Unintrusive multi-cancer detection by circulating cell-free DNA methylation sequencing (THUNDER): development and independent validation studies.

BACKGROUND: Early detection of cancer offers the opportunity to identify candidates when curative treatments are achievable. The THUNDER study (THe UNintrusive Detection of EaRly-stage cancers, NCT04820868) aimed to evaluate the performance of enhanced linear-splinter amplification sequencing, a previously described cell-free DNA (cfDNA) methylation-based technology, in the early detection and localization of six types of cancers in the colorectum, esophagus, liver, lung, ovary, and pancreas. PATIENTS AND METHODS: A customized panel of 161 984 CpG sites was constructed and validated by public and in-house (cancer: n = 249; non-cancer: n = 288) methylome data, respectively. The cfDNA samples from 1693 participants (cancer: n = 735; non-cancer: n = 958) were retrospectively collected to train and validate two multi-cancer detection blood test (MCDBT-1/2) models for different clinical scenarios. The models were validated on a prospective and independent cohort of age-matched 1010 participants (cancer: n = 505; non-cancer: n = 505). Simulation using the cancer incidence in China was applied to infer stage shift and survival benefits to demonstrate the potential utility of the models in the real world. RESULTS: MCDBT-1 yielded a sensitivity of 69.1% (64.8%-73.3%), a specificity of 98.9% (97.6%-99.7%), and tissue origin accuracy of 83.2% (78.7%-87.1%) in the independent validation set. For early-stage (I-III) patients, the sensitivity of MCDBT-1 was 59.8% (54.4%-65.0%). In the real-world simulation, MCDBT-1 achieved a sensitivity of 70.6% in detecting the six cancers, thus decreasing late-stage incidence by 38.7%-46.4%, and increasing 5-year survival rate by 33.1%-40.4%, respectively. In parallel, MCDBT-2 was generated at a slightly low specificity of 95.1% (92.8%-96.9%) but a higher sensitivity of 75.1% (71.9%-79.8%) than MCDBT-1 for populations at relatively high risk of cancers, and also had ideal performance. CONCLUSION: In this large-scale clinical validation study, MCDBT-1/2 models showed high sensitivity, specificity, and accuracy of predicted origin in detecting six types of cancers.

[In vitro activity of ß-lactamase inhibitors avibanvctam and relebactam in combination with ß-lactams against multidrug-resistant Mycobacterium tuberculosis and mutations of resistance genes].

Objective: To evaluate the activity of six ß-lactams in combination with three ß-lactamase inhibitors against mycobacterium tuberculosis(MTB) in vitro. Methods: A total of 105 multidrug-resistant tuberculosis (MDR-TB) strains from different regions of Henan province from January to September 2020 were included in this study. Drug activity of six ß-lactams (biapenem, meropenem, imipenem, doripenem, ertapenem and tebipenem) alone or in combination with ß-lactamase inhibitors (clavulanic acid, avibactam and relebactam) was examined by minimum inhibitory concentration method (MICs) against 105 clinical isolates. Mutations of blaC, ldtmt1 and ldtmt2 were analyzed by PCR and DNA sequencing. Chi-square test was used to compare the antimicrobial activities of different ß-lactam drugs. Results: Out of the ß-lactams used herein, tebipenem was the most effective against MDR-TB and had an MIC50 value of 8 mg/L(χ2=123.70,P=0.001). Besides, after the addition of ß-lactamase inhibitors, the MICs of most ß-lactam drugs were reduced more evidently in the presence of avibactam and relebactam compared to clavulanic acid.Especially, relebactam decreased both the MIC50 and MIC90 of telbipenem by 16-fold, and diluted the MIC of 23 (21.90%) and 41 (39.04%) isolatesby 32-fold and 16-fold.In addition, a total of 13.33% (14/105) of isolates harbored mutations in the blaC gene, with three different nucleotide substitutions: AGT333AGG, AAC638ACC and ATC786ATT. For the strains with Ser111Arg and Asn213Thr substitution in BlaC, the MIC values of the meropenem-clavulanate combination were reduced compared with a synonymous single nucleotide polymorphism (SNP) group. Conclusions: Both avibactam and relebactam had better synergistic effects on ß-lactams than clavulanic acid. The combination of tebipenem and relebactam showed the most potent activity against MDR-TB isolates. In addition, the Ser111Arg and Asn213Thr substitution of BlaC may be associated with an increased susceptibility of MDR-TB isolates to meropenem in the presence of clavulanate.

[Sub-health status of middle school teachers and its correlation analysis with occupational stress].

Objective: To study the distribution of sub-health and occupational stress as well as their correlation among middle school teachers in Tianjin, then provide evidences for prevention and control of the status of sub-health. Methods: A total of 3 522 middle school teachers from six districts of Tianjin were recruited with stratified cluster sampling strategy for the investigation of Sub-Health Measurement Scale version 1.0 (SHMS V1.0) and Occupational Stress Inventory-Revised Edition (OSI-R) . Results: Detection rate of sub-health status among Tianjin middle school teachers was 58.55%. Men had significantly lower sub-health detection rate (55.19%) than women (59.71%) . Sub-health detection rate increased with age (P<0.05) , the sub-health detection rate among middle school teachers more than 50 years old was the highest (66.84%) . The mean score of OSI-R was 403.18±41.80 with the scores of 176.00±21.05, 103.17±17.53, and 124.02±20.28 for ORQ, PSQ, PRQ, respectively, which showed significantly difference compared with the occupational stress norm of China (P<0.001) . The mean scores of OSI-R, ORQ, PSQ, PRQ in different health status were significantly different (P<0.001) . The partial correlation analysis between the scores of sub-health and occupational stress of middle school teachers showed that the scores of occupational role and personal strain were negatively correlated with the scores of sub-health state (P< 0.001) , while, there was significantly positive correlation between the scores of personal resource and the scores of sub-health state (P<0.001) . Conclusion: Sub-health detection rate of middle school teachers in Tianjin is higher. Effective measures should be taken to appropriately mitigate the occupational stress level of middle school teachers, increase personal resources, and scientific and effective health guidance and education should be strengthened.

A Salvia miltiorrhiza ethanol extract ameliorates tissue destruction caused by experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: Salvia miltiorrhiza Bunge (Labiatae), or Danshen, is a Chinese medicine used for treatment of cardiac diseases by improving blood circulation and inhibiting inflammatory responses. In this study, we aimed to determine whether an ethanol extract of S. miltiorrhiza can ameliorate tissue damage caused by periodontitis. MATERIAL AND METHODS: An ethanolic extract of S. miltiorrhiza roots was prepared, and its major constituents were determined by HPLC analysis, by comparison with known standards for the major bioactive components. The activity of the extract was evaluated in a rat model in which periodontitis was induced by ligation of a silk suture around the neck of molar teeth. The effects of the S. miltiorrhiza extract on periodontitis were assessed by dental radiography, micro-computed tomography and histology. RESULTS: The cemento-enamel junction-bone distances among the four different groups of rats were significantly different: the distance was shorter in groups treated with ligation + S. miltiorrhiza extract than in the group treated with ligation only, but was longer than in the nonligated group, regardless of the radiographic methods used. Histology and histometry also indicated a similar trend of less gingival inflammation and alveolar bone destruction in the histological sections from the S. miltiorrhiza extract groups than in those from the ligation group. CONCLUSION: Because the S. miltiorrhiza extract reduced tissue damage and bone loss caused by ligation-induced periodontitis in rats, we suggest that the S. miltiorrhiza extract might have an ameliorative effect on periodontal tissue destruction during the process of periodontitis.

LITAF, HHEX, and DUSP1 expression in mesenchymal stem cells from patients with psoriasis.

Psoriasis is a common chronic relapsing inflammatory skin disease, in which mesenchymal stem cells (MSCs) have been hypothesized to play an important role in abnormal localized inflammation and vascular proliferation observed in skin lesions. Previous studies have revealed abnormal gene expression patterns, DNA methylation status, and cytokine secretion of MSCs in psoriatic skin lesions, as well as some gene expression abnormalities related to inflammation and angiogenesis. We further verified the gene and protein expressions of inflammation-related lipopolysaccharide-induced tumor necrosis factor-alpha transcription factor (LITAF), dual-specificity protein phosphatase 1 (DUSP1), and angiogenesis-related hematopoietically expressed homeobox (HHEX) in MSCs derived from the skin lesions of psoriasis patients. The gene expression of LITAF, DUSP1, and HHEX in dermal MSCs was measured at the mRNA level using reverse transcription-polymerase chain reaction and the corresponding protein expression levels were analyzed by western blotting analysis. The gene and protein expression levels of LITAF, HHEX, and DUSP1 in dermal MSCs were significantly lower in psoriasis patients compared to controls. Amplification and western blotting results were consistent with our previously reported gene chip data. Our results suggest that dermal MSCs in psoriatic skin lesions may be involved in the development, progression, and regulation of localized inflammatory abnormalities by reducing the expression of LITAF, HHEX, and DUSP1, which are related to inflammation and angiogenesis.

Prospective analysis of quality of life in elderly patients treated with adjuvant chemotherapy for non-small-cell lung cancer.

BACKGROUND: Given the more comorbidities with a decline in physiologic reserve, it can be challenging to make appropriate treatment decisions in the elderly. PATIENTS AND METHODS: Here, we prospectively evaluated and compared the health-related quality of life (HRQOL) of patients aged ≥ 65 with aged <65 who were treated with a postoperative chemotherapy for completely resected stage Ib, II or IIIa non-small-cell lung cancer (NSCLC). Either four cycles of paclitaxel (Taxol)-carboplatin (PC) or vinorelbine-cisplatin (NP) was used. The HRQOL was assessed with EORTC QLQ-C30 and EORTC QLQ-LC13. RESULTS: Between October 2008 and October 2011, a total of 139 patients (aged <65, n = 73; ≥ 65, n = 66) were enrolled, and 127 (91.4%) completed the questionnaire. Overall, the quality of life (QOL) in elderly patients did not significantly deteriorate with adjuvant chemotherapy and the time trend of QOL in elderly patients was similar to that of younger patients. Although the elderly suffered from increased treatment-related adverse events involving sore mouth, peripheral neuropathy and alopecia compared with the baseline, the same time trends were also observed in younger group. The mean dose intensities (MDIs) for PC and NP regimen were not significantly different between the two age groups. CONCLUSIONS: Postoperative chemotherapy did not substantially reduce HRQOL in elderly NSCLC patients, and HRQOL during and after adjuvant chemotherapy did not significantly differ by age.

Development of a micro-electrochemical machining nanosecond pulse power supply.

Micro-electrochemical machining (micro-ECM) has been widely used for microscale and nanoscale processing of materials. The performance of the nanosecond pulse power supply is directly related to the precision of micro-ECM, which is one of the core technologies for micro-ECM. In this work, a nanosecond pulse power supply, with adjustable pulse frequency, duty cycle, and voltage, was designed with an STM32F103VET6 single-chip microcomputer as the control core and a metal-oxide-semiconductor field-effect transistor as the chopper switch component. The performance test has shown that the power supply can produce a continuous pulse with the highest frequency of 8 MHz, the shortest pulse width of 50 ns, the maximum peak current of 12 A, and the maximum voltage of 10 V. As compared with the power supply reported in the literature, the present power supply demonstrated the enhanced output current and improved waveform of the nanosecond pulse output, which could result in better machining accuracy and efficiency for micro-ECM.

Prevalence of antibiotics resistance and OXA carbapenemases genes in multidrug-resistant Acinetobacter baumannii isolates in central Taiwan.

This study analyzed the prevalence of antibiotics resistance and the distribution of genes responsible for carbapenems resistance in Acinetobacter baumannii isolates. Clinical A. baumannii isolates were cultured, identified, and collected during the period from May 2007 to February 2009. Antibiotics resistance rates of the clinical isolates were analyzed by antimicrobial susceptibility testing. The distribution of carbapenemase alleles were investigated in the multidrug-resistant (MDR) A. baumannii isolates by multiplex polymerase chain reaction (PCR) techniques. A total of 1,265 independent A. baumannii isolates were identified. Approximately 70% of the clinical isolates were resistant to ampicillin/sulbactam, followed by imipenem, meropenem, cefepime, piperacillin/tazobactam, ceftazidime, and cefoperazone. Overall, 15.18% (192/1,265) of the isolates were characterized as MDR strains. All of the MDR A. baumannii isolates carried the bla (OXA51-like) allele. The detection rate of the bla (OXA23-like) and bla (OXA24-like) alleles was 96.35% (185/192) and 0.52% (1/192), respectively. Most of the isolates (185/192, 96.35%) carried genes which encode more than one carbapenemase. This report demonstrated that approximately 15% of A. baumannii clinical isolates in central Taiwan are MDR strains, with most of them harboring multiple carbapenemases. This study provides updated data regarding the prevalence of beta-lactam resistance and genotyping information of carbapenems resistance of A. baumannii in central Taiwan.

[Synchronous robotic resection for colorectal liver metastasis].

Surgical resection remains the only curative therapy for colorectal adenocarcinoma and liver metastasis. Synchronous robotic resection for colorectal liver metastasis (CRLM) offers the advantage of avoiding double surgical stress, while providing the benefits of small incision, quicker recovery, shorter hospital stay and faster postoperative adjuvant therapy. Compared with the laparoscopic approach, robotic approach is mostly suitable for rectal cancer liver metastasis, which is associated with low conversion rate, good nerve protection, high success rate for major hepatectomy and resection of difficult segments. Appropriately selected patients, multidisciplinary cooperation and skillful robotic surgeons are the key to success. Current data have demonstrated the feasibility and safety of synchronous robotic resection for CRLM. With the coming randomized controlled trial data and evolution of robotic surgical system, the future of synchronous robotic resection for colorectal liver metastasis is promising.

[Experience of teaching and training for medical students at gastrointestinal surgery department under COVID-19 epidemic situation].

In hospitals and medical schools as densely populated sites with high risk of coronavirus disease 2019 (COVID-19), it is vital to adjust the teaching and training strategy for medical students to ensure curriculum completion with safety. This article aims to introduce the experience of teaching and training for medical students under the epidemic situation at Department of Surgery, Shanghai Medical College, Fudan University and Zhongshan Hospital. The content includes exploring diversified online teaching models for undergraduate surgery courses and clinical practice, carrying out online graduate education and dissertation plans, and strengthening comprehensive education of medical humanity combined with knowledge of COVID-19 prevention. Through implementation of the above teaching strategies, scheduled learning plans of medical students can be well completed in an orderly, safe and quality-ensured manner. Our experience provides practical solution of medical teaching and could be advisable for other medical colleges and teaching hospitals.

[Risk factors of anastomotic leakage after robotic surgery for low and mid rectal cancer].

Objective: To investigate the risk factors associated with anastomotic leakage after robotic surgery in mid-low rectal cancer. Methods: A retrospective case-control study method was conducted. Inclusion criteria: (1) 18 to 80 years old; (2) pathologically confirmed rectal cancer; (3) distance <10 cm from tumor to anal margin; (4) robotic anterior rectal resection. Patients with previous history of colorectal cancer surgery, distant metastases or other malignant tumors, undergoing emergency surgery, with severe abdominal adhesions or those receiving combined organ resection were excluded. Based on the above criteria, 636 patients undergoing robotic radical sphincter-preserving surgery for mid-low rectal cancer in Zhongshan Hospital from January 2015 to December 2018 were included in this study, including 398 males (62.6%) and 238 females (37.4%) with a mean age of (61.9±11.3) years. Sixty-eight cases (10.7%) received neoadjuvant chemoradiotherapy. Amony the 636 included patients, 123(19.3%) underwent natural orifice specimen extraction surgery (NOSES) and 15 (2.3%) underwent preventive stoma. According to the cirteria developed by the International Rectal Cancer Research Group in 2010, the anastomotic leakage was classified as grade A (no requirement of intervention), B (requirement of intervention), and C (requirement of operation). Logistic regression was used to analyze the relationship between anastomotic leakage and clinicopathological factors. Factors in univariate analysis with P<0.05 were included in the multivariate analysis. Results: Anastomotic leakage occurred in 38 cases (6.0%). The grading of anastomotic leakage was grade A in 13 cases (2.0%), grade B in 19 cases (3.0%), and grade C in 6 cases (0.9%). The 3-year disease-free survival rate of patients with anastomotic leakage and without anastomotic leakage was 83.5% and 83.6% respectively (P=0.862); the 3-year overall survival rate of the two group was 85.1% and 87.5% respectively (P=0.296). The results of univariate logistic regression analysis showed that male (P=0.011), longer operation time (P=0.042), distance ≤5 cm from tumor to anal margin (P=0.012), more intraoperative blood loss (P=0.048) were associated with anastomotic leakage (all P<0.05). NOSES was not associated with anastomotic leakage (P=0.704). Multivariate analysis confirmed that male (OR=3.03, 95%CI: 1.37 to 7.14, P=0.010), operation time ≥180 minutes (OR=2.04, 95%CI: 1.03 to 3.99, P=0.040), distance ≤5 cm from tumor to anal margin (OR=2.56, 95%CI:1.28 to 5.26, P=0.008) were independent risk factors for anastomotic leakage. Conclusion: Male, short distance from tumor to anal margin, and long operation time are independent risk factors for anastomotic leakage in patients undergoing robotic mid-low rectal cancer radical surgeries. These patients need to be cautiously treated during surgery.

Lowering the cholesterol content of MA104 cells inhibits receptor-mediated transport of folate.

The folate receptor is clustered on the surface of MA104 cells in association with caveolae. This relationship is thought to be essential for the proper internalization and recycling of the receptor during the delivery of 5-methyltetrahydrofolate to the cytoplasm of folate-depleted cells. Both the clustered organization of the receptor and the integrity of caveolae are disrupted when cells are deprived of cholesterol. We now show that cholesterol depletion of MA104 cells markedly reduces the rate of 5-methyltetrahydrofolate internalization and causes a 70% decline in the number of receptors present in the internal, recycling compartment. This effect is consistent with morphologic data showing that cholesterol-depleted MA104 cells have a reduced number of caveolae as well as fewer receptors per caveolae.

Purification and characterization of smooth muscle cell caveolae.

Plasmalemmal caveolae are a membrane specialization that mediates transcytosis across endothelial cells and the uptake of small molecules and ions by both epithelial and connective tissue cells. Recent findings suggest that caveolae may, in addition, be involved in signal transduction. To better understand the molecular composition of this membrane specialization, we have developed a biochemical method for purifying caveolae from chicken smooth muscle cells. Biochemical and morphological markers indicate that we can obtain approximately 1.5 mg of protein in the caveolae fraction from approximately 100 g of chicken gizzard. Gel electrophoresis shows that there are more than 30 proteins enriched in caveolae relative to the plasma membrane. Among these proteins are: caveolin, a structural molecule of the caveolae coat; multiple, glycosylphosphatidylinositol-anchored membrane proteins; both G alpha and G beta subunits of heterotrimeric GTP-binding protein; and the Ras-related GTP-binding protein, Rap1A/B. The method we have developed will facilitate future studies on the structure and function of caveolae.

Electronic structure of pyrochlore Cd(2)Re(2)O(7).

Detailed band structure calculations have been performed for Cd(2)Re(2)O(7) in high-, middle- and low-temperature (T) phases. The calculations are based on the observed lattice structures from x-ray diffraction measurements. The spin-orbit interaction is incorporated self-consistently in both the generalized gradient approximation (GGA) and the GGA plus Hubbard U (GGA+U) approaches. It is found that the on-site U has negligible effects on the Re 5d band structures; therefore both the GGA and GGA+U Re 5d band energies agree well with the observed O K-edge x-ray absorption spectroscopy (XAS) spectrum, whereas the Cd 4d band energy observed from photoemission spectroscopy can only be correctly reproduced by GGA+U calculations, indicating the relatively itinerant Re 5d and localized Cd 4d electrons. On the other hand, the spin-orbit coupling gives rise to nontrivial spin and orbital magnetic moments for the middle- T phase. Most unexpectedly, we found that the low- T phase exhibits quasi-two-dimensional Fermi surfaces. The calculated carrier numbers for the three phases are, at least qualitatively, consistent with the measured Hall coefficient.

Effect of damping properties on fracture resistance of root filled premolar teeth: a dynamic finite element analysis.

AIM: To evaluate the ex vivo effects of damping on stress concentration in root filled premolar teeth. METHODOLOGY: Damping ratios of maxillary premolar teeth that had undergone root canal treatment were tested in a laboratory model. In addition, two-dimensional finite element (FE) models were established for dynamic analysis. RESULTS: The mean-damping ratio was significantly lower in premolar teeth that had undergone root canal preparation (8.50 +/- 0.53%) than in unprepared teeth (14.42 +/- 2.17%) (P < 0.05). However, root filling had a significant positive effect on the damping ratio of the tooth (10.84 +/- 1.70%) (P < 0.05). When the damping ratio was taken into consideration, FE analysis revealed that peak stresses in the apical one-third of the root on the buccal side were reduced by 31.8% when mastication forces were applied on the palatal cusp and occlusal fossa. CONCLUSION: Pulp tissue plays an important role in providing protective effects when teeth are subjected to a dynamic load. However, root filled teeth do not provide such protective effects.

Detection of the furcation involvement of a multi-rooted molar using natural frequency analysis: a numerical approach.

The aim of this study was to evaluate the potential for using the natural frequency (NF) as a parameter to detect vertical bone loss at the furcation of human molars as well as to assess the role that the surrounding bone plays in maintaining molar stability. A three-dimensional finite element model of the human maxillary molar was built. The NF values of the molar modal were calculated with one-sided, two-sided, and three-sided vertical bone loss. It was found that the change in the NF was less than 25 per cent in molars with a one-sided defect when the bone level varied by 10 mm from the cementoenamel junction. However, when a three-sided bony defect was simulated, the change in the NF ranged from 40 to 60 per cent. In addition, it was found that bone loss that had reached the furcation entrance (4 mm) resulted in a sharp change in the NF value. Furthermore, it was found that bone loss involving the mesial and distal surfaces resulted in a larger decrease in the NF value compared with bone loss involving the buccal and palatal surfaces. These results demonstrated that the bone surrounding the mesial and distal sides plays a more important role in maintaining molar stability than does the bone surrounding the buccal and palatal sides.

Reductions in the effects of damping on stress concentration in premolars by post-endodontic restorations: a non-linear finite element study.

The aim of this study was to measure the structural damping constants of premolars after treatment with a cast Co-Cr post-core system or permanent root filling, and to evaluate the stress damping effects of these restored premolars. Both the damping ratio and the natural frequency (NF) of the cast Co-Cr post-core restored premolars and the permanent root-filled premolars were detected by in-vitro NF testing experiments. Unprepared premolars served as the control. The damping constants beta of the samples were calculated from the measured damping ratios and natural frequencies. The measured damping constants beta of the test premolars were then used for dynamic finite element (FE) analyses. Stress contours and damping effects of stresses in each treated type of premolar were computed and compared using ANSYS. The measured damping constants beta were 0.75 x 10(-5) for the unprepared premolars, 0.69 x 10(-5) for the root-filled premolars with coronal restoration, and 0.72 x 10(-5) for the cast Co-Cr post-core restored premolars. The unprepared intact premolars demonstrated the highest stress dissipation effects with a ratio of 29.3 per cent at the middle root opposite to the loading side. However, no stress dissipation effects were found in the premolars that had been restored with the cast Co-Cr post-core system. The FE analysis showed that metallic post treatment attenuated the damping properties of the premolar. The effects of damping on stress concentration were significantly lower in restored premolars than in untreated vital premolars. These findings suggest that future research on post material should take the damping property into consideration.

Secreted protein acidic and rich in cysteine expression in human colorectal cancer predicts postoperative prognosis.

OBJECTIVE: Secreted protein acidic and rich in cysteine (SPARC) is an extracellular matrix glycoprotein involved in cell proliferation, migration and angiogenesis. The aim of this study was to assess its expression in colorectal cancer, see whether and how it correlates with clinicopathological features, and evaluate its potential prognostic significance. PATIENTS AND METHODS: SPARC expression was detected by microarrays containing 847 immunohistochemically stained specimens, and further correlated with the clinicopathological and prognostic data. The prognostic significance of its expression was assessed using Kaplan-Meier survival with log-rank tests. Multivariate regression utilizing Cox's proportional hazard model was used to evaluate prognostic factors. RESULTS: SPARC expression in the normal colorectal mucosa and colorectal cancer tissue was significantly different (p < 0.001). Low SPARC expression was found to be associated with poor prognosis, and it was unfavorably correlated with overall survival and disease-free survival in colorectal cancer patients. In addition, SPARC expression in surrounding mesenchymal and stromal cells, bowel wall invasion, lymph node metastasis, and distant metastasis were independent prognostic factors for overall survival and disease-free survival. CONCLUSIONS: Reduced expression of SPARC in colorectal cancer tissue is associated with poor prognosis and aggressive clinicopathological features. Therefore, SPARC expression could potentially be used as a prognostic predictor for colorectal cancer patients.

Skeletal muscle contractions stimulate cGMP formation and attenuate vascular smooth muscle myosin phosphorylation via nitric oxide.

Nitric oxide generated by neuronal nitric oxide synthase in contracting skeletal muscle fibers may regulate vascular relaxation via a cGMP-mediated pathway. Neuronal nitric oxide synthase content is greatly reduced in skeletal muscles from mdx mice. cGMP formation increased in contracting extensor digitorum longus muscles in vitro from C57 control, but not mdx mice. The increase in cGMP content was abolished with NG-nitro-L-arginine. Sodium nitroprusside treatment increased cGMP levels in muscles from both C57 and mdx mice. Skeletal muscle contractions also inhibited phenylephrine-induced phosphorylation of smooth muscle myosin regulatory light chain. Arteriolar dilation was attenuated in contracting muscles from mdx but not C57 mice. NO generated in contracting skeletal muscle may contribute to vasodilation in response to exercise.

The structure and function of acid proteases. V. Comparative studies on the specific inhibition of acid proteases by diazoacetyl-DL-norleucine methyl ester, 1,2-epoxy-3-(p-nitrophenoxy) propane and pepstatin.

Comparative studies have been made on the effects of diazoacetyl-DL-norleucine methyl ester (DAN), 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and pepstatin on acid proteases, including those from Acrocylindrium sp., Aspergillus niger, Aspergillus saitoi, Mucor pusillus, Paecilomyces varioti, Rhizopus chinensis, and Trametes sanguinea, and also porcine pepsin [EC 3.4.23.1] and calf rennin [EC 3.4.23.4] for comparative purposes. These enzymes were rapidly inactivated at similar rates and in 1:1 stiochiometry by reaction with DAN in the presence of cupric ions. The pH profiles of inactivation of these enzymes were similar and had optima at pH 5.5 to 6. They were also inactivated at similar rates by reaction with EPNP, with concomitant incorporation of nearly 2 EPNP molecules per molecule of enzyme. The pH profiles of inactivation were again similar and maximal inactivation was observed at around pH 3 to 4. Some of the EPNP-inactivated enzymes were treated with DAN and shown still to retain reactivity toward DAN. All these enzymes were inhibited strongly by pepstatin, and the reactions of DAN and EPNP with them were also markedly inhibited by prior treatment with pepstatin. These results indicate that the active sites of these enzymes are quite similar and that they presumably have at least two essential carboxyl groups at the active site in common, one reactive with DAN in the presence of cupric ions and the other reactive with EPNP, as has already been demonstrated for porcine pepsin and calf rennin. Pepstatin appears to bind at least part of the active site of each enzyme in a simmilar manner.