Protease Concentration Research Articles

Rehydrated corn ensiled with different concentrations of protease in the diet of dairy cows: impacts on intake, digestibility, ruminal and blood parameters, and milk yield and composition

ABSTRACT The objective of this work was to evaluate the protease levels in rehydrated corn silage and the impacts of its inclusion in the diet of dairy cows on intake, nutrient digestibility, ruminal and blood parameters, fatty acid profile, and animal performance. The study included eight cows with an initial body weight (BW) of 432.9 ± 15 kg and 45 ± 5 days of lactation at the beginning of the experiment. The experimental design was in two 4 × 4 Latin squares, simultaneous, each composed of four animals, four treatments, and four experimental periods. The experimental diets were as follows: Control – basal diet; SGR – rehydrated corn grain silage without added protease; SGR – 500, rehydrated corn grain silage with inclusion of 500 g t−1 of protease; SGR – 1000, rehydrated corn grain silage with inclusion of 1,000 g t−1 of protease. Protease activity was 600 IU g−1. Cows fed the SGR diet with proteases showed a 9.64% higher milk yield compared to those fed the control diet (average 17.93 kg day−1). The use of protease at up to 1,000 g t−1 in rehydrated corn silage improved the ruminal and productive parameters of lactating Jersey cows.

Profiling of charge characteristics and effect of pH on charge dynamics in cyprinid milt

Electrostatic properties of milt have a huge bearing on the reproductive success of externally fertilizing freshwater fish species. Considering the global significance of carp in aquaculture, we explored the charge profile of milt of ten commercially important carp species for the first time. Mean zeta potential of milt ranged from (−) 33.86 mV in Cirrhinus mrigala to (−) 56.34 mV in Systomus sarana at pH 7.0. The effect of pH on milt characteristics was studied with Labeo rohita as the model species. Zeta potential reduced significantly upon exposure to low pH (4.0) and increased at alkaline pH (10.0). Fertilization rate was significantly reduced at acidic pH. Our results indicate that changes in milt zeta potential due to acidic pH is a significant factor in reduced reproductive success of externally fertilizing freshwater fish. While low concentration of proteases were detected in milt, it remained in a stable colloidal form even after incubation for 18 h at room temperature with no significant difference in the mean zeta potential at the end of incubation. This seminal work on milt zeta potential of carp will open several avenues in aquaculture and fisheries for productivity enhancement and conservation. Study of the electrostatic properties of milt can also help explain puzzling questions of ecology, such as migratory behavior.

Yarrowia lipolytica growth, lipids, and protease production in medium with higher alkanes and alkenes

Two strains of Yarrowia lipolytica (CBS 2075 and DSM 8218) were first studied in bioreactor batch cultures, under different controlled dissolved oxygen concentrations (DOC), to assess their ability to assimilate aliphatic hydrocarbons (HC) as a carbon source in a mixture containing 2 g·L−1 of each alkane (dodecane and hexadecane), and 2 g·L−1 hexadecene. Both strains grew in the HC mixture without a lag phase, and for both strains, 30 % DOC was sufficient to reach the maximum values of biomass and lipids. To enhance lipid-rich biomass and enzyme production, a pulse fed-batch strategy was tested, for the first time, with the addition of one or three pulses of concentrated HC medium. The addition of three pulses of the HC mixture (total of 24 g·L−1 HC) did not hinder cell proliferation, and high protease (> 3000 U·L−1) and lipids concentrations of 3.4 g·L−1 and 4.3 g·L−1 were achieved in Y. lipolytica CBS 2075 and DSM 8218 cultures, respectively. Lipids from the CBS 2075 strain are rich in C16:0 and C18:1, resembling the composition of palm oil, considered suitable for the biodiesel industry. Lipids from the DSM 8218 strain were predominantly composed of C16:0 and C16:1, the latter being a valuable monounsaturated fatty acid used in the pharmaceutical industry. Y. lipolytica cells exhibited high intrinsic surface hydrophobicity (> 69 %), which increased in the presence of HC. A reduction in surface tension was observed in both Y. lipolytica cultures, suggesting the production of extracellular biosurfactants, even at low amounts. This study marks a significant advancement in the valorization of HC for producing high-value products by exploring the hydrophobic compounds metabolism of Y. lipolytica.

Inclusion of sorghum (Sorghum bicolor (L). Moench) with protease and NSP-ase increases jejunal histomorphology and barrier function gene expression in broiler chickens

The objective of this study was to investigate the effect of sorghum with or without protease and NSP-ase on performance, jejunal histomorphology, caecal short-chain fatty acids (SCFA), and gene expression of barrier function in the broiler chickens. A total of 240 males New Lohmann strain MB 202 broiler chicks (1-day-old) were randomly placed into 12 treatments with 4 replications, 5 birds per replicate for 35 d. The diets were a factorial combination of 3 levels of sorghum (0, 10, and 20%), 2 concentrations of protease (with and without protease), and 2 concentrations of NSP-ase (with and without NSP-ase). Results showed that villus height (VH) was improved (P < 0.05) and villus to crypt ratio (VH:CD) tended (P = 0.05) to increase with inclusion of sorghum. Inclusion of sorghum upregulated (P < 0.05) expression of the gene anti-inflammatory IL-13, and downregulated (P < 0.05) gene of toll receptor (TLR) (TLR-3 and TLR-4), pro-inflammatory (IL-18, IL-6, tumor necrosis factor-alpha (TNF-α) and tight junction zona occluding-1 (ZO-1). The result further indicated a decrease (P < 0.05) in the concentration of SCFA. The birds fed diets with protease had lower (P < 0.05) expression of gene TLR-4 and TLR-3, pro-inflammatory (IL-6 and TNF-α), and anti-inflammatory IL-10. Inclusion of sorghum markedly (P < 0.05) interacted with protease, increasing the total of SCFA, acetate, and VH in the broiler. The bird offered diets with NSP-ase had higher (P < 0.05) expression genes of pro-inflammatory IL-18 and TNF-α. Inclusion of sorghum and NSP-ase significantly (P < 0.05) interacted, upregulating gene of tight junction ZO-1 and anti-inflammatory IL-13 but downregulating gene TLR-3, and pro-inflammatory IL-6, while the SCFA production was decreased. Inclusion of sorghum in the diet with or without enzymes did not affect broiler performance (P > 0.05). In conclusion, a diet with sorghum improved jejunal histomorphology without negatively affecting growth performance. Additionally, supplementation with enzymes enhanced intestinal immune responses by up-regulating anti-inflammatory, and downregulating pro-inflammatory gene expression.

A homo-FRET assay for patatin-specific proteolytic activity

The potato protein patatin embeds bioactive peptides that require targeted hydrolysis to be released as promising food additives. This study presents a patatin-specific protease assay for assessing a wide range of protease activities in high-throughput format. Conjugating patatin to the amine reactive fluorogenic BODIPY FL dye provided a stable protease substrate with efficient homo-FRET quenching at a low degree (7–8) of labeling. Compared to commercial BODIPY-casein, BODIPY-patatin provided higher fluorescence enhancement (by de-quenching) at high protease concentrations, while the sensitivity was generally comparable for both highly specific (e.g. Trypsin) and industrial relevant proteases (e.g. Alcalase and Neutrase) at low doses. For Chymotrypsin, BODIPY-patatin provided a 39 % response improvement at 5 ng dose. A peptide-centric analysis of mass spectrometry-based bottom-up proteomics data identified several BODIPY-labeling sites with varying occupancies in patatin, indicating heterogenous labeling under the applied conjugation conditions. BODIPY-labeled patatin complements commercial BODIPY-labeled casein as a globular, plant-based alternative for screening of proteolytic activity.

Physicochemical Features of Thrombin Binding to Platelet Membrane

Thrombin is a key enzyme of the blood coagulation system, which has been actively studied since the beginning of the last century. The formation of thrombin from prothrombin in the area of vessel injury leads not only to the formation of fibrin – an important structural component of the hemostatic clot – but also to the activation of platelets, endothelium and immune system cells. The binding of thrombin to the platelet surface is thought to play a critical role in the process of platelet activation and may also ensure the maintenance of a high concentration of thrombin within the thrombus due to the concentration of protease on the platelet surface. To date, all major thrombin receptors on platelets have been thoroughly characterized: through various experimental methods, the physicochemical parameters of the corresponding intermolecular interactions have been established. Since the interaction of thrombin with platelets leads to their activation, which includes changes in the number of receptors as a result of granule secretion, the interpretation of the observed kinetic binding curves faces a number of difficulties. It is known that some receptors as a result of platelet activation are able to redistribute on the membrane and form dimers and clusters, which makes the kinetics of thrombin binding to platelets an extremely complex process depending on many factors, such as activator concentrations, platelet state, and other local parameters of the system. This review aims to describe the current understanding of the interaction of thrombin with the platelet membrane and to outline important unresolved issues in this area of research. The survey provides not only information on structural and kinetic features of thrombin binding to individual platelet membrane proteins, but also analyzes the relationship between the relevant interaction parameters and previously obtained data on the integral kinetics of protease binding to the platelet surface.

Ultrasound-assisted enzymatic extraction of soluble dietary fiber from Hericium erinaceus and its in vitro lipid-lowering effect

Dietary fiber (DF) is an important active polysaccharide in Hericium erinaceus. Obesity can lead to a wide range of diseases. In this work, we investigated the in vitro lipid-lowering effect of soluble dietary fiber (SDF) from H. erinaceus, aiming to provide a basis for the subsequent development of lipid-lowering products. Ultrasound-assisted enzymatic extraction (UAEE) of SDF from H. erinaceus was performed. The optimal extraction parameters determined via single-factor experiments and response surface methodology (RSM) were as follows: Lywallzyme concentration, 1.0%; complex protease concentration, 1.2%; ultrasonication time, 35 min; and ultrasonication power, 150 W. In vitro lipid-lowering experiments revealed that the adsorption amount of cholesterol micelles by H. erinaceus SDF was 11.91 mg/g. The binding amount and binding rate of sodium taurocholate were 3.73 mg/g and 42.47%, respectively, and those of sodium glycocholate were 3.43 mg/g and 39.12%, respectively. The pancreatic lipase inhibition rate reached 52.11%, and the type of inhibition was competitive. Therefore, H. erinaceus SDF has good in vitro lipid-lowering ability.

Analysis of the expression of the Serpina1 gene in SARS-CoV-2 infection: study of a new biomarker

IntroductionThe SERPINA1 gene encodes the protein Alpha-1 Antitrypsin (AAT1). Possible imbalances between the concentrations of proteases and antiproteases (AAT1) can lead to the development of serious pulmonary and extrapulmonary pathologies. In this work we study the importance of this possible imbalance in patients with COVID-19. ObjectivesTo correlate the severity of the symptoms of SARS-COV-2 infection with the AAT1 concentrations at diagnosis of the disease. MethodsAn observational, prospective, cross-sectional, non-interventional, analytical study was carried out where 181 cases with COVID-19 admitted to the “Lozano Blesa” University Clinical Hospital of Zaragoza were selected. The concentration of AAT1 was studied in all of them and this was correlated with the clinical aspects and biochemical parameters at hospital admission. Results141 cases corresponded to patients with severe COVID and 40 patients with mild COVID. AAT1 levels were positively correlated with the days of hospitalization, severity, C-Reactive Protein, ferritin, admission to Intensive Care, and death, and presented a negative correlation with the number of lymphocytes/mm3. AAT1 concentrations higher than 237.5 mg/dL allowed the patient to be classified as “severe” (S72%; E78%) and 311.5 mg/dL were associated with the risk of admission to Intensive Care or Exitus (S67%; E79%). ConclusionsLevels of the SERPINA1 gene expression product, AAT1, correlate with the severity of COVID-19 patients at diagnosis of the disease, being useful as a prognostic biomarker.

Insight into the Antioxidant Activity of Ascorbic Acid-Containing Gelatin Nanoparticles in Simulated Chronic Wound Conditions.

Chronic wounds differ from acute wounds by remaining in the inflammatory phase for a long time. This chronic inflammation confers a high concentration of inflammatory cytokines, proteases, and ROS. Likewise, the pH environment of chronic wounds has been recorded within the range of 7.2-8.9 due to the alkaline by-products of bacterial proliferation. In this work, differences in pH between healthy skin and chronic cutaneous wounds have been used for the design and development of pH-responsive gelatin-based nanoparticles (NPs). Ascorbic acid (AA), as an antioxidant compound that can neutralize reactive oxygen species (ROS), has been the therapeutic model compound included in these NPs. The goal of the present work has been the preparation and characterization (physicochemical and biological properties) of NPs for the effective release of AA under simulated chronic wound conditions. In vitro experiments demonstrated total AA release at pH corresponding to the chronic wounds. The biocompatible character of these gelatin-based NPs based on their hemolytic and cytotoxicity responses has been highlighted under in vitro conditions. The reversible and protective antioxidant properties of the AA-including NPs in erythrocytes and skin cell lines, respectively, have been confirmed to be modulated by the gelatin A gel strength.

Development of a mini pig model of peanut allergy.

The prevalence of peanut allergies is increasing, emphasizing the need for an animal model to enhance our understanding of peanut allergy pathogenesis and to advance diagnostic tools and therapeutic interventions. While mice are frequently used as model organisms, their allergic responses do not fully mirror those observed in humans, warranting the exploration of a higher animal model. The porcine gastrointestinal system closely resembles that of humans, and exhibits allergy symptoms akin to human responses, making pigs a promising model for peanut allergy research. In this study we compared two allergen sensitization protocols involving either topical allergen application after repeated tape stripping (TS) or intraperitoneal (IP) injections to induce peanut-specific allergy and anaphylaxis reactions in mini pigs. Mini pigs sensitized with a combination of peanut protein extract (PE) and cholera toxin (CT) through either the IP or the TS route. Sensitized pigs via both methods developed systemic PE-specific IgG and IgE responses. Following peanut challenge via the IP route, both TS- and IP-sensitized pigs displayed allergy symptoms, including lethargy, skin rashes, vomiting, and a drop in body temperature. However, respiratory distress was observed exclusively in pigs sensitized through the TS route and not in those sensitized through the IP route. However, it is noteworthy that both groups of sensitized pigs maintained peanut hypersensitivity for up to two months post-sensitization, albeit with a reduction in the severity of allergy symptoms. Importantly, both groups exhibited sustained levels of PE-specific IgG, IgE, and elevated concentrations of mast cell protease in their blood following the IP challenges. Overall, this study reports TS and IP as two different modes of sensitization leading to onset of peanut specific allergic reactions in mini pigs, but only the TS-sensitization led to systemic anaphylaxis (simultaneous presence of symptoms: breathing difficulty, intense skin rash, and impaired mobility). A distinctive feature of these sensitization protocols is the 100% success rate (N = 4 pigs per group) in sensitizing the subjects.

Activation of renal epithelial Na+ channels (ENaC) in infants with congenital heart disease.

This study was designed to measure the concentration and activity of urinary proteases that activate renal epithelial sodium channel (ENaC) mediated Na+ transport in infants with congenital heart disease, a potential mechanism for fluid retention. Urine samples from infants undergoing cardiac surgery were collected at three time points: T1) pre-operatively, T2) 6-8 h after surgery, and T3) 24 h after diuretics. Urine was collected from five heathy infant controls. The urine was tested for four proteases and whole-cell patch-clamp testing was conducted in renal collecting duct M-1 cells to test whether patient urine increased Na+ currents consistent with ENaC activation. Heavy chain of plasminogen, furin, and prostasin were significantly higher in cardiac patients prior to surgery compared to controls. There was no difference in most proteases before and after surgery. Urine from cardiac patients produced a significantly greater increase in Na+ inward currents compared to healthy controls. Urine from infants with congenital heart disease is richer in proteases and has the potential to increase activation of ENaC in the nephron to enhance Na+ reabsorption, which may lead to fluid retention in this population.

Effects of Bacillus subtilis as a single strain probiotic on growth, disease resistance and immune response of striped catfish (Pangasius hypophthalmus).

The present study investigated the potential role of Bacillus subtilis as probiotic in striped catfish (Pangasius hypophthalmus). Fish (initial weight = 150.00±2.63g n = 180) were stocked in circular tanks. Four isonitrogenous (30%) and isolipidic (3.29%) diets were formulated having supplementation of B. subtilis at four different levels (P0; 0, P1: 1×106, P2: 1×108 and P3: 1×1010 CFU/g). Each treatment had three replicates, while each replicate had fifteen fish. The trial started on second week of July and continued for eight weeks. Growth, feed conversion ratio, crude protein content, the concentration of amylase and protease, the profile of both dispensable and non-dispensable amino acids in all four dietary groups increased with a gradual increase of B. subtilis in the diet. At the end of growth experiment, fish in all four groups were exposed to Staphylococcus aureus (5×105 CFU/ml). After S. aureus challenge, fish fed with B. subtilis responded better to damage caused by reactive oxygen species and lipid peroxidation and better survival rate. The catalase and superoxide dismutase level also increased in response to bacterial challenge in B. subtilis fed groups. On the other hand, the concentration of malondialdehyde gradually decreased in these groups (+ve P0 >P1>P2>P3). It is concluded that supplementation of B. subtilis as a probiotic improved the growth, protein content, antioxidant response and immunocompetency against S. aureus in striped catfish. The optimum dosage of B. subtilis, at a concentration of 1×1010 CFU/g, resulted in the most favorable outcomes in striped catfish. This single bacterial strain can be used as an effective probiotic in large scale production of aquafeed for striped catfish. Future studies can investigate this probiotic's impact in the intensive culture of the same species.

Effect of ultrasound combined with pineapple protease treatment on the tenderness of dried shrimp.

In order to improve the tenderness of dried shrimp products as well as to reduce the hardness of the meat during the drying process, shrimp were treated with ultrasound combined with pineapple protease and the tenderization condition was optimized by measuring the texture and shear force of dried shrimp. In addition, the sulfhydryl content, myofibril fragmentation index (MFI) and microstructure were also examined to clarify the mechanisms of shrimp tenderization. The results showed UB1 group withultrasonic power of 100 W, heating temperature of 50 °C and pineapple protease concentration of 20 U mL-1 were the optimum tenderization conditions, where shrimp showed the lowest hardness (490.76 g) and shear force (2006.35 gf). Microstructure as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis results suggested that during the tenderization process the muscle segments of shrimps were broken, degradation of myofibrillar proteins occurred, and MFI values and total sulfhydryl content increased significantly (P < 0.05) (MFI value = 193.6 and total sulfhydryl content = 93.93 mmol mg-1 protein for UB 1 group). Ultrasound combined with bromelain could be used as a simple and effective tenderization method for the production of tender dried shrimp. The best conditions were 100 W ultrasonic power, 50 °C ultrasonic temperature, and 20 U mL-1 bromelain. © 2024 Society of Chemical Industry.

Surface Cross-Linking by Macromolecular Tethers Enhances Virus-like Particles' Resilience to Mucosal Stress Factors.

Virus-like particles (VLPs) are emerging as nanoscaffolds in a variety of biomedical applications including delivery of vaccine antigens and cargo such as mRNA to mucosal surfaces. These soft, colloidal, and proteinaceous structures (capsids) are nevertheless susceptible to mucosal environmental stress factors. We cross-linked multiple capsid surface amino acid residues using homobifunctional polyethylene glycol tethers to improve the persistence and survival of the capsid to model mucosal stressors. Surface cross-linking enhanced the stability of VLPs assembled from Acinetobacter phage AP205 coat proteins in low pH (down to pH 4.0) and high protease concentration conditions (namely, in pig and mouse gastric fluids). Additionally, it increased the stiffness of VLPs under local mechanical indentation applied using an atomic force microscopy cantilever tip. Small angle X-ray scattering revealed an increase in capsid diameter after cross-linking and an increase in capsid shell thickness with the length of the PEG cross-linkers. Moreover, surface cross-linking had no effect on the VLPs' mucus translocation and accumulation on the epithelium of in vitro 3D human nasal epithelial tissues with mucociliary clearance. Finally, it did not compromise VLPs' function as vaccines in mouse subcutaneous vaccination models. Compared to PEGylation without cross-linking, the stiffness of surface cross-linked VLPs were higher for the same length of the PEG molecule, and also the lifetimes of surface cross-linked VLPs were longer in the gastric fluids. Surface cross-linking using macromolecular tethers, but not simple conjugation of these molecules, thus offers a viable means to enhance the resilience and survival of VLPs for mucosal applications.

Морфофункциональные, молекулярные и иммунологические изменения в плаценте и периферической крови при патологическом прикреплении плаценты

Introduction. Placenta accreta spectrum is an abnormal invasion of villous trophoblast into the myometrium and one of the most severe complications of pregnancy. We aimed to study morphofunctional, molecular, and immunological changes in the placenta and peripheral blood in various types of placenta accreta spectrum.Materials and methods. The study involved 45 pregnant women who underwent ultrasound examination at weeks 35–38. According to the ultrasound data, 15 women had placenta accreta and 15 women developed placenta increta. The comparison group included 15 pregnant women without placenta accreta spectrum with a uterine scar after cesarean section. Histological and immunohistochemical studies were performed on paraffin sections of the placentas. We used mouse antibodies to insulin-like growth factor-binding protein-1, pregnancy-specific β-1 glycoprotein, and human placental lactogen, proteinase 3 and measured them using ELISA. We also studied tumor necrosis factor. Immunophenotyping of peripheral blood lymphocytes was performed using flow cytometry. Results. Рlacenta accreta spectrum is accompanied by increased intensity of IHC staining of placental villi for all the studied markers, in contrast to those in the comparison group. The maximum intensity of insulin-like growth factor-binding protein-1 staining was found in placenta increta. Increased serum concentration of proteinase 3 and placental lactogen (placenta increta) and decreased level of insulin-like growth factor-binding protein-1 were found in placenta accreta spectrum.Conclusion. In placenta accreta spectrum, there is a functional redundancy of placental proteins and an increased pro-inflammatory status. An imbalance between aggressive factors are likely to contribute to pathological villous invasion. Keywords: placenta accreta spectrum, immune status, placental proteins, proteinase 3

Box-Behnken Design for Optimizing Ultrasonic-Assisted Enzymatic Extraction of Soluble Dietary Fiber from Pleurotus citrinopilestus

Pleurotus citrinopilestus contains a variety of physiologically and pharmacologically active compounds. A key active component among these compounds is dietary fiber, a polysaccharide that exhibits several biological properties. The objective of this study was to assess how soluble dietary fiber (SDF) from Pleurotus citrinopilestus responded to ultrasonic-assisted enzymatic (UAE) extraction. The response surface method (RSM) combined with the Box-Behnken design method (BBD) was used to optimize the yield of SDF. The effects of the liquid-solid ratio (35–55 mL/g), α-amylase concentration (0.5–2.5%), complex protease concentration (0.4–2.0%), and ultrasonication time (15–55 min) on the yield of SDF were examined. The RSM results revealed the optimal liquid-solid ratio (45 mL/g), α-amylase concentration (1.5%), complex protease concentration (1.2%), and ultrasonic time (35 min). The SDF yield was 10.25%, which is close to the predicted value (10.08%).

DNA‐Barcoded Plasmonic Nanostructures for Activity‐Based Protease Sensing

AbstractWe report the development of a new class of protease activity sensors called DNA‐barcoded plasmonic nanostructures. These probes are comprised of gold nanoparticles functionalized with peptide‐DNA conjugates (GPDs), where the peptide is a substrate of the protease of interest. The DNA acts as a barcode identifying the peptide and facilitates signal amplification. Protease‐mediated peptide cleavage frees the DNA from the nanoparticle surface, which is subsequently measured via a CRISPR/Cas12a‐based assay as a proxy for protease activity. As proof‐of‐concept, we show activity‐based, multiplexed detection of the SARS‐CoV‐2‐associated protease, 3CL, and the apoptosis marker, caspase 3, with high sensitivity and selectivity. GPDs yield &gt;25‐fold turn‐on signals, 100‐fold improved response compared to commercial probes, and detection limits as low as 58 pM at room temperature. Moreover, nanomolar concentrations of proteases can be detected visually by leveraging the aggregation‐dependent color change of the gold nanoparticles. We showcase the clinical potential of GPDs by detecting a colorectal cancer‐associated protease, cathepsin B, in three different patient‐derived cell lines. Taken together, GPDs detect physiologically relevant concentrations of active proteases in challenging biological samples, require minimal sample processing, and offer unmatched multiplexing capabilities (mediated by DNA), making them powerful chemical tools for biosensing and disease diagnostics.

DNA-Barcoded Plasmonic Nanostructures for Activity-Based Protease Sensing.

We report the development of a new class of protease activity sensors called DNA-barcoded plasmonic nanostructures. These probes are comprised of gold nanoparticles functionalized with peptide-DNA conjugates (GPDs), where the peptide is a substrate of the protease of interest. The DNA acts as a barcode identifying the peptide and facilitates signal amplification. Protease-mediated peptide cleavage frees the DNA from the nanoparticle surface, which is subsequently measured via a CRISPR/Cas12a-based assay as a proxy for protease activity. As proof-of-concept, we show activity-based, multiplexed detection of the SARS-CoV-2-associated protease, 3CL, and the apoptosis marker, caspase 3, with high sensitivity and selectivity. GPDs yield >25-fold turn-on signals, 100-fold improved response compared to commercial probes, and detection limits as low as 58 pM at room temperature. Moreover, nanomolar concentrations of proteases can be detected visually by leveraging the aggregation-dependent color change of the gold nanoparticles. We showcase the clinical potential of GPDs by detecting a colorectal cancer-associated protease, cathepsin B, in three different patient-derived cell lines. Taken together, GPDs detect physiologically relevant concentrations of active proteases in challenging biological samples, require minimal sample processing, and offer unmatched multiplexing capabilities (mediated by DNA), making them powerful chemical tools for biosensing and disease diagnostics.

Ethanol-salt based two-phase partitioning for concentration of milk clotting proteases from Sweet Indrajao (Wrightia tinctoria R. Br.) fruit

Cheese is the naturally fermented dairy product beloved for its wide variety of textures and other nutritional qualities. Animal rennet, microbial and fermentation produced chymosin are generally considered for commercial level cheese preparation. In order to meet the growing global demand observed in cheese market, milk clotting proteases from diverse alternative sources are under exploration. Sweet Indrajao (Wrightia tinctoria R. Br.) fruit extract is known for its utility in traditional cheese preparation. Organic solvent-salt based aqueous two-phase system (ATPS) using ethanol and salt was employed to partially purify milk clotting proteases from W. tinctoria fruit. The system with ethanol-25.90% (w/w) and dipotassium phosphate-13.60% (w/w) was found favorable with yield of 96.33%, milk clotting index of 77.85, purification factor of 2.42, partition coefficient of 458.80 and selectivity of 345.58. Tricine SDS-PAGE and zymogram revealed presence of two prominent protein bands between 66 and 97.4 kDa with caseinolytic activity. Whole casein and κ- casein hydrolysis pattern was analyzed by tricine SDS-PAGE. The optimized system was scaled up to 100 g and its performance was found comparable to its 10 g counterpart. Characteristics of the cheese prepared using partitioned enzyme was comparable to that of the positive control used (Enzeco®).

SUMO in the regulation of DNA repair and transcription at nuclear pores.

Two related post-translational modifications, the covalent linkage of Ubiquitin and the Small Ubiquitin-related MOdifier (SUMO) to lysine residues, play key roles in the regulation of both DNA repair pathway choice and transcription. Whereas ubiquitination is generally associated with proteasome-mediated protein degradation, the impact of sumoylation has been more mysterious. In the cell nucleus, sumoylation effects are largely mediated by the relocalization of the modified targets, particularly in response to DNA damage. This is governed in part by the concentration of SUMO protease at nuclear pores [Melchior, F etal. (2003) Trends Biochem Sci 28, 612-618; Ptak, C and Wozniak, RW (2017) Adv Exp Med Biol 963, 111-126]. We review here the roles of sumoylation in determining genomic locus positioning relative to the nuclear envelope and to nuclear pores, to facilitate repair and regulate transcription.