The evaluation of red blood cell folate and methotrexate levels during protocol M in childhood acute lymphoblastic leukemia.

BACKGROUND: After High-Dose Methotrexate (HD-MTX), folinic acid rescue therapy (Leucovorin) is administered to reduce side effects in pediatric acute lymphoblastic leukemia (ALL) patients. Leucovorin and MTX are structural analogues, possibly competing for cellular transport and intracellular metabolism. We hypothesize that Leucovorin accumulates during consecutive courses, which might result in a lower MTX uptake. METHODS: We prospectively measured red blood cell (RBC) folate and MTX levels during four HD-MTX and Leucovorin courses in 43 patients treated according the DCOG ALL-11 protocol with 2-weekly HD-MTX (5 g/m2/dose) and Leucovorin (15 mg/m2/dose) using LC-MS/MS. We estimated a linear mixed model to assess the relationship between these variables over time. RESULTS: Both RBC MTX-PG and folate levels increased significantly during protocol M. MTX-PG2-5 levels increased most substantially after the first two HD-MTX courses (until median 113.0 nmol/L, IQR 76.8-165.2) after which levels plateaued during the 3d and 4th course (until median 141.3 nmol/L, IQR 100.2-190.2). In parallel, folate levels increased most substantially after the first two HD-MTX courses (until median 401.6 nmol/L, IQR 163.3-594.2) after which levels plateaued during the 3d and 4th course (until median 411.5 nmol/L, IQR 240.3-665.6). The ratio folate/MTX-PG decreased significantly over time, which was mostly due to the relatively higher increase (delta) of MTX-PG. CONCLUSION: These results suggest that the increase in RBC folate levels does not seem to have a large effect on RBC MTX levels. Future studies, assessing competition of Leucovorin and MTX on other cellular mechanisms which might negatively affect treatment efficacy, are necessary.

HIV in practice: current approaches and challenges in the diagnosis, treatment and management of HIV infection in Australia.

As treatment improves, people living with HIV (PLWHIV) can now expect to live longer, which means that the foci of HIV-related care for them and their medical practitioners continue to change. With an increasingly older cohort of patients with HIV infection, practitioners' key considerations are shifting from issues of acute treatment and patient survival to multiple comorbidities, toxicities associated with chronic therapy, and ongoing health maintenance. Within this context, this paper explores the current standard of practice for the management of HIV infection in Australia. We surveyed 56 Australian practitioners currently involved in managing HIV infection: 'HIV section 100' (HIV therapy-prescribing) general practitioners (s100 GPs; n = 26), sexual health physicians (SHPs; n = 24) and hospital-based physicians (HBPs; n = 6). Survey results for practice approaches and challenges were broadly consistent across the three practitioner specialties, apart from a few key areas. s100 GPs reported less prophylaxis use among patients whom they deemed at risk of HIV infection in comparison with SHPs, which may reflect differences in patient populations. Further, a higher proportion of s100 GPs nominated older HIV treatment regimens as their preferred therapy choices compared with the other specialties. In contrast with SHPs, s100 GPs were less likely to switch HIV therapies to simplify the treatment protocol, and to immediately initiate treatment upon patient request in those newly diagnosed with HIV infection. Considerably lower levels of satisfaction with current HIV practice guidelines were also reported by s100 GPs. It appears that greater support for s100 GPs may be needed to address these identified challenges and enhance approaches to HIV practice. Across all specialties, increasing access to mental health services for patients with HIV infection was reported as a key management issue. A renewed focus on providing improved mental health and wellbeing supports is recommended, particularly in the face of an ageing HIV-infected population.

In-flight characterization of the lunar orbiter laser altimeter instrument pointing and far-field pattern.

The Lunar Orbiter Laser Altimeter (LOLA) aboard the Lunar Reconnaissance Orbiter (LRO) has collected nearly seven billion measurements of surface height on the Moon with an absolute accuracy of ∼1 m and a precision of ∼10 cm. Converting time-of-flight laser altimeter measurements to topographic elevations requires accurate knowledge of the laser pointing with respect to the spacecraft body-fixed coordinate system. To that end, we have utilized altimetric crossovers from LOLA, as well as bidirectional observations of the LOLA laser and receiver boresight via an Earth-based laser tracking ground station. Based on a sample of ∼780,000 globally distributed crossovers from the circular-orbit phase of LRO's mission (∼27 months), we derive corrections to the LOLA laser boresight. These corrections improve the cross-track and along-track agreement of the crossovers by 24% and 33%, respectively, yielding RMS residuals of ∼10 m. Since early in the LRO mission, the bidirectional laser tracking experiments have confirmed a pointing anomaly when the LOLA instrument is facing toward deep space or the night side of the Moon and have allowed the reconstruction of the laser far-field pattern and receiver telescope pointing. By conducting such experiments shortly after launch and nearly eight years later, we have directly measured changes in the laser characteristics and obtained critical data to understand the laser behavior and refine the instrument pointing model. The methods and results presented here are also relevant to the design, fabrication, and operation of future planetary laser altimeters and their long-term behavior in the space environment.

Ice cover extent drives phytoplankton and bacterial community structure in a large north-temperate lake: implications for a warming climate.

Mid-winter limnological surveys of Lake Erie captured extremes in ice extent ranging from expansive ice cover in 2010 and 2011 to nearly ice-free waters in 2012. Consistent with a warming climate, ice cover on the Great Lakes is in decline, thus the ice-free condition encountered may foreshadow the lakes future winter state. Here, we show that pronounced changes in annual ice cover are accompanied by equally important shifts in phytoplankton and bacterial community structure. Expansive ice cover supported phytoplankton blooms of filamentous diatoms. By comparison, ice free conditions promoted the growth of smaller sized cells that attained lower total biomass. We propose that isothermal mixing and elevated turbidity in the absence of ice cover resulted in light limitation of the phytoplankton during winter. Additional insights into microbial community dynamics were gleaned from short 16S rRNA tag (Itag) Illumina sequencing. UniFrac analysis of Itag sequences showed clear separation of microbial communities related to presence or absence of ice cover. Whereas the ecological implications of the changing bacterial community are unclear at this time, it is likely that the observed shift from a phytoplankton community dominated by filamentous diatoms to smaller cells will have far reaching ecosystem effects including food web disruptions.

Effect of testing experience and profession on provider acceptability of rapid HIV testing after implementation in public sexual health clinics in Sydney.

OBJECTIVES: Rapid HIV testing (RHT) is well established in many countries, but it is new in Australia since a policy change in 2011. We assessed service provider acceptability of RHT before and after its implementation in four Sydney public sexual health clinics. METHODS: Service providers were surveyed immediately after training in RHT and again 6-12 months later. Differences in mean scores between survey rounds were assessed via t-tests, with stratification by profession and the number of tests performed. RESULTS: RHT was rated as highly acceptable among staff at baseline and acceptability scores improved between survey rounds. Belief in being sufficiently skilled and experienced to perform RHT (P = 0.004) and confidence in the delivery of nonreactive results increased (P = 0.007), while the belief that RHT was disruptive declined (P = 0.001). Acceptability was higher for staff who had performed a greater number of tests regarding comfort with their role in RHT (P = 0.004) and belief that patients were satisfied with RHT (P = 0.007). Compared with nurses, doctors had a stronger preference for a faster rapid test (P = 0.027) and were more likely to agree that RHT interfered with consultations (P = 0.014). CONCLUSIONS: Differences in responses between professions may reflect differences in staff roles, the type of patients seen by staff and the model of testing used, all of which may affect the number of tests performed by staff. These findings may inform planning for how best to implement RHT in clinical services.

Improved serological response to H1N1 monovalent vaccine associated with viral suppression among HIV-1-infected patients during the 2009 influenza (H1N1) pandemic in the Southern Hemisphere.

OBJECTIVES: Patients infected with HIV-1 were targeted for vaccination against H1N1 influenza because of their anticipated increased risk of mortality associated with H1N1 infection. Reports regarding the efficacy of vaccination in HIV-1-infected patients have suggested a reduced immunogenic response compared with the general population. Hence, the study aimed to determine the serological response to pandemic H1N1 influenza vaccine in HIV-1-infected patients in a clinical setting. METHODS: A retrospective review of all HIV-1-infected patients who attended mass H1N1 vaccination between October 2009 and March 2010 at an Australian HIV clinic was carried out. Pre- and post-vaccination H1N1 antibody titres were measured. The main outcome measure was response to the vaccination, which was defined as an H1N1 antibody titre of ≥ 1:40 using a haemagglutination inhibition (HI) assay. RESULTS: Baseline blood samples were collected from 199 patients, of whom 154 agreed to receive vaccination; of these, 126 had pre- and post-vaccination HI titres measured. Seventy-seven of 199 patients (38.7%) showed a baseline antibody titre of ≥ 1:40. Eighty-five (67.4%) showed a fourfold or greater increase in titre and 109 of 126 (86.5%) achieved an antibody titre of ≥ 1:40 after vaccination. The serum HI H1N1 antibody geometric mean titre (GMT) for the 126 paired samples was 39.32 ± 3.46 pre-vaccination and increased to 237.36 ± 3.94 [standard deviation (SD)] post-vaccination (P<0.001). In a binary logistic regression analysis, HIV viral load and baseline HI antibody titre were significantly associated with post-vaccination increase in HI H1N1 antibody titre. CONCLUSIONS: A high prevalence of HI H1N1 antibodies was found before vaccination in the cohort, consistent with previous exposure to H1N1 influenza virus. The response to vaccination was considered adequate, as more than two-thirds of patients achieved a fourfold or more increase in antibody titre after vaccination. The response to vaccination was significantly greater in those patients who were aviraemic for HIV, suggesting that antiretroviral therapy improves the humoral response, which is important in optimizing vaccine effectiveness.

IL-33: a tissue derived cytokine pathway involved in allergic inflammation and asthma.

Besides classic T cell-derived T-helper type 2 (Th2) cytokines such as IL-4, IL-5 and IL-13, tissue-produced cytokines such as thymic stromal lymphopoietin, IL-25 and IL-33 are now recognized as important contributors to allergic inflammation. IL-33 is produced by various tissue dwelling cells and broadly enhances allergic inflammation through its effects on hematopoietic cell types. The environmental or endogenous triggers that provoke IL-33 cellular release may be associated with infection, inflammation or tissue damage. This review summarizes the wide range of documented IL-33 activities on human cellular mediators of inflammation as well as genetic evidence that IL-33 contributes to disease. Finally, there will be a discussion of still unanswered questions regarding the mechanisms by which cytokine activity is generated and IL-33's relationship with other Th2-associated cytokines.

Biotechnical development of genetic addiction risk score (GARS) and selective evidence for inclusion of polymorphic allelic risk in substance use disorder (SUD).

Research into the neurogenetic basis of addiction identified and characterized by Reward Deficiency Syndrome (RDS) includes all drug and non-drug addictive, obsessive and compulsive behaviors. We are proposing herein that a new model for the prevention and treatment of Substance Use Disorder (SUD) a subset of RDS behaviors, based on objective biologic evidence, should be given serious consideration in the face of a drug epidemic. The development of the Genetic Addiction Risk Score (GARS) followed seminal research in 1990, whereby, Blum's group identified the first genetic association with severe alcoholism published in JAMA. While it is true that no one to date has provided adequate RDS free controls there have been many studies using case -controls whereby SUD has been eliminated. We argue that this deficiency needs to be addressed in the field and if adopted appropriately many spurious results would be eliminated reducing confusion regarding the role of genetics in addiction. However, an estimation, based on these previous literature results provided herein, while not representative of all association studies known to date, this sampling of case- control studies displays significant associations between alcohol and drug risk. In fact, we present a total of 110,241 cases and 122,525 controls derived from the current literature. We strongly suggest that while we may take argument concerning many of these so-called controls (e.g. blood donors) it is quite remarkable that there are a plethora of case -control studies indicating selective association of these risk alleles ( measured in GARS) for the most part indicating a hypodopaminergia. The paper presents the detailed methodology of the GARS. Data collection procedures, instrumentation, and the analytical approach used to obtain GARS and subsequent research objectives are described. Can we combat SUD through early genetic risk screening in the addiction field enabling early intervention by the induction of dopamine homeostasis? It is envisaged that GARS type of screening will provide a novel opportunity to help identify causal pathways and associated mechanisms of genetic factors, psychological characteristics, and addictions awaiting additional scientific evidence including a future meta- analysis of all available data -a work in progress.

Interconversion of Drosophila nuclear lamin isoforms during oogenesis, early embryogenesis, and upon entry of cultured cells into mitosis.

Two isoforms of a single nuclear lamin, distinguishable on one-dimensional SDS-polyacrylamide gels, have previously been identified in Drosophila nuclei during interphase. A third species, designated lamin Dmmit, has now been identified as soluble in extracts of Drosophila tissue culture cells blocked in mitosis by drugs. An apparently identical form is the only lamin species detectable in late-stage egg chambers and early embryos. Phosphoamino acid analyses suggest that the conversion of lamins Dm1 and Dm2 to lamin Dmmit is brought about by a specific rearrangement of phosphate groups rather than by dramatic net changes in the levels of lamin phosphorylation. The residues involved in these phosphorylation/dephosphorylation reactions have been tentatively mapped to a 17.8-kD cyanogen bromide fragment containing amino acids 385-547. This represents a potential "hinge" domain in the lamin structure between the end of coil 2 and the globular COOH terminus. These results have implications for understanding the regulation of nuclear envelope breakdown during mitosis and karyoskeletal dynamics during oogenesis and early embryogenesis.

Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots.

An affinity-purification method has been developed for the rapid, efficient, and precise elution of antibodies specifically bound to antigens immobilized on nitrocellulose after blot transfer from SDS polyacrylamide gels. The applicability of this technology has been demonstrated using antisera raised against the nuclear matrix-pore complex-lamina fraction prepared from Drosophila melanogaster embryos. In so doing, we have established the existence in whole embryo lysates, of two nearly identical forms of the predominant 74-kilodalton polypeptide previously identified in lower resolution studies of the nuclear matrix-pore complex-lamina fraction. These species, distinguishable on the basis of a slight difference in SDS PAGE mobilities on low concentration polyacrylamide gels, are immunochemically cross-reactive and have been localized exclusively to the nuclear periphery (nuclear envelope) by indirect immunofluorescence analyses of cryosections. The steady-state levels of these two polypeptides have been examined in total embryo lysates both as a function of embryogenesis and in response to heat shock. The larger species is not detectable in early embryos but approaches levels approximately equal to that of the smaller form by about the temporal midpoint of embryonic development. In response to heat shock, this larger form appears to be converted nearly quantitatively into the lower molecular weight polypeptide. These results, as well as the general reliability of the nitrocellulose blot immunoaffinity-purification methodology, have been substantiated through the use of monoclonal antibodies.

Biosynthesis and interconversion of Drosophila nuclear lamin isoforms during normal growth and in response to heat shock.

Two major immunocross-reactive polypeptides of the Drosophila nuclear envelope, distinguishable in interphase cells on the basis of one-dimensional SDS-PAGE mobility, have been localized to the nuclear lamina by immunoelectron microscopy. These have been designated lamins Dm1 and Dm2. Both lamins are apparently derived posttranslationally from a single, primary translation product, lamin Dm0. A pathway has been established whereby lamin Dm0 is processed almost immediately upon synthesis in the cytoplasm to lamin Dm1. Processing occurs posttranslationally, is apparently proteolytic, and has been reconstituted from cell-free extracts in vitro. Processing in vitro is ATP dependent. Once assembled into the nuclear envelope, a portion of lamin Dm1 is converted into lamin Dm2 by differential phosphorylation. Throughout most stages of development and in Schneider 2 tissue culture cells, both lamin isoforms are present in approximately equal abundance. However, during heat shock, lamin Dm2 is converted nearly quantitatively into lamin Dm1. Implications for understanding the regulation of nuclear lamina plasticity through normal growth and in response to heat shock are discussed.

Heat shock-induced changes in the structural stability of proteinaceous karyoskeletal elements in vitro and morphological effects in situ.

Karyoskeletal protein fractions prepared from Drosophila melanogaster embryos contain morphologically identifiable remnants of nuclear pore complexes and peripheral lamina as well as what appears to be an internal nuclear "matrix" (Fisher, P. A., M. Berrios, and G. Blobel, 1982, J. Cell Biol., 92: 674-686). Structural stability of these proteinaceous assemblies is dependent on thermal incubation in vitro (37 degrees C, 15 min) before subfractionation of nuclei. In the absence of such incubation, greater than 90% of the total karyoskeletal protein including major polypeptide components of internal "matrix," pore complexes, and the peripheral lamina, is solubilized by 1 M NaCl. In vivo heat shock induces karyoskeletal stabilization resembling that resulting from thermal incubation in vitro. Immunocytochemical studies have been used to establish the effects of heat shock on the organization and distribution of major karyoskeletal marker proteins in situ. Taken together, these results are consistent with the notion that in vivo, regulation of karyoskeletal plasticity (and perhaps form) may be a functionally significant component of the Drosophila heat shock response. They also have broad practical implications for studies pertaining to the structure and function of karyoskeletal protein (nuclear "matrix") fractions isolated from higher eukaryotic cells.

Drosophila nuclear lamin precursor Dm0 is translated from either of two developmentally regulated mRNA species apparently encoded by a single gene.

A cDNA clone encoding a portion of Drosophila nuclear lamins Dm1 and Dm2 has been identified by screening a lambda-gt11 cDNA expression library using Drosophila lamin-specific monoclonal antibodies. Two different developmentally regulated mRNA species were identified by Northern blot analysis using the initial cDNA as a probe, and full-length cDNA clones, apparently corresponding to each message, have been isolated. In vitro transcription of both full-length cDNA clones in a pT7 transcription vector followed by in vitro translation in wheat germ lysate suggests that both clones encode lamin Dm0, the polypeptide precursor of lamins Dm1 and Dm2. Nucleotide sequence analyses confirm the impression that both cDNA clones code for the identical polypeptide, which is highly homologous with human lamins A and C as well as with mammalian intermediate filament proteins. The two clones differ in their 3'-untranslated regions. In situ hybridization of lamin cDNA clones to Drosophila polytene chromosomes shows only a single locus of hybridization at or near position 25F on the left arm of chromosome 2. Southern blot analyses of genomic DNA are consistent with the notion that a single or only a few highly similar genes encoding Drosophila nuclear lamin Dm0 exist in the genome.

Single polymer dynamics in an elongational flow.

The stretching of individual polymers in a spatially homogeneous velocity gradient was observed through use of fluorescently labeled DNA molecules. The probability distribution of molecular extension was determined as a function of time and strain rate. Although some molecules reached steady state, the average extension did not, even after a approximately 300-fold distortion of the underlying fluid element. At the highest strain rates, distinct conformational shapes with differing dynamics were observed. There was considerable variation in the onset of stretching, and chains with a dumbbell shape stretched more rapidly than folded ones. As the strain rate was increased, chains did not deform with the fluid element. The steady-state extension can be described by a model consisting of two beads connected by a spring representing the entropic elasticity of a worm-like chain, but the average dynamics cannot.

Single-polymer dynamics in steady shear flow.

The conformational dynamics of individual, flexible polymers in steady shear flow were directly observed by the use of video fluorescence microscopy. The probability distribution for the molecular extension was determined as a function of shear rate, gamma;, for two different polymer relaxation times, tau. In contrast to the behavior in pure elongational flow, the average polymer extension in shear flow does not display a sharp coil-stretch transition. Large, aperiodic temporal fluctuations were observed, consistent with end-over-end tumbling of the molecule. The rate of these fluctuations (relative to the relaxation rate) increased as the Weissenberg number, gamma;tau, was increased.

Direct observation of tube-like motion of a single polymer chain.

Tube-like motion of a single, fluorescently labeled molecule of DNA in an entangled solution of unlabeled lambda-phage DNA molecules was observed by fluorescence microscopy. One end of a 16- to 100-micrometer-long DNA was attached to a 1-micrometer bead and moved with optical tweezers. The molecule was stretched into various conformations having bends, kinks, and loops. As the polymer relaxed, it closely followed a path defined by its initial contour. The relaxation time of the disturbance caused by the bead was roughly 1 second, whereas tube-like motion in small loops persisted for longer than 2 minutes. Tube deformation, constraint release, and excess chain segment diffusion were also observed. These observations provide direct evidence for several key assumptions in the reptation model developed by de Gennes, Edwards, and Doi.

Lateral hypothalamic control of killing: evidence for a cholinoceptive mechanism.

In rats that would not ordinarily kill mice, lateral hypothalamic injection of crystalline carbachol, a cholinomimetic, elicited killing. Norepinephrine, amphetamine, serotonin, and sodium salts were ineffective at the same site. Carbachol was ineffective when injected into the medial, dorsal, or ventral hypothalamus. As additional evidence for a cholinoceptive mechanism, neostigmine elicited killing, and, in spontaneous killers, methyl atropine blocked it. The results indicate that the lateral hypothalamus contains a cholinoceptive component of an innate system that activates killing, and anticholinergic treatment can be used as a means of suppressing killing.

Seasonal variations of snow depth on Mars.

Using topography collected over one martian year from the Mars Orbiter Laser Altimeter on the Mars Global Surveyor (MGS) spacecraft, we have measured temporal changes in the elevation of the martian surface that correlate with the seasonal cycle of carbon dioxide exchange between the surface and atmosphere. The greatest elevation change (1.5 to 2 meters) occurs at high latitudes ( above 80 degrees ), whereas the bulk of the mass exchange occurs at lower latitudes (below 75 degrees N and below 73 degrees S). An unexpected period of sublimation was observed during northern hemisphere autumn, coincident with dust storms in the southern hemisphere. Analysis of MGS Doppler tracking residuals revealed temporal variations in the flattening of Mars that correlate with elevation changes. The combined changes in gravity and elevation constrain the average density of seasonally deposited carbon dioxide to be 910 +/- 230 kilograms per cubic meter, which is considerably denser than terrestrial snow.

Stretching of a single tethered polymer in a uniform flow.

The stretching of single, tethered DNA molecules by a flow was directly visualized with fluorescence microscopy. Molecules ranging in length (L) from 22 to 84 micrometers were held stationary against the flow by the optical trapping of a latex microsphere attached to one end. The fractional extension x/L is a universal function of eta vL 0.54 +/- 0.05, where eta and v are the viscosity and velocity of the flow, respectively. This relation shows that the DNA is not "free-draining" (that is, hydrodynamic coupling within the chain is not negligible) even near full extension (approximately 80 percent). This function has the same form over a long range as the fractional extension versus force applied at the ends of a worm-like chain. For small deformations (< 30 percent of full extension), the extension increases with velocity as x approximately v0.70 +/- 0.08. The relative size of fluctuations in extension decreases as sigma x/x approximately equal to 0.42 exp (-4.9 x/L). Video images of the fluctuating chain have a cone-like envelope and show a sharp increase in intensity at the free end.

The shape and internal structure of the moon from the clementine mission.

Global topographic and gravitational field models derived from data collected by the Clementine spacecraft reveal a new picture of the shape and internal structure of the moon. The moon exhibits a 16-kilometer range of elevation, with the greatest topographic excursions occurring on the far side. Lunar highlands are in a state of near-isostatic compensation, whereas impact basins display a wide range of compensation states that do not correlate simply with basin size or age. A global crustal thickness map reveals crustal thinning under all resolvable lunar basins. The results indicate that the structure and thermal history of the moon are more complex than was previously believed.