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Tropical Medicine and Infectious Disease May 2024Diverse larval habitats significantly influence female mosquito oviposition. Utilizing traps that simulate these habitats is helpful in the study of the bioecology and...
Applicability of Traps for Collecting Mosquito Immatures (Diptera: Culicidae) for Entomological Surveillance of Arbovirus Vectors in a Remnant of the Atlantic Forest, Rio de Janeiro State, Brazil.
Diverse larval habitats significantly influence female mosquito oviposition. Utilizing traps that simulate these habitats is helpful in the study of the bioecology and characteristics of pathogen-transmitting species during oviposition. This study evaluated the feasibility of different traps in natural environments by comparing sampling methods and detecting the oviposition of epidemiologically important mosquitoes, with emphasis on species, in a fragment of the Atlantic Forest in Silva Jardim, Rio de Janeiro State, Brazil. Monthly collections were conducted from March 2021 to October 2023 using four types of traps: plastic containers, tires, bamboo, and sapucaia. Immatures were collected from these traps using a pipette, placed in plastic bags, and transported to the laboratory. Tire was the most efficient trap, showing the highest mosquito abundance (n = 1239) and number of species (S = 11). Conversely, the plastic container trap exhibited the lowest diversity (H = 0.43), with only two species and a low mosquito abundance (n = 26). The bamboo trap captured six species and recorded the second-highest diversity index (H = 1.04), while the sapucaia trap captured five species and had the third-highest diversity index (H = 0.91). Of the total immatures collected, 1817 reached adulthood, comprising 13 species, two of which are vectors of the sylvatic yellow fever virus: and . In conclusion, detecting key vectors of the sylvatic yellow fever virus in Brazil highlights the need for ongoing entomological and epidemiological surveillance in the study area and its vicinity. These efforts are crucial for monitoring vector presence and activity, identifying potential transmission hotspots, and devising effective control and prevention strategies.
PubMed: 38922037
DOI: 10.3390/tropicalmed9060125 -
APL Bioengineering Jun 2024Micropipette aspiration (MPA) is one of the gold standards for quantifying biological samples' mechanical properties, which are crucial from the cell membrane scale to...
Micropipette aspiration (MPA) is one of the gold standards for quantifying biological samples' mechanical properties, which are crucial from the cell membrane scale to the multicellular tissue. However, relying on the manipulation of individual home-made glass pipettes, MPA suffers from low throughput and no automation. Here, we introduce the sliding insert micropipette aspiration method, which permits parallelization and automation, thanks to the insertion of tubular pipettes, obtained by photolithography, within microfluidic channels. We show its application both at the lipid bilayer level, by probing vesicles to measure membrane bending and stretching moduli, and at the tissue level by quantifying the viscoelasticity of 3D cell aggregates. This approach opens the way to high-throughput, quantitative mechanical testing of many types of biological samples, from vesicles and individual cells to cell aggregates and explants, under dynamic physico-chemical stimuli.
PubMed: 38894959
DOI: 10.1063/5.0193333 -
BioRxiv : the Preprint Server For... May 2024Sperm cryopreservation is important for individuals undergoing infertility treatment, and for those who wish to preserve fertility potential, prior to treatments like...
Sperm cryopreservation is important for individuals undergoing infertility treatment, and for those who wish to preserve fertility potential, prior to treatments like chemotherapy, radiation therapy, gender-affirming medical interventions, elective fertility delay, or individuals in high-risk professions such as the military. Current methods for sperm cryopreservation result in approximately 30-50% decrease in sperm motility. However, recent studies have shown that ultra-rapid freezing (vitrification) is a valuable approach for maintaining sperm quality after freeze-thawing processes in the clinical laboratory setting and requires submicroliter to microliter volumes. A major challenge for the adoption of vitrification in fertility laboratories is the ability to pipette small volumes of sample. Here, we present a method that leverages open-channel droplet microfluidics to autonomously generate sub-microliter to microliter volumes of purified human sperm samples. Using a novel, open-channel droplet generator, we found no change in sperm movement and kinematic data after exposure to device and reagents in our platform. We conclude that our platform is compatible with human sperm, an important foundation for future implementation of vitrification in fertility laboratories.
PubMed: 38798664
DOI: 10.1101/2024.05.09.593416 -
Analytical Chemistry Jun 2024We describe micro- and nanoelectrode array analysis with an automated version of the array microcell method (AMCM). Characterization of hundreds of electrodes, with...
We describe micro- and nanoelectrode array analysis with an automated version of the array microcell method (AMCM). Characterization of hundreds of electrodes, with diameters ranging from 100 nm to 2 μm, was carried out by using AMCM voltammetry and chronoamperometry. The influence of solvent evaporation on mass transport in the AMCM pipette and the resultant electrochemical response were investigated, with experimental results supported by finite element method simulations. We also describe the application of AMCM to high-throughput single-entity electrochemistry in measurements of stochastic nanoparticle impacts. Collision experiments recorded 3270 single-particle events from 671 electrodes. Data collection parameters were optimized to enable these experiments to be completed in a few hours, and the collision transient sizes were analyzed with a U-Net deep learning model. Elucidation of collision transient sizes by histograms from these experiments was enhanced due to the large sample size possible with AMCM.
PubMed: 38780285
DOI: 10.1021/acs.analchem.4c01092 -
BioRxiv : the Preprint Server For... May 2024Extracellular vesicles (EVs) are particles secreted by all cells that carry bioactive cargo and facilitate intercellular communication with roles in normal physiology...
Extracellular vesicles (EVs) are particles secreted by all cells that carry bioactive cargo and facilitate intercellular communication with roles in normal physiology and disease pathogenesis. EVs have tremendous diagnostic and therapeutic potential and accordingly, the EV field has grown exponentially in recent years. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are undermined by complicated detection schemes often coupled with prohibitive instrumentation. To address these issues, we propose a microfluidic technique for EV characterization called 'tch and isplay for iquid iopsy (CAD-LB)'. CAD-LB rapidly captures fluorescently labeled EVs in the similarly-sized pores of an ultrathin silicon nitride membrane. Minimally processed sample is introduced pipette injection into a simple microfluidic device which is directly imaged using fluorescence microscopy for a rapid assessment of EV number and biomarker colocalization. In this work, nanoparticles were first used to define the accuracy and dynamic range for counting and colocalization by CAD-LB. Following this, the same assessments were made for purified EVs and for unpurified EVs in plasma. Biomarker detection was validated using CD9 in which Western blot analysis confirmed that CAD-LB faithfully recapitulated differing expression levels among samples. We further verified that CAD-LB captured the known increase in EV-associated ICAM-1 following the cytokine stimulation of endothelial cells. Finally, to demonstrate CAD-LB's clinical potential, we show that EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients undergoing immune checkpoint blockade.
PubMed: 38746341
DOI: 10.1101/2024.04.29.589900 -
Neuroscience Research May 2024The gramicidin-perforated patch-clamp technique is indispensable for recording neuronal activities without changing the intracellular Cl concentration. Conventionally,...
The gramicidin-perforated patch-clamp technique is indispensable for recording neuronal activities without changing the intracellular Cl concentration. Conventionally, gramicidin contained in the pipette fluid is delivered to the cell membrane by passive diffusion. Gramicidin deposited on the pipette orifice sometimes hampers giga-seal formation, and perforation progresses only slowly. These problems may be circumvented by delivering a high concentration of gramicidin from an intra-pipette capillary after a giga-seal is formed. We herein describe the detailed protocol of this improved method. This protocol would greatly facilitate the investigation of Cl gradient-dependent neuronal activities.
PubMed: 38740268
DOI: 10.1016/j.neures.2024.05.002 -
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 -
Micromachines Apr 2024A high porosity micropore arrayed parylene membrane is a promising device that is used to capture circulating and exfoliated tumor cells (CTCs and ETCs) for liquid...
A high porosity micropore arrayed parylene membrane is a promising device that is used to capture circulating and exfoliated tumor cells (CTCs and ETCs) for liquid biopsy applications. However, its fabrication still requires either expensive equipment or an expensive process. Here, we report on the fabrication of high porosity (>40%) micropore arrayed parylene membranes through a simple reactive ion etching (RIE) that uses photoresist as the etching mask. Vertical sidewalls were observed in etched parylene pores despite the sloped photoresist mask sidewalls, which was found to be due to the simultaneous high DC-bias RIE induced photoresist melting and substrate pedestal formation. A theoretical model has been derived to illustrate the dependence of the maximum membrane thickness on the final pore-to-pore spacing, and it is consistent with the experimental data. A simple, yet accurate, low number (<50) cell counting method was demonstrated through counting cells directly inside a pipette tip under phase-contrast microscope. Membranes as thin as 3 μm showed utility for low number tumor cell capture, with an efficiency of 87-92%.
PubMed: 38675332
DOI: 10.3390/mi15040521 -
MicroPublication Biology 2024In , avoidance behaviors are vital for the nematode's ability to respond to noxious environmental stimuli, including the odorant 1-octanol. To test avoidance to...
In , avoidance behaviors are vital for the nematode's ability to respond to noxious environmental stimuli, including the odorant 1-octanol. To test avoidance to 1-octanol, researchers expose to this odorant and determine the time taken to initiate backward locomotion. However, the 1-octanol avoidance assay is sensitive to sensory adaptation, where the avoidance response is reduced due to overexposure to the odorant. Here, we examined two methods to expose nematodes to 1-octanol, using an eyelash hair or a p10 pipette tip, to compare their susceptibility to cause sensory adaptation.
PubMed: 38660566
DOI: 10.17912/micropub.biology.001177 -
BMC Chemistry Apr 20246-mercaptopurine (6-MP) is a chemotherapy drug mainly used to treat leukemia. It is a persistent organic pollutant and can remain in the environment for a long period of...
BACKGROUND
6-mercaptopurine (6-MP) is a chemotherapy drug mainly used to treat leukemia. It is a persistent organic pollutant and can remain in the environment for a long period of time. The presence of 6-MP in the environment poses a number of hazards and needs to be assessed to monitor its potential risk to human health and the environment. However, due to its trace amount in complicated matrices, a clean-up and preconcentration step before its determination is compulsory.
RESULTS
As a highly efficient adsorbent for the extrication of 6-mercaptopurine (6-MP), a novel carbon nanotube doped with camphor: decanoic acid deep eutectic solvent was synthesized and applied as a packing material for the pipette-tip micro solid phase extraction sorbent of 6-MP from tap, wastewater and seawater samples before its spectrophotometric determination. Characteristics and structure of this adsorbent was fully investigated. Factors affecting extraction, including type and volume of the eluent, ionic strength and pH of the sample solution, amount of adsorbent, and number of extraction and elution cycles were optimized using one-factor-at-a-time and response surface methodologies. The method was found to be linear in the range of 1 to 1000 µg/L with a limit of detection and quantification of 0.2 and 0.7 µg/L, respectively. Reproducibility as relative standard deviation was better than 4.6%.
CONCLUSION
Application of deep eutectic solvent modified carbon nanotube indicated suitable microextraction results and good potential for rapid extraction of trace amounts of 6-MP from different aqueous samples. The amount of sample required for the analysis was less than 10 mL and only 1.5 mg of the adsorbent was used. The total analysis time, including extraction was less than 15 min and the adsorbent could be used for at least 10 times, without significantly losing its adsorption ability. Compared to using unmodified usual carbon nanotubes, deep eutectic solvent doped carbon nanotubes showed 19.8% higher extraction ability.
PubMed: 38654336
DOI: 10.1186/s13065-024-01199-y