[Development and validation of a prognostic prediction model for patients with stage â to â ¢ colon cancer incorporating high-risk pathological features].

Objective: To examine a predictive model that incorporating high risk pathological factors for the prognosis of stage Ⅰ to Ⅲ colon cancer. Methods: This study retrospectively collected clinicopathological information and survival outcomes of stage Ⅰ~Ⅲ colon cancer patients who underwent curative surgery in 7 tertiary hospitals in China from January 1, 2016 to December 31, 2017. A total of 1 650 patients were enrolled, aged (M(IQR)) 62 (18) years (range: 14 to 100). There were 963 males and 687 females. The median follow-up period was 51 months. The Cox proportional hazardous regression model was utilized to select high-risk pathological factors, establish the nomogram and scoring system. The Bootstrap resampling method was utilized for internal validation of the model, the concordance index (C-index) was used to assess discrimination and calibration curves were presented to assess model calibration. The Kaplan-Meier method was used to plot survival curves after risk grouping, and Cox regression was used to compare disease-free survival between subgroups. Results: Age (HR=1.020, 95%CI: 1.008 to 1.033, P=0.001), T stage (T3:HR=1.995,95%CI:1.062 to 3.750,P=0.032;T4:HR=4.196, 95%CI: 2.188 to 8.045, P<0.01), N stage (N1: HR=1.834, 95%CI: 1.307 to 2.574, P<0.01; N2: HR=3.970, 95%CI: 2.724 to 5.787, P<0.01) and number of lymph nodes examined (≥36: HR=0.438, 95%CI: 0.242 to 0.790, P=0.006) were independently associated with disease-free survival. The C-index of the scoring model (model 1) based on age, T stage, N stage, and dichotomous variables of the lymph nodes examined (<12 and ≥12) was 0.723, and the C-index of the scoring model (model 2) based on age, T stage, N stage, and multi-categorical variables of the lymph nodes examined (<12, 12 to <24, 24 to <36, and ≥36) was 0.726. A scoring system was established based on age, T stage, N stage, and multi-categorical variables of lymph nodes examined, the 3-year DFS of the low-risk (≤1), middle-risk (2 to 4) and high-risk (≥5) group were 96.3% (n=711), 89.0% (n=626) and 71.4% (n=313), respectively. Statistically significant difference was observed among groups (P<0.01). Conclusions: The number of lymph nodes examined was an independent prognostic factor for disease-free survival after curative surgery in patients with stage Ⅰ to Ⅲ colon cancer. Incorporating the number of lymph nodes examined as a multi-categorical variable into the T and N staging system could improve prognostic predictive validity.

[Research progress and future development prospect of transanal total mesorectal excision].

Transanal total mesorectal excision (taTME) is one of the hotspots in colorectal surgery in recent years. Although most studies confirm that taTME is safe and feasible, some studies still showed that the morbidity of complication and local recurrence rate of taTME were higher than traditional laparoscopic surgery. This article reviews and analyzes the short-term and long-term outcomes of taTME and the related progress of postoperative function. The results showed that there were no significant differences in the main short-term and long-term efficacy between taTME and traditional laparoscopic TME, but taTME had potential advantages in postoperative functional recovery. The results of case study after passing the learning curve suggested that taTME had better short-term and long-term efficacy. Moreover, with the maturity of taTME technology, transanal endoscopic surgery has gradually shown its advantages in the treatment of complex pelvic diseases. In the future, the application of single-port robot will further promote the development of natural orifice transluminal endoscopic colorectal surgery.

[Efficacy of transanal hand-sewn reinforcement in low rectal stapled anastomosis in preventing anastomotic leak after transanal total mesorectal excision].

Objective: To explore the efficacy and feasibility of transanal hand-sewn reinforcement of low stapled anastomosis in preventing anastomotic leak after transanal total mesorectal excision (taTME). Methods: A descriptive cohort study was conducted. Clinical data of 51 patients with rectal cancer who underwent taTME with transanal hand-sewn reinforcement of low stapled anastomosis at Department of Colorectal Surgery, the Sixth Affiliated Hospital of Sun Yat-sen University from January 2019 to December 2020 were retrospectively collected. Inclusion criteria: (1) age >18 years old; (2) rectal cancer confirmed by preoperative pathology; (3) distance from tumor to anal verge ≤ 8 cm according to pelvic MR; (4) the lesion was evaluated to be resectable before operation; (5) with or without neoadjuvant chemotherapy and radiotherapy; (6) taTME, end-to-end stapled anastomosis, and reinforcement in the anastomosis with absorbable thread intermittently were performed, and the distance between anastomosis and anal verge was ≤ 5 cm. Exclusion criteria: (1) previous history of colorectal cancer surgery; (2) emergency surgery due to intestinal obstruction, bleeding or perforation; (3) patients with local recurrence or distant metastasis; (4) the period of postoperative follow-up less than 3 months. The procedure of transanal hand-sewn reinforcement was as follows: firstly, no sign of bleeding was confirmed after checking the anastomosis. Then, the anastomosis was reinforced by suturing the muscle layer of rectum intermittently in a figure-of-eight manner using 3-0 single Vicryl. The entry site of the next suture was close next to the exit site of the last one. Any weak point of the anastomosis could also be reinforced according to the specimen from the circular stapler. The primary outcome were the incidence of anastomotic leak, methods of the secondary operation, anastomotic infection, anastomotic stricture, and conditions of Intraoperative and postoperative. Results: All the 51 enrolled patients completed surgery successfully without any conversion to open surgery. The median operative time was 169 (109-337) minutes, and the median intraoperative blood loss was 50 (10-600) ml. The median postoperative hospital stay was 8 (5-16) days. The mssorectum was complete and distal resection margin was negative in all patients. Postive circumferential resection margin was observed in 1 patients (2.0%). Twelve (23.5%) patients underwent prophylactic ileostomy. One patient developed anastomosis stricture which was cured by digital dilatation of the anastomosis. ISREC grade C anastomotic leak was observed in 3 (5.9%) male patients, of whom 2 cases did not received prophylactic ileostomy during the operation, and were cured by a second operation with the ileostomy and anastomotic repair. The other one healed by transanal repair of the anastomosis and anti-infection therapy. One (2.0%) patient suffered from perianal infection and healed by sitz bath and anti-infection therapy. No death was reported within 30 days after operation. Conclusion: Transanal hand-sewn reinforcement in low rectal stapled anastomosis in preventing anastomotic leak after taTME is safe and feasible.

[Clinical phenotype and immunological features of a patient with A20 haploinsufficiency].

Objective: To explore the clinical phenotype, immunological features, pathogenesis and gene variation of a case with A20 haploinsufficiency (HA20). Methods: A patient diagnosed with tumor necrosis factor α-induced protein 3 (TNFAIP3) mutated HA20 was admitted into Shenzhen Children's Hospital in May,2019.The clinical data was analyzed. Flow cytometry was used to detect the patient's peripheral blood lymphocyte subsets, and also, the percentage of follicular helper T cell (TFH) cells in the patient and thirteen healthy controls. After the construction of empty vector, wild-type and mutant plasmid vectors, a wild-type or mutant overexpression system of the TNFAIP3 gene was established in 293T cells and Hela cells. Then, the expression level of A20 in 293T cells and the expression of inhibitor K binding α (IKBα) in green fluorescent protein (GFP)+Hela cells before and after tumor necrosis factor α (TNF-α) stimulation were measured, to verify the pathogenicity of this variation. Results: A 5 years and 11 months old boy, presented with recurrent oral ulcer, abdominal pain, joint swelling and arthralgia. Oral ulcer, chronic skin rashes, knee joint swelling were observed. The levels of inflammatory markers were increased. Colonoscopy showed congestion of mucosa and multiple ulcers in terminal ileum and ileocecus. The absolute number of naive B cells was 124×10(6) cells/L (reference range 147×10(6)-431×10(6) cells/L), accounting for 0.430 of the total B cells (reference range 0.484-0.758). Compared to healthy controls (0.016-0.071), the percentage of TFH cells in CD4(+)T cells was much lower (0.008).A heterozygous mutation of TNFAIP3 gene (c.909_913 del, p.L303fs) was identified by genetic analysis. In vitro study showed that truncated A20 protein was expressed in TNFAIP3 mutant overexpressed 293T cells, which verified the pathogenicity of this variation. Besides, after TNF-α stimulation, the degradation rate of IkBα protein in mutant overexpressed Hela cells (35%) was between the other two groups (15% in the wild-type group and 57% in the non-loaded group). Conclusions: This case with HA20 due to a de novo TNFAIP3 gene mutation presents with early onset Behcet-like autoinflammatory syndrome. This variation leads to expression of truncated A20 protein, enhanced degradation of IkBα, and further activation of nuclear factor κB signaling pathway.

[Transanal lateral lymph node dissection surgery for 5 cases of mid-low rectal cancer].

Objective: To evaluate the feasibility and safety of transanal lateral lymph node dissection for mid-low rectal cancer. Methods: A descriptive case series research method was used. Clinical and pathological data of 5 mid-low rectal cancer patients who underwent transanal lateral lymph node dissection at Department of Colorectal Surgery, the Sixth Affiliated Hospital of Sun Yat-sen University from November 2018 to May 2019 were retrospectively collected and analyzed. Of 5 cases, 4 were male and 1 was female with mean age of (43.2±13.2) years and mean body mass index of (21.2±2.6) kg/m(2); the mean diameter of tumor was (3.2±2.4) cm; the mean distance between tumor and anus was (6.3±2.5) cm; 3 received preoperative neoadjuvant chemotherapy. In preoperative TNM staging, 2 cases were T3N1M0, 1 was T3cN2aM0, 1 was T3cN2bM0, and 1 was T2N1M0. All the patients had no intestinal obstruction before operation. Surgical methods: (1) total mesorectal excision: using general transanal and transabdominal methods to mobilize and resect total mesorectum, and dissect No.252, No.253 lymph nodes; (2) transanal lateral lymph node dissection: dissect the adipose lymphoid tissue on the surface of the iliococcygeal muscle, the coccygeal muscle, and the obturator muscle (the No.283 lymph nodes) upward, and dissect No.263d and No.263p lymph nodes with fat tissue sequentially till the bifurcation of the internal and external iliac artery; (3) take out the specimen from anus, and make anastomosis between proximal colon and anal canal. Intraoperative and postoperative variables was observed. Results: All the 5 patients completed surgery successfully, and no patient needed to convert to open approach. The mean operative time was (295.6±97.7) minutes, and the median intraoperative blood loss was 70 (50-500) ml. The mean length of specimen was (12.9±3.0) cm, and the mean number of harvested lymph node was 30.4±9.9. The positive lateral lymph nodes were founder in 4 patients. The median distance between tumor and distal resection margin was 1.5 (1.2-8.0) cm. The resection margin in all the patients was negative. The mean time to postoperative flatus was (4.2±1.6) days, the mean postoperative spontaneous urination was (3.0±1.9) days, time to drainage tube removal was (5.6±1.9) days, and the mean postoperative hospital stay was (9.4±2.1) days. The postoperative TNM staging by pathology was 1 case with T1N0M0, 1 with T2N1M0, 1 with T3N2bM0, and 2 with T3N2M0. Five patients were moderately differentiated adenocarcinoma. Only 1 patient developed postoperative abdominal bleeding, who was healed after conservative treatment. The other 4 patients did not develop any perioperative complications, such as incision infection, presacral abscess, pelvic abscess, anastomotic leakage, or anastomotic stricture. Four patients underwent postoperative chemotherapy. All the patients were followed up for 2 to 28 weeks after surgery and they all felt well. The patients with stoma had fluent bowel. Conclusions: Transanal lateral lymph node dissection is feasible and safe in the treatment of mid-low rectal cancer, which can achieve the purpose of extended radical resection of mid-low movement rectal cancer. Moreover, this procedure is a new method to treat rectal cancer patients with lateral lymph node metastasis.

[Study on the pharyngeal constriction and function of cricopharyngeal muscle in normal persons using dynamic magnetic resonance imaging].

OBJECTIVE: To investigate the superiority of dynamic magnetic resonance imaging as an assessment method of pharyngeal constriction and cricopharyngeal muscle function, and introduce a new way to measure the pharyngeal constriction ratio, anteroposterior diameter of esophageal entrance and thickness of cricopharyngeal muscle in healthy volunteers. METHODS: Twenty volunteers who were assessed as normal by fiberoptic endoscopic evaluation of swallowing and videofluoroscopic swallowing study were included in this study. With the use of 3.0T magnetic resonance imaging, Fiesta sequence and Asset technique, the median sagittal plane and intervertebral transverse plane from C1 to C5 were scanned repeatedly. Pharyngeal constriction ratio in the median sagittal and intervertebral transverse plane, anteroposterior diameter of esophageal entrance and thickness of cricopharyngeal muscle were assessed and the data were analysed by SPSS13.0. RESULTS: The intervertebral transverse plane between C1 and C2 was superior in observation of pharyngeal constriction ratio in the same way that the plane between C4 and C5 was superior in cricopharyngeal evaluation. The average of sagittal and intervertebral pharyngeal constriction ratio were estimated as 0.08±0.02, 0.09±0.04 respectively and the average of thickness of cricopharyngeal muscle and anteroposterior diameter of esophageal entrance were (6.50±1.69) mm, (1.99±0.76) mm respectively. CONCLUSION: Dynamic magnetic resonance imaging was superior in assessing the function of pharyngeal constriction and cricopharyngeal muscle by scanning the swallowing process in the median sagittal and intervertebral transverse plane.

Transanal total mesorectal excision assisted by single-port laparoscopic surgery for low rectal cancer.

INTRODUCTION: We have combined the minimally invasive single-port laparoscopic surgery and the transanal total mesorectal excision (TaTME) for rectal cancer with the goal to standardize the approach and improve the quality of rectal cancer resection. METHODS: By using two single-port platforms, selected patients were first operated by TaTME, and then a single-port laparoscopic surgery was introduced to assist and complete the abdominal portion. Short-term outcomes including perioperative outcome and pathologic results of these patients were evaluated. RESULTS: Between July 2014 and March 2015, six patients with low rectal cancer (five males and one female) at a median age of 68 years were successfully operated in a median time of 360 min (range 310-420). The median estimated blood loss was 150 ml (range 50-800). In one patient, the spleen was removed because of a lesion identified preoperatively. Their postoperative recovery was uneventful except one acute myocardial infarction on postoperative day 3. Pathologic specimens showed negative margins and a complete excision of the mesorectum in all cases. The median number of harvested lymph nodes was 11.5 (range 4-12). At a median follow-up of 4 months (range 3-9), after ileostomy closure, none of the patients suffered from fecal incontinence. CONCLUSION: TaTME assisted by abdominal single-port may be safely achieved in selected rectal cancer patients.

Cloning and heterologous expression of UDP-glycosyltransferase genes from Bacillus subtilis and its application in the glycosylation of ginsenoside Rh1.

UNLABELLED: Bacillus subtilis CCTCC AB 2012913 can transform ginsenoside Rh1 to 3-O-ß-D-glucopyranosyl-6-O-ß-D-glucopyranosyl-20(S)-protopanaxatriol. Based on its genome sequence, strain B. subtilis 168 contains three UDP-glycosyltransferase genes. Here, we cloned the three UDP-glycosyltransferase genes (ydhE1, yojK1 and yjiC1) from B. subtilis CCTCC AB 2012913 and expressed in Escherichia coli BL21 (DE3) with His-tag. The crude enzyme extracts were assayed, respectively, for their activities to transform ginsenoside Rh1. Extracts containing enzymes YojK1 and YjiC1 could use ginsenoside Rh1 as a substrate to produce 3-O-ß-D-glucopyranosyl-6-O-ß-D-glucopyranosyl-20(S)-protopanaxatriol, which had an additional glucopyranosyl linked with C-3 over the substrate. Enzyme YjiC1 was purified by affinity chromatography on Ni-NTA His Binding resin. The molecular mass of purified YjiC1 was c. 47 kDa as determined by SDS-PAGE. This is the first report of an in vitro biotransformation of ginsenoside Rh1 to 3-O-ß-D-glucopyranosyl-6-O-ß-D-glucopyranosyl-20(S)-protopanaxatriol using the recombinant UDP-glycosyltransferase. SIGNIFICANCE AND IMPACT OF THE STUDY: The Chinese traditional medicinal plant Panax is reported to have multiple health benefits. Its main active ingredient is saponin, and different saponins have different activity spectrum. In the study, three UDP-glycosyltransferase genes, ydhE1, yojK1 and yjiC1, were cloned from Bacillus subtilis CCTCC AB2012913 and the three genes were expressed in Escherichia coli BL21 (DE3). The enzyme YjiC1 was purified and converted ginsenoside Rh1 to 3-O-ß-D-glucopyranosyl-6-O-ß-D-glucopyranosyl-20(S)-protopanaxatriol in vitro. The compound is the first saponin possessing ß-glucopyranosyl at both C-3 and C-6 sites. We showed that the in vitro biotransformation was effective, and the reaction condition was easy to control. Our research suggests that a diversity of saponins could be generated through efficient and directed enzymatic biotransformation.

Regulation different network analysis of rheumatoid arthritis (RA) and osteoarthritis (OA).

BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic synovitis that progresses to destruction of cartilage and bone. AIM: The purpose of this study was to employ microarray analysis combined with bioinformatics techniques to evaluate differential gene expression in BM-derived mononuclear cells obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) to study the pathogenesis of this disease. MATERIALS AND METHODS: Gene expression profiles in BM-derived mononuclear cells from 9 RA and 10 OA patients were obtained from GEO. RESULTS: The bone marrow (BM) mononuclear cells showed 2581 up-regulated and 649 down-regulated genes in RA patients relative to the OA group: Our analysis indicated that several differentially expressed genes might play crucial roles in RA development, including SP1, RARA, ETS1, ETS2, FOS and ESR1. CONCLUSIONS: Further analysis predicted these genes might be involved in RA through cancer related pathways and immunity related pathways. Furthermore, these genes may serve as novel therapeutic targets for the treatment of RA.

Identification of molecular genetic markers tightly linked to downy mildew resistant genes in grape.

Bulk segregant analysis (BSA), randomly amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) methods were used to tag the downy mildew-resistant genes of grape with molecular markers. Parents and their 60 individuals of an F1 progeny resulting from a cross 88-110 between 83-4-96 (Vitis quinquangularis, downy mildew-resistant) and Muscat Rose (V. vinifera, downy mildew-susceptible), three F2 progenies resulting from three crosses of self-cross and inter-cross of 88-110 F1 seedlings, as well as another interspecific F1 hybrids of 88-84 cross [Xun-3 (V. quinquangularis, downy mildew-resistant) x Ugni Blanc (V. vinifera, downy mildew-susceptible)] and the wild grapes native to China were used for the study. Among 280 Operon primers, 160 gave distinct band patterns. One RAPD marker OPO06-1500 was tightly linked to a major gene resistant to Plasmopara viticola (RPv-1). Based on Mapmaker software analysis, the map distance between RPv-1 and OPO06-1500 was 1.7 cM. Marker OPO06-1500 was cloned and sequenced. According to the sequence, two specific primers were designed to amplify all plant materials. RAPD marker was converted into SCAR marker (SCO06-1500). One distinct single band only in resistant plants was amplified, whose size was the same as that of the RAPD marker. The SCAR marker's popularity was confirmed, and it could be used for the identification of hybrid resistant to P. viticola and will be potentially useful in the development of new resistant grape cultivars.

Affinity purification of Schistosoma japonicum glutathione-S-transferase and its site-directed mutants with glutathione affinity chromatography and immobilized metal affinity chromatography.

A C-terminally polyhistidine-tagged protein of Schistosoma japonicum glutathione-S-transferase, named as SjGST/His, and its Cys85-->Ser, Cys138-->Ser, and Cys178-->Ser site-directed mutants were prepared and highly expressed in Escherichia coli. Both immobilized metal affinity chromatography (IMAC) and glutathione (GSH) affinity chromatography were used to purify these four enzymes. All of them were purified with equal efficiency by Ni2+-chelated nitrilotriacetic acid agarose gel, but not by GSH Sepharose 4B gel. The protein amounts of wild-type and Cys85-->Ser enzymes purified by the latter gel were three to seven-fold greater than those of the other two enzymes purified by the same gel, while their specific activities were two-fold lower, presumably because of the occurrence of noncovalent aggregation. Both purification methods yielded highly pure enzymes, while there were minor amounts of inter- and intra-disulfide forms in the IMAC purified enzymes except for the Cys85-->Ser mutant. Addition of dithiothreitol to GSH-affinity purified enzymes shifted all of their mass spectra of matrix-assisted laser desorption/ionization-time of flight mass spectrometry toward low molecular-mass regions, while addition of GSH to IMAC purified enzymes shifted the spectra toward high molecular-mass regions. The shift values of wild-type enzyme were larger than those of the three mutants, indicating that the Cys85, Cys138, and Cys178 residues were S-thiolated by GSH during the GSH-affinity purification. This result was confirmed by isoelectric focusing. These findings suggest that IMAC is more efficient than the conventional GSH-affinity system for the purification of SjGST/His enzyme, especially for its mutants and fusion proteins.

Epidemic non-A, non-B hepatitis in Xinjiang. Clinical and pathologic observations.

131 cases of epidemic hepatitis from two villages in Xinjiang, were admitted to the provisional infectious diseases hospital from February to April 1987. In 125 (95%) patients neither anti-HAV IgM nor anti-HBc IgM were positive. 118 (90%) were drinking water from a local stream of the Hotan River. The study was conducted on 125 patients aged from 5 to 90 years, with a mean of 26.4 years. There were slightly more males than females, a ratio of 1.5 to 1. The incubation period was roughly estimated to be 17 to 37 days (mean 24.8 days). All subjects were jaundiced. 12 patients (including 6 pregnant women) had cholestatic symptoms. Except one woman who died of postpartum bleeding, all patients ran a benign course without fulminant hepatic failure. Pathologic changes of liver biopsies from 35 cases included lobular damage and inflammation, ballooned hepatocytes with clear cytoplasm and delineated cell membrane which were named "clear cell". There were cholestasis of different degrees in the acute icteric stage and fatty changes in the convalescent stage. It was seen under the electron microscope that the livers of 8 cases had neither tubular structure nor sponge-like inclusions, but had nuclear changes including curled edge of nuclear membrane and nuclear heterochromatin condensed in masses. Liver biopsies were repeated in 7 cases followed up for 6-13 months, and hepatohistology was normal in 5. Of the other two cases there was one of each.