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Cyclic oligoadenylates (cOAs) are small second messenger molecules produced by the type III CRISPR-Cas system as part of the prokaryotic immune response. The role of cOAs is to allosterically activate downstream effector proteins that induce dormancy or cell death, and thus abort viral spread through the population. Interestingly, different type III systems have been reported to utilize different cOA stoichiometries (with 3 to 6 adenylate monophosphates). However, so far, their characterization has only been possible in bulk and with sophisticated equipment, while a portable assay with single-molecule resolution has been lacking. Here, we demonstrate the label-free detection of single cOA molecules using a simple protein nanopore assay. It sensitively identifies the stoichiometry of individual cOA molecules and their mixtures from synthetic and enzymatic origin. To achieve this, we trained a convolutional neural network (CNN) and validated it with a series of experiments on mono- and polydisperse cOA samples. Ultimately, we determined the stoichiometric composition of cOAs produced enzymatically by the CRISPR type III-A and III-B variants of Thermus thermophilusand confirmed the results by liquid chromatography–mass spectroscopy (LC-MS). Interestingly, both variants produce cOAs of nearly identical composition (within experimental uncertainties), and we discuss the biological implications of this finding. The presented nanopore-CNN workflow with single cOA resolution can be adapted to many other signaling molecules (including eukaryotic ones), and it may be integrated into portable handheld devices with potential point-of-care applications.

The molecular apparatus behind biological photosynthesis retains its long-term functionality through enzymatic repair. However, bioinspired molecular devices designed for artificial photosynthesis, consisting of a photocentre, a bridging ligand and a catalytic centre, can become unstable and break down when their individual modules are structurally compromised, halting their overall functionality and operation. Here we report the active repair of such an artificial photosynthetic molecular device, leading to complete recovery of catalytic activity. We have identified the hydrogenation of the bridging ligand, which inhibits the light-driven electron transfer between the photocentre and catalytic centre, as the deactivation mechanism. As a means of repair, we used the light-driven generation of singlet oxygen, catalysed by the photocentre, to enable the oxidative dehydrogenation of the bridging unit, which leads to the restoration of photocatalytic hydrogen formation.

The isolation of biomolecules in a high vacuum enables experiments on fragile species in the absence of a perturbing environment. Since many molecular properties are influenced by local electric fields, here we seek to gain control over the number of charges on a biopolymer by photochemical uncaging. We present the design, modeling, and synthesis of photoactive molecular tags, their labeling to peptides and proteins as well as their photochemical validation in solution and in the gas phase. The tailored tags can be selectively cleaved off at a well-defined time and without the need for any external charge-transferring agents. The energy of a single or two green photons can already trigger the process, and it is soft enough to ensure the integrity of the released biomolecular cargo. We exploit differences in the cleavage pathways in solution and in vacuum and observe a surprising robustness in upscaling the approach from a model system to genuine proteins. The interaction wavelength of 532 nm is compatible with various biomolecular entities, such as oligonucleotides or oligosaccharides.

Supplemental Digital Content is available in the text.Extraction of data from the electronic medical record is becoming increasingly important for quality improvement initiatives such as the American Society of Anesthesiologists Perioperative Surgical Home. To meet this need, the authors have built a robust and scalable data mart based on their implementation of EPIC containing data from across the perioperative period. The data mart is structured in such a way so as to first simplify the overall EPIC reporting structure into a series of Base Tables and then create several Reporting Schemas each around a specific concept (operating room cases, obstetrics, hospital admission, etc.), which contain all of the data required for reporting on various metrics. This structure allows centralized definitions with simplified reporting by a large number of individuals who access only the Reporting Schemas. In creating the database, the authors were able to significantly reduce the number of required table identifiers from >10 to 3, as well as to correct errors in linkages affecting up to 18.4% of cases. In addition, the data mart greatly simplified the code required to extract data, making the data accessible to individuals who lacked a strong coding background. Overall, this infrastructure represents a scalable way to successfully report on perioperative EPIC data while standardizing the definitions and improving access for end users.

Good’s syndrome is a relatively rare immunodeficiency condition that presents in the fourth or fifth decade of life and is defined by hypogammaglobulinemia in the setting of a thymoma. The humoral defect may be severe enough to cause an absence in B cells, with a consequent recurrence of sinopulmonary disease, chronic non-infectious diarrhea and opportunistic infections. The prognosis in patients with Good’s syndrome appears to be worse than in those with X-linked agammaglobulinemia (XLA) and common variable immune deficiency (CVID). There have only been three cases of Good’s syndrome associated with mycobacterium, and only one case with a cavitary lesion in the lungs. We present here a unique case of Good’s syndrome with a non-mycobacterial cavitary lesion.

Background:All usual daily doses of plain aspirin inhibit thromboxane B2 (TXB2) as well as prostaglandin E2 (PGE2). The role of 81-mg enteric-coated aspirin (ECA) is controversial. Method:In a randomized, double-blind trial, 37 patients (25 men and 12 women) with chronic stable coronary disease taking ECA 81 mg at baseline were assigned to plain aspirin 81, 162.5, 325, 650 or 1300 mg daily for 12 weeks. At baseline and 12 weeks, blood was tested for TXB2and PGE2. Results:All doses of plain aspirin produced virtually identical reductions in TXB2and PGE2. For all doses combined, the mean ratio of the 12-week to baseline value was 0.03 for TXB2(p < 0.001) and 0.63 for PGE2(p < 0.001). Conclusion:These data indicate that ECA 81 mg daily does not inhibit TXB2and PGE2, markers of acute and systemic responses to aspirin. Randomized trials designed a priorito test this hypothesis are necessary.

Abstract: We conducted a trenching experiment in a mountain forest in order to assess the contribution of the autotrophic respiration to total soil respiration and evaluate trenching as a technique to achieve it. We hypothesised that the trenching experiment would alter both microbial biomass and microbial community structure and that fine roots (less than 2 mm diameter) would be decomposed within one growing season. Soil CO2 efflux was measured roughly biweekly over two growing seasons. Root presence and morphology parameters, as well as the soil microbial community were measured prior to trenching, 5 and 15 months after trenching. The trenched plots emitted about 20 and 30% less CO2 than the control plots in the first and second growing season, respectively. Roots died in trenched plots, but root decay was slow. After 5 and 15 months, fine root biomass was decreased by 9% (not statistically different) and 30%, (statistically different) respectively. When we corrected for the additional trenched-plot CO2 efflux due to fine root decomposition, the autotrophic soil respiration rose to ~26% of the total soil respiration for the first growing season, and to ~44% for the second growing season. Soil microbial biomass and community structure was not altered by the end of the second growing season. We conclude that trenching can give accurate estimates of the autotrophic and heterotrophic components of soil respiration, if methodological side effects are accounted for, only.

Die Technologie räumlicher elektronischer Schaltungsträger (Molded Interconnect Devices – MID) bietet mit der hohen Gestaltungsfreiheit, Umweltverträglichkeit sowie dem umfangreichen Rationalisierungspotenzial vielfältige Möglichkeiten zur Realisierung innovativer Produkte und effizienter Produktionsprozesse. Charakteristisch ist die direkte stoffliche Integration von Mechanik und Elektronik auf einem Bauteil. Die Umsetzung innovativer Produktideen erfordert umfangreiches Entwicklungs-Know-how und die entsprechende Rechnerunterstützung. Dabei genügt der Einsatz klassischer Konstruktionssoftware für Mechanik und Schaltungsentwurf nicht den Anforderungen von MID. Neben einer dreidimensionalen Entwicklungsumgebung für die Darstellung des räumlichen Schaltungsträgers ist die Integration von Konstruktionsbefehlen aus verschiedenen domänenspezifischen Software-Paketen notwendig. Bereits in der frühen Konstruktionsphase müssen die spezifischen Eigenschaften der MID-Technologie während des Fertigungsprozesses berücksichtigt werden. Da MID keine Standardlösungen wie elektronische Baugruppen in planarer Leiterplattentechnik sind, kommt der Kenntnis aller Prozessschritte (elektronisches und mechanisches Design, Werkstoffauswahl, Spritzguss, Metallisierung und AVT) hohe Bedeutung zu.

ZusammenfassungIn der Prozess- und Qualitätskontrolle spielen die Erkennung und Einstufung von Oberflächenstrukturen und Defekten eine immer wichtigere Rolle. Mit der Methode des Makyoh-Imaging (“Ma-Kyoh”, jap. für magischer Spiegel) steht ein leistungsstarkes, hochempfindliches Verfahren zur Verfügung, bei dem lokale konvexe und konkave Strukturen unter kollimierter Beleuchtung die Intensitätsverteilung im Reflex beeinflussen. Anhand einer am Fraunhofer IOF realisierten Messanordnung wird gezeigt, dass über den Reflex sowohl zuverlässig als auch schnell qualitative und quantitative Rückschlüsse auf Oberflächenstrukturen und Defekte möglich sind. Ergebnisse untersuchter Feinstrukturen auf Wafern und polierten Glasoberflächen werden vorgestellt.

The purpose of this study was to identify and validate novel serological protein biomarkers of human colorectal cancer (CRC). Proteins from matched CRC and adjacent normal tissue samples were resolved by two-dimensional gel electrophoresis. From each gel all spots were excised, and enveloped proteins were identified by MS. By comparison of the resulting protein profiles, dysregulated proteins can be identified. A list of all identified proteins and validation of five exemplarily selected proteins, elevated in CRC was reported previously (Roessler, M., Rollinger, W., Palme, S., Hagmann, M. L., Berndt, P., Engel, A. M., Schneidinger, B., Pfeffer, M., Andres, H., Karl, J., Bodenmuller, H., Ruschoff, J., Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W., and Tacke, M. (2005) Identification of nicotinamide N-methyltransferase as a novel serum tumor marker for colorectal cancer. Clin. Cancer Res.11, 6550–6557). Here we describe identification and initial validation of another potential marker protein for CRC. Comparison of tissue protein profiles revealed strong elevation of proteasome activator complex subunit 3 (PSME3) expression in CRC tissue. This dysregulation was not detectable based on the spot pattern. The PSME3-containing spot on tumor gels showed no visible difference to the corresponding spot on matched control gels. MS analysis revealed the presence of two proteins, PSME3 and annexin 4 (ANXA4) in one and the same spot on tumor gels, whereas the matched spot contained only one protein, ANXA4, on control gels. Therefore, dysregulation of PSME3 was masked by ANXA4 and could only be recognized by MS-based analysis but not by image analysis. To validate this finding, antibody to PSME3 was developed, and up-regulation in CRC was confirmed by Western blot analysis and immunohistochemistry. Finally by developing a highly sensitive immunoassay, PSME3 could be detected in human sera and was significantly elevated in CRC patients compared with healthy donors and patients with benign bowel disease. We propose that PSME3 be considered a novel serum tumor marker for CRC that may have significance in the detection and in the management of patients with this disease. Further studies are needed to fully assess the potential clinical value of this marker candidate.

Microbial biomass, respiratory activity, and in-situ substrate decomposition were studied in soils from humid temperate forest ecosystems in SW Germany. The sites cover a wide range of abiotic soil and climatic properties. Microbial biomass and respiration were related to both soil dry mass in individual horizons and to the soil volume in the top 25 cm. Soil microbial properties covered the following ranges: soil microbial biomass: 20 µg C g–1–8.3 mg C g–1 and 14–249 g C m–2, respectively; microbial C–to–total organic C ratio: 0.1%–3.6%; soil respiration: 109–963 mg CO2-C m–2 h–1; metabolic quotient (qCO2): 1.4–14.7 mg C (g Cmic)–1 h–1; daily in-situ substrate decomposition rate: 0.17%–2.3%. The main abiotic properties affecting concentrations of microbial biomass differed between forest-floor/organic horizons and mineral horizons. Whereas microbial biomass decreased with increasing soil moisture and altitude in the forest-floor/organic horizons, it increased with increasing Ntot content and pH value in the mineral horizons. Quantities of microbial biomass in forest soils appear to be mainly controlled by the quality of the soil organic matter (SOM), i.e., by its C : N ratio, the quantity of Ntot, the soil pH, and also showed an optimum relationship with increasing soil moisture conditions. The ratio of Cmic to Corg was a good indicator of SOM quality. The quality of the SOM (C : N ratio) and soil pH appear to be crucial for the incorporation of C into microbial tissue. The data and functional relations between microbial and abiotic variables from this study provide the basis for a valuation scheme for the function of soils to serve as a habitat for microorganisms.

Abstract. Optimisation of the separation of a synthetic drug mixture by HPLC is performed by changing both continuous variables, i.e. mobile phase composition and temperature, and categorical variables, here the stationary phase. The retention of solutes is described on the basis of a general linear model in which the different columns are modelled by indicator variables. From the solute-specific retention models the global separation optimum is evaluated on the basis of multidimensional window diagrams using relative retentions of all peak pairs as the figure-of-merit.

Important gas exchange characteristics of C4 plants depend on the properties of phophoenolpyruvate carboxylase (PEPC), the enzyme catalysing the primary fixation of CO2 during C4 photosynthesis. In this study, the relationship between intracellular resistance for CO2 fixation (ri) at high photosynthetically active photon flux densities (PPFD) and maximum PEPC activity in vitro (Vpm) was examined in leaves of Zea mays L. The analysis allowed the estimation of the Michaelis constant Kpof the enzyme for CO2 (or the equivalent number for bicarbonate) in vivo. At low PPFD (below 100 μmol m-2 s-1) the initial slopes of the curves describing net CO2 uptake rate A as a function of intercellular CO2 concentration ciincreased with increasing PPFD. The increase (i. e. a decrease in ri) was interpreted as due to a reversible activation of PEPC by light. Including this assumption into a model of C4 photosynthesis enabled us to reproduce A(ci) response curves measured at low levels of PPFD. Fitting the model to experimental data resulted in values for KI, the PPFD at which PEPC reaches half of its full activation, of about 200 μmol m-2 s-1. Similar results were derived from the dependence of rion PPFD. The analysis of the relationships between riand Vpmand between riand PPFD, as well as fitting of the model to gas exchange data all gave rise to estimates for the resistance for CO2 transfer within mesophyll cells that are comparable with those known from C3 plants.

An improved method for producing fiber tips for scanning near-field optical microscopy is presented. The improvement consists of chemically etching quartz optical fibers through their acrylate jacket. This new method is compared with the previous one in which bare fibers were etched. With the new process the meniscus formed by the acid along the fiber does not move during etching, leading to a much smoother surface of the tip cone. Subsequent metallization is thus improved, resulting in better coverage of the tip with an aluminum opaque layer. Our results show that leakage can be avoided along the cone, and light transmission through the tip is spatially limited to an optical aperture of a 100-nm dimension.

Leaf conductance gLis strongly influenced by environmental factors like CO2, irradiance and air humidity. According to Ball et al. (1987), gLis correlated with an index calculated as the product of net CO2exchange rate A and ambient water vapour concentration Wa, divided by ambient CO2concentration ca. However, this empirical model does not apply to high values of gLobserved at cabelow CO2compensation concentration Γ. Therefore, we applied modified indices in which A is replaced by estimates for the rate of carboxylation. Such estimates, P1and P2, were determined by adding to A the quotient of Γ and the sum of gas phase resistance rgand intracellular resistance for CO2exchange ri, P1= A+Γ/(rg+ ri), or the quotient of Γ and ri, P2= A + Γ/ri. If P2is chosen, cain the Ball index has to be replaced by the intercellular CO2concentration ci. By using the modified indices P1·Wa/caand P2·Wa/ci, we analysed data from the C3species Nicotiana tabacum and Nicotiana plumbaginifolia, the C3–C4intermediate species Diplotaxis tenuifolia, and the C4species Zea mays. The data were collected at widely varying levels of irradiance and CO2concentration. For all species uniform relationships between gLand the new indices were found for the whole range of CO2concentrations below and above Γ. Correlations between gLand P1·Wa/cawere closer than those between gLand P2·Wa/cibecause P1/caimplicitly contains gL. Highly significant correlations were also obtained for the relationships between gLand the ratios P1/caand P2/ci.

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