Dealing with Dust Entrained in the Nitrogen Plume Demonstration.

Water condensation plumes produced by the addition of iron powder to liquid nitrogen can be contaminated with small quantities of particulate matter. Variations on the plume demonstration, including those using noisemakers, are described to help minimize the release of particulates into the air.

Plumes from Using Iron to Boil Liquid Nitrogen to Illustrate the Importance of Surface Area.

The relationship between surface area and dynamics of processes can be demonstrated by adding iron at room temperature to liquid nitrogen. The rate at which the liquid nitrogen boils to produce gas is related to the surface area of the iron. Adding iron in the form of consistent units that have measurable sizes can be readily connected to observable differences in rates of nitrogen gas production. For example, samples of smaller iron spheres with their greater surface area transfer heat more quickly than do larger spheres of the same volume to liquid nitrogen causing it to boil faster, but more briefly, and produce larger plumes of nitrogen gas from a container vent. The plumes are essentially comprised of nitrogen and water, which make them potentially safer than plumes from other demonstrations such as the "genie in a bottle", based on hydrogen peroxide decomposition. These simple activities can be used as stand-alone demonstrations or as the basis of laboratory activities.

Infrared Spectroscopic Analysis of the Adsorption of Pyridine Carboxylic Acids on Colloidal Ceria.

Surface adsorption of a homologous series of pyridine carboxylic acids on a hydrated colloidal cerium dioxide (ceria) film is characterized using the combination of experimental and computationally determined infrared (IR) spectra. Experimental analyses employ attenuated total reflectance (ATR) IR spectroscopy of deposited colloidal ceria thin films equilibrated with three pyridine carboxylic acids at pH 3.0, 5.5, and 8.5. The corresponding computational IR spectra for the energy-minimized intermediate and base forms of the pyridine carboxylic acids use density functional theory calculations at the B3LYP/6-311++G** level of theory. Solvent effects are modeled using both the COSMO implicit solvation model and the inclusion of explicit water molecules. Experimental IR spectra show that the adsorptive interactions between the pyridine carboxylic acids and ceria surface are due to the outer-sphere coordination of cerium ions in the films. Vibrational assignments based on combined experimental and computational results indicate that both pyridyl ring nitrogen and carboxylate functional groups account for the interaction of pyridine carboxylic acids at ceria surfaces. Experimentally determined Langmuir constants point to the intermediate form of picolinic acid (pyridine-2-carboxylic acid) as having the strongest adsorption to ceria compared to the other pyridine carboxylic acids investigated. The enhanced adsorption of picolinic acid is attributed to the adjacency of the protonated pyridyl nitrogen and the carboxylate group relative to nicotinic acid (pyridine-3-carboxylic acid) and isonicotinic acid (pyridine-4-carboxylic acid).

Myeloid and dendritic cells enhance therapeutics-induced cytokine release syndrome features in humanized BRGSF-HIS preclinical model.

Objectives: Despite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings. Methods: BRGSF mice reconstituted with human umbilical cord blood CD34+ cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells' contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed. Results: OKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment's effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds. Conclusions: These data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

Structure-guided engineering of human thymidine kinase 2 as a positron emission tomography reporter gene for enhanced phosphorylation of non-natural thymidine analog reporter probe.

Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-ß-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.

Requirement for deoxycytidine kinase in T and B lymphocyte development.

Deoxycytidine kinase (dCK) is a rate-limiting enzyme in deoxyribonucleoside salvage, a metabolic pathway that recycles products of DNA degradation. dCK phosphorylates and therefore activates nucleoside analog prodrugs frequently used in cancer, autoimmunity, and viral infections. In contrast to its well established therapeutic relevance, the biological function of dCK remains enigmatic. Highest levels of dCK expression are found in thymus and bone marrow, indicating a possible role in lymphopoiesis. To test this hypothesis we generated and analyzed dCK knockout (KO) mice. dCK inactivation selectively and profoundly affected T and B cell development. A 90-fold decrease in thymic cellularity was observed in the dCK KO mice relative to wild-type littermates. Lymphocyte numbers in the dCK KO mice were 5- to 13-fold below normal values. The severe impact of dCK inactivation on lymphopoiesis was unexpected given that nucleoside salvage has been thought to play a limited, "fine-tuning" role in regulating deoxyribonucleotide triphosphate pools produced by the de novo pathway. The dCK KO phenotype challenges this view and indicates that, in contrast to the great majority of other somatic cells, normal lymphocyte development critically requires the deoxyribonucleoside salvage pathway.

Noninvasive prediction of tumor responses to gemcitabine using positron emission tomography.

Gemcitabine (2',2'-difluorodeoxycytidine, dFdC) and cytosine arabinoside (cytarabine, ara-C) represent a class of nucleoside analogs used in cancer chemotherapy. Administered as prodrugs, dFdC and ara-C are transported across cell membranes and are converted to cytotoxic derivatives through consecutive phosphorylation steps catalyzed by endogenous nucleoside kinases. Deoxycytidine kinase (DCK) controls the rate-limiting step in the activation cascade of dFdC and ara-C. DCK activity varies significantly among individuals and across different tumor types and is a critical determinant of tumor responses to these prodrugs. Current assays to measure DCK expression and activity require biopsy samples and are prone to sampling errors. Noninvasive methods that can detect DCK activity in tumor lesions throughout the body could circumvent these limitations. Here, we demonstrate an approach to detecting DCK activity in vivo by using positron emission tomography (PET) and (18)F-labeled 1-(2'-deoxy-2'-fluoroarabinofuranosyl) cytosine] ([(18)F]FAC), a PET probe recently developed by our group. We show that [(18)F]FAC is a DCK substrate with an affinity similar to that of dFdC. In vitro, accumulation of [(18)F]FAC in murine and human leukemia cell lines is critically dependent on DCK activity and correlates with dFdC sensitivity. In mice, [(18)F]FAC accumulates selectively in DCK-positive vs. DCK-negative tumors, and [(18)F]FAC microPET scans can predict responses to dFdC. We suggest that [(18)F]FAC PET might be useful for guiding treatment decisions in certain cancers by enabling individualized chemotherapy.

Epithelial uptake of [18F]1-(2'-deoxy-2'-arabinofuranosyl) cytosine indicates intestinal inflammation in mice.

BACKGROUND & AIMS: Uptake of [18F]1-(2'-deoxy-2'-arabinofuranosyl)cytosine (D-FAC) is a trait of activated lymphocytes; its biodistribution predominates in the spleen, thymus, and bone marrow. In addition, D-FAC is taken up at high levels by the intestine. We analyzed the regional specificity of uptake and cell types that mediate it. METHODS: In mice, 3-dimensional isocontour regions of interest were drawn based on computed tomographic images to quantify intestinal signals from micro-positron emission tomography scans. To ascertain the cell type responsible, intestinal epithelium and immune cells were isolated and D-FAC uptake was analyzed in vitro. Mice deficient in mucosal homing (beta7 integrin-/-), enteric microbiota (germ-free), or active for immune colitis (G alpha i2-/- CD3+ transferred into Rag-/- recipients) were studied. RESULTS: Strong uptake of D-FAC was detected throughout the intestine, with greatest signal per region of interest in the duodenum. Fractionation of intestinal cell types after in vivo uptake revealed that the signal was almost entirely from epithelial cells. Among resident immune cell types, CD4+ T cells showed the greatest per-cell and total uptake. D-FAC uptake increased in both intestinal and systemic lymphoid sites during colitis. Compared with fluorodeoxyglucose, increased uptake of D-FAC in the small and large intestine occurred at an earlier stage of disease development. CONCLUSIONS: Uptake of D-FAC is a prominent trait of normal mouse intestinal epithelial cells, which is useful for their noninvasive visualization by positron emission tomography. Increased uptake of D-FAC reflects the activity of the epithelium and lymphocytes, providing a unique early marker of intestinal inflammation.

Foxc1 Ablated Mice Are Anhidrotic and Recapitulate Features of Human Miliaria Sweat Retention Disorder.

Sweat glands are critical for thermoregulation. The single tubular structure of sweat glands has a lower secretory portion and an upper reabsorptive duct leading to the secretory pore in the skin. Genes that determine sweat gland structure and function are largely unidentified. Here we report that a Fox family transcription factor, Foxc1, is obligate for appreciable sweat duct activity in mice. When Foxc1 was specifically ablated in skin, sweat glands appeared mature, but the mice were severely hypohidrotic. Morphologic analysis revealed that sweat ducts were blocked by hyperkeratotic or parakeratotic plugs. Consequently, lumens in ducts and secretory portions were dilated, and blisters and papules formed on the skin surface in the knockout mice. The phenotype was strikingly similar to the human sweat retention disorder miliaria. We further show that Foxc1 deficiency ectopically induces the expression of keratinocyte terminal differentiation markers in the duct luminal cells, which most likely contribute to keratotic plug formation. Among those differentiation markers, we show that Sprr2a transcription is directly repressed by overexpressed Foxc1 in keratinocytes. In summary, Foxc1 regulates sweat duct luminal cell differentiation, and mutant mice mimic miliaria and provide a possible animal model for its study.

Canonical Notch signaling plays an instructive role in auditory supporting cell development.

The auditory sensory epithelium, composed of mechano-sensory hair cells (HCs) and highly specialized glial-like supporting cells (SCs), is critical for our ability to detect sound. SCs provide structural and functional support to HCs and play an essential role in cochlear development, homeostasis and repair. Despite their importance, however, surprisingly little is known about the molecular mechanisms guiding SC differentiation. Here, we provide evidence that in addition to its well-characterized inhibitory function, canonical Notch signaling plays a positive, instructive role in the differentiation of SCs. Using γ-secretase inhibitor DAPT to acutely block canonical Notch signaling, we identified a cohort of Notch-regulated SC-specific genes, with diverse functions in cell signaling, cell differentiation, neuronal innervation and synaptogenesis. We validated the newly identified Notch-regulated genes in vivo using genetic gain (Emx2(Cre/+); Rosa26(N1ICD/+)) and loss-of-function approaches (Emx2(Cre/+); Rosa26(DnMAML1/+)). Furthermore, we demonstrate that Notch over-activation in the differentiating murine cochlea (Emx2(Cre/+); Rosa26(N1ICD/+)) actively promotes a SC-specific gene expression program. Finally, we show that outer SCs -so called Deiters' cells are selectively lost by prolonged reduction (Emx2(Cre/+); Rosa26(DnMAML1/+/+)) or abolishment of canonical Notch signaling (Fgfr3-iCreER; Rbpj(-/Δ)), indicating a critical role for Notch signaling in Deiters' cell development.

Preclinical Evaluation of an Anti-Nectin-4 ImmunoPET Reagent in Tumor-Bearing Mice and Biodistribution Studies in Cynomolgus Monkeys.

PURPOSE: Nectin-4 is selectively overexpressed in a variety of cancers and is currently under clinical investigation as a therapeutic target. A monoclonal antibody against nectin-4 (AGS-22M6) was evaluated as an Immuno-positron emission tomography (ImmunoPET) reagent. Its ability to assay nectin-4 expression as well as detect nectin-4 positive tumors in the liver and bone was evaluated using mouse models. PROCEDURES: The biodistribution of [(89)Zr]AGS-22M6 was evaluated in mice bearing tumors with varying levels of nectin-4 expression. An isogenic breast cancer tumor line was used to model metastatic liver and bone disease in mice. The biodistribution of [(18)F]AGS-22M6 in cynomolgus monkeys was evaluated. RESULTS: A positive correlation was demonstrated between tumor nectin-4 expression and [(89)Zr]AGS-22M6 uptake. Tumors in the liver and bone were detected and differentiated based on nectin-4 expression. [(18)F]AGS-22M6 showed limited uptake in cynomolgus monkey tissues. CONCLUSIONS: [(89)Zr]AGS-22M6 is a promising ImmunoPET reagent that can assay nectin-4 expression in both primary and metastatic lesions.

Defining, Treating, and Understanding Chronic Kidney Disease--A Complex Disorder.

Chronic kidney disease (CKD) is prevalent in more than 20 million people in the United States. The majority of care provided to patients with this disease comes from primary care physicians, although it is often poorly understood. After an extensive literature review, it is clear that it can be difficult to classify and there are many barriers to care. Risk factors for both incident CKD and disease progression include hypertension, poor glycemic control, sociodemographic factors, acute kidney injury, metabolic acidosis, and possibly hyperuricemia and dietary factors. Treatment of patients with CKD should focus on mitigating risk factors, as well as common comorbidities such as cardiovascular disease, anemia, and bone mineral disease. Novel therapies such as pirfenidone, pentoxifylline, and endothelin-1 antagonists are being investigated with promising results.

Characterization and spatial distribution of heavy metals in sediment from Cedar and Ortega rivers subbasin.

The Cedar and Ortega rivers subbasin is a complex environment where both natural and anthropogenic processes influence the characteristics and distributions of sediments and contaminants, which in turn is of importance for maintenance, dredging and pollution control. This study investigated the characteristics and spatial distribution of heavy metals, including lead (Pb), copper (Cu), zinc (Zn) and cadmium (Cd), from sediments in the subbasin using field measurements and three-dimensional kriging estimates. Sediment samples collected from three sampling depth intervals (i.e., 0-0.10, 0.11-0.56 and 0.57-1.88 m) in 58 locations showed that concentrations of Pb ranged from 4.47 to 420.00 mg/kg dry weight, Cu from 2.30 to 107.00 mg/kg dry weight, Zn from 9.75 to 2,050.00 mg/kg dry weight and Cd from 0.07 to 3.83 mg/kg dry weight. Kriging estimates showed that Pb, Cu and Cd concentrations decreased significantly from the sediment depth of 0.10 to 1.5 m, whereas Zn concentrations were still enriched at 1.5 m. It further revealed that the Cedar River area was a potential source area since it was more contaminated than the rest of the subbasin. Comparison of aluminum (Al)-normalized metal concentrations indicated that most of the metals within the top two intervals (0-0.56 m) had concentrations exceeding the background levels by factors of 2-10. A three-dimensional view of the metal contamination plumes showed that all of the heavy metals, with concentrations exceeding the threshold effect level (TEL) that could pose a threat to the health of aquatic organisms, were primarily located above the sediment depth of 1.5 m.

Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication.

Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.

Septicemia and Aortic Valve Endocarditis due to Erysipelothrix rhusiopathiae in a Homeless Man.

We report a case of bacterial endocarditis due to Erysipelothrix rhusiopathiae in a homeless man with no animal exposure. His course was complicated by an allergic reaction to ampicillin, urinary bladder infection, respiratory failure, and acute kidney injury. He recovered completely after aortic valve replacement and a 6-week course of intravenous ceftriaxone.

A 32-Year-Old Female with AIDS, Pneumocystis jiroveci Pneumonia, and Methemoglobinemia.

We report a case of methemoglobinemia with significant hemoglobin desaturation in a young female with AIDS who was being treated for Pneumocystis jiroveci pneumonia. A review of the etiology, pathophysiology, and treatment of methemoglobinemia is presented.

Application of a rapid, simple, and accurate adenovirus-based method to compare PET reporter gene/PET reporter probe systems.

PURPOSE: This study aims to use a simple, quantitative method to compare the HSV1sr39TK/(18) F-FHBG PET reporter gene/PET reporter probe (PRG/PRP) system with PRGs derived from human nucleoside kinases. PROCEDURES: The same adenovirus vector is used to express alternative PRGs. Equal numbers of vectors are injected intravenously into mice. After PRP imaging, quantitative hepatic PET signals are normalized for transduction by measuring hepatic viral genomes. RESULTS: The same adenovirus vector was used to express equivalent amounts of HSV1sr39TK, mutant human thymidine kinase 2 (TK2-DM), and mutant human deoxycytidine kinase (dCK-A100VTM) in mouse liver. HSV1sr39TK expression was measured with (18) F-FHBG, TK2-DM and dCK-A100VTM with (18) F-L-FMAU. TK2-DM/(18) F-L-FMAU and HSV1sr39TK/(18) F-FHBG had equivalent sensitivities; dCK-A100VTM/(18) F-L-FMAU was twice as sensitive as HSV1sr39TK/(18) F-FHBG. CONCLUSIONS: The human PRG/PRP sensitivities are comparable and/or better than HSV1sr39TK/(18) F-FHBG. However, for clinical use, identification of the best PRP substrate for each enzyme, characterization of probe distribution, and consequences of overexpressing nucleoside kinases must be evaluated.

Fast metabolic response to drug intervention through analysis on a miniaturized, highly integrated molecular imaging system.

UNLABELLED: We report on a radiopharmaceutical imaging platform designed to capture the kinetics of cellular responses to drugs. METHODS: A portable in vitro molecular imaging system comprising a microchip and a ß-particle imaging camera permitted routine cell-based radioassays of small numbers of either suspended or adherent cells. We investigated the kinetics of responses of model lymphoma and glioblastoma cancer cell lines to (18)F-FDG uptake after drug exposure. Those responses were correlated with kinetic changes in the cell cycle or with changes in receptor tyrosine kinase signaling. RESULTS: The platform enabled direct radioassays of multiple cell types and yielded results comparable to those from conventional approaches; however, the platform used smaller sample sizes, permitted a higher level of quantitation, and did not require cell lysis. CONCLUSION: The kinetic analysis enabled by the platform provided a rapid (≈ 1 h) drug screening assay.

Development of new deoxycytidine kinase inhibitors and noninvasive in vivo evaluation using positron emission tomography.

Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest, and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using (18)F-L-1-(2'-deoxy-2'-FluoroArabinofuranosyl) Cytosine ((18)F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC50 = ∼1-12 nM) using the (18)F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy.