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Context Acanthamoeba is increasingly implicated in causing keratitis in patients wearing contact lens or ocular trauma and has a poor prognosis. Establishment of an animal model is critical to study the disease pathology, pathogenesis and to evaluate anti-amoebic drugs. Some studies have used contact lenses to establish Acanthamoeba keratitis (AK) in a mouse model, which is expensive and not very successful as lenses get dislodged. Objective To assess the feasibility of using parafilm (Bemis Company Inc., USA) as an alternative to contact lens for the establishment of AK in the mouse model. Methods Thirty-six Balb/c mice in three groups of six mice each for two strains of Acanthamoeba were used to induce AK. Three experimental approaches used were; i) Acanthamoeba impregnated contact lens, ii) Acanthamoeba impregnated parafilm and iii) scratching followed by inoculation of Acanthamoeba suspension. In all three models, tarsorrhaphy was performed. Infection was evaluated by clinical examination and also through microscopic examination of corneal scrapings and corneal sections. Results AK model was successfully established with parafilm whereas only one mouse developed AK with the use of contact lens and none with scratching and Acanthamoeba inoculation. Conclusion The use of parafilm is convenient, reliable and cheaper and can be considered an alternative to contact lenses to induce AK in a mouse model.

In this study, the effects of alkaline stress on germination growth of grass pea (Lathyrus sativus L.) were investigated. As plant material landraces 2006, 4403 and 1603 and the registered varieties GAP Blue and Iptas were used. Alkali stress was generated using Na2CO3 and applied at 6 concentrations 0, 10, 20, 30, 40 ve 50 mM). The study was arranged as split plot design with 3 replications, and 20 seeds from each genotype were placed in a petri dish and 10 ml of Na2CO3 solution was added to the petri dishes. Then, the petri dishes were tightly covered with parafilm and allowed to germinate in the dark environment at 20 ± 1 ° C for 7 days in the climate room. In the experiment, germination speed, germination rate, root length, shoot length, root fresh and dry weights, seedling vigour index and ODAP content were examined. it was determined that 1603 local populations respond well to alkali stress as well as varietie in terms of the investigated traits. The 1603 local populations responded well to alkali stress as well as varietie in terms of the investigated traits. It was observed that the plants had an acceptable germination development even at 20 mM alkali doseBesides, it was determined that the ODAP content decreased with increasing doses and was below the level that would adversely affect animal health in all genotypes and doses. Accordingly, it was determined that the grass pea was positively affected by mild alkali stress, and the results obtained from the study shed light on future studies.

Bu çalışma in-vitro koşullarda 2 farklı besi ortamında [patates dekstroz agar (PDA) ve malt ekstrakt agar (MEA)] ve 5 farklı sıcaklıkta (10, 15, 20, 25 ve 30°C) shii-take mantar (Lentinula edodes) miselinin gelişim süresi ve hızını belirlemek amacıyla yürütülmüştür. Araştırmada kullanılan tohumluk miseller Atatürk Bahçe Kültürleri Merkez Araştırma Enstitüsü Müdürlüğü (Yalova)’ndan, klon olarak kullanılan anaçlık mantar AgroMantar (Denizli)’den temin edilmiştir. Hazırlanan MEA (48 g/L) ve PDA (39 g/L) besi ortamları 121°C’de 20 dk otoklavlandıktan sonra ılıkken steril kabin içerisinde UV ışık altında petri kaplarına doldurulmuş ve soğumaya bırakılmıştır. Anaçlık özellikteki shii-take mantarı alkolle dezenfekte edildikten sonra klonlar alınmış, tohumluk misellerden ise tek dane alınarak petri kaplarında besi ortamları üzerine yerleştirilmiş ve petri kapları parafilm ile kapatılmıştır. Her konudan 15 adet petri kabı hazırlanmış ve petriler konusuna uygun sıcaklıkta inkübatöre yerleştirilmiştir. Düzenli olarak farklı iki noktadan günlük misel uzunlukları ölçülmüş; petri kapları miseller ile tam sarıldığında ölçümler bitirilmiştir. Elde edilen verilerden misel gelişim süresi (gün) ve hızı (mm/gün) hesaplanmıştır. Uygulamaların teksel, ikili ve üçlü interaksiyonları misel gelişimini istatistiksel önem derecesinde etkilemiştir. Kullanılan besi ortamları içerisinde MEA, PDA’ya göre, dane misel aşılaması doku aşılamasına göre, 25°C diğer sıcaklık derecelerine göre daha kısa sürede daha hızlı misel gelişimi sağlamıştır. Araştırma sonuçlarına göre 25°C x MEA x dane misel aşılaması shii-take mantar miseli üretiminde uygun bulunmuştur.

Soft, flexible and conductive interfaces are in high demand in wearable electronics where it can be used as electrode material integrated with commercial electronic components and living system. In...

The chlorophyll derivative copper chlorophyllin and related chlorins have promise as environmentally friendly agricultural chemicals, however, spray application is hindered by the propensity of dried spray deposits to wash off leaf surfaces during rain or irrigation. Hypothesis Polyelectrolyte complexes formed between anionic carboxymethyl cellulose and cationic polyamidoamine-epichlorohydrin can prevent the release of copper chlorophyllin from dried spray deposits on leaf surfaces when exposed to water. Experiments Sessile drops on parafilm and containing polyelectrolyte complex and copper chlorophyllin or Brilliant Sulfaflavine, an anionic water-soluble dye, were dried to form deposits that were physical models for crop spray drop deposits on hydrophobic leaf surfaces. Larger buffer drops were placed on the dried deposits and the release of copper chlorophyllin or the dye were measured. Findings Copper chlorophyllin showed some immediate (burst) release upon exposure to buffer whereas the remainder was immobilized on the parafilm. By contrast, Brilliant Sulfaflavine displayed rapid release following square root time dependence, typical of a diffusion-controlled process. The unusual behavior of copper chlorophyllin is attributed to the presence of CuChl nanoparticles when dispersed in water. The nanoparticles are encased in the polyelectrolyte complex that adheres to parafilm. The fraction of the added copper chlorophyllin lost in the burst release can be controlled by varying the polyelectrolyte complex composition and concentration.

A fast and low-cost fabrication process of flexible hanging drop chips for 3D spheroid cultures was proposed by cutting and bonding Parafilm®, a cohesive thermoplastic. The Parafilm® Hanging Drop Chip (PHDC) was assembled by two-layer of Parafilm® sheet with different sizes of holes. The hole on the upper layer of the Parafilm® is smaller than the hole on the bottom layer. The impact of hole size and sample volume on hanging drop formation and 3D spheroid formations in the hanging drop were investigated. The results showed that 20 µL solution on PHDC with a 3 mm hole could form stabile drop and facilitate spheroid formation. The initial cell number determinates the size of the formed spheroids. Exchanging liquid from the upper hole of the PHDC enables the co-culture of two types of cells in one spheroid and drug efficacy testing in hanging drops. The relative expression of cell adhesion and hypoxia-related genes from spheroids in hanging drop and conventional culture plate suggested the relevance of 3D spheroids and in vivo tumor tissue. The economical hanging drop chip can be fabricated without wet chemistry or expensive fabrication equipment, strengthening its application potential in conventional biological laboratories.

Preclinical research to predict the effects of drugs and chemicals on humans is commonly carried out either by cell culture studies in vitro condition or on animals in vivo condition. While drug studies tested on cells cultured as a monolayer in plastic flasks are incompatible with realistic results, falsifying findings can also be achieved from in vivo studies performed on different species. In recent years, research on drug tests using spheroid cultures formed by growing cells in three-dimensional (3D) in vitro has attracted great interest. 3D spheroid structures are formed by growing the cells in a drop suspended on superhydrophobic surfaces. In this study, HEK-293 cells were investigated on parafilm surfaces displaying superhydrophobic properties by growing in 2 µl volume using hanging drop culture method in terms of spheroid formation. Light microscopy images from spheroid structures were taken on different incubation days and the area of spheroids was measured using the ImageJ program. Our study holds important findings for a chip platform that can be developed for use in vitro drug tests.

Background/ Objectives: Biosurfactants are extracellular surface-active compounds produced by bacteria, fungi and yeast. Interest in biosurfactant has been increasing due to their unique properties. The objective of this study is to screen for potential biosurfactant producing yeasts from mangrove areas of Gujarat in India. Methodology: The biosurfactant producing yeasts isolated from five different mangrove sites i.e. Hazira, Mandroi, Mirzapur, Kantiyajal and Machhad of Surat, Gujarat during summer season of 2015. Isolated yeasts were screened for the production of biosurfactant by Parafilm M, Oil displacement and Emulsification index method in which cottonseed oil was used as a substrate. The type of biosurfactant produced was identified by Phenol-sulfuric acid test, Biuret test and phosphate test. Findings: Total twenty four yeasts were isolated from soils of sampling sites. Yeasts were initially screened by Parafilm M and oil displacement method. Out of these 24 isolate, 6 isolates namely, Ky-48, Ky-53, Ky-54, Ky-84, Ky-86 and Ky-87 showed promising biosurfactant activity. These six isolates were further subjected to secondary screening method: Emulsification test, to identify biosurfactant production. Two isolates, namely Ky-46 (emulsification index: 61.53 %) and Ky-86 (emulsification index: 46.66 %) showed maximum biosurfactant production. All six isolates showed positive result for phenol- sulfuric acid method. This indicated that the isolated biosurfactant was Glycolipid in nature. Novelty/Applications: Few mangrove yeasts have potential to produce high amount of glycolipid that can be used in food processing and detergents, healthcare and cosmetics industries. Keywords: Biosurfactant; emulsification index; oil displacement; glycolipid

A series of meglumine-based polysiloxane surfactants, (PSim-n) with different numbers of meglumine branches were prepared. The formed surfactants were characterized by FT-IR and NMR techniques. Physiochemical properties, such as surface activity, aggregation behavior, and wettability, were closely examined and used to differentiate the various samples. The findings demonstrate that PSim-n surfactants possess high surface activity and can wet-out a parafilm surface at all but extremely low concentrations. Furthermore, PSi6-4, the surfactant with the highest meglumine concentration, can form larger complex aggregates in aqueous solutions.

Objective Due to different concerns in using appropriate mosquito blood feeding techniques, this work focused on evaluating the effectiveness of two artificial mosquito feeding systems (Rutledge and Hemotek) and three different membranes (Parafilm, mouse and chicken skins). Female mosquitoes from an An. coluzzii strain aged between 2 and 5 days were exposed to blood with the two systems at time intervals (5, 10, 15, 20, 25 and 30 min) with blood used on the day of collection, the next day and 2 days after. Results Our results showed that the Hemotek system gave better blood feeding rates than the Rutledge system. Among the three membranes, the blood feeding rates with chicken and mouse skins were higher than those provided by the Parafilm membrane. Likewise, blood stored 1 day after collection gave higher levels than blood used on the day of collection and 2 days after. Regardless of the system, the lowest blood feeding rates were observed at 5 min compared to the other exposure times.

In this paper, we demonstrate an easy-to-use and inexpensive stamping method that can be used to generate hydrophobic paraffin surface patterns on a variety of materials and surface geometries, including flat and curved surfaces. In this method, a waxy Parafilm sheet is placed on the back of a polydimethylsiloxane (PDMS) stamp containing microscale surface features; when heated above the melting temperature, the paraffin liquefies and diffuses into the stamp, which functions as a paraffin reservoir. The directional diffusion of the paraffin allows only a thin layer of liquid to be present at the micropatterned stamp surface. Hence, when it is put into contact with a target surface, it transfers a paraffin pattern with submicron lateral resolution and sub-100 nm thickness. By using XPS analysis to track paraffin diffusion process within the PDMS stamp, the diffusion coefficient in PDMS is estimated to be 5.3 x 10-11 m2/s at 65°C. In addition to improved patterning, the paraffin-saturated PDMS stamp removes the need for re-inking for consecutive patterning. In general, our process allows for rapid deposition of complex, multi-layer paraffin patterns on a variety of surfaces.

The scions belonging to 'Black Magic' grape cultivar were grafted on ‘41B’, ‘1103 P’, ‘Fercal’ and ‘SO4’ American rootstock cuttings using omega grafting method in the experiment. Paraffin was applied to one half of the grafted cuttings. The other half was wrapped with parafilm and then paraffin was applied. Grafted cuttings were planted in perlite medium at 3.0 x 3.5 cm intervals inside plastic buckets and kept in the stratification room for 4 weeks. Grafted cuttings, which had been maintained under room conditions for one week, were then grown for two months under unheated greenhouse conditions. In order to determine rootstock and application effects, bud burst ratio (%), callus formation rate (%), callus formation degree (0-4), rooting ratio (%), rooting degree (0-4), shoot length (cm), shoot diameter (mm) and grafted vine ratio (%) were examined. According to the results of the study, it was determined that paraffin+parafilm application yielded better results than paraffin application in terms of bud burst ratio (respectively 70.83%, 53.33%), callus formation ratio (respectively 83.75%, 69.58%) and yield of grafted-vines (respectively 68.33%, 51.67%). In terms of the rootstocks, bud burst ratio was determined to be the highest in SO4 rootstock (71.67%) and the lowest in Fercal (54.17%). 1103 P rootstock yielded a highest value than other rootstocks in terms of shoot length (26.17 cm), callus rate at grafting surface (88.33%), and rooting ratio (97.50%). The effect of rootstocks on shoot diameter, rooting degree, and grafted vine ratio was found to be similar.

ObjectiveParafilm M®is a moisture-resistant thermoplastic commonly used to seal Nematode Growth Media (NGM) agar plates on which the nematodeCaenorhabditis elegansis cultured. This practice reduces media dehydration and microbial contamination. However, the effects onC. elegansindividuals of placing this barrier between the external environment and the interior of the NGM plate are currently unknown. Our research aims to determine if this common practice engenders developmental changes, such as growth, that could subsequently and unintentionally alter experimental data. We compared the larval growth over 48 h of animals cultured on Parafilm-wrapped and unwrapped control NGM plates.ResultsWrapping culture plates with Parafilm significantly accelerated and increased larval growth, with a 0.87 μm/h increase in growth rate (~ 6%) and a 37.90 μm increase in the change in growth (Δgrowth; ~ 5%) over 48 h. Therefore,C. elegansinvestigators should be aware that wrapping their experimental cultures with Parafilm may result in statistically detectable changes in worm growth and possibly other developmental processes.

This study investigated the uniqueness of advancing contact angle (CA) by examining the evaporation process of a sessile drop of surfactant solution [sodium dodecyl sulfate (SDS), hexaethylene glycol monododecyl ether (C12E6) and Triton X-100 (TX-100)] on parafilm. The sessile drop was formed from a pendant drop that had reached its equilibrium surface tension in an atmosphere with ∼100% humidity. The relaxation of the drop profile and wetting diameter were recorded from the moment the drop touched the parafilm, and the data were then analyzed to determine the advancing CA. The results showed that for SDS solution drops, only one advancing CA was observed at each SDS concentration. However, the experimental data of TX-100 and C12E6 solution drops indicated the presence of two distinct regions that showed a near-constant CA with a quasi-equilibrium advance of the triple line; hence matching the definition of an advancing CA. The possibility of the existence of two advancing CA was further explored and the presence of two advancing CA for the sessile drops of TX-100 and C12E6 solution was verified by closely examining the advance of the triple line.

Duddingtonia flagrans is a nematode-trapping fungus that has shown promising results as a tool to combat parasitic nematode infections in livestock. The fungus interrupts the parasitic lifecycle by trapping and killing larval stages on pasture to prevent re-infection of animals. One barrier to the fungus’ commercial use is scaling up production of the fungus, and specifically of chlamydospores, which survive the digestive tract to grow in fecal pats on pasture, thus have potential as a feed through anthelmintic. The purpose of this study was to evaluate the effect of dehydration on sporulation of the fungus. Disks of Duddingtonia flagrans type strain (ATCC® 13423™) were grown on 17% cornmeal agar for 26 days at 30 °C, then split into three groups; dried quickly at 38 °C and 37% humidity over 48 h (“incubated”), dried more slowly at 24 °C and 55% humidity over 10 days (“air-dried”), or kept at 30 °C and sealed with parafilm to prevent loss of moisture as a control (“wet”). Half of each dried culture was resuspended in water, then heated to liquify and homogenized through vortexing. Spores were then counted in a Neubauer hematocytometer. Both the “air-dried” and “incubated” drying techniques yielded significantly more spores than the “wet” control (Welch’s two sample t test p values of .0359 and .0411, respectively). The difference in average chlamydospores per milliliter was insignificant between the two drying techniques, although a visual representation of the data shows less spore count variability in the “air-dried” technique.

This paper describes a technique for small-scale rearing of a phytoseiid mite, Gynaeseius liturivorus (Ehara) (Acari: Phytoseiidae), to facilitate studies on the use of this predator in the biological control of pest insects. The rearing arena consists of a Parafilm floor and concertina-folded roof. At 4 days after the introduction of 20 adults at 25 °C, most adults and immatures colonized the folded roof, and 60% of eggs were laid on it. Many individuals favored the underside. At 5–9 days, about 80–90 individuals were present, and at 14 days, 125 individuals and 105 eggs were present. Most newly hatched immatures climbed to the underside of the roof, where emerged adults also sheltered. Individuals can be easily separated from the smooth Parafilm at any developmental stage on a fine-point brush. The results show that an artificial arena with a folded Parafilm roof is suitable for rearing G. liturivorus. The method may also be suitable for use in risk assessment of chemical pesticides with G. liturivorus.

The wettability of phosphonium benzene sulfonate on parafilm has been investigated by means of dynamic contact angle measurement. Based on the surface tension data, the spreading coefficient, adhesion tension (γlg cosθ), and adhesion work (WA) were further analyzed. It was revealed that the surface tension and contact angle decreased with the increase in concentration, and remained constant when the concentration was higher than the critical micelle concentration (CMC). Furthermore, the spreading coefficient increased with increase in concentration then stays unchanged. In the studied concentration range, the value of spreading coefficient is negative, indicating that it cannot rapidly spread the surfactant on the parafilm surface to an uniform film. The measurements show a linear relationship between the adhesion tension γlg cosθ and the surface tension in the concentration range of 2 × 10−6 mol L−1 to 1 × 10−4 mol L−1. The adsorbed amount of surfactant molecules at the parafilm-solution interface is lower than that at the gas-liquid interface. The value of WA was initially decreased then increased as the concentration was increasing and reaches a minimum value at the concentration near the CMC.

This paper describes the development of two microfluidic paper-based analytical devices (μPADs), one well-based and the other based on a lateral flow assay (LFA) configuration, to detect glucose via a colorimetric assay using the solid metal-organic framework (MOF) Zr-PCN-222(Fe), to encapsulate glucose oxidase (GOx). The well-based platform consisted of laminate sheets and multiple layers of wax-printed chromatography paper. Solutions of KI and glucose placed into the well flowed through the device and reacted with the GOx@MOF species sandwiched between the paper layers realizing a yellow-brown color. The LFA platform consisted of chromatography paper between parafilm and polyvinyl acetate (PVA) layers. GOx@MOFs spotted on the paper subjected to solutions of KI and glucose yielded a brown color. The devices were then dried, scanned, and analyzed yielding a correlation between average inverse yellow intensity and glucose concentrations. The development of these devices employing MOFs as biomimetic catalysts should further expand the applications of microfluidic technologies for sensors a variety of analytes.

This study investigated the effect of surface wettability, hardness and surface contact on the germination and subsequent development of Botryosphaeriaceae species conidia. Mean percent germination for isolates Neofusicoccum luteum MM558, N. luteum CC445, N. parvum G652 and Botryosphaeria dothidea 007 was highest on cellulose which was the most hydrophilic surface (72.2%, 76.9%, 52.4% and 74.9%, respectively), but did not differ between the other surfaces tested (glass, polystyrene and Parafilm M™). Germ tube growth for all isolates was least on cellulose and similar on the other surfaces. Cellulose agar assays indicated that mycelia could be utilising the cellulose as a food source. Germination assays on surfaces of different hardness showed no discernible patterns of germination or growth related to hardness. Further, there was no effect on germination of surface contact versus continuous shaking of conidial suspensions for isolate N. luteum MM558. These assays demonstrated that conidia of the Botryosphaeriaceae were able to germinate on a variety of surfaces with this flexibility indicative of their reported pathogenicity of different host tissues.

Allelopathy plays crucial roles in invasive plant viability and agricultural production systems. However, there is no well-established hands-on learning activity to teach the concept of allelopathy. Nor is there an activity which allows students to gain knowledge about glucosinolates and their corresponding enzyme, myrosinase, which are present in almost all Brassica crops. Lettuce germination was counted by the students from three different treatments including water treated with Parafilm sealing, horseradish treated with Parafilm sealing, and horseradish treated without Parafilm sealing. Additionally, lettuce root length was measured by students using ImageJ software from each treatment using pictures captured by students' smartphones. Students took an identical quiz as a pre-laboratory and a post-laboratory assignment. Their average scores on the pre-laboratory and post-laboratory quizzes were 3.14 and 6.56 out of 10, respectively, indicating the lab activity significantly improved students' understanding of allelopathy and glucosinolate-myrosinase system. In addition, students (n = 76) completed a survey post-laboratory to assess their self-efficacy. This simple and cost-effective laboratory activity improved students' knowledge and skill development as it made learning more inviting, meaningful, and fun. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):333-340, 2019.

Tissue culture and micrografting techniques are known as effective procedures to produce healthy rose rootstocks and high quality scions. In order to establish successful micrografting protocol for roses, several factors including different concentrations of Benzylamino-purine (BA) (0, 1, 1.5, 2 mg l−1) + 0.1 mg l−1 α-Naphthaleneacetic acid (NAA), sucrose concentrations (30, 50, 70 g l−1), types of grafting devices (aluminium foil, parafilm, paper bridge), and types of rose scions cultivars (Rosa hybrida cv. Red One, R. hybrida cv. Samurai) on two different types of native (Rosa canina) and commercial rose rootstocks (Rosa multiflora cv. Natal Briar) were examined. The high micrografting success (100%) was micrografting R. hybrida cv. Samurai scion on R. mutiflora cv. Natal Briar rootstock with the paper bridge as the best grafting device in the liquid Van der salm (VS) medium containing 2 mg l−1 BA + 0.1 mg l−1 NAA + 300 mg l−1 Casein Hydrolyzate + 2 g l−1 myo-inositol + 50 g l−1 sucrose. Treating the wounded explants (scions and rootstocks) with silver nitrate (50 mg l−1) during the micrografting process, increased the survival of micrografts. The micrografted plants were successfully rooted and well acclimatized. The present results provide the first efficient protocol to produce high successful micrografted rose plantlets in tissue culture. It is the first research protocol for micrografting of native and commercial roses by considering important factors relating to successful micrografting such as plant growth regulators, growth adjuvants, grafting devices, and medium ingredients.

Phoebe sheareri (Hemsl.) Gamble is a high-value timber tree species with a wide range of uses. It can also be applied for landscape afforestation considering its beautiful shape and luxuriant branches. Previous studies disclosed that Neofusicum parvum can cause ulceration and necrosis on the main stems of P. sheareri (Chen et al. 2019), and branch wilt of P. zhennan (Zhu et al. 2019). In September 2019, anthracnose was observed from P. sheareri leaves in Lishui, Zhejiang province, China. The diseased leaves are characterized by dark brown lesions. The infection usually starts from the leaf tip or edge, then the infected leaves turn yellow, wither and fall finally. The infection sometimes occurrs on the small twigs, causing the whole branch to wither. Plants from 15 plantations were surveyed, and the disease incidence was about 30%. A total number of 15 freshly infected leaves were collected and cut into small pieces (5×5 mm), sterilized in 75% ethanol solution for 30 s, and washed with sterile water three times. They were further sanitized in 2% sodium hypochlorite solution for 2 to 3 minutes and dried in sterile filter paper after being washed with sterile water another three times. Leaf pieces were transferred to potato dextrose agar (PDA) plates and incubated at 25℃ in the dark for 3 days. The fungal cultures were purified and the typical isolate (TJ01) was selected for further morphological characterization and DNA sequence comparison. The strain was initially grayish white, villous, and posteriorly grayish green, and produced orange red spore clumps. The spores are single celled after maturation, oblong, colorless, and the size ranges from 8.5-19.5 × 4.5-5.5 μm. Morphological characteristics of the obtained isolate are consistent with those in the genus of Colletotrichum. DNA of the isolate was extracted and sequence data obtained and compared with those downloaded from GenBank. The internal transcribed spacer (ITS), intronic glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), beta-tubulin (TUB2), and calmodulin (CAL) genes were partially amplified and sequenced using the primer pairs ITS-1 + ITS-4, GDF + GDR, ACT-512F + ACT-783R, T1 + Bt2b, CL1C + Cl2C, respectively (Fu et al. 2019). The resulted nucleotide sequences were individually subjected to BLAST searche in GenBank. The nucleotide sequences of ITS (MZ088144), GAPDH (MZ133607), ACT (MZ133608), TUB2 (MZ133609), CAL (MZ133610) of the isolate showed 99.83% similarity to ITS sequence (MN829453), 98.24% similarity to GAPDH sequence (MN525875), 98.94% similarity to ACT sequence (MK341539), 100% similarity to TUB2 sequence (MG657352), and 100% similarity to CAL sequence (MN525839). The phylogenetic tree was constructed using MEGA 7 based on the tandem of five genes (ITS-GAPDH-ACT-TUB2-CAL). The result revealed that the isolate resides in the clade of C. fructicola species. Inoculation was done on leaves of ten P. sheareri plants in the field to verify its pathogenicity. Three healthy leaves of each plant were surface sterilized with 75% ethanol and dried up. The leaves were punctured using a sterilized needle, inoculated with 5-mm-diameter PDA plugs excised from 7-day-old cultures, and wrapped with parafilm. Nine pieces of healthy leaves inoculated with sterilized PDA plugs were served as controls. Disease symptoms developed on all the C. fructicola-inoculated leaves 5 days after inoculation, and a yellow brown lesion became apparent 16 days later, whereas the control leaves remained asymptomatic. C. fructicola was reisolated from the lesions, but not from the control leaves, fulfilling the Koch's postulates. This fungus is a well-known pathogen and has led to anthracnose on many plant species globally. However, our study represents the first report of C. fructicola causing anthracnose on P. sheareri worldwide and its potential threat should be evaluated.

Mosquito colony maintenance in the laboratory is essential for research but presents logistical and ethical problems with the use of live animals for bloodfeeding. The Glytube is an artificial bloodfeeding system for mosquitoes that uses Parafilm-M® membrane and human blood to feed Aedes aegypti. This study evaluated the efficiency of Glytube with different types of membranes and chicken blood to feed Ae. aegypti and Ae. albopictus. We evaluated 2 artificial (thread seal tape [TST], Parafilm-M) and 2 natural membranes (pork, sheep intestine). The results for Ae. aegypti suggest that TST was the best membrane because it presented a high percentage of fed females (63%), a high average number of eggs per female (54.65), and an egg viability rate significantly similar to control (mouse). For Ae. albopictus, there was no significant difference between the membranes and the control; however, the use of TST is suggested due to the low cost and easy manipulation. The treatments that used chicken blood did not present significant differences in the egg viability when compared with the control. The Glytube functionality can be increased by replacing the Parafilm-M membrane by TST and human to chicken blood.

Free-standing films of graphene oxide (GO) are regarded as potential materials for numerous applications in different fields. However, fabricating macroscopic free-standing GO films with high throughput is a bottleneck in large-scale application of these films. Here we introduce a method suitable for the mass-scale fabrication of free-standing GO films by exploring paraffin film (Parafilm M®) as a sacrificial substrate. The method involves deposition of GO on a paraffin film and peeling of the paraffin film after swelling it with toluene to obtain free-standing film of GO. Furthermore, the GO films were reduced into highly conductive RGO films using a green reducing agent ascorbic acid. A strategy of treating GO films with CuSO4 solution prior to reduction is also designed to avoid the possible disintegration of GO films in ascorbic acid solution. A detailed comparative study on the properties of RGO films prepared via thermal and chemical treatment and through the combination of both is also reported. An investigation of the variation in the electrical conductivity of the RGO films based on the reduction method is also carried out. The hydrophobic nature of the thermally reduced RGO films is explored for the oil/water separation.

A reusable and simple to fabricate electrochemical sensor for the detection of glucose and acetylthiocholine using thread-based electrodes and nylon thread is described. The fabrication of the device consisted of two steps. First, three nylon-based electrodes (reference, working, and counter) were painted with one layer of conductive inks (silver and carbon ink, or silver/silver chloride ink). The electrodes were taped onto parafilm, and a piece of white nylon thread was wrapped around each electrode connecting the three electrodes. For the glucose system, a PBS solution containing glucose oxidase (GOx) (10 mg/mL), and potassium ferricyanide (K3 [Fe(CN)6 ]) (10 mg/mL) as mediator, was dried onto the thread, and increasing concentrations of glucose (0-15 mM) was added to the thread and measured by cyclic voltammetry (CV). The current output from the glucose oxidation was proportional to the concentration of glucose. For the second system, a solution of acetylcholinesterase (AChE) (0.08 U/mL) in PBS was added to the nylon thread, and increasing concentrations of acetylthiocholine (ATC) (0-9.84 mg/mL) was added and measured by CV. The current output from the oxidation of thiocholine (produced by AChE reacting with ATC) was proportional to the concentrations of ATC added to the thread. From both systems, a graph of current output versus substrate concentration was produced and fitted with a linear regression line that gave R2 values of 0.985 (GOX /glucose) and 0.995 (AChE/ATC).

This study screened four Rhizopus species as biosurfactant producers using different markers. First of all, Rhizopus spp. UCP 1607 was identified as Rhizopus arrhizus by morphological and molecular methods. The production of biosurfactant/bioemulsifier was investigated by submerged fermentation using soybean post-frying oil (5% v/v) and sodium glutamate (1% w/v) medium. The primary markers’ hemolysis and parafilm M tests showed that R. arrhizus UCP1607 strain exhibited higher hemolytic activity (49 mm of clear zone) on sheep blood agar and a larger drop diameter (12 mm) on parafilm hydrophobic surface. The experimental results showed the most promising biosurfactant production by R. arrhizus UCP 1607 strain led to a reduction of surface tension (31.8 mN/m) and the diameter of the oil-spreading covered an area of 66.4 cm2. The strains Rhizopus microsporus var. chinensis UCP1296, R. microsporus var. microsporus UCP1304, and R. arrhizus UCP1607 were capable of forming stable emulsions corresponding to 91.7, 94.8, and 82.6%, respectively in crude oil. Key words: Tensio-active agent, bioemulsifier, screening of Rhizopus strains, submerged fermentation.

Numerous fabrication methods have been reported for microfluidic paper-based analytical devices (μPADs) using barrier materials ranging from photoresist to wax. While these methods have been used with wide success, consistently producing small, high-resolution features using materials and methods that are compatible with solvents and surfactants remains a challenge. Two new methods are presented here for generating μPADs with well-defined, high-resolution structures compatible with solvents and surfactant-containing solutions by partially or fully fusing paper with Parafilm® followed by cutting with a CO2 laser cutter. Partial fusion leads to laminated paper (l-paper) while the complete fusion results in infused paper (i-paper). Patterned structures in l-paper were fabricated by selective removal of the paper but not the underlying Parafilm® using a benchtop CO2 laser. Under optimized conditions, a gap as small as 137 ± 22 μm could be generated. Using this approach, a miniaturized paper 384-zone plate, consisting of circular detection elements with a diameter of 1.86 mm, was fabricated in 64 × 43 mm2 area. Furthermore, these ablation-patterned substrates were confirmed to be compatible with surfactant solutions and common organic solvents (methanol, acetonitrile and dimethylformamide), which has been achieved by very few μPAD patterning techniques. Patterns in i-paper were created by completely cutting out zones of the i-paper and then fixing pre-cut paper into these openings similar to the strategy of fitting a jigsaw piece into a puzzle. Upon heating, unmodified paper was readily sealed into these openings due to partial reflow of the paraffin into the paper. This unique and simple bonding method was illustrated by two types of 3D μPADs, a push-on valve and a time-gated flow distributor, without adding adhesive layers. The free-standing jigsaw-patterned sheets showed good structural stability and solution compatibility, which provided a facile alternative method for fabricating complicated μPADs.

This paper presents a study aiming to verify the accuracies of various bulk density measurement methods for open-graded and gap-graded asphalt mixtures using X-ray Computed Tomography (CT) scanning. The industrial X-ray CT scanning was performed on laboratory prepared open-graded and gap-graded mixture specimens. The histograms of gray values from the CT scanning images showed distinct peaks of air voids and asphalt materials, based on which the threshold value for calculating the voxels of air voids was determined by the ISO50 threshold method. In this way, the actual air void contents of the testing specimens were determined successfully and then compared to the values calculated from the bulk densities measured using the selected methods, including the dimensional analysis, automatic vacuum sealing, Parafilm coating and saturated surface dry methods. The results from the statistical analyses showed that for the open-graded mixture, only the automatic vacuum sealing method produced air void contents, which were not significantly different from the CT scanning results. For the gap-graded mixture, neither the results from the automatic vacuum sealing method nor those from the Parafilm coating method showed significant difference compared with the CT scanning results.

Sacha inchi (Plukenetia volubilis L.) belongs to the family Euphorbiaceae. It is a perennial wooden oilseed crop, and also exhibits a good source of polyunsaturated fatty acids, protein and other bioactive compounds, such as tocopherols, carotenes and phytosterols (Chirinos et al. 2013). During 2017-2018 survey, canker disease showing greyish-brown sunken lesions was observed on the branches of sacha inchi in Danzhou campus, Hainan University, China. The disease incidence is less than 5%. However, it can lead to leaf yellowing, wilt, and eventually the whole plant death. In Nov. 2017, twelve branches showing the typical canker symptoms were collected and covered with parafilm at both ends of all samples to prevent desiccation and placed in black plastic bags keeping at 4°C until isolations were made. Samples were rinsed with tap water and dried with paper towels. Fragments, 5mm in length and cut from the junction of diseased and healthy parts of branches, were surface-sterilized with 2% sodium hypochlorite solution for 2 min, rinsed with sterilized distilled water for 5 times, dried by sterilized filter paper, plated on PDA medium amended with 100 μg/mL streptomycin (PDA-str) and incubated in the dark for 4 days at 28°C. Pure cultures of fungal isolates were obtained by transferring mycelial fragments from colony margins onto fresh PDA plates and incubated as described before. The colonies of cultures were initially white, and eventually turned black after 4 days on PDA medium (Fig S1A). The morphology characterization of conidia produced by the isolates was initially hyaline and aseptate (Fig S1B), and a single median septum formed in the mature conidia (Fig S1B). The average size of 50 conidia was 16.39±1.46ⅹ 8.52±0.92μm for J6, and 15.64±1.73ⅹ 8.94±0.86μm for J3. Three genes were used for phylogenetic analysis (Alves et al. 2006). ITS regions and the partial of TUB (β-tubulin gene) were amplified using the primer pairs ITS1 and ITS4 (White et al. 1990), Bt2a and Bt2b (Glass and Donaldson 1995), respectively, and EF1-688F/EF1-1251R for J3 and EF1-728F/EF1-986R for J6 were used to amplify TEF (translation elongation factor 1-alpha) (Alves et al. 2008). The sequences of ITS, TUB and TEF from J3 and J6 were deposited in Gene-Bank (Table S1). The blast searches in Gene-Bank with ITS, TUB and TEF amplified from isolates J3, respectively, revealed 100, 99, and 100% identities with L. pseudotheobromae, and isolate J6 showed 100, 100 and 99% of identity with L. theobromae. The phylogenetic analysis of the combined ITS, TUB and TEF sequences of J3, J6 and 28 reference strains retrieved from Gene-Bank was performed using the program MEGA 6.0 evaluated by 1000 bootstrap replications, and the result was consistent with the conclusion above (Fig S2). With the phylogenic studies supported by morphological characters, J3 was identified as L. pseudotheobromae and J6 was L. theobromae. For the pathogenicity test, J3 and J6 were used to inoculate 4-week-old healthy sacha inchi potted seedlings. One wound about 5 mm in depth per seedling stem was made using a sterile blade. A 5-mm-diameter mycelium plug of each isolate taken from the edge of 4-day-old culture growing on PDA was placed to the freshly wound of each plant stem and the inoculated area was wrapped with Parafilm. Sterile PDA plugs were placed onto the wounds of control seedlings. Nine healthy seedlings were inoculated with each isolate or PDA plugs in a completely randomized design. After inoculation, plants were placed in a greenhouse at room temperature (26 to 30°C, 80% RH) and were irrigated when needed. The experiment was conducted twice. Five days later, black or dark-brown canker lesions formed on the stems of inoculated plants, and expended upward and downward from the inoculation points. Pycnidia produced on the necrotic regions and were used to to observe the morphology of conidia (Fig S3). The fungus L. pseudotheobromae or L. theobromae can be re-isolated from the inoculated plants, but not from the control ones. L. pseudotheobromae was recorded to be collected from dead leaves of P. volubilis in Yunnan Province, China, but did not prove this fungus to be pathogenic (Tennakoon et al. 2016). This is the first report that L. theobromae and L. pseudotheobromae causing stem canker in sacha inchi in Hainan, China. The results pave the way for the development of management strategies for canker disease in sacha inchi.