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We present proof-of-concept experiments for developing a highly-sensitive and fast-response miniaturized single-walled carbon nanotube field-effect transistor (SWNT-FET) biosensor for electrically detecting adenovirus using ligand-receptor-protein specificity. SWNTs are mildly oxidized to form carboxylic groups on the surfaces without compromising the electronic integrity of the nanotubes. Then the human coxsackievirus and adenovirus receptor (CAR) is covalently functionalized onto the nanotube surface via diimide-activated amidation process. Upon exposure of the device to adenovirus protein, Ad12 Knob (Knob), specific binding of Knob to CAR decreases the current that flows through the SWNT-FET device. For control experiment, the CAR-SWNT device is exposed to YieF, which is a virus protein that does not bind specifically to CAR, and no current change is observed. The biological activity of the CAR and Knob proteins that are immobilized on SWNTs has been confirmed by previous fluorescence studies [1]. AFM analysis is done to show height increase of a few nanometers at specific spots where the CAR-Knob complex are covalently linked to the nanotube surface. Therefore, our results show that the human receptor protein CAR does immobilize on SWNT surface while fully retains its biological activity. Moreover, the specific binding of CAR to its complementary adenovirus Knob can be electrically detected using individual SWNT-FET devices. These findings suggest that CAR-functionalized SWNT-FETs can ably serve as biosensors for detection of environmental adenoviruses.

High grade serous ovarian cancer is typified by p53 mutation, high degrees of genomic instability and the development of chemo-resistance. Genomic translocations result from incorrectly repaired DNA double strand breaks (DSBs). DNA-PKcs is a central catalytic component of the error prone non-homologous end joining (NHEJ) DSB repair mechanism. We report here that DNA-PKcs is frequently amplified in copy number in HGSC and that amplification correlates with higher DNA-PKcs gene expression and poorer patient outcome using both in-house and publicly available datasets. Targeting DNA-PKcs, pharmacologically or by RNAi, enhances apoptosis in response to platinum treatment in platinum resistant cell lines and primary models and inhibition of DNA-PK restores response to cisplatin in chemoresistant ovarian cancer cells in vivo. SKOV-3 tumor xenografts were implanted subcutaneously into Balb/c Nu/Nu mice and treated with DNA-PKcs inhibitor NU7441 for 2 weeks alone or in combination with cisplatin. DNA-PK inhibition or cisplatin treatment alone were ineffective however in combination they decreased tumor growth at 14 days by 90%, relative to platinum only treatment. On investigating the mechanism of DNA-PKcs mediated chemoresistance we revealed that, in response to DNA damage, DNA-PKcs phosphorylates AKT on serine residue 473 in platinum resistant ovarian tumor cells, but not sensitive cells from the same patient. Phosphorylation at AKT threonine 308 is unaffected and DNA-PK inhibition does not interfere with insulin-mediated phosphorylation of AKT S473. DNA-PK mediated AKT activation in chemoresistant cells results in inhibitory phosphorylation of the pro-apoptotic protein BAD and stabilization of the anti-apoptotic Bcl-2 protein resulting in the observed attenuation of apoptotic response to platinum treatment. DNA-PK inhibition is an attractive therapeutic strategy for resensitizing resistant tumors to platinum based therapy, directly linking the DNA damage caused by platinum to pro-survival AKT pathway signaling. Citation Format: Edward Curry, Azadeh Cheraghchi-Bashi-Astaneh, Michelle Chen, Paula Cunnea, De Sousa Camila, Elaina Maginn, Yan Dai, Edison Liu, Harpreet Wasan, Gordon B. Mills, David Bowtell, Hani Gabra, Euan A. Stronach. DNA-PKcs is amplified in high-grade serous ovarian cancer (HGSC), correlates with poor outcome and drives resistance to platinum therapy via the AKT signaling pathway. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A47.

We demonstrate a versatile class of nanoscale sensors based on single-stranded DNA (ss-DNA) as the chemical recognition site and single-walled carbon nanotube field effect transistors (swCN-FETs) as the electronic readout component. Coating swCN-FETs with ss-DNA causes a current change when exposed to gaseous analytes, whereas bare swCN-FETs show no detectable change. The responses differ in sign and magnitude depending both on the type of gaseous analyte and the sequence of DNA. Our results suggest that the conformation of ss-DNA on swCN-FET plays a role in determining the sensor response to gaseous analytes. The conformation depends not only on the base content of the oligomer, but also on the specific arrangement of the bases in the ss-DNA. We compare our results with the molecular dynamic simulations for understanding of the sensing mechanisms. SsDNA/swCN-FETs possess rapid recovery and self-regenerating ability, which could lead to realization of large arrays for sensitive electronic olfaction and disease diagnosis.

Improving models of human stroke by the use of aged animals has been advocated; however the commonly used rat middle cerebral artery thread-occlusion model has produced suboptimal stroke induction and excess mortality in aged rats. We report the development of a modified method for silicone-coating the tip of occluding threads which produces a malleable silicone-coated tip which is firmly bonded and of highly consistent diameter, and overcomes problems of thread insertion through the narrowed carotid canal found in aged animals. Comparison of stroke outcomes and mortality were made between these threads and heat-treated poly-L-lysine coated threads. The rate of successful stroke induction in aged rats was significantly improved (from 14% to 86%). Similarly, mortality fell from 21-31% to 3-7% or less in both young and old rats with or without diabetes and hypertension. An occluding thread tip diameter of 0.35-0.38 mm was optimal for induction of mid-sized strokes in both young and old rats. This method of thread manufacture overcomes problems of inconsistency of diameter and bonding of the silicone-coated tip, and these threads produce significant improvements in stroke induction by MCA occlusion, particularly in aged animals and those with co-morbidities.

We demonstrate a new, versatile class of nanoscale chemical sensors based on single-stranded DNA (ss-DNA) as the chemical sensors recognition site and single-walled carbon nanotube field effect transistors (swCN-FET) as the electronic read-out component. swCN-FETs with a nanoscale coating of ss-DNA respond to gas odors that do not cause a detectable conductivity change in bare devices. Responses of ss-DNA/swCN-FETs differ in sign and magnitude for different gases, and can be tuned by choosing the base sequence of the ss-DNA. ss-DNA/swCN-FET sensors detect a variety of odors, with rapid response and recovery times on the scale of seconds. The sensor surface is self-regenerating: samples maintain a constant response with no need for sensor refreshing through at least 50 gas exposure cycles. This very remarkable set of attributes makes sensors based on ss-DNA decorated nanotubes very promising for "electronic nose" and "electronic tongue" applications ranging from homeland security to disease diagnosis., Comment: 9 pages, 5 figures, Nano Letters web release: 23-Aug-2005

We demonstrate that single walled carbon nanotube field effect transistors (swCN-FET) decorated with single stranded DNA (ss-DNA) form a new, versatile, and highly sensitive class of nanoscale chemical sensors. These sensors are based on ss-DNA as the chemical recognition site and swCN-FET as the electronic read-out component. The ss-DNA decorated swCN-FETs are sensitive to chemical species (odors) that do not cause a detectable response in bare, as-fabricated swCN-FETs. Furthermore, odor responses ss-DNA decorated FETs differ in sign and magnitude for different odors, and the response characteristics depend on the base sequence of the ss-DNA used to decorate the swCN. The sensor surface is self-regenerating: samples maintain a constant response with no need for sensor refreshing through at least several dozens gas exposure cycles. These remarkable attributes suggest that DNA-decorated swCN-FET sensors could be created with sensitivity to a large variety of compounds, as required for “electronic-nose” and “electronic tongue” applications in medicine and homeland security.

Orientadores: Elias Basile Tambourgi, Luiz Carlos Bertevello Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica Resumo: Os sistemas bifásicos aquosos têm sido muito utilizados nas pesquisas bioquímicas para separação e purificação de células, macromoléculas e partículas celulares. A partição em sistemas de duas fases aquosas é uma alternativa que vem sendo muito utilizada nos últimos anos. Neles ambas as fases são compatíveis com quase todas as proteínas conhecidas. Neste trabalho, estudou-se a aplicação da extração líquido-líquido na separação da bromelina presente no caldo do abacaxi, utilizando sistemas de duas fases aquosas compostos por polietileno glicol (PEG) e polissacarídeo da goma do cajueiro (POLlCAJU). O POLlCALU é obtido do exsudado de Anarcadium occidentale L.. Consiste em um polissacarídeo ácido largamente encontrado no Nordeste brasileiro, onde é empregado como substituto da goma arábica para uso farmacêutico. A goma bruta foi coletada como exsudado natural de árvores cultivadas no estado de Pernambuco. Nódulos livres de cascas foram selecionados para processo de isolamento da goma do cajueiro via precipitação por etanol. O comportamento da partição da bromelina em sistema de duas fases aquosas foi verificado estudando-se a influência da massa molar do PEG, pH e a concentração das fases (linha de amarração) no sistema, a fim de se obter o melhor sistema para a separação em descontínuo. Nestes ensaios foi medido o coeficiente de partição entre a fase aquosa superior (PEG) e a fase aquosa inferior (POLlCAJU), utilizando o caldo do abacaxi e a Bromelina P.A.. Observou-se que a proteína se particionou predominantemente na fase inferior, rica em POLlCAJU, nas condições experimentais testadas. Foram encontrados valores de coeficiente de partição da ordem de 0.119 e 0.200, o que demonstra uma boa separação Abstract: The aqueous two-phase systems have found widespread use in biochemical research for separation and purification of macromolecules, cells and call particles. The partition in aqueous two-phase systems is an alternative for the separation processes that has been studied extensively late years. In these works both phases are compatible with almost ali known proteins. In this work, separation of bromelain present in pineapple's juice was studied using liquid-liquid extraction in aqueous two phase based on polyethylene glycol (PEG) and cashew-nut tree gum (POLlCAJU). The exsudate gum from Anarcadium occidentale L. is a branched acidic heteropolysaccharide natural from Brazilian agreste, where it is employed as a substitute for Arabic gum for pharmaceutical use. Crude gum was collected as natural exsudate from cultivated trees in Pernambuco state. Clear nodules free of bark were selected to be isolated via ethanol precipitation. The partition behavior of bromelain in aqueous two-phase systems was investigated studying the influence of molar mass of PEG, pH and phases concentration (ti e line) in the system in order to obtain the best system to the separation. In these trials, the partition coefficient was measured between the higher phase (PEG) and the lower phase (POLlCAJU). It was observed that the protein partitioned almost in the bottom gum-rich phase for the experimental conditions tested. They were found values of partition coefficient of the order of 0.119 and 0.200, what demonstrates a good separation Mestrado Sistemas de Processos Químicos e Informática Mestre em Engenharia Química

Structural, electrical and thermal methods are applied to characterize single-walled carbon nanotube (SWNT) fibers with post-extrusion stretching as the independent variable. HiPco SWNTs are dispersed in water using sodium dodecyl sulfate (SDS), and then co-extruded with polyvinyl alcohol (PVA)/water through a long syringe into a rotating water/PVA coagulation bath. Partial axial alignment is thereby achieved, and further enhanced by applying tension to the flexible green fibers in the coagulation bath. Our findings include: (1) X-ray diffraction shows that the full width at half maximum (FWHM) of the Bragg peaks decreases from 55 (as-extruded) to less than 30 degrees by 80% elongation. That is, SWNT alignment increases linearly with stretch (up to 80%). (2) In resistivity at room temperature vs. stretch ratio, result shows an initial rapid decrease followed by saturation; essentially all the improvement in electronic transport is obtained once alignment reached 40° FWHM. (3) Annealing in hydrogen at 1000°C is performed to drive out PVA, to improve inter-tube and inter-bundle contacts, and to heal damage on the tube walls. Such annealing is found to increase conductivity by at least 4 orders of magnitude. (4) Below 25 K, resistivity vs. temperature of the annealed fiber is well-represented by Coulomb gap variable range hopping (CG-VRH). It is rationalized that the Coulomb interactions in disordered systems open a gap at the Fermi energy. Above 25 K, the thermal energy is greater than the Coulomb gap, so thermal activation is more probable than correlated electron hops. (5) Finally, a measurable thermal conductivity is observed as stretch alignment increases.

We report on the dramatic evolution of the quantum Hall ferromagnet in the fractional quantum Hall regime at $\nu = 2/5$ filling. A large enhancement in the characteristic timescale gives rise to a dynamical transition into a novel quantized Hall state. The observed Hall state is determined to be a zero-temperature phase distinct from the spin-polarized and spin-unpolarized $\nu = 2/5$ fractional quantum Hall states. It is characterized by a strong temperature dependence and puzzling correlation between temperature and time., Comment: 4 pages, 3 figures. to appear in Phys. Rev. Lett

First-generation adenovectors have been developed for gene therapy and vaccine applications. The construction of these adenovectors has entailed the use of numerous types of expression cassettes. It has long been known that first-generation adenovectors can be rescued more easily and to higher titers with some transgenes than with others. This study has systematically shown that there can be marked differences in growth properties of recombinant adenovectors attributable to the use of promoters, the orientation of the transgene within the E1A/E1B-deleted region, and the inclusion of the E3 region. In addition, we had demonstrated the benefit of extending the packaging signal region to include elements V, VI, and VII. The effects of the complete packaging region were studied by plasmid competition studies between original and modified adenovectors. Similar competition studies between E3(+) and E3(-) adenovectors were performed and showed that the E3(+) vector had a growth advantage over its E3(-) counterpart. By making various changes, we have enhanced the growth capacity of our recombinant adenovector by more than 3-fold under serum-free and cell suspension growth conditions. Along with this enhanced growth, our adenovectors have maintained their genetic stability after 21 successive passages in cell culture. This increased robustness will be critical when adapting first-generation recombinant adenovectors to commercial production.

Self-annealing properties of electroplated and sputtered copper films at room temperature were investigated in this study, in particular, the effect of copper film thickness, electrolyte systems used, as well as their level of organic additives for electroplating. Real-time grain growth was observed by transmission electron microscopy. Sheet resistance and X-ray diffraction measurements further confirmed the recrystallization of the electroplated copper film with time. The recrystallization of electroplated films was then compared with that of sputtered copper films.

We present integration of single-stranded DNA (ss-DNA)-decorated single-walled carbon nanotubes (SWNTs) onto complementary metal oxide semiconductor (CMOS) circuitry as nanoscale chemical sensors. SWNTs were assembled onto CMOS circuitry via a low voltage dielectrophoretic (DEP) process. Besides, bare SWNTs are reported to be sensitive to various chemicals, and functionalization of SWNTs with biomolecular complexes further enhances the sensing specificity and sensitivity. After decorating ss-DNA on SWNTs, we have found that the sensing response of the gas sensor was enhanced (up to approximately 300% and approximately 250% for methanol vapor and isopropanol alcohol vapor, respectively) compared with bare SWNTs. The SWNTs coupled with ss-DNA and their integration on CMOS circuitry demonstrates a step towards realizing ultra-sensitive electronic nose applications.

Infants presenting with ALTE are frequently discharged with home cardiorespiratory monitoring. While reassuring to many parents, home monitoring may increase psychosocial and financial stress. Although not proven to predict adverse outcomes, pneumogram evaluation is often used to select patients for monitoring. Accordingly, we sought to determine the clinical and pneumogram characteristics of ALTE patients discharged with and without home monitoring. Data was collected on 32 consecutive infants with ALTE who had 6 channel pneumogram evaluations. Pneumogram evidence of central apnea was defined as an event >15 seconds during the 12 hour period. Monitoring was recommended in 19 (59%) infants including 4 infants whose ALTE required CPR. Clinical and 12 hour pneumogram characteristics of infants with and without monitors are shown. Table

11β-Hydroxysteroid dehydrogenase type 1 is a homodimer where the carboxyl terminus of one subunit covers the active site of the dimer partner. Based on the crystal structure with CHAPS, the carboxyl terminal tyrosine 280 (Y280) has been postulated to interact with the substrate/inhibitor at the binding pocket of the dimer partner. However, the co-crystal structure with carbenoxolone argues against this role. To clarify and reconcile these findings, here we report our mutagenesis data and demonstrate that Y280 is not involved in substrate binding but rather plays a selective role in inhibitor binding. The involvement of Y280 in inhibitor binding depends on the inhibitor chemical structure. While Y280 is not involved in the binding of carbenoxolone, it is critical for the binding of glycyrrhetinic acid.

Recent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism. Structural analysis of the SARS-CoV S and MERS-CoV S glycoproteins in complex with neutralizing antibodies from human survivors sheds light into the mechanisms of membrane fusion and neutralization