Vascular Endothelial Cell-Derived Exosomal Sphingosylphosphorylcholine Attenuates Myocardial Ischemia-Reperfusion Injury through NR4A2-Mediated Mitophagy.

Cardiomyocyte survival is a critical contributing process of host adaptive responses to cardiovascular diseases (CVD). Cells of the cardiovascular endothelium have recently been reported to promote cardiomyocyte survival through exosome-loading cargos. Sphingosylphosphorylcholine (SPC), an intermediate metabolite of sphingolipids, mediates protection against myocardial infarction (MI). Nevertheless, the mechanism of SPC delivery by vascular endothelial cell (VEC)-derived exosomes (VEC-Exos) remains uncharacterized at the time of this writing. The present study utilized a mice model of ischemia/reperfusion (I/R) to demonstrate that the administration of exosomes via tail vein injection significantly diminished the severity of I/R-induced cardiac damage and prevented apoptosis of cardiomyocytes. Moreover, SPC was here identified as the primary mediator of the observed protective effects of VEC-Exos. In addition, within this investigation, in vitro experiments using cardiomyocytes showed that SPC counteracted myocardial I/R injury by activating the Parkin and nuclear receptor subfamily group A member 2/optineurin (NR4A2/OPTN) pathways, in turn resulting in increased levels of mitophagy within I/R-affected myocardium. The present study highlights the potential therapeutic effects of SPC-rich exosomes secreted by VECs on alleviating I/R-induced apoptosis in cardiomyocytes, thereby providing strong experimental evidence to support the application of SPC as a potential therapeutic target in the prevention and treatment of myocardial infarction.

Highly sensitive and stable fluorescent aptasensor based on an exonuclease III-assisted amplification strategy for ATP detection.

Fluctuations in intracellular adenosine triphosphate (ATP) concentration are closely associated with some cancer diseases. Thus, it is a worthwhile undertaking to predict sickness by monitoring changes in ATP levels. However, the detection limits of current fluorescent aptamer sensors for ATP detection are in the range of nmol L-1 to µmol L-1. It has become crucial to employ amplification strategies to increase the sensitivity of fluorescent aptamer sensors. In the current paper, a duplex hybrid aptamer probe was developed based on exonuclease III (Exo III)-catalyzed target recycling amplification for ATP detection. The target ATP forced the duplex probe configuration to change into a molecular beacon that can be hydrolyzed with Exo III to achieve the target ATP cycling to amplify the fluorescence signal. Significantly, many researchers ignore that FAM is a pH-sensitive fluorophore, leading to the fluorescence instability of FAM-modified probes in different pH buffers. The negatively charged ions on the surface of AuNPs were replaced by new ligands bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt (BSPP) to improve the drawback of FAM instability in alkaline solutions in this work. The aptamer probe was designed to eliminate the interference of other similar small molecules, showing specific selectivity and providing ultra-sensitive detection of ATP with detection limits (3σ) as low as 3.35 nM. Such detection limit exhibited about 4-500-fold better than that of the other amplification strategies for ATP detection. Thus, a relatively general high sensitivity detection system can be established according to the wide target adaptability of aptamers, which can form specific binding with different types of targets.

Toxicity of polymer-modified CuS nanoclusters on zebrafish embryo development.

Despite the vast amount of research on the toxicity of copper-based nanoparticles, the toxicity of CuS nanoparticles is still largely unknown. Due to the application of CuS-based nanomaterials in biomedical engineering, it is necessary to study their potential toxicity and biological effects. In this study, we evaluated the toxicity of polymer-modified CuS nanoclusters (PATA3-C4@CuS) on embryo development through exposing zebrafish embryos to 1, 2.5, 5, 7.5, and 10 mg/L PATA3-C4@CuS at 0.75-h post-fertilization. The morphological results demonstrated that PATA3-C4@CuS at concentrations greater than 1 mg/L PATA3-C4@CuS induced abnormal phenotypes including smaller heads and eyes, pericardial edema, and epiboly retardation and it increased mortality, lowered the hatching rate, and inhibited swim bladder inflation. In situ hybridization and quantitative reverse transcription polymerase chain reaction showed that PATA3-C4@CuS could alter the expression patterns of tbxta, dlx3, and cstlb and increase the expression levels of wnt5 and wnt11, which suggested that PATA3-C4@CuS disrupts cell migration by increasing the levels of wnt5 and wnt11 during gastrulation. It was also discovered that PATA3-C4@CuS exposure caused a slow heart rate and smaller ventricles in zebrafish larvae. Immunofluorescence and behavioral analyses showed that PATA3-C4@CuS could damage the ventral projection of the primary motor neurons CaP, which was in accordance with the reduction in locomotion ability. Together, our data demonstrated that functional PATA3-C4@CuS could disrupt cell migration during gastrulation, affect cardiac development and function, and decrease locomotive activity.

Plasma Membrane Targeting of Protocadherin 15 Is Regulated by the Golgi-Associated Chaperone Protein PIST.

Protocadherin 15 (PCDH15) is a core component of hair cell tip-links and crucial for proper function of inner ear hair cells. Mutations of PCDH15 gene cause syndromic and nonsyndromic hearing loss. At present, the regulatory mechanisms responsible for the intracellular transportation of PCDH15 largely remain unknown. Here we show that PIST, a Golgi-associated, PDZ domain-containing protein, interacts with PCDH15. The interaction is mediated by the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI) of PCDH15. Through this interaction, PIST retains PCDH15 in the trans-Golgi network (TGN) and reduces the membrane expression of PCDH15. We have previously showed that PIST regulates the membrane expression of another tip-link component, cadherin 23 (CDH23). Taken together, our finding suggests that PIST regulates the intracellular trafficking and membrane targeting of the tip-link proteins CDH23 and PCDH15.

HT-NMR Studies of the Be-F Coordination Structure in FNaBe and FLiBe Mixed Salts.

Molten NaF-BeF2 salt is widely considered a promising candidate to replace FLiBe in molten salt reactor applications, which is crucial to reducing the operating costs of the molten salt reactor. Studies on beryllium compounds are rarely conducted due to their volatility and high toxicity. Herein, the Be-F coordination structure of NaF/BeF2 mixed salts was investigated in-depth through various HT-NMR and solid-state NMR methods, which are optimized to be appropriate for the detection of beryllium compounds. It was found that Na2BeF4 and NaBeF3 crystals were transformed into amorphous tetrahedral coordinated networks when there was an increase in the BeF2 concentration in the mixed salts. The main coordinate structure comparisons between FNaBe and FLiBe were analyzed, which exhibit high similarity due to the covalent effect of Be-F bonding, demonstrating the theoretical feasibility of applying FNaBe salts as a substitute for FLiBe in MSR systems. In addition, the transition from the crystal phase to the amorphous phase occurred at a lower BeF2 concentration for FNaBe than that for FLiBe. This was further verified by the results of ab initio molecular dynamics (AIMD) simulation that FNaBe melts had more disordered structures, thus causing slight changes in their physical properties.

RBP4 promotes denervation-induced muscle atrophy through STRA6-dependent pathway.

BACKGROUNDS: Fat infiltration of skeletal muscle has been recognized as a common feature of many degenerative muscle disorders. Retinol binding protein 4 (RBP4) is an adipokine that has been demonstrated to be correlated with the presence and severity of sarcopenia in the elderly. However, the exact role and the underlying mechanism of RBP4 in muscle atrophy remains unclear. METHODS: Denervation-induced muscle atrophy model was constructed in wild-type and RBP4 knockout mice. To modify the expression of RBP4, mice were received intramuscular injection of retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) or oral gavage of RBP4 inhibitor A1120. Holo-RBP4-stimulated C2C12 myotubes were treated with siRNAs or specific inhibitors targeting signalling receptor and transporter of retinol 6 (STRA6)/Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway. Fat accumulation, myofibre cross-sectional area, myotube diameter and the expression of muscle atrophy markers and myogenesis markers were analysed. RESULTS: The expression levels of RBP4 in skeletal muscles were significantly up-regulated more than 2-fold from 7 days and sustained for 28 days after denervation. Immunofluorescence analysis indicated that increased RBP4 was localized in the infiltrated fatty region in denervated skeletal muscles. Knockout of RBP4 alleviated denervation-induced fatty infiltration and muscle atrophy together with decreased expression of atrophy marker Atrogin-1 and MuRF1 as well as increased expression of myogenesis regulators MyoD and MyoG. By contrast, injection of retinol-bound holo-RBP4 aggregated denervation-induced ectopic fat accumulation and muscle atrophy. Consistently, holo-RBP4 stimulation also had a dose-dependent effect on the reduction of C2C12 myotube diameter and myofibre cross-sectional area, as well as on the increase of Atrogin-1and MuRF1 expression and decrease of MyoD and MyoG expression. Mechanistically, holo-RBP4 treatment increased the expression of its membrane receptor STRA6 (>3-fold) and promoted the phosphorylation of downstream JAK2 and STAT3. Inhibition of STRA6/JAK2/STAT3 pathway either by specific siRNAs or inhibitors could decrease the expression of Atrogin-1 and MuRF1 (>50%) and decrease the expression of MyoD and MyoG (>3-fold) in holo-RBP4-treated C2C12 myotube. RBP4 specific pharmacological antagonist A1120 significantly inhibited the activation of STRA6/JAK2/STAT3 pathway, ameliorated ectopic fat infiltration and protected against denervation-induced muscle atrophy (30% increased myofibre cross-sectional area) in mice. CONCLUSIONS: In conclusion, our data reveal that RBP4 promotes fat infiltration and muscle atrophy through a STRA6-dependent and JAK2/STAT3 pathway-mediated mechanism in denervated skeletal muscle. Our results suggest that lowering RBP4 levels might serve as a promising therapeutic approach for prevention and treatment of muscle atrophy.

A novel predictive model for optimizing diabetes screening in older adults.

INTRODUCTION: The fasting blood glucose test is widely used for diabetes screening. However, it may fail to detect early-stage diabetes characterized by elevated postprandial glucose levels. Hence, we developed and internally validated a nomogram to predict the diabetes risk in older adults with normal fasting glucose levels. MATERIALS AND METHODS: This study enrolled 2,235 older adults, dividing them into a Training Set (n = 1,564) and a Validation Set (n = 671) based on a 7:3 ratio. We employed the least absolute shrinkage and selection operator regression to identify predictors for constructing the nomogram. Calibration and discrimination were employed to assess the nomogram's performance, while its clinical utility was evaluated through decision curve analysis. RESULTS: Nine key variables were identified as significant factors: age, gender, body mass index, fasting blood glucose, triglycerides, alanine aminotransferase, the ratio of alanine aminotransferase to aspartate aminotransferase, blood urea nitrogen, and hemoglobin. The nomogram demonstrated good discrimination, with an area under the receiver operating characteristic curve of 0.824 in the Training Set and 0.809 in the Validation Set. Calibration curves for both sets confirmed the model's accuracy in estimating the actual diabetes risk. Decision curve analysis highlighted the model's clinical utility. CONCLUSIONS: We provided a dynamic nomogram for identifying older adults at risk of diabetes, potentially enhancing the efficiency of diabetes screening in primary healthcare units.

DNA methylation of skeletal muscle function-related secretary factors identifies FGF2 as a potential biomarker for sarcopenia.

BACKGROUND: Sarcopenia is characterized by progressive loss of muscle mass and function due to aging. DNA methylation has been identified to play important roles in the dysfunction of skeletal muscle. The aim of our present study was to explore the whole blood sample-based methylation changes of skeletal muscle function-related factors in patients with sarcopenia. METHODS: The overall DNA methylation levels were analysed by using MethlTarget™ DNA Methylation Analysis platform in a discovery set consistent of 50 sarcopenic older adults (aged ≥65 years) and 50 age- and sex-matched non-sarcopenic individuals. The candidate differentially methylated regions (DMRs) were further validated by Methylation-specific PCR (MSP) in another two independent larger sets and confirmed by pyrosequencing. Receiver operating characteristic (ROC) curve analysis was used to determine the optimum cut-off levels of fibroblast growth factor 2 (FGF2)_30 methylation best predicting sarcopenia and area under the ROC curve (AUC) was measured. The correlation between candidate DMRs and the risk of sarcopenia was investigated by univariate analysis and multivariate logistic regression analysis. RESULTS: Among 1149 cytosine-phosphate-guanine (CpG) sites of 27 skeletal muscle function-related secretary factors, 17 differentially methylated CpG sites and 7 differentially methylated regions (DMRs) were detected between patients with sarcopenia and control subjects in the discovery set. Further methylation-specific PCR identified that methylation of fibroblast growth factor 2 (FGF2)_30 was lower in patients with sarcopenia and the level was decreased as the severity of sarcopenia increased, which was confirmed by pyrosequencing. Correlation analysis demonstrated that the methylation level of FGF2_30 was positively correlated to ASMI (r = 0.372, P < 0.001), grip strength (r = 0.334, P < 0.001), and gait speed (r = 0.411, P < 0.001). ROC curve analysis indicated that the optimal cut-off value of FGF2_30 methylation level that predicted sarcopenia was 0.15 with a sensitivity of 84.6% and a specificity of 70.1% (AUC = 0.807, 95% CI = 0.756-0.858, P < 0.001). Multivariate logistic regression analyses showed that lower FGF2_30 methylation level (<0.15) was significantly associated with increased risk of sarcopenia even after adjustment for potential confounders including age, sex, and BMI (adjusted OR = 9.223, 95% CI: 6.614-12.861, P < 0.001). CONCLUSIONS: Our results suggest that lower FGF2_30 methylation is correlated with the risk and severity of sarcopenia in the older adults, indicating that FGF2 methylation serve as a surrogate biomarker for the screening and evaluation of sarcopenia.

Association of AST/ALT (De Ritis) ratio with sarcopenia in a Chinese population of community-dwelling elderly.

Background: The aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio, also known as De Ritis ratio, has been reportedly associated with malnutrition which plays a crucial role in sarcopenia. The aim of this study was to examine the relationship between AST/ALT ratio and sarcopenia in the Chinese community-dwelling elderly. Methods: A cross-sectional study with 2751 participants (1343 men and 1408 women) aged ≥60 years was performed. Appendicular skeletal muscle mass index (ASMI), grip strength, and gait speed were measured to diagnose sarcopenia according to the latest Asian Working Group for Sarcopenia (AWGS) consensus. The association of AST/ALT ratio with sarcopenia was examined using logistic regression analysis. Results: The prevalence of sarcopenia in the present study was 4.4%. AST/ALT ratio was higher in the sarcopenia group than in the non-sarcopenia group (1.30 ± 0.33 vs. 1.16 ± 0.62, P = 0.010). AST/ALT ratio was negatively correlated with the components of sarcopenia, including ASMI, grip strength, and gait speed. Logistic regression analysis indicated that high AST/ALT ratio (>1.20) was associated with increased risk of sarcopenia even after adjustment for potential confounders (adjusted OR = 2.33, 95%CI = 1.48-3.68, P < 0.001). Stratification analyses indicated that the association of high AST/ALT ratio with high risk of sarcopenia was more significant in males and the elderly with ≥70 years. Conclusions: Our findings demonstrate that high AST/ALT ratio is associated with increased risk of sarcopenia in a Chinese population of community-dwelling elderly.

Membrane occupation and recognition nexus (MORN) motif controls protein localization and function.

The membrane occupation and recognition nexus (MORN) motif was first defined in 2000, when it was identified in the junctophilin protein family. Dozens of studies have been published ever since, mainly focusing on the function of a given MORN motif-containing protein in parasites, plants or animal cells. Proteins with MORN motifs are not only expressed in most animal and plant cell types, but also significantly differ in their intracellular localization, suggesting that the MORN motifs may fulfill multiple physiological functions. Recent studies have found that MORN motif-containing proteins junctophilin-1/2 and MORN3 play a role in cardiac hypertrophy, skeletal muscle fiber stability and cancer. Hence, MORN motif-containing proteins may be exploited to develop improved treatments for various pathological conditions, such as cardiovascular diseases. Here, we review current research on MORN motif-containing proteins in different organisms and provide both ideas and approaches for follow-up exploration of their functions and applications.

A Multicenter, Randomized, Double-Blind, Positive-Controlled, Non-Inferiority, Phase III Clinical Trial Evaluating the Efficacy and Safety of Emulsified Isoflurane for Anesthesia Induction in Patients.

BACKGROUND: Emulsified isoflurane was designed to circumvent the deficiencies of inhalation anesthetics, which have a longer time to onset, result in a higher drug consumption, and for which a specific anesthesia machine is required for clinical use. The aim of this study was to compare the efficacy and safety of emulsified isoflurane with propofol for anesthesia induction in adults patients. METHODS: This multicenter, randomized, double-blind, positive-controlled, non-inferiority, phase III clinical trial compared the efficacy and safety of emulsified isoflurane with propofol for anesthesia induction. Each patient in the emulsified isoflurane group received a single bolus injection of 12% emulsified isoflurane at a dose of 30 mg/kg, and each patient in the propofol group received a single bolus injection of 0.8% propofol at a dose of 2 mg/kg. The primary outcome of the efficacy evaluation was the proportion of participants with successful anesthesia induction, which was regarded as a Modified Observer's Assessment of Alertness/Sedation (MOAA/S) score of < 1 and lack of use of other sedative drugs. A number of secondary efficacy outcomes were also assessed. Safety was monitored based on (1) adverse events, (2) repeated measurement of vital signs; (3) physical examination, (4) routine laboratory examinations of hematology, biochemistry, urine, coagulation function, and (5) 12-lead electrocardiogram. RESULTS: A total of 416 patients were enrolled (n = 208 in each group) and 398 patients were administered study drug. The proportion of participants with successful anesthesia induction was 100% with a 95% confidence interval of - 1.9% to + 1.9% for the emulsified isoflurane and propofol groups, which met the predesigned non-inferiority criteria of 5%. The study demonstrated the non-inferiority of sedation produced by emulsified isoflurane compared to propofol. Among the secondary efficacy outcomes, emulsified isoflurane showed a better cardiovascular stability than propofol. The number of patients from the emulsified isoflurane group who experienced drug-related adverse events was significantly higher than that of patients from the propofol group. However, there was no significant difference between the two groups in terms of adverse events or drug-related adverse events of grades 3-5. CONCLUSIONS: Emulsified isoflurane exhibited non-inferiority of anesthesia/sedation compared to propofol in patients undergoing anesthesia induction. CLINICAL TRIAL REGISTRATION: ChiCTR2000038185, registered on 12 December, 2020 ( www.chictr.org.cn ).

[Effect of ulinastatin preconditioning on gene expression profile of kidney tissue in a rat sepsis model].

OBJECTIVE: To investigate the modulation effect of ulinastatin (UTI) preconditioning on gene expression of kidney tissue in septic rats by DNA microarrays. METHODS: Forty-five male Wistar rats were divided into control group, sepsis group and UTI group, with 15 rats in each group by means of random number table. Cecal ligation and puncture (CLP) was used to reproduce rat sepsis model. The control group only experienced a simulated operation without CLP. In UTI group the rats were treated with intramuscular injection of UTI (100 kU/kg). In sepsis group and control group intramuscular balanced solution (5 ml/kg) was given. Gene expression spectrum was studied with oligonucleotide gene expression profile microarray that contained 22 523 rat cDNA clones to detect the changes in gene expression pattern of rat kidney tissue 24 hours after CLP. Genes with fluorescent signal of Cy3/Cy5 of ratio average (RA)>2.0 or RA<0.5 were identified as differential genes, then those highly correlated to sepsis and UTI were screened by means of related computer software, and their relationship was analyzed. RESULTS: Three hundred and twenty-seven differential genes were found in sepsis group/control group, accounting for 1.45%, and among them 181 genes showed up-regulation,with 78 known functional genes, and 146 genes showed down-regulation, with 51 known functional genes. One hundred and twenty-seven differential genes were found in UTI group/sepsis group, accounting for 0.56%, and among them 41 genes showed up-regulation, with 14 known functional genes, and 86 genes showed down-regulation, with 37 known functional genes. Twenty-two genes were down-regulated in sepsis group/control group but up-regulated in UTI group/sepsis group, with 11 known functional genes, 51 genes were up-regulated in sepsis group/control group but down-regulated in UTI group/sepsis group, with 24 known functional genes. CONCLUSION: UTI preconditioning can alleviate the damage of kidney tissue in rat sepsis model, thus showing a protective effect on kidney, and the mechanism may be attributable to effect of UTI on modulation of immune reaction, energy metabolism, inflammatory reaction, signal transduction, defense reaction, oxidation-reduction reaction, DNA replication, and transcription related genes.

Ptc, Smo, Sufu, and the Hedgehog signaling pathway in amphioxus.

The Hedgehog (Hh) signaling pathway regulates many developmental processes both in vertebrates and in invertebrates. However, little is known about this pathway in the cephalochordate amphioxus. In this paper, we focus on the Ptc, Smo, and Sufu homologs in amphioxus, which are the key members of the Hh signaling pathway. Their genomic structures show their comparability with homologs in vertebrates. In situ hybridization reveals that amphioxus Ptc, Smo, and Sufu have similar expression patterns in embryogenesis. They are expressed in the neural plate at early neurula stage, and then down-regulated in dorsal neural ectoderm. During development, their transcripts appear and persist in the notochord, the wall of the head cavity, the epithelium of the pharynx, and the gut. The data show that the expression patterns of these three genes are overlapping with Hh and Gli during the embryonic development in amphioxus. Moreover, injection of amphioxus Hh RNA into zebrafish-fertilized eggs can expand the expression domains of Ptc1 and Nk2.2a, the target genes of the Hh signaling pathway, which is similar to the injection of zebrafish Sonic hh a (zShha) and Sonic hh b (zShhb). Our results suggest that amphioxus may possess a conserved and functional Hh signaling pathway similar to that of vertebrates.

Characterization of SoxB2 and SoxC genes in amphioxus (Branchiostoma belcheri): implications for their evolutionary conservation.

Most Sox genes directly affect cell fate determination and differentiation. In this study, we isolated two Sox genes: SoxB2 and SoxC from amphioxus (Branchiostoma belcheri), the closest living invertebrate relative of the vertebrates. Alignments of SoxB2 and SoxC protein sequences and their vertebrate homologs show high conservation of their HMG domains. Phylogenic analysis shows that amphioxus SoxB2 and SoxC fall out of the vertebrate branches, suggesting that vertebrate homologs might arise from gene duplications during evolution. The two genes possess similar spatial and temporal expression patterns during embryogenesis and in adults. They are both maternally inherited. During neurulation, they are expressed in the neural ectoderm and archenterons. In adults, they are expressed not only in the nerve cord, but also in the gut, midgut diverticulum, gill and oocytes. These results suggest that amphioxus SoxB2 and SoxC might co-function and have conserved functions in the nervous system and gonads as their vertebrate homologs.

[Isolation, phylogenetic analysis and developmental expression parttern of AmphiRab23b in amphioxus].

The hedgehog (Hh) pathway plays an important role during the embryonic development and is related to the progression of cancers. Rab23 is a crucial functional molecule in Hh pathway. However, there is no report about amphioxus Rab23 up to now except the annotations of two isoforms in the genome of Florida lancelet (Branchiostoma floridae). Here a 2062 bp full-length cDNA sequence of the Rab23, AmphiRab23b, was isolated from Chinese amphioxus (Branchiostoma belcheri), which included the UTRs and an open reading frame of 714 bp, encoding a protein of 237 amino acids. Phylogenetic analysis suggested that AmphiRab23b falled outside the vertebrate clade. But sequence analysis indicated that this putative AmphiRab23b protein contained a specific Rab23_lke domain, which implied that Rab23 gene was functional conservative during evolution. And its developmental expression pattern showed that AmphiRab23b was expressed in the differentiating neural plate and alimentary canal, as the same as the expression pattern of the homologous vertebrate genes, which suggested that AmphiRab23b may function in the development of nervous system and alimentary canal.

Characterization of AGAT, GAMT and CT1 in amphioxus: implications for the evolutionary conservation of creatine metabolism related molecules at the invertebrate-to-vertebrate transition.

In vertebrates, glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and creatine transporter (CT1) are three proteins involved in creatine synthesis. To provide insight into the molecular evolution mechanism of creatine metabolism, we have cloned and identified BbAGAT, BbGAMT, and BbCT1 homologous genes in amphioxus (Branchiostoma belcheri), whose predicted proteins show high identities with AGAT, GAMT, and CT1 proteins in vertebrates. The phylogenetic analysis indicates that amphioxus AGAT, GAMT, and CT1 are branched off at the base of the vertebrate homologous clade, respectively. Genomic structures of BfAGAT, BfGAMT, and BfCT1 show their comparability with the homologs in vertebrate and original characteristic of cephalochordate, which is consistent with animal classification. To determine the expression patterns of BbAGAT, BbGAMT, and BbCT1, whole-mount and section in situ hybridizations are carried out in embryos and adults of amphioxus. During embryogenesis, they are all expressed mainly in mesendoderm and late somites, but BbCT1 is also expressed in differentiating notochord and digestive tract, as well as in the cytoplasm of zygotes and the blastomeres at cleavage stage. In adult, the transcripts of BbAGAT and BbGAMT are detected in the neural cord, gill, nephridia, endostyle, gut, and gonads, while BbCT1 is expressed mainly in the epithelium of gut. The expression pattern of these three genes is similar to their vertebrate homologs. The result reveals that AGAT, GAMT, and CT1, the primary elements of vertebrate creatine metabolism, exist in cephalochordate amphioxus, and are highly conserved during evolution. It also suggests that similar mechanism of creatine synthesis in vertebrate may occur in amphioxus.

Characterization and expression of two amphioxus DDAH genes originating from an amphioxus-specific gene duplication.

Two dimethylarginine dimethylaminohydrolase (DDAH) homologous genes in amphioxus are identified and cloned. The phylogenetic analysis indicates two DDAHs in amphioxus originated by independent duplication specific in cephalochordate lineage. Analysis of AmphiDDAHa and AmphiDDAHb genomic structure shows their comparability with the DDAH in vertebrate and aboriginality which is consistent with animal classification. To explore the function relationship of AmphiDDAHa and AmphiDDAHb with AmphiNOS, the nitric oxide synthase homologue in amphioxus, we investigate the three genes expression patterns in embryos and adult tissues. The results indicate that these three genes possess different spatial and temporal expression patterns during embryogenesis. AmphiDDAHa transcripts are detected extensively in the differentiating ectoderm, mesendoderm in gastrula/early neurula, the developing and newly formed neural tube, somites, notochord, alimentary canal and epidermis at neurula stage, as well as in the differentiating pharynx and tailbud at early larval stage. While AmphiDDAHb is expressed in the differentiating non-neural ectoderm at the gastrla/early neurula stage, then locates abroad in the developing and formed neural tube, notochord, somites and alimentary canal. AmphiNOS is expressed weakly but widely from early neurula stage to at least 72-h larva stage. In adult, AmphiDDAHa and AmphiNOS share similar expression patterns in diverse adult tissues and cells such as the neural cord, gut, midgut diverticulum, wheel organ, gill, blood vessels, endostyle, oocytes and macrophages. But no expression of AmphiDDAHb is detected in tissues mentioned above. The results suggest that AmphiDDAHa and AmphiDDAHb should be two homologous genes with different functions in amphioxus. AmphiDDAHa may play a conserved role in the regulation of NO synthesis and immune defense, whereas AmphiDDAHb may mainly play the roles in the cell movement or differentiation of embryonic non-neural ectoderm cells during gastrulation and early neurulation.

Embryonic lethality in mice lacking Trim59 due to impaired gastrulation development.

TRIM family members have been implicated in a variety of biological processes such as differentiation and development. We here found that Trim59 plays a critical role in early embryo development from blastocyst stage to gastrula. There existed delayed development and empty yolk sacs from embryonic day (E) 8.5 in Trim59-/- embryos. No viable Trim59-/- embryos were observed beyond E9.5. Trim59 deficiency affected primary germ layer formation at the beginning of gastrulation. At E6.5 and E7.5, the expression of primary germ layer formation-associated genes including Brachyury, lefty2, Cer1, Otx2, Wnt3, and BMP4 was reduced in Trim59-/- embryos. Homozygous mutant embryonic epiblasts were contracted and the mesoderm was absent. Trim59 could interact with actin- and myosin-associated proteins. Its deficiency disturbed F-actin polymerization during inner cell mass differentiation. Trim59-mediated polymerization of F-actin was via WASH K63-linked ubiquitination. Thus, Trim59 may be a critical regulator for early embryo development from blastocyst stage to gastrula through modulating F-actin assembly.

Changes of serum beta-endorphin by programmed exercise training are correlated with improvement of clinical symptoms and quality of life in female mitral valve prolapse syndrome.

BACKGROUND: Mitral valve prolapse (MVP) is a common entity in female population. Although this is a minor disease, it may cause annoying symptoms that impair quality of life (QOL), and no established therapy for this problem. The aim of this study isto examine whether programmed exercise training by treadmill in female MVP syndrome would improve clinical symptoms and QOL. METHODS: An interventional study of 39 females with MVP syndrome with treadmill exercise endurance training for 12 weeks. Every individual received training for 30 min a day, thrice a week for 12 weeks. Baseline and post-exercise at 12 weeks serum beta-endorphins were measured. Symptom improvement was assessed by the MVP symptom checklist questionnaire and the Euro-QOL-5D was used to measure QOL improvement in these females. RESULTS: The mean serum beta-endorphin increased from 0.5 to 1.68 ng/ml (p = 0.001) in the exercise group (n = 18) after 12 weeks exercise, whereas the control group (n = 21) did not show any significant changes (0.44 vs. 0.43 ng/ml). Major symptoms of MVP such as chest pain, palpitation, fatigue were improved significantly by the assessment of MVP symptom checklist. The QOL of the exercised females also showed significant changes. CONCLUSIONS: Through programmed exercise training in these MVP females, the improvement of symptoms and QOL is parallel to the increase of serum beta-endorphin. This result implicates that MVP females should initiate exercise to tackle this annoying problem.

Phylogenetic analysis and expression pattern of the AmphiCaBP-like gene from amphioxus, encoding a novel member of the calmodulin-like subfamily.

We characterized a novel gene, AmphiCaBP-like, encoding a putative Ca(2 + )-binding protein (CaBP) with three EF-hand motifs from amphioxus (Branchiostoma belcheri tsingtauense). It exhibits significant similarities with the calmodulin and troponin C proteins from other species. Results of phylogenetic analysis indicated that amphioxus AmphiCaBP-like falls on the base of the troponin C clade. It may be a novel member of the calmodulin-like subfamily. In situ hybridization and RT-PCR analysis showed that AmphiCaBP-like is expressed throughout early development except the stages around the blastula. From neurula stage onward, transcripts of the AmphiCaBP-like are detected in the neural plate, the neural tube, the differentiating somites and the splanchnopleure, as well as the epithelium of the pharynx and gut. It is also expressed in the presumptive, but not the well-developed notochord. In adult amphioxus, the transcripts are found in the epithelial cells of the gut and midgut diverticulus, the wall of coelom, the branchia, the neural cord and gonads.