Classification of marine microalgae using low-resolution Mueller matrix images and convolutional neural network.

In this paper, we used a convolutional neural network to study the classification of marine microalgae by using low-resolution Mueller matrix images. Mueller matrix images of 12 species of algae from 5 families were measured by a Mueller matrix microscopy with an LED light source at 514 nm wavelength. The data sets of seven resolution levels were generated by the bicubic interpolation algorithm. We conducted two groups of classification experiments; one group classified the algae into 12 classes according to species category, and the other group classified the algae into 5 classes according to family category. In each group of classification experiments, we compared the classification results of the Mueller matrix images with those of the first element (M11) images. The classification accuracy of Mueller matrix images declines gently with the decrease of image resolution, while the accuracy of M11 images declines sharply. The classification accuracy of Mueller matrix images is higher than that of M11 images at each resolution level. At the lowest resolution level, the accuracy of 12-class classification and 5-class classification of full Mueller matrix images is 29.89% and 35.83% higher than those of M11 images, respectively. In addition, we also found that the polarization information of different species had different contributions to the classification. These results show that the polarization information can greatly improve the classification accuracy of low-resolution microalgal images.

Polarimetric learning: a Siamese approach to learning distance metrics of algal Mueller matrix images.

Polarimetric measurements are becoming increasingly accurate and fast to perform in modern applications. However, analysis on the polarimetric data usually suffers from its high-dimensional nature spatially, temporally, or spectrally. This paper associates polarimetric techniques with metric learning algorithms, namely, polarimetric learning, by introducing a distance metric learning method called Siamese network that aims to learn good distance metrics of algal Mueller matrix images in low-dimensional feature spaces. As an experimental example, 12,162 Mueller matrix images of eight algal species are measured via a forward Mueller matrix microscope. Eight classical metric learning algorithms, including principle component analysis, multidimensional scaling, isometric feature mapping, t-distributed stochastic neighbor embedding, Laplacian eigenmaps, locally linear embedding, linear discriminant analysis, and metric learning to rank, are considered, by which the algal Mueller matrix images are mapped to two-dimensional (2D) feature spaces with different distance metrics. Support-vector-machine-based holdout sample classification accuracies of the 2D feature vectors are provided in a supervised manner for quantitative comparisons of the low-dimensional distance metrics, including the results of the eight metric learning algorithms and 16 Siamese architectures with varying convolution, inception, and full connection modules. This study shows that the Siamese approach is an effective metric learning algorithm that can adaptively extract features exhibiting empirical correlations with the fast-axis-orientation-dependent and spatially variant algal retardance induced by the algal microstructures.

Classification of morphologically similar algae and cyanobacteria using Mueller matrix imaging and convolutional neural networks.

We present the Mueller matrix imaging system to classify morphologically similar algae based on convolutional neural networks (CNNs). The algae and cyanobacteria data set contains 10,463 Mueller matrices from eight species of algae and one species of cyanobacteria, belonging to four phyla, the shapes of which are mostly randomly oriented spheres, ovals, wheels, or rods. The CNN serves as an automatic machine with learning ability to help in extracting features from the Mueller matrix, and trains a classifier to achieve a 97% classification accuracy. We compare the performance in two ways. One way is to compare the performance of five CNNs that differ in the number of convolution layers as well as the classical principle component analysis (PCA) plus the support vector machine (SVM) method; the other way is to quantify the differences of scores between full Mueller matrix and the first matrix element m11, which does not contain polarization information under the same conditions. As the results show, deeper CNNs perform better, the best of which outperforms the conventional PCA plus SVM method by 19.66% in accuracy, and using the full Mueller matrix earns 6.56% increase of accuracy than using m11. It demonstrates that the coupling of Mueller matrix imaging and CNN may be a promising and efficient solution for the automatic classification of morphologically similar algae.

Temperature-dependent physiological and biochemical responses of the marine medaka Oryzias melastigma with consideration of both low and high thermal extremes.

This study aimed to investigate temperature effect on physiological and biochemical responses of the marine medaka Oryzias melastigma larvae. The fish were subjected to a stepwise temperature change at a rate of 1 °C/h increasing or decreasing from 25 °C (the control) to six target temperatures (12, 13, 15, 20, 28 and 32 °C) respectively, followed by a 7-day thermal acclimation at each target temperature. The fish were fed ad libitum during the experiment. The results showed that cumulative mortalities were significantly increased at low temperatures (12 and 13 °C) and at the highest temperature (32 °C). For the survivors, their growth profile closely followed the left-skewed 'thermal performance curve'. Routine oxygen consumption rates of fish larvae were significantly elevated at 32 °C but suppressed at 13 and 15 °C (due to a high mortality, larvae from 12 °C were not examined). Levels of heat shock proteins and activities of malate dehydrogenase and lactate dehydrogenase were also measured in fish larvae exposed at 15, 25 and 32 °C. The activities of both enzymes were significantly increased at both 15 and 32 °C, where the fish larvae probably suffered from thermal discomfort and increased anaerobic components so as to compensate the mismatch of energy demand and supply at these thermal extremes. Coincidently, heat shock proteins were also up-regulated at both 15 and 32 °C, enabling cellular protection. Moreover, the critical thermal maxima and minima of fish larvae increased significantly with increasing acclimation temperature, implying that the fish could develop some degrees of thermal tolerance through temperature acclimation.

Isoform-specific responses of metallothioneins in a marine pollution biomonitor, the green-lipped mussel Perna viridis, towards different stress stimulations.

Metallothioneins (MTs) are commonly used as biomarker for metal pollution assessment in marine ecosystems. Using integrated genomic and proteomic analyses, this study characterized two types of MT isoform in the digestive gland of a common biomonitor, the green-lipped mussel Perna viridis, towards the challenges of a metal (cadmium; Cd) and a non-metal oxidant (hydrogen peroxide; H2 O2 ) respectively. The two isoforms differed in their deduced protein sequences, with 73 amino acids for MT10-I and 72 for MT10-II (a novel type), but both consisted of a high percentage (27.4 to 29.2%) of cysteine. Two-dimensional gel and Western blot showed that the MT proteins were present in multiple isoform spots, and they were further validated to be MT10-I and MT10-II using MS analysis coupled with unrestricted modifications searching. Expression of mRNA revealed that MT10-I responded promptly to Cd but had a lagged induction to H2 O2 treatments, while MT10-II was exclusively induced by Cd treatment over the course of exposure. Expression of the MT proteins also showed a delayed response to H2 O2 , compared to Cd treatments. This study uncovered the potential different functional roles of various MTs isoforms in P. viridis and thus advances the resolution of using MTs as biomarkers in future applications.

De novo transcriptome analysis of Perna viridis highlights tissue-specific patterns for environmental studies.

BACKGROUND: The tropical green-lipped mussel Perna viridis is a common biomonitor throughout the Indo-Pacific region that is used for environmental monitoring and ecotoxicological investigations. However, there is limited molecular data available regarding this species. We sought to establish a global transcriptome database from the tissues of adductor muscle, gills and the hepatopancreas of P. viridis in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses in this sentinel species. RESULTS: Illumina sequencing results yielded 544,272,542 high-quality filtered reads. After de novo assembly using Trinity, 233,257 contigs were generated with an average length of 1,264 bp and an N50 length of 2,868 bp; 192,879 assembled transcripts and 150,111 assembled unigenes were obtained after clustering. A total of 93,668 assembled transcripts (66,692 assembled genes) with putative functions for protein domains were predicted based on InterProScan analysis. Based on similarity searches, 44,713 assembled transcripts and 25,319 assembled unigenes were annotated with at least one BLAST hit. A total of 21,262 assembled transcripts (11,947 assembled genes) were annotated with at least one well-defined Gene Ontology (GO) and 5,131 assembled transcripts (3,181 assembled unigenes) were assigned to 329 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The quantity of assembled unigenes and transcripts obtained from male and female mussels were similar but varied among the three studied tissues, with the highest numbers recorded in the gills, followed by the hepatopancreas, and then the adductor muscle. Multivariate analyses revealed strong tissue-specific patterns among the three different tissues, but not between sexes in terms of expression profiles for annotated genes in various GO terms, and genes associated with stress responses and degradation of xenobiotics. The expression profiles of certain selected genes in each tissue type were further validated using real-time quantitative polymerase chain reaction assays and a similar tissue-specific trend was seen. CONCLUSIONS: The extensive sequence data generated from this study will provide a valuable molecular resource for facilitating environmental studies with P. viridis, and highlight the importance of tissue-specific approaches in the future.

Temperature-dependent toxicities of four common chemical pollutants to the marine medaka fish, copepod and rotifer.

We hypothesize that chemical toxicity to marine ectotherms is the lowest at an optimum temperature (OT) and it exacerbates with increasing or decreasing temperature from the OT. This study aimed to verify this hypothetical temperature-dependent chemical toxicity (TDCT) model through laboratory experiments. Acute toxicity over a range of temperatures was tested on four commonly used chemicals to three marine ectotherms. Our results confirmed that toxicities, in terms of 96-h LC50 (median lethal concentration; for the marine medaka fish Oryzias melastigma and the copepod Tigriopus japonicus) and 24-h LC50 (for the rotifer Brachionus koreanus), were highly temperature-dependent, and varied between test species and between study chemicals. The LC50 value of the fish peaked at 20 °C for copper (II) sulphate pentahydrate and triphenyltin chloride, and at 25 °C for dichlorophenyltrichloroethane and copper pyrithione, and decreased with temperature increase or decrease from the peak (i.e., OT). However, LC50 values of the copepod and the rotifer generally showed a negative relationship with temperature across all test chemicals. Both copepod and rotifer entered dormancy at the lowest temperature of 4 °C. Such metabolic depression responses in these zooplanktons could reduce their uptake of the chemical and hence minimize the chemical toxicity at low temperatures. Our TDCT model is supported by the fish data only, whereas a simple linear model fits better to the zooplankton data. Such species-specific TDCT patterns may be jointly ascribed to temperature-mediated changes in (1) the physiological response and susceptibility of the marine ectotherms to the chemical, (2) speciation and bioavailability of the chemical, and (3) toxicokinetics of the chemical in the organisms.

Proteomic response of marine invertebrate larvae to ocean acidification and hypoxia during metamorphosis and calcification.

Calcifying marine invertebrates with complex life cycles are particularly at risk to climate changes as they undergo an abrupt ontogenetic shift during larval metamorphosis. Although our understanding of the larval response to climate changes is rapidly advancing, the proteome plasticity involved in a compensatory response to climate change is still unknown. In this study, we investigated the proteomic response of metamorphosing larvae of the tubeworm Hydroides elegans, challenged with two climate change stressors, ocean acidification (OA; pH 7.6) and hypoxia (HYP; 2.8 mg O2 l(-1)), and with both combined. Using a two-dimensional gel electrophoresis (2-DE)-based approach coupled with mass spectrometry, we found that climate change stressors did not affect metamorphosis except under OA, but altered the larval proteome and phosphorylation status. Metabolism and various stress and calcification-related proteins were downregulated in response to OA. In OA and HYP combined, HYP restored the expression of the calcification-related proteins to the control levels. We speculate that mild HYP stress could compensate for the negative effects of OA. This study also discusses the potential functions of selected proteins that might play important roles in larval acclimation and adaption to climate change.

Extreme cold or warm events can potentially exacerbate chemical toxicity to the marine medaka fish Oryzias melastigma.

Marine ecosystems are currently subjected to dual stresses of chemical pollution and climate change. Through a series of laboratory experiments, this study investigated the impact of exposure to chemical contaminant such as DDT or copper (Cu), in combination with cold or warm temperature extremes on the marine medaka fish Oryzias melastigma. The results showed that extreme seawater temperatures (i.e., 15 and 32 °C in sub-tropical Hong Kong) exacerbated adverse chemical impacts on the growth performance of O. melastigma, in particular at the high thermal extreme. This was likely associated with an interruption of oxygen consumption and aerobic scope. Most importantly, the results of acclimation experiments, as reflected by thermal tolerance polygons, showed that chemical exposure substantially narrowed the thermal tolerance of the medaka, making them more vulnerable to temperature changes and extreme thermal events. Under dual stresses of thermal extremes and chemical exposure, the medaka switched their metabolic pathway to anaerobic respiration that might deplete their energy reserve for chemical detoxification. Although stress proteins such as heat shock proteins (HSP90) were up-regulated for cellular protection in the fish, such a defensive mechanism was repressed with intensifying dual stresses at high temperature and high chemical concentration. Bioconcentration of DDT or Cu generally increased with increasing temperature and its exposure concentration. Overall, these complex chemical-temperature interactions concomitantly exerted a concerted adverse impact to O. melastigma. The temperature-dependent toxicity of DDT or Cu shown in this study clearly demonstrated the potential challenge brought by the risk of chemical pollution under the impact of global climate change.

Toxicity effects of hydrophilic algal lysates from Coolia tropicalis on marine medaka larvae (Oryzias melastigma).

Coolia tropicalis is a species of benthic and epiphytic toxic algae, which can produce phycotoxins that intoxicate marine fauna. In this study, the potential toxic effects of C. tropicalis on fish were investigated using larval marine medaka (Oryzias melastigma) as a model to evaluate fish behavior, physiological performance, and stress-induced molecular responses to exposure to two sublethal concentrations (LC10 and LC20) of hydrophilic algal lysates. Exposure to C. tropicalis lysates inhibited swimming activity, activated spontaneous undirected locomotion, altered nerve length ration, and induced early development abnormalities, such as shorter eye diameter, body as well as axon length. Consistent with these abnormalities, changes in the expression of genes associated with apoptosis (CASPASE-3 and BCL-2), the inflammatory response (IL-1ß and COX-2), oxidative stress (SOD), and energy metabolism (ACHE and VHA), were also observed. This study advances our understanding of the mechanisms of C. tropicalis toxicity in marine fish in the early life stages and contributes to future ecological risk assessments of toxic benthic dinoflagellates.

Littoral Water in Hong Kong as a Potential Transient Habitat for Juveniles of a Temperate Deepwater Gnomefish, Scombrops boops (Acropomatiformes: Scombropidae).

A total of 40 juveniles belonging to a temperate deepwater gnomefish species, Scombrops boops, were sampled from littoral habitats (2-5 m depth) of eastern Hong Kong waters in April and May 2017 and March 2019. The presence of gnomefish juveniles in subtropical southern China is reported for the first time at a record low latitude of 22°11'-22°21'N. The specimens were identified based on the COI gene sequence. The genetic composition between Japan and Hong Kong gnomefish populations were compared by sequencing the mitochondrial Cytb gene, which showed no genetic differentiation. The juveniles ranged from 3.5-10.1 cm (n = 40) in total length, with 35 individuals caught from Sargassum beds and five from rocky reefs. Our findings highlighted that the littoral habitats in Hong Kong waters, in particular the seasonal Sargassum beds, are important for small juveniles of S. boops.

Transcriptomics reveal triphenyltin-induced molecular toxicity in the marine mussel Perna viridis.

Triphenyltin (TPT) is widely used as an active ingredient in antifouling paints and fungicides, and continuous release of this highly toxic endocrine disruptor has caused serious pollution to coastal marine ecosystems and organisms worldwide. Using bioassays and transcriptome sequencing, this study comprehensively investigated the molecular toxicity of TPT chloride (TPTCl) to the marine mussel Perna viridis which is a commercially important species and a common biomonitor for marine pollution in Southeast Asia. Our results indicated that TPTCl was highly toxic to adult P. viridis, with a 96-h LC10 and a 96-h EC10 at 18.7 µg/L and 2.7 µg/L, respectively. A 21-day chronic exposure to 2.7 µg/L TPTCl revealed a strong bioaccumulation of TPT in gills (up to 36.48 µg/g dry weight) and hepatopancreas (71.19 µg/g dry weight) of P. viridis. Transcriptome analysis indicated a time course dependent gene expression pattern in both gills and hepatopancreas. Higher numbers of differentially expressed genes were detected at Day 21 (gills: 1686 genes; hepatopancreas: 1450 genes) and at Day 28 (gills: 628 genes; hepatopancreas: 238 genes) when compared with that at Day 7 (gills: 104 genes, hepatopancreas: 112 genes). Exposure to TPT strongly impaired the endocrine system through targeting on nuclear receptors and putative steroid metabolic genes. Moreover, TPT widely disrupted cellular functions, including lipid metabolism, xenobiotic detoxification, immune response and endoplasmic-reticulum-associated degradation expression, which might have caused the bioaccumulation of TPT in the tissues and aggregation of peptides and proteins in cells that further activated the apoptosis process in P. viridis. Overall, this study has advanced our understanding on both ecotoxicity and molecular toxic mechanisms of TPT to marine mussels, and contributed empirical toxicity data for risk assessment and management of TPT contamination.

Toxicities of nano zinc oxide to five marine organisms: influences of aggregate size and ion solubility.

Nano zinc oxide (nZnO) is increasingly used in sunscreen products, with high potential of being released directly into marine environments. This study primarily aimed to characterize the aggregate size and solubility of nZnO and bulk ZnO, and to assess their toxicities towards five selected marine organisms. Chemical characterization showed that nZnO formed larger aggregates in seawater than ZnO, while nZnO had a higher solubility in seawater (3.7 mg L(-1)) than that of ZnO (1.6 mg L(-1)). Acute tests were conducted using the marine diatoms Skeletonema costatum and Thalassiosia pseudonana, the crustaceans Tigriopus japonicus and Elasmopus rapax, and the medaka fish Oryzias melastigma. In general, nZnO was more toxic towards algae than ZnO, but relatively less toxic towards crustaceans and fish. The toxicity of nZnO could be mainly attributed to dissolved Zn(2+) ions. Furthermore, molecular biomarkers including superoxide dismutase (SOD), metallothionein (MT) and heat shock protein 70 (HSP70) were employed to assess the sublethal toxicities of the test chemicals to O. melastigma. Although SOD and MT expressions were not significantly increased in nZnO-treated medaka compared to the controls, exposure to ZnO caused a significant up-regulation of SOD and MT. HSP70 was increased two to fourfold in all treatments indicating that there were probably other forms of stress in additional to oxidative stress such as cellular injury.

Effects of dietary exposure to ciguatoxin P-CTX-1 on the reproductive performance in marine medaka (Oryzias melastigma).

Ciguatoxins are natural compounds produced by benthic dinoflagellates Gambierdiscus and Fukuyoa spp., which cause fish intoxication by ciguatera fish poisoning. This study aimed to assess the dietary exposure effects of ciguatoxin P-CTX-1 on the reproductive performance in marine medaka (Oryzias melastigma). Fish which ingested >1.16 pg·day-1 for 21 days exhibited abnormal behaviors including diarrhea, abnormal swimming, loss of appetite and decreased egg production. After 7-day exposure to P-CTX-1 at a dose of 1.93 pg·day-1, significant gender-specific differences in reproductive performance and decreased hatching rate of the offspring were observed. Chemical analysis of P-CTX-1 showed that the P-CTX-1 accumulation rates were 24.1 ± 1.4% in female fish and 9.9 ± 0.4% in male fish, and 0.05 pg·egg-1 was detected. The results illustrate that dietary exposure to P-CTX-1 affected the reproductive performance and survival of offspring, and caused bioaccumulation and maternal transfer of P-CTX-1 in marine medaka.

Population Genetic Structure of A Marine Pelagic Egg Producer and Popular Marine Aquarium Species, the Mandarinfish Synchiropus splendidus.

The mandarinfish Synchiropus splendidus is extensively collected in Southeast Asia (mainly in the Philippines) and highly favoured for the marine aquarium trade. Males are more popular than females for their large first dorsal fins and the fishery is not managed. To examine possible population replenishment dynamics arising as a result of selective fishing, the effects of sex-selective fishing on sex ratios and population connectivity were considered. This study determined the sex ratios and analyzed the population genetic structure from mandarinfish collected at six locations: one from Palau, where the species is not exploited, and five from Bohol in the Philippines, where the species has long been heavily fished. The findings reported very low male to female ratios (0.12 to 0.30) from four of the five locations in Bohol, with relatively more males to females in the specimens collected from Palau (2.3). The analyses from allozymes (43 alleles from 10 loci) and microsatellites (118 alleles from 5 loci) revealed that genetic connectivity was high among the five locations in the Philippines as well as with the specimens collected from the more-distant Palau. The genetic homogeneity observed across the geographical range considered is inconsistent with the hypothesized limited dispersal ability of the species and could be explained by recent species range expansion associated with sea level rise in the region. The results suggest that the present genetic structure, at least in the geographic region considered, may not be determined by current patterns of gene flow, but may, instead, be driven by recent sea-level changes associated with periods of glaciation. Caution is suggested to ensure that heavily localized fishing does not produce excessively biased adult sex ratios.

Sunscreens containing zinc oxide nanoparticles can trigger oxidative stress and toxicity to the marine copepod Tigriopus japonicus.

The study, for the first time, evaluated the leaching rate of zinc oxide nanoparticles (nZnO) from human skins which were applied with three commercial sunscreens containing nZnO as an active ingredient. The leaching rate of nZnO varied greatly among the sunscreens, with a range of 8-72% (mean ± SD: 45% ± 33%). We further investigated their toxicities to the marine copepod Tigriopus japonicus. We found that 96-h median lethal concentrations of the three sunscreens to T. japonicus were > 5000, 230.6, and 43.0 mg chemical L-1, respectively, equivalent to Zn2+ concentrations at >82.5, 3.2, and 1.2 mg Zn L-1, respectively. Exposure to the individual sunscreens at environmentally realistic concentrations for 96 h led to up-regulation of antioxidant genes in T. japonicus, while they triggered the release of reactive oxygen species based on the results of in vivo assays. Evidently, these nZnO-included sunscreens can cause oxidative stress and hence pose risk to marine organisms.

The effect of temperature on physiology, toxicity and toxin content of the benthic dinoflagellate Coolia malayensis from a seasonal tropical region.

Coolia malayensis is one of the commonly found benthic dinoflagellates in Hong Kong which can produce biotoxins and threaten the early life stages of marine invertebrates. Seawater temperature has been recognized as one of the primary environmental factors that affect the formation of harmful algal blooms. The present study evaluated the responses of C. malayensis, including growth, toxicity and toxin content (putative analogues of okadaic acid and azaspiracids), after exposure to a range of seven different temperatures (i.e., 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, and 28°C). The highest algal density and specific growth rate were recorded at 24°C. Significantly higher Fv/Fm (maximum quantum yield of PSII) and total phaeo-pigment values were observed in the exponential growth phase at 28°C. The toxicity of the algal extract, which was assessed by the lethality rate of Artemia larvae, increased with temperature. The highest toxin content was detected at the second highest temperature treatment, i.e., 26°C. Overall, temperature had significant effects on the physiological activities and toxicity of C. malayensis. This study has raised attention to the potentially increasing risks posed by toxic benthic dinoflagellates during heat waves in coastal waters.

Hemolysis associated toxicities of benthic dinoflagellates from Hong Kong waters.

Benthic dinoflagellates produce a diverse range of phycotoxins, which are responsible for intoxication events in marine fauna. This study assessed the hemolysis associated toxicities of six species of benthic dinoflagellates from the genera Coolia, Fukuyoa, Amphidinium and Prorocentrum. Results demonstrated that Amphidinium carterae, Coolia tropicalis and Fukuyoa ruetzleri were the three most toxic species, while Prorocentrum cf. lima did not have significant hemolytic effect. Grouper samples (Cephalopholis boenak) were more tolerant to the hemolytic algae than the blackhead seabream (Acanthopagrus schlegelii), with decreased heart rate and blood flow being observed in medaka larvae after exposure to toxic algal extracts. LC-MS/MS analysis detected a gambierone analogue called 44-methylgambierone produced by the C. tropicalis isolate. This analogue was also detected in the F. ruetzleri isolate. This study provided new information on the hemolysis associated toxicities of local toxic benthic dinoflagellates, which contributes to better understanding of their emerging threats to marine fauna and reef systems in Hong Kong.

Identification and characterization of proteins interacting with SIRT1 and SIRT3: implications in the anti-aging and metabolic effects of sirtuins.

Sirtuins are a family of NAD(+)-dependent protein deacetylases that regulate cellular functions through deacetylation of a wide range of protein targets. Overexpression of Sir2, the first gene discovered in this family, is able to extend the life span in various organisms. The anti-aging effects of human homologues of sirtuins, SIRT1-7, have also been suggested by animal and human association studies. However, the precise mechanisms whereby sirtuins exert their anti-aging effects remain elusive. In this study, we aim to identify novel interacting partners of SIRT1 and SIRT3, two human sirtuins ubiquitously expressed in many tissue types. Our results demonstrate that SIRT1 and SIRT3 are localized within different intracellular compartments, mainly nuclei and mitochondria, respectively. Using affinity purification and MALDI-TOF/TOF-MS/MS analysis, their potential interacting partners have been identified from the enriched subcellular fractions and specific interactions confirmed by co-immunoprecipitation and Western blotting experiment. Further analyses suggest that overexpression of SIRT1 or SIRT3 in HEK293 cells could induce hypoacetylation and affect the intracellular localizations and protein stabilities of their interacting partners. Taken together, the present study has identified a number of novel SIRT protein interacting partners, which might be critically involved in the anti-aging and metabolic regulatory activities of sirtuins.

Overexpression of angiopoietin-like protein 4 alters mitochondria activities and modulates methionine metabolic cycle in the liver tissues of db/db diabetic mice.

Angiopoietin-like protein 4 (ANGPTL4) is a circulating protein predominantly produced from fat tissue and liver. Recent data from others and our laboratory have demonstrated this protein to be an important player in energy metabolism and insulin sensitivity. However, the molecular mechanisms underlying its metabolic actions remain elusive. In this study, we have employed a two-dimensional fluorescence difference gel electrophoresis technique to study the protein profiles in the livers of db/db mice treated with or without ANGPTL4. When compared with those of lean mice, 118 proteins were found to be up- or down-regulated in db/db mice. Adenovirus-mediated overexpression of ANGPTL4 could reverse a large portion of the up- or down-regulated proteins to control levels. Especially, a number of mitochondria proteins were down-regulated by ANGPTL4 to a great extent. Chronic treatment with ANGPTL4 resulted in an elevated activity of mitochondria respiratory chain complexes II-III and IV in db/db mice. Additionally, several key enzymes in the methionine/homocysteine metabolic cycle were found to be increased in db/db diabetic mice but decreased by ANGPTL4 treatment. HPLC analysis consistently revealed that ANGPTL4 could significantly restore the augmented S-adenosylmethionine levels and S-adenosylmethionine/S-adenosylhomocysteine ratios in livers of db/db mice. In summary, our results suggest that ANGPTL4 might elicit its metabolic effects through modulating the mitochondria functions and methionine metabolic cycles in the liver tissue.