[Effect of sleep spindle density on memory function in patients with obstructive sleep apnea hypopnea syndrome].

Objective: To explore the characteristics of sleep spindle density in nonrapid eye movement (NREM) stage 2 (N2) sleep and its effect on memory function in patients with obstructive sleep apnea hypopnea syndrome (OSAHS). Methods: Patients who underwent polysomnography (PSG) examination due to snoring in the Second Affiliated Hospital of Soochow University from January to December 2021 were prospectively collected. A total of 119 male patients, aged 23-60 (37.4±7.3) years, were enrolled finally. According to the apnea hyponea index (AHI), the subjects were divided into a control group (AHI<15 times/h) of 59 cases and an OSAHS group (AHI≥15 times/h) of 60 cases. The basic information, general clinical data and PSG parameters were collected. Memory function scores were evaluated by using logical memory test (LMT), digit ordering test (DOT) and pattern recognition memory (PRM), spatial recognition memory (SRM) and spatial working memory (SWM) in CANTAB test. The number of N2 sleep spindles in leads left central area (C3) and right central area (C4) was counted by hand and the sleep spindle density (SSD) was calculated. The differences in the above indexes and N2 SSD were compared between the two groups. Shapiro-Wilk method, chi-squared test, Spearman correlation analysis and stepwise multivariate logistic regression analysis were used to investigate the influencing factors of memory scores in patients with OSAHS. Results: Compared with the control group, the proportion of the slow-wave sleep, the minimum blood oxygen saturation, the SSD in C3 of NREM2 stage and the SSD in C4 of NREM2 stage were lower in the OSAHS group. The body mass index (BMI), proportion of N2 sleep, oxygen reduction index, percentage of time with oxyhemoglobin saturation below 90% (TS90), maximum duration of apnea and respiratory effort-related arousal(RERA) were higher in the OSAHS group (all P<0.05). Compared with the control group, the immediate LMT score was lower, while the time for immediately completing PRM test, the total time for immediately completing SRM test and the time for delayed completing PRM test were longer in the OSAHS group, suggesting that the immediate logical memory, immediate visual memory, spatial recognition memory and delayed visual memory were worse in the OSAHS group. Stepwise multivariate logistic regression analysis revealed that the number of years of education (OR=0.744, 95%CI 0.565-0.979, P=0.035), maximum duration of apnea (OR=0.946, 95%CI 0.898-0.997, P=0.038) and N2-C3 SSD (OR=0.328, 95%CI 0.207-0.618, P=0.012) and N2-C4 SSD (OR=0.339, 95%CI 0.218-0.527, P=0.017) were independent factors affecting the immediate visual memory. The AHI (OR=1.449, 95%CI 1.057-1.985, P=0.021), N2-C3 SSD (OR=0.377, 95%CI 0.246-0.549, P=0.009), and N2-C4 SSD (OR=0.400, 95%CI 0.267-0.600, P=0.010) were independent factors affecting delayed visual memory. Conclusions: The decrease in SSD is associated with impaired memory function in patients with moderate-severe OSAHS, which is manifested as impairment of immediate visual memory and delayed visual memory. This suggests that changes of sleep spindle wave in N2 may be an electroencephalographic biomarker for assessing cognitive impairment in OSAHS patients.

[Analysis of bronchoscope-guided tracheal intubation in 12 cases with coronavirus disease 2019 under the personal protective equipment with positive pressure protective hood].

Endotracheal intubation is an independent risk factor for respiratory infectious diseases. We conducted a retrospective study in 12 cases with COVID-19 who underwent endotracheal intubation at ICU of the Guangzhou eighth hospital from January 20 to February 10, 2020. The intubation procedure, anesthetic regimen, and complication were collected and analyzed. The 9 healthcare workers who involved in intubation received virus nucleic acid test and 14 days temperature monitoring. All 12 patients were successfully intubated under the guidance of bronchoscope, without any complications. Midazolam, Propofol and Morphine or fentanyl were used for sedation and analgesia, avoiding patients cough and agitated during the procedure. The 9 healthcare workers were protected under the Personal Protective Equipment(PPE) with positive pressure protective hood. The detection of oropharyngeal swab virus nucleic acid were negative in all 9 healthcare workers, none of them had fever or any respiratory symptoms. The PPE with positive pressure protective hood should be needed to perform bronchoscope-guided endotracheal intubation in patients with COVID-19, it could strengthen to protect healthcare workers from virus exposure.

Association between a point mutation at the -743-bp region of the transthyretin (TTR) gene and familial vitreous amyloidosis.

The aim of this study was to identify changes in the base sequence of the upstream regulatory region of the transthyretin (TTR) gene. Whole-blood DNA was extracted from ten subjects belonging to a family with familial amyloidosis vitreoretinopathy; the upstream regulatory sequence was amplified by polymerase chain reaction, detected by gel electrophoresis, and sequenced. The DNA sequence of the upstream regulatory region of the TTR gene was successfully sequenced, and a point mutation (-743A→T) was identified in six of the ten blood samples: four patients and two family members without disease incidence. Therefore, a point mutation was identified in the upstream regulatory region of the TTR gene in a Han Chinese family with familial vitreous amyloidosis.

Establishment of a blue light damage model of human retinal pigment epithelial cells in vitro.

To establish a blue-light damage model of human retinal pigment epithelium (RPE). Fourth-generation human RPE cells were randomly divided into two groups. In group A, cells were exposed to blue light (2000 ± 500 lux) for 0 (control), 3, 6, 9, and 12 h, and cell culture was stopped after 12 h. In group B, cells were exposed to blue light at the same intensity and time periods, but cell culture was stopped after 24 h. TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed to determine the most suitable illuminating time with apoptotic index. Flow cytometry was used to determine apoptotic ratio of RPEs. In group A, the apoptotic index of cells that received 6, 9 and 12 h of blue light was higher than that of control. The apoptotic index of cells receiving 9 and 12 h was higher than that of 6 h (P = 0.000). In group B, the apoptotic index and RPE cell apoptosis ratio of cells exposed to 6, 9 and 12 h of blue light were higher than that of 3 h (P = 0.000); and cells receiving 9 and 12 h had higher values than that of 6 h. This study demonstrated that the best conditions to establish a blue light damage model of human retinal pigment epithelial cells in vitro are 2000 ± 500 lux light intensity for 6 h, with 24 h of cell culture post-exposure.

Omni Scale Perceived Exertion responses in obese and normal weight male adolescents during cycle exercise.

AIM: The Children's OMNI scale of perceived exertion (OMNI Scale) has been validated with children. However, the scale's validity to assess exertional perceptions in obese (OB) adolescents has not been investigated. The aim of this paper was to validate and compare exertional perceptions using the OMNI Scale between OB and normal weight (NW) adolescent males. METHODS: Eighteen OB and 23 NW adolescent males (age: 12-14) participated in a perceptual estimation paradigm with a load incremented cycle ergometer protocol. Heart rate (HR;beats·min-1), oxygen uptake (VO2 mL·min-1 and mL·kg-1·min-1) and rating of perceived exertion (RPE) for the overall body (RPE-O), legs (RPE-L), and chest (RPE-C) were determined at the end of each continuously administered 3-min power output stage as subjects cycled stage-wise incrementally to exhaustion beginning with 4 sub-maximal stages. RESULTS: For OB, 17 completed 4 stages and 3 attained maximal stage 7. For NW, all 18 completed stage 5, and 8 reached maximal stage 7. RPE ranges (i.e. stage 1 to stage 7) for OB and NW were: Stage 1: RPE-O (1.0±1.6, 1.2±0.6), RPE-L (0.9±1.4, 0.9±0.8) and RPE-C (0.9±1.1, 0.9±0.7). Stage 7: RPE-O (7.3±2.3, 8.8±1.4), RPE-L (8.0±2.0, 8.8±1.4) and RPE-C (7.7±2.1, 7.6±3.0). Linear regression for all three RPEs correlated significantly (P<0.001) with HR and VO2 (0.61 to 0.79) for both cohorts. ANOVA indicated no differences between RPE-O and differentiated RPE (RPE-C and RPE-L) at each stage. The RPE responses between OB and NW adolescent males were analyzed by the Bland Altman plot, which indicated significant agreement (95% CI) between the two groups. CONCLUSION: The OMNI Scale is valid for use with 12-14 year-old OB and NW adolescent males. The scale seems to be robust and RPE remained similar for the two cohorts with the same fat free mass but with different fat mass. Hence OMNI scale RPE seems to be able to reflect effort delivered by metabolically active muscle during cycle exercise.

[The diagnostic value of lung ultrasound in children with community-acquired pneumonia].

Objective: To investigate the diagnostic value of lung ultrasound in hospitalized children with community-acquired pneumonia (CAP). Methods: In the cross-sectional study, a total of 422 children with CAP who were hospitalized in the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, from February 2021 to August 2022 and completed lung ultrasound examination within 48 hours after admission were enrolled. The clinical characteristics, lung ultrasound and chest CT were collected. The patients were divided into two groups according to the signs of pneumonia indicated by chest CT, and the signs of lung ultrasound with diagnostic value were screened according to the signs of pneumonia indicated by chest CT by least absolute shrinkage and selection operator (Lasso) regression. According to severity of the disease, the children were divided into the severe group and the mild group, and the differences of lung ultrasound signs between the two groups were compared. Kruskal-Wallis test, Fisher's exact test was selected for comparison between groups. Random forest classifier wes used to evaluate the value of lung ultrasound in the diagnosis of CAP and prediction of severe pneumonia in children. The receiver operating characteristic curve was used to evaluate the prediction effect. Use DeLong test to compare the area under the curve. Results: Among the 422 cases of CAP, there were 258 males and 164 females, and the age of onset was 2.8 (1.3, 4.3) years. The confluent B-line, consolidation and pleural effusion detected by lung ultrasound were 309 cases (73.2%), 232 cases (55.0%) and 16 cases (3.8%), respectively, and the size of consolidation was 3.0 (0, 11.0) mm. One hundred and ten children (26.1%) with CAP completed chest CT. There were 90 cases with signs of pneumonia in chest CT and 20 cases without signs of pneumonia. Lasso was used for feature selection.Lung consolidation (OR=2.46), bilateral lung consolidation (OR=1.16) and confluent B-line (OR=1.34) were the main index. With random forest classifier, the accuracy of models using full variables and Lasso-selected variables were 0.79 (95%CI 0.70-0.86) and 0.79 (95%CI 0.70-0.86), the sensitivity were 0.81 and 0.81, and the specificity were 0.75 and 0.70, and the area under curve were 0.87 (95%CI 0.81-0.94, P<0.001) and 0.84 (95%CI 0.76-0.91, P<0.001), respectively. There were 97 cases in severe group and 325 cases in mild group. Compared with the mild group, the detection rate of consolidation, multiple consolidation, the size of consolidation and the size of consolidation was adjusted by body surface area (consolidation size/body surface area) in severe group were higher (66 cases (68.0%) vs. 166 cases (51.1%), 42 cases (43.3%) vs. 93 cases (28.6%), 8.0 (0, 17.0) vs. 1.0 (0, 9.0) mm, 12.5 (0, 24.6) vs. 2.1 (0, 17.6), χ2=8.59, 9.98, Z=14.40, 12.79, all P<0.05). Using lung ultrasound lung consolidation size and consolidation size/body surface area to predict the severe CAP, the optimal cut-off value were 6.7 mm and 10.2, the accuracy was 0.80 (95%CI 0.75-0.83) and 0.89 (95%CI 0.86-0.92), the sensitivity was 0.99 and 0.99, the specificity was 0.14 and 0.56, respectively, and the area under the curve was 0.66 (95%CI 0.60-0.72, P<0.001) and 0.76 (95%CI 0.70-0.83, P<0.001), respectively. The area under the curve of consolidation size/body surface area was higher than that of consolidation size (Z=5.50, P<0.001). Conclusions: Consolidation and confluent B-line, are important index for lung ultrasound diagnosis of CAP in children. The actual consolidation size adjusted by body surface area is superior to the size of consolidation in predicting severe CAP.

[Short-course radiotherapy combined with CAPOX and PD-1 inhibitor for the total neoadjuvant therapy of locally advanced rectal cancer: the preliminary single-center findings of a prospective, multicentre, randomized phase II trial (TORCH)].

Objective: Total neoadjuvant therapy has been used to improve tumor responses and prevent distant metastases in patients with locally advanced rectal cancer (LARC). Patients with complete clinical responses (cCR) then have the option of choosing a watch and wait (W&W) strategy and organ preservation. It has recently been shown that hypofractionated radiotherapy has better synergistic effects with PD-1/PD-L1 inhibitors than does conventionally fractionated radiotherapy, increasing the sensitivity of microsatellite stable (MSS) colorectal cancer to immunotherapy. Thus, in this trial we aimed to determine whether total neoadjuvant therapy comprising short-course radiotherapy (SCRT) combined with a PD-1 inhibitor improves the degree of tumor regression in patients with LARC. Methods: TORCH is a prospective, multicenter, randomized, phase II trial (TORCH Registration No. NCT04518280). Patients with LARC (T3-4/N+M0, distance from anus ≤10 cm) are eligible and are randomly assigned to consolidation or induction arms. Those in the consolidation arm receive SCRT (25Gy/5 Fx), followed by six cycles of toripalimab plus capecitabine and oxaliplatin (ToriCAPOX). Those in the induction arm receive two cycles of ToriCAPOX, then undergo SCRT, followed by four cycles of ToriCAPOX. Patients in both groups undergo total mesorectal excision (TME) or can choose a W&W strategy if cCR has been achieved. The primary endpoint is the complete response rate (CR, pathological complete response [pCR] plus continuous cCR for more than 1 year). The secondary endpoints include rates of Grade 3-4 acute adverse effects (AEs) etc. Results: Up to 30 September 2022, 62 patients attending our center were enrolled (Consolidation arm: 34, Induction arm:28). Their median age was 53 (27-69) years. Fifty-nine of them had MSS/pMMR type cancer (95.2%), and only three MSI-H/dMMR. Additionally, 55 patients (88.7%) had Stage III disease. The following important characteristics were distributed as follows: lower location (≤5 cm from anus, 48/62, 77.4%), deeper invasion by primary lesion (cT4 7/62, 11.3%; mesorectal fascia involved 17/62, 27.4%), and high risk of distant metastasis (cN2 26/62, 41.9%; EMVI+ 11/62, 17.7%). All 62 patients completed the SCRT and at least five cycles of ToriCAPOX, 52/62 (83.9%) completing six cycles of ToriCAPOX. Finally, 29 patients achieved cCR (46.8%, 29/62), 18 of whom decided to adopt a W&W strategy. TME was performed on 32 patients. Pathological examination showed 18 had achieved pCR, four TRG 1, and 10 TRG 2-3. The three patients with MSI-H disease all achieved cCR. One of these patients was found to have pCR after surgery whereas the other two adopted a W&W strategy. Thus, the pCR and CR rates were 56.2% (18/32) and 58.1% (36/62), respectively. The TRG 0-1 rate was 68.8% (22/32). The most common non-hematologic AEs were poor appetite (49/60, 81.7%), numbness (49/60, 81.7%), nausea (47/60, 78.3%) and asthenia (43/60, 71.7%); two patients did not complete this survey. The most common hematologic AEs were thrombocytopenia (48/62, 77.4%), anemia (47/62, 75.8%), leukopenia/neutropenia (44/62, 71.0%) and high transaminase (39/62, 62.9%). The main Grade III-IV AE was thrombocytopenia (22/62, 35.5%), with three patients (3/62, 4.8%) having Grade IV thrombocytopenia. No Grade V AEs were noted. Conclusions: SCRT-based total neoadjuvant therapy combined with toripalimab can achieve a surprisingly good CR rate in patients with LARC and thus has the potential to offer new treatment options for organ preservation in patients with MSS and lower-location rectal cancer. Meanwhile, the preliminary findings of a single center show good tolerability, the main Grade III-IV AE being thrombocytopenia. The significant efficacy and long-term prognostic benefit need to be determined by further follow-up.

Effects of nano-selenium on performance, meat quality, immune function, oxidation resistance, and tissue selenium content in broilers.

This study was conducted to investigate the effect of nano-selenium (nano-Se) on performance, meat quality, immune function, oxidation resistance, and tissue selenium content in broilers. A total of five hundred forty 1-d-old male Arbor Acres broilers were randomly allotted to 1 of 5 treatments with each treatment being applied to 6 replicates of 18 chicks. The 5 treatments consisted of corn-soybean meal-based diets supplemented with 0.0, 0.3, 0.5, 1.0, or 2.0 mg/kg of nano-Se. The selenium content of the unsupplemented control diet was 0.09 mg/kg for the starter phase (0 to 21 d) and 0.08 mg/kg for the grower phase (22 to 42 d). There were no significant differences (P > 0.05) in performance, meat color, or immune organ index (thymus, bursa, and spleen) due to supplementation with nano-Se. On d 42, a significant quadratic effect of nano-Se was observed on glutathione peroxidase activity, free radical inhibition, contents of IgM, glutathione, and malondialdehyde in serum, on glutathione peroxidase activity, free radical inhibition in liver, and on glutathione peroxidase activity in muscle, with birds fed 0.30 mg/kg of nano-Se exhibiting the best effect and birds fed 2.0 mg/kg of nano-Se showing the worst effect on these parameters. Liver and muscle selenium content increased linearly and quadratically as the dietary nano-Se level increased (P < 0.01), and reached the highest value when 2.0 mg/kg of nano-Se was fed. Based on a consideration of all experiment indexes, 0.3 to 0.5 mg/kg is suggested to be the optimum level of supplementation of nano-Se, and the maximum supplementation of nano-Se could not be more than 1.0 mg/kg in broilers.

Interaction between synchronous bilateral prophylactic oophorectomy and adjuvant chemotherapy in female patients with locally advanced colorectal cancer.

AIM: In this study we explored the prognostic impact of synchronous bilateral prophylactic oophorectomy in female patients with primary colorectal cancer undergoing radical surgery. METHOD: From 1991 to 2000, 267 female patients with stage II or stage III colorectal cancer, who had undergone curative resection, were retrospectively reviewed. In 224 patients, the ovaries were preserved. The other 43 patients underwent synchronous bilateral prophylactic oophorectomy. Univariate and multivariate analyses (Kaplan-Meier and Cox regression, respectively) were used to evaluate the effect of prophylactic oophorectomy and other clinical factors on the prognosis of patients. RESULTS: Both univariate and multivariate analyses showed that tumor stage and adjuvant chemotherapy were the only two significant clinical factors that affected the 5-year overall survival of patients (P < 0.01). There was no significant difference in the 5-year overall survival between patients who had, or had not, undergone prophylactic oophorectomy (75%vs 73%, P > 0.05). In the subgroup analysis by adjuvant chemotherapy, the 5-year overall survival in patients receiving adjuvant chemotherapy was similar between nonoophorectomy and oophorectomy groups. However, in patients without adjuvant chemotherapy, the oophorectomy group was shown to have a significantly better 5-year overall survival than the nonoophorectomy group (76%vs 51%, P = 0.047). CONCLUSION: Prophylactic oophorectomy may improve the overall survival of female patients with locally advanced colorectal cancer without adjuvant chemotherapy, but its survival benefit vanished in patients receiving adjuvant chemotherapy. The role of prophylactic oophorectomy may be substituted by adjuvant chemotherapy, which makes prophylactic oophorectomy unnecessary during surgery for locally advanced colorectal cancer.

Apolipoprotein B-48 is the product of a messenger RNA with an organ-specific in-frame stop codon.

The primary structure of human apolipoprotein (apo) B-48 has been deduced and shown by a combination of DNA excess hybridization, sequencing of tryptic peptides, cloned complementary DNAs, and intestinal messenger RNAs (mRNAs) to be the product of an intestinal mRNA with an in-frame UAA stop codon resulting from a C to U change in the codon CAA encoding Gln2153 in apoB-100 mRNA. The carboxyl-terminal Ile2152 of apoB-48 purified from chylous ascites fluid has apparently been cleaved from the initial translation product, leaving Met2151 as the new carboxyl-terminus. These data indicate that approximately 85% of the intestinal mRNAs terminate within approximately 0.1 to 1.0 kilobase downstream from the stop codon. The other approximately 15% have lengths similar to hepatic apoB-100 mRNA even though they have the same in-frame stop codon. The organ-specific introduction of a stop codon to a mRNA appears unprecedented and might have implications for cryptic polyadenylation signal recognition and RNA processing.

Rapid identification of subtype-selective agonists of the somatostatin receptor through combinatorial chemistry.

Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.

[Adjuvant and perioperative neoadjuvant therapy for colorectal cancer].

Colorectal cancer is one of the most common malignant tumors in the world, threatening human health. The treatment strategy of stage II and stage III colorectal cancer has changed from surgery alone to multidisciplinary mode emphasizing perioperative treatment. The indication of adjuvant chemotherapy for stage II colon cancer is still defined by high-risk factors, but only microsatellite status and BRAF gene mutation can help predict efficacy of chemotherapy. Combined chemotherapy is the main adjuvant therapy for stage III colon cancer. The recommended course of adjuvant chemotherapy is 6 months. Based on the results of the IDEA study, the three-month CapeOX regimen (oxaliplatin and capecitabine) is recommended for the treatment of patients with T1-3 and N1 tumors. Neoadjuvant chemotherapy for locally advanced colon cancer is still in the exploratory stage of clinical trials. The difference between the treatment of rectal cancer and colon cancer lies in the application of radiotherapy. Chemoradiotherapy combined with TME (total mesorectal excision) surgery and adjuvant chemotherapy has become the standard treatment for locally advanced rectal cancer. Nowadays, the research hotspots in neoadjuvant therapy of rectal cancer include neoadjuvant chemotherapy and total neoadjuvant therapy (TNT). This article will review the progress of perioperative treatment for colorectal cancer.

Role of pelvic radiotherapy for locally advanced rectal cancer and synchronous unresectable distant metastases.

PURPOSE: To evaluate the efficacy and safety of pelvic irradiation combined systematic chemotherapy in patients with locally advanced (cT3-T4 and/or cN+) rectal cancer and synchronous unresectable distant metastases. PATIENTS AND METHODS: A total of 76 eligible patients who received pelvic radiotherapy and concurrent capecitabine-based chemotherapy were retrospectively reviewed. Patients survival curves were constructed using the Kaplan-Meier method, and a multivariate analysis was performed to identify independent prognostic factors. RESULTS: Most of the adverse events were mild during the period of combined chemoradiotherapy. Twenty-two patients experienced resection of primary tumour and 16 patients underwent radical surgery of all lesions. Only five patients had pelvic progression during the follow-up period. The median progression-free survival and median overall survival were 13 and 30 months, respectively. Radical surgery of all lesions following chemoradiotherapy was found to be an independent prognostic factor according to multivariate analysis. CONCLUSIONS: Pelvic irradiation combined with systematic chemotherapy in patients with locally advanced rectal cancer and synchronous unresectable distant metastases is effective and tolerable, both for pelvic and distant control. A curative resection following chemoradiotherapy was associated with prolonged survival.

Integrative vectors for heterologous gene expression in Streptomyces spp.

Integrative expression vectors for heterologous expression of the genes in Streptomyces were developed. The vectors are comprised of a strong constitutive promoter, PE, a synthetic ribosome-binding site, ATG start codon, multiple cloning site, transcription terminator and hygromycin-resistance-encoding gene. The vectors also contain a ColE1 replicon for propagation in Escherichia coli and a wide-host-range Streptomyces integration element, the mini-circle, to direct the insertion of the vectors into the Streptomyces genome at the mini-circle attachment site. HyR transformants are stable in the absence of drug selection. Conjugative derivatives were also constructed by incorporating oriT, the origin of transfer of the IncP plasmid RK2, into these vectors, and conjugal transfer was demonstrated from an appropriate E. coli donor to Steptomyces lividans (Sl). Derivatives of these vectors potentially useful for gene disruption, as well as complementation, are also described. Replicative forms of the constructed mini-circle-based vectors in Sl, that co-exist with the integrated copy of the vector, were also present without any apparent instability problems. The utility of the vectors was demonstrated by expression of the gene encoding 31-O-methyltransferase, which is involved in methylation at position 31 of the immunosuppressive drug FK506, in Sl.

Mobile phase effects on membrane protein elution during immobilized artificial membrane chromatography.

The eluotropic strength of different mobile phases for eluting membrane proteins from immobilized artificial membrane (IAM) chromatography surfaces was studied. Two protein mixtures containing bovine pancreatic PLA2 were used in this study. Protein mixture I was PLA2 obtained from Sigma which contained approximately 5-10 major protein bands in electrophoretic gels. Protein mixture II was obtained from flesh bovine pancreatic tissue and contained > 100 proteins including the target protein, PLA2. After adsorbing Sigma PLA2 to IAM columns, the elution conditions common to conventional chromatographic methods were evaluated for their ability to selectively purify PLA2. Elution conditions tested were (i) detergent gradients, (ii) salt gradients used during ion-exchange chromatography, (iii) salt conditions used during hydrophobic interaction chromatography, (iv) acetonitrile gradients used during reversed-phase chromatography, and (v) a two-step gradient consisting of first a detergent gradient followed by an acetonitrile gradient. Based on silver-stained electrophoretic protein gels. PLA2 from protein mixture I was purified to electrophoretic homogeneity with 417-fold increase in specific activity in one step using elution condition (v), and PLA2 from protein mixture II was purified in one step (660-fold increase in specific activity) using elution condition (iv). Total protein recovery from IAM columns is 70-100%.

Rapid purification of cotton seed membrane-bound N-acylphosphatidylethanolamine synthase by immobilized artificial membrane chromatography.

N-Acylphosphatidylethanolamine synthase (NAPES) is a membrane-bound enzyme present in cotton seedlings at a concentration of < or = 0.02% of the total protein. NAPES was purified to electrophoretic homogeneity in a single chromatographic step using immobilized artificial membrane (IAM) chromatography. The IAM column used for NAPES purification was etherIAM.PEC10/C3 and this surface contains a monolayer of immobilized phosphatidyl-ethanolamine (PE). Since PE is an analogue of the natural substrate for NAPES, etherIAM.PEC10/C3 columns function as an affinity column for this enzyme. Detergent-solubilized microsomal proteins from cotton were loaded on to the etherIAM.PEC10/C3 column and eluted with buffered mobile phases containing 0.2 mM dimyristoylphosphatidylethanolamine (DMPE) and 2 mM dodecylmaltoside. Little NAPES functional activity eluted if DMPE was removed from the mobile phase. Mobile phase DMPE is also a substrate for NAPES, both the mobile phase and IAM surface contains NAPES substrates. Mobile phase DMPE may function as both a surfactant-type affinity displacing ligand effecting protein elution and also a stabilizing factor of NAPES functional activity. The loading capacity on semi-preparative etherIAM.PEC10/C3 (6.5 x 1.0 cm) columns was ca. 5 mg of total detergent solubilized microsomal proteins, and protein recovery was quantitative. This one-step IAM purification of NAPES resulted in a single band on silver-stained polyacrylamide gels, and 3940 fold increase in NAPES specific activity. The molecular mass of the purified NAPES protein is 64,000. 125I labeled [12-(4-azidosalicyl)amino]dodecanoic acid is a photoreactive fatty acid substrate of NAPES that was used to confirm protein purity.

Clinicopathological and molecular genetic analysis of 4 typical Chinese HNPCC families.

AIM: To study the clinicopathological and molecular genetic characteristics of typical Chinese hereditary nonpolyposis cotorectal cancer (HNPCC) families. METHODS: Four typical Chinese HNPCC families were analyzed using microdissection, microsatellite instability analysis, immunostaining of hMSH2 and hMLH1 proteins and direct DNA sequencing of hMSH2 and hMLH1 genes. RESULTS: All five tumor tissues of 4 probands from the 4 typical Chinese HNPCC families showed microsatellite instability at more than two loci (MSI-H or RER+ phenotype). Three out of the 4 cases lost hMSH2 protein expression and the other case showed no hMLH1 protein expression. Three pathological germline mutations (2 in hMSH2 and 1 in hMLH1), which had not been reported previously, were identified. The same mutations were also found in other affected members of two HNPCC families,respectively. CONCLUSION: Typical Chinese HNPCC families showed relatively frequent germline mutation of mismatch repair genes. High-level microsatellite instability and loss of expression of mismatch repair genes correlated closely with germline mutation of mismatch repair genes. Microsatellite instability analysis and immunostaining of mismatch repair gene might serve as effective screening methods before direct DNA sequencing. It is necessary to establish clinical criteria and molecular diagnostic strategies more suitable for Chinese HNPCC families.

Clinicopathological features and prognostic factors of rectal gastrointestinal stromal tumors.

AIM: Rectal gastrointestinal stromal tumor (GIST) was a rare disease. This study sought to summarize clinicopathological features and prognostic factors of rectal GISTs. METHODS: Clinicopathological characteristics and prognostic factors of rectal GISTs were investigated by reviewing patients undergoing curative resection at the Fudan University Shanghai Cancer Center between 1986 and 2010. RESULTS: Twenty-one patients, 15 male and 6 female, were included. The mean age of onset was 51 years. The most common initial presentation was hematochezia (7 cases). Eleven patients underwent radical resection, and the other 10 received local resection. No lymph node metastases were identified. All cases were positive for CD117. Seventeen patients were classified as high National Institutes of Health (NIH) risk category. The 5-year disease-free survival (DFS) and overall survival were 33 and 46 %, respectively. Fifteen cases had recurrence postoperatively. Both univariate and multivariate analyses demonstrated the NIH risk category (p = 0.028) and hematochezia symptom (p = 0.014) were independent prognostic factors of the DFS of patients. CONCLUSIONS: Hematochezia was the most common initial symptom. Over 50 % of patients received radical surgery. More than 80 % of patients were at high NIH risk of recurrence. Hematochezia symptom and high NIH risk category indicated poor prognosis of rectal GISTs.

Selective estrogen receptor-beta (SERM-beta) compounds modulate raphe nuclei tryptophan hydroxylase-1 (TPH-1) mRNA expression and cause antidepressant-like effects in the forced swim test.

Estrogen acts through two molecularly distinct receptors termed estrogen receptor alpha (ERα) and estrogen receptor beta (ERß) which bind estradiol with similar affinities and mediate the effects of estrogen throughout the body. ERα plays a major role in reproductive physiology and behavior, and mediates classic estrogen signaling in such tissues as the uterus, mammary gland, and skeleton. ERß, however, modulates estrogen signaling in the ovary, the immune system, prostate, gastrointestinal tract, and hypothalamus, and there is some evidence that ERß can regulate ERα activity. Moreover, ERß knockout studies and receptor distribution analyses in the CNS suggest that this receptor may play a role in the modulation of mood and cognition. In recent years several ERß-specific compounds (selective estrogen receptor beta modulators; SERM-beta) have become available, and research suggests potential utility of these compounds in menopausal symptom relief, breast cancer prevention, diseases that have an inflammatory component, osteoporosis, cardiovascular disease, and inflammatory bowel disease, as well as modulation of mood, and anxiety. Here we demonstrate an antidepressant-like effect obtained using two SERM-beta compounds, SERM-beta1 and SERM-beta2. These compounds exhibit full agonist activity at ERß in a cell based estrogen response element (ERE) transactivation assay. SERM-beta1 and 2 are non-proliferative with respect to breast as determined using the MCF-7 breast cancer cell-based assay and non-proliferative in the uterus as determined by assessing the effects of SERM-beta compounds on immature rat uterine weight and murine uterine weight. In vivo SERM-beta1 and 2 are brain penetrant and display dose dependent efficacy in the murine dorsal raphe assays for induction of tryptophan hydroxylase mRNA and progesterone receptor protein. These compounds show activity in the murine forced swim test and promote hippocampal neurogenesis acutely in rats. Taken together these data suggest that ERß may play an important role in modulating mood and the ERß specific compounds described herein will be useful tools for probing the utility of an ERß agonist for treating neuroendocrine-related mood disturbance and menopausal symptoms.