The surface macromolecules of probiotic bacteria play crucial roles in modulating immune responses in the host.Exopolysaccharide(EPS)from lactic acid bacteria(LAB)have been widely reported to exhibit immunomodulatory ...The surface macromolecules of probiotic bacteria play crucial roles in modulating immune responses in the host.Exopolysaccharide(EPS)from lactic acid bacteria(LAB)have been widely reported to exhibit immunomodulatory activity.In this study,the EPS biosynthesis gene cluster of Lacticaseibacillus paracasei S-NB was analyzed and a deletion mutant S-NBΔ7576(two genes S-NB_2175/wze and S-NB_2176/wzd were responsible for the chain length determination and export of EPS)was successfully constructed,resulting a 40.02%decrease in the production of EPS.The deletion of wze and wzd had little effect on the monosaccharide composition and major groups of the two EPS fractions(BEPS1 and BEPS2).Both BEPS1 and BEPS2 could inhibit the transcriptional level of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),inducible nitric oxide synthase(i NOS)and cyclooxygenase-2(COX-2)mRNA in RAW 264.7 cells induced by lipopolysaccharide(LPS),and enhance host immune tolerance via suppressing NF-κB and MAPK signaling.Notably,the S-NBΔ7576 mutant supplied with the BEPS1/BEPS2 exhibited more significant inhibition of cytokines production and the phosphorylation of p65 and c-Jun N-terminal kinase(JNK)in LPS-stimulated cells compared with the S-NBΔ7576 mutant alone.Our study provided the immunomodulatory effect of BEPS1 and BEPS2 from L.paracasei S-NB,in which the wze and wzd genes associated with EPS biosynthesis may play an important role.展开更多
Food allergy(FA) is an aberrant immune response triggered by the ingestion of a food antigen.Ovalbumin(OVA)-sensitized and challenged BALB/c mice were orally administered heat-killed(HK)-Lactobacillus paracasei JY56.I...Food allergy(FA) is an aberrant immune response triggered by the ingestion of a food antigen.Ovalbumin(OVA)-sensitized and challenged BALB/c mice were orally administered heat-killed(HK)-Lactobacillus paracasei JY56.In this work,HK-L.paracasei JY56 alleviated the FA-induced decrease in body weight and rectal temperature and reduced the allergy score.Serum analysis showed that HK-L.paracasei JY56 reduced the levels of specific antibodies(OVA-specific Immunoglobulin E(sIgE) and OVA-specific Immunoglobulin G(sIgG)) and allergic mediators(histamine and mast cell protease) in FA mice.In addition,HK-L.paracasei JY56 also could alleviate OVA-induced FA by suppressing T helper(Th)2 and Th17-type immune responses,which was evidenced by the regulation of splenic lymphocyte subpopulations and associated cytokine secretion.Moreover,jejunal histological analysis and intestinal barrier function related gene expression measurement were performed to verify the intestinal barrier repair of HK-L.paracasei JY56.Meanwhile,the TLR4/NF-κB inflammatory pathway activation was inhibited by HK-L.paracasei JY56 at gene and protein levels.Finally,HK-L.paracasei JY56 was performed to modulate the gut microbiota structure and increase the levels of short-chain fatty acids.In conclusion,HK-L.paracasei JY56 could alleviate OVA-induced FA in multiple ways,and this study provides a theoretical basis for the application of inactivated probiotics in functional foods for FA.展开更多
Many studies have shown that the development of allergic asthma is associated with intestinal microbiota dysbiosis.Based on the gut-lung axis theory,probiotic intervention may be a potential strategy for respiratory d...Many studies have shown that the development of allergic asthma is associated with intestinal microbiota dysbiosis.Based on the gut-lung axis theory,probiotic intervention may be a potential strategy for respiratory diseases.Lacticaseibacillus paracasei K56 was reported to regulate gut microbiota in mice.However,its effect on allergic asthma has not been reported.In this study,we investigated the effect of the K56 on ovalbumininduced asthma and its possible mechanisms.Our results showed that K56 reduced asthma symptoms and inflammatory cell infiltration in the lung of asthmatic mice.And K56 regulated the differentiation of helper T cells in the lung and intestine.Results from 16S r RNA gene sequencing showed that K56 prophylaxis significantly elevated the richness of Akkermansia and Burkholderia.Meanwhile,K56 abrogated the decrease in the levels of short-chain fatty acids(SCFAs)in the feces.In addition,a rebound in the m RNA expression of the SCFA receptors was observed after K56 prophylaxis.Notably,the regulatory T cell(Treg)frequencies and the FFAR3 levels were positively correlated.These results suggest that K56 could attenuate asthma,possibly by modulating the intestinal microbiota and regulating Treg differentiation through SCFA metabolized.Our study showed that K56 may be used as a probiotic to prevent pulmonary inflammation and dysbiosis of the intestinal microbiota in allergic asthma.展开更多
Compared with the rice-acid soup inoculated with single starter,the synergistically intensifi ed rice-acid soup inoculated with Lactobacillus paracasei H4-11(L.paracasei H4-11)and Kluyveromyces marxianus L1-1(K.marxia...Compared with the rice-acid soup inoculated with single starter,the synergistically intensifi ed rice-acid soup inoculated with Lactobacillus paracasei H4-11(L.paracasei H4-11)and Kluyveromyces marxianus L1-1(K.marxianus L1-1)contained more fl avor compounds.Organic acids mainly included L-lactic acid and the main volatile fl avor component was ethyl acetate.Moreover,the signal intensity of astringency and bitterness and the total concentration of volatile sulfur compounds were reduced.The combined analysis results of RNA sequencing(RNA-seq)technology and 4D label-free quantitative(4D LFQ)proteomics explained the fl avor formation pathways in rice-acid soup inoculated with L.paracasei H4-11 and K.marxianus L1-1.In L.paracasei H4-11,L-lactate dehydrogenase,phosphoglucomutase,acetate kinase,alcohol dehydrogenase and acetyl-CoA were up-regulated and D-lactate dehydrogenase and N-Acetyltransferase were down-regulated.In K.marxianus L1-1,Acetyl-CoA,acetaldehyde dehydrogenase,acyl-coenzyme A,N-acetyltransferase,and L-lactate dehydrogenase were up-regulated and hexokinase,alcohol dehydrogenase,and alcohol O-acetyltransferase were down-regulated.The above up-regulation and down-regulation synergistically promoted the formation of characteristic fl avor compounds(mainly L-lactic acid and ethyl acetate).Enzyme-linked immunoassay(ELISA)and parallel reaction monitoring(PRM)quantitative analysis respectively verifi ed that 5 key metabolic enzymes and 27 proteins in L.paracasei H4-11 and K.marxianus L1-1 were associated with the characteristic fl avor of rice-acid soup,as confi rmed by the quantitative results of 4D LFQ.展开更多
AIM： To investigate in symptomatic uncomplicated di- verticular disease the efficacy of symbiotics associated with a high-fibre diet on abdominal symptoms. METHODS： This study was a multicentre, 6-too ran- domized, ...AIM： To investigate in symptomatic uncomplicated di- verticular disease the efficacy of symbiotics associated with a high-fibre diet on abdominal symptoms. METHODS： This study was a multicentre, 6-too ran- domized, controlled, parallel-group intervention with a preceding 4-wk washout period. Consecutive out- patients with symptomatic uncomplicated diverticular disease, aged 40-80 years, evaluated in 4 Gastroenterology Units, were enrolled. Symptomatic uncomplicated diverticular disease patients were randomized to two treatment arms A or B. Treatment A （n = 24 patients） received 1 symbiotic sachet Flortec~ （Lactobacillus paracasei B21060） once daily plus high-fibre diet for 6 mo. Treatment B （n = 21 patients） received high-fibre diet alone for 6 rno. The primary endpoint was regres- sion of abdominal symptoms and change of symptom severity after 3 and 6 mo of treatment. RESULTS： In group A, the proportion of patients with abdominal pain 〈 24 h decreased from 100% at base- line to 35% and 25% after 3 and 6 mo, respectively （P 〈 0.001）. In group B the proportion of patients with this symptom decreased from 90.5% at baseline to 61.9% and 38.1% after 3 and 6 mo, respectively （P = 0.001）. Symptom improvement became statistically sig- nificant at 3 and 6 mo in group A and B, respectively. The proportion of patients with abdominal pain 〉24 h decreased from 60% to 20% then 5% after 3 and 6 too, respectively in group A （P 〈 0.001） and from 33.3% to 9.5% at both 3 and 6 mo in group B （P = 0.03）. In group A the proportion of patients with ab- dominal bloating significantly decreased from 95% to 60% after 3 mo, and remained stable （65%） at 6-mo follow-up （P = 0.005） while in group B, no significant changes in abdominal bloating was observed （P = 0.11）. After 6 mo of treatment, the mean visual analogic scale （VAS） values of both short-lasting abdominal pain （VAS, mean ± SD, group A： 4.6 ± 2.1 vs 2.2 ± 0.8, P = 0.02; group B： 4.6 ± 2.9 vs 2.0 ± 1.9, P = 0.03） and abdom- inal bloating （VAS, mean ± SD, group A： 5.3 ± 2.2 vs 3.0 ± 1.7, P = 0.005; group B： 5.3 ±3.2 vs 2.3 ± 1.9, P = 0.006） decreased in both groups, whilst the VAS values of prolonged abdominal pain decreased in the Flortec group, but remained unchanged in the high-fibre diet group （VAS, mean ± SD, group A： 6.5 ± 1.5 vs 4.5 ± 2.1, P = 0.052; group B： 4.5 ± 3.8 vs 5.5 ± 3.5）. CONCLUSION： A high-fibre diet is effective in relievingabdominal symptoms in symptomatic uncomplicated di- verticular disease. This treatment may be implemented by combining the high-fibre diet with Flortec~.展开更多
Background:Esophageal cancer(EC)is the seventh most prevalent cancer and the sixth most common cause of cancer-related mortalities worldwide.Camrelizumab,a monoclonal antibody,has demonstrated moderate efficacy in eso...Background:Esophageal cancer(EC)is the seventh most prevalent cancer and the sixth most common cause of cancer-related mortalities worldwide.Camrelizumab,a monoclonal antibody,has demonstrated moderate efficacy in esophageal squamous cell carcinoma(ESCC).Lactobacillus paracasei,a probiotic bacterium,has a complementary effect in immunotherapy.This study aimed to evaluate the combination of camrelizumab and L.paracasei for advanced ESCC.Methods:This single-arm,single-center,exploratory trial was conducted at the First Affiliated Hospital of Zhengzhou University,Zhengzhou,China.Eligible patients received 200 mg camrelizumab biweekly and two bags of L.paracasei twice daily.The primary endpoint was progression-free survival(PFS),and the secondary endpoints were disease control rate(DCR),overall survival(OS),objective response rate(ORR),and adverse events(AEs).Results:From May 2020 to October 2022,ten patients with advanced ESCC who did not respond to first-line therapy were admitted.At the data cutoff date(August 9,2023),the median follow-up duration was 12 months.Two of 10(20%)achieved objective responses.The median survival was 7.5 months and the median OS was not reached.Grade 3 treatment-related AEs occurred in two of the 10 patients(20%).No serious treatmentrelated AEs or deaths occurred.Conclusions:Camrelizumab combined with L.paracasei showed favorable anticancer activity and may be a viable second-line treatment for patients with ESCC.展开更多
Lacticaseibacillus paracasei is a food-grade lactic acid bacteria(LAB)that plays an important role in improving the human intestinal tract.However,effective gene modification tools are not reported in L.paracasei CGMC...Lacticaseibacillus paracasei is a food-grade lactic acid bacteria(LAB)that plays an important role in improving the human intestinal tract.However,effective gene modification tools are not reported in L.paracasei CGMCC4691.Here,we constructed clustered regularly interspaced short palindromic repeat(CRISPR)genetic editing tools by screening and optimizing promoters of Cas9 and sgRNA,respectively.To verify the availability of this system,single gene mutants(4691ΔAF91_08090 and 4691ΔAF91_05150)and double gene mutants(4691ΔAF91_08090ΔAF91_05150)were obtained,the editing efficiency was 54.1%and 90%after replacing P1 promoter,respectively.In addition,the addition of 5-fluorouracil(5-Fu)was lethal to wild strains compared to mutants,therefore,the gene function of mutants was verified by growth phenotype.The study realizes efficient application of the Cas9 system in L.paracasei CGMCC4691,provides a feasible optimization method for gene editing,and lays the foundation for the investigation of genetic function mechanism.展开更多
基金co-financed by the International Science&Technology Cooperation Program of Hainan Province(GHYF2023009)Jiangsu Provincial Key Research and Development Program(BE2022325)+1 种基金Postgraduate Research&Practice Innovation Program of Jiangsu Province(KYCX23_0777)Qing Lan Project of Jiangsu Province and Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)。
文摘The surface macromolecules of probiotic bacteria play crucial roles in modulating immune responses in the host.Exopolysaccharide(EPS)from lactic acid bacteria(LAB)have been widely reported to exhibit immunomodulatory activity.In this study,the EPS biosynthesis gene cluster of Lacticaseibacillus paracasei S-NB was analyzed and a deletion mutant S-NBΔ7576(two genes S-NB_2175/wze and S-NB_2176/wzd were responsible for the chain length determination and export of EPS)was successfully constructed,resulting a 40.02%decrease in the production of EPS.The deletion of wze and wzd had little effect on the monosaccharide composition and major groups of the two EPS fractions(BEPS1 and BEPS2).Both BEPS1 and BEPS2 could inhibit the transcriptional level of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),inducible nitric oxide synthase(i NOS)and cyclooxygenase-2(COX-2)mRNA in RAW 264.7 cells induced by lipopolysaccharide(LPS),and enhance host immune tolerance via suppressing NF-κB and MAPK signaling.Notably,the S-NBΔ7576 mutant supplied with the BEPS1/BEPS2 exhibited more significant inhibition of cytokines production and the phosphorylation of p65 and c-Jun N-terminal kinase(JNK)in LPS-stimulated cells compared with the S-NBΔ7576 mutant alone.Our study provided the immunomodulatory effect of BEPS1 and BEPS2 from L.paracasei S-NB,in which the wze and wzd genes associated with EPS biosynthesis may play an important role.
基金supported by the Natural Science Foundation of Heilongjiang Province of China (LH2023C033)。
文摘Food allergy(FA) is an aberrant immune response triggered by the ingestion of a food antigen.Ovalbumin(OVA)-sensitized and challenged BALB/c mice were orally administered heat-killed(HK)-Lactobacillus paracasei JY56.In this work,HK-L.paracasei JY56 alleviated the FA-induced decrease in body weight and rectal temperature and reduced the allergy score.Serum analysis showed that HK-L.paracasei JY56 reduced the levels of specific antibodies(OVA-specific Immunoglobulin E(sIgE) and OVA-specific Immunoglobulin G(sIgG)) and allergic mediators(histamine and mast cell protease) in FA mice.In addition,HK-L.paracasei JY56 also could alleviate OVA-induced FA by suppressing T helper(Th)2 and Th17-type immune responses,which was evidenced by the regulation of splenic lymphocyte subpopulations and associated cytokine secretion.Moreover,jejunal histological analysis and intestinal barrier function related gene expression measurement were performed to verify the intestinal barrier repair of HK-L.paracasei JY56.Meanwhile,the TLR4/NF-κB inflammatory pathway activation was inhibited by HK-L.paracasei JY56 at gene and protein levels.Finally,HK-L.paracasei JY56 was performed to modulate the gut microbiota structure and increase the levels of short-chain fatty acids.In conclusion,HK-L.paracasei JY56 could alleviate OVA-induced FA in multiple ways,and this study provides a theoretical basis for the application of inactivated probiotics in functional foods for FA.
基金supported by grants from the National Natural Science Foundation of China(82174113,81473656)National Center of Technology Innovation for Dairy(No.2023-JSGG-15)。
文摘Many studies have shown that the development of allergic asthma is associated with intestinal microbiota dysbiosis.Based on the gut-lung axis theory,probiotic intervention may be a potential strategy for respiratory diseases.Lacticaseibacillus paracasei K56 was reported to regulate gut microbiota in mice.However,its effect on allergic asthma has not been reported.In this study,we investigated the effect of the K56 on ovalbumininduced asthma and its possible mechanisms.Our results showed that K56 reduced asthma symptoms and inflammatory cell infiltration in the lung of asthmatic mice.And K56 regulated the differentiation of helper T cells in the lung and intestine.Results from 16S r RNA gene sequencing showed that K56 prophylaxis significantly elevated the richness of Akkermansia and Burkholderia.Meanwhile,K56 abrogated the decrease in the levels of short-chain fatty acids(SCFAs)in the feces.In addition,a rebound in the m RNA expression of the SCFA receptors was observed after K56 prophylaxis.Notably,the regulatory T cell(Treg)frequencies and the FFAR3 levels were positively correlated.These results suggest that K56 could attenuate asthma,possibly by modulating the intestinal microbiota and regulating Treg differentiation through SCFA metabolized.Our study showed that K56 may be used as a probiotic to prevent pulmonary inflammation and dysbiosis of the intestinal microbiota in allergic asthma.
基金funded by National Natural Science Foundation of China(32060530)Guizhou University,Gui Da Te Gang He Zi(2022)39,Science and Technology Project of Guizhou Province,Qian Ke He Zhicheng[2022]Zhongdian 001-2,Qian Ke He Zhicheng[2022]Zhongdian 003-3+1 种基金Industry-University-Research Cooperation Project of Guizhou University(701/700465172217)China Scholarship Council(201906670006).
文摘Compared with the rice-acid soup inoculated with single starter,the synergistically intensifi ed rice-acid soup inoculated with Lactobacillus paracasei H4-11(L.paracasei H4-11)and Kluyveromyces marxianus L1-1(K.marxianus L1-1)contained more fl avor compounds.Organic acids mainly included L-lactic acid and the main volatile fl avor component was ethyl acetate.Moreover,the signal intensity of astringency and bitterness and the total concentration of volatile sulfur compounds were reduced.The combined analysis results of RNA sequencing(RNA-seq)technology and 4D label-free quantitative(4D LFQ)proteomics explained the fl avor formation pathways in rice-acid soup inoculated with L.paracasei H4-11 and K.marxianus L1-1.In L.paracasei H4-11,L-lactate dehydrogenase,phosphoglucomutase,acetate kinase,alcohol dehydrogenase and acetyl-CoA were up-regulated and D-lactate dehydrogenase and N-Acetyltransferase were down-regulated.In K.marxianus L1-1,Acetyl-CoA,acetaldehyde dehydrogenase,acyl-coenzyme A,N-acetyltransferase,and L-lactate dehydrogenase were up-regulated and hexokinase,alcohol dehydrogenase,and alcohol O-acetyltransferase were down-regulated.The above up-regulation and down-regulation synergistically promoted the formation of characteristic fl avor compounds(mainly L-lactic acid and ethyl acetate).Enzyme-linked immunoassay(ELISA)and parallel reaction monitoring(PRM)quantitative analysis respectively verifi ed that 5 key metabolic enzymes and 27 proteins in L.paracasei H4-11 and K.marxianus L1-1 were associated with the characteristic fl avor of rice-acid soup,as confi rmed by the quantitative results of 4D LFQ.
基金Supported by The study was in part supported by Bracco Spa(Milan,Italy)
文摘AIM： To investigate in symptomatic uncomplicated di- verticular disease the efficacy of symbiotics associated with a high-fibre diet on abdominal symptoms. METHODS： This study was a multicentre, 6-too ran- domized, controlled, parallel-group intervention with a preceding 4-wk washout period. Consecutive out- patients with symptomatic uncomplicated diverticular disease, aged 40-80 years, evaluated in 4 Gastroenterology Units, were enrolled. Symptomatic uncomplicated diverticular disease patients were randomized to two treatment arms A or B. Treatment A （n = 24 patients） received 1 symbiotic sachet Flortec~ （Lactobacillus paracasei B21060） once daily plus high-fibre diet for 6 mo. Treatment B （n = 21 patients） received high-fibre diet alone for 6 rno. The primary endpoint was regres- sion of abdominal symptoms and change of symptom severity after 3 and 6 mo of treatment. RESULTS： In group A, the proportion of patients with abdominal pain 〈 24 h decreased from 100% at base- line to 35% and 25% after 3 and 6 mo, respectively （P 〈 0.001）. In group B the proportion of patients with this symptom decreased from 90.5% at baseline to 61.9% and 38.1% after 3 and 6 mo, respectively （P = 0.001）. Symptom improvement became statistically sig- nificant at 3 and 6 mo in group A and B, respectively. The proportion of patients with abdominal pain 〉24 h decreased from 60% to 20% then 5% after 3 and 6 too, respectively in group A （P 〈 0.001） and from 33.3% to 9.5% at both 3 and 6 mo in group B （P = 0.03）. In group A the proportion of patients with ab- dominal bloating significantly decreased from 95% to 60% after 3 mo, and remained stable （65%） at 6-mo follow-up （P = 0.005） while in group B, no significant changes in abdominal bloating was observed （P = 0.11）. After 6 mo of treatment, the mean visual analogic scale （VAS） values of both short-lasting abdominal pain （VAS, mean ± SD, group A： 4.6 ± 2.1 vs 2.2 ± 0.8, P = 0.02; group B： 4.6 ± 2.9 vs 2.0 ± 1.9, P = 0.03） and abdom- inal bloating （VAS, mean ± SD, group A： 5.3 ± 2.2 vs 3.0 ± 1.7, P = 0.005; group B： 5.3 ±3.2 vs 2.3 ± 1.9, P = 0.006） decreased in both groups, whilst the VAS values of prolonged abdominal pain decreased in the Flortec group, but remained unchanged in the high-fibre diet group （VAS, mean ± SD, group A： 6.5 ± 1.5 vs 4.5 ± 2.1, P = 0.052; group B： 4.5 ± 3.8 vs 5.5 ± 3.5）. CONCLUSION： A high-fibre diet is effective in relievingabdominal symptoms in symptomatic uncomplicated di- verticular disease. This treatment may be implemented by combining the high-fibre diet with Flortec~.
基金supported by the Henan Provincial Health Commission Program for Young and Middle-aged Discipline Leaders(No.HNSWJW-2022026)the Outstanding Youth Talent Program of the Henan Provincial Health Commission(No.YXKC2022039).
文摘Background:Esophageal cancer(EC)is the seventh most prevalent cancer and the sixth most common cause of cancer-related mortalities worldwide.Camrelizumab,a monoclonal antibody,has demonstrated moderate efficacy in esophageal squamous cell carcinoma(ESCC).Lactobacillus paracasei,a probiotic bacterium,has a complementary effect in immunotherapy.This study aimed to evaluate the combination of camrelizumab and L.paracasei for advanced ESCC.Methods:This single-arm,single-center,exploratory trial was conducted at the First Affiliated Hospital of Zhengzhou University,Zhengzhou,China.Eligible patients received 200 mg camrelizumab biweekly and two bags of L.paracasei twice daily.The primary endpoint was progression-free survival(PFS),and the secondary endpoints were disease control rate(DCR),overall survival(OS),objective response rate(ORR),and adverse events(AEs).Results:From May 2020 to October 2022,ten patients with advanced ESCC who did not respond to first-line therapy were admitted.At the data cutoff date(August 9,2023),the median follow-up duration was 12 months.Two of 10(20%)achieved objective responses.The median survival was 7.5 months and the median OS was not reached.Grade 3 treatment-related AEs occurred in two of the 10 patients(20%).No serious treatmentrelated AEs or deaths occurred.Conclusions:Camrelizumab combined with L.paracasei showed favorable anticancer activity and may be a viable second-line treatment for patients with ESCC.
基金supported by the National Natural Science Foundation of China(32272361)National Science Fundation for Distinguished Young Scholars(32025029).
文摘Lacticaseibacillus paracasei is a food-grade lactic acid bacteria(LAB)that plays an important role in improving the human intestinal tract.However,effective gene modification tools are not reported in L.paracasei CGMCC4691.Here,we constructed clustered regularly interspaced short palindromic repeat(CRISPR)genetic editing tools by screening and optimizing promoters of Cas9 and sgRNA,respectively.To verify the availability of this system,single gene mutants(4691ΔAF91_08090 and 4691ΔAF91_05150)and double gene mutants(4691ΔAF91_08090ΔAF91_05150)were obtained,the editing efficiency was 54.1%and 90%after replacing P1 promoter,respectively.In addition,the addition of 5-fluorouracil(5-Fu)was lethal to wild strains compared to mutants,therefore,the gene function of mutants was verified by growth phenotype.The study realizes efficient application of the Cas9 system in L.paracasei CGMCC4691,provides a feasible optimization method for gene editing,and lays the foundation for the investigation of genetic function mechanism.
