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NASA NTRS1985

Evidence that Resorption of Bone by Rat Peritoneal Macrophages Occurs in an Acidic Environment

Skeletal loss in space, like any form of osteoporosis, reflects a relative imbalance of the activities of cells resorbing (degrading) or forming bone. Consequently, prevention of weightlessness induced bone loss may theoretically be accomplished by (1) stimulating bone formation or (2) inhibiting bone resorption. This approach, however, requires fundamental understanding of the mechanisms by which cells form or degrade bone, information not yet at hand. An issue central to bone resorption is the pH at which resorption takes place. The pH dependent spectral shift of a fluorescent dye (fluorescein isothiocyanate) conjugated to bone matrix was used to determine the pH at the resorptive cell bone matrix interface. Devitalized rat bone was used as the substrate, and rat peritoneal macrophages were used as the bone resorbing cells. The results suggest that bone resorption is the result of generation of an acidic microenvironment at the cell matrix junction.

Blair, H. C.↗

NASA NTRS1985

Characterization of flow in a scroll duct

A quantitative, flow visualization study was made of a partially elliptic cross section, inward curving duct (scroll duct), with an axial outflow through a vaneless annular cutlet. The working fluid was water, with a Re(d) of 40,000 at the inlet to the scroll duct, this Reynolds number being representative of the conditions in an actual gas turbine scroll. Both still and high speed moving pictures of fluorescein dye injected into the flow and illuminated by an argon ion laser were used to document the flow. Strong secondary flow, similar to the secondary flow in a pipe bend, was found in the bottom half of the scroll within the first 180 degs of turning. The pressure field set up by the turning duct was strong enough to affect the inlet flow condition. At 90 degs downstream, the large scale secondary flow was found to be oscillatory in nature. The exit flow was nonuniform in the annular exit. By 270 degs downstream, the flow appeared unorganized with no distinctive secondary flow pattern. Large scale structures from the upstream core region appeared by 90 degs and continued through the duct to reenter at the inlet section.

Begg, E. K.↗

NASA NTRS1993

Cosmos 2229 immunology study (Experiment K-8-07)

The purpose of the current study was to further validate use of the rhesus monkey as a model for humans in future space flight testing. The areas of immunological importance examined in the Cosmos 2229 flight were represented by two sets of studies. The first set of studies determined the effect of space flight on the ability of bone marrow cells to respond to granulocyte/monocyte colony stimulating factor (GM-CSF). GM-CSF is an important regulator in the differentiation of bone marrow cells of both monocyte/macrophage and granulocyte lineages and any change in the ability of these cells to respond to GM-CSF can result in altered immune function. A second set of studies determined space flight effects on the expression of cell surface markers on both spleen and bone marrow cells. Immune cell markers included in this study were those for T-cell, B-cell, natural killer cell, and interleukin-2 populations. Variations from a normal cell population percentage, as represented by these markers, can be correlated with alterations in immunological function. Cells were stained with fluorescein-labelled antibodies directed against the appropriate antigens, and then analyzed using a flow cytometer.

Sonnenfeld, Gerald↗

NASA NTRS1996

Modification of reproductive development in Arabidopsis thaliana under spaceflight conditions

Reproductive development in Arabidopsis thaliana (L.) Heynh. cv. Columbia plants was investigated under spaceflight conditions on shuttle mission STS-51. Plants launched just prior to initiation of the reproductive phase developed flowers and siliques during the 10-d flight. Approximately 500 flowers were produced in total by the 12 plants in both the ground control and spaceflight material, and there was no significant difference in the number of flowers in each size class. The flower buds and siliques of the spaceflight plants were not morphologically different from the ground controls. Pollen viability tests immediately post-flight using fluorescein diacetate indicated that about 35% of the pollen was viable in the spaceflight material. Light-microscopy observations on this material showed that the female gametophytes also had developed normally to maturity. However, siliques from the spaceflight plants contained empty, shrunken ovules, and no evidence of pollen transfer to stigmatic papillae was found by light microscopy immediately post-flight or by scanning electron microscopy on fixed material. Short stamen length and indehiscent anthers were observed in the spaceflight material, and a film-like substance inside the anther that connected to the tapetum appeared to restrict the release of pollen from the anthers. These observations indicate that given appropriate growing conditions, early reproductive development in A. thaliana can occur normally under spaceflight conditions. On STS-51, reproductive development aborted due to obstacles in pollination or fertilization.

manned↗

NASA NTRS1998

Detection and quantification of Epstein-Barr virus EBER1 in EBV-infected cells by fluorescent in situ hybridization and flow cytometry

A rapid and highly sensitive fluorescent in situ hybridization (FISH) assay was developed to detect Epstein Barr virus (EBV)-infected cells in peripheral blood. Multiple fluorescein-labeled antisense oligonucleotide probes were designed to hybridize to the EBER1 transcript, which is highly expressed in latently infected cells. After a rapid (30 min) hybridization, the cells were analyzed by flow cytometry. EBER1 was detected in several positive control cell lines that have variable numbers of EBV genome copies. No EBER1 was detected in two known EBV-negative cell lines. Northern blot analyses confirmed the presence and quantity of EBER1 transcripts in each cell line. This method was used to quantify the number of EBV-infected cells in peripheral blood from a patient with chronic mononucleosis. These results indicate that EBV-infected cells can be detected at the single cell level, and that this assay can be used to quantify the number of EBV-infected cells in clinical samples.

NASA Discipline Regulatory Physiology↗

NASA NTRS1998

The effect of track structure on the induction of chromosomal aberrations in murine cells

PURPOSE: To measure chromosome aberrations in C3H 10T1/2 mouse fibroblasts using FISH painting at the first mitosis following exposure to 30 keV/microm hydrogen or neon ions. MATERIALS AND METHODS: Cells in plateau-phase were irradiated with 0.86 MeV protons at the TTT-3 Tandem accelerator in Naples (Italy), or with 400 MeV/n Ne ions at the HIMAC accelerator in Chiba (Japan). Colcemid-blocked cells were harvested at the first mitosis following exposure, and chromosome spreads were hybridized in situ with a fluorescein-labelled composite mouse DNA probe specific for chromosomes 2 and 8. RESULTS: Protons were more efficient than neon ions at the same LET in the induction of chromosome interchanges and breaks. Yields of complex exchanges were similar for both particles at the same dose, but protons produced mostly insertions, while with Ne exposure non-reciprocal exchanges were the most frequent complex-type exchange. CONCLUSIONS: Charged particles with the same LET produce different yields of chromosome aberrations, and some observed differences can be explained based on the available track-structure models.

NASA Center JSC↗

NASA NTRS1992

Identification of a nuclear localization sequence in the polyomavirus capsid protein VP2

A nuclear localization signal (NLS) has been identified in the C-terminal (Glu307-Glu-Asp-Gly-Pro-Gln-Lys-Lys-Lys-Arg-Arg-Leu318) amino acid sequence of the polyomavirus minor capsid protein VP2. The importance of this amino acid sequence for nuclear transport of newly synthesized VP2 was demonstrated by a genetic "subtractive" study using the constructs pSG5VP2 (expressing full-length VP2) and pSG5 delta 3VP2 (expressing truncated VP2, lacking amino acids Glu307-Leu318). These constructs were transfected into COS-7 cells, and the intracellular localization of the VP2 protein was determined by indirect immunofluorescence. These studies revealed that the full-length VP2 was localized in the nucleus, while the truncated VP2 protein was localized in the cytoplasm and not transported to the nucleus. A biochemical "additive" approach was also used to determine whether this sequence could target nonnuclear proteins to the nucleus. A synthetic peptide identical to VP2 amino acids Glu307-Leu318 was cross-linked to the nonnuclear proteins bovine serum albumin (BSA) or immunoglobulin G (IgG). The conjugates were then labeled with fluorescein isothiocyanate and microinjected into the cytoplasm of NIH 3T6 cells. Both conjugates localized in the nucleus of the microinjected cells, whereas unconjugated BSA and IgG remained in the cytoplasm. Taken together, these genetic subtractive and biochemical additive approaches have identified the C-terminal sequence of polyoma-virus VP2 (containing amino acids Glu307-Leu318) as the NLS of this protein.

Non-NASA Center↗

NASA NTRS2010

Oscillation of Angiogenesis with Vascular Dropout in Diabetic Retinopathy by VESsel GENeration Analysis (VESGEN)

PURPOSE. Vascular dropout and angiogenesis are hallmarks of the progression of diabetic retinopathy (DR). However, current evaluation of DR relies on grading of secondary vascular effects, such as microaneurysms and hemorrhages, by clinical examination instead of by evaluation of actual vascular changes. The purpose of this study was to map and quantify vascular changes during progression of DR by VESsel GENeration Analysis (VESGEN). METHODS. In this prospective cross-sectional study, 15 eyes with DR were evaluated with fluorescein angiography (FA) and color fundus photography, and were graded using modified Early Treatment Diabetic Retinopathy Study criteria. FA images were separated by semiautomatic image processing into arterial and venous trees. Vessel length density (L(sub v)), number density (N(sub v)), and diameter (D(sub v)) were analyzed in a masked fashion with VESGEN software. Each vascular tree was automatically segmented into branching generations (G(sub 1)...G(sub 8) or G(sub 9)) by vessel diameter and branching. Vascular remodeling status (VRS) for N(sub v) and L(sub v) was graded 1 to 4 for increasing severity of vascular change. RESULTS. By N(sub v) and L(sub v), VRS correlated significantly with the independent clinical diagnosis of mild to proliferative DR (13/15 eyes). N(sub v) and L(sub v) of smaller vessels (G(sub >=6) increased from VRS1 to VRS2 by 2.4 X and 1.6 X, decreased from VRS2 to VRS3 by 0.4 X and 0.6X, and increased from VRS3 to VRS4 by 1.7 X and 1.5 X (P < 0.01). Throughout DR progression, the density of larger vessels (G(sub 1-5)) remained essentially unchanged, and D(sub v1-5) increased slightly. CONCLUSIONS. Vessel density oscillated with the progression of DR. Alternating phases of angiogenesis/neovascularization and vascular dropout were dominated first by remodeling of arteries and subsequently by veins.

Parsons-Wingerter, Patricia↗

NASA NTRS2011

Flight Testing of the Forward Osmosis Bag for Water Recovery on STS-135

The Forward Osmosis Bag (FOB) is a personal water purification device for recovery of potable liquid from almost any non-potable water source. The FOB experiment was flown as a sortie mission on STS-135/ULF7 using flight-certified materials and a design based on the X-Pack(TradeMark) from Hydration Technology Innovations (Albany, OR). The primary objective was to validate the technology for use under microgravity conditions. The FOB utilizes a difference in solute concentration across a selectively permeable membrane to draw water molecules from the non-potable water while rejecting most chemical and all microbial contaminants contained within. Six FOB devices were tested on STS-135 for their ability to produce a potable liquid permeate from a feed solution containing 500 mL potassium chloride (15 g/L) amended with 0.1% methyl blue dye (w:v) tracer against an osmotic gradient created by addition of 60 mL of concentrate containing the osmolytes fructose and glucose, and 0.01% sodium fluorescein (w:v) tracer. Three FOB devices were physically mixed by hand for 2 minutes by a crewmember after loading to augment membrane wetting for comparison with three unmixed FOB devices. Hydraulic flux rate and rejection of salt and dye in microgravity were determined from a 60-mL sample collected by the crew on orbit after 6 hours. Post-flight analysis of samples collected on orbit demonstrated that the Forward Osmosis Bag achieved expected design specifications in microgravity. The hydraulic flux rate of water across the membrane was reduced approximately 50% in microgravity relative to ground controls that generated an average of 50 mL per hour using the same water and osmolyte solutions. The membrane rejected both potassium and chloride at >92% and methyl blue dye at >99.9%. Physical mixing of the FOB during water recovery did not have any significant effect on either flux rate or rejection of solutes from the water solution. The absence of buoyancy-driven convection in microgravity suggests that mass transport was dominated by diffusion, slowing the rate of permeate production across the membrane. It is possible that a predicted reduction in concentration polarization at the membrane surface that may have acted to increase the rate of permeate production in microgravity was negligible under the described test conditions.

Roberts, Michael S.↗

NASA NTRS2005

Radiation Dosimetry via Automated Fluorescence Microscopy

A developmental instrument for assessment of radiation-induced damage in human lymphocytes includes an automated fluorescence microscope equipped with a one or more chargecoupled- device (CCD) video camera(s) and circuitry to digitize the video output. The microscope is also equipped with a three-axis translation stage that includes a rotation stage, and a rotary tray that holds as many as thirty specimen slides. The figure depicts one version of the instrument. Once the slides have been prepared and loaded into the tray, the instrument can operate unattended. A computer controls the operation of the stage, tray, and microscope, and processes the digital fluorescence-image data to recognize and count chromosomes that have been broken, presumably by radiation. The design and method of operation of the instrument exploit fluorescence in situ hybridization (FISH) of metaphase chromosome spreads, which is a technique that has been found to be valuable for monitoring the radiation dose to circulating lymphocytes. In the specific FISH protocol used to prepare specimens for this instrument, metaphase lymphocyte cultures are chosen for high mitotic index and highly condensed chromosomes, then several of the largest chromosomes are labeled with three of four differently colored whole-chromosome-staining dyes. The three dyes, which are used both individually and in various combinations, are fluorescein isothiocyanate (FITC), Texas Red (or equivalent), and Cy5 (or equivalent); The fourth dye 4',6-diamidino- 2-phenylindole (DAPI) is used as a counterstain. Under control by the computer, the microscope is automatically focused on the cells and each slide is scanned while the computer analyzes the DAPI-fluorescence images to find the metaphases. Each metaphase field is recentered in the field of view and refocused. Then a four-color image (more precisely, a set of images of the same view in the fluorescent colors of the four dyes) is acquired. By use of pattern-recognition software developed specifically for this instrument, the images in the various colors are processed to recognize the metaphases and count the chromosome fragments of each color within the metaphases. The intermediate results are then further processed to estimate the proportion of cells that have suffered genetic damage. The prototype instrument scans at an average areal rate of 4.7 mm2/h in unattended operation, finding about 14 metaphases per hour. The false-alarm rate is typically less than 3 percent, and the metaphase-miss rate has been estimated to be less than 5 percent. The counts of chromosomes and fragments thereof are 50 to 70 percent accurate.

Castleman, Kenneth R.↗

NASA NTRS2012

Nano Sponges for Drug Delivery and Medicinal Applications

This invention is a means of delivering a drug, or payload, to cells using non-covalent associations of the payload with nano-engineered scaffolds; specifically, functionalized single-walled carbon nanotubes (SWNTs) and their derivatives where the payload is effectively sequestered by the nanotube's addends and then delivered to the site (often interior of a cell) of interest. Polyethylene glycol (PEG) and other water-soluble organic molecules have been shown to greatly enhance the solubility of SWNTs in water. PEG groups and other water-solubilizing addends can act to sequester (sponge) molecules and deliver them into cells. Using PEG that, when attached to the SWNTs, the SWNT/PEG matrix will enter cells has been demonstrated. This was visualized by the addition of fluorescein isothiocyanate (FITC) to the SWNT/PEG matrix. Control studies showed that both FITC alone and FITC/PEG did not enter the cells. These observations suggest that the FITC is highly associated with the SWNT/PEG matrix that brings the FITC into the cells, allowing visualization of SWNTs in cells. The FITC is not covalently attached, because extended dialysis in hot DMF will remove all fluorescence quickly (one week). However, prolonged dialysis in water (1-2 months) will only slowly diminish the fluorescence. This demonstrates that the SWNT/PEG matrix solubilizes the FITC by sequestering it from the surrounding water and into the more solubilizing organic environment of the SWNT/PEG matrix of this type. This can be extended for the sequestering of other molecules such as drugs with PEG and other surfactants.

Tour, James M.↗

NASA NTRS2015

Selection of Leafy Green Vegetable Varieties for a Pick-and-Eat Diet Supplement on ISS

Several varieties of leafy vegetables were evaluated with the goal of selecting those with the best growth, nutrition, and organoleptic acceptability for ISS. Candidate species were narrowed to commercially available cultivars with desirable growth attributes for space (e.g., short stature and rapid growth). Seeds were germinated in controlled environment chambers under conditions similar to what might be found in the Veggie plant growth chamber on ISS. Eight varieties of leafy greens were grown: 'Tyee' spinach, 'Flamingo' spinach, 'Outredgeous' Red Romaine lettuce, 'Waldmann's Dark Green' leaf lettuce, 'Bull's Blood' beet, 'Rhubarb' Swiss chard, 'Tokyo Bekana' Chinese cabbage, and Mizuna. Plants were harvested at maturity and biometric data on plant height, diameter, chlorophyll content, and fresh mass were obtained. Tissue was ground and extractions were performed to determine the tissue elemental content of Potassium (K), Magnesium (Mg), Calcium (Ca) and Iron (Fe). Following the biometric/elemental evaluation, four of the eight varieties were tested further for levels of anthocyanins, antioxidant (ORAC-fluorescein) capacity, lutein, zeaxanthin, and Vitamin K. For sensory evaluation, 'Outredgeous' lettuce, Swiss chard, Chinese cabbage, and Mizuna plants were grown, harvested when mature, packaged under refrigerated conditions, and sent to the JSC Space Food Systems Laboratory. Tasters evaluated overall acceptability, appearance, color intensity, bitterness, flavor, texture, crispness and tenderness. All varieties received acceptable scores with overall ratings greater than 6 on a 9-point hedonic scale. Chinese cabbage was the highest rated, followed by Mizuna, 'Outredgeous' lettuce, and Swiss chard. Based on our results, the selected varieties of Chinese cabbage, lettuce, Swiss chard and Mizuna seem suitable for a pick-and-eat scenario on ISS with a ranking based on all factors analyzed to help establish priority.

Nutrition↗

NASA NTRS1962

Effect of Alpha-Particle Irradiation on Brain Glycogen in the Rat

The studies of Klatzo, Miquel, Tobias and Haymaker (1961) have shown that one of the earliest and most sensitive indications of the effects of alpha-particle irradiation on rat bran is the appearance of glycogen granules mainly in the neuroglia of the exposed area of the brain. Periodic acid-Schiff (PAS) positive, alpha-amylase soluble granules were demonstrated within 12 hr after irradiation, preceding by approximately 36 hr the first microscopically detectable vascular permeability disturbances, as shown by the fluorescein labeled serum protein technique. These studies suggested that the injurious effects of alpha-particle energy were on cellular elements primarily, according to the physical properties and distribution of the radiation in the tissue, and that the vascular permeability disturbances played a secondary role in pathogenesis. The purpose of this study was to correlate the histochemical observations on glycogen with a quantitative assessment of the glycogen in the irradiated brain tissue. It is felt that such a study may contribute to the understanding of radiation injury at the molecular level. A practical aspect of this problem is that the information on biological radiation effects due to accelerated particles from the cyclotron source, is employed in this study, is applicable to radiation from cosmic particles both in free space and entrapped in the Van Allen belts.

Wolfe, L. S.↗

NASA NTRS2016

Impact of The Protective Renin-Angiotensin System (RAS) on The Vasoreparative Function of CD34+ CACs in Diabetic Retinopathy

Purpose: In diabetes, the impaired vasoreparative function of Circulating Angiogenic Cells (CACs) is believed to contribute to the progression of diabetic retinopathy (DR). Accumulating evidence suggests that the protective arm of renin-angiotensin system (RAS) ACE2 Angiotensin-(1-7) Mas plays an important role in restoring the function of diabetic CACs. We examined the protective RAS in CACs in diabetic individuals with different stages of retinopathy. Methods: Study subjects (n43) were recruited as controls or diabetics with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fundus photography and fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ CACs were isolated from peripheral blood of diabetics and control subjects. RAS gene expressions in CACs were measured by qPCR. The vasoreparative function of CACs was assessed by migration ability toward CXCL12 using the QCM 5M 96-well chemotaxis cell migration assay. Results: ACE2 gene is a key enzyme converting the deleterious Angiotensin II to the beneficial Angiotensin-(1-7). ACE2 expression in CACs from diabetic subjects without DR was increased compared to controls, suggestive of compensation (p0.0437). The expression of Mas (Angiotensin-(1-7) receptor) in CACs was also increased in diabetics without DR, while was reduced in NPDR compared to controls (p0.0002), indicating a possible loss of compensation of the protective RAS at this stage of DR. The presence of even mild NPDR was associated with CD34+ CAC migratory dysfunction. When pretreating CACs of DR subjects with Angiotensin-(1-7), migratory ability to a chemoattractant CXCL12 was restored (p0.0008). By VESGEN analysis, an increase in small vessel density was observed in NPDR subjects when compared with the controls. Conclusions: These data suggest the protective RAS axis within diabetic CACs may help maintain their vasoreparative potential. The VESGEN analysis supports the presence of retinal repair in small vessels. The loss of the protective arm of RAS may predict the progression of DR.

diabetes↗

NASA NTRS2017

Association Between Vascular Density and Loss of Protective RAS During Early NPDR by Fractal Dimension

Purpose: Our hypothesis predicts that blood vessels within the retina increase in density during early-stage nonproliferative diabetic retinopathy (NPDR), based on previous results of a small retrospective study. For the current prospective study, the remodeling of arteries and veins during progression of early NPDR is assessed by a repertoire of parameters that includes the fractal dimension (D(sub f) ). In complex structures such as branching vascular trees, D(sub f) is a sensitive measure of space-filing capacity. The renin-angiotensin system (RAS) is implicated in DR pathogenesis and the function of circulating angiogenic cells (CACs), a critical bone marrow-derived population instrumental in vascular repair. Methods: Arterial and venous branching patterns were extracted from images of 6 normal controls and 3 early NPDR subjects (2 moderate, 1 mild) acquired by Heidelberg Spectralis (Registered Trademark) OCT following fluorescein angiography (FA). The vascular branching patterns were analyzed by NASAs VESsel GENeration Analysis (VESGEN) software, in which skeletonized representations were generated automatically to yield D(sub f) by the box-counting method. For binary 2D images, D(sub f) varies between limiting Euclidean dimensions of 1 and 2. Peripheral blood of diabetics and controls was collected for CD34+ CAC isolation. The gene expression of RAS in CACs was assessed by qPCR for Mas receptor to Ang-(1-7). The vasoreparative function of the CACs was measured by migration ability toward CXCL12 (SDF-1). Results: By D(sub f), venous and arterial densities were 1.370 +/- 0.006 and 1.329 +/- 0.016 for early NPDR, compared to 1.318 +/- 0.012 and 1.320 +/- 0.036 for control. The space filling capacity in early NPDR measured by D(sub f), a sensitive parameter, therefore demonstrated a pronounced increase for veins, but not for arteries. Mas receptor mRNA in CACs was increased in diabetics without DR but reduced with onset of NPDR, indicating possible loss of compensation of protective RAS during early DR. Migratory dysfunction of CD34+ cells was further associated with DR. Conclusions: As assessed by the fractal dimension in our preliminary study, the space-filling capacity of veins, but not arteries, was greater in early NPDR than in control. Larger patient populations will be examined as we complete our ongoing longitudinal study. Results further suggest the protective RAS axis within diabetic CACs is lost early in DR and is associated with increased vascular remodeling as evidenced by VESGEN analysis.

vascular↗

NASA NTRS2017

Sample Processor for Life on Icy Worlds (SPLIce): Design and Test Results

We report the design, development, and testing of the Sample Processor for Life on Icy Worlds (SPLIce) system, a microfluidic sample processor to enable autonomous detection of signatures of life and measurements of habitability parameters in Ocean Worlds. This monolithic fluid processing-and-handling system (Figure 1; mass 0.5 kg) retrieves a 50-L-volume sample and prepares it to supply a suite of detection instruments, each with unique preparation needs. SPLIce has potential applications in orbiter missions that sample ocean plumes, such as found in Saturns icy moon Enceladus, or landed missions on the surface of icy satellites, such as Jupiters moon Europa. Answering the question Are we alone in the universe? is captivating and exceptionally challenging. Even general criteria that define life very broadly include a significant role for water [1,2]. Searches for extinct or extant life therefore prioritize locations of abundant water whether in ancient (Mars), or present (Europa and Enceladus) times. Only two previous planetary missions had onboard fluid processing: the Viking Biology Experiments [3] and Phoenixs Wet Chemistry Laboratory (WCL) [4]. SPLIce differs crucially from those systems, including its capability to process and distribute L-volume samples and the integration autonomous control of a wide range of fluidic functions, including: 1) retrieval of fluid samples from an evacuated sample chamber; 2) onboard multi-year storage of dehydrated reagents; 3) integrated pressure, pH, and conductivity measurement; 4) filtration and retention of insoluble particles for microscopy; 5) dilution or vacuum-driven concentration of samples to accommodate instrument working ranges; 6) removal of gas bubbles from sample aliquots; 7) unidirectional flow (check valves); 8) active flow-path selection (solenoid-actuated valves); 9) metered pumping in 100 nL volume increments. The SPLIce manifold, made of three thermally fused layers of precision-machined cyclo-olefin polymer, supports all fluidic components (Figure 1) and integrated microchannels (125 x 250 m). Fluid is pumped by a stepper-motor-driven pump (Lee Co.). The functionality of the integrated MEMS pressure sensor (Honeywell) and passive check valves (Figure 2) were tested in conjunction with our newly designed integral bubble traps (Figure 3) and hydrophobic membrane-based concentrator (Figure 4). The concentrator (initially tested as a standalone component) demonstrated 5-fold vacuum-evaporative concentration. Polyethylene fused bead beds (PEFBBs; 50 porosity) store drylyophilized buffers, calibrants, and fluorescent dyes, and also promote mixing of sample with calibrant, dye, or H2O. Software-controlled automated tests demonstrated successful 1) fluid delivery to each component 2) valve and pump synchronization 3) sample aliquot delivery to instrument interface ports, and 4) rehydration of vacuum-dried fluorescent dye. In Figure 5, fluorescein on PEFBBs was rehydrated for 15 min using a pump-delivered water aliquot; it is displaced as H2O enters the bottom of the channel and pushes the dye into a check valve. Ultimately, SPLIce will fluorescently label amino acids in the sample for microchip-based electrophoretic (MCE) chiral separation and detection to seek and quantify key organic bio-signatures [5]; it will also deliver sample to a microfluidic version of WCL (mWCL) to measure soluble ions and redox-active species.

Life detection↗

NASA NTRS2017

Renin-Angiotensin System (RAS) in Hematopoietic Stem/Progenitor Cells (HS/PC) Predicts Vaso-Reparative Dysfunction and Progression of Diabetic Retinopathy (DR)

Purpose: We tested the hypothesis that loss of angiotensin converting enzyme 2 (ACE2) within diabetic HS/PCs (Hematopoietic Stem/Progenitor Cells) would be detrimental to HS/PC reparative function, and alter their ability to contribute to vascular remodeling in human subjects and rodent models of DR (Diabetic Retinopathy). Methods: Subjects (n52) were recruited as controls (n13) or diabetics (n39) with either no DR, mild non-proliferative DR (NPDR), moderate NPDR, severe NPDR or proliferative DR (PDR). Fluorescein angiograms were analyzed using Vessel Generation Analysis (VESGEN) software in a cohort of subjects. CD34+ HS/PCs were isolated from peripheral blood. RAS (Renin-Angiotensin System) gene expression and migration was measured. Diabetic ACE2 knockout (KO)C57BL6-Ins2 (Akita) mice at 3, 6 and 9 months of diabetes were compared to age-matched controls. Bone marrow HS/PC populations were analyzed by flow cytometry and migration and proliferation studies performed. Results: ACE2 gene expression in human CD34+ cells from diabetics without DR was increased compared to controls (p0.0437). Mas receptor mRNA was also increased in diabetics without DR, but reduced with the onset of NPDR (p0.0002), suggesting a loss of compensation. DR was associated with CD34+ cell migratory dysfunction. By VESGEN analysis, vessel density measured by several confirming parameters in early NPDR (n3) was greater than in normal retina (n6) in both arteries and veins, which suggests active retinal remodeling. ACE2KO-Akita and Akita cohorts showed reduced retinal thickness by OCT (Optical Coherence Tomography) at 9 months of diabetes. Absence of ACE2 in 9-month Akita mice led to an accelerated increase in acellular capillaries compared to diabetic alone. Electroretinogram (ERG) in ACE2KO-Akita mice resulted in persistent deterioration of the neural retina. Reparative function studies showed that ACE2KO exacerbated diabetes-induced impairment of LK (Low Potassium) cell migration and proliferative functions as early as 3-month of diabetes (p0.0019). Conclusions: Retinopathy and adverse vascular remodeling in subjects with diabetes was associated with a loss of the protective arm of RAS in HS/PCs. Loss of ACE2 exacerbated vascular dysfunction in diabetic mice.

retina↗

NASA NTRSDate not supplied

Retinal Image Quality Assessment for Spaceflight-Induced Visual Impairment Study

Medical reports have identified visual impairments as a risk associated with extended exposure to microgravity. Etiology of these ocular changes is currently unknown. Current hypotheses propose cephalad fluid shifts resulting from microgravity as the primary cause of ocular damage. One approach to studying ocular response to microgravity is by examining possible changes in retinal blood vessels using a NASA model of microgravity, the head-down tilt (HDT) of human subjects undergoing prolonged bed rest (BR). Retinal vessels in astronauts and BR subjects are monitored by Heidelberg Spectralis infrared (IR) imaging, in which retinal image quality is limited by insufficient resolution of small vessels. Yet small vessels respond and remodel most actively to physiological stress.For our NASA study of BR subjects, we identify retinal image quality as thecapability to capture vascular detail to acceptable resolution of small vessels. We therefore are analyzing Spectralis images acquired with fluorescein angiography(FA), where increased contrast significantly improves image resolution. The FA images are of normal subjects participating in a clinical study on diabeticretinopathy (US National Institutes of Health). We hypothesize that FA Spectralis images are of superior quality compared to non-FA Spectralis IR images.

Rodrigo Rene Rai Munoz Abujder↗