TPH1, TPH2, KAT1, KAT2 and IDO1 polymorphisms and the risk of stroke development.The following 10 polymorphisms of the genes encoding enzymes of the TRYCATs pathway were selected: c.804-7C > A (rs10488682), c.-1668T > A (rs623580), c.803+221C > A (rs1800532), c.-173A > T (rs1799913) – TPH1, c.-1449C > A (rs7963803), and c.-844G > T (rs4570625) – TPH2. c.*456G > A of KAT1 (rs10988134), c.975-7T > C of KAT2 (rs1480544), c.-1849C > A (rs3824259) and c. -1493G > C (rs10089084) of IDO1. The study was carried out on DNA isolated from the peripheral blood taken from 107 patients after a stroke and 107 healthy volunteers. All DNA samples were genotyped using TaqMan probes. The genotypes of eight studied polymorphisms modulated the risk of stroke. No significant difference in genotype and allele frequencies of the c.804-7C > A –TPH1 (rs10488682) and c.*456G > A – KAT1 (rs10988134) polymorphisms were found between patients and controls. Having performed haplotype and gen-gen analyses, it was possible to determine that patients after a stroke and controls differed in terms of the frequency of selected genotypes and haplotypes. Among the studied polymorphisms, eight SNPs were linked with stroke risk modulation. The results obtained confirmed our hypothesis regarding the involvement of the TRYCATs pathway in the pathogenesis of stroke.The abnormal activation of the tryptophan catabolites pathway (TRYCATs) is observed in patients suffering from cerebrovascular disease, including stroke. A previous study confirmed that lower bioavailability of tryptophan for serotonin synthesis was characterized in the patients during the acute stroke phase. Interestingly, according to various studies, polymorphisms of the genes involved in the TRYCATs pathway may modulate the risk of stroke occurrence. Therefore, this study aimed to investigate the association between the occurrence of  Stroke is considered and classified as a cardiovascular disease, next to coronary artery diseases, heart failure, hypertensive heart disease, valvular heart disease, and carditis . The Wor2-dependent oxidation, whereas KAT catalyses irreversible transamination of kynurenine acid in the brain and peripheral tissues [IDO is a rate-limiting enzyme, which initiates tryptophan metabolism through O tissues ,13,14. F tissues . Interes tissues ,17. Thus tissues . Additio tissues ,20.Moreover, Ormstad et al.  revealedIn addition, an animal-based study showed that the level of kynurenine (a neurotoxic metabolite of tryptophan) during ischemia was increased in the brain . InteresWe recruited 107 patients with stroke and 107 age- and sex-matched control subjects (without stroke) in our case-control study. The case group comprised patients after an ischemic stroke hospitalized at the Neurorehabilitation Department of the 3rd General Hospital in Lodz, Poland, in the years 2015–2017. The cerebral ischemic event in each patient had been documented using computed tomography (CT) of the brain. Neurological and CT findings were interpreted by two or more independent and experienced neurologists. All the patients had been diagnosed with an ischemic stroke; we did not include patients having other types of strokes.Among the selected individuals, the people with a history of cranial trauma, cerebral haemorrhage, atrial fibrillation, other major sources of cardioembolism, coagulation disorders, tumours, chronic inflammatory diseases, and autoimmune diseases were excluded from the study. Having excluded all such cases, 107 subjects  with a clinically overt stroke were selected and classified into the study group. Paresis of the left part of the body was observed more frequently (68 vs. 39).A complete medical history was collected for all the individuals enrolled in the study; the medical history included smoking habits, alcohol consumption, presence of diabetes, and drug treatment. Hypertension was defined as either  systolic blood pressure > 140 mm Hg, diastolic pressure > 90 mm Hg, or current treatment using antihypertensive drugs. Hypercholesterolemia was defined as either a need for statins or total plasma cholesterol level >200 mg/L. Diabetes was diagnosed when a given subject had a fasting glucose level >120 mg/L.The healthy volunteer’s group included 107 healthy individuals with no history or prior diagnosis of any cardiovascular disease. The exclusion criteria were the same as those in the aforementioned study group. The controls were not related to the patients, and no history of stroke in their family members was confirmed.All the patients were Caucasian and were recruited simultaneously from the same demographic area . The full characteristics of the two groups are presented in All blood samples were collected in the morning (between 7 am and 9 am) in fasting status and stored using the same protocol. All procedures were done according to the Helsinki Declaration for Human Research and were approved by the Committee on the Ethics of Research in Human Experimentation at the University of Lodz with Resolution No. 28/2015. Informed consent was obtained from all participants and/or their legal guardians.http://www.ncbi.nlm.nih.gov/snp .All studied polymorphisms were chosen using the Database of Single Nucleotide Polymorphisms (dbSNP) of the National Center for Biotechnology Information (NCBI) available at a minor allele frequency of 0.05 or greater in the European populationlocation at the regulatory region of genes, i.e., the 5′ near a gene, the 5′ UTR, the 3′ UTR and intron. All studied polymorphisms are presented in Our searches were conducted based on the following criteria: Genomic DNA was extracted from venous blood according to the SaMag™ Blood DNA Extraction Kit protocol and using the commercially available SaMag™ System . Then, the concentration and purity of DNA samples were determined by a spectrophotometer, where a 260/280 nm optical density (OD) ratio in range 1.8–2.0 was considered high quality and stored at −20 °C until use. the Genotyping.® SNP Genotyping Assays  and TaqMan™ Universal Master Mix II, no UNG  were used to perform the genotyping of the selected SNPs. The Taq-Man Assay IDs are presented in During our study, we investigated 10 SNPs of the genes encoding enzymes of the TRYCATs pathway. TaqManp < 0.05 were considered statistically significant.A statistical analysis of the results was performed in Statistica, version 12 for Windows , and SigmaPlot, version 11.0 for Windows . Calculations of the association between case/control and each studied polymorphism were performed in accordance with the unconditional multiple logistic regression model. The odds ratios (ORs) with 95% confidence interval (95% CI) served to assess the strength of association. Haplotypic frequencies were estimated using MedCalc software, version 15.0 for Windows . The values of – TPH1 (rs1799913) were associated with a reduced risk of stroke occurrence, and the homozygote A/A and the A allele of the same SNP decreased this risk. A statistical analysis of disturbances in the genotypes and alleles of the c.803 + 221C > A – TPH1 (rs1800532) polymorphism confirmed that the C/C and A/A homozygotes were linked with an increased frequency of stroke occurrence by 20 times, while the heterozygote of the same SNP showed a protective character and reduced the risk. The results demonstrated that the T/T genotype and T allele of c.-1668T > A – TPH1 (rs623580) were negatively correlated with stroke, whereas the A/A, T/A and A allele of the same polymorphism were positively correlated with the disease. Interestingly, the analysis of distribution of the genotypes and alleles of the c.-844G > T – TPH2 (rs4570625) polymorphism showed that the G/G homozygote and the G allele increased the risk of stroke by more than 30 times (p < 0.001), while the G/T genotype of the same SNP decreased the risk by nearly 27 times (p < 0.001). In case of c.-1449C > A – TPH2 (rs7963803), the C/C genotype and the C allele were positively correlated with stroke occurrence, whereas the heterozygote and the A allele protected against the onset of stroke. Additionally, the c.975-7T > C – KAT2 (rs1480544) polymorphism modulated the risk of stroke development. The C/C genotype and the C allele of the SNP increased the risk of the disease, while the T/C and the T allele reduced the risk. Moreover, in the case of the c.-1493G > C – IDO1 (rs10089084) polymorphism, the G/G genotype and the G allele were linked with an increased frequency of stroke occurrence as compared to the control subjects, whereas the C/C homozygote and the C allele reduced this risk. No correlation was found between genotypes/alleles of the c.-173A > T – TPH1 (rs10488682), c.*456G > A – KAT1 (rs10988134), c.-1849C > A – IDO1 (rs3824259) polymorphisms and stroke development.Among the studied SNPs , the hetTPH1, c.-1449C > A (rs7963803) and c.-844G > T (rs4570625) SNPs of the TPH2 gene, c.-1849C > A (rs3824259) and c.-1493G > C (rs10089084) SNP of the IDO1 gene. In case of c.804-7C > A (rs1799913) and c.803+221C > A (rs1800532), haplotype AC decreased the risk of stroke by more than seventy-six times (p < 0.0001), while haplotype AA of the same SNPs combination only doubled this risk. Moreover, the CT haplotype of c.804-7C > A (rs1799913) and c.-173A > T (rs10488682) SNPs reduced the risk of stroke occurrence, whereas AT of the same combination of polymorphisms increased this risk. The obtained results showed that the TT and AT haplotypes of the c.804-7C > A (rs1799913) and c.-1668T > A (rs623580) polymorphisms were negatively correlated with stroke development, whereas the CA haplotype of the same combined SNPs increased the risk of the disease. Additionally, the AA haplotype of the c.-173A > T (rs10488682) and c.803+221C > A (rs1800532) combined SNPs reduced the frequency of stroke in the Polish population as compared to healthy volunteers. In the case of the c.-1668T > A (rs623580) and c.803+221C > A (rs1800532) polymorphism, the TC haplotype was linked with the reduced risk of stroke development, while the AC haplotype of the same combination increased the frequency of stroke occurrence. Moreover, the AA and TA haplotypes of c.-173A > T (rs10488682) and c.-1668T > A (rs623580) were positively correlated with stroke, whereas the AT and TT haplotypes negatively correlated with the disease. In case of combined polymorphisms encoding TPH2 – i.e., c.-1449C > A (rs7963803) and c.-844G > T (rs4570625) – the CG increased the risk of stroke, while the CT, AG and AT haplotypes reduced this risk. Besides, the CG haplotype of c.-1849C > A (rs3824259) and c.-1493G > C (rs10089084) was linked with an increase in the risk of stroke occurrence, whereas the CC haplotype of the same SNPs combination reduced the risk.Our team also investigated the correlation between the occurrence of stroke and haplotypes  of the c– TPH1 (rs1799913) and c.-844G > T – TPH2 (rs4570625) polymorphisms were associated with an increased risk of stroke. Interestingly, the presence of the C/A-G/T genotype of the same polymorphism combination reduced the risk of stroke development 100 times (p < 0.001), whereas the A/A-G/G combined polymorphisms elevated this risk more than 50 times (p < 0.001). There was a link between an increased risk of stroke and the frequency of the C/C-C/C, A/A-C/C genotypes of c.804-7C > A – TPH1 (rs1799913) and c.-1449C > A – TPH2 (rs7963803), while the C/A-C/C and A/A-C/A genotypes reduced this risk. In the case of c.803 + 221C > A – TPH1 (rs1800532) and c.-844G > T – TPH2 (rs4570625), the C/C-G/G and C/A-G/G were linked with the risk of stroke elevated by more than 10 times (p < 0.001). On the other hand, the C/A-G/T genotypes of the same combined genotypes reduced this risk. Moreover, we observed that the C/C-C/C combined genotype of the c.803 + 221C > A – TPH1 (rs1800532) and c.-1449C > A – TPH2 (rs7963803) polymorphisms were associated with an increased risk of stroke, whereas the C/A-C/A genotypes reduced the risk. Interestingly, the A/A-C/C genotypes of the same polymorphism combination increased the occurrence of stroke by more than 18 times (p = 0.005). The T/T-G/G and A/T-G/G genotypes of c.-173A > T – TPH1 (rs10488682) and c.-844G > T – TPH2 (rs4570625) SNPs caused an above 30-fold (p < 0.001) and a nearly 70-fold (p < 0.001) increase of the risk in the Polish population, respectively. However, the T/T-G/T and A/T-G/T genotypes of the same SNP combination were associated with a diminished risk of stroke occurrence. Moreover, we found that the T/T-C/C genotype of c.-173A > T – TPH1 (rs10488682) and c.-1449C > A – TPH2 (rs7963803) was linked with an elevated risk of stroke development, whereas the A/T-C/A genotype caused a decrease of this risk by nearly fifteen times. In the case of c.-1668T > A – TPH1 (rs623580) and c.-844G > T – TPH2 (rs4570625), combined genotypes T/T-G/G and T/A-G/G were associated with a more than 10-fold (p < 0.001) and eleven-fold (p < 0.001) increase of the DD risk, respectively. On the other hand, the T/T-G/T and T/A-G/T genotypes of the same combinations diminished the risk of the disease more than 20-fold (p < 0.001) and more than twofold (p = 0.004), respectively Additionally, the presence of the T/T-C/C and T/A-C/A combined genotypes of the c.-1668T > A – TPH1 (rs623580) and c.-1449C > A – TPH2 (rs7963803) polymorphisms reduced the risk of stroke development, whereas the T/A-C/C and A/A-C/C genotypes were linked with an increased risk of the disease. Furthermore, we revealed that the G/G-C/A genotype of c.*456G > A (rs10988134) – KAT1 and c. -1849C > A – IDO1 (rs3824259) increased the risk of stroke. In addition, we observed that the C/C-C/A genotype of c.975-7T > C– KAT2 (rs1480544) and– c.-1849C > A IDO1 (rs3824259) SNPs was associated with an increased risk of stroke. However, the T/C-C/A and T/C-A/A genotypes of the same combination caused an increase of stroke occurrence by more than 30 times (p < 0.001) and six times (p = 0.005), respectively. In the case of the c.-1493G > C – IDO1 (rs10089084) and c.*456G > A– KAT1 (rs10988134) combined genotypes, we found that G/G-G/G and G/G-G/A were associated with a more than fourfold increase of the stroke risk, while the C/C-G/G combined genotype of the same polymorphisms was associated with an increase of this risk by about 10 times (p < 0.001). The link between the reduced risk of stroke and the frequency of the G/G-T/C and G/A-T/C genotypes of the c.*456G>A– KAT1 (rs10988134) and c.975-7T>C– KAT2 (rs1480544) polymorphisms were confirmed in our study. On the other hand, we found that the G/G-C/C and G/A-C/C genotypes of the same SNP combination caused an increase in the frequency of stroke occurrence. Moreover, we found that the G/G-T/T, G/G-C/C and G/C-C/C genotypes of c.-1493G>C – IDO1 (rs10089084) and c.975-7T > C– KAT2 (rs1480544) increased the risk of stroke about four times (p = 0.009), 18 times (p < 0.001) and three times (p < 0.001), respectively. On the other hand, the G/C-T/C genotype of the same polymorphism caused a drop in the risk just about fourteen times (p < 0.001). In the case of the c.-1849C > A – IDO1 (rs3824259) and c.804-7C > A – TPH1 (rs1799913) polymorphisms, the C/C-C/A and C/A-C/A combined polymorphisms were linked with a reduction of stroke risk about 20 times (p = 0.004) and 10 times (p < 0.001), respectively. The C/C-G/T, C/A-G/T and A/A-G/T combined genotypes of c.-1849C>A – IDO1 (rs3824259) and c.-844G > T – TPH2 (rs4570625) caused a reduction of the stroke risk in the Polish population. However, the C/C-G/G, C/A-G/G and A/A-G/G genotypes of the same combined SNPs led to an increase of this risk about five times (p = 0.003), 66 times (p < 0.001) and 10 times (p < 0.001), respectively. In the case of the next combined polymorphism, i.e., c.-1849C > A – IDO1 (rs3824259) and c.803+221C>A – TPH1 (rs1800532), we confirmed that the C/C-C/A and A/A-C/A genotypes decreased the risk of stroke occurrence, while the C/A-C/C and A/A-A/A genotypes increased this risk in the studied population. Moreover, the frequency of the C/C-C/A genotype of c.-1849C > A – IDO1 (rs3824259) and c.-1449C>A - TPH2 (rs7963803) was decreased, while the C/A-C/C genotype was increased in the patients after a stroke as compared to the healthy volunteers. Additionally, we found that the only C/A-T/T genotype of the c.-1849C > A – IDO1 (rs3824259) and c.-173A>T – TPH1 (rs10488682) combined polymorphisms elevated the risk of stroke development. The C/C-T/T and C/A-T/T genotypes of c.-1849C>A – IDO1 (rs3824259) and c.-1668T > A – TPH1 (rs623580) were associated with a reduced risk of stroke, whereas the C/A-T/A and C/A-A/A genotypes caused an increase of the risk in the Polish population. Interestingly, the G/G-C/C, G/G-A/A as well as G/C-A/A of the c.-1493G > C – IDO1 (rs10089084) and c.804-7C > A – TPH1 (rs1799913) combined polymorphisms caused an increased frequency of stroke occurrence in the Polish population more than 20 times (p = 0.003), five times (p < 0.001), and nearly four times (p < 0.001), respectively. However, the G/C-C/A genotype of the same polymorphism combination was associated with increased stroke risk in this population by about eight times (p < 0.001). On the one hand, we observed that the G/C-G/G combined genotype of the c.-1493G > C – IDO1 (rs10089084) and c.-844G > T – TPH2 (rs4570625) polymorphisms were associated with an increased risk of stroke. On the other hand, we found that the G/C-G/T and C/C-G/T genotypes of the same combined SNPs caused a reduction of this risk about six times (p < 0.001) and 17 times (p < 0.001), respectively. Interestingly, in the case of c.-1493G>C – IDO1 (rs10089084) and c.803+221C > A – TPH1 (rs1800532), we observed that the presence of the G/G-C/C combined genotype elevated the risk of stroke development more than 22 times (p = 0.003), whereas the C/C-C/A genotype was associated with a nearly 18-fold increase of the risk in the Polish population (p < 0.001). Moreover, an elevated risk of stroke for the G/G-C/G genotypes and a reduced risk for the G/C-C/A and C/C-C/C genotypes of the same polymorphisms were confirmed in our study. In our study, we confirmed the link between a reduced risk of stroke and the frequency of the C/C-C/C genotype of the c.-1493G>C – IDO1 (rs10089084) and c.-1449C > A – TPH2 (rs7963803) polymorphisms. However, the G/G-C/C and G/C-C/C genotypes of the same polymorphism combination increased this risk in the studied polymorphism. The G/G-A/T and G/G-T/T genotypes of c.-1493G>C – IDO1 (rs10089084) and c.-173A > T – TPH1 (rs10488682) caused an increase of the risk of stroke, whereas the C/C-A/T and C/C-T/T genotypes of the same combined polymorphisms brought about a reduction of this risk. We found that the G/C-T/T, C/C-T/T and C/C-T/A genotypes of c.-1493G > C – IDO1 (rs10089084) and c.-1668T>A – TPH1 (rs623580) reduced the risk of stroke, whereas the G/G-T/A genotype increased this risk. Moreover, the frequency of the G/G-C/A and G/A-C/A genotypes of the c.*456G > A (rs10988134) – KAT1 and c.804-7C > A – TPH1 (rs1799913) combined polymorphisms was decreased in the patients after a stroke as compared to the controls by about 12 times (p < 0.001) and 25 times (p = 0.002), respectively. However, in the case of the same polymorphisms, the patients with stroke were characterized by an increased frequency of occurrence of the G/G-A/A, G/A-C/C and G/A-A/A genotypes. Moreover, the G/G-G/G combined genotypes of c.*456G > A (rs10988134) – KAT1 and c.-844G > T – TPH2 (rs4570625) caused a more than elevenfold increase of the risk in the Polish population (p < 0.001), whereas the G/G-G/T and G/A-G/T genotypes caused a reduction of stroke occurrence. We also confirmed that the G/G-C/A and G/A-C/A combined genotypes of c.*456G > A (rs10988134) – KAT1 and c.803+221C > A – TPH1 (rs1800532) were related with a decreased risk of stroke, but the G/G-A/A and G/A-C/C combined genotypes of the same polymorphism increased this risk. The G/G-C/C, G/A-C/C combined genotypes of c.*456G>A (rs10988134) – KAT1 and c.-1449C > A – TPH2 (rs7963803) were linked with elevated occurrence of stroke, while the G/G-C/A genotype of the same polymorphism combination decreased this risk more than thirty times (p < 0.001). Additionally, the presence of the G/G-T/T and G/A-T/T combined genotypes of the c.*456G > A (rs10988134) – KAT1 and c.-1668T > A – TPH1 (rs623580) polymorphisms reduced the risk of stroke development, while the G/G-T/A genotype increased this risk in the studied population. The T/T-A/A, C/C-C/C and C/C-A/A genotypes of c.975-7T > C– KAT2 (rs1480544) and c.804-7C > A - TPH1 (rs1799913) caused an increase of the risk of stroke. Interestingly, the presence of the T/T-G/G and C/C-G/G combined genotypes of the c.975-7T > C– KAT2 (rs1480544) and c.-844G>T – TPH2 (rs4570625) polymorphisms elevated the risk of stroke development more than 30 times (p = 0.001). On the other hand, the T/T-G/T genotype of the same combined SNPs caused a decrease of this risk. Moreover, the T/C-C/A combined genotypes c.975-7T > C– KAT2 (rs1480544) and c.803+221C > A – TPH1 (rs1800532) caused a more than 20-fold reduction of the stroke risk in the Polish population (p = 0.001), whereas the C/C-C/C genotype increased this risk about ten times (p = 0.001). The T/T-C/C and C/C-C/C genotypes of c.975-7T > C – KAT2 (rs1480544) and c.-1449C > A – TPH2 (rs7963803) caused an increase of the risk of DD by just about three times (p = 0.003) and 11 times (p < 0.001), respectively. However, the T/C-C/C and C/C/C/A genotypes of the same combined polymorphisms brought about a reduction of this risk by 16 times (p < 0.001) and 13 times (p = 0.014), respectively. The T/T-T/T, C/C-A/T, C/C-T/T combined genotypes of c.975-7T > C– KAT2 (rs1480544) and c.-173A > T – TPH1 (rs10488682) were linked with an increased risk of stroke occurrence, while the T/C-T/T genotype of the same polymorphism combination decreased this risk. Moreover, we found that the T/T-T/A, C/C-T/A, C/C-A/A genotypes of c.975-7T > C– KAT2 (rs1480544) and c.-1668T > A – TPH1 (rs623580) increased the risk of stroke about threefold (p = 0.016), fourfold (p < 0.001) and 27-fold (p < 0.001), respectively. On the other hand, the T/C-T/T and T/C-T/A genotypes of the same combined SNPs caused a drop in stroke occurrence by more than 37 times and 15 times, respectively. No statistical correlation was found between combined genotypes of the c.*456G > A (rs10988134) – KAT1 and c.-173A > T – TPH1 (rs10488682) polymorphisms and the development of stroke.We also investigated the correlation between stroke occurrence and combined genotypes of all the studied polymorphisms . We founA growing body of evidence suggests that abnormal tryptophan metabolism may play a crucial role in stroke development. Our results presented in this paper confirmed this hypothesis. This is the first study to show that all chosen polymorphisms of the genes encoding enzymes of the TRYCATs pathway  may modulate the risk of stroke occurrence.TPH1 is located at chromosome 11p15.3 – 14, and comprises 11 exons, while the human TPH2 gene is located at chromosome 12q15 and comprises 11 exons. Both isoforms are highly homologous and exhibit 71% of amino acid identity [TPH1 and two SNPs of TPH2. One of them – c.804 – 7C > A – TPH1 (rs1799913) – is localised at intron 7 and the polypyrimidine stretch immediately upstream of the 3′acceptor splice site. This substitution of pyrimidine for purine in the polypyrimidine consensus sequence may decrease the fidelity of splicing. Indeed, sequencing of TPH1 cDNA revealed no evidence of abnormal splicing [TPH1 polymorphism, we confirmed that the A/A genotype increased the risk of stroke. Moreover, the frequency of A allele was high in suicide attempters as compared to healthy volunteers in a Turkish population [TPH1 polymorphism studied during this experiment was c.-173A > T (rs10488682). The SNP is localized in the promoter region of TPH1 and may reduce promoter activity, changing the transcription level of TPH1 [TPH1 may contribute to the development of stroke. We found that the polymorphism was not associated with the occurrence of stroke. Likewise, the case-control study revealed no statistically significant association between TPH1 SNPs and progression of idiopathic scoliosis in an Eastern European population [Tryptophan hydroxylase, which exists in two isoforms: TPH1 and TPH2, is one of the key enzymes of tryptophan metabolism. Human identity . TPH conidentity . Thus, asplicing . So far,splicing . Thus fasplicing ,31. By csplicing . The A/Apulation . Howeverpulation . Interespulation . Another of TPH1 . So far,pulation . On the pulation . Moreovepulation  showed tThe next studied polymorphism was c.-1668T > A SNP (rs623580). The SNP is localised in the exon 1c/intron 1 region and is within the 5′UTR region . The T/TTPH1, we also studied TPH2 polymorphisms, and c.-844G>T (rs4576025) was one of them. This polymorphism is associated with a change of DNA-protein interactions, ultimately affecting transcription of the TPH2 gene. Indeed, the presence of the T allele may lead to a reduction of TPH2 promoter activity and may cause inhibition of serotonin synthesis [Besides the SNP localised in ynthesis ,41,42,43844G>T rs76025 wasynthesis . Interesynthesis ,48. Thusynthesis . SimilarTPH2 polymorphism was associated with the occurrence of post-stroke anxiety [TPH2 gene was associated with the development of paranoid schizophrenia, multiple sclerosis, and panic disorders [TPH2 was c.-1449C > A (rs7963803). Moreover, the C/C homozygote and C allele of this SNP decreased the risk of development of depressive disorders [Interestingly, stroke survivors were characterized by the occurrence of affective disorders both in the acute period and follow-ups, which may adversely impact rehabilitation and prognosis . Thus, p anxiety . Likewisisorders ,46,53. Tisorders . On the KAT1 and stroke occurrence. This SNP causes a transition in the 3′UTR region, which may affect KAT1 transcript stability [KAT1, which are known to influence substrate specificity, have been described. We found no link between polymorphism occurrence and the risk of stroke. Interestingly, apart from our team, no one has been concerned with this polymorphism so far. KAT2 is another isoform of KAT presented in this paper. The gene of this enzyme is localized on chromosome 4q33, while the studied c.975-71T > C (rs1480544) polymorphism is found in putative exonic splicing silencers (ESSs). The SNP may cause quantitative changes in the production of canonical mRNAs and peptide generation, and, consequently, may contribute significantly to inter-individual phenotypic variability [Kynurenine aminotransferase is the next key enzyme of the TRYCATs pathway, described in this paper. KAT catalyses irreversible transamination of kynurenine acid in the brain and peripheral tissues ,14. Fourtability . To dateiability . Moreoveiability . Thus, iiability .IDO1, i.e., c.-1849C > A (rs3824259) and c.-1493G > C (rs10089084), are localized in 5′ region of the gene. A previous study showed no association between the two polymorphisms and IFN-α-related depression [Indoleamine 2,3-dioxygenase, which catalyses the first and rate-limiting step in the TRYCATs pathway leading to the initiation of N-formylkynurenine, is yet another enzyme involved in the transformation of tryptophan. The gene encoding this enzyme is localized at chromosome 8p11 ,59,60. Fpression . SimilarThe findings of this work cast a new light on the pathogenesis of stroke; however, the present study had some potential limitations. The size of the studied group was relatively small and consequently of low power, which could lead to both false negative as well as false-positive findings. Thus, received results should be interpreted with caution and considered preliminary. Moreover, the studied population included patients and controls from Poland only, which reduces the possibility of confounding from ethnicity, so it does not permit any extrapolation of the results to other ethnic groups. Therefore, some additional larger case-control studies on different population groups and functional experiments are necessary before the final resolution about/findings as to the role of the TRYCATs pathway in the development of this disease. Moreover, the next limitations included no matching of the control group and patients in terms of vascular risk factors. In the future, the analysis should be extended to both the control group/patients with and without this risk. To conclude, the presented study showed a significant association between the tryptophan catabolism pathway and the occurrence of stroke, but the results obtained should be interpreted with great caution.TPH1 (rs10488682) and c.*456G > A – KAT1 (rs10988134). This study confirms the hypothesis that the TRYCATs pathway may be involved in the pathogenesis of stroke development. Thus, these polymorphisms may be considered independent markers of stroke. SNPs as biomarkers will allow the use of preventive measures in people at increased risk of stroke occurrence. Moreover, these markers will allow quick and new therapeutic strategies. In the future, further molecular studies of the underlying causes of stroke, including analyzing the level of expression of the studied genes, are necessary.In the presented paper, we confirmed that the selected SNPs of the genes involved in the tryptophan catabolites pathway might influence the risk of depression occurrence. Among the studied SNPs, we found no link between the risk of stroke occurrence and only two polymorphisms, c.-173A > T –


Escherichia coli O157:H7-specific immune responses have been shown to reduce E. coli O157:H7 shedding in cattle. Although E. coli O157:H7 colonization is correlated with perturbations in intestinal microbial diversity, it is not yet known whether vaccination against E. coli O157:H7 could cause shifts in bovine intestinal microbiota. To understand the impact of E. coli O157:H7 vaccination and colonization on intestinal microbial diversity, cattle were vaccinated with two doses of different E. coli O157:H7 vaccine formulations. Six weeks post-vaccination, the two vaccinated groups (Vx-Ch) and one non-vaccinated group (NonVx-Ch) were orally challenged with E. coli O157:H7. Another group was neither vaccinated nor challenged (NonVx-NonCh). Fecal microbiota analysis over a 30-day period indicated a significant  association of bacterial community structure with vaccination until E. coli O157:H7 challenge. Shannon diversity index and species richness were significantly lower in vaccinated compared to non-vaccinated groups after E. coli O157:H7 challenge (p < 0.05). The Firmicutes:Bacteroidetes ratio (p > 0.05) was not associated with vaccination but the relative abundance of Proteobacteria was significantly lower (p < 0.05) in vaccinated calves after E. coli O157:H7 challenge. Similarly, Vx-Ch calves had higher relative abundance of Paeniclostridium spp. and Christenellaceae R7 group while Campylobacter spp., and Sutterella spp. were more abundant in NonVx-Ch group post-E. coli O157:H7 challenge. Only Vx-Ch calves had significantly higher (p < 0.001) E. coli O157:H7-specific serum IgG but no detectable E. coli O157:H7-specific IgA. However, E. coli O157:H7-specific IL-10-producing T cells were detected in vaccinated animals prior to challenge, but IFN-γ-producing T cells were not detected. Neither E. coli O157:H7-specific IgG nor IgA were detected in blood or feces, respectively, of NonVx-Ch and NonVx-NonCh groups prior to or post vaccinations. Both Vx-Ch and NonVx-Ch animals shed detectable levels of challenge strain during the course of the study. Despite the lack of protection with the vaccine formulations there were detectable shifts in the microbiota of vaccinated animals before and after challenge with E. coli O157:H7.Vaccination-induced  Escherichia coli O157:H7 is a food borne pathogen acquired by ingestion of contaminated food, water or through direct contact with infected cattle or fomites [E. coli O157:H7, which preferentially colonizes at the rectoanal junction (RAJ) [E. coli O157:H7 is not pathogenic to adult cattle [E. coli O157:H7 by cattle [on (RAJ) . Althougt cattle , in humat cattle , 5. A may cattle .E. coli O157:H7 by cattle could be reduced by 50% [E. coli O157:H7 vaccination data also suggested that vaccination is an effective strategy for reducing fecal shedding of E. coli O157:H7 by cattle [E. coli O157:H7 shedding were significantly reduced in cattle vaccinated with either a siderophore receptor and porin protein (SRP)- or type III secreted proteins (T3SS)-based vaccine [E. coli O157:H7  [E. coli O157:H7 shedding by an average of 1.7 log10 units in cattle feces [hha deletion mutant vaccine reduced the duration of fecal shedding of E. coli O157:H7 [E. coli O157:H7 shedding in cattle feces [hha mutant strain of E. coli O157:H7) and a water-in-oil adjuvant [E. coli O157:H7 in three weeks after a challenge dose of 1010 colony forming units (CFU) of E. coli O157:H7 [E. coli O157:H7 in cattle.A mathematical model predicted an 80% reduction in the number of human illnesses if fecal shedding of  vaccine , 10. Altstrains) , a threele feces . Similar O157:H7 . Previoule feces . This vaadjuvant . Calves  O157:H7 . While pThe native gut microbiota plays a significant role in the development and regulation of the immune response to pathogens, and an altered gastrointestinal (GIT) microbiota (dysbiosis) may affect the immunological outcome of vaccination , 16. AnaBacteroidetes, Firmicutes, and Proteobacteria [Firmicutes relative to Bacteroidetes and Proteobacteria is an indicator of GIT microbiota dysbiosis [Firmicutes-to-Bacteroidetes (F:B) ratio was strongly correlated with daily milk-fat yield and parity of cows in milk, but the F:B ratio was not correlated with milk fat percentage or milk protein percentage [E. coli O157:H7 on cattle GIT microbiota, and subsequent shifts in microbiota following E. coli O157:H7 challenge. Vaccine immunogenicity and efficacy of the formulations were also assessed, and results integrated with microbiota shifts. Collectively, vaccination was associated with shifts in the Paeniclostridium spp. and Christenellaceae R7 group of the GIT microbiota. Vaccination induced a peripheral anti-E. coli O157:H7 antibody response and E. coli O157:H7-specific T cell response dominated by IL-10 producers. Following challenge, peripheral T cells did produce E. coli O157:H7-specific IFN-γ, but it was not associated with protection.The three dominant phyla that comprise the GIT microbiota in cattle and almost all mammals are bacteria . Lower aysbiosis , 26. In rcentage , 27. In E-Ch); 3) Vaccinated (Carbigen adjuvanted vaccine) and Challenged (VxC-Ch); and 4) Non-Vaccinated and Not-Challenged (NonVx-NonCh). Adjuvants mentioned above were purchased from a commercial supplier . All the calves had free access to feed (pasture and hay) and water while housed outside in a pasture. A day before the challenge, calves were relocated from the pasture into a climate controlled BSL2 facility at the National Animal Disease Center (NADC) and housed in separate pens . Animals were fed twice daily with a maintenance diet of pelleted feed and alfalfa hay cubes and ad libitum access to water. Fecal samples (~10 grams) were collected from each animal by rectal palpation on day 0 (week 0) before vaccination, day 1 (week 1) at priming, day 21 (week 4) at boosting, day 42 (week7) at challenge, and once during weeks 8 to 12  post-challenge . All calves remained clinically healthy throughout the course of the study, except one calf developing an upper respiratory issue that was noticeable as a wheezing during the breathing. This calf was treated with an antibiotic (Draxxin) and an analgesic (Banamine) on the day the symptoms were first noticed while the analgesic was administered for the additional two days. On the third day following the treatment, calf’s symptoms were marked resolved.Standard husbandry practices and veterinary care were applied to animals in the study. The research protocols used were approved by the USDA-ARS-NADC Institutional Animal Care and Use Committee. Jersey calves (6–8 months old) (n = 16) were tagged with a unique identification number and randomly assigned to one of the following treatment groups (n = 4 per group): 1) Non-Vaccinated and Challenged (NonVx-Ch); 2) Vaccinated (Emulsigen-D Adjuvanted vaccine) and Challenged  E. coli O157:H7 strain NADC 6564 deleted of stx2 and hha genes (NADC 6597) was used as a vaccine strain in the current study [600 of about 1.2. The bacterial culture was inactivated by adding formaldehyde (37% w/v) to a final concentration of 1.5% and incubation for 60 min at 37°C with intermittent shaking as described previously [10 cells in 1. 2 mL of phosphate-buffered saline (PBS)) was mixed with 0.8 mL of Emulsigen-D or Carbigen adjuvant and the resulting emulsions were used for vaccinating calves assigned to VxE-Ch and VxC-Ch groups, respectively. The vaccine formulation (2 mL) was injected in the muscle mass of the neck in front of the shoulder. Calves in the NonVx-Ch and NonVx-NonCh groups were mock vaccinated with 2 mL of PBS. Animals were boosted with the same preparations at day 21 (week 4) on the contralateral side of neck.Streptomycin-resistant  of E. coli O157:H7 strain NADC 6564, and calves from the NonVx-NonCh group were mock-challenged with an equal volume of sterile PBS .On day 42 (week 7), twelve calves, belonging to the Vx-1) and potassium tellurite (2.5 μg mL-1) (SMAC-ST), directly or after incubation for 18 to 24 h, static at 37°C (enrichment), to quantify fecal shedding of E. coli O157:H7. Fecal samples collected before the E. coli O157:H7 challenge were also serially-diluted in TSB with or without overnight enrichment and plated on sorbitol-MacConkey agar containing only potassium tellurite (2.5 μg mL-1) to detect the naturally present E. coli O157:H7 in calves. Detection sensitivity for direct plating (without enrichment) was 103 colony-forming units (CFU) per 100 ml of fecal suspension (ten-gram feces in 90-mL of TSB). For enrichment plating, fecal samples were either assigned a value of one-log CFU or a value of 0 if these produced or did not produce E. coli O157:H7-sepcific colonies, respectively, on SMAC-ST plates.Ten-grams of fecal samples (n = 144) after serial dilution in trypticase soy broth (TSB)  were plated on sorbitol-MacConkey agar containing streptomycin , NonVx-Ch , and NonVx-NonCh . After 60 min incubation, the wells were washed with a kit-supplied washing buffer and then filled with 100 μl aliquots of a 10−4-diluted HRP-conjugated anti-bovine IgG. The plates were washed after 60 min of incubation with HRP-conjugate and wells reacted with 100 μl of a two-component TMB. The reaction was stopped after 5 min of color development by adding 100 μl of stopping buffer and plates were read for absorbance at 405 nm wavelength (OD405) in a SpectraMax Spectrophotometer . A commercially available Bovine IgA Quantitation kit  and the manufacturer’s recommended procedure were used for detecting relative amounts of E. coli O157:H7-specific fecal IgA. Fecal IgA was detected in fecal extracts that were prepared as described previously [E. coli O157:H7 challenge: Vx-Ch group , NonVx-Ch group , and NonVx-NonCh group . Fecal extracts were analyzed against killed challenge strain NADC 6564 or a nonpathogenic commensal E. coli strain NADC 479 coated at 108 cells per well onto ELISA Immulon High Binding 2 plates . After blocking the coated plate wells with a blocking buffer, 100 μl of a 10-fold diluted fecal extracts was added to blocked plates. Plates were washed five times with PBS containing 0.05% Tween-20 and anti-bovine IgA-HRP  was added to plate wells as per manufacturer’s instructions. Ultra-TMB (Thermo Fisher Scientific) was added to plates after washing ten times with PBS+0.05% Tween-20. Plates were allowed to develop for 15 min and reaction stopped by adding 1M HCl. Plates were immediately read for absorbance at 450 nm (OD450) on a SpectraMax Spectrophotometer.eviously  and it eADC 6564 . The weleviously . Before 6 cells/well into 96-well, tissue culture-treated, round bottom plates. Cells were then stimulated for 3 days with 5 μg of heat-inactivated, sonicated E. coli O157:H7 strain NADC 6564 lysate. Cells stimulated with 5 μg of pokeweed mitogen (Sigma Aldrich) were used as a positive control.Peripheral blood mononuclear cells (PBMCs) were isolated from 10 mL of blood as previously described . IsolateCells were stained as previously described . BrieflyAccuPrime Pfx SuperMix , 5.0 μM each of the primers, and 25 ng of the template DNA. PCR settings included denaturation at 95°C for 2 min and 22 cycles of  amplification followed by final extension at 72°C for 10 min. PCR amplicons were normalized using the SequalPrep™ Normalization Plate (96) Kit . Normalized amplicons were pooled and quantified using Kapa SYBR Fast qPCR  and sequenced on a MiSeq Instrument using a MiSeq Reagent Kit v2 following manufacturer’s instructions . DNA from a mock community with defined composition [DNA was extracted from fecal samples (n = 144) using the DNeasy PowerSoil kit . DNA yield and purity were evaluated on a Nanodrop  and by electrophoresis on a 0.8% agarose gel. Previously described primers and conditions were used to amplify and sequence the V4 region of the 16S rRNA gene . Each PCposition  was alsohttps://www.qiagenbioinformatics.com/solutions/microbial-genomics-solution/)  following the manufacturer’s protocol for clustering of operational taxonomic units (OTUs). Specifically, the paired-end read data (forward and reverse sequences) were merged to create the highest quality sequences (trimmed to a fixed length of 250 bp) for clustering. The alignment settings were set as 1 for mismatch cost, 40 for minimum score, 4 for gap cost, and 5 as the maximum unaligned end mismatches. The OTUs were clustered at 97% similarity against the SILVA 16S rRNA small subunit reference database [The sequences were analyzed using the Microbial Genomics Module (MGM) 1.6.1  of calves after the E. coli O157:H7 challenge was analyzed by one-way ANOVA and a cutoff value of 0.05 (p <0.05) was selected to determine statistical significance  of the output data. To determine the taxa that significantly differed between groups, we used differential abundance analysis (DAA) with the OTU table  as the input data. Prior to the analysis, OTU tables were rarefied to the sample containing the lowest number of sequences in each analysis. OTUs were assigned at the genus level, and genera with relative abundance of more than 0.1% of the total were used in DAA analysis. Post-hoc test  was used to determine significantly different  taxa between groups.The curated sequences were aligned in the MGM module using MUSCLE by the neighbor joining method and following the Jukes-Cantor model. This alignment was used to create a maximum likelihood phylogenetic tree. The phylogenetic tree and the OTU table describing the taxonomic differences among treatments and between weighted groups were used to calculate the Bray-Curtis dissimilarity and generate the PCoA plot. The difference in beta diversity among the treatment groups was analyzed using the Permutation Multivariate Analysis of Variance (PERMANOVA) in the MGM module. This distance-based method tests the association of microbiome composition with any covariates of interest. The analysis and comparisons of alpha diversity (represented by measuring Shannon diversity index and Chao 1 species richness) between groups was carried out after the OTU tables were rarefied to the sample containing the lowest number of sequences . Alpha-diversity measures were calculated in the MGM 1.6.1 and compared between vaccinated and non-vaccinated calves, using the Student’s T-test. The difference in alpha diversity among the three groups  groups throughout the study, so both were combined into a single group, called as a vaccinated-challenged group (Vx-Ch). No significant differences  were observed in bacterial community structure between the vaccinated and non-vaccinated groups at week 0, week 1 (samples collected before the vaccination) and at week 4 sampling (sampled before the booster-dose vaccine was given) . But at week 5, one-week after the booster-dose was administered, the bacterial community structure was significantly different   between vaccinated and non-vaccinated groups . Also, the bacterial community structure was different  (S1 Table) between vaccinated and non-vaccinated groups on week 7 of sampling before the E. coli O157:H7 challenge . After oral challenge of calves with E. coli O157:H7 or PBS, the bacterial community structure was different between Vx-Ch and NonVx-Ch, and between Vx-Ch and NonVx-NonCh groups  .Sequencing of the V4 region of the 16S rRNA gene in all 144 fecal samples resulted in 2.63 million reads, which after filtering and removing chimeras yielded 3,974 predicted Operational Taxonomic Units (OTUs). The OTU table and the sample information (metadata) was used to determine statistical differences in bacterial community structure p-value < 0.05) lower than that of NonVx-Ch and NonVx-NonCh groups . Chao 1 species richness was numerically lower in Vx-Ch group for all but weeks 7 and 8  reduction in species richness in Vx-Ch group at week 9 , week 10  and week 11  of sampling . The results obtained with Shannon index and species richness corroborate the dynamic changes seen in the community structure  wherein the Vx-Ch calves showed a marked change in microbiome after oral challenge with E. coli O157:H7. Interestingly, there were no significant differences in the Shannon index or species richness between NonVx-Ch and NonVx-NonCh groups from week 7–11 indicating that oral challenge with E. coli O157:H7 alone did not cause significant changes in microbial community structure or alpha-diversity measures.The analysis and comparisons of alpha-diversity  among the three groups of calves showed that the Shannon diversity index was numerically higher in Vx-Ch group up to week 4 of sampling but this trend was reversed from week 5 and at weeks 10 and 11, and the Shannon diversity index of Vx-Ch calves was significantly  to create the taxonomic profile of GIT microbiota of all four groups of calves and at all sampling weeks . The type of adjuvant ) used in vaccinating calves did not yield significant differences in the bacterial community structure  and bacterial community structures were not significantly different between vaccinated and non-vaccinated groups at sampling weeks 0, 1 and 4 . However, fecal samples collected after the second booster dose of vaccine (at week 5) indicated significant differences in community structure between vaccinated and non-vaccinated groups  . We investigated which members of the bacterial community were associated with the significant changes in microbiota due to vaccination at week 5 of sampling. To get the useful information, we selected bacterial genera with overall abundance more than 0.1% so as to avoid bias, sequencing errors, or any genera for which taxonomy was not available. The differential abundance analysis (DAA) of OTU table at the genus level of taxonomy indicated higher relative abundance of Rikenellaceae dgA-11 gut group and Ruminococcaceae UCG-013 in samples from the vaccinated group and Anaerorhabdus furcosa group and Acetitomaculum spp. in samples from the non-vaccinated animals .The sequencing data generated on IlluminaE. coli O157:H7 alone did not cause significant changes in microbial community structure , S2 Fig, we compared the taxonomic profiles of GIT microbiota of Vx-Ch and NonVx-Ch groups from week 7 to week 11 by DAA of OTU table at genus level of taxonomy to assess differential impact of vaccination on microbiota composition of these two calf groups. The results of this comparison indicated that a higher number of bacterial genera were differentially abundant between the two groups from week 9 to 11 compared to weeks 7 or 8. These results were in accordance with the observations made about bacterial community structure during these sampling weeks . The Vx-Ch calves had higher relative abundance of Christensenellaceae R-7 group while Sutterella was relatively more abundant in samples from the NonVx-Ch group . The DAA analysis at the genus level is more informative compared to the family level because certain members of one bacterial family can be differentially abundant in two groups. For example, Lachnospiraceae NK3A20 group is relatively more abundant in Vx-Ch calves at week 9, while Lachnospiraceae NK4A136 group is relatively more abundant in NonVx-Ch group at week 10 of sampling . After the 12 calves  were challenged with E. coli O157:H7, we observed higher abundance of Campylobacter in NonVx-Ch group (from week 8 to 11) while Romboutsia, Paeniclostridium and Methanobrevibacter were more abundant in Vx-Ch group (from week 9 to 11) . These results indicated that although there was only a slight change in bacterial community structure due to vaccination, the relative abundance of certain genera was significantly different between the Vx-Ch and the NonVx-Ch calves, after calves received oral challenge of E. coli O157:H7.Since oral challenge of calves with . The Vx-Ch group represents two vaccinated and challenged (VxE-Ch and VxC-Ch) calf groups. Higher F:B ratio at up to week 5 of sampling could be associated with the housing of these calves allowing pasture grazing during this time. All 16 calves were moved in to a BSL2 building a day before week 7 sampling and were kept there until the end of this study. We did not observe a significant difference in F:B ratio due to vaccination or E. coli O157:H7 challenge, except for week 0 sampling (before primary vaccination) when the two vaccinated and challenged (VxE-Ch and VxC-Ch) calf groups had higher F:B ratio compared to non-vaccinated but challenged (NonVx-Ch) calves  .We observed a decrease in F:B ratio over the course of this study in all three  calf groups Proteobacteria (measured as Log10 of number of OTUs) showed the effect of both time and vaccination. We observed increase in relative abundance of Proteobacteria over the course of study for both NonVx-Ch and NonVx-NonCh calves, but for the Vx-Ch groups of calves, the Proteobacteria population relatively increased up to week 5 and then decreased from week 7 to 11 . The abundance of Proteobacteria was higher in Vx-Ch compared to NonVx-NonCh calves (significantly higher at weeks 4 (2.26 vs 1.83) and 5 (2.21 vs 1.77) of sampling). However, once the vaccinated animals received oral challenge with E. coli O157:H7, the relative abundance of Proteobacteria was reduced compared to both NonVx-Ch and NonVx-NonCh calves . We did not observe a significant difference in relative abundance of Proteobacteria due to E. coli O157:H7 challenge alone, except for week 0 sampling when Vx-Ch calves had higher Proteobacteria compared to the NonVx-NonCh group.The relative abundance of members of the phylum E. coli O157:H7 colonization on their arrival as E. coli O157:H7 was not cultured from any animal feces prior to challenge. After oral challenge with E. coli O157:H7 strain NADC 6564, fecal samples were analyzed for the shedding of the challenge strain. Since we did not observe any significant difference in bacterial community structure between samples from the two vaccinated (VxE-Ch and VxC-Ch) groups throughout the study, both vaccinated groups  were combined in to a single group, called as a vaccinated-challenged group (Vx-Ch), for analysis of the fecal shedding data. All 12 calves in challenged groups (Vx-Ch and NonVx-Ch) shed detectable amounts (≥ 102 CFU/g feces) of the challenge strain in feces during the course of the study although the concentration (CFU/g feces) was one-log lower in Vx-Ch group of calves compared to the NonVx-Ch calves on day 43 of week 7 , day 49 of week 8 , and day 56 of week 9 , of sampling  after E. coli O157:H7 challenge .All animals were determined negative for E. coli O157:H7-specific antibodies in periphery and feces are important indicators of vaccine immunogenicity and are associated with reduction of E. coli O157:H7 fecal shedding in vaccinated cattle [E. coli-specific IgG and fecal E. coli-specific IgA. We first tested sera of four animal groups  by ELISA to determine differences in the level of induction of E. coli O157:H7-specific serum IgG and cross-reactivity of these antibodies to commensal E. coli. A preliminary screening of 10-fold serial dilutions (10−1 to 10−4) of sera collected from each animal of the four study groups on day 42 of week 7 showed that only sera from the two vaccinated and challenged animal groups (VxE-Ch and VxC-Ch) had significantly higher reactivity to the vaccine strain (NADC 6597) compared to sera of NonVx-Ch and NonVx-NonCh animals at all tested serum dilutions . We then tested the pooled sera  at 10−3 dilution   to determine the specificity of serum IgG response and its cross-reactivity to E. coli O157:H7 challenge strain (NADC 6564) and non-pathogenic commensal E. coli (NADC 479), respectively. As shown in Fig 6A, the pooled sera of Vx-Ch animal groups showed about 9-fold higher reactivity with E. coli O157:H7 challenge strain NADC 6564 (green bars) compared to the commensal strain NADC 479 (red bars). The pooled sera of NonVx-Ch and NonVx-NonCh animals, on the other hand, reacted poorly with strain NADC 6564 and commensal strain NADC 479 indicting that the vaccination-induced IgG was specific to E. coli O157:H7 and did not cross-react to the nonpathogenic commensal E. coli NADC 479. For determining fecal IgA response, we tested pooled fecal extracts  of vaccinated and non-vaccinated animals  using a commercially available kit enabling detection of bovine IgA. As shown in Fig 6B, minimal IgA to E. coli was detected in the feces, and for the minimal detected, there was no difference in reactivity to E. coli O157:H7 (green bars) and generic E. coli (red bars).Vaccine induced d cattle , 34–37. E. coli O157:H7-specific T cell interferon (IFN)-γ responses, which associated with reduced fecal shedding of E. coli O157:H7 from calves [E. coli O157:H7-specific CD4+IFN-γ+ cells were not detected after vaccination. Prior to challenge, there were minimal E. coli O157:H7-specific T cells responses detected. Primarily CD4+ and CD8+ cells responded to E. coli O157:H7-restimulation with the production of IL-10, but not IFN-γ . The percentage of CD4+IL-10+ cells was significantly greater at week 4 and 7 in Vx-Ch calves compared to NonVx-Ch and NonVx-NonCh groups  , Panel A. The percentage of CD8+ IL-10+ was significantly greater at week 7 in Vx-Ch and NonVx-Ch calves compared to NonVx-NonCh calves (p<0.05) , Panel B.Previously, vaccination with adjuvanted NADC 6597 induced peripheral m calves . HoweverE. coli O157:H7-specific γδ T cell responses, as percentages of cytokine producing cells were similar between Vx-Ch and NonVx-Ch compared to NonVx-NonCh group. Specifically, the percentage of CD4+ cells producing IFN-γ+ after in vitro stimulation with E. coli O157:H7 antigen was significantly greater in Vx-Ch and NonVx-Ch calf groups compared to the NonVx-NonCh group  , Panel A. Following challenge, the percentage of CD4+IFN-γ+IL-10+ cells was significantly greater in Vx-Ch and NonVx-Ch calves compared to the NonVx-NonCh calves  , Panel A. The percentage of CD8+IFN-γ+ was significantly greater at week 8 in NonVx-Ch (p<0.01) and Vx-Ch calves (p<0.05) compared to the NonVx-Non-Ch animals , Panel B. The percentage of CD4+IFN-γ+IL-10+ cells was significantly greater at week 8 for the Vx-Ch and NonVx-Ch groups of calves compared to NonVx-NonCh calves (p<0.05) , Panel B.Challenge, but not prior vaccination, impacted peripheral in vitro restimulation were impacted by prior vaccination. The percentage of γδTCR+ intracellular IFN-γ was significantly greater in Vx-Ch calves at week 8 (p < 0.0001) and week 10 (p < 0.001) compared to NonVx-Ch and NonVx-NonCh calves , Panel C. However, the percentage of γδTCR+IL-10+ cells was significantly greater at week 8 in both Vx-Ch and NonVx-Ch calves (p<0.05) compared to NonVx-NonCh calves , Panel C. Overall, IL-10 producing cells were prominent both before challenge and after, and vaccination only impacted IFN-γ producing γδ T cells, but only after challenge.Unlike γδ T cells, the percentage of γδ T cells producing IFN-γ upon E. coli (STEC) cause 2.8 million illnesses globally [E. coli O157:H7 alone. In addition, a combined economic loss of about $993 million per year to public health, agriculture and meat industry has been attributed to STEC (mainly E. coli O157:H7) contamination and infections [E. coli O157:H7 reservoirs [E. coli O157:H7 can be a commensal organism such as in cattle or a pathogen when it infects humans. Vaccination itself may alter the GIT microbiota, and would be important to understand. Vaccine formulations that do not adversely affect or do constructively affect GIT microbiota may minimize other impacts on animal health. In the current study, we evaluated the effect of vaccination and E. coli O157:H7 challenge on the GIT bacterial community structure of calves and immunogenicity of the vaccine formulation immune responses targeting E. coli O157:H7.Shiga toxin producing globally , more thglobally  and 36% fections , 41. Catservoirs . UnfortuE. coli O157:H7 challenge altered the fecal microbiota but the two vaccine formulations used did not cause changes in the fecal microbiota. Thus, data from the two vaccinated groups were pooled for the analyses. Genera like Paeniclostridium and Chritensenellaceae R-7 group were relatively more abundant in GIT microbial communities of calves vaccinated and challenged (Vx-Ch) with E. coli O157:H7. At week 7 (on or before day 42) of sampling, which was prior to challenge, a higher relative abundance of Rikenellaceae dgA-11 gut group was detected in vaccinated calves compared to non-vaccinated calf groups. Following challenge (week 10 of study) a higher relative abundance of Romboutsia and Paeniclostridium was detected in vaccinated (Vx-Ch) calves while challenged non-vaccinated calves (NonVx-Ch) had higher abundance of Sutterella and Campylobacter. Thus, vaccination alone did shift microbial communities, and challenge also had an impact.Vaccination and Romboutsia, Paeniclostridium, Methanobrevibacter, and Turicibacter) associated with vaccination which will be useful in future studies for identifying association of microbiota with vaccination in cattle. Specifically, we observed higher relative abundance of Turicibacter in Vx-Ch calves at week 9  without noticing any significant effect on F:B ratio. Previous studies have demonstrated higher abundance of Turicibacter in the GIT of cattle that were fed feedlot ration and had higher F:B ratio [Turicibacter are reportedly also enriched in the large intestine of cattle which contained relatively lower abundance of Proteobacteria [Sutterella and Campylobacter in NonVx-Ch calves. Further studies involving cattle are needed to determine the functional profile of the GIT microbiota due to vaccination.The role and impact of GIT microbiota composition in domestic animals in bacterial colonization/pathogenesis is largely unknown, as evidenced by the relatively small number of studies compared to the ones evaluating the human microbiome , 25. We :B ratio , 20. Turbacteria . We alsoE. coli O157:H7 by itself did not cause significant changes in microbial community structure. Change in the bacterial community structure was significantly associated with vaccination but independent of the adjuvants used in the vaccine formulations. The significant differences in the GIT bacterial community structure and taxonomic profiles were between Vx-Ch and NonVx-Ch calves, but we did not follow these animals for an extended period to see if the GIT microbiota of Vx-Ch animals would become similar to that of the NonVx-Ch animals over time. A slight reduction in the F:B ratio was observed over the course of the study, regardless of the vaccination/challenge status of calves. However, the abundance of Proteobacteria was significantly greater before and lower after challenge with E. coli O157:H7 in the vaccinated group (Vx-Ch) compared to the non-vaccinated but challenged group (NonVx-Ch).Oral challenge with E. coli O157:H7 proteins, SRP vaccine based on siderophore receptor and porin proteins of E. coli O157:H7, culture supernatants containing type III-secreted proteins of E. coli O157:H7, or bacterial ghosts of E. coli O157:H7 have shown induction of E. coli O157:H7-specific IgG, IgA, or both IgG and IgA [E. coli O157:H7-specific IgG appeared to be the major circulating immune response generated by vaccinated animals that also showed reduction in the fecal shedding of experimental E. coli O157:H7 challenge strain or naturally infecting strain of E. coli O157:H7. In a previous study, we demonstrated the induction of both E. coli O157:H7-specific serum IgG and fecal IgA in animals vaccinated with a vaccine formulation containing the inactivated vaccine strain (E. coli O157:H7 NADC 6597) and ISA61 Montenide adjuvant [E. coli O157:H7-specific IgA exerted inhibitory effect on the adherence of E. coli O157:H7 to cultured epithelial cells, thus demonstrating functional specificity of this IgA against E. coli O157:H7. However, in the current study examining the effects of vaccination on intestinal microbiota diversity, we observed that the two vaccine formulations prepared by mixing the inactivated E. coli O157:H7 strain NADC 6597 with adjuvant Emulsigen-D or Carbigen only induced higher levels of E. coli O157:H7-specific IgG but not E. coli O157:H7-specific fecal IgA. It could be hypothesized that the increases in E. coli O157:H7-specific IgG might be important considering that after oral challenge with E. coli O157:H7 strain NADC 6564, all 12 calves in the three challenged groups  continued to shed the challenge strain in feces, but the amount (CFU/g feces) of E. coli O157:H7 shed in feces was one-log lower on week 7 (day 43), week 8 (day 49), and week 9 (day 56) in Vx-Ch group  of calves compared to the NonVx-Ch calves. Several studies have reported that the circulating IgG could cross transluminal barriers to reach intestinal mucosal surfaces where these could interact with specific pathogenic entities and provide protection against the targeted pathogen [E. coli O157:H7 and intestinal microbial community structure and diversity.A variety of vaccine candidates including subunit vaccines prepared from specific purified  and IgA , 36, 43.adjuvant . The E. pathogen . Since wE. coli O157:H7 resulted in additional changes in the GIT microbiota community structure that were not seen in the unvaccinated but E. coli O157:H7-exposed animals. Since vaccination did transiently reduce shedding of E. coli O157:H7 for three weeks post-E. coli O157:H7 exposure of vaccinated animals, the microbiota community structure could be considered constructive but suboptimal in their ability to repress fecal shedding in vaccinated animals for longer time periods. It is also possible that besides inducing specific microbiota shifts, vaccination must also induce both E. coli O157:H7-sepcific antibody and cell-mediated immune responses in order to provide optimal protection in terms of reducing the magnitude of E. coli O157:H7 fecal shedding for longer time periods. In the current study, we were able to show that the vaccine formulations that we used had no adverse vaccine site reactivity in animals, were able to induce significantly high levels of peripheral IgG response, but generated no fecal IgA response, did not induce peripheral E. coli O157:H7-specific CD4+ IFN-γ+ cells, and produced IL-10 upon restimulation but no IFN-γ. In a previous study, we demonstrated that a vaccine formulation formulated with the same vaccine strain that was used in the current study but mixed with a different adjuvant induced significantly high levels of E. coli O157:H7-specific peripheral IgG, fecal IgA, cell-mediated immune cells (CD4+ IFN-γ+ cells), and immune markers (IFN-γ+) and these vaccinated animals also showed significant reduction in fecal shedding over the duration of the study [E. coli O157:H7.In conclusion, we have demonstrated that vaccination altered the composition of the GIT microbial population community structure and subsequent experimental exposure of vaccinated animals to he study . To streS1 TableDifferences in bacterial community structure were examined by PERMANOVA analysis of the bacterial community structure (Beta-diversity).(DOCX)Click here for additional data file.S1 FigE. coli O157:H7 challenge was given.PCoA was performed for comparing bacterial community structure between vaccinated and non-vaccinated groups at sampling weeks 0, 1, 4 and 5, before the (TIF)Click here for additional data file.S2 Fig10 CFU/g feces) and compared by ANOVA for difference between groups over a period of 12 weeks of sampling.Number of bacteria shed was first Log transformed (represented as Log (TIF)Click here for additional data file.S3 FigE-Ch, VxC-Ch, NonVx-Ch, and pooled sera of the four calves of NonVx-NonCh groups to the E. coli O157:H7 vaccine strain NADC 6597. Serum IgG levels are represented as Mean ± SD (shown as bars) of three replicate wells of the serum samples. Statistical analysis was performed using one way-ANOVA with multiple comparison of means.The serum IgG induced after vaccination was determined by reacting 10-fold serial dilutions of serum of each of the four calves from Vx(TIF)Click here for additional data file.


Glycyrrhiza species. Cytochrome P450 monooxygenases (P450s) are important for generating triterpene chemodiversity by catalyzing site-specific oxidation of the triterpene scaffold. CYP72A154 was previously identified from the glycyrrhizin-producing plant Glycyrrhiza uralensis as a C-30 oxidase in glycyrrhizin biosynthesis, but its regioselectivity is rather low. In contrast, CYP72A63 from Medicago truncatula showed superior regioselectivity in C-30 oxidation, improving the production of glycyrrhizin aglycone in engineered yeast. The underlying molecular basis of C-30 product regioselectivity is not well understood. Here, we identified two amino acid residues that control C-30 product regioselectivity and contribute to the chemodiversity of triterpenes accumulated in legumes. Amino acid sequence comparison combined with structural analysis of the protein model identified Leu149 and Leu398 as important amino acid residues for C-30 product regioselectivity. These results were further confirmed by mutagenesis of CYP72A154 homologs from glycyrrhizin-producing species, functional phylogenomics analyses, and comparison of corresponding residues of C-30 oxidase homologs in other legumes. These findings could be combined with metabolic engineering to further enhance the production of high-value triterpene compounds.The triterpenes are structurally diverse group of specialized metabolites with important roles in plant defense and human health. Glycyrrhizin, with a carboxyl group at C-30 of its aglycone moiety, is a valuable triterpene glycoside, the production of which is restricted to legume medicinal plants belonging to the  The triterpenoids are a large group of plant specialized metabolites consisting of six isoprene units. Plants produce structurally diverse triterpenoids that often have important roles in plant defense . MoreoveDecoration of the triterpene scaffold catalyzed by cytochrome P450 monooxygenases (P450s) is the second step of triterpene biosynthesis . GeneralGlycyrrhiza plants ; Glycyrrhiza lepidota and Glycyrrhiza macedonica were obtained from Osaka University of Pharmaceutical Science , and Glycyrrhiza glabra was obtained from Health Sciences University of Hokkaido .The seed plants used in this experiment are listed in β-Amyrin was purchased from Extrasynthese . Sophoradiol, 30-hydroxy-β-amyrin, and 11-deoxoglycyrrhetinic acid were synthesized as described in our previous report .M. truncatula genome project Mt4.0v1 proteins (M. truncatula Hapmap (CYP72A62 (Medtr8g042060), CYP72A63 (Medtr8g042040), CYP72A64 (Medtr8g042020), and CYP72A65 (Medtr8g042000) (obtained from M. truncatula genome database) as queries. Amino acid sequences were predicted according to the known coding sequences of gene references in M. truncatula. CYP72A63 homologs from other legumes were mined from publicly available genomic sequences in the 1KP database (www.clovergarden.jp/) (www.coolseasonfoodlegume.org/)  , Vigna Gden.jp/) , Cool Seme.org/) , and Legme.org/) .RNA preparation and cloning methods are explained briefly in the GmaxCYP72A141), MK534533 (GgCYP72A154), MK534534 (GpCYP72A154), MK534535 (LcCYP72A698), MK534536 (PsCYP72A698), MK534537 (CYP72A302), MK534538 (CYP72A694), MK534539 (CYP72A697), MK534540 (CYP72A336v2), MK534541 (CYP72A70), MK534542 (GsojaCYP72A141), MK534543 (CYP72A337v2), MK534544 (CYP72A557), MK534545 (CYP72A558), MK534546 (CYP72A559), MK534547 (CYP72A560), MK534548 (CYP72A64v2), MK534549 (CYP72A699), MK792941 (CYP72A66v2).Sequence data from this experiment have been submitted to the DNA DataBank of Japan (DDBJ), the European Nucleotide Archive (ENA), and GenBank databases under the following accession numbers: MK534530 (CYP72A695), MK534531 (CYP72A696), MK534532 (In vivo enzymatic assay in yeast was performed by co-expression of Lotus japonicus cytochrome P450 reductase (CPR) and β-amyrin synthase (OSC1), with three yeast expression clones for each of the CYP72A subfamily genes. Each corresponding set of pELC-CPR-CYP72A, pYES2-DEST52-CYP72A, and pESC-HIS-CYP72A was transformed into Saccharomyces cerevisiae INVSc1  carrying pYES3-ADH-OSC1 , and a yeast strain harboring the three empty vectors  was used as a control. All genes tested in this experiment are listed in In vivo enzymatic assay in yeast was performed as reported previously (N-methyl-N-(trimethylsilyl)trifluoroacetamide . GC-MS analysis was performed using a gas chromatograph  connected to a mass spectrometer  and HP-5MS capillary column  (Agilent Technologies). The initial oven temperature was 150°C with a hold time of 1 min, increasing from 150°C to 260°C at 30°C/min and 260°C to 300°C at 1°C/min. Samples were injected in splitless mode with an injection temperature of 250°C, with helium as the carrier gas at a flow rate of 1.0 ml/min. Comparison of retention times and mass fragmentation patterns of detectable compounds with those of authentic standards was performed to assign the peaks.ADH-OSC1  using Freviously  with somM. truncatula CYP72A subfamily were predicted as described previously . Yeast metabolites were obtained by saponification prior to extraction with n-hexane three times. Yeast extracts were evaporated and the residues were applied to silica gel chromatography  . Hexane:ethyl acetate (1:9) was used as the mobile phase, and afforded about 8 mg of compound 2. Nuclear magnetic resonance (NMR) data were recorded on a Bruker Avance III 600 MHz spectrometer  using CDCl3 as the solvent.A yeast strain carrying β-amyrin synthase co-expressed together with CPR and CYP72A63M. truncatula genome. CYP72A59-like6, CYP72A59-like7, and CYP72A68-like showed shorter sequences and no transcripts in transcriptome data  were functionally characterized  were reported in this study.Using representatives of each subgroup in the CYP72A subfamily described previously , we idencterized . In summMedicago truncatula Mt4.01 genome database using JBrowser  and 30-hydroxy-β-amyrin on GC-MS analysis  was not achieved due to incomplete removal of impurities. However, the data indicated the presence of 29-hydroxy-β-amyrin  and non-glycyrrhizin-producing species  were investigated. As the full-length amino acid sequences of the three non-glycyrrhizin-producing species were identical, we selected GpCYP72A154 as a representative species. Sequence alignment of CYP72A154 variants showed that amino acid residue #149 (numbering based on CYP72A63) is Val149 for both types, but amino acid residue #398 (numbering based on CYP72A63) differs between them, i.e., Gly398 for glycyrrhizin-producing species and Ala398 for non-glycyrrhizin-producing species  and 30-hydroxy-β-amyrin as the main products and 29-hydroxy-β-amyrin as a trace product  and 29-hydroxy-β-amyrin as the main products and 30-hydroxy-β-amyrin as a trace product  and 29-hydroxy-β-amyrin as main products  showed oxidation activity mainly at the C-30 position, producing 30-hydroxy-β-amyrin as the main product, while CYP72A63L149V/L398A (mimicking non-glycyrrhizin-producing variants) showed oxidation activity mainly at the C-29 position producing 29-hydroxy-β-amyrin as the main product. Mutagenesis of CYP72A63 mimicking CYP72A154 variants showed good agreement with the product regioselectivity of CYP72A154 variants from glycyrrhizin-producing and non-producing species. These results suggested that differences in amino acid residue #398 may be involved in generating triterpene chemodiversity in Glycyrrhiza species by conferring variable product regioselectivity.To investigate whether the differences in product regioselectivity of CYP72A154 variants are associated with divergence of amino acid residue #398, we generated mutant in vivo enzymatic assays. CYP72A63 homologs were searched from publicly available genomic information and transcriptome databases of legume plants. Selected CYP72A63 homologs were cloned, confirmed by DNA sequencing, and submitted to the P450 Committee for naming , Glycine max Gmax CYP72A141 , and Lotus japonicus LjCYP72A697 , but in vivo enzymatic assays showed that they have regioselectivity in the C-29 position. Trifolium pratense TpCYP72A699 and Phaseolus vulgaris PvCYP72A302 have a combination of amino acid residues, Val149 and Val398, as seen in Lotus japonicus LjCYP72A697, but in vivo enzymatic assay showed that they differed in product regioselectivity; however, regioselectivity in the C-29 position was common among them. These results support the important roles of amino acid residues #149 and #398 in determining regioselective oxidation activity.To further analyze the roles of amino acid residues #149 and #398 in CYP72A63 homologs from other legumes, we performed phylogenomic analyses of CYP72A63 homologs by constructing a phylogenetic tree, comparing amino acid residues #149 and #398, and performing M. truncatula identified two amino acid residues, Leu149 and Leu398, responsible for C-30 regioselective oxidation activity.In the absence of protein structures for P450s involved in triterpene biosynthesis, comprehensive analyses of the structure–function relationships could not be performed. Recent studies successfully demonstrated that comparative functional analyses of natural variants or evolutionarily related enzymes could be useful in narrowing down important amino acid residues, such as those involved in substrate and product specificity . ElucidaM. truncatula , showing sweetness 600 times greater than sucrose. In Thai folk medicine, the stem of A. myriophylla has been used as a substitute for licorice due to its sweetness . Similarly, M. truncatula CYP72A65v2, which catalyzed oxidation at putative C-21 (peak 1), also has a short side chain amino acid at both residues #149 and #398 . The substitutions Leu149Ala and Leu398Val in CYP72A63 did not alter the product regioselectivity resembling CYP72A65v2 (data not shown), suggesting that C-21β product regioselectivity may be controlled by amino acid residues other than residues #149 and #398. The amino acid residues involved in product regioselectivity of CYP72A65v2 remain to be determined; doing so would lead to a better understanding of the evolutionary diversification of product regioselectivity of tandem array on chromosome 8.A recent study showed that CYP72A69 from soybean, ynthesis . G. max V149L/V398L produced only trace amounts of C-30 carboxylated product , toward the C-30 position by rational protein engineering. The application of protein engineering in combination with metabolic engineering has been shown to significantly improve the production of natural products. Our findings will provide opportunities to further enhance the production of the valuable triterpene glycyrrhizin through rational protein engineering of C-30 oxidase.We identified the key amino acid residues, Leu149 and Leu398, controlling C-30 product regioselectivity in CYP72A63. The results reported here will enable us to improve the product specificity and produce desired compounds more efficiently. Rational engineering of C-30 oxidase by site saturation mutagenesis amino acid residues #149 and #398 may be useful for fine tuning of the methyl-30 functional group to a favorable position for enzyme catalysis, to improve product specificity and production yield. Alternatively, the results presented here suggested that it may be possible to redirect the product regioselectivity of ncbi.nlm.nih.gov/genbank/.The nucleotide sequences isolated in this study have been submitted to the GenBank at NCBI, MF, EF, and SS designed experiments. MF, SS, JT, MI, HSu, KO, and HSe performed experiments. MF, EF, SS, KO, and HSe wrote the article. EF, KS, and TM supervised the research. All authors discussed the results and approved the article.This study was supported in part by the Grants-in-Aid for Scientific Research of the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP19H02921; the Scientific Technique Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry, Japan; The Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN); The Special Fund from the Director of RIKEN Yokohama Institute; The RIKEN Rijicho Fund; and the Monbukagakusho Scholarship.Authors HSu and SS were employed by company Tokiwa Phytochemical Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The modular structural domains of multidomain RNA enzymes can often be dissected into separate domain RNAs and their noncovalent assembly can often reconstitute active enzymes. These properties are important to understand their basic characteristics and are useful for their application to RNA-based nanostructures. Bimolecular forms of bacterial RNase P ribozymes consisting of S-domain and C-domain RNAs are attractive as platforms for catalytic RNA nanostructures, but their S-domain/C-domain assembly was not optimized for this purpose. Through analysis and engineering of bimolecular forms of the two bacterial RNase P ribozymes, we constructed a chimeric ribozyme with improved catalytic ability and S-domain/C-domain assembly and developed a pair of bimolecular RNase P ribozymes the assembly of which was considerably orthogonal to each other. In the molecular evolution of protein enzymes, the emergence of more complex structures through gene fusion and less complex structures through gene fission are important events in the expansion of the diversity of protein structures and functions ,2. Gene In natural evolution as well as artificial engineering of functional RNAs with three-dimensional (3D) structures, fusion or fission of the RNA strands must generate multidomain RNAs or smaller structural RNAs, respectively. Smaller structural RNAs can also reconstitute the parent multidomain structures and their functions through noncovalent assembly ,13,14,15Escherichia coli and Bacillus subtilis ribozymes ..E. coli ucidated . TherefoT. thermophilus sys1 bimolecular ribozyme with that of the E. coli sys1 ribozyme  pairs of S-domain and C-domain RNAs (Ec-Sd/Tth-Cd and Tth-Cd/Ec-Sd chimeric ribozymes). Among the four sys1 bimolecular ribozymes, the E. coli ribozyme was poorly active and the Ec-Sd/Tth-Cd chimeric ribozyme showed no activity  makes a positive contribution to the catalytic activity of sys1 bimolecular ribozymes. We then performed an EMSA to examine whether the S-domain RNA and the C-domain RNA, each of which showed folding uniformity to yield a single band  and the Tth-Sd/Ec-Cd ribozyme (lanes 11 and 12) exhibited no stable S-domain/C-domain complexes, suggesting that the two active bimolecular sys1 ribozymes were structurally unstable  and T. thermophilus sys2 ribozyme (lanes 9 and 10) formed complexes between their S-domain and C-domain based on mobility shift of their S-domain RNA in the presence of the C-domain RNA (E. coli and Ec-Sd/Tth-Cd ribozymes) showed no mobility shift in S-domain RNA (lanes 5 and 7), suggesting no stable complex formation  receptor motif. The R(11nt) receptor motif specifically and strongly recognizes a GAAA loop . In the E. coli sys1 bimolecular ribozyme, the variant bearing the GAAA–R(11nt) pair cleaved 58% of the substrate pre-tRNA in the initial burst phase  pair also significantly improved the activities of the remaining three sys1 ribozymes . In the st phase B althougst phase B. The L9st phase C. Modulast phase  20,21].,21.E. coIn EMSA, complex formation of S-domain/C-domain bearing GAAA/R(11nt) pair was confirmed by the mobility shift of the labeled RNA component in the presence of the partner RNA , whereas mismatched P6 base pairs significantly reduced the activity. Therefore, we introduced the P6m1 base pairs into the Tth-Sd/Ec-Cd sys2 ribozyme  and ebRz-β (0.098 min−1), respectively  to determine whether its bimolecular format is promising as a platform for the development of supramolecular ribozyme polymers. Comparative analysis of the bimolecular T. thermophilus and E. coli ribozymes indicated that a chimeric bimolecular ribozyme consisting of the T. thermophilus S-domain RNA and E. coli C-domain (Tth-Sd/Ec-Cd sys2 ribozyme) had promising properties related to catalytic ability and complex formation. In EMSA analysis, no S-domain/C-domain complex of the E. coli sys2 ribozyme was observed (E. coli sys2 ribozyme may be induced by the association with pre-tRNA. The complex was detected in the T. thermophilus sys2 ribozyme (T. thermophilus sys2 ribozyme was less efficient than that of the E. coli sys2 especially under conditions of substrate excess (E. coli ribozyme at 50–60 °C but they were often less active than the E. coli ribozyme at 30–40 °C. The relatively poor activities of thermophilic RNase P ribozymes at 30–40 °C are partially due to slow product release [This study was performed to investigate A-type bacterial RNase P ribozyme from a thermophilic bacterium (observed . This obe excess B. These T. thermophilus ribozyme formed a stable S-domain/C-domain complex, while the E. coli ribozyme was distinctly more active at 37 °C. This was largely resolved in the chimeric Tth-Sd/Ec-Cd sys2 ribozyme because it formed a stable complex and exhibited promising catalytic activity. Although we did not exploit the molecular mechanism of action of the Tth-Sd/Ec-Cd sys2 ribozyme to exhibit promising structural and catalytic properties, these properties may be due to the T. thermophilus S-domain, which is rigid at 37 °C, and the E. coli C-domain, which must adapt its catalytic ability at 37 °C. Further improvement of the bimolecular Tth-Sd/Ec-Cd sys2 ribozyme was achieved by optimizing the interdomain tetraloop/receptor interaction between L9 in the S-domain and P1 in the C-domain. Modulation of tetraloop/receptor interactions has been employed successfully to redesign the structural and catalytic properties of group I intron ribozymes [The ibozymes . This stibozymes . This stInstallation of orthogonality to the RNA–RNA interface between the S-domain and C-domain relied mainly on the substitution of base pairs in the P6 element and was also assisted by the two sets of tetraloop/receptor interactions . The resT. thermophilus S-domain RNA and E. coli C-domain showed better properties with regard to assembly and catalysis than homologous S-domain/C-domain combinations. The assembly and catalytic properties of the chimeric ribozyme were improved by installation of the evolved form of GNRA/receptor interaction. Based on these modifications, a pair of orthogonal bimolecular RNase P ribozyme derivatives was developed. Although problems remain to be resolved, these derivatives would serve as advanced forms of RNase P ribozyme for the construction of catalytic RNA nanostructures. The strategies described in this study will be applicable to the engineering of other functional RNAs to elucidate their structure–function relationships and also for their application as RNA nanobiotechnology tools.A heterologous S-domain/C-domain combination consisting of