Point Absorber Limits to Future Gravitational-Wave Detectors.

High-quality optical resonant cavities require low optical loss, typically on the scale of parts per million. However, unintended micron-scale contaminants on the resonator mirrors that absorb the light circulating in the cavity can deform the surface thermoelastically and thus increase losses by scattering light out of the resonant mode. The point absorber effect is a limiting factor in some high-power cavity experiments, for example, the Advanced LIGO gravitational-wave detector. In this Letter, we present a general approach to the point absorber effect from first principles and simulate its contribution to the increased scattering. The achievable circulating power in current and future gravitational-wave detectors is calculated statistically given different point absorber configurations. Our formulation is further confirmed experimentally in comparison with the scattered power in the arm cavity of Advanced LIGO measured by in situ photodiodes. The understanding presented here provides an important tool in the global effort to design future gravitational-wave detectors that support high optical power and thus reduce quantum noise.

Considerations on nonclinical approaches to modeling risk factors of suicidal ideation and behavior.

Given the serious nature of suicidal ideation and behavior (SIB) and the possibility of treatment-emergent SIB, pharmaceutical companies are now applying more proactive approaches in clinical trials and are considering the value of nonclinical models to predict SIB. The current review summarizes nonclinical approaches to modeling three common risk factors associated with SIB: aggression, impulsivity, and anhedonia. For each risk factor, a general description, advantages and disadvantages, species considerations, nonclinical to clinical translation, and pharmacological validation with respect to treatments associated with SIB are summarized. From this review, several gaps were identified that need to be addressed before use of these nonclinical models can be considered a viable option to predict the relative risk for SIB. Other future directions that may compliment these nonclinical approaches, including the use of selectively-bred or genetically-modified rodent models, transgenic models, gene expression profiling, and biomarker analysis, are discussed. This article was developed with the support of the DruSafe Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ, www.iqconsortium.org).

Earth-like Habitable Environments in the Subsurface of Mars.

In Earth's deep continental subsurface, where groundwaters are often isolated for >106 to 109 years, energy released by radionuclides within rock produces oxidants and reductants that drive metabolisms of non-photosynthetic microorganisms. Similar processes could support past and present life in the martian subsurface. Sulfate-reducing microorganisms are common in Earth's deep subsurface, often using hydrogen derived directly from radiolysis of pore water and sulfate derived from oxidation of rock-matrix-hosted sulfides by radiolytically derived oxidants. Radiolysis thus produces redox energy to support a deep biosphere in groundwaters isolated from surface substrate input for millions to billions of years on Earth. Here, we demonstrate that radiolysis by itself could produce sufficient redox energy to sustain a habitable environment in the subsurface of present-day Mars, one in which Earth-like microorganisms could survive wherever groundwater exists. We show that the source localities for many martian meteorites are capable of producing sufficient redox nutrients to sustain up to millions of sulfate-reducing microbial cells per kilogram rock via radiolysis alone, comparable to cell densities observed in many regions of Earth's deep subsurface. Additionally, we calculate variability in supportable sulfate-reducing cell densities between the martian meteorite source regions. Our results demonstrate that martian subsurface groundwaters, where present, would largely be habitable for sulfate-reducing bacteria from a redox energy perspective via radiolysis alone. We present evidence for crustal regions that could support especially high cell densities, including zones with high sulfide concentrations, which could be targeted by future subsurface exploration missions.

Defective antigen processing in GILT-free mice.

Processing of proteins for major histocompatibility complex (MHC) class II-restricted presentation to CD4-positive T lymphocytes occurs after they are internalized by antigen-presenting cells (APCs). Antigenic proteins frequently contain disulfide bonds, and their reduction in the endocytic pathway facilitates processing. In humans, a gamma interferon-inducible lysosomal thiol reductase (GILT) is constitutively present in late endocytic compartments of APCs. Here, we identified the mouse homolog of GILT and generated a GILT knockout mouse. GILT facilitated the processing and presentation to antigen-specific T cells of protein antigens containing disulfide bonds. The response to hen egg lysozyme, a model antigen with a compact structure containing four disulfide bonds, was examined in detail.

Code status discussions and goals of care among hospitalised adults.

BACKGROUND AND OBJECTIVE: Code status discussions may fail to address patients' treatment-related goals and their knowledge of cardiopulmonary resuscitation (CPR). This study aimed to investigate patients' resuscitation preferences, knowledge of CPR and goals of care. Design, setting, patients and measurements: 135 adults were interviewed within 48 h of admission to a general medical service in an academic medical centre, querying code status preferences, knowledge about CPR and its outcome probabilities and goals of care. Medical records were reviewed for clinical information and code status documentation. RESULTS: 41 (30.4%) patients had discussed CPR with their doctor, 116 (85.9%) patients preferred full code status and 11 (8.1%) patients expressed code status preferences different from the code status documented in their medical record. When queried about seven possible goals of care, patients affirmed an average of 4.9 goals; their single most important goals were broadly distributed, ranging from being cured (n = 36; 26.7%) to being comfortable (n = 8; 5.9%). Patients' mean estimate of survival to discharge after CPR was 60.4%. Most patients believed it was helpful to discuss goals of care (n = 95; 70.4%) and the chances of surviving in hospital CPR (n = 112; 83.0%). Some patients expressed a desire to change their code status after receiving information about survival following in hospital CPR (n = 11; 8.1%) or after discussing goals of care (n = 2; 1.5%). CONCLUSIONS: Doctors need to address patients' knowledge about CPR and take steps to avoid discrepancies between treatment orders and patients' preferences. Addressing CPR outcome probabilities and goals of care during code status discussions may improve patients' knowledge and influence their preferences.

[14C]acetate metabolism in the peripheral nervous system.

Lipid biosynthesis was studied by incorporation of [14C]acetate into different compartments of rat sciatic nerve during development, degeneration and regeneration. Acetate incorporation was over three times higher in the sciatic endoneurium (desheathed nerve) than in epi- and perineurium. The endoneurium contained much higher contents of radioactively labeled membrane lipids (cholesterol and phospholipids) than did the epi- and perineurium (mainly triacylglycerol), indicating a benefit of utilization of endoneurium in the study of the metabolic derangements of peripheral nerve lipids. When 3H2O was used as a precursor, no incorporation was found. Endoneurial lipid biosynthesis from [14C]acetate decreased rapidly as myelination proceeded. After 4 months, the decrease continued but at a much slower rate. The total acetate incorporation found in endoneurial lipids of 6-month-old rats was predominantly in the free fatty acid fraction (40%), but was only 5% of that found in 10-day-old rats, demonstrating the importance of age-matched controls for metabolic studies of diseased nerve. During Wallerian degeneration, a decreased acetate incorporation into endoneurial lipids was observed as early as 2 days after crush injury. The profile of labeled lipids in developing and degenerating nerve revealed that the rate of lipogenesis did not change to the same extent for each lipid subclass. Cholesterol biosynthesis appeared to be the most sensitive. During regeneration, an increase in the uptake of [14C]acetate and an altered profile of labeled lipids demonstrated that the metabolic state of adult peripheral nerve, which is normally relatively inactive, can be modified by an exogenous factor such as crush injury.

Effects of polyunsaturated fatty acid diets on plasma lipids of patients with adrenomultineuronal degeneration, hepatosplenomegaly and fatty acid derangement.

The effect on plasma fatty acid composition of 3-6 weeks feeding of standard diets supplemented with various omega 6 polyenoic fatty acids (18:2, 18:3, 20:3 or 20:4) was studied in two young brothers with multineuronal degeneration plus. These boys had mental retardation or maldevelopment, neurosensory hearing loss, retinitis pigmentosa, progressive muscular atrophy, hepatosplenomegaly and adrenal failure. The study objectives were to localize the site of metabolic block and to assess the safety and short-term clinical effect of dietary treatment. Our studies have shown that the low plasma levels of 20:4 omega 6 can be corrected by feeding ethyl arachidonate and that no adverse effects were experienced. A diet enriched in ethyl linoleate produced no obvious increases of 18:2 omega 6 metabolites, indicating that these patients do not have a linoleate deficiency in their omega 6 polyenoic fatty acid pathway. Lack of incorporation of 20:4 omega 6 and a retroconversion of 20:3 omega 6 to 18:2 omega 6 after a dihomo-gamma-linolenate-enriched diet suggest that a defect of delta 5 desaturase may be involved.

Prospective randomized evaluation of two methods of drawing coagulation studies from heparinized arterial lines.

To establish a standardized protocol of obtaining coagulation studies from arterial lines without heparin contamination, two methods were evaluated in a prospective randomized fashion for accuracy by comparing PT and aPTT results with simultaneous samples drawn by venipuncture. Fifty paired samples were obtained, 25 by method A and 25 by method B, from critically ill patients in the surgical intensive care unit at City of Memphis Hospital. Analyses of PT and aPTT from these samples revealed a close statistical correlation with the venous controls and no clinically significant difference between the samples.

Evaluation of coagulation studies from heparinized arterial lines with use of Lab-Site high-pressure tubing.

Heparinized arterial catheters are commonly used in critically ill patients to monitor pressures and to collect blood for laboratory analysis. To remove the heparinized fluid used to keep these lines patent large volumes of blood are often withdrawn and discarded or calculations of tube volume must be made. Repeated violation of stopcocks may lead to contamination and infection of arterial lines. In addition, discarded blood has become an increasing concern as a source of infection for health care personnel. This study evaluates the efficacy of noncompliant arterial line tubing that contains two polymer sampling ports permanently placed at prefixed distances such that if blood is withdrawn to the distal port, undiluted arterial blood can then be withdrawn from the proximal port. Blood from arterial lines that consisted of 20-gauge catheters connected to Lab-Site tubing was withdrawn in the method suggested by the manufacturer with no removal or wasting of excess blood from the system. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) studies performed on this sample were compared with those performed on a simultaneously drawn venous sample. Regression analysis showed a correlation coefficient of 0.966 for PT and 0.935 for aPTT, demonstrating an excellent correlation for the technique. The average arterial PT was 0.12 seconds less than venous control and the average arterial aPTT was 0.49 seconds greater than control. Neither of these differences was significant. We conclude that this type of high-pressure tubing allows accurate blood samples to be obtained from arterial lines without the necessity of precise calculations or blood wastage.

Validation of the NFL-225 test for predicting 1-RM bench press performance in college football players.

BACKGROUND: The purpose of this study was to evaluate the accuracy of repetitions-to-fatigue (RTF) using an absolute load of 102.3 kg (225 lbs) to estimate one-repetition maximum (1-RM) bench press performance in college football players using various prediction equations. EXPERIMENTAL DESIGN: a prospective study on the association between muscular endurance and muscular strength. PARTICIPANTS: 260 players from NCAA Division IA (n=43), IAA (n=63), II (n=129), and red-shirts (n=25) were evaluated at the conclusion of a minimum of eight weeks of heavy-resistance training during the off-season. MEASURES: all subjects performed a 1-RM bench press and RTF using an absolute load of 102.3 kg. RESULTS: The Mayhew et al. NFL-225 equation nonsignificantly overestimated 1-RM from RTF by 0.5 kg, while the Chapman et al. NFL-225 equation significantly underpredicted by 3.2 kg, although both equations were comparable in the number of players predicted within +/-4.5 kg of actual 1-RM (52% vs 51%, respectively). Only two of nine RTF equations currently in use produced predicted 1-RM values that were not significantly different from actual 1-RM performance. CONCLUSIONS: Specific NFL-225 equations are more accurate in estimating 1-RM bench press from absolute muscle endurance in college football players than previous published RTF equations. The accuracy of prediction decreases at higher repetitions.

Does ultrasonic energy for surgical dissection reduce the incidence of renal transplant lymphocele?

OBJECTIVE: To determine the difference in post-renal transplant lymphocele rate based on the surgical dissection technique for control of lymphatics by examining the historical case group under the direction of a single, university-based surgeon in a retrospective, cohort study. PATIENTS: Five hundred thirty-two consecutive renal transplant patients from January 1994 to December 2009. FINDINGS: Of the 532 cases studied, 259 (48.7%) had suture ligation and 273 (51.3%) employed ultrasonic dissection (UD) for control of lymphatics during renal transplantation. There was no difference found in the rate of lymphocele formation, requiring either percutaneous or surgical drainage, when surgical ties (8.9%) were compared to UD (9.2%; P=.999). Logistic regression analysis showed that the odds ratio for developing a lymphocele was independent of surgical dissection technique. Within the logistic analysis, the prediction for lymphocele was increased 3.29 times for pediatric patients (P=.002) and increased 2.97 times for those who received a living donor graft (P=.001), and there was a trend for those with a history of more than one renal transplant of 2.01 times (P=.079). SUMMARY: Surgical dissection technique was not a factor in the development of post-renal transplant lymphocele. Younger age, living donor transplant, and repeat transplant status were found to be predictive variables for symptomatic lymphoceles requiring drainage, which may be considered when patients present for posttransplant evaluations for laboratory alterations.

Upper limits on a stochastic background of gravitational waves.

The Laser Interferometer Gravitational-Wave Observatory has performed a third science run with much improved sensitivities of all three interferometers. We present an analysis of approximately 200 hours of data acquired during this run, used to search for a stochastic background of gravitational radiation. We place upper bounds on the energy density stored as gravitational radiation for three different spectral power laws. For the flat spectrum, our limit of omega0 < 8.4 x 10(-4) in the 69-156 Hz band is approximately 10(5) times lower than the previous result in this frequency range.

Trimming and readdition of glucose to N-linked oligosaccharides determines calnexin association of a substrate glycoprotein in living cells.

To analyze the role of glucose trimming and reglucosylation in the binding of substrate proteins to calnexin in the endoplasmic reticulum (ER) of living cells, we made use of the thermosensitive vesicular stomatitis virus tsO45 glycoprotein (G protein). At nonpermissive temperature the G protein failed to fold completely and remained bound to calnexin. When the cells were shifted to permissive temperature, complete folding occurred accompanied by glucosidase-mediated elimination of calnexin-G protein complexes. If release from calnexin was blocked during the temperature shift by inhibiting the glucosidases, folding occurred, albeit at a reduced rate. In contrast, when unfolded by a shift from permissive to nonpermissive temperature, the G protein was reglucosylated rapidly and became capable of rebinding to calnexin. The rate at which calnexin binding occurred showed a 20-min delay that was explained by accumulation of the G protein in calnexin-free exit sites of the ER. These contained the glucosyltransferase responsible for reglucosylation of misfolded glycoproteins but had little or no calnexin. After unfolding and reglucosylation, the G proteins moved slowly from these structures back to the ER where they reassociated with the chaperone. Taken together, these results in live cells fully supported the lectin-only model of calnexin function. The ER exit sites emerged as a potentially important location for components of the quality control system.

Quality control of transmembrane domain assembly in the tetraspanin CD82.

Retention of misfolded proteins in the endoplasmic reticulum (ER) is a primary mechanism of quality control. To discover whether quality control can monitor assembly inside the hydrophobic ER membrane, we characterized the folding and transport of the tetraspanin glycoprotein CD82. Truncated forms of CD82 that are missing one or more transmembrane segments remain in the ER. A construct (TM 2-4) that is missing the first transmembrane segment remains in the ER, even though its extracellular domain, which is facing the ER lumen, has folded to the native structure. Transport to the cell surface is restored by co-expressing the missing segment (TM 1) as a separate polypeptide. Prior to leaving the ER, CD82 transiently associates with the membrane-bound chaperone calnexin but not with its soluble homolog calreticulin. TM 2-4, in contrast, remains in a prolonged interaction with calnexin that is partially reversed by co-expressing TM 1. These findings establish a simple system to study transmembrane domain assembly, show that ER quality control can directly monitor assembly inside the lipid bilayer and suggest that calnexin may play a role in this process.

Moloney murine leukemia virus protease expressed in bacteria is enzymatically active.

Replication of Moloney murine leukemia virus requires a readthrough translation mechanism to generate the Gag-Pol polyprotein. One of the final products of this polyprotein is the protease (PR), which is required to generate the mature virion proteins. The assembly of Gag and Gag-Pol polyprotein into a virion followed by activation of the viral protease is necessary to produce a mature, infectious particle. These events are believed to occur near the cell membrane just prior to the budding of the virion. We report here the autoproteolytic activity of the viral PR when a Gag-PR fusion protein is expressed in E. coli. Efficient cleavage at the p12/CA, CA/NC and NC/PR junctions was observed. Thus the Moloney murine leukemia virus PR is capable of cleaving its substrates in the absence of specific host factors.

Glycan-dependent and -independent association of vesicular stomatitis virus G protein with calnexin.

Calnexin (CNX) is a membrane-bound molecular chaperone that associates with newly synthesized proteins in the endoplasmic reticulum. Although several studies have indicated that it interacts exclusively with glycoproteins that carry monoglucosylated N-linked oligosaccharides, others have reported that it can bind to proteins that have no glycans. To address this discrepancy, we translated wild-type vesicular stomatitis virus G protein and nonglycosylated mutant forms in the presence of microsomes and examined their association with CNX. Individual G protein molecules were found to efficiently associate with CNX when both glycans were present and less efficiently if there was only a single glycan. Nonglycosylated G protein also interacted with CNX, but only when misfolded and present in high molecular weight aggregates. The results indicated that CNX can interact with G protein in two ways: through an oligosaccharide-dependent mechanism that involves individual substrate proteins; and in an oligosaccharide-independent association with large aggregates.

Product of a new gene, syd, functionally interacts with SecY when overproduced in Escherichia coli.

A mutant form of SecY, SecY-d1, was previously suggested to sequester a component(s) of the protein translocator complex. Its synthesis from a plasmid leads to interference with protein export in Escherichia coli. SecE is a target of this sequestration, and its overproduction cancels the export interference. We now report that overexpression of another gene, termed syd, also suppresses secY-d1. The nucleotide sequence of syd predicted that it encodes a protein of 181 amino acid residues, which has been identified by overproduction, purification, and determination of the amino-terminal sequence. Cell fractionation experiments suggested that Syd is loosely associated with the cytoplasmic surface of the cytoplasmic membrane. SecY may be involved in the membrane association of Syd since the association is saturable, the extent of which depends on the overproduction of SecY. SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available. Overproduction of Syd was found to stabilize oversynthesized SecY. However, Syd cannot stabilize the SecY-d1 form of SecY. Thus, in the presence of both secY+ and secY-d1, Syd increases the effective SecY+/SecY-d1 ratio in the cell and cancels the dominant interference by the latter. We also found that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been weakened. These results indicate that Syd, especially when it is overproduced, has abilities to interact with SecY. Possible significance of such interactions is discussed in conjunction with the apparent lack of phenotypic consequences of genetic disruption of syd.

Antiretroviral effect of a gag-RNase HI fusion gene.

We have previously shown that a molecule consisting of a fusion of a Ca(2+)-dependent nuclease (from Staphylococcus aureus) to a retroviral coat protein specifies a potent antiviral specific for that retrovirus. Genes specifying such fusion proteins can be delivered to virus-susceptible cells, providing an antiviral gene therapy aimed at limiting virus spread. We report here the results of experiments to vary the nuclease moiety of such fusion proteins. We found that one nuclease. Serratia marcescens nuclease, was extremely toxic to host cells and hence not likely to be useful for therapeutic purposes. A second nuclease, Escherichia coli RNase Hl was found to be nontoxic and highly effective against a murine leukemia virus when it was fused to the leukemia virus coat protein. The fusion protein was enzymatically active and stably expressed, without apparent toxicity to host cells. Reduction in infectious virus output was as high as 97-99%. These studies provide a model system for the development of gene therapeutic agents aimed at combating retroviral infections in vivo.

Therapeutic effect of a Gag-nuclease fusion protein against retroviral infection in vivo.

Recently, remarkable progress has been made in developing effective combination drug therapies that can control but not cure retroviral replication. Even when effective, these drug regimens are toxic, they require demanding administration schedules, and resistant viruses can emerge. Thus the need for new gene-based therapies continues. In one such approach, capsid-targeted viral inactivation (CTVI), nucleases fused to viral coat proteins are expressed in infected cells and become incorporated during virion assembly. CTVI can eliminate infectious murine retrovirus titer in tissue culture. Here we describe transgenic mice expressing fusions of the Moloney murine leukemia virus (Mo-MuLV) Gag protein to staphylococcal nuclease. This work tests the protective effect and demonstrates in vivo proof-of-principle of CTVI in transgenic mice expressing endogenous proviral copies of Mo-MuLV. The antiviral protein-expressing mice are phenotypically normal, attesting to the lack of toxicity of the fusion protein. The Mo-MuLV infection was much less virulent in transgenic littermates than in nontransgenic littermates. Gag-nuclease expression reduced infectious titers in blood up to 10-fold, decreased splenomegaly and leukemic infiltration, and increased life spans up to 2.5-fold in transgenic relative to nontransgenic infected animals. These results suggest that gene therapies based on similar fusion proteins, designed to attack human immunodeficiency virus or other retroviruses, could provide substantial therapeutic benefits.