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BMC Biotechnology Aug 2023The in-vitro scratch assay is a useful method in wound healing research to assess cell migration. In this assay, a scratch is created in a confluent cell layer by...
BACKGROUND
The in-vitro scratch assay is a useful method in wound healing research to assess cell migration. In this assay, a scratch is created in a confluent cell layer by mechanically removing cells through manual scraping with a sharp-edged tool. This step is traditionally done with pipette tips and is unsuitable for high-throughput assays, as the created scratches are highly variable in width and position. Commercially available solutions are often expensive, and require specific cultureware which might not be suitable for all studies.
RESULTS
In this study, we have developed a flexible cell scratch device comprising a single wounding tool, a guide and an imaging template for consistent and reproducible scratch assays in 96-well plates. Our results showed that the device produced a more consistent scratch profile compared to the conventional method of using pipette tips. The imaging template also allowed operators to easily locate and image the same region of interest at different time points, which potentially could be used for other assays.
CONCLUSIONS
Our flexible yet effective scratch device thus enables robust scratch assays that can be applied to different experimental needs, providing researchers with an easy and reliable tool for their studies.
Topics: Research Design; Biological Assay; High-Throughput Screening Assays; Wound Healing
PubMed: 37641063
DOI: 10.1186/s12896-023-00806-5 -
Bio-protocol Nov 2023Measuring the action potential (AP) propagation velocity in axons is critical for understanding neuronal computation. This protocol describes the measurement of...
Measuring the action potential (AP) propagation velocity in axons is critical for understanding neuronal computation. This protocol describes the measurement of propagation velocity using a combination of somatic whole cell and axonal loose patch recordings in brain slice preparations. The axons of neurons filled with fluorescent dye via somatic whole-cell pipette can be targeted under direct optical control using the fluorophore-filled pipette. The propagation delays between the soma and 5-7 axonal locations can be obtained by analyzing the ensemble averages of 500-600 sweeps of somatic APs aligned at times of maximal rate-of-rise (dV/dtmax) and axonal action currents from these locations. By plotting the propagation delays against the distance, the location of the AP initiation zone becomes evident as the site exhibiting the greatest delay relative to the soma. Performing linear fitting of the delays obtained from sites both proximal and distal from the trigger zone allows the determination of the velocities of AP backward and forward propagation, respectively. Key features • Ultra-thin axons in cortical slices are targeted under direct optical control using the SBFI-filled pipette. • Dual somatic whole cell and axonal loose patch recordings from 5-7 axonal locations. • Ensemble averaging of 500-600 sweeps of somatic APs and axonal action currents. • Plotting the propagation delays against the distance enables the determination of the trigger zone's position and velocities of AP backward and forward propagation.
PubMed: 37969753
DOI: 10.21769/BioProtoc.4876 -
Light, Science & Applications Aug 2023The dynamics and structure of mixed phases in a complex fluid can significantly impact its material properties, such as viscoelasticity. Small-angle X-ray Photon...
The dynamics and structure of mixed phases in a complex fluid can significantly impact its material properties, such as viscoelasticity. Small-angle X-ray Photon Correlation Spectroscopy (SA-XPCS) can probe the spontaneous spatial fluctuations of the mixed phases under various in situ environments over wide spatiotemporal ranges (10-10 s /10-10 m). Tailored material design, however, requires searching through a massive number of sample compositions and experimental parameters, which is beyond the bandwidth of the current coherent X-ray beamline. Using 3.7-μs-resolved XPCS synchronized with the clock frequency at the Advanced Photon Source, we demonstrated the consistency between the Brownian dynamics of ~100 nm diameter colloidal silica nanoparticles measured from an enclosed pendant drop and a sealed capillary. The electronic pipette can also be mounted on a robotic arm to access different stock solutions and create complex fluids with highly-repeatable and precisely controlled composition profiles. This closed-loop, AI-executable protocol is applicable to light scattering techniques regardless of the light wavelength and optical coherence, and is a first step towards high-throughput, autonomous material discovery.
PubMed: 37596264
DOI: 10.1038/s41377-023-01233-z -
Neuroprotection Sep 2023Intracerebral delivery of agents in liquid form is usually achieved through commercially available and durable metal needles. However, their size and texture may...
OBJECTIVE
Intracerebral delivery of agents in liquid form is usually achieved through commercially available and durable metal needles. However, their size and texture may contribute to mechanical brain damage. Glass pipettes with a thin tip may significantly reduce injection-associated brain damage but require access to prohibitively expensive programmable pipette pullers. This study is to remove the economic barrier to the application of minimally invasive delivery of therapeutics to the brain, such as chemical compounds, viral vectors, and cells.
METHODS
We took advantage of the rapid development of free educational online resources and emerging low-cost 3D printers by designing an affordable pipette puller (APP) to remove the cost obstacle.
RESULTS
We showed that our APP could produce glass pipettes with a sharp tip opening down to 20 μm or less, which is sufficiently thin for the delivery of therapeutics into the brain. A pipeline from pipette pulling to brain injection using low-cost and open-source equipment was established to facilitate the application of the APP.
CONCLUSION
In the spirit of frugal science, our device may democratize glass pipette-puling and substantially promote the application of minimally invasive and precisely controlled delivery of therapeutics to the brain for finding more effective therapies of brain diseases.
PubMed: 37771648
DOI: 10.1002/nep3.20 -
Longitudinal diffusion barriers imposed by myofilaments and mitochondria in murine cardiac myocytes.The Journal of General Physiology Oct 2023Using optical and electrical methods, we document that diffusion in the cytoplasm of BL6 murine cardiomyocytes becomes restricted >20-fold as molecular weight increases...
Using optical and electrical methods, we document that diffusion in the cytoplasm of BL6 murine cardiomyocytes becomes restricted >20-fold as molecular weight increases from 30 to 2,000, roughly as expected for pores with porin channel dimensions. Bodipy-FL ATP diffuses >40-fold slower than in free water at 25°C. From several fluorophores analyzed, bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. Unbound fluorophores diffuse at 0.5-8 × 10-7 cm2/s (5-80 μm2/s). Analysis of Na/K pump and veratridine-modified Na channel currents suggests that Na diffusion is nearly unrestricted at 35°C (time constant for equilibration with the pipette tip, ∼20 s). Using multiple strategies, we estimate that at 35°C, ATP diffuses four to eight times slower than in free water. To address whether restrictions are caused more by protein or membrane networks, we verified first that a protein gel, 10 g% gelatin, restricts diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is less restricted than in intact myocytes. Notably, myofilaments extracted similarly from skeletal (diaphragm) myocytes are less restrictive. Solute diffusion in myocytes with sarcolemma permeabilized by β-escin (80 µM) is similar to diffusion in intact myocytes. Restrictions are strain-dependent, being twofold greater in BL6 myocytes than in CD1/J6/129svJ myocytes. Furthermore, longitudinal diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, VDAC1, than in WT CD1/J6/129svJ myocytes. Thus, mitochondria networks restrict long-range diffusion while presumably optimizing nucleotide transfer between myofilaments and mitochondria. We project that diffusion restrictions imposed by both myofilaments and the outer mitochondrial membrane are important determinants of total free cytoplasmic AMP and ADP (∼10 μM). However, the capacity of diffusion to deliver ATP to myofilaments remains ∼100-fold greater than ATP consumption.
Topics: Mice; Animals; Myocytes, Cardiac; Myofibrils; Mitochondria; Diffusion; Voltage-Dependent Anion Channels; Adenosine Triphosphate; Water
PubMed: 37555782
DOI: 10.1085/jgp.202213329 -
Journal of Mass Spectrometry and... Aug 2023Free thyroxine (FT4) measurement is one of the most requested tests in patient care for diagnosing and treating thyroid-related illnesses. Equilibrium dialysis (ED) is...
BACKGROUND
Free thyroxine (FT4) measurement is one of the most requested tests in patient care for diagnosing and treating thyroid-related illnesses. Equilibrium dialysis (ED) is considered the "gold standard" for FT4 measurement; however, several factors have a profound effect on the reliability of FT4 assays and require special consideration.
METHODS
In the current study, we focused on evaluating critical factors that could contribute to reporting errors, such as adsorption of thyroxine (T4) to labware surfaces, stability of serum samples, stock solutions, and calibrator storage conditions, as well as the solvents used to prepare T4 solutions.
RESULTS
The adsorption of T4 in ethanolic solutions and dialysates to labware surfaces can be reduced with the careful selection of pipette tips, test tubes, and 96-well plates. Adding pH modifiers to neat T4 solutions can improve its stability. FT4 in serum samples remains stable after exposure to four freeze-thaw cycles, 5 °C for 18-20 h, or -70 °C for a minimum of three years.
CONCLUSION
The presented study has demonstrated that the loss of analyte due to pre-analytical and analytical factors during operation of the FT4 reference measurement procedure (RMP) can be minimized by careful selection of all labware for sample preparation. It was found that the accuracy and imprecision of FT4 assays can be influenced by different types of dialysis devices, but acceptable alternatives to ED membranes were identified. This study demonstrates approaches to establish a FT4 method that is independent from specific suppliers and addresses critical pre-analytical and analytical factors important for FT4 measurements.
PubMed: 37449264
DOI: 10.1016/j.jmsacl.2023.06.001 -
Plant Disease Aug 2023Quinoa (Chenopodium quinoa Willd.) is a traditional food originally from the Andes Mountains in South America. It was first planted in China in 1987 and is grown in...
Quinoa (Chenopodium quinoa Willd.) is a traditional food originally from the Andes Mountains in South America. It was first planted in China in 1987 and is grown in Tibet, Gansu, and Qinghai provinces. In May 2021, 40% of 2-month-old quinoa plants in the 3.4 hm² experimental base of Qinghai University (36.7262° N, 101.7487° E) were found to have leaves with grey-brown subcircular spots (about 0.4 to 0.7 cm) with black dots (acervuli). Severely infected plants exhibited symptoms such as withered and stunted growth. The diseased-healthy junctions of infected leaves (0.5 cm) were cut out, disinfected with 3% NaClO for 1.5 min, washed three times with sterile water, dried, placed on water agar, and incubated at 25°C for 48 h. After sporulation was seen on the leaf surface, spore suspensions were prepared by placing conidia in sterile water using a pipette. Next, 200 μl of each spore suspension was spread on the surface of water agar and incubated at 25°C for 12 h. Single spores were selected under a stereomicroscope and cultured on potato dextrose agar (PDA) (Qi et al. 2022). The mycelium of two representative isolates (20DLMF-5-4-1 and 20DLMF-7-4-1) was grey-black with white edges and included a fluffy aerial mycelium. Conidia were unicellular, colorless, long ellipsoid or curved moon shaped, averaging 14.3 × 1.8 to 20.2 × 2.2 μm (n=100). The light brown appressoria were ovoid, averaging 8.5 × 5.2 to 7.7 × 4.1 μm (n=20). Spherical, dark brown acervuli were observed on the leaves, averaging 160 to 200 μm (n=20), and there were dark brown spiny bristles. The ITS, partial ACT, CHS, GAPDH and TUB2 genes were amplified from genomic DNA of the two isolates (Weir et al. 2012). Sequences were deposited in GenBank (accession no. OQ871595 to OQ871602 for ACT, CHS, GAPDH, and TUB2, and OQ860235 to OQ860236 for ITS) and showed over 99% identities with the corresponding sequences of C. spinaciae CBS125347 and CBS128.57 (Vu et al. 2019; Damm et al. 2009). Both isolates clustered with the type culture of C. spinaciae (CBS125347, CBS128.57), with 100% bootstrap support in the phylogenetic tree. Thus, according to the morphological and molecular characteristics, the two isolates were identified as C. spinaciae. Pathogenicity tests were conducted on 24 healthy, tender leaves of six 1-month-old quinoa plants, with three replicates (Yang et al. 2021). The leaves were gently scratched in 3-4 areas with a sterile needle. A conidial suspension (105 conidia/ml) of the two isolates was sprayed on these wounds. The control group was unscratched and sprayed with sterile water. The plants were incubated in a greenhouse at 25°C for 24 h in the dark and 7 days in the light. Tiny grey-brown spots appeared on day 3 (about 0.4 to 0.6 cm) and continued to enlarge until perforations and ruptures developed on day 7. Subsequently, acervuli were observed on the surface of the leaves. The control leaves remained healthy. Isolates were reisolated from the symptomatic leaves and they had the same morphological and molecular characteristics as the original isolates, confirming Koch's postulates. To our knowledge, this is the first report of C. spinaciae causing quinoa leaf anthracnose in China. C. spinaciae seriously affects the yield and quality of quinoa and has been previously reported to cause anthracnose of Vicia sativa in China (Wang et al. 2019). The results provide a basis for the study and control of quinoa leaf anthracnose.
PubMed: 37622274
DOI: 10.1094/PDIS-07-23-1285-PDN -
Scientific Reports Oct 2023In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk:...
In this study, our primary objective was to develop an effective analytical method for studying trypsin-digested peptides of two proteins commonly found in cow's milk: β-casein (βCN) and β-lactoglobulin (βLG). To achieve this, we employed two distinct approaches: traditional in-gel protein digestion and protein digestion using immobilized enzyme microreactors (μ-IMER). Both methods utilized ZipTip pipette tips filled with C18 reverse phase media for sample concentration. The μ-IMER was fabricated through a multi-step process that included preconditioning the capillary, modifying its surface, synthesizing a monolithic support, and further surface modification. Its performance was evaluated under HPLC chromatography conditions using a small-molecule trypsin substrate (BAEE). Hydrolysates from both digestion methods were analyzed using MALDI-TOF MS. Our findings indicate that the μ-IMER method demonstrated superior sequence coverage for oxidized molecules in βCN (33 ± 1.5%) and βLG (65 ± 3%) compared to classical in-gel digestion (20 ± 2% for βCN; 49 ± 2% for βLG). The use of ZipTips further improved sequence coverage in both classical in-gel digestion (26 ± 1% for βCN; 60 ± 4% for βLG) and μ-IMER (41 ± 3% for βCN; 80 ± 5% for βLG). Additionally, phosphorylations were identified. For βCN, no phosphorylation was detected using classical digestion, but the use of ZipTips showed a value of 27 ± 4%. With μ-IMER and μ-IMER-ZipTip, the values increased to 30 ± 2% and 33 ± 1%, respectively. For βLG, the use of ZipTip enabled the detection of a higher percentage of modified peptides in both classical (79 ± 2%) and μ-IMER (79 ± 4%) digestions. By providing a comprehensive comparison of traditional in-gel digestion and μ-IMER methods, this study offers valuable insights into the advantages and limitations of each approach, particularly in the context of complex biological samples. The findings set a new benchmark in protein digestion and analysis, highlighting the potential of μ-IMER systems for enhanced sequence coverage and post-translational modification detection.
Topics: Enzymes, Immobilized; Caseins; Lactoglobulins; Trypsin; Peptides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 37783762
DOI: 10.1038/s41598-023-43521-z -
Plant Disease Aug 2023Multiple Diaporthe spp. cause root and fruit rots or stem lesions on Cucumis spp.: (syn. ), , and (Broge et al. 2020; Fukada et al. 2018; Udayanga et al. 2012, 2015)....
Multiple Diaporthe spp. cause root and fruit rots or stem lesions on Cucumis spp.: (syn. ), , and (Broge et al. 2020; Fukada et al. 2018; Udayanga et al. 2012, 2015). From May-August 2021, cucumbers () 'Katrina' and 'Alcazar' were grown in a 24-plant, commercial Bato bucket system with rockwool blocks on a perlite substrate in a research greenhouse in Wooster, Ohio. At maturity, plants collapsed rapidly from stem lesions without foliar chlorosis (25% of 'Katrina' and 17% of 'Alcazar'). Lesions were 7.5 to 15 cm in length, tan to golden-brown with black pycnidia and located 5 to 15 cm above the crown. Stems shredded easily with vascular discoloration around the lesion. Two identical fungal strains were isolated on ½ acidified potato dextrose agar (APDA) following surface disinfestation with 0.6% sodium hypochlorite for 30 s and sterile water rinse. Fungal cultures were floccose, white to tan mycelia with pycnidia. Oblong, elliptical, biguttulate, aseptate alpha conidia were observed with mean dimensions: 8.0 µm (5.2-9.8 µm) by 3.1 µm (2.5-3.8 µm) on ½ APDA and 9.8 µm (6.6-12.4 µm) by 3.0 µm (1.9-5.3 µm) on petioles. On prune extract agar, beta conidia mean dimensions were: 19.7 µm (12.0-27.7 µm) by 1.2 µm (0.8-1.8 µm). Fungal DNA was amplified and sequenced bidirectionally with ITS (ITS4/ITS5), CAL (CAL228F/737R), HIS (CYLH3F/H3-1B), TEF1 (EF1-728f/EF1-986R), and TUB2 (Bt1a/Bt1b) primers (Carbone and Kohn 1999; Glass and Donaldson 1995) (GenBank: OP265712-13, OP288460-65, OQ418506-07). Based on a maximum likelihood phylogenetic tree of concatenated genes, this novel sp., most closely related to D. stewartii, has not been reported on Cucumis spp. Strains were deposited in the USDA-ARS Culture Collection (NRRL# 64461-62). Koch's postulates were conducted in a greenhouse with mean day temperature of 25°C and 12 hr supplemental lighting. One-month old cucumbers 'Katrina,' grown in rockwool cubes (5 plants per isolate) and potting mix (6 plants per isolate), were inoculated with a one-week-old culture of either strain. The second true leaf was cut and a pipette tip containing an inoculated plug of ½ APDA was placed on the remaining petiole (Mathew et al. 2018). Non-inoculated ½ APDA was used for controls. Plants were tarped for 24 hours to increase humidity and pipette tips removed after one week. After two weeks, petioles were shrunken, tan to golden brown with pycnidia. After 3-4 weeks, stem lesions matching those above were observed on inoculated plants, and plants collapsed. For fruit rot, three Beit Alpha cucumbers were rinsed with tap water, dried, a 5 mm plug was removed from the fruit and replaced with a 5 mm plug of one-week-old fungus on ½ APDA. After 3 days, fruits were water soaked and soft. For root rot, two plates of one-week-old cultures were macerated in 500 mL of sterile water and mixed with 1500 mL of vermiculite. Two seeds of cucumber 'Katrina' were planted into three reps of each isolate and control. All control seeds germinated, but all inoculated seeds experienced pre- or post-emergence damping off. No symptoms were ever observed on any controls. Fungi were isolated from all inoculated tissues as described above. Based on morphology, Diaporthe sp. was isolated from all inoculated plants but never from controls. This Diaporthe sp. may be a new constraint to hydroponic cucumber production, but incidence needs to be determined globally.
PubMed: 37578364
DOI: 10.1094/PDIS-06-23-1214-PDN -
Plant Disease Apr 2024Anthracnose fruit rot affecting field peppers ( L.) has been reported in Ontario, Canada, leading to significant crop losses of up to 80% over the past three years. Ten...
Anthracnose fruit rot affecting field peppers ( L.) has been reported in Ontario, Canada, leading to significant crop losses of up to 80% over the past three years. Ten symptomatic fruits per field, exhibiting one or more soft, sunken lesions covered with salmon-colored spore masses (Fig. S1), were collected from one and two Banana pepper fields in August 2022 and 2023, respectively, all located in southwestern Ontario. Small sections of diseased tissue (0.5 cm in length) from lesion edges underwent surface sterilization and plated on 2% potato dextrose agar (PDA, Difco) supplemented with kanamycin (50 mg liter), neomycin sulfate (12 mg liter) and streptomycin sulfate (100 mg liter), and incubated at 22°C for 7 days in the dark. Fifteen fungal colonies were isolated and purified using the hyphal tipping method. All fungal isolates showed a pale gray colony morphology with a faint salmon tint on PDA (Fig. S1). Conidia, produced on PDA after incubating the 15 isolates at 22°C for 17 days in the dark, were hyaline, aseptate, smooth-walled, cylindrical with obtuse ends (Fig. S1), and measured 9.4 to 15.0 × 2.7 to 4.8 µm (mean ± standard deviation of 145 conidia = 11.3 ± 1.2 μm × 3.7 ± 0.5 μm), the typical morphology of species (Damm et al. 2012). Internal transcribed spacer (ITS), actin (ACT), chitin synthase 1 (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), histone H3 (HIS3) and beta-tubulin 2 (TUB2) gene regions of all isolates were amplified and sequenced with primers ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF1/GDR1, GSF1/GSR1, CYLH3F/CYLH3R and Bt2a/Bt2b and deposited in GenBank (Accession Nos. ITS: PP060584 to PP060596; ACT, CHS-1, GAPDH, GS, HIS3 and TUB2: PP085919 to PP086005), respectively. The sequences were 100% identical to Colletotrichum scovillei strains from different hosts and countries (ITS: PP079643; ACT: MN718468; CHS-1: MN718466, GAPDH: MN718465.1, HIS3: MT592502, TUB2: MK462971). The maximum likelihood-based phylogenetic analysis of ITS, ACT, CHS-1, GAPDH, GS, HIS3, and TUB2 concatenated sequences was conducted using IQ-TREE 2.2.2.7 (Minh et al. 2020). All isolates from this study were grouped with high bootstrap support values with the holotype CBS 126529 (Fig. S2). Living cultures of these isolates were deposited in the Canadian Collection of Fungal Cultures (DAOMC 252833 to 252847). Pathogenicity was tested by inoculating 4 Banana (cv. Jumbo Stuff) and 4 Bell (cv. Archimedes) pepper fruits with 10 μl droplet of a 1 × 10 conidia ml suspension of each isolate onto a wound made with a sterile pipette tip. Eight control fruits were mock-inoculated with sterilized water. Nine days post-inoculation, necrotic lesions measuring 24.7 ± 0.3 mm on Bell and 27.9 ± 0.2 mm on Banana peppers were observed. was re-isolated from all symptomatic fruits, and its species identity was confirmed through morphology, fulfilling Koch's postulates. Control fruits remained symptom-free, and no fungi were isolated from them. This is the first report of in Canada. Previously identified as a pathogen causing anthracnose on peppers in eastern Asia, the United States, Brazil, and Kosovo (Farr and Rossman 2024; Xhemali et al. 2023), its emergence in Ontario raises significant concerns for pepper crops. Additional research is essential to better understand the epidemiology of the disease and develop effective phytosanitary strategies for control.
PubMed: 38679597
DOI: 10.1094/PDIS-02-24-0373-PDN