Clean Bench Research Articles

Assessing the stability of daptomycin in icodextrin-based peritoneal dialysis solution.

The stability of antimicrobials in peritonitis during peritoneal dialysis (PD) solutions is a critical factor influencing treatment success. This study investigated the stability of daptomycin (DAP) when combined with icodextrin-based PD solution, by measuring DAP concentrations and observing any structural changes. A dose of DAP (350 mg) was dissolved in 7 mL of saline in a clean bench. The solution was then injected into the large compartment (1,260 mL) of NICOPELIQ® Peritoneal Dialysis Solution and thoroughly mixed. Samples were collected at intervals ranging from 0 to 336 h (7 points in total). The concentration of DAP was quantified using high-performance liquid chromatography (HPLC). The structure of any unidentified peaks was determined using HPLC coupled with electrospray ionization tandem mass spectrometry. DAP maintained 90% of the initial concentration in NICOPELIC® for 72 h at room temperature and 12 h at 37 °C. Unidentified peaks, distinct from DAP, were detected during analysis. Further investigation indicated that these peaks corresponded to anhydrated DAP. The findings from this stability study are expected to enhance the effectiveness of outpatient management and preparation for treating peritonitis using DAP.

Sorting differentiated mammalian cells using deterministic lateral displacement microfluidic devices.

Separation of differentiated and undifferentiated cells without labeling is required for cell analyses and clinical application of cultured differentiated cells in vitro. To proceed with the passive separation of differentiated cells inside a clean bench, we developed a system of deterministic lateral displacement (DLD) microfluidic devices and applied this system to sort differentiated cells in vitro. The fluid flow is driven by compressed air to the buffer. Priming and sorting can be completed by air pressure control. We use this system to separate C2C12 mononuclear myocytes from multinuclear myotubes. Additionally, using a DLD microfluidic channel of Dc = 20μm, multinuclear myotubes can be effectively sorted as larger particles. We prepared differentiated adipocytes from mouse embryonic fibroblast (MEF) cells and sorted those containing lipid droplets. The diameters of these sorted adipocytes considered larger particles, exceeded 20μm, similar to the Dc of the DLD microfluidic channel. Differentiated cell sorting by cell size will contribute to single-cell analyses and in vitro tissue model preparation for drug discovery.

First Report of Brown Rot Disease on Cultivated Auricularia cornea Caused by Trichoderma pleuroticola in Sichuan Province, China.

First Report of Brown Rot Disease on Cultivated Auricularia cornea Caused by Trichoderma pleuroticola in Sichuan Province, China.

First Report of Pulp Rot in Externally Asymptomatic Pomegranate Fruit Caused by Talaromyces albobiverticillius in Henan Province, China.

As a popular deciduous fruit tree, pomegranate (Punica granatum L.) is grown from tropical to temperate zones worldwide, therein China has at least 120000 hm2 cultivation area. In August 2020, severe pulp rot occurred in the externally asymptomatic pre-harvest pomegranate fruit on a 3-year-old soft-seeded variety (Tunisia) in the Zhanghe village (32º40´34˝N, 111º44´20˝E) of Jiuchong township, Xichuan county in Henan province, China with 6.4-20 (av. 12.6) % pulp rot incidence evaluated from 11 freshly sampled fruits (360 pulps per fruit investigated). The fruits showed no external symptoms, however, browning occurred on part of their pulps before harvest compared to the normal ones with white or pink color. The surface of the externally asymptomatic fruits was sterilized with 75% ethanol, and air-dried in a clean bench. The surface-disinfected fruits were dissected with a sterilized knife. Brown pulps from the fruits were picked up using flame-sterilized tweezers and placed on potato dextrose agar (PDA) plates. After five days of incubation at 28 °C, pure fungal cultures with similar phenotypic features developed from the affected pulps. Two randomly selected isolates Tp-2 and Tp-8 were used for the study. The colony surface of the isolates was greyish-green with claret-red exudates. Claret-red pigments were commonly secreted into the medium from the colonies. Conidia were unicellular, hyaline to greyish, mostly rugby ball-shaped with a dimension of 2.2-3.5 (2.7) µm × 1.6-2.0 (1.8) µm (n=50) for Tp-2, and 2.2-3.1 (2.6) µm × 1.6-2.2 (1.8) µm (n=50) for Tp-8. The rDNA internal transcribed spacer (ITS) and β-tubulin gene sequences of the isolates were amplified with primers ITS1/ITS4 and Bt2a/Bt2b, respectively. Sequences were submitted to GenBank with accession numbers MW132153 and MW132077 for the rDNA-ITS sequences, and MW507822 and MW507823 for the β-tubulin gene sequences of Tp-2 and Tp-8, respectively, with a maximal sequence identity greater than 99 % to multiple strains of Talaromyces albobiverticillius (TA) based on BLAST analyses. In the Neighbor-joining phylogenetic trees constructed using rDNA-ITS and β-tubulin gene sequences, both Tp-2 and Tp-8 formed a clade with mutiple strains of TA, clearly separated from other Talaromyces spp. Conidial suspensions (106 spores ml-1) of Tp-2 and Tp-8 were separately injected into five pomegranate fruits (Tunisia) sampled from an orchard free of the disease with a sterilized syringe. Five fruits inoculated with sterilized water were used as control (CK). The inoculated fruits were incubated at 25 °C for 10 days and cut out through the inoculated sites. Pulp rot symptoms occurred in the Tp-2/Tp-8-inoculated fruits, being similar to the naturally affected pulps. The CK pulps remained symptomless during the inoculation tests. Fungal cultures with the same phenotypic features as the inocula were constantly isolated from the brown pulps of the inoculated fruits, verifying both Tp-2 and Tp-8 as the causal agents of the disease based on Koch's postulates. During a long-term (30-40 days) storage at ambient conditions, fruits sampled from affected orchards developed brown lesions on their peels from which TA cultures could be isolated. TA was reported as the pathogen causing postharvest fruit rot on pomegranate in Italy (Mincuzzi et al. 2017). This is the first report of TA causing pulp rot in the externally asymptomatic pomegranate fruit in China.

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Measurement of lactose concentration in milk by using engineered bacteria producing lycopene

Lycopene is an orange–red carotenoid, which confers a visual phenotype to assess genetic transformation of microorganisms. In this study, the lycopene synthesis pathway was constructed in engineered Escherichia coli BL21 (DE3) by transforming plasmid pET-15b-crtBEI, wherein crtB, crtE, and crtI could be expressed under the control of the T7 promoter and lacO operator and lycopene could be accumulated in the engineered bacteria upon induction by lactose. A good linear relationship was observed between the lycopene content in engineered bacterial culture and lactose concentration in the range of 4–52 g/L; using this relation, the lactose concentration in milk could be determined. This method could be used to overcome several limitations of the high-performance liquid chromatography (HPLC) method for lactose detection, such as cumbersome sample preparation and expensive detection equipment. Moreover, this method required only a clean bench, shaker, and spectrophotometer for lactose analysis. Additionally, no significant difference was observed between this method and HPLC in terms of lactose measurement in milk, indicating that this method is reasonable and simple.

Bacteria in Surgical Smoke: A Self-controlled Laboratory Study Using Porcine Spinal Tissues.

A laboratory study performed in a self-controlled fashion. The aim of this study was to examine whether "surgical smoke" generated by using the electrocautery on porcine spinal tissues could contain viable bacteria. The year 2020 has seen a surge in legislation regarding surgical smoke evacuation in the operating room across the United States. Surgical smoke contains numerous toxic and carcinogenic substances, which spine surgeons and operating room staff are regularly exposed to. Although the smoke's chemical toxicity has been extensively studied, little is known about its biohazard level. The electrocautery was used to "operate on" 20 segments of porcine spinal tissues on a clean bench. In each operation, the generated smoke was captured with a swab, which was positioned above the surgical site and within the smoke, to obtain bacteria cultures. Two other swabs-a tissue swab (swabbing the porcine tissue itself) and a blank swab (swabbing an empty sterile bowl)-were collected as controls. The culture results were compared among the three groups. An additional experiment was conducted to see whether using a mask can reduce the "infection rate" of the swab. Although all blank swabs tested negative, 95% (19/20) of the smoke swabs tested positive, with a total of nine bacteria species identified. The most prevalent species observed in the smoke swabs included Serratia liquefaciens, Lactococcus garvieae, and Hafnia alvei, and so on. Among the positive smoke swabs, 84% (16/19) had one or more bacteria species that were consistent with the cultures of their corresponding tissue swabs. Using a surgical or N95 mask reduced the "infection rate" of the swab. "Surgical smoke" generated by electrocauterization of porcine spinal tissues contains viable bacteria. Further research in actual spine surgery is needed next.Level of Evidence: N/A.

Evaluation of the Usefulness of Postoperative Analgesic Solution Preparation by Pharmacists in the Surgical Department

Pharmacists began preparing drug solutions intraoperatively for postoperative analgesia in the Department of Surgery at Chiba University Hospital from May 2014. To verify the usefulness of pharmacists preparing these drug solutions, we conducted a questionnaire survey among 51 anesthesiologists and received 44 responses (recovery rate 86.3%). Burden on the anesthesiologists was significantly reduced both temporally and mentally when the pharmacists prepared the drug solutions compared with when the anesthesiologists did (p<0.01). The anesthesiologists' degree of anxiety about sometimes having to prepare drug solutions alone without any confirmation was also significantly reduced when pharmacists prepared them (p<0.01), which implies the need for a double-check system. In addition, 88.6% of anesthesiologists said that they were reassured with preparations done by the pharmacists under a sterile environment using a clean bench. Overall, 88.6% of anesthesiologists responded that they were satisfied with the preparation of drug solutions by pharmacists. Based on the results of this survey, pharmacists' preparation of drug solutions for postoperative analgesia is considered to be useful in ensuring the quality and safety of medical care because it reduced anesthesiologists' work to prepare the drug solutions, allowing them to concentrate on anesthesia and related work, it established a double-check system between the two staff teams, and it was done under a sterile environment.

Extemporaneous compounding: Selective pharmacists with separate skill

Extemporaneous compounding takes place in community and hospital pharmacies. There are usually specialist compounding pharmacies in major towns and cities, but any pharmacy may undertake compounding as long as they have appropriate facilities according to state-based legislation (e.g. allocated clean bench, specific compounding equipment). Although development is a continuous process, companies are customizing features to meet the majority of patient needs, but the very nature of the process cannot meet all patient needs. The risk-benefit ratio of using traditionally compounded medicines is favorable for patients who require specialized medications that are not commercially available, as they would otherwise not have access to suitable treatment. However, if an FDA-approved drug is commercially available, the use of an unapproved compounded drug confers additional risk with no commensurate benefit. Published reports of independent testing by the FDA, state agencies, and others consistently show that compounded drugs fail to meet specifications at a considerably higher rate than FDA-approved drugs. Compounded sterile preparations pose the additional risk of microbial contamination to patients. In the last 11 years, three separate meningitis outbreaks have been traced to purportedly ‘sterile’ steroid injections contaminated with fungus or bacteria, which were made by compounding pharmacies. The 2012 outbreak has resulted in intense scrutiny of pharmacy compounding practices and increased recognition of the need to ensure that compounding is limited to appropriate circumstances. Purpose of the Study: The article aims to physico-chemical and economic considerations before compounding; factors and quality control issues; compounding support, training, chemical supplies, types of compounding (specially in hospital and ambulatory care compounding). It should aid to practice the extemporaneous preparation of basic and advanced formulations including pharmacopoeial and

Effect of Pulse Interval on Promoting Secretion of Neurite-Inducing Factor from Glioma Cells in the Application of Nanosecond High Electric Field Pulses

1. Purpose We previously discovered and reported that the application of nanosecond high electric field pulses transiently increased intracellular Ca2+ concentration in glioma cells to promote the secretion of neurite-inducing factor. Moreover, it has been clarified that the electric field strength, the number of pulse application, and the pulse width have a strong effect on the secretory ability as a condition for promoting the secretion of these neurite-inducing factors. In this presentation, we further report that even nanosecond pulses with a weak electric field strength which does not cause the increase of intracellular Ca2+ concentration with a single pulse can induce the intracellular Ca2+ increase and neurite-inducing factor secretion from glioma cells by applying multiple pulses with short intervals. The considered mechanism also will be discussed in this presentation. さらに表示 2. Experimental method Rat glioma C6 cells were seeded on an ITO electrode and cultured overnight to adhere. To monitor intracellular Ca2+ concentration the cells were treated with a medium containing Fura-2 AM for 30 minutes to introduce the Ca2+ indicator into the cells. After washing the cells on the electrode, Hanks Balanced Salt Solution (HBSS) was put in the chamber, and an aluminum plate electrode serving as a counter electrode was faced to ITO electrode with a spacer in thickness of 30 µm, and set on the stage of an inverted fluorescence microscope. After two minutes from the start of measurement, nanosecond electric field pulse was applied in various voltage and pulse interval, and changes in intracellular Ca2+ concentration were observed. Furthermore, thapsigargin and 2-aminoethoxydiphenylborane (2-APB) were used to confirm that the increase in intracellular Ca2+ concentration may be due to Ca2+ release from the endoplasmic reticulum via IP3 receptors. In addition, after applying a nanosecond high electric field pulse to C6 cells in the same manner in a clean bench, the HBSS was immediately replaced with DMEM medium containing 10% FBS, and the cells were cultured for 1 day or 2 days. The culture supernatant (Conditioned Medium: CM) of the C6 cells was then collected, and exchanged completely with the medium of PC12 cells, which is a neural progenitor cell model. The morphological change of PC12 cells was observed and recorded for 6 days. The PC12 cells which extended the neurites more than twice as the cell body were recognized as neuronal differentiated cells. By calculating the differentiation rate of PC12 cells, it was evaluated how the secretion of the neurite-inducing factor from C6 cells was promoted or not. 3. Results and discussion Three pulses with a pulse width of 140 ns and an electric field strength of 23 kV / cm were applied at intervals of 2 seconds, 30 seconds, and 1 minute to C6 cells and intracellular Ca2+ concentration changes and neurite-inducing factor secretion was examined. It was demonstrated that intracellular Ca2+ gradually increased at shorter intervals of 2 seconds and 30 seconds, but not increased at intervals of 1 minute. In addition, intracellular Ca2+ concentration increase was observed even when pulse application was done in the absence of extracellular Ca2+. It indicated that this increase in intracellular Ca2+ concentration is due to Ca2+ release from organelles. Furthermore, intracellular Ca2+ concentration elevation was not induced by the same pulse application after treatment with thapsigargin or 2-APB. Therefore, it was considered that Ca2+ was released from the endoplasmic reticulum via IP3 receptors by the pulse application in this condition. Figure 1 (a) and (b) show images of PC12 cells cultivated in the CM of C6 cells which were exposed to three pulses with 1 minute or 2 seconds intervals, respectively. From these results, it was demonstrated that the secretion of the neurite-inducing factor was promoted by the multi-pulse application with short intervals. Furthermore, it was found that the Ca2 + release via IP3 receptors of the endoplasmic reticulum and following promotion of the secretion of neurite-inducing factor could be induced by shortening the nanosecond multi-pulse interval in the application of weak electric field. Now, we are planning to clarify what kind of gene expression in C6 cells is induced by pulse application to identify and quantify the secreted neurite-inducing factor. Figure 1

First Report of Fusarium fujikuroi Causing Leaf spot of Dalbergia odorifera T.Chen in China.

Dalbergia odorifera T. Chen is a national second-grade protected and one of the four famous trees in China, with high medicinal and economic value. Leaf spot disease in this plant can cause the leaves to dry up, perforate or even fall off, which affects the growth and development, and also has a great influence on its products. In May 2019, the leaf spot of Dalbergia odorifera T. Chen was found and observed in Chengmai County (N19°40', E110°0'), Hainan Province, China, and the symptomatic leaves were brought back to the laboratory for research; According to our survey at that time, the incidence of the disease was between 10% and 15%. A sterile stainless-steel scalpel was used to cut the tissues at the junction of the leaf lesions and placed on a clean bench, soaked in alcohol (75 %) for 30 s, and rinsed thrice with sterile water. Then it was inserted obliquely onto lactic acid-containing potato dextrose agar (PDA) and incubated at 28 °C for 5 days. The growing prominent colonies were singled out and re-inoculated on PDA and SNA plates. Preliminary identification was based on morphological characteristics, followed by molecular identification of strains by evaluating genes for translation elongation factor-1α(TEF-1α), beta-tubulin, mitochondrial small subunit (mtSSU)( Duan et al. 2019; Cao et al.2019; Stenglein et al.2010), and histone H3 (Jacobs, et al. 2010) . Through morphological observation, the isolate was identified as Fusarium fujikuroi. At the initial stage of growth on PDA, the strain produced a large number of white hyphae, followed by pink and purple-brown hyphae in the center of the colony which spread to the surrounding area. The microspores were abundant, colorless, elliptic or clavate, without septum or at 1-2 septate, and the size was about 3.3 to 13.5 × 1.2 to 3.2 µm. After nine days of culturing on SNA medium, few, large conidia were observed, typically sickle-like, with 3-4 septa with a size of about 20 to 40.2 × 2.3 to 4.4 μm. The identity of the strains was determined by comparing the gene sequences of TEF-1α, mtSSU, beta-tubulin and histone H3 by NCBI BLAST. The results showed that TEF-1 α (MN958396), mtSSU (MN958394), β - tubulin (MN958395), and histone H3 (MN958397) from the target strain (jxht0302) had 100% sequence homology with F. fujikuroi (GenBank, accession numbers KF604040.1, MF984420.1, XM023575231.1, and MF356523.1 respectively). Next, the infection of D. odorifera T. Chen seedlings with and without injury was studied using a fungus block, with PDA as a control. Two days after inoculation with injury, obvious lesions were observed on the leaves, which appeared at least 5 days post- inoculation without injury, with no lesions in the control group. F. fujikuroi could be re-isolated from the leaves with lesions, but not from the control group. F. fujikuroi causes Black Rot of Macleaya cordata and maize ear rot (Yull et al.2019; Duan et al. 2019). As far as we know, this is the first report of F. fujikuroi causing leaf spot disease of D. odorifera T. Chen. Given the importance of D. odorifera T. Chen products, this disease needs more attention to tackle it.

Analysis of the influence of flame sterilization included in sampling operations on shake-flask cultures of microorganisms

Shake-flask cultures of microorganisms involve flame sterilization during sampling, which produces combustion gas with high CO2 concentrations. The gaseous destination has not been deeply analyzed. Our aim was to investigate the effect of flame sterilization on the headspace of the flask and on the shake-flask culture. In this study, the headspace CO2 concentration was found to increase during flame sterilization ~0.5–2.0% over 5–20 s empirically using the Circulation Direct Monitoring and Sampling System. This CO2 accumulation was confirmed theoretically using Computational Fluid Dynamics; it was 9% topically. To evaluate the influence of CO2 accumulation without interference from other sampling factors, the flask gas phase formed by flame sterilization was reproduced by aseptically supplying 99.8% CO2 into the headspace, without sampling. We developed a unit that can be sampled in situ without interruption of shaking, movement to a clean bench, opening of the culture-plug, and flame sterilization. We observed that the growth behaviour of Escherichia coli, Pelomonas saccharophila, Acetobacter pasteurianus, and Saccharomyces cerevisiae was different depending on the CO2 aeration conditions. These results are expected to contribute to improving microbial cell culture systems.

Minimal microfabrication required digital microfluidic system toward point-of-care nucleic acid amplification test application for developing countries

Digital Microfluidics (DMF) has potentially been a favorable platform for point-of-care molecular diagnostics. However, its fabrication process normally relies heavily on expensive microfabrication facilities and equipment. In this work, we developed a portable, small footprint DMF device that requires minimal microfabrication work (only a small clean bench and a DIY spin coater were required) but is still capable of performing nucleic acid amplification tests. The DMF system implemented in this work could perform successfully the Loop-mediated isothermal Amplification (LAMP) reaction with samples containing extracted DNA of Plasmodium falciparum that causes Malaria in human. The LAMP reaction was conducted at the optimal temperature of 65 °C in 45 min with the total reaction volume of 1.5 µL. The results of LAMP were designed to be observable to naked eyes via the color change from pink to yellowish orange, which simplifies the readout step and eliminates the necessity for external and bulky result-reading equipment.

Extemporaneous Compounding: Selective Pharmacists with Separate Skill.

Extemporaneous compounding takes place in community and hospital pharmacies. There are usually specialist compounding pharmacies in major towns and cities, but any pharmacy may undertake compounding as long as they have appropriate facilities according to state-based legislation (e.g. allocated clean bench, specific compounding equipment). Although development is a continuous process, companies are customizing features to meet the majority of patient needs, but the very nature of the process cannot meet all patient needs. The risk-benefit ratio of using traditionally compounded medicines is favorable for patients who require specialized medications that are not commercially available, as they would otherwise not have access to suitable treatment. However, if an FDA-approved drug is commercially available, the use of an unapproved compounded drug confers additional risk with no commensurate benefit. Published reports of independent testing by the FDA, state agencies, and others consistently show that compounded drugs fail to meet specifications at a considerably higher rate than FDA-approved drugs. Compounded sterile preparations pose the additional risk of microbial contamination to patients. In the last 11 years, three separate meningitis outbreaks have been traced to purportedly 'sterile' steroid injections contaminated with fungus or bacteria, which were made by compounding pharmacies. The 2012 outbreak has resulted in intense scrutiny of pharmacy compounding practices and increased recognition of the need to ensure that compounding is limited to appropriate circumstances.

Continuous cell culture monitoring using a compact microplate reader with a silicone optical technology-based spatial filter.

Continuous cell monitoring is very important for the maintenance and control of cell multiplication and differentiation. This paper presents a compact microplate reader that is able to continuously measure a 24-well microplate (6 × 4 wells) using the optical absorption measurement method. The 24-channel plate reader consisted of a spatial filter, light emitting diode light source, and color sensors and was similarly sized with the cell culture microwell plates. A spatial filter was previously fabricated by our group using silicone optical technology (SOT). This SOT-based spatial filter has an excellent noise reduction effect. Light reflection at the optical path interface can be absorbed and only forward light can be transmitted; accordingly, a larger S/N ratio than that of conventional optical systems is expected. The fabricated 24-channel plate reader permits real-time cell monitoring during cultivation on the clean bench and in cell culture conditions by incorporating the SOT spatial filter. Using the device, it was possible to continuously evaluate the concentration and pH of reagents in the 24 wells in real time. Moreover, cell activity and protein production were detectable using the device. These results suggest that the newly fabricated device is a promising tool for the evaluation of cell behaviors for cell management.

In Vitro and In Vivo Evaluation of Titanium Surface Modification for Biological Aging by Electrolytic Reducing Ionic Water

In this study, using electrolytic reducing ionic water (S-100®), a novel surface treatment method safely and easily modifying the surface properties was evaluated in vitro and in vivo. Ti-6Al-4V disks were washed and the disks were kept standing on a clean bench for one and four weeks for aging. These disks were immersed in S-100® (S-100 group), immersed in ultra-pure water (Control group), or irradiated with ultraviolet light (UV group), and surface analysis, cell experiment, and animal experiment were performed using these disks. The titanium surface became hydrophilic in the S-100 group and the amount of protein adsorption and cell adhesion rate were improved in vitro. In vivo, new bone formation was noted around the disk. These findings suggested that surface treatment with S-100® adds bioactivity to the biologically aged titanium surface. We are planning to further investigate it and accumulate evidence for clinical application.

Rapid one-pot fabrication of magnetic calcium phosphate nanoparticles immobilizing DNA and iron oxide nanocrystals using injection solutions for magnetofection and magnetic targeting

This is the first report on rapid (5–30 min) one-pot fabrication of magnetic calcium phosphate (CaP) nanoparticles (NPs) co-immobilizing DNA and iron oxide (IO) nanocrystals with high immobilization efficiencies (DNA (∼90%) and IO (∼70%)). NPs were fabricated via coprecipitation under clean bench in supersaturated CaP solutions using DNA, IO-based MRI contrast agent, and infusion quality source solutions (even the water used was of injectable quality) that ensured high safety level of the fabrication process and product. Prepared DNA-IO-CaP NPs exhibited strong magnetic property allowing their noncontact manipulation by external magnet. These NPs were smaller than 500 nm, had relatively large negative zeta potential showing stable dispersion without any additional surfactant, and exhibited no significant cytotoxicity under the tested transfection conditions. The IO content of these NPs was significantly varied and by adjusting the initial IO concentration, DNA-IO-CaP NPs with gene delivery capability to CHO-K1 cells as high as that of the IO-free DNA-CaP NPs without external magnetic field were prepared. Fabricated DNA-IO-CaP NPs showed significantly improved gene delivery capability under external magnetic field compared to the IO-free DNA-CaP NPs. Thus, the present coprecipitation process can be considered as a novel technique to fabricate multifunctional CaP-based NPs to achieve magnetofection and targeted delivery.

Assuring quality in microplastic monitoring: About the value of clean-air devices as essentials for verified data

Avoiding aerial microfibre contamination of environmental samples is essential for reliable analyses when it comes to the detection of ubiquitous microplastics. Almost all laboratories have contamination problems which are largely unavoidable without investments in clean-air devices. Therefore, our study supplies an approach to assess background microfibre contamination of samples in the laboratory under particle-free air conditions. We tested aerial contamination of samples indoor, in a mobile laboratory, within a laboratory fume hood and on a clean bench with particles filtration during the examining process of a fish. The used clean bench reduced aerial microfibre contamination in our laboratory by 96.5%. This highlights the value of suitable clean-air devices for valid microplastic pollution data. Our results indicate, that pollution levels by microfibres have been overestimated and actual pollution levels may be many times lower. Accordingly, such clean-air devices are recommended for microplastic laboratory applications in future research work to significantly lower error rates.

Corynebacterium guangdongense sp. nov., isolated from a contaminated plate

A novel Gram-reaction-positive, non-motile and facultatively anaerobic bacterium, designated strain S01T, was isolated from a nutrient agar plate kept on a laboratory clean bench at Guangdong Institute of Microbiology, PR China, which was contaminated from an unknown source. Strain S01T was found to be catalase-positive and oxidase-negative. Similarity searches revealed that the strain shared the highest 16S rRNA gene similarity with Corynebacterium humireducens MFC-5T (95.9 %). However, phylogenetic analysis based on the 16S rRNA gene sequences showed that strain S01T was closely related to Corynebacteriumdoosanense JCM 17317T (94.8 %) and Corynebacterium maris JCM 17018T (94.8 %). The major fatty acids were C18:1ω9c, C16:0, 10-methyl C18:0 and C18:0. The respiratory quinones predominantly consisted of MK-8(H2), with small amounts of MK-8 and MK-9(H2). Polar lipids contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, an unidentified aminolipid, two unidentified glycolipids and two unidentified lipids. Mycolic acids were present. The cell-wall peptidoglycan contained meso-diaminopimelic acid and the major cell-wall sugars were galactose, arabinose and glucose. The genomic DNA G+C content of strain S01T was 70.7±0.1 mol%. The results of phenotypic, phylogenetic and chemotaxonomic analyses indicated that strain S01T represents a novel species of the genus Corynebacterium, for which the name Corynebacterium guangdongense sp. nov. is proposed. The type strain is S01T (=GDMCC 1.1022T=CCTCC AB 2015423T=KCTC 39608T).

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.