[Research progress and prospects of artificial intelligence in diagnosis and treatment of colorectal cancer].

Colorectal cancer is one of the most common malignant tumors worldwide. Due to the heterogeneity in patient outcomes and treatment responses to standard therapy regimens, personalized diagnostic and therapeutic strategies have remained a focus of sustained interest in research. In recent years, with the rapid progression of artificial intelligence (AI) technology in the medical field, an abundance of phased research results has emerged in the decision-making for preoperative, intraoperative, and postoperative diagnostic and therapeutic plans for colorectal cancer, demonstrating great potential for application. This new and efficient solution provides for the personalized evaluations and auxiliary diagnoses and treatments of patients with colorectal cancer. In the future, AI systems may continue to advance towards multimodal, multi-omics, and real-time directions. This paper aims to explore the current state of research on the multi-faceted auxiliary applications of AI in the diagnosis and treatment of colorectal cancer, as well as to present a prospective view of the innovations that AI technology could bring to personalized colorectal cancer treatment in the future and the challenges it may face.

[Importance of comprehensive management of anastomotic site after ultra-low anal sphincter-preservation surgery].

Intersphincteric resection (ISR) surgery increases the rate of anal sphincter preservation in patients with ultra-low rectal cancers. However, the anastomotic site of ISR surgery is at risk for structural healing complications such as anastomotic leakage, anastomotic dehiscence, secondary anastomotic stenosis, chronic presacral sinus, rectovaginal fistula, and rectourethral fistula, which can lead to a persistent defunctioning ostomy or a secondary permanent colostomy. This article systematically describes the preoperative high-risk factors and characteristics of anastomotic site structural healing complications after ISR surgery, as well as the management of the anastomotic site during various stages including hospitalization, from discharge to one month after surgery, from one month after surgery to before stoma reversal, and after stoma reversal. This is to provide a clearer understanding of the risks associated with the anastomotic site at different stages of the healing process and to timely detect and actively manage related complications, thereby reducing the rate of permanent colostomy and truly achieving the dual goals of "survival benefit" and "quality of life improvement" in ISR surgery.

[Interpretation of Chinese expert consensus on the pelvic exenteration for primary rectal cancer beyond total mesorectal excision planes and locally recurrent rectal cancer (2023 edition)].

The China PelvEx Collaborative, under the direction of Colorectal Cancer Committee of the Chinese Medical Doctor Association, Gastrointestinal Surgery Committee of China International Exchange and Promotive Association for Medical and Health, has formulated and issued the Chinese expert consensus for primary rectal cancer beyond total mesorectal excision planes and locally recurrent rectal cancer (2023 edition) , with the academic support of the Chinese Journal of Gastrointestinal Surgery and Chinese Journal of Colorectal Disease (Electronic Edition). This Consensus refers to the expert consensus developed by the International PelvEx Collaboration, incorporates the latest international multi-center research results and combines the latest research results in China. The Consensus unifies some definitions, clarifies the surgical indications, and puts forward the definition and preventive measures of "empty pelvic syndrome" earlier. For the controversial classification of local recurrent rectal cancer, the Chinese classification was proposed for the first time in Consensus. At the same time, the definition of pelvic exenteration is controversial, and a more consistent cognition is proposed. It is believed that, with the in-depth research on complicated rectal cancer, C-PelvEx will gather more higher-level data from clinical research in several domestic centers, so as to further enrich the content of the updated Consensus.

[Laparoscopic versus open pelvic exenteration for locally advanced rectal cancer: analysis of short- and long-term effects].

Objective: To explore the feasibility, safety, and short- and long-term efficacy of laparoscopic pelvic exenteration (LPE) in treating locally advanced rectal cancer. Methods: The clinical data of 173 patients who had undergone pelvic exenteration (PE) for locally advanced rectal cancer that had been shown by preoperative imaging or intraoperative exploration to have invaded beyond the mesorectal excision plane and adjacent organs in the Cancer Hospital, Chinese Academy of Medical Sciences (n=64) and Peking University First Hospital (n=109) from 2010 January to 2021 December were collected retrospectively. Laparoscopic PE (LPE) had been performed on 82 of these patients and open PE (OPE) on 91. Short- and long-term outcomes (1-, 3-, and 5-year overall and disease-free survival and 1- and 3-year cumulative local recurrence rates) were compared between these groups. Results: The only statistically significant difference in baseline data between the two groups (P>0.05) was administration of neoadjuvant therapy. Compared with OPE, LPE had a significantly shorter operative time (319.3±129.3 minutes versus 417.3±155.0 minutes, t=4.531, P<0.001) and less intraoperative blood loss (175 [20-2000] ml vs. 500 [20-4500] ml, U=2206.500, P<0.001). The R0 resection rates were 98.8% and 94.5%, respectively (χ2=2.355, P=0.214). At 18.3% (15/82), and the incidence of perioperative complications was lower in the LPE group than in the OPE group (37.4% [34/91], χ2=7.727, P=0.005). The rates of surgical site infection were 7.3% (6/82) and 23.1% (21/91) in the LPE and OPE group, respectively (χ2=8.134, P=0.004). The rates of abdominal wound infection were 0 and 12.1% (11/91) (χ2=10.585, P=0.001), respectively, and of urinary tract infection 0 and 6.6% (6/91) (χ2=5.601, P=0.030), respectively. Postoperative hospital stay was shorter in the LPE than OPE group (12 [4-60] days vs. 15 [7-87] days, U=2498.000, P<0.001). The median follow-up time was 40 (2-88) months in the LPE group and 59 (1-130) months in the OPE group. The 1-, 3-, and 5-year overall survival rates were 91.3%, 76.0%, and 62.5%, respectively, in the LPE group, and 91.2%, 68.9%, and 57.6%, respectively, in the OPE group. The 1, 3, and 5-year disease-free survival rates were 82.8%, 64.9%, and 59.7%, respectively, in the LPE group and 76.9%, 57.8%, and 52.7%, respectively, in the OPE group. The 1- and 3-year cumulative local recurrence rates were 5.1% and 14.1%, respectively, in the LPE group and 8.0% and 15.1%, respectively, in the OPE group (both P>0.05). Conclusions: In locally advanced rectal cancer patients, LPE is associated with shorter operative time, less intraoperative blood loss, fewer perioperative complications, and shorter hospital stay compared with OPE. It is safe and feasible without compromising oncological effect.

[Risk factor and nomogram for predicting the probability of a permanent stoma after laparoscopic intersphincteric resection for ultralow rectal cancer].

Objective: A permanent stoma can seriously affect patients' quality of life. Clinicians need to consider the risk of a permanent stoma when making clinical decisions. This study analyzed preoperative predictors of a permanent stoma after laparoscopic intersphincteric resection for low rectal cancer (LISR), and a prediction model was constructed validated. Methods: This was a retrospective study that analyzed clinical data of 331 ultralow rectal cancer patients who were diagnosed with primary rectal adenocarcinoma by endoscopy and pathology, including 218 males and 113 female, (58.8±11.2) years and (23.7±3.1) kg/m2. The patients underwent LISR with a preventive stoma from January 2012 to December 2020. Patients with multiple primary colorectal cancers, who underwent emergency surgery for intestinal obstruction or bleeding or perforation, and did not complete 18 months follow up were exclucled. R software was used to randomly select 234 patients as the modeling group with a ratio of approximately 7:3, and the remaining 97 patients comprised the validation group. The stoma site was determined by the surgeon before the operation, and the ileum 30 cm from the ileocecal valve was selected. The rates of a permanent stoma for the entire group and the preoperative clinical factors that may affect the permanency of a stoma in the modeling group were determined. A permanent stoma was defined as failure to close the stoma at 18 months after surgery. Multivariate logistic regression analysis was used to analyze the preoperative independent risk factors for a permanent stoma after LISR. R software was used to create the nomogram model, and the predictive ability of the nomogram model was evaluated by receiver operating characteristic (ROC) curve analysis. Results: Among the 331 patients who underwent LISR, 37 (26 cases in the modeling group and 11 cases in the validation group, 11.2%) developed a permanent stoma for the following reasons: anastomotic stenosis due to leakage (16 cases, 43.2%), distant metastasis (16 cases, 43.2%), intolerant to stoma closure surgery (3 cases, 8.1%), stenosis due to postoperative radiation (1 case, 2.7%), and poor recovery of anorectal function (1 case, 2.7%). Univariate analysis showed that preoperative neoadjuvant chemoradiotherapy, poorly differentiated tumor, cT3 stage, and distant metastasis were associated with a permanent stoma. Multivariate logistic regression analysis showed that neoadjuvant chemoradiotherapy [OR=3.078, 95% confidence interval (CI): 1.326-7.147; P=0.009], cT3 stage (OR=2.257, 95%CI: 1.001-5.091; P=0.049), and stage IV cancer (OR=16.180, 95%CI: 2.753-95.102; P=0.002) were independent risk factors for permanent stoma after LISR. Based on the selected risk factors, a nomogram model for predicting permanent stoma was constructed. The area under the ROC curve of the modeling group was 0.793, the optimal cut-off value was 0.890, the sensitivity was 0.577, and the specificity was 0.885. The area under the ROC curve of the validation group was 0.953. The corrected curves of the modeling group and the validation group showed a good degree of fit. Conclusion: Neoadjuvant chemoradiotherapy, cT3 stage, and distant metastasis are independent predictors of a permanent stoma after LISR, and the nomogram model is helpful to predict the probability of a permanent stoma. Patients with high-risk factors should be adequately informed of the risk of a permanent stoma before colorectal surgery.

High-affinity chromodomains engineered for improved detection of histone methylation and enhanced CRISPR-based gene repression.

Histone methylation is an important post-translational modification that plays a crucial role in regulating cellular functions, and its dysregulation is implicated in cancer and developmental defects. Therefore, systematic characterization of histone methylation is necessary to elucidate complex biological processes, identify biomarkers, and ultimately, enable drug discovery. Studying histone methylation relies on the use of antibodies, but these suffer from lot-to-lot variation, are costly, and cannot be used in live cells. Chromatin-modification reader domains are potential affinity reagents for methylated histones, but their application is limited by their modest affinities. We used phage display to identify key residues that greatly enhance the affinities of Cbx chromodomains for methylated histone marks and develop a general strategy for enhancing the affinity of chromodomains of the human Cbx protein family. Our strategy allows us to develop powerful probes for genome-wide binding analysis and live-cell imaging. Furthermore, we use optimized chromodomains to develop extremely potent CRISPR-based repressors for tailored gene silencing. Our results highlight the power of engineered chromodomains for analyzing protein interaction networks involving chromatin and represent a modular platform for efficient gene silencing.

[Safety and efficacy of laparoscopic distal rectal transection by transanterior obturator nerve gateway].

Objective: To evaluate the safety and efficacy of distal rectal transection by using transanterior obturator nerve gateway (TANG) in laparoscopic radical resection for lower rectal cancers. Methods: A descriptive case series study was performed. Inclusion criteria: (1) patients with primary rectal adenocarcinoma, with the distance of 3-5 cm from tumor to anal verge, with normal anal function before surgery and a desire to preserve anus; (2) laparoscopic radical resection of rectal cancer was performed and the distal rectum was transected using TANG approach. Exclusion criteria: (1) patients with distant metastasis or receiving palliative surgery; (2) the distal rectum was transected using non-TANG approach; (3) patients receiving combined multiple organs resection; (4) patients complicated with other tumors requiring additional treatment during the study. Clinicopathological data of 50 patients with low rectal cancer undergoing laparoscopic resection using TANG approach between January 2019 and December 2020 in Peking University First Hospital were retrospectively collected. Perioperative conditions, length of specific pelvic lines, additional angle and postoperative short-term outcomes were observed and described. Additional angle was defined as the angle between the simulated stapling line with the traditional approach and the real stapling line with the TANG approach. Data following normal distribution were presented as Mean±SD, or M [quartile range (Q(R))] otherwise. Results: All the patients successfully completed laparoscopic surgery without transferring to open or transanal surgery. The median operative time was 193 (80) min and blood loss was 50 (58) ml. All tumors received R0 resection with the distance from the tumor to distal resection margin of 1.7 (0.4) cm and the anastomotic height of 2.0 (0.1) cm. Rectal transection was completed by one cartridge in 52.0% of the cases (26/50) and two cartridges in 48.0% (24/50). Length of the stapling line was 6.6 (1.5) cm. The time to construct the gateway was 8.0 (6.0) min. The vessel damage occurred in 4.0% of the cases (2/50) and none of the cases encountered obturator nerve damage. Inlets of the pelvis in TANG and traditional approach were (9.9±1.3) cm vs. (7.2±1.1) cm (t=24.781, P<0.001). Additional angle of TANG was (15±2) °. The transecting positions on the midline and right edge of the rectum specimen by TANG were 0.6 (0.2) cm and 1.0 (0.2) cm lower than those by the traditional approach. One case (2.0%) died of pulmonary infection on the 17th day after surgery, 2 cases (4.0%) received re-operation and 14 cases (28.0%) had postoperative complications, including anastomotic leakage (7/50, 14.0%), urinary retention (6/50, 12.0%), pelvic infection (2/50, 4.0%) and ileus (2/50, 4.0%). The median postoperative hospital stay was 12 (6) days. Conclusions: Laparoscopic distal rectal transection by using TANG approach is safe and effective in the treatment of low rectal cancer. As an alternative rectal transecting method, TANG has advantages especially for the obese and those with a contracted pelvis and ultralow rectal cancers.

[Thirty years' changes of the strategy of lateral lymph node dissection in low rectal cancer: treatment experience and prognostic analysis of 289 cases in one single center].

Objective: The surgical indications, resection extent and management principle of lateral lymph node dissection (LLND) in lower rectal cancer have been controversial between Eastern and Western countries. This study aims to provide a theoretical basis for the rational implementation of LLND by reviewing the changes of LLND strategy over the past 30 years in a single-center, and analyzing prognostic factors for the survival outcomes of patients with lateral lymph node metastasis (LLNM). Methods: A retrospective observational study was performed. Clinical data of 289 patients with rectal cancer who received LLND at the Department of General Surgery of Peking University First Hospital from 1990 to 2019 were collected. Patients were divided into three groups based on decades. There were 89 cases in 1990-1999 group, 92 cases in the 2000-2009 group, and 108 cases in the 2010-2019 group. Data analyzed: (1) patient baseline data; (2) surgery and postoperative recovery; (3) lateral lymph node dissection; (4) postoperative survival and prognosis of patients with positive lateral lymph nodes. The surgical methods and pathological results of LLND were compared between groups, and the prognostic risk factors of patients with LLNM were analyzed. Results: A total of 289 patients underwent radical resection with LLND' accounting for 6.3% of the 4542 patients with rectal cancer during the same period in our hospital. Except decade-by-decade increase in tumors with distance from anal verge ≤ 7 cm, the proportion of ulcerated tumors, and the proportion of neoadjuvant radiochemotherapy, the differences in other baseline data were not statistically significant among 3 decade groups (all P>0.05). The proportion of LLND in the 3 groups decreased decade by decade [9.9% (89/898) vs. 8.0% (92/1154) vs. 4.3% (108/2490), χ(2)=40.159, P<0.001]. The proportion of laparoscopic surgery and unilateral LLND increased, while the mean operative time, intraoperative blood loss, surgical complications above grade III and postoperative hospital stay decreased decade by decade. These 289 patients completed a total of 483 lateral dissections, including 95 cases of the unilateral dissection and 194 cases of the bilateral dissection. The proportion of LLND in the 3 groups decreased decade by decade [9.9% (89/898) vs. 8.0% (92/1154) vs. 4.3% (108/2510), P<0.001]. The median number of dissected lymph nodes in the internal iliac artery and obturator regions increased (2 vs. 3 vs. 3, P<0.001), but those in the common iliac and external iliac regions decreased significantly (4 vs. 3 vs. 2, P=0.014). A total of 71 patients with LLNM were identified. The rate of LLNM in the 2010-2019 group was significantly higher than that in the previous two groups [37.0% (40/108) vs. 16.9% (15/89) vs. 17.4% (16/92), P=0.001]. The patients with LLNM showed a poorer overall survival (OS) and disease-free survival (DFS) compared with negative lateral lymph nodes (P<0.001). There were statistically significant differences in 5-year OS rate (30.9% vs. 27.2% vs. 0, P=0.028) and 5-year DFS rate (28.3% vs. 16.0% vs. 0, P=0.038) among patients with only internal iliac lymph node metastasis, patients with only obturator lymph node metastasis, and patients with external iliac or common iliac lymph node metastasis. Multivariate analysis of prognostic factors showed that external iliac or common iliac lymph node metastasis was an independent risk factor for OS (HR=1.649, 95%CI: 1.087-2.501) and DFS (HR=1.714, 95%CI: 1.173-2.504) in patients with LLNM (all P<0.05) . The OS and DFS were not significant different in patients with LLNM among 3 decade groups. Conclusions: In the past decade, the proportion of LLND in rectal cancer has decreased significantly. However, LLNM rate has been significantly increased due to preoperative imaging assessments focusing on suspicious LLNM without compromising the survival. Internal iliac artery and obturator lymph nodes can be regarded as regional lymph nodes with a satisfactory prognosis after LLND. For suspected external iliac or common iliac lymph node metastasis, the significance of LLND remains to be further evaluated.

[Analysis of risk factors of anastomotic leakage after laparoscopic intersphincteric resection for low rectal cancer and construction of a nomogram prediction model].

Objectives: To examine the risk factors of anastomotic leakage for low rectal cancers undergoing laparoscopic intersphincteric resection (ISR), and to construct a nomogram prediction model for it. Methods: The perioperative data of 302 low rectal cancer patients undergoing laparoscopic ISR by the same surgical team of Department of General Surgery, Peking University First Hospital between January 2012 and January 2019 were retrospectively reviewed. There were 190 males and 112 females, aging 60(14) years (range: 20 to 84 years). χ2 test, independent sample t test, U test and Logistic regression analysis were used to analyze the risk factors for anastomotic leakage. R software was used to complete the drawing of the nomogram prediction model, and the receiver operating characteristic curve was used to evaluate the predictive ability of the nomogram prediction model. Results: There were 24 patients (7.9%) had anastomotic leakage among the 302 patients enrolled, including 10 cases of grade A leakage, 9 cases of grade B leakage, and 5 cases of grade C leakage. Out of the 24 patients, 2 patients (8.3%) died, 3 patients (12.5%) received leakage-related reoperation. Median healing time of the anastomotic leakage was 74 (58) days (range: 14 to 180 days). Univariate analysis showed male gender (P=0.009), preoperative serum albumin concentration (P=0.004), neoadjuvant radiochemotherapy (P=0.017), preserving left colonic artery (P=0.002) and performing a diverting ileostomy (P=0.015) were significantly correlated with anastomotic leakage. Logistic multivariate analysis showed male gender (OR=6.052, 95%CI: 1.535 to 23.860, P=0.010), neoadjuvant radiochemotherapy (OR=4.098, 95%CI: 1.318 to 12.821, P=0.015), no preserving left colonic artery (OR=16.699, 95%CI: 3.051 to 91.406, P=0.001) and not performing a diverting ileostomy (OR=21.218, 95%CI: 4.341 to 103.710, P<0.01) were independent risk factors for anastomotic leakage. According to the results of multi-factor regression analysis, the nomogram prediction model was constructed. The area under the curve of the nomogram prediction model was 0.840 (95%CI: 0.766 to 0.914). After internal verification, the concordance index value of the model was 0.840. Conclusion: Male gender, neoadjuvant radiochemotherapy, no preserving left colonic artery and not performing a diverting ileostomy are independent risk factors for anastomotic leakage for low rectal cancers undergoing laparoscopic ISR.

[Analysis on risk factors of the degree of radical resection and prognosis of patients with locally recurrent rectal cancer].

Objective: To evaluate the factors affecting the degree of radical resection and the prognosis of patients with locally recurrent rectal cancer (LRRC). Methods: A retrospective case-control study was performed. Clinical data of 111 patients with LRRC undergoing operation at the General Surgery Department of Peking University First Hospital from January 2009 to August 2019 were analyzed retrospectively. The "Peking University First Hospital F typing" was performed according to the preoperative images of the pelvic involvement. The pelvis was assigned into four directions: the front wall, lateral sides of the pelvic wall and the sacrum. According to the degree of pelvic wall involvement, F typing included F0 type (no involvement of the pelvic wall, the cancer only involved the adjacent organs or invaded conteriorly the urinary tract, genital organs or small intestine), F1 type (cancer involved the pelvic wall in one direction, such as the sacrum, or one side of the pelvic wall), F2 type (cancer involved the pelvic wall in two directions) and F3 type (cancer involved the pelvic wall in three directions). Case inclusion criteria: (1) LRRC was confirmed by imaging and pathological examination of samples (puncture or endoscopic biopsy); (2) complete clinical and follow-up data; (3) informed consent of patient. Those with dysfunction of heart, lung, etc., intolerance of operation, F3 type indicated by image, and distant metastasis were excluded. The degree of radical resection was evaluated according to the postoperative pathological results. Patients were followed up every 12 months and related examinations were arranged. The univariate analysis of radical resection was performed by χ(2) test, and the multivariate analysis was performed by logistic methods. The survival rate was calculated by Kaplan-Meier method and the survival curve was drawn. The survival rate was compared by log-rank test. Cox proportional hazards model was used to analyze the factors affecting the prognosis of patients with LRRC. Results: A total of 111 patients were included in this study. Of 111 patients, 59 were male and 52 were female; recurrent age of 36 cases was ≥ 65 years old; CEA level of 48 cases was ≥15 µg/L. According to the "Peking University First Hospital F typing", 70 cases were F0 type, 38 F1 type and 3 F2 type. Surgical procedures were abdominoperineal resection (n=28), posterior pelvic exenteration (n=32), and total pelvic exenteration (n=51, including 1 case of TPE combined with sacrectomy). According to the postoperative pathological results, R0, R1 and R2 resections were 83, 20 and 8 cases, respectively. Univariate analysis showed that the degree of radical resection was associated with the secondary surgical procedure, F typing and lymph node metastasis (all P<0.05). Multivariate analysis showed that F typing (F1-F2) was an independent risk factor for non- R0 resection (OR=37.256, 95%CI:8.572 to 161.912, P<0.001). The morbidity of operative complications was 22.5% (25/111); the perioperative mortality was 1.8% (2/111); the local recurrence rate after the second operation was 37.8% (42/111). The 3- and 5-year overall survival rates were 41.2% and 21.9% respectively. The 3-year survival rates of patients with and without postoperative chemotherapy were 52.7% and 32.4% respectively (P=0.005). The 3-year survival rates of patients with lower (<15 µg/L) and higher CEA level (≥15 µg/L) were 52.9% and 24.3% respectively (P<0.001). The 3-year survival rates of patients with R0, R1 and R2 resection were 49.8%, 21.3% and 8.5% respectively (P=0.002). The 3-year survival rates of patients with F0, F1 and F2 type were 52.7%, 22.0% and 0 respectively (P<0.001). Cox analysis confirmed that the degree of radical resection (HR=2.088, 95%CI:1.095 to 3.979, P=0.025), the CEA level before the secondary operation (HR=1.857, 95%CI:1.157 to 2.980, P=0.010) and postoperative chemotherapy (HR=1.826, 95%CI:1.137 to 2.934, P=0.013) were independent factors affecting the prognosis. Conclusions: The indication of LRRC surgical treatments must be strictly limited. Evaluation of the fixation site to the pelvic wall is helpful for improving the rate of R0 resection. Lower preoperative CEA level, radical resection and postoperative chemotherapy are protective factors of prolonged overall survival time of patients with LRRC.

[Mechanism of cross talk between tissue factor/active coagulation factor VII and epidermal growth factor receptor signalings in colon cancer cells in culture].

OBJECTIVE: To preliminarily verify the cross talk between tissue factor/active coagulation factor VII (TF/FVIIa) and epidermal growth factor receptor (EGFR) pathways in human colon cancer cells in culture. METHODS: FVIIa was treated to HT-29 (KRAS-wild type) and LoVo (KRAS-mutant) colon cancer cells to activate TF/FVIIa pathway, qRT-PCR and Western blot were used to detect the expressions of amphiregulin (AREG) and epiregulin (EREG), ligands of EGFR on mRNA and protein levels, respectively. After knocking down expression of TF by TF-targeted siRNA transfection, FVIIa was treated and mRNA expressions of AREG and EREG were detected to see whether the FVIIa-induced effects were dependent on TF. Expressions of mRNA of TF and FVII were detected by qRT-PCR following the activation of EGFR pathway by treatment with epidermal growth factor (EGF) to HT-29 and LoVo cells. RESULTS: After TF/FVIIa pathway was activated, for HT-29 cells, expressions of AREG (on mRNA level) and EREG (both on mRNA and protein level) were significantly down-regulated versus those of control group, gene expressions of AREG and EREG were 0.55±0.09 vs.0.99 ±0.09, 0.67±0.10 vs.1.02±0.02, protein expressions of EREG were 0.54±0.09 vs.1.04±0.13, all P<0.05. For LoVo cells, expressions of AREG (both on mRNA and protein level) and EREG (on protein level) were significantly up-regulated versus those of control group, gene expression of AREG were 1.87±0.39 vs. 0.93±0.23, protein expressions of AREG and EREG were 3.09±0.73 vs.1.11±0.21, 1.53±0.19 vs.0.97±0.23, all P<0.05. The regulating effect of AREG and EREG mRNA expression by FVIIa in HT-29 and LoVo cells could both be partly blocked by knocking down TF expression. For HT-29 cells, activation of EGFR pathway induced no significant TF mRNA expression, FVII mRNA expression was not detected. However,for LoVo cells, activation of EGFR pathway induced significantly higher mRNA expressions of both TF and FVII, expressions were 1.53±0.23 vs.1.00±0.23, 53.20±6.08 vs.1.00±0.15, all P<0.05. CONCLUSION: In colon cancer cell LoVo, when activated, TF/FVIIa pathway and EGFR pathway could interact through upregulating the other pathway's effectors, and mutant KRAS might play a critical role in the two pathways' cross talk.

Direct visualization of the novel pathogen, Spiroplasma eriocheiris, in the freshwater crayfish Procambarus clarkii (Girard) using fluorescence in situ hybridization.

Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans. It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and in some other crustaceans. The monitoring of the pathogen in crustacean populations and study of its behaviour in the laboratory require the development of reliable diagnostic tools. In this article, we improved microscopic identification of S. eriocheiris by combining in situ hybridization with specific fluorescently labelled oligonucleotide probes. The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S. eriocheiris in the tissues of diseased crayfish P. clarkii and exhibited low background autofluorescence and ideal signal-to-noise ratio. With the advantages of better tissue penetration, potentially more specific and stable, we designed three species-specific oligonucleotide probes utilizing the sequences of 16S-23S rRNA intergenic spacer regions (ISRs) of S. eriocheiris. Positive hybridization signals were visualized in haemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish. This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S. eriocheiris probably spread throughout the tissues in P. clarkii by hemokinesis. This assay will facilitate our understanding of the pathogenesis of S. eriocheiris and enhance the early diagnosis of the novel pathogen.

[A multiple-channel system for synchronous acquisition and analysis of neural electrophysiological signals].

The technique for synchronous electrophysiological recording from multiple single cells has been widely applied, but there is still a shortage of suitable data acquisition and analysis systems to fit electrophysiological equipment made in China or Japan. Recently we have developed an acquisition and analysis system for multiple-channel electrophysiological recording. In this paper, we present the pivotal technique of the system and some examples of applications.

Overall informativity, OI, in DNA polymorphisms revealed by inter-Alu PCR: detection of genomic rearrangements.

We studied two systems of multilocus markers revealed by PCR using primers directing amplification between Alu repeats in a tail-to-tail orientation. Genomic polymorphisms were detected as the presence or absence of the electrophoretic bands representing DNA fragments of a given length. A total of 104 such fragments segregating as Mendelian markers in a panel of eight CEPH families were analyzed by two-point linkage analysis. Fifty-one of these fragments were localized with respect to CEPH markers; they represented 33 loci, 7 of which were multiallelic. Locus-specific oligonucleotides were developed and used as hybridization probes to identify the mapped loci within a complex pattern of inter-Alu PCR products. A great proportion of inter-Alu PCR polymorphisms represented length variants within amplified DNA segments, while others were presumably due to mutations within the priming sites. To describe the expected number of informative loci per typing experiment we introduced a parameter called overall informativity (OI), which provides a single measure of the multiplex ratio and the informativity of markers contributing to a multilocus system (OI of a single locus is equivalent to its heterozygosity and cannot exceed 0.5 for a biallelic codominant marker). High OI values (5.8 and 11.5) of the two presented systems of inter-Alu PCR markers of random chromosomal distribution render them suitable for mapping genomic rearrangements such as genomic deletions in tumoral tissues. This was illustrated by the detection of loss of heterozygosity in the 9q22-qter region in sporadic colon cancer.

Alu-PCR combined with non-Alu primers reveals multiple polymorphic loci.

A marker suitable for genetic mapping and genomic fingerprinting is characterized by high polymorphic information content (PIC) and high "multiplex ratio" (defined as the number of loci that can be simultaneously typed). Towards this goal, we combined an Alu-specific with a non-Alu primer in a single PCR amplification targeting genomic regions where length polymorphisms are abundant. Three loci were revealed with the variable number of (AAT), (TAAA), (AG), and/or (AAAGG) motifs, and PIC values between 0.7 and > 0.94. Their location on Chromosomes (Chrs) 19q12, 17q12-q24, and 5q31.2-33.3 was determined by multipoint analysis with markers from CEPH database. The most common genotype for this three-locus marker, estimated from the occurrence of the most frequent alleles, is of the order of 2 x 10(-4), while the combined PIC value of a single typing experiment is 2.37. The use of a similar primer pair, as well as examples from the literature, indicates the general nature of this approach when a non-Alu oligonucleotide, presumably with "random" priming sites downstream of Alu repeats, is combined with an Alu-specific one. Clustering of DNA length variants in the regions adjacent to interspersed repeats provides opportunity to develop other highly informative multiple-locus markers similar to that described here.