[Advances in three-dimensional tumor models for colorectal cancer].

Conventional tumor culture models include two-dimensional tumor cell cultures and xenograft models. The former has disadvantages including lack of tumor heterogeneity and poor clinical relevance, while the latter are limited by the slow growth, low engraftment successful rate, and high cost. In recent years, in vitro three-dimensional (3D) tumor models have emerged as the tool to better recapitulate the spatial structure and the in vivo environment of tumors. In addition, they preserve the pathological and genetic features of tumor cells and reflect the complex intracellular and extracellular interactions of tumors, which have become a powerful tool for investigating the tumor mechanism, drug screening, and personalized cancer treatment. 3D tumor model technologies such as spheroids, organoids, and microfluidic devices are maturing. Application of new technologies such as co-culture, 3D bioprinting, and air-liquid interface has further improved the clinical relevance of the models. Some models recapitulate the tumor microenvironment, and some can even reconstitute endogenous immune components and microvasculature. In recent years, some scholars have combined xenograft models with organoid technology to develop matched in vivo/in vitro model biobanks, giving full play to the advantages of the two technologies, and providing an ideal research platform for individualized precision therapy for specific molecular targets in certain subtypes of tumors. So far, the above technologies have been widely applied in the field of colorectal cancer research. Our research team is currently studying upon the application of patient-derived tumor cell-like clusters, a self-assembly 3D tumor model, in guiding the selection of postoperative chemotherapy regimens for colorectal cancer. A high modeling success rate and satisfactory results in the drug screening experiments have been achieved. There is no doubt that with the advancement of related technologies, 3D tumor models will play an increasingly important role in the research and clinical practice of colorectal cancer.

[Risk factor and early diagnosis of anastomotic leakage after rectal cancer surgery].

Anastomotic leakage (AL) is one of the most serious complications after sphincter- preserving surgery for rectal cancer, which can significantly prolong the length of stay of patients, increase perioperative mortality, cause dysfunction, shorten overall survival and recurrence-free survival of patients. In order to reduce the serious consequences caused by AL, prediction of AL through preoperative and intraoperative risk factors are of great importance. However, the influences of neoadjuvant chemoradiotherapy, protective stoma, laparoscopic surgery and some intraoperative manipulations on AL are still controversial. Through the auxiliary judgment of anastomotic blood supply during operation, such as indocyanine green imaging, hemodynamic ultrasound, etc., it is expected to achieve the source control of AL. Early diagnosis of AL can be achieved by attention to clinical manifestations and drainage, examination of peripheral blood, drainage and intestinal flora, identification of high risk factors such as fever, diarrhea and increased infectious indicators, and timely administration of CT with contrast enema.

[Quality of life of patients with locally advanced rectal cancer after neoadjuvant therapy and sphincter-preserving surgery].

Objective: To investigate quality of life (QoL) of patients with locally advanced rectal cancer (LARC) who underwent low anterior resection with protective stoma under neoadjuvant therapy mode, and to explore the changes of QoL of patients from before neoadjuvant therapy to 12 months after stoma reversal. Methods: A descriptive case series study was carried out. A retrospective study was performed on patients with mid and low LARC who received complete neoadjuvant long course radiotherapy and chemotherapy, followed by radical low anterior resection (LAR) combined with protective stoma at Peking Union Medical College Hospital from December 2017 to January 2020. Inclusion criteria: (1) patients with rectal MRI assessment of mT3-4b or mN1-2 without distant metastasis (M0) before neoadjuvant therapy; (2) distance from tumor lower margin to the anal verge <12 cm; (3) rectal adenocarcinoma confirmed by biopsy before neoadjuvant therapy; (4) complete cycle of neoadjuvant therapy; (5) patients undergoing radical LAR with sphincter preservation and protective ostomy; (6) patients receiving follow-up for more than 12 months after stoma reversal. Exclusion criteria: (1) patients as grade Ⅳ to Ⅴclassified by the American Society of Anesthesiologists (ASA); (2) patients with multiple primary colorectal cancer; (3) patients with history of other malignant tumors in the past 5 years; (4) patients of emergency surgery; (5) pregnant or lactating women; (6) patients with history of severe mental illness; (7) patients with contraindication of MRI, radiotherapy, chemotherapy, or surgical treatment. A total of 83 patients were enrolled, including 51 males and 28 females with median age of 59 years and mean BMI of (24.4±3.1) kg/m(2). EORTC QLQ-CR29, international erectile function index (IIEF), Wexner constipation score and low anterior resection syndrome (LARS) score were applied to investigate the QoL of the patients before neoadjuvant therapy, 3 and 12 months after ostomy reversal, including rectal anal function and sexual function. M (P25, P75) was used for the scores of the scale. Results: (1) EORTC QLQ-CR29 score showed that before neoadjuvant therapy, before surgery, 3 months and 12 months after ostomy reversal, anxiety [64.4 (52, 0, 82.5), 75.3 (66.0, 89.5), 82.6 (78.5, 90.0), 83.6 (78.0, 91.0)] and concern about body image [76.8 (66.0, 92.0), 81.1 (76.5, 91.5), 85.5 (82.5, 94.0), 86.1 (82.0, 92.0)] were improved (all P<0.01); pelvic pain [5.4 (2.0, 8.0), 5, 0 (2.0, 7.8), 3.9 (1.0, 5.0), 3.0 (1.0, 5.0)], urinary incontinence [15.7 (7.0, 22.0), 11.1 (0, 17.5), 10.0 (0, 17.0), 9.9 (0, 16.0)], impotence [14.3 (4.2, 19.0), 12.2 (0, 16.8), 5.6 (0, 10.0), 5.2 (0.2, 8.0)], urinate [26.4 (13.0, 38.5), 13.9 (0, 20.0), 13.4 (2.5, 21.5), 13.2 (2.0, 20.0)] and mucous bloody stool [4.7 (3.0, 6.0), 2.6 (0, 5.0), 2.2 (0, 5.0), 1.9 (0, 4.0)] were improved as well (all P<0.01). The scores fluctuated in the improvement of male sexual function, abdominal pain, dry mouth, worry about body mass change, skin pain and dyspareunia, but the symptoms were significantly improved after ostomy reversal compared with before neoadjuvant therapy (all P<0.05). There were no significant changes in female sexual function, dysuria, dysgeusia and fecal incontinence after ostomy reversal compared with before neoadjuvant therapy (all P>0.05). (2) IIEF scale showed that all scores were similar before and after neoadjuvant therapy (all P>0.05). (3) Rectal and anal function scale revealed that before neoadjuvant therapy, before operation, 3 months and 12 months after stoma reversal, gas incontinence [3.1 (0, 4.0), 2.3 (0, 4.0), 1.8 (0, 4.0), 1.2 (0, 3.0)] and urgent defecation [7.2 (0, 11.0), 5.2 (0, 11.0), 2.9 (0, 9.0), 1.7 (0, 0)] were improved (all P<0.001). In terms of improving incomplete emptying sensation, the symptoms fluctuated, but the symptoms improved significantly after ostomy reversal compared with before neoadjuvant therapy (all P<0.05). While the symptoms of assistance with defecation [0 (0, 0), 0.7 (0, 1.0), 0.6 (0, 1.0), 0.7 (0, 1.0)] and defecation failure [0.2 (0, 0), 1.0 (0, 2.0), 0.8 (0, 1.5), 0.8 (0, 1.0)] showed a worsening trend (all P<0.001). Stratified analysis was performed on patients with different efficacy of neoadjuvant therapy to compare the changes in QoL before and after neoadjuvant therapy. Patients with less sensitive and more sensitive neoadjuvant therapy showed similar changes in function and symptoms. Patients with less sensitive therapy showed significant improvement in dysuria, urinary incontinence, skin pain and dyspareunia (all P<0.05), and the symptom of defecation frequency in more sensitive patients was significantly improved (P<0.05). Conclusions: For patients with LARC, neoadjuvant radiochemotherapy combined with radical LAR and protective stoma can improve QoL in many aspects. It is noted that patients show a worsening trend in the need for assistance with defecation and in defecation failure.

[Protective colostomy and protective ileostomy for the prevention of anastomotic leak in patients with rectal cancer after neoadjuvant chemoradiotherapy and radical surgery].

Objective: To investigate whether protective colostomy and protective ileostomy have different impact on anastomotic leak for rectal cancer patients after neoadjuvant chemoradiotherapy (nCRT) and radical surgery. Methods: A retrospectively cohort study was conducted. Inclusion criteria: (1) Standard neoadjuvant therapy before operation; (2) Laparoscopic rectal cancer radical resection was performed; (3) During the operation, the protective enterostomy was performed including transverse colostomy and ileostomy; (4) The patients were followed up regularly; (5) Clinical data was complete. Exclusion criteria: (1) Colostomy and radical resection of rectal cancer were not performed at the same time; (2) Intestinal anastomosis is not included in the operation, such as abdominoperineal resection; (3) Rectal cancer had distant metastasis or multiple primary colorectal cancer. Finally 208 patients were included in this study. They suffered from rectal cancer and underwent protective stoma in radical surgery after nCRT at our hospital from January 2014 to December 2018. There were 148 males and 60 females with age of (60.5±11.1) years. They were divided into protective transverse colostomy group (n=148) and protective ileostomy group (n=60). The main follow up information included whether the patient has anastomotic leak and the type of leak according to ISREC Grading standard. Besides, stoma opening time, stoma flow, postoperative hospital stay, stoma related complications and postoperative intestinal flora were also collected. Results: A total of 28 cases(13.5%) suffered from anastomotic leak and 26 (92.9%) of them happened in the early stage after surgery (less than 30 days) . As for these early-stage leak, ISREC Grade A happened in 11 cases(42.3%), grade B in 15 cases(57.7%) and no grade C occurred. There was no significant difference in the incidence [12.8% (19/148) vs. 15.0% (9/60) , χ(2)=0.171, P=0.679] or type [Grade A: 5.4%(8/147) vs. 5.1%(3/59); Grade B: 6.8%(10/147) vs. 8.5%(5/59), Z=0.019, P=1.000] of anastomotic leak between the transverse colostomy group and ileostomy group (P>0.05), as well as operation time, postoperative hospital stay, drainage tube removal time or stoma reduction time (P>0.05). There were 10 cases (6.8%) and 24 cases (40.0%) suffering from intestinal flora imbalance in protective transverse colostomy and protective ileostomy group, respectively (χ(2)=34.503, P<0.001). Five cases (8.3%) suffered from renal function injury in the protective ileostomy group, while protective colostomy had no such concern (P=0.002). The incidence of peristomal dermatitis in the protective colostomy group was significantly lower than that in the protective ileostomy group [12.8% (9/148) vs. 33.3%(20/60), χ(2)=11.722, P=0.001]. Conclusions: It is equally feasible and effective for rectal cancer patients after nCRT to carry out protective transverse colostomy or ileostomy in radical surgery. However, we should pay more attention to protective ileostomy patients, as they are at high risk of intestinal flora imbalance, renal function injury and peristomal dermatitis.

[Prognosis and complications of local excision for rectal cancer after neoadjuvant chemoradiotherapy].

Objective: To investigate the prognosis and postoperative complications of local excision for rectal cancer after neoadjuvant chemoradiotherapy (nCRT). Methods: A descriptive case series study was carried out. Patient inclusion criteria: (1) patients who underwent local excision by transanal endoscopic microsurgery (TEM) after nCRT; (2) magnetic resonance evaluated tumor regression grade (mrTRG) as 1, 2 after nCRT;(3) American Society of Anesthesiologists class I to III. Patient exclusion criteria: (1) with multiple primary colorectal cancers; (2) with other malignant tumors within five years; (3) with emergency surgicery indications like digestive tract obstruction, perforation or bleeding. Clinicopathological and follow-up data of rectal cancer patients with obvious tumor regression after nCRT who underwent local excision in Peking Union Medical College Hospital from January 2010 to August 2019 were retrospectively collected. Outcome measures included disease-free survival (DFS), short-term postoperative complications, and at postoperative 1-year during follow up, gas continence, fecal continence, and quality of life (using the EORTC QLQ-CR29 scale, higher score indicated worse quality of life) at postoperative 1-year. Results: A total of 40 patients were included in this study. There were 27 males and 13 females with an average age of (66.7±12.3) years. Preoperative rectal ultrasound and other imaging examinations indicated that the tumor was located in the anterior wall in 16 cases, the lateral wall in 12 cases, and the posterior wall in 12 cases. The distance between the lower margin of the tumor and the anal verge was (4.3±1.2) cm before nCRT and (5.1±0.9) cm after nCRT. According to mrTRG, 31 cases were assessed as mrTRG 1 and 9 cases as mrTRG 2. All the patients received local extended excision of rectal cancer using TEM platform. A total of 19 cases(47.5%) suffered from complications within one month postoperatively. Clavien-Dindo grade I complications happened in 14 cases, grade II in 3 patients, and grade III in 2 cases, who all were healed by conservative treatment. Except that 2 patient presented severe low anterior resection syndrome (LARS) at 1 year postoperatively, no severe anal dysfunction was found in this cohort patients. EORTC QLQ CR29 scale results for quality of life showed that at 1 year after TEM excision, except taste (Z=-1.968, P=0.049), anxiety (Z=-3.624, P<0.001) and skin irritation (Z=-2.420, P=0.023) were worse than the situation before neoadjuvant therapy, there were no statistically significant differences in other assessment results between pre-operation and post-operation (all P>0.05). Postoperative pathological results indicated complete tumor regression (pTRG0) in 17 cases, moderate remission (pTRG1) in 13, and mild remission (pTRG2) in 10. During the follow-up of (49.1±29.6) months, 3 patients had local recurrence and 4 had distant metastasis (3 patients with liver metastasis and 1 patient with lung metastasis followed by liver metastasis). No death was found and the 5-year disease-free survival (DFS) was 84.3%. Conclusions: Local excision through TEM following nCRT not only can be adopted as an important means to accurately determine complete clinical remission (cCR), but also has high therapeutic value for rectal cancer patients presenting cCR or near cCR, with little impact on defecatory function and quality of life. However, the morbidity of complication of TEM excision after nCRT is relatively high and there is a risk of recurrence and metastasis. Therefore, it is still necessary to strictly select the indications of local excision.

[Correlative factors of sigmoid sinus diverticulum formation in individuals without pulsatile tinnitus based on CT angiography].

Objective: To analyze the correlative factors of sigmoid sinus diverticulum(SSD) formation in individuals without pulsatile tinnitus based on computed tomography angiography (CTA). Methods: A hospital-based 1∶2 matched case-control study was performed on 42 cases with unilateral SSD and 84 controls for age, gender, and side-matched in Beijing Friendship Hospital affiliated to the Capital Medical University from January 2018 to December 2018.The signs including sigmoid sinus wall dehiscence, venous outflow dominance, transverse sinus stenosis, high jugular bulb, large emissary veins, degree of mastoid pneumatization (grade Ⅰ-Ⅳ) and empty sella, were detected and analyzed. Results: In SSD group and control group, there were statistically significant differences in the incidence of the sigmoid sinus wall dehiscence (66.7%(28/42) vs 19.0%(16/84)), ipsilateral transverse sinus stenosis on the dominant side of venous outflow (26.2%(11/42) vs 4.8%(4/84)), and empty sellae (19.0%(8/42) vs 7.1%(6/84))(all P<0.05).The degree of mastoid pneumatization was worse in SSD group (P<0.05). After stepwise logistic regression analysis,the sigmoid sinus wall dehiscence (P<0.01,OR=6.794,95%CI 2.530-18.245), ipsilateral transverse sinus stenosis on the dominant side (P=0.001, OR=13.293, 95%CI 2.841-62.194), and degree of mastoid pneumatization (P<0.01, OR=0.289, 95%CI 0.156-0.536) were found independently correlated with SSD. Conclusion: Sigmoid sinus wall dehiscence, ipsilateral transverse sinus stenosis on the dominant side of venous outflow and mastoid pneumatization may be some of the independent correlative factors of SSD.

[Analysis on current status and influential factors of occupational stress among couriers].

Objective: To investigate the current status and influencing factors of occupational stress among couriers. Methods: Couriers (n=925) were selected on this study used cluster sampling method from January to March 2018. They were from SF and Zhongtong Express Co., Ltd., on the Wechat platform, and surveyed by a job stress questionnaire based on a job demand-control model.Valid questionnaires(n=617) were obtained. Results: A total of 418 workers were occupational stress positive (67.7%). The results of Chi-square analysis showed that there were significant differences in occupational stress among workers categorized by job position, working years, mealtime, sleeping time, and weekly work time (P<0.05). The multivariate logistic analysis indicated that non-regular meals, short-term sleep and less than 0.5 working years were risk factors for occupational stress(P<0.05). Conclusion: Couriers generally have occupational stress. The main influencing factors are job position, working years mealtime, sleeping time, and weekly work time. It is necessary to guide healthy lifestyle, rationally organize labor and assign tasks, and improve working environment to relieve their occupational stress.

[Influence of TNF-α on the immunomodulatory property of laryngeal mucosa mesenchymal stromal cells'].

Objective: To investigate the effect of tumor necrosis factor-alpha(TNF-α)on the immunoregulatory capacity of laryngeal mucosal mesenchymal stromal cells (LM-MSCs) and its potential molecular mechanism, and provide a theoretical basis for the study of chronic laryngitis. Methods: LM-MSCs were separated from epiglottal mucosa. The LM-MSCs cells were directly co-cultured with T cells in vitro to detect the immunomodulatory property of LM-MSCs. After long-term stimulation with inflammatory factors TNF-α in vitro, the differences were compared in the immunomodulatory ability of LM-MSCs between normal LM-MSCs and TNF-α stimulated LM-MSCs. The expression of general control non-repressed protein5(GCN5), FAS, FASL in normal LM-MSCs and TNF-α stimulated LM-MSCs was detected by Western blot and quantitative real-time RT-PCR(RT-qPCR). Results: After chronic stimulation of TNF-α, the RNA relative expression of GCN5 was 0.31±0.03 (3 days) and 0.53±0.06 (7 days) compared with control group, showing significant difference (F=13.45, P<0.05). The percentage of LM-MSC-induced T cell apoptosis was 6.27%±0.81% (3 days) and 4.99%±0.52% (7 days) in chronic stimulation group compared with control group 10.02%±1.02%. There is a significant difference among these groups (F=11.13, P<0.05). Moreover, the ability of LM-MSCs to induce T cell apoptosis is regulated by GCN5. Conclusion: With the chronic stimulation of TNF-α, the expression of GCN5 in LM-MSCs is decreased, thus impairing its immunoregulatory capacity.

Systematic review with meta-analysis: thiopurines decrease the risk of colorectal neoplasia in patients with inflammatory bowel disease.

BACKGROUND: Patients with inflammatory bowel disease (IBD) have a high risk of developing colorectal neoplasia. AIM: To investigate whether thiopurines can decrease the risk of developing colorectal neoplasia in patients with ulcerative colitis (UC) or Crohn's disease (CD). METHODS: We conducted a meta-analysis of 24 observational studies involving 76,999 participants to evaluate the risks of developing colorectal neoplasia in IBD patients receiving thiopurine treatment. Pooled odds ratios (ORs) and 95% confidence intervals (CIs) for the risks of colorectal neoplasia were calculated using a random-effects model. RESULTS: The overall pooled estimate revealed a protective effect of thiopurine use on colorectal neoplasia in patients with IBD (OR = 0.63, 95% CI 0.46-0.86). The effect was significant in UC patients (OR = 0.67, 95% CI 0.45-0.98), but was not significant in CD patients (OR = 1.06, 95% CI 0.54-2.09). Thiopurines exposure significantly decreased the risk of colorectal cancer (CRC) (OR = 0.65, 95% CI 0.45-0.96) and advanced colorectal neoplasia (CRC and/or high-grade dysplasia) (OR = 0.62, 95% CI 0.44-0.89), but did not decrease the risk of dysplasia alone (OR = 0.90, 95% CI 0.37-2.21). Tendencies towards the protective effect of thiopurines were distinct in clinic-based studies (OR = 0.59, 95% CI 0.42-0.82) and case-control studies (OR = 0.40, 95% CI 0.26-0.62), but not in population-based studies (OR = 0.95, 95% CI 0.55-1.62) and cohort studies (OR = 0.98, 95% CI 0.81-1.18). Interestingly, studies conducted in Europe (OR = 0.48, 95% CI 0.31-0.77), rather than in North America (OR = 0.91, 95% CI 0.67-1.24), showed the protective effect of thiopurines. CONCLUSIONS: This meta-analysis revealed an antineoplastic effect of thiopurines on colorectal neoplasia in patients with IBD, particularly amongst patients with UC.

[Primary investigation of immunoglobulin A synthesis and secretion in human mesangial cells].

OBJECTIVE: To investigate the expression of immunoglobulin A (IgA) in human mesangial cells (HMCs). METHODS: The HMCs were cultured. The subcellular location of IgA was detected by immunofluorescence staining; the transcripts of Igα,Igκ and IgΛ constant region were detected by reverse transcription-polymerase chain reaction (RT-PCR) and further analyzed by DNA sequencing. The expressions of Igα and IgΛ were detected at transcription level by Western blot after the cytoplasmic protein extraction. The culture supernatant was collected to explore whether IgA could be secreted out of the cell and the protein was further analyzed by mass spectrometry after being purified by affinity chromato-graphy with jacalin-sepharose. The results of DNA sequencing and mass spectrometry were aligned with the mRNA and amino acid sequences in the National Center of Biotechnology Information (NCBI) database. RESULTS: By immunofluorescence staining, we detected the presence of IgA heavy chain Igα, light chain, both Igκ and IgΛ in expressions of transcripts of Igα1, Igα2, Igκ and IgΛ in the HMCs and the alignment of the sequences of the RT-PCR products with those of the IgCα1, IgCα2, Igκ and IgΛ mRNA in the NCBI database exhibited that the similarities were 99%, 97%, 98% and 97%, respectively. Western blot showed Igα and IgΛ expressions in the cell lysate and secretion of Igα1 and Igα2 heavy chains in cell culture supernatant. To further explore the protein that secreted into the supernatant, after supernatant affinity chromatography with jacalin-sepharose, the proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and the band approximating to 65 000 was cut and sent to mass spectrometry. The results were aligned with the amino acid sequences of Igα1 and Igα2 constant region in NCBI database, showing that amino acids between No.52 and No.104, amino acids between No.154 and No.221, amino acids between No.276 and No.327 from IgCα1 and amino acids between No.52 and No.113, amino acids between No.151 and No.204, amino acids between No.251 and No.314 from IgCα2 were the same with those derived from B cells. CONCLUSION: Our fin-dings suggested that HMCs could synthesize and secret IgA.

[Tumor derived IgG suppress the proliferation of T cells in cord blood].

OBJECTIVE: To explore the function of tumor derived IgG (tIgG) and whether the tIgG can inhibit T cells activity. METHODS: The tIgG was purified from ovarian cancer tissue. The cord blood monocyte cells (CBMC) and cord blood lymphocyte (CBL) were isolate from human umbilical cord blood. The CBMC and CBL were stimulated with phytohaemagg lutinin (PHA) in order to let the CBMC and CBL in the state of proliferation. Carboxyfluorescein succinimidyl amino ester (CFSE) was cultured with CBMC and CBL. CFSE had no cell toxicity, which could penetrate through the cell membrane and combine the intracellular protein. The fluorescence intensity decreased with the proliferation of cells step by step, so the proliferation of these cells could be detected in flow ctytometry. The tIgG which was purified from ovarian cancer tissue was divided into three groups, 1 mg/L group, 10 mg/L group, and 100 mg/L group, and the intravenous immunoglobulin (IVIG) was also divided into three groups too. The CBMC and CBL were treated by tIgG with 1 mg/L, 10 mg/L, and 100 mg/L in order to observe the proliferation of T cells. The cells were treated with IVIG as a positive control group, and the cells were treated with phosphate buffer saline (PBS) as a negative control. The proliferation of CD4+ or CD8+ T cells were detected in CBMC and CBL. The proliferation of the T cells in CBMC and CBL after 64 h and 86 h were detected. RESULTS: In the system of CBMC, the tIgG could suppress the proliferation of CD4+ or CD8+ T cells. The results could also be found in the system of CBL. The CD4+ or CD8+ T cells in the group which were treated with PBS were more active than those in the group which were treated with tIgG and IVIG. The suppression in the group which were treated with tIgG, was stronger than that in the group treated with IVIG. In addition, the suppression of T cells in the group which were stimulated with tIgG as 100 mg/L was more effective than that in the group which were stimulated with tIgG as 10 mg/L. This could prove that tIgG had the function of immunomodulation. CONCLUSION: The tIgG can be involved in immune escape of cancer.

[Expression of enterotoxigenic Bacteroides fragilis and polyketide synthase gene-expressing Escherichia coli in colorectal adenoma patients].

OBJECTIVE: To investigate the distribution of various bacteria in adenoma tissue of colorectal adenoma (T/CRA), normal colonic mucosa tissue adjacent to the adenoma (N/CRA), and healthy colonic mucosa tissue (N/H) by comparing the number of total bacteria, Bacteroides fragilis (BF), enterotoxigenic Bacteroides fragilis (ETBF), polyketide synthase (pks) gene-expressing Escherichia coli(E.coli)(pks(+) E. coli)among the above 3 types of tissues. METHODS: A total of 36 patients diagnosed with colorectal adenoma by colonoscopy and pathology in Department of Gastroenterology, Peking University People's Hospital from September 2011 to September 2013 were selected into this study. T/CRA and N/CRA tissues from the 36 patients and N/H tissues from 18 healthy controls were collected for DNA extraction. The number of total bacteria, BF, ETBF, pks(+) E. coli was detected by quantitative real time PCR, and their correlation with colorectal adenoma was analyzed. RESULTS: (1) The number of total bacteria decreased gradually from N/H, N/CRA, to T/CRA, with the median values being 3.18×10(8,) 1.57×10(8,) and 7.91×10(7) copies/g, respectively, and with significant difference among the three groups and between each two groups (all P<0.01). (2) The content of BF decreased gradually from N/H, N/CRA, to T/CRA, the median values being 6.03×10(5,) 4.28×10(4,) and 5.48×10(3) copies/g, respectively, and with significant difference among the three groups and between each two groups (all P<0.01). (3) The toxin content produced by ETBF increased from N/H, N/CRA, to T/CRA, the relative expression being 1.73±0.30, 6.15±1.52, and 8.54±1.80, respectively. Significant difference was found between the T/CRA and N/H tissue (P=0.003), but not between any other two groups. (4) The expression of clbB in pks(+) E.coli was highest in T/CRA colonic tissue (2.96±0.28), followed by the N/CRA (2.79±0.19) and N/H tissue (1.06±0.08). Significant difference was found between T/CRA and N/H tissues, as well as between N/CRA and N/H tissues (both P<0.001), but not between T/CRA and N/CRA tissues. CONCLUSIONS: The number of total bacteria is markedly reduced in the colonic mucosa of CRA patients compared to normal people, while the expressions of ETBF and pks(+) E.coli are significantly increased. Such changes in total bacterial, ETBF and pks(+) E.coli concentrations in colonic mucosa may be related to the tumorigenesis of colorectal adenoma.

Desmoglein 4 diversity and correlation analysis with coat color in goat.

Desmoglein 4 (DSG4) has an important role in the development of wool traits in domestic animals. The full-length DSG4 gene, which contains 3918 bp, a complete open-reading-frame, and encodes a 1040-amino acid protein, was amplified from Liaoning cashmere goat. The sequence was compared with that of DSG4 from other animals and the results show that the DSG4 coding region is consistent with interspecies conservation. Thirteen single-nucleotide polymorphisms (SNPs) were identified in a highly variable region of DSG4, and one SNP (M-1, G>T) was significantly correlated with white and black coat color in goat. Haplotype distribution of the highly variable region of DSG4 was assessed in 179 individuals from seven goat breeds to investigate its association with coat color and its differentiation among populations. However, the lack of a signature result indicates DGS4 haplotypes related with the color of goat coat.

Dynamic comparison of genetic diversity in a Small Tail Han sheep population using meta-analysis.

The aim of this research was to identify the dynamic diversity of Small Tail Han sheep in its main producing areas between different years, and provide a basis for a breeding and genetic resources conservation strategy. For this purpose, 15 microsatellites were genotyped for Small Tail Han Sheep sampled in 2014 from Heze, China, and a comparative analysis of these data with those from a previous study was undertaken using meta-analysis. The results reveal that inbreeding has caused a reduction in diversity of Small Tail Han Sheep from 2008 to 2014. Overall, our results are helpful in understanding the dynamic change in diversity, as well as providing information for a conservation strategy for this population.

Expressional analysis of immune-related miRNAs in breast milk.

Immune-related miRNAs in breast milk are extracellular miRNAs that are related to immune organ development and regulation of the immune function in infants and young animals. The goal of this study was to compare the expression levels of five immune-related miRNAs in breast milk in black goats, humans, and dairy cattle. The miRNAs from milk were extracted and the expression levels were assessed using quantitive RT-PCR methods. MiR-146, miR-155, miR-181a, miR-223, and miR-150 were all detected in Dazu black goat milk, and these miRNAs were significantly more highly expressed in colostrum than in mature milk of goats (P < 0.01), except for miR-150. Further, all five miRNAs were expressed in human colostrum, but patterns differed from those in goats: miR-146 and miR-155 were highly expressed (P < 0.01) in human colostrum, whereas miR-223 was abundant in goat colostrum (P < 0.01). In addition, five miRNAs were significantly higher in bovine mature milk than in goat milk (P < 0.01). Taken together, these results confirm that immune-related miRNAs are rich in breast milk with different expression levels depending on the lactation phase and species.

Brief Note :Variability in the cathelicidin 6 (CATHL-6) gene in Tianzhu white yak from Tibetan area in China.

Cathelicidins are a major family of antimicrobial peptides (AMPs), an important component of innate immune system, playing a critical role in host defense and disease resistance in virtually all living species. Polymorphism and functional studies on cathelicidin of Tianzhu white yak contribute to understanding the specific innate immune mechanism in animals living at high altitudes in comparison to cattle and domesticated white yak. Thirty-six individuals of Tianzhu white yak, originating from the area of three ecotypes (Gansu in China), were investigated. The total length of the aligned Yak cathelicidin 6 (CATHL-6) sequences was 1923 bp, including six single nucleotide polymorphisms and one indel. Ten haplotypes were identified, and phylogenetic analyses resolved those 10 haplotypes in two clusters. The results indicate that the white yak originated from two domestication sites. In addition, lack of significant pairwise difference between sequences (Tajima's D = 0.92865, P > 0.10) in the CATHL-6 region indicates absence of population size expansion in current white yak population.

Association of ABCB1, CYP3A4*18B and CYP3A5*3 genotypes with the pharmacokinetics of tacrolimus in healthy Chinese subjects: a population pharmacokinetic analysis.

WHAT IS KNOWN AND OBJECTIVE: Tacrolimus (TAC) is metabolized mainly by the CYP3A subfamily and extruded into the intestine by P-glycoprotein, which is encoded by the ABCB1 gene. Several studies have suggested that the CYP3A5*3 genotype influenced the pharmacokinetics (PK) of TAC. The CYP3A4*18B and CYP3A5*3 alleles are clinically important in Chinese subjects because of their relatively high frequency. The present study aimed at evaluating the effects of ABCB1 (C1236T-G2677T/A-C3435T), CYP3A4*18B and CYP3A5*3 genetic polymorphisms on TAC PK in healthy Chinese subjects. METHODS: Data were obtained from a comparative bioavailability study of oral TAC formulations (n = 22). TAC whole blood concentrations were measured by LC-MS/MS. Genetic polymorphisms were determined using a direct sequencing method. Nonlinear mixed-effects modelling (NONMEM) was performed to assess the effect of genotypes and demographics on TAC PKs. RESULTS AND DISCUSSION: Both CYP3A4*18B and CYP3A5*3 polymorphisms affected the TAC PK, whereas ABCB1 genetic polymorphisms and other demographic characteristics did not. The combined genotypes of CYP3A4*18B and CYP3A5*3 had a greater impact than either genotype alone, and they were estimated to account for 28·4% of the inter-subject variability of apparent clearance (CL/F) by NONMEM. The CL/F in subjects with CYP3A4*1/*1-CYP3A5*3/*3 was 10·3 L/h and was 48·5% in those not carrying CYP3A4*1/*1-CYP3A5*3/*3. WHAT IS NEW AND CONCLUSION: This is the first study to extensively explore the influence of CYP3A4*18B, CYP3A5*3 and ABCB1 genetic polymorphisms on TAC PK in healthy Chinese subjects. The results demonstrated that subjects with a combined genotype of CYP3A4*1/*1-CYP3A5*3/*3 may require lower TAC doses to achieve target concentration levels and further investigation is needed in larger populations to confirm the clinical benefits.

Detection and quantification of copper-denitrifying bacteria by quantitative competitive PCR.

We developed a quantitative competitive PCR (QC-PCR) system to detect and quantify copper-denitrifying bacteria in environmental samples. The primers were specific to copper-dependent nitrite reductase gene (nirK). We were able to detect about 200 copeis of nirK in the presence of abundant non-specific target DNA and about 1.2 x 10(3)Pseudomonas sp. G-179 cells from one gram of sterilized soil by PCR amplification. A 312-bp nirK internal standard (IS) was constructed, which showed very similar amplification efficiency with the target nirKfragment (349 bp) over 4 orders of magnitude (10(3)-10(6)). The accuracy of this system was evaluated by quantifying various known amount of nirK DNA. The linear regressions were obtained with a R(2) of 0.9867 for 10(3)copies of nirK, 0.9917 for 10(4) copies of nirK, 0.9899 for 10(5) copies of nirK and 0.9846 for 10(6) copies of nirK. A high correlation between measured nirK and calculated nirK (slope of 1.0398, R(2)=0.9992) demonstrated that an accurate measurement could be achieved with this system. Using this method, we quantified nirK in several A-horizon and stream sediment samples from eastern Tennessee. In general, the abundance of nirK was in the range of 10(8)-10(9) copies g soil(-1) dry weight. The nirK content in the soil samples appeared correlated with NH(4)(N) content in the soil. The activities of copper-denitrifying bacteria were evaluated by quantifying cDNA of nirK. In most of sample examined, the content of nirK cDNA was less than 10(5) copies g soil(-1) dry weight. Higher nirK cDNA content (>10(6) copies g soil(-1) dry weight) was detected from both sediment samples at Rattlebox Creek and the Walker Branch West Ridge. Although the stream sediment samples at the Walker Branch West Ridge contained less half of the nirK gene content as compared to A-horizon sample, the activities of copper-denitrifying bacteria were almost 600 times higher than in the A-horizon sample.

Increment of hFIX expression with endogenous intron 1 in vitro.

This paper probes into the feasibility of increasing expression level of hFIX gene with endogenous intron 1 sequence. hFIX minigene was obtained with middle sequence truncated intron 1 inserted into the relative site of hFIX cDNA, and plasmid vector pKG5i'IX, retroviral vector GINaCi'IX were constructed. These vectors were transduced into target cells of PA317, C2C12, primary rabbit skin fibroblasts (RSF) and primary human skin fibroblasts (HSF). The expression level of mixed colonies are PA317/pKGoi'IX, 151 ng/10(6) cells/24h; PA317/G1NaCi'IX, 308 ng/10(6) cells/24 h; C2C12/G1 NaCi'IX, 188 ng/10(5) cells/24 h; RSF/G1NaCi'IX, 1929 ng/10(5) cells/24 h; HSF/G1NaCi'IX, 1646 ng/10(6) cells/24 h. These results indicated that hFIX minigene with intron 1 is able to increase the expression level to about 3 times of that of hFIX cDNA. Meanwhile, in order to study the application of hFIX minigene in the retroviral-mediated gene transfer system and refrain from intron splicing during viral production, a retroviral vector G1NaCi'IXR with reversely inserted hFIX minigene expression cassette was constructed. The expression level of reverse constructor in PA317 cells was 390 ng/10(6) cells/24 h with 79% of bioactivity. PCR detection of HT/G1NaCi'IXR cells infected with PA317/G1NaCi'IXR supernatant confirmed the existence of intron 1 sequence. These results suggested that expression vector with forward-inserted intron1-carrying hFIX expression cassette can be used in directed gene transfer, but when using the retroviral-mediated gene transfer system, reversely-inserted intronl-carrying hFIX expression cassette should be considered.