The cerebellum is responsible for coordinating motor functions and has a unique laminated architecture. Purkinje cells are inhibitory neurons and represent the only output from the cerebellar cortex. Tyrosine hydroxylase (TH) is the key enzyme for the synthesis of catecholamines, including dopamine and noradrenaline, and it is normally not expressed in cerebellar neurons.

BACKGROUND The epithelial-mesenchymal transition (EMT) has been shown to be involved in the process of invasion and metastasis of prostate cancer. SIRT1 is the mammalian homologue of the silent information regulator 2 (Sir2) gene, and is abnormally expressed in prostate cancer cells. Therefore, it is hypothesized that SIRT1 mediates the invasion/metastatic ability of prostate cancer via EMT regulation. This study thus investigated the effect of SIRT1 gene on the invasion and migration of prostate cancer cell line PC-3 via the small interference RNA (siRNA) against SIRT1. MATERIAL AND METHODS SiRNA construct was transfected into PC-3 cells, which were tested for the cell migration and invasion ability by scratch assay and Transwell migration assay, respectively. Expression levels of vimentin, E-cadherin, and N-cadherin were further quantified by Western blotting and RT-PCR. RESULTS Both mRNA and protein levels of SIRT1 were depressed after siRNA transfection, along with weakened migration and invasion ability of PC-3 cells. Elevated E-cadherin and suppressed N-cadherin and vimentin were observed in those transfected cells. CONCLUSIONS The silencing of SIRT1 gene in PC-3 cells can suppress the movement, migration, and invasion functions of prostate cancer cells, possibly via the down-regulation of mesenchymal markers vimentin and N-cadherin accompanied with up-regulation of epithelial marker N-cadherin, thus reversing the EMT process.

Engineered nucleases have transformed biological research and offer great therapeutic potential by enabling the straightforward modification of desired genomic sequences. While many nuclease platforms have proven functional, all can produce unanticipated off-target lesions and have difficulty discriminating between homologous sequences, limiting their therapeutic application. Here we describe a multi-reporter selection system that allows the screening of large protein libraries to uncover variants able to discriminate between sequences with substantial homology. We have used this system to identify zinc-finger nucleases that exhibit high cleavage activity (up to 60% indels) at their targets within the CCR5 and HBB genes and strong discrimination against homologous sequences within CCR2 and HBD. An unbiased screen for off-target lesions using a novel set of CCR5-targeting nucleases confirms negligible CCR2 activity and demonstrates minimal off-target activity genome wide. This system offers a straightforward approach to generate nucleases that discriminate between similar targets and provide exceptional genome-wide specificity.

Cis-element encyclopedias provide information on phenotypic diversity and disease mechanisms. Although cis-element polymorphisms and mutations are instructive, deciphering function remains challenging. Mutation of an intronic GATA motif (+9.5) in GATA2, encoding a master regulator of hematopoiesis, underlies an immunodeficiency associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Whereas an inversion relocalizes another GATA2 cis-element (-77) to the proto-oncogene EVI1, inducing EVI1 expression and AML, whether this reflects ectopic or physiological activity is unknown. We describe a mouse strain that decouples -77 function from proto-oncogene deregulation. The -77(-/-) mice exhibited a novel phenotypic constellation including late embryonic lethality and anemia. The -77 established a vital sector of the myeloid progenitor transcriptome, conferring multipotentiality. Unlike the +9.5(-/-) embryos, hematopoietic stem cell genesis was unaffected in -77(-/-) embryos. These results illustrate a paradigm in which cis-elements in a locus differentially control stem and progenitor cell transitions, and therefore the individual cis-element alterations cause unique and overlapping disease phenotypes.

Positional information derived from local morphogen concentration plays an important role in patterning. A key question is how morphogen diffusion and gene expression regulation shape positional information into an appropriate profile with suitably low noise. We address this question using a model system--the C. elegans germline--whose regulatory network has been well characterized genetically but whose spatiotemporal dynamics are poorly understood. We show that diffusion within the germline syncytium is a critical control of stem cell differentiation and that semi-permeable diffusion barriers present at key locations make it possible--in combination with a feedback loop in the germline regulatory network--for mitotic zone size to be robust against spatial noise in Notch signaling. Spatial averaging within compartments defined by diffusion barriers is an advantageous patterning strategy, which attenuates noise while still allowing for sharp transitions between compartments. This strategy could apply to other organs.

Prader-Willi syndrome (PWS) is the predominant genetic cause of obesity in humans. Recent clinical reports have suggested that micro-deletion of the Snord116 gene cluster can lead to PWS, however, the extent of the contributions of the encoded snoRNAs is unknown. Here we show that mice lacking Snord116 globally have low birth weight, increased body weight gain, energy expenditure and hyperphagia. Consistent with this, microarray analysis of hypothalamic gene expression revealed a significant alteration in feeding related pathways that was also confirmed by in situ hybridisation. Importantly, selective deletion of Snord116 only from NPY expressing neurons mimics almost exactly the global deletion phenotype including the persistent low birth weight, increased body weight gain in early adulthood, increased energy expenditure and hyperphagia. Mechanistically, the lack of Snord116 in NPY neurons leads to the upregulation of NPY mRNA consistent with the hyperphagic phenotype and suggests a critical role of Snord116 in the control of NPY neuronal functions that might be dysregulated in PWS.

The plant height is an important trait in fruit tree. However, the molecular mechanism on dwarfism is still poorly understood. We found that colchicine-induced autotetraploid apple plants (Malus × domestica) exhibited a dwarf phenotype. The vertical length of cortical parenchyma cells was shorter in autotetraploids than in diploids, by observing paraffin sections. Hormone levels of indoleacetic acid (IAA) and brassinosteroid (BR) were significantly decreased in 3- and 5-year-old autotetraploid plants. Digital gene expression (DGE) analysis showed that the differentially expressed genes were mainly involved in IAA and BR pathways. microRNA390 was significantly upregulated according to microarray analysis. Exogenous application of IAA and BR promoted stem elongation of both apple plants grown in medium. The results show that dwarfing in autotetraploid apple plants is most likely regulated by IAA and BR. The dwarf phenotype of autotetraploid apple plants could be due to accumulation of miR390 after genome doubling, leading to upregulation of apple trans-acting short-interfering RNA 3 (MdTAS3) expression, which in turn downregulates the expression of MdARF3. Overall, this leads to partial interruption of the IAA and BR signal transduction pathway. Our study provides important insights into the molecular mechanisms underlying dwarfism in autopolyploid apple plants.

Dysregulated microRNAs play important pathological roles in carcinogenesis that are yet to be fully elucidated. This study was performed to investigate the biological functions of microRNA-320a (miR-320a) in breast cancer and the underlying mechanisms. Function analyses for cell proliferation, cell cycle, and cell invasion/migration, were conducted after miR-320a silencing and overexpression. The specific target genes of miR-320a were predicted by TargetScan algorithm and then determined by dual luciferase reporter assay and rescue experiment. The relationship between miR-320a and its target genes was explored in human breast cancer tissues. We found that miR-320a overexpression could inhibit breast cancer invasion and migration abilities in vitro, while miR-320a silencing could enhance that. In addition, miR-320a could suppress activity of 3'-untranslated region luciferase of metadherin (MTDH), a potent oncogene. The rescue experiment revealed that MTDH was a functional target of miR-320a. Moreover, we found that MTDH was negatively correlated with miR-320a expression, and it was related to clinical outcomes of breast cancer. Further xenograft experiment also showed that miR-320a could inhibit breast cancer metastasis in vivo. Our findings clearly demonstrate that miR-320a suppresses breast cancer metastasis by directly inhibiting MTDH expression. The present study provides a new insight into anti-oncogenic roles of miR-320a and suggests that miR-320a/MTDH pathway is a putative therapeutic target in breast cancer.

Hand, foot, and mouth disease (HFMD) is a common childhood illness caused by serotypes of the Enterovirus A species in the genus Enterovirus of the Picornaviridae family. The disease has had a substantial burden throughout East and Southeast Asia over the past 15 y. China reported 9 million cases of HFMD between 2008 and 2013, with the two serotypes Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CV-A16) being responsible for the majority of these cases. Three recent phase 3 clinical trials showed that inactivated monovalent EV-A71 vaccines manufactured in China were highly efficacious against HFMD associated with EV-A71, but offered no protection against HFMD caused by CV-A16. To better inform vaccination policy, we used mathematical models to evaluate the effect of prospective vaccination against EV-A71-associated HFMD and the potential risk of serotype replacement by CV-A16. We also extended the model to address the co-circulation, and implications for vaccination, of additional non-EV-A71, non-CV-A16 serotypes of enterovirus.

Trimetazidine, as an anti-ischemic and antioxidant agent, has been demonstrated to have many cardioprotective effects. However, whether early administration of trimetazidine has an effect on diabetic cardiomyopathy and the mechanisms underlying the effect have not yet been elucidated.

Tibetan Plateau is one of the largest and most unique habitats for organisms including arbuscular mycorrhizal fungi (AMF). However, it remains unclear how AMF communities respond to key environmental changes in this harsh environment. To test if precipitation could be a driving force in shaping AMF community structures at regional scale, we examined AMF communities associated with dominant plant species along a precipitation gradient in Tibetan alpine steppe. Rhizosphere soils were collected from five sites with annual precipitation decreasing from 400 to 50 mm. A total of 31 AMF operational taxonomic units (OTUs) were identified. AMF community composition varied significantly among sites, whereas AMF community composition did not vary among plant species. Path analysis revealed that precipitation directly affected AMF hyphal length density, and indirectly influenced AMF species richness likely through the mediation of plant coverage. Our results suggested that water availability could drive the changes of AMF communities at regional scale. Given the important roles AMF could play in the dynamics of plant communities, exploring the changes of AMF communities along key environmental gradients would help us better predict the ecosystem level responses of the Tibetan vegetation to future climate change.

The ribozyme-sensitive element NHE-III1 in the P1 promoter region of the important proto-oncogene c-myc contains many guanine (G)-rich sequences. Induction and stabilization of the G-quadruplex formed by NHE-III1 can downregulate c-myc expression. In the present study, we found that QPB-15e, a quinazoline derivative designed and synthesized by our laboratory, binds to and stabilizes the c-myc G-quadruplex in vitro, thereby inhibiting double-stranded DNA replication, downregulating c-myc gene expression and arresting cancer cell proliferation. PCR termination experiments showed that QPB-15e blocked double-stranded DNA replication by inducing or stabilizing the c-myc G-quadruplex. FRET-melting further confirmed that QPB-15e improved the stability of the G-quadruplex, and CD spectroscopy indicated that the compound interacted directly with the G-rich sequence. In competitive dialysis experiments, QPB-15e bound preferentially to quadruplex DNA in various structures, especially the G-quadruplex within the c-myc promoter region. Moreover, QPB-15e reduced the weights and volumes of tumors transplanted into nude mice. These findings strongly suggest that QPB-15e is a c-myc G-quadruplex ligand with anti-tumor properties, and may be efficacious for treating cancer in humans.

Accumulating evidence shows that large tumor suppressor 1 (LATS1) as a novel resident governor of cellular homeostasis is implicated in multiple tumorigenic properties including cell growth, apoptosis and metastasis. However, the contribution of LATS1 to gastric carcinoma (GC) remains unclear. The correlation of LATS1 expression with clinicopathologic characteristics, GC prognosis and recurrence was analyzed by immunohistochemistry, Univariate and Kaplan-Meier analysis. Functional experiments were performed to investigate biological behaviors of GC cells and underlying molecular mechanisms. Tumor growth and metastasis was assessed in vivo using orthotopic implantation GC models in severe combined immune deficiency (SCID) mice. Consequently, decreased LATS1 expression was significantly associated with the lymph node metastasis, poor prognosis and recurrence. Ectopic expression of LATS1 decreased GC cell proliferation and invasion in vitro and inhibited tumor growth and liver metastasis in vivo, but depletion of LATS1 expression restored the invasive phenotype. Further observation indicated that YAP pathway was required for LATS1-induced inhibition of cell growth and invasion, and LATS1 restrained nuclear transfer of YAP, downregulated YAP, PCNA, CTGF, MMP-2, MMP-9, Bcl-2 and CyclinD1 expression and upregulated p-YAP and Bax expression. Our findings suggest that LATS1 is a potential candidate tumor suppressor and inhibits the growth and metastasis of GC cells via downregulation of the YAP signaling.

Schizophrenia is a persistent chronic mental illness with an unknown pathogenic mechanism; no empirical laboratory-based tests are available to support the diagnosis of schizophrenia or to identify biomarkers correlated with the therapeutic effect of olanzapine. For this study, 15 female first-episode, drug-naïve patients with schizophrenia and 15 healthy female volunteers were recruited. Tests for blood glucose and lipids were conducted at baseline and after 4 weeks of treatment with olanzapine. UPLC-MS based metabonomic analysis was performed on both case and control groups to identify biomarkers of schizophrenia at baseline and to explore which biomarkers correlated with the therapeutic effect of olanzapine after a 4-week treatment. Compared with the control group, the case group showed significant changes in plasma metabolites. Thirteen distinct metabolites were identified. Among all the therapeutically effective cases, levels of these metabolites appeared to shift towards the normal trend; 8 of the identified 13 metabolites changed dramatically. The metabolites that we found are potential biomarkers for the diagnosis and treatment of schizophrenia.

Serotonin 1a-receptor (5-HT1aR) has been specifically implicated in the pathogenesis of anxiety. However, the mechanism underlying the role of 5-HT1aR in anxiety remains poorly understood. Here we show in mice that the transcription factor cAMP response element binding protein (CREB) in the hippocampus functions as an effector of 5-HT1aR in modulating anxiety-related behaviors. We generated recombinant lentivirus LV-CREB133-GFP expressing a dominant negative CREB which could not be phosphorylated at Ser133 to specifically reduce CREB activity, and LV-VP16-CREB-GFP expressing a constitutively active fusion protein VP16-CREB which could be phosphorylated by itself to specifically enhance CREB activity. LV-CREB133-GFP neutralized 5-HT1aR agonist-induced up-regulation of synapse density, spine density, dendrite complexity, neurogenesis, and the expression of synapsin and spinophilin, two well-characterized synaptic proteins, and abolished the anxiolytic effect of 5-HT1aR agonist; whereas LV-VP16-CREB-GFP rescued the 5-HT1aR antagonist-induced down-regulation of synapse density, spine density, dendrite complexity, neurogenesis and synapsin and spinophilin expression, and reversed the anxiogenic effect of 5-HT1aR antagonist. The deletion of neurogenesis by irradiation or the diminution of synaptogenesis by knockdown of synapsin expression abolished the anxiolytic effects of both CREB and 5-HT1aR activation. These findings suggest that CREB-mediated hippoacampus structural plasticity is crucial for the role of 5-HT1aR in modulating anxiety-related behaviors.

Apolipoprotein E (ApoE) is a major lipid carrier that supports lipid transport and injury repair in the brain. The APOE ε4 allele is associated with depression, mild cognitive impairment (MCI) and dementia; however, the precise molecular mechanism through which ApoE4 influences the risk of disease development remains unknown. To address this gap in knowledge, we investigated the potential effects of chronic unpredictable mild stress (CUMS) on ApoE3 and ApoE4 target replacement (ApoE3-TR and ApoE4-TR) mice.

Cisplatin-based treatment has been considered the standard treatment regimen of HNSCC. Cetuximab is an emerging target therapy that has potential therapeutic benefits over cisplatin. Nevertheless, curative effects of cisplatin-based chemoradiotherapy (CRT) versus cetuximab-based bioradiotherapy (BRT) are still controversial.

X-linked inhibitor of apoptosis protein (XIAP) and second mitochondrial-derived activator of caspase (Smac) are two important prognostic biomarkers for cancer. They are negatively correlated in many types of cancer. However, their relationship is still unknown in lung cancer. In the present study, we found that there was a negative correlation between Smac and XIAP at the level of protein but not mRNA in NSCLC patients. However, XIAP overexpression had no effect on degrading endogenous Smac in lung cancer cell lines. Therefore, we constructed plasmids with full length of Smac (fSmac) and mature Smac (mSmac) which located in cytoplasm instead of original mitochondrial location, and was confirmed by immunofluorescence. Subsequently, we found that mSmac rather than fSmac was degraded by XIAP and inhibited cell viability. CHX chase assay and ubiquitin assay were performed to illustrate XIAP degraded mSmac through ubiquitin pathway. Overexpression of XIAP partially reverted apoptotic induction and cell viability inhibition by mSmac, which was due to inhibiting caspase-3 activation. In nude mouse xenograft experiments, mSmac inhibited Ki-67 expression and slowed down lung cancer growth, while XIAP partially reversed the effect of mSmac by degrading it. In conclusion, XIAP inhibits mature Smac-induced apoptosis by degrading it through ubiquitination in NSCLC.

Crohn disease (CD) is a chronic relapsing disease. Imaging modalities are essential for the diagnosis and assessment of CD. Small intestine contrast ultrasonography (SICUS) is a well-tolerated, noninvasive and radiation-free modality and has shown potential in CD assessment. We aimed at evaluating the diagnostic accuracy of SICUS in the detection and assessment of small-bowel lesions and complications in CD.

Renal tubule cells can recover after they undergo AKI (acute kidney injury). An incomplete repair of renal tubules can result in progressive fibrotic CKD (chronic kidney disease). Studies have revealed the relationship between tubular epithelial cells and kidney fibrogenesis. However, the underlying mechanism remains unclear. Hippo pathway components were evaluated in complete/incomplete repair of I/R (ischaemia/reperfusion) AKI rat models, HK-2 cells and AKI human renal biopsy samples. We found that the expression levels of the Hippo pathway components changed dynamically during kidney regeneration and fibrogenesis in rat models of I/R-induced AKI and human renal biopsy samples. The transcription cofactor YAP (Yes-associated protein) might be a key effector of renal regeneration and fibrogenesis. Our results showed further that YAP might elicit both beneficial and detrimental effects on I/R AKI. After I/R injury occurred, YAP could promote the repair of the injured epithelia. The constant YAP increase and activation might be related to interstitial fibrosis and abnormal renal tubule differentiation. These results indicate that the proper modulation of the Hippo pathway, specifically the transcription cofactor YAP, during repair might be a potent therapeutic target in AKI-CKD transition after I/R injury.