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Supplementary Figures 1-3, Table 1 from AZD1152 Rapidly and Negatively Affects the Growth and Survival of Human Acute Myeloid Leukemia Cells In vitro and In vivo

Soft tissues rely on the incredible complexity of their microstructure for proper function. Local variations in material properties arise as tissues develop and adapt, often in response to changes in loading. A barrier to investigating the heterogeneous nature of soft tissues is the difficulty of developing experimental protocols and analysis tools that can accurately capture spatial variations in mechanical behavior. In this article, we detail protocols enabling mechanical characterizations of anisotropic, heterogeneous soft tissues or tissue analogs. We present a series of mechanical tests designed to maximize inhomogeneous strain fields and in-plane shear forces. A customized, 3D-printable gripping system reduces tissue handling and enhances shear. High-resolution imaging and laser micrometry capture full-field displacement and thickness, respectively. As the equipment necessary to conduct these protocols is commercially available, the experimental methods presented offer an accessible route toward addressing heterogeneity. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Unique biaxial testing of soft tissues and tissue analogs Basic Protocol 2: Full-field thickness measurement of soft tissues and tissue analogs Support Protocol 1: Creating and speckling cruciform-shaped samples for mechanical testing Support Protocol 2: Creating custom gripping system to minimize sample handling.

Common genetic variants can have profound effects on cellular function, but studying these effects in primary human tissue samples and during development is challenging. Human induced pluripotent stem cell (iPSC) technology holds great promise for assessing these effects across different differentiation contexts. Here, we use an efficient pooling strategy to differentiate 215 iPS cell lines towards a midbrain neural fate, including dopaminergic neurons, and profile over 1 million cells sampled across three differentiation timepoints using single cell RNA sequencing. We find that the proportion of neuronal cells produced by each cell line is highly reproducible over different experimental batches, and identify robust molecular markers in pluripotent cells that predict line-to-line differences in cell fate. We identify expression quantitative trait loci (eQTL) that manifest at different stages of neuronal development, and in response to oxidative stress, by exposing cells to rotenone. We find over one thousand eQTL that colocalise with a known risk locus for a neurological trait, nearly half of which are not found in GTEx. Our study illustrates how coupling single cell transcriptomics with long-term iPSC differentiation can profile mechanistic effects of human trait-associated genetic variants in otherwise inaccessible cell states.

This protocol describes a method for the production of dopaminergic neurons from human iPSCs, using a 52-day long differentiation method adapted from doi.org/10.1038/nature10648. Steps in this protocol assume the use of a single 12 well plate for the purposes of differentiation; adjust volumes accordingly depending on labware used.

This protocol outlines a method for dissociating a human pluripotent stem cell-derived neuronal culture to single cells for loading onto a Chromium 10x chip for single cell RNA-sequencing. This protocol makes a distinction between early neuronal progenitors and mature neuronal cultures, as additional steps and reagents are required in order to sufficiently dissociate the latter. These include: - DNase Vial (D2) - PDS Kit Papain Vial Note: In our labs, iPS cells were undergoing a 52-day long differentiation process to dopaminergic neurons (adapted from doi.org/10.1038/nature10648), and were treated under the 'mature' conditions when harvested from day 20 onward, and as 'early neuronal progenitors' on day 11. Example images of neuronal culture from our labs can be found in the Guidelines of this protocol for reference. This protocol assumes use of: - 12-well tissue culture plates (3.8 cm2 surface area per well ) for samples/wells being harvested for dissociation (see Materials). - A NucleoCounter®NC-200™ for purposes of cell counting.

HIV infects and destroys CD4+ T cells leading to a compromised immune system. In a double-blinded study, a group of HIV-infected individuals with CD4+ T cell counts below 350 cells/mm3 were given either an empty liposomal supplement or a liposomal glutathione (L-GSH) supplement to take over a 3-month period. Baseline measurements in HIV-positive subjects show a significant decrease in levels of interleukin (IL)-12, IL-2, and interferon (IFN)-γ, along with a substantial increase in the levels of IL-6, IL-10, transforming growth factor (TGF)-β, and free radicals, compared to healthy individuals. Supplementation of HIV-positive subjects with L-GSH for 3 months resulted in a notable increase in the levels of IL-12, IL-2, and IFN-γ, with a concomitant decrease in the levels of IL-6, IL-10, and free radicals, and stabilization in the levels of TGF-β, IL-1, and IL-17, compared to their placebo counterparts. Levels of free radicals in CD4+ T cells stabilized, while GSH levels increased in the treatment group. Those in the placebo group showed no significant difference throughout the study. In summary, supplementation with L-GSH in HIV-infected individuals with CD4+ T cell counts below 350 cells/mm3 can help restore redox homeostasis and cytokine balance, therefore aiding the immune system to control opportunistic infections.

This paper demonstrates how a phased site investigation approach complements the design process for offshore wind farms. Examples are given showing how three-dimensional (3D) engineering ground models and real-time data analysis have been utilized by integrated project teams to maximize efficiencies, achieve cost and program savings, and reduce risk. In general, the aim of a site investigation should be to ensure that: Sufficient ground data is obtained for design, certification, and construction;Data collected is fit for purpose and fully integrated; andA comprehensive interpretation can be carried out with consideration of engineering design and construction requirements. In the authors’ experience, offshore site investigations are most effective when a ‘geoteam’ (an integrated team of ground specialists including geologists, geotechnical engineers, geophysicists, and others) is assembled at an early stage in the project and carries out early optioneering studies followed by scoping, supervision, and interpretation of a phased site investigation program. Crucially, the geoteam must be able to read and communicate the ground from a designer's perspective and appreciate the Client's constraints and requirements. If they do this effectively, the geoteam will be able to scope a more efficient, targeted site investigation, and provide almost live updates to the engineering ground model. The model can in turn be used to inform real-time decisions being made by the developer, their offshore site investigation representative (Client Representative), and the wind farm's designer. Consideration of the 3D ground conditions is paramount and should always be combined with an appreciation of the site's geological history and earth surface processes to assess how geological variability and ground hazards may affect design. This will provide strong justification for the scope of site investigation works. While the UK and European wind farm market is maturing, with the first offshore wind farm in the UK constructed in the year 2000, the US market is currently in a period of rapid early development. Drawing on considerable experience gained from work in the UK and Europe, the authors present a designer's perspective of site investigation, describing lessons learnt from the scoping, execution, and interpretation of numerous site investigations for offshore wind farms.

The Glasgow s17 syn+ strain of herpes simplex virus 1 (HSV1) is arguably the best characterised strain and has provided the reference sequence for HSV1 genetic studies. Here we show that our original s17 syn+ stock was a mixed population from which we have isolated a minor variant that, unlike other strains in the laboratory, fails to be efficiently released from infected cells and spreads predominantly by direct cell-to-cell transmission. Analysis of other s17-derived viruses that had been isolated elsewhere revealed a number with the same release phenotype. Second generation sequencing of eight plaque-purified s17-derived viruses revealed sequences that vary by 50 SNPs including approximately 10 coding SNPs. This compared to interstrain variations of around 800 SNPs in strain Sc16, of which a quarter were coding changes. Amongst the variations found within s17, we identified thirteen variants of glycoprotein C within the original stock of virus which were predominantly a consequence of altered homopolymeric runs of C residues. Characterisation of seven isolates coding for different forms of gC indicated that all were expressed, despite six of them lacking a transmembrane domain. While the release phenotype did not correlate directly with any of these identified gC variations, further demonstration that nine clinical isolates of HSV1 also fail to spread through extracellular release raises the possibility that propagation in tissue culture had altered the HSV1 s17 transmission phenotype. Hence, this s17 intrastrain variation identified here offers an excellent model for understanding both HSV1 transmission and tissue culture adaptation.

Cavitation is a phenomenon affected considerably by the underlying pressure waves that occur on similar timeand length scales as the bubble dynamics. Thus appropriate representation of wave dynamics within numericalframeworks is of paramount importance for the prediction of the phase change process in the nozzle as well as thesubsequent spray formation. In this paper we focus on investigating the sensitivity of the wave dynamics within acompressible Large Eddy Simulation framework with regards to downstream geometry and boundary representation.Diesel was used as working fluid and was injected at various pressures through a micro-channel. Results interms of vapour fraction, velocity and pressure are compared with the experimental data of Winklhofer [30, 31]. Thedownstream domain length and reflectivity properties are shown to exert a significant effect on in-nozzle processes.DOI: http://dx.doi.org/10.4995/ILASS2017.2017.4600

With the publication of this, the 2012 volume, the online Africa Bibliography will hold just over 88,000 entries, dating from between 1990 and 2012. Nearly 55,000 additional archival entries remain in production, which together promise to enrich the online Bibliography's coverage of the period 1990–96 as well as take it back to 1984 and the origins of the print series. The transition of these records from the printed page to the online database involves a painstaking process – with each entry scanned, copyedited and individually tagged to support searching – but this work is providing a multiplicity of opportunities to enhance the intellectual value of the Bibliography and better achieve, in a contemporary context, the principles on which the Bibliography was launched thirty years ago.

Background: Antiretroviral treatment is imperative for increasing survival among HIV positive individuals. Treatment success is assured by strict adherence to antiretroviral therapy (ART) resulting in improved quality and quantity of life. Aim: To compare visual analogue scale (VAS) (self-report) adherence with pill counts adherence reports of individuals receiving HIV treatment in a rural faith-based clinic while evaluating the relationships between those two adherence measures with medication side effects, food insecurity, demographic characteristics, and socialpsychological constructs. Methods: This is a non-experimental cross-sectional study of a convenience sample of 200 reproductive age HIV positive women on ART at a faith-based clinic in Makwasa, Thyolo District, Malawi. Pill counts, VAS adherence, and other constructs measurements were taken from November to December 2013. Bivariate analysis was used to test the association between the two adherence outcomes and background factors and univariate logistic regression (ULR) models were used to explore the association of each variable to the two adherence outcomes; and multivariate logistic regression (MLR) was used to examine the association between outcome variables and adherence determinants. Results: Mean pill count adherence was 79.00 ± 29.66 compared to 96.55 ± 14.21 for VAS adherence. Household food insecurity (OR=1.40; P=0.01), individual food insecurity (OR=1.54; P=0.00), and self-efficacy (OR=2.93; P≤0.00) were significantly associated with pill count in the ULR. Household (OR=0.44; P=0.00) and individual food insecurity (OR=0.38; P=0.003), self-efficacy (OR=0.35; P=0.04), subjective norms (OR=0.24; P=0.02), and attitude (OR=0.34; P=0.04) were associated with VAS adherence in the ULR. In the multivariate, self-efficacy (adjusted) was associated with pill count, while attitude (adjusted) was associated with VAS adherence. Conclusions: The study showed that a gap existed between VAS adherence and pill counts adherence indicating the participants verbally overestimated their adherence. Additionally, intervention focusing on food supplements would be useful for improving poor adherence associated with food insecurity.

Few studies have explored hospitalization outcome differences between patients who are seropositive for human immunodeficiency virus (HIV) compared to HIV-seronegative patients with acute myocardial infarctions (AMIs). The aim of this study was to explore in-hospital AMI mortality risk in seropositive and seronegative patients. A secondary analysis of the Nationwide Inpatient Sample from 1997 to 2006 was conducted. This sample allows the approximation of all United States hospitalizations. All AMI encounters with and without co-occurring HIV were identified using appropriate International Classification of Diseases and procedure codes. Descriptive and Cox proportional-hazards analyses were then conducted to estimate mortality differences between seropositive and seronegative patients while adjusting for demographic, clinical, hospital, and care factors. The results demonstrated higher AMI hospitalization mortality hazard in seropositive compared to seronegative patients after adjustment for age, gender, ethnicity, medical co-morbidities, hospital type, and number of in-hospital procedures (HR 1.38, 95% confidence interval 1.01 to 1.87, p = 0.04). Stratified analysis demonstrated greater although not statistically significant mortality hazard for non-ST-segment elevation myocardial infarction and ST-segment elevation myocardial infarction in seropositive compared to seronegative patients. Typical AMI care procedures occurred at significantly lower rates in seropositive versus seronegative patients, including thrombolytic and anticoagulant agents (18% vs 22%), coronary arteriography (48% vs 63%), left cardiac catheterization (52% vs 66%), and coronary artery bypass graft (6% vs 14%). In conclusion, additional mortality burden and lower procedure rates occur for HIV-seropositive patients receiving AMI care. Health care providers should be alert to the increased mortality burden when treating seropositive patients with AMI.

Pegylated-interferon (PEG-IFN) and ribavirin (RBV), current standard treatment for hepatitis C virus (HCV) infection, are frequently associated with neutropenia and anemia, leading to high treatment discontinuation rates in HIV/HCV-coinfected patients. Our objective was to compare the effectiveness of intervening with hematologic growth factors versus dose reductions of standard HCV therapy for the management of treatment-induced hematologic disorders.Ninety-two HIV/HCV-coinfected, therapy-naive subjects received PEG-IFN alfa-2b 1.5 μg·kg⁻¹·wk⁻¹ and RBV 13 ± 2 mg·kg⁻¹·d⁻¹ for up to 48 weeks. Before treatment initiation, subjects were randomized to subsequently receive growth factors, recombinant human erythropoietin (rHuEPO) and/or granulocyte colony-stimulating factor, or dose reduction (RBV and/or PEG-IFN) for anemia and neutropenia management, respectively. We analyzed the ability of each management strategy to control anemia and neutropenia and the percentage of subjects who achieved a successful treatment outcome according to the different management strategies.During treatment, 43 subjects developed anemia (human erythropoietin, n = 24; dose reduction, n = 19), whereas 25 subjects developed neutropenia (granulocyte colony-stimulating factor, n = 10; dose reduction, n = 15). After the intervention, the increase in both hemoglobin and absolute neutrophil counts did not differ between the 2 side effect management strategies. Sustained response percentages were similar comparing anemic and neutropenic subjects regardless of management strategy (anemia: recombinant human erythropoietin, 29% versus dose reduction, 21%, P = 0.92; neutropenia: granulocyte colony-stimulating factor, 40% versus dose reduction, 20%, P = 0.46).Growth factor supplementation and dose reduction do not seem to differ as management strategies for anemia and neutropenia in HIV/HCV-coinfected individuals treated with PEG-IFN/RBV.

Objective: To assess the effects of decreased antiretroviral therapy exposure on body fat and metabolic parameters. Design: Substudy of the Strategies for Management of Anti-Retroviral Therapy study, in which participants were randomized to intermittent CD4-guided [Drug Conservation (DC) group] or to continuous [Viral Suppression (VS) group] antiretroviral therapy. Methods: Participants at 33 sites were coenrolled in the Strategies for Management of Anti-Retroviral Therapy Body Composition substudy. Regional fat was assessed annually by whole-body dual-energy X-ray absorptiometry and abdominal Computed tomography. Fasting metabolic parameters were assessed at months 4, 8, and annually. Treatment groups were compared for changes in fat and metabolic markers using longitudinal mixed models. Results: Two hundred and seventy-five patients were randomized to the DC (n = 142) or VS (n = 133) group and followed for a median of 2.0 years. By month 12, limb fat (DC-VS difference 9.8%, 95% confidence interval 3.5-16.1; P=0.003) and subcutaneous abdominal fat (DC-VS difference 14.3 cm(2), 95% confidence interval -0.1 to 28.7; P=0.05) increased in the DC group. There was no treatment difference in visceral abdominal fat (DC-VS difference -2.1%, 95% confidence interval -13.5 to 9.4; P=0.72). Lipids significantly decreased in the DC group by month 4 and treatment differences persisted throughout follow-up (P

The measurement of the rate of fuel injection using a constant volume, fluid filled chamber and measuring the pressure change as a function of time due to the injected fluid (the so called “Zeuch” method) is an industry standard due to its simple theoretical underpinnings. Such a measurement device is useful to determine key timing and quantity parameters for injection system improvements to meet the evolving requirements of emissions, power and economy. This study aims to further the understanding of the nature of cavitation which could occur in the near nozzle region under these specific conditions of liquid into liquid injection using high pressure diesel injectors for heavy duty engines. The motivation for this work is to better understand the temporal signature of the pressure signals that arise in a typical injection cycle. A preliminary CFD study was performed, using OpenFOAM, with a transient (Large Eddy Simulation -LES), multiphase solver using the homogenous equilibrium model for the compressibility of the liquid/vapour. The nozzle body was modelled for simplicity without the nozzle needle using a nozzle hole of 200μm diameter and the body pressurised to values typical for common rail engines. Temperature effects were neglected and the wall condition assumed to be adiabatic. The chamber initial static pressure was varied between 10 and 50 bar to reflect typical testing conditions. Results indicate that vapour formation could occur in areas 10-30mm distant from the nozzle itself. The cavitation was initiated around 100 μs after the jet had started for low ΔP cases and followed the development period required for the formation of vortices associated with the vortex roll up of this jet. These vortices had localised sites, in their core region, below the vapour pressure and were convected downstream of their initial formation location. It was also found that vapour formation could occur at chamber static pressures up to 50 bar (the highest tested) due to cavitation in the shear layer and this vortex effect. The pressure signal received at the chamber would therefore be more difficult to interpret with additional error components.

Cytokines are signaling biomolecules that serve as key regulators of our immune system. CD4(+) T-cells can be grouped into 2 major categories based on their cytokine profile: T-helper 1 (TH1) subset and T-helper 2 (TH2) subset. Protective immunity against HIV infection requires TH1-directed CD4 T-cell responses, mediated by cytokines, such as interleukin-1β (IL-1β), IL-12, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α). Cytokines released by the TH1 subset of CD4 T-cells are considered important for mediating effective immune responses against intracellular pathogens such as Mycobacterium tuberculosis (M. tb). Oxidative stress and redox imbalance that occur during HIV infection often lead to inappropriate immune responses. Glutathione (GSH) is an antioxidant present in nearly all cells and is recognized for its function in maintaining redox homeostasis. Our laboratory previously reported that individuals with HIV infection have lower levels of GSH. In this study, we report a link between lower levels of GSH and dysregulation of TH1- and TH2-associated cytokines in the plasma samples of HIV-positive subjects. Furthermore, we demonstrate that supplementing individuals with HIV infection for 13 weeks with liposomal GSH (lGSH) resulted in a significant increase in the levels of TH1 cytokines, IL-1β, IL-12, IFN-γ, and TNF-α. lGSH supplementation in individuals with HIV infection also resulted in a substantial decrease in the levels of free radicals and immunosuppressive cytokines, IL-10 and TGF-β, relative to those in a placebo-controlled cohort. Finally, we determined the effects of lGSH supplementation in improving the functions of immune cells to control M. tb infection by conducting in vitro assays using peripheral blood mononuclear cells collected from HIV-positive individuals at post-GSH supplementation. Our studies establish a correlation between low levels of GSH and increased susceptibility to M. tb infection through TH2-directed response, which may be relieved with lGSH supplementation enhancing the TH1 response.

The impact of an adherence enhancement program for low income HIV-infected Spanish-speaking Latinos on health literacy, patient-provider relationships, and adherence to HAART was examined. Evaluations were conducted at baseline, 6 weeks, and 6 months for participants (n = 85) randomly assigned to either the intervention group or a comparison group; 69 (81%) remained in the study for the entire 6-month duration. The intervention group scored significantly better than the comparison group on 3 of 5 measures of HIV health literacy at 6 weeks and on 2 of 5 measures, at 6 months. While there was a weak trend for the intervention group to report an increase in self-efficacy of medication adherence management, baseline to 6 weeks, no other changes were significant. Perceptions of the quality of relationship and communications with their HIV-treating physicians improved both at 6 weeks (p = 0.04) and at 6 months (p < 0.001). The comparison group showed little change baseline to 6 weeks and baseline to 6 months. While there was a trend for the pilot group to report better medication adherence, these differences were not statistically significant. Further evaluation of the impact of this adherence enhancement program is needed.

The term electrosurgery (also called radiofrequency surgery) refers to the passage of high-frequency alternating electrical current through the tissue in order to achieve a specific surgical effect. Although the mechanism behind electrosurgery is not completely understood, heat production and thermal tissue damage is responsible for at least the majority--if not all--of the tissue effects in electrosurgery. Adjacent to the active electrode, tissue resistance to the passage of current converts electrical energy to heat. The only variable that determines the final tissue effects of a current is the depth and the rate at which heat is produced. Electrocoagulation occurs when tissue is heated below the boiling point and undergoes thermal denaturation. An additional slow increase in temperature leads to vaporization of the water content in the coagulated tissue and tissue drying, a process called desiccation. A sudden increase in tissue temperature above the boiling point causes rapid explosive vaporization of the water content in the tissue adjacent to the electrode, which leads to tissue fragmentation and cutting.

Electrosurgical currents can be delivered to tissue in monopolar or bipolar and monoterminal or biterminal modes, with the primary difference between these modes being their safety profiles. A monopolar electrosurgical circuit includes an active electrode and a dispersive (return) electrode, while there are 2 active electrodes in bipolar mode. In monoterminal mode, there is an active electrode, but there is no dispersive electrode connected to the patient's body and instead the earth acts as the return electrode. Biterminal mode uses a dispersive electrode connected to the patient's body, has a higher maximum power, and can be safer than monoterminal mode in certain situations. Electrosurgical units have different technologies for controlling the output power and for providing safety. A thorough understanding of these technologies helps with a better selection of the appropriate surgical generator and modes.

Objectives: Many US HIV-positive patients are unaware of their infection. Although there are multiple studies assessing the acceptance of testing, there are none that assess the patient’s expectations of routine HIV testing. Methods: Through a prospective, cross-sectional, unfunded, convenience sample survey, we assessed the patient’s expectation of testing at a regional medical center serving an indigent population. Also, we compared the providers’ predictions of the proportion of patients expecting a test. Results: Of the 69 patients, 23% expected a test. The only factor that correlated with this was their desire to be tested ( P = .004). Providers’ (N = 59) mean prediction was that 21% of the patients expected a test (range: 0%-100%). The proportion of emergency department (ED) patients wanting a test was 52% (of 30) and internal medicine inpatients were 44% (of 39). Conclusions: Nearly a quarter of patients expected routine HIV testing. This finding should encourage a review of policies promoting HIV testing, especially within the ED.

Emtricitabine is a new, once-daily nucleoside reverse transcriptase inhibitor (NRTI) with potent activity against human immunodeficiency virus (HIV).To assess the efficacy and safety of emtricitabine as compared with stavudine when used with a background regimen of didanosine and efavirenz.Randomized, double-blind, double-dummy study conducted at 101 research clinics in North America, Latin America, and Europe. The first patient was enrolled on August 21, 2000; no investigator or patient was unblinded until the last patient randomized completed the week 48 visit on October 24, 2002. Analyses were based on data collected in a double-blind setting with a median follow-up of 60 weeks. Patients were 571 antiretroviral-naive, HIV-1-infected adults aged 18 years or older with viral load levels greater than or equal to 5000 copies/mL.Receipt of either 200 mg of emtricitabine once daily (plus stavudine placebo twice daily) (n = 286) or stavudine at standard doses twice daily (plus emtricitabine placebo once daily) (n = 285) plus open-label didanosine and efavirenz, once daily.Persistent virological response, defined as achieving and maintaining viral load at or below the limit of assay quantification (or =400 or 50 copies/mL).At the interim analysis on June 14, 2002, when the last patient randomized completed 24 weeks of double-blind treatment (median follow-up time of 42 weeks), patients in the emtricitabine group had a higher probability of a persistent virological responseor =50 copies/mL vs the stavudine group (85% vs 76%, P =.005). This was associated with a higher mean CD4 cell count change from baseline for the emtricitabine group (156 cells/ microL vs 119 cells/microL, P =.01 [of note, there was no statistical difference at 48 weeks [P =.15], although a sensitivity analysis, using an intent-to-treat population with the last CD4 cell count observation carried forward to week 48 showed a difference [P =.02]]). The independent data and safety monitoring board recommended offering open-label emtricitabine based on the interim analysis. The probability of persistent virological responseor =50 copies/mL through week 60 was 76% for the emtricitabine group vs 54% for the stavudine group (P.001). The probability of virological failure through week 60 was 4% in the emtricitabine group and 12% in the stavudine group (P.001). Patients in the stavudine group had a greater probability of an adverse event that led to study drug discontinuation through week 60 than did those in the emtricitabine group (15% vs 7%, P =.005).Once-daily emtricitabine appeared to demonstrate greater virological efficacy, durability of response, and tolerability compared with twice-daily stavudine when used with once-daily didanosine and efavirenz.

Adult stem cells are finding increased therapeutic potential not least in tissue regeneration protocols. The cell sources being proposed for such protocols include embryonic, umbilical cord blood (CB) and adult bone marrow (BM). Although embryonic sources are controversial, CB and marrow are available immediately. The appropriate cells of use in these sources are considered to be extremely rare and a characterisation of the starting cell source is important for the development of adult stem cell protocols and ex vivo expansion. Umbilical CB and BM mononuclear cells were labelled for the antigens CD34, CD133, CD117, CD164, Thy-1 or CD38, and additional intracellular CD34 antigen. Three dimensional flow-cytometric analyses were carried out together with dual laser confocal microscope analysis for antigen profile expression. Variable levels of immaturity were detected on CB and BM populations using internal and external CD34 antigen. For CB and BM cells, internal CD34 (intCD34+) could be detected on co-expressing CD133+ cells before expression of external CD34 antigen (extCD34+). CD38 co-expression analysis also showed that a small but distinct group of cells expressing low CD38 and no external CD34 antigen could be detected. Additional phenotyping of these cells using CD117, Thy-1, CD164 and CD133 demonstrated variable primitive status detectable within the external CD34- population. Newly harvested primary CB and BM populations were shown to contain not only cellular populations of known standard sequential maturity but also populations of more extreme rarity. The presence of cells which lacked extracellular CD34 antigen, in both CB and BM, but which possessed CD133, has important implications for purification of human stem cells in clinical applications.

Determining the cellular distribution of key adhesion molecules may aid in understanding haematopoietic progenitor/stem cell (HPSC) homing to bone marrow (BM). CD34, a well-characterized marker for blast-like HPSC, is widely used for the isolation and enumeration of HPSC. Functional studies have yet to identify a ligand for CD34. However, growing evidence suggests that CD34 may aid the regulation of HPSC differentiation and modulate the expression of other HPSC adhesion molecules necessary for homing. This study aimed to determine CD34 distribution on umbilical cord blood (CB) and BM. CD34-selected cells were adhered to positively charged gold slides at room temperature, before indirect fluorescent antibody labelling with fluorescein isothiocyanate. Fluorescent distribution was determined by 1-microm interval, confocal laser scanning microscope Z-sections. Initial analysis showed CD34 distributed within peripheral halos and dense pocket regions. The development of three-dimensional imaging software enabled spatial visualization of CD34 distribution on CB and BM in association with differential interference contrast cell image. This showed that CD34 was distributed within peripheral halos, with magnupodia-associated 'meridian-shaped crescents', extending from points of cell-slide adhesion towards the top of the cell. True CD34 distribution, not previously discernible by confocal laser scanning microscopy alone, suggests a possible role in cell adhesion/homing revealed by three-dimensional imaging.

Magnetic resonance spectroscopy (MRS) provides a noninvasive means of assessing in vivo tissue biochemistry. N-Acetyl aspartate (NAA) is a major brain metabolite, and its presence is used increasingly in clinical and experimental MRS studies as a putative neuronal marker. A reduction in NAA levels as assessed by in vivo 1H MRS has been suggested to be indicative of neuronal viability. However, temporal observations of brain pathologies such as multiple sclerosis, mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), and hypothyroidism have shown reversibility in NAA levels, possibly reflecting recovery of neuronal function. A knowledge of the cellular localisation of NAA is critical in interpreting these findings. The assumption that NAA is specific to neurones is based on previous immunohistochemical studies on whole brain using NAA-specific antibodies. The neuronal localisation was further substantiated by cell culture experiments in which its presence in the oligodendrocyte-type 2 astrocyte progenitors and immature oligodendrocytes, but not in the mature oligodendrocytes, was observed. More recently, studies on oligodendrocyte biology have revealed the requirement for trophic factors to promote the generation, maturation, and survival of oligodendrocytes in vitro. Here, we have used this new information to implement a more pertinent cell cultivation procedure and demonstrate that mature oligodendrocytes can express NAA in vitro. This observation brings into question whether the NAA changes observed in clinical in vivo 1H MRS studies reflect neuronal function alone. The data presented here support the hypothesis that oligodendrocytes may express NAA in vivo and contribute to the NAA signal observed by 1H MRS.

AZD1152 is a specific Aurora kinase inhibitor with selectivity for Aurora B kinase, designed to target cell division in proliferating tumour cells. It has been shown in model systems that inhibition of Aurora B kinase with AZD1152 reduces histone H3 phosphorylation (phH3) and inhibits cytokinesis, thus inducing cellular multi-nucleation and polyploidy, leading to apoptosis and cell death. Here, we present results of preliminary studies on the in vivo action of AZD1152 on the growth and development of both a human acute myeloid leukaemia (AML) cell line (HL-60) and primary AML samples in the NOD/SCID xeno-transplantation model. Two weeks of AZD1152 at 25 mg/kg/day is well tolerated in NOD/SCID mice. HL-60 cells were injected iv into sub-lethally irradiated NOD/SCID mice (n=24) and allowed to engraft in the bone marrow. Three weeks later, when malignant cell growth was well established in the bone marrow, mice were split into treatment groups. Either vehicle (n=12) or AZD1152 (25 mg/kg/day via minipump, n=12) was administered for 7 days. One AZD1152-treated group was sacrificed immediately post-treatment (n=9) and another was left for a further 2 weeks before bone marrow analysis for HL-60 cell content (defined by human CD45+/CD33+/CD19− expression). The mean percentage HL-60 cell content in the marrow of untreated mice was 64.5 ± 19.0 (range 14.0–86.6, n=11), whereas in mice that had received AZD1152 and were sacrificed at the end of dosing period, the mean percentage was 0.29 ± 0.74 (range 0–2.26, n=9; p Similar experiments were performed with two primary AML samples. AML-1 is from a patient with French-American-British (FAB)-type M1 AML and AML-2 is from a FAB-type M2 AML patient. Ten million cells from both AML samples were injected into NOD/SCID mice (n=12 for AML-1; n=6 for AML-2). Ten weeks later, mice were split into treatment groups and dosed with either control vehicle or 25 mg/kg/day of AZD1152 via minipumps for 7 days. All mice were sacrificed after 1 week of treatment in these initial experiments. The mean percentage engraftment in untreated mice was 59.7 ± 36.0 (range 6.2–84.0, n=4) for AML-1 and 21.8 ± 22.5 (range 5.9–37.7, n=2) for AML-2. Whereas, in mice that had received 25 mg/kg/day of AZD1152 for 7 days, the percentage of engraftment was 1.8 ± 1.7 (range 0.38–4.53, n=5; p These data indicate that AZD1152 has a profound effect on the engraftment of human AML cell lines and primary AML samples in an in vivo model. Furthermore, initial results may indicate a long-term effect of AZD1152 treatment in vivo. AZD1152 is currently undergoing Phase I clinical trials in AML patients.

ContextEmtricitabine is a new, once-daily nucleoside reverse transcriptase inhibitor (NRTI) with potent activity against human immunodeficiency virus (HIV).ObjectiveTo assess the efficacy and safety of emtricitabine as compared with stavudine when used with a background regimen of didanosine and efavirenz.Design, Setting, and PatientsRandomized, double-blind, double-dummy study conducted at 101 research clinics in North America, Latin America, and Europe. The first patient was enrolled on August 21, 2000; no investigator or patient was unblinded until the last patient randomized completed the week 48 visit on October 24, 2002. Analyses were based on data collected in a double-blind setting with a median follow-up of 60 weeks. Patients were 571 antiretroviral-naive, HIV-1–infected adults aged 18 years or older with viral load levels greater than or equal to 5000 copies/mL.InterventionsReceipt of either 200 mg of emtricitabine once daily (plus stavudine placebo twice daily) (n = 286) or stavudine at standard doses twice daily (plus emtricitabine placebo once daily) (n = 285) plus open-label didanosine and efavirenz, once daily.Main Outcome MeasurePersistent virological response, defined as achieving and maintaining viral load at or below the limit of assay quantification (≤400 or 50 copies/mL).ResultsAt the interim analysis on June 14, 2002, when the last patient randomized completed 24 weeks of double-blind treatment (median follow-up time of 42 weeks), patients in the emtricitabine group had a higher probability of a persistent virological response ≤50 copies/mL vs the stavudine group (85% vs 76%, P = .005). This was associated with a higher mean CD4 cell count change from baseline for the emtricitabine group (156 cells/µL vs 119 cells/µL, P = .01 [of note, there was no statistical difference at 48 weeks {P = .15}, although a sensitivity analysis, using an intent-to-treat population with the last CD4 cell count observation carried forward to week 48 showed a difference {P = .02}]). The independent data and safety monitoring board recommended offering open-label emtricitabine based on the interim analysis. The probability of persistent virological response ≤50 copies/mL through week 60 was 76% for the emtricitabine group vs 54% for the stavudine group (P

A steady incompressible three-dimensional viscous flow analysis has been conducted for the Space Shuttle External Tank/Orbiter propellant feed line disconnect flapper valves with upstream elbows. The full Navier-Stokes code, INS3D, is modified to handle interior obstacles. Grids are generated by SVTGD3D code. Two dimensional initial grids in the flow cross section with and without the flappers are improved by elliptic smoothing to provide better orthogonality, clustering and smoothness to the three dimensional grid. The flow solver is tested for stability and convergence in the presence of interior flappers. An under-relaxation scheme has been incorporated to improve the solution stability. Important flow characteristics such as secondary flows, recirculation, vortex and wake regions, and separated flows are observed. Computed values for forces, moments, and pressure drop are in satisfactory agreement with water flow test data covering a maximum tube Reynolds number of 3.5 x 10(exp 6). The results will serve as a guide to improved design and enhanced testing of the disconnect.