An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice

Background & Aims

The brain dopaminergic (DA) system is involved in fine tuning many behaviors and several human diseases are associated with pathological alterations of the DA system such as Parkinson’s disease (PD) and drug addiction. Because of its complex network integration, detailed analyses of physiological and pathophysiological conditions are only possible in a whole organism with a sophisticated tool box for visualization and functional modification.

Methods & Results

Here, we have generated transgenic mice expressing the tetracycline-regulated transactivator (tTA) or the reverse tetracycline-regulated transactivator (rtTA) under control of the tyrosine hydroxylase (TH) promoter, TH-tTA (tet-OFF) and TH-rtTA (tet-ON) mice, to visualize and genetically modify DA neurons. We show their tight regulation and efficient use to overexpress proteins under the control of tet-responsive elements or to delete genes of interest with tet-responsive Cre. In combination with mice encoding tet-responsive luciferase, we visualized the DA system in living mice progressively over time.

Conclusion

These experiments establish TH-tTA and TH-rtTA mice as a powerful tool to generate and monitor mouse models for DA system diseases.     

The zebrafish mutant bumper shows a hyperproliferation of lens epithelial cells and fibre cell degeneration leading to functional blindness

The development of the eye lens is one of the classical paradigms of induction during embryonic development in vertebrates. But while there have been numerous studies aimed at discovering the genetic networks controlling early lens development, comparatively little is known about later stages, including the differentiation of secondary lens fibre cells. The analysis of mutant zebrafish isolated in forward genetic screens is an important way to investigate the roles of genes in embryogenesis. In this study we describe the zebrafish mutant bumper (bum), which shows a transient, tumour-like hyperproliferation of the lens epithelium as well as a progressively stronger defect in secondary fibre cell differentiation, which results in a significantly reduced lens size and ectopic location of the lens within the neural retina. Interestingly, the initial hyperproliferation of the lens epithelium in bum spontaneously regresses, suggesting this mutant as a valuable model to study the molecular control of tumour progression/suppression. Behavioural analyses demonstrate that, despite a morphologically normal retina, larval and adult bum^?/? zebrafish are functionally blind. We further show that these fish have defects in their craniofacial skeleton with normal but delayed formation of the scleral ossicles within the eye, several reduced craniofacial bones resulting in an abnormal skull shape, and asymmetric ectopic bone formation within the mandible. Genetic mapping located the mutation in bum to a 4 cM interval on chromosome 7 with the closest markers located at 0.2 and 0 cM, respectively.     

2-Propanol degradation by Sulfolobus solfataricus

Sulfolobus solfataricus used 2-propanol and 2-propanone (acetone) when grown in static cultures at 78 °C with or without glucose at 10 g l^–1. The presence of 3.92 g 2-propanol l^–1 in both cases inhibited growth. However, acetone accumulation following 2-propanol depletion suggested that 2-propanol was co-metabolized via the acetone metabolic pathway. Glucose at 10 g l^–1 increased 2-propanol and acetone utilization from 0.93 g l^–1 to 1.77 g l^–1 and from 0.11 g l^–1 to 1.62 g l^–1, respectively. Without glucose, immobilized S. solfataricus cells increased the 2-propanol removal rate to 0.035 g l^–1 h^–1, compared to 0.0012 g l^–1 h^–1 by its suspended counterpart. The results suggest the establishment of an immobilized reactor configuration is preferential for the treatment of high temperature solvent waste streams by this acidothermophilic Crenarchaeon.     

3‐Aryl Coumarin Derivatives Bearing Aminoalkoxy Moiety as Multi‐Target‐Directed Ligands against Alzheimer's Disease

Two series of novel coumarin derivatives, substituted at 3 and 7 positions with aminoalkoxy groups, are synthesized, characterized, and screened. The effect of amine substituents and the length of cross‐linker are investigated in acetyl‐ and butyrylcholinesterase (AChE and BuChE) inhibition. Target compounds show moderate to potent inhibitory activities against AChE and BuChE. 3‐(3,4‐Dichlorophenyl)‐7‐[4‐(diethylamino)butoxy]‐2H‐chromen‐2‐one ( 4y ) is identified as the most potent compound against AChE (IC₅₀=0.27 μm ). Kinetic and molecular modeling studies affirmed that compound 4y works in a mixed‐type way and interacts simultaneously with the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. In addition, compound 4y blocks β‐amyloid (Aβ) self‐aggregation with a ratio of 44.11 % at 100 μm and significantly protects PC12 cells from H₂O₂‐damage in a dose‐dependent manner.     

Quercetin can be a more reliable treatment for metastatic prostate cancer than the localized disease: An in vitro study

Quercetin is a plant flavonoid that has been recognized to have anti-inflammatory, antioxidant and anti-proliferative activities. This study aims to evaluate the inhibitory effects of quercetin against prostate malignancy in vitro and the underlying resistance mechanism. IC₅₀ values of quercetin were determined by MTT assay. Annexin-V/PI staining was used to measure the rate of apoptosis. DNA cell cycle was analysed by PI staining method. Real-time PCR was performed to assess mRNA levels of OPN isoforms, VEGF isoforms, P53 and KLK2. Migration potential, proliferative capability and nucleus morphology of cells were evaluated by the scratch-wound assay, colony-forming assay and Hoechst staining, respectively. Quercetin significantly increased the apoptosis rate of PC-3 and LNCaP cell lines, arrested the cell cycle at the sub-G1/G1 phase, and reduced the migration potential and colony-forming capability. Moreover, upregulation of apoptosis-related genes and downregulation of genes involved in proliferation and angiogenesis was also observed. Although our results elucidated that quercetin has antitumor effects on PC-3 and LNCaP, for the first time, we showed that quercetin treatment causes alterations in the expression of OPN and VEGF isoforms, which are cancer-promoting modulators through various processes such as angiogenesis and drug-resistance. Prostate malignant cells can dodge the anti-carcinogenic properties of quercetin via modulation of OPN and VEGF isoforms in vitro. Therefore, quercetin acts as a double-edged sword in prostate cancer treatment.