HS‐SPME‐GC‐FID method for detection and quantification of Bacillus cereus ATCC 10702 mediated 2‐acetyl‐1‐pyrroline

A rapid micro‐scale solid‐phase micro‐extraction (SPME) procedure coupled with gas‐chromatography with flame ionized detector (GC‐FID) was used to extract parts per billion levels of a principle basmati aroma compound “2‐acetyl‐1‐pyrroline” (2‐AP) from bacterial samples. In present investigation, optimization parameters of bacterial incubation period, sample weight, pre‐incubation time, adsorption time, and temperature, precursors and their concentrations has been studied. In the optimized conditions, detection of 2‐AP produced by Bacillus cereus ATCC10702 using only 0.5 g of sample volume was 85 μg/kg. Along with 2‐AP, 15 other compounds produced by B. cereus were also reported out of which 14 were reported for the first time consisting mainly of (E)?2‐hexenal, pentadecanal, 4‐hydroxy‐2‐butanone, n‐hexanal, 2–6‐nonadienal, 3‐methoxy‐2(5H) furanone and 2‐acetyl‐1‐pyridine and octanal. High recovery of 2‐AP (87 %) from very less amount of B. cereus samples was observed. The method is reproducible fast and can be used for detection of 2‐AP production by B. cereus. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1356–1363, 2014     

Hydrogel‐based three‐dimensional cell culture for organ‐on‐a‐chip applications

Recent studies have reported that three‐dimensionally cultured cells have more physiologically relevant functions than two‐dimensionally cultured cells. Cells are three‐dimensionally surrounded by the extracellular matrix (ECM) in complex in vivo microenvironments and interact with the ECM and neighboring cells. Therefore, replicating the ECM environment is key to the successful cell culture models. Various natural and synthetic hydrogels have been used to mimic ECM environments based on their physical, chemical, and biological characteristics, such as biocompatibility, biodegradability, and biochemical functional groups. Because of these characteristics, hydrogels have been combined with microtechnologies and used in organ‐on‐a‐chip applications to more closely recapitulate the in vivo microenvironment. Therefore, appropriate hydrogels should be selected depending on the cell types and applications. The porosity of the selected hydrogel should be controlled to facilitate the movement of nutrients and oxygen. In this review, we describe various types of hydrogels, external stimulation‐based gelation of hydrogels, and control of their porosity. Then, we introduce applications of hydrogels for organ‐on‐a‐chip. Last, we also discuss the challenges of hydrogel‐based three‐dimensional cell culture techniques and propose future directions. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:580–589, 2017     

Application of NMR spectroscopy for assignment of the absolute configuration of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one

In order to assign the absolute configurations of 8‐tert‐butyl‐2‐hydroxy‐7‐methoxy‐8‐methyl‐9‐oxa‐6‐azaspiro[4.5]dec‐6‐en‐10‐one ( 2a , 2b ), their esters ( 5a , 5b , 5c , 5d ) with (R)‐ or (S)‐2‐methoxyphenylacetic acid ( 4a , 4b ) have been synthesized. The absolute configurations of these compounds have been determined on the basis of NOESY correlations between the protons of the tert‐butyl group and the cyclopentane fragment of the molecules. The crucial part of this analysis was assignment of the absolute configuration at C‐5. Additionally, by calculation of the chemical shift anisotropy, δ^RS, for the relevant protons, it was also possible to confirm the absolute configurations at the C‐2 centres of compounds 2a , 2b and 5a , 5b , 5c , 5d . Chirality, 25:422–426, 2013.© 2013 Wiley Periodicals, Inc.     

Validated spectrofluorimetric method for the determination of carbamazepine in pharmaceutical dosage forms after reaction with 4‐chloro‐7‐‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl)

A sensitive and simple spectrofluorimetric method has been developed and validated for the determination of the anti‐epileptic drug carbamazepine (CBZ) in its dosage forms. The method was based on a nucleophilic substitution reaction of CBZ with 4‐chloro‐7‐nitrobenzo‐2‐ oxa‐1,3‐diazole (NBD‐Cl) in borate buffer (pH 9) to form a highly fluorescent derivative that was measured at 530 nm after excitation at 460 nm. Factors affecting the formation of the reaction product were studied and optimized, and the reaction mechanism was postulated. The fluorescence–concentration plot is rectilinear over the range of 0.6–8 µg/mL with limit of detection of 0.06 µg/mL and limit of quantitation of 0.19 µg/mL. The method was applied to the analysis of commercial tablets and the results were in good agreement with those obtained using the reference method. Validation of the analytical procedures was evaluated according to ICH guidelines. Copyright © 2015 John Wiley & Sons, Ltd.     

Establishment and clinical application of a highly sensitive enzyme immunoassay for determination of N‐acetyl‐seryl‐aspartyl‐lysyl‐proline

N‐acetyl‐seryl‐aspartyl‐lysyl‐proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is hydrolyzed by the N‐terminal active site of angiotensin converting enzyme and partially eliminated in urine, its plasma level is a result of a complex balance between its production, hydrolysis by ACE, and renal elimination. In this study, we attempted to establish an enzyme immunoassay (EIA) for quantifying AcSDKP‐like immunoreactive substance (IS), which is applicable for monitoring plasma AcSDKP levels in healthy subjects and patients with chronic renal failure. Using β‐ d ‐galactosidase‐labeled Gly‐γAbu‐SDKP as a marker antigen, an anti‐rabbit IgG‐coated immunoplate as a bound/free separator and 4‐methylumbelliferyl‐β‐ d ‐galactopyranoside as a fluorogenic substrate, a highly sensitive and specific EIA was developed for the quantification of AcSDKP‐IS in human plasma. The lower limit of quantification was 0.32 fmol/well, and the sharp inhibition competitive EIA calibration curve obtained was linear between 8.0 and 513 fmol/ml. This EIA was so sensitive that only 10 µl plasma sample was required for a single assay. The coefficients of variation (reproducibility) for human plasma concentrations of 0.2 and 2.1 pmol/ml were 7.2 and 7.7%, respectively, for inter‐assay and 13.3 and 7.8% for intra‐assay comparisons. Plasma AcSDKP‐IS level was significantly higher in patients with chronic renal failure (0.92 ± 0.39 pmol/ml) compared with healthy subjects (0.29 ± 0.07 pmol/ml). These results suggest that our EIA may be useful to evaluate plasma AcSDKP level as a biomarker in various patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

The Mycobacterium tuberculosis complex has a pathway for the biosynthesis of 4‐formamido‐4,6‐dideoxy‐d‐glucose

Recent studies have demonstrated that the O‐antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N‐formylated sugars (3‐formamido‐3,6‐dideoxy‐d ‐glucose or 4‐formamido‐4,6‐dideoxy‐d ‐glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6‐dehydratase, a pyridoxal 5'‐phosphate or PLP‐dependent aminotransferase, and an N‐formyltransferase. To date, there have been no published reports of N‐formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N‐formyltransferase. Given that M. tuberculosis produces l ‐rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6‐dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N‐formylated sugar in M. tuberculosis, namely a PLP‐dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.     

Low‐Cost N,N′‐Bicarbazole‐Based Dopant‐Free Hole‐Transporting Materials for Large‐Area Perovskite Solar Cells

Despite the recent unprecedented development of efficient dopant‐free hole transporting materials (HTMs) for high‐performance perovskite solar cells (PSCs) on small‐area devices (≤0.1 cm²), low‐cost dopant‐free HTMs for large‐area PSCs (≥1 cm²) with high power conversion efficiencies (PCEs) have rarely been reported. Herein, two novel HTMs, 3,3′,6,6′ (or 2,2′,7,7′)‐tetrakis(N,N′‐di‐p‐methoxyphenylamine)‐N,N′‐bicarbazole (3,6 BCz‐OMeTAD or 2,7 BCz‐OMeTAD), are synthesized via an extremely simple route from very cheap raw materials. Owing to their excellent film‐forming abilities and matching energy levels, 3,6 BCz‐OMeTAD and 2,7 BCz‐OMeTAD can be successfully employed as a perfect ultrathin (≈30 nm) hole transporting layer in large‐area PSCs up to 1 cm². The 3,6 BCz‐OMeTAD and 2,7 BCz‐OMeTAD based large‐area PSCs show highest PCEs up to 17.0% and 17.6%, respectively. More importantly, high performance large‐area PSCs based on 2,7 BCz‐OMeTAD retain 90% of the initial efficiency after 2000 h storage in an ambient environment without encapsulation.     

High‐Voltage Oxygen‐Redox‐Based Cathode for Rechargeable Sodium‐Ion Batteries

Recently, anionic‐redox‐based materials have shown promising electrochemical performance as cathode materials for sodium‐ion batteries. However, one of the limiting factors in the development of oxygen‐redox‐based electrodes is their low operating voltage. In this study, the operating voltage of oxygen‐redox‐based electrodes is raised by incorporating nickel into P2‐type Na_2/3[Zn_0.3Mn_0.7]O₂ in such a way that the zinc is partially substituted by nickel. As designed, the resulting P2‐type Na_2/3[(Ni_0.5Zn_0.5)_0.3Mn_0.7]O₂ electrode exhibits an average operating voltage of 3.5 V and retains 95% of its initial capacity after 200 cycles in the voltage range of 2.3–4.6 V at 0.1C (26 mA g^?1). Operando X‐ray diffraction analysis reveals the reversible phase transition: P2 to OP4 phase on charge and recovery to the P2 phase on discharge. Moreover, ex situ X‐ray absorption near edge structure and X‐ray photoelectron spectroscopy studies reveal that the capacity is generated by the combination of Ni²⁺/Ni⁴⁺ and O^2?/O^1? redox pairs, which is supported by first‐principles calculations. It is thought that this kind of high voltage redox species combined with oxygen redox could be an interesting approach to further increase energy density of cathode materials for not only sodium‐based rechargeable batteries, but other alkali‐ion battery systems.     

Thieno[3,4‐c]Pyrrole‐4,6‐Dione‐Based Polymer Acceptors for High Open‐Circuit Voltage All‐Polymer Solar Cells

While polymer acceptors are promising fullerene alternatives in the fabrication of efficient bulk heterojunction (BHJ) solar cells, the range of efficient material systems relevant to the “all‐polymer” BHJ concept remains narrow, and currently limits the perspectives to meet the 10% efficiency threshold in all‐polymer solar cells. This report examines two polymer acceptor analogs composed of thieno[3,4‐c ]pyrrole‐4,6‐dione (TPD) and 3,4‐difluorothiophene ([2F]T) motifs, and their BHJ solar cell performance pattern with a low‐bandgap polymer donor commonly used with fullerenes (PBDT‐TS1; taken as a model system). In this material set, the introduction of a third electron‐deficient motif, namely 2,1,3‐benzothiadiazole (BT), is shown to (i) significantly narrow the optical gap (E _opt) of the corresponding polymer (by ≈0.2 eV) and (ii) improve the electron mobility of the polymer by over two orders of magnitude in BHJ solar cells. In turn, the narrow‐gap P2TPDBT[2F]T analog (E _opt = 1.7 eV) used as fullerene alternative yields high open‐circuit voltages (V _OC) of ≈1.0 V, notable short‐circuit current values (J _SC) of ≈11.0 mA cm⁻², and power conversion efficiencies (PCEs) nearing 5% in all‐polymer BHJ solar cells. P2TPDBT[2F]T paves the way to a new, promising class of polymer acceptor candidates.     

Three‐dimensional ring‐oven washing technique for a paper‐based immunodevice

Washing is a standard step for enzyme‐linked immunosorbent assays (ELISA) performed on a paper‐based chip, in which nonspecific‐binding antibodies and antigens should be removed completely from the paper surface. In this study, a novel three‐dimensional (3D) washing strategy using a heating ring‐oven was carried out on a paper‐based chip. Compared with a plane washing mode by a ring‐oven, this 3D washing strategy obtained a lower background, as gravity played an important role in the washing step. The paper‐based chip was placed on a 3D plastic holder and the waste area was connected to a heating ring. Use of a heating waste area meant that the nonspecific‐binding protein was continuously carried to the waste area through gravity and capillary action. The angle between the plastic holder and the ring plane was carefully selected. The effect of washing on different parts of the detection area was investigated by upconversion fluorescence and chemiluminescence (CL). This novel 3D washing strategy was performed for carcinoembryonic antigen detection through CL and a lower detection limit of 2 pg ml^?1 was obtained. This approach provides an effective washing strategy to remove nonspecific‐binding antibody from a paper‐based immunodevice.     

Chemical de‐O‐glycosylation of glycoproteins for application in LC‐based proteomics

We describe a cyclic on‐column procedure for the sequential degradation of complex O‐glycans on proteins or peptides by periodate oxidation of sugars and cleavage of oxidation products by elimination. Desialylated glycoproteins were immobilized to alkali‐stable, reversed‐phase Poros 20 beads followed by two degradation cycles and the eluted apoproteins were either separated by SDS gel electrophoresis or digested with trypsin prior to LC/ESI‐MS. We demonstrate on the peptide and protein level that even complex glycan moieties are removed under mild conditions with only minimal effects on structural integrity of the peptide core by fragmentation, dehydration or by racemization of the Lys/Arg residues. The protocol is applicable on gel‐immobilized glycoproteins after SDS gel electrophoresis. Conversion of O‐glycoproteins into their corresponding apoproteins should result in facilitated accessibility of tryptic cleavage sites, increase the numbers of peptide fragments, and accordingly enhance protein coverage and identification rates within the subproteome of mucin‐type O‐glycoproteins.     

Optofluidic single‐cell absorption flow analyzer for point‐of‐care diagnosis of malaria

In this work, an optofluidic flow analyzer, which can be used to perform malaria diagnosis at the point‐of‐care is demonstrated. The presented technique is based on quantitative optical absorption measurements carried out on a single cell level for a given population of Human Red Blood Cells (RBCs). By measuring the optical absorption of each RBC, the decrease in the Hemoglobin (Hb) concentration in the cytoplasm of the cell due to the invasion of malarial parasite is detected. Cells are assessed on a single cell basis, as they pass through a microfluidic channel. The proposed technique has been implemented with inexpensive off‐the‐shelf components like laser diode, photo‐detector and a micro‐controller. The ability of the optofluidic flow analyzer to asses about 308,049 cells within 3 minutes has been demonstrated. The presented technique is capable of detecting very low parasitemia levels with high sensitivity.

     

The Skp1‐like protein SSK1 is required for cross‐pollen compatibility in S‐RNase‐based self‐incompatibility

The self‐incompatibility (SI) response occurs widely in flowering plants as a means of preventing self‐fertilization. In these self/non‐self discrimination systems, plant pistils reject self or genetically related pollen. In the Solanaceae, Plantaginaceae and Rosaceae, pistil‐secreted S‐RNases enter the pollen tube and function as cytotoxins to specifically arrest self‐pollen tube growth. Recent studies have revealed that the S‐locus F‐box (SLF) protein controls the pollen expression of SI in these families. However, the precise role of SLF remains largely unknown. Here we report that PhSSK1 (Petunia hybrida SLF‐interacting Skp1‐like1), an equivalent of AhSSK1 of Antirrhinum hispanicum, is expressed specifically in pollen and acts as an adaptor in an SCF(Skp1‐Cullin1‐F‐box)^SLF complex, indicating that this pollen‐specific SSK1‐SLF interaction occurs in both Petunia and Antirrhinum, two species from the Solanaceae and Plantaginaceae, respectively. Substantial reduction of PhSSK1 in pollen reduced cross‐pollen compatibility (CPC) in the S‐RNase‐based SI response, suggesting that the pollen S determinant contributes to inhibiting rather than protecting the S‐RNase activity, at least in solanaceous plants. Furthermore, our results provide an example that a specific Skp1‐like protein other than the known conserved ones can be recruited into a canonical SCF complex as an adaptor.     

Widespread evidence for incipient ecological speciation: a meta‐analysis of isolation‐by‐ecology

Ecologically mediated selection has increasingly become recognised as an important driver of speciation. The correlation between neutral genetic differentiation and environmental or phenotypic divergence among populations, to which we collectively refer to as isolation‐by‐ecology (IBE), is an indicator of ecological speciation. In a meta‐analysis framework, we determined the strength and commonality of IBE in nature. On the basis of 106 studies, we calculated a mean effect size of IBE with and without controlling for spatial autocorrelation among populations. Effect sizes were 0.34 (95% CI 0.24–0.42) and 0.26 (95% CI 0.13–0.37), respectively, indicating that an average of 5% of the neutral genetic differentiation among populations was explained purely by ecological contrast. Importantly, spatial autocorrelation reduced IBE correlations for environmental variables, but not for phenotypes. Through simulation, we showed how the influence of isolation‐by‐distance and spatial autocorrelation of ecological variables can result in false positives or underestimated correlations if not accounted for in the IBE model. Collectively, this meta‐analysis showed that ecologically induced genetic divergence is pervasive across time‐scales and taxa, and largely independent of the choice of molecular marker. We discuss the importance of these results in the context of adaptation and ecological speciation and suggest future research avenues.     

Exploiting Lithium–Ether Co‐Intercalation in Graphite for High‐Power Lithium‐Ion Batteries

The intercalation of lithium ions into graphite electrode is the key underlying mechanism of modern lithium‐ion batteries. However, co‐intercalation of lithium‐ions and solvent into graphite is considered undesirable because it can trigger the exfoliation of graphene layers and destroy the graphite crystal, resulting in poor cycle life. Here, it is demonstrated that the [lithium–solvent]⁺ intercalation does not necessarily cause exfoliation of the graphite electrode and can be remarkably reversible with appropriate solvent selection. First‐principles calculations suggest that the chemical compatibility of the graphite host and [lithium–solvent]⁺ complex ion strongly affects the reversibility of the co‐intercalation, and comparative experiments confirm this phenomenon. Moreover, it is revealed that [lithium–ether]⁺ co‐intercalation of natural graphite electrode enables much higher power capability than normal lithium intercalation, without the risk of lithium metal plating, with retention of ≈87% of the theoretical capacity at current density of 1 A g^?1. This unusual high rate capability of the co‐intercalation is attributed to the (i) absence of the desolvation step, (ii) negligible formation of the solid–electrolyte interphase on graphite surface, and (iii) fast charge‐transfer kinetics. This work constitutes the first step toward the utilization of fast and reversible [lithium–solvent]⁺ complex ion intercalation chemistry in graphite for rechargeable battery technology.     

Blood‐Capillary‐Inspired,Free‐Standing,Flexible, and Low‐Cost Super‐Hydrophobic N‐CNTs@SS Cathodes for High‐Capacity,High‐Rate,and Stable Li‐Air Batteries

With the rising demand for flexible and wearable electronic devices, flexible power sources with high energy densities are required to provide a sustainable energy supply. Theoretically, rechargeable, flexible Li‐O₂/air batteries can provide extremely high specific energy densities; however, the high costs, complex synthetic methods, and inferior mechanical properties of the available flexible cathodes severely limit their practical applications. Herein, inspired by the structure of human blood capillary tissue, this study demonstrates for the first time the in situ growth of interpenetrative hierarchical N‐doped carbon nanotubes on the surface of stainless‐steel mesh (N‐CNTs@SS) for the fabrication of a self‐supporting, flexible electrode with excellent physicochemical properties via a facile and scalable one‐step strategy. Benefitting from the synergistic effects of the high electronic conductivity and stable 3D interconnected conductive network structure, the Li‐O₂ batteries obtained with the N‐CNTs@SS cathode exhibit superior electrochemical performance, including a high specific capacity (9299 mA h g^?1 at 500 mA g^?1), an excellent rate capability, and an exceptional cycle stability (up to 232 cycles). Furthermore, as‐fabricated flexible Li‐air batteries containing the as‐prepared flexible super‐hydrophobic cathode show excellent mechanical properties, stable electrochemical performance, and superior H₂O resistibility, which enhance their potential to power flexible and wearable electronic devices.     

Dual labeling with 5‐bromo‐2'‐deoxyuridine and 5‐ethynyl‐2'‐deoxyuridine for estimation of cell migration rate in the small intestinal epithelium

Small intestinal epithelium is a self‐renewing system in which the entire sequence of cell proliferation, differentiation, and removal is coupled to cell migration along the crypt‐villus axis. We examined whether dual labeling with different thymidine analogues, 5‐bromo‐2'‐deoxyuridine (BrdU) and 5‐ethynyl‐2'‐deoxyuridine (EdU), can be used to estimate cell migration rates on the villi of small intestines in rats. Rats received a single intraperitoneal injection of BrdU and EdU within a time interval, and signals in tissue sections were examined by immunohistochemistry and the “click” reaction, respectively. We successfully observed BrdU‐ and EdU‐positive cells on the epithelium with no cross‐reaction. In addition, we observed an almost complete overlapping of BrdU‐ and EdU‐positive cells in rats administered simultaneously with BrdU and EdU. By calculating the cell migration rate by dividing the distance between the median cell positions of the distribution of BrdU‐ and EdU‐positive cells by the time between the injection of BrdU and EdU, we estimated approximately 9 and 5 μm/h for the cell migration rates on the villi in the jejunum and ileum, respectively. We propose that dual labeling with BrdU and EdU within a time interval, followed by detecting with immunohistochemistry and the click reaction, respectively, is useful to estimate accurately the cell migration rate in the intestinal epithelium in a single animal.