Extensive Steps Research Articles

CB2 Cannabinoid Receptors Promote Neural Progenitor Cell Proliferation via mTORC1 Signaling

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.

Reviewing -Gal in valve immunology

Calcification of bioprosthetic heart valves is an unsolved problem ever since. Lim et al. [1] recently made another attempt to improve tissue performance by applying the novel fixation agent genipin. Numerous efforts of chemical tissue pretreatment have been tested, but up to now, the longevity of biovalves has not substantially increased, especially not in young recipients. Our group has shown that the implantation of biovalves causes an increase in anti-Gal immunoglobulin M and immunoglobulin G (IgG) titers in patients [2, 3]. Thus, we suggested a mechanism of chronic xenograft rejection and consecutive calcification via that anti-Gal response. The α-Gal barrier is known to prevent xenotransplantation from suitable animals to humans, because humans and old world monkeys are the sole mammals which produce anti-Gal antibodies in high titers and lack the α-Gal epitope [4]. Since recently, biovalve researchers perceive the problem of α-Gal antigenicity in glutaraldehyde-fixed biovalves, and α-Gal knockout pigs are proposed as a possible source for valve tissue [5, 6]. Lim et al. [1] delivered a concise report on the use of the cross-linking agent genipin in the pretreatment of bovine pericardium and evaluated calcification in a rabbit intramuscular transplantation model. They proved the superiority of genipin over glutaraldehyde to prevent tissue calcification in a clear sequence of experiments. However, their interpretation of the α-Gal barrier in their setup is disconcerting. They implanted bovine tissue into a rabbit muscle and measured anti-Gal IgG antibodies in plasma before, 12 and 60 days after implantation. Genipin-treated tissue recipients showed significantly lower anti-Gal IgG titers than glutaraldehyde-treated tissue recipients. Furthermore, they showed that decellularization of tissue before implantation abolished this titer increase. But rabbits do not have any anti-α-Gal antibodies, as they express the α-Gal epitope themselves; at least according to the present opinion. How are these results possible then? The authors do not give a clear statement; they indicate an explanation by not correctly citing Macher and Galili [4], saying ‘the differences in the fine specificity of natural anti-Gal in various species may cause the multiple B-cell clones to produce anti-Gal antibodies which have specificities that differ slightly from each other, and thus recognize various facets of the a-Gal epitope in its three-dimensional form’. But Macher and Galili never mention ‘various species’, they merely speak of anti-Gal varieties among individuals. Do the authors suggest differing α-Gal epitopes among different species? This is unlikely, as the sequence homology of α1,3GT is very high among a wide range of species [4]. If uniform Gal epitopes are assumed, one would expect autoimmunologic affection in animals with elevated anti-Gal titers; did the transplanted animals show any signs of systemic inflammation? Most of the researchers who measure anti-Gal antibodies use self-established ELISAs with internal or no standards. Such ELISAs require extensive titration steps to yield a reliable technique that is suitable for publication. Reevaluation and verification of the results of Lim et al. should be considered.

Development of immunosensors for direct detection of three wound infection biomarkers at point of care using electrochemical impedance spectroscopy

A method for label-free, electrochemical impedance immunosensing for the detection and quantification of three infection biomarkers in both buffer and directly in the defined model matrix of mock wound fluid is demonstrated. Triggering Receptor-1 Expressed on Myeloid cells (TREM-1) and Matrix MetalloPeptidase 9 (MMP-9) are detected via direct assay and N-3-oxo-dodecanoyl-l-HomoSerineLactone (HSL), relevant in bacterial quorum sensing, is detected using a competition assay. Detection is performed with gold screen-printed electrodes modified with a specific thiolated antibody. Detection is achieved in less than 1h straight from mock wound fluid without any extensive sample preparation steps. The limits of detection of 3.3pM for TREM-1, 1.1nM for MMP-9 and 1.4nM for HSL are either near or below the threshold required to indicate infection. A relatively large dynamic range for sensor response is also found, consistent with interaction between neighbouring antibody–antigen complexes in the close-packed surface layer. Together, these three novel electrochemical immunosensors demonstrate viable multi-parameter sensing with the required sensitivity for rapid wound infection detection directly from a clinically relevant specimen.

Control of cell attachment on pH-responsive chitosan surface by precise adjustment of medium pH

The purpose of this study is to demonstrate pH-responsive chitosan is able to control cell behavior in response to small changes in environmental pH, which is at useful pH suitable for recovering cultured cells without additional enzymatic treatment and extensive washing steps. HeLa cells attached and spread well on chitosan at pH 6.99 and 7.20. When the pH was increased to 7.65, over 90% of cells would rapidly detached from chitosan surface within 1 h. Similarly, fibronectin adsorbed on chitosan at pH 7.20 also rapidly desorbed after increasing the medium pH. Most importantly and interestingly, medium pH adjustment could be facilitated by altering environment pCO2. It was found over 80% of HeLa cells could be recovered from chitosan surface within 1 h and the viability of detached cells was more than 95% by transferring the culture plate from incubator to atmospheric condition. Additionally, chitosan substrate could effectively control attachment/detachment of various types of cells including cell lines HaCaT, H1299, NIH-3T3, and primary corneal fibroblasts, indicating the technology described here is easily reproducible and should be promising for controlling rapid fibronectin adsorption/desorption and cell attachment/detachment for tissue engineering applications.

Synthesis of Complex Multiblock Copolymers via a Simple Iterative Cu(0)-Mediated Radical Polymerization Approach

Controlled/living radical polymerization is an efficient technique for the synthesis of well-defined polymeric architectures, including copolymers, block copolymers, stars, graft, and variations thereof. In this article, we report for the first time the synthesis of a decablock copolymer via a simple and efficient iterative Cu(0)-mediated radical polymerization technique. In this approach, purification is not required between the iterative chain extension steps, as each block formation is taken to full conversion. The final decablock copolymer can be obtained with a yield in mass of ∼90%. Using traditional controlled/living radical polymerization techniques, including reversible addition–fragmentation chain transfer (RAFT) polymerization, nitroxide-mediated polymerization (NMP), or atom transfer radical polymerization (ATRP), synthesis of decablock copolymer in such high yield is very difficult (and may be impossible) and requires numerous purification steps. The synthesis of the final complex copolymers required the concomitant synthesis of 16 architecturally discrete block copolymers.

In Vitro Replication Studies of Carboxymethylated DNA Lesions with Saccharomyces cerevisiae Polymerase η

Humans are exposed to N-nitroso compounds (NOCs) both endogenously and exogenously from a number of environmental sources, and NOCs are both mutagenic and carcinogenic. After metabolic activation, some NOCs can induce carboxymethylation of nucleobases through a diazoacetate intermediate, which could give rise to p53 mutations similar to those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase η-deficient human cells was inhibited by treatment with azaserine, a DNA carboxymethylation agent, suggesting the importance of this polymerase in bypassing the azaserine-induced carboxymethylated DNA lesions. In this study, we examined how carboxymethylated DNA lesions, which included N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA), N(4)-carboxymethyl-2'-deoxycytidine (N(4)-CMdC), N3-carboxymethylthymidine (N3-CMdT), and O(4)-carboxymethylthymidine (O(4)-CMdT), perturbed the efficiency and fidelity of DNA replication mediated by Saccharomyces cerevisiae polymerase η (pol η). Our results from steady-state kinetic assay showed that pol η could readily bypass and extend past N(6)-CMdA and incorporated the correct nucleotides opposite the lesion and its neighboring 5'-nucleoside with high efficiency. By contrast, the polymerase could bypass N(4)-CMdC inefficiently, with substantial misincorporation of dCMP followed by dAMP, though pol η could extend past the lesion with high fidelity and efficiency when dGMP was incorporated opposite the lesion. On the other hand, yeast pol η experienced great difficulty in bypassing O(4)-CMdT and N3-CMdT, and the polymerase inserted preferentially the incorrect dGMP opposite these two DNA lesions; the extension step, nevertheless, occurred with high fidelity and efficiency when the correct dAMP was opposite the lesion, as opposed to the preferentially incorporated incorrect dGMP. These results suggest that these lesions may contribute significantly to diazoacetate-induced mutations and those in the p53 gene observed in human gastrointestinal tumors.

Effect of subjective knee-joint pain on the laterality of knee extension strength and gait in elderly women

This study aimed to examine the effect of subjective knee-joint pain on the laterality of knee extension strength and gait in elderly women. The subjects were 144 elderly women (62–94 years old; mean age 76.2±6.0 years; ±S.D.) who were divided into the following groups: 81 persons without knee-pain (no knee-pain group), 39 persons with the subjective pain in right or left knee (single knee-pain group), and 24 persons with the subjective pain in both knees (double knee-pain group). The subjects took a knee extension strength test and a 12m maximum effort walk test. Knee extension strength, stance time, swing time, stride length, step length and swing speed were selected as parameters. A significant laterality was found in knee extension strength only in the one knee-pain group. The laterality of gait parameters was not found in all groups. In conclusion, elderly women who can perform daily living activity independently, even though having subjective pain in either knee or laterality in knee extension strength exertion show little laterality of gait during short distance walking.

Real time electrochemical monitoring of PCR amplicons using electroactive hydrolysis probe

A novel electrochemical DNA detection principle based on a hydrolysis DNA probe labeled with an electro-active indicator was developed. This strategy was further utilized to realize an electrochemical real-time PCR (ERT-PCR) technique which requires no probe immobilization step. Inspired by the Taqman® probe based fluorescence assay, our approach utilizes sequence-specific hydrolysis oligonucleotide probe modified with electroactive tags (also called eTaq probe). Upon the nucleotide extension step during the PCR, E-tagged nucleotides closer to the 5′ end of the hydrolysis probe would be cleaved away one-by-one as a result of the 5′ to 3′ exonuclease activity of the DNA polymerase. Unlike the negatively-charged hydrolysis probe, the released e-tagged mono- or short-nucleotide, because of the less extent of electrostatic repulsion with a negatively charged substrate surface, would diffuse freely to the negatively charged electrode (e.g. indium tin oxide) contributing to an increase of electrochemical signal. Experimental investigations demonstrate the cycle-by-cycle electrochemical signal enhancement. This new ERT-PCR method provides higher specificity and multiplexing capability and is a significant advancement towards the application of ERT-PCR in portable DNA-analysis.

Die Realisierung von anatomischen Felsenbeinfaksimilemodellen mit cochleären Hohlraumstrukturen

Microsurgical dissection exercises are essential in otosurgery training. Human temporal bone specimens are rarely available for necessary extensive preparation steps up to the cochlea. This requires the development of new Anatomical Facsimile Models (AFM) of the temporal bone with its diffizil internal structures. The construction of AFM was realized by rapid prototyping technologies. Data for processing come from high resolution CT-scans. With the production of AFM true to the original structures of the temporal bone by rapid prototyping methods it was possible to reproduce the very small cavity structures of the inner ear (cochlea, semicircular canals). All cavity structures of the temporal bone, including the middle ear, are constructed without solid support material. This allows the introduction of Cochlea-Implant electrodes into the cochlea. The use of modern rapid prototyping technologies enables us to produce any number of identical models of an original specimen. The preparation steps and the material properties correspond to those of the original temporal bone. Therefore AFM are excellent preparation models.

Occurrence of Grapevine leafroll-associated virus 1 in Two Ornamental Grapevine Cultivars in Washington State

Roger's Red, an interspecific hybrid between wild grape (Vitis californica, native to northern California) and the V. vinifera cv. Alicante Bouschet (1), and Claret Vine (V. vinifera cv. Purpurea Nana) are grown for their ornamental value in home gardens and other settings. We collected potted grapevines of Roger's Red and Claret Vine showing dull green-to-scarlet red leaves from two different retail nurseries in the Richland-Kennewick area and Prosser, WA, respectively. Since these symptoms 'mimic' grapevine leafroll disease, we tested petiole samples from four grapevines per cultivar for a panel of grapevine-infecting viruses by single-tube one-step reverse transcription (RT)-PCR (4). All samples tested positive only for Grapevine leafroll-associated virus 1 (GLRaV-1; genus Ampelovirus, family Closteroviridae). To further confirm these results, total RNA was subjected to RT-PCR to amplify a portion of the heat shock protein 70 homolog (HSP70h), coat protein duplicate 2 (CPd2), and ORF 9 (p24) of GLRaV-1. RT was performed at 52°C for 60 min, followed by 35 cycles of PCR (30 s denaturation at 94°C, 45 s annealing at 55°C, and 30 s extension at 72°C) and a 5 min final extension step at 72°C. Primers specific to HSP70h (HSP70h/416F: 5'-CAGGCGTCGTTTGTACTGTG and HSP70h/955R: 5'-TCGGACAGCGTTTAAGTTCC), CPd2 (CPd2/F: 5'-GTTACGGCCCTTTGTTTATTATGG and CPd2/R: 5'-CGACCCCTTTATTGTTTGAGTATG) and ORF 9 (p24/F: 5'-CGATGGCGTCACTTATACCTAAG and p24/R (5'-CACACCAAATTGCTAGCGATAGC) were designed based on GLRaV-1 sequence (GenBank Accession No. AF195822) to amplify 540, 398, and 633 base pair (bp) DNA fragments, respectively. To verify that the amplified products were specific to the genome of GLRaV-1, the amplicons were cloned into pCR2.1 vector (Invitrogen Corp, Carlsbad, CA) and three independent clones for each amplicon were sequenced in both directions. Pairwise comparison of HSP70h (Accession Nos. HQ833472 and HQ833473), CPd2 (Accession Nos. HQ833474 and HQ833475), and p24 (Accession Nos. HQ833476 and HQ833477) sequences from Roger's Red and Claret Vine showed 100, 96, and 99% identities, respectively, between them, and 86 to 100, 80 to 97, and 86 to 90% nucleotide sequence identities, respectively, with corresponding sequences of GLRaV-1 isolates deposited in GenBank. We further confirmed the presence of GLRaV-1 in these two ornamental grape cultivars by double antibody sandwich-ELISA using commercially available antibodies (Bioreba AG, Reinach, Germany). Previous studies have reported the presence of GLRaV-2 and -3 (1,3) and Grapevine virus A and B (2,3) in Roger's Red. To our knowledge, this study represents the first report of the occurrence of GLRaV-1 in two Vitis species distributed as ornamental grapes. It is important to prevent virus spread via the supply of virus-tested ornamental grapevines by commercial nurseries.

Fundamental study of the radiation monitoring system based on evaluation of DNA lesions

The biological dosemeter that measures biological responses to ionising radiation is useful for radiation protection. This paper presents the development and characterisation of a gamma ray irradiation dosimetry system based on real-time PCR (polymerase chain reaction) methodology. Real-time PCR is used to amplify and simultaneously quantify a targeted DNA molecule. If there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment. The essential point of this assay is that DNA lesions caused by ionising radiation block DNA synthesis by DNA polymerase, resulting in a decrease in the amplification of a damaged DNA template compared with that of non-damaged DNA templates.

Molecular Biology Techniques Q&A

Molecular Biology Techniques Q&A

Hematogenous and lymphatic tumor cell dissemination may be detected in patients diagnosed with ductal carcinoma in situ of the breast

Tumor cell dissemination in bone marrow (BM) and lymph nodes is considered an important step in systemic disease progression and is associated with poor prognosis. Only invasive cancers are assumed to shed isolated tumor cells (ITC) into the bloodstream and infiltrate lymph nodes. However, latest studies indicate that tumor cell dissemination may occur before stroma invasion, i.e., in ductal carcinoma in situ (DCIS). Therefore, the purpose of this study was to examine the incidence of ITC in bone marrow and sentinel lymph nodes (SN) in patients diagnosed with DCIS and its correlation with clinicopathological factors. 266 patients who were treated at the Department of Gynecology and Obstetrics (University Hospital Tuebingen, Germany) between 2003 and 2009 with DCIS were included into this study. BM aspirates were analyzed by immunocytochemistry (pancytokeratin antibody A45-B/B3) using ACIS system (Chromavision) according to the ISHAGE evaluation criteria. SN were analyzed in 221 of these patients by extensive step sectioning and hematoxylin-eosin staining. In 34 of 266 patients (13%), ITC in BM could be detected. There was no correlation found between tumor size, grading, histology, or Van Nuys Prognostic Index and tumor cell dissemination. In two cases, metastatic spread into lymph nodes was observed (pN1mi), whereas in one case, ITC in lymph nodes were detected; however, additional sectioning and immunohistochemical staining of the primary lesion in the cases with positive SN did not reveal invasive cancer. Interestingly, all the three patients were BM negative. Tumor cell dissemination may be detected in patients diagnosed with DCIS. Either these cells have started already to disseminate from preinvasive mammary lesions or from occult invasive tumors or represent the earliest step of microinvasion in a preinvasive lesion. The clinical relevance of these cells has to be further evaluated.

Deformation mechanisms in gold nanowires and nanoporous gold

We present a study of the deformation of gold nanowires of diameter 30–70 nm and nanoporous gold specimens with ligament diameter 5–10 nm, both produced by electrodeposition into anodised aluminium oxide templates. The nanowires show extensive surface slip steps and low dislocation densities with a few perfect dislocation loops, Shockley partial dislocations and microtwins observed after deformation. Nanoporous specimens show deformation localised to the nodes between the ligaments of the foamed structure, with high densities of microtwins and Shockley partial dislocations in these regions. Similar dislocation structures are seen in larger nanowires deformed in bending. This is shown to be consistent with a strain gradient plasticity model for the deformation of nanoporous gold, with the strain gradient accommodated by geometrically necessary twins and partial dislocations.

Large-Scale Manufacture and Characterization of a Lentiviral Vector Produced for ClinicalEx VivoGene Therapy Application

From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.

Windsor, Ontario Exposure Assessment Study: Design and Methods Validation of Personal, Indoor, and Outdoor Air Pollution Monitoring

ABSTRACT The Windsor, Ontario Exposure Assessment Study evaluated the contribution of ambient air pollutants to personal and indoor exposures of adults and asthmatic children living in Windsor, Ontario, Canada. In addition, the role of personal, indoor, and outdoor air pollution exposures upon asthmatic children's respiratory health was assessed. Several active and passive sampling methods were applied, or adapted, for personal, indoor, and outdoor residential monitoring of nitrogen dioxide, volatile organic compounds, particulate matter (PM; PM ≤ 2.5 μm [PM2.5] and ≤ 10 μm [PM10] in aerodynamic diameter),elemental carbon, ultrafine particles, ozone, air exchange rates, allergens in settled dust, and particulate-associated metals. Participants completed five consecutive days of monitoring during the winter and summer of 2005 and 2006. During 2006, in addition to undertaking the air pollution measurements, asthmatic children completed respiratory health measurements (including peak flow meter tests and exhaled breath condensate) and tracked respiratory symptoms in a diary. Extensive quality assurance and quality control steps were implemented, including the collocation of instruments at the National Air Pollution Surveillance site operated by Environment Canada and at the Michigan Department of Environmental Quality site in Allen Park, Detroit, MI. During field sampling, duplicate and blank samples were also completed and these data are reported. In total, 50 adults and 51 asthmatic children were recruited to participate, resulting in 922 participant days of data. When comparing the methods used in the study with standard reference methods, field blanks were low and bias was acceptable, with most methods being within 20% of reference methods. Duplicates were typically within less than 10% of each other, indicating that study results can be used with confidence. This paper covers study design, recruitment, methodology, time activity diary, surveys, and quality assurance and control results for the different methods used. IMPLICATIONS It is important to obtain data to identify any factors that can influence the relationships among personal, indoor, and outdoor concentrations for a range of air pollutants. Ensuring that the methods used are valid and comparable to reference methods used in typical air pollution, monitoring is crucial for data to be of use to regulators. These exposure data can then be used to develop risk management policies that reduce personal and indoor exposures to air pollutants.

Microdisplay-Based Intraoral 3D Scanner for Dentistry

Up to now, Microdisplays are mainly used in multimedia applications or head-mounted displays. Due to their interesting properties, these displays open more and more alternative application fields, for example, in optical metrology. Projection lenses for this application area have to be specially designed, because the requirements for these systems differ completely from those for multimedia applications. The lenses must have very low geometrical image distortion and they have to be adapted to small objects and/or image distances. On the other hand, they often work with light sources with small spectral bandwidths; consequently they do not need to be corrected for chromatic aberrations. In addition, the numerical aperture (NA) has to be large enough to collect and transfer as much light as possible. Secondary the size of the projection lens has to be as small as possible to ensure compact measurement systems. All these requirements lead to a compromise in optical lens and system design. Within this paper, the development and realization of a 3D-scanner for the registration of dental surfaces directly inside the patient's mouth is presented. The advantages of such an intraoral scanning system are the reduced pain level for the patient and the absence of extensive intermediate steps. The production of prosthesis can be performed directly after measurement. Thus a quality improvement can be obtained as well as a reduction of the efforts in time and costs.

Windsor, Ontario Exposure Assessment Study: Design and Methods Validation of Personal, Indoor, and Outdoor Air Pollution Monitoring

ABSTRACT The Windsor, Ontario Exposure Assessment Study evaluated the contribution of ambient air pollutants to personal and indoor exposures of adults and asthmatic children living in Windsor, Ontario, Canada. In addition, the role of personal, indoor, and outdoor air pollution exposures upon asthmatic children's respiratory health was assessed. Several active and passive sampling methods were applied, or adapted, for personal, indoor, and outdoor residential monitoring of nitrogen dioxide, volatile organic compounds, particulate matter (PM; PM ≤2.5 μm [PM2.5] and ≤ 10 μm [PM10] in aerodynamic diameter), elemental carbon, ultrafine particles, ozone, air exchange rates, allergens in settled dust, and particulate-associated metals. Participants completed five consecutive days of monitoring during the winter and summer of 2005 and 2006. During 2006, in addition to undertaking the air pollution measurements, asthmatic children completed respiratory health measurements (including peak flow meter tests and exhaled breath condensate) and tracked respiratory symptoms in a diary. Extensive quality assurance and quality control steps were implemented, including the collocation of instruments at the National Air Pollution Surveillance site operated by Environment Canada and at the Michigan Department of Environmental Quality site in Allen Park, Detroit, MI. During field sampling, duplicate and blank samples were also completed and these data are reported. In total, 50 adults and 51 asthmatic children were recruited to participate, resulting in 922 participant days of data. When comparing the methods used in the study with standard reference methods, field blanks were low and bias was acceptable, with most methods being within 20% of reference methods. Duplicates were typically within less than 10% of each other, indicating that study results can be used with confidence. This paper covers study design, recruitment, methodology, time activity diary, surveys, and quality assurance and control results for the different methods used. IMPLICATIONS It is important to obtain data to identify any factors that can influence the relationships among personal, indoor, and outdoor concentrations for a range of air pollutants. Ensuring that the methods used are valid and comparable to reference methods used in typical air pollution, monitoring is crucial for data to be of use to regulators. These exposure data can then be used to develop risk management policies that reduce personal and indoor exposures to air pollutants.

Assembly of uniform photoluminescent microcomposites using a novel micro‐fluidic‐jet‐spray‐dryer

AbstractGeneration of uniform micro‐particles containing (i) pure lactose; (ii) silica nanoparticles and lactose; (iii) silica nanoparticles/lactose doped with Eu(III) have been successfully achieved using a novel spray dryer with a uniquely designed microfluidic aerosol nozzle as the monodisperse droplet generator. Here we investigate the impacts of precursor compositions and concentrations, as well as the drying temperature profile on particle size, morphology, and surface element distribution. Distinct morphologies are observed with different precursor compositions, ranging from smooth spherical lactose microparticles to the buckled shape for composites containing silica nanoparticles. The formation of such morphology is qualitatively interpreted by using Peclet number, indicating that the presence of the suspended silica nanoparticles facilitates shell formation at the early stage of the drying process. As the drying continue, such shell is subject to buckling, induced by the capillary force due to the lower mechanical integrity inside the droplet. Post calcination, transmission electron micrographs of Eu(III)/silica nanoparticles/lactose microcomposites confirm the formation of nano‐sized Eu2O3 homogeneously embedded on the silica shell. Photoluminescence spectra of these particles indicate that enhancement of photoluminescence intensity is directly related to the europium loading, which could be adjusted from the precursor composition. This work demonstrates a scalable route to assemble relatively complex composites with uniform properties, without extensive conjugation or purification steps commonly required in wet chemistry‐based processes. © 2011 American Institute of Chemical Engineers AIChE J, 2011

BioSpotlight

BioTechniquesVol. 50, No. 1 BioSpotlightOpen AccessBioSpotlightNathan S. Blow & Patrick C.H. LoNathan S. BlowSearch for more papers by this author & Patrick C.H. LoSearch for more papers by this authorPublished Online:28 Jun 2018https://doi.org/10.2144/000113580AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Automatic for the PeopleWhen it comes to studying gene or microRNA expression patterns, quantitative PCR (qPCR) is still the method of choice. While in a low-throughput format the equipment to perform qPCR and the downstream analysis tools are readily available, when it comes to higher-throughput applications and studies, automating the assay can be challenging. One of the major stumbling blocks comes from the rigid robotic scripts that are often supplied with liquid handling robots, making optimization for complicated applications such as qPCR intricate and time-consuming. In the current issue of BioTechniques, S. Callejas and colleagues from the Centro Nacional de Investigaciones Cardiovasculares in Madrid describe a new program they developed called Automatic Genomics which can be used for designing medium-throughput qPCR experiments and load 96- and 384-well plates. The software is based on Microsoft Excel with a Visual Basic interface and requires only a limited programming background to execute. Subapplications give users the opportunity to automate steps such as sample transfer, reverse transcription, and volume calculations according to their defined parameters without the need for specific vendor-supplied scripts. To validate their software, the authors tested technical reproducibility for three genes with 88 cDNA samples, with each sample-gene combination loaded in triplicate. The final data showed that 96% of the sample-gene combinations passed the technical quality control metric cutoff of 0.5 cycles (the maximum allowable difference in cycles between the technical replicates) and 61.5% passed a cutoff of 0.2 cycles. The authors note that this level of reproducibility is higher than what could have been achieved by hand. Although designed for use specifically with the Freedom EVO series of liquid handling robots, this software will enable researchers using those platforms to more easily perform larger scale qPCR experiments, with high reproducibility, without the need for complicated modifications to robotics scripts for each experiment.See “Automatic Genomics: a user-friendly program for the automatic designing and plate loading of medium-throughput qPCR experiments”Convection, ‘Vection, What's Your Function?Continuous improvements in PCR thermocycler designs have significantly reduced instrument size, cost, complexity, and amplification times, facilitating the development of simplified platforms suitable for point-of-care applications. One alternative to traditional PCR is convective PCR, where the sample is repeatedly circulated by convection through different temperature zones corresponding to the denaturation, annealing, and extension steps. Previous convective PCR systems, however, have relied on sophisticated temperature controllers and complex designs requiring tubing and chambers to circulate samples, which has limited their widespread usage. In this issue, P.J. Chen and colleagues at the National Taiwan University (Taipei, Taiwan) describe a greatly simplified convective PCR platform that dispenses with these requirements by carrying out amplification in a single capillary tube placed on a dry heat bath set at 95°C. In their capillary convective PCR (CCPCR) approach, the heated base acts to both denature the DNA sample at the bottom of the tube and also cause it to rise by convection. As this portion of the sample rises, it is cooled by the air surrounding the upper part of the tube and undergoes the annealing and extension steps of PCR, after which it sinks down to begin another cycle of convective PCR. Complete amplification can be achieved in as little as 30–40 minutes and does not require any of the complicated manipulations necessary with previous convective PCR systems. The authors demonstrate that CCPCR can amplify fragments up to 500 bp starting from as few as 30 copies, although proper design of amplicons and primers is required such that their Td and Tm, respectively, are optimized for convective PCR. The hardware requirements of the CCPCR platform can be further reduced by using a basic water bath or the heating plate contained in a portable scent-based mosquito repellent. The low cost and ease of operation of CCPCR makes it ideally suited for use in less-developed countries for the rapid detection of disease outbreaks and point-of-care applications.Schematic diagram of CCPCR illustrating the locations of the different steps of PCR along the temperature gradient of the capillary tube heated at 95°C from the bottom.See “Rapid amplification in a capillary tube by natural convection with a single isothermal heater”FiguresReferencesRelatedDetails Vol. 50, No. 1 Follow us on social media for the latest updates Metrics History Published online 28 June 2018 Published in print January 2011 Information© 2011 Author(s)PDF download