Through-space H-F coupling in a series of 4-substituted-1H-1,2,3-triazoles.

In the 1H-NMR spectra of a series of N-1 substituted 4-substituted-1H-1,2,3-triazoles that have been prepared, the lone heterocyclic ring hydrogen (H-5) appears as a singlet in all cases except those compounds that contain a 2-fluorophenyl moiety at Position 4. In those cases, H-5 is a doublet with J ~3.7 Hz. Based on computational chemistry results and geometric considerations, we attribute this splitting to through-space H-F coupling.

Differential Susceptibility to Benzo[a]pyrene Exposure during Gestation and Lactation in Mice with Genetic Variations in the Aryl Hydrocarbon Receptor and Cyp1 Genes.

Polycyclic aromatic hydrocarbons are ubiquitous air pollutants, with additional widespread exposure in the diet. PAH exposure has been linked to adverse birth outcomes and long-term neurological consequences. To understand genetic differences that could affect susceptibility following developmental exposure to polycyclic aromatic hydrocarbons, we exposed mice with variations in the aryl hydrocarbon receptor and the three CYP1 enzymes from gestational day 10 (G10) to weaning at postnatal day 25 (P25). We found unexpectedly high neonatal lethality in high-affinity AhrbCyp1b1(-/-) knockout mice compared with all other genotypes. Over 60% of BaP-exposed pups died within their first 5 days of life. There was a significant effect of BaP on growth rates in surviving pups, with lower weights observed from P7 to P21. Again, AhrbCyp1b1(-/-) knockout mice were the most susceptible to growth retardation. Independent of treatment, this line of mice also had impaired development of the surface righting reflex. We used high-resolution mass spectrometry to measure BaP and metabolites in tissues from both dams and pups. We found the highest BaP levels in adipose from poor-affinity AhrdCyp1a2(-/-) dams and identified three major BaP metabolites (BaP-7-OH, BaP-9-OH, and BaP-4,5-diol), but our measurements were limited to a single time point. Future work is needed to understand BaP pharmacokinetics in the contexts of gestation and lactation and how differential metabolism leads to adverse developmental outcomes.

Investigation on the Synthesis, Application and Structural Features of Heteroaryl 1,2-Diketones.

A set of unsymmetrical heteroaryl 1,2-diketones were synthesized by a heteroarylation/oxidation sequence with up to 65% isolated yields. Palladium catalyst XPhos Pd G4 and SeO2 were the key reagents used in this methodology, and microwave irradiation was utilized to facilitate an efficient and ecofriendly process. The application of heteroaryl 1,2-diketones is demonstrated through the synthesis of an unsymmetrical 2-phenyl-3-(pyridin-3-yl)quinoxaline (5a) from 1-phenyl-2-(pyridin-3-yl)ethane-1,2-dione (4a). The lowest energy conformations of 4a and 5a were located using Density Functional Theory (DFT) at the M06-2X/def2-TZVP level of theory. Two lowest energy conformations of 4a differ with respect to the position of the N atom in the pyridyl ring and 0.27 kcal/mol energy difference between them corresponds to 60.4 and 39.6% at 50 °C in toluene. Four lowest energy conformations for 5a have the energy differences of 0.01, 0.03 and 0.07 kcal/mol that corresponds to 26.0, 25.7, 24.9 and 23.4%, respectively. A comparison of 4a and 5a to the less hindered analogs (oxalyl chloride and oxalic acid) is used to investigate the structural features and bonding using Natural Bond Orbital (NBO) analysis.

Solvent- and Wavelength-Dependent Photolysis of Estrone.

The direct photolysis of estrone in solvents ranging from water to cyclohexane is reported. The photodegradation is dominated by lumiestrone, an epimer of estrone resulting from the inversion of the methyl group at carbon 13, regardless of solvent and photolysis wavelength in the range 254-320 nm. Solvent addition products are also observed in lesser amounts. The photodegradation rate in water is an order of magnitude slower than in nonaqueous solvents. Short wavelength excitation enhances photodegradation. Together, these results suggest complicated photophysics underlie the photochemistry with implications for the remediation of environmental estrogens.

Novel inactivation of the causative fungal pathogen of white-nose syndrome with methoxsalen plus ultraviolet A or B radiation.

White-nose syndrome is a fungal disease responsible for the rapid decline of North American bat populations. This study addressed a novel method for inactivating Pseudogymnoascus destructans, the causative agent of WNS, using ultraviolet A (UVA) or B (UVB) radiation in combination with methoxsalen, a photosensitizer from the furanocoumarin family of compounds. Fungal spore suspensions were diluted in micromolar concentrations of methoxsalen (50-500 µM), then exposed to fixed doses of UVA radiation (500-5000 mJ/cm2), followed by plating on germination media. These plates were examined for two to four weeks for evidence of spore germination or inactivation, along with resultant growth or inhibition of P. destructans colonies. Pretreatment of fungal spores with low doses of methoxsalen resulted in a UVA dose-dependent inactivation of the P. destructans spores. All doses of methoxsalen paired with 500 mJ/cm2 of UVA led to an approximate two-log10 (~99%) reduction in spore viability, and when paired with 1000 mJ/cm2, a four-log10 or greater (>99.99%) reduction in spore viability was observed. Additionally, actively growing P. destructans colonies treated directly with methoxsalen and either UVA or UVB radiation demonstrated UV dose-dependent inhibition and termination of colony growth. This novel approach of using a photosensitizer in combination with UV radiation to control fungal growth may have broad, practical application in the future.

Chemotherapy-Induced Tunneling Nanotubes Mediate Intercellular Drug Efflux in Pancreatic Cancer.

Intercellular communication plays a critical role in the ever-evolving landscape of invasive cancers. Recent studies have elucidated the potential role of tunneling nanotubes (TNTs) in this function. TNTs are long, filamentous, actin-based cell protrusions that mediate direct cell-to-cell communication between malignant cells. In this study, we investigated the formation of TNTs in response to variable concentrations of the chemotherapeutic drug doxorubicin, which is used extensively in the treatment of cancer patients. Doxorubicin stimulated an increased formation of TNTs in pancreatic cancer cells, and this occurred in a dose-dependent fashion. Furthermore, TNTs facilitated the intercellular redistribution of this drug between connected cells in both pancreatic and ovarian cancer systems in vitro. To provide supportive evidence for the relevance of TNTs in pancreatic cancer in vivo, we performed multiphoton fluorescence microscopy and imaged TNTs in tumor specimens resected from three human patients with pancreatic adenocarcinoma, and one with neuroendocrine carcinoma. In sum, TNT formation was upregulated in aggressive forms of pancreatic carcinoma, was further stimulated after chemotherapy exposure, and acted as a novel method for drug efflux. These findings implicate TNTs as a potential novel mechanism of drug resistance in chemorefractory forms of cancer.

A transwell assay that excludes exosomes for assessment of tunneling nanotube-mediated intercellular communication.

BACKGROUND: Tunneling nanotubes (TNTs) are naturally-occurring filamentous actin-based membranous extensions that form across a wide spectrum of mammalian cell types to facilitate long-range intercellular communication. Valid assays are needed to accurately assess the downstream effects of TNT-mediated transfer of cellular signals in vitro. We recently reported a modified transwell assay system designed to test the effects of intercellular transfer of a therapeutic oncolytic virus, and viral-activated drugs, between cells via TNTs. The objective of the current study was to demonstrate validation of this in vitro approach as a new method for effectively excluding diffusible forms of long- and close-range intercellular transfer of intracytoplasmic cargo, including exosomes/microvesicles and gap junctions in order to isolate TNT-selective cell communication. METHODS: We designed several steps to effectively reduce or eliminate diffusion and long-range transfer via these extracellular vesicles, and used Nanoparticle Tracking Analysis to quantify exosomes following implementation of these steps. RESULTS: The experimental approach outlined here effectively reduced exosome trafficking by >95%; further use of heparin to block exosome uptake by putative recipient cells further impeded transfer of these extracellular vesicles. CONCLUSIONS: This validated assay incorporates several steps that can be taken to quantifiably control for extracellular vesicles in order to perform studies focused on TNT-selective communication.

Lost in translation: applying 2D intercellular communication via tunneling nanotubes in cell culture to physiologically relevant 3D microenvironments.

Tunneling nanotubes (TNTs) are membranous conduits for direct cell-to-cell communication. Until the past decade, little had been known about their composite structure, function, and mechanisms of action in both normal physiologic conditions as well as in disease states. Now TNTs are attracting increasing interest for their key role(s) in the pathogenesis of disease, including neurodegenerative disorders, inflammatory and infectious diseases, and cancer. The field of TNT biology is still in its infancy, but inroads have been made in determining potential mechanisms and function of these remarkable structures. For example, TNTs function as critical conduits for cellular exchange of information; thus, in cancer, they may play an important role in critical pathophysiologic features of the disease, including cellular invasion, metastasis, and emergence of chemotherapy drug resistance. Although the TNT field is still in a nascent stage, we propose that TNTs can be investigated as novel targets for drug-based treatment of cancer and other diseases.

Healthcare experiences of lesbian, gay, and bisexual college students: recommendations for the clinical nurse specialist.

PURPOSE/AIMS: The purpose of this study was to describe the healthcare experiences of lesbian, gay, and bisexual college students (ages 18-24 years) in the local college community. A specific aim of the study was to describe the factors (eg, healthcare system, patient, provider, clinical encounter) that influence this experience. DESIGN: This qualitative descriptive study used a community-based participatory research approach. SETTING: This study was conducted at a local college consortium in New England that consisted of 13 private and public colleges and universities. SAMPLE: A total of 19 college students who self-identified as lesbian (n = 7), gay (n = 7), and female bisexual (n = 4) were included. The mean (SD) age of the sample was 20.7 (1.2) years (range, 19-24 years), and the mean (SD) number of completed college years was 2.4 (1.2) (range, 1-5). METHODS: Three online synchronous focus groups were conducted. RESULTS: Qualitative content analysis revealed 1 overarching theme (not all the same), 1 main theme (comfort during the clinical encounter), and 3 subthemes (personalizing the clinical encounter, deciding to disclose and social stigma, and seeking support of self-identified sexual orientation). CONCLUSIONS: Participants provided recommendations that are helpful to clinical nurse specialists to promote positive clinical encounters. Implications for clinical nurse specialist practice and recommendations for further research are addressed.

Solvent-dependent fluorescence lifetimes of estrone, 17ß-estradiol and 17α-ethinylestradiol.

The fluorescence lifetimes of the estrogens, estrone, 17ß-estradiol and 17α-ethinylestradiol, were studied in various solvents. The fluorescence lifetimes of 17ß-estradiol and 17α-ethinylestradiol decreased from 4.7 to 0.9 ns as the solvent hydrogen bond accepting ability increased, in good agreement with other phenolic molecules. Estrone's two fluorescence bands had distinct lifetimes, with the 304 nm band having a lifetime shorter than 200 ps, reflecting efficient energy transfer to the carbonyl group, which had lifetimes ranging from 4.4 to 4.9 ns depending on the solvent. Solvent effects on the (1) ππ*, (1) πσ* and (1) nπ* states that are relevant to estrogen photophysics can adequately explain these trends. The solvent dependence on the excited states of these potent endocrine disruptors has significant implications for their photochemistry.

Solvent effects on the steady state photophysics of estrone and 17ß-estradiol.

Absorption and emission yields for estrone and 17ß-estradiol were measured in a variety of room temperature solvents. Molar extinction coefficients were found to not vary as a function of solvent, while fluorescence yields were found to be significantly affected by the polarity and hydrogen-bond accepting ability of the solvent, with the yield for 17ß-estradiol being highest in nonpolar, hydrogen-bond donating solvents, and lowest in the nonpolar, hydrogen-bond accepting solvent ethyl acetate. Estrone's emission yield was found to be a factor of ten smaller than 17ß-estradiol's. Strong solvent and excitation wavelength dependences were found for the relative amounts of emission between estrone's two emission bands, with increased relative emission occurring in nonpolar aprotic solvents, and under higher excitation energies. These results are interpreted with the aid of vertical excitation energies from time-dependent density functional calculations using both explicit and implicit solvation models.

Solvated electron extinction coefficient and oscillator strength in high temperature water.

The decadic extinction coefficient of the hydrated electron is reported for the absorption maximum from room temperature to 380 degrees C. The extinction coefficient is established by relating the transient absorption of the hydrated electrons in the presence of a scavenger to the concentration of stable product produced in the same experiment. Scavengers used in this report are SF(6,) N(2)O, and methyl viologen. The room temperature value is established as 22,500 M(-1) cm(-1), higher by 10-20% than values used over the last several decades. We demonstrate how previous workers arrived at a low value by incorrect choice of a radiolysis yield value. With this revision, the integrated oscillator strength, corrected by refractive index, is definitely (ca. 10%) larger than unity. This result is fully consistent with EPR and resonance Raman results which indicate mixing of the hydrated electron wave function with solvent electronic orbitals. Oscillator strength appears to be conserved vs temperature.

Hydrated electron extinction coefficient revisited.

The extinction coefficient of the hydrated electron (e(-))aq generated by pulse radiolysis is evaluated relative to the methyl viologen radical cation (*)MV(+), whose extinction coefficient at 605 nm has been carefully measured in the past. We find that the room temperature (e(-))aq extinction coefficients reported in the literature are underestimated by 10-20%. We obtain = 22,700 M(-1) cm(-1) for the 20 degrees C hydrated electron at 720 nm, assuming the (*)MV(+) extinction is 13,700 M(-1) cm(-1) at 605 nm. This has implications both for second-order reaction rate measurements of (e(-))aq and for the estimate of its integrated oscillator strength.

Time-resolved infrared spectroscopy of the lowest triplet state of thymine and thymidine.

Vibrational spectra of the lowest energy triplet states of thymine and its 2'-deoxyribonucleoside, thymidine, are reported for the first time. Time-resolved infrared (TRIR) difference spectra were recorded over seven decades of time from 300 fs - 3 micros using femtosecond and nanosecond pump-probe techniques. The carbonyl stretch bands in the triplet state are seen at 1603 and ~1700 cm(-1) in room-temperature acetonitrile-d(3) solution. These bands and additional ones observed between 1300 and 1450 cm(-1) are quenched by dissolved oxygen on a nanosecond time scale. Density-functional calculations accurately predict the difference spectrum between triplet and singlet IR absorption cross sections, confirming the peak assignments and elucidating the nature of the vibrational modes. In the triplet state, the C4=O carbonyl exhibits substantial single-bond character, explaining the large (~70 cm(-1)) red shift in this vibration, relative to the singlet ground state. Femtosecond TRIR measurements unambiguously demonstrate that the triplet state is fully formed within the first 10 ps after excitation, ruling out a relaxed (1)npi* state as the triplet precursor.

Internal conversion to the electronic ground state occurs via two distinct pathways for pyrimidine bases in aqueous solution.

The femtosecond transient absorption technique was used to study the relaxation of excited electronic states created by absorption of 267-nm light in all of the naturally occurring pyrimidine DNA and RNA bases in aqueous solution. The results reveal a surprising bifurcation of the initial excited-state population in <1 ps to two nonradiative decay channels within the manifold of singlet states. The first is the subpicosecond internal conversion channel first characterized in 2000. The second channel involves passage through a dark intermediate state assigned to a lowest-energy (1)npi* state. Approximately 10-50% of all photoexcited pyrimidine bases decay via the (1)npi* state, which has a lifetime of 10-150 ps. Three- to 6-fold-longer lifetimes are seen for pyrimidine nucleotides and nucleosides than for the corresponding free bases, revealing an unprecedented effect of ribosyl substitution on electronic energy relaxation. A small fraction of the (1)npi* population is proposed to undergo intersystem crossing to the lowest triplet state in competition with vibrational cooling, explaining the higher triplet yields observed for pyrimidine versus purine bases at room temperature. Some simple correlations exist between yields of the (1)npi* state and yields of some pyrimidine photoproducts, but more work is needed before the photochemical consequences of this state can be definitively determined. These findings lead to a dramatically different picture of electronic energy relaxation in single pyrimidine bases with important ramifications for understanding DNA photostability.

Solvent-dependent photophysics of 1-cyclohexyluracil: ultrafast branching in the initial bright state leads nonradiatively to the electronic ground state and a long-lived 1npi* state.

The modified nucleic acid base, 1-cyclohexyluracil, was studied by femtosecond transient absorption spectroscopy in protic and aprotic solvents of varying polarity. UV excitation at 267 nm populates the lowest-energy bright state, a (1)pipi* state, which has a lifetime of 120-270 fs, depending on the solvent. In all solvents, this initial bright state population bifurcates with approximately 60% undergoing subpicosecond nonradiative decay to the electronic ground state and the remaining population branching to a singlet dark state. The latter absorbs between 340 and 450 nm. The latter state is assigned to the lowest-energy (1)npi* state. It decays to the electronic ground state with a lifetime that varies from 26 ps in water to at least several nanoseconds in aprotic solvents. The results suggest that the two nonradiative decay pathways identified for photoexcited uracil in recent quantum chemical calculations (Matsika, S. J. Phys. Chem. A. 2004, 108, 7584) are simultaneously operative in a wide variety of solvent environments. The lowest-energy triplet state was also detected by transient absorption. The triplet population appears in a few picoseconds and is not formed from the thermalized (1)npi* state. It is suggested that high spin-orbit coupling is found only along initial segments of the nonradiative decay pathways. Efficient intersystem crossing prior to vibrational cooling offers a possible explanation for the wavelength-dependent triplet yields seen in single DNA bases.

Pay for performance: will your hospital be ready?

With increased payment-or, in some cases, even the ability to contract with a payer-likely to be tied to the achievement of quality measures, providers need to: work with physicians in developing associated care delivery protocols, establish compliance mechanisms that are acceptable to clinicians and meet appropriate regulatory and legal standards.

Beyond saber rattling.

New legislative proposals are threatening aggressive regulation of tax-exempt healthcare providers. The proposals pertain to operations, governance, additional tax filing requirements, and enforcement. The proposals signal the determination of Congress to eliminate any perceived abuses among exempt healthcare organizations through increased scrutiny and regulation. Healthcare financial managers should monitor any developments related to the proposed legislation and participate in hearings and lobbying regarding these issues.

Singlet excited-state dynamics of 5-fluorocytosine and Cytosine: an experimental and computational study.

The photophysics of singlet excited 5-fluorocytosine (5FC) was studied in steady-state and time-resolved experiments and theoretically by quantum chemical calculations. Femtosecond transient absorption measurements show that replacement of the C5 hydrogen of cytosine by fluorine increases the excited-state lifetime by 2 orders of magnitude from 720 fs to 73 +/- 4 ps. Experimental evidence indicates that emission in both compounds originates from a single tautomeric form. The lifetime of 5FC is the same within experimental uncertainty in the solvents ethanol and dimethyl sulfoxide. The insensitivity of the S(1) lifetime to the protic nature of the solvent suggests that proton transfer is not the principal quenching mechanism for the excited state. Excited-state calculations were carried out for the amino-keto tautomer of 5FC, the dominant species in polar environments, in order to understand its longer excited-state lifetime. CASSCF and CAS-PT2 calculations of the excited states show that the minimum energy path connecting the minimum of the (1)pi,pi state with the conical intersection responsible for internal conversion has essentially the same energetics for cytosine and 5FC, suggesting that both bases decay nonradiatively by the same mechanism. The dramatic difference in lifetimes may be due to subtle changes along the decay coordinate. A possible reason may be differences in the intramolecular vibrational redistribution rate from the Franck-Condon active, in-plane modes to the out-of-plane modes that must be activated to reach the conical intersection region.

Implementing a smallpox vaccination program aboard an aircraft carrier.

OBJECTIVE: To determine the feasibility of implementing a smallpox vaccination program aboard an aircraft carrier in conjunction with anthrax vaccination. METHODS: Retrospective review of smallpox vaccination program conducted from January 17, 2003 to February 19, 2003. Morbidity and loss of manpower were the major endpoints. RESULTS: There were 5,204 sailors available for vaccination. There were 243 (4.7%) medical exemptions and 24 administrative exemptions. During the program, 4,931 sailors were vaccinated. There were five reportable complications. Three sailors had autoinoculation, one sailor had localized cellulitis, and one patient had a positive beta human chorionic gonadotropin during vaccination. None of the complications required medical evacuation. Only two sailors required time off from duty. CONCLUSIONS: Smallpox vaccination can be accomplished rapidly and safely aboard an aircraft carrier. There was not an increase in adverse events compared to historical data despite the close-quarter conditions. Smallpox and anthrax vaccinations can be completed simultaneously with minimal morbidity.