Assistive device utilization among disabled elderly in China: A cross-sectional study.

OBJECTIVE: This study aimed to examine the characteristics of assistive device users and influencing factors among disabled elderly in China. METHODS: A total of 13,510 disabled elderly in Sichuan Province were surveyed. Disability was assessed using the Barthel Activities of Daily Living Scale, mental status, sensory perception, and social engagement evaluation. Univariate analysis and logistic regression analysis were employed to identify the impact factors. RESULTS: The prevalence of assistive device utilization among participants was 79.2% (10,700/13,510, 95% CI 78.5%-79.9%), with the wheelchair being the most commonly used device. Various factors were found to influence the usage of the device, including disability level, somatic disability, age, caregivers, income, caregiver fees, and living situation (p < .05). Additionally, several factors were identified associated with the frequency of device usage, such as somatic disability, education background, income, caregiver fees, living situation, access to acquire assistive devices, duration of assistive device usage, education on assistive devices, and satisfaction level (p < .05). CONCLUSIONS: The use of assistive devices among elderly individuals in China is prevalent. There are many factors that affect the use of assistive devices, which can provide a reference for the formulation of policies in the field of assistive devices.

Comparison of the efficacy of fish oil and probiotic supplementation on glucose and lipid metabolism in patients with type 2 diabetes: a systematic review and network meta-analysis.

BACKGROUND: Abnormalities in glucose and lipid metabolism contribute to the progression and exacerbation of type 2 diabetes mellitus (T2DM). Fish oil and probiotics are dietary supplements that have the potential to improve glucose and lipid metabolism. However, their efficacy remains unclear in T2DM patients. METHODS: PubMed, Embase, and the Cochrane Library were retrieved to collect randomized controlled trials (RCTs) on the efficacy of fish oil or probiotic supplementation in T2DM patients from the database inception to December 13, 2023. Primary outcome indicators encompassed glycated hemoglobin (HbA1c), homeostatic model assessment for insulin resistance (HOMA-IR) and blood lipid profile (triglyceride (TG) and total cholesterol (TC). Secondary outcome indicators included inflammatory markers such as tumor necrosis factor -α (TNF-α) and adipocytokine (including leptin and adiponectin). The R software was used for statistical analysis, and GraphPad Prism was used for figure rendering. RESULTS: A total of 60 RCTs involving 3845 T2DM patients were included in the analysis. The results showed that the probiotics (Bifidobacterium, Lactobacillus, Lactococcus, Propionibacterium, etc.) were more effective in reducing HOMA-IR than fish oil (Surca = 0.935). Bifidobacterium demonstrated the highest efficacy in reducing HbA1c levels (Surca = 0.963). Regarding lipid metabolism, fish oil was superior to probiotics in lowering TG and TC levels (Surca values of 0.978 and 0.902, respectively). Furthermore, fish oil outperformed probiotics in reducing TNF-α (Surca = 0.839) and leptin (Surca = 0.712), and increasing adiponectin levels (Surca = 0.742). Node-splitting analysis showed good consistency (P > 0.05 for direct, indirect, and network comparison across various interventions). CONCLUSIONS: In T2DM patients, fish oil was more effective than probiotics in regulating lipid metabolism. Probiotics outperformed fish oil in regulating glucose metabolism particularly; specifically, Bifidobacterium showed higher efficacy in reducing blood glucose.

Prognostic Value of Transglutaminase 2 in Patients with Solid Tumors: A Meta-analysis.

Background: Transglutaminase 2 (TG2), a member of the transglutaminase family, also known as tissue transglutaminase, plays a fundamental role in cancer growth and progression. In this study, we aimed to comprehensively review the evidence of TG2 as a prognostic biomarker in solid tumors. Methods: PubMed, Embase, and Cochrane databases were searched for human studies with clearly described cancer types if they presented the relationship between TG2 expression and prognostic indicators from inception to February 2022. Two authors independently screened the eligible studies and extracted the relevant data. The association between TG2 and overall survival (OS), disease-free survival (DFS), and relapse-free survival (RFS) were described as hazard ratios (HR) and their corresponding 95% confidence intervals (CIs). Statistical heterogeneity was assessed using Cochrane Q-test and Higgins I-squared statistic. A sensitivity analysis was conducted by sequentially omitting the impact of each study. Publication bias was assessed by Egger's funnel plot. Results: A total of 2864 patients with various cancers from 11 individual studies were enrolled. Results showed that elevated TG2 protein expression and mRNA expression predicted a shorter OS, with a combined HR of 1.93 (95% CI: 1.41-2.63) or HR of 1.95 (95% CI: 1.27-2.99), respectively. Moreover, data suggested that elevated TG2 protein expression was correlated with a shorter DFS (HR = 1.76, 95% CI: 1.36-2.29); whereas elevated TG2 mRNA expression was associated with a shorter DFS (HR = 1.71, 95% CI: 1.30-2.24). Conclusions: Our meta-analysis indicated that TG2 might serve as a promising biomarker of cancer prognosis.

The Platelet-to-Lymphocyte Ratio Predicts Diabetic Retinopathy in Type 2 Diabetes Mellitus.

Introduction: As a severe and specific neurovascular complication of type 2 diabetes mellitus (T2DM), diabetic retinopathy (DR) remains the leading cause of vision loss and preventable blindness in adults aged 20 to 74. The pathogenesis of DR is not completely understood, however, studies indicate that chronic inflammation plays a significant role. Emerging evidence suggests that the neutrophil-to-lymphocyte ratio (NLR), the platelet-to-lymphocyte ratio (PLR), and the monocyte-to-lymphocyte ratio (MLR) are novel potential inflammatory response markers. The purpose of this study was to investigate the relationships between the NLR, PLR, MLR, and DR. Patients and Methods: 290 patients who had been diagnosed with T2DM participated in the study. Patients were categorized into three groups: 142 control subjects with T2DM, 124 subjects with nonproliferative diabetic retinopathy (NPDR), and 24 patients with proliferative diabetic retinopathy (PDR). Characteristics, laboratory data, as well as NLR, PLR and MLR levels of the study groups were compared. Results: In patients with DR, the median NLR, PLR, and MLR were significantly higher than in patients without DR (p = 0.012, p < 0.001, and p = 0.043, respectively). In the post hoc analysis, there was no correlation between the severity of retinopathy and the increase in NLR or PLR. Multiple logistic regression revealed that the PLR was an independent risk factor for DR (odds ratio [OR]: 1.020, 95% confidence interval [CI]: 1.010-1.029 p = 0.026). Based on the receiver operating characteristic (ROC) curve, the cutoff value of PLR as an indicator for diagnosing DR was estimated to be 129.65, with a sensitivity and specificity of 53.4% and 76.1%, respectively, and an area under the curve of 0.668 (95% CI: 0.605-0.730, p < 0.001). Conclusion: Our findings suggest that PLR may be an independent risk factor for evaluating DR in type 2 diabetes patients.

18F-FDG PET as an imaging biomarker for the response to FGFR-targeted therapy of cancer cells via FGFR-initiated mTOR/HK2 axis.

Rationale: The overall clinical response to FGFR inhibitor (FGFRi) is far from satisfactory in cancer patients stratified by FGFR aberration, the current biomarker in clinical practice. A novel biomarker to evaluate the therapeutic response to FGFRi in a non-invasive and dynamic manner is thus greatly desired. Methods: Six FGFR-aberrant cancer cell lines were used, including four FGFRi-sensitive ones (NCI-H1581, NCI-H716, RT112 and Hep3B) and two FGFRi-resistant ones (primary for NCI-H2444 and acquired for NCI-H1581/AR). Cell viability and tumor xenograft growth analyses were performed to evaluate FGFRi sensitivities, accompanied by corresponding 18F-fluorodeoxyglucose (18F-FDG) uptake assay. mTOR/PLCγ/MEK-ERK signaling blockade by specific inhibitors or siRNAs was applied to determine the regulation mechanism. Results: FGFR inhibition decreased the in vitro accumulation of 18F-FDG only in four FGFRi-sensitive cell lines, but in neither of FGFRi-resistant ones. We then demonstrated that FGFRi-induced transcriptional downregulation of hexokinase 2 (HK2), a key factor of glucose metabolism and FDG trapping, via mTOR pathway leading to this decrease. Moreover, 18F-FDG PET imaging successfully differentiated the FGFRi-sensitive tumor xenografts from primary or acquired resistant ones by the tumor 18F-FDG accumulation change upon FGFRi treatment. Of note, both 18F-FDG tumor accumulation and HK2 expression could respond the administration/withdrawal of FGFRi in NCI-H1581 xenografts correspondingly. Conclusion: The novel association between the molecular mechanism (FGFR/mTOR/HK2 axis) and radiological phenotype (18F-FDG PET uptake) of FGFR-targeted therapy was demonstrated in multiple preclinical models. The adoption of 18F-FDG PET biomarker-based imaging strategy to assess response/resistance to FGFR inhibition may benefit treatment selection for cancer patients.

The High-Effective Catalytic Degradation of Benzo[a]pyrene by Mn-Corrolazine Regulated by Oriented External Electric Field: Insight From DFT Study.

The degradation of BaP into hydroxybenzo[a]pyrene by Mn-corrolazine and its regulation by an oriented external electronic field (OEEF) were systematically studied using first-principle calculations. Extensive density function calculations showed that the degradation of BaP into hydroxybenzo[a]pyrene by Mn-corrolazine occurs via a three-step process in the absence of OEEF, in which a more toxic and stable epoxide intermediate is generated. However, upon application of OEEF along the intrinsic Mn-O reaction axis, the degradation of BaP into hydroxybenzo[a]pyrene is greatly simplified. The negative charge on the terminal O atom of Mn-OO corrolazine increases with an increase in the OEEF intensity. As the intensity of the OEEF increases over 0.004 a.u., the negatively charged terminal O atom has the ability to directly abstract the positively charged H atom of BaP and the degradation of BaP into hydroxybenzo[a]pyrene can be completed via a one-step process, avoiding the production of more toxic epoxide intermediates.

Dolabellane Diterpenoids from the Xisha Soft Coral Clavularia viridis.

Twelve new members (1-12) of the dolabellane family, co-occurring with three related known diterpenoids (13-15), were isolated from the Xisha soft coral Clavularia viridis. Their structures were determined by extensive spectroscopic analysis, modified Mosher's method, and X-ray diffraction analysis. Clavuperoxylides A (3) and B (4) represent the first examples of dolabellanes containing peroxyl groups, especially the novel peroxide bridge in 4, whereas clavufuranolides A-C (9-11) are the first example of dolabellane diterpenoids comprising a tetrahydrofuran ring. The possible biogenetic relationship of all the isolates was proposed. In bioassay, several compounds exhibited considerable cytotoxicity against A549 and P388 cell lines. Compound 7 exhibited inhibitory activity against protein tyrosine phosphatases 1B (PTP1B), an anti-diabetic target, representing the first report of PTP1B inhibitory activity for dolabellane diterpenoids.

Predictive value of novel inflammatory markers platelet-to-lymphocyte ratio, neutrophil-to-lymphocyte ratio, and monocyte-to-lymphocyte ratio in arterial stiffness in patients with diabetes: A propensity score-matched analysis.

Background: Increased arterial stiffness is common in patients with diabetes, and inflammation is one of the main causes of increased arterial stiffness. Platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), and monocyte-to-lymphocyte ratio (MLR) are novel inflammatory markers that are reproducible, widely available, and easy to measure, and are associated with low costs. This study sought to investigate the predictive value of these novel inflammatory markers in patients with diabetes having arterial stiffness. Methods: We retrospectively included inpatients with diabetes mellitus from the Endocrinology Department of the Chengdu Fifth People's Hospital from June 2021 to May 2022 and collected data on their general information, biochemical indicators, and brachial-ankle pulse wave velocity (baPWV). After propensity matching, the risk relationship between PLR, NLR, and MLR and arterial stiffness was assessed in the recruited patients. Results: A total of 882 hospitalized patients with diabetes were included in this study and categorized into the low baPWV (507 cases) or high baPWV group (375 cases) based on the baPWV. After propensity matching, there were 180 patients in all in the high and low baPWV groups. Univariate and multivariate logistic regression analyses revealed that high PLR, NLR, and MLR were independently associated with an increased risk of arterial stiffness in patients with diabetes. In the receiver operating characteristic curve analysis, the NLR area under the curve (AUC) was 0.7194 (sensitivity = 84.4%, specificity = 51.1%) when distinguishing low baPWV and high baPWV in patients with diabetes, which was higher than that for PLR AUC (0.6477) and MLR AUC (0.6479), and the combined diagnosis for AUC. Conclusions: NLR was superior to PLR, and MLR and combined diagnosis have certain predictive values that indicate the increase in arterial stiffness in patients with diabetes. These predictive values can help with the early identification of increased arterial stiffness in patients with diabetes.

Vitexin, a fenugreek glycoside, ameliorated obesity-induced diabetic nephropathy via modulation of NF-κB/IkBα and AMPK/ACC pathways in mice.

Obesity is one of the most critical risk factors for diabetes mellitus and plays a significant role in diabetic nephropathy (DN). The present investigation aimed to evaluate the possible mechanism of action of vitexin on obesity-induced DN in a high-fat diet (HFD)-fed experimental C57BL/6 mice model. Obesity was induced in male C57BL/6 mice by chronic administration of HFD, and mice were concomitantly treated with vitexin (15, 30, and 60 mg/kg, p.o.). HFD-induced increased renal oxido-nitrosative stress and proinflammatory cytokine levels were significantly inhibited by vitexin. The Western blot analysis suggested that alteration in renal NF-κB, IκBα, nephrin, AMPK, and ACC phosphorylation levels was effectively restored by vitexin treatment. Histological aberration induced in renal tissue after chronic administration of HFD was also reduced by vitexin. In conclusion, vitexin suppressed the progression of obesity-induced DN via modulation of NF-κB/IkBα and AMPK/ACC pathways in an experimental model of HFD-induced DN in C57BL/6J mice.

Clindamycin-induced Kidney Diseases: A Retrospective Analysis of 50 Patients.

Objective There are many adverse reactions due to clindamycin, but kidney diseases (acute kidney injury, AKI) are uncommon. However, in recent years, the rate of clindamycin-induced kidney diseases has increased. We analyzed 50 patients with clindamycin-induced kidney diseases retrospectively, and investigated the characteristics of these kidney diseases in order to provide a reference for rational clinical drug use and to reduce drug-induced organ damage. Methods We investigated 50 patients diagnosed with clindamycin-induced kidney diseases retrospectively at the Department of Nephrology, Shandong University Qilu Hospital, from January 2009 to December 2013. The parameters included in our study were age, sex, clinical manifestations, efficacy and prognosis. Results All patients were diagnosed with clindamycin-induced kidney diseases within 48 hours of the application of clindamycin at 1.0-2.0 g/day. The patients included 29 women and 21 men. Most of the enrolled patients were 20-59 years old. Fifty-one patients were diagnosed with AKI stage 3 upon admission. Thirty-three had episodes of gross hematuria, but fever, skin rash and eosinophilia were rare. Urine analysis revealed mild proteinuria and severe tubular dysfunction. In the majority of patients, AKI was severe and required renal replacement therapy, but renal function in all patients had recovered significantly two months after discharge. Conclusion Clindamycin-induced AKI is largely reversible and is associated with episodes of gross hematuria. Clinicians should use clindamycin rationally and reduce the incidence of adverse reactions.

Prostaglandin signalling regulates ciliogenesis by modulating intraflagellar transport.

Cilia are microtubule-based organelles that mediate signal transduction in a variety of tissues. Despite their importance, the signalling cascades that regulate cilium formation remain incompletely understood. Here we report that prostaglandin signalling affects ciliogenesis by regulating anterograde intraflagellar transport (IFT). Zebrafish leakytail (lkt) mutants show ciliogenesis defects, and the lkt locus encodes an ATP-binding cassette transporter (ABCC4). We show that Lkt/ABCC4 localizes to the cell membrane and exports prostaglandin E2 (PGE2), a function that is abrogated by the Lkt/ABCC4(T804M) mutant. PGE2 synthesis enzyme cyclooxygenase-1 and its receptor, EP4, which localizes to the cilium and activates the cyclic-AMP-mediated signalling cascade, are required for cilium formation and elongation. Importantly, PGE2 signalling increases anterograde but not retrograde velocity of IFT and promotes ciliogenesis in mammalian cells. These findings lead us to propose that Lkt/ABCC4-mediated PGE2 signalling acts through a ciliary G-protein-coupled receptor, EP4, to upregulate cAMP synthesis and increase anterograde IFT, thereby promoting ciliogenesis.

Zip1, Zip2, and Zip8 mRNA expressions were associated with growth hormone level during the growth hormone provocation test in children with short stature.

Short stature of children is affected by multiple factors. One of them is growth hormone (GH) deficiency. Growth hormone therapy can increase the final height of children with growth hormone deficiency. Zinc is found to induce dimerization and to enhance the bioactivity of human GH. Two gene families have been identified involved in zinc homeostasis. Previous studies in our laboratory have shown that Zip1, Zip2, Zip6, and ZnT1 mRNA were associated with zinc level in established human breast cancer in nude mice model; Zip8 was significantly lower in zinc-deficient Wistar rats in kidney. In this study, five zinc transporters: Zip1, Zip2, Zip6, Zip8, and ZnT1 were chosen. We aimed to investigate the mRNA expression of zinc transporters and to explore the relationship between zinc transporters and growth hormone in short stature children. Growth hormone provocation test is used to confirm the diagnosis of growth hormone deficiency. Six short children for the test were enrolled. At the same time, 15 sex- and age-matched normal children were enrolled as control. The expression levels of zinc transporters in peripheral blood mononuclear cells were determined by quantitative real-time PCR. Zip1 and Zip2 mRNA expression positively correlated with growth hormone level (r = 0.5133, P = 0.0371; r = 0.6719, P = 0.0032); Zip8 mRNA expression negatively correlated with growth hormone level (r = -0.5264, P = 0.0285) during the test in short stature children. The average expression level of Zip2 was significantly higher and Zip6, Zip8 mRNA levels were significantly lower in short stature children than in health controls at 0 min (P < 0.05, P < 0.05).

Pre-gastrula expression of zebrafish extraembryonic genes.

BACKGROUND: Many species form extraembryonic tissues during embryogenesis, such as the placenta of humans and other viviparous mammals. Extraembryonic tissues have various roles in protecting, nourishing and patterning embryos. Prior to gastrulation in zebrafish, the yolk syncytial layer - an extraembryonic nuclear syncytium - produces signals that induce mesoderm and endoderm formation. Mesoderm and endoderm precursor cells are situated in the embryonic margin, an external ring of cells along the embryo-yolk interface. The yolk syncytial layer initially forms below the margin, in a domain called the external yolk syncytial layer (E-YSL). RESULTS: We hypothesize that key components of the yolk syncytial layer's mesoderm and endoderm inducing activity are expressed as mRNAs in the E-YSL. To identify genes expressed in the E-YSL, we used microarrays to compare the transcription profiles of intact pre-gastrula embryos with pre-gastrula embryonic cells that we had separated from the yolk and yolk syncytial layer. This identified a cohort of genes with enriched expression in intact embryos. Here we describe our whole mount in situ hybridization analysis of sixty-eight of them. This includes ten genes with E-YSL expression (camsap1l1, gata3, znf503, hnf1ba, slc26a1, slc40a1, gata6, gpr137bb, otop1 and cebpa), four genes with expression in the enveloping layer (EVL), a superficial epithelium that protects the embryo (zgc:136817, zgc:152778, slc14a2 and elovl6l), three EVL genes whose expression is transiently confined to the animal pole (elovl6l, zgc:136359 and clica), and six genes with transient maternal expression (mtf1, wu:fj59f04, mospd2, rftn2, arrdc1a and pho). We also assessed the requirement of Nodal signaling for the expression of selected genes in the E-YSL, EVL and margin. Margin expression was Nodal dependent for all genes we tested, including the concentrated margin expression of an EVL gene: zgc:110712. All other instances of EVL and E-YSL expression that we tested were Nodal independent. CONCLUSION: We have devised an effective strategy for enriching and identifying genes expressed in the E-YSL of pre-gastrula embryos. To our surprise, maternal genes and genes expressed in the EVL were also enriched by this strategy. A number of these genes are promising candidates for future functional studies on early embryonic patterning.

Vertebrate heart growth is regulated by functional antagonism between Gridlock and Gata5.

Embryonic organs attain their final dimensions through the generation of proper cell number and size, but the control mechanisms remain obscure. Here, we establish Gridlock (Grl), a Hairy-related basic helix-loop-helix (bHLH) transcription factor, as a negative regulator of cardiomyocyte proliferative growth in zebrafish embryos. Mutations in grl cause an increase in expression of a group of immediate-early growth genes, myocardial genes, and development of hyperplastic hearts. Conversely, cardiomyocytes with augmented Grl activity have diminished cell volume and fail to divide, resulting in a marked reduction in heart size. Both bHLH domain and carboxyl region are required for Grl negative control of myocardial proliferative growth. These Grl-induced cardiac effects are counterbalanced by the transcriptional activator Gata5 but not Gata4, which promotes cardiomyocyte expansion in the embryo. Biochemical analyses show that Grl forms a complex with Gata5 through the carboxyl region and can repress Gata5-mediated transcription via the bHLH domain. Hence, our studies suggest that Grl regulates embryonic heart growth via opposing Gata5, at least in part through their protein interactions in modulating gene expression.

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter.

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development.

[Short-term effect of paclitaxel-eluting stent in the treatment of coronary heart disease due to coronary arteriosclerosis].

OBJECTIVE: To evaluate the short-term safety and efficacy of paclitaxel-eluting stent (TAXUS stent, Boston Scientific) in the treatment of coronary heart disease (CHD) due to coronary arteriosclerosis. METHODS: From July 2003 to November 2004, 300 consecutive patients with CHD due to coronary arteriosclerosis were admitted for selective percutaneous coronary intervention (PCI) and paclitaxel-eluting stent implantation in the coronary arteries. The immediate effects after PCI and follow-up results were investigated. RESULTS: Altogether 350 lesions were treated and 355 paclitaxel-eluting stents implanted in the 300 cases. Of these lesions, 248 (70.9%) was complicated lesions of B2 type or worse, 94 (26.5%) small-caliber stents (2.50-2.75 mm) and 130 (36.6%) long stents (>20 mm) were implanted, without occurrence of severe intra-operative complications. Follow-up study of 250 cases (83.3%) lasting for 1 to 15 months was conducted, and chest pain was reported in 8 cases, 2 of which underwent coronary artery angiography and no in-stent restenosis was found. One patient developed myocardial infarction 5 months after PCI, and 2 died for non-cardiogenic factors. CONCLUSION: Paclitaxel-eluting stent implantation in patients undergoing PCI is safe approach with good short-term efficacy.

Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system.

To test the Cre/loxP recombination system in zebrafish, a stable transgenic zebrafish line was developed by using a floxed (loxP flanked) gfp (green fluorescent protein) gene construct under the muscle-specific mylz2 promoter. Like our previous non-floxed gfp transgenic line under the same promoter, the new transgenic line expresses GFP reporter faithfully in fast skeletal muscles to the same intensity. To demonstrate the excision of floxed gfp transgene, in vitro synthesized Cre RNA was injected into embryos of floxed gfp transgenic zebrafish and we found a dramatic reduction of GFP expression. To confirm the excision, PCR was performed and a DNA fragment of correct size was amplified as predicted from the Cre/loxP mediated excision. Finally, we cloned the fragment and sequence information confirmed that the excision occurred at the precise site as predicted. Our experiments demonstrated that the Cre/loxP system can function efficiently and accurately in the zebrafish system.

Expression analyses of zebrafish transferrin, ifabp, and elastaseB mRNAs as differentiation markers for the three major endodermal organs: liver, intestine, and exocrine pancreas.

In the present work, three zebrafish cDNA clones encoding transferrin, intestinal fatty acid binding protein (IFABP), and elastaseB were cloned and their expression patterns in early zebrafish development were characterized as differentiation markers for the three major endoderm organs: liver, intestine, and exocrine pancreas. transferrin and ifabp mRNAs exhibit a biphasic expression pattern during early development. transferrin mRNAs were first expressed at approximately 7 hours postfertilization (hpf) in the yolk syncytial layer (YSL) and later in the liver rudiment (from approximately 48 hpf) and in the esophagus transiently (72-96 hpf). Ifabp mRNAs were initially expressed in the YSL at the ventral side during late epiboly (8-9 hpf), spread throughout the YSL of later stage embryos, and appeared in the intestine rudiment at approximately 36 hpf. In contrast to the transferrin and ifabp mRNAs, elastaseB mRNAs were not expressed in the yolk sac or YSL, and these transcripts were detected exclusively in the exocrine pancreas after approximately 56 hpf.

Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle.

In the present study, new applications of the transgenic technology in developing novel varieties of ornamental fish and bioreactor fish were explored in a model fish, the zebrafish (Danio rerio). Three "living color" fluorescent proteins, green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP or dsRed), were expressed under a strong muscle-specific mylz2 promoter in stable lines of transgenic zebrafish. These transgenic zebrafish display vivid fluorescent colors (green, red, yellow, or orange) visible to unaided eyes under both daylight and ultraviolet light in the dark. The level of foreign protein expression is estimated between 3% and 17% of total muscle proteins, equivalent to 4.8-27.2mg/g wet muscle tissue. Thus, the fish muscle may be explored as another useful bioreactor system for production of recombinant proteins. In spite of the high level of foreign protein expression, the expression of endogenous mylz2 mRNAs was not negatively affected. Furthermore, compared to the wild-type fish, these fluorescent transgenic fish have no advantage in survival and reproduction.

Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter.

A 1,934-bp muscle-specific promoter from the zebrafish mylz2 gene was isolated and characterized by transgenic analysis. By using a series of 5' promoter deletions linked to the green fluorescent protein (gfp) reporter gene, transient transgenic analysis indicated that the strength of promoter activity appeared to correlate to the number of muscle cis-elements in the promoter and that a minimal -77-bp region was sufficient for a relatively strong promoter activity in muscle cells. Stable transgenic lines were obtained from several mylz2-gfp constructs. GFP expression in the 1,934-bp promoter transgenic lines mimicked well the expression pattern of endogenous mylz2 mRNA in both somitic muscle and nonsomitic muscles, including fin, eye, jaw, and gill muscles. An identical pattern of GFP expression, although at a much lower level, was observed from a transgenic line with a shorter 871-bp promoter. Our observation indicates that there is no distinct cis-element for activation of mylz2 in different skeletal muscles. Furthermore, RNA encoding a dominant negative form of cAMP-dependent protein kinase A was injected into mylz2-gfp transgenic embryos and GFP expression was significantly reduced due to an expanded slow muscle development at the expense of GFP-expressing fast muscle. The mylz2-gfp transgene was also transferred into two zebrafish mutants, spadetail and chordino, and several novel phenotypes in muscle development in these mutants were discovered.