Protein Solutions Research Articles

A multi-scale numerical approach to study monoclonal antibodies in solution.

Developing efficient and robust computational models is essential to improve our understanding of protein solution behavior. This becomes particularly important to tackle the high-concentration regime. In this context, the main challenge is to put forward coarse-grained descriptions able to reduce the level of detail, while retaining key features and relevant information. In this work, we develop an efficient strategy that can be used to investigate and gain insight into monoclonal antibody solutions under different conditions. We use a multi-scale numerical approach, which connects information obtained at all-atom and amino-acid levels to bead models. The latter has the advantage of reproducing the properties of interest while being computationally much faster. Indeed, these models allow us to perform many-protein simulations with a large number of molecules. We can, thus, explore conditions not easily accessible with more detailed descriptions, perform effective comparisons with experimental data up to very high protein concentrations, and efficiently investigate protein-protein interactions and their role in phase behavior and protein self-assembly. Here, a particular emphasis is given to the effects of charges at different ionic strengths.

Suppression of Coffee-ring Effect (CRE) in the Development of Low-cost Diagnostic Kit

One of the applications of membrane technology is using the polymeric membrane as an adsorber or assay-capturing matrix in the diagnostic kits' assembly. This study explores the addition of NaCl into a protein solution to suppress the coffee-ring effect (CRE) in developing a low-cost diagnostic kit. The highest concentration of NaCl addition shows the optimum results with no formation of CRE and high color intensity (low grey scale value). Adding NaCl into the protein solution is a safe and cheap alternative for lowering the cost of assembly, benefiting people in low-resource places.

Desalting Plasma Protein Solutions by Membrane Capacitive Deionization.

Plasma protein therapies are used by millions of people across the globe to treat a litany of diseases and serious medical conditions. One challenge in the manufacture of plasma protein therapies is the removal of salt ions (e.g., sodium, phosphate, and chloride) from the protein solution. The conventional approach to remove salt ions is the use of diafiltration membranes (e.g., tangential flow filtration) and ion-exchange chromatography. However, the ion-exchange resins within the chromatographic column as well as filtration membranes are subject to fouling by the plasma protein. In this work, we investigate the membrane capacitive deionization (MCDI) as an alternative separation platform for removing ions from plasma protein solutions with negligible protein loss. MCDI has been previously deployed for brackish water desalination, nutrient recovery, mineral recovery, and removal of pollutants from water. However, this is the first time this technique has been applied for removing 28% of ions (sodium, chloride, and phosphate) from human serum albumin solutions with less than 3% protein loss from the process stream. Furthermore, the MCDI experiments utilized highly conductive poly(phenylene alkylene)-based ion exchange membranes (IEMs). These IEMs combined with ionomer-coated nylon meshes in the spacer channel ameliorate Ohmic resistances in MCDI improving the energy efficiency. Overall, we envision MCDI as an effective separation platform in biopharmaceutical manufacturing for deionizing plasma protein solutions and other pharmaceutical formulations without a loss of active pharmaceutical ingredients.

Correlating Surface Activity with Interface-Induced Aggregation in a High-Concentration mAb Solution.

Interface-induced aggregation resulting in protein particle formation is an issue during the manufacturing and storage of protein-based therapeutics. High-concentration formulations of therapeutic proteins are even more prone to protein particle formation due to increased protein-protein interactions. However, the dependence of interface-induced protein particle formation on bulk protein concentration is not understood. Furthermore, the formation of protein particles is often mitigated by the addition of polysorbate-based surfactants. However, the details of surfactant-protein interactions that prevent protein particle formation at high concentrations remain unclear. In this work, a tensiometer technique was used to evaluate the surface pressure of an industrially relevant mAb at different bulk concentrations, and in the absence and presence of a polysorbate-based surfactant, polysorbate 20 (PS20). The adsorption kinetics was correlated with subvisible protein particle formation at the air-water interface and in the bulk protein solution using a microflow imaging technique. Our results showed that, in the absence of any surfactant, the number of subvisible particles in the bulk protein solutions increased linearly with mAb concentration, while the number of protein particles measured at the interface showed a logarithmic dependence on bulk protein concentration. In the presence of surfactants above the critical micelle concentration (CMC), our results for low-concentration mAb solutions (10 mg/mL) showed an interface that is surfactant-dominated, and particle characterization results showed that the addition of the surfactant led to reduced particle formation. In contrast, for the highest concentration (170 mg/mL), coadsorption of proteins and surfactants was observed at the air-water interface, even for surfactant formulations above CMC and the surfactant did not mitigate subvisible particle formation. Our results taken together provide evidence that the ratio between the surfactant and mAb molecules is an important consideration when formulating high-concentration mAb therapeutics to prevent unwanted aggregation.

Facile Preparation of β-Cyclodextrin-Modified Polysulfone Membrane for Low-Density Lipoprotein Adsorption via Dopamine Self-Assembly and Schiff Base Reaction.

A facile method for the immobilization of β-cyclodextrin on polysulfone membranes with the aim of selectively adsorbing low-density lipoprotein (LDL) was established, which is based on the self-assembly of dopamine on the membrane followed by the Schiff base reaction with mono-(6-ethanediamine-6-deoxy)-β-cyclodextrin. The surface modification processes were validated using X-ray photoelectron spectroscopy and attenuated total reflectance Fourier-transform infrared spectroscopy. Surface wettability and surface charge of the membranes were investigated through the water contact angle and zeta potential analysis. The cyclodextrin-modified polysulfone membrane (PSF-CD) showed good resistance to protein solutions, as shown by the measurement of BSA adsorption. The assessment of BSA adsorption revealed that the cyclodextrin-modified polysulfone membrane (PSF-CD) exhibited excellent resistance to protein solutions. To investigate the adsorption and desorption behaviors of the membranes in single-protein or binary-protein solutions, an enzyme-linked immunosorbent assay was employed. The results revealed that the PSF-CD possessed remarkable adsorption capacity and higher affinity for LDL in both single-protein and binary-protein solutions, rendering it a suitable material for LDL apheresis.

The β-Hairpins peptides affect Alzheimer’s Disease by Regulating theInflammation of Astroglial and Microglial Cell

Alzheimer’s disease is the most common type of brain disease now present in the world. Patients with Alzheimer’s disease will experience memory loss, trouble with daily tasks, and other cognitive difficulties. In Alzheimer’s disease, neuroinflammation and inflammation of astroglial and microglial cells occur. Inflammation of these two cells will damage the neurons in the brain, causing the number of synapses to decrease, which impairs cognitive function. With time, the number of neuroinflammations will increase. β-Hairpins peptides are found in proteins; they play an important role in the self-assembly of peptides and proteins. To further explore the functional mechanism of B-bairn, Adam G Kreutzer and James S Nowick assembled the Aβ-hairpins structure. They found that the synthetic Aβ-hairpins structure could produce toxicity to nerve cells, but the effect on astrocytes and microglia was not further explored. In the present study, we isolate and culture the astroglial cell and microglia cultures from newborn mice’s stripped brains. To simulate the beta-hairpin’s environment, we will put different concentrations of protein solutions into the cell cultures. To investigate the effects of the designed β-hairpin peptide on astrocytes and microglia, as well as its potential role in modulating the progression of Alzheimer’s disease, we conducted experiments to examine the expression of inflammation and cellular viability after the addition of the β-hairpin peptide. This paper only provides theoretical experiment design and possible results about the relationship between the β-Hairpins peptides and the inflammation process of astroglial and microglial cells, which needs further research in the pathology of atherosclerosis. It also provided the possibility that β-hairpin peptides regulate Alzheimer’s disease and provided the chance to understand the molecular basis of amyloid diseases.

Advancing injection force modeling and viscosity-dependent injectability evaluation for prefilled syringes

The development of PFS requires a detailed understanding of the forces occurring during the drug administration process and patient’s capability. This research describes an advanced mathematic injection force model that consisting hydrodynamic force and friction force. The hydrodynamic force follows the basic law of Hagen-Poiseuille but refines the modeling approach by delving into specific properties of drug viscosity (Newtonian and Shear-thinning) and syringe shape constant, while the friction force was accounted from empty barrel injection force. Additionally, we take actual temperature of injection into consideration, providing more accurate predication. The results show that the derivation of the needle dimension constant and the rheological behavior of the protein solutions are critical parameters. Also, the counter pressure generated by the tissue has been considered in actual administration to address the issue of the inaccuracies of current injection force evaluation preformed in air, especially when the viscosity of the injected drug solution is below 9.0 cP (injecting with 1 mL L PFS staked with 29G ½ inch needle). Human factor studies on patients’ capability against medication viscosity filled the gap in design space of PFS drug product and available viscosity data in very early phase.

Combination Therapy of Autologous Protein Solution and Extracorporeal Shock Wave Therapy for Severe Knee Osteoarthritis

Knee osteoarthritis (OA) is a multifaceted metabolic disorder influenced by biomechanical, inflammatory, and immune system factors. Although autologous protein solution (APS) and extracorporeal shock wave therapy (ESWT) have shown promise for treating mild-to-moderate knee OA, their efficacy for severe cases remains limited when administered individually. Thus, we examined the combination effects of APS and ESWT for severe knee OA. Twenty-four cases (33 knees) of Kellgren–Lawrence grade 4 knee OA with bone marrow lesions and synovitis detected via magnetic resonance imaging from December 2019 to November 2022 were included. All patients underwent an ESWT session before the APS injection. The study included 20 knees in the APS + ESWT group, which underwent an average of 4.5 ESWT sessions, and 13 in the APS-alone group. We evaluated both groups’ Knee Injury and Osteoarthritis Outcome Scores (KOOSs) before the APS injection at 3 and 6 months. Additionally, we compared the mean KOOS changes between the APS + ESWT and APS-alone groups at 3 months (Pre-3M) and from 3 to 6 months (3–6M). Pre-3M showed no significant difference in the KOOSs between the two groups; however, there was a significant change in 3–6M (p < 0.05). Combining APS therapy with ESWT in severe knee OA was more effective and durable than APS alone.

Use of quantitative mass spectrometry-based proteomics and ELISA to compare the alpha 2 macroglobulin concentration in equine blood-based products processed by three different orthobiologic devices.

Alpha 2 macroglobulin (A2M), a multi-functional protein in the plasma protease inhibitor class, regulates proinflammatory cytokines and the clearance of chondrodestructive enzymes in cases of joint injury and osteoarthritis (OA). The purpose of this study was to compare A2M concentrations in equine plasma samples processed by three commercial devices developed for stall-side regenerative joint therapy. Plasma samples were obtained from healthy adult horses (N = 13). Mass spectrometry analysis was used to determine the concentration of protein analytes in each sample. Selected reaction monitoring measured a specific A2M peptide as a surrogate of the whole A2M protein. A2M concentrations produced by each test device were compared for two sample types: a pre-concentrate or platelet-poor (PP) component and a final component for use in the horse. There was no significant difference (p > 0.05) in the geometric mean (GM) concentration of A2M in the final concentration samples produced by the Alpha2EQ® device (N horses = 13) and the single-centrifugation PP samples produced by the Pro-Stride® APS (autologous protein solution) device (N = 13) and the Restigen® PRP (platelet-rich plasma) device (N = 11). When A2M content in final concentration samples produced by each device was compared, the Pro-Stride APS and Restigen PRP samples had significantly greater GM A2M content (p < 0.0001) compared to the Alpha2EQ samples, and the Pro-Stride APS final concentration samples had significantly greater GM A2M concentration (p < 0.0001) versus that for the Restigen PRP final samples. This comparison demonstrated that the volume and A2M concentration of an Alpha2EQ final concentrate are no different than the volume and concentration of A2M in the PP from Pro-Stride or Restigen devices.

Autologous Protein Solution (APS) and Osteoarthritis of the Knee: A Scoping Review of Current Clinical Evidence.

Knees are the most regularly affected weight-bearing joints in osteoarthritis (OA), impacting millions of individuals across the globe. The incidence of knee OA will further rise with increasing rates of obesity and lifespan, resulting in a significant increase in the worldwide socioeconomic burden. Conventional therapies used to manage the symptoms associated with knee OA have limitations. Lately, there has been an increased interest in the use of autologous peripheral blood-derived orthobiologics (APBO), including autologous protein solution (APS), for the management of knee OA. Here, the primary objective is to summarize the outcomes of clinical studies involving APS for the treatment of knee OA. Several databases (Embase, Scopus, PubMed, and Web of Science) were searched using terms for the intervention "APS" and treatment "knee OA" for articles published in English until January 21, 2024. All clinical studies using APS as an intervention for the treatment of knee OA were included. Studies not utilizing APS alone or not aiming at the management of knee OA were excluded. Six clinical studies that met our predefined search terms and inclusion and exclusion criteria were included in this study. The results demonstrated that the intra-articular administration of APS is safe and efficacious in reducing pain and/or improving function in patients suffering from knee OA. However, more multicenter, randomized controlled trials involving active comparators, with adequate power and long-term follow-up along with post-market real-world studies in clinical practice are required to further assess the efficacy of APS and justify its regular clinical use for the management of knee OA.

One-step prepared of e-Gelatin-CNTs nano-composite fiber modified carbon fiber microelectrode for continuous dopamine monitoring: Characterization, biocompatibility evaluation and in vivo experiment

Fabricating electrochemical sensor capable of stable sensing in complex organism's environment will undoubtedly underpin key in the development of implanted electrochemical sensor. However, nonspecific protein adsorption can greatly reduce sensing sensitivity of electrode due to the abundance of protein in vivo. Herein, we aim to fabricating a modified layer of good protein adsorption resistance. The nano-composite fibers from gelatin with good hydrophily and multiwall carbon nanotubes (CNTs) for constructing on the surface of carbon fiber microelectrode using one-step electrospinning technology (e-Gelatin-CNTs/CFME). A comprehensive discussing on the protein adsorption resistance of e-Gelatin-CNTs/CFME that are, through the use of SEM, AFM, water contact angle, UV–vis, cyclic voltammetry and I-T. The resulting electrochemical sensor revealed stable sensing sensitivity in protein solution, a slightly decrease (0.02nA/μM). Furthermore, the sensor was successfully used for the detection of DA in human blood. More remarkable, e-Gelatin-CNTs nano-composite fibers presented excellent biocompatibility, which could meet the biosafety as implantation materials in vivo. This work provided a platform for the development of implantable electrochemical sensor.

Study on the Modification and Properties of Silk Sericin Protein/Nano-Titanium Dioxide Composite for Textile Applications

Abstract Silk sericin protein is a natural high-molecular-weight compound that contains eighteen types of amino acids. It is non-toxic, biodegradable, and biocompatible, with simple preparation methods and low cost. It finds widespread application in functional clothing, medical and pharmaceutical fields, tissue engineering, and more. Nano-titanium dioxide, on the other hand, possesses non-toxic, self-cleaning, antibacterial, and deodorizing properties. To develop multifunctional textiles with deodorization, UV protection, and good thermal and mechanical properties, this study utilized a compounding method to modify citric acid-pre-treated cotton fabrics through a two-dipping and two-padding process using a blend finishing solution of silk sericin protein and nano-titanium dioxide. Observations of the microstructure before and after fabric finishing, along with evaluations of deodorization, UV protection, and thermal properties, revealed that controlling the proportion of the silk sericin protein/nano-titanium dioxide blend finishing solution can result in a smooth surface of the modified cotton fabric. This modification not only enhances the fabric’s UV protection and tensile strength but also improves its thermal properties while imparting certain deodorization capabilities. Comprehensive analysis concludes that using silk sericin protein and nano-titanium dioxide for modifying cotton fabric to prepare multifunctional textiles with deodorization, UV protection, and good thermal and mechanical performance is feasibly viable.

Quality characteristics of plant-based whipped cream with ultrasonicated pea protein

The rise in popularity of vegetarian and plant-based diets has led to extensive research into plant-based whipped creams. Whipped cream is an oil-in-water emulsion that creates foam through whipping, stabilizing the foam with proteins and fats. Pea protein is an excellent emulsifier and foaming agent among plant-based proteins, but its application in whipped cream is currently limited. The objective of this study was to investigate the quality characteristics of plant-based whipped cream made with ultrasonicated pea protein. The whipped creams were evaluated based on their quality characteristics. A commercially available dairy whipped cream (CON) was used as a control. Plant-based creams were evaluated using pea protein solution, cocoa butter, and canola oil to produce un-ultrasonicated pea protein whipped cream (PP) and ultrasonicated pea protein whipped cream (UPP) at 360 W for 6 min. UPP significantly reduced whipping time and foam drainage compared with CON and PP, resulting in significantly increased overrun, fat destabilization, and hardness. Optical microscopy showed that UPP had smaller fat globules and bubble size than PP. The fat globules of UPP and CON were mostly below 5 μm, whereas those of PP were distributed at 5-20 μm. Finally, ultrasonication significantly improved the overrun, foam drainage, fat destabilization, and hardness of UPP, which are significant quality characteristics of whipped creams. Therefore, ultrasonicated plant-based pea protein whipped cream is believed to be a viable alternative to dairy whipped cream.

Computational Procedure for Predicting Excipient Effects on Protein-Protein Affinities.

Protein-protein interactions lie at the center of many biological processes and are a challenge in formulating biological drugs, such as antibodies. A key to mitigating protein association is to use small-molecule additives, i.e., excipients that can weaken protein-protein interactions. Here, we develop a computationally efficient model for predicting the viscosity-reducing effect of different excipient molecules by combining atomic-resolution MD simulations, binding polynomials, and a thermodynamic perturbation theory. In a proof of principle, this method successfully ranks the order of four types of excipients known to reduce the viscosity of solutions of a particular monoclonal antibody. This approach appears useful for predicting the effects of excipients on protein association and phase separation, as well as the effects of buffers on protein solutions.

Monitoring polysorbate 80 degradation in protein solutions using Total Holographic Characterization

The degradation of polysorbate surfactants can limit the shelf life of biologic pharmaceutical products. Polysorbate is susceptible to degradation via either oxidation or hydrolysis pathways which releases free fatty acids (FFA) and other complex polymers. Degradants from Polysorbate 80 (PS80) can form particles and impact drug product quality. PS80 degradation products appear at low concentrations, and their refractive indexes are similar to that of the buffer, making them very challenging to detect. Furthermore, aggregates of FFA are similar in size and refractive index to protein aggregates adding complexity to characterizing these particles in protein solutions. Total Holographic Characterization (THC) is used in this work to characterize FFA particles of oleic acid and linoleic acid, the two most common degradation products of PS80. We demonstrate that the characteristic THC profile of the FFA oleic acid emulsion droplets can be used to monitor the degradation of PS80. THC can detect oleic acid at a concentration down to less than 100 ng/mL. Using the characteristic THC signal of oleic acid as a marker, the degradation of PS80 in protein solutions can be monitored quantitatively even in the presence of other contaminants of the same size, including silicone oil emulsion droplets and protein aggregates.

PEGDA hydrogel microspheres with encapsulated salt for versatile control of protein crystallization

Due to their biocompatibility and adjustable chemical structure and morphology, hydrogels have great potential in many applications, and can be used to enhance protein crystal quality and crystallization efficiency, contributing to biomedicine manufacturing. Monodispersed PEGDA hydrogel microspheres (HMSs) were synthesized using a Lego-inspired microfluidic device. The generated droplets were then UV polymerized, partially hydrolyzed with 0.1 M NaOH solution to improve their absorption capacity, and soaked in a buffer solution containing 0, 0.5, 1, 2, and 4 M NaCl. Salt-loaded HMSs were used as the medium for the enhanced crystallization of hen egg white lysozyme from aqueous solutions. Different supersaturations were achieved in the protein solutions by releasing NaCl of different concentrations from HMSs, as confirmed by electrical conductivity measurements. HMSs with or without NaCl can both provide heterogeneous nucleation sites due to their nano-porous structure and wrinkled surface. The addition of NaCl-loaded HMSs to the protein solution can also increase or decrease the supersaturation in the whole solution or locally near the HMS, leading to controllable nucleation time and crystal size distribution dependent on the NaCl concentration loaded into HMSs.

Electron beam irradiation induced aggregation, structural and functional changes of soybean 11S globulin

This study investigated the effects of different irradiation doses of an electron beam (e-beam) (0, 2, 4, 6, 8, and 10 kGy) on the structure, emulsification, foaming, and rheological and gel properties of soybean 11S globulin. The irradiation treatment at 4 and 6 kGy significantly increased the solubility, surface hydrophobicity, disulfide bonding, and ζ-potential of 11S globulin, decreased the particle size of the protein solution, and effectively improved the emulsifying activity and foaming stability of the protein solution. Moreover, irradiation induced moderate cross-linking and aggregation of the proteins, thereby increasing the apparent viscosity and shear stress of the protein solution. In addition, the low-field NMR and microstructure analysis results revealed that protein gels formed a dense and homogeneous three-dimensional mesh structure after irradiation (6 kGy), along with increased content of bound water (T2b) and water not readily flowable (T21) and a decrease content of free water (T22). Overall, our results confirmed that e-beam irradiation could significantly improve the physicochemical properties of soybean 11S globulin. Our study thus provides a new technical means for the application of electron beam irradiation technology toward protein modification and broadens the high-value utilization of soybean 11S globulin in the food processing industry.

Poly(glutamic acid)-Based Viscosity Reducers for Concentrated Formulations of a Monoclonal IgG Antibody.

Above a concentration threshold, the viscosity of solutions of proteins increases abruptly, which hampers the injectability of therapeutic formulations. Concentrations above 200 g/L are an ideal goal for subcutaneous application of antibodies. Molecular additives, such as amino acids (e.g., arginine) help decrease the viscosity, but they are used at concentrations as high as about 200 mmol/L. We addressed the question of whether poly(amino acids) could be more efficient than small molecular additives. We observed marked fluidification of a model therapeutic monoclonal antibody (mAb) solution by poly(d,l-glutamic acid) and poly(l-glutamic acid) derivatives added at concentrations of <6.5 g/L (i.e., a mAb/polymer chain molar ratio between 4:1 and 1:1 mol/mol). The bare poly(glutamate) parent chains were compared with polyethylene glycol-grafted chains as PEGylation is a common way to enhance stability. Viscosity could be decreased to ∼20 mPa s as compared to values of ∼100 mPa s in the absence of polymers at 200 g/L mAb. Formation of complexes between the mAb and the polyglutamates was characterized by capillary electrophoresis analysis in dilute solutions (1 g/L mAb) and by observation of phase separation at higher concentrations, suggesting tight association at about 2:1 mol/mol mAb/polymer. Altogether, these results show that polyglutamate derivatives hold an untapped potential as an excipient for fluidification of concentrated protein solutions.

SPiRIT study protocol (Shoulder Pain: Randomised trial of Injectable Treatments): a randomised feasibility and pilot study of autologous protein solution (APS) vs corticosteroids for treating subacromial shoulder pain

BackgroundThe management of subacromial shoulder pain represents a significant challenge and is typically managed through either physiotherapy, joint injection or surgical intervention. Recent surgical trials have questioned the efficacy and there is a need to improve the evidence base for the non-surgical management of this condition.The study aims to provide evidence of the feasibility of conducting a randomised controlled trial to compare the efficacy of autologous protein solution (APS) against the current standard of care, corticosteroid injection (CSI) for subacromial shoulder pain. Autologous protein solution (APS) is a blood-derived biological injection which has been shown to have anti-inflammatory effects.MethodsA parallel-group two-arm randomised control trial will be conducted, comparing APS and CSI for shoulder pain. Fifty patients will be recruited. Feasibility will be assessed by examination of the conversion rate of eligible participants to the total number of participants recruited, whether it is possible to collect the appropriate outcome measures and the levels of retention/data compliance at follow-up dates.DiscussionCSI is the mainstay of conservative management of subacromial shoulder pain. Trials and systematic reviews have reported differing conclusions, but the consensus view is that any benefits seen from CSI use are most likely to be short-term and there remains a significant number of patients who go on to have surgical intervention despite CSI.Biological injections, such as APS are being increasingly used, in the anticipation they may offer improved longer lasting outcomes for shoulder pain. However, the evidence to demonstrate the comparative efficacy of CSI versus APS does not currently exist. If feasible, a fully powered study will offer clarity to the treatment pathway of thousands of patients each year with subacromial pain.Trial registrationThe study is funded by the National Institute for Health Research–Research for Patient Benefit, NIHR 201473, Trial Registration Number (ISRCTN12536844: SPiRIT. Shoulder pain: randomised trial of injectable treatments–date of Registration 15/9/2021). Protocol Version V1.0_30Jul2021. IRAS Project ID: 294,982.

Diabrotica v. virgifera Seems Not Affected by Entomotoxic Protease Inhibitors from Higher Fungi.

Certain soil insects, such as the root-damaging larvae of the maize pest Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), are increasingly difficult to control because of recent bans of some insecticides. An alternative and safer approach may be the development of biopesticides based on entomotoxic defense proteins of higher fungi. Many of these potentially interesting proteins are protease inhibitors, and some have been shown to adversely affect insects. We examined the effects of the cysteine protease inhibitors macrocypin 1, 3, and 4 from Macrolepiota procera, clitocypin from Clitocybe nebularis, and cocaprin 1 and the serine protease inhibitor cospin 1 from Coprinopsis cinerea on D. v. virgifera. We confirmed the inhibition by mycocypins of the cysteine catalytic-type proteolytic activities in gut extracts of larvae and adults. The inhibition of pGlu-Phe-Leu-hydrolyzing activity was stronger than that of Z-Phe-Arg-hydrolyzing activity. Mycocypins and cospin resisted long-term proteolytic digestion, whereas cocaprin 1 was digested. Bioassays with overlaid artificial diet revealed no effects of proteins on neonatal mortality or stunting, and no effects on adult mortality. Immersion of eggs in protein solutions had little effect on egg hatching or mortality of hatching neonates. Microscopic analysis of the peritrophic matrix and apical surface of the midguts revealed the similarity between larvae of D. v. virgifera and the chrysomelid Leptinotarsa decemlineata, which are sensitive to these inhibitors. The resistance of D. v. virgifera to fungal protease inhibitors is likely due to effective adaptation of digestive enzyme expression to dietary protease inhibitors. We continue to study unique protein complexes of higher fungi for the development of new approaches to pest control.