Mixing Experiments Research Articles

Revisiting Thorpe scale analysis and diapycnal diffusivities in Drake Passage

Ship heave effects in the swell-prevalent Southern Ocean induce frequent false overturns in density profiles, and conventional Thorpe scale analysis leads to unrealistically large diapycnal diffusivities. Another critical factor causing large positive biases in Thorpe scale analysis concerns the Ozmidov to Thorpe scales ratio α often considered constant and equal to 0.8. A revised Thorpe scale analysis is proposed with an innovative segment-by-segment approach to circumvent heave-induced false overturns together with best-fitting α functions to Diapycnal and Isopycnal Mixing Experiment in the Southern Ocean (DIMES) data, which yields realistic diffusivities in the Drake Passage region. The revised Thorpe scale method applied to a finely-resolved CTD section across the Drake Passage yields typical diffusivities of O (10-5-10-3 m2 s-1), with the largest values being preferentially concentrated close to the bottom at major circumpolar fronts, consistent with nearby DIMES microstructure observations. In the southern Drake Passage the method highlights a middepth (500-1500 m) mixing maximum of 10-4-10-3 m2 s-1 in the Lower Circumpolar Deep Water (LCDW) layer due to large intrusions of Warm Deep Water (a cold/fresh variety of LCDW) of Weddell Gyre origin. The inferred middepth mixing maximum in intrusive regions, which is associated with isopycnal eddy stirring rather than caused by internal waves or double diffusive mixing, supports the short-circuiting paradigm of meridional overturning circulation in the southwestern Scotia Sea region and has important climatic implications in a warming climate.

Novel Gas Supply System for Multi-Chamber Tri-Gas Cell Culture: Low Gas Consumption and Wide Concentration Range

Gas plays a crucial role in cell culture as cells require a specific gas environment to maintain their growth, reproduction, and function. Here, we propose a gas supply system for tri-gas multi-channel cell incubators to meet the specific needs of various cells. The system utilizes a circulating gas supply method powered by air pumps for each chamber. Gas inflow from the cylinder is independently controlled by Mass Flow Controllers (MFCs), and a quantitative step-by-step adjustment control strategy is employed to calculate the volume of different gases being introduced. Through mixing simulations and experiments, we identified the SV static mixer with an L/D ratio of 2.5 as the optimal choice. To evaluate the concentration accuracy and gas consumption of the gas system, we conduct gas mixing and distribution experiments under different conditions. The results show that the system could achieve a concentration range of 0–100% for O2 with an accuracy of ±0.5%, and a concentration range of 0–10% for CO2 with an accuracy of ±0.1%. The daily gas consumption during cultivation is 3570 mL of N2, 330 mL of CO2, and 115 mL of O2, significantly lower than conventional incubators. Overall, our system can effectively manage dynamic gas concentration changes, particularly in high O2 concentration environments. It offers advantages such as low gas consumption, a wide concentration range, and high accuracy compared to existing incubators.

Three-wave mixing experiments in indium–tin–oxide thin-films with no phase matching

Abstract One challenge of using nonlinear optical phenomena for practical applications is the need to perform phase-matching. Recently, epsilon-near-zero materials have been shown to demonstrate strong optical nonlinearities, in addition to their other unique properties. As suggested by their name, the permittivity of the material is close to zero for a certain wavelength range. We demonstrate that this small permittivity allows for efficient three-wave mixing interactions to take place in indium–tin–oxide thin films without the need for phase matching the pump and signal beams. The efficiency of the second-order nonlinear interactions is characterized, and cascaded three-wave mixing is demonstrated.

Neuropathologically directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases,<1% were transmitted by misfolded PrP, ~ 15% are inherited, and ~ 85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate localized initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of > 5000× across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a localized presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Chaotic Mixing Experiments: Unravelling Contamination Processes on Dacitic Melts

Magmas are the ultimate source of volcanism on Earth. At high-temperatures these complex silicate melts may easily chaotically mix, on their way towards the surface of the planet.The Paraná-Etendeka Magmatic Province (PEMP), one of the largest volcanic provinces of our planet, serves as the focus of this experimental study. The investigation simulates the contamination of high-Ti dacites passing through the continental crust. Our final aim is to contribute to the origin of the high-Ti Chapecó dacites from PEMP, with particular attention to the genesis of Chapecó-Ourinhos dacites (COD).The experimental apparatus is based on the Journal Bearing System. All experimental runs have been operated at atmospheric pressure and a temperature of 1500 °C. A dacitic magma chamber under the assimilation of crustal material has been simulated. The resulting experimental products (dry/bubble free glasses) exhibit concentric folded and stretched filaments of contrasting compositions. The calculated fractal dimension for a representative section is Dbox = 1.60(3), confirming the achievement of chaotic dynamics. Electron Microprobe Analysis (EMPA) from the experimental glasses clearly shows SiO2-contamination, while Al2O3-data indicate lower contamination levels. As a consequence, different elements depict dissimilar curves in chemical transects. We used the parameter σn2 (normalized variance) to quantify the mixing efficiency and differential elemental mobilities. In addition, our data reproduced the chemical behaviour of some elements in COD- dacites, especially the network formers such as Fe, Al, Ti and Si. EMPA reveals clear deviations from the linear mixing model. Our experiments and data replicate a selective contamination process. Differential elemental mobilities could be calculated. Comparisons with natural data depict how the mixing involving Chapecó-Guarapuava (CGD) and Chapecó-Ourinhos (COD) dacitic end-members may have contributed to the generation of more evolved COD from the interaction of CGD pre-existing melts with crustal rocks from the PEMP basement via chaotic dynamics.

Two‐way feedback between the Madden–Julian Oscillation and diurnal warm layers in a coupled ocean–atmosphere model

AbstractDiurnal warm layers develop in the upper ocean on sunny days with low surface wind speeds. They rectify intraseasonal sea‐surface temperatures (SSTs), potentially impacting intraseasonal weather patterns such as the Madden–Julian Oscillation (MJO). Here we analyse 15‐lead‐day forecast composites of coupled ocean–atmosphere and atmosphere‐only numerical weather prediction (NWP) models of the UK Met Office to reveal that the presence of diurnal warming of SST (dSST) leads to a faster MJO propagation in the coupled model compared with the atmosphere‐only model. To test the feedback between the MJO and the dSST, we designed a set of experiments with instantaneous vertical mixing over the top 5 or of the ocean component of the coupled model. Weaker dSST in the mixing experiments leads to a slower MJO over 15 lead days. The dSST produces a increase in the MJO phase speed between the coupled and the atmosphere‐only model. An additional increase is found for other coupling effects, unrelated to the dSST. A two‐way feedback manifests in the coupled model over the 15 lead days of the forecast between the MJO and the dSST. The MJO regime dictates the strength of the dSST and the dSST rectifies the intraseasonal anomalies of SST in the coupled model. Stronger dSST in the coupled model leads to stronger intraseasonal anomalies of SST. The MJO convection responds to these SSTs on a seven‐lead‐day timescale, and feeds back into the SST anomalies within the next three lead days. Overall, this study demonstrates the importance of high vertical resolution in the upper ocean for predicting the eastward propagation of the MJO in an NWP setting, which is potentially impactful for seasonal predictions and climate projections, should this feedback be unrepresented in the models.

Neuropathologically-directed profiling of PRNP somatic and germline variants in sporadic human prion disease.

Creutzfeldt-Jakob Disease (CJD), the most common human prion disease, is associated with pathologic misfolding of the prion protein (PrP), encoded by the PRNP gene. Of human prion disease cases, ~1% were transmitted by misfolded PrP, ~15% are inherited, and ~85% are sporadic (sCJD). While familial cases are inherited through germline mutations in PRNP, the cause of sCJD is unknown. Somatic mutations have been hypothesized as a cause of sCJD, and recent studies have revealed that somatic mutations accumulate in neurons during aging. To investigate the hypothesis that somatic mutations in PRNP may underlie sCJD, we performed deep DNA sequencing of PRNP in 205 sCJD cases and 170 age-matched non-disease controls. We included 5 cases of Heidenhain variant sporadic CJD (H-sCJD), where visual symptomatology and neuropathology implicate focal initiation of prion formation, and examined multiple regions across the brain including in the affected occipital cortex. We employed Multiple Independent Primer PCR Sequencing (MIPP-Seq) with a median depth of >5,000X across the PRNP coding region and analyzed for variants using MosaicHunter. An allele mixing experiment showed positive detection of variants in bulk DNA at a variant allele fraction (VAF) as low as 0.2%. We observed multiple polymorphic germline variants among individuals in our cohort. However, we did not identify bona fide somatic variants in sCJD, including across multiple affected regions in H-sCJD, nor in control individuals. Beyond our stringent variant-identification pipeline, we also analyzed VAFs from raw sequencing data, and observed no evidence of prion disease enrichment for the known germline pathogenic variants P102L, D178N, and E200K. The lack of PRNP pathogenic somatic mutations in H-sCJD or the broader cohort of sCJD suggests that clonal somatic mutations may not play a major role in sporadic prion disease. With H-sCJD representing a focal presentation of neurodegeneration, this serves as a test of the potential role of clonal somatic mutations in genes known to cause familial neurodegeneration.

Modeling the geochemical evolution of mine waters during mixing

This study focuses on assessing the hydrogeochemical processes influencing the mobility of dissolved metal and metalloid species during mine effluent mixing. Field samples were collected to characterize effluents at an active gold mine located in the Abitibi Greenstone belt in western Québec, Canada. Controlled laboratory mixing experiments were further performed with real effluents. In situ physicochemical parameters, concentrations of major dissolved ions and trace elements were analyzed. Mineralogical analyses were also performed on precipitates from the laboratory mixtures. The data were used for statistical analyses and for modeling the geochemical evolution of effluents using PHREEQC with the wateq4f.dat database (with modifications). The results suggest that the formation of secondary minerals such as schwertmannite, Fe(OH)3, and jarosite could significantly affect the concentrations of trace elements in effluents. The precipitation of secondary minerals immobilized trace elements through coprecipitation and sorption processes. The main limitations of the modeling approach used here include the evaluation of the ion balance for low pH samples with high Fe and Al concentrations and the omission of biological processes. The approach provides insights into the geochemical evolution of mine effluents and could be adapted to several mining sites as a tool for improving water management.

Experimental and Numerical Investigations of Mixing Performance of Mixing Agitators of Deep Cement Mixing Ships

Recent decades have witnessed the increasing usage of deep cement mixing (DCM) mixers in the field of marine infrastructure construction. The mixing performance, including the torque history, can be helpful for structural safety evaluation, design, and the optimization of agitators, which is of engineering significance. However, to the best of the authors’ knowledge, there are no related publications that have reported the mixing behaviors of deep cement mixing agitators. In light of this, the present work conducts experimental and numerical investigations of the mixing behaviors of a DCM ship mixing agitator. To achieve this end, a model test device is established, and mixing experiments using two- and three-blade mixers are respectively conducted. Silt and clay soils are considered in the experiments with a three-blade mixer, while clay soils are used for those with a two-blade mixer. In addition, this work designs a torque transducer placed inside the rotating rod to accurately measure the torque history of the agitator during model test experiments. The experimental results show that, when mixing clay using agitators with different blades, the average torque value required for a two-blade agitator is slightly larger than that for a three-blade one. This study also presents a computational framework based on the arbitrary Lagrangian–Eulerian (ALE) method for an efficient and accurate modeling of the soil-mixing behaviors of the agitator. The numerical results are found to be in good agreement with the experimental data from model tests in terms of torque history, which demonstrates the effectiveness and capacity of our presented computational framework. The numerical results show that the average torque value is smaller at a higher rotational speed during the mixing of clay using a two-bladed agitator, but the effect of rotational speed on the torque history is small. The experimental and numerical methods introduced in the present work can act as a useful tool for investigations of mixing behaviors of DCM agitators.

Research on the thermal conductivity and mechanical properties of carbon fiber reinforced thermoplastic composites doped with different sizes carbon nanotubes: A combination of experiments, numerical simulations and multi‐objective optimization

AbstractWhile improving the thermal conductivity of composites, single‐size carbon nanotubes (CNT) often degrade their mechanical properties due to the agglomerates. To overcome this limitation, this study reported thermoplastic resin matrix composites synergistically reinforced with small, medium, and large‐size carbon nanotubes (S‐CNT, M‐CNT, and L‐CNT). Using the thermal conductivity (λ) and mechanical properties (flexural strength [F], compressive strength [C] and tensile strength [T]) as the response values, we conducted 21 sets of mixing design experiments and developed the associated quadratic term models. Further multi‐objective optimization was carried out for the above response values. Optimized multiscale CNT synergistically modified composites (S‐CNT, M‐CNT, and L‐CNT contents of 1.25, 7.02, and 2.56 wt%) exhibited a λ value of 1.168 W/mK, which was 121% higher than that of pure composite and also superior to the same content of S‐CNT‐ (1.031 W/mK), M‐CNT‐ (1.016 W/mK), and L‐CNT‐modified composites (0.983 W/mK). Additionally, the optimized composite demonstrated excellent mechanical properties (F = 1482 MPa, C = 848 MPa, and T = 1759 MPa), which were 13%, 11%, and 13% higher than those of the pure composites, respectively, and also surpassed those of the S‐CNT‐, M‐CNT‐and L‐CNT‐modified composites.Highlights Using three different scales of carbon nanotubes modified CF/PPBESK composites. Polynomial modeling of composite's thermal conductivity and mechanical properties. Synergistic interactions between carbon nanotubes to improve the composite's thermal conductivity and mechanical properties.

Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

An asymmetric orifice-based active micromixer in the microfluidic chip with 3D microelectrode

The fluid mixing in microscale is central to the development of flow patterns in microfluidic chips for particles separation and biological analysis. Yet, as the size of the fluid decreases, the mixing becomes challenging to change this state of flow. In this work, a novel active micromixer for enhancing the mixing performance in the micromixer with asymmetric orifices is presented. To induce the electric-driven micromixing, an AC electric field is employed to the inserted 3D microelectrodes to produce the local electric field through the asymmetric orifices located on the opposite channel walls, which is perpendicular to the main microchannel. Then the mixing experiments were conducted in a microfluidic chip comprising microelectrode with asymmetric orifices, specifically exploiting the mixing efficiency varying with the asymmetric orifice pairs, applied electric potential and frequency of the AC electric field, and flow rate. The bending degree of the mixing interface was calculated and found increasing as a function of applied electric voltage at a given AC frequency, thus enabling effective mixing crossing the microelectrode's area. The predictions are confirmed by optical microscopy experiments and the micromixing can be significantly improved as they experience longer-exposed electrical forces. The integration of multiple asymmetric orifices leads a larger zone to increase mixing efficiency and makes it a promising method for rapid and stable fluid mixing in microscale.

Experimental and theoretical study of single bubble formation at the nozzle in a liquid oxygen crossflow

In this paper, the condensation bubbles generated by the mixing and condensation of gaseous and liquid oxygen crossflow experimentally and theoretically. The mixing and condensation experiment of the bubbles was carried out, employing gaseous and liquid oxygen as working fluids in vertical tubes. Three successive flow patterns were observed: initial gas plume, condensation oscillation, and separation condensation. The corresponding characteristics include gas mass aggregation, bubble surface smoothness and wrinkling alternately, and bubble disappearance after separation and condensation. Then, a Nusselt number (Nu) heat transfer correlation formula is fitted with a corresponding fitting error in the range of −31% ∼ 35%. In addition, bubble force balance and conservation equations were employed to establish a mixing condensation bubble model suitable for gaseous and liquid oxygen in vertical tubes, and the error between theoretical and experimental results was in the range of −25% ∼ 20%. Furthermore, the effects of working conditions and forces on bubble condensation length has been analyzed. The results showed that increasing the liquid oxygen flow rate, reducing the gas oxygen volume flow rate, or appropriately reducing the gas inlet diameter led to reducing the bubble condensation length. It was shown that the bubble drag and shear lift forces affect the condensation length mostly.

An Efficient Probe-Based Quantitative PCR Assay Targeting Human-Specific DNA in ST6GALNAC3 for the Quantification of Human Cells in Preclinical Animal Models.

Precise quantification of human cells in preclinical animal models by a sensitive and specific approach is warranted. The probe-based quantitative PCR (qPCR) assay as a sensitive and swift approach is suitable for the quantification of human cells by targeting human-specific DNA sequences. In this study, we developed an efficient qPCR assay targeting human-specific DNA in ST6GALNAC3 (termed ST6GAL-qPCR) for the quantification of human cells in preclinical animal models. ST6GAL-qPCR probe was synthesized with FAM and non-fluorescent quencher-minor groove binder conjugated to the 5' and 3' end of the probe, respectively. Genomic DNA from human, rhesus monkeys, cynomolgus monkeys, New Zealand White rabbits, SD rats, C57BL/6, and BALB/c mice were utilized for analyzing the specificity and sensitivity of the ST6GAL-qPCR assay. The ST6GAL-qPCR assay targeted human-specific DNA was cloned to pUCM-T vector and released by EcoR I/Hind III digestion for generating a calibration curve. Cell mixing experiment was performed to validate the ST6GAL-qPCR assay by analysis of 0.1%, 0.01%, and 0.001% of human leukocytes mixed with murine thymocytes. The ST6GAL-qPCR assay detected human DNA rather than DNA from the tested animal species. The amplification efficiency of the ST6GAL-qPCR assay was 93% and the linearity of calibration curve was R2 = 0.999. The ST6GAL-qPCR assay detected as low as 5 copies of human-specific DNA and is efficient to specially amplify as low as 30-pg human DNA in the presence of 1μg of DNA from the tested species, respectively. The ST6GAL-qPCR assay was able to quantify as low as 0.01% of human leukocytes within murine thymocytes. This ST6GAL-qPCR assay can be used as an efficient approach for the quantification of human cells in preclinical animal models.

Strategy on rapid discrimination of different varieties based on the combination of HS-GC-IMS and DNA Mini-barcode, spore powder of Ganoderma as a case study

The demand for the consumption of Ganoderma (LingZhi) is increasing significantly nowadays. While the wild resources of Ganoderma lucidum and Ganoderma sinense have been reduced severely, and artificially cultivated products are gradually occupying the market. However, it is usually difficult to distinguish the different species of Ganoderma spore powder (GSP). In this research, a rapid and accurate identification method which combined headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) with the DNA mini-barcode was successfully developed to discriminate different GSP. The feasibility of the method was evaluated by both GSP and its Chinese patent medicines. HS-GC-IMS was used to characterize GSP volatile organic compounds (VOCs) and a total of seventy-one VOCs were identified. Principal component analysis could be utilized to show the significantly difference between Korean LingZhi (KL) and American LingZhi (AL) or Taishan LingZhi (TL). Then, we clarified the species information of all samples based on the internal transcribed spacer (ITS) regions. The identification results showed that TL and KL were Ganoderma lucidum, and the AL was Ganoderma resinaceum. Furthermore, according to the mutation sites of the above ITS sequences, the specialized DNA mini-barcode was successfully developed and applied. The artificial mixtures of the three types of GSP and Chinese patent medicines containing GSP were used to authenticate the barcode capability. The mixing experiment found that there was a linear relationship between the sequence numbers and the mixed biomass, indicating that the qualitative and quantitative ability of DNA mini-barcode performed well. The investigation of the ingredients of commercially available GSP products found that the commercial products maybe contain Ganoderma resinaceum, Ganoderma applanatum and Daedaleopsis confragosa, except for Ganoderma lucidum. Therefore, the results suggested that the available GSP products might be adulterated according to Chinese Pharmacopoeia. In this paper, we established a novel approach to distinguish the different GSP samples based on HS-GC-IMS and DNA mini-barcode, and the results indicated that our strategy could not only discriminate different GSP samples, but also identify the origins in their related products.

All-optical flip-flop by carbon/metal nanostructures in a multi-wave mixing

This work presents a design of a flip-flop controlled by the nonlinear optical response exhibited by a dynamic distribution of nanostructures in a liquid. Silver decorated carbon nanotubes suspended in ethanol were employed. The flip-flop proposed corresponds to a probabilistic NAND latch. The nonlinear nanofluids were studied by two-wave mixing experiments with nanosecond pulses at 532 nm wavelength. The behavior of the system is based on the Brownian motion with influence over the nonlinear transmittance of the nanostructures associated with a probabilistic optical bit. The optical response of the latch system output states was defined by the irradiance dependent on nonlinear optical absorption of the nanofluid. Specifically, the optical logical outputs depend on the state present at the optical inputs and the previous optical output states regulated by the feedback of the system. The nonlinear optical evolution of the effects in the nanofluids was explored as probabilistic phenomena governed by a concentration gradient of nanoparticles. The evaluation of the stability of a nanofluid defines the variables in the probabilistic system proposed. This work highlights the potential of nanohybrid materials for processing probabilistic functions, switching quantum effects, and for developing optical sensors.

Vortex-Enhanced Microfluidic Chip for Efficient Mixing and Particle Capturing Combining Acoustics with Inertia.

Vortex-based microfluidics has received significant attention for its unique characteristics of high efficiency, flexible control, and label-free properties for the past decades. Herein, we present a vortex-based acousto-inertial chip that allows both fluid and particle manipulation within a significantly wider flow range and lower excitation voltage. Composed of contraction-expansion array structures and vibrating microstructures combined with bubbles and sharp edges, such a configuration results in more vigorous vortical fluid motions. The overall improvement in device performance comes from the synergistic effect of acoustics and inertia, as well as the positive feedback loop formed by vibrating bubbles and sharp edges. We characterize flow patterns in the microchannels by fluorescence particle tracer experiments and uncover single- and double-vortex modes over a range of sample flow rates and excitation voltages. On this basis, the ability of rapid and efficient sample homogenization up to a flow rate of 200 μL/min under an excitation voltage of 15 Vpp is verified by a two-fluid fluorescence mixing experiment. Moreover, the recirculation motion of particles in microvortices is investigated by using a high-speed imaging system. We also quantitatively measure the particle velocity variation on the trajectory and illustrate the capturing mechanism, which results from the interaction of the microvortices, particle dynamics, and composite microstructure perturbations. Further utilizing the shear forces derived by microvortices, our acousto-inertial chip is demonstrated to lysis red blood cells (RBCs) in a continuous, reagent-free manner. The high controllability and multifunction of this technology allow for the development of multistep miniaturized "lab-on-chip" analytical systems, which could significantly broaden the application of microvortex technology in biological, chemical, and clinical applications.

Rapid and non-destructive nanofiber diameter measurement using Spontaneous Four Wave Mixing

We will present a rapid and non-destructive technique for the measurement of the mean diameter of a silica nanofiber using the wavelength position of the signal peak in a spontaneous four wave mixing experiment. The nanofiber diameter can be characterized in a range going at least from 650 to 1250nm. Several nanofibers were characterized, and the measured diameter show a good accordance with the one obtained using a Scanning Electron Microscope. The technique is simple to use and has even the potential to be implemented in situ in order to realize a diameter measurement during the pulling of the nanofiber for a better control of the final diameter of the nanofiber.

Understanding molecular and cellular mechanisms of APOE4‐mediated susceptibility to tau‐related cognitive impairments in the miBrain model

AbstractBackgroundAPOE4 is the strongest genetic risk factor for Alzheimer’s disease with Tau pathology, namely hyperphosphorylation (p‐Tau) and aggregation of Tau into neurofibrillary tangles (NFTs) being correlated with poor cognitive outcomes in AD. Instances of mild traumatic brain injury (TBI) have been associated with increased susceptibility to neurodegenerative diseases such as AD and related dementias. We hypothesize that human APOE4 carriers’ brains are more susceptible to increased tau pathology with instances of TBI further increasing the severity.MethodUsing the 3D multi‐cellular integrated human brain (miBrain) model, which recapitulates key brain tissues through the co‐culturing of all hiPSC‐derived major brain cell‐types, Tau pathology can be induced with treatment of Tau fibrils or by increasing endogenous phosphorylated Tau with methotrexate.ResultAPOE4/4 miBrain cultures exhibit increased Tau pathology compared to isogenic APOE3/3 miBrains. Combinatorial genetic mixing experiments revealed that the increase of Tau hyperphosphorylation is dependent on the presence of APOE4 microglia.ConclusionData suggests that microglia facilitate the accumulation of p‐tau in APOE4 genetic backgrounds in an in vitro 3D model.

Two Players, One Goal: BiTE ® Vs CART Targeting FLT3 in AML

Translation of the success of bispecific T cell engagers (BiTE®) and CAR T cells from B-cell to myeloid malignancies has been challenging. Identifying suitable target antigens in myeloid malignancies has been hampered by expected on-target-off leukemia toxicity. FLT3 presents with a rather favorable expression profile due to broad AML expression and limited expression within the normal hematopoietic compartment (Brauchle et al., 2020). Importantly, expression on AML bulk cells and leukemic stem cells is observed independent of FLT3 mutational status. Here, we compared two T-cell based immunotherapy approaches, BiTE® vs CART, in targeting FLT3 in AML. We tested BiTE® and CAR-mediated cytotoxicity against several AML cell lines and primary AML (pAML) cells in vitro. On-target off-leukemia toxicity was evaluated in mixing assays of pAML cells and healthy donor bone marrow (hBM). The impact of positive costimulation on both approaches was tested using our previously established Ba/F3 model system (Marcinek et al., 2023), which is devoid of any human costimulatory molecules. As a surrogate for synapse formation, conjugate formation as well as consecutive T-cell degranulation were studied. Further, we studied the cell death pathway mediating cytotoxicity against AML target cells, e.g. FAS/FASL vs perforin/granzyme B. The impact of continuous stimulation by BiTE®-and CART on evolving T-cell exhaustion was tested using our previously established 28-day long-term culture assay (Philipp et al., 2022). Finally, we compared BiTE® and CAR in an in vivo xenograft AML model. In a second in vivo experiment, all mice were harvested 18 days after T-cell injection, followed by T-cell isolation from the bone marrow and subsequent bulk RNA sequencing. BiTE®- and CAR-redirected T cells led to comparable effector-to-target (E:T) ratio-dependent specific lysis of various AML cell lines and pAML cells. Using our in vitro mixing experiments, we could show that BiTE®- and CAR-redirected T cells efficiently killed pAML cells while mostly sparing the hBM. Using the Ba/F3 model system, we observed that the expression of the costimulatory molecule CD86 significantly increased the cytotoxicity of BiTE®-redirected T cells while CAR-mediated lysis was unaltered. Furthermore, we were able to show that the percentage of T cell-AML conjugates formed as well as degranulation of CD107a was significantly higher when using CART compared to BiTE® molecules. Interestingly, when we added a FAS-blocking antibody to our cocultures, we could show that CART-mediated lysis of the target cells was significantly reduced. This was not observed when using the BiTE® construct. Using our long-term culture system, we found that T-cell proliferation and cytokine secretion decreased with both constructs over time. The cytotoxic capacity of BiTE®-redirected T cells seemed to decrease faster compared to the CART construct. Moving to an in vivo AML xenograft model, we observed that CART-treated mice had an improved OS compared to the BiTE®-treated mice (Fig.1). This was accompanied by enhanced T-cell proliferation and splenic homing of the CAR T cells. Bulk RNA sequencing of T cells isolated from the murine bone marrow 18 days post T-cell injection revealed signs of exhaustion in BiTE®-redirected T cells compared to CAR T cells. We found an upregulation of several inhibitory immune receptors and transcription factors (Fig.2) that have been well described to promote T-cell exhaustion. Furthermore, gene set enrichment analysis revealed that genes upregulated in effector vs exhausted were also upregulated in CAR T cells. This was accompanied by an upregulated glycolysis and fatty acid metabolism in CAR T cells. We conclude, that FLT3 is a promising target antigen with limited on-target off-leukemia toxicity. Our preclinical in vitro data demonstrate similar cytotoxicity of both platforms against AML cell lines and pAML cells. Utilizing a xenograft mouse model, CAR T cells provided a significant survival benefit over BiTE® molecules. Our data support the hypothesis that positive costimulation integrated within the CAR construct provided an advantage in T-cell activation and might lead to better T-cell homing, resulting in improved efficacy and delayed T-cell exhaustion in vivo. Future studies will be needed to further dissect differences between BiTE® vs CAR T cell-based immunotherapy platforms and identify suitable combination partners.