Stimulating bioelectric generation and recovery of toxic metals through benthic microbial fuel cell driven by local sago (Cycas revoluta) waste.

Benthic microbial fuel cell (BMFC) is the most promising type of bioelectrochemical approach for producing electrons and protons from natural organic waste. In the present work, a single-chamber BMFC was used, containing sago (Cycas revoluta) waste as the organic feed for microorganisms. The local wastewater was supplemented with heavy metal ions (Pb2+, Cd2+, Cr3+, Ni2+, Co2+, Ag+, and Cu2+) and used as an inoculation source to evaluate the performance of BMFC against the toxic metal remediations. According to the experimental results, the maximum power density obtained was 42.55 mW/m2 within 25 days of the BMFC operation. The maximum remediation efficiency of the metal ion removal from the wastewater was found to be 99.30% (Ag+). The conductive pili-type bacteria species (Acinetobacter species, Leucobacter species, Bacillus species, Proteus species. and Klebsiella pneumoniae) were found in the present study during isolation and identification processes. This study's multiple parameter optimization revealed that pH 7 and room temperature is the best condition for optimal performance. Finally, this study included the mechanism, future recommendations, and concluding remarks.

Effects of heat shock on photosynthesis-related characteristics and lipid profile of Cycas multipinnata and C. panzhihuaensis.

BACKGROUND: Cycas multipinnata and C. panzhihuaensis are two attractive ornamental tree species. With the global climate change, the temperature in the natural habitats of both the species shows a marked rising trend. However, how the two species respond to extreme high temperatures are not clear. Chlorophyll fluorescence parameters, chlorophyll content, chloroplast ultrastructure and lipid metabolism in the two species were determined following plant exposure to heat stress. RESULTS: The results demonstrated that the photosynthetic efficiency decreased significantly in both the species following heat shock and recovery, but to a greater extent in C. panzhihuaensis. Compared to the control, chlorophyll content of C. multipinnata did not change significantly following heat stress and recovery. However, chlorophyll content of C. panzhihuaensis increased significantly after 1 d of recovery in comparison with the control. Chloroplast ultrastructures of C. panzhihuaensis were more severely affected by heat shock than C. multipinnata. C. multipinnata and C. panzhihuaensis followed a similar change trend in the amounts of most of the lipid categories after heat stress. However, only the amounts of lysophospholipids and fatty acyls differed significantly between the two species following heat treatment. Additionally, the unsaturation levels of the major lipid classes in C. multipinnata were significantly lower than or equal to those in C. panzhihuaensis. CONCLUSIONS: C. multipinnata was less affected by extremely high temperatures than C. panzhihuaensis. The differential stability of chlorophyll and chloroplast ultrastructure and the differential adjustment of lipid metabolism might contribute to the different responses to heat shock between the two species.

A chemogenomic approach is required for effective treatment of amyotrophic lateral sclerosis.

ALS is a fatal untreatable disease involving degeneration of motor neurons. Μultiple causative genes encoding proteins with versatile functions have been identified indicating that diverse biological pathways lead to ALS. Chemical entities still represent a promising choice to delay ALS progression, attenuate symptoms and/or increase life expectancy, but also gene-based and stem cell-based therapies are in the process of development, and some are tested in clinical trials. Various compounds proved effective in transgenic models overexpressing distinct ALS causative genes unfortunately though, they showed no efficacy in clinical trials. Notably, while animal models provide a uniform genetic background for preclinical testing, ALS patients are not stratified, and the distinct genetic forms of ALS are treated as one group, which could explain the observed discrepancies between treating genetically homogeneous mice and quite heterogeneous patient cohorts. We suggest that chemical entity-genotype correlation should be exploited to guide patient stratification for pharmacotherapy, that is administered drugs should be selected based on the ALS genetic background.

Differences in lipid homeostasis and membrane lipid unsaturation confer differential tolerance to low temperatures in two Cycas species.

BACKGROUND: C. panzhihuaensis is more tolerant to freezing than C. bifida but the mechanisms underlying the different freezing tolerance are unclear. Photosynthesis is one of the most temperature-sensitive processes. Lipids play important roles in membrane structure, signal transduction and energy storage, which are closely related to the stress responses of plants. In this study, the chlorophyll fluorescence parameters and lipid profiles of the two species were characterized to explore the changes in photosynthetic activity and lipid metabolism following low-temperature exposure and subsequent recovery. RESULTS: Photosynthetic activity significantly decreased in C. bifida with the decrease of temperatures and reached zero after recovery. Photosynthetic activity, however, was little affected in C. panzhihuaensis. The lipid composition of C. bifida was more affected by cold and freezing treatments than C. panzhihuaensis. Compared with the control, the proportions of all the lipid categories recovered to the original level in C. panzhihuaensis, but the proportions of most lipid categories changed significantly in C. bifida after 3 d of recovery. In particular, the glycerophospholipids and prenol lipids degraded severely during the recovery period of C. bifida. Changes in acyl chain length and double bond index (DBI) occurred in more lipid classes immediately after low-temperature exposure in C. panzhihuaensis compare with those in C. bifida. DBI of the total main membrane lipids of C. panzhihuaensis was significantly higher than that of C. bifida following all temperature treatments. CONCLUSIONS: The results of chlorophyll fluorescence parameters confirmed that the freezing tolerance of C. panzhihuaensis was greater than that of C. bifida. The lipid metabolism of the two species had differential responses to low temperatures. The homeostasis and plastic adjustment of lipid metabolism and the higher level of DBI of the main membrane lipids may contribute to the greater tolerance of C. panzhihuaensis to low temperatures.

Arabinogalactan-proteins from non-coniferous gymnosperms have unusual structural features.

Arabinogalactan-proteins (AGPs), important signalling molecules of the plant cell wall, are structurally extensively investigated in angiosperms, but information on AGPs in gymnosperms is still limited. We characterized AGPs from the gymnosperms Ginkgo biloba, Ephedra distachya, Encephalartos longifolius and Cycas revoluta. The protein contents are comparable to that of angiosperm AGPs. Hydroxyproline is the site of linking the carbohydrate part and was detected in all AGPs with highest concentration in Cycas AGP (1.1 % of the AGP). Interestingly, with the exception of Cycas, all AGPs contained the monosaccharide 3-O-methylrhamnose not present in angiosperm polysaccharides. The carbohydrate moieties of Cycas and Ephredra showed the main components 1,3,6-linked galactose and terminal arabinose typical of angiosperm AGPs, whereas that of Ginkgo AGP was unique with 1,4-linked galactose as dominant structural element. Bioinformatic search for glycosyltransferases in Ginkgo genome also revealed a lower number of galactosyltransferases responsible for biosynthesis of the 1,3-Gal/1,6-Gal AGP backbone.

Physicochemical and structural properties of sago starch.

A comprehensive study was conducted to elucidate physicochemical and structural properties of sago starches. Two sago starch granules were oval in shape with an average diameter of 34.41 µm and had C-type polymorph with a crystallinity of about 28.13%. The amylose and resistant starch (RS) contents of two sago starches were higher than those of corn and potato starches. The two sago starches had a large amount of A and B1 chains (DP 6-24) which could form double helix structures. FTIR exhibited that the structure of two sago starches had a lower degree of order. The peak viscosity and breakdown of sago starch 2 were lower than corn starch, and the setback was higher than potato starch. Additionally, sago starches had lower gelatinization enthalpy and higher regeneration tendency. According to rheological results, sago starches showed lower shear thinning degree and thixotropy compared to corn and potato starches. Sago starch 1 gels represented the highest hardness, adhesiveness, springiness and cohesiveness, which could be used as potential food stabilizer. This study revealed the characteristics of two sago starches compared with other starches. The results indicated that the amylose content and amylopectin structures had significant influence on the physicochemical properties of sago starch.

Identification and characterization of caleosin in Cycas revoluta pollen.

Oil bodies are essential energy storage organelles that are generally present in the seeds of plants. Caleosin protein has been identified in the seed oil bodies of Cycas revolutaseed. In this study, mature Cycas revoluta pollen grains were collected from cycad elliptical cones. However, the isolation and identification of oil bodies protein from mature Cycas revoluta grains have never been experimentally determined. Ultrastructural studies have shown that the oil bodies were present in pollen Cycas revoluta grains. Lipid analysis showed that oil bodies are predominantly composed of triacylglycerol. Complete cDNA fragments encoding the caleosin were obtained by PCR cloning. Phylogenetic tree analyzes showed that cycad pollen caleosin is closely related to the caleosin of Cycas revoluta seeds. Fresh Cycas revoluta pollen grains were allowed to germinate for 48 h in a germination medium containing 2% sucrose and 0.01% H3BO3. Pollen germination were recorded periodically from day one to day 10 and the results showed that the Cycas revoluta pollen elongate the tube and increasing of triacylglycerol(TAG) after 4 days.

Stem CO2 efflux of Cycas micronesica is reduced by chronic non-native insect herbivory.

Carbon dioxide efflux (Es) from the base of Cycas micronesica K.D. Hill stems was quantified in four locations containing healthy populations and in one location with populations threatened by non-native insect herbivores in order to determine the influence of reduced plant health on Es. Minimal variation of Es occurred among the four locations with healthy plants, and Es ranged from 1.68 to 1.79 µmol·m-2·s-1. The threatened in situ populations were on the island of Guam, where recent invasions of non-native insects have caused epidemic plant mortality, and the Es was 0.59 µmol·m-2·s-1. This is the first known report of Es for any cycad species, and the values for the unique pachycaulous stem form fit in the lower half of the range of published Es for woody trees. The results illuminate the potential for using Es to screen in situ C. micronesica populations to identify the individual trees with the greatest likelihood of surviving conservation measures.

Metabolite profiling and in-vitro colon cancer protective activity of Cycas revoluta cone extract.

The methanolic extract of Cycas revoluta cone (MECR) was analyzed by GC-MS and UHPLC for metabolite profiling and was evaluated for anti-colon cancer property by using in vitro assays like Cell Viability Assay, Colony Formation Assay, ROS Determination, Flowcytometry, DAPI staining assay, Tunel assay. GC-MS and HPLC analysis confirmed the presence of different phytochemicals in the extract of Cycas revoluta cone. In-vitro studies showed MECR extract showed significant anti-colon cancer activity by reducing proliferation and inducing apoptosis in colon cancer cell (HCT-8) line, but no such activity was seen in normal colon cell (CCD-18Co) line. The investigation confirms that MECR may be a promising candidate in colon cancer protection.

Metabolite profiling of Cycas revoluta leaf extract and docking studies on alpha-glucosidase inhibitory molecular targets by phytochemicals.

The leaves of Cycas revoluta were explored for their antioxidant, α-glucosidase and α-amylase inhibitory properties to develop safe and diet based therapeutic treatment of diabetes. In optimized fractionation, 60% ethanol provided the highest extract yield of 19.35±0.05a%, TPC 95.70±1.60a mg GAE/g and TFC 55 .60 ± 1.20a mg Rutin/g extract. The antioxidant and anti α-glucosidase activities of 60% ethanolic extracts were also promising and statistically significant as compared with remaining plant extracts. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) analysis of the leaf extract revealed the presence of three apigenin derivatives, kaempferol derivative, hexadecenoic acid and citric acid. The binding energy values of molecular docking studies supported the synergistic behavior of leaf extract to inhibit α-glucosidase activity. The leaves of Cycas revoluta were proved to be apigenin rich natural pool of metabolites of antidiabetic importance to improvise food functionalities.

Metabolic solutions to the biosynthesis of some diaminomonocarboxylic acids in nature: Formation in cyanobacteria of the neurotoxins 3-N-methyl-2,3-diaminopropanoic acid (BMAA) and 2,4-diaminobutanoic acid (2,4-DAB).

The non-encoded diaminomonocarboxylic acids, 3-N-methyl-2,3-diaminopropanoic acid (syn: α-amino-ß-methylaminopropionic acid, MeDAP; ß-N-methylaminoalanine, BMAA) and 2,4-diaminobutanoic acid (2,4-DAB), are distributed widely in cyanobacterial species in free and bound forms. Both amino acids are neurotoxic in whole animal and cell-based bioassays. The biosynthetic pathway to 2,4-DAB is well documented in bacteria and in one higher plant species, but has not been confirmed in cyanobacteria. The biosynthetic pathway to BMAA is unknown. This review considers possible metabolic routes, by analogy with reactions used in other species, by which these amino acids might be biosynthesised by cyanobacteria, which are a widespread potential environmental source of these neurotoxins. Where possible, the gene expression that might be implicated in these biosyntheses is discussed.

Selection and Validation of Reference Genes for qRT-PCR in Cycas elongata.

Quantitative reverse transcription PCR (qRT-PCR) is a sensitive technique used in gene expression studies. To achieve a reliable quantification of transcripts, appropriate reference genes are required for comparison of transcripts in different samples. However, few reference genes are available for non-model taxa, and to date, reliable reference genes in Cycas elongata have not been well characterized. In this study, 13 reference genes (ACT7, TUB, UBQ, EIF4, EF1, CLATHRIN1, PP2A, RPB2, GAPC2, TIP41, MAPK, SAMDC and CYP) were chosen from the transcriptome database of C. elongata, and these genes were evaluated in 8 different organ samples. Three software programs, NormFinder, GeNorm and BestKeeper, were used to validate the stability of the potential reference genes. Results obtained from these three programs suggested that CeGAPC2 and CeRPB2 are the most stable reference genes, while CeACT7 is the least stable one among the 13 tested genes. Further confirmation of the identified reference genes was established by the relative expression of AGAMOUSE gene of C. elongata (CeAG). While our stable reference genes generated consistent expression patterns in eight tissues, we note that our results indicate that an inappropriate reference gene might cause erroneous results. Our systematic analysis for stable reference genes of C. elongata facilitates further gene expression studies and functional analyses of this species.

Selective quantitation of the neurotoxin BMAA by use of hydrophilic-interaction liquid chromatography-differential mobility spectrometry-tandem mass spectrometry (HILIC-DMS-MS/MS).

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been reported in cyanobacteria and shellfish, raising concerns about widespread human exposure. However, inconsistent results for BMAA analysis have led to controversy. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the most appropriate method for analysis of BMAA, but the risk of interference from isomers, other sample components, and the electrospray background is still present. We have investigated differential mobility spectrometry (DMS) as an ion filter to improve selectivity in the hydrophilic interaction liquid chromatographic (HILIC)-MS/MS determination of BMAA. We obtained standards for two BMAA isomers not previously analyzed by HILIC-MS, ß-amino-N-methylalanine and 3,4-diaminobutanoic acid, and the typically used 2,4-diaminobutanoic acid and N-(2-aminoethyl)glycine. DMS separation of BMAA from these isomers was achieved and optimized conditions were used to develop a sensitive and highly selective multidimensional HILIC-DMS-MS/MS method. This work revealed current technical limitations of DMS for trace quantitation, and practical solutions were implemented. Accurate control of low levels of DMS carrier gas modifier was essential, but required external metering. The linearity of our optimized method was excellent from 0.01 to 6 µmol L(-1). The instrumental LOD was 0.4 pg BMAA injected on-column and the estimated method LOD was 20 ng g(-1) dry weight for BMAA in sample matrix. The method was used to analyze cycad plant tissue, a cyanobacterial reference material, and mussel tissues, by use of isotope-dilution quantitation with deuterated BMAA. This confirmed the presence of BMAA and several of its isomers in cycad and mussel tissues, including commercially available mussel tissue reference materials certified for other biotoxins. Graphical Abstract Differential Mobility Spectrometry is used to increases the selectivity of BMAA analysis by HILIC-MS/MS.

Cycas micronesica (Cycadales) plants devoid of endophytic cyanobacteria increase in beta-methylamino-L-alanine.

Cycads are among the most ancient of extant Spermatophytes, and are known for their pharmacologically active compounds. beta-methylamino-l-alanine (BMAA) is one metabolite that been implicated as causal of human neurodegenerative diseases in Guam. We grew Cycas micronesica seedlings without endophytic cyanobacteria symbiosis, and quantified initial and ending BMAA in various plant tissues. BMAA increased 79% during nine months of seedling growth, and root tissue contained 75% of the ultimate BMAA pool. Endophytic cyanobacteria symbionts were not the source of BMAA increase in these seedlings, which contradicts previously reported claims that biosynthesis of this toxin by cyanobacteria initiates its accumulation in the Guam environment. The preferential loading of root tissue with BMAA does not support earlier reports that this toxin serves a defensive role against herbivory of leaf or seed tissues. The long history of conflicting results in Guam's cycad toxin research continues, and recent developments underscore the sense of urgency in continued research as this endangered cycad population approaches extirpation from the island.

A plant class V chitinase from a cycad (Cycas revoluta): biochemical characterization, cDNA isolation, and posttranslational modification.

Chitinase-A (CrChi-A) was purified from leaf rachises of Cycas revoluta by several steps of column chromatography. It was found to be a glycoprotein with a molecular mass of 40 kDa and an isoelectric point of 5.6. CrChi-A produced mainly (GlcNAc)(3) from the substrate (GlcNAc)(6) through a retaining mechanism. More interestingly, CrChi-A exhibited transglycosylation activity, which has not been observed in plant chitinases investigated so far. A cDNA encoding CrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction procedures. It consisted of 1399 nucleotides and encoded an open reading frame of 387-amino-acid residues. Sequence analysis indicated that CrChi-A belongs to the group of plant class V chitinases. From peptide mapping and mass spectrometry of the native and recombinant enzyme, we found that an N-terminal signal peptide and a C-terminal extension were removed from the precursor (M1-A387) to produce a mature N-glycosylated protein (Q24-G370). This is the first report on a plant chitinase with transglycosylation activity and posttranslational modification of a plant class V chitinase.

Serine proteinase inhibitors in seeds of Cycas siamensis and other gymnosperms.

Seeds of 32 species selected from two of the four major groups of gymnosperms, the ancient Cycadales and the economically important Coniferales, were analysed for inhibitors (I) of the serine proteinases trypsin (T), chymotrypsin (C), subtilisin (S) and elastase (E) using isoelectric focusing (IEF) combined with gelatin replicas. Subtilisin inhibitors were detected in 17 species, being particularly active in the Cycadales. Several species of the genera Cephalotaxus, Pseudotsuga and Cycas contained inhibitors active against elastase while strong CSTIs and CSIs were also present in Cycas pectinata and C. siamensis. No inhibitors were detected in seeds of Chamaecyparis, Thuja, Abies, Larix, Picea and Pinus spp. Serine proteinase inhibitors were purified from seeds of C. siamensis by affinity chromatography using trypsin and chymotrypsin, IEF and SDS-PAGE. Several CSTI components with M(r) ranging from 4000 to 18,000 were partially sequenced using Edman degradation and mass spectrometry. Most of the sequences were similar to a hypothetical protein encoded by an mRNA from sporophylls of C. rumphii which in turn was similar to Kunitz-type proteinase inhibitors from flowering plants. Analysis of expressed sequence tag (EST) databases confirmed the presence of mRNAs encoding Kunitz-type inhibitors in the Cycadales and Coniferales and also demonstrated their presence in a third major group of gymnosperms, the Ginkgoales. This is the first report of Kunitz-type serine proteinase inhibitors from plants other than Angiosperms.

Cloning and expression of a mannose-binding jacalin-related lectin from leaves of Japanese cycad (Cycas revoluta Thunb.).

Cycad leaf lectin (CRLL), a mannose-recognizing jacalin-related lectin (mJRL), was first cloned as a gymnosperm lectin and expressed. The cDNA sequence of CRLL (DDBJ, accession no. AB198328), coding 291 amino acid residues, has a tandem repeat of about 150 amino acids divided into N- and C-terminal domains as Japanese chestnut mJRL. Sequence alignment showed deletion and insertion of the sequence, and its putative carbohydrate-binding sites showed some differences from other JRLs. PCR analysis showed that this lectin was expressed in the cycad leaf but not in the root or seed. Recombinant CRLL (rCRLL) was expressed in Escherichia coli and purified by affinity chromatography after refolding procedures. Properties of active rCRLL appeared to be almost the same as those of native CRLL.

Conservation of class C function of floral organ development during 300 million years of evolution from gymnosperms to angiosperms.

Flower development in angiosperms is regulated by the family of MADS-box transcription factors. MADS-box genes have also been reported from gymnosperms, another major group of seed plants. AGAMOUS (AG) is the class C MADS-box floral organ identity gene controlling the stamen and carpel development in Arabidopsis. We report the characterization of an ortholog of the AG gene, named Cycas AGAMOUS (CyAG), from the primitive gymnosperm Cycas edentata. The expression pattern of CyAG in Cycas parallels that of AG in Arabidopsis. Additionally, the gene structure, including the number and location of the introns, is conserved in CyAG and other AG orthologs known. Most importantly, functional analysis shows that CyAG driven by the AG promoter can rescue the loss-of-function ag mutant of Arabidopsis. However, the ectopic expression of CyAG in ag mutant Arabidopsis cannot produce the carpeloid and stamenoid organs in the first and second whorls, although the stamen and carpel are rescued in the third and fourth whorls of the transformants. These observations show that the molecular mechanism of class C function controlling reproductive organ identity (stamen and carpel of angiosperms or microsporophyll and megasporophyll of gymnosperms) arose before the divergence of angiosperms and gymnosperms, and has been conserved during 300 million years of evolution thereafter.

Expressed sequence tag analysis in Cycas, the most primitive living seed plant.

BACKGROUND: Cycads are ancient seed plants (living fossils) with origins in the Paleozoic. Cycads are sometimes considered a 'missing link' as they exhibit characteristics intermediate between vascular non-seed plants and the more derived seed plants. Cycads have also been implicated as the source of 'Guam's dementia', possibly due to the production of S(+)-beta-methyl-alpha, beta-diaminopropionic acid (BMAA), which is an agonist of animal glutamate receptors. RESULTS: A total of 4,200 expressed sequence tags (ESTs) were created from Cycas rumphii and clustered into 2,458 contigs, of which 1,764 had low-stringency BLAST similarity to other plant genes. Among those cycad contigs with similarity to plant genes, 1,718 cycad 'hits' are to angiosperms, 1,310 match genes in gymnosperms and 734 match lower (non-seed) plants. Forty-six contigs were found that matched only genes in lower plants and gymnosperms. Upon obtaining the complete sequence from the clones of 37/46 contigs, 14 still matched only gymnosperms. Among those cycad contigs common to higher plants, ESTs were discovered that correspond to those involved in development and signaling in present-day flowering plants. We purified a cycad EST for a glutamate receptor (GLR)-like gene, as well as ESTs potentially involved in the synthesis of the GLR agonist BMAA. CONCLUSIONS: Analysis of cycad ESTs has uncovered conserved and potentially novel genes. Furthermore, the presence of a glutamate receptor agonist, as well as a glutamate receptor-like gene in cycads, supports the hypothesis that such neuroactive plant products are not merely herbivore deterrents but may also serve a role in plant signaling.

Biomagnification of cyanobacterial neurotoxins and neurodegenerative disease among the Chamorro people of Guam.

We here report biomagnification (the increasing accumulation of bioactive, often deleterious molecules through higher trophic levels of a food chain) of the neurotoxic nonprotein amino acid beta-methylamino-l-alanine (BMAA) in the Guam ecosystem. Free-living cyanobacteria produce 0.3 microg/g BMAA, but produce 2-37 microg/g as symbionts in the coralloid roots of cycad trees. BMAA is concentrated in the developing reproductive tissues of the cycad Cycas micronesica, averaging 9 microg/g in the fleshy seed sarcotesta and a mean of 1,161 microg/g BMAA in the outermost seed layer. Flying foxes (Pteropus mariannus), which forage on the seeds, accumulate a mean of 3,556 microg/g BMAA. Flying foxes are a prized food item of the indigenous Chamorro people who boil them in coconut cream and eat them whole. Chamorros who die of amyotrophic lateral sclerosis/parkinsonism-dementia complex (AL-SPDC), a neurodegenerative disease with symptoms similar to amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease, have an average of 6.6 microg/g BMAA in their brain tissues. The biomagnification of BMAA through the Guam ecosystem fits a classic triangle of increasing concentrations of toxic compounds up the food chain. This may explain why the incidence of ALS-PDC among the Chamorro was 50-100 times the incidence of amyotrophic lateral sclerosis elsewhere. Biomagnification of cyanobacterial BMAA may not be unique to Guam; our discovery of BMAA in the brain tissue from Alzheimer's patients from Canada suggests alternative ecological pathways for the bioaccumulation of BMAA in aquatic or terrestrial ecosystems.