Proposal of 28 GHz In Vitro Exposure System Based on Field Uniformity for Three‐Dimensional Cell Culture Experiments

This paper proposes a novel in vitro exposure system operating at millimeter‐wave (mmWave) 28 GHz, one of the frequency bands under consideration for fifth generation (5G) communication. We employed the field uniformity concept along cross‐sectional observation planes at shorter distances from the radiation antenna for better efficiency and a small‐size system. A choke‐ring antenna was designed for this purpose in consideration of a wider beamwidth (BW) and a symmetric far‐field pattern across three principal planes. The permittivity of Dulbecco's modified Eagle's medium solution was measured to examine the specific absorption rate (SAR) of the skin cell layer inside a Petri dish model for a three‐dimensional (3D) cell culture in vitro experiment. The best deployment of Petri dishes, taking into account a geometrical field symmetry, was proposed. Local SAR values within the cell layer among the Petri dishes were determined with different polarization angles. It was determined that this polarization effect should be considered when the actual exposure and deployment were conducted. We finally proposed an in vitro exposure system based on the field uniformity including downward exposure from an antenna for 3D cell culture experiments. A small‐size chamber system was obtained, and the size was estimated using the planar near‐field chamber design rule. Bioelectromagnetics. 2019;40:445–457. © 2019 Bioelectromagnetics Society     

Numerical modeling and dosimetry of the 35 mm Petri dish under 46 GHz millimeter wave exposure

Experimental studies on effects of millimeter wave (MMW) exposure on cells cultured in Petri dishes have attracted interest in recent decades. To improve the quantification of the biological responses toward the MMW energy, an accurate and precise MMW dosimetry is to be provided. By using the finite difference time domain (FDTD) method, the numerical dosimetry is performed for a typical 35 mm Petri dish under 46 GHz continuous MMW exposure from an irradiator of a specified power pattern. With the aim of building a precise model, the meniscus at the interface between the culture solution and the Petri dish sidewall is taken into account, followed by the modeling of smooth edges of the Petri dish. The trilinear interpolation is introduced to assist the FDTD method to obtain a more precise dosimetric assessment. The specific absorption rate (SAR) distributions in the cornea cells covered by culture solution in the Petri dish are calculated and compared to display the effects of using Petri dish models of various precision and the trilinear interpolation on dosimetry results. In addition, the SAR distribution in the cells is analyzed to study its homogeneity. The results indicate that the precise Petri dish model and the application of the trilinear interpolation are helpful in improving the precision of numerical dosimetry. It is also revealed that the inhomogeneity of the SAR distribution is well beyond neglect, which deserves cautious consideration in experiments investigating MMW effects on cells in vitro.     

SAR and efficiency evaluation of a 900 MHz waveguide chamber for cell exposure

In this work we present the results of numerical and experimental dosimetry carried out for an in vitro exposure device to irradiate sample groups at 900 MHz. The cells are kept in 8 and 15 ml cell cultures, contained, respectively in T25 and T75 rectangular flasks. The dosimetric assessment of the distribution of the specific absorption rate (SAR) is performed for both the bottom of the flask and the whole volume of the sample to provide results for experiments on either the cell layer or the cell suspension. The irradiating chamber is a rectangular waveguide (WG). Different configurations are considered to assess the optimum orientation and positioning of the cell cultures inside the WG. The system performance is optimal when the electric field is parallel to the sample and the WG is terminated by a matched load. In this condition two 15 or four 8 ml cells cultures can be exposed. The efficiency (ratio between the power absorbed by the sample and the incident power) and the non-uniformity degree (ratio between the standard deviation of SAR values and the average SAR over the sample) are calculated and successfully verified through measurements of the scattering parameters and local temperature increases. In the chosen exposure configuration, the efficiency is 0.40 and the non-uniformity degree is 39% for the 15 ml samples. For the 8 ml samples, the efficiency is 0.19 and a low non-uniformity degree (15%) is found.     

Millimeter‐Wave Heating in In Vitro Studies: Effect of Convection in Continuous and Pulse‐Modulated Regimes

Shallow penetration of millimeter waves (MMW) and non‐uniform illumination in in vitro experiments result in a non‐uniform distribution of the specific absorption rate (SAR). These SAR gradients trigger convective currents in liquids affecting transient and steady‐state temperature distributions. We analyzed the effect of convection on temperature dynamics during MMW exposure in continuous‐wave (CW) and pulsed‐wave (PW) amplitude‐modulated regimes using micro‐thermocouples. Temperature rise kinetics are characterized by the occurrence of a temperature peak that shifts to shorter times as the SAR of the MMW exposure increases and precedes initiation of convection in bulk. Furthermore, we demonstrate that the liquid volume impacts convection. Increasing the volume results in earlier triggering of convection and in a greater cooling rate after the end of the exposure. In PW regimes, convection strongly depends on the pulse duration that affects the heat pulse amplitude and cooling rate. The latter results in a change of the average temperature in PW regime. Bioelectromagnetics. 2019;40:553–568. © 2019 Bioelectromagnetics Society.     

Complex permittivity of representative biological solutions in the 2–67 GHz range

The main purpose of this study is to provide experimental data on the complex permittivity of some biological solutions in the 2–67 GHz range at room and human body temperatures. The permittivity measurements are performed using an open‐ended coaxial probe. Permittivity spectra of several representative monomolecular solutions of proteins, amino acids, nucleic acids, and carbohydrates are analyzed and compared. Furthermore, measurements have also been performed for complex biomolecular solutions, including bovine serum albumin (BSA)–DNA–glucose mixture, culture medium, and yeast extract solution. The results demonstrate that for concentrations below 1%, the permittivity spectra of the solutions do not substantially differ from that of distilled water. Measurements carried out for 4% and 20% BSA solutions show that the presence of proteins results in a decrease in permittivity. For highly concentrated RNA solutions (3%), a slight increase in the imaginary part of the permittivity is observed below 10 GHz. Experimental data show that free water permittivity can be used for modeling of the culture medium above 10 GHz. However, at lower frequencies a substantial increase in the imaginary part of the permittivity due to ionic conductivity should be carefully taken into account. A similar increase has also been observed for the yeast extract solution in the lower frequency region of the considered spectrum. Above 10 GHz, the high concentration of proteins and other low‐permittivity components of the yeast extract solution results in a decrease in the complex permittivity compared to that of water. Obtained data are of utmost importance for millimeter‐wave dosimetry studies. Bioelectromagnetics 33:346–355, 2012. © 2011 Wiley Periodicals, Inc.     

Effect of the meniscus at the solid/liquid interface on the SAR distribution in Petri dishes and flasks

Several authors have reported that the meniscus as occurring at the walls of Petri dishes and flasks (solid-liquid interface) can significantly affect the SAR distribution in the entire dish and flask, while others have ignored this effect. In this study, this effect has been comprehensively analyzed by numerical means supported by theoretical considerations. The focus is on E polarization, which results in the most homogeneous exposure for monolayer cell cultures and therefore is the most often applied polarization in exposure setups for risk assessment studies. This includes setups based on TEM cells, waveguides, radial transmission lines (RTL), and HF chambers. The conclusion of this study is that the meniscus and its size have a significant effect on the strength and homogeneity of the induced SAR distribution. Hence, the meniscus needs to be accounted for in the determination of the averaged SAR as well as in the uncertainty assessment. It has also been demonstrated that a voxel size of less than 0.2 mm is needed to predict the SAR values for a monolayer of cells accurately.     

Establishment and Characterization of a New Muscle Cell Line of Zebrafish (Danio rerio) as an In Vitro Model for Gene Expression Studies

A new continuous fibroblast cell line was established from the muscle tissue of healthy juvenile Danio rerio (Zebrafish) through explant method. Fish cell lines serve as useful tool for investigating basic fish biology, as a model for bioassay of environmental toxicant, toxicity ranking, and for developing molecular biomarkers. The cell line was continuously subcultured for a period of 12 months (61 passages) and maintained at 28 °C in L-15 medium supplemented with 10% FBS and 10 ng/mL of basic fibroblastic growth factor (bFGF) without use of antibiotics. Its growth rate was proportional to the FBS concentration, with optimum growth at 15% FBS. DNA barcoding (16SrRNA and COX1) was used to authenticate the cell line. Cells were incubated with propidium iodide and sorted via flow cytometry to calculate the DNA content to confirm the genetic stability. Significant green fluorescent protein (GFP) signals confirmed the utility of cell line in transgenic and genetic manipulation studies. In vitro assay was performed with MTT to examine the growth potential of the cell line. The muscle cell line would provide a novel invaluable in vitro model to identify important genes to understand regulatory mechanisms that govern the molecular regulation of myogenesis and should be useful in biomedical research.     

Co‐localization of β1,6‐branched Oligosaccharides and Coarse Melanin in Macrophage–Melanoma Fusion Hybrids and Human Melanoma Cells In Vitro

Fusion hybrids between normal macrophages and Cloudman S91 melanoma cells were shown earlier to have increased metastatic potential, along with high expression of β1,6‐N‐acetylglucosaminyltransferase  V and β1,6‐branched oligosaccharides. Curiously, hybrids, but not parental melanoma cells, also produced ‘coarse melanin’– autophagic vesicles with multiple melanosomes. As β1,6‐branched oligosaccharides were known to be associated with metastasis, and coarse melanin had been described in invasive human melanomas, we looked for potential relationships between the two. Using lectin‐ and immunohistochemistry, we analyzed cell lines producing coarse melanin for β1,6‐branched oligosaccharides: gp100/pmel‐17 (a melanosomal structural component) and CD63 (a late endosome/lysosome component associated with melanoma and certain other human cancers). Cell lines used in this study were (i) hybrid 94‐H48, a highly metastatic, macrophage–melanoma experimental fusion hybrid; (ii) 6^neo mouse melanoma cells, the weakly metastatic, parental fusion partner; and (iii) SKmel‐23, a human melanoma cell line derived from a metastasis. Coarse melanin granules were prominent both in hybrids and in SKmel‐23 cells, and co‐localized with stains for β1,6‐branched oligosaccharides, gp100/pmel 17, and CD63. This is the first report of this phenotype being expressed in vitro, although co‐expression of β1,6‐branched oligosaccharides and coarse melanin was recently shown to be a common and pervasive characteristic in archival specimens of human melanomas, and was most prominent in metastases. The results suggest that pathways of melanogenesis in melanoma may differ significantly from those in normal melanocytes. In vitro expression of this phenotype provides new biological systems for more detailed analyses of its genesis and regulation at the molecular genetic level.     

In vitro culture of secondary follicles and prematuration of cumulus–oocyte complexes from antral follicles increase the levels of maturation‐related transcripts in bovine oocytes

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.     

In vitro evidence for the participation of Drosophila melanogaster sperm β‐N‐acetylglucosaminidases in the interactions with glycans carrying terminal N‐acetylglucosamine residues on the egg's envelopes

Fertilization is a complex and multiphasic process, consisting of several steps, where egg‐coating envelope's glycoproteins and sperm surface receptors play a critical role. Sperm‐associated β‐N‐acetylglucosaminidases, also known as hexosaminidases, have been identified in a variety of organisms. Previously, two isoforms of hexosaminidases, named here DmHEXA and DmHEXB, were found as intrinsic proteins in the sperm plasma membrane of Drosophila melanogaster. In the present work, we carried out different approaches using solid‐phase assays in order to analyze the oligosaccharide recognition ability of D. melanogaster sperm hexosaminidases to interact with well‐defined carbohydrate chains that might functionally mimic egg glycoconjugates. Our results showed that Drosophila hexosaminidases prefer glycans carrying terminal β‐N‐acetylglucosamine, but not core β‐N‐acetylglucosamine residues. The capacity of sperm β‐N‐acetylhexosaminidases to bind micropylar chorion and vitelline envelope was examined in vitro assays. Binding was completely blocked when β‐N‐acetylhexosaminidases were preincubated with the glycoproteins ovalbumin and transferrin, and the monosaccharide β‐N‐acetylglucosamine. Overall, these data support the hypothesis of the potential role of these glycosidases in sperm–egg interactions in Drosophila.     

In Vitro and In Vivo Evaluation of N‐{2‐[4‐(3‐Cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐[11C]methoxybenz­amide,a Positron Emission Tomography (PET) Radioligand for Dopamine D4 Receptors,in Rodents

The D₄ dopamine receptor belongs to the D₂‐like family of dopamine receptors, and its exact regional distribution in the central nervous system is still a matter of considerable debate. The availability of a selective radioligand for the D₄ receptor with suitable properties for positron emission tomography (PET) would help resolve issues of D₄ receptor localization in the brain, and the presumed diurnal change of expressed protein in the eye and pineal gland. We report here on in vitro and in vivo characteristics of the high‐affinity D₄ receptor‐selective ligand N‐{2‐[4‐(3‐cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐[¹¹C]methoxybenzamide ([¹¹C] 2 ) in rat. The results provide new insights on the in vitro properties that a brain PET dopamine D₄ radioligand should possess in order to have improved in vivo utility in rodents.     

Lack of Enantioselectivity in the SULT1A3‐catalyzed Sulfoconjugation of Normetanephrine Enantiomers: An In Vitro and Computational Study

(1R)‐Normetanephrine is the natural stereoisomeric substrate for sulfotransferase 1A3 (SULT1A3)‐catalyzed sulfonation. Nothing appears known on the enantioselectivity of the reaction despite its potential significance in the metabolism of adrenergic amines and in clinical biochemistry. We confronted the kinetic parameters of the sulfoconjugation of synthetic (1R)‐normetanephrine and (1S)‐normetanephrine by recombinant human SULT1A3 to a docking model of each normetanephrine enantiomer with SULT1A3 and the 3′‐phosphoadenosine‐5′‐phosphosulfate cofactor on the basis of molecular modeling and molecular dynamics simulations of the stability of the complexes. The K_M, V_max, and k_cat values for the sulfonation of (1R)‐normetanephrine, (1S)‐normetanephrine, and racemic normetanephrine were similar. In silico models were consistent with these findings as they showed that the binding modes of the two enantiomers were almost identical. In conclusion, SULT1A3 is not substrate‐enantioselective toward normetanephrine, an unexpected finding explainable by a mutual adaptability between the ligands and SULT1A3 through an “induced‐fit model” in the catalytic pocket. Chirality, 25:28‐34, 2012.© 2012 Wiley Periodicals, Inc.     

Characterization of H5N6 highly pathogenic avian influenza viruses isolated from wild and captive birds in the winter season of 2016–2017 in Northern Japan

On 15 November 2016, a black swan that had died in a zoo in Akita prefecture, northern Japan, was strongly suspected to have highly pathogenic avian influenza (HPAI); an HPAI virus (HPAIV) belonging to the H5N6 subtype was isolated from specimens taken from the bird. After the initial report, 230 cases of HPAI caused by H5N6 viruses from wild birds, captive birds, and domestic poultry farms were reported throughout the country during the winter season. In the present study, 66 H5N6 HPAIVs isolated from northern Japan were further characterized. Phylogenetic analysis of the hemagglutinin gene showed that the H5N6 viruses isolated in northern Japan clustered into Group C of Clade 2.3.4.4 together with other isolates collected in Japan, Korea and Taiwan during the winter season of 2016–2017. The antigenicity of the Japanese H5N6 isolate differed slightly from that of HPAIVs isolated previously in Japan and China. The virus exhibited high pathogenicity and a high replication capacity in chickens, whereas virus growth was slightly lower in ducks compared with that of an H5N8 HPAIV isolate collected in Japan in 2014. Comprehensive analyses of Japanese isolates, including those from central, western, and southern Japan, as well as rapid publication of this information are essential for facilitating greater control of HPAIVs.

     

Monoamine Oxidase-Inhibiting Properties of SR 95191, a New Pyridazine Derivative, in the Rat: Evidence for Selective and Reversible Inhibition of Monoamine Oxidase Type A In Vivo but Not In Vitro

In rodents, SR 95191 [3-(2-morpholinoethylamino)-4-cyano-6-phenylpyridazine] has been shown to be active in animal models of depression. The profile of activity of SR 95191 suggests that the compound is a selective and short-acting type A monoamine oxidase (MAO) inhibitor (MAOI) in vivo. In the present study, the interaction of SR 95191 with MAO-A and MAO-B activity was further examined in vivo and in vitro. In brain, liver, and duodenum of pretreated rats, SR 95191 selectively inhibited MAO-A (ED50 = 3-5 mg/kg, p.o.), whereas MAO-B was only weakly inhibited for doses as high as 300 mg/kg, p.o. In vivo, SR 95191 (1-100 mg/kg, p.o.) antagonized, in a dose-dependent fashion, the irreversible inhibition of brain and liver MAO-A induced by phenelzine. Finally, dopamine and 5-hydroxytryptamine depleted from their striatal stores by tetrabenazine were able to displace SR 95191 from the active site of MAO-A. However, ex vivo, kinetic studies showed that the inhibitory effect of SR 95191 (1-10 mg/kg) towards MAO-A was noncompetitive and was unchanged after dilution or dialysis. In vitro, the inhibition of brain MAO-A, but not MAO-B, by SR 95191 was time dependent, with a 19-fold decrease in the IC50 values being observed over a 30-min incubation period (140 to 7.5 microM). At this time, the SR 95191-induced inhibition of MAO-A was not removed by repeated washings. When the reaction was started by adding the homogenate without prior preincubation with SR 95191, the inhibition of brain MAO-A was fully competitive (Ki = 68 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

A plea for ‘genealogical thinking’ in comparative biology – a rebuttal to the reply of Szucsich,Wirkner, and Pass to my article ‘Deconstructing Morphology’

Scholtz G. in press. A plea for ‘genealogical thinking’ in comparative biology – a rebuttal to the reply of Szucsich, Wirkner, and Pass to my article ‘Deconstructing Morphology’. —Acta Zoologica (Stockholm) 00 : 1–4. Szucsich et al. (in press) claim that – in contrast to my statement – morphological thinking has to be ‘cladistic.’ Based on this premise, they stress the difference between the relationships among states of characters versus those among structures assigned to the same character state as implemented in numerical cladistic reasoning. SEA claim that my approach to the homology concept only deals with the problem of the integration of various character states into the same character, whereas the necessary relationships among structures assigned to the same state are not covered. Based on this distinction, SEA also criticise the application of similarity in my definition of homology. Furthermore, they address the issue of evolutionarily independent units.     

The role of bioenergy and biochemicals in CO2 mitigation through the energy system – a scenario analysis for the Netherlands

Bioenergy as well as bioenergy with carbon capture and storage are key options to embark on cost‐efficient trajectories that realize climate targets. Most studies have not yet assessed the influence on these trajectories of emerging bioeconomy sectors such as biochemicals and renewable jet fuels (RJFs). To support a systems transition, there is also need to demonstrate the impact on the energy system of technology development, biomass and fossil fuel prices. We aim to close this gap by assessing least‐cost pathways to 2030 for a number of scenarios applied to the energy system of the Netherlands, using a cost‐minimization model. The type and magnitude of biomass deployment are highly influenced by technology development, fossil fuel prices and ambitions to mitigate climate change. Across all scenarios, biomass consumption ranges between 180 and 760 PJ and national emissions between 82 and 178 Mt CO₂. High technology development leads to additional 100–270 PJ of biomass consumption and 8–20 Mt CO₂ emission reduction compared to low technology development counterparts. In high technology development scenarios, additional emission reduction is primarily achieved by bioenergy and carbon capture and storage. Traditional sectors, namely industrial biomass heat and biofuels, supply 61–87% of bioenergy, while wind turbines are the main supplier of renewable electricity. Low technology pathways show lower biochemical output by 50–75%, do not supply RJFs and do not utilize additional biomass compared to high technology development. In most scenarios the emission reduction targets for the Netherlands are not met, as additional reduction of 10–45 Mt CO₂ is needed. Stronger climate policy is required, especially in view of fluctuating fossil fuel prices, which are shown to be a key determinant of bioeconomy development. Nonetheless, high technology development is a no‐regrets option to realize deep emission reduction as it also ensures stable growth for the bioeconomy even under unfavourable conditions.     

High‐throughput method for the analysis of venlafaxine in pharmaceutical formulations and biological fluids,using a tris(2,2′‐bipyridyl) ruthenium(II)–peroxydisulphate chemiluminescence system in a two‐chip device

A simple, rapid and sensitive chemiluminescent (CL) method for the assay of venlafaxine (VEN) in pharmaceutical formulations and serum samples by a two‐chip device is proposed. The method is based on the reaction of this drug with a tris(2,2′‐bipyridyl) ruthenium(II)–peroxydisulphate CL system. The optimum chemical conditions for CL emission were investigated. The calibration graph was linear for the concentration range 0.02–8.0 µg/mL. The detection and quantification limits were found to be 0.006 and 0.018 µg/mL, respectively, while the relative standard deviation (RSD) was <2.0%. The present CL procedure was applied to the determination of VEN in pharmaceutical formulations and serum samples; the recovery levels were in the range 96.5–101.2%. The results suggest that the method is unaffected by the presence of common formulation excipients found in these samples. Copyright © 2012 John Wiley & Sons, Ltd.