Effect of Sintering Temperature on Microstructure Characteristics of Porous NiTi Alloy Fabricated via Elemental Powder Sintering.

To investigate the effect of the sintering temperature on the microstructure characteristics of porous NiTi alloys, two types of porous NiTi alloys with equal atomic ratios were fabricated via elemental powder sintering at 950 °C and 1000 °C. Afterwards, optical microscopy (OM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were collectively applied to investigate the pore characteristics and microstructure of the fabricated porous NiTi alloy. The results show that when the sintering temperature increases from 950 °C to 1000 °C, the average pore size increases from 36.00 µm to 181.65 µm, owing to the integration of these newly formed small pores into these pre-existing large-sized pores. The measured density increases from 2.556 g/cm3 to 3.030 g/cm3, while the porosity decreases from 60.4% to 51.8%. This is due to the occurrence of shrinkage after the sufficient diffusion of atoms. Furthermore, the characterization results confirm that a change in the sintering temperature would not change the phase types within a porous NiTi alloy; namely, the matrix consists primarily of B2 NiTi, with a significant amount of Ni4Ti3 precipitates and a small amount of Ni3Ti precipitates and Ti2Ni precipitates. However, as the sintering temperature increases, the number of Ni4Ti3 precipitates decreases significantly. The formation of a Ni4Ti3 phase in the present study is closely related to the enrichment of Ni content in the matrix owing to the diffusion rate difference between Ni atoms and Ti atoms and the absence of a transient liquid phase (TLP) during the sintering process owing to the relatively low sintering temperature (lower than the eutectic temperature). Moreover, the increasing sintering temperature speeds up the atom diffusion, which contributes to a reduction in the enrichment of Ni as well as the number of formed Ni4Ti3 precipitates.

Pediatric Crohn's disease diagnosis aid via genomic analysis and machine learning.

Introduction: Determination of pediatric Crohn's disease (CD) remains a major diagnostic challenge. However, the rapidly emerging field of artificial intelligence has demonstrated promise in developing diagnostic models for intractable diseases. Methods: We propose an artificial neural network model of 8 gene markers identified by 4 classification algorithms based on Gene Expression Omnibus database for diagnostic of pediatric CD. Results: The model achieved over 85% accuracy and area under ROC curve value in both training set and testing set for diagnosing pediatric CD. Additionally, immune infiltration analysis was performed to address why these markers can be integrated to develop a diagnostic model. Conclusion: This study supports further clinical facilitation of precise disease diagnosis by integrating genomics and machine learning algorithms in open-access database.

The Allocation Method for Personal Protective Equipment in the Emerging Infectious Disease Environment.

The COVID-19 pandemic gives humankind a lesson that the outbreak of an emerging infectious disease (EID) is sudden and uncertain. Accurately mastering its dynamics and putting forward an efficient and fair humanitarian logistics plan for personal protective equipment (PPE) remains difficult. This study examines the decision making for humanitarian logistics to answer the question that how to coordinate fairness and efficiency when facing supply-demand imbalance during humanitarian logistics planning in an EID environment. The main contributions include two aspects: (1) The victims' losses in terms of fairness and efficiency in receiving PPE are jointly explored by evaluating their bearing capacity evolution, and then a novel loss function is built to search for a reasonable compromise between fairness and efficiency. (2) A multi-objective optimization model is built, which is solved using the combined use of goal programming approach and improved branch and bound method. Finally, the practicability of the proposed model is tested by an EID case study. The potential advantages of the proposed model and improved approach are discussed.

ß-Boswellic Acid Suppresses Breast Precancerous Lesions via GLUT1 Targeting-Mediated Glycolysis Inhibition and AMPK Pathway Activation.

Breast carcinoma is a multistep progressive disease. Precancerous prevention seems to be crucial. ß-Boswellic acid (ß-BA), the main component of the folk medicine Boswellia serrata (B. serrata), has been reported to be effective in various diseases including tumors. In this work, we demonstrated that ß-BA could inhibit breast precancerous lesions in rat disease models. Consistently, ß-BA could suppress proliferation and induce apoptosis on MCF-10AT without significantly influencing MCF-10A. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that ß-BA may interfere with the metabolic pathway. Metabolism-related assays showed that ß-BA suppressed glycolysis and reduced ATP production, which then activated the AMPK pathway and inhibited the mTOR pathway to limit MCF-10AT proliferation. Further molecular docking analysis suggested that GLUT1 might be the target of ß-BA. Forced expression of GLUT1 could rescue the glycolysis suppression and survival limitation induced by ß-BA on MCF-10AT. Taken together, ß-BA could relieve precancerous lesions in vivo and in vitro through GLUT1 targeting-induced glycolysis suppression and AMPK/mTOR pathway alterations. Here, we offered a molecular basis for ß-BA to be developed as a promising drug candidate for the prevention of breast precancerous lesions.

[~1H-NMR-based metabonomics analysis of the acute hepatotoxicity mechanism of Artemisia argyi essential oil].

This study based on~1H-NMR urine metabolomics technique combined with biochemical indicators to focus on studying the acute hepatotoxicity mechanism of Artemisia argyi essential oil( AAEO). In order to further explore the acute hepatotoxicity mechanism of AAEO,the researchers collected the urine nuclear magnetic data of rats in different periods of high and low doses of olive oil and AAEO group. Using the principal component analysis( PCA) and orthogonal partial least squares-discrimination analysis( OPLSDA) to analyze the endogenous small molecule metabolites in rat urine to study the effects of AAEO on the metabolic process of normal rats. The results showed there was a significant difference between the olive oil group and the AAEO group,the PCA scores chart demonstrated that there was no obvious separation tendency in the urine of olive oil group rats 0-6,6-12,12-24 h,and the metabolic components were distributed in aggregation pattern. The urinary metabolic trajectory of the rats in the AAEO group was conspicuously separated at 0-6,6-12,12-24 h. The experiments proved that the analysis of metabolites by~1H-NMR found that AAEO caused metabolic disorders in rats and produced acute hepatotoxicity. After metabolite differential comparison,it was speculated that the mechanism of acute hepatotoxicity may be involved in the tricarboxylic acid cycle and energy metabolism,while the citrate and oleanolic acid would be the potential biomarkers. This study discussed that the acute hepatotoxicity mechanism of AAEO was used to provide the experimental data for the clinical prescription of Artemisia argyi.

Effectiveness and safety of acupuncture combined with Madopar for Parkinson's disease: a systematic review with meta-analysis.

OBJECTIVE: To evaluate the effectiveness and safety of acupuncture combined with Madopar for the treatment of Parkinson's disease (PD), compared to the use of Madopar alone. METHODS: A systematic search was carried out for randomised controlled trials (RCTs) of acupuncture and Madopar for the treatment of PD published between April 1995 and April 2015. The primary outcome was total effectiveness rate and secondary outcomes included Unified Parkinson's Disease Rating Scale (UPDRS) scores. Data were pooled and analysed with RevMan 5.3. Results were expressed as relative ratio (RR) with 95% confidence interval (CIs). RESULTS: Finally, 11 RCTs with 831 subjects were included. Meta-analyses showed that acupuncture combined with Madopar for the treatment of PD can significantly improve the clinical effectiveness compared with Madopar alone (RR=1.28, 95% CI 1.18 to 1.38, P<0.001). It was also found that acupuncture combined with Madopar significantly improved the UPDRS II (SMD=-1.00, 95% CI -1.71 to -0.29, P=0.006) and UPDRS I-IV total summed scores (SMD=-1.15, 95% CI -1.63 to -0.67, P<0.001) but not UPDRS I (SMD=-0.37, 95% CI -0.77 to 0.02, P=0.06), UPDRS III (SMD=-0.93, 95% CI -2.28 to 0.41, P=0.17) or UPDRS IV (SMD=-0.78, 95% CI -2.24 to 0.68, P=0.30) scores. Accordingly, acupuncture combined with Madopar appeared to have a positive effect on activities of daily life and the general condition of patients with PD, but was not better than Madopar alone for the treatment of mental activity, behaviour, mood and motor disability. In the safety evaluation, it was found that acupuncture combined with Madopar was associated with significantly fewer adverse effects including gastrointestinal reactions (RR=0.38, 95% CI 0.23 to 0.65, P<0.001), on-off phenomena (RR=0.27, 95% CI 0.11 to 0.66, P=0.004) and mental disorders (RR=0.24, 95% CI 0.06 to 0.92, P=0.04) but did not significantly reduce dyskinesia (RR=0.64, 95% CI 0.35 to 1.16, P=0.14). CONCLUSION: Acupuncture combined with Madopar appears, to some extent, to improve clinical effectiveness and safety in the treatment of PD, compared with Madopar alone. This conclusion must be considered cautiously, given the quality of most of the studies included was low. Therefore, more high-quality, multicentre, prospective, RCTs with large sample sizes are needed to further clarify the effect of acupuncture combined with Madopar for PD.

Molecular network-based analysis of the mechanism of liver injury induced by volatile oils from Artemisiae argyi folium.

BACKGROUND: Volatile oils from Artemisiae argyi folium (VOAAF) is reported with hepatotoxicity, but the underlying mechanism is still unclear. METHODS: In the present study this molecular mechanism was explored with the Ingenuity Pathway Analysis (IPA). The chemical components of the VOAAF were searched in the database, and their target proteins were all identified in the PubChem, while drug-induced liver injury (DILI) genes were searched in the PubMed gene databases. The molecular network of protein targets for VOAAF and DILI genes was built with the IPA. The canonical pathways between the 2 networks were compared to decipher the molecular mechanisms of the liver injury induced by VOAAF. RESULTS: There were 159 target proteins for VOAAF and 338 genes related to DILI identified, which were further analyzed in the IPA. The canonical pathway comparison showed that VOAAF and DILI both worked on aryl hydrocarbon receptor (AHR), lipopolysaccharide (LPS)/interleukin 1 (IL-1) mediated inhibition of retinoid X receptor (RXR) function, pregnane X receptor (PXR)/RXR activation, xenobiotic metabolism, peroxisome proliferator-activated receptor (PPAR), hepatic cholestasis, farnesoid X receptor (FXR)/RXR activation, and glucocorticoid receptor. CONCLUSION: VOAAF-induced liver injury may be involved in many pathways in which the AHR signaling and LPS/IL-1 mediated inhibition of RXR function pathways could be the most vital.

To Set Up a Logistic Regression Prediction Model for Hepatotoxicity of Chinese Herbal Medicines Based on Traditional Chinese Medicine Theory.

Aims. To establish a logistic regression (LR) prediction model for hepatotoxicity of Chinese herbal medicines (HMs) based on traditional Chinese medicine (TCM) theory and to provide a statistical basis for predicting hepatotoxicity of HMs. Methods. The correlations of hepatotoxic and nonhepatotoxic Chinese HMs with four properties, five flavors, and channel tropism were analyzed with chi-square test for two-way unordered categorical data. LR prediction model was established and the accuracy of the prediction by this model was evaluated. Results. The hepatotoxic and nonhepatotoxic Chinese HMs were related with four properties (p < 0.05), and the coefficient was 0.178 (p < 0.05); also they were related with five flavors (p < 0.05), and the coefficient was 0.145 (p < 0.05); they were not related with channel tropism (p > 0.05). There were totally 12 variables from four properties and five flavors for the LR. Four variables, warm and neutral of the four properties and pungent and salty of five flavors, were selected to establish the LR prediction model, with the cutoff value being 0.204. Conclusions. Warm and neutral of the four properties and pungent and salty of five flavors were the variables to affect the hepatotoxicity. Based on such results, the established LR prediction model had some predictive power for hepatotoxicity of Chinese HMs.

SeGSTo, a novel glutathione S-transferase from the beet armyworm (Spodoptera exigua), involved in detoxification and oxidative stress.

Members of the glutathione S-transferase superfamily can protect organisms against oxidative stress. In this study, we characterized an omega glutathione S-transferase from Spodoptera exigua (SeGSTo). The SeGSTo gene contains an open reading frame (ORF) of 744 nucleotides encoding a 248-amino acid polypeptide. The predicted molecular mass and isoelectric point of SeGSTo are 29007 Da and 7.74, respectively. Multiple amino acid sequence alignment analysis shows that the SeGSTo sequence is closely related to the class 4 GSTo of Bombyx mori BmGSTo4 (77 % protein sequence similarity). Homologous modeling and molecular docking reveal that Cys35 may play an essential role in the catalytic process. Additionally, the phylogenetic tree indicates that SeGSTo belongs to the omega group of the GST superfamily. During S. exigua development, SeGSTo is expressed in the midgut of the fifth instar larval stage, but not in the epidermis or fat body. Identification of recombinant SeGSTo via SDS-PAGE and Western blot shows that its molecular mass is 30 kDa. The recombinant SeGSTo was able to protect super-coiled DNA from damage in a metal-catalyzed oxidation (MCO) system and catalyze the 1-chloro-2,4-dinitrobenzene (CDNB), but not 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrophenethyl bromide (4-NPB), or 4-nitrobenzyl chloride (4-NBC). The optimal reaction pH and temperature were 8 and 50 °C, respectively, in the catalysis of CDNB by recombinant SeGSTo. The mRNA expression of SeGSTo was up-regulated by various oxidative stresses, such as CdCl2, CuSO4, and isoprocarb, and the catalytic activity of recombinant SeGSTo was noticeably inhibited by heavy metals (Cu(2+) and Cd(2+)) and various pesticides. Taken together, these results indicate that SeGSTo plays an important role in the antioxidation and detoxification of pesticides.

Peroxiredoxin 5 from common cutworm (Spodoptera litura) acts as a potent antioxidant enzyme.

In this study, we describe the cloning and characterization of a Prx from the common cutworm Spodoptera litura (SlPrx5). The SlPrx5 cDNA contains an open reading frame of 477 bp encoding a predicted protein of 159 amino acid residues, 16.902 kDa, and an isoelectric point of 7.68. Furthermore, the deduced amino acid sequence of the SlPrx5 cDNA showed 86% identity to Papilio xuthus Prx5, 72% to Aedes aegypti Prx5, and 64-67% to other insect Prxs. A phylogenetic analysis further revealed that the deduced amino acid sequence of SlPrx5 groups within the atypical 2-Cys Prx cluster. Recombinant SlPrx5 (20 kDa) purified from baculovirus-infected insect cells was found to reduce H2O2 in the presence of electrons donated by dithiothreitol and protect super-coiled DNA from damage by metal-catalyzed oxidation in vitro. During S. litura development, SlPrx5 is constitutively expressed in the epidermis, fat body, and midgut, with the highest expression occurring in the sixth-instar larval stage in the fat body and midgut. Additionally, SlPrx5 mRNA expression was up-regulated after injection with H2O2, cumene hydroperoxide, indoxacarb, and metaflumizone. A disc diffusion assay indicated that recombinant SlPrx5 can play a functional role in protecting cells from oxidative stress in vivo. These results provide insight into the role of SlPrx5 during development and the oxidative stress response of S. litura.

Facile synthesis and enhanced visible-light photocatalytic activity of Ag2S nanocrystal-sensitized Ag8W4O16 nanorods.

Narrow band-gap (NBG) Ag2S nanocrystals (NCs) attaching on the surface of wide band-gap (WBG) Ag8W4O16 nanorods were prepared by employing a facile in situ anion exchange method with the reaction between S(2)(-) and WO4(2-), and the photocatalytic activity was evaluated by the photocatalytic decolorization of methyl orange solution under visible-light irradiation. It was found that in situ anion exchange could uniformly deposit Ag2S NCs on the surface of Ag8W4O16 nanorods, controllably adjust the size, distribution and amount of Ag2S NCs, and solidly connect Ag2S NCs to the Ag8W4O16 nanorods via the replacement of S(2)(-) in the solution with lattice WO4(2-) on the Ag8W4O16 surface. The photocatalytic results indicated that the as-prepared Ag2S/Ag8W4O16 composite photocatalysts exhibited obviously higher activity compared with the pure Ag8W4O16 and N-TiO2 photocatalysts. On the basis of band structures of Ag2S and Ag8W4O16 semiconductors and the quantum size effect of Ag2S NCs, a possible photocatalytic mechanism about the Ag2S nanocrystal-sensitized Ag8W4O16 nanorods was proposed to account for the effective visible-light photocatalytic activities. This present work may provide some insight into the design of novel and high-efficiency NBG semiconductor NCs coupled with WBG semiconductor composite photocatalysts.

Selection and evaluation of potential reference genes for gene expression analysis in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae) using reverse-transcription quantitative PCR.

The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.

Selection and evaluation of reference genes for expression analysis using qRT-PCR in the beet armyworm Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae).

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. The most common method for analyzing qRT-PCR data is to normalize mRNA levels of target genes to internal reference genes. Evaluating and selecting stable reference genes on a case-by-case basis is critical. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for normalization of mRNA expression in qRT-PCR analysis of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). For this purpose, three software tools (geNorm, NormFinder and BestKeeper) were used to investigate 10 candidate reference genes in nine developmental stages and five different tissues (epidermis, head, midgut, fat body and hemolymph) in three larval physiological stages (molting, feeding and wandering stages) of, S. exigua. With the exception of 18S ribosomal RNA (18S), all other candidate genes evaluated, ß-actin1(ACT1), ß-actin2 (ACT2), elongation factor1(EF1), elongation factor 2 (EF2), Glyceralde hyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (SOD), α-tubulin (TUB),proved to be acceptable reference genes. However, their suitability partly differed between physiological stages and different tissues. L10, EF2 and L17A ranked highest in all tissue sample sets. SOD, ACT2, GAPDH, EF1 and ACT1 were stably expressed in all developmental stage sample sets; ACT2, ACT1 and L10 for larvae sample sets; GAPDH, ACT1 and ACT2 for pupae and adults; SOD and L17A for males; and EF2 and SOD for females. The expression stability of genes varied in different conditions. The findings provided here demonstrated, with a few exceptions, the suitability of most of the 10 reference genes tested in tissues and life developmental stages. Overall, this study emphasizes the importance of validating reference genes for qRT-PCR analysis in S. exigua.

Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae).

Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ΔCt method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms.

Developmental regulation and antifungal activity of a growth-blocking peptide from the beet armyworm Spodoptera exigua.

Insect cytokine growth-blocking peptides (GBPs) are involved in growth regulation and the innate immune response. However, the microbial binding and antimicrobial activities of GBPs remain unclear. Here, we investigate the developmental role and antifungal activity of a GBP from the beet armyworm Spodoptera exigua (SeGBP). Sequence analysis predicted that mature SeGBP consists of 24 amino acid residues, including 2 cysteine residues. During S. exigua development, SeGBP is constitutively expressed in the fat body during the larval and adult stages but not in pupae. SeGBP expression is up-regulated by 20-hydroxyecdysone and down-regulated by juvenile hormone analog. Recombinant SeGBP purified from baculovirus-infected insect cells retards the growth of S. exigua larvae. Additionally, SeGBP expression is acutely induced in the fat body after injection with Escherichia coli, Bacillus thuringiensis, or Beauveria bassiana. Recombinant SeGBP can bind to B. bassiana but not to E. coli or B. thuringiensis. Consistent with these findings, SeGBP shows antifungal activity against B. bassiana. Therefore, these results provide insight into the role of SeGBP during the innate immune response following microbial infection, and furthermore, they suggest a novel function for SeGBP as a direct antifungal agent against entomopathogenic fungi, such as B. bassiana.

A spider (Araneus ventricosus) chymotrypsin inhibitor that acts as an elastase inhibitor and a microbial serine protease inhibitor.

Spider-derived Kunitz-type serine protease inhibitors have been shown to exhibit plasmin and elastase inhibition activity and potassium channel blocking activity, but thus far, no additional roles for spider-derived chymotrypsin inhibitors have been elucidated. In this study, a spider (Araneus ventricosus) chymotrypsin inhibitor (AvCI) that acts as an elastase inhibitor and a microbial serine protease inhibitor was identified. AvCI is a 70-amino acid mature peptide that displays eight conserved cysteine residues and a P1 lysine residue. Recombinant AvCI expressed in baculovirus-infected insect cells demonstrated inhibitory activity against chymotrypsin (Ki 49.85 nM), but not trypsin, which defines a role for AvCI as a spider-derived chymotrypsin inhibitor. AvCI also exhibited inhibitory activity against microbial serine proteases such as subtilisin A (Ki 20.51 nM) and proteinase K (Ki 65.42 nM). Furthermore, AvCI exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or plasmin; however, AvCI strongly inhibited human neutrophil elastase (Ki 8.74 nM) and porcine pancreatic elastase (Ki 11.32 nM), indicating that AvCI acts as an anti-elastolytic factor. These findings constitute molecular evidence that AvCI acts as an inhibitor against chymotrypsin, microbial serine proteases, and elastases. This paper provides a novel view of the functions of a spider-derived chymotrypsin inhibitor.