Flexible electrochemical aptasensor for cortisol detection in human sweat.

This communication demonstrates an electrochemical DNA aptasensor for the detection of cortisol in human sweat. The aptasensor was fabricated via layer-by-layer assembly on stretchable polydimethylsiloxane (PDMS) coated with conductive nanoporous carbon nanotube-cellulose nanocrystals (CNC/CNT) film using a linker to a cortisol specific DNA aptamer. The flexible cortisol aptasensor had a dynamic range of 2.5-35 ng mL-1. The aptasensor precision was determined to be 2.7% relative standard deviation (%RSD) across the concentration dynamic range. The aptasensor was determined to have a limit of detection (LOD) of ∼ 1.8 ng mL-1. The aptasensor was demonstrated to have high selectivity to cortisol and was unresponsive to interfering species including glucose, sodium lactate, and ß-estradiol. The aptasensor was successfully evaluated for the detection of cortisol in human sweat indicative of its high specificity.

Using anticipated learning outcomes for backward design of a molecular cell biology Course-based Undergraduate Research Experience.

Anticipated learning outcomes (LOs) were defined and used for the backward design of a Course-based Undergraduate Research Experience (CURE). These LOs reflect the inquiry-based nature of CUREs and capture key knowledge and skills inherent to scientific practice and essential in research. The LOs were used to plan a formative and summative assessment strategy to support and evaluate student achievement. A research question was identified that aligned with the learning goals of the course, provided an opportunity for discovery and iteration, and introduced a variety of molecular, cellular, and biochemical techniques. The course is offered to students in the final year of their degree and delivered over a 12-week period with two 3-hr labs each week. These LOs, and the rigorous assessment strategy used to support them, could be adapted to different projects. Likewise, the laboratory exercises are presented as a series of modules highlighting opportunities for adaptation to a variety of schedules.

Metasurface Broadband Solar Absorber.

We demonstrate a broadband, polarization independent, wide-angle absorber based on a metallic metasurface architecture, which accomplishes greater than 90% absorptance in the visible and near-infrared range of the solar spectrum, and exhibits low absorptivity (emissivity) at mid- and far-infrared wavelengths. The complex unit cell of the metasurface solar absorber consists of eight pairs of gold nano-resonators that are separated from a gold ground plane by a thin silicon dioxide spacer. Our experimental measurements reveal high-performance absorption over a wide range of incidence angles for both s- and p-polarizations. We also investigate numerically the frequency-dependent field and current distributions to elucidate how the absorption occurs within the metasurface structure.

Structural basis for the phosphatase activity of polynucleotide kinase/phosphatase on single- and double-stranded DNA substrates.

Polynucleotide kinase/phosphatase (PNKP) is a critical mammalian DNA repair enzyme that generates 5'-phosphate and 3'-hydroxyl groups at damaged DNA termini that are required for subsequent processing by DNA ligases and polymerases. The PNKP phosphatase domain recognizes 3'-phosphate termini within DNA nicks, gaps, or at double- or single-strand breaks. Here we present a mechanistic rationale for the recognition of damaged DNA termini by the PNKP phosphatase domain. The crystal structures of PNKP bound to single-stranded DNA substrates reveals a narrow active site cleft that accommodates a single-stranded substrate in a sequence-independent manner. Biochemical studies suggest that the terminal base pairs of double-stranded substrates near the 3'-phosphate are destabilized by PNKP to allow substrate access to the active site. A positively charged surface distinct from the active site specifically facilitates interactions with double-stranded substrates, providing a complex DNA binding surface that enables the recognition of diverse substrates.

Molecular characterization of Plasmodium falciparum putative polynucleotide kinase/phosphatase.

Polynucleotide kinase/phosphatase (PNKP) is a bifunctional enzyme that can phosphorylate the 5'-OH termini and dephosphorylate the 3'-phosphate termini of DNA. It is a DNA repair enzyme involved in the processing of strand break termini, which permits subsequent repair proteins to replace missing nucleotides and rejoin broken strands. Little is known about DNA repair in Plasmodium falciparum, including the roles of PNKP in repairing parasite DNA. We identified a P. falciparum gene encoding a protein with 24% homology to human PNKP and thus suggestive of a putative PNKP. In this study, the PNKP gene of P. falciparum strain K1 (PfPNKP) was successfully cloned and expressed in E. coli as a GST-PfPNKP recombinant protein. MALDI-TOF/TOF analysis of the protein confirmed the identity of PfPNKP. Assays for enzymatic activity were carried out with a variety of single- and double-stranded substrates. Although 3'-phosphatase activity was detected, PfPNKP was observed to dephosphorylate single-stranded substrates or double-stranded substrates with a short 3'-single-stranded overhang, but not double-stranded substrates that mimicked single-strand breaks. We hypothesize that unlike human PNKP, PfPNKP may not be involved in single-strand break repair, since alternative terminal processing mechanisms can substitute for PfPNKP, and that PfPNKP DNA repair actions may be confined to overhanging termini of double-strand breaks.

[Non-contact monitoring of heart and lung activity using magnetic induction measurement in a neonatal animal model]. / Kontaktlose Uberwachung von Atemtätigkeit und Herzaktion mittels magnetischer Bioimpedanzmessung in einem neonatalen Tiermodell.

BACKGROUND: Magnetic induction measurement (MIM) allows the identification of resistance in biologic tissues by alternating magnetic fields. These occur when well-conducting (blood) and poor-conducting matter (air) is moved through the thorax during heart and lung activity. As a result, allocation of the resistance changes and the total resistance of the thorax is shifted. By using coils, these changes can be registered in a non-contact manner and recorded. To date, this measuring principle was employed only in adult volunteers or in full-grown pigs. A neonatal animal model has not yet been described. The aim of this study was to test the hypothesis that non-contact monitoring of heart and lung activity using MIM in a porcine newborn piglet model can be applied in order to evaluate neonatal disorders of heart and lung activity in the future. MATERIALS AND METHODS: By using five coils (three measurement and two excitation coils), placed at the bottom of an experimental incubator, magnetic induction changes, depending on the heart and lung activity in 16 analgosedated piglets, were simultaneously measured and compared with pulse oximetry and airflow detection (flow resistance and pressure differential sensor) as reference signals. In addition, spontaneous breathing, including apnea, CPAP (continuous positive airway pressure to prevent end-expiratory alveolar collapse, flow 8 l/min; pressure 5 cm H(2)O), mechanical ventilation (inspiratory pressure 14 cm H(2)O; frequency 40/min) and high frequency oxygenation ventilation (HFOV, ventilation method in lung failure) (frequency 10 Hz, mean pressure 10 cm H(2)O, amplitude 1.5) were performed. Lung activity with MIM compared with the reference signal was estimated with a detection rate (%) of "correct registered lung activity". To quantify the analogy between MIM and reference signal for heart activity, the concordance correlation coefficient after Lin (95% confidence interval) and the Bland-Altman plot were calculated. RESULTS AND DISCUSSION: The detection rate for breathing [%] of MIM compared with the reference signal under CPAP was 88% [95% CI: (87.1%; 88.5%)], mechanical ventilation 91% [95% CI: (90.3%; 91.2%)] and under HFOV 95% [95% CI: (94.7%; 94.9%)]. For heart activity, during apnea the difference between MIM and reference signal was 1.1 bpm (+/-11.3 SD) in apnea and during HFOV 5.3 bpm (+/-26.4 SD). Under spontaneous breathing it was not possible to achieve a correlation. Owing to interference problems, registration of heart activity with MIM during simultaneous breathing activity (CPAP, conventional mechanical ventilation, HFOV) was insufficient. CONCLUSION: Non-contact monitoring of lung activity using MIM in a neonatal piglet model is possible under specific conditions. These results might be a basis for the development of non-invasive parameters in neonatology. It also provides the possibility of obtaining more information about the characteristics of lung activity of the newborn.

Multichannel simultaneous magnetic induction measurement system (MUSIMITOS).

Non-contact heart and lung activity monitoring would be a desirable supplement to conventional monitoring techniques. Based on the potential of non-contact magnetic induction measurements, requirements for an adequate monitoring system were estimated. This formed the basis for the development of the presented extendable multichannel simultaneous magnetic induction measurement system (MUSIMITOS). Special focus was given to the dynamic behaviour and simultaneous multichannel measurements, so that the system allows for up to 14 receiver coils working simultaneously at 6 excitation frequencies. Moreover, a real-time software concept for online signal processing visualization in combination with a fast software demodulation is presented. Finally, first steps towards a clinical application are pointed out and technical performance as well as first in vivo measurements are presented. This paper covers some aspects previously presented in Steffen and Leonhardt (2007 Proc. 13th Int. Conf. on Electrical Bioimpedance and the 8th Conf. on Electrical Impedance Tomography, Graz 2007).

The nuclear PP1 interacting protein ZAP3 (ZAP) is a putative nucleoside kinase that complexes with SAM68, CIA, NF110/45, and HNRNP-G.

The targeting of protein kinases and phosphatases is fundamental to their roles as cellular regulators. The type one serine/threonine protein phosphatase (PP1) is enriched in the nucleus, yet few nuclear PP1 targeting subunits have been described and characterized. Here we show that the human protein, ZAP3 (also known as ZAP), is localized to the nucleus, that it is expressed in all mammalian tissues examined, and docks to PP1 through an RVRW motif located in its highly conserved carboxy-terminus. Proteomic analysis of a ZAP3 complex revealed that in addition to binding PP1, ZAP3 complexes with CIA (or nuclear receptor co-activator 5) and the RNA binding proteins hnRNP-G, SAM68 and NF110/45, but loses affinity for SAM68 and hnRNP-G upon digestion of endogenous nucleic acid. Bioinformatics has revealed that the conserved carboxy-terminus is orthologous to T4- and mammalian polynucleotide kinases with residues necessary for kinase activity maintained throughout evolution. Furthermore, the substrate binding pocket of uridine-cytidine kinase (or uridine kinase) has localized sequence similarity with ZAP3, suggesting uridine or cytidine as possible ZAP3 substrates. Most polynucleotide kinases have a phosphohydrolase domain in conjunction with their kinase domain. In ZAP3, although this domain is present, it now appears degenerate and functions to bind PP1 through an RVRW docking site located within the domain.

Structural basis for phosphorylation-dependent signaling in the DNA-damage response.

The response of eukaryotic cells to DNA damage requires a multitude of protein-protein interactions that mediate the ordered repair of the damage and the arrest of the cell cycle until repair is complete. Two conserved protein modules, BRCT and forkhead-associated (FHA) domains, play key roles in the DNA-damage response as recognition elements for nuclear Ser/Thr phosphorylation induced by DNA-damage-responsive kinases. BRCT domains, first identified at the C-terminus of BRCA1, often occur as multiple tandem repeats of individual BRCT modules. Our recent structural and functional work has revealed how BRCT repeats recognize phosphoserine protein targets. It has also revealed a secondary binding pocket at the interface between tandem repeats, which recognizes the amino-acid 3 residues C-terminal to the phosphoserine. We have also studied the molecular function of the FHA domain of the DNA repair enzyme, polynucleotide kinase (PNK). This domain interacts with threonine-phosphorylated XRCC1 and XRCC4, proteins responsible for the recruitment of PNK to sites of DNA-strand-break repair. Our studies have revealed a flexible mode of recognition that allows PNK to interact with numerous negatively charged substrates.

The molecular architecture of the mammalian DNA repair enzyme, polynucleotide kinase.

Mammalian polynucleotide kinase (PNK) is a key component of both the base excision repair (BER) and nonhomologous end-joining (NHEJ) DNA repair pathways. PNK acts as a 5'-kinase/3'-phosphatase to create 5'-phosphate/3'-hydroxyl termini, which are a necessary prerequisite for ligation during repair. PNK is recruited to repair complexes through interactions between its N-terminal FHA domain and phosphorylated components of either pathway. Here, we describe the crystal structure of intact mammalian PNK and a structure of the PNK FHA bound to a cognate phosphopeptide. The kinase domain has a broad substrate binding pocket, which preferentially recognizes double-stranded substrates with recessed 5' termini. In contrast, the phosphatase domain efficiently dephosphorylates single-stranded 3'-phospho termini as well as double-stranded substrates. The FHA domain is linked to the kinase/phosphatase catalytic domain by a flexible tether, and it exhibits a mode of target selection based on electrostatic complementarity between the binding surface and the phosphothreonine peptide.

Structural insights into the activation of P. vivax plasmepsin.

The malarial aspartic proteinases (plasmepsins) have been discovered in several species of Plasmodium, including all four of the human malarial pathogens. In P.falciparum, plasmepsins I, II, IV and HAP have been directly implicated in hemoglobin degradation during malaria infection, and are now considered targets for anti-malarial drug design. The plasmepsins are produced from inactive zymogens, proplasmepsins, having unusually long N-terminal prosegments of more than 120 amino acids. Structural and biochemical evidence suggests that the conversion process of proplasmepsins to plasmepsins differs substantially from the gastric and plant aspartic proteinases. Instead of blocking substrate access to a pre-formed active site, the prosegment enforces a conformation in which proplasmepsin cannot form a functional active site. We have determined crystal structures of plasmepsin and proplasmepsin from P.vivax. The three-dimensional structure of P.vivax plasmepsin is typical of the monomeric aspartic proteinases, and the structure of P.vivax proplasmepsin is similar to that of P.falciparum proplasmepsin II. A dramatic refolding of the mature N terminus and a large (18 degrees ) reorientation of the N-domain between P.vivax proplasmepsin and plasmepsin results in a severe distortion of the active site region of the zymogen relative to that of the mature enzyme. The present structures confirm that the mode of inactivation observed originally in P.falciparum proplasmepsin II, i.e. an incompletely formed active site, is a true structural feature and likely represents the general mode of inactivation of the related proplasmepsins.