Total Protease Research Articles

Exploring The Protease Diversity of Psychrophilic Yeast, Glaciozyma antarctica Through Genome Mining Analysis

Proteases are one of the most significant classes of enzymes, holding immense physiological relevance and extensive industrial applications. The genome of Glaciozyma antarctica was fully sequenced, showing 7,857 open reading frames that offer an intriguing opportunity to investigate its proteolytic repertoire. This study aims to unveil the protease landscape of G. antarctica, a psychrophilic yeast that produces cold-active enzymes that offer remarkable benefits, particularly in the food and pharmaceutical industries. In this work, we performed a comprehensive analysis to identify the diverse families of proteases encoded within the G. antarctica genome and compare them with proteases from other mesophilic and thermophilic fungi in the MEROPS database. The sequence similarity searches resulted in the identification of 195 open reading frames predicted to encode for proteases in G. antarctica with a high number of intracellular proteases. These findings suggest an evolved system for protein quality control and turnover, essential for cell viability and adaptation to environmental stressors. The MEROPS classification analysis showed an abundance of metalloproteases, constituting 38% of the total protease genes, a proportion surpassing that found in other yeast and fungal genomes studied. This reflects the vital role of metalloproteases in the cold adaptation of microbes in the Antarctic region. This unique profile not only sheds light on the adaptive mechanisms of psychrophilic organisms but also presents a rich reservoir of potential cold-active proteases for various applications. The findings of this study provide a foundation for targeted enzyme discovery and engineering, unlocking new frontiers in industrial biotechnology and extremophile biology.

Skin mucosal immune parameters and expression of the immune-relevant genes in Danio rerio treated by white button mushroom (Agaricus bisporus).

This study evaluates using different levels of the white button mushroom powder (WBMP) on some mucosal innate immune parameters (lysozyme, protease, esterase, alkaline phosphatase activities, and total immunoglobulin levels), and the relative expression of some principal immune-relevant genes (lysozyme, TNF-α, and IL-1β) in the zebra danio intestine. Zebrafish specimens (1.75 ± 0.25g) were divided into experimental units based on the additives to a diet including 5, 10, and 20g of WBMP per kilogram of food weight, alone or in conjunction with the antibiotic (10mg/kg BW), and the AGRIMOS (1g/kg food weight). Following the 11-day experimental duration, the skin mucus and intestine were sampled. To assess the immune gene expression, the real-time PCR detection system was conducted according to the ΔΔCt method using the IQ5 software (Bio-RAD). Results showed that all groups had a significant increase in terms of mucosal lysozyme activity compared to the control group. Examination of total immunoglobulin, protease, esterase, and ALP activity in fish under experimental treatment showed that there was no significant difference between the trial groups and the control groups. The most expression of the lysozyme gene was related to the group that was separately taken the lower concentration (5g per kg of FW) of WBMP. In conclusion, the amount of 1% mushroom powder in the diet can improve its immune function. Our recommendation is that given the positive effects that mushroom powder added on the diet alone, avoid taking antibiotics for this purpose.

Betaine and the probiotic, Lactobacillus rhamnosus in the diet of the Common carp, Cyprinus carpio: Effects on growth, digestive enzyme activities, antioxidant system, humoral and mucosal immunity and resistance to Streptococcus iniae

The probiotic, Lactobacillus rhamnosus (LAC-R) and Betaine (BT) alone and combined were used in the diet of the common carp, Cyprinus carpio to evaluate their potentials in improving growth, immunity and disease resistance. Seven experimental treatments (LAC-R: 1 × 108 CFU/g LAC-R; BT1: 0.5 % BT; BT2: 1 % BT; BT3: 2 % BT; LAC-R + BT1; LAC-R + BT2; LAC-P + BT3) and one control group (control: non-supplemented fish) were designed in three replicates and fish (13.3 ± 1.5 g) fed with the test diets for two months. After the feeding experiment, fish were exposed to Streptococcus iniae for 14 days to evaluate the fish disease resistance. The fish supplemented with BT and LAC-R alone or combined exhibited better growth compared to control (P<0.01), however, fish of LAC-R + BT had the highest growth performance (P<0.01). Digestive enzyme activities increased in response to LAC-R, BT and their combinations, in which the highest activities were for LAC-R + BT2 and LAC-R + BT3 (P<0.01). The lactic acid bacteria (LAB) populations in intestine increased after the probiotic supplementation (P<0.01), while the results were non-significant for total bacteria count (P>0.01). The total antioxidant capacity (T-AOC), antioxidant enzymes [serum catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx)] and immune components [lysozyme, total immunoglobulin (Ig), myeloperoxidase (MPO), alternative complement activity (ACH50), Nitroblue tetrazolium (NBT) and total protein in blood and total Ig, lysozyme, alkaline phosphatase (ALP) and protease in mucus] were mostly enhanced by LAC-R, BT2, BT3 and their combinations (P<0.01). The oxidative stress was ameliorated by LAC-R, BT and their combinations, as the levels of malondialdehyde (MDA) were lower in these treatments (P<0.01). Fish supplemented with LAC-R, BT and LAC-R + BT had lower mortality rate compared to control after challenge with S. Iniae (P<0.01). In conclusion, dietary LAC-R and BT improved growth and immune performance in the fish. Also, the best performance was found for fish supplemented with a combination of LAC-R and 1–2 % BT.

Assessment of Encapsulated Probiotic Lactococcus lactis A12 Viability Using an In Vitro Digestion Model for Tilapia.

Probiotics face harsh conditions during their transit through the gastrointestinal tract (GIT) of fish because of low-pH environments and intestine fluid. Therefore, the evaluation of probiotic viability under simulated gastrointestinal conditions is an important step to consider for probiotic supplementation in fish feed prior to in vivo trials. Therefore, this study aimed to evaluate the effect of stomach and intestinal simulated conditions on the viability of encapsulated Lactococcus lactis A12 using an in vitro digestion model for tilapia. A Box Behnken design was used to evaluate the potential effect of three factors, namely stomach pH, residence time in the stomach, and enzyme quantity, on the viability of encapsulated Lactococcus lactis A12. As the main results, low pH (4.00), long residence time (4 h), and enzyme quantity (2.68 U of total protease activity) led to lower final cell counts after the phases of the stomach and intestine. Encapsulated probiotic bacteria showed higher viability (p < 0.05) and antibacterial activity (p < 0.05) against the pathogen Streptococcus agalactiae than non-encapsulated bacteria. The results suggest that L. lactis A12 survives in GIT conditions and that the proposed in vitro model could be used to explore the viability of probiotic bacteria intended for fish feed supplementation.

The effects of protein hydrolysates from rainbow trout by-products on growth, digestive and antioxidant enzymes, and liver lysozyme activity in sobaity (Sparidentex hasta) and Arabian yellowfin (Acanthopagrus arabicus) seabream juveniles

An eight-week feeding trial was conducted to elucidate the effects of rainbow trout by-products protein hydrolysates (FPH, 92 % peptides <1 kDa, ∼78.1 % crude protein, ∼2.7 % lipid) on growth and some selected physiological responses in sobaity (Sparidentex hasta) (1.7 ± 0.0 g) and Arabian yellowfin (Acanthopagrus arabicus) (0.9 ± 0.0 g) seabream. Fish meal in a basal diet (∼58 % crude protein, ∼12 % lipid) was replaced with FPH at four levels, including 0 (control), 1 % (FPH1), 2.5 % (FPH2.5), and 5 % (FPH5). Each experimental diet was offered thrice daily to fish in triplicate. At the end of the feeding trial, S. hasta fed with FPH diets showed positive linear and quadratic trends in growth performance, and those fed with FPH5 had the highest final weight (FBW; 9.8 ± 0.4) and better feed conversion ratio (FCR; 0.9 ± 0.0). Regarding A. arabicus it showed positive quadratic trend and fish fed FPH2.5 diet had the highest FBW (6.2 ± 0.3), and better FCR (1.2 ± 0.0) value. Trypsin and alkaline phosphatase (ALP) activities showed both positive linear and quadratic trends in S. hasta. In A. arabicus, both linear and quadratic trends were observed in the activities of trypsin and total proteases (P = 0.001), while positive quadratic trends in pepsin and ALP activities were noticed. Lysozyme activity significantly increased by supplementing the diet with FPH and showed both linear and quadratic trends in S. hasta, but it did not change in A. arabicus. Total antioxidant capacity (TAC) in A. arabicus increased and showed both linear and quadratic responses. The catalase and glutathione peroxidase activities in both species increased significantly in response to dietary FPH. This study showed that FPH effects in diet were species-specific and its supplementation levels at 2.5 and 5 % could be recommended in A. arabicus and S. hasta juveniles, respectively.

Citral and triclosan synergistically silence quorum sensing and potentiate antivirulence response in Pseudomonas aeruginosa.

Among the ESKAPE pathogens, Pseudomonas aeruginosa is an extensively notorious superbug that causes difficult-to-treat infections. Since quorum sensing (QS) directly promotes pseudomonal virulence, targeting QS circuits is a promising approach for disarming phenotypic virulence. Hence, this study scrutinizes the anti-QS, antivirulence, and anti-biofilm potential of citral (CiT; phytochemical) and triclosan (TcN; disinfectant), alone and in combination, against P. aeruginosa PAO1/PA14. The findings confirmed synergism between CiT and TcN and revealed their quorum quenching (QQ) potential. At sub-inhibitory levels, CiT-TcN combination significantly impeded pyocyanin, total bacterial protease, hemolysin, and pyochelin production alongside inhibiting biofilm formation in P. aeruginosa. Moreover, the QQ and antivirulence potential of CiT and TcN was positively correlated by molecular docking studies that predicted strong associations of the drugs with QS receptors of P. aeruginosa. Collectively, the study identifies CiT-TcN as an effective drug combination that harbors QQ, antivirulence, and anti-biofilm prospects against P. aeruginosa.

Safflower yellow in Carthami Flos is responsible for Xuebijing Injection-induced immediate hypersensitivity reaction through activating complement C3

ObjectiveXuebijing Injection (XBJI) is mainly used for treating sepsis in China, and even COVID-19 recently. This study aimed to clarify the molecular mechanism(s) and identify the potential “common culprit(s)” for XBJI-caused immediate hypersensitivity reaction (IHR) which is the main type of its adverse reactions. MethodsAntiserum against XBJI was prepared by intraperitoneal immunization in combination with aluminum adjuvant for five weeks. Antagonistic experiments were performed by using several antagonists against different mediators in Evans Blue leakage model. Propranolol-pretreated mice were used to determine the capacity of XBJI to trigger systemic IHR. Serum total IgE (tIgE) and mouse mast cell protease 1 (MCPT-1) levels, complement activation, and the levels of supernatant inflammatory mediators were determined by ELISAs. Lipopolysaccharide (LPS)-activated RAW264.7 macrophages were used for evaluating the anti-inflammatory activity of XBJI, while human mast cells (LAD2) were used for assessing the effect of XBJI on mast cell degranulation. ResultsContinuous treatment (i.p.) with XBJI along with aluminum adjuvant did not elevate the levels of serum tIgE and MCPT-1. In vitro, XBJI could not directly cause the degranulation of LAD2 cells. It induced a robust Evans Blue leakage after the first injection in mouse paw. Mechanism study demonstrated that antagonists for histamine H1/H2 receptors and complement C3a receptor counteracted XBJI-induced IHR. XBJI also directly activated complement C3 in human serum. Through screening five herbs of XBJI and the constituents, only safflower yellow (SY) in Carthami Flos was able to induce IHR. The discolored-XBJI not only did not induce IHR locally and systemically, but also could suppressing the production of proinflammatory mediators in LPS-activated RAW264.7 macrophages. ConclusionXBJI failed to induce immune IHR, but potently triggered non-immune IHR through direct activating complement C3 to provoke histamine release. SY in Carthami Flos was the underlying “common culprit” responsible for XBJI-caused IHR. The anti-inflammatory action of XBJI can be retained after decolorization. Our study provides a scientific basis for not only preventing and treating XBJI-caused IHR clinically, but also improving its production process.

Fermentation Products, Microbial Protein Synthesis, and Hydrolytic Enzymes Activity of Rumen Fluids Local Beef Cattle Supplemented by Direct-Fed Microbials and Waru Leaf Flour

The aim of this study was to evaluate the effect of Waru Leaf Flour (WLF) and Direct-Fed Microbials (DFM) supplementation on volatile fatty acid (VFA) and ammonia (N-NH3) concentrations, total protozoa, microbial protein synthesis (MPS), and the specific activity of cellulase, amylase, protease in rumen fluids of local beef cattle. The study was conducted as an experimental investigation using a factorial Completely Randomized Design (FCRD). The first factor were three levels of DFM (P) mixed with rice straw (0%, 0.5%, and 1% dry matter of rice straw). The second factor consisted of three levels of WLF supplementation (D) in concentrates (0%, 0.24%, and 0.48% of dry matter concentrate). This resulted nine treatment groups, each it replicated three times.The diet composition consisted of rice straw and concentrate at 40:60 ratio (% dry matter). The interaction between DFM and WLF had a statistically significant impact (p&lt;0.01) on the rumen fluid's total protozoa, N-NH3, and protein content. However, this interaction was not statistically significant (P&gt;0.05) in relation to VFA, MPS, cellulase, amylase, and protease activity. While no significant interaction were observed for total VFA, cellulase, amylase, and protease, DFM supplementation had a statistically significant effect (P&lt;0.01) on reducing enzymes activity, and total VFA (P&lt;0.05). SDS-PAGE and zymography analyzes showed two protease molecules at all treatments with molecular weight of 144 kDa and 133 kDa, cellulase molecules at 62-67 kDa and 19-21 kDa respectively. The addition of DFM showed a new protease band, they had a measurement of 14 and 25 kDa.This study provides valuable insights into enzyme activities in rumen, particularly protease, cellulase, and amylase. The results suggest that the combination of 0.24% WLF and 0.5% DFM supplementation holds promise for optimizing rumen conditions and improving the performance of local beef cattle.

Effect of solid-state fermentation of Brewer's spent grain on digestibility and digestive function of european seabass (Dicentrarchus labrax) juveniles

This study assessed the effects of dietary inclusion of solid-state fermented (SSF) brewer’s spent grain (BSG) with Aspergillus ibericus on nutrient and energy digestibility, digestive enzyme activity, and intestinal histomorphology of juvenile European seabass (Dicentrarchus labrax). Five diets (18 % crude lipids and 45 % crude protein) were formulated, including a control diet (without BSG), two diets with 10 % and 20 % unfermented BSG (10BSG and 20BSG), and two other diets with 10 % and 20 % fermented BSG (10BSG-SSF and 20BSG-SSF). SSF affected the BSG's nutritional composition, including a 21 % increase in protein content and reductions in lipid (49 %), cellulose (30 %), hemicellulose (34 %), and lignin (7.3 %) content. Antioxidant activity and total phenolic compounds were minimal before SSF but significantly increased after SSF. Dietary incorporation of 20 % BSG-SSF increased the digestibility of dry matter (p<0.01), protein (p=0.03), isoleucine (p=0.03), glutamate (p=0.02), lipids (p=0.04) and energy (p=0.04) compared to the 20BSG diet. Moreover, SSF also modulated digestive enzyme activity, reducing total protease (p=0.03) and trypsin (p=0.01) activities in fish fed the 10BSG-SSF diet compared to the control diet. Fish fed the 20BSG diet showed changes in intestinal histomorphology compared to those fed the control diet, and SSF of BSG appeared to mitigate these effects.Overall, these results indicate that SSF is a promising technique for enhancing the nutritional quality of low-value agro-industrial by-products, such as BSG, increasing their potential use as feed ingredients and contributing to reducing the climate and environmental footprint of aquaculture production.

Rapid Colorimetric Quantita2tive Portable Platform for Detection of Brucella melitensis Based on a Fluorescence Resonance Energy Transfer Assay and Nanomagnetic Particles.

Brucellosis is a bacterial zoonotic disease that requires major attention for both health and financial facilities in many parts of the world including the Mediterranean and the Middle East. The existing gold standard diagnosis relies on the culturing technique, which is costly and time-consuming with a duration of up to 45 days. The Brucella protease biosensor represents a new detection approach that will lead to low-cost point-of-care devices for sensitive Brucella detection. In addition, the described diagnostic device is portable and simple to operate by a nurse or non-skilled clinician making it appropriate for the low-resource setting. In this study, we rely on the total extracellular protease proteolytic activity on specific peptide sequences identified using the FRET assay by high-throughput screening from the library of peptide (96 short peptides such as dipeptides and tripeptides) substrates for Brucella melitensis (B. melitensis). The B. melitensis-specific protease substrate was utilized in the development of the paper-based colorimetric assay. Two specific and highly active dipeptide substrates were identified (FITC-Ahx-K-r-K-Ahx-DABCYL and FITC-Ahx-R-r-K-Ahx-DABCYL). The peptide-magnetic bead nanoprobe sensors developed based on these substrates were able to detect the Brucella with LOD as low as 1.5 × 102 and 1.5 × 103 CFU/mL using K-r dipeptide and R-r dipeptide substrates, respectively, as the recognition element. The samples were tested using this sensor in few minutes. Cross-reactivity studies confirmed that the other proteases extracted from closely related pathogens have no significant effect on the sensor. To the best of our knowledge, this assay is the first assay that can be used with low-cost, rapid, direct, and visual detection of B. melitensis.

Bioprospecting of serratiopeptidase-producing bacteria from different sources.

Anti-inflammatory enzymes have wide applications in the pharmaceutical industry. The objective of this study was to find new and efficient strains for the commercial production of serratiopeptidase enzyme. Vast number of samples were processed for the isolation of potent strains. The experimental treatment includes processing of twenty soil samples, silkworm gut, and sugarcane stem. The total protein and protease activity was estimated by Lowry's method and casein hydrolysis. The HRBC stabilization assay was performed for finding the anti-inflammatory potential of all strains. The serratiopeptidase production was confirmed by HPLC with the standard. Molecular characterization of selected potent strains was done by 16S rDNA and confirmed the taxonomy. The one step rapid purification of serratiopeptidase was performed by Ultra three phase partitioning method. The clot lysis potential of the Serratia marcescens VS56 was observed by modified Holmstorm method. The results of the study revealed that among the 60 strains, 12 strains were protease-positive on skim milk agar plates and showed significant protease activity. All 12 strains were screened for serratiopeptidase using high-performance liquid chromatography (HPLC) and VS56, VS10, VS12 and VS18 showed a similar retention time (4.66 ± 0.10 min) with standard. The selected potent strain, Serratia marcescens VS56 showed a proteolytic activity of 21.30 units/mL and produced a total protein of 102 mg/mL. The HRBC suspension results also showed a percentage of 94.6 ± 1.00 protection, which was compared to the standard diclofenac. The clot lysis potential of Serratia marcescens VS56 was 53% in 4 h. Furthermore, the molecular weight of the protein was identified to confirm the presence of serratiopeptidase. The study hence contributed successfully to isolating, screening, and identifying a potent producer for serratiopeptidase from an environmental source. This inherent advantage of the strain will undoubtedly contribute much to the coco comm commercial production of serratiopeptidase in the near future.

Enzymatic and Algebraic Methodology to Determine the Contents of Kunitz and Bowman-Birk Inhibitors and Their Contributions to Total Trypsin or Chymotrypsin Inhibition in Soybeans.

Soybeans are the number one source of plant proteins for food and feed, but the natural presence of protein protease inhibitors (PIs), namely, the Kunitz trypsin inhibitor (KTI) and the Bowman-Birk inhibitor (BBI), exerts antinutritional effects. This communication describes a new methodology for simultaneously quantitating all parameters of PIs in soybeans. It consists of seven steps and featured enzymatically measuring trypsin and chymotrypsin inhibitory activities, respectively, and subsequently determining the contents of reactive KTI and BBI and the contributions of each toward total PI mass and total trypsin or chymotrypsin inhibition by solving a proposed system of linear equations with two variables (C = dB + eK and T = xB + yK). This enzymatic and algebraic (EA) methodology was based on differential inhibitions of KTI and BBI toward trypsin and chymotrypsin and validated by applications to a series of mixtures of purified KTI and BBI, two KTI-null and two conventional soybeans, and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The EA methodology allowed calculations of PI composition and the contributions of individual inhibitors toward total inhibition with ease. It was first found that although BBI constituted only about 30% of the total PI mass in conventional raw soybeans, it contributed about 80% toward total chymotrypsin inhibitor activity and about 45% toward trypsin inhibitor activity. Therefore, BBI caused more total protease inhibitions than those of KTI. Furthermore, the so-called KTI-null soybean mutants still contained measurable KTI content and thus should be named KTI-low soybeans.

Anti-microbial, anti-diabetic and anti-quorum sensing activity of the essential oil of Dialium guineense Willd. grown in South-West Nigeria

Dialium guineense essential oil possesses various biological and biochemical activities. The present study is based on determination of antimicrobial, anti-diabetic, anti-cholinesterase, anti-biofim, anti-antibiotic resistance modifying activities and anti-quorum sensing potential of essential oil. Chemical compounds in essential oil were recognised by retention indices using gas chromatograph-mass spectroscopy (GC-MS). A total of 45 compounds were present in the essential oil of which 12 are most active. Essential oil of D. guineense showed highest anti-bacterial activity in the form of zone of inhibition, produced 23 mm zone against Staphylococcus aureus and 15 mm against Escherichia coli. D. guineense oil at various concentrations showed excellent anti-diabetic and anti-cholinesterase activities. Since many active compounds were present in D. guineense oil so authors of this study planned to observe effect of D. guineense oil on quorum sensing regulated virulence factors and thus bio-film formation in pathogens Essential oil reduced bio-film formation considerably at all concentrations against E. coli, Staphylococcus aureus and Pseudomonas aeruginosa. Determination of quorum sensing (QS) inhibitory activity was demonstrated by reduction in pyocyanin (73.93%), total protease (68.59%) and chitinase activity (66.18%) in Pseudomonas aeruginosa at 1 mg/mL of D. guineense oil compared to control. Essential oil at all concentrations tested showed synergistic effect. Since essential oil showed anti-microbial, anti-biofilm and quorum sensing regulated anti-pathogenic activities. Based on results mentioned above, it is concluded that bioactive chemicals from D. guineense oil can act as an anti-infective agent by controlling growth, bio-film formation and signalling pathways of pathogenic microbes as well as other diseases.

Proteolytic system parameters in the brain of rats with hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) is now being actively studied as a potential risk factor and/or biomarker for numerous pathological conditions, including brain diseases. This study aimed to analyze the proteolytic processes in the brains of rats with HHcy. Total proteolytic activity, metal-dependent, and serine proteases activities, the content of matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases-1, cytokines, serine proteases, total protein and medium and low molecular-weight substances (MLMWS), were evaluated. HHcy was induced by DL-homocysteine thiolactone (HTL) daily intragastric administration (200 mg·kg–1 of body weight) to young and adult albino non-linear male rats for 8 weeks following rat sacrifice and brain harvesting. It was established that HHcy causes an increase in total proteolytic activity and a rise in MLMWS levels in rat brains. Serine protease activity increased to a greater extent compared to metal-dependent one, and bigger changes were observed in young rats. Rise in MMP-9 and -10 levels (in young animals), a decline in MMP-3 and -8 levels, and a decrease in the content of interleukin-1β, interferon-γ, interleukin-4 and tumor necrosis factor-α (the last two in young animals) was also detected. No significant changes were found in serine protease content. Therefore, proteolysis intensification in the brain of rats with HHcy is more likely caused by protease up-regulation through mechanisms stimulated by homocysteine, HTL, and oxidative stress, without involving pro-inflammatory signaling pathways.

Growth performance of gilthead sea bream (Sparus aurata) fed a mixture of single cell ingredients for organic diets

The aim of this study was to investigate the growth performance of gilthead sea bream (Sparus aurata) fed with a mixture of a single cell ingredient (SCI) (bacterial protein, yeast protein and algae) for organic aquaculture. Sea bream with an initial mean body weight of 6.87±0.07 g were randomly allocated to 250-litre tanks. Four isonitrogenous, isolipidic and isoenergetic diets were formulated by replacing organic fishmeal trimmings with 0% (control), 12% (SCI12), 15% (SCI15) and 18% (SCI18) of the SCI mixture. Each diet was distributed to the groups in triplicate. The fish were fed three times daily until satiation and the experimental period was 60 days. Survival rates and voluntary feed intake were similar in the fish with the different feed treatments (p > 0.05). Similar (p > 0.05) growth performance (final weight, SGR) and feed conversion ratio (FCR) were observed in fish fed all SCI-based diets, which was superior to the control diet treatment. Apparent protein digestibility was similar among treatments, but lipid digestibility was significantly reduced in the SCI15 and SCI18 groups compared to the SCI12 group. The specific activities of trypsin, total alkaline protease and lipase were increased in fish fed the SCI15 diet (p < 0.05). The protein content in the fillet was significantly increased and the lipid content was decreased in the SCI12 and SCI18 groups compared to the other groups. No adverse effects on liver and intestinal histology were observed in the different groups. The results suggest that organic fishmeal trimmings can be successfully replaced by innovative SCIs based on microbial protein, yeast meal and algae, and that this SCI mixture could be a candidate for organic aqua feed for gilthead sea bream.

Digestive proteases of Morelet’s crocodile (Crocodylus moreletii) in three life stages

Introduction: Morelet’s crocodile (Crocodylus moreletii) is a species distributed in the Mexican southeast and threatened due to multiple pressures. Objective: To characterize the digestive proteases in the acid phase (stomach) and alkaline phase (intestine) of three life stages of C. moreletii in captivity (hatchling, juvenile, and adult). Methods: Total alkaline and acid protease activities were quantified using casein and haemoglobin as substrates. Trypsin, chymotrypsin, leucine aminopeptidase, and elastase activities were quantified using synthetic substrates. Protease profiles were analysed by SDS-PAGE and Native-PAGE. Results: The specific activity of acid and alkaline proteases showed differences between the three stages, finding the highest activity in the juveniles. Trypsin, chymotrypsin, leucine aminopeptidase, and elastase activities were higher in hatchlings. There were differences in optimum pH and temperature of acid and alkaline proteases, trypsin, and leucine aminopeptidase between the three stages, demonstrating the diversification of the enzymes according to different stages, as well as the presence of specific isoforms in each stage of C. moreletii. The acid phase zymogram showed four bands with pepsin-like acid activity in the hatchling and juvenile crocodile, while in the adult only two of the four bands were detected. The alkaline zymogram showed that the hatchling had the highest number of activity bands compared to the other stages, corresponding to the high specific activity reported in the alkaline phase. Conclusions: Digestive proteases of Morelet’s crocodile differ in their biochemical characteristics and the number of proteases between hatchling, juvenile, and adult. This could help in the future design of balanced diets as well to the sustainable management and production of this species.

The effect of the probiotic drug "Ecoflor" on the proteolytic activity of the sterlet intestine

The effect of the probiotic preparation "Ecoflor" on proteolytic activity of the intestinal mucosa and chyme of sterlet was studied. This probiotic is a consortium of strains of lacto- and bifidobacteria: B. bifidum, B. longum and L. acidophilus, L. casei, L. plantarum, immobilized on carbon-mineral sorbent SUMS-1. For the experiment, fish were divided into 4 groups: control and three experimental groups. Experimental groups of fish were given feed with probiotic in the amount of 2, 4, 6 g/kg of feed. Sampling was carried out before the beginning of the experiment, on 7, 14, and 21 days. Material for the study was taken from 5 individuals from each group. To obtain enzymatically active preparations, fish intestines were freed from fat and dried on ice bath glass. After incision, the chyme was collected with a spatula and scraper, then the mucous membrane was removed. The total proteinase activity was calculated by the ratio of the difference between the spectrophotometer readings of the sample with substrate and the blank sample to the product of the intestinal weight (g) by the incubation time (min) EA = ΔE425/(g×min). Determinations were performed in three repetitions. Data analysis showed the stimulating effect of the preparation on proteolytic activity. The maximum increase in enzymatic activity in the experimental groups compared to the control group was recorded on the 7th day of the experiment. A more significant increase in proteolytic activity of the intestinal mucosa compared to chyme was found. At the beginning of feeding, the stimulating effect of the preparation was found in all experimental groups compared to the control. In further observation periods the effectiveness of the probiotic significantly decreased.

Abstract 7178: Neuronal cells derived from iPSCs cell to evaluate neurotoxicity after 48 or 72 hours in high-through put screening format

Abstract Neurotoxicity is a major concern in central nervous system drug discovery and a frequent cause of attrition in clinical trials or approved drug withdrawal. Neuropathy is a side effect defined by the injury of peripheral nerves causing a loss of sensation/motion. Drug development pipelines typically involve testing in cell lines followed by animal investigations with translation to humans. The induced pluripotent stem cells (iPSCs) have widened new model systems to study adverse toxicities. With evolution in regulatory guidance the use of human- iPSC-derived tissue provides high-throughput access and a more relevant testing environment. Neuropathy is one of the most chemotherapy adverse effects. In this study we have screened 8 compounds. Bortezomib (proteasome inhibitor), gemcitabine (approved ovarian cancer treatment), ivermectin (parasitic diseases treatment), flavopiridol (approved CDKs inhibitors), SNS-032 (approved CDKs inhibitor), mefloquine (anti-malaria drug), digitonin (nonionic detergent) and tamoxifen (hormone receptor-positive breast cancer treatment). These molecule toxicities were tested on neuronal-derived iPSCs Dopaneurone, GABAneurone, Glutaneurone, Motorneurone, Microglya and Astrocytes in high throughput screening. For the project, cells were plated in 384 well/plates. DMSO tolerance was performed (up to 10%). Drugs were added, then, after 48h or 72h, total protease and protease activities were measured using CytoTox-GloTM. Compounds were tested at 7 concentrations (from 10−4 to 10−9M).In this study we have validated cell culture and the assay conditions. The signal window range was good after 48h and 72h of treatment. No toxicity was found until 10% DMSO. A specific neurotoxicity signature was measurable for each drug tested. Digitonin, our positive control, induced toxicity in all IPSCs after 48h and 72h of incubation. On the contrary, Tamoxifen, our negative control, did not induce any neurotoxicity. The other molecules generated a signature of severe to mild neurotoxicity. These results are in agreement with their known adverse side-effects. In conclusion, this panel is a good tool to anticipate possible neurotoxicity within the 3Rs respect. It can be used in early drug de-risking or neuroprotection screening, with the aim of preventing/reducing/curing neuropathy in at-risk populations. Citation Format: Fabienne Sabatier, Justine Chaigne, Maryse Martin, Rodolphe Barbeau, Luisa Houssin, Stephane Bonnin, Alastair King, Vanessa Jahnke. Neuronal cells derived from iPSCs cell to evaluate neurotoxicity after 48 or 72 hours in high-through put screening format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7178.

Abstract 1021: Defining total protease activity profiles in pancreatic cancer using a FRET substrate array

Abstract Proteases are an important component of many biological processes in the human body, and as such makes them a well-known subject to explore when investigating a variety of disease states from cancer to cardiovascular disease. In all stages of cancer, including early development, proteases play critical roles in almost every aspect for processes such as invasion, promotion of tumor growth, and metastasis. This makes screening of circulating proteases a potentially powerful tool for early detection and prognosis for a variety of cancers. The human proteome, however, is very complex, with around 600 proteases in total, and at least 234 of them found in circulation. Additionally, proteases often act on more than one substrate, cross reactively cleaving several substrates at different rates, further adding to the complexity of cleavage patterns of the human proteome. Previous liquid biopsies for cancer detection based on protease cleavage patterns focused mainly on detecting the activities of specific proteases, using single substrates to examine the effects of well-studied cancer-related proteases such as MMPs and cathepsins. In this study, we developed a method for screening cleavage patterns of proteases active in plasma samples that deviated from this norm. Due to the complexity of the human proteome, rather than developing a specific substrate for a specific protease, we utilized a large array of substrates to screen for cleavage patterns in plasma samples of cancer patients aimed at not one specific protease, but a wide range that may be present. In this proof of concept study, we used a commercially available internally quenched set of 360 substrates composed of 8 amino acids and a FRET pair. Initially, we optimized this assay to measure protease activity in the complex plasma environment. Next, we screened several plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC) or pancreatitis, and screen negative samples using the 360 substrate set assay and identified 5 top substrates that were proficient in identifying PDAC from other conditions. Finally, we performed a validation study with a larger cohort (45 screen negative, 26 PDAC, 19 pancreatitis) using the selected substrates to determine the diagnostic performance of the assay. All five of the selected substrates showed 1.3-1.8-fold increase in fluorescence signal in PDAC compared to screen negative and pancreatitis. Amongst the cancer samples, the substrates showed no significant difference between stage, supporting possible applications for early detection. Additionally, area under the curve analysis of the five substrates gave a score of 0.8. In summary, in this study, we reported a novel approach for the detection of cancer exploiting the protease cleavage patterns in plasma samples. We believe that this approach holds great potential for detecting substrates and proteases for early detection of PDAC and other cancers. Citation Format: Morgan Stewart, Arnaud Quentel, Jose Montoya Mira, Elise Manalo-Hall, Srivathsan Ranganathan, Bruce Branchaud, Fehmi Civitci, Eugene Tu, Jared Fischer, Yu-Jui Chiu, Adem Yildirim. Defining total protease activity profiles in pancreatic cancer using a FRET substrate array [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1021.

Mitigation of cadmium toxicity stress by magnetopriming during germination of soybean

Cadmium (Cd) is a highly toxic heavy metal that poses a significant threat to food safety and agricultural production worldwide. Its solubility enables easy entry into plants, leading to reductions in seed germination, growth and crop yield. Thus, it is crucial to discover methods to alleviate the harmful impact of Cd on plant growth. Magnetopriming (MP) is a non-invasive and cost-effective technique that has been proposed to improve plant growth under abiotic stress conditions. The current study investigated the potential of MP to improve the seed germination, early seedling growth and biochemical responses of soybean under Cd toxicity. Soybean seeds were magnetoprimed with static magnetic field (SMF) strength of 200 mT for 1 hour before germination. The unprimed and magnetoprimed seeds were grown under different concentrations of cadmium chloride (0, 25, 50, 75, 100, 200 and 300 μM CdCl2) in Petri plates for five days in the dark at 25 + 1°C. The results revealed that Cd toxicity significantly reduced the germination percentage and inhibited the seedling growth parameters and increased oxidative stress, as determined by malondialdehyde (MDA) content in soybean seedlings from unprimed seeds. The inhibitory effect was increased with increasing concentration of Cd. However, MP remarkably increased the germination percentage, seedling growth parameters, activities of total amylase and protease, and hydrogen peroxide (H2O2) content and decreased MDA and proline content in germinating soybean seedlings at all concentrations of Cd. These findings suggest that MP can alleviate the adverse effects of Cd stress in soybean seedlings and increase the tolerance index towards Cd toxicity by enhancing the activity of amylotic and proteolytic enzymes and reducing oxidative stress.