Testing the NASA BioSentinel Pixel Dosimeter Using Gamma-ray and Neutron Sources at the LLNL Calibration Lab.

ABSTRACT: The objective of this paper is to evaluate the accuracy of the NASA BioSentinel Pixel Dosimeter (BPD) using gamma-ray and neutron sources in a standard calibration lab. The dosimeter tested here is the ground-based version of the BPD that will be onboard the BioSentinel mission. The BPD was exposed to radiation from 60Co, 137Cs, and 252Cf at selected distances (dose rates) at the Lawrence Livermore National Laboratory (LLNL) Radiation Calibration Laboratory (RCL), and the results were compared with NIST traceable benchmark values. It is recognized that these sources are not analogs for the space environment but do provide direct comparisons between BPD response and well characterized calibration lab values. For gamma rays, the BPD measured absorbed dose agrees to ≤ 3.8% compared with RCL benchmark values. For neutrons, the results show that the BPD is insensitive, i.e., the BPD detected only the gamma-ray dose component from 252Cf. The LET spectra obtained for gamma rays from 60Co and 252Cf are consistent with expectations for these gamma-ray energies, but the LET spectrum from the 137Cs gamma rays differs substantially. The potential causes for this difference are the high dose rate from 137Cs and the lower secondary electron energy produced by 137Cs gamma rays. However, neither of these results in errors in the absorbed dose. Based on comparisons with NIST-traceable standards, it is evident that the BPD can measure absorbed dose accurately from low LET charged particles. The sensor's insensitivity to neutrons is unlikely to be a limitation for the BioSentinel mission due to the expected low secondary neutron fluence.

Tapering and discontinuation of thrombopoietin receptor agonists in immune thrombocytopenia: Real-world recommendations.

Thrombopoietin receptor agonists (TPO-RAs) are currently indicated for continuous treatment of chronic primary immune thrombocytopenia (ITP). However, there is growing evidence that TPO-RAs can also trigger sustained response in 10-30% of cases after treatment tapering and discontinuation. Therefore, at least for selected responding patients, it might be rational to plan TPO-RA interruption to exploit off-treatment response. Intriguingly, complete or partial responses with TPO-RAs are frequently observed when treatments are initiated early, suggesting that unknown immune-related mechanisms may be involved in this phenomenon. The sustained responses observed after interruption of TPO-RAs may be interpreted as a recovery of immunological tolerance; thus, the re-establishment of immunological equilibrium might be primarily responsible for the observed off-treatment effect. Importantly, these findings may indicate that anticipated TPO-RA usage can lead to improved responses, and that optimized tapering and interruption in selected patients can furthermore improve prognoses. On the base of this rationale, a series of real-life considerations have been generated by a panel of Experts to elucidate possible novel criteria and modalities to identify subgroups of patients who can benefit from tapering and/or discontinuation of TPO-RAs. Towards this aim, the results of a survey of ITP experts are herein reported, reflecting a snapshot of current real-life experience on early discontinuation of TPO-RA-based therapy. The present manuscript also highlights the importance of future translational studies on novel prognostic and predictive biomarkers that can stratify patients and facilitate the clinical choice for second-line treatment of ITP.

An autonomous lab on a chip for space flight calibration of gravity-induced transcellular calcium polarization in single-cell fern spores.

This report describes the development of lab-on-a-chip device designed to measure changes in cellular ion gradients that are induced by changes in gravitational (g) forces. The bioCD presented here detects differential calcium ion concentrations outside of individual cells. The device includes sufficient replicates for statistical analysis of the gradients around multiple single cells and around control wells that are empty or include dead cells. In the data presented, the degree of the cellular response correlates with the magnitude of the g-force applied via rotation of the bioCD. The experiments recorded the longest continuous observation of a cellular response to hypergravity made to date, and they demonstrate the potential utility of this device for assaying the threshold of cells' g-force responses in spaceflight conditions.

Dynamic platelet function on von Willebrand factor is different in preterm neonates and full-term neonates: changes in neonatal platelet function.

Essentials It is unclear if platelet function differs between preterm and full-term neonates. Platelet behavior was characterized using a flow-based assay on von Willebrand Factor (VWF). Preterms had increased platelet interaction with VWF and glycoprotein Ibα expression. Platelets from preterm neonates behave differently on VWF compared to full-term neonates. SUMMARY: Background Very low birth weight (VLBW) preterm neonates have an increased risk of hemorrhage-related morbidity and mortality as compared with their full-term counterparts. It is unclear whether platelet function differs between preterm and full-term neonates. This is partly because of the large volumes of blood required to perform standard platelet function tests, and the difficulty in obtaining such samples in neonates. Objectives This study was designed to characterize platelet behavior in neonates with a physiologic flow-based assay that quantifies platelet function in microliter volumes of blood under arterial shear. Methods Blood from VLBW preterm neonates of ≤ 32 weeks' gestation (n = 15) and full-term neonates (n = 13) was perfused under arterial shear over surface-immobilized von Willebrand factor (VWF). Platelet behavior was recorded by digital-image microscopy and analyzed. Surface expression of platelet glycoprotein (GP) Ibα and GPIIIa of VLBW preterm and full-term neonates was also measured. Results VLBW preterm neonates had increased numbers of platelets interacting with VWF, and increased GPIbα expression on the platelet surface. Despite the increased numbers of VWF interactions as reflected by flow-driven platelet translocation along the protein surface, no significant differences were observed in the numbers of platelets that adhered in a stationary fashion to VWF. Platelets from VLBW preterm neonates and those from full-term neonates behaved differently on VWF. Conclusions These differences in platelet function may contribute to the higher incidence of bleeding observed in VLBW preterm neonatal populations, or may represent a compensatory mechanism to counteract this risk of bleeding.

Accuracy of physician assessment of treatment preferences and health status in elderly patients with higher-risk myelodysplastic syndromes.

Higher-risk myelodysplastic syndromes (MDS) are rarely curable and have a poor prognosis. We investigated the accuracy of physicians' perception of patients' health status and the patients' preferences for involvement in treatment decisions. We examined 280 newly diagnosed higher-risk elderly MDS patients paired with their physicians. Survey tools included the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) and the Control Preference Scale. Overall concordance was 49% for physician perception of patient preferences for involvement in treatment decisions. In 36.4% of comparisons there were minor differences and in 14.6% there were major differences. In 44.7% of the patients preferring a passive role, physicians perceived them as preferring an active or collaborative role. Absence of the patient's request for prognostic information (P=0.001) and judging the patient as having a poor health status (P=0.036) were factors independently associated with the physicians' attitude toward a lower degree of patient involvement in clinical decisions. Agreement on health status was found in 27.5% of cases. Physicians most frequently tended to overestimate health status of patients who reported low-level health status. The value of decision aid-tools in the challenging setting of higher-risk MDS should be investigated to further promote patient-centered care.

Fabrication and characterisation of spin coated oxidised PMMA to provide a robust surface for on-chip assays.

A method to prepare a highly stable carboxylic acid functional surface on various substrates for use in bioassays is reported. A thin layer of a poly(methylmethacrylate) (PMMA) is achieved by spin coating dissolved PMMA onto a variety of underlying substrates at various thicknesses in a range of c. 5-27 nm. Varying the PMMA concentration, time and spinning speed controls the thickness of the spin coated layer. The root-mean-squared roughness values of the spin coated PMMA are less than 1.5 nm, resulting in smooth and uniform layer. Substrate functionalisation is carried out by either ultraviolet/ozone (UV/O3) or oxygen plasma oxidation. Both techniques result in initially stable, highly functional films as demonstrated by the covalent attachment of amino-modified oligonucleotides, however longevity studies comparing the stability of films attached following oxidative activation show better stability for UV/O3 activated substrates when compared to oxygen plasma activated substrates. PMMA films activated by UV/O3 yield highly stable (for up to 24 days) functional surfaces that retain immobilised biomolecules after several extended wash steps. In contrast, films attached to surfaces pre-treated with oxygen plasma discharge lose their functionality within 5 days of oxidation. Direct DNA and sandwich antibody assays were successfully demonstrated on the UV/O3 functionalised surfaces, showing a low level of non-specific binding. Furthermore, the quenching of fluorescently labelled biomolecules bound to PMMA-coated gold-coated slides is shown to be dependent on the PMMA thickness, indicating potential usage in surface-plasmon resonance-based assays.

Single-step separation of platelets from whole blood coupled with digital quantification by interfacial platelet cytometry (iPC).

We report the efficient single-step separation of individual platelets from unprocessed whole blood, enabling digital quantification of platelet function using interfacial platelet cytometry (iPC) on a chip. iPC is accomplished by the precision micropatterning of platelet-specific protein surfaces on solid substrates. By separating platelets from whole blood using specific binding to protein spots of a defined size, iPC implements a simple incubate-and-rinse approach, without sample preparation, that enables (1) the study of platelets in the physiological situation of interaction with a protein surface, (2) the choice of the number of platelets bound on each protein spot, from one to many, (3) control of the platelet-platelet distance, including the possibility to study noninteracting single platelets, (4) digital quantification (counting) of platelet adhesion to selected protein matrices, enabling statistical characterization of platelet subpopulations from meaningfully large numbers of single platelets, (5) the study of platelet receptor expression and spatial distribution, and (6) a detailed study of the morphology of isolated single platelets at activation levels that can be manipulated. To date, we have demonstrated 1-4 of the above list. Platelets were separated from whole blood using iPC with fibrinogen, von Willebrand factor (VWF), and anti-CD42b antibody printed "spots" ranging from a fraction of one to several platelet diameters (2-24 µm). The number of platelets captured per spot depends strongly on the protein matrix and the surface area of the spot, together with the platelet volume, morphology, and activation state. Blood samples from healthy donors, a May-Hegglin-anomaly patient, and a Glanzmann's Thrombasthenia patient were analyzed via iPC to confirm the specificity of the interaction between protein matrices and platelets. For example, the results indicate that platelets interact with fibrinogen spots only through the fibrinogen receptor (αIIbß3) and, relevant to diagnostic applications, platelet adhesion correlates strongly with normal versus abnormal platelet function. A critical function of platelets is to adhere to regions of damage on blood vessel walls; in contrast to conventional flow cytometry, where platelets are suspended in solution, iPC enables physiologically relevant platelet bioassays based on platelet/protein-matrix interactions on surfaces. This technology should be inexpensive to implement in clinical assay format, is readily integrable into fluidic microdevices, and paves the way for high-throughput platelet assays from microliter volumes of whole blood.

[Applicability of the new law of State and Regional Agreement on management of swimming pools]. / Applicabilità del nuovo Atto di Intesa Stato-Regioni per la gestione degli impianti natatori.

The Authors carried out a survey on private and public managers of 80 Italian swimming pools to evaluate the hygienic aspects and safety of the swimming pools. Participants were submitted to a set of questions about the modifications recently brought to the Act on management of the swimming pools during 2003. The study showed a poor knowledge about the hygienic-safety parameters, in particular of chlorine doses, range of temperature and frequency of daily turnover of the swimming pool water. The respect of chemical-physical parameters is necessary not only to assure an adequate microbial control of the water, but also to reduce the production of irritant and potentially toxic substances. The Authors pointed out the need of greater attention to hygienic aspects in order to reduce health risks, deriving from an uncorrected application of the laws, and to provide a greater comfort to the users of swimming pools.

Application of disposable plastic microfluidic device arrays with customized chemistries to multiplexed biochemical assays.

Plastic microfluidic array platforms and synergistic multiplexed assay chemistries are under development for a variety of applications, including assays of gene expression, proteomics, genotyping, DNA sequencing and fragment analysis, sample preparation and high-throughput pharmaceutical discovery. The low production costs of plastic substrates makes possible economical single-use device arrays, eliminating cleaning and sample-to-sample carryover contamination. Hundreds of microchannels and reservoirs are readily included on a single microtitre-plate-size substrate, enabling the manufacture of highly parallel fluidic array systems to increase throughput and speed.

Use of linear solvation energy relationships for modeling responses from polymer-coated acoustic-wave vapor sensors.

The applicability and performance of linear solvation energy relationships (LSERs) as models of responses from polymer-coated acoustic-wave vapor sensors are critically examined. Criteria for the use of these thermodynamic models with thickness-shear-mode resonator (TSMR) and surface-acoustic-wave (SAW) vapor sensors are clarified. Published partition coefficient values derived from gas-liquid chromatography (GLC) are found to be consistently lower than those obtained gravimetrically, in accordance with previous reports, suggesting that LSERs based on GLC-derived partition coefficients will not provide accurate estimates of acoustic-wave sensor responses. The development of LSER models directly from polymer-coated TSMR vapor sensor response data is demonstrated and a revised model developed from SAW vapor sensor response data, which takes account of viscoelastic changes in polymeric coating films, is presented and compared to those developed by other methods.

Molecular cytogenetic resources for chromosome 4 and comparative analysis of phylogenetic chromosome IV in great apes.

We have generated a panel of 55 somatic cell hybrids retaining fragments of human chromosome 4. Each hybrid has been characterized cytogenetically by FISH and molecularly by 37 STSs, evenly spaced along the chromosome. The panel can be exploited to map subregionally DNA sequences on chromosome 4 and to generate partial chromosome paints useful in the characterization of chromosomal rearrangements involving this chromosome. Furthermore, a panel of 84 YACs mapping on chromosome 4 has been characterized by FISH. A subset of this panel is recognized by STSs used in the somatic cell hybrid characterization. In this way a correlation between the genetic and the physical maps can be established. These resources have been used to investigate the conservation of the phylogenetic chromosome IV in great apes. The results indicate that all the pericentric inversions that differentiate chromosome IV in these species are distinct and that one of the breakpoints frequently lies very close to the centromere. In 4 instances, the YAC containing the breakpoint was identified. The breakpoint in IVq of PTR and MMU lies in the same YAC, suggesting that this breakpoint has been utilized twice in the evolutionary history of this chromosome.

Micromanipulation of cryopreserved embryos and cryopreservation of micromanipulated embryos in PGD.

The possibility to employ cryopreservation in Preimplantation Genetic Diagnosis (PGD) should enlarge the opportunities for research and clinical activity. For these purposes, we tried three kinds of approaches on human abnormal embryos: (1) cryopreservation of biopsied embryos; (2) biopsy of thawed embryos; and (3) biopsy of embryos derived from thawed oocytes. Our preliminary results show that: (1) biopsy of thawed embryos is feasible and FISH analysis is possible on both survived and lysed cells; (2) Optimization of freezing/thawing procedures are necessary to obtain better survival rate after thawing of biopsied embryos; (3) Biopsy and FISH are feasible on embryos derived from thawed oocytes and they could be a good way to study the chromosomal arrangement of these poorly investigated embryos.

Characteristics of acoustic plate modes on rotated Y-cuts of quartz utilized for biosensing applications.

Acoustic plate modes (APM) on various quartz substrates have been investigated in order to determine their usefulness for liquid-sensing applications. The modes have been characterized in terms of their mass sensitivity, mode separation, temperature sensitivity, and reproducibility of the experimental results. Promising characteristics are found for rotated Y-cuts of quartz with the direction of acoustic mode propagation being perpendicular to the X-axis of the quartz crystal. Experiments on the detection of immunochemical reactions are performed using different quartz APM sensors, and the results are compared to similar experiments utilizing APM devices on ZX-LiNbO3.

Molecular cytogenetic resources specific for chromosome 12.

We have generated a panel of 20 somatic cell hybrids retaining fragments of human chromosome 12. Each hybrid was characterized cytogenetically by reverse fluorescence in situ hybridization (FISH) and molecularly by 24 sequence tagged sites (STSs) spaced evenly along the chromosome. The panel can be exploited to map subregionally DNA sequences on chromosome 12 and to generate partial chromosome paints useful in the characterization of chromosomal rearrangements involving this chromosome. Furthermore, a panel of 58 yeast artificial chromosomes (YACs) mapping to chromosome 12 was characterized by FISH experiments on normal human metaphases. A subset of this panel is recognized by the STSs used in the somatic cell hybrid characterization. In this way a correlation between the genetic and the physical maps of this chromosome can be established.

Effective use of molecular recognition in gas sensing: results from acoustic wave and in situ FT-IR measurements.

To probe directly the analyte/film interactions that characterize molecular recognition in gas sensors, we recorded changes to the in situ surface vibrational spectra of specifically functionalized surface acoustic wave (SAW) devices concurrently with analyte exposure and SAW measurement of the extent of sorption. Fourier transform infrared external-reflectance spectra (FT-IR-ERS) were collected from operating 97-MHz SAW delay lines during exposure to a range of analytes as they interacted with thin-film coatings previously shown to be selective: cyclodextrins for chiral recognition, nickel camphorates for Lewis bases such as pyridine or organophosphonates, and phthalocyanines for aromatic compounds. In most cases where specific chemical interactions--metal coordination, "cage" compound inclusion, or pi-stacking--were expected, analyte dosing caused distinctive changes in the IR spectra, together with anomalously large SAW sensor responses. In contrast, control experiments involving the physisorption of the same analytes by conventional organic polymers did not cause similar changes in the IR spectra, and the SAW responses were smaller. For a given conventional polymer, the partition coefficients (or SAW sensor signals) roughly followed the analyte fraction of saturation vapor pressure. These SAW/FT-IR results support earlier conclusions derived from thickness-shear mode resonator data.

Sequences flanking the centromere of human chromosome 10 are a complex patchwork of arm-specific sequences, stable duplications and unstable sequences with homologies to telomeric and other centromeric locations.

Little is known about sequence organization close to human centromeres, despite empirical and theoretical data which suggest that it may be unusual. Here we present maps which physically define large sequence duplications flanking the centromeric satellites of human chromosome 10, together with a fluorescence in situ hybridization (FISH) analysis of pericentromeric sequence stability. Our results indicate that the duplications on each chromosome arm are organized into two blocks of approximately 250 and 150 kb separated by approximately 300 kb of non-duplicated DNA. The larger proximal blocks, containing ZNF11A, ZNF33A and ZNF37A (10p11) and ZNF11B, ZNF33B and ZNF37B (10q11), are inverted. However, the smaller distal blocks, containing D10S141A (10p11) and D10S141B (10q11), are not. A primate FISH analysis indicates that these loci were duplicated before the divergence of orang-utans from other Great Apes, that a cytogenetically cryptic pericentric inversion may have been involved in the formation of the flanking duplications and that they have undergone further rearrangement in other primate species. More surprising is the fact that sequences across the entire pericentromeric region appear to have undergone unprecedented levels of duplication, transposition, inversion and either deletion or sequence divergence in all primate species analysed. Extrapolating our data to the whole genome suggests that a minimum of 50 Mb of DNA in centromere-proximal regions is subject to an elevated level of mechanistically diverse sequence rearrangements compared with the bulk of genomic DNA.

A panel of partial chromosome paints and YAC probes specific for human chromosome 2.

Twenty nine hybrids retaining fragments of human chromosome 2 were characterized by reverse-FISH and by a panel of 106 STSs. Most of the hybrids are radiation hybrids retaining fragments of chromosome 2 as the only human contribution. The hybrid panel dissected chromosome 2 in 69 distinct physical regions, allowing a fine mapping of the sequences. These hybrids are particularly useful as starting points for generation, via Alu-PCR, of specific partial chromosome paints (PCP). We also report the mapping by FISH of 60 YACs located on chromosome 2. These resources can be advantageously used in cytogenetic investigations, with particular reference to cancer cytogenetics, as illustrated with the renal carcinoma cell line KRC/Y.

Evolution of chromosome Y in primates.

We have investigated, by fluorescence in situ hybridization (FISH), the cytogenetic evolution of the Y chromosome in primates using 17 yeast artificial chromosomes, representative of the Y-specific euchromatic region of the human chromosome Y. The FISH experiments were performed on great apes (Homo sapiens, Pan troglodytes, Gorilla gorilla and Pongo pygmaeus pygmaeus), and on two Old World monkeys species as an outgroup (Cercopitecidae Macaca fascicularis and Papio anubis). The results showed that this peculiar chromosome has undergone rapid and unconstrained evolution both in sequence content and organization.

Lipoblastoma: a case with t(7;8)(q31;q13).

Lipoblastoma is a rare benign adipose tumor which, in all of the cases so far described, presents an involvement of chromosome 8 in the region 8q11-13. We hereby report the results of the second case of lipoblastoma studied by fluorescence in situ hybridization (FISH), in a 13-month-old boy. An abnormal karyotype 46,XY,t(7;8)(q31;q13) was found in 90% of the metaphases examined, in agreement with the previously reported observations. We suggest the region 8q11-13 may contain a relevant locus for lipoblastoma origin.