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OBJECTIVES/SPECIFIC AIMS: A small molecule therapy is within reach to treat a molecular mechanism known to result in thousands of fatal diseases. For 10% of patients with a genetic disease, a nonsense/STOP mutation/premature termination codon (PTC) is the underlying cause of their malady. PTCs prematurely stop protein synthesis and yield truncated proteins. Truncated proteins typically provide little to no proper function or activity and are rapidly degraded; thus, disease is imminent. Recent work has demonstrated that small molecules including aminoglycosides can cause the ribosome to readthrough these PTCs. Thus, PTC readthrough with small molecules is a very attractive approach for treating diseases caused by PTCs. Small molecules that promote readthrough act on the ribosome and induce a ribosomal conformational change. In this conformation, the PTC is not recognized by the translational machinery and an amino acid is incorporated into the growing peptide chain, thus protein synthesis continues and does not stop. The use of a single small molecule to readthrough various PTC mutations has been repeatedly effective for in vitro studies and some of these have progressed to clinical trials. Although there has been success in defining these small molecules, the field has discovered that every PTC is unique and likely requires a different small molecule. Thus, developing a cell culture model to test read-through of Lafora PTCs and the functionality of the protein product is the first step to developing a readthrough therapy for a LD. METHODS/STUDY POPULATION: Method for in vitro quantification of readthrough: 24 hours before transfection, HEK293 cells were split in 6-well plates. On the following day, approximately 60% confluence, the cells were transiently transfected with the WT or PTC mutated constructs using Polyethylenimine HCl MAX. Cells were transfected with a total amount of 0.35 μg DNA/well and 2 μl Polyethylenimine HCl MAX/well. Four hours later, the transfection medium was removed and replaced with fresh medium, without streptomycin and penicillin. The fresh media contained gentamicin diluted to the indicated concentration per well. Fresh gentamicin-containing medium was replaced after 24 hours. After 48 hours, lysates were collected in 100 μL mRIPA supplemented with protease inhibitors for each construct. The lysates were run on a western blot and the N-terminal was probed with anti-FLAG. A malachite green phosphatase assay to measure inorganic phosphate release from phospho-glucans, that is glycogen or LBs. Glycogen is used in this laforin bioassay as the biologically relevant substrate in order to determine the specific activity of the readthrough products. All reactions are incubated for 40 minute the absorbance is measured at 620 nm and the pmoles of phosphate released/min/nmol protein was calculated using a standard curve. RESULTS/ANTICIPATED RESULTS: HEK293 cells were transfected with MeCP2 R241X, laforin R241X, or laforin WT NT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. HEK293 cells were transfected with WT laforin or a laforin PTC CT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. B. Quantification of read-through for PTC experiments. *p-value≤0.001. #p-value≤0.001. Schematic of laforin bioassay. The assay has been performed with human and mouse tissue as well as cultured cells. B. Laforin bioassay results using laforin from PTC experiment. **p-value≤0.001. *p-value≤0.01. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that gentamicin is not only responsible for inducing readthrough of the PTC mutations, but also for promoting translation of fully functional laforin. Therefore, our in vitro system for the analysis of PTC readthrough of laforin will be useful for determining which PTC mutations are suppressible with gentamicin or other small molecules, in what quantities laforin is recovered from PTC mutations, and if the protein products possess the appropriate enzymatic function.
Recent estimates of total pre-weaning piglet mortality range between 16-19% (MLC 2006). With environmental modification using the farrowing crate reaching its potential to decrease mortality, as well as raising serious welfare concerns, a different approach to effectively address piglet survival is needed. Genetic breeding programmes implemented in alternative farrowing systems could prove a viable option.
Despite its extreme geographical isolation, numerous expeditions have surveyed the marine flora and fauna of Johnston Atoll. However, historical information about the marine biodiversity of Johnston is mostly limited to SCUBA surveys in shallow-waters (<30 m), and submersible observations in deeper waters (100–500 m). Extensive coral reefs, known as mesophotic coral ecosystems, exist between these two depth ranges at Johnston, but have remained largely unexplored. We used closed-circuit rebreathers to survey eleven sites at mesophotic depths (32–78 m) surrounding Johnston Atoll. A total of 130 species were recorded, including 99 species of fish, 15 species of corals, nine species of macroalgae, three species of echinoderms, three species of sponges and one species of squat lobster. Most species recorded during our mesophotic surveys have previously been recorded on shallow-water (<30 m) reefs at Johnston, with the exception of one black coral, one zoanthid, one squat lobster, two macroalgae, three sponges, and 22 fish, which represent new records for the atoll. As noted in previous studies, our surveys found a near absence of endemism, and recorded high proportions of species that are also known from the Hawaiian Archipelago. The similarity between the mesophotic biodiversity of Johnston Atoll and Hawaiʻi provides further support for the strong connectivity between these two locations highlighted in previous studies.
A new solid-state embedding approach has been developed which focuses on modelling the surfaces of polar materials. The method is applied to investigate the chemisorption of pre- methanol species on the polar (000-1) surface of zincite (a major phase of zinc oxide having the wurtzite structure). Initial results include the geometries of active sites and adsorbates in different charge states.
The interactions of YBa2Cu3O7-δ type high Tc superconductors with other metals and oxides are of significant technical importance because of the need for i) proper stabilizing normal metal for composite superconductor wire, ii) nonreactive crucible materials for melt processing or crystal growth, and iii) suitable nonpoisonous substrate materials for thin film/thick film superconducting devices. For these reasons, and also for the purpose of exploring possible improvements in Tc, Jc and mechanical properties, the effects of various metal and oxide additions (1–40% by weight) have been investigated. It is shown that many of the elements in the periodic table deteriorate the superconducting properties to a various degree ranging from a broadened transition or reduced Tc to a complete elimination of the superconducting behavior. However, silver, gold and cadmium were relatively benign or slightly improved the properties. These benign materials have potential for practical application in superconducting composites.
Carbon aggregates of various nuclearities have been recently the subject of renewed interest . Despite an intense research, effort no molecular level characterization of C60 and related molecules has been reported yet [1,2]. Moreover, the chemical properties of these materials is virtually unknown. In this paper we report briefly the preparation, electrochemical and structural characterization of crystalline C60/70 solvates .
The transport Jc of the polycrystalline YBa2Cu3O7-δ superconductor seems to be dominated by weak links between high Jc regions as evidenced by low Jc values and their strong field dependence. The possible effects of thermal expansion-contraction and the tetragonal-orthorhombic transformation on the weak links and the Jc values were investigated by repeated thermal cycling of sintered pellets between -196°C and various high temperatures (600–850°C) using a furnace heating and cooling in an oxygen atmosphere. While more than a five-fold decrease in Jc from -400 to -70 A/cm2 (at 77K in zero field) is observed after 5 temperature cycles between -196'C and 850°C, only a slight decrease (to -370 Atm2) is noticed after 5 cycles between -196*C to 600°C, the temperature span of which is not all that much smaller than the former cycles. Transmission electron microscopy analysis clearly indicates that the drastic deterioration in Jc by repeated phase transformation is caused by increased amount of microcracks on (001) basal planes near the grain boundaries. The results will be discussed in terms of the large thermal expansion anisotropy of this layer-structured compound.
In this paper we will describe the production, separation and characterization of the new all carbon molecules, C60 and C70. High performance liquid chromatography HPLC is used to obtain purified samples of C60 and C70, which are subsequently characterized by electron impact and chemical ionization mass spectrometry, IR and UV-visible absorption spectroscopy, NMR, ESR, scanning tunnelling microscopy, transmission electron microscopy and x-ray absorption fine structure measurements.
The recent discovery of the YBa2Cu3O7-δ type high Tc superconductors stimulated worldwide R&D interest in this field. However, the relatively low critical current density (Jc) in the polycrystalline, bulk superconductors (as well as its significant deterioration in weak magnetic fields) has been a major roadblock to the rapid technical advancement toward applications. In this paper, we investigated the effect of processing and microstructural control on Jc of the superconductor. Improved Jc values of -3100 A/cm2 at 77K with somewhat reduced field dependence have been obtained through appropriate microstructural modifications.
The nucleation and cluster growth of C60 and C70 crystallites on various substrates at ambient temperature have been investigated using electron microscopy. It was found that the initial nucleation is closely associated with surface defects, and the fullerenes are much more strongly bonded to each other than to the substrate. Sublimed C60 or C70 crystallites nucleate at the step edge in the liquid state and are aligned with the step walls and terraces through the process of coalescence. Reflection Electron Microscopy (REM) studies have shown an abnormal profile of C60 grown crystals as a result of the interaction of C60 molecules with the surface strain field during crystal growth. Transmission electron diffraction patterns reveal a twin structure with (110) habit plane for the low temperature ordered phase.
The synthesis and properties of two polycarbosilanes that have essentially a
“SiH2CH2” composition is described. One of these
polymers is a highly branched hydridopolycarbosilane (HPCS) derived from
Grignard coupling of CI3SiCH2CI followed by
LiAIH4 reduction. This synthesis is amenable to large scale
production and we are exploring applications of HPCS as a source of SiC
coatings and its allyl-derivative, AHPCS, as a matrix source for SiC- and
C-fiber-reinforced composites. These polymers thermoset on heating at
200-400 °C (or at 100 °C with a catalyst) and give near stoichiometric SiC
with low O content in ca. 80% yield on pyrolysis to 1000 °C. The second
method involves ring-opening polymerization of
1,1,3,3-tetrachlorodisilacyclobutane and yields a high molecular weight,
linear polymer that can be reduced to
[SiH2CH2]n (PSE), the monosilicon analog
of polyethylene. In contrast to high density polyethylene which melts at 135
°C, PSE is a liquid at room temperature which crystallizes at ca. 5 °C. On
pyrolysis to 1000 °C, PSE gives stoichiometric, nanocrystalline, SiC in
virtually quantitative yield. The polymer-to-ceramic conversion was examined
for PSE by using TGA, mass spec, solid state NMR, and IR methods yielding
information regarding the cross-linking and structural evolution processes.
The results of these studies of the polymer-to-ceramic conversion process
and our efforts to employ the AHPCS polymer as a source of SiC matrices are
The macromolecular matrix present in the composite shell of the blue mussel, Mytilus edulis, accounts for less than 1% of the shell by weight but is theorized to play a significant role in controlling the growth, morphology, and orientation of the CaCO3 that makes up the shell. The presence of several proteins in this matrix, only some of which have affinity for calcium, suggests a hierarchical structural model for the shell. Proteins were isolated under denaturing, reducing conditions and separated by centrifugation, gel electrophoresis, and high performance liquid chromatography. The major matrix proteins, both soluble and insoluble, were evaluated for amino acid composition, calcium binding, and glycosylation. Some N-terminal sequence data was collected. Non-proteinaceous components of the matrix were also analyzed. Comparison of the mussel shell matrix with the protein matrix of other molluscan systems suggests that this complexity is not unique to the mussel and may provide a key to the understanding of more generic biomineralization processes necessary for such applications as biomimetic ceramics.
Transparent, nanophase-separated, inorganic-organic hybrid polymers with dielectric constants below 3.0 have been prepared from reactively functionalized poly(amic ester) derivatives and substituted, oligomeric silsesquioxanes. These hybrid materials are stable to 400 °C and above and form tough, crack-free films. Induced cracking and crack propagation studies performed with the application of external stress suggest a maximum critical film thickness of at least 2.0 μm under severe stress conditions. These hybrid materials appear to be significantly toughened by the chemical incorporation of the polyimides relative to organically modified silicates and spin-on-glasses without significantly effecting other important polymer properties of the silicates.
Porous organosilicates useful for on-chip insulator applications can be prepared by templating the vitrification of low molecular weight silsesquioxanes (SSQs) using highly branched, thermally labile macromolecules which are subsequently removed in a thermal process to generate porosity. The process involves spin coating a mixture of the matrix material and the porogen (pore generator) followed by thermal curing to initiate vitrification and decomposition of the porogen. The morphology is fixed during the formation of the nanoscopic inorganicorganic hybrid and is maintained during foaming. This process generates controllable and stable morphologies where the void volume is determined by the porogen loading level. The porous materials are thermally robust and intrinsically hydrophobic without subsequent chemical treatment. Dielectric constants of < 2.2 are easily achieved for pore volumes of only 20%, and this porosity appears to be predominately closed cell in nature. These materials display a number of thermal mechanical and electric properties consistent with the requirements for on-chip insulator applications.
The commercial development of low-power electronics and electro-optics based on antimonides demands a better understanding of the mechanical properties of ternary and quaternary thin-film alloys fabricated from the InGaAlAsSbP material system. Of particular importance is the determination of Young's modulus of these materials. In this paper, a technique for studying the mechanical behavior of these thin films was developed by using microbeam bending and finite element modeling. The technique was successfully applied to investigate the mechanical properties of GaSb. A test structure consisting of an array of gallium antimonide microbeams was fabricated with lengths ranging from 50 to 500 μm long. The microbeams were deflected using a calibrated nanoprobe, thereby generating load-displacement curves. Young's modulus was then extracted from the data using beam bending theory and a finite element simulation of the structures under load. A total of five microbeams with the same trapezoidal cross-section and lengths of 80, 85, 200, 250 and 500 μm were tested to study the technique applicability and size scaling effects on the mechanical properties. It was observed that the 80 and 85 μm beams exhibited linear elastic behavior and the 200, 250, and 500 μm microbeams exhibited non-linear elastic behavior.
1. The use of Esch. coli alone as an index of faecal pollution for shellfish, and the correlation between the 44° C. MacConkey test and citrate tests are discussed.
2. The mercury-toluene thermo-regulator used in these experiments, which gives a maximum variation of ± 0.1° C., is discussed briefly and illustrated.
3. Experiments are described in which 522 colonies from polluted shellfish were isolated, inoculated into MacConkey's broth and incubated at temperatures of 37° C. and at successive 1° intervals from 41 to 46° C., in accurately controlled water-baths. An almost perfect negative correlation was found to exist between 44° C. incubation and the citrate test.
4. It appeared that temperatures above 44° C. are detrimental to the growth of Esch. coli.
5. Certain cultures of citrate-negative lactose fermenters at 37° C., which were inhibited at 44° C., were found on further investigation to be mostly intermediate types.
It appears that one of the first things that occurred to Felix d'Herelle when he discovered bacteriophages in 1917 was that these mysterious objects might provide a means of killing bacteria that are pathogenic to humans (Summers, 1999). The still ongoing story of phage therapy, as this approach was called, has been told elsewhere and will not be retold here, but it serves to point out that scientists have been interested in the effects of phages on their hosts since their discovery. d'Herelle believed, and eventually established, that phages are viruses that infect bacteria. However, it was not until the experimental investigations of phages at the dawn of molecular biology in the 1940s and 1950s that it became clear that phages - and for that matter their bacterial hosts - are genetic organisms (Luria and Delbrück, 1943; Hershey and Rotman, 1949; Hershey and Chase, 1952; Stent, 1963), just like fruit flies, corn, and humans, and so could be expected to mutate and evolve.
Although some work was done on the evolution of phages in the 1960s, 1970s, and 1980s, a more detailed understanding of the genetic mechanisms of phage evolution had to wait until the advent of high-throughput DNA sequencing in the 1990s. This is because the genetic history of a phage, while it is to a significant extent encoded in the phage's genome sequence, is largely invisible to our analysis until we can compare that sequence to the genome sequences of other phages.
The aim was to determine the prevalence of chronic fatigue syndrome (CFS), chronic fatigue and fibromyalgia in UK military personnel after the Gulf War 1990–1991.
A two-phase cohort study was used. Three randomly selected subsamples identified from a population-based cross-sectional postal survey of over 10 000 current and ex-service UK military personnel (Gulf veterans were those deployed to the Gulf War 1990–1991; non-Gulf veterans were Bosnia peacekeepers 1992–1997 and those on active duty during the Gulf War 1990–1991 but not deployed) were recruited. Their disability status was assessed using the Short Form 36 physical functioning scale; Gulf veterans who reported physical disability (n=111) were compared with non-Gulf (n=133) veterans who reported similar levels of physical disability. Screening for known medical and psychiatric conditions was conducted to exclude medical explanations for disability and symptomatic distress. Standardised criteria for CFS, chronic fatigue and fibromyalgia were used.
Disabled Gulf veterans were more likely to be overweight, have elevated γ-glutamyl transferase levels and screen positive for hypertension. There were no other clinically significant differences in clinical markers for medically explainable conditions. Disabled Gulf veterans were more likely than similarly disabled Bosnia and Era veterans (adjusted odds ratio 7.8, 95% confidence interval 2.5–24.5) to meet the criteria for CFS. Rates for other medically unexplained conditions were not significantly increased.
Symptoms in keeping with CFS account for a significant part of the symptomatic distress in Gulf veterans.
Pre-weaning piglet mortality is currently 11.8% of piglets born alive in indoor units (MLC, 2005) and is a major welfare concern and a continuing production problem within the pig industry. The farrowing crate was implemented with some success to decrease the amount of crushing of piglets (Edwards & Fraser, 1997). However, this system is restrictive, limits the behaviour and compromises the welfare of the sow (Jarvis et al., 2001). There is growing pressure to abolish this technology in favour of less restrictive systems. It is therefore vital to identify behavioural and physiological characteristics relating to piglet survival, which can then be influential in alternative systems. Important factors in relation to piglet survival include birth weight, birth order, and adequate thermoregulation (Tuchscherer et al. 2001). The aim of this study was to identify additional behavioural and physiological indicators, which could predict piglet survival.
This letter carries the name of the Senior Editor of Word of God Books (incorporating Jamnia Press Ltd, T&T Muratori and Oxbridge University Press) to contributors of The Bible. We are unsure whether this letter was actually sent, or the exact date of writing (some of the instructions in the letter seem not to have been carried out). The present draft is apparently a verbal transcription of a dictated text, as indicated by the appended note from the secretary, who has scribbled ‘He pronounced all these words unto me with his mouth, and I wrote them with ink in the book.’
I am writing to update you on the progress of The Bible, our flagship project. Many thanks to those of you who have already submitted the first drafts of their contracted chapters. May I remind the remainder that deadlines are pressing, and that, due to the joint pressures of market and mortality, we are absolutely unable to accommodate some of the requests for extended revision periods. (Some manuscripts were originally commissioned in the Iron Age and have still not been completed centuries later, and it is now time for closure [Note to Daphne: Please underline this sentence on the copy that goes to Isaiah]).
Could we also direct you to that part of the contract that says that ‘I represent that the chapter was written entirely by me, that publication of my chapter will not violate or infringe on the rights of any other individual or company’.