Genome characteristics reveal the impact of lichenization on lichen-forming fungus Endocarpon pusillum Hedwig (Verrucariales, Ascomycota).

BACKGROUND: Lichen is a classic mutualistic organism and the lichenization is one of the fungal symbioses. The lichen-forming fungus Endocarpon pusillum is living in symbiosis with the green alga Diplosphaera chodatii Bialsuknia as a lichen in the arid regions. RESULTS: 454 and Illumina technologies were used to sequence the genome of E. pusillum. A total of 9,285 genes were annotated in the 37.5 Mb genome of E. pusillum. Analyses of the genes provided direct molecular evidence for certain natural characteristics, such as homothallic reproduction and drought-tolerance. Comparative genomics analysis indicated that the expansion and contraction of some protein families in the E. pusillum genome reflect the specific relationship with its photosynthetic partner (D. chodatii). Co-culture experiments using the lichen-forming fungus E. pusillum and its algal partner allowed the functional identification of genes involved in the nitrogen and carbon transfer between both symbionts, and three lectins without signal peptide domains were found to be essential for the symbiotic recognition in the lichen; interestingly, the ratio of the biomass of both lichen-forming fungus and its photosynthetic partner and their contact time were found to be important for the interaction between these two symbionts. CONCLUSIONS: The present study lays a genomic analysis of the lichen-forming fungus E. pusillum for demonstrating its general biological features and the traits of the interaction between this fungus and its photosynthetic partner D. chodatii, and will provide research basis for investigating the nature of its drought resistance and symbiosis.

The absence of the ERBB4 hotspot mutations in melanomas in patients from southern China.

V-erb-a erythroblastic leukemia viral oncogene homolog 4 (ERBB4) has been reported to be somatically mutated in 19% of melanoma cases. To investigate the prevalence of ERBB4 mutations in melanoma patients from southern China, we analyzed 117 formalin-fixed, paraffin-embedded melanoma samples archived in the Sun Yat-sen University Cancer Center. A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform was used to screen for mutations. No ERBB4 hotspot mutations were detected. Our results indicate that ERBB4 mutations may play a limited role in melanomas in China; therefore, targeting the ERBB4 mutation in melanoma patients from southern China may not be a promising strategy.

Research progress on the relationship between Paneth cells-susceptibility genes, intestinal microecology and inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a disorder of the immune system and intestinal microecosystem caused by environmental factors in genetically susceptible people. Paneth cells (PCs) play a central role in IBD pathogenesis, especially in Crohn's disease development, and their morphology, number and function are regulated by susceptibility genes. In the intestine, PCs participate in the formation of the stem cell microenvironment by secreting antibacterial particles and play a role in helping maintain the intestinal microecology and intestinal mucosal homeostasis. Moreover, PC proliferation and maturation depend on symbiotic flora in the intestine. This paper describes the interactions among susceptibility genes, PCs and intestinal microecology and their effects on IBD occurrence and development.

Extraction optimization and identification of four advanced glycation-end products inhibitors from lotus leaves and interaction mechanism analysis.

Previous research indicated lotus leaves extract could effectively inhibit advanced glycation end-products (AGEs) formation, but the optimal extraction condition, bio-active compounds and interaction mechanism remain unclear. The current study was designed to optimize the extraction parameters of AGEs inhibitors from lotus leaves by bio-activity-guided approach. The bio-active compounds were enriched and identified, the interaction mechanisms of inhibitors with ovalbumin (OVA) were investigated by fluorescence spectroscopy and molecular docking. The optimum extraction parameters were solid-liquid ratio of 1:30, ethanol concentration of 70 %, ultrasonic time of 40 min, temperature of 50 °C, and power of 400 W. Isoquercitrin, hyperoside, astragalin, and trifolin were identified from the 80 % ethanol fraction of lotus leaves (80HY). Hyperoside and isoquercitrin were dominant AGEs inhibitors and accounted for 55.97 % of 80HY. Isoquercitrin, hyperoside, trifolin interacted with OVA via the same mechanism, hyperoside exhibited the strongest affinity, trifolin caused the most conformational changes.

[Value of CIK in the treatment of TACE combined with RFA for HCC in long-term survival and prognostic analysis].

OBJECTIVE: To assess the long-term efficacy and investigate the prognostic factors of cytokine-induced killer (CIK) combined with the sequential transarterial chemoembolization (TACE) and radiofrequency ablation (RFA) on hepatocellular carcinoma (HCC). METHOD: A total of 95 HCC patients with the informed consents received TACE combined with RFA, 48 cases of which accepted the CIK via intravenous drop infusion for more than 6 times (study group) while the other 47 cases were enrolled in control group. The following-up duration was more than 3 years. Primary endpoint was the overall survival (OS) and the secondary endpoint was the disease-free survival (DFS). RESULTS: 76 patients in all (38 in study group, 38 in control group) complied with the study and follow-up (44 months in median, 10-88 months). No mortality and serve complications were observed in both groups. The ratio for patients with DFS over 1-year, 3-year and 5-year were 79%, 26% and 16% (28 months in median and 32.3 months in mean) while 71%, 21% and 8% (22 months in median and 23.1 months in mean) for the control group. There was significant difference between the two groups (P=0.001). For the OS, the ratio for 1-year, 3-year and 5-year in the study group were 92%, 53% and 26% (38 months in median and 42.5 in mean) and 89%, 42% and 24% (35 months in median and 37 in mean). No significant difference was observed in both groups. ECOG performance status, Hepatitis B virus infection and treatment were the prognostic factors for DFS while ECOG performance status was the only prognosis for OS. CONCLUSION: CIK infusion basing on the TACE combined with RFA can control the recurrence of HCC, decrease the times of TACE or RFA.

The density of macrophages in the invasive front is inversely correlated to liver metastasis in colon cancer.

BACKGROUND: Although an abundance of evidence has indicated that tumor-associated macrophages (TAMs) are associated with a favorable prognosis in patients with colon cancer, it is still unknown how TAMs exert a protective effect. This study examined whether TAMs are involved in hepatic metastasis of colon cancer. MATERIALS AND METHODS: One hundred and sixty cases of pathologically-confirmed specimens were obtained from colon carcinoma patients with TNM stage IIIB and IV between January 1997 and July 2004 at the Cancer Center of Sun Yat-Sen University. The density of macrophages in the invasive front (CD68TFHotspot) was scored with an immunohistochemical assay. The relationship between the CD68TFHotspot and the clinicopathologic parameters, the potential of hepatic metastasis, and the 5-year survival rate were analyzed. RESULTS: TAMs were associated with the incidence of hepatic metastasis and the 5-year survival rate in patients with colon cancers. Both univariate and multivariate analyses revealed that the CD68TFHotspot was independently prognostic of survival. A higher 5-year survival rate among patients with stage IIIB after radical resection occurred in patients with a higher macrophage infiltration in the invasive front (81.0%) than in those with a lower macrophage infiltration (48.6%). Most importantly, the CD68TFHotspot was associated with both the potential of hepatic metastasis and the interval between colon resection and the occurrence of hepatic metastasis. CONCLUSION: This study showed evidence that TAMs infiltrated in the invasive front are associated with improvement in both hepatic metastasis and overall survival in colon cancer, implying that TAMs have protective potential in colon cancers and might serve as a novel therapeutic target.

Serum alpha-fetoprotein measurement in predicting clinical outcome related to autologous cytokine-induced killer cells in patients with hepatocellular carcinoma undergone minimally invasive therapy.

BACKGROUND AND OBJECTIVE: In patients with hepatocellular carcinoma (HCC) receiving potentially curative minimally invasive therapy, autologous cytokine-induced killer (CIK) cells were used to reduce recurrence. In this study we observed the changes in serum alpha-fetoprotein (AFP) after the treatment with CIK cells to explore if AFP could serve as a marker for predicting immunotherapeutic clinical outcome. METHODS: A total of 122 patients with HCC and elevated AFP (>25 ng/mL) received a curative treatment of transcatheter arterial chemoembolization (TACE) plus radiofrequency ablation (RFA) at the Sun Yat-sen University Cancer Center. Of these patients, 83 patients without residual tumor or extrahepatic metastasis and with AFP level less than 1.5 times the normal range (AFP<37.5 ng/mL) were randomly assigned to the study group (n=42) and the control group (n=41). In the study group, CIK cells were transfused intravenously or via common hepatic arteries every week for at least 4 times, and the T-lymphocyte subset data before and after CIK cell infusions was examined by flow cytometry. All the two groups of patients were screened by tomography every 2 months to observe tumor recurrence. Serum AFP was collected at baseline and at different time points after treatment in parallel with radiologic response and clinical outcome. RESULTS: Two patients in the control group were lost to follow-up after treatment. After CIK cell infusions, the downtrend of the AFP level was observed in the study group and not in the control group. There was a significant difference in the level of AFP between different time points after CIK infusions in both groups. The 1-year recurrence rate was 7.14% for the study group and 23.1% for the control group (P=0.044). In subgroup analysis, for patients with a slightly high level of AFP (25 ng/mL

Mitigation of isoquercitrin on ß-lactoglobulin glycation: Insight into the mechanisms by mass spectrometry and interaction analysis.

Formation of advanced glycation end products (AGEs) on foods imposes threats to human health after intaking. This research firstly evaluated the inhibition of isoquercitrin on ß-lactoglobulin (ß-Lg) glycation, the mechanisms were elucidated by fluorescence spectroscopy, Orbitrap MSn and molecular docking. Fluorescence spectra indicated that isoquercitrin effectively alleviated the formation of AGEs, it could stabilize the conformation structure of glycated ß-Lg (G-ß-Lg), change the micro-environment in the vicinity of chromophores. SDS-PAGE analysis revealed the suppressed cross-linking of G-ß-Lg induced by isoquercitrin. The number of glycation site detected on G-ß-Lg was reduced from ten to eight after the addition of isoquercitrin, and the relative glycation degree of substitution of per site (RGDSP) of most glycation sites were also greatly decreased. As indicated by intermolecular interaction, isoquercitrin quenched the fluorescence of ß-Lg via a static mechanism, and their combination is an endothermic processing mainly derived by hydrophobic interaction, hydrogen bonds, and van der Waals forces. Isoquercitrin interacted with ß-Lg to form an equimolar complex, and one hydrogen bond was formed between isoquercitrin and Lys69 (4.96 Å). Above results proved that isoquercitrin can be a promising anti-glycation agent used in food system to prevent the formation of harmful glycation products.

[Determination of trace elements in electrical absorption prospecting polyform sample by inductively coupled plasma mass spectrometry].

An ICP-MS method was established for the determination of sixteen trace elements, V, Cr, Mn, Co, Ni, Cu, Zn, Ga, Nb, Mo, Ag, Cd, Au, Tl, Pb and Bi in electrical absorption prospecting polyform. Three methods for polyform samples (ashing method, extraction by HNO3 + H2O2 and digestion with aqua regia) were compared and the results showed that the second method is the best one. The best operational paramenters of X series ICP-MS were confirmed, the inner standard 103Rh and 185Re were selected for the determination of elements, and analysis of isotopes interference correction equations was established. Satisfactory linearity of working curves of the sixteen trace elements was obtained, giving all their correlation coefficients over 0.999 8. The determination limit of the analytes was in the range of 0.001-2.2 microg x g(-1). The precision was 1.39%-4.84%, and the recoveries were between 94.86% and 105.2%. The method is sensitive, quick and simple and has been applied to the analysis of a great number of polyform samples.

A study of the genetic variability of Rhizoplaca chrysoleuca using DNA sequences and secondary metabolic substances.

Specimens of Rhizoplaca chrysoleuca from Mount Wuling can be divided into two distinct groups based on obvious differences in morphological characters. Here we investigated 26 specimens of R. chrysoleuca from Mount Wuling, 10 specimens of this species from other areas and seven specimens of other Rhizoplaca species by analyzing morphology, chemistry and genetics. Nine chemotypes were detected among the specimens of R. chrysoleuca from Mount Wuling, and five of them were reported for the first time. Based on the ITS phylogenetic analysis, the chemotypes and the insertion distribution patterns in SSU rDNA, the samples of R. chrysoleuca from Mount Wuling were grouped in two distinct clades corresponding to two phenotypic groups and no gene flow was detected between these two groups. Our results establish all individuals of Rhizoplaca chrysoleuca are conspecific although some populations have been isolated on Mount Wuling, indicating that they are in the process of speciation. Our study also reveals that the relationships between genotypes and chemotypes are complicated and should be avoided, and we instead recommend using single individuals or few individuals from the same site to represent the population or whole species in systematics study. The results also indicate that Rhizoplaca chrysoleuca might provide a good model for studying the speciation of saxicolous lichenized fungi.

[Cytokine-induced killer cell fusion to lower recurrence of hepatocellular carcinoma after transcatheter arterial chemoembolization sequentially combined with radiofrequency ablation: a randomized trial].

OBJECTIVE: To ed evaluate the clinical effects of autologous cytokine-induced killer cell (CIK) fusion to lower recurrence of primary hepatocellular carcinoma (HCC) and the anti-hepatitis B virus (HBV) effect after transcatheter arterial chemoembolization (TACE) sequentially combined with radiofrequency ablation (RFA). METHODS: Sixty-four HCC patients underwent TACE sequentially combined with RFA without residual tumor or extrahepatic metastasis were randomly divided into 2 groups: study group (n = 33), receiving autologous CIK fusion of the dose of (1.1 - 1.5) x 10(10) via the peripheral vein or hepatic artery, firstly once every 3 - 4 weeks for 4 times, and then once every 4 weeks for 4 times; and control group (n = 31). All patients were followed up for 1 year. RESULTS: In the study group, 29 patients (29/33) were recurrence-free during the 1 year follow-up, 3 had recurrence in the liver 5, 6, and 7 months later respectively, and 1 patient had lymphoadenopathy in the hepatic portal 9 months later. In the control group, 23 patients (68.01%) were recurrence-free, 7 had recurrence in the liver within 1 year, and 1 had lung metastasis 11 months later. In the study group, the number of the patients with the HBV DNA content < 1 x 10(3) before treatment was 19, and increased to 29 after the treatment; 2 of the 19 patients who were HBsAg positive before the treatment became HBsAg negative after the treatment, and number of the patients with the HBV DNA content of 10(3) - 10(4) was 3 before the treatment, and became 13 after the treatment among which 1 patient had his HBV DNA content dropping from 1.6 x 10(7) to 1.6 x 10(4). In the control group, only 1 patient showed his HBV DNA content dropping from 1.1 x 10(5) to below 10(3). CONCLUSION: Capable of reducing recurrence, prolonging the recurrence-free span, and attacking HBV, autologous CIK fusion after TACE sequentially combined with RFA is an effective novel therapeutic strategy for HCC.

The complete mitochondrial genome of Bombyx mori strain Huayu (Lepidoptera: Bombycidae).

The complete mitochondrial genome of Bombyx mori strain Huayu (Lepidoptera: Bombycidae) is determined in this study. The genome was 15,666 bp long, with 37 typical animal mitochondrial genes and 1 non-coding A + T-rich region. Its gene content and order were identical to those of other lepidopteran mitochondrial genomes. All protein-coding genes (PCGs) were initiated by ATN codons except for the COI gene, which began with uncertained codon. Eleven PCGs stopped with termination codon TAA, whereas the COI and COII genes ended with single T. All tRNAs have typical structures of insect mitochondrial tRNAs. The 494 bp AT-rich region contains several features common to other lepidopterans, such as the motif ATAGA followed by an 18 bp poly-T stretch and an 11 bp poly-A element upstream of transfer RNA M (trnM) gene.

The complete mitochondrial genome of Bombyx mori strain Yu5 (Lepidoptera: Bombycidae).

The complete mitochondrial genome of Bombyx mori strain Yu5 (Lepidoptera: Bombycidae) is a circular molecule of 15,644 bp in length, containing 37 typical coding genes and one non-coding AT-rich region. The overall composition of the mitogenome was A (43.07%), G (7.34%), C (11.33%), and T (38.26%). Its gene order and content were identical to the common type found in most insect mitogenomes. All protein coding genes (PCGs) started with a typical ATN initiation codon, except for the cox1 gene, which began with CGA codon. Eleven genes used standard complete termination codon TAA, whereas the cox1 and cox2 genes ended with single T. All tRNA genes displayed typical secondary cloverleaf structures as those of other insects. Additionally, the 494 bp long AT-rich region contained several structures common to the other lepidopterons, such as some structures of repeated motifs and microsatellite-like elements and a poly-A element upstream of transfer RNA M (trnM) gene.

The complete mitochondrial genome of Bombyx mori strain Jin6 (Lepidoptera: Bombycidae).

In this study, the complete mitochondrial genome of Bombyx mori strain Jin6 (Lepidoptera: Bombycidae) has been reported for the first time. It was a circular molecule of 15,648 bp in length, containing 37 typical coding genes and one non-coding AT-rich region. The overall composition of the mitogenome was A (43.05%), G (7.30%), C (11.35%), and T (38.29%). Its gene order and content were identical to the common type found in most insect mitogenomes. All protein coding genes (PCGs) started with a typical ATN initiation codon, except for the cox1 gene, which began with CGA codon. Eleven genes used standard complete termination codon TAA, whereas the cox1 and cox2 genes ended with single T. All tRNA genes displayed typical secondary cloverleaf structures as those of other insects. Additionally, the 494 bp long AT-rich region contained several structures common to the other lepidopterons, such as some structures of repeated motifs and microsatellite-like elements.

The complete mitochondrial genome of Bombyx mori strain Yu39 (Lepidoptera: Bombycidae).

The complete mitochondrial genome of Bombyx mori strain Yu39 (Lepidoptera: Bombycidae) is a circular molecule of 15,652 bp in length, containing 37 typical mitochondrial genes: 13 protein-coding genes (PCGs), 22 transfer RNAs, 2 ribosomal RNAs and a non-coding AT-rich region. Its gene order and arrangement are identical to the common type found in most insect mitogenomes. All PCGs start with a typical ATN codon, except for the cox1 gene, which begins with uncertained codon. All PCGs terminate in the common stop codon TAA, except for the cox1 and cox2, which use single T as their stop codons. The non-coding AT-rich region is 494-bp long, located between rrnS and trnM genes. It contains some structures of repeated motifs and microsatellite-like elements characteristic of the other lepidopterons.

The complete mitochondrial genome of Antheraea pernyi strain Yu6 (Lepidoptera: Saturniidae).

In this study, the complete mitochondrial genome of Antheraea pernyi strain Yu6 (Lepidoptera: Saturniidae) has been reported for the first time. It is a circular molecule of 15,569 bp in length, containing 37 typical coding genes and 1 non-coding AT-rich region. The overall composition of the mitogenome is A (39.27%), G (7.71%), C (12.01%), and T (41.01%). Its gene order and content are identical to the common type found in most insect mitogenomes. All protein coding genes (PCGs) start with a typical ATN initiation codon, except for the cox1 gene, which begins with TTAG codon. Nine genes used standard complete termination codon TAA, whereas the cox1, cox2, nad3, and nad5 genes end with single T. All tRNAs display typical secondary cloverleaf structures as those of other insects. Additionally, the 552 bp long AT-rich region contained several structures common to the other lepidopterons, such as some structures of repeated motifs and microsatellite-like elements. Phylogenetic analysis showed that the Antheraea pernyi Yu6 was close to Saturniidae.

Salvage therapy of gemcitabine plus endostar significantly improves progression-free survival (PFS) with platinum-resistant recurrent epithelial ovarian cancer.

Anti-angiogenic agents have played crucial roles in the treatment of ovarian cancer in recent years, but potential benefits of endostatin have been largely unexplored. The present retrospective study evaluated its efficacy and toxicity with two cohorts of patients with platinum-resistant recurrent ovarian cancer. One cohort received gemcitabine plus endostar (rh-endostatin), and the second cohort received gemcitabine regimen alone, with totals of 31 and 27 patients, respectively. The main endpoints were disease control rate (DCR), PFS, overall survival (OS) and safety. There were statistically significant differences in DCR (70.9% vs. 40.7%; P = 0.02) and PFS (6.3 months vs. 3.2 months, P = 0.001) between the two cohorts. Though the endostar cohort also improved median OS by 2.1 months, there was no statistically significant difference compared with gemcitabine alone cohort in this case (12.5 months vs. 10.4 months, P = 0.201). Treatment was well tolerated for most patients, and toxicity of endostar was negligible. Gemcitabine plus endostar significantly improved the prognosis in patients with platinum-resistant recurrent ovarian cancer, especially in those with malignant effusion. The endostar- containing regimen is recommended in this setting.

Cytokine-induced killer cells in combination with transcatheter arterial chemoembolization and radiofrequency ablation for hepatocellular carcinoma patients.

This study evaluated the clinical efficacy of autologous cytokine-induced killer (CIK) cell transfusion in combination with transcatheter arterial chemoembolization (TACE) and radiofrequency ablation (RFA), compared to sequential therapy with TACE and RFA, for the treatment of hepatocellular carcinoma (HCC). We retrospectively studied 2 groups of HCC patients: 85 patients in the TACE+RFA+CIK group were treated with adoptive autologous CIK cell transfusion in combination with minimally invasive therapy, 89 patients in the TACE+RFA group were treated with minimally invasive therapy alone. The overall response rate was 76.5% in the TACE+RFA+CIK group and 79.8% in the TACE+RFA group. The disease control rate was higher in the TACE+RFA+CIK group than that in the TACE+RFA group (95.3% vs. 88.8%), but the difference was not significant (P=0.113). Kaplan-Meier analysis showed that the patients in the TACE+RFA+CIK group had significantly longer overall survival (56 vs. 31 mo, P=0.001) and progression-free survival (17 vs. 10 mo, P=0.001) than those in the TACE+RFA group. No severe side effects occurred in the CIK cell transfusion patients. In conclusion, CIK cell immunotherapy may be a valuable therapeutic strategy to prevent recurrence and metastasis in HCC patients after TACE and RFA, and to improve patient prognosis and quality of life. Combined CIK immunotherapy and minimally invasive therapies represent a safe, potential treatment modality for HCC. However, because patient assignment to the 2 treatments was not randomized, any conclusions concerning improvements in survival must be interpreted with great caution.

Identification of anaplastic lymphoma kinase break points and oncogenic mutation profiles in acral/mucosal melanomas.

Acral and mucosal melanomas, the two most common subtypes of melanoma in China, exhibit different genetic alterations and biologic behavior compared with other subtypes of melanomas. The purpose of this study was to identify the genetic alterations in patients with acral or mucosal melanomas in southern China. Fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) analysis, polymerase chain reaction (PCR), and quantitative real-time reverse transcriptase PCR (qRT-PCR) were used to assess the anaplastic lymphoma kinase (ALK) break points. Furthermore, a mass spectrometry-based genotyping platform was used to analyze 30 acral melanomas and 28 mucosal melanomas to profile 238 known somatic mutations in 19 oncogenes. ALK break points were identified in four acral cases (6.9%). Eight (13.8%) cases harbored BRAF mutations, six (10.3%) had NRAS mutations, four (6.9%) had KIT mutations, two (3.5%) had EGFR mutations, two (3.5%) had KRAS mutations, two (3.5%) had MET mutations, one (1.7%) had an HRAS mutation, and one (1.7%) had a PIK3CA mutation. Two cases exhibited co-occurring mutations, and one case with a BRAF mutation had a translocation in ALK. This study represents a comprehensive and concurrent analysis of the major recurrent oncogenic mutations involved in melanoma cases from southern China. These data have implications for both clinical trial designs and therapeutic strategies.

PPIase domain of trigger factor acts as auxiliary chaperone site to assist the folding of protein substrates bound to the crevice of trigger factor.

Trigger factor (TF) is the first chaperone encountered by nascent chains in bacteria, which consists of two modules: peptidyl-prolyl-cis/trans-isomerase (PPIase) domain and a crevice built by both N- and C-terminal domains. While the crevice is suggested to provide a protective space over the peptide exit site of ribosome for nascent polypeptides to fold, it remains unclear whether PPIase domain is directly involved in assisting protein folding. Here, we introduced structural change into different regions of TF, and investigated their influence on the chaperone function of TF in assisting the folding of various substrate proteins, including oligomeric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and monomeric carbonic anhydrase II (CA II) and lysozyme. Results showed that structural disturbances by site-specific mutations in the PPIase active site or by deletion of the PPIase domain from TF affected the chaperone activity of TF toward CA II and GAPDH but had no effect on TF-assisted lysozyme refolding, suggesting PPIase domain is involved in assisting the folding of substrates larger than lysozyme. Mutants with the structural disturbances in the crevice totally lost the chaperone activity toward all the substrates we used in this investigation. These results provide further evidence to confirm that the crevice is the major chaperone site of TF, and the hydrophobic pocket in PPIase domain acts as an auxiliary site to assist the folding of substrate proteins bound to the crevice in a substrate-dependent manner, which is beneficial for TF to provide appropriate assistance for protein folding by changing protective space and binding affinity.