Intermittent fasting in type 2 diabetes: from fundamental science to clinical applications.

Type 2 diabetes mellitus (T2DM) is a huge challenge for global public health systems. Currently, healthcare policies advocate the prevention of the onset and progression of T2DM by improving individual lifestyles. The increasing benefits of intermittent fasting (IF) as a dietary intervention have been elucidated. However, the beneficial effects of IF in T2DM remain inconclusive. We demonstrated the physiological mechanisms underlying the positive effects of IF in T2DM. IF could trigger metabolic transformation to improve systemic metabolism and induce tissue-specific metabolic adaptations through alterations in the gut microbiota, adipose tissue remodeling, correction of circadian rhythm disturbances, and increased autophagy in peripheral tissues. The efficacy and safety of IF regimens in clinical applications carry a risk of hypoglycemia and require monitoring of blood glucose and timely adjustment of medications. However, there is limited evidence of a positive effect of IF in weight loss and improvement of glycemic variables. Overall, IF serves as a promising therapeutic target for T2DM and needs to be established by a large randomized controlled trial.

MiRNA-15a-3p inhibits the metastasis of hepatocellular carcinoma by interacting with HMOX1.

OBJECTIVE: To uncover the relationship between microRNA-15a-3p (miRNA-15a-3p) level and clinical features of hepatocellular carcinoma (HCC), and to explore the influence of miRNA-15a-3p on metastasis of HCC cells. PATIENTS AND METHODS: HCC and paracancerous tissues were surgically resected from 44 HCC patients. Their clinical data and follow-up files were recorded. Differential expressions of miRNA-15a-3p in HCC samples were determined. The relationship between miRNA-15a-3p level and clinical features of HCC patients was analyzed. Changes in proliferative, migratory and invasive potentials in Huh7 and HepG2 cells overexpressing miRNA-15a-3p were examined. The downstream gene of miRNA-15a-3p and its involvement in HCC development were finally explored. RESULTS: MiRNA-15a-3p was downregulated in HCC tissues. High metastasis rate and poor prognosis were observed in HCC patients expressing a low level of miRNA-15a-3p. Overexpression of miRNA-15a-3p attenuated proliferative, migratory and invasive potentials in HCC. Protein levels of HMOX1, CD31, c-Myc, MMP-2 and MMP-9 were downregulated in HCC cells after overexpression of miRNA-15a-3p. HMOX1 was the downstream gene of miRNA-15a-3p, which was upregulated in HCC samples. Highly expressed HMOX1 was unfavorable to the prognosis in HCC. Overexpression of HMOX1 abolished the regulatory effects of miRNA-15a-3p on HCC cell phenotypes. CONCLUSIONS: MiRNA-15a-3p is closely linked to lymphatic metastasis, distant metastasis and poor prognosis in HCC. It inhibits the malignant development of HCC by interacting with HMOX1.

Knockdown of lncRNA XIST inhibits hypoxia-induced glycolysis, migration and invasion through regulating miR-381-3p/NEK5 axis in nasopharyngeal carcinoma.

OBJECTIVE: Hypoxia is an important feature of nasopharyngeal carcinoma (NPC). Growing evidence demonstrated that long non-coding RNAs (lncRNAs) could participate in cancer progression and hypoxia regulation. However, the exact roles and underlying mechanism of lncRNA X-inactive specific transcript (XIST) in NPC under hypoxia are still unclear. MATERIALS AND METHODS: The expressions of XIST, microRNA-381-3p (miR-381-3p) and NIMA related kinase 5 (NEK5) were detected by quantitative Real-time polymerase chain reaction (qRT-PCR). The glucose consumption and lactate production were measured using the glucose assay kit and lactate assay kit, respectively. Western blot assay was used to determine the protein levels of hexokinase II (HK2) and NEK5. Transwell assay was employed to evaluate cell migration and invasion. The interaction between miR-381-3p and XIST or NEK5 was predicted by bioinformatics analysis and verified by dual-luciferase reporter assay. The mice xenograft model was established to investigate the roles of XIST in NPC progression in vivo. RESULTS: XIST and NEK5 were highly expressed while miR-381-3p was lowly expressed in NPC (tissues and cells) and hypoxia-induced NPC cells. Deficiency of XIST or NEK5 suppressed hypoxia-induced glycolysis and metastasis in NPC cells. Moreover, miR-381-3p could directly bind to XIST and its inhibition reversed the inhibitory effects of XIST knockdown on glycolysis and metastasis under hypoxia. NEK5 was a direct target of miR-381-3p and its interference attenuated the promotive effects of miR-381-3p downregulation on glycolysis and metastasis under hypoxic conditions. Besides, interference of XIST decreased tumor growth by upregulating miR-381-3p and downregulating NEK5. CONCLUSIONS: XIST knockdown inhibited glycolysis and metastasis in hypoxia-induced NPC cells through regulating miR-381-3p/NEK5 axis, providing new insights into the pathogenesis of NPC.

MiR-135a inhibitor alleviates pulmonary arterial hypertension through ß-Catenin/GSK-3ß signaling pathway.

OBJECTIVE: The aim of this study was to investigate the regulatory role of micro-ribonucleic acid (miR)-135a in monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) in rats, and to analyze the possible regulatory mechanism. MATERIALS AND METHODS: A total of 30 Sprague-Dawley rats were randomly divided into three groups, including the blank control group, model group and miR-135a inhibitor intervention group. The right ventricular systolic pressure (RVSP) and right ventricle hypertrophy index (RVHI) were measured in rats of each group. Hematoxylin and eosin (HE) staining was adopted to detect the pathological changes in lung tissues of rats. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of interleukin-6 (IL-6) and IL-1ß in lung tissues. Meanwhile, the messenger RNA (mRNA) and protein levels of ß-catenin and glycogen synthase kinase-3ß (GSK-3ß) in lung tissues of rats were determined via Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Western blotting assay, respectively. RESULTS: Compared with the blank control group, RVSP and RVHI increased significantly in the model group. The pathological morphology of the lung tissues was poor, and the content of IL-6 and IL-1ß was markedly up-regulated in the model group. Meanwhile, the mRNA and protein levels of ß-catenin and GSK-3ß were notably elevated in the model group than the blank control group. In the miR-135a inhibitor intervention group, RVSP and RVHI decreased significantly, and the pathological morphology of lung tissues was evidently improved when compared with the blank control group. Furthermore, the content of IL-6 and IL-1ß was remarkably reduced, and the mRNA and protein levels of ß-catenin and GSK-3ß were significantly declined in the miR-135a inhibitor intervention group. CONCLUSIONS: MiR-135a inhibitor significantly alleviates inflammatory response in the lung tissues and ameliorates damage to the pathological morphology. The possible underlying mechanism may be associated with the ß-catenin/GSK-3ß signaling pathway.

High levels of long non-coding RNA DICER1-AS1 are associated with poor clinical prognosis in patients with osteosarcoma.

OBJECTIVE: Long noncoding RNA DICER1-AS1 (DICER1-AS1) has been reported to be upregulated in osteosarcoma cells and to serve as a tumor promoter. However, the clinical significance of DICER1-AS1 in osteosarcoma remains unclear. The aim of this study was to investigate the association of DICER1-AS1 expression with prognosis of osteosarcoma. PATIENTS AND METHODS: The expression of DICER1-AS1 was measured in 214 osteosarcoma samples and normal bone samples by using Real-time PCR. The correlations between DICER1-AS1 expression and clinical features were statistically analyzed. Overall survival (OS) and disease-free survival (DFS) were examined using Kaplan-Meier curves. Multivariate analyses were performed to analyze the prognostic significance of DICER1-AS1 expression. RESULTS: It was found that the expression levels of DICER1-AS1 in osteosarcoma tissues were significantly higher than those in corresponding noncancerous bone tissues (p < 0.01). Higher DICER1-AS1 had significant association with clinical stage (p = 0.005) and distant metastasis (p = 0.000). Kaplan-Meier survival analysis showed that patients with high DICER1-AS1 expression had a shorter OS and DFS compared with the low DICER1-AS1 expression group (p = 0.007 and p < 0.0001). In a multivariate Cox model, our results showed that DICER1-AS1 expression was an independent poor prognostic factor for both 5-year OS (HR = 3.236, 95% CI: 1.148-5.347; p = 0.004) and 5-year DFS (HR = 3.935, 95% CI: 1.556-6.349; p = 0.001). CONCLUSIONS: DICER1-AS1 is up-regulated in osteosarcoma and may serve as a potential prognostic biomarker for osteosarcoma.

Myeloablative conditioning regimens with combined of haploidentical and cord blood transplantation for myelodysplastic syndrome patients.

The purpose of this study was to evaluate the strategy of haploidentical (HID) stem cell combined with a small doses of umbilical cord blood (UCB) from a third-party donor transplantation (haplo-cord transplant) for treatment of myelodysplastic syndromes (MDS), by comparing with identical-sibling donor (ISD) transplantation. Eighty-five patients were included between January 2012 and December 2015, with a median 40 years old. Forty-eight patients received haplo-cord transplant and 37 patients received ISD transplant. Haplograft engraftment succeeded in all haplo-cord patients. For haplo-cord and ISD transplantation, adjusted cumulative incidences of grades 2-4 acute GvHD at 100 days were 27 and 11% (P=0.059); adjusted cumulative incidences of chronic GvHD at 2 years were 22 and 34% (P=0.215). The 2-year adjusted probabilities of overall survival were 64 and 70% (P=0.518), and of relapse-free survival were 56 and 66% (P=0.306). The 2-year adjusted cumulative incidences of relapse were 12 and 14% (P=0.743), and of non-relapse mortality were 33 and 23% (P=0.291). In conclusion, haplo-cord-HSCT achieves outcomes similar to those of ISD-HSCT for MDS and the haplo-cord-HSCT may potentially improve the outcome of HID- and UCB-HSCT alone. Thus, the haplo-cord transplantation may be a better valid alternative for MDS when an ISD is not available.

Sumoylation in Lens Differentiation and Pathogenesis.

Sumoylation, a post-translational modification discovered over a decade ago, turns out to be a very important regulatory mechanism mediating multiple cellular processes. Recent studies from our laboratory and others also revealed that it plays a crucial role in regulating both differentiation and pathogenesis of the ocular lens. This review will summarize these progresses.

Molecular Cloning and Developmental Expression Patterns of the Striatin Gene Encoding A Member of the Regulatory Subunits for the Protein Serine/Threonine Phosphatase-2A in Fish.

PURPOSE: The protein phosphatase-2A (PP-2A) is one of the most important serine/threonine phosphatases in eukaryotes. The holoenzyme of PP-2A consists of three subunits: a scaffold A subunit, a catalytic C subunit and a regulatory B subunit. While both A and C subunits are coded by two different genes, the B subunits exist in 26 or more isoforms which are encoded by at least 15 different genes. Previous studies have shown that besides regulating specific PP-2A activity, various B subunits may have other functions. To explore the possible roles of the regulatory subunits of PP-2A in vertebrate development, we have cloned the gene encoding goldfish striatin, a member of the B'" family regulatory subunits for PP-2A, and determined their tissue-specific and temporal expression patterns. METHODS: The cDNA cloning was conducted with RT-PCR-based RACE. The mRNA expression levels for the goldfish striatin were analyzed with RT-PCR. The expression levels of the striatin protein from goldfish were determined with Western blot analysis. The semi-quantitation of the mRNA and protein expression levels was conducted with the software of U-scanning. RESULTS: Our study revealed that the full length cDNA for striatin consists of 2965 bp coding for a deduced protein of 769 amino acids, which bears a very high level of amino acid sequence identity with the homolog protein from other species. The striatin mRNA is highly expressed in the kidney, to a less degree in brain, fin, muscle, liver, ovary and gill, and the lowest in testis and heart. Similar pattern of protein expression is detected in the above 9 tissues. During the development of goldfish, the striatin mRNA maintains a relatively high level at the 2-cell, multiple cell and blastula stages. Then, it drops down substantially at gastrula stage and fluctuates around this level in the next 8 different stages. At the protein level, the striatin maintained higher level from 2-cell to gastrula stages, then decreased at neurula and optic vesicle stages, and gradually increased again to peak at eye pigmentation stage, then slightly decreased in the next few stages of development. CONCLUSIONS: Our results suggest that the striatin may play an important role in regulating goldfish development and adult tissue homeostasis. While the former function may or may not occur through PP- 2A functions, the later function appears to occur via PP-2A activity.

Regulation of CREB Functions by Phosphorylation and Sumoylation in Nervous and Visual Systems.

CREB is an ubiquitous transcription factor regulating diverse cellular responses. Its phosphorylation at S133 is an essential event for its activation in both nervous and visual systems. The activated CREB is implicated in the regulation of development, protection, learning, memory and plasticity in the nerve system. Moreover, sumoylation, an important post-translational modification of protein, plays a key role in sustaining CREB activation in the rat hippocampus in order to enhance the long-term memory and other aspects. In the visual system, although the CREB activation by phosphorylation at S133 is similar to that as observed in the nervous system, the role of CREB sumoylation remains to be explored. This review will discuss the aspects of CREB functions and their regulation by phosphorylation and sumoylation in both systems.

The Male Abnormal Gene Family 21 (Mab21) Members Regulate Eye Development.

The male abnormal gene family contains 3 members, named mab21l1, mab21l2 and mab21l3. Since their first discovery in C. elegans, homologues of mab21l1 and mab21l2 have been found in Drosophila, Zebrafish, Xenopus, chicken, mouse and human. A number of studies have revealed that mab21 gene family members, mab21l1 and mab21l2, play important roles in regulating eye development. Here, we review the functions of the mab genes in regulating ocular development.

Shorter hemodialysis duration is a risk factor for the recurrence of urothelial carcinoma of the bladder in patients on maintenance hemodialysis.

PURPOSE: To investigate the association of hemodialysis duration with the recurrence of urothelial carcinoma (UC) of the bladder and overall survival in patients undergoing maintenance hemodialysis (MHD). PATIENTS AND METHODS: 52 bladder cancer patients who underwent MHD at the Xiangya Hospital of The Central South University between 2001 and 2011 were enrolled in the study. The patients were divided into three groups according to hemodialysis duration, and patient mortality and tumor recurrence rates were analyzed. The association of hemodialysis duration with occurrence and recurrence of UC of the bladder was analyzed by Cox regression analysis. Survival was evaluated by the Kaplan-Meier method. RESULTS: Out of 6266 chronic hemodialysis patients, 52 patients had UC of the bladder after the initiation of hemodialysis for 6 months. The mean age at hemodialysis onset was 55 years (IQR 36, 71). The major complaints were painless gross hematuria and urethral bloody discharge. Tumors were generally large and multifocal. The standardized incidence ratio of UC of the bladder was 43.9 compared with general population, and it was higher in women (76.7) and in the age group 61-65 years (186.6). The mean hemodialysis duration before the diagnosis of bladder cancer was 32 months. 30 (57.7 %) patients received hemodialysis no more than 3 years, 10 (19.2 %) patients received hemodialysis between 3 and 6 years, and 12 (23.1 %) patients received hemodialysis for more than 6 years. CONCLUSION: Preoperative shorter hemodialysis duration is a risk factor for the occurrence and recurrence of UC of the bladder in patients undergoing MHD.

Regulation of Eye Development by Protein Serine/Threonine Phosphatases-1 and -2A.

The protein serine/threonine phosphatases-1 and -2A are major cellular phosphatases, playing a fundamental role in organisms from prokaryotes to eukaryotes. They contribute to 90% dephosphorylation in eukaryote proteins. In the eye, both phosphatases are highly expressed and display important functions in regulating normal eye development. Moreover, they are implicated in pathogenesis through modulation of stress-induced apoptosis. Here we review the recent progresses on these aspects.

The tumor suppressor, p53 regulates the γA-crystallin gene during mouse lens development.

The tumor suppressor, p53 regulates a large number of target genes to control cell proliferation and apoptosis. In addition, it is also implicated in the regulation of cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates several genes including c-Maf and Prox1, two important transcription factors for lens differentiation, and αA and ßA3/A1, the lens differentiation markers. In the present study, we present evidence to show that the γA-crystallin gene distal promoter and the first intron also contain p53 binding sites and are capable of mediating p53 control during mouse lens development. First, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from human lens epithelial cells (HLE) directly binds to the p53 binding sites present in the γA-crystallin gene. Second, the exogenous wild type p53 induces the dose-dependent expression of the luciferase reporter gene driven by the basic promoter containing the γA-crystallin gene p53 binding site. In contrast, the exogenous dominant negative mutant p53 causes a dose-dependent inhibition of the same promoter. Third, ChIP assays revealed that p53 binds to the γA-crystallin gene promoter in vivo. Finally, in the p53 knockout mouse lenses, the expression level of the γAcrystallin gene was found attenuated in comparison with that in the wild type mouse lenses. Together, our results reveal that p53 regulates γA-crystallin gene expression during mouse lens development. Thus, p53 directly regulates all 3 types of crystallin genes to control lens differentiation.

Enhanced stability and activity with Pd-O junction formation and electronic structure modification of palladium nanoparticles supported on exfoliated montmorillonite for the oxygen reduction reaction.

Palladium has been the focus of recent research on alternative Pt catalysts for the oxygen reduction reaction (ORR). We show that the activity and stability of Pd toward the ORR can be enhanced by Pd-O-oxide covalent bonding when Pd is supported on exfoliated montmorillonite (ex-MMT) nanoplatelets.

Imaging findings of solitary fibrous tumor in the abdomen and pelvis.

PURPOSE: The purpose of this study was to investigate the imaging characteristics of solitary fibrous tumor (SFT) in the abdomen and pelvis. METHODS: Nine cases of SFT confirmed by surgery and pathology were retrospectively analyzed in terms of computed tomography (CT, eight cases) and magnetic resonance imaging (MRI, one case). RESULTS: SFT were located in the retroperitoneum (4/9), abdominal cavity (1/9), pelvis (4/9). Eight cases were single (8/9) and one case (1/9) with three tumors. The average tumor size of 11 lesions was 9.7 cm (4.7-20 cm). Nine tumors were round or ovoid, and two lesions were irregular. The CT value of the plain scans ranged from 33 to 43 Hounsfield units (HU, mean 37.6 HU) in five cases. Arterial-phase CT found solid parts demonstrate avid enhancement (eight cases) and five of them presented with multiple circuitous vessels along the periphery with a CT value of 68-89 HU (mean 76.6 HU). In the venous and delayed phases, enhancement was strengthened progressively. The CT values at venous (eight cases) and delayed phases (five cases) were 108-115 and 112-123 HU respectively, with averages of 109.8 and 114.8 HU. Patch or nodular no-enhanced areas were observed in eight cases during the enhanced phases. One case showed isointensity on T1-weighted images and high signal intensity on T2-weighted images accompanied by linear or curvilinear hypointense lines. Intense enhancements along with linear no-enhancement areas are seen in the arterial and venous phases. CONCLUSION: The possibility of SFT should be considered when a single or multiple masses with sharp border, inhomogeneous density or signal are detected, especially, with inhomogeneous intense enhancement in the arterial phase being maintained in the venous and delayed phases.

Protein serine/threonine Phosphotase-2A is differentially expressed and regulates eye development in vertebrates.

Protein serine/threonine phosphatase-2A (PP-2A) is one of the key enzymes responsible for dephosphorylation in vertebrates. PP-2A-mediated dephosphorylation participates in many different biological processes including cell proliferation, differentiation, transformation, apoptosis, autophage and senescence. However, whether PP-2A directly controls animal development remains to be explored. Here, we present direct evidence to show that PP-2A displays important functions in regulating eye development of vertebrates. Using goldfish as a model system, we have demonstrated the following novel information. First, inhibition of PP-2A activity leads to significant death of the treated embryos, which is derived from blastomere apoptosis associated with enhanced phosphorylation of Bcl-XL at Ser-62, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of PP-2A with morpholino oligomers leads to significant death of the injected embryos. The survived embryos from PP-2A knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each catalytic subunit of PP-2A also causes death of majority of the injected embryos and leads to absence of goldfish eye lens or severely disturbed differentiation. Together, our results provide direct evidence that protein phosphatase-2A is important for normal eye development in goldfish.

ERK signaling pathway regulates embryonic survival and eye development in goldfish, Carassius auratus.

The extracellular signal-regulated kinase (ERK) is one of the three major types of mitogen-activated protein kinases. Previous studies showed that ERKs mediate various signaling pathways for cell proliferation, differentiation, survival and transformation in mammals. In the present study, we use goldfish as a model system and demonstrate that ERK kinases play important roles in promoting embryonic survival and regulate development of eye and trunk in vertebrates. ERKs are highly expressed in multiple tissues including lens epithelial cells, lens fiber cells, retina, brain, muscle and heart of adult goldfish. Injection of the dominant negative ERK mutant (DNM-ERK) into the fertilized eggs of goldfish significantly inhibited ERK activity at blastula stage, and completely blocked ERK activity at gastrula and later stages. As a result, the blastula cells were induced into apoptosis, and majority of the injected embryos were lethal at embryonic stages. At the molecular level, inhibition of ERK activity by DNM-ERKs suppressed phosphorylation of Bad at Ser-112 to promote apoptosis. Similar results were observed when MEK activity was inhibited by U0126 treatment. The survived embryos display significant abnormality in the phenotypes of both eye and trunk. Associated with the abnormality in the eye development, phosphorylation in Pax-6 and expression of HSF4 were significantly decreased and expression of the ß-crystallin gene was also downregulated. These results provide novel information regarding the roles of ERKs in regulating vertebrate development.

p53 directly regulates αA- and ßA3/A1-crystallin genes to modulate lens differentiation.

It is well established that the tumor suppressor p53 plays major roles in regulating apoptosis and cell cycle progression. In addition, recent studies have demonstrated that p53 is actively involved in regulating cell differentiation in muscle, the circulatory system and various carcinoma tissues. We have recently shown that p53 also controls lens differentiation. Regarding the mechanism, we reveal that p53 directly regulates c-Maf and Prox1, two important transcription factors to control cell differentiation in the ocular lens. In the present study, we present further evidence to show that p53 can regulate lens differentiation by controlling expression of the differentiation genes coding for the lens crystallins. First, the αA and ßA3/A1 gene promoters or introns all contain putative p53 binding sites. Second, gel mobility shifting assays revealed that the p53 protein in nuclear extracts from lens epithelial cells directly binds to the p53 binding sites found in these crystallin gene promoters or introns. Third, exogenous wild type p53 induces dose-dependent expression of the luciferase reporter gene driven by different crystallin gene promoters and the exogenous dominant negative mutant p53 causes dose-dependent inhibition of the same crystallin genes. Fourth, ChIP assays revealed that p53 binds to crystallin gene promoters in vivo. Finally, in the p53 knockout mouse lenses, expression levels of various crystallins were found down-regulated in comparison with those from the wild type mouse lenses. Together, our results reveal that p53 directly regulates expression of different sets of genes to control lens differentiation.

PP-1α and PP-1γ display antagonism and differential roles in tumorigenicity of lung cancer cells.

Protein serine/threonine phosphatases are important cellular signaling molecules and play major roles in regulating many different functions including cell proliferation, senescence, programmed cell death, and oncogenic cell transformation. Among different serine/threonine phosphatases, PP-1 and PP-2A contribute to more than 90% phosphatase activities in eukaryotes. While the functions of PP-2A in cell transformation and tumorigenesis have been well established, the role of PP-1 in carcinogenesis remains to be further explored. Moreover, PP-1 exists in different isoforms, whether these isoforms have differential functions in tumorigenesis remains to be examined. In the present study, we demonstrated that in lung cancer 1299 cells, PP1α and PP- 1 & γ exist in an antagonizing balance. In the parent H1299 cells, PP-1γ is dominant, about 4-fold higher than that of PP-1α. Overexpression of PP-1α significantly down-regulates PP-1γ at both mRNA and protein levels. In contrast, knockdown of PP-1α leads to upregulation of PP-1γ. Moreover, overexpression of PP-1α significantly attenuates the ability of the H1299 cells in promoting tumorigenicity as tested in immuno-deficient nude mice. This attenuation is derived from the halted cell cycle progression, which is largely attributed by the changed RB-E2F activity. Together, our results demonstrate that PP-1α and PP-1γ not only antagonize each other in lung cancer cells, but also display differential functions in tumorigenicity.

Protein serine/threonine phosphotase-1 is essential in governing normal development of vertebrate eye.

Protein serine/threonine phosphatase-1 (PP-1) is one of the key enzymes responsible for dephosphorylation in vertebrates. Protein dephosphorylation via PP-1 is implicated in many different biological processes including gene expression, cell cycle control, transformation, neuronal transmission, apoptosis, autophage and senescence. However, whether PP-1 directly controls animal development remains to be investigated. Here, we present direct evidence to show that PP-1 plays an essential role in regulating eye development of vertebrates. Using goldfish as a model system, we have shown the following novel results. First, inhibition of PP-1 activity leads to death of a majority of the treated embryos, and the survived embryos displayed severe phenotype in the eye. Second, knockdown of each catalytic subunit of PP-1 with morpholino oligomers leads to partial (PP-lα knockdown) or complete (PP-lß or PP-lγ knockdown) death of the injected embryos. The survived embryos from PP-1α knockdown displayed clear retardation in lens differentiation. Finally, overexpression of each subunit of PP-1 also causes death of majority of the injected embryos and leads to abnormal development of goldfish eye. Mechanistically, Pax-6 is one of the major downstream targets mediating the effects of PP-1 function since the eye phenotype in Pax-6 knockdown fish is similar to that derived from overexpression of PP-1. Together, our results for the first time provide direct evidence that protein phosphatase-1 plays a key role in governing normal eye formation during goldfish development.