Generation of an external guide sequence library for a reverse genetic screen in Caenorhabditis elegans

<p>Abstract</p> <p>Background</p> <p>A method for inhibiting the expression of particular genes using external guide sequences (EGSs) has been developed in bacteria, mammalian cells and maize cells.</p> <p>Results</p> <p>To examine whether EGS technology can be used to down-regulate gene expression in <it>Caenorhabditis elegans </it>(<it>C. elegans</it>), we generated EGS-Ngfp-lacZ and EGS-Mtgfp that are targeted against <it>Ngfp-lacZ </it>and <it>Mtgfp </it>mRNA, respectively. These EGSs were introduced, both separately and together, into the <it>C. elegans </it>strain PD4251, which contains <it>Ngfp-lacZ </it>and <it>Mtgfp</it>. Consequently, the expression levels of <it>Ngfp-lacZ </it>and <it>Mtgfp </it>were affected by EGS-Ngfp-lacZ and EGS-Mtgfp, respectively. We further generated an EGS library that contains a randomized antisense domain of tRNA-derived EGS ("3/4 EGS"). Examination of the composition of the EGS library showed that there was no obvious bias in the cloning of certain EGSs. A subset of EGSs was randomly chosen for screening in the <it>C. elegans </it>strain N2. About 6% of these EGSs induced abnormal phenotypes such as P0 slow postembryonic growth, P0 larval arrest, P0 larval lethality and P0 sterility. Of these, EGS-35 and EGS-83 caused the greatest phenotype changes, and their target mRNAs were identified as ZK858.7 mRNA and <it>Lin-13 </it>mRNA, respectively.</p> <p>Conclusion</p> <p>EGS technology can be used to down-regulate gene expression in <it>C. elegans</it>. The EGS library is a research tool for reverse genetic screening in <it>C. elegans</it>. These observations are potentially of great importance to further our understanding and use of <it>C. elegans </it>genomics.</p

The molecular record of metabolic activity in the subsurface of the Río Tinto Mars analog

In the subsurface, the interplay between microbial communities and the surrounding mineral substrate, potentially used as an energy source, results in different mineralized structures. The molecular composition of such structures can record and preserve information about the metabolic pathways that have produced them. To characterize the molecular composition of the subsurface biosphere, we have analyzed some core samples by time-of-flight secondary ion mass spectrometry (ToF-SIMS) that were collected in the borehole BH8 during the operations of the Mars Analog and Technology Experiment (MARTE) project. The molecular analysis at a micron-scale mapped the occurrence of several inorganic complexes bearing PO3-, SOx(2to4)-, NOx(2,3)-, FeOx(1,2)- SiO2-, and Cl-. Their distribution correlates with organic molecules that were tentatively assigned to saturated and monounsaturated fatty acids, polyunsaturated fatty acids, saccharides, phospholipids, sphingolipids, and potential peptide fragments. SOx- appear to be mineralizing some microstructures larger than 25 microns, which have branched morphologies, and that source SO3-bearing adducts. PO3-rich compounds occur in two different groups of microstructures which size, morphology, and composition are different. While a group of >40-micron sized circular micronodules lacks organic compounds, an ovoidal microstructure is associated with m/z of other lipids. The NO2-/NO3- and Cl- ions occur as small microstructure clusters (<20 microns), but their distribution is dissimilar to the mineralized microstructures bearing PO3-, and SO3-. However, they have a higher density in areas with more significant enrichment in iron oxides that are traced by different Fe-bearing anions like FeO2-. The distribution of the organic and inorganic negative ions, which we suggest, resulted from the preservation of at least three microbial consortia (PO4-, and NO2-/NO3-mineralizers PO4-lipid bearing microstructures), would have resulted from different metabolic and preservation pathway

Sleeping mattress determinants and evaluation: a biomechanical review and critique

Background Sleeping mattress parameters significantly influence sleeping comfort and health, as reflected by the extensive investigations of sleeping support biomechanics to prevent sleep-related musculoskeletal problems. Methodology Herein, we review the current trends, research methodologies, and determinants of mattress biomechanics research, summarizing evidence published since 2008. In particular, we scrutinize 18 articles dealing with the development of new designs, recommendation criteria, instruments/methods of spine alignment evaluation, and comparative evaluation of different designs. Results The review demonstrated that mattress designs have strived for customization, regional features, and real-time active control to adapt to the biomechanical features of different body builds and postures. However, the suggested threshold or target values for desirable spine alignment and body pressure distribution during sleep cannot yet be justified in view of the lack of sufficient evidence. Conclusions It is necessary to formulate standard objectives and protocols for carrying out mattress evaluation

High-power mid-infrared femtosecond master oscillator power amplifier Er:ZBLAN fiber laser system

High-power femtosecond mid-infrared (MIR) lasers are of vast importance to both fundamental research and applications. We report a high-power femtosecond master oscillator power amplifier laser system consisting of a single-mode Er:ZBLAN fiber mode-locked oscillator and pre-amplifier followed by a large-mode-area Er:ZBLAN fiber main amplifier. The main amplifier is actively cooled and bidirectionally pumped at 976 nm, generating a slope efficiency of 26.9%. Pulses of 8.12 W, 148 fs at 2.8 μm with a repetition rate of 69.65 MHz are achieved. To the best of our knowledge, this is the highest average power ever achieved from a femtosecond MIR laser source. Such a compact ultrafast laser system is promising for a wide range of applications, such as medical surgery and material processing

Chromodomain Helicase DNA-Binding Protein 5 Inhibits Renal Cell Carcinoma Tumorigenesis by Activation of the p53 and RB Pathways

Chromodomain helicase DNA-binding protein 5 (CHD5) plays a crucial tumor suppressor role in multiple types of tumors. For this study, we investigated its clinical significance and the molecular mechanism(s) underlying tumorigenesis in renal cell carcinoma (RCC). Initially, CHD5 expression was assessed in primary tumor tissue and in tissue array. Correlations among CHD5 expression and clinicopathological characteristics were analyzed. Next, lentivirus-mediated CHD5 overexpression in the ACHN and 769-P cells was used to assess effects on proliferation, migration, invasion ability, and the regulation of the p14ARF/p53 and p16INK4a/RB signaling pathways. Finally, a xenograft mouse model was used to verify its impact on tumor growth in vivo. Results demonstrated that CHD5 was downregulated in tumor tissues and that low CHD5 expression was correlated with advanced TNM stage, high Fuhrman grade, lymph node metastasis, and poor survival. Overexpression of CHD5 inhibited proliferation, migration, and invasion in vitro; prompted cell cycle G1 phase arrest; induced apoptosis; and suppressed tumor growth in vivo. Furthermore, we confirmed that CHD5 activates the p53 and RB pathways to inhibit tumorigenesis in RCC. In summary, CHD5 is involved in the initiation and progression of RCC and may serve as a diagnostic biomarker and a potential therapeutic target for RCC

Ordered arrays of Ag nanodendrite clusters as effective surface-enhanced Raman scattering substrates

We report a cheap synthetic approach to large-area ordered Ag nanodendrite cluster arrays as effective surface-enhanced Raman scattering (SERS) substrates, on a Zn plate coated with periodical porous photoresist via galvanic replacement reaction of Zn and AgNO3 in aqueous solution. Due to the geometrical confinement of the porous photoresist template coated on the Zn plate, the well-positioned Ag nanodendrite clusters are distributed on the Zn plate in ordered arrays. It is demonstrated that citric acid is essential to the formation of the nanodendrite cluster arrays. The resultant Ag nanodendrite cluster arrays not only have high SERS-activity to rhodamine 6G with good signal reproducibility but also can identify thiram (a widely-used pesticide) even at 0.1 mu M level, showing potential in SERS-based detection of pesticide remnants in the environmentclose

Silencing of <i>CHD5</i> Gene by Promoter Methylation in Leukemia

<div><p>Chromodomain helicase DNA binding protein 5 (CHD5) was previously proposed to function as a potent tumor suppressor by acting as a master regulator of a tumor-suppressive network. <i>CHD5</i> is down-regulated in several cancers, including leukemia and is responsible for tumor generation and progression. However, the mechanism of <i>CHD5</i> down-regulation in leukemia is largely unknown. In this study, quantitative reverse-transcriptase polymerase chain reaction and western blotting analyses revealed that <i>CHD5</i> was down-regulated in human leukemia cell lines and samples. Luciferase reporter assays showed that most of the baseline regulatory activity was localized from 500 to 200 bp upstream of the transcription start site. Bisulfite DNA sequencing of the identified regulatory element revealed that the <i>CHD5</i> promoter was hypermethylated in human leukemia cells and samples. Thus, <i>CHD5</i> expression was inversely correlated with promoter DNA methylation in these samples. Treatment with DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates <i>CHD5</i> expression in human leukemia cell lines. <i>In vitro</i> luciferase reporter assays demonstrated that methylation of the CHD5 promoter repressed its promoter activity. Furthermore, a chromatin immunoprecipitation assay combined with qualitative PCR identified activating protein 2 (AP2) as a potential transcription factor involved in <i>CHD5</i> expression and indicated that treatment with DAC increases the recruitment of AP2 to the <i>CHD5</i> promoter. <i>In vitro</i> transcription-factor activity studies showed that AP2 over-expression was able to activate <i>CHD5</i> promoter activity. Our findings indicate that repression of <i>CHD5</i> gene expression in human leukemia is mediated in part by DNA methylation of its promoter.</p></div

The methylation status of the –560 to –240 region of the <i>CHD5</i> promoter.

<p>The CpG island, located at –560 to –240, was analyzed by BGS. Methylation data from BGS were analyzed by BiQ Analyzer software to generate the lollipop diagram and to calculate the efficiency of bisulfite conversion. Analysis of non-CpG cytosines indicated the efficiency of bisulfite conversion at ∼99%. The lollipop diagram presented the percentage of methylation of each CpG. The overall methylation percentage indicates the total proportion of methylated CpGs in this region taking into account all sequenced alleles.</p

A Distributed Parallel Algorithm Based on Low-Rank and Sparse Representation for Anomaly Detection in Hyperspectral Images

Anomaly detection aims to separate anomalous pixels from the background, and has become an important application of remotely sensed hyperspectral image processing. Anomaly detection methods based on low-rank and sparse representation (LRASR) can accurately detect anomalous pixels. However, with the significant volume increase of hyperspectral image repositories, such techniques consume a significant amount of time (mainly due to the massive amount of matrix computations involved). In this paper, we propose a novel distributed parallel algorithm (DPA) by redesigning key operators of LRASR in terms of MapReduce model to accelerate LRASR on cloud computing architectures. Independent computation operators are explored and executed in parallel on Spark. Specifically, we reconstitute the hyperspectral images in an appropriate format for efficient DPA processing, design the optimized storage strategy, and develop a pre-merge mechanism to reduce data transmission. Besides, a repartitioning policy is also proposed to improve DPA&#8217;s efficiency. Our experimental results demonstrate that the newly developed DPA achieves very high speedups when accelerating LRASR, in addition to maintaining similar accuracies. Moreover, our proposed DPA is shown to be scalable with the number of computing nodes and capable of processing big hyperspectral images involving massive amounts of data