High-yield Method Research Articles

High-Yield Bioproduction of Extracellular Vesicles from Stem Cell Spheroids via Millifluidic Vortex Transport.

Extracellular vesicles (EVs) are emerging as novel therapeutics, particularly in cancer and degenerative diseases. Nevertheless, from both market and clinical viewpoints, high-yield production methods using minimal cell materials are still needed. Herein, a millifluidic cross-slot chip is proposed to induce high-yield release of biologically active EVs from less than three million cells. Depending on the flow rate, a single vortex forms in the outlet channels, exposing transported cellular material to high viscous stresses. Importantly, the chip accommodates producer cells within their physiological environment, such as human mesenchymal stem cells (hMSCs) spheroids, while facilitating their visualization and individual tracking within the vortex. This precise control of viscous stresses at the spheroid level allows for the release of up to 30000 EVs per cell at a Reynolds number of ≈400, without compromising cellular integrity. Additionally, it reveals a threshold initiating EV production, providing evidence for a stress-dependent mechanism governing vesicle secretion. EVs mass-produced at high Reynolds displayed pro-angiogenic and wound healing capabilities, as confirmed by proteomic and cytometric analysis of their cargo. These distinct molecular signatures of these EVs, compared to those derived from monolayers, underscore the critical roles of the production method and the 3D cellular environment in EV generation.

Modular Micromotor Fabrication with Self-Focusing Lithography.

Synthetic Janus micro/nanomotors can efficiently convert ambient energy into asymmetrical self-propulsive force, overcoming random thermal fluctuations and enabling autonomous migration. Further modifications to the motors can equip them with different functional modules to meet different needs. However, developing a versatile and high-yield fabrication method for multifunctional Janus micromotors remains challenging. In this study, a modular fabrication approach for micromotors with a particle-tip structure based on the self-focusing lithography induced by an array of TiO2 microspheres is presented. By adjusting the tip composition or loading, precise programming of motor functionality is achieved, allowing for various capabilities such as photoredox reaction-induced propulsion, fluorescent imaging, electric and magnetic navigation. Furthermore, the flexibility of this fabrication method by selectively loading materials onto two tips is demonstrated to achieve multifunctionality within a micromotor unit. This study proposes a straightforward and versatile approach for modular functional micromotors.

New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies.

Mild Catalyst- and Additive-Free Three-Component Synthesis of 3-Thioisoindolinones and Tricyclic γ-Lactams Accelerated by Microdroplet Chemistry.

Isoindolinones, bearing both γ-lactam and aromatic rings, draw extensive interest in organic, pharmaceutical, and medicinal communities as they are important structural motifs in many natural products, bioactive compounds, and pharmaceuticals. As the main contributor to isoindolinone synthesis, metal catalysis is associated with many drawbacks including essential use of toxic/precious metals and excessive additives, high reaction temperatures, specially predesigned starting materials, and long reaction times (typically 8-30 h). In this study, we developed a catalyst- and additive-free, minute-scale, and high-yield microdroplet method for tricomponent isoindolinone synthesis at mild temperatures. By taking advantage of the astonishing reaction acceleration (1.9 × 102-9.4 × 103 acceleration factor range with a typical rate acceleration factor of 1.51 × 103 for the prototype reaction as the ratio of rate constants by microdroplet and bulk phase), 12 3-thioisoindolinones and two tricyclic γ-lactams were synthesized using various 2-acylbenzaldehydes, amines, and thiols with satisfactory yields ranging from 85% to 97% as well as a scale-up rate of 3.49 g h-1. Because of the advantages (no use of any catalysts or additives, mild temperature, rapid and satisfactory conversion, broad substrate scope, and gram scalability), the microdroplet method represents an attractive alternative to metal catalysis for laboratory synthesis of isoindolinones and their derivatives.

An environmentally sustainable ultrasonic-assisted exfoliation approach to graphene and its nanocompositing with polyaniline for supercapacitor applications

In the present work, a green high-yielding method for the preparation of graphene is introduced via ultrasonic-assisted liquid phase exfoliation (LPE) of graphite in a green solvent medium, since the common preparation method of graphene via graphite oxide is hazardous. A high concentration of 3.2 mg/ml graphene is achieved here in a comparatively short duration of 3 h ultrasonication. By using a mixed solvents strategy (acetophenone and isopropyl alcohol, 1:19 V/V), surface energy requirements needed for the exfoliation of graphite are satisfied here with acetophenone, where isopropyl alcohol further facilitated the exfoliation via non-conventional CH-π and OH-π interactions. Turbostratic graphene in high-yield (16 %) in a simple means of ultrasonic assisted LPE is the added attraction of the present procedure. The less-defective structure of graphene, its few-layered turbostratic nature, and edge functionalization of the sheets are evident from the material characterization via Raman spectroscopy, XRD, TEM-SAED, and XPS analyses. Here, we report a combination of the attractive conducting polymer polyaniline (PANI) with the as-prepared graphene for supercapacitor applications, where the PANI/graphene nanocomposites with different aniline concentrations (PANI1.125/G, PANI4.5/G, and PANI9/G) have been prepared via in-situ polymerization of aniline in the graphene dispersion. The structure and morphology of the nanocomposites are investigated using different characterization techniques which revealed that the molecular structure of the PANI is retained in the nanocomposites even with a strong interaction with graphene. FESEM and TEM images revealed the good coverage of graphene sheets with PANI that limit the volume change of PANI during the repeated charge-discharge processes. Electrochemical studies showed that PANI4.5/G has the highest specific capacitance of 126.16 mF/cm2 at a current density of 1 mA/cm2, resulting from the perfect combination of the pseudocapacitance behavior of the PANI along with the electrical double layer capacitance of graphene. A symmetric supercapacitor device is also fabricated with PANI4.5/G, which showed the highest areal capacitance of 116.38 mF/cm2 similar to that with three-electrode studies and also good cycling stability with 87 % capacitance retention in the specific capacitance after 6000 cycles. It also exhibited an energy density of 16 µWh/cm2 (0.29 Wh/kg) and a power density of 3.99 mW/cm2 (72.72 W/kg).

Transition-Metal-Free C-Diarylations to Reach All-Carbon Quaternary Centers.

Herein, we disclose a convenient protocol for the α-diarylation of carbon nucleophiles to yield heavily functionalized quaternary products. Diaryliodonium salts are utilized to transfer both aryl groups under transition-metal-free conditions, which enables an atom-efficient and high-yielding method with broad functional group tolerance. The methodology is amenable to a wide variety of carbon nucleophiles and can be utilized in late-stage functionalization of complex arenes. Furthermore, it is compatible with a new class of zwitterionic iodonium reagents, which gives access to phenols with an ortho-quaternary center. The diarylated products bear an ortho-iodo substituent that can be utilized in further transformations, including the formation of novel, functionalized six-membered cyclic iodonium salts.

Facile Synthesis of Silanol Group Rich Sn‐Beta for Highly Efficient Isomerization of Glucose to Fructose

AbstractThe isomerization of glucose to fructose is an important process in the conversion of biomass into valuable fuels and chemicals. Development of efficient catalyst for this reaction has attracted much attention. In this study, Sn‐Beta zeolites with rich silanols were synthesized for glucose isomerization by solid‐state ion‐exchange method, where Si‐Beta zeolites with different amounts of silanols were applied as the precursors. It is confirmed that open Sn sites show much higher catalytic performance than closed Sn sites for glucose isomerization (about 2.1 times). The silanol amount in Si‐Beta not only affects the formation of framework Sn sites in Sn‐Beta but also influences the isomerization reaction. More silanols in the catalyst can promote the 1,2‐H shift in isomerization and reduce the side reactions of formation of mannose and C3 products. Over 3Sn‐Beta‐3h with rich silanols, 64 % of fructose yield was obtained. Furthermore, the prepared Sn‐Beta catalyst is stable and recyclable. High fructose yield and facile synthesis method of Sn‐Beta reported in this work will contribute to the development of biomass and sugar industry.

Novel Polypeptide Automated Synthesis Devices: A Review

Peptides are a unique class of proteins and one-dimensional biological nanomaterials that play an exceptionally important role in life science and industrial applications. At present, peptide synthesis equipment encounters issues like low synthetic efficiency, challenges in scaling up, and limited automation. This article provides an overview of the key technologies in peptide synthesis equipment, covering aspects such as peptide information screening, peptide synthesis, and detection. It analyzes and summarizes the principles, methods, and critical challenges of traditional solid-phase peptide synthesis technology, microwave-assisted solid-phase peptide synthesis technology, and continuous-flow solid-phase peptide synthesis technology. It also discusses microfluidic solid-phase peptide synthesis technology and future research trends, offering insights into the search for an efficient, high-yield, high-purity, scalable, and intelligent peptide synthesis method, which holds significant research significance.

Tailoring silk fibroin fibrous architecture by a high-yield electrospinning method for fast wound healing possibilities.

In this study, a novel array electrospinning collector was devised to generate two distinct regenerated silk fibroin (SF) fibrous membranes: ordered and disordered. Leveraging electrostatic forces during the electrospinning process allowed precise control over the orientation of SF fiber, resulting in the creation of membranes comprising both aligned and randomly arranged fiber layers. This innovative approach resulted in the development of large-area membranes featuring exceptional stability due to their alternating patterned structure, achievable through expansion using the collector, and improving the aligned fiber membrane mechanical properties. The study delved into exploring the potential of these membranes in augmenting wound healing efficiency. Conducting in vitro toxicity assays with adipose tissue-derived mesenchymal stem cells (AD-MSCs) and normal human dermal fibroblasts (NHDFs) confirmed the biocompatibility of the SF membranes. We use dual perspectives on exploring the effects of different conditioned mediums produced by cells and structural cues of materials on NHDFs migration. The nanofibers providing the microenvironment can directly guide NHDFs migration and also affect the AD-MSCs and NHDFs paracrine effects, which can improve the chemotaxis of NHDFs migration. The ordered membrane, in particular, exhibited pronounced effectiveness in guiding directional cell migration. This research underscores the revelation that customizable microenvironments facilitated by SF membranes optimize the paracrine products of mesenchymal stem cells and offer valuable physical cues, presenting novel prospects for enhancing wound healing efficiency.

Isolation of stilbenoids from fresh knotwood of Scots pine using a high yield method

Abstract Scots pine knotwood contains valuable amount of stilbenoids such as pinosylvin (Ps) and pinosylvin monomethyl ether (PsMME). The stilbenoids have numerous biological activities. Traditional methods of extracting stilbenoids often yield low quantities and involve complex processes. In the present study, a simple and high-yield method was proposed for the extraction of stilbenoids from fresh Scots pine knotwood using ethanol taking a total of three days. In addition, the impact of drying process, extraction time, extraction solvents (acetone and ethanol) and storage time were evaluated on the yield of the stilbenoids. The increased yield and improved extraction efficiency make this method a promising advancement for promoting the utilization of stilbenoids for their valuable bioactive properties.

Gas-Flow-Rate Inversion Based on Experiments and Simulation of Flame Combustion Characteristics in a Drilling Blowout

Accurately estimating the gas-flow rate at a wellhead to invert the formation pressure and production capacity information can be the basis for subsequent well-killing parameter design following oil and gas-well drilling blowout and ignition. Based on the multicomponent characteristics of the blowout gas and the turbulence intensity of the blowout flame, as well as the effects of complex factors such as environmental wind direction, wind speed, and wellhead structure, a numerical model for actual drilling blowout ignition is established. Jet-flame experiments are conducted under blowout conditions to verify the accuracy of the model. The temperature and radiation fields and flame morphologies of the well jet flow flame under different lateral wind speeds, well jet flow rates, and wellhead diameters are analyzed. Previous studies have found that as the lateral wind speed increases, the maximum temperature and maximum thermal radiation intensity of the blowout flame first decrease and then increase. However, as the amount of well jet increases, although the flame influence area increases, the maximum temperature does not increase significantly, and the maximum thermal radiation intensity actually decreases. Based on experimental and numerical simulation datasets, a high-yield gas-well jet volume prediction method based on the flame height and thermal radiation intensity of the well jet flow is constructed, which has important engineering application value for achieving successful well killing following well jet ignition.

Integration of resol/block-copolymer carbonization and machine learning: A convenient approach for precise monitoring of glycan-associated disorders

Ensuring the timely and precise monitoring of severe liver diseases is crucial for guiding effective therapies and significantly extending overall quality of life. However, this remains a worldwide challenge, given the high incidence rate and the presence of strong confounding clinical symptoms. Herein, we applied a convenient and high-yield method to prepare the magnetic mesoporous carbon (MMC-Fe), guided by a composite of resol and triblock copolymer. With the combination of MMC-Fe, high-throughput mass spectrometry, and a simple machine learning algorithm, we extracted N-glycan profiles from various serum samples, including healthy controls, liver cirrhosis, and liver cancer, and from which we screened specific N-glycans. Specifically, the selected N-glycans demonstrate exceptional performance with area under the curve (AUC) values ranging from 0.948 to 0.993 for the detection of liver diseases, including alpha fetoprotein (AFP)-negative liver cancer. Among them, five N-glycans holds potential in monitoring distinctions between liver cirrhosis and AFP-negative liver cancer (AUC values of 0.827–0.842). This study is expected to promote the glycan-based precise monitoring of diseases, not limited to liver disease.

Optimization of an efficient hydroponic cultivation method for high yield of strawberry plants

The widely grown octoploid strawberry (2n = 8x = 56) (Fragaria × Ananassa Duch.), cherished for its delectable flavour, rich nutritional content, and pleasing aroma has consistently held a significant position in global fruit market. Despite of rising demand, strawberry plants are not grown universally, likely due to variations in weather conditions across regions, available of infectious soil pathogen and insufficient knowledge of soilless hydroponic cultivation. The current research aims to establish an efficient nutrient medium that enhances the growth and yield of strawberry (Fragaria × Ananassa Duch.) Cv. Winter Dawn, using soilless hydroponic and semi-hydroponic cultivation methods. The fruit fresh weight and total fruit yield of formulation 2 treated plants (EC 2.41 mScm−1) in hydroponic condition exhibited a 1.73-fold and 2.18-fold higher respectively as compared to soil control plants. Among the five different growing medium treatments in semi-hydroponic condition, the fresh weight and total strawberry fruit yield was found to be maximum in treatment 3 and observed 2.04-fold and 3.03-fold increase respectively as compared to plants grown in soil control condition. The high yield may be due to higher uptake of macronutrients (NPK) by the plants supplemented with F2 formulation and T3 substrate medium the plants. As a result, the strawberry fruits of these plants accumulated higher amount of anthocyanin, which ultimately enhanced the fruit quality. The studies concluded that formulation 2 in hydroponic and treatment 3 in semi-hydroponic were most effective in terms of growth, nutrient uptake, physiology, fruit yield and anthocyanin content. The result of this study gives an alternative to the farmers for cultivation of strawberry plants in soilless hydroponic and semi-hydroponic conditions.

High-yield and rapid isolation of extracellular vesicles by flocculation via orbital acoustic trapping: FLOAT

Extracellular vesicles (EVs) have been identified as promising biomarkers for the noninvasive diagnosis of various diseases. However, challenges in separating EVs from soluble proteins have resulted in variable EV recovery rates and low purities. Here, we report a high-yield ( > 90%) and rapid ( < 10 min) EV isolation method called FLocculation via Orbital Acoustic Trapping (FLOAT). The FLOAT approach utilizes an acoustofluidic droplet centrifuge to rotate and controllably heat liquid droplets. By adding a thermoresponsive polymer flocculant, nanoparticles as small as 20 nm can be rapidly and selectively concentrated at the center of the droplet. We demonstrate the ability of FLOAT to separate urinary EVs from the highly abundant Tamm-Horsfall protein, addressing a significant obstacle in the development of EV-based liquid biopsies. Due to its high-yield nature, FLOAT reduces biofluid starting volume requirements by a factor of 100 (from 20 mL to 200 µL), demonstrating its promising potential in point-of-care diagnostics.

Polydopamine-Based Resveratrol-Hyaluronidase Nanomedicine Inhibited Pancreatic Cancer Cell Invasive Phenotype in Hyaluronic Acid Enrichment Tumor Sphere Model.

The dense storm microenvironment formed by an excessively cross-linked extracellular matrix, such as hyaluronic acid and collagens, serves as a major barrier that prevents drugs from reaching the deeper tumor. Current traditional two-dimensional (2D) cultures are not capable of modeling this drug delivery barrier in vitro. Thus, tumor spheroids have become increasingly important in cancer research due to their three-dimensional structure. Currently, various methods have been developed to construct tumor spheroids. However, there are still challenges, such as lengthy construction time, complex composition of added growth factors, and high cultivation costs. To address this technical bottleneck, our study combined the GelMA hydrogel system to develop a rapid and high-yield method for tumor spheroids generation. Additionally, we proposed an evaluation scheme to assess the effects of drugs on tumor spheroids. Building on the hyaluronic acid-rich pathological tumor microenvironment, we constructed a resveratrol-loaded nano-drug delivery system with tumor stroma modulation capability and used a three-dimensional (3D) tumor sphere model to simulate in vivo tumor conditions. This process was utilized to completely evaluate the ability of the nano-drug delivery system to enhance the deep penetration of resveratrol in the tumor microenvironment, providing new insights into future oncology drug screening, efficacy assessment, and drug delivery methods.

Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.

Analytical Validation Study with Uncertainty Evaluation of the Method for Quantitative Determination of Hesperidin in the Extracted Product and Mandarin Processing Waste

The aim of this study was to develop and validate an alternative, selective, reproducible, and high-yield extraction-based method by ultrasound-assisted technique combined with an effective, specific, sensitive, and rapid analytical HPLC procedure for obtaining hesperidin from mandarin (Citrus unshiu) peel as waste material in citrus processing industries and its quantitative determination in the obtained extracted product and citrus waste materials as well. The evaluation of the uncertainty of the developed method is also described in the present work. The proposed method is focused not only on obtaining hesperidin but also provides the possibility for obtaining other valuable bioactive compounds within the single stepwise extraction process. The proposed method was validated concerning the following validation parameters: robustness, system suitability test, specificity, linearity range, precision, accuracy, sensitivity, limits of quantification (LOQ), and detection (LOD). A design of experiments using the Placket-Burman approach was used for the robustness study of the combined method. The measurement uncertainty of the proposed method was evaluated based on the four-step process using a combination of two appropriate - bottom-up and top-down approaches using the method validation data. The LOD and LOQ of the analytical procedure were 0.00001 mg/mL and 0.000025 mg/mL, respectively. The calibration curve (0.000025-0.5 mg/mL) is linear and the square of the correlation coefficient is equal to 0.99992. The determined average amount of hesperidin in the dry citrus peel samples is equal to 35.36 ± 3.14 mg/g (k=2, 95 % level of confidence). The mean recovery of the combined method is 91.48 %. The purity of the dry-extracted product of hesperidin is not less than 90 %.

Expression, purification, and biochemical analysis of the RNA-DNA hybrid helicase Sen1/SETX from Chaetomium thermophilum.

Yeast Sen1 and its vertebrate ortholog Senataxin (also known as SETX) are RNA-DNA resolving helicases. Sen1 and SETX are implicated in multiple critical nuclear functions not limited to but including DNA replication and repair, RNA processing, and transcription. These>200kDa helicases have a two-domain architecture with an N-terminal regulatory helical repeat array linked to an SF1b helicase motor core via a variable sized central linker of low complexity sequence. Given the size of these proteins, production of milligram quantities of protein that is suitable for biochemical, biophysical, and protein structural analysis has been challenging. To overcome these limitations, we developed a robust selectable high-yield YFP-fusion protein expression method for Sen1 production in mammalian cells, followed by purification on a high-affinity YFP-binding camelid nanobody support. Herein, we detail methods and protocols for the expression and purification of recombinant Sen1 from the thermophilic fungus Chaetomium thermophilum, and the quantitative characterization of its RNA-DNA duplex resolution activity.

The Use of Descriptive Written Feedback for Enhancing Students’ Mathematics Achievement

Giving learners high-quality feedback as part of formative assessment is a high-yield method teachers can use to support learning and raise academic achievement. Despite its significance, the influence of Descriptive Written Feedback (DWF) on students’ mathematics achievement has yet to be widely studied. This study, therefore, investigated the influence of the teachers’ descriptive written feedback on mathematics achievement in selected secondary schools in Konoin Sub-County of Bomet County, Kenya. The study adopted a quasi-experimental design to establish a cause-and-effect relationship between the independent (Descriptive Written Feedback) and dependent variable (Mathematics Achievement). The Student Achievement Test (SAT) scores attained by the students right after covering the topic of the gradient and the equation of a straight line were used to measure the students’ mathematics achievement. A simple random sampling method was employed to select the ten schools used in this study. ANOVA was used to analyse the data. The study findings showed that descriptive written feedback significantly influences mathematics achievement. The study suggested that mathematics teachers should provide students with descriptive written feedback for improved attainment of expected learning outcomes and enhanced student performance.

Spheroidization of Nd–Fe–B particles

Spherical powders are required for many advanced manufacturing techniques due to their inherent requirement of flowability, either within feed tubes or during powder spreading. As advanced manufacturing of magnets continues to develop, new production methods for feedstocks are also sought. Plasma spheroidization is a high-yield method to produce spherical Nd–Fe–B powders from irregularly shaped particles, with advantages including high throughput and a well-controlled size distribution. Highly spherical Nd–Fe–B powders with large scale production (i.e., kg) have been demonstrated using an inductively coupled thermal plasma system; however, the magnetic properties of the output powder display significant degradation. The coercivity was decreased from the initial 8 kOe (636 kA/m) of the as-received to 0.7 kOe (55 kA/m) for spheroidized powders. Microstructural investigation reveals 6% Nd depletion caused by the extreme temperatures of the plasma, leading to the formation of low-coercivity α-Fe and a subsequent decrease in energy product. Post-spheroidization heat treatments with Nd can partially mitigate the coercivity degradation, increasing to 1.7 kOe (135 kA/m), potentially offering a pathway toward spherical powders for a range of applications.