Fatigue in U.S. Astronauts Onboard the International Space Station: Environmental Factors, Operational Impacts, and Implementation of Countermeasures

Since 2000, US astronauts have been supporting missions up to a six month duration on the International Space Station (ISS). Crewmembers have experienced fatigue for reasons similar to military deployments. Astronauts experience psychological stressors such as heavy workloads, extended duty periods, circadian misalignment, inadequate/ineffective sleep, and loss of the environmental cues of a gravity environment. Complicating the psychological stressors are environmental factors; distracting background noise, unexpected and variable mission schedules, unfavorable thermal control, elevated CO2 levels, and an unusual sleep environment with schedules that impinge on presleep periods. Physiological contributors to poor sleep and fatigue include a cephalad fluid shift and back pain. Restful sleep is further challenged due to a lack of gravityrelated proprioceptive cues and need for restraints. The term "space fog" has been used by astronauts to describe a phenomenon of forgetfulness, slowed reaction time and transient confusion while trying to complete tasks. There is a distinct temporal correlation with arrival on the Space Station and the onset of slowed cognitive skills and a spontaneous resolution that may take up to 6 weeks. The Genesis of this phenomenon may be chronic fatigue secondary to transitioning from a planar environment to a 360deg microgravity perspective. Recently, countermeasures to improve sleep duration and quality in astronauts on the ISS have been instituted with moderate degrees of success as measured by selfreaction time (psychomotor vigilance task testing), actigraphy, and subjective reports. Judicious use of stimulants and hypnotics, light therapy, controlled sleep periods and sleep shifting and reducing ambient CO2 levels are a few of the most promising countermeasures being used in space to improve sleep and reduce fatigue

Fatigue in U.S. Astronauts Onboard the International Space Station: Environmental factors, Operational Impacts, and Implementation of Countermeasures

Crewmembers have experienced fatigue for reasons similar to military deployments. Astronauts experience psychological stressors such as: heavy workloads, extended duty periods, circadian misalignment, inadequate/ineffective sleep, distracting background noise, unexpected and variable mission schedules, unfavorable thermal control, unusual sleep environment with schedules that impinge on presleep periods

Three-dimensional structure of an immunoglobulin light-chain dimer with amyloidogenic properties

The X-ray structure of an immunoglobulin light-chain dimer isolated from the urine as a 'Bence-Jones protein' from a patient with multiple myeloma and amyloidosis (Sea) was determined at 1.94 Angstrom resolution and refined to R and R-free factors of 0.22 and 0.25, respectively. This 'amyloidogenic' protein crystallized in the orthorhombic P2(1)2(1)2(1) space group with unit-cell parameters a=48.28, b=83.32, c=112.59 Angstrom as determined at 100 K. In the vital organs (heart and kidneys), the equivalent of the urinary protein produced fibrillar amyloid deposits which were fatal to the patient. Compared with the amyloidogenic Mcg light-chain dimer, the Sea protein was highly soluble in aqueous solutions and only crystallized at concentrations approaching 100 mg ml(-1). Both the Sea and Mcg proteins packed into crystals in highly ordered arrangements typical of strongly diffracting crystals of immunoglobulin fragments. Overall similarities and significant differences in the three-dimensional structures and crystalline properties are discussed for the Sea and Mcg Bence-Jones proteins, which together provide a generalized model of abnormalities present in lambda chains, facilitating a better understanding of amyloidosis of light-chain origin (AL)

Promiscuous Binding in a Selective Protein: The Bacterial Na+/H+ Antiporter

The ability to discriminate between highly similar substrates is one of the remarkable properties of enzymes. For example, transporters and channels that selectively distinguish between various solutes enable living organisms to maintain and control their internal environment in the face of a constantly changing surrounding. Herein, we examine in detail the selectivity properties of one of the most important salt transporters: the bacterial Na/H antiporter. Selectivity can be achieved at either the substrate binding step or in subsequent antiporting. Surprisingly, using both computational and experimental analyses synergistically, we show that binding per se is not a sufficient determinant of selectively. All alkali ions from Li to Cs were able to competitively bind the antiporter's binding site, whether the protein was capable of pumping them or not. Hence, we propose that NhaA's binding site is relatively promiscuous and that the selectivity is determined at a later stage of the transport cycle

Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

Ruiz JC, D'Afonseca V, Silva A, et al. Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains. PLoS ONE. 2011;6(4): e18551.Background: Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829

Hybridization Assays Using an Expressible DNA Fragment Encoding Firefly Luciferase as a Label

We report the use of a new label, an expressible enzymecoding DNA fragment, for nucleic acid hybridization assays. The DNA label contains a firefly luciferase coding sequence downstream from a T7 RNA polymerase promoter. The target DNA (200 bp) is denatured and hybridized simultaneously with two oligonucleotide probes. One of the probes is immobilized in microtiter wells, via the digoxigenin/anti-digoxigenin interaction, and the other probe is biotinylated. After completion of the hybridization, the hybrids are reacted with a streptavidin-luciferase DNA complex. Subsequently, the solid-phase bound DNA is expressed by coupled transcription/ translation. The synthesized luciferase catalyzes the luminescent reaction of luciferin with O 2 and ATP. The luminescence is linearly related to the amount of target DNA in the range of 5-5000 amol. The CVs obtained for 20 and 100 amol of target are 6.5% and 10.8%, respectively (n ) 4). The specific and strong interaction between two complementary nucleic acid strands forms the basis for the development of hybridization assays. Hybridization methodology is emerging as the most promising area in laboratory medicine and has transformed the way clinical testing is realized. Previous tests have been based on the monitoring of gene products, i.e., phenotypic markers, such as oncoproteins, viral antigens, etc. In contrast, current laboratory tests that are based on hybridization allow the analysis of disease at the nucleic acid level. Thus, pre-or postnatal diagnosis of genetic disease can be accomplished by hybridization of the patient's DNA with allele-specific oligonucleotide probes that recognize mutations, deletions, or insertions causing the disease. Also, the various infectious agents can be measured in biological fluids by hybridization with specific probes. In forensic science, hybridization of DNA with minisatellite probes allows the unique identification of individuals (DNA fingerprinting). 1,2 Radioactive probes (usually labeled with 32 P), in combination with autoradiographic detection, dominated in the field of hybridization assays for more than 2 decades and provide the highest sensitivities. However, the short half-life of 32 P, the health hazards and problems associated with its use and disposal, and the long exposure times (many hours to days) required for detection have placed limitations on the routine use of hybridization assays in the clinical laboratory. The current trend in this area is toward novel nonradioactive alternatives. 2,3 The labels can be incorporated into the probes either enzymatically (e.g., using DNA polymerase or deoxynucleotidyl transferase and modified deoxynucleoside triphosphates) or by chemical conjugation (e.g., introduction of NH 2 groups into the probe via cytidine transamination and then conjugation to the reporter molecule). 4 Nonisotopic hybridization assays based on fluorescent, chemiluminescent, or enzyme labels have been developed. Generally, there are two strategies for the analysis of hybrids. Either the reporter molecule is directly conjugated to the probe, 5,6 or a ligand is attached to the probe and the hybrids are measured in a subsequent step by adding a specific, labeled binding protein. The ligand may be biotin or a hapten (e.g., digoxigenin). Labeled (strept)avidin or antihapten antibodies may then be employed for detection. 7,8 Enzymes (such as alkaline phosphatase and horseradish peroxidase) are the most widely used nonradioisotopic labels because they provide amplification through the high turnover of their substrates to detectable products. 2 Recently, we reported 9 that a DNA fragment (DNA template) coding for an enzyme can be used as a novel label for the development of highly sensitive immunoassays (expression immunoassays). In these assays, after completion of the immunoreaction, the DNA template (a luciferase-coding DNA) is expressed by in vitro transcription/translation and the activity of the synthesized enzyme is measured. Furthermore, it was estimated that 12-14 luciferase molecules were synthesized from each DNA template molecule. In the present work, we extend our investigation in the area of hybridization assays. EXPERIMENTAL SECTION Instrumentation. Luminescence measurements were carried out using a liquid scintillation counter (Model LS-6500, Beckman Instruments Inc., Fullerton, CA) in the single photon monitoring mode. Fluorescence measurements were performed with th