The Paleocene cephalopod fauna from pebble point, Victoria (Australia)-fulcrum between two Eras

Three species belonging to three genera of early Cenozoic nautilid cephalopods are described from Paleocene aged beds of the Pebble Point and Dilwyn formations, Victoria, Australia: Aturoidea distans Teichert, Eutrephoceras victorianum Teichert, both previously known from these deposits, and Nautilus praepompilius Shimansky, previously unknown from Australia. Here we present new occurrence and paleoecological information about these three taxa based on previously and newly collected specimens from Pebble Point Formation beds, as well as presenting the first paleotemperature analyses for Australian nautiloid cephalopods of any age. We sampled for shell carbonates from the single known specimen of Nautilus praepompililus, as well as from a specimen of Aturoidea distans from the Pebble Point beds. The A. distans samples showed temperature of calcification to have been between 20 and 25°C; and for N. praepompilius the temperatures were between 18 and 21°C. There were too few samples to provide statistical analyses, yet the implication is that these two taxa inhabited different depths during calcification. For comparison, we have sampled shell carbonates from the only known extant site where two different nautilid genera coexist, Manus Island, Papua New Guinea. There, specimens of Nautilus pompilius and Allonautilus scrobiculatus calcified (1984 specimens) at temperatures of 12 to 17°C, conforming to previous measurements in the literature and significantly colder than any of the Paleocene specimens sampled here

Feasibility test for a V-slit star mapper for pioneer spacecraft terminal navigation

A laboratory demonstration of the feasibility of using a V-slit star mapper to meet the sensitivity and accuracy of on-board navigational requirements for future Pioneer Missions to the outer planets was conducted by the Control and Sensors Laboratory of TRW. The breadboard was extremely simple in configuration, consisting of an end-on photomultiplier tube and a V-slit reticle located at the focal plane of the objective lens. In addition, a plano-convex lens was used between the reticle and the PMT in a Fabry-Perot configuration. The analytical effort indicated that the sensor should easily meet the requirements. The Pioneer SRA test set was examined to determine its basic accuracy and modify it where necessary to bring its accuracy into the 1-3 arc second range. The test results show that it is feasible to use this type of star mapper in the 10 arc second accuracy range. The test equipment accuracy (approximately 5 arc Sec) was sufficient to bound the sensor errors at less than 10 arc seconds

In vitro analysis of promoter activity in Müller cells

PurposeRational modification of promoter architecture is necessary for manipulation of transgene activity and requires accurate deciphering of regulatory control elements. Identification of minimally sized promoters is critical to the design of viral vectors for gene therapy. To this end, we evaluated computational methods for predicting short DNA sequences capable of driving gene expression in Müller cells.MethodsWe measured enhanced green fluorescent protein (eGFP) expression levels driven by "full-length" promoters, and compared these data with computationally identified shorter promoter elements from the same genes. We cloned and screened over 90 sequences from nine Müller cell-associated genes: CAR2, CD44, GFAP, GLUL, PDGFRA, RLBP1, S100B, SLC1A3, and vimentin (VIM). We PCR-amplified the "full-length" promoter (~1500 bp), the proximal promoter (~500 bp), and the most proximal evolutionarily conserved region (ECR; 95-871 bp) for each gene, both with and without their respective 5' untranslated regions (UTRs), from C57BL/6J mouse genomic DNA. We selected and cloned additional ECRs from more distal genomic regions (both 5' and 3') of the VIM and CD44 genes, using both mouse and rat (Sprague-Dawley) genomic DNA as templates. PCR products were cloned into the pFTMGW or pFTM3GW lentiviral transfer vectors. Plasmid constructs were transfected into rat (wMC) or human (MIO-M1) Müller cells, and eGFP expression levels were evaluated by fluorescence microscopy and flow cytometry. Selected constructs were also examined in NIH/3T3 and Neuro-2a cells.ResultsSeveral ECRs from the nine Müller cell-associated genes were able to drive reporter gene expression as well as their longer counterparts. Preliminary comparisons of ECRs from the VIM and CD44 genes suggested that inclusion of UTRs in promoter constructs resulted in increased transgene expression levels. Systematic comparison of promoter activity from nine Müller cell-expressed genes supported this finding, and characteristic regulation profiles were evident among the different genes tested. Importantly, individual cloned promoter sequences were capable of driving distinct levels of transgene expression, resulting in up to eightfold more cells expressing eGFP with up to 3.8-fold higher mean fluorescence intensity (MFI). Furthermore, combining constructs into single regulatory "units" modulated transgene expression, suggesting that secondary gene sequences provided in cis may be used to fine-tune gene expression levels.ConclusionsIn this study, we demonstrate that computational and empirical methods, when used in combination, can efficiently identify short promoters that are active in cultured Müller cells. In addition, the pFTM3GW vector can be used to study the effects of combined promoter elements. We anticipate that these methods will expedite the design and testing of synthetic/chimeric promoter constructs that should be useful for both in vitro and in vivo applications

Voltage dip generator for testing wind turbines connected to electrical networks

This paper describes a new voltage dip generator that allows the shape of the time profile of the voltage generated to be configured. The use of this device as a tool to test the fault ride-through capability of wind turbines connected to the electricity grid can provide some remarkable benefits: First, this system offers the possibility of adapting the main features of the time–voltage profile generated (dip depth, dip duration, the ramp slope during the recovery process after clearing fault, etc.) to the specific requirements set forth by the grid operation codes, in accordance with different network electrical systems standards. Second, another remarkable ability of this system is to provide sinusoidal voltage and current wave forms during the overall testing process without the presence of harmonic components. This is made possible by the absence of electronic converters. Finally, the paper includes results and a discussion on the experimental data obtained with the use of a reduced size laboratory prototype that was constructed to validate the operating features of this new device

Functional promoter testing using a modified lentiviral transfer vector

PurposeThe importance of retinal glial cells in the maintenance of retinal health and in retinal degenerations has not been fully explored. Several groups have suggested that secretion of neurotrophic proteins from the retina's primary glial cell type, the Müller cell, holds promise for treating retinal degenerations. Tight regulation of transgene expression in Müller cells is likely to be critical to the efficacy of long-term neuroprotective therapies, due to the genetic heterogeneity and progressive nature of retinal disease. To this end, we developed a modified lentiviral (LV) transfer vector (pFTMGW) to accelerate the testing and evaluation of novel transcriptional regulatory elements. This vector facilitates identification and characterization of regulatory elements in terms of size, cell specificity and ability to control transgene expression levels.MethodsA synthetic multiple cloning site (MCS) which can accept up to five directionally cloned DNA regulatory elements was inserted immediately upstream of an enhanced green fluorescent protein (eGFP) reporter. A cytomegalovirus (CMV) promoter, required for tat-independent viral packaging, is located around 2 kb upstream of the eGFP reporter and is capable of directing transgene expression. A synthetic transcription blocker (TB) was inserted to insulate the MCS/eGFP from the CMV promoter. We evaluated eGFP expression from pFTMGW and control constructs using flow cytometry and quantitative reverse transcriptase polymerase chain reaction (RT-PCR). We also tested and compared the activity and cell specificity of a computationally identified promoter fragment from the rat vimentin gene (Vim409) in transfection and lentiviral infection experiments using fluorescence microscopy.ResultsTransfection data, quantitative RT-PCR, and flow cytometry show that around 85% of expression from the CMV promoter was blocked by the TB element, allowing direct evaluation of expression from the Vim409 candidate promoter cloned into the MCS. Lentiviruses generated from this construct containing the Vim409 promoter (without the TB element) drove robust eGFP expression in Müller cells in vitro and in vivo.ConclusionsThe TB element efficiently prevented eGFP expression by the upstream CMV promoter and the novel MCS facilitated testing of an evolutionarily conserved regulatory element. Additional sites allow for combinatorial testing of additional promoter, enhancer, and/or repressor elements in various configurations. This modified LV transfer vector is an effective tool for expediting functional analysis of gene regulatory elements in Müller glia, and should prove useful for promoter analyses in other cell types and tissues