Rapamycin and CCI-779 inhibit the mammalian target of rapamycin signalling in hepatocellular carcinoma

Background: The mammalian target of rapamycin (mTOR), which phosphorylates p70S6K and 4EBP1 and activates the protein translation process, is upregulated in cancers and its activation may be involved in cancer development. Aims: In this study, we investigated the tumour-suppressive effects of rapamycin and its new analogue CCI-779 on hepatocellular carcinoma (HCC). Methods: Rapamycin and its new analogue CCI-779 were applied to treat HCC cells. Cell proliferation, cell cycle profile and tumorigenicity were analysed. Results: In human HCCs, we observed frequent (67%, 37/55) overexpression of mTOR transcripts using real-time reverse transcriptasepolymerase chain reaction. Upon drug treatment, PLC/PRF/5 showed the greatest reduction in cell proliferation using the colony formation assay, as compared with HepG2, Hep3B and HLE. Rapamycin was a more potent antiproliferative agent than CCI-779 in HCC cell lines. Proliferation assays by cell counting showed that the IC50 value of rapamycin was lower than that of CCI-779 in PLC/PRF/5 cells. Furthermore, flow cytometric analysis showed that both drugs could arrest HCC cells in the G1 phase but did not induce apoptosis of these cells, suggesting that these mTOR inhibitors are cytostatic rather than cytotoxic. Upon rapamycin and CCI-779 treatment, the phosphorylation level of mTOR and p70S6K in HCC cell lines was significantly reduced, indicating that both drugs can suppress mTOR activity in HCC cells. In addition, both drugs significantly inhibited the growth of xenografts of PLC/PRF/5 cells in nude mice. Conclusions: Our findings indicate that rapamycin and its clinical analogue CCI-779 possess tumour-suppressive functions towards HCC cells. © 2009 John Wiley & Sons A/S.postprin

Systematic review of the current psychosocial interventions for people with moderate to severe dementia

Objective: Dementia, a global epidemic, currently affects 50 million individuals worldwide. There are currently limited effective treatments for moderate to severe dementia, and most treatments focus on reducing symptoms rather than improving positive factors. It is unclear if improvements are not possible due to disease severity. This review examines the efficacy of the current psychosocial interventions for people with moderate to severe dementia, focusing on improving cognition and quality of life (QoL) to evaluate what treatments are working and whether improvements are possible. / Methods: A systematic search was conducted using six key databases to identify psychosocial interventions for people with moderate to severe dementia, measuring cognition or QoL in randomized controlled trials (RCTs), published between 2000 and 2020. / Results: The search identified 4193 studies, and 74 articles were assessed for full‐text review. Fourteen RCTs were included and appraised with the Physiotherapy Evidence Database Scale. The included RCTs were moderate in quality. / Conclusions: Aromatherapy and reminiscence therapy showed the strongest evidence in improving QoL. There was some evidence that aerobic exercise enhanced cognition, and a multicomponent study improved QoL. However, a quality assessment, using pre‐specified criteria, indicated many methodological weaknesses. While we found improvements in cognition and QoL for moderate to severe dementia, results must be interpreted with caution. Future interventions with rigorous study designs are a pressing need and required before we can recommend specific interventions

Systematic Review and Meta-Analysis of Brief Cognitive Instruments to Evaluate Suspected Dementia in Chinese-Speaking Populations

Background: Chinese is the most commonly spoken world language; however, most cognitive tests were developed and validated in the West. It is essential to find out which tests are valid and practical in Chinese speaking people with suspected dementia. Objective: We therefore conducted a systematic review and meta-Analysis of brief cognitive tests adapted for Chinese-speaking populations in people presenting for assessment of suspected dementia. Methods: We searched electronic databases for studies reporting brief (≤20 minutes) cognitive test's sensitivity and specificity as part of dementia diagnosis for Chinese-speaking populations in clinical settings. We assessed quality using Centre for Evidence Based Medicine (CEBM) criteria and translation and cultural adaptation using the Manchester Translation Reporting Questionnaire (MTRQ), and Manchester Cultural Adaptation Reporting Questionnaire (MCAR). We assessed heterogeneity and combined sensitivity in meta-Analyses. Results: 38 studies met inclusion criteria and 22 were included in meta-Analyses. None met the highest CEBM criteria. Five studies met the highest criteria of MTRQ and MCAR. In meta-Analyses of studies with acceptable heterogeneity (I2 <  75%), Addenbrooke's Cognitive Examination Revised III (ACE-R ACE-III) had the best sensitivity and specificity; specifically, for dementia (93.5% 85.6%) and mild cognitive impairment (81.4% 76.7%). Conclusions: Current evidence is that the ACE-R and ACE-III are the best brief cognitive assessments for dementia and mild cognitive impairment in Chinese-speaking populations. They may improve time taken to diagnosis, allowing people to access interventions and future planning

Differential levels of glutamate dehydrogenase 1 (GLUD1) in Balb/c and C57BL/6 mice and the effects of overexpression of the Glud1 gene on glutamate release in striatum

We have previously shown that overexpression of the Glud1 (glutamate dehydrogenase 1) gene in neurons of C57BL/6 mice results in increased depolarization-induced glutamate release that eventually leads to selective neuronal injury and cell loss by 12 months of age. However, it is known that isogenic lines of Tg (transgenic) mice produced through back-crossing with one strain may differ in their phenotypic characteristics from those produced using another inbred mouse strain. Therefore, we decided to introduce the Glud1 transgene into the Balb/c strain that has endogenously lower levels of GLUD1 (glutamate dehydrogenase 1) enzyme activity in the brain as compared with C57BL/6. Using an enzyme-based MEA (microelectrode array) that is selective for measuring glutamate in vivo, we measured depolarization-induced glutamate release. Within a discrete layer of the striatum, glutamate release was significantly increased in Balb/c Tg mice compared with wt (wild-type) littermates. Furthermore, Balb/c mice released approx. 50–60% of the amount of glutamate compared with C57BL/6 mice. This is similar to the lower levels of endogenous GLUD1 protein in Balb/c compared with C57BL/6 mice. The development of these Glud1-overexpressing mice may allow for the exploration of key molecular events produced by chronic exposure of neurons to moderate, transient increases in glutamate release, a process hypothesized to occur in neurodegenerative disorders

The Nereid on the rise: Platynereis as a model system.

The Nereid Platynereis dumerilii (Audouin and Milne Edwards (Annales des Sciences Naturelles 1:195-269, 1833) is a marine annelid that belongs to the Nereididae, a family of errant polychaete worms. The Nereid shows a pelago-benthic life cycle: as a general characteristic for the superphylum of Lophotrochozoa/Spiralia, it has spirally cleaving embryos developing into swimming trochophore larvae. The larvae then metamorphose into benthic worms living in self-spun tubes on macroalgae. Platynereis is used as a model for genetics, regeneration, reproduction biology, development, evolution, chronobiology, neurobiology, ecology, ecotoxicology, and most recently also for connectomics and single-cell genomics. Research on the Nereid started with studies on eye development and spiralian embryogenesis in the nineteenth and early twentieth centuries. Transitioning into the molecular era, Platynereis research focused on posterior growth and regeneration, neuroendocrinology, circadian and lunar cycles, fertilization, and oocyte maturation. Other work covered segmentation, photoreceptors and other sensory cells, nephridia, and population dynamics. Most recently, the unique advantages of the Nereid young worm for whole-body volume electron microscopy and single-cell sequencing became apparent, enabling the tracing of all neurons in its rope-ladder-like central nervous system, and the construction of multimodal cellular atlases. Here, we provide an overview of current topics and methodologies for P. dumerilii, with the aim of stimulating further interest into our unique model and expanding the active and vibrant Platynereis community

A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice, however until recently a major limitation to performing screens on this scale has been the cost effective isolation and sequencing of insertion sites. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA isolation, DNA digestion, linker or splinkerette ligation, primary and secondary PCR amplification, and sequencing. This method, which takes about 1 week to perform, has allowed us to isolate hundreds of thousands of insertion sites from mouse tumours and, unlike other methods, has been specifically optimised for the isolation of insertion sites generated with the murine leukaemia virus (MuLV), and can easily be performed in 96 well plate format for the efficient multiplex isolation of insertion sites

Differentiating Non-Motor Symptoms in Parkinson's Disease from Controls and Hemifacial Spasm

10.1371/journal.pone.0049596PLoS ONE82

Restriction Site Extension PCR: A Novel Method for High-Throughput Characterization of Tagged DNA Fragments and Genome Walking

BACKGROUND: Insertion mutant isolation and characterization are extremely valuable for linking genes to physiological function. Once an insertion mutant phenotype is identified, the challenge is to isolate the responsible gene. Multiple strategies have been employed to isolate unknown genomic DNA that flanks mutagenic insertions, however, all these methods suffer from limitations due to inefficient ligation steps, inclusion of restriction sites within the target DNA, and non-specific product generation. These limitations become close to insurmountable when the goal is to identify insertion sites in a high throughput manner. METHODOLOGY/PRINCIPAL FINDINGS: We designed a novel strategy called Restriction Site Extension PCR (RSE-PCR) to efficiently conduct large-scale isolation of unknown genomic DNA fragments linked to DNA insertions. The strategy is a modified adaptor-mediated PCR without ligation. An adapter, with complementarity to the 3' overhang of the endonuclease (KpnI, NsiI, PstI, or SacI) restricted DNA fragments, extends the 3' end of the DNA fragments in the first cycle of the primary RSE-PCR. During subsequent PCR cycles and a second semi-nested PCR (secondary RSE-PCR), touchdown and two-step PCR are combined to increase the amplification specificity of target fragments. The efficiency and specificity was demonstrated in our characterization of 37 tex mutants of Arabidopsis. All the steps of RSE-PCR can be executed in a 96 well PCR plate. Finally, RSE-PCR serves as a successful alternative to Genome Walker as demonstrated by gene isolation from maize, a plant with a more complex genome than Arabidopsis. CONCLUSIONS/SIGNIFICANCE: RSE-PCR has high potential application in identifying tagged (T-DNA or transposon) sequence or walking from known DNA toward unknown regions in large-genome plants, with likely application in other organisms as well

Working Memory Impairment in Fibromyalgia Patients Associated with Altered Frontoparietal Memory Network

BACKGROUND: Fibromyalgia (FM) is a disorder characterized by chronic widespread pain and frequently associated with other symptoms. Patients with FM commonly report cognitive complaints, including memory problem. The objective of this study was to investigate the differences in neural correlates of working memory between FM patients and healthy subjects, using functional magnetic resonance imaging (MRI). METHODOLOGY/PRINCIPAL FINDINGS: Nineteen FM patients and 22 healthy subjects performed an n-back memory task during MRI scan. Functional MRI data were analyzed using within- and between-group analysis. Both activated and deactivated brain regions during n-back task were evaluated. In addition, to investigate the possible effect of depression and anxiety, group analysis was also performed with depression and anxiety level in terms of Beck depression inventory (BDI) and Beck anxiety inventory (BAI) as a covariate. Between-group analyses, after controlling for depression and anxiety level, revealed that within the working memory network, inferior parietal cortex was strongly associated with the mild (r = 0.309, P = 0.049) and moderate (r = 0.331, P = 0.034) pain ratings. In addition, between-group comparison revealed that within the working memory network, the left DLPFC, right VLPFC, and right inferior parietal cortex were associated with the rating of depression and anxiety? CONCLUSIONS/SIGNIFICANCE: Our results suggest that the working memory deficit found in FM patients may be attributable to differences in neural activation of the frontoparietal memory network and may result from both pain itself and depression and anxiety associated with pain

Epidermal growth factor receptor dimerization status determines skin toxicity to HER-kinase targeted therapies

Skin toxicity, a common drug-related adverse event observed in cancer patients treated with epidermal growth factor receptor (EGFR)-directed therapies is rarely seen with therapies targeting HER2. This study reports the significance of the EGFR and HER2 dimerization status in skin with regard to these dermatologic side effects. We demonstrate the differential effect of HER-directed therapies on the ligand driven activation status of EGFR, HER2 and MAPK in normal human epidermal keratinocytes. EGFR-directed therapies, such as gefitinib and cetuximab, inhibited ligand-induced activation of EGFR and MAPK in human keratinocytes. Pertuzumab, an antibody interfering with functional HER2 heterodimerization, failed to block ligand-induced HER signaling in primary keratinocytes. Using a novel proximity-based dimerization assay (eTag™) we show that EGFR homodimers are the predominant HER dimer pair in normal primary kertinocytes and in normal skin tissue from 16 patients with solid malignancies. The presence of [p]EGFR and [p]MAPK, but the absence of [p]HER2, demonstrates productive signaling via EGFR but not HER2 in human skin. These data illustrate the importance of the EGFR dimerization partner in human skin and suggests that inhibition of EGFR homodimer signaling rather than EGFR/HER2 heterodimer signaling maybe the key molecular event determining dermatologic toxicity discrepancies observed between EGFR-targeted versus HER2-targeted therapies