Trypsin-encoding gene function of efficient star polycation nanomaterial-mediated dsRNA feeding delivery system of Grapholita molesta.

BACKGROUND: Grapholita molesta is an important and harmful fruit pest worldwide, with widespread feeding hosts. Trypsin, an indispensable hydrolytic digestive protease in the insect gut, is crucial in digestion, growth and development. We analyzed the characteristics of the trypsin-encoding genes, screened for the optimal dose of RNAi mediated by nanocarriers, and investigated various indices of larval growth and development of G. molesta. RESULTS: Gut content (GC) and RNase A degraded double-stranded RNA (dsRNA), with a faster degradation rate at higher concentrations. Star polycation (SPc) nanomaterials protected dsGFP from degradation by anion-cation binding and did not migrate through agarose gel. The key conserved motifs of the trypsin-encoding genes were similar, exhibiting high homology with those in other lepidopteran insects. An interference efficiency of ≈70% was achieved with SPc nanomaterial-mediated RNA interference with 0.05 µg dsRNA. The efficiency of continuous interference was stable. Trypsin activity, body weight of 8-day-old larvae, pupal weight and emergence rate were significantly reduced, and the larval stage was significantly prolonged. CONCLUSION: The investigated trypsin gene is a key target gene in the growth and development of G. molesta. We investigated the efficiency and convenience of feeding SPc nanomaterials in a functional study of insects. Our results provide valuable data for the development of efficient trypsin-targeting pesticides. © 2024 Society of Chemical Industry.

A comprehensive physiological and -Omic analysis of trypsin-mediated protection of green pepper fruits from chilling injury.

Chilling injury (CI) in green pepper fruits during low-temperature storage causes a significant decline in quality. The present study utilized physiological, transcriptomic, and metabolomic analyses to idneitfy the mechanisms by which trypsin mitigates CI in green peppers stored at 4 °C for 8 days, followed by 3 days of shelf life. Results indicated that the trypsin treatment significantly reduced electrolyte leakage and the CI index in peppers, effectively extending their shelf life and preserving postharvest quality. After 4 days of storage, comparative -omic analyses identified 2514 differentially expressed genes (DEGs) and 397 differentially abundant metabolites (DAMs) between trypsin-treated and control peppers. The trypsin treatment induced changes in sugar metabolism, modulating the expression of HK, SUS, INV, and GLGC, which affected the abundance of metabolites such as CDP-glucose and α-D-p-glucose. Trypsin also enhanced carotenoid metabolism, altering the abundance of rhodopinal glucoside, 1'-hydroxyl-γ-carotene glucoside, and farnesyl 1-PP, and influencing the expression of PDS, CRTH, CRTB, and LUT5. Notably, the trypsin treatment activated the mitogen-activated protein kinase (MAPK) pathway that plays an integral role in the signal transduction of abiotic stress. Differential expression of FLS2, ELF18, PTO, PR1, PTI5, WPKY, MEKK1, and MPK6 genes in the MAPK pathway was observed, which was correlated with CI mitigation in green peppers during cold storage. In conclusion, trypsin is an effective treatment for reducing CI in green peppers during cold storage. The present study provides valuable insights into its physiological and molecular impact on green pepper fruit.

Abamectin exposure causes chronic toxicity and trypsin/chymotrypsin damages in Chironomus kiiensis Tokunaga (Diptera: Chironomidae).

Abamectin has been extensively used in paddy fields to control insect pests. However, little information is available regarding its effects on non-target insects. In this study, we performed acute (3rd instar larvae) and chronic toxicity (newly hatched larvae <24 h) to determine the toxicity effects of abamectin on Chironomus kiiensis. The median lethal concentration (LC50) values of 24 h and 10 d were 0.57 mg/L and 68.12 µg/L, respectively. The chronic exposure significantly prolonged the larvae growth duration and inhibited pupation and emergence. The transcriptome and biochemical parameters were measured using 3rd instar larvae exposed to acute LC10 and LC25 for 24 h. Transcriptome data indicated that five trypsin and four chymotrypsin genes were downregulated, and RT-qPCR verified a significant expression decrease in trypsin3 and chymotrypsin1 genes. Meanwhile, abamectin could significantly inhibit the activities of the serine proteases trypsin and chymotrypsin. RNA interference showed that silencing trypsin3 and chymotrypsin1 genes led to higher mortality of C. kiiensis to abamectin. In conclusion, these findings indicated that trypsin and chymotrypsin are involved in the abamectin toxicity against C. kiiensis, which provides new insights into the mechanism of abamectin-induced ecotoxicity to chironomids.

Bowman-Birk Inhibitor Mutants of Soybean Generated by CRISPR-Cas9 Reveal Drastic Reductions in Trypsin and Chymotrypsin Inhibitor Activities.

Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman-Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts the quality of seed proteins. As an alternative approach, we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild-type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays, and qRT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss-of-function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed-specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities.

Heterozygous Spink1 Deficiency Promotes Trypsin-dependent Chronic Pancreatitis in Mice.

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.

Characteristics of Trypsin genes and their roles in insecticide resistance based on omics and functional analyses in the malaria vector Anopheles sinensis.

Trypsin is one of the most diverse and widely studied protease hydrolases. However, the diversity and characteristics of the Trypsin superfamily of genes have not been well understood, and their role in insecticide resistance is yet to be investigated. In this study, a total of 342 Trypsin genes were identified and classified into seven families based on homology, characteristic domains and phylogenetics in Anopheles sinensis, and the LY-Domain and CLECT-Domain families are specific to the species. Four Trypsin genes, (Astry2b, Astry43a, Astry90, Astry113c) were identified to be associated with pyrethroid resistance based on transcriptome analyses of three field resistant populations and qRT-PCR validation, and the knock-down of these genes significantly decrease the pyrethroid resistance of Anopheles sinensis based on RNAi. The activity of Astry43a can be reduced by five selected insecticides (indoxacarb, DDT, temephos, imidacloprid and deltamethrin); and however, the Astry43a could not directly metabolize these five insecticides, like the trypsin NYD-Tr did in earlier reports. This study provides the overall information frame of Trypsin genes, and proposes the role of Trypsin genes to insecticide resistance. Further researches are necessary to investigate the metabolism function of these trypsins to insecticides.

Characterization and Hydrolysis Mechanism Analysis of a Cold-Adapted Trypsin-Like Protease from Antarctic Krill.

Cold-adapted proteases are capable of efficient protein hydrolysis at reduced temperatures, which offer significant potential applications in the area of low temperature food processing. In this paper, we attempted to characterize cold-adapted proteases from Antarctic krill. Antarctic krill possesses an extremely active autolytic enzyme system in their bodies, and the production of peptides and free amino acids accompanies the rapid breakdown of muscle proteins following the death. The crucial role of trypsin in this process is recognized. A cold-adapted trypsin named OUC-Pp-20 from Antarctic krill genome was cloned and expressed in Pichia pastoris. Recombinant trypsin is a monomeric protein of 26.8 ± 1.0 kDa with optimum reaction temperature at 25 °C. In addition, the catalytic specificity of OUC-Pp-20 was assessed by identifying its hydrolysis sites through LC-MS/MS. OUC-Pp-20 appeared to prefer Gln and Asn at the P1 position, which is an amino acid with an amide group in its side chain. Hydrolysis reactions on milk and shrimp meat revealed that it can effectively degrade allergenic components in milk and arginine kinase in shrimp meat. These findings update the current knowledge of cold-adapted trypsin and demonstrate the potential application of OUC-Pp-20 in low temperature food processing.

Pathogen-associated molecular patterns (PAMPs) derived from Leishmania and bacteria increase gene expression of antimicrobial peptides and gut surface proteins in sand flies.

The interaction between pathogens and vectors' physiology can impact parasite transmission. Studying this interaction at the molecular level can help in developing control strategies. We study leishmaniases, diseases caused by Leishmania parasites transmitted by sand fly vectors, posing a significant global public health concern. Lipophosphoglycan (LPG), the major surface glycoconjugate of Leishmania, has been described to have several roles throughout the parasite's life cycle, both in the insect and vertebrate hosts. In addition, the sand fly midgut possesses a rich microbiota expressing lipopolysaccharides (LPS). However, the effect of LPG and LPS on the gene expression of sand fly midgut proteins or immunity effectors has not yet been documented. We experimentally fed Lutzomyia longipalpis and Phlebotomus papatasi sand flies with blood containing purified LPG from Leishmania infantum, Leishmania major, or LPS from Escherichia coli. The effect on the expression of genes encoding gut proteins galectin and mucin, digestive enzymes trypsin and chymotrypsin, and antimicrobial peptides (AMPs) attacin and defensins was assessed by quantitative PCR (qPCR). The gene expression of a mucin-like protein in L. longipalpis was increased by L. infantum LPG and E. coli LPS. The gene expression of a galectin was increased in L. longipalpis by L. major LPG, and in P. papatasi by E. coli LPS. Nevertheless, the gene expression of trypsins and chymotrypsins did not significantly change. On the other hand, both L. infantum and L. major LPG significantly enhanced expression of the AMP attacin in both sand fly species and defensin in L. longipalpis. In addition, E. coli LPS increased the expression of attacin and defensin in L. longipalpis. Our study showed that Leishmania LPG and E. coli LPS differentially modulate the expression of sand fly genes involved in gut maintenance and defence. This suggests that the glycoconjugates from microbiota or Leishmania may increase the vector's immune response and the gene expression of a gut coating protein in a permissive vector.

Genetic Factors Associated With Adverse Pregnancy Outcomes in Chronic Pancreatitis.

INTRODUCTION: The effects of genetic factors on pregnancy outcomes in chronic pancreatitis (CP) patients remain unclear. We evaluated the impacts of clinical features and mutations in main CP-susceptibility genes ( SPINK1 , PRSS1 , CTRC , and CFTR ) on pregnancy outcomes in Chinese CP patients. METHODS: This was a prospective cohort study with 14-year follow-up. The sample comprised female CP patients with documented pregnancy and known genetic backgrounds. Adverse pregnancy outcomes were compared between patients with and without gene mutations. Univariate and multivariate analyses were performed to determine the impact factors for adverse pregnancy outcomes. RESULTS: Totally, 160 female CP patients with a pregnancy history were enrolled; 59.4% of patients carried pathogenic mutations in CP-susceptibility genes. Adverse pregnancy outcomes occurred in 38 patients (23.8%); the prevalence of adverse outcomes was significantly higher in those harboring gene mutations than those without (30.5% vs 13.8%, P = 0.015). Notably, the rates of preterm delivery (12.6% vs 3.1%, P = 0.036) and abortion (17.9% vs 4.6%, P = 0.013) were remarkably higher in patients with gene mutations (especially SPINK1 mutations) than those without. In multivariate analyses, both CP-susceptibility gene mutations (odds ratio, 2.52; P = 0.033) and SPINK1 mutations (odds ratio, 2.60; P = 0.037) significantly increased the risk of adverse pregnancy outcomes. Acute pain attack during pregnancy was another risk factor for adverse pregnancy outcomes. DISCUSSION: Pathogenic mutations in CP-susceptibility genes, especially SPINK1 , were independently related to adverse pregnancy outcomes in CP patients. Significant attention should be paid to pregnant females harboring CP-susceptibility gene mutations (ClinicalTrials.gov: NCT06055595).

MiR-2b-3p Downregulated PxTrypsin-9 Expression in the Larval Midgut to Decrease Cry1Ac Susceptibility of the Diamondback Moth, Plutella xylostella (L.).

Crystal (Cry) toxins, produced by Bacillus thuringiensis, are widely used as effective biological pesticides in agricultural production. However, insects always quickly evolve adaptations against Cry toxins within a few generations. In this study, we focused on the Cry1Ac protoxin activated by protease. Our results identified PxTrypsin-9 as a trypsin gene that plays a key role in Cry1Ac virulence in Plutella xylostella larvae. In addition, P. xylostella miR-2b-3p, a member of the micoRNA-2 (miR-2) family, was significantly upregulated by Cry1Ac protoxin and targeted to PxTrypsin-9 downregulated its expression. The mRNA level of PxTrypsin-9, regulated by miR-2b-3p, revealed an increased tolerance of P. xylostella larvae to Cry1Ac at the post-transcriptional level. Considering that miR-2b and trypsin genes are widely distributed in various pest species, our study provides the basis for further investigation of the roles of miRNAs in the regulation of the resistance to Cry1Ac and other insecticides.

Trypsin-induced aggregation of transthyretin Valine 30 variants associated with hereditary amyloidosis.

Amyloid fibrils of transthyretin (TTR) consist of full-length TTR and C-terminal fragments starting near residue 50. However, the molecular mechanism underlying the production of the C-terminal fragment remains unclear. Here, we investigated trypsin-induced aggregation and urea-induced unfolding of TTR variants associated with hereditary amyloidosis. Trypsin strongly induced aggregation of variants V30G and V30A, in each of which Val30 in the hydrophobic core of the monomer was mutated to less-bulky amino acids. Variants V30L and V30M, in each of which Val30 was mutated to bulky amino acids, also exhibited trypsin-induced aggregation. On the other hand, pathogenic variant I68L as well as the nonpathogenic V30I did not exhibit trypsin-induced aggregation. The V30G variant was extremely unstable compared with the other variants. The V30G mutation caused the formation of a cavity and the rearrangement of Leu55 in the hydrophobic core of the monomer. These results suggest that highly destabilized transthyretin variants are more susceptible to trypsin digestion.

Genetic Investigations in Turkish Idiopathic Pancreatitis Patients Show Unique Characteristics.

BACKGROUND/AIMS: Pancreatitis is one of the leading causes of digestive system-related hospital admissions, and it has a genetic background in a considerable portion of the patients. In this study, we aimed to investigate the genetic risk factors of idiopathic pancreatitis in Turkish patients and the contribution of copy number variations to the pathogenesis. MATERIALS AND METHODS: Idiopathic pancreatitis is defined as failure to detect risk factors despite comprehensive clinical assessments. Next-generation sequencing and multiple ligand-dependent probe amplification of PRSS1, SPINK1, CTRC, and CFTR were performed. For further genotype-phenotype correlations, patients were also questioned for the age of onset, family history, and pancreatic divisum. RESULTS: A total of 68 idiopathic pancreatitis cases were enrolled. Variants with potential clinical significance of PRSS1 were identified in 13.4%, SPINK1 in 6.3%, CTRC in 4.7%, and CFTR in 26.5% of the patients. No copy number variants were seen in any of these genes. At least 7.4% of the participants had complex genetic etiology involving 2 genes. CONCLUSIONS: At least 42.6% of the participants had a potential genetic risk factor. Five novel genetic variants were identified, and distinctive genetic risk factors of Turkish population were shown. The results showed that genetic etiology was frequent in pancreatitis and it was even more prominent in patients with early-onset disease. Considering that genetic risk factors may be informative for decisionmaking in the treatment options in addition to providing extensive prognostic value and familial genetic consultation; clinicians need to be more eager to offer genetic tests to pancreatitis patients.

Classification of PRSS1 variants responsible for chronic pancreatitis: An expert perspective from the Franco-Chinese GREPAN study group.

BACKGROUND: PRSS1 was the first reported chronic pancreatitis (CP) gene. The existence of both gain-of-function (GoF) and gain-of-proteotoxicity (GoP) pathological PRSS1 variants, together with the fact that PRSS1 variants have been identified in CP subtypes spanning the range from monogenic to multifactorial, has made the classification of PRSS1 variants very challenging. METHODS: All currently reported PRSS1 variants (derived primarily from two databases) were manually reviewed with respect to their clinical genetics, functional analysis and population allele frequency. They were classified by variant type and pathological mechanism within the framework of our recently proposed ACMG/AMP guidelines-based seven-category system. RESULTS: The total number of distinct germline PRSS1 variants included for analysis was 100, comprising 3 copy number variants (CNVs), 12 5' and 3' variants, 19 intronic variants, 5 nonsense variants, 1 frameshift deletion variant, 6 synonymous variants, 1 in-frame duplication, 3 gene conversions and 50 missense variants. Based upon a combination of clinical genetic and functional analysis, population data and in silico analysis, we classified 26 variants (all 3 CNVs, the in-frame duplication, all 3 gene conversions and 19 missense) as "pathogenic", 3 variants (missense) as "likely pathogenic", 5 variants (four missense and one promoter) as "predisposing", 13 variants (all missense) as "unknown significance", 2 variants (missense) as "likely benign", and all remaining 51 variants as "benign". CONCLUSIONS: We describe an expert classification of the 100 PRSS1 variants reported to date. The results have immediate implications for reclassifying many ClinVar-registered PRSS1 variants as well as providing optimal guidelines/standards for reporting PRSS1 variants.

Clinical and Genetic Description of Hereditary Chronic Pancreatitis in Pakistani Children.

BACKGROUND/AIMS: The purpose of this study was to identify the spectrum and frequency of pathogenic variants as well as the clinical and genetic insight of hereditary chronic pancreatitis in Pakistani children. MATERIALS AND METHODS: The deoxyribonucleic acid of affected probands of 44 unrelated Pakistani families, having hereditary chronic pancreatitis-affected children, were subjected to massive parallel sequencing for candidate reported genes (SPINK1, PRSS1, CFTR, CPA1, CTRC, CBS, AGL, PHKB, and LPL). Data were analyzed using different bioinformatics tools for the variants and in-silico analysis. All the identified variants were validated by direct sequencing of the targeted exons in the probands and their parents. RESULTS: There were 50 patients included in this study with confirmed hereditary chronic pancreatitis. Nine known mutations in SPINK1, PRSS1, CFTR, CTRC, CBS, and AGL genes, and 10 novel variants in LPL, CFTR, CTR, and PHKB genes were identified. The identified variants were found in heterozygous, compound heterozygous, and trans-heterozygous forms, with rare allele frequency in the normal population. The novel variants were [c.378C>T(p.Lys126Asn) and c.719G>A(p.Arg240Gln) in CTRC, c.586-3C>A and c.763A>G(p.Arg255Gly) in CPA1, c.1160_1161insT(p.Lys387Asnfs*26), c.784C>T(p.Gln262*), c.1139+1G>A, c.175G>A(p.Gly59Arg) in LPL, c.388C>G(p.leu130val) in CFTR, and c.2327G>A(p.Arg776His in PHKB)]. The phenotypic characteristics were variable and correlated with the relevant variant. CONCLUSIONS: The genetic composition plays a significant role in the predisposition of hereditary chronic pancreatitis. The clinical presentation varies with the genetic determinant involved. This information would help in building up a diagnostic algorithm for our population that can be used for genetic screening services in affected cohorts.

Spectrum of PRSS1, SPINK1, CTRC, CFTR, and CPA1 Gene Variants in Chronic Pancreatitis Patients in Russia.

The aim of the study was to define the spectrum of genetic risk factors of chronic pancreatitis (CP) development in patients living in the European part of the Russian Federation. Materials and Methods: The study group included 105 patients with CP, with the age of the disease onset under 40 years old (the average age of onset was 26.9 years). The control group consisted of 76 persons without clinical signs of pancreatitis. The diagnosis of chronic pancreatitis in patients was made on the basis of clinical manifestations and the results of laboratory and instrumental investigations. Genetic examination of patients was conducted using the next-generation sequencing (NGS) technology and included targeted sequencing of all exons and exon-intron boundaries of the PRSS1, SPINK1, CTRC, CFTR, and CPA1 genes. The genotyping of the rs61734659 locus of the PRSS2 gene was also conducted. Results: Genetic risk factors of the CP development were found in 61% of patients. Pathogenic and likely-pathogenic variants associated with the risk of CP development were identified in the following genes: CTRC (37.1% of patients), CFTR (18.1%), SPINK1 (8.6%), PRSS1 (8.6%), and CPA1 (6.7%). The frequent gene variants in Russian patients with CP were as follows: CTRC gene - c.180C>T (rs497078), c.760C>T (rs121909293), c.738_761del24 (rs746224507); cumulative odds ratio (OR) for all risk alleles was 1.848 (95% CI: 1.054-3.243); CFTR gene - c.3485G>T (rs1800120), c.1521_1523delCTT (p.Phe508del, rs113993960), and c.650A>G (rs121909046); OR=2.432 (95% CI: 1.066-5.553). In the SPINK1, PRSS1, and CPA1 genes, pathogenic variants were found only in the group of patients with CP. The frequent variants of the SPINK1 gene include c.101A>G (p.Asn34Ser, rs17107315) and c.194+2T>C (rs148954387); of the PRSS1 gene - c.86A>T (p.Asn29Ile, rs111033566); of the CPA1 gene - c.586-30C>T (rs782335525) and c.696+23_696+24delGG. The OR for the CP development for the c.180TT genotype (rs497078) CTRC according to the recessive model (TT vs. CT+CC) was 7.05 (95% CI: 0.86-263, p=0.011). In the CTRC gene, the variant c.493+49G>C (rs6679763) appeared to be benign, the c.493+51C>A (rs10803384) variant was frequently detected among both the diseased and healthy persons and did not demonstrate a protective effect. The protective factor c.571G>A (p.Gly191Arg, rs61734659) of the PRSS2 gene was detected only in the group of healthy individuals and confirmed its protective role. 12.4% of the patients with CP had risk factors in 2 or 3 genes. Conclusion: Sequencing of the coding regions of the PRSS1, SPINK1, CTRC, CFTR, and CPA1 genes allowed to identify genetic risk factors of the CP development in 61% of cases. Determining the genetic cause of CP helps to predict the disease course, perform preventive measures in the proband's relatives, and facilitate a personalized treatment of the patient in future.

Risk of chronic pancreatitis in carriers of the c.180C>T (p.Gly60=) CTRC variant: case-control studies and meta-analysis.

Chymotrypsin C (CTRC) is a digestive serine protease produced by the pancreas that regulates intrapancreatic trypsin activity and provides a defensive mechanism against chronic pancreatitis (CP). CTRC exerts its protective effect by promoting degradation of trypsinogen, the precursor to trypsin. Loss-of-function missense and microdeletion variants of CTRC are found in around 4% of CP cases and increase disease risk by approximately 3-7-fold. In addition, a commonly occurring synonymous CTRC variant c.180C>T (p.Gly60=) was reported to increase CP risk in various cohorts but a global analysis of its impact has been lacking. Here, we analyzed the frequency and effect size of variant c.180C>T in Hungarian and pan-European cohorts, and performed meta-analysis of the new and published genetic association data. When allele frequency was considered, meta-analysis revealed an overall frequency of 14.2% in patients and 8.7% in controls (allelic odds ratio (OR) 2.18, 95% confidence interval (CI) 1.72-2.75). When genotypes were examined, c.180TT homozygosity was observed in 3.9% of CP patients and in 1.2% of controls, and c.180CT heterozygosity was present in 22.9% of CP patients and in 15.5% of controls. Relative to the c.180CC genotype, the genotypic OR values were 5.29 (95% CI 2.63-10.64), and 1.94 (95% CI 1.57-2.38), respectively, indicating stronger CP risk in homozygous carriers. Finally, we obtained preliminary evidence that the variant is associated with reduced CTRC mRNA levels in the pancreas. Taken together, the results indicate that CTRC variant c.180C>T is a clinically relevant risk factor, and should be considered when genetic etiology of CP is investigated.

Direct pathway cloning and expression of the radiosumin biosynthetic gene cluster.

Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 µM to >7.2 µM). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Inhibition of mouse trypsin isoforms by SPINK1 and effect of human pancreatitis-associated mutations.

Serine protease inhibitor Kazal type 1 (SPINK1) is a trypsin-selective inhibitor protein secreted by the exocrine pancreas. Loss-of-function SPINK1 mutations predispose to chronic pancreatitis through either reduced expression, secretion, or impaired trypsin inhibition. In this study, we aimed to characterize the inhibitory activity of mouse SPINK1 against cationic (T7) and anionic (T8, T9, T20) mouse trypsin isoforms. Kinetic measurements with a peptide substrate, and digestion experiments with ß-casein indicated that the catalytic activity of all mouse trypsins is comparable. Human SPINK1 and its mouse ortholog inhibited mouse trypsins with comparable efficiency (KD range 0.7-2.2 pM), with the sole exception of T7 trypsin, which was inhibited less effectively by the human inhibitor (KD 21.9 pM). Characterization of four chronic pancreatitis-associated human SPINK1 mutations in the context of the mouse inhibitor revealed that the reactive-loop mutations R42N (human K41N) and I43M (human I42M) impaired SPINK1 binding to trypsin (KD 60 nM and 47.5 pM, respectively), whereas mutations D35S (human N34S) and A56S (human P55S) had no impact on trypsin inhibition. Our results confirmed that high-affinity trypsin inhibition by SPINK1 is conserved in the mouse, and the functional consequences of human pancreatitis-associated SPINK1 mutations can be replicated in the mouse inhibitor.

Establishment of reverse genetics for genotype VII Newcastle disease virus and altering the cell tropism by inserting TMPRSS2 into the viral genome.

Newcastle disease (ND) is the most important infectious disease in poultry, which is caused by avian orthoavulavirus type 1 (AOAV-1), previously known as Newcastle disease virus (NDV). In this study, an NDV strain SD19 (GenBank accession number OP797800) was isolated, and phylogenetic analysis suggested the virus belongs to the class II genotype VII. After generating wild-type rescued SD19 (rSD19), the attenuating strain (raSD19) was generated by mutating the F protein cleavage site. To explore the potential role of the transmembrane protease, serine S1 member 2 (TMPRSS2), the TMPRSS2 gene was inserted into the region between the P and M genes of raSD19 to generate raSD19-TMPRSS2. Besides, the coding sequence of the enhanced green fluorescent protein (EGFP) gene was inserted in the same region as a control (rSD19-EGFP and raSD19-EGFP). The Western blot, indirect immunofluorescence assay (IFA), and real-time quantitative PCR were employed to determine the replication activity of these constructs. The results reveal that all the rescued viruses can replicate in chicken embryo fibroblast (DF-1) cells; however, the proliferation of raSD19 and raSD19-EGFP needs additional trypsin. We next evaluated the virulence of these constructs, and our results reveal that the SD19, rSD19, and rSD19-EGFP are velogenic; the raSD19 and raSD19-EGFP are lentogenic; and the raSD19-TMPRSS2 are mesogenic. Moreover, due to the enzymatic hydrolysis of serine protease, the raSD19-TMPRSS2 can support itself to proliferate in the DF-1 cells without adding exogenous trypsin. These results may provide a new method for the NDV cell culture and contribute to ND's vaccine development.

High expression of serine protease 2 (PRSS2) associated with invasion, metastasis, and proliferation in gastric cancer.

BACKGROUND: Accumulating evidence indicates that the occurrence and development of tumors are related to the activation of oncogenes and the inactivation of tumor suppressor genes caused by epigenetic mechanisms. However, the function of serine protease 2 (PRSS2) in gastric cancer (GC) is still unknown. Our study aimed to find a regulation network involved in GC. METHODS: The mRNA data (GSE158662 and GSE194261) of GC and normal tissues were downloaded from the Gene Expression Omnibus (GEO) dataset. Differential expression analysis was performed using R software, and Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was conducted by using Xiantao software. Besides, we used Quantitative Real-time PCR (qPCR) to verify our conclusions. After gene knockdown, cell migration and CCK-8 experiment were carried out to detect the effect of gene on cell proliferation and invasion. RESULTS: Totally, 412 differentially expressed genes (DEGs) were identified from GSE158662 and 94 DEGs were identified from GSE196261. Km-plot database results indicated that PRSS2 exhibited high diagnosis worth for GC. Gene functional annotation enrichment analysis revealed that these hub mRNAs were mainly taken part in the process of tumorigenesis and development. Besides, vitro experiments showed that down-regulation of PRSS2 gene reduced the proliferation and invasion ability of GC cells. CONCLUSIONS: Our results indicated that PRSS2 may play vital roles in the carcinogenesis and progression of GC and can be potential biomarkers for patients with GC.