarker change patterns that reflect the physiological or pathological status of an individual patient. Metabolomics has the ultimate potential to be useful in a clinical context, where it could be used to predict treatment response and survival and for early disease diagnosis. During drug treatment, an individual's metabolic status could be monitored and used to predict deleterious effects. Therefore, metabolomics has the potential to improve disease diagnosis, treatment, and follow-up care. In this chapter, we demonstrate how a metabolomics study can be used to diagnose a disease by classifying patients as either healthy or pathological, while accounting for individual variation.
The most economically important form of aquaculture is fish farming, which is an industry that accounts for an ever increasing share of world fishery production. Molecular markers can be used to enhance the productivity of the aquaculture and fish industries to meet the increasing demand. Molecular markers can be identified via a DNA test regardless of the developmental stage, age or environmental challenges experienced by the organism. The application of 16s and cytochrome b markers has enabled rapid progress in investigations of genetic variability and inbreeding, parentage assignments, species and strain identification and the construction of high resolution genetic linkage maps for aquaculture fisheries. In this review, the advantages of principles and potential power tools of 16s and cytochrome b markers are discussed. Main findings in term of trend, aspects and debates on the reviewed issue made from the model of aquatic species for the benefit of aquaculture genomics and aquaculture genetics research are discussed. The concepts in this review are illustrated with various research examples and results that relate theory to reality and provide a strong review of the current status of these biotechnology topics.
Over the last decade, metabolomics has continued to grow rapidly and is considered a dynamic technology in envisaging and elucidating complex phenotypes in systems biology area. The advantage of metabolomics compared to other omics technologies such as transcriptomics and proteomics is that these later omics only consider the intermediate steps in the central dogma pathway (mRNA and protein expression). Meanwhile, metabolomics reveals the downstream products of gene and expression of proteins. The most frequently used tools are nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). Some of the common MS-based analyses are gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). These high-throughput instruments play an extremely crucial role in discovery metabolomics to generate data needed for further analysis. In this chapter, the concept of metabolomics in the context of systems biology is discussed and provides examples of its application in human disease studies, plant responses towards stress and abiotic resistance and also microbial metabolomics for biotechnology applications. Lastly, a few case studies of metabolomics analysis are also presented, for example, investigation of an aromatic herbal plant, Persicaria minor metabolome and microbial metabolomics for metabolic engineering applications.
Gas chromatography mass spectrometry (GC-MS) and headspace gas chromatography mass spectrometry (HS/GC-MS) were used to study metabolites produced by Lactococcus lactis subsp. cremoris MG1363 grown at a temperature of 30 °C with and without agitation at 150 rpm, and at 37 °C without agitation. It was observed that L. lactis produced more organic acids under agitation. Primary alcohols, aldehydes, ketones and polyols were identified as the corresponding trimethylsilyl (TMS) derivatives, whereas amino acids and organic acids, including fatty acids, were detected through methyl chloroformate derivatization. HS analysis indicated that branched-chain methyl aldehydes, including 2-methylbutanal, 3-methylbutanal, and 2-methylpropanal are degdradation products of isoleucine, leucine or valine. Multivariate analysis (MVA) using partial least squares discriminant analysis (PLS-DA) revealed the major differences between treatments were due to changes of amino acids and fermentation products.
In this study, we report the starvation effect and vibriosis infection on a tropical fish, the tiger grouper (Epinephelus fuscoguttatus). The tiger groupers were infected with Vibrio vulnificus for 21 days. Gas chromatography-mass spectrometry combined with multivariate analysis was used to assess the variation in metabolite profiles of E. fuscoguttatus. Metabolite productions in infected fishes were significantly influenced by fatty acid production. The Omega 9 (ω-9) was abundant under the challenged conditions compared to Omega 3 (ω-3) and Omega 6 (ω-6). A total of six fatty acids from the ω-9 group were detected in high concentration in the infected fishes compared to the control groupers. These metabolites are Oleic acid, Palmitoleic acid, 6,9-Octadecenoic acid, 8,11-Eicosadienoic acid, cis-Erucic acid and 5,8,11-Eicosatrienoic acid. The production of ω-9 differed significantly (p ≤ 0.001) in the challenged samples. The detected ω-9 compounds were quantified based on three different extraction techniques with Supelco 37-component FAME mix (Supelco, USA). The highest concentration of ω-9 groups compared to the other fatty acids detected is 1320.79 mg/4 g and the lowest is 939 mg/4 g in challenged-starved; meanwhile, in challenged-fed, the highest concentration detected is 1220.87 mg/4 g and the lowest is 917.25 mg/4 g. These changes demonstrate that ω-9 can be used as a biomarker of infection in fish.
Mass mortality resulting from bacterial infection poses a major problem in the grouper aquaculture industry. The purpose of this study was to profile the metabolites released in challenged fish and to reconstruct the metabolic pathways of brown marble grouper (Epinephelus fuscoguttatus) in response to Vibrio vulnificus infection. Metabolite profiles from control and challenged treatment groups after feeding were determined using gas chromatography-mass spectrometry (GC-MS). Forty metabolites were identified from the GC-MS analysis. These metabolites comprised of amino acids, fatty acids, organic acids and carbohydrates. The profiles showed the highest percent area (33.1%) for leucine from the amino acid class in infected fish compared to the control treatment group (12.3%). Regarding the fatty acid class, a higher percent area of the metabolite 8,11-eicosadienoic acid (27.04%) was observed in fish infected with V. vulnificus than in the control treatment group (22.5%). Meanwhile, in the carbohydrate class, glucose (47.0%) was the metabolite in the carbohydrate class present at highest percentage in the control treatment group compared to infected fish (30.0%). Our findings highlight the importance of a metabolic analysis for understanding the changes of metabolites in E. fuscoguttatus in response to bacterial infections.
With the advent of synthetic biology, scientists are increasingly relying on digital sequence information, instead of physical genetic resources. This article examines the potential impact of this shift on the access and benefit-sharing (ABS) regime of the Convention on Biological Diversity (CBD) and the Nagoya Protocol. These treaties require benefit-sharing with the owners of genetic resources. However, whether "genetic resources" include digital sequence information is unsettled. The CBD conceives genetic resources as genetic material containing functional units of heredity. "Material" implies tangibility, and for some scholars, "functional units of heredity," undefined in both treaties, mean full-coding sequences. This article argues that digital sequence information obtained from physical genetic resources, full-coding or not, should be treated as genetic resources. Literal construction of the CBD risks eroding its usefulness and the ABS regime. This is because through bioinformatics, sequence information can easily be obtained from genetic resources for utilization, without physically moving them or concluding ABS agreement with owners. The CBD must evolve with scientific progress also because sequence functionality depends on the state of knowledge. These arguments are vindicated by domestic ABS laws equating genetic information with genetic resources; Nagoya Protocol provisions deeming research exploiting the genetic composition of genetic resources as utilization of genetic resources; and CBD provisions requiring the sharing of benefits from the utilization of genetic resources. Moreover, treaty interpretation and case law demand that generic, scientific terms, such as "genetic resources" and "functional units of heredity" be interpreted in an evolutionary manner to capture scientific developments.
Epinephelus fuscoguttatus is a commercially important marine fish species in southeast Asia. Due to overfishing and water pollution, this species has been declared as near-threatened. Thus, to provide information to help maintain and preserve the species, microsatellites were developed, using an enriched genomic library method. Thirty individuals were collected from the hatchery of the Fishery Research Institute, Terengganu, Malaysia. These individuals, from four to six years old, originated from Sabah and are maintained in captive culture as broodstock. Genomic DNA was extracted from the fins of selected individuals that weighed 3-8 kg. Ten microsatellite loci were found to be polymorphic in this population, with 5 to 21 alleles per locus. Observed and expected heterozygosities ranged from 0.53 to 0.97 and 0.59 to 0.95, respectively. Only one locus deviated significantly from Hardy-Weinberg equilibrium and no significant linkage disequilibrium was found among the pairs of loci. These polymorphic microsatellite loci will be used by the Malaysian Fishery Research Institute for investigating genetic diversity and for developing breeding strategies.
In this study, an organic solvent tolerant bacterial strain was isolated. This strain was identified as Pseudomonas sp. strain S5, and was shown to degrade BTEX (Benzene, Toluene, Ethyl-Benzene, and Xylene). Strain S5 generates an organic solvent-tolerant lipase in the late logarithmic phase of growth. Maximum lipase production was exhibited when peptone was utilized as the sole nitrogen source. Addition of any of the selected carbon sources to the medium resulted in a significant reduction of enzyme production. Lower lipase generation was noted when an inorganic nitrogen source was used as the sole nitrogen source. This bacterium hydrolyzed all tested triglycerides and the highest levels of production were observed when olive oil was used as a natural triglyceride. Basal medium containing Tween 60 enhanced lipase production to the most significant degree. The absence of magnesium ions (Mg2+) in the basal medium was also shown to stimulate lipase production. Meanwhile, an alkaline earth metal ion, Na+, was found to stimulate the production of S5 lipase.
An organic solvent-tolerant S5 lipase was purified by affinity chromatography and anion exchange chromatography. The molecular mass of the lipase was estimated to be 60 kDa with 387 purification fold. The optimal temperature and pH were 45 degrees C and 9.0, respectively. The purified lipase was stable at 45 degrees C and pH 6-9. It exhibited the highest stability in the presence of various organic solvents such as n-dodecane, 1-pentanol, and toluene. Ca2+ and Mg2+ stimulated lipase activity, whereas EDTA had no effect on its activity. The S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride. It showed random positional specificity on the thin-layer chromatography.
Temperature is one of the key factors in limiting the distribution of plants and controlling major metabolic processes. A series of simulated reciprocal transplant experiments were performed to investigate the effect of temperature on plant chemical composition. Polygonum minus of different lowland and highland origin were grown under a controlled environment with different temperature regimes to study the effects on secondary metabolites. We applied gas chromatography-mass spectrometry and liquid chromatography time-of-flight mass spectrometry to identify the chemical compounds. A total of 37 volatile organic compounds and 85 flavonoids were detected, with the largest response observed in the compositional changes of aldehydes and terpenes in highland plants under higher temperature treatment. Significantly less anthocyanidin compounds and larger amounts of flavonols were detected under higher temperature treatment. We also studied natural variation in the different plant populations growing under the same environment and identified compounds unique to each population through metabolite fingerprinting. This study shows that the origin of different plant populations influences the effects of temperature on chemical composition.
Increasing demands for the supply of biopharmaceuticals have propelled the advancement of metabolic engineering and synthetic biology strategies for biomanufacturing of bioactive natural products. Using metabolically engineered microbes as the bioproduction hosts, a variety of natural products including terpenes, flavonoids, alkaloids, and cannabinoids have been synthesized through the construction and expression of known and newly found biosynthetic genes primarily from model and non-model plants. The employment of omics technology and machine learning (ML) platforms as high throughput analytical tools has been increasingly leveraged in promoting data-guided optimization of targeted biosynthetic pathways and enhancement of the microbial production capacity, thereby representing a critical debottlenecking approach in improving and streamlining natural products biomanufacturing. To this end, this mini review summarizes recent efforts that utilize omics platforms and ML tools in strain optimization and prototyping and discusses the beneficial uses of omics-enabled discovery of plant biosynthetic genes in the production of complex plant-based natural products by bioengineered microbes.
Numerous potyvirus studies, including virus biology, transmission, viral protein function, as well as virus-host interaction, have greatly benefited from the utilization of reverse genetic techniques. Reverse genetics of RNA viruses refers to the manipulation of viral genomes, transfection of the modified cDNAs into cells, and the production of live infectious progenies, either wild-type or mutated. Reverse genetic technology provides an opportunity of developing potyviruses into vectors for improving agronomic traits in plants, as a reporter system for tracking virus infection in hosts or a production system for target proteins. Therefore, this review provides an overview on the breakthroughs achieved in potyvirus research through the implementation of reverse genetic systems.
Rice tungro disease was discovered in Malaysia in the 1930s. The first and only genome of Rice tungro bacilliform virus (RTBV) isolated from rice in Malaysia was sequenced in 1999. After nearly two decades, here, we present the complete genome sequence of an RTBV isolate in rice from Seberang Perai, Malaysia.
Expansin is a cell wall loosening protein without hydrolytic activity, which allows cell expansion by influencing cell wall extensibility. Previous studies showed that the suppression of expansin genes (EXPA1, EXPA3, EXPA5 and EXPA10) resulted in defective organ growth and altered cell wall chemical composition [1,2]. However, the molecular mechanism on how the suppression of non-enzymatic expansin expression can result in widespread effects on plant cell wall and organ growth is still unclear. In this study, we performed transcriptomic analysis on the hypocotyls of previously reported transgenic Arabidopsis line [1] to investigate the effects of expansin gene suppression on the global gene expression pattern, particularly on the cell wall related genes.
Callus was induced from mangosteen (Garcinia mangostana L.) young purple-red leaves on Murashige and Skoog basal medium with various combinations of plant growth regulators. Murashige and Skoog medium with 4.44 µM 6-benzylaminopurine and 4.52 µM 2,4-dichlorophenoxyacetic acid was the best for friable callus induction. This friable callus was used for the initiation of cell suspension culture. The effects of different combinations of 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid, carbon sources and inoculum sizes were tested. It was found that combination of 2.22 µM 6-benzylaminopurine + 2.26 µM 2,4-dichlorophenoxyacetic acid, glucose (30 g/l) and 1.5 g/50 ml inoculum size was the best for cell growth. Callus and cell suspension cultures were then treated either with 100 µM methyl jasmonate as an elicitor for 5 days, or 0.5 g/l casein hydrolysate as an organic supplement for 7 days. Metabolites were then extracted and profiled using liquid chromatography-time of flight mass spectrometry. Multivariate discriminant analyses revealed significant metabolite differences (P ≤ 0.05) for callus and suspension cells treated either with methyl jasmonate or casein hydrolysate. Based on MS/MS data, methyl jasmonate stimulated the production of an alkaloid (thalsimine) and fatty acid (phosphatidyl ethanolamine) in suspension cells while in callus, an alkaloid (thiacremonone) and glucosinolate (7-methylthioheptanaldoxime) was produced. Meanwhile casein hydrolysate stimulated the production of alkaloids such as 3ß,6ß-dihydroxynortropane and cis-hinokiresinol and triterpenoids such as schidigerasaponin and talinumoside in suspension cells. This study provides evidence on the potential of secondary metabolite production from in vitro culture of mangosteen.
Discovery of species-specific interaction between the host and virus has drawn the interest of many researchers to study the evolution of the newly emerged virus. Comparative genome analysis provides insights of the virus functional genome evolution and the underlying mechanisms of virus-host interactions. The analysis of nucleotide composition signified the evolution of nodavirus towards host specialization in a host-specific mutation manner. GC-rich genome of betanodavirus was significantly deficient in UpA and UpU dinucleotides composition, whilst the AU-rich genome of gammanodavirus was deficient in CpG dinucleotide. The capsid of MrNV and PvNV of gammanodavirus retains the highest abundance of adenine and uracil at the second codon position, respectively, which were found to be very distinctive from the other genera. ENC-GC3 plot inferred the influence of natural selection and mutational pressure in shaping the evolution of MrNV RdRp and capsid, respectively. Furthermore, CAI/eCAI analysis predicts a comparable adaptability of MrNV in squid, Sepia officinalis than its natural host, Macrobrachium rosenbergii. Thus, further study is warranted to investigate the capacity of MrNV replication in S. officinalis owing to its high codon adaptation index.
Hybridisation plays a significant role in the evolution and diversification of plants. Hybridisation among Nepenthes species is extensive, either naturally or man-made. To investigate the effects of hybridisation on the chemical compositions, we carried out metabolomics study on pitcher tissue of Nepenthes ampullaria, Nepenthes rafflesiana and their hybrid, Nepenthes × hookeriana. Pitcher samples were harvested and extracted in methanol:chloroform:water via sonication-assisted extraction before analysed using LC-TOF-MS. MS data were analysed using XCMS online version 2.2.5. This is the first MS data report towards the profiling, identification and comprehensive comparison of metabolites present in Nepenthes species.
Expansin increases cell wall extensibility to allow cell wall loosening and cell expansion even in the absence of hydrolytic activity. Previous studies showed that excessive overexpression of expansin gene resulted in defective growth (Goh et al., 2014; Rochange et al., 2001) [1,2] and altered cell wall chemical composition (Zenoni et al., 2011) [3]. However, the molecular mechanism on how the overexpression of non-enzymatic cell wall protein expansin can result in widespread effects on plant cell wall and organ growth remains unclear. We acquired transcriptomic data on previously reported transgenic Arabidopsis line (Goh et al., 2014) [1] to investigate the effects of overexpressing a heterologus cucumber expansin gene (CsEXPA1) on the global gene expression pattern during early and late phases of etiolated hypocotyl growth.