Nephroprotective effects of coriander (Coriandrum sativum) leaves aqueous extracts in aristolochic acid-intoxicated zebrafish embryos.

Coriander is a notable medicinal plant known for its diverse properties, including anti-inflammatory, antioxidant, anticancer, analgesic, and anti-diabetic effects. Despite its recognized health benefits, research on its nephroprotective properties is limited. This study aimed to investigate the potential nephroprotective properties of an aqueous extract derived from coriander leaves using an aristolochic acid-intoxicated zebrafish model. To assess kidney abnormalities induced by aristolochic acid (AA), we utilized the transgenic line Tg(wt1b:egfp), which expresses green fluorescent protein (GFP) in the kidney. Our previous report indicated that AA exposure leads to acute renal failure in zebrafish characterized by kidney malformation and impaired renal function. However, pretreatment of coriander extract (CE) can mitigate kidney malformations induced by AA. In addition, CE pretreatment reduces the accumulation of red blood cells in the glomerular region. To verify the nephroprotective effects of CE, we analyzed renal function by measuring the glomerular filtration rate in zebrafish embryos. Results indicate that CE partially mitigates renal function impairment caused by AA exposure, suggesting its potential to attenuate AA-induced renal failure. Mechanistically, pretreatment with CE reduces the expression of proinflammatory and proapoptotic genes induced by AA. This suggests that CE likely alleviates acute renal failure by reducing inflammation and apoptosis. As a result, we regard zebrafish as a valuable model for screening natural compounds that have the potential to alleviate AA-induced nephrotoxicity.

Combination treatment of Src inhibitor Saracatinib with GMI, a Ganoderma microsporum immunomodulatory protein, induce synthetic lethality via autophagy and apoptosis in lung cancer cells.

Saracatinib is an oral Src-kinase inhibitor and has been studied in preclinical models and clinical trials of cancer therapy. GMI, a fungal immunomodulatory protein from Ganoderma microsporum, possesses antitumor capacity. The aim of this study is to evaluate the cytotoxic effect of combination treatment with saracatinib and GMI on parental and pemetrexed-resistant lung cancer cells. Cotreatment with saracatinib and GMI induced synergistic and additive cytotoxic effect in A549 and A400 cells by annexin V/propidium iodide assay and combination index. Using western blot assay, saracatinib, and GMI combined treatment synergistically induced caspase-7 activation in A549 cells. Different from A549 cells, saracatinib and GMI cotreatment markedly increased LC3B-II in A400 cells. ATG5 silencing abolished the caspase-7 activation and reduced cell death in A549 cells after cotreatment. This is the first study to provide a novel strategy of treating lung cancer with or without drug resistance via combination treatment with GMI and saracatinib.

Regulation of Adipogenesis and Lipid Deposits by Collapsin Response Mediator Protein 2.

As emerging evidence suggesting neurodegenerative diseases and metabolic diseases have common pathogenesis, we hypothesized that the neurite outgrowth-controlling collapsin response mediator protein 2 (CRMP2) was involved in energy homeostasis. Therefore, putative roles of CRMP2 in adipocyte differentiation (adipogenesis) and lipid metabolism were explored and addressed in this study. CRMP2 expression profiles were in vitro and in vivo characterized during adipogenic process of 3T3-L1 pre-adipocytes and diet-induced obese (DIO) mice, respectively. Effects of CRMP2 on lipid metabolism and deposits were also analyzed. Our data revealed that CRMP2 expression pattern was coupled with adipogenic stages. CRMP2 overexpression inhibited cell proliferation at MCE phase, and significantly reduced lipid contents by down-regulating adipogenesis-driving transcription factors and lipid-synthesizing enzymes. Interestingly, GLUT4 translocation and the lipid droplets fusion were disturbed in CRMP2-silencing cells by affecting actin polymerization. Moreover, adipose CRMP2 was significantly increased in DIO mice, indicating CRMP2 is associated with obesity. Accordingly, CRMP2 exerts multiple functions in adipogenesis and lipid deposits through mediating cell proliferation, glucose/lipid metabolism and cytoskeleton dynamics. The present study identifies novel roles of CRMP2 in mediating adipogenesis and possible implication in metabolic disorders, as well as provides molecular evidence supporting the link of pathogenesis between neurodegenerative diseases and metabolic abnormalities.

Oxygenated Water Inhibits Adipogenesis and Attenuates Hepatic Steatosis in High-Fat Diet-Induced Obese Mice.

The expansion of adipose tissue mass is the primary characteristic of the process of becoming obesity, which causes chronic adipose inflammation and is closely associated with type 2 diabetes mellitus (T2DM). Adipocyte hypertrophy restricts oxygen availability, leading to microenvironmental hypoxia and adipose dysfunction. This study aimed at investigating the effects of oxygenated water (OW) on adipocyte differentiation (adipogenesis) and the metabolic function of mature adipocytes. The effects of OW on adipogenesis and the metabolic function of mature adipocytes were examined. Meanwhile, the in vivo metabolic effects of long-term OW consumption on diet-induced obesity (DIO) mice were investigated. OW inhibited adipogenesis and lipid accumulation through down-regulating critical adipogenic transcription factors and lipogenic enzymes. While body weight, blood and adipose parameters were not significantly improved by long-term OW consumption, transient circulatory triglyceride-lowering and glucose tolerance-improving effects were identified. Notably, hepatic lipid contents were significantly reduced, indicating that the DIO-induced hepatic steatosis was attenuated, despite no improvements in fibrosis and lipid contents in adipose tissue being observed in the OW-drinking DIO mice. The study provides evidence regarding OW's effects on adipogenesis and mature adipocytes, and the corresponding molecular mechanisms. OW exhibits transient triglyceride-lowering and glucose tolerance-improving activity as well as hepatic steatosis-attenuating functions.

Interleukin-4 Promotes Myogenesis and Boosts Myocyte Insulin Efficacy.

Anti-inflammatory cytokine interleukin-4 (IL-4) promotes glucose tolerance and insulin sensitivity while reduces lipid deposits. However, the effects of IL-4 on energy metabolism in muscle, the largest insulin-targeting organ, remain obscure. The study aimed at addressing the roles of IL-4 in myocyte differentiation (myogenesis) and energy metabolism of muscle cells. Effects of IL-4 on myogenesis, and interaction between IL-4 and insulin on glucose metabolism of C2C12 myoblasts and the terminal differentiated myocytes were analyzed. IL-4 improved GLUT4 translocation and tended to elevate glucose uptake by boosting insulin signaling. In diabetic mice, transient and long-term IL-4 showed differential effects on insulin signaling and efficacy. The study provides evidence to address the roles of IL-4 in mediating whole-body muscle reservoir and glucose metabolism, as well as the interaction between immune responses and energy homeostasis. IL-4 has dual potential to act as an adjuvant therapeutic target for sarcopenia to preserve muscle mass and insulin resistance to improve insulin sensitivity, which implicates the regulation of immune system to the muscle differentiation and exercise performance.

Embryonic exposure to 4-methylimidazole leads to zebrafish myofibril misalignment.

4-methylimidazole (4-MI) is an imidazole-derived organic chemical compound that can be used as a raw material in the manufacture of diverse chemicals and has been identified as an ingredient of caramel color in soybean sauce, beers, and other soft drinks. The aim of the present study was to investigate the teratogenic effects of 4-MI during zebrafish embryogenesis. Zebrafish embryos were treated with different dosages of 4-MI (0-120 mM) for different exposure durations (12-60 hours). The percentages of embryos with malformed phenotypes increased as the exposure dosages and duration time of 4-MI increased. We also used immunofluorescence and transmission microscopy to evaluate the subtle changes in the myofibril alignment and ultrastructure of muscle organization. Our data showed that 4-MI treatment disturbs muscle fiber alignment. Electron microscopy data indicated that Z-lines were undetectable in the 4-MI-treated embryos. Although the thick and thin filaments were visible, they were all disorganized. In addition, zebrafish embryos treated by 4-MI exhibited aberrant expression of 2 muscle-specific genes, myod and myogenin. Taken together, we concluded that early exposure to 4-MI affects zebrafish myogenesis, especially in myofibril alignment.

N-Glycosylation of Human R-Spondin 1 Is Required for Efficient Secretion and Stability but Not for Its Heparin Binding Ability.

R-spondin 1 (Rspo1) plays an essential role in stem cell biology by potentiating Wnt signaling activity. Despite the fact that Rspo1 holds therapeutic potential for a number of diseases, its biogenesis is not fully elucidated. All Rspo proteins feature two amino-terminal furin-like repeats, which are responsible for Wnt signal potentiation, and a thrombospondin type 1 (TSR1) domain that can provide affinity towards heparan sulfate proteoglycans. Using chemical inhibitors, deglycosylase and site-directed mutagenesis, we found that human Rspo1 and Rspo3 are both N-glycosylated at N137, a site near the C-terminus of the furin repeat 2 domain, and Rspo2 is N-glycosylated at N160, a position near the N-terminus of TSR1 domain. Elimination of N-glycosylation at these sites affects their accumulation in media but have no effect on the ability towards heparin. Introduction of the N-glycosylation site to Rspo2 mutant at the position homologous to N137 in Rspo1 restored full glycosylation and rescued the accumulation defect of nonglycosylated Rspo2 mutant in media. Similar effect can be observed in the N137 Rspo1 or Rspo3 mutant engineered with Rspo2 N-glycosylation site. The results highlight the importance of N-glycosylation at these two positions in efficient folding and secretion of Rspo family. Finally, we further showed that human Rspo1 is subjected to endoplasmic reticulum (ER) quality control in N-glycan-dependent manner. While N-glycan of Rspo1 plays a role in its intracellular stability, it had little effect on secreted Rspo1. Our findings provide evidence for the critical role of N-glycosylation in the biogenesis of Rspo1.

Knocking down 10-Formyltetrahydrofolate dehydrogenase increased oxidative stress and impeded zebrafish embryogenesis by obstructing morphogenetic movement.

BACKGROUND: Folate is an essential nutrient for cell survival and embryogenesis. 10-Formyltetrahydrofolate dehydrogenase (FDH) is the most abundant folate enzyme in folate-mediated one-carbon metabolism. 10-Formyltetrahydrofolate dehydrogenase converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2, the only pathway responsible for formate oxidation in methanol intoxication. 10-Formyltetrahydrofolate dehydrogenase has been considered a potential chemotherapeutic target because it was down-regulated in cancer cells. However, the normal physiological significance of 10-Formyltetrahydrofolate dehydrogenase is not completely understood, hampering the development of therapeutic drug/regimen targeting 10-Formyltetrahydrofolate dehydrogenase. METHODS: 10-Formyltetrahydrofolate dehydrogenase expression in zebrafish embryos was knocked-down using morpholino oligonucleotides. The morphological and biochemical characteristics of fdh morphants were examined using specific dye staining and whole-mount in-situ hybridization. Embryonic folate contents were determined by HPLC. RESULTS: The expression of 10-formyltetrahydrofolate dehydrogenase was consistent in whole embryos during early embryogenesis and became tissue-specific in later stages. Knocking-down fdh impeded morphogenetic movement and caused incorrect cardiac positioning, defective hematopoiesis, notochordmalformation and ultimate death of morphants. Obstructed F-actin polymerization and delayed epiboly were observed in fdh morphants. These abnormalities were reversed either by adding tetrahydrofolate or antioxidant or by co-injecting the mRNA encoding 10-formyltetrahydrofolate dehydrogenase N-terminal domain, supporting the anti-oxidative activity of 10-formyltetrahydrofolate dehydrogenase and the in vivo function of tetrahydrofolate conservation for 10-formyltetrahydrofolate dehydrogenase N-terminal domain. CONCLUSIONS: 10-Formyltetrahydrofolate dehydrogenase functioned in conserving the unstable tetrahydrofolate and contributing to the intracellular anti-oxidative capacity of embryos, which was crucial in promoting proper cell migration during embryogenesis. GENERAL SIGNIFICANCE: These newly reported tetrahydrofolate conserving and anti-oxidative activities of 10-formyltetrahydrofolate dehydrogenase shall be important for unraveling 10-formyltetrahydrofolate dehydrogenase biological significance and the drug development targeting 10-formyltetrahydrofolate dehydrogenase.

Pro-Angiogenic Effects of Chalcone Derivatives in Zebrafish Embryos in Vivo.

The aim of this study was to investigate novel chalcones with potent angiogenic activities in vivo. Chalcone-based derivatives were evaluated using a transgenic zebrafish line with fluorescent vessels to real-time monitor the effect on angiogenesis. Results showed that the chalcone analogues did not possess anti-angiogenic effect on zebrafish vasculatures; instead, some of them displayed potent pro-angiogenic effects on the formation of the sub-intestinal vein. Similar pro-angiogenic effects can also be seen on wild type zebrafish embryos. Moreover, the expression of vegfa, the major regulator for angiogenesis, was also upregulated in their treatment. Taken together, we have synthesized and identified a series of novel chalcone-based derivatives as potent in vivo pro-angiogenic compounds. These novel compounds hold potential for therapeutic angiogenesis.

Exposure to benzidine caused apoptosis and malformation of telencephalon region in zebrafish.

Exposure to benzidine has been known to induce human cancers, particularly bladder carcinomas. In this study, the zebrafish model was used to investigate the developmental toxicity of benzidine. Embryos at 6 h postfertilization (hpf) that were exposed to benzidine exhibited embryonic death in a dose- and time-dependent manner. Benzidine induced malformations in zebrafish, such as small brain development, shorter axes, and a slight pericardial edema. High concentrations (50, 100, and 200 µM) of benzidine triggered widespread apoptosis in the brain and dorsal neurons, as evidenced by acridine orange and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays. Real-time polymerase chain reaction analysis also showed that benzidine treatment affected p53, bax, and noxa expression. Decreases in specific brain markers, such as emx1 in the telencephalon, ngn1 in differentiated neurons, and otx2 in the midbrain, were observed in benzidine-treated embryos at 24 hpf. Conversely, no overt changes to pax2.1 expression in the midbrain-hindbrain boundary were found. Moreover, the use of Tg(HuC:GFP) zebrafish showed that benzidine caused a malformation of the telencephalon region. Our findings show that benzidine exposure triggers widespread apoptosis in the zebrafish brain and dorsal neurons, resulting in the development of an abnormal telencephalon.

Caffeine treatment disturbs the angiogenesis of zebrafish embryos.

Caffeine is a widely consumed substance that occurs in numerous dietary sources, but teratogenic effects of caffeine intake during embryonic development are still not clear. In the present study, we used the zebrafish as a model to assess caffeine-induced toxicity on embryonic vascular development. A green fluorescent vascular endothelium transgenic line, Tg(fli1:egfp), was utilized for the sensitive detection of vascular development, including vasculo- and angiogenesis. Caffeine-treated embryos showed no defects in vasculogenesis, but revealed dose-dependent (250-350 ppm) developmental defects in intersegmental vessels, dorsal longitudinal anastomotic vessels, and subintestinal vein sprouting. Further, real-time polymerase chain reaction analysis of caffeine-treated embryos showed an upregulation of nrp1a along with a downregulation of sema3aa and sema3c. In conclusion, caffeine treatment induces defects of angiogenesis in zebrafish embryos.

Zebrafish cyclin-dependent protein kinase-like 1 (zcdkl1): identification and functional characterization.

The cyclin-dependent protein kinase family regulates a wide range of cellular functions such as cell cycle progression, differentiation, and apoptosis. In this study, we identified a zebrafish cyclin-dependent protein kinase-like 1 protein called zebrafish cdkl1 (zcdkl1), which shared a high degree of homology and conserved synteny with mammalian orthologs. zcdkl1 exhibited abilities for phosphorylation of myelin basic protein and histone H1. RT-PCR analysis revealed that zcdkl1 was expressed starting from fertilization and continuing thereafter. In adult tissues, zcdkl1 was predominantly detected in brain, ovary, and testis, and was expressed at low levels in other tissues. At 50% epiboly stage, zcdkl1 was widely expressed. At 12 to 48 h post-fertilization, zcdkl1 was predominantly expressed in the hypochord, the medial and lateral floor plate, and the pronephric duct. Interference of zcdkl1 expression resulted in abnormalities, such as brain and eye malformation, pericardial edema, and body axis curvature. Disruption of zcdkl1 reduced neurogenin-1 in the brain and sonic hedgehog expression in the floor plate region. These deformities were apparently rescued by co-injection of zcdkl1 mRNA. Findings of this study indicate that zcdkl1 plays an essential role in zebrafish development.

Administration of Lactobacillus reuteri Combined with Clostridium butyricum Attenuates Cisplatin-Induced Renal Damage by Gut Microbiota Reconstitution, Increasing Butyric Acid Production, and Suppressing Renal Inflammation.

Cisplatin-induced nephrotoxicity is associated with gut microbiota disturbance. The present study aimed to investigate whether supplementation of Lactobacillus reuteri and Clostridium butyricum (LCs) had a protective effect on cisplatin-induced nephrotoxicity through reconstruction of gut microbiota. Wistar rats were given different treatments: control, cisplatin (Cis), cisplatin + C. butyricum and L. reuteri (Cis+LCs), and C. butyricum and L. reuteri (LCs). We observed that cisplatin-treated rats supplemented with LCs exhibited significantly decreased renal inflammation (KIM-1, F4/80, and MPO), oxidative stress, fibrosis (collagen IV, fibronectin, and a-SMA), apoptosis, concentration of blood endotoxin and indoxyl sulfate, and increased fecal butyric acid production compared with those without supplementation. In addition, LCs improved the cisplatin-induced microbiome dysbiosis by maintaining a healthy gut microbiota structure and diversity; depleting Escherichia-Shigella and the Enterobacteriaceae family; and enriching probiotic Bifidobacterium, Ruminococcaceae, Ruminiclostridium_9, and Oscillibacter. Moreover, the LCs intervention alleviated the cisplatin-induced intestinal epithelial barrier impairment. This study indicated LCs probiotic serves as a mediator of the gut-kidney axis in cisplatin-induced nephrotoxicity to restore the intestinal microbiota composition, thereby suppressing uremic toxin production and enhancing butyrate production. Furthermore, the renoprotective effect of LCs is partially mediated by increasing the anti-inflammatory effects and maintaining the integrity of the intestinal barrier.

A novel zebrafish model to emulate lung injury by folate deficiency-induced swim bladder defectiveness and protease/antiprotease expression imbalance.

Lung injury is one of the pathological hallmarks of most respiratory tract diseases including asthma, acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD). It involves progressive pulmonary tissue damages which are usually irreversible and incurable. Therefore, strategies to facilitate drug development against lung injury are needed. Here, we characterized the zebrafish folate-deficiency (FD) transgenic line that lacks a fully-developed swim bladder. Whole-mount in-situ hybridization revealed comparable distribution patterns of swim bladder tissue markers between wild-type and FD larvae, suggesting a proper development of swim bladder in early embryonic stages. Unexpectedly, neutrophils infiltration was not observed in the defective swim bladder. Microarray analysis revealed a significant increase and decrease of the transcripts for cathepsin L and a cystatin B (CSTB)-like (zCSTB-like) proteins, respectively, in FD larvae. The distribution of cathepsin L and the zCSTB-like transcripts was spatio-temporally specific in developing wild-type embryos and, in appropriate measure, correlated with their potential roles in maintaining swim bladder integrity. Supplementing with 5-formyltetrahydrofolate successfully prevented the swim bladder anomaly and the imbalanced expression of cathepsin L and the zCSTB-like protein induced by folate deficiency. Injecting the purified recombinant zebrafish zCSTB-like protein alleviated FD-induced swim bladder anomaly. We concluded that the imbalanced expression of cathepsin L and the zCSTB-like protein contributed to the swim bladder malformation induced by FD and suggested the potential application of this transgenic line to model the lung injury and ECM remodeling associated with protease/protease inhibitor imbalance.

Characterization and comparative studies of zebrafish and human recombinant dihydrofolate reductases--inhibition by folic acid and polyphenols.

Dihydrofolate reductase (DHFR) catalyzes folic acid reduction and recycles dihydrofolate generated during dTMP biosynthesis to tetrahydrofolate. DHFR is the main target of methotrexate, the most widely used agent for antifolate therapy. Nevertheless, the emergence of methotrexate-resistance has greatly impeded the curative potential of this drug. Therefore, drugs with improved efficacy are still in demand, as well as an efficient in vitro assay system and animal model for antifolate drug discovery. The aim of this study is to evaluate the suitability of using zebrafish DHFR as an alternative assay system for antifolate drug discovery. The cDNAs encoding zebrafish and human DHFR were cloned, overexpressed, and purified. Similar structural and kinetic properties were revealed between zebrafish and human recombinant DHFRs. The susceptibilities of both enzymes to known DHFR inhibitors, including methotrexate and trimethoprim, and compounds with antifolate potential, such as polyphenols, are also comparable. In addition, the DHFR-mediated dihydrofolate reduction was significantly inhibited by its own substrate folic acid. An unexpected tissue-specific distribution of DHFR was observed with the highest level present in ova and brains of zebrafish. DHFR is also abundant in zebrafish embryos of early stages and decreased abruptly after 3 days postfertilization. The substantial resemblance between zebrafish and human DHFRs, as demonstrated in this study, provides compelling evidence supporting the use of zebrafish DHFR as an in vitro assay system for folate-related studies and drug discovery.

Glycogen synthase kinase 3 alpha and 3 beta have distinct functions during cardiogenesis of zebrafish embryo.

BACKGROUND: Glycogen synthase kinase 3 (GSK3) encodes a serine/threonine protein kinase, is known to play roles in many biological processes. Two closely related GSK3 isoforms encoded by distinct genes: GSK3alpha (51 kDa) and GSK3beta (47 kDa). In previously studies, most GSK3 inhibitors are not only inhibiting GSK3, but are also affecting many other kinases. In addition, because of highly similarity in amino acid sequence between GSK3alpha and GSK3beta, making it difficult to identify an inhibitor that can be selective against GSK3alpha or GSK3beta. Thus, it is relatively difficult to address the functions of GSK3 isoforms during embryogenesis. At this study, we attempt to specifically inhibit either GSK3alpha or GSK3beta and uncover the isoform-specific roles that GSK3 plays during cardiogenesis. RESULTS: We blocked gsk3alpha and gsk3beta translations by injection of morpholino antisense oligonucleotides (MO). Both gsk3alpha- and gsk3beta-MO-injected embryos displayed similar morphological defects, with a thin, string-like shaped heart and pericardial edema at 72 hours post-fertilization. However, when detailed analysis of the gsk3alpha- and gsk3beta-MO-induced heart defects, we found that the reduced number of cardiomyocytes in gsk3alpha morphants during the heart-ring stage was due to apoptosis. On the contrary, gsk3beta morphants did not exhibit significant apoptosis in the cardiomyocytes, and the heart developed normally during the heart-ring stage. Later, however, the heart positioning was severely disrupted in gsk3beta morphants. bmp4 expression in gsk3beta morphants was up-regulated and disrupted the asymmetry pattern in the heart. The cardiac valve defects in gsk3beta morphants were similar to those observed in axin1 and apcmcr mutants, suggesting that GSK3beta might play a role in cardiac valve development through the Wnt/beta-catenin pathway. Finally, the phenotypes of gsk3alpha mutant embryos cannot be rescued by gsk3beta mRNA, and vice versa, demonstrating that GSK3alpha and GSK3beta are not functionally redundant. CONCLUSION: We conclude that (1) GSK3alpha, but not GSK3beta, is necessary in cardiomyocyte survival; (2) the GSK3beta plays important roles in modulating the left-right asymmetry and affecting heart positioning; and (3) GSK3alpha and GSK3beta play distinct roles during zebrafish cardiogenesis.

One crisis, diverse impacts-Tissue-specificity of folate deficiency-induced circulation defects in zebrafish larvae.

Folate (vitamin B9) is an essential nutrient required for cell survival, proliferation, differentiation and therefore embryogenesis. Folate deficiency has been associated with many diseases, including congenital heart diseases and megaloblastic anemia, yet the mechanisms underlying these remains elusive. Here, we examine the impact of folate deficiency on the development of the circulation system using a zebrafish transgenic line which displays inducible folate deficiency. Impaired hematopoiesis includes decreased hemoglobin levels, decreased erythrocyte number, increased erythrocyte size and aberrant c-myb expression pattern were observed in folate deficient embryos. Cardiac defects, including smaller chamber size, aberrant cardiac function and cmlc2 expression pattern, were also apparent in folate deficient embryos. Characterization of intracellular folate content in folate deficiency revealed a differential fluctuation among the different folate derivatives that carry a single carbon group at different oxidation levels. Rescue attempts by folic acid and nucleotides resulted in differential responses among affected tissues, suggesting that different pathomechanisms are involved in folate deficiency-induced anomalies in a tissue-specific manner. The results of the current study provide an explanation for the inconsistent outcome observed clinically in patients suffering from folate deficiency and/or receiving folate supplementation. This study also supports the use of this model for further research on the defective cardiogenesis and hematopoiesis caused by folate deficiency.

The Transgenic Zebrafish Display Fluorescence Reflecting the Expressional Dynamics of Dihydrofolate Reductase.

Dihydrofolate reductase (DHFR) reduces folic acid and recycles dihydrofolate generated during dTMP biosynthesis to tetrahydrofolate. DHFR is upregulated in rapidly proliferating cells and hence a favored target of antifolate drug against cancers, autoimmune diseases, and microbial infections. However, increased expression of dhfr contributed to the often emerging drug resistance and impeded the therapeutic efficacy of antifolate drugs. Therefore, comprehensive knowledge on the expressional control of dhfr becomes crucial. We generated two zebrafish transgenic lines, Tg(zdhfr+91:EGFP) and Tg(zdhfr+79:EGFP), which express green fluorescent protein driven by two zebrafish dhfr promoter fragments separately. The fluorescence intensity displayed in these transgenic embryos recapitulated the expressional dynamics of endogenous dhfr and reflected changes in dhfr mRNA and protein levels. The fluorescence intensity of these transgenic embryos was responsive to both genetic and environmental factors potentially modulating dhfr promoter activity. Sequence analyses revealed partial conservation on the landscape of transcription factor arrangement between zebrafish and human dhfr promoters. A noncanonical and inhibitory Sp1 site was identified 170 base-pair upstream to the conserved Sp1 site in close proximity to the translation initiation codon. Our results supported the potential use of these transgenic embryos for studying the expressional dynamics of dhfr and preliminary screening for dhfr promoter modulators.

Mycobacterial Prevalence and Antibiotic Resistance Frequency Trends in Taiwan of Mycobacterial Clinical Isolates From 2002 to 2014.

Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC) infections, is one of the most widespread infectious diseases worldwide. Nontuberculous mycobacteria (NTM) also cause chronic pulmonary infections, however, NTM infection is generally overlooked.This study analyzed the frequencies of MTBC and NTM clinical isolates from 181,132 specimens obtained from patients in Taiwan suspected of having a pulmonary mycobacterial infection from 2002 to 2014. The resistant rates to 4 first-line antibiotics (isoniazid, ethambutol, rifampicin, and streptomycin) of 9079 clinical MTBC isolates were also examined by the modified agar proportion method.Overall, the mycobacterial isolation rate was 8.65%, and this consisted of MTBC isolation rate of 5.01% and NTM isolation rate of 3.63%. The prevalence of MTBC isolates among the identified mycobacterial strains could be seen to decrease significantly from 82.5% in 2002 to 41.18% in 2014. Notably, the corresponding NTM prevalence increased 3.36 fold from 17.54% in 2002 to 58.82% in 2014. The frequencies of MTBC and NTM isolates showed a reciprocal trend with the crossing over occurring in the years 2010 and 2011. Although the resistance rates of the MTBC isolates to isoniazid and streptomycin were relatively stable over the study period, resistance rates of the MTBC isolates against rifampicin and ethambutol fluctuated across the study period. Overall, the incidence of multidrug resistance was relatively consistent at about 1.74%.The diagnosis, identification, and susceptibility tests for NTM should be standardized and integrated into appropriate clinical settings to cope with the increase in NTM infections. In addition, the documentation of the antibiotic resistance rates of MTBC clinical isolates to the antibiotic treatments most often clinically prescribed over a decade provides valuable clues and reference points for effective mycobacterial control.

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages.

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca(2+)-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-kappaB (NF-kappaB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca(2+)-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca(2+)-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC-alpha and -beta activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-kappaB-specific DNA-protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-kappaB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-kappaB activation. These data suggest that persistent inhibition of ER Ca(2+)-ATPase by TG would influence calcium release from ER Ca2+ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-kappaB activation that induces iNOS expression and NO production.