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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 -
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 -
BioTechniques Feb 2024Additive manufacturing (3D printing) has been deployed across multiple platforms to fabricate bioengineered tissues. We demonstrate the use of a Thermal Inkjet Pipette...
Additive manufacturing (3D printing) has been deployed across multiple platforms to fabricate bioengineered tissues. We demonstrate the use of a Thermal Inkjet Pipette System (TIPS) for targeted delivery of cells onto manufactured substrates to design bio-bandages. Two cell lines - HEK 293 (kidney) and K7M2 wt (bone) - were applied using TIPS. We demonstrate a novel means for targeted cell delivery to a hydrogel support structure. These cell/support constructs (bio-bandages) had a high viability for survival and growth over extended periods. Combining a flexible biosupport with application of cells via TIPS printing now for the first time allows for custom cell substrate constructs with various densities to be deployed for regenerative medicine applications.
Topics: Humans; Hydrogels; Tissue Engineering; HEK293 Cells; Printing, Three-Dimensional; Tissue Scaffolds; Bioprinting
PubMed: 38084384
DOI: 10.2144/btn-2023-0052 -
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 -
Botanical Studies Jan 2024The bimolecular fluorescence complementation (BiFC) assay is commonly used for investigating protein-protein interactions. While several BiFC detection systems have been...
BACKGROUND
The bimolecular fluorescence complementation (BiFC) assay is commonly used for investigating protein-protein interactions. While several BiFC detection systems have been developed, there is a limited amount of research focused on using laser scanning confocal microscope (LSCM) techniques to observe protoplasts. Protoplasts are more susceptible to damage and instability compared to their original cell state due to the preparation treatments they undergo, which makes it challenging for researchers to manipulate them during observation under LSCMs. Therefore, it is crucial to utilize microscope techniques properly and efficiently in BiFC assays.
RESULTS
When the target fluorescence is weak, the autofluorescence of chloroplast particles in protoplasts can interfere with the detection of BiFC signals localized in the nuclear region. Spectrum analysis revealed that chloroplast autofluorescence can be excited by lasers of various types, with the highest fluorescence signal observed at around 660 nm. Furthermore, our investigation into the impact of different pipette tips on the integrity of protoplast samples indicated that the utilization of cut tips with larger openings can mitigate cell breakage. We presented a workflow of LSCM techniques for investigating protoplast BiFC and discussed the microscopic manipulation involved in sample preparation and image capturing.
CONCLUSION
When the BiFC signals are weak, they may be affected by chloroplast autofluorescence. However, when used properly, the autofluorescence of chloroplasts can serve as an excellent internal marker for effectively distinguishing other signals. In combination with other findings, this study can provide valuable reference for researchers conducting BiFC assays and related studies.
PubMed: 38194078
DOI: 10.1186/s40529-024-00409-z -
Proceedings. Biological Sciences Feb 2024Sabotaging milkweed by monarch caterpillars () is a famous textbook example of disarming plant defence. By severing leaf veins, monarchs are thought to prevent the flow...
Sabotaging milkweed by monarch caterpillars () is a famous textbook example of disarming plant defence. By severing leaf veins, monarchs are thought to prevent the flow of toxic latex to their feeding site. Here, we show that sabotaging by monarch caterpillars is not only an avoidance strategy. While young caterpillars appear to avoid latex, late-instar caterpillars actively ingest exuding latex, presumably to increase sequestration of cardenolides used for defence against predators. Comparisons with caterpillars of the related but non-sequestering common crow butterfly () revealed three lines of evidence supporting our hypothesis. First, monarch caterpillars sabotage inconsistently and therefore the behaviour is not obligatory to feed on milkweed, whereas sabotaging precedes each feeding event in caterpillars. Second, monarch caterpillars shift their behaviour from latex avoidance in younger to eager drinking in later stages, whereas caterpillars consistently avoid latex and spit it out during sabotaging. Third, monarchs reared on detached leaves without latex sequestered more cardenolides when caterpillars imbibed latex offered with a pipette. Thus, we conclude that monarch caterpillars have transformed the ancestral 'sabotage to avoid' strategy into a 'sabotage to consume' strategy, implying a novel behavioural adaptation to increase sequestration of cardenolides for defence.
Topics: Animals; Larva; Latex; Butterflies; Asclepias; Cardenolides
PubMed: 38378155
DOI: 10.1098/rspb.2023.2721 -
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 -
ACS Omega Jan 2024The ability of the centrifugal Lab-on-a-Disc (LoaD) platform to closely mimic the "on bench" liquid handling steps (laboratory unit operations (LUOs)) such as metering,...
Low-High-Low Rotationally Pulse-Actuated Serial Dissolvable Film Valves Applied to Solid Phase Extraction and LAMP Isothermal Amplification for Plant Pathogen Detection on a Lab-on-a-Disc.
The ability of the centrifugal Lab-on-a-Disc (LoaD) platform to closely mimic the "on bench" liquid handling steps (laboratory unit operations (LUOs)) such as metering, mixing, and aliquoting supports on-disc automation of bioassay without the need for extensive biological optimization. Thus, well-established bioassays, normally conducted manually using pipettes or using liquid handling robots, can be relatively easily automated in self-contained microfluidic chips suitable for use in point-of-care or point-of-use settings. The LoaD's ease of automation is largely dependent on valves that can control liquid movement on the rotating disc. The optimum valving strategy for a true low-cost and portable device is rotationally actuated valves, which are actuated by changes in the disc spin-speed. However, due to tolerances in disc manufacturing and variations in reagent properties, most of these valving technologies have inherent variation in their actuation spin-speed. Most valves are actuated through stepped increases in disc spin-speed until the motor reaches its maximum speed (rarely more than 6000 rpm). These manufacturing tolerances combined with this "analogue" mechanism of valve actuation limits the number of LUOs that can be placed on-disc. In this work, we present a novel valving mechanism called low-high-low serial dissolvable film (DF) valves. In these valves, a DF membrane is placed in a dead-end pneumatic chamber. Below an actuation spin-speed, the trapped air prevents liquid wetting and dissolving the membrane. Above this spin-speed, the liquid will enter and wet the DF and open the valve. However, as DFs take ∼40 s to dissolve, the membrane can be wetted, and the disc spin-speed reduced before the film opens. Thus, by placing valves in a series, we can govern on which "digital pulse" in spin-speeding a reagent is released; a reservoir with one serial valve will open on the first pulse, a reservoir with two serial valves on the second, and so on. This "digital" flow control mechanism allows the automation of complex assays with high reliability. In this work, we first describe the operation of the valves, outline the theoretical basis for their operation, and support this analysis with an experiment. Next, we demonstrate how these valves can be used to automate the solid-phase extraction of DNA on on-disc LAMP amplification for applications in plant pathogen detection. The disc was successfully used to extract and detect, from a sample lysed off-disc, DNA indicating the presence of thermally inactivated , a bacterial pathogen on tomato leaf samples.
PubMed: 38284094
DOI: 10.1021/acsomega.3c05117 -
Cureus Feb 2024This case report explores the application of mechanical hatching as a successful intervention in the treatment of primary infertility for a couple with a consanguineous...
This case report explores the application of mechanical hatching as a successful intervention in the treatment of primary infertility for a couple with a consanguineous marriage history and recurrent implantation failure. A 32-year-old female patient and her 37-year-old spouse, after six years of unsuccessful attempts to conceive, underwent multiple intrauterine insemination (IUI) and in vitro fertilization (IVF) and embryo transfer (ET) cycles without success. Normal parameters were observed in semen analysis and hormone tests for the male and female partners, respectively. Despite a series of failed assisted reproductive technology (ART) procedures, the implementation of mechanical hatching using partial zona dissection (PZD) pipettes led to a positive pregnancy outcome. The case underscores the potential efficacy of individualized approaches, specifically mechanical hatching, in addressing challenges associated with implantation failure, offering hope to couples facing infertility issues.
PubMed: 38455805
DOI: 10.7759/cureus.53709 -
Sensors (Basel, Switzerland) Mar 2024This paper presents high-sensitivity sensors based on an open complementary square split-ring resonator and a modified open complementary split-ring resonator operating...
This paper presents high-sensitivity sensors based on an open complementary square split-ring resonator and a modified open complementary split-ring resonator operating at 4.5 GHz and 3.4 GHz, respectively. The sensors are designed for the detection of multiple liquid materials, including distilled water, methanol, and ethanol. The liquid under test is filled in a glass container loaded using a pipette. Compared to the conventional OCSSRR, the modified OCSSRR with multiple rings exhibits a higher frequency shift of 1200 MHz, 1270 MHz, and 1520 MHz for ethanol, methanol, and distilled water, respectively. The modified sensor also demonstrates a high sensitivity of 308 MHz/RIU for ethanol concentration which is the highest among the existing microwave sensors. The sensors in this manuscript are suitable for multiple liquid-material-sensing applications.
PubMed: 38544102
DOI: 10.3390/s24061840