A field method for cryopreservation of whole blood from a finger prick for later analysis with flow cytometry.

OBJECTIVES: Flow cytometry is a powerful tool for investigating immune function, allowing for the quantification of leukocytes by subtype. Yet it has not been used extensively for field work due to perishable reagents and the need for immediate analysis of samples. To make flow cytometry more accessible, we devise and evaluate a field protocol for freezing capillary blood. MATERIALS AND METHODS: We collected finger prick blood samples from 110 volunteers, age 18 to 42. Blood samples were analyzed immediately for 18 cell surface markers. Aliquots of whole blood were frozen in the vapor phase of a liquid nitrogen tank with 10% dimethyl sulfoxide in medium. Samples were analyzed on a Guava EasyCyte HT flow cytometer after 2, 4, or 14 weeks. RESULTS: Major lymphocyte fractions in frozen samples were correlated with fresh values (T-cells: r = 0.82; Natural Killer [NK] cells: r = 0.64; CD4: r = 0.67; CD8: r = 0.82; Naïve CD4: r = 0.73, Naïve CD8: r = 0.71; B-cells: r = 0.73; all p < 0.001), and mean values were similar to those from fresh samples. However, correlations for smaller subsets of CD4 and B cells were generally poor. Some differences resulted from changes in non-specific binding for some antibody-conjugate pairs. Cryopreservation also resulted in a reduction in granulocytes more than lymphocytes. DISCUSSION: Our results suggest that antibody/fluorochrome combinations should be validated before use on frozen samples, and that functional changes in cells may affect some cell markers. However, this simple freezing protocol utilizing finger pricks, whole blood, and a liquid nitrogen shipping tank is viable for obtaining samples for flow cytometry under field conditions.

Disruption of the ciliary GTPase Arl13b suppresses Sonic hedgehog overactivation and inhibits medulloblastoma formation.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor, and overactivation of the Sonic Hedgehog (Shh) signaling pathway, which requires the primary cilium, causes 30% of MBs. Current treatments have known negative side effects or resistance mechanisms, so new treatments are necessary. Shh signaling mutations, like those that remove Patched1 (Ptch1) or activate Smoothened (Smo), cause tumors dependent on the presence of cilia. Genetic ablation of cilia prevents these tumors by removing Gli activator, but cilia are a poor therapeutic target since they support many biological processes. A more appropriate strategy would be to identify a protein that functionally disentangles Gli activation and ciliogenesis. Our mechanistic understanding of the ciliary GTPase Arl13b predicts that it could be such a target. Arl13b mutants retain short cilia, and loss of Arl13b results in ligand-independent, constitutive, low-level pathway activation but prevents maximal signaling without disrupting Gli repressor. Here, we show that deletion of Arl13b reduced Shh signaling levels in the presence of oncogenic SmoA1, suggesting Arl13b acts downstream of known tumor resistance mechanisms. Knockdown of ARL13B in human MB cell lines and in primary mouse MB cell culture decreased proliferation. Importantly, loss of Arl13b in a Ptch1-deleted mouse model of MB inhibited tumor formation. Postnatal depletion of Arl13b does not lead to any overt phenotypes in the epidermis, liver, or cerebellum. Thus, our in vivo and in vitro studies demonstrate that disruption of Arl13b inhibits cilia-dependent oncogenic Shh overactivation.

Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression.

As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.

Temporal deletion of Arl13b reveals that a mispatterned neural tube corrects cell fate over time.

Cilia are necessary for sonic hedgehog (Shh) signaling, which is required to pattern the neural tube. We know that ventral neural cell fates are defined by a specific cohort of transcription factors that are induced by distinct thresholds of Shh activity mediated by opposing gradients of Gli activator (GliA) and Gli repressor (GliR). Despite this understanding, the role of Shh as an instructive morphogen is viewed as increasingly complex, with current models integrating positive inputs in terms of ligand concentration and time, along with negative feedback via the downstream gene regulatory network. To investigate the relative contributions of the positive and negative inputs from Shh signaling in neural patterning, we took advantage of a protein that uncouples the regulation of GliA and GliR: the cilia protein ADP-ribosylation factor-like 13b (Arl13b). By deleting Arl13b in mouse, we induced low-level constitutive GliA function at specific developmental stages and defined a crucial period prior to E10.5 when shifts in the level of GliA cause cells to change their fate. Strikingly, we found that improperly patterned cells in these mice converted to the wild-type pattern by E12.5. We further showed that the recovery of patterning did not occur when we also deleted Gli3, the primary GliR in the neural tube, revealing a crucial role of Gli3 in the maintenance of neural patterning.

The Ugi four-component reaction as a concise modular synthetic tool for photo-induced electron transfer donor-anthraquinone dyads.

Phenothiazinyl and carbazolyl-donor moieties can be covalently coupled to an anthraquinone acceptor unit through an Ugi four-component reaction in a rapid, highly convergent fashion and with moderate to good yields. These novel donor-acceptor dyads are electronically decoupled in the electronic ground state according to UV-vis spectroscopy and cyclic voltammetry. However, in the excited state the inherent donor luminescence is efficiently quenched. Previously performed femtosecond spectroscopic measurements account for a rapid exergonic depopulation of the excited singlet states into a charge-separated state. Calculations of the Gibbs energy of photo-induced electron transfer from readily available UV-vis spectroscopic and cyclovoltammetric data applying the Weller approximation enables a quick evaluation of these novel donor-acceptor dyads. In addition, the X-ray structure of a phenothiazinyl-anthraquinone dyad supports short donor-acceptor distances by an intramolecular π-stacking conformation, an important assumption also implied in the calculations of the Gibbs energies according to the Weller approximation.

Using Technology to Affect Influenza Vaccine Coverage Among Children With Chronic Respiratory Conditions.

INTRODUCTION: Influenza presents additional burdens for children with chronic respiratory conditions. Influenza vaccinations may reduce complications, yet approximately half of children remain unprotected. Evidence supports integration of text and e-mail into multicomponent strategies to increase influenza vaccination rates among children with chronic respiratory conditions. METHODS: A single text and e-mail message was sent to those with enabled preferences in the patient portal. A follow-up survey assessed aspects of message receipt. Surveys were completed without collection of demographics. RESULTS: A total of 3,206 messages were successfully delivered. Surveys were initiated by 107 recipients. Frequency analysis showed that text and e-mail messages were preferred forms of communication. A statistically significant relationship was found between receiving a message and receiving an influenza vaccination (p = .027). DISCUSSION: Text and e-mail messaging are cost effective and well received, and they can be easily integrated into existing systems. These methods are translatable across populations and can convey various types of messages.

Compensatory mutations in predicted metal transporters modulate auxin conjugate responsiveness in Arabidopsis.

Levels of the phytohormone indole-3-acetic acid (IAA) can be altered by the formation and hydrolysis of IAA conjugates. The isolation and characterization of Arabidopsis thaliana mutants with reduced IAA-conjugate sensitivity and wild-type IAA responses is advancing the understanding of auxin homeostasis by uncovering the factors needed for conjugate metabolism. For example, the discovery that the IAA-Ala-resistant mutant iar1 is defective in a protein in the ZIP family of metal transporters uncovered a link between metal homeostasis and IAA-conjugate sensitivity. To uncover additional factors impacting auxin conjugate metabolism, we conducted a genetic modifier screen and isolated extragenic mutations that restored IAA-amino acid conjugate sensitivity to the iar1 mutant. One of these suppressor mutants is defective in a putative cation diffusion facilitator, MTP5 (At3g12100; formerly known as MTPc2). Loss of MTP5 function restored IAA conjugate sensitivity to iar1 but not to mutants defective in IAA-amino acid conjugate amidohydrolases. Our results are consistent with a model in which MTP5 and IAR1 transport metals in an antagonistic fashion to regulate metal homeostasis within the subcellular compartment in which the IAA-conjugate amidohydrolases reside, and support previous suggestions that the ion composition in this compartment influences hydrolase activity.

Efficient conversion of arylene precursors into photoluminescent phosphonates for surface modification of metal oxides.

Three highly fluorescent phosphonates have been prepared in good yields from different arylene bridged 5-iodothiophenes by following an optimized four-step procedure. The compounds have been immobilised on mesoporous zirconia, alumina and titania particles in order to probe their luminescence properties on the surfaces. The organic compounds as well as the obtained hybrid materials have been characterised using liquid phase or MAS NMR spectroscopy, N2 physisorption measurements, ATR-IR spectroscopy and elemental analysis. The luminescence intensity of the organic molecules was found to be in general much more dependent on the surface used for grafting rather than on the dye loading obtained for different metal oxides powders. The luminescence extinction, which has been detected for all coated TiO2 samples, is most likely due to the different electronic properties of this semiconducting support compared to the other surfaces. The method described shows how fluorescent tracers can be easily synthesised and applied in surface analytics after exploring the interaction of the corresponding organophosphonates with several ceramic interfaces.

What are those cilia doing in the neural tube?

Primary cilia are present on almost all vertebrate cells, and they have diverse functions in distinct tissues. Cilia are important for sensation in multiple capacities in contexts as different as the retina, kidney, and inner ear. In addition to these roles, cilia play a critical part in various developmental processes. Of particular importance is the development of the neural tube, where cilia are essential for the transduction of the Sonic Hedgehog (Shh) signaling pathway that specifies neuronal cell fates. This relationship is well established and is the most recognizable function for cilia in the neural tube, but it may be part of a larger picture. Here, we discuss the links between cilia and Shh signaling, as well as suggesting additional roles for cilia, and mechanisms for their placement, in the neural tube.

Novel acridone-modified MCM-41 type silica: Synthesis, characterization and fluorescence tuning.

A Mobil Composition of Matter (MCM)-41 type mesoporous silica material containing N-propylacridone groups has been successfully prepared by co-condensation of an appropriate organic precursor with tetraethyl orthosilicate (TEOS) under alkaline sol-gel conditions. The resulting material was fully characterized by means of X-ray diffraction (XRD), N(2)-adsorption-desorption, transmission electron microscopy (TEM), IR and UV-vis spectroscopy, as well as (29)Si and (13)C CP-MAS NMR techniques. The material features a high inner surface area and a highly ordered two-dimensional hexagonal pore structure. The fluorescence properties of the organic chromophore can be tuned via complexation of its carbonyl group with scandium triflate, which makes the material a good candidate for solid state sensors and optics. The successful synthesis of highly ordered MCM materials through co-condensation was found to be dependent on the chemical interaction of the different precursors.

Redox active mesoporous hybrid materials by in situ syntheses with urea-linked triethoxysilylated phenothiazines.

Triethoxysilyl functionalized phenothiazinyl ureas were synthesized and immobilized by in situ synthesis into mesoporous hybrid materials. The designed precursor molecules influence the structure of the final materials and the intermolecular distance of the phenothiazines. XRD and N(2) adsorption measurements indicate the presence of highly ordered two-dimensional hexagonally structured functional materials, while the incorporation of the organic compounds in the solid materials was proved by means of (13)C and (29)Si solid state NMR spectroscopy as well as by FT-IR spectroscopy. Upon oxidation with (NO)BF(4) or SbCl(5), stable phenothiazine radical cations were generated in the pores of the materials, which was detected by means of UV/Vis, emission, and EPR spectroscopies.