SLAM-family receptors promote resolution of ILC2-mediated inflammation.

Type 2 innate lymphoid cells (ILC2) initiate early allergic inflammation in the lung, but the factors that promote subsequent resolution of type 2 inflammation and prevent prolonged ILC2 activation are not fully known. Here we show that SLAM-family receptors (SFR) play essential roles in this process. We demonstrate dynamic expression of several SFRs on ILC2s during papain-induced type 2 immunity in mice. SFR deficiency exacerbates ILC2-driven eosinophil infiltration in the lung, and results in a significant increase in IL-13 production by ILC2s exclusively in mediastinal lymph nodes (MLN), leading to increased dendritic cell (DC) and TH2 cell numbers. In MLNs, we observe more frequent interaction between ILC2s and bystander T cells, with T cell-expressed SFRs (especially SLAMF3 and SLAMF5) acting as self-ligands to suppress IL-13 production by ILC2s. Mechanistically, homotypic engagement of SFRs at the interface between ILC2s and T cells delivers inhibitory signaling primarily mediated by SHIP-1. This prevents activation of NF-κB, driven by IL-7 and IL-33, two major drivers of ILC2-mediated type 2 immunity. Thus, our study shows that an ILC2-DC-TH2 regulatory axis may promote the resolution of pulmonary type 2 immune responses, and highlights SLAMF3/SLAMF5 as potential therapeutic targets for ameliorating type 2 immunity.

A novel ACE inhibitory peptide from Pelodiscus sinensis Wiegmann meat water-soluble protein hydrolysate.

Pelodiscus sinensis meat is a nutritional food and tonic with angiotensin-converting enzyme (ACE) inhibitory activities. To identify the bioactive substances responsible, several bioinformatics methods were integrated to enable a virtual screening for bioactive peptides in proteins identified within a water-soluble protein fraction of Pelodiscus sinensis meat by Shotgun proteomics. The peptides were generated from the identified proteins by in silico proteolysis using six proteases. A comparison of the numbers of proteins suitable for digestion with each enzyme and the iBAQ (intensity-based absolute quantification) values for these proteins revealed that bromelain and papain were the most suitable proteases for this sample. Next, the water solubility, toxicity, and ADMET (absorption/distribution/metabolism/excretion/toxicity) properties of these peptides were evaluated in silico. Finally, a novel ACE inhibitory peptide IEWEF with an IC50 value of 41.33 µM was identified. The activity of the synthesized peptide was verified in vitro, and it was shown to be a non-competitive ACE inhibitor. Molecular docking revealed that IEWEF could tightly bind to C-ACE, and N-ACE with energies less than 0 kJ mol-1, and the peptide IEWEF can form hydrogen bonds with C-ACE and N-ACE respectively. These results provide evidence that bioactive peptides in the water-soluble protein fraction account for (at least) some of the ACE inhibitory activities observed in Pelodiscus sinensis meat. Furthermore, our research provides a workflow for the efficient identification of novel ACE inhibitory peptides from complex protein mixtures.

Impact of papain on the treatment of raw diluted dromedary semen.

A variety of parameters, including liquefaction and semen viscosity, affect the sperm's ability to travel and reach the egg for fertilization and conception. Given that the details behind the viscosity of the semen in male camels have not yet been fully clarified, the purpose of this study was to ascertain how the addition of papain affected the viscosity of fresh diluted camel semen. The study examined semen samples derived from camels that had distinct viscosities. Sperm motility, viability, abnormal sperm percentage, concentration, viscosity, morphometry, acrosome integrity and liquefaction were among the evaluations following 0, 5, 10, 20 or 30 min of incubation at 37°C with papain (0.004 mg/mL, 0.04 mg/mL or 0.4 mg/mL; a semen sample without papain was used as a control). A statistically significant interaction between the effects of papain concentrations and incubation time was found (F = 41.68, p = .0001). Papain concentrations (p = .0001) and incubation times (p = .0001) both had a statistically significant impact on viscosity, according to a simple main effects analysis. A lower viscosity was found (p < .05) at 0.04 mg/mL (0.1 ± 0.0) after 10 min of incubation. A simple main effects analysis showed that papain concentrations and incubation time have a statistically significant effect on sperm motility (p = .0001). At 0.04 mg/mL papain, the sperm motility % was higher (p < .05) after 10 min (64.4 ± 4.8), 20 min (68.4 ± 6.2), and 30 min incubation (72.2 ± 6.6) compared to 0, 5 min (38.3 ± 4.1 and 51.6 ± 5.0, respectively). In conclusion, the fresh diluted camel semen had the lowest viscosity properties after 10 min of incubation with 0.04 mg/mL papain, without compromising sperm motility, viability, acrosome integrity and sperm morphology.

Microhardness and elemental analysis of ion-releasing restoration/ dentin interface following enzymatic chemomechanical caries excavation.

BACKGROUND: This study was conducted to compare chemical, elemental and surface properties of sound and carious dentin after application of two restorative materials resin-modified glassionomer claimed to be bioactive and glass hybrid restorative material after enzymatic chemomechanical caries removal (CMCR) agent. METHODS: Forty carious and twenty non-carious human permanent molars were used. Molars were randomly distributed into three main groups: Group 1 (negative control) - sound molars, Group 2 (positive control) - molars were left without caries removal and Group 3 (Test Group) caries excavated with enzymatic based CMCR agent. After caries excavation and restoration application, all specimens were prepared Vickers microhardness test (VHN), for elemental analysis using Energy Dispersive Xray (EDX) mapping and finally chemical analysis using Micro-Raman microscopy. RESULTS: Vickers microhardness values of dentin with the claimed bioactive GIC specimens was statistically higher than with glass hybrid GIC specimens. EDX analysis at the junction estimated: Calcium and Phosphorus of the glass hybrid GIC showed insignificantly higher mean valued than that of the bioactive GIC. Silica and Aluminum mean values at the junction were significantly higher with bioactive GIC specimens than glass hybrid GIC specimen. Micro-raman spectroscopy revealed that bioactive GIC specimens showed higher frequencies of v 1 PO 4, which indicated high level of remineralization. CONCLUSIONS: It was concluded that ion-releasing bioactive resin-based restorative material had increased the microhardness and remineralization rate of carries affected and sound dentin. In addition, enzymatic caries excavation with papain-based CMCR agent has no adverse effect on dentin substrate.

Management of severity lesions of hypomineralized molars (MIH) with different treatment alternatives: 9-month results of a clinical trial.

The aim of this study was to evaluate the 9-month clinical performance of different materials and treatment procedures in teeth with MIH in children, and to evaluate the effectiveness of Papacarie gel as a deproteinization agent. The study included 90 children (aged 8-15) who had 189 first permanent molars with MIH were restored randomly with 4 different materials/methods. Equia Forte HT (GC, Tokyo, Japan) was used in Group 1; In Group 2, G-eanial composite (GC, Tokyo, Japan) was used with a Fuji IX (GC, Tokyo, Japan) base; In Group 3 and Group 4, EverX Posterior (GC, Tokyo, Japan) base and G-eanial composite (GC, Tokyo, Japan) were used. In group 4, deproteinization was performed with Papacarie Duo gel (F&A, Sao Paulo, Brazil). The restorations were evaluated at 3-month intervals for 9 months using modified United States Public Health Service (USPHS) criteria. The overall recall rate was 94.1% for every 3-month clinical evaluation over 9 months. A total of 9 restorations were unsuccessful. Surface roughness of Group 1 was statistically different from all other groups in all control periods (p < 0.05). Marginal adaptation of Group 2 was found to be significantly different from Groups 3 and 4 at the both of 6th and 9th month controls. There was no significant difference between the groups in terms of retention, color match, marginal discoloration and secondary caries in all control months. Restoration of MIH with Equia Forte HT is almost as successful as composites. The use of dentin replacement materials instead of glass ionomer cements as a base in composite restorations shows better results. Papacarie deproteinization showed similar success with other composite groups. This study was the first clinical study in which Papacarie was used for deproteinization in teeth with MIH and will thus contribute to the literature.

Evaluation of Papacarie®, Carie-Care™, BRIX3000™ and conventional hand instrumentation for caries removal in primary teeth: a randomized control study.

In the current odontological era, carious lesions are removed while tooth tissue is preserved. Most of these ideals are met by chemomechanical caries removal (CMCR) methods, which are easy and comfortable to use, differentiate and eliminate infected tissues, minimize pressure, vibration and heat, and are cost-effective. This study examines the efficacy of commercially available CMCR agents, namely Papacarie®, Carie-Care™ and BRIX3000™, and a conventional hand instrumentation method for caries removal in deciduous molars in terms of time consumption, ease of application, and pain perception. For this randomized clinical trial, 120 children aged 4 to 9 years were selected and randomly allocated to four groups of 30 patients each. Time consumption, ease of application, and pain perception were evaluated at three intervals: pre-, during- and post-caries removal, using Wong-Baker FACES (WBF) Pain Rating Scale and the Face, Legs, Activity, Cry, Consolability (FLACC) scale. The results showed that among the compared materials and conventional hand instrumentation technique, Carie-Care™ was statistically found to be the least time-consuming with a p-value of 0.019, have the least pain perception with a p-value of 0.02, and was clinically the best with respect to manipulation and handling. While all three CMCR agents aid in the removal of carious tissue, Carie-Care™ was the most effective based on time consumption, pain perception and simplicity of administration.

A papain-based colorimetric catalytic sensing system for immunoassay detection of carcinoembryonic antigen.

A colorimetric immunoassay was built for determination of carcinoembryonic antigen (CEA) based on papain-based colorimetric catalytic sensing system through the use of glucose oxidase (GOx). In the presence of GOx, glucose was catalytically oxidized to produce H2O2. Through the assistance of papain (as a peroxide mimetic enzyme), the signal came from the oxidative color development of 3,3',5,5'-tetramethylbenzidine (TMB, from colorless to blue) catalyzed by the generated H2O2. Herein, a sandwich-type immunoassay was built based on GOx as labels. As the concentration of CEA increased, more GOx-labeled antibodies specifically associate with target, which leaded to more H2O2 generation. Immediately following this, more TMB were oxidized with the addition of papain. Accordingly, the absorbance increased further. As a result, the concentration of CEA is positively correlated with the change in absorbance of the solution. Under optimal conditions, the CEA concentration was linear in the range of 0.05-20.0 ng/mL, and the limit of detection (LOD) reached 37 pg/mL. The papain-based colorimetric immunoassay also exhibited satisfactory repeatability, stability, and selectivity.

Enzymatic Hydrolysis Optimization of Yak Whey Protein Concentrates and Bioactivity Evaluation of the Ultrafiltered Peptide Fractions.

Yak whey protein concentrates (YWPCs) have good functional properties, but there is still a gap in the study of their peptides. In this study, peptides were obtained by enzymatic hydrolysis, and the bioactivity of each ultrafiltration fraction was evaluated using an optimal process. YWPCs were isolated and purified from yak milk as the raw material. Alkaline protease, trypsin, and papain were used to hydrolyze YWPCs. The protease with the highest degree of hydrolysis (DH) and peptide concentration was selected as the most suitable enzyme. The effects of pH, temperature, time, and the enzyme-to-substrate ratio (E/S) on the DH and peptide concentration were investigated, and response surface methodology was utilized to optimize the hydrolysis process. The hydrolysate was separated using ultrafiltration membranes with molecular weight cut-offs of 10 kDa, 5 kDa, 3 kDa, and 1 kDa. The bioactivity of each ultrafiltration component was analyzed, including the inhibition rates of α-amylase and xanthine oxidase (XOD) activities and the scavenging rates of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radicals. The results indicated that alkaline protease was the best enzyme for hydrolyzing YWPCs. The peptide concentration in the YWPC hydrolysate was the highest (17.21 mg/mL) at a pH of 8 and a concentration of 7500 U/g, after 2.5 h at 62 °C. The enzymatic hydrolysate was ultrafiltered to yield four peptide fractions, of which the <1 kDa peptides exhibited the highest α-amylase inhibitory activity (22.06%), XOD inhibitory activity (17.15%), and ABTS cationic free radical scavenging rate (69.55%). This demonstrates the potential of YWPC hydrolyzed peptides for hypoglycemic, uric acid-lowering, and antioxidant applications, providing a theoretical basis for the high-value utilization of YWPCs.

Effects of different concentrations of bromelain and papain enzymes on shear bond strength of composite resin to deep dentin using an etch-and-rinse adhesive system.

BACKGROUND: The dentin substrate can be modified by proteolytic agents, which may affect the bonding strength of adhesive systems to the treated dentin surface. Papain, a cysteine protease enzyme with antibacterial and anti-inflammatory properties, can be used for deproteinization of dentin. An alternative deproteinizing enzyme is bromelain. OBJECTIVES: This study aimed to evaluate the impact of deproteinization on the shear bond strength (SBS) of composite resin to deep dentin using different concentrations of bromelain and papain. MATERIAL AND METHODS: Sixty upper premolars were extracted and randomly divided into 5 groups (n = 12 per group). In all groups, the dentin surface was etched with 37% phosphoric acid. Group 1 did not receive any enzyme treatment, group 2 was treated with a 10% papain solution, group 3 was treated with a 15% papain solution, group 4 was treated with a 6% bromelain solution, and group 5 was treated with a 10% bromelain solution. After applying an etch-and-rinse adhesive system, the specimens were restored with composite resin and the SBS was measured. RESULTS: Statistically significant differences were found between groups 2 and 3 (10% papain and 15% papain, p = 0.004), groups 2 and 4 (10% papain and 6% bromelain, p = 0.017), groups 4 and 5 (6% bromelain and 10% bromelain, p = 0.021), and groups 3 and 5 (15% papain and 10% bromelain, p = 0.005). CONCLUSIONS: Deproteinization with papain and bromelain at different concentrations after acid etching did not affect the SBS of composite resin to deep dentin when using an etch-and-rinse adhesive system. However, the group deproteinized with 15% papain demonstrated a higher SBS than the group deproteinized with 10% papain, and the group deproteinized with 6% bromelain showed a higher SBS compared to the group deproteinized with 10% bromelain.

Effects of Leukocyte-rich platelet-rich plasma and Leukocyte-poor platelet-rich plasma on cartilage in a rabbit osteoarthritis model.

Osteoarthritis is a prevalent chronic disease. One of its primary pathological processes involves the degeneration of articular cartilage. Platelet-rich plasma (PRP) contains cytokines and growth factors that can stimulate the repair and regeneration of articular cartilage tissues. PRP may also slow the progression of osteoarthritis. The purpose of this experiment is to compare the efficacy of Leukocyte poor (LP) - PRP and Leukocyte rich (LR) - PRP in treating rabbit osteoarthritis and to investigate their mechanisms of action. Analyzing the impact of leukocytes on PRP therapeutic effectiveness will provide a valuable clinical reference for the choice of which PRP is better for the treatment of osteoarthritis. A rabbit osteoarthritis model was established by injecting papain into the knee joint cavity, and LP-PRP and LR-PRP were prepared through different centrifugation methods for injection into the knee joint cavity. Eight weeks after injection, rabbit knee cartilage specimens were observed for gross changes, HE staining, senna O-solid green staining, and immunohistochemistry of type II collagen and were quantitatively compared using Pelletier's score, Mankin's pathology score, and ImageJ image processing software. Injection of papain into the knee joint cavity successfully established a rabbit model of osteoarthritis. All three evaluation indexes differed significantly from those of the blank group (P<0.05). LP-PRP and LR-PRP exhibited therapeutic effects when compared with the model group. The two PRP groups had similar gross tissue appearance and pathology (P>0.05). The LR-PRP group had higher collagen type-II expression (P < 0.05) than the LP-PRP group. Both LP-PRP and LR-PRP proved therapeutic for the rabbit papain osteoarthritis model. The difference in leukocyte content between the two groups did not yield different cartilage morphology or other factors by 8 weeks posttreatment. LR-PRP displayed the ability to release more factors relevant to the metabolism of type II collagen than LP-PRP, enabling the preservation of into cartilage collagen content of type II collagen and delaying osteoarthritis progression.

Evaluation of Stain Removal Efficacy and Color Stability of Three Different Dentifrices on Artificially Stained Enamel Surface-An In Vitro Study.

AIM: The aim of the present study was to assess the stain removal ability and color stability of three distinct dentifrices on artificially stained enamel surface. MATERIALS AND METHODS: This study included 75 intact, healthy premolars free of dental caries that were extracted during orthodontic therapy. The samples were allowed to dry for 6 hours after being submerged in the prepared tea solution for roughly 18 hours every day. Then this procedure was repeated for seven successive days. All samples were randomly divided into three experimental groups with 25 samples in each group. Group I: control dentifrice, group II: dentifrice containing hydrogen peroxide, group III: dentifrice containing papain and bromelain. A specially designed toothbrushing simulator was used to brush every sample in the relevant group. Using a spectrophotometer and a measurement program, color measurement was evaluated after staining process after 4 weeks and 8 weeks of teeth cleaning. Using a profilometer, the surface roughness values (Ra) were assessed. RESULTS: After 8 weeks of brushing of stained samples, the color stability was better in dentifrice containing hydrogen peroxide (1.14 ± 0.11) followed by dentifrice containing papain and bromelain (1.22 ± 0.08) and control group (1.30 ± 0.09). And after 8 weeks of brushing of stained samples, the surface roughness was more in dentifrice containing hydrogen peroxide (0.237 ± 0.02) followed by dentifrice containing papain and bromelain (0.229 ± 0.13) and control group (0.207 ± 0.05). CONCLUSION: The present study concluded that the dentifrice containing hydrogen peroxide showed a superior whitening effect on the stained enamel surface than dentifrice containing papain and bromelain and control dentifrice. CLINICAL SIGNIFICANCE: The development of various dentifrice products has been greatly aided by the increased demand for an improved esthetic appearance. Teeth's natural color and any external stains that could accumulate on the tooth surface combine to determine a tooth's color. Additionally, the use of whitening dental pastes to remove external stains has grown in favor. With the development of these whitening toothpastes, dentifrices' ability to lessen or eliminate extrinsic dental stains has increased. How to cite this article: Mishra D, Kamath DG, Alagla M, et al. Evaluation of Stain Removal Efficacy and Color Stability of Three Different Dentifrices on Artificially Stained Enamel Surface-An In Vitro Study. J Contemp Dent Pract 2024;25(1):68-71.

Novel nitric oxide donors are coronary vasodilators that also bind to the papain-like protease of SARS-CoV-2.

Several investigational nitric oxide donors were originally created to correct vascular endothelial dysfunction in cardiovascular diseases. These 48 compounds contain an urea-like moiety attached to the well-known NO donors isosorbide 2- and 5-mononitrate. CR-0305 and CR-0202 were synthesized and found to be nontoxic in the cell lines HMEC-1, A549/hACE2 and VeroE6. CR-0305 induced vasodilation in human coronary arteries ex vivo. Since NO can also have antiviral properties, a study of drug-protein interactions with SARS-CoV-2 was undertaken using in silico modeling. CR-0305 experimentally outperformed the other compounds, including CR-0202, in binding the catalytic site of SARS-CoV-2 papain-like protease (PLpro). PLpro is a primary target for therapeutic inhibition of SARS-CoV-2 as it mediates viral replication and modulates host innate immune responses. CR-0305 is predicted to sit firmly in the PLpro catalytic pocket as confirmed by molecular dynamics simulations, wherein stability of binding to the catalytic site of PLpro induces a conformational change in the BL2 loop to a more closed conformation as observed previously with GRL0617. Surface plasmon resonance was performed with CR-0305 and CR-0202 to characterize binding affinity to purified SARS-CoV-2 PLpro protein. CR-0305 and CR-0202 also inhibited SARS-CoV-2 infection compared to vehicle as measured by virus N protein staining with a specific antibody in A549-ACE2 and VeroE6 cells at 20 µM. CR-0305 is a coronary vasodilator that appears to bind to the catalytic site of the PLpro of SARS-CoV-2 while targeting delivery of antiviral NO to cells infected by SARS-CoV-2, suggesting multiple indications for future development.

Investigation of the peptides with calcium chelating capacity in hydrolysate derived from spent hen meat.

Calcium peptide chelates are developed as efficient supplements for preventing calcium deficiency. Spent hen meat (SHM) contains a high percentage of proteins but is generally wasted due to the disadvantages such as hard texture. We chose the underutilized SHM to produce peptides to bind calcium by proteolysis and aimed to investigate chelation between calcium and peptides in hydrolysate for a sustainable purpose. The optimized proteolysis conditions calculated from the result of response surface methodology for two-step hydrolysis were 0.30% (wenzyme/wmeat) for papain with a hydrolysis time of 3.5 h and 0.18% (wenzyme/wmeat) for flavourzyme with a hydrolysis time of 2.8 h. The enzymatic hydrolysate (EH) showed a binding capacity of 63.8 ± 1.8 mg calcium/g protein. Ethanol separation for EH improved the capacity up to a higher value of 68.6 ± 0.6 mg calcium/g protein with a high association constant of 420 M-1 (25°C) indicating high stability. The separated fraction with a higher amount of Glu, Asp, Lys, and Arg had higher calcium-binding capacity, which was related to the number of ─COOH and ─NH2 groups in peptide side chains according to the result from amino acid analysis and Fourier transform infrared spectroscopy. Two-step enzymatic hydrolysis and ethanol separation were an efficient combination to produce peptide mixtures derived from SHM with high calcium-binding capacity. The high percentage of hydrophilic amino acids in the separated fraction was concluded to increase calcium-binding capacity. This work provides foundations for increasing spent hen utilization and developing calcium peptide chelates based on underutilized meat.

Structural analyzes suggest that MiSSP13 and MiSSP16.5 may act as proteases inhibitors during ectomycorrhiza establishment in Laccaria bicolor.

•The signaling process during mycorrhiza establishment involves intense molecular communication between symbionts. It has been suggested that a group of protein effectors, the so-called MiSSPs, plays a broader function in the symbiosis metabolism, however, many of these remain uncharacterized structurally and functionally. •Herein we used three-dimensional protein structure modeling methods, ligand analysis, and molecular docking to structurally characterize and describe two protein effectors, MiSSP13 and MiSSP16.5, with enhanced expression during the mycorrhizal process in Laccaria bicolor. •MiSSP13 and MiSSP16.5 show structural homology with the cysteine and aspartate protease inhibitor, cocaprin (CCP1). Through structural analysis, it was observed that MiSSP13 and MiSSP16.5 have an active site similar to that observed in CCP1. The protein-protein docking data showed that MiSSP13 and MiSSP16.5 interact with the papain and pepsin proteases at sites that are near to where CCP1 interacts with these same targets, suggesting a function as inhibitor of cysteine and aspartate proteases. The interaction of MiSSP13 with papain and MiSSP16.5 with pepsin was stronger than the interaction of CCP1 with these proteases, suggesting that the MiSSPs had a greater activity in inhibiting these classes of proteases. Based on the data supplied, a model is proposed for the function of MiSSPs 13 and 16.5 during the symbiosis establishment. Our findings, while derived from in silico analyses, enable us formulate intriguing hypothesis on the function of MiSSPs in ectomycorrhization, which will require experimental validation.

Airway inflammation in a novel mouse model of asthma-COPD overlap induced by co-exposure to papain and tobacco smoke.

Asthma and chronic obstructive pulmonary disease (COPD) are respiratory diseases associated with airway inflammation, which is the main pathogenesis. Although their causes and characteristics differ, in some cases, asthma and COPD may coexist in the same patient in a condition called asthma-COPD overlap (ACO). The prognosis of ACO is more unfavourable than those of asthma or COPD alone, without any treatment strategies demonstrating efficacy. Owing to its intricate spectrum of features, the detailed pathogenesis of how ACO exacerbates respiratory features remains unclear. In this study, we exposed papain-induced asthma model mice to tobacco smoke to establish an ACO mouse model, in which features of airway inflammation observed in both asthma and COPD were incorporated. This model exhibited distinctive mixed and corticosteroid-resistant airway inflammation and emphysematous changes that are characteristic of ACO. The novel mouse model established here is expected to significantly contribute to elucidating the mechanisms of the broad pathologies of ACO and identifying potential therapeutic targets.

Drug deconjugation-assisted peptide mapping by LC-MS/MS to identify conjugation sites and quantify site occupancy for antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are a heterogeneous mixture of conjugated species with varied drug loadings. Depending on conjugation sites, linkers and drugs can exhibit different stability as influenced by the solvent-accessibility and local charge, resulting in different ADC efficacy, pharmacokinetics, and toxicity. Conjugation site analysis is critical for ADC structural characterization to assure product quality and consistency. It enables early conjugation studies at site-specific levels, confirms the absence of unexpected products to support conjugation process development, and aids in ensuring lot-to-lot consistency for comparability studies. Peptide mapping using liquid chromatography-tandem mass spectrometry is the industry standard method for analyzing conjugation sites. However, some concerns remain for this approach as the large and hydrophobic drug moieties often result in poor MS/MS fragmentation quality and impede the identification of conjugation sites. Additionally, the ionization discrepancy between conjugated and unconjugated peptides can lead to a relatively large bias for site occupancy calculation. In this work, we present a simple drug deconjugation-assisted peptide mapping method to identify and quantify the drug conjugation for ADCs with protease-cleavable linkers. Papain-based drug deconjugation was used to remove the highly hydrophobic drug moiety, which significantly improved the quantitation accuracy of conjugation level and the fragmentation quality. Sample preparation conditions were optimized to avoid introducing artificial modifications, allowing the tracking of initial sample status and subsequent changes of quality attributes during process development and stability assessment. This method was applied to analyze thermally-stressed ADC samples to monitor changes of site-specific conjugation levels, DAR, succinimide hydrolysis of the linker, and various PTMs. We believe this is an effective and straightforward tool for conjugation site analysis while simultaneously monitoring multiple quality attributes for ADCs with protease-cleavable linkers.

Deubiquitinating activity of SARS-CoV-2 papain-like protease does not influence virus replication or innate immune responses in vivo.

The coronavirus papain-like protease (PLpro) is crucial for viral replicase polyprotein processing. Additionally, PLpro can subvert host defense mechanisms by its deubiquitinating (DUB) and deISGylating activities. To elucidate the role of these activities during SARS-CoV-2 infection, we introduced mutations that disrupt binding of PLpro to ubiquitin or ISG15. We identified several mutations that strongly reduced DUB activity of PLpro, without affecting viral polyprotein processing. In contrast, mutations that abrogated deISGylating activity also hampered viral polyprotein processing and when introduced into the virus these mutants were not viable. SARS-CoV-2 mutants exhibiting reduced DUB activity elicited a stronger interferon response in human lung cells. In a mouse model of severe disease, disruption of PLpro DUB activity did not affect lethality, virus replication, or innate immune responses in the lungs. This suggests that the DUB activity of SARS-CoV-2 PLpro is dispensable for virus replication and does not affect innate immune responses in vivo. Interestingly, the DUB mutant of SARS-CoV replicated to slightly lower titers in mice and elicited a diminished immune response early in infection, although lethality was unaffected. We previously showed that a MERS-CoV mutant deficient in DUB and deISGylating activity was strongly attenuated in mice. Here, we demonstrate that the role of PLpro DUB activity during infection can vary considerably between highly pathogenic coronaviruses. Therefore, careful considerations should be taken when developing pan-coronavirus antiviral strategies targeting PLpro.

Identification of Phytochemicals from Arabian Peninsula Medicinal Plants as Strong Binders to SARS-CoV-2 Proteases (3CLPro and PLPro) by Molecular Docking and Dynamic Simulation Studies.

We provide promising computational (in silico) data on phytochemicals (compounds 1-10) from Arabian Peninsula medicinal plants as strong binders, targeting 3-chymotrypsin-like protease (3CLPro) and papain-like proteases (PLPro) of SARS-CoV-2. Compounds 1-10 followed the Lipinski rules of five (RO5) and ADMET analysis, exhibiting drug-like characters. Non-covalent (reversible) docking of compounds 1-10 demonstrated their binding with the catalytic dyad (CYS145 and HIS41) of 3CLPro and catalytic triad (CYS111, HIS272, and ASP286) of PLPro. Moreover, the implementation of the covalent (irreversible) docking protocol revealed that only compounds 7, 8, and 9 possess covalent warheads, which allowed the formation of the covalent bond with the catalytic dyad (CYS145) in 3CLPro and the catalytic triad (CYS111) in PLPro. Root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and radius of gyration (Rg) analysis from molecular dynamic (MD) simulations revealed that complexation between ligands (compounds 7, 8, and 9) and 3CLPro and PLPro was stable, and there was less deviation of ligands. Overall, the in silico data on the inherent properties of the above phytochemicals unravel the fact that they can act as reversible inhibitors for 3CLPro and PLPro. Moreover, compounds 7, 8, and 9 also showed their novel properties to inhibit dual targets by irreversible inhibition, indicating their effectiveness for possibly developing future drugs against SARS-CoV-2. Nonetheless, to confirm the theoretical findings here, the effectiveness of the above compounds as inhibitors of 3CLPro and PLPro warrants future investigations using suitable in vitro and in vivo tests.

Targeting Magnaporthe oryzae effector MoErs1 and host papain-like protease OsRD21 interaction to combat rice blast.

Effector proteins secreted by plant pathogenic fungi are important artilleries against host immunity, but there is no precedent of such effectors being explored as antifungal targets. Here we demonstrate that MoErs1, a species-specific effector protein secreted by the rice blast fungus Magnaporthe oryzae, inhibits the function of rice papain-like cysteine protease OsRD21 involved in rice immunity. Disrupting MoErs1-OsRD21 interaction effectively controls rice blast. In addition, we show that FY21001, a structure-function-based designer compound, specifically binds to and inhibits MoErs1 function. FY21001 significantly and effectively controls rice blast in field tests. Our study revealed a novel concept of targeting pathogen-specific effector proteins to prevent and manage crop diseases.

Production of polyunsaturated fatty acids in pork backfat fermented by Mucor circinelloides.

Pork backfat (PB) contains excessive saturated fatty acids (SFAs), but lacks polyunsaturated fatty acids (PUFAs). Excessive SFAs can be used as a substrate for the growth of certain microorganisms that convert them into PUFAs and monounsaturated fatty acids (MUFAs), and the added value of PB can be enhanced. In this study, Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189 were co-cultured for conversion of PB into fermented pork backfat (FPB) with high level of PUFAs. Our results showed that the content of γ-linolenic acid (GLA) and linoleic acid (LA) in the surface of FPB reached 9.04 ± 0.14 mg/g and 107.31 ± 5.16 mg/g for 7-day fermentation, respectively. To convert the internal SFAs of PB, ultrasound combined with papain was used to promote the penetrative growth of M. circinelloides into the internal PB, and the GLA level in the third layer of fat reached 2.58 ± 0.31 mg/g FPB. The internal growth of M. circinelloides in PB was promoted by adjusting the oxygen rate and ventilation rate through the wind velocity sensor. When the oxygen rate is 2 m/s and the ventilation rate is 18 m3/h, the GLA level in the third layer of fat reached 4.13 ± 1.01 mg/g FPB. To further improve the level of PUFAs in PB, FPB was produced by M. circinelloides at 18 °C. The GLA content on the surface of FPB reached 15.73 ± 1.13 mg/g FPB, and the GLA yield in the second and third layers of fat reached 8.68 ± 1.77 mg/g FPB and 6.13 ± 1.28 mg/g FPB, the LA yield in the second and third layers of fat reached 105.45 ± 5.01 mg/g FPB and 98.46 ± 4.14 mg/g FPB, respectively. These results suggested that excessive SFAs in PB can be converted into PUFAs and provided a new technique for improving PUFAs in FPB. KEY POINTS: • This article achieved the conversion of PUFAs in pork backfat by Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189. • This article solved the internal growth of M. circinelloides CBS277.49 in pork backfat by ultrasound combined with papain. • This article proposed an innovative of promoting the internal growth of M. circinelloides and increasing the PUFAs production by oxygen ventilation in pork backfat.