Subsequent Washing Research Articles

P0178 Lack of DNA methyltransferase DNMT3A in fibroblasts impairs cell growth and wound healing

Abstract Background Fibrosis in Crohn’s disease (CD) is a complication of chronic inflammatory processes that may result in impaired gastrointestinal function and bowel strictures. However, exact pathomechanisms leading to intestinal fibrosis in CD are not fully understood. Since the de novo DNA methyltransferase DNMT3A is involved in cellular differentiation processes and genetic variants have been associated with increased risk for CD, we sought to characterize how DNMT3A loss might affect fibroblasts, the key mesenchymal cells involved in fibrosis. Methods We stably deleted Dnmt3a in 3T3 fibroblast cell line using CRISPR-Cas9 technology and subsequently selected GPF-positive clones using FACS sorting. Cell cultures were performed in cell culture plates with or without 0.2% gelatin coat. Reverse transcription quantitative PCR, western blot, and immunofluorescence were used to confirm Dnmt3a gene deletion and characterize wild-type (WT) and knockout (KO) cells. Cell adhesion was evaluated by fluorescence levels of calcein acetoxymethyl ester (CAM)-labelled cells that attached to cell culture plates after 1h of culture and subsequent wash. Collagen lattice assays in presence of proinflammatory and profibrotic stimuli served to assess fibroblast contractibility. Results 3T3 fibroblasts lacking Dnmt3a acquire an activated myofibroblast phenotype (increased a-SMA expression) that also overexpress tissue remodeling matrix metalloproteinase Mmp3, and the inflammation-induced necroptosis marker RIPK3 when compared to their WT counterparts. In addition, Dnmt3a-depleted 3T3 cells adhere more strongly to cell culture plates. In presence of transforming growth factor (TGF)-b, cells embedded in a 3D collagen matrix and lacking Dnmt3a build an interconnected cellular network and contract up to a 77.78% (compared to interconnected cells that reach up to 13.73% contraction by WT cells). This contraction was independent of the presence of interleukin (IL)-11. Profibrotic factors such as bleomycin prevent intercellular network formation in both WT and KO cells; hence, incapacitating cells to contract the collagen matrix. Conclusion We conclude that Dnmt3a is crucial for fibroblast homeostasis, including cell activation and functionality. Furthermore, TGF-b induces higher fibroblast contractibility when Dnmt3a is lacking. Thus, additional characterization of Dnmt3a depletion in fibroblasts will deepen the understanding of fibrosis and may point towards new therapeutic approaches for this untreatable complication.

A structural investigation on the interactions of cotton fabric cellulose with olive oil and water

The cotton fabric consists of cellulose arranged in a complex structure with multiple levels of organization at different length scales. Understanding this structure and its interactions with water and oil is essential for developing efficient and environmentally friendly methods of cotton washing. In this study, the structure of raw cotton fabric cellulose and the effects of water and oil were examined across a broad range of length scales using spatially resolved synchrotron small-angle X-ray scattering (SAXS) and auxiliary techniques.Water was observed to penetrate the cotton fabric and interact across nearly all length scales. Although a certain amount of the material was not affected by water as seen by intact distance between microfibrils, fractal analysis of the scattering data indicated a loosening of the microfibril arrangement after contact with water. This process was hindered if the material had been pre-treated with oil and was not seen after subsequent washing with water or surfactant solution. Analyzing spatially resolved SAXS data using a bi-sinusoidal model and 2D maps of the oil-to-cotton ratio facilitates understanding the structure of the material and its interactions with oil on the molecular, nano and macrolevels.

Portable label-free electrochemical aptasensor for sensitive detection of paraquat herbicide mediated by oxygen reduction reaction

Paraquat (PQ) is a highly toxic herbicide that has been prohibited in almost 70 countries, but remains in use worldwide. Thus, routine on-site PQ monitoring is a key mechanism to ensure safety and efficiently enforce regulations. Herein, a label-free portable electrochemical aptasensor for the detection of PQ was developed by utilizing aptamer designed to specifically recognize PQ. The aptasensor employs square-wave voltammetry (SWV) to quantify PQ binding on the aptamer-functionalized electrode surface by tracking the downstream oxygen reduction reaction. It provided a detection range spanning from 0.01 to 100.0 μg mL−1 PQ with a limit of detection (LOD) of 8.9 ng mL−1. Validation against spiked tap water, pomegranate juice, and orange juice revealed recovery rate performances of 75 %–130 %. The aptasensor demonstrates promising feasibility for PQ detection in real-world applications, offering remarkable portability and operational simplicity. Notably, it can operate without supplementary redox agents, requiring only sample incubation and subsequent washing steps.

Oxygen-rich 3D hierarchical porous MXene prepared by Zn powder reduction for flexible supercapacitors

Ti3C2Tx MXene is highly compelling as an energy storage material, but 2D MXene sheets experience significant stacking due to hydrogen bonding and van der Waals forces. This blocks active sites and impedes fast electrolyte ion transport. The pseudocapacitance of MXene materials is highly reliant on their terminal groups. Therefore, an effective strategy was designed herein to prepare an oxygen-rich 3D hierarchical porous MXene (P-OR-Ti3C2Tx). First, Zn powder was used as a template to construct three-dimensional hierarchical porous structures that spanned the microporous, mesoporous, and macroporous scales. Second, Zn powder was used as a metal reducing agent in a reaction at 500 °C to eliminate most −F terminal groups on the MXene. Then, a subsequent acid washing step was used to introduce numerous −O terminal groups. The energy storage process of the prepared MXene was investigated by in situ Raman. More redox active sites (−O terminal groups) are exposed by the constructed oxygen-rich 3D hierarchical porous MXene structure, which also provides a shorter pathway for electrolyte ion transport. P-OR-Ti3C2Tx displays superb rate performance (88.19 % retention at 100Ag−1) and ultra-high capacitance (676.7F/g at 2 mV/s and 698.7Fg−1 at 1 Ag−1) when used as a supercapacitor electrode. Moreover, a symmetric flexible supercapacitor device prepared using P-OR-Ti3C2Tx and carbon cloth achieves a superb energy density of 32.8Whkg−1 at a power density of 236.5Wkg−1. This study offers insight into the design of MXenes that exhibit both high capacity and excellent rate performance.

Flux synthesis of two-dimensional covalent organic frameworks.

Covalent organic frameworks (COFs) are crystalline porous polymers constructed from organic building blocks into ordered two- or three-dimensional networks through dynamic covalent bonds. Attributed to their high porosity, well-defined structure, tailored functionality and excellent chemical stability, COFs have been considered ideal sorbents for various separation applications. The synthesis of COFs mainly employs the solvothermal method, which usually requires organic solvents in sealed Pyrex tubes, resulting in unscalable powdery products and environmental pollution that seriously limits their practical applications. Herein, our protocol focuses on an emerging synthesis method for COFs based on organic flux synthesis without adding solvents. The generality of this synthesis protocol has been applied in preparing various types of COFs, including olefin-linked, imide-linked, Schiff-based COFs on both gram and kilogram scales. Furthermore, organic flux synthesis avoids the disadvantages of solvothermal synthesis and enhances the crystallization and porosity of COFs. Typically, COF synthesis takes 3-5 d to complete, and subsequent washing procedures leading to pure COFs need 1 d. The procedure for kilogram-scale production of COFs with commercially available monomers is also provided. The resulting COFs are suitable for separation applications, particularly as adsorbent materials for industrial gas separation and water treatment applications. The protocol is suited for users with prior expertise in the synthesis of inorganic materials and porous organic materials.

Fibre fragment pollution: source directed intervention through design

This study analyses the difference in fibre fragment pollution generated during the laundering of woven and knit fabrics for clothing and the influence of fabric parameters on the amount of pollution released. 100% polyester single jersey knit and 2/2 twill woven fabrics were created and washed according to AATCC TM212-2021. Results indicated more tightly woven structures released over three times less pollution than looser knit structures, releasing 6.41 mg of fibre fragments per kg of woven fabric (mean ± 1.50 SD, n = 8) compared to 21.21 mg of fibre fragments per kg of knit fabric (mean ± 1.80 SD, n = 8). The first wash for both knit and woven fabrics shed significantly more fibre fragments than subsequent washes. By the fifth wash both fabrics released under a tenth of the amount of pollution released in the first wash. To mitigate fibre fragment pollution that is released by fabrics designed for clothing this work recommends designing out pollution using more tightly constructed fabrics. Furthermore, the implementation of technology in fabric and garment manufacturing processes (e.g. fibre catching devices) should be utilised specifically during first washes allowing pollution to be filtered from the wastewater to stem the flow of contaminants into the environment.

Direct Recycling at the Material Level: Unravelling Challenges and Opportunities through a Case Study on Spent Ni‐Rich Layered Oxide‐Based Cathodes

AbstractDirect recycling is a key technology for enabling a circular economy of spent lithium ion batteries (LIBs). For cathode active materials (CAMs), it is regarded as the tightest closed‐loop and most efficient approach among current recycling techniques as it simply proceeds via re‐lithiation and reconstruction of aged CAMs instead of separating them into elemental components. In this work, spent, i.e., morphologically and structurally decomposed CAM based on LiNi0.83Co0.12Mn0.05O2 (NCM‐831205) is restored by mimicking conditions of original CAM synthesis. After evaluating and optimizing the high‐temperature duration for CAM restoration and subsequent washing procedure, the recycled CAM is shown to maintain poly‐crystallinity and tap density, successfully recover specific surface area, lithium content, crystal structure in surface and bulk, while, however, only partly the original secondary particle size and shape. Though, comparable in initial 100 charge/discharge cycles with pristine CAM in lithium ion‐cells, the subsequent increase in resistance and capacity fading remains a challenge. High temperature during recycling can be regarded as a key challenge on material level, as it not only promotes detrimental surface carbonate species from residual carbon black but also enhances cation disorder and micro‐/nanoscopic porosity through oxygen release, likely in de‐lithiated, thus less thermally stable regions of cycled NCM.

From Extraction to Stabilization: Employing a 22 Experimental Design in Developing Nutraceutical-Grade Bixin from Bixa orellana L.

Bixin is the main carotenoid found in the outer portion of the seeds of Bixa orellana L., commercially known as annatto. This compound is industrially employed in pharmaceutical, cosmetic, and food formulations as a natural dye to replace chemical additives. This study aimed to extract bixin from annatto seeds and obtain encapsulated bixin in a powder form, using freeze-drying encapsulation and maltodextrin as encapsulating agent. Bixin was extracted from annatto seeds employing successive washing with organic solvents, specifically hexane and methanol (1:1 v/v), followed by ethyl acetate and dichloromethane for subsequent washes, to effectively remove impurities and enhance bixin purity, and subsequent purification by crystallization, reaching 1.5 ± 0.2% yield (or approximately 15 mg of bixin per gram of seeds). Bixin was analyzed spectrophotometrically in different organic solvents (ethanol, isopropyl alcohol, dimethylsulfoxide, chloroform, hexane), and the solvents chosen were chloroform (used to solubilize bixin during microencapsulation) and hexane (used for spectrophotometric determination of bixin). Bixin was encapsulated according to a 22 experimental design to investigate the influence of the concentration of maltodextrin (20 to 40%) and bixin-to-matrix ratio (1:20 to 1:40) on the encapsulation efficiency (EE%) and solubility of the encapsulated powder. Higher encapsulation efficiency was obtained at a maltodextrin concentration of 40% w/v and a bixin/maltodextrin ratio of 1:20, while higher solubility was observed at a maltodextrin concentration of 20% w/v for the same bixin/maltodextrin ratio. The encapsulation of this carotenoid by means of freeze-drying is thus recognized as an innovative and promising approach to improve its stability for further processing in pharmaceutical and food applications.

Removal of silica in rice husk derived Ni/biochar modifies reaction intermediates formed and minimizes coking in steam reforming of glycerol

Metal/biochar catalyst features with the advantages for easy recovery of chemical energy of biochar carrier and metal species via simply combustion. Another unique feature of biochar carrier is intrinsic inorganic species, which might also interact with metal species in resulting catalyst. This was investigated herein by synthesis of the Ni/biochar with rice husk-derived biochar carrier pretreated with NaOH for removal of SiO2 and evaluation of the resulting catalysts in steam reforming of glycerol. The solid phase reactions between NaOH and biochar (mass ratio: 2) plus subsequent washing removed 94.6% of ash (mainly SiO2). This formed Ni/biochar of fragmented surface with specific surface area increasing from 207.8 to 457.4 m2g-1 and simultaneously increasing mesopore percentage from 15.5% to 24.0%. NaOH treatment also effectively removed significant portion of both Lewis and Brønsted acidic sites but created abundant alkaline sites. These drastically enhanced nickel dispersion and activity for steam reforming. In-situ IR characterization suggested enhanced adsorption/activation of CO2 and H2O over Ni/biochar treated with NaOH (Ni/biochar-NaOH), which mitigated accumulation of coke precursors bearing CO or CC via accelerated gasification reactions. This led to the reduced amount of coke (mass ratio to catalyst) from 193.4% to 94.7% over Ni/biochar treated with 2-fold of NaOH or to 111.3% over Ni/biochar treated with 1-fold of NaOH. The coke formed was mainly catalytic type in form of carbon nanotube over Ni/biochar-NaOH. The superior resistivity to coking originated from developed pores, enhanced Ni dispersion and abundant alkaline sites from the NaOH treatment.

A 3-step method for preparing cryopreserved samples of apheresis products for post-thaw analysis yields a higher percentage of viable cells.

Standard flow cytometry protocols for CD34+ cell enumeration designed for fresh samples are not appropriate for cryopreserved products. Special protocols have been developed to remove the cryoprotectant by quickly washing a freshly thawed sample. Exposing cells to a large volume of hypotonic solution and subsequent washing process was hypothesized to cause lab-induced cell death. Moreover, standard gating strategies must be altered to avoid reporting falsely high viabilities. We developed a novel method whereby thawed samples were diluted step-wise to 1:2 by 3 additions of 1/3 sample volume using 1% Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. An additional 1:10 dilution was required to obtain a desired cell concentration for flow cytometry testing resulting in a 1:20 dilution. Twenty samples were tested simultaneously in a method comparison; the new method demonstrated significant increases in mean cell viabilities for white blood cells, hematopoietic progenitor cells, and T cells as well as reduced standard deviations for each parameter. Slow, step-wise dilutions of freshly thawed samples of cryopreserved apheresis products to 1:20 yielded higher and more precise viability measurements compared to quickly washing samples to remove DMSO.

Attachment of Salmonella Typhimurium to Fish Surfaces from a Suspension in Seawater and the Effect of Subsequent Washing

The article is about cooling (with cold water) of the lower abdomen (which is most sensitive to cold) for: relief of abdominal pain, boosting the immune system, handling hot flashes during menopause and better sleep. The article also explains how it works – it is done through the biofield seen as aura, which rules and regulates everything in the body. I found it to be Nonlinear Electromagnetic Field (NEMF), which being nonlinear is not dissipating and can imprint information. The waves of the biofield (aura) scan the new environment and bring the information to the material body telling it how to change to adapt to the new environment. If the environment is drastically new, it handles the evolution to new species. In the process of adaptation, first the biofield (aura) NEMF is changed and if the drastically new environment stays the same for a long time, it evolves into DNA changes and evolution to new species. A proof that the evolution to new species is done through the biofield is the fact that the evolution to new species is done through a quantum jump – there are no intermediate forms.

Attachment of Salmonella Typhimurium to Fish Surfaces from a Suspension in Seawater and the Effect of Subsequent Washing

ackground: Salmonella contamination of seafood is an important consumer health concern. Anthropogenic contamination of coastal-marine water is an important source of seafood contamination with Salmonella. In this study, we aimed to investigate the extent of Salmonella attachment when fish is exposed to contaminated seawater and also, the effect of washing on the removal of Salmonella attached to fish surface. Methods and Findings: Bombay duck (Harpadon nehereus) fish was dipped in seawater artificially contaminated with 5 log CFU/ ml Salmonella Typhimurium for 30, 45 and 60 seconds and the number of bacteria present on the fish surface was quantified by surface plating. Separately, the effect of rinsing Salmonella-contaminated fish for 30, 45 and 60 seconds on the removal of Salmonella from the fish surface was investigated. The results showed attachment of bacteria in significant numbers, in the range of 3.6 – 4.16 log CFU/g, within 60 seconds of exposure to Salmonella-contaminated seawater. However, no significant reduction in the Salmonella counts was observed after 30 to 60 seconds of rinsing of fish in freshwater. Conclusion: The results suggest that Salmonella Typhimurium can readily attach to the fish surface from a seawater medium and resist detachment by routine washing procedures.

A simple and effective method to remove pigments from heterologous secretory proteins expressed in Pichia pastoris

Pichia pastoris is a popular yeast host for high-level heterologous expression of proteins on an industrial scale owing to its reliable expression, robust growth, high fermentation density, and easy genetic manipulation and cultivation at a relatively low cost. Of particular interest is its high secretion efficiency for small proteins including insulin, human serum albumin, vaccines, enzymes, and llama-derived heavy-chain only antibodies (nanobodies) for pharmaceutical and research applications. However, a recurring challenge in using P. pastoris heterologous secretory proteins is the co-purification of a sticky, yellow pigment which has been identified as a tetra-benzoyl disaccharide. Current methods for pigment removal involve crystallization of the heterologous secretory protein, active carbon absorption, and chromatography using cation exchange and hydrophobic interaction. Here, we present a simple and effective method to remove the yellow pigment, demonstrated with divalent nanobodies targeting SARS-CoV-2. The method entails capturing the nanobody on an affinity column and subsequent washing with the zwitterionic detergent lauryldimethylamine N-oxide (LDAO). We anticipate the method become generally useful to remove pigments from secretion proteins produced in P. pastoris, offering a practical solution to enhance the purity of heterologous proteins in various biotechnological applications.

Extraction of proteinaceous components and biominerals from cold water fish fileting side streams: a review

Fileting is a popular form of processing methods and in addition to being sold fresh or frozen, filets are used for preparation products battered and breaded filets. This generates considerable amount of side streams like skin, frames, and cut-offs which forms around 30–70% of the total body weight. The European Waste Framework Directive 2008 and recent amendments [EU WFD (2018/851)] stipulates comprehensive regulations which the manufacturers must follow while handling the side streams generated during the processing. This demands a detailed compilation of information regarding the yield, classification and valorization potential of side streams associated with the fileting operations of the cold-water finfishes. The side streams are a rich source of proteins including structural proteins like collagen and biominerals such as hydroxyapatite which find multiple application in food and pharmaceutical industry. Previously, the recovery of these components was performed by extensive chemical treatment with acids or bases, including subsequent washing steps. Nowadays, green extraction methods, defined as technologies with reduced energy and chemical consumption, should be considered to achieve a green shift in the food industry. Nevertheless, industrial upscaling of green extraction methods and subsequent refinement of the isolated compounds must be further evaluated and improved in order to achieve a green shift in food industry by using side-stream derived compounds as ingredients. Moreover, legislations as well as national and international regulations must be considered and evaluated. Even though a number of articles are recently available regarding seafood side stream valorization, this review focus on side streams generated predominantly from cold water fish species and also discusses sustainable green technologies to be included during the recovery process.

Development of a Novel Energy Saving and Environmentally Friendly Starch via a Graft Copolymerization Strategy for Efficient Warp Sizing and Easy Removal.

Warp sizing is a key process in textile production. However, before the yarn/fabric finishing, such as dyeing, the paste adhering to the warp must be eliminated to ensure optimal dyeing properties and the flexibility of the fabric. Therefore, the sizing will often consume a lot of energy and produce a lot of industrial wastewater, which will cause serious harm to the environment. In this study, we have developed an energy saving and environmentally friendly starch-based slurry by modifying natural starch with acrylamide. The paste has excellent viscosity stability and fiber adhesion, and exhibits excellent performance during warp sizing. In addition, the slurry has good water solubility at 60-70 °C, so it is easy to desize at low temperatures. Because of this, the sizing of the warp can be deslimed directly from the yarn during subsequent washing processes. This work can not only reduce some costs for the textile industry, but also achieve the purpose of energy conservation and emission reduction.

Tonicity-induced cargo loading into extracellular vesicles.

The current challenge in using extracellular vesicles (EVs) as drug delivery vehicles is to precisely control their membrane permeability, specifically in the ability to switch between permeable and impermeable states without compromising their integrity and functionality. Here, we introduce a rapid, efficient, and gentle loading method for EVs based on tonicity control (TC) using a lab-on-a-disc platform. In this technique, a hypotonic solution was used for temporarily permeabilizing a membrane ("on" state), allowing the influx of molecules into EVs. The subsequent isotonic washing led to an impermeable membrane ("off" state). This loading cycle enables the loading of different cargos into EVs, such as doxorubicin hydrochloride (Dox), ssDNA, and miRNA. The TC approach was shown to be more effective than traditional methods such as sonication or extrusion, with loading yields that were 4.3-fold and 7.2-fold greater, respectively. Finally, the intracellular assessments of miRNA-497-loaded EVs and doxorubicin-loaded EVs confirmed the superior performance of TC-prepared formulations and demonstrated the impact of encapsulation heterogeneity on the therapeutic outcome, signifying potential opportunities for developing novel exosome-based therapeutic systems for clinical applications.

Enhancing filtration flux and antifouling performance of PVDF membrane constructed from semi-interpenetrating network and block copolymers

Organic fouling causes the filtration property and serve life decrease of the PVDF membrane and is the challenge for filtration applications. Therefore, it is an urgency desire to construct a PVDF membrane with good filtration and antifouling for researchers. To this end, a based PVDF and poly(N-isopropyl acrylamide)(PNIPA) semi-interpenetrating network polymer was synthesized and used as membrane matrix for preparing semi-interpenetrating network/block copolymers blend membrane. The membrane composition, structure and performance were systematically characterized and investigated by FTIR spectrum, XPS, SEM, contact angle instrument and the other experimental techniques etc. Results indicate that the prepared membrane contains PVDF, PNIPA and block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b- poly(ethylene oxide) (F127) components, shows a porous cross section comprising a thin top surface layer with small pores, a finger-shape lower layer and a sponge-shape pore bottom layer. When the membrane are used for the filtration of bovine serum albumin(BSA) in aqueous solution at 25 ℃, the rejection can exceed 95.0% at a high flux of 307 L/(m2·h·bar). Simply raising filtration temperature to 60 °C, the flux can reach 606 L/(m2·h·bar) while the rejection barely changes, significantly improving the filtration ability of the membrane. Furthermore, the flux can recover above 93% of the original value after a BSA filtration and a subsequent water washing. Undoubtedly, the prepared membrane displays great potential for filtering BSA aqueous solution and the reason can be ascribed to the synergy between PNIPA and F127 of the membrane.

Optimization of Sirius Red-Based Microplate Assay to Investigate Collagen Production In Vitro.

Tissue fibrosis is characterized by chronic fibroblast activation and consequently excessive accumulation of collagen-rich extracellular matrix. In vitro microplate-based assays are essential to investigate the underlying mechanism and the effect of antifibrotic drugs. In this study, in the absence of a gold-standard method, we optimized a simple, cost-effective, Sirius Red-based colorimetric measurement to determine the collagen production of fibroblasts grown on 96-well tissue culture plates. Based on our findings, the use of a serum-free medium is recommended to avoid aspecific signals, while ascorbate supplementation increases the collagen production of fibroblasts. The cell-associated collagens can be quantified by Sirius Red staining in acidic conditions followed by alkaline elution. Immature collagens can be precipitated from the culture medium by acidic Sirius Red solution, and after subsequent centrifugation and washing steps, their amount can be also measured. Increased attention has been paid to optimizing the assay procedure, including incubation time, temperature, and solution concentrations. The resulting assay shows high linearity and sensitivity and could serve as a useful tool in fibrosis-related basic research as well as in preclinical drug screening.

Microfluidic Assessment of T Cell Deformability and Capillary Network Occlusion

Introduction: Adoptive T cell therapies offer a new frontier of cancer treatment that disrupts the conventional chemotherapy paradigm. Naïve T cells can be genetically modified to directly target cancerous cells and have proven to be aggressively effective in fighting cancers resistant to chemotherapies. A major challenge that adoptive T cell therapies face is successfully delivering the cell products to the location of the target cells, which is particularly challenging in the case of solid tumors. A complete characterization of the biomechanical properties of T cell products would help researchers gain insight into the capacity of these engineered cells to navigate and extravasate through vasculature and tumor microenvironments. Previous studies have found that T cell volume, cortical tension, and dynamic resistance increase with activation (Waugh et al. Front Bioeng Biotechnol. 2023). A simple and reproducible method to easily quantify and assess such cellular properties has yet to be established. Presently, we describe a microfluidic assay for the rapid and robust characterization of the mechanical changes of T cells upon activation. Our assay models the microcapillaries through a gradient of micropillar arrays, which allows for the evaluation of T cells perfused through the network. The spacing between pillars is 12 µm at the first array, similar to small arterioles, and incrementally decreases to 3 µm spacing in the last array, mimicking small capillaries ( Figure 1A). Trafficking of adoptive T cell therapies through the microvasculature networks of this scale into their target tissues represents a persistent and major challenge facing these therapies (Marofi et al. Stem Cell Res. Ther. 2021). Methods: The microfluidic devices were fabricated by casting polydimethylsiloxane (PDMS) on a silicone wafer fabricated through photolithographic protocols. Devices contain arrays of pillars measuring 20 µm x 10 µm x 12 µm with gradient spacing beginning at 12 µm at the inlet and 3 µm at the outlet. Jurkat T cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and used in a naïve state or activated via antibody stimulation of CD3 and CD28. T cells were then suspended in growth media at 1x10 6 cells/mL. Microchannels were wetted and cleaned with 100% ethanol perfused at 10 µL/min for 15 minutes followed by cell culture media at 10 µL/min for 15 minutes to block the non-specific binding sites on the PDMS and to create a biocompatible environment for the cell sample. The devices were connected to a digital pneumatic pressure-based flow pump system. The cell sample was perfused at 100 mBar for 7 minutes and a subsequent wash process with growth media at 100 mBar for 7 minutes was performed to flush non-occluded cells. Upon completion of the experiment, the channel was scanned using Phase and DAPI filters with an Olympus IX83 inverted motorized microscope, and occluded T cells were quantified using ImageJ. Results: Our assay showed distinct levels of T cell occlusion upon activation through CD3 and CD28 stimulation. Naïve T cells showed lower levels of occlusion in each of the first five arrays compared to activated T cells, while both groups showed high levels of occlusion in the final 3 µm array. Activated T cells showed approximately three times the amount of occlusion in the second through fifth arrays. To assess overall occlusion, we use the Occlusion Index (OI), which considers the density of pillars and the number of cells caught in each array. We found that activated T cells had a significantly higher OI when compared to non-activated T cell samples (p < 0.05, unpaired t-test). Naïve T cells had an OI mean of 24.4 ± 5.0 while activated T cells had a mean of 42.5 ± 5.6 ( Figure 1B). Conclusions: Our results show that there is a significant increase in resistance of activated T cells through a microvasculature-modeled environment compared to their naïve counterparts. As adoptive T cell therapies increase in popularity, a complete understanding of the biomechanical properties of these cell products will be necessary for effective design. Our microfluidic assay mimics the narrowing channels of the microvasculature and permits the study of how naïve and engineered T cells travel through these mechanically challenging spaces.

Research on the performance of the active vibration cutting system for the rotary cylinder of a tunneling machine

High-efficiency rock breaking technology is an important problem to be solved urgently in deep mining. The existing auxiliary rock breaking technology for coal mine excavation has problems such as polluting coal, high requirements for tool materials, and difficulty in subsequent washing. In this paper, an active excitation cutting system for rotary cylinder of cantilever roadheader based on alternating valve is proposed. According to the classical calculation formula of cutting load of roadheader, considering the stiffness-damping characteristics of cutting part and cutting cylinder, the simulation algorithm of cutting load is compiled based on MATLAB simulation analysis software. The excitation cutting experiments of different cutting depths are carried out on the cutting test bench, and compared with the simulation algorithm of cutting load. Taking the cutting load as the discriminant index, the influence of oil supply pressure and excitation frequency of rotary cylinder on the cutting load is analyzed based on the simulation algorithm of cutting load. The results show that the error between the simulation results and the experimental results of the active excitation state is less than 12%, and the two are in good agreement. Under the condition of 30 HZ, when the excitation amplitude is 8 Mpa, 10 Mpa, 12 Mpa, 14 Mpa, when the rotary cylinder excitation amplitude is 10 Mpa, the minimum is 131.42 KN. Compared with the rotary cylinder excitation amplitude under the condition of 10 Mpa, when the excitation frequency is 30 HZ, 35 HZ, 40 HZ, 45 HZ, when the excitation frequency is 40 HZ, the minimum is 83.08 KN, indicating that changing the excitation amplitude or oil supply pressure of the rotary cylinder is helpful to adjust the cutting performance.