Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of familial Parkinson's disease (PD). Although biochemical studies have shown that certain PD mutations confer elevated kinase activity in vitro on LRRK2, there are no methods available to directly monitor LRRK2 kinase activity in vivo. We demonstrate that LRRK2 autophosphorylation on Ser(1292) occurs in vivo and is enhanced by several familial PD mutations including N1437H, R1441G/C, G2019S, and I2020T. Combining two PD mutations together further increases Ser(1292) autophosphorylation. Mutation of Ser(1292) to alanine (S1292A) ameliorates the effects of LRRK2 PD mutations on neurite outgrowth in cultured rat embryonic primary neurons. Using cell-based and pharmacodynamic assays with phosphorylated Ser(1292) as the readout, we developed a brain-penetrating LRRK2 kinase inhibitor that blocks Ser(1292) autophosphorylation in vivo and attenuates the cellular consequences of LRRK2 PD mutations in vitro. These data suggest that Ser(1292) autophosphorylation may be a useful indicator of LRRK2 kinase activity in vivo and may contribute to the cellular effects of certain PD mutations.

In this work, we propose Adversarial Complementary Learning (ACoL) to automatically localize integral objects of semantic interest with weak supervision. We first mathematically prove that class localization maps can be obtained by directly selecting the class-specific feature maps of the last convolutional layer, which paves a simple way to identify object regions. We then present a simple network architecture including two parallel-classifiers for object localization. Specifically, we leverage one classification branch to dynamically localize some discriminative object regions during the forward pass. Although it is usually responsive to sparse parts of the target objects, this classifier can drive the counterpart classifier to discover new and complementary object regions by erasing its discovered regions from the feature maps. With such an adversarial learning, the two parallel-classifiers are forced to leverage complementary object regions for classification and can finally generate integral object localization together. The merits of ACoL are mainly two-fold: 1) it can be trained in an end-to-end manner; 2) dynamically erasing enables the counterpart classifier to discover complementary object regions more effectively. We demonstrate the superiority of our ACoL approach in a variety of experiments. In particular, the Top-1 localization error rate on the ILSVRC dataset is 45.14%, which is the new state-of-the-art.

The residual defects and groups in chemically reduced graphene oxide cannot only improve the impedance match characteristic and prompt energy transition from contiguous states to Fermi level, but also introduce defect polarization relaxation and groups’ electronic dipole relaxation, which are all in favor of electromagnetic wave penetration and absorption. The chemically reduced graphene oxide shows enhanced microwave absorption compared with graphite and carbon nanotubes, and can be expected to display better absorption than high quality graphene, exhibiting a promising prospect as microwave absorbing material.

Yin Yang 1 (YY1) is a zinc finger-containing transcription factor and a target of viral oncoproteins. To determine the biological role of YY1 in mammalian development, we generated mice deficient for YY1 by gene targeting. Homozygosity for the mutated YY1 allele results in embryonic lethality in the mouse. YY1 mutants undergo implantation and induce uterine decidualization but rapidly degenerate around the time of implantation. A subset of YY1 heterozygote embryos are developmentally retarded and exhibit neurulation defects, suggesting that YY1 may have additional roles during later stages of mouse embryogenesis. Our studies demonstrate an essential function for YY1 in the development of the mouse embryo.Yin Yang 1 (YY1) is a multifunctional transcription factor that can act as a transcriptional repressor, an activator, or an initiator element-binding protein that directs and initiates transcription in vitro (8,12,22,25,27). Recent studies have focused on mechanisms by which YY1 regulates transcription and have identified repression and activation domains in YY1 (2, 9, 17, 27) as well as interactions of YY1 with coactivators and corepressors (16,34). These findings have suggested potential molecular mechanisms that may underlie the ability of YY1 to regulate transcription but have not elucidated how these molecular events contribute to the biological activities of YY1.Previous studies have shown that YY1 is a target of the adenovirus E1A oncoprotein (27). Mutations that abrogate the ability of E1A to induce oncogenic transformation also disrupt the ability of E1A to regulate YY1 (16), suggesting that YY1 is likely to play a role in cell proliferation. Studies performed with cell culture systems suggest that YY1 might also play a role in differentiation in multiple cell types (reviewed in references 26 and 28). In addition, although YY1 appears to regulate many genes that encode proteins with diverse biological activities, the genes that have been shown to be repressed by YY1 are largely associated with differentiation (reviewed in references 26 and 28). Taken together, these in vitro studies suggest a global role of YY1 in the regulation of differentiation and cell proliferation, possibly in a variety of cell types. These studies further predict that YY1 might play a crucial and exciting role in the development of higher organisms such as the mouse. However, the in vivo function of mammalian YY1 remains unclear to date.To address the role of YY1 in vivo, we disrupted one YY1 allele in mouse embryonic stem (ES) cells by homologous recombination and generated mice harboring the mutant YY1 allele. Homozygosity for the mutant YY1 allele results in embryonic lethality in the mouse. By genotyping embryos at different gestational times, we identified YY1 Ϫ/Ϫ embryos at the blastocyst stage. The YY1-deficient embryos were implanted in the uterine tissue but failed to develop to the gastrulation stage, resulting in embryonic death around the time of implantation. These findings suggest that YY1 plays an indispensable rol...

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BackgroundThe anaplastic lymphoma kinase (ALK) gene is frequently involved in translocations that lead to gene fusions in a variety of human malignancies, including lymphoma and lung cancer. Fusion partners of ALK include NPM, EML4, TPM3, ATIC, TFG, CARS, and CLTC. Characterization of ALK fusion patterns and their resulting clinicopathological profiles could be of great benefit in better understanding the biology of lung cancer.ResultsRACE-coupled PCR sequencing was used to assess ALK fusions in a cohort of 103 non-small cell lung carcinoma (NSCLC) patients. Within this cohort, the EML4-ALK fusion gene was identified in 12 tumors (11.6%). Further analysis revealed that EML4-ALK was present at a frequency of 16.13% (10/62) in patients with adenocarcinomas, 19.23% (10/52) in never-smokers, and 42.80% (9/21) in patients with adenocarcinomas lacking EGFR and KRAS mutations. The EML4-ALK fusion was associated with non-smokers (P = 0.03), younger age of onset (P = 0.03), and adenocarcinomas without EGFR/KRAS mutations (P = 0.04). A trend towards improved survival was observed for patients with the EML4-ALK fusion, although it was not statistically significant (P = 0.20). Concurrent deletion in EGFR exon 19 and fusion of EML4-ALK was identified for the first time in a Chinese female patient with an adenocarcinoma. Analysis of ALK expression revealed that ALK mRNA levels were higher in tumors positive for the EML-ALK fusion than in negative tumors (normalized intensity of 21.99 vs. 0.45, respectively; P = 0.0018). However, expression of EML4 did not differ between the groups.ConclusionsThe EML4-ALK fusion gene was present at a high frequency in Chinese NSCLC patients, particularly in those with adenocarcinomas lacking EGFR/KRAS mutations. The EML4-ALK fusion appears to be tightly associated with ALK mRNA expression levels. RACE-coupled PCR sequencing is a highly sensitive method that could be used clinically for the identification of EML4-ALK-positive patients.

ObjectivePatients with renal failure suffer from symptoms caused by uraemic toxins, possibly of gut microbial origin, as deduced from studies in animals. The aim of the study is to characterise relationships between the intestinal microbiome composition, uraemic toxins and renal failure symptoms in human end-stage renal disease (ESRD).DesignCharacterisation of gut microbiome, serum and faecal metabolome and human phenotypes in a cohort of 223 patients with ESRD and 69 healthy controls. Multidimensional data integration to reveal links between these datasets and the use of chronic kidney disease (CKD) rodent models to test the effects of intestinal microbiome on toxin accumulation and disease severity.ResultsA group of microbial species enriched in ESRD correlates tightly to patient clinical variables and encode functions involved in toxin and secondary bile acids synthesis; the relative abundance of the microbial functions correlates with the serum or faecal concentrations of these metabolites. Microbiota from patients transplanted to renal injured germ-free mice or antibiotic-treated rats induce higher production of serum uraemic toxins and aggravated renal fibrosis and oxidative stress more than microbiota from controls. Two of the species, Eggerthella lenta and Fusobacterium nucleatum, increase uraemic toxins production and promote renal disease development in a CKD rat model. A probiotic Bifidobacterium animalis decreases abundance of these species, reduces levels of toxins and the severity of the disease in rats.ConclusionAberrant gut microbiota in patients with ESRD sculpts a detrimental metabolome aggravating clinical outcomes, suggesting that the gut microbiota will be a promising target for diminishing uraemic toxicity in those patients.Trial registration numberThis study was registered at ClinicalTrials.gov (NCT03010696).

Demonstrating hybridization between an antisense oligodeoxynucleotide and its mRNA target has proven to be extremely difficult in living cells. To address this fundamental problem in antisense research, we synthesized ''molecular beacon'' (MB) reporter oligodeoxynucleotides with matched f luorescent donor and acceptor chromophores on their 5 and 3 ends. In the absence of a complementary nucleic acid strand, the MB remains in a stem-loop conformation where f luorescence resonance energy transfer prevents signal emission. On hybridization with a complementary sequence, the stem-loop structure opens increasing the physical distance between the donor and acceptor moieties thereby reducing f luorescence resonance energy transfer and allowing a detectable signal to be emitted when the beacon is excited by light of the appropriate wavelength. Solution hybridization studies revealed that in the presence of a complementary strand targeted MB could yield up to a 60-fold increase in f luorescence intensity in comparison to control MB. By using a f luorescence microscope fitted with UV f luoride lenses, the detection limit of preformed MB͞target sequence duplexes microinjected into cells was found to be >1 ؋ 10 ؊1 ag of MB, or ϳ10 molecules of mRNA. On the basis of this exquisite sensitivity, real-time detection of MB͞target mRNA hybridization in living cells was attempted by microinjecting MB targeted to the vav protooncogene, or control MB, into K562 human leukemia cells. Within 15 min, confocal microscopy revealed f luorescence in cells injected with targeted, but not control, MB. These studies suggest that realtime visualization and localization of oligonucleotide͞mRNA interactions is now possible. MB could find utility in studying RNA processing, trafficking, and folding in living cells. We hypothesize that MB may also prove useful for finding targetable mRNA sequence under physiologic conditions.