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BACKGROUND & AIMS: Heterozygous SPINK1 mutations are strong risk factors for chronic pancreatitis in humans, yet heterozygous disruption of mouse Spink1 yielded no pancreatic phenotype. To resolve this contradiction, we used CRISPR/Cas9-mediated genome editing to generate heterozygous Spink1-deleted mice (Spink1-KOhet) in the C57BL/6N strain and studied the effect of this allele in trypsin-independent and trypsin-dependent pancreatitis models. METHODS: We investigated severity of acute pancreatitis and progression to chronic pancreatitis in Spink1-KOhet mice after transient (10 injections) and prolonged (2 × 8 injections) cerulein hyperstimulation. We crossed Spink1-KOhet mice with T7D23A and T7D22N,K24R mice that carry strongly autoactivating trypsinogen mutants and exhibit spontaneous chronic pancreatitis. RESULTS: Prolonged but not transient cerulein stimulation resulted in increased intrapancreatic trypsin activity and more severe acute pancreatitis in Spink1-KOhet mice relative to the C57BL/6N control strain. After the acute episode, Spink1-KOhet mice developed progressive disease with chronic pancreatitis-like features, whereas C57BL/6N mice recovered rapidly. Trypsinogen mutant mice carrying the Spink1-KOhet allele exhibited strikingly more severe chronic pancreatitis than the respective parent strains. CONCLUSIONS: Heterozygous Spink1 deficiency caused more severe acute pancreatitis after prolonged cerulein stimulation and promoted chronic pancreatitis after the cerulein-induced acute episode, and in two strains of trypsinogen mutant mice with spontaneous disease. In contrast, acute pancreatitis induced with limited cerulein hyperstimulation was unaffected by heterozygous Spink1 deletion, in agreement with recent observations that trypsin activity does not mediate pathologic responses in this model. Taken together, the findings strongly support the notion that loss-of-function SPINK1 mutations in humans increase chronic pancreatitis risk in a trypsin-dependent manner.

Moringa oleifera Lam. (horseradish tree) leaves demonstrate high nutritional value, are rich in proteins, and are widely used in folk medicine and food. This study investigated the presence of secondary metabolites and antinutritional proteins in leaf extract (LE) and the protein-rich fraction (PRF) derived from M. oleifera leaves, as well as the cytotoxicity to human cells, hemolytic activity, and in vivo acute toxicity and genotoxicity in mice. The flavonoids rutin and vitexin as well as trypsin inhibitors and lectins were detected in LE and PRF. Neither sample demonstrated toxicity against human peripheral blood mononuclear cells and both showed low hemolytic action. In vivo, LE and PRF did not show antinutritional effects and caused no death. The hematological parameters of the animals in the treated group were similar to those of the control. A significant increase in the serum levels of alanine aminotransferase and a discrete leukocyte infiltration with cytoplasmic vacuolization of the hepatocytes in the liver were detected in LE-treated animals. The preparations were not genotoxic or mutagenic. This study shows that LE and PRF are not antinutritional agents and presented low acute toxicity and no genotoxicity or mutagenicity. The present study contributes to the determination of the safety of using M. oleifera leaf proteins. [ABSTRACT FROM AUTHOR]

BACKGROUND: There is growing evidence that unfavorable neighborhood contexts may influence prostate cancer progression. Whether these associations may be explained in part by differences in tumor-level somatic alterations remain unclear. METHODS: Data on tumor markers (PTEN, p53, ERG, and SPINK1) were obtained from 1,157 participants with prostate cancer in the Health Professionals Follow-up Study. Neighborhood greenness, socioeconomic status, and the income Index of Concentration at Extremes were obtained from satellite and census data and linked to participants address at diagnosis and at study enrollment. Exposures were scaled to an interquartile range and modeled as tertiles. Bivariate associations between tertiles of neighborhood factors and tumor markers were assessed in covariate adjusted logistic regression models to estimate ORs and 95% confidence intervals. RESULTS: There was no association between any of the neighborhood contextual factors and PTEN, p53, ERG, or SPINK1 in bivariate or multivariable adjusted models. Results were generally consistent when modeling exposure using exposure at diagnosis or at study enrollment. CONCLUSIONS: In this multilevel study of men with prostate cancer, we found no evidence of associations between neighborhood context and tumor tissue markers. IMPACT: Our results provide some of the first empirical data in support of the hypothesis that prostate cancer risk conferred by tumor tissue markers may arise independently of underlying neighborhood context. Prospective studies in more diverse populations are needed to confirm these findings.

Recovering valuable active substances from the by-products of agricultural processing is a crucial concern for scientific researchers. This paper focuses on the enrichment of soybean trypsin inhibitor (STI) from soybean whey wastewater using either ammonium sulfate salting or ethanol precipitation, and discusses their physicochemical properties. The results show that at a 60% ethanol content, the yield of STI was 3.983 mg/mL, whereas the yield was 3.833 mg/mL at 60% ammonium sulfate saturation. The inhibitory activity of STI obtained by ammonium sulfate salting out (A-STI) was higher than that obtained by ethanol precipitation (E-STI). A-STI exhibited better solubility than E-STI at specific temperatures and pH levels, as confirmed by turbidity and surface hydrophobicity measurements. Thermal characterization revealed that both A-STI and E-STI showed thermal transition temperatures above 90 °C. Scanning electron microscopy demonstrated that A-STI had a smooth surface with fewer pores, while E-STI had a rough surface with more pores. In conclusion, there was no significant difference in the yield of A-STI and E-STI (p < 0.05); however, the physicochemical properties of A-STI were superior to those of E-STI, making it more suitable for further processing and utilization. This study provides a theoretical reference for the enrichment of STI from soybean whey wastewater. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever recorded in human history, causing significant losses to both life and economies globally. However, the available evidence is currently insufficient to establish the effectiveness and safety of antiviral drugs or vaccines. The entry of the virus into host cells involves binding to angiotensin-converting enzyme 2 (ACE2), a cell surface receptor, via its spike protein. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a host surface protease, cleaves and activates the virus's S protein, thus promoting viral infection. Plant protease inhibitors play a crucial role in protecting plants against insects and/or microorganisms. The major storage proteins in sweet potato roots include sweet potato trypsin inhibitor (SWTI), which accounts for approximately 60% of the total water-soluble protein and has been found to possess a variety of health-promoting properties, including antioxidant, anti-inflammatory, ACE-inhibitory, and anticancer functions. Our study found that SWTI caused a significant reduction in the expression of the ACE2 and TMPRSS2 proteins, without any adverse effects on cells. Therefore, our findings suggest that the ACE2 and TMPRSS2 axis can be targeted via SWTI to potentially inhibit SARS-CoV-2 infection. [ABSTRACT FROM AUTHOR]

SUMMARY: Modifying pellet die thickness (PDT) has been shown to affect the frictional heat exposure of feed. The inclusion of Azomite (AZM) in broiler diets containing dicalcium phosphate has been shown to maintain apparent ileal amino acid digestibility (AIAAD). This is likely due to AZM's proposed die-scouring and lubrication properties that decreased the frictional heat exposure of feed which can change protein conformation and reduce AIAAD. Therefore, it was hypothesized that PDT and AZM would interact to influence AIAAD and broiler performance. The objective of the study was to determine the effect of AZM (0.0% or 0.25%) and PDT (32 and 45 mm; with a constant pellet diameter of 4.5 mm) on broiler performance and AIAAD from 0 to 21 d of age using a corn and soybean meal-based diet. Live performance was not influenced by AZM, PDT, or their 2-way interaction (P > 0.05). However, AIAAD was affected by AZM and PDT interactions (P < 0.05), with 11 amino acids demonstrating increased AIAAD in only the 45 mm control treatment. The AIAAD increase was likely not enough to influence performance. It was presumed that increased frictional heat deactivated trypsin inhibitors (TI) and chymotrypsin inhibitors (CTI), ultimately increasing AIAAD. Quantitative analysis of TI and CTI activity, utilizing a novel assay based on the current American Oil Chemists’ Society (Ba 12–75) and the American Association of Cereal Chemists International (22-40.01) accepted procedures, showed no practically influential amount of either inhibitor before or after pelleting. The authors, therefore, speculate that the increased AIAAD was due to corn aleurone layer cell wall lysis via increased frictional heat exposure of the 45 mm PDT and the absence of AZM.

We present a numerical simulation method for designing a buffer system in microchip electrophoresis (MCE) equipped with capacitively-coupled-contactless-conductivity detection (C4D). One of the key design considerations for MCE-C4D is background electrolyte (BGE). This is because a C4D typically exhibits low sensitivity, and optimizing BGE conditions (e.g., base and acid species, pH, and ionic strength) can improve its sensitivity. However, BGE has been traditionally designed through experience or trial and error, which is time- and reagent-consuming. In this study, we employ Simul 5, an open-source electrophoresis simulation software, for rational BGE design. Four BGEs including trimethylamine (TEA)/acetic acid (AcOH, pH 10.6), MES/His (pH 6.1), MES/TRIS (pH 8.1), and TRIS/HCl (pH 7.4), previously used in electrophoresis-C4D of amino acids and protein, were selected for evaluation of our numerical method. Glutamic acid (Glu) was selected as a model analyte for initial simulation verification. Our numerical simulation revealed that the best achievable detection sensitivity was 1.046 × 10–5 S/(m µM) in the TRIS/HCl buffer because anionic Glu species with a low mobility (27 × 10–9 m2/Vs) replaced Cl− co-ion of a high mobility (79.1 × 10–9 m2/Vs) in the analyte zone, leading to a significant negative conductivity peak. TEA/AcOH, MES/His, and MES/TRIS buffers exhibited progressively lower sensitivity. After the initial evaluation, trypsin inhibitor (TI), a more complex proteinous analyte was tested in the MES/His and MES/TRIS BGEs. The best detection sensitivity was 1.032 × 10–4 S/(m µM) in the MES/TRIS buffer because counter-ionic species TRIS+ of a high mobility (29.5 × 10–9 m2/Vs) was replaced by the ionic TI, characterized by a large charge (− 11.5) and a low mobility (8.08 × 10–9 m2/Vs), resulting in a strong negative peak. Based on a comprehensive analysis of the impacts of compositional changes in each ionic species of the analyte zone on conductivity-peak height, we propose a BGE design guideline for enhanced sensitivity. Subsequent MCE-C4D confirmation experiments demonstrated excellent qualitative agreement with the simulation results for the Glu and TI analytes. We anticipate that our numerical analysis method will find wide application in designing BGEs for portable MCE-C4D systems by enhancing sensitivity. [ABSTRACT FROM AUTHOR]

In order to evaluate the influence of different sowing dates on soybean yield and seed quality, ten genotypes from three different maturity groups were studied at Research and Development Station for Agriculture Turda and provided results from two different sowing dates. The laboratory analysis included the examination of various parameters and assessed the several quality parameters; these analyses were conducted non-destructively using NIR spectrometry, ensuring the integrity of the grains. The trypsin inhibitor activity was determined using benzoyl-DL-arginine-p-nitranilide. The obtained results revealed that sowing date had a profound impact on: number of pods, grains and weight per plant. Notably, a substantial reduction in yield was observed when soybeans were sown later compared to the optimal sowing date. Moreover, a substantial decrease in oil content was noted with late sowing dates, while a significant increase in protein content was achieved. Furthermore, the trypsin inhibitor activity exhibited lower levels across all studied cultivars during late sowing experiments compared to the optimal sowing date. It's also worth noting that the fatty acid profile displayed variations dependent on both the genotype and sowing date. [ABSTRACT FROM AUTHOR]

This study was aimed at determining the mineral and anti-nutritional properties of naturally fermented millet and pumpkin leaf flour blends. The millet grains were allowed to ferment spontaneously for 24 hrs and 48 hrs and were processed into flour. Dried pumpkin leaves were blended into flour and substituted using D-optimal mixture design, which resulted in ten experimental runs. The mineral content and the anti-nutritional properties of the flour blend formulation were analysed. Duncan’s multiple range test was used to evaluate the mean at p < 0.05 with SPSS version 21.0. Significant differences were observed in the mineral and anti-nutritional composition of the fermented millet and pumpkin leaf flour blends at 24 hrs and 48 hrs of fermentation time respectively. Calcium, potassium, and iron content increased significantly (p < 0.05) with increasing the amount of pumpkin leaf flour in the flour blends. The values for tannins and total phenolic composition ranged from 0.089 to 0.162% and from 0.075 to 0.120% for 24 hrs and from 0.080 to 0.141% and from 0.060 to 0.120% for 48 hrs of fermentation time respectively. Results showed that fermentation technique could be used to enrich the nutritional and bioactive potential of millet.

Moringa oleifera Lam. (horseradish tree) leaves demonstrate high nutritional value, are rich in proteins, and are widely used in folk medicine and food. This study investigated the presence of secondary metabolites and antinutritional proteins in leaf extract (LE) and the protein-rich fraction (PRF) derived from M. oleifera leaves, as well as the cytotoxicity to human cells, hemolytic activity, and in vivo acute toxicity and genotoxicity in mice. The flavonoids rutin and vitexin as well as trypsin inhibitors and lectins were detected in LE and PRF. Neither sample demonstrated toxicity against human peripheral blood mononuclear cells and both showed low hemolytic action. In vivo, LE and PRF did not show antinutritional effects and caused no death. The hematological parameters of the animals in the treated group were similar to those of the control. A significant increase in the serum levels of alanine aminotransferase and a discrete leukocyte infiltration with cytoplasmic vacuolization of the hepatocytes in the liver were detected in LE-treated animals. The preparations were not genotoxic or mutagenic. This study shows that LE and PRF are not antinutritional agents and presented low acute toxicity and no genotoxicity or mutagenicity. The present study contributes to the determination of the safety of using M. oleifera leaf proteins.

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Background & Aims: Heterozygous SPINK1 mutations are strong risk factors for chronic pancreatitis in humans, yet heterozygous disruption of mouse Spink1 yielded no pancreatic phenotype. To resolve this contradiction, we used CRISPR/Cas9-mediated genome editing to generate heterozygous Spink1-deleted mice (Spink1-KOhet) in the C57BL/6N strain and studied the effect of this allele in trypsin-independent and trypsin-dependent pancreatitis models. Methods: We investigated severity of acute pancreatitis and progression to chronic pancreatitis in Spink1-KOhet mice after transient (10 injections) and prolonged (2 × 8 injections) cerulein hyperstimulation. We crossed Spink1-KOhet mice with T7D23A and T7D22N,K24R mice that carry strongly autoactivating trypsinogen mutants and exhibit spontaneous chronic pancreatitis. Results: Prolonged but not transient cerulein stimulation resulted in increased intrapancreatic trypsin activity and more severe acute pancreatitis in Spink1-KOhet mice relative to the C57BL/6N control strain. After the acute episode, Spink1-KOhet mice developed progressive disease with chronic pancreatitis-like features, whereas C57BL/6N mice recovered rapidly. Trypsinogen mutant mice carrying the Spink1-KOhet allele exhibited strikingly more severe chronic pancreatitis than the respective parent strains. Conclusions: Heterozygous Spink1 deficiency caused more severe acute pancreatitis after prolonged cerulein stimulation and promoted chronic pancreatitis after the cerulein-induced acute episode, and in two strains of trypsinogen mutant mice with spontaneous disease. In contrast, acute pancreatitis induced with limited cerulein hyperstimulation was unaffected by heterozygous Spink1 deletion, in agreement with recent observations that trypsin activity does not mediate pathologic responses in this model. Taken together, the findings strongly support the notion that loss-of-function SPINK1 mutations in humans increase chronic pancreatitis risk in a trypsin-dependent manner.

Recovering valuable active substances from the by-products of agricultural processing is a crucial concern for scientific researchers. This paper focuses on the enrichment of soybean trypsin inhibitor (STI) from soybean whey wastewater using either ammonium sulfate salting or ethanol precipitation, and discusses their physicochemical properties. The results show that at a 60% ethanol content, the yield of STI was 3.983 mg/mL, whereas the yield was 3.833 mg/mL at 60% ammonium sulfate saturation. The inhibitory activity of STI obtained by ammonium sulfate salting out (A-STI) was higher than that obtained by ethanol precipitation (E-STI). A-STI exhibited better solubility than E-STI at specific temperatures and pH levels, as confirmed by turbidity and surface hydrophobicity measurements. Thermal characterization revealed that both A-STI and E-STI showed thermal transition temperatures above 90 °C. Scanning electron microscopy demonstrated that A-STI had a smooth surface with fewer pores, while E-STI had a rough surface with more pores. In conclusion, there was no significant difference in the yield of A-STI and E-STI (p < 0.05); however, the physicochemical properties of A-STI were superior to those of E-STI, making it more suitable for further processing and utilization. This study provides a theoretical reference for the enrichment of STI from soybean whey wastewater.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic. Known as COVID-19, it has affected billions of people worldwide, claiming millions of lives and posing a continuing threat to humanity. This is considered one of the most extensive pandemics ever recorded in human history, causing significant losses to both life and economies globally. However, the available evidence is currently insufficient to establish the effectiveness and safety of antiviral drugs or vaccines. The entry of the virus into host cells involves binding to angiotensin-converting enzyme 2 (ACE2), a cell surface receptor, via its spike protein. Meanwhile, transmembrane protease serine 2 (TMPRSS2), a host surface protease, cleaves and activates the virus’s S protein, thus promoting viral infection. Plant protease inhibitors play a crucial role in protecting plants against insects and/or microorganisms. The major storage proteins in sweet potato roots include sweet potato trypsin inhibitor (SWTI), which accounts for approximately 60% of the total water-soluble protein and has been found to possess a variety of health-promoting properties, including antioxidant, anti-inflammatory, ACE-inhibitory, and anticancer functions. Our study found that SWTI caused a significant reduction in the expression of the ACE2 and TMPRSS2 proteins, without any adverse effects on cells. Therefore, our findings suggest that the ACE2 and TMPRSS2 axis can be targeted via SWTI to potentially inhibit SARS-CoV-2 infection.

Abstract Edamame milk is a protein‐dense milk derived from green soybeans harvested before they mature. Being a legume of soy origin, it contains antinutritional factors for example, serine protease inhibitors, which hinder its in vitro digestibility. The objective of this study was to evaluate the effect of microwave processing techniques in improving the in vitro digestibility (IVPD %) of edamame milk protein by varying processing time and temperature. Conventional and microwave‐assisted processing was employed to investigate the effect on in vitro protein digestibility (IVPD %), using temperatures 70°Ϲ, 85°Ϲ, and 100°Ϲ for 5, 10, and 15 min, respectively. Fourier‐transform infrared (FTIR) data showed microwave and conventional treatments significantly modified the Amide I region of the edamame milk protein and the extent of modification varied with variation in the treatment temperature. In the FTIR analysis β‐sheet content was observed to change little with an increase in the temperature, suggesting similarity in the surface hydrophobicity of the protein leading to similar IVPD % values for all treatment temperatures. The experiment resulted in increased in vitro digestibility with increasing time and temperature during microwave processing conditions and conventional thermal conditions. It was also observed that the trypsin inhibitor activity decreased with an increase in processing time and temperature.

Objective: To purify and characterize a water-soluble protein from Pueraria lobata and to fabricate its nanoparticles by heating-induced assembly. Methods: Pueraria lobata protein was purified by anion exchange chromatography High Q, and its molecular weight and amino acid sequence were determined by SDS-PAGE and N-terminal sequencing. The particle size, optical dispersion intensity and Zeta potential of protein nanoparticles were measured by laser-scattering particle analyzer. The drug loading efficiency of the nanocarrier was determined by chromatography. Results: A major water-soluble protein, named PP, was purified from Pueraria lobata and sequenced with a N-terminal amino acid sequence of DFVYDMCGNVLNGGTYYIL. PP was identified as a novel trypsin inhibitor by NCBI database searching and rypsin inhibitory assay. PP was also well-stabilized in the pH2~10 and 20~50 °C ranges. After heating PP solution (0.1 mg/mL, pH6.0) for 60 minutes at 100 ℃, homogenous nanoparticles (PP-NPs) were harvested. These PP-NPs had a particle size of 172.78 nm and a Zeta potential of −25.40 mV. The puerarin and berberine were effectively loaded onto PP-NPs, with loading efficiency of 33.83% and 24.61%, respectively. Conclusion: The major water-soluble Pueraria lobata protein PP can be fabricated into protein nanoparticles by heating-induced assembly, indicating a potential as drug carriers.

Soymilk was subjected to high pressure processing (HPP) at different pressure levels (300–600 MPa) and time (5, 15 and 25 min) intervals. After the HPP treatment, a mild reduction was observed in soymilk pH and conductivity values; however, a sudden surge in the protein content (17.34±0.10 mg/ml) was recorded at 600 MPa (5 min) treatment. When soymilk proteins were evaluated for their structural changes, FTIR (amide regions), CD spectra, and UV inflorescence showed the positive responses and as the result, soy Kunitz trypsin inhibitor (SKTI) antigen binding ability reduced from 100% to 44.39±8.50% at 300 MPa (5 min). However, prolong exposure at 300 MPa had a negative effect on allergenicity. Nevertheless, HPP treatment reduced the lipoxygenase (100.00% to 68.13±12.80%) and trypsin inhibitor (100.00% to 91.30±1.45%) activities. In addition, the treatment also slightly altered the viscosity, whiteness, and volatile aroma components of soymilk after HPP treatment. Principal component analysis revealed that among 22 selected volatiles, only those belong to PC1 and PC2 caused 68% variance in aroma changes. Overall, HPP treatment reduced allergenicity, trypsin inhibitor activity and lipoxygenase activity without modifying the soymilk nature. Further, it also reduced the beany flavor inducing hexanal volatile content after the treatment.

Soymilk contained serine protease enzymes with inhibitory activity against trypsin, causing a negative effect on nutritional absorption. This project aimed to investigate the effects of conventional heating (100 °C/20 min) and microwave heating (360–900 W/1–6 min) on trypsin inhibitor, protein digestibility, and odor profiles. Soymilk contained 46–47% protein, regardless of heating conditions. Using scanning electron microscopy, it can be seen that the conventionally heated sample had a smooth surface and a porous structure, whereas microwave-heated samples contained a protein matrix in clusters with rough surfaces. The molecular weight of proteins in SDS-PAGE was reduced to 19 kDa and <16 kDa after conventional and microwave heating, respectively, resulting in the reduction of trypsin inhibitor from 36.66 to 0.91 and 0.56–0.66 mg/g, respectively. Protein digestibility was significantly improved when either the conventional heating at 100 °C for 20 min or the microwave heating at 900 W for 6 min was applied. From e-nose analysis, the beany flavor was reduced after the microwave heating for 3–6 min, and the highest intensity of the sweet odor compounds were observed after the microwave heating at 600 W for 1 min. Due to the short processing time, microwave heating was then recommended for inhibition of trypsin inhibitors and improving protein digestibility. [ABSTRACT FROM AUTHOR]

IntroductionLiver cancer-secreted serine protease inhibitor Kazal (LC-SPIK) is a protein that is specifically elevated in cases of hepatocellular carcinoma (HCC). We assessed the performance of LC-SPIK in detecting HCC, including its early stages, in patients with cirrhosis, hepatitis B virus (HBV), and hepatitis C virus (HCV).MethodsWe enrolled 488 patients, including 164 HCC patients (81 early HCC) and 324 controls in a blinded, prospective, case-control study. Serum LC-SPIK levels were determined by an enzyme-linked immunosorbent assay-based assay. The performance of serum LC-SPIK and α-fetoprotein (AFP), including area under the curve (AUC), sensitivity, and specificity, are compared. The performance of LC-SPIK was evaluated in an independent validation cohort with 102 patients.ResultsIn distinguishing all HCC patients from those with cirrhosis and chronic HBV/HCV, LC-SPIK had an AUC of 0.87, with 80% sensitivity and 90% specificity using a cutoff of 21.5 ng/mL. This is significantly higher than AFP, which had an AUC of 0.70 and 52% sensitivity and 86% specificity using a standard cutoff value of 20.0 ng/mL. For early-stage HCC (Barcelona Clinic Liver Cancer stage 0 and A), LC-SPIK had an AUC of 0.85, with 72% sensitivity and 90% specificity, compared with AFP, which had an AUC of 0.61, with 42% sensitivity and 86% specificity. In addition, LC-SPIK accurately detected the presence of HCC in more than 70% of HCC patients with false-negative AFP results.DiscussionThe study provided strong evidence that LC-SPIK detects HCC, including early-stage HCC, with high sensitivity and specificity, and might be useful for surveillance in cirrhotic and chronic HBV/HCV patients, who are at an elevated risk of developing HCC.

Climate variability has major implications for agriculture due to the increase in the frequency and intensity of simultaneous abiotic, namely water-stress, and biotic stresses to crops. Plant water-stress alone harms crops but also can attract outbreaks of herbivores with varied host specialization, and plants succumb to further yield losses dealing with multiple stressors. Host plant resistance provides a route to lessen yield losses from herbivory; however, our knowledge of the interactions between water-stress and pest resistance is limited, especially for mite herbivores of maize including the generalist two-spotted spider mite (Tetranychus urticae, TSM) and the specialist Banks grass mite (Oligonychus pratensis, BGM). We conducted parallel greenhouse and field experiments whereby a susceptible line (B73) and two TSM-resistant lines (B75 and B96) were subjected to either optimal irrigation or water-stress [50–60% and 5–10% volumetric water content (VWC), and 25–32% and 10–15% VWC, in the greenhouse and field, respectively] to test whether pest-resistant lines maintain their resistance when exposed to water-stress. We found that under optimal irrigation, TSM and BGM populations increased readily on B73, while B75 and B96 were largely resistant to the TSM but not BGM. While plant water-stress increased the susceptibility of B73 to both mite species, water-stress did not disrupt initial resistance levels of B75 and B96 maize for either mite species. Elevated protease activity was found in B75 and B96 and may contribute to maize resistance. Our findings that B75 and B96 are highly resistant to the TSM, and maintain resistance to both mite species with water-stress, highlight the importance of including the nuances of multiple stressors within the framework of host plant resistance. [ABSTRACT FROM AUTHOR]

Edamame milk is a protein-dense milk derived from green soybeans harvested before they mature. Being a legume of soy origin, it contains antinutritional factors for example, serine protease inhibitors, which hinder its in vitro digestibility. The objective of this study was to evaluate the effect of microwave processing techniques in improving the in vitro digestibility (IVPD %) of edamame milk protein by varying processing time and temperature. Conventional and microwave-assisted processing was employed to investigate the effect on in vitro protein digestibility (IVPD %), using temperatures 70°C, 85°C, and 100°C for 5, 10, and 15min, respectively. Fourier-transform infrared (FTIR) data showed microwave and conventional treatments significantly modified the Amide I region of the edamame milk protein and the extent of modification varied with variation in the treatment temperature. In the FTIR analysis ß-sheet content was observed to change little with an increase in the temperature, suggesting similarity in the surface hydrophobicity of the protein leading to similar IVPD % values for all treatment temperatures. The experiment resulted in increased in vitro digestibility with increasing time and temperature during microwave processing conditions and conventional thermal conditions. It was also observed that the trypsin inhibitor activity decreased with an increase in processing time and temperature. [ABSTRACT FROM AUTHOR]

Green bean (Phaseolus vulgaris) is a valuable vegetable that has a high amount of functional nutrients and bioactive compounds. The use of zinc (Zn) fertilizer to improve green bean productivity and nutrient value is limited. A study was performed to evaluate plant growth, productivity and harvest quality of different pole and bush green bean (Phaseolus vulgaris) varieties as affected by zinc sulphate and Lys-Zn. Results showed that green bean shoot fresh and dry weights increased by the two sources of Zn fertilizer in pole varieties compared with controls. The bush types, 'Burpees' and 'Black valentine' showed higher yield with Lys-Zn application compared with the control. Antioxidant activity was enhanced by Lys-Zn in 'Blv". The 'Burpees', 'Dragon' and 'Black valentine' indicated highest antioxidant activity. Results indicated that regardless of the source of Zn fertilizer, all the green bean varieties were rich in calcium, magnesium, and potassium. 'Dragon' under ZnSO4 had the highest Zn content. Also, foliar application with Lys-Zn enhanced protein, starch, calcium, auxin, and folate mostly in 'Burpees', 'Dragon', and 'Black valentine'. Moreover, total essential and non-essential amino acids were improved by Lys-Zn in 'Sunray' and 'Burpees'. A real-time PCR indicated that Lys-Zn increased Bowman–Birk inhibitor (BBI) gene expression in'Sunray', 'Burpees' and 'Cherokee' compared with the others. Moreover, the interaction effect of variety and zinc gave the highest yield, antioxidant capacity, nutrient elements, and folate contents in 'Burpees' under the Lys-Zn treatment. [ABSTRACT FROM AUTHOR]

Piriformospora indica (P. indica) is a mutualistic endophyte that colonizes plant roots. In this study, effects of P. indica on rice resistance against rice leaffolder (Cnaphalocrocis medinalis Guenée) were investigated. Growth inhibition and leaf injury caused by leaffolder larvae infestation in P. indica-colonized rice was significantly alleviated. Moreover, growth retardation was observed in larvae which fed on P. indica-colonized plants. JA-Ile levels and trypsin inhibitor activities in leaf tissues of P. indica-colonized plants were higher than those in noncolonized plants under leaffolder-infested conditions. P. indica effects on increasing trypsin inhibitor expression and reducing larval growth were repressed by aspirin. JA signaling drives P. indica-enhanced insect resistance in rice, and trypsin inhibitors might be candidates involved in this mechanism. Changes in antioxidant enzyme activities and malondialdehyde levels in P. indica-inoculated plants were observed, and it was demonstrated that oxidative stress induced by insect infestation could be reduced in P. indica-colonized plants.

Fungal infections are a growing public health concern worldwide and the emergence of antifungal resistance has limited the number of therapeutic options. Therefore, developing novel strategies for identifying and developing new antifungal compounds is an active area of research in the pharmaceutical industry. In this study, we purified and characterized a trypsin protease inhibitor obtained from Yellow Bell Pepper (Capsicum annuum L.) seeds. The inhibitor not only showed potent and specific activity against the pathogenic fungus Candida albicans, but was also found to be non-toxic against human cells. Furthermore, this inhibitor is unique in that it also inhibits α-1,4-glucosidase, positioning it as one of the first plant-derived protease inhibitors with dual biological activity. This exciting discovery opens new avenues for the development of this inhibitor as a promising antifungal agent and highlights the potential of plant-derived protease inhibitors as a rich source for the discovery of novel multifunctional bioactive molecules. [ABSTRACT FROM AUTHOR]

Soybeans are characterized by high functionality and nutrients; however, they contain unpleasant tastes and odors, as well as nutritional inhibitors that are difficult to inactivate, hindering their use. Therefore, to inactive the heat-resistant antinutritional trypsin inhibitor (TI), we investigated the protein transformation effect of alcohol on inactivation. In addition, we examined whether heat-resistant urease (UA) and TI inactivation are compatible 6). The change in TI activity of soybean flour liquid and edamame in a boiling water bath was investigated over time. As a result, the TI activity of soybean flour was hardly inactivated. Edamame showed a gradual decrease in TI activity with elution of TI into the boiling water. In addition, soybean TI was hardly inactivated by 70 % alcohol treatment and normal pressure heating. Therefore, soybean flour and alcohol were placed in a sealed can, and inactivation by pressure heating was studied. As a result, obvious TI inactivation was observed by this treatment. The inactivation of UA and TI by pressurized heated alcohol was dependent on the alcohol concentration and treatment temperature and time. The inactivation rate of TI was 1/10, and a 50 % or more ethanol concentration was required with heating at 110 °C. UA was almost completely inactivated at 105 °C with an ethanol concentration of 20 % or more. Thus, it was confirmed that simultaneous inactivation of both proteins was possible. [ABSTRACT FROM AUTHOR]

Cyclotides are cysteine-rich cyclic peptides that have various activities and applications. Mining at the whole-genome scale facilitates the profiling and development of cyclotide-based markers. However, the characterization of cyclotide subfamilies at a genome-scale in plants is an untapped area that needs more studies. Here we developed four degenerate cyclotide subfamily-specific (CSS) markers based on their subfamily's semi-conserved sequence for identifying each subfamily separately. Initially, an in silico cyclotide mining analysis was performed on 15 plant genomes/transcriptomes. The in silico PCR produced 476 amplicons in the Bracelet, Moebius, Hybrid and Trypsin Inhibitor (TI) subfamilies. In silico profiling results identified 903 cyclotide mature domains (CMDs) in the analyzed genomes/transcriptomes for the four subfamilies. In addition, the prevalence of each subfamily correlated positively with the genome size, particularly in Hybrid, followed by TI and Bracelet subfamilies. The CSS markers were validated in vitro on 19 genotypes/cultivars. The CSS markers generated a total of 175 bands, with a polymorphic percentage of 98%–100%. Cluster analysis showed distinct clusters for cultivars/genotypes of each plant species. The marker efficiency parameters demonstrated the capability of CSS markers to determine the genetic diversity among the studied plant species. This study represents the first report to perform genome/transcriptome-wide mining to characterize the Cyclotide subfamilies in plants. This study proposed potential applications of the novel CSS markers as an effective system for screening cyclotide subfamilies across multiple plant species with no prior sequencing knowledge requirement. Additionally, it proposes an innovative approach for identifying the appropriate cyclotide subfamily used in drug design. [ABSTRACT FROM AUTHOR]

Bamboo shoot is well known for the high nutritive values it possesses. People in Northeastern India, the majority of which are tribal and lives in rural area consume bamboo shoot extensively without knowing its benefits and demerits. The present study was undertaken in Arunachal Pradesh, India considering the possible threats it may pose to the people of the region in particular by estimating the antinutritional elements such as Phytic acid and Trypsin inhibitor from bamboo shoots of seven species and its three processed forms that are widely consume. Phytic acid content was observed within a range of 0.81 to 0.31 Pmg/100g and 0.75 to 0.38 Pmg/100g in bamboo shoots and processed products respectively whereas trypsin inhibitor in tender shoots of all the seven species (0.53-0.24 TIU mg/protein) and processed products (0.37-0.19 TIU mg/protein) was also recorded. Similarly, there were also significant variations in phytic acid (F=41.509, P<0.001) and trypsin inhibitor (F=16.631, P< 0.001) concentration between the fresh shoots and processed forms. The amount of antinutrients estimated from these samples are considerably less and far from any harm. [ABSTRACT FROM AUTHOR]

Chemotherapy and radiation not only trigger cancer cell apoptosis but also damage stromal cells in the tumour microenvironment (TME), inducing a senescence-associated secretory phenotype (SASP) characterized by chronic secretion of diverse soluble factors. Here we report serine protease inhibitor Kazal type I (SPINK1), a SASP factor produced in human stromal cells after genotoxic treatment. DNA damage causes SPINK1 expression by engaging NF-κB and C/EBP, while paracrine SPINK1 promotes cancer cell aggressiveness particularly chemoresistance. Strikingly, SPINK1 reprograms the expression profile of cancer cells, causing prominent epithelial-endothelial transition (EET), a phenotypic switch mediated by EGFR signaling but hitherto rarely reported for a SASP factor. In vivo, SPINK1 is expressed in the stroma of solid tumours and is routinely detectable in peripheral blood of cancer patients after chemotherapy. Our study substantiates SPINK1 as both a targetable SASP factor and a novel noninvasive biomarker of therapeutically damaged TME for disease control and clinical surveillance.

Piriformospora indica (P. indica) is a mutualistic endophyte that colonizes plant roots. In this study, effects of P. indica on rice resistance against rice leaffolder (Cnaphalocrocis medinalis Guenée) were investigated. Growth inhibition and leaf injury caused by leaffolder larvae infestation in P. indica-colonized rice was significantly alleviated. Moreover, growth retardation was observed in larvae which fed on P. indica-colonized plants. JA-Ile levels and trypsin inhibitor activities in leaf tissues of P. indica-colonized plants were higher than those in noncolonized plants under leaffolder-infested conditions. P. indica effects on increasing trypsin inhibitor expression and reducing larval growth were repressed by aspirin. JA signaling drives P. indica-enhanced insect resistance in rice, and trypsin inhibitors might be candidates involved in this mechanism. Changes in antioxidant enzyme activities and malondialdehyde levels in P. indica-inoculated plants were observed, and it was demonstrated that oxidative stress induced by insect infestation could be reduced in P. indica-colonized plants. [ABSTRACT FROM AUTHOR]

A novel low molecular mass ginkgo biloba trypsin inhibitor (GBTI) was isolated from ginkgo fruits (GF) by trypsin inhibitory activity-guided fractionation by using ammonium sulphate precipitation, followed by ultra-filtration, affinity chromatography and RP-HPLC. The molecular mass and amino acid sequence of GBTI was determined using ESI-MS and ESI-MS/MS, respectively. The structure of GBTI was identified as MKNLTVIPPICLKFPN, with a molecular mass of 1826 Da. GBTI was stable in the pH range of 4–8 and in the temperature range of 0–80 °C for 30 min. However, the inhibitory activity of the GBTI reduced when incubated with various metalions (K+, Na+, Fe2+, Mg2+ and Ca2+). Finally, GBTI exhibited significant antiproliferative effect in human MDA-MB-231 and mouse 4 T-1 triple-negative breast cancer cells and without toxicity to MCF-10A normal breast cells. Our results suggest that GBTI could be exploited as a natural and hyperstable anticancer agent for triple-negative breast cancer patients. [ABSTRACT FROM AUTHOR]

Tamarind is a fruit with a wide variety of uses, and the presence of enzyme inhibitors in Tamarind seeds gives the fruit bioactive activities. Protease inhibitors have been recognized as molecules that can potentially contribute to health. Trypsin inhibitors present different physiological effects, such as anti-inflammatory and satietogenic effects, which make them potent candidates for the treatment and prevention of chronic diseases, despite the challenging methodologies in characterizing these new molecules. This paper aims to address the activities and health application perspectives of Tamarind enzyme inhibitors using a systematic integrative literature review. [ABSTRACT FROM AUTHOR]

The study aimed to evaluate the nanoparticles (ECW) containing tamarind trypsin inhibitor (TTI) concerning the storage effect under different conditions on antitrypsin activity and the bioactive potential in a preclinical model. ECW was exposed to different pH and temperatures to evaluate the interaction between TTI and its encapsulating agents, monitored by antitrypsin activity. Wistar rats (n = 25) with obesity induced by diet were divided into groups: untreated; treatment with nutritionally adequate diet; treatment with nutritionally adequate diet and ECW/12.5 mg/kg; treatment with ECW/12.5 mg/kg; and treatment with TTI/25 mg/kg. The groups were evaluated over ten days with regards to satiety, zoometric, biochemical, and inflammatory parameters, using ten times less TTI (2.5 mg/kg) contained in ECW. TTI was protected and encapsulated in ECW without showing residual inhibitory activity. Only at gastric pH did ECW show antitrypsin activity. At different temperatures, it showed high antitrypsin activity, similar to TTI. The animals treated with ECW had significantly reduced body weight variation (p < 0.05), and only TTI treatment reduced the inflammatory parameters significantly (p < 0.05). The study showed that by using lower concentrations of TTI in ECW it was possible to perceive promising effects with perspectives of use in functional products for managing obesity and its complications. [ABSTRACT FROM AUTHOR]

Kunitz (KTI) and Bowman-Birk (BBI) trypsin inhibitors were characterized in soybean seeds. Cultivars having KTI/BBI (Pannónia Kincse, PK) or lacking KTI (Aries; Hilario; Bahia) were assessed with well-characterized soybean varieties having Ti-a or ti types of KTI mobility. The TIA values of Pannónia Kincse (9.8 ± 0.48 mg/g) were not significantly different (p ≤ 0.05) from Ti-a samples (10.07 ± 1.86 mg/g), while of Aires, Bahia, Hilario (6.19 ± 1.89) were identical (p ≤ 0.05) with ti samples (6.63 ± 1.99). Radiofrequency heat treatment (RF) decreased TIA values (p ≤ 0.05) at ≥ 100 °C. However, in the traditional soybean variety, the RF at 110 °C was more effective in eliminating the residual KTI activity. The remaining or the disapperaing bioactive form of trypsin inhibitors were succesfully characterized by the means of a standardized in vitro digestion model. It showed that residual BBI-originated trypsin inhibitor activity was in the stomach even after RF at 110 °C, whereas its chymotrypsin inhibitor activity was not detectable at all. Although PK and KTI null types of soybean seeds still required an energy-saving, gentle heat treatment to inactive the trypsin inhibitors before using them as food or feed, the physicochemical properties and processing quality of soybean products were protected, improved. [ABSTRACT FROM AUTHOR]

In this study, the in vitro apparent rumen degradability of organic matter (ARDOM) and plant secondary metabolites (ARDPSM) of three tropical legumes (Mucuna pruriens, Canavalia ensiformis, and Leucaena leucocephala) were assessed. For this, 3 experiments were set up, i.e., single end-point incubations (24 h) with ruminal inoculum from either Belgian or Cuban sheep, as well as kinetic assessments (0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, and 24 h) inoculum from Belgian sheep. L-mimosine, L-canavanine, Concanavalin A (Con A), and trypsin inhibitor (TI) were the plant secondary metabolites (PSM) targeted in this study. In all three experiments, both beans, as well as forage/bean meals of M. pruriens and C. ensiformis and their PSM, were extensively degraded during 24 h incubation, irrespective of the inoculum source (0.44 to 0.70 and 0.43 to 0.78 g/g of organic matter (OM) for ARDOM, respectively, and > 0.80 g/g for L-canavanine, > 0.76 TIU/TIU for TI, and > 0.95 g/g for Con A, for both legumes). Forage meal of L. leucocephala was considerably less degraded, with apparent ruminal degradabilities of 0.20 g/g OM and 0.35 g/g OM after 24 h incubation with Belgian or Cuban sheep inoculum, respectively. This could – at least partially – be related to L-mimosine, present in L. leucocephala, which was hardly degraded in the Belgian incubation, while a more extensive ruminal breakdown was observed under the Cuban conditions (0.05 g/g PSM vs. 0.78 g/g PSM, respectively). The negative effect of L-mimosine on OM degradability was supported in an additional in vitro experiment with straw and inoculum from Belgian sheep, as ruminal degradation of straw was 31% lower when pure L-mimosine was supplemented. [ABSTRACT FROM AUTHOR]

Soybean trypsin inhibitor (STI) was obtained from simulated soybean whey wastewater through a sustainable method consisting of isoelectric precipitation, ammonium sulfate salting out, and gel filtration chromatography, and the effect of temperature, pH, and pepsin on the stability of STI was also discussed. The results showed that the recovery rate of the trypsin inhibitory activity was 89.47%, the purity and the specific activity of STI were 71.11%, and 1442.5 TIU/mg in the conditions of pH 4.0 and 40% ammonium sulfate saturation. The soybean Kunitz trypsin inhibitor (KTI) and soybean Bowman–Brik trypsin inhibitor (BBI) were obtained via gel filtration chromatography, and their specific activity levels were 1733.5 TIU/mg and 2588.3 TIU/mg, respectively. The STI displayed good stability over a wide temperature and pH range. The STI, KTI, and BBI were all resistant to pepsin hydrolysis, and their ability was ranked as BBI > STI > KTI. These findings will provide a theoretical basis for recycling STI from soybean whey wastewater and promoting better active compound utilization. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the expression of serine protease inhibitor kazal type 1 (SPINK1) and its carcinogenic effect in oral tongue squamous cell carcinoma (OTSCC). Design: Initially, bioinformatics analysis was conducted using data from The Cancer Genome Atlas and Gene Expression Omnibus to compare SPINK1 mRNA expression between malignant and adjacent tissues. Subsequently, the impact of differential expression on survival and other clinical variables was examined. Additionally, histology microarray analysis was performed to assess SPINK1 protein expression in 35 cases of malignant and adjacent tissues. Finally, alterations in SPINK1 expression were evaluated to determine its biological phenotypes in OTSCC, including proliferation, apoptosis, invasion, and metastasis. Results: OTSCC tissues exhibit higher levels of SPINK1 compared to surrounding cancerous tissues. Notably, increased SPINK1 expression correlates with the pathological N stage and independently predicts overall survival among patients with OTSCC. Conclusion: Suppression of SPINK1 inhibited OTSCC cell proliferation, invasion, and motility while promoting apoptosis. These findings suggest that SPINK1 may serve as a prognostic biomarker as well as a potential therapeutic target for managing OTSCC. • High SPINK1 expression in oral tongue squamous cell carcinoma (OTSCC) is related with the pathological N stage. • SPINK1 serves as a crucial independent factor influencing the overall survival of patients with OTSCC. • SPINK1 promotes proliferation, invasion, and motility of OTSCC cells while suppressing apoptosis. [ABSTRACT FROM AUTHOR]

Legumes and cereals account for the vast proportion of people's daily intake of plant‐based foods. Meanwhile, a large number of antinutritional factors in legumes and cereals hinder the body absorption of nutrients and reduce the nutritional value of food. In this paper, the antinutritional effects, determination, and passivation methods of thermosensitive antinutritional factors such as trypsin inhibitors, urease, lipoxygenase, and lectin were reviewed to provide theoretical help to reduce antinutritional factors in food and improve the utilization rate of plant‐based food nutrition. Since trypsin inhibitors and lectin have been more extensively studied and reviewed previously, the review mainly focused on urease and lipoxygenase. This review summarized the information of thermosensitive antinutritional factors, trypsin inhibitors, urease, lipoxygenase, and lectin, in cereals and legumes. The antinutritional effects, and physical and chemical properties of trypsin inhibitors, urease, lipoxygenase, and lectin were introduced. At the same time, the research methods for the detection and inactivation of these four antinutritional factors were also summarized in the order of research conducted time. The rapid determination and inactivation of antinutrients will be the focus of attention for the food industry in the future to improve the nutritional value of food. Exploring what structural changes could passivation technologies bring to antinutritional factors will provide a theoretical basis for further understanding the mechanisms of antinutritional factor inactivation. Practical applications: Antinutritional factors in plant‐based foods hinder the absorption of nutrients and reduce the nutritional value of the food. Among them, thermosensitive antinutritional factors, such as trypsin inhibitors, urease, lipoxygenase, and lectins, have a high proportion among the antinutritional factors. In this paper, we investigate thermosensitive antinutritional factors from three perspectives: the antinutritional effect of thermosensitive antinutritional factors, determination, and passivation methods. The current passivation methods for thermosensitive antinutritional factors revolve around biological, physical, and chemical aspects, and their elimination mechanisms still need further research, especially at the protein structure level. Reducing the level of antinutritional factors in the future food industry while controlling the loss of other nutrients in food is a goal that needs to be balanced. This paper reviews the antinutritional effects of thermosensitive antinutritional factors and passivation methods, expecting to provide new research ideas to improve the nutrient utilization of food. [ABSTRACT FROM AUTHOR]

The objectives of this study were to evaluate the hypoglycemic effect of the trypsin inhibitor isolated from tamarind seeds (TTI) in an experimental model of T2DM and the in silico interaction between the conformational models of TTI 56/287 and the insulin receptor (IR). After inducing T2DM, 15 male Wistar rats were randomly allocated in three groups (n = 5): 1—T2DM group without treatment; 2—T2DM group treated with adequate diet; and 3—T2DM treated with TTI (25 mg/kg), for 10 days. Insulinemia and fasting glucose were analyzed, and the HOMA-IR and HOMA-β were calculated. The group of animals treated with TTI presented both lower fasting glucose concentrations (p = 0.0031) and lower HOMA-IR indexes (p = 0.0432), along with higher HOMA-β indexes (p = 0.0052), than the animals in the other groups. The in silico analyses showed that there was an interaction between TTIp 56/287 and IR with interaction potential energy (IPE) of −1591.54 kJ mol−1 (±234.90), being lower than that presented by insulin and IR: −894.98 kJ mol−1 (±32.16). In addition, the presence of amino acids, type of binding and place of interaction other than insulin were identified. This study revealed the hypoglycemic effect of a bioactive molecule of protein origin from Tamarind seeds in a preclinical model of T2DM. Furthermore, the in silico analysis allowed the prediction of its binding in the IR, raising a new perspective for explaining TTI's action on the glycemic response. [ABSTRACT FROM AUTHOR]

Cysteine-rich trypsin inhibitor-like domain (TIL)-harboring proteins are broadly distributed in nature but remain understudied in vector mosquitoes. Here we have explored the biology of a TIL domain-containing protein of the arbovirus vector Aedes aegypti, cysteine-rich venom protein 379 (CRVP379). CRVP379 was previously shown to be essential for dengue virus infection in Ae. aegypti mosquitoes. Gene expression analysis showed CRVP379 to be highly expressed in pupal stages, male testes, and female ovaries. CRVP379 expression is also increased in the ovaries at 48 h post-blood feeding. We used CRISPR-Cas9 genome editing to generate two mutant lines of CRVP379 with mutations inside or outside the TIL domain. Female mosquitoes from both mutant lines showed severe defects in their reproductive capability; mutant females also showed differences in their follicular cell morphology. However, the CRVP379 line with a mutation outside the TIL domain did not affect male reproductive performance, suggesting that some CRVP379 residues may have sexually dimorphic functions. In contrast to previous reports, we did not observe a noticeable difference in dengue virus infection between the wild-type and any of the mutant lines. The importance of CRVP379 in Ae. aegypti reproductive biology makes it an interesting candidate for the development of Ae. aegypti population control methods. [ABSTRACT FROM AUTHOR]

Introduction: Melon seeds, as an excellent source of protease inhibitors, may have a protective role against tumor progression and angiogenesis. However, their effects on angiogenesis and the mechanism of their action against cancer progression remain elusive. This study aimed to investigate the effect of bioactive compounds of melon seed on the expression of angiogenesis genes in BALB/c mice with breast cancer. Material and methods: Trypsin inhibitor (TI) was purified from the seed powder of Cucumis melo. Halfmaximal inhibitory concentration was determined for TI, extract of melon seed powder (EXT), and tamoxifen (TAM) by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. Also, breast tumor was induced by subcutaneous injection of MC4-L2 cells in BALB/c inbred mice breast tissue. After tumor growth, mice were treated with TI, EXT, and TAM to examine their effects on the tumor characteristics and expression of angiogenesis-related genes including MMP-2, MMP-9, and vascular endothelial growth factor (VEGF) using the reverse transcription polymerase chain reaction method. Results: Trypsin inhibitor, EXT, TAM, and adjuvant treatment of TI + TAM resulted a reduction in expression of MMP-2, MMP-9, and VEGF. All treatments improved the breast tumor characteristics and the necrosis. The real-time polymerase chain reaction method verified the positive effects of the treatments on the breast cancer cell line and tumors. Conclusions: The results indicated that treatments with TI purified from Cucumis melo seeds and also combination therapy of TI and TAM can be considered as an alternative therapy in breast cancer patients. Further studies are warranted. [ABSTRACT FROM AUTHOR]

Introduction: Obesity has emerged as one of the main public health problems. This condition triggers a series of hormonal and metabolic changes related to a low-grade chronic inflammatory condition. The trypsin inhibitor purified from tamarind (TTIp) seeds is a promising anti-inflammatory molecule, but its safety needs to be evaluated. This study aimed to evaluate TTIp bioactive dose effects on organs involved in its metabolism (liver and pancreas) and affected tissues (small intestine and perirenal adipose tissue) in an obesity model. Methods: Three groups of adult male Wistar rats were used (n = 5). Two of these groups had diet-induced obesity, and a third group was eutrophic. TTIp was administered by gavage in one of the obese groups for 10 days, while the remaining groups received a vehicle. The chromatographic profile and the inhibition assay corroded the purification of the inhibitor. Physical and behavioral changes, liver enzymes, and stereological and histopathological analyses of tissues were evaluated. Results: TTIp did not cause visible signs of toxicity, nor caused changes in liver enzymes, the liver, and pancreatic tissues. TTIp did not cause changes in the intestinal mucosa, showing improvement in the villi’s histopathological characteristics compared to the group of animals with obesity without treatment with TTIp (p = 0.004). The analysis of perirenal adipose tissue showed that the average sectional area of animals with obesity that received TTIp did not differ from the control. There was a difference between the high glycemic load diet group and the group treated with the inhibitor (351.8 ± 55.5) (p = 0.016). In addition, the group that received TTIp had no inflammatory infiltrates. Conclusion: Based on histological and stereological analysis, the use of TTIp is potentially safe and anti-inflammatory in the evaluated obesity model and can be investigated as a possible adjuvant in obesity therapy.

Fibers of potato protein and polysaccharides were obtained by needleless electrospinning. Mixtures of maltodextrin DE2 (dextrose equivalent) (0.8 g/mL), DE21 (0.1 g/mL), and different concentrations of potato protein (0.05, 0.1, 0.15, and 0.2 g/mL) were used for fiber production. Glycation was performed via the Maillard reaction after thermal treatment (0/6/12/24/48 h, 65 °C, 75% relative humidity). The effects of electrospinning and heating on trypsin inhibitor activity (IA) were studied. The results of the IA assay showed that electrospinning and glycation caused significant differences in IA among blends, heating times, and the interaction of blend and heating time (p < 0.001). The higher the protein content in the fibers, the higher the IA. The lowest IA was found in the mixture with the lowest protein content after 48 h. In other blends, the minimum IAs were found between 6 and 12 h of heating. The determination of the free lysine groups showed a nonsignificant decrease after heating. However, higher free lysine groups per protein (6.3–9.5 g/100 g) were found in unheated fibers than in the potato protein isolate (6.0 ± 0.5 g/100 g). The amide I and amide II regions, detected by the Fourier transform infrared spectra, showed only a slight shift after heating. [ABSTRACT FROM AUTHOR]

Anterior segment dysgeneses (ASDs) comprise a spectrum of developmental disorders affecting the anterior segment of the eye. Here, we describe three unrelated families affected by a previously unclassified form of ASD. Shared ocular manifestations include bilateral iris hypoplasia, ectopia lentis, corectopia, ectropion uveae, and cataracts. Whole-exome sequencing and targeted Sanger sequencing identified mutations in CPAMD8 (C3 and PZP-like alpha-2-macroglobulin domain-containing protein 8) as the cause of recessive ASD in all three families. A homozygous missense mutation in the evolutionarily conserved alpha-2-macroglobulin (A2M) domain of CPAMD8, c.4351T>C (p. Ser1451Pro), was identified in family 1. In family 2, compound heterozygous frameshift, c.2352_2353insC (p.Arg785Glnfs∗23), and splice-site, c.4549-1G>A, mutations were identified. Two affected siblings in the third family were compound heterozygous for splice-site mutations c.700+1G>T and c.4002+1G>A. CPAMD8 splice-site mutations caused aberrant pre-mRNA splicing in vivo or in vitro. Intriguingly, our phylogenetic analysis revealed rodent lineage-specific CPAMD8 deletion, precluding a developmental expression study in mice. We therefore investigated the spatiotemporal expression of CPAMD8 in the developing human eye. RT-PCR and in situ hybridization revealed CPAMD8 expression in the lens, iris, cornea, and retina early in development, including strong expression in the distal tips of the retinal neuroepithelium that form the iris and ciliary body, thus correlating CPAMD8 expression with the affected tissues. Our study delineates a unique form of recessive ASD and defines a role for CPAMD8, a protein of unknown function, in anterior segment development, implying another pathway for the pathogenicity of ASD.

The loss-of-function mutations of serine protease inhibitor, Kazal type 1 (SPINK1) gene are associated with human chronic pancreatitis, but the underlying mechanisms remain unknown. We previously reported that mice lacking Spink3, the murine homologue of human SPINK1, die perinatally due to massive pancreatic acinar cell death, precluding investigation of the effects of SPINK1 deficiency. To circumvent perinatal lethality, we have developed a novel method to integrate human SPINK1 gene on the X chromosome using Cre-loxP technology and thus generated transgenic mice termed "X-SPINK1". Consistent with the fact that one of the two X chromosomes is randomly inactivated, X-SPINK1 mice exhibit mosaic pattern of SPINK1 expression. Crossing of X-SPINK1 mice with Spink3+/- mice rescued perinatal lethality, but the resulting Spink3-/-;XXSPINK1 mice developed spontaneous pancreatitis characterized by chronic inflammation and fibrosis. The results show that mice lacking a gene essential for cell survival can be rescued by expressing this gene on the X chromosome. The Spink3-/-;XXSPINK1 mice, in which this method has been applied to partially restore SPINK1 function, present a novel genetic model of chronic pancreatitis.

ABSTRACT: The present study investigated the effect of varying trypsin inhibitor activity (TIA) in differently processed soybean expellers on apparent prececal amino acid (AA) digestibility in male broiler chickens. Two different raw soybean batches were treated using varying processing techniques and intensities. In this way, 45 expeller extracted soybean meal (ESBM) variants were created. The processed soybean variants were then merged into a basal diet (160 g/kg crude protein [CP]) at 2 inclusion levels (15%, 30%) resulting in 90 different diets plus one basal diet (0.4 mg/g–8.5 mg/g TIA). All diets contained 0.5% titanium dioxide. A total of 5,460-day-old male broilers (Ross 308) were allocated on d 14 to 546 pens (10 birds/pen) after a starter phase (CP 215 g/kg, 14 g/kg Lysine, 12.5 MJ ME/kg). The 91 experimental diets were fed ad libitum until d 22. Subsequently, birds were euthanized and digesta of the terminal ileum was collected for determination of AA digestibility. TIA depressed the prececal digestibility of every single AA significantly in a straight linear fashion (P < 0.001). Sulfur-containing AA expressed the strongest suppression by TIA with cystine showing the lowest apparent prececal digestibility measured (10.6% at 23.6 mg/g TIA in raw ESBM). The present data demonstrate that TIA severely depresses digestibility of essential and nonessential AA in a straight linear fashion. On the one hand, this questions the usefulness of defined upper limits of TIA in soy products whereas on the other hand, TIA must be considered when testing raw components for their feed protein value in vivo.

Auricularia auricula fermentation was performed to reduce anti-nutritional factors, improve nutritional components, and enhance biological activity of soybean. Results showed that the contents of raffinose, stachyose, and trypsin inhibitor were significantly decreased from initial 1.65 g L−1, 1.60 g L−1, and 284.67 μg g−1 to 0.14 g L−1, 0.35 g L−1, and 4.52 μg g−1 after 144 h of fermentation, respectively. Simultaneously, the contents of polysaccharide, total phenolics, and total flavonoids were increased, and melanin was secreted. The isoflavone glycosides were converted to their aglycones, and the contents of glyctin and genistin were decreased from initial 1107.99 μg g−1 and 2852.26 μg g−1 to non-detection after 72 h of fermentation, respectively. After 96 h of fermentation, the IC 50 values of samples against DPPH and ABTS radicals scavenging were decreased from 17.61 mg mL−1 and 3.43 mg mL−1 to 4.63 mg mL−1 and 0.89 mg mL−1, and those of samples inhibiting α-glucosidase and angiotensin I-converting enzyme were decreased from 53.89 mg mL−1 and 11.27 mg mL−1 to 18.24 mg mL−1 and 6.78 mg mL−1, respectively, indicating the significant increase in these bioactivities. These results suggested A. auricula fermentation can enhance the nutritional quality and biological activity of soybean, and the fermented soybean products have the potential to be processed into health foods/food additives. • Soybean was fermented by edible fungus Auricularia auricula. • The flatulence factor (stachyose and raffinose) and trypsin inhibitor were decreased. • The contents of polysaccharide, total phenolics and total flavonoids were increased. • The glycosylated isoflavones were converted into their aglycones. • The antioxidant and inhibitory activities against ACE and α-glucosidase were significantly enhanced. [ABSTRACT FROM AUTHOR]

Computer simulation of proteins in aqueous solution at the atomic level of resolution is still limited in time span and system size due to limited computing power available and thus employs a variety of time‐saving techniques that trade some accuracy against computational effort. An example of such a time‐saving technique is the application of constraints to particular degrees of freedom when integrating Newton's or Langevin's equations of motion in molecular dynamics (MD) or stochastic dynamics (SD) simulations, respectively. The application of bond‐length constraints is standard practice in protein simulations and allows for a lengthening of the time step by a factor of three. Applying recently proposed algorithms to constrain bond angles or dihedral angles, it is investigated, using the protein trypsin inhibitor as test molecule, whether bond angles and dihedral angles involving hydrogen atoms or even stiff proper (torsional) dihedral angles as well as improper ones (maintaining particular tetrahedral or planar geometries) may be constrained without generating too many artificial side effects. Constraining the relative positions of the hydrogen atoms in the protein allows for a lengthening of the time step by a factor of two. Additionally constraining the improper dihedral angles and the stiff proper (torsional) dihedral angles in the protein does not allow for an increase of the MD or SD time step. [ABSTRACT FROM AUTHOR]