In Vitro Organoid-Based Assays Reveal SMAD4 Tumor-Suppressive Mechanisms for Serrated Colorectal Cancer Invasion.

Colon cancer is the third most prominent cancer and second leading cause of cancer-related deaths in the United States. Up to 20% of colon cancers follow the serrated tumor pathway driven by mutations in the MAPK pathway. Loss of SMAD4 function occurs in the majority of late-stage colon cancers and is associated with aggressive cancer progression. Therefore, it is important to develop technology to accurately model and better understand the genetic mechanisms behind cancer invasion. Organoids derived from tumors found in the Smad4KO BRAFV600E/+ mouse model present multiple phenotypes characteristic of invasion both in ex vivo and in vivo systems. Smad4KO BRAFV600E/+ tumor organoids can migrate through 3D culture and infiltrate through transwell membranes. This invasive behavior can be suppressed when SMAD4 is re-expressed in the tumor organoids. RNA-Seq analysis reveals that SMAD4 expression in organoids rapidly regulates transcripts associated with extracellular matrix and secreted proteins, suggesting that the mechanisms employed by SMAD4 to inhibit invasion are associated with regulation of extracellular matrix and secretory pathways. These findings indicate new models to study SMAD4 regulation of tumor invasion and an additional layer of complexity in the tumor-suppressive function of the SMAD4/Tgfß pathway.

SMAD4 is critical in suppression of BRAF-V600E serrated tumorigenesis.

BRAF-driven colorectal cancer is among the poorest prognosis subtypes of colon cancer. Previous studies suggest that BRAF-mutant serrated cancers frequently exhibit Microsatellite Instability (MSI) and elevated levels of WNT signaling. The loss of tumor-suppressor Smad4 in oncogenic BRAF-V600E mouse models promotes rapid serrated tumor development and progression, and SMAD4 mutations co-occur in human patient tumors with BRAF-V600E mutations. This study assesses the role of SMAD4 in early-stage serrated tumorigenesis. SMAD4 loss promotes microsatellite stable (MSS) serrated tumors in an oncogenic BRAF-V600E context, providing a model for MSS serrated cancers. Inactivation of Msh2 in these mice accelerated tumor formation, and whole-exome sequencing of both MSS and MSI serrated tumors derived from these mouse models revealed that all serrated tumors developed oncogenic WNT mutations, predominantly in the WNT-effector gene Ctnnb1 (ß-catenin). Mouse models mimicking the oncogenic ß-catenin mutation show that the combination of three oncogenic mutations (Ctnnb1, Braf, and Smad4) are critical to drive rapid serrated dysplasia formation. Re-analysis of human tumor data reveals BRAF-V600E mutations co-occur with oncogenic mutations in both WNT and SMAD4/TGFß pathways. These findings identify SMAD4 as a critical factor in early-stage serrated cancers and helps broaden the knowledge of this rare but aggressive subset of colorectal cancer.

Effect of dietary ascorbate on lipogenesis and lipolysis activities in black sea bream, Acanthopagrus schlegelii.

To assess the effect of dietary ascorbate on lipid metabolism, 1-year black sea bream (Acanthopagrus schlegelii) were reared on a casein-based purified diet and an ascorbate fortified diet (1,100 mg of L: -ascorbyl-2- monophosphate-Mg/kg diet). The fortified ascorbate was effectively incorporated into the fish body and elevated muscle carnitine content. Fortifications of dietary ascorbate depressed activities of glucose-6-phosphate dehydrogenase and NADP-isocitrate dehydrogenase as lipogenic enzymes in the hepatopancreas and intraperitoneal fat body. Starvation after feeding experiment activated carnitine palmitoyltransferase as a lipolysis enzyme in the hepatopancreas in both control and vitamin C(VC) groups, while the lipolysis activity was significantly higher in VC group. These results confirmed that dietary ascorbate depressed lipogenesis and activated lipolysis, i.e., influenced the lipid metabolism of black sea bream.

A quantitative structure-activity relationship model for radical scavenging activity of flavonoids.

A quantitative structure-activity relationship (QSAR) study has been carried out for a training set of 29 flavonoids to correlate and predict the 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (RSA) values obtained from published data. Genetic algorithm and multiple linear regression were employed to select the descriptors and to generate the best prediction model that relates the structural features to the RSA activities using (1) three-dimensional (3D) Dragon (TALETE srl, Milan, Italy) descriptors and (2) semi-empirical descriptor calculations. The predictivity of the models was estimated by cross-validation with the leave-one-out method. The result showed that a significant improvement of the statistical indices was obtained by deleting outliers. Based on the data for the compounds used in this study, our results suggest a QSAR model of RSA that is based on the following descriptors: 3D-Morse, WHIM, and GETAWAY. Therefore, satisfactory relationships between RSA and the semi-empirical descriptors were found, demonstrating that the energy of the highest occupied molecular orbital, total energy, and energy of heat of formation contributed more significantly than all other descriptors.

Comparison of the isolation rates and characteristics of Salmonella isolated from antibiotic-free and conventional chicken meat samples.

Salmonella contamination in chicken samples can cause major health problems in humans. However, not only the effects of antibiotic treatment during growth but also the impacts of the poultry slaughter line on the prevalence of Salmonellae in final chicken meat sold to consumers are unknown. In this study, we compared the isolation rates and antimicrobial resistance of Salmonellae among antibiotic-free, conventional, conventional Korean native retail chicken meat samples, and clonal divergence of Salmonella isolates by multilocus sequence typing. In addition, the distribution of extended-spectrum ß-lactamase (ESBL) genes in ESBL-producing Salmonella isolates was analyzed. A total of 72 retail chicken meat samples (n = 24 antibiotic-free broiler [AFB] chickens, n = 24 conventional broiler [CB] chickens, and n = 24 conventional Korean native [CK] chickens) was collected from local retail markets in Seoul, South Korea. The isolation rates of Salmonellae were 66.6% in AFB chickens, 45.8% in CB chickens, and 25% in CK chickens. By analyzing the minimum inhibitory concentrations of ß-lactam antibiotics with the disc-diffusion test, we found that 81.2% of Salmonella isolates from AFB chickens, 63.6% of isolates from CB chickens, and 50% of isolates from CK chickens were ESBL producers; all ESBL-positive isolates had the CTX-M-15 genotype. Interestingly, all ESBL-producing Salmonellae were revealed as ST16 by multilocus sequence typing and had the genetic platform of blaCTX-M gene (IS26-ISEcp1-blaCTX-M-15-IS903), which was first reported in Salmonellae around the world. The Salmonella ST33 strain (S. Hadar) isolated in this study has never been reported in South Korea. In conclusion, our findings showed that antibiotic-free retail chicken meat products were also largely contaminated with ESBL-producing Salmonellae and that their ESBL genes and genetic platforms were the same as those isolated from conventional retail chicken meat products.

Degree of Tissue Differentiation Dictates Susceptibility to BRAF-Driven Colorectal Cancer.

Oncogenic mutations in BRAF are believed to initiate serrated colorectal cancers; however, the mechanisms of BRAF-driven colon cancer are unclear. We find that oncogenic BRAF paradoxically suppresses stem cell renewal and instead promotes differentiation. Correspondingly, tumor formation is inefficient in BRAF-driven mouse models of colon cancer. By reducing levels of differentiation via genetic manipulation of either of two distinct differentiation-promoting factors (Smad4 or Cdx2), stem cell activity is restored in BRAFV600E intestines, and the oncogenic capacity of BRAFV600E is amplified. In human patients, we observe that reduced levels of differentiation in normal tissue is associated with increased susceptibility to serrated colon tumors. Together, these findings help resolve the conditions necessary for BRAF-driven colon cancer initiation. Additionally, our results predict that genetic and/or environmental factors that reduce tissue differentiation will increase susceptibility to serrated colon cancer. These findings offer an opportunity to identify susceptible individuals by assessing their tissue-differentiation status.

Direct determination of verapamil in rat plasma by coupled column microbore-HPLC method.

This report describes an automated coupled column microbore-high-performance liquid chromatography (HPLC) with fluorescence detection for direct determination of verapamil in small volume of rat plasma. We used HPLC system consisting of three columns such as precolumn, intermediate and analytical column and six-port switching valve and injected small volume of rat plasma to the system without sample preparation. An aliquot of sample was directly injected into Capcell Pak MF Ph precolumn for clean-up and enrichment, 35 mm Capcell Pak C18, intermediate column for concentration of compounds and 250 mm Capcell Pak C18 analytical column for separation of compounds and two mobile phases are used as mobile phase A (50mM ammonium phosphate, pH 4.5) and B (50mM ammonium phosphate:acetonitrile=70:30 v/v). Analysis of verapamil and internal standard, propranolol was performed with direct injection of 10 microl of rat plasma to the system and were eluted at 22 and 12 min, respectively, at a mobile phase flow rate of 0.5 (mobile phase A) and 0.15 ml/min (mobile phase B). The peaks of verapamil and internal standard were good shapes and well separated from any interfering endogenous peaks during a total run time of 25 min. The calibration curve for verapamil showed good linearity (r(2)=0.9997) over the concentration range of 0.01-2.50 microg/ml. The mean RSD (%) values of intra-day (n=5) and inter-day (n=5) variability of verapamil ranged from 1.96 to 9.06 and 0.62 to 3.08%, respectively. The LOD and LOQ were 0.01 and 0.025 microg/ml, respectively, for verapamil using 10 microl of rat plasma. An automated coupled column microbore-HPLC method was successfully applied to a pharmacokinetic study after intravenous injection of 3mg/kg of verapamil to the normal and dimethylnitrosamine (DMN)-induced hepatofibrotic rats.

Use of anti-skeletal muscle antibody from myasthenic patients in the diagnosis of childhood rhabdomyosarcomas.

Rhabdomyosarcoma (RMS), a common soft tissue tumor in children, may often be difficult to distinguish from Ewing's sarcoma, neuroblastoma, and malignant lymphomas. Confirmation of the skeletal muscle origin of RMS depends partly on the demonstration of striations in tumor cells that are usually undetectable in poorly differentiated tumors. A number of tissue markers (e.g., myoglobin and desmin) are currently being used to establish the origin of RMS. However, most of these markers lack specificity and have relatively low sensitivity. We have investigated the specificity and sensitivity of anti-skeletal muscle antibody (ASMA) from patients with myasthenia gravis in the diagnosis of childhood RMS. Out of eight cases of childhood RMS (four embryonal and four alveolar) examined, two showed striations with hematoxylin and eosin and four with phosphotungstic acid hematoxylin. Myoglobin was detected in five tumors; only well-differentiated tumor cells contained myoglobin. Anti-desmin antibody and ASMA reacted with cells in all the eight tumors whether or not the tumor cells were well differentiated. Anti-skeletal muscle antibody did not react with nine lymphomas, four Ewing's sarcomas, four neuroblastomas, four osteogenic sarcomas, four lipomas, eight duct carcinomas of the breast, and eight squamous cell carcinomas of the lung. Eight leiomyomas and four leiomyosarcomas of the uterus were compared for their reactivity with anti-desmin antibody and ASMA. All the tumors stained with anti-desmin antibody and none with ASMA. The results show that ASMA is useful in the diagnosis of childhood RMS and is a more sensitive reagent than anti-myoglobin antibody. Unlike anti-desmin antibody, it can distinguish skeletal muscle tumors from smooth muscle tumors.

Frequency of coronary artery disease and left ventricle dysfunction in cocaine users.

To evaluate the spectrum of coronary artery disease (CAD) in cocaine users, coronary angiograms obtained from 33 patients (26 men [79%] and 7 women [21%], mean age 37 years) with history of cocaine use and cardiac symptoms were retrospectively reviewed. Clinical indications for coronary angiograms included chest pain (n = 28), congestive failure (n = 4) and complete heart block (n = 1). Coronary angiograms were reviewed independently by 2 angiographers unaware of patient's clinical status. Thirteen patients (40%) had normal coronary angiograms, and 20 (60%) had CAD; 7 (21%) had mild CAD (less than or equal to 70% diameter stenosis), and 13 (40%) had significant CAD (greater than 70% diameter stenosis). Of 13 patients with significant CAD, 7 had 1-vessel, 4 had 2-vessel and 2 had 3-vessel CAD. There was enzymatic evidence of myocardial infarction in 12 of 33 patients (36%); all 12 had CAD (10 with significant and 2 with mild CAD). Mean age and number of risk factors (serum total cholesterol, cigarette smoking, systemic hypertension, diabetes mellitus, family history of CAD, and obesity) in patients with CAD (mild or significant) and with normal coronary angiograms were not statistically different. Left ventricular ejection fraction was normal in 15 patients (45%) and depressed in 18 (55%). All patients with CAD and low ejection fractions (n = 12) had regional wall motion abnormalities, whereas all those with normal coronary arteries and low ejection fraction (n = 6) had global hypokinesia.

Reversal of severe cardiac systolic dysfunction caused by pheochromocytoma in a heart transplant candidate.

Whenever a patient is evaluated as a possible candidate for heart transplantation, potential causes of reversible cardiomyopathy must always be considered. Although rate, it is well-known that pheochromocytoma can result in a dilated cardiomyopathy, which can be partially or completely reversible. We report a case of a 33-year-old woman with heart failure that was caused by a severe dilated cardiomyopathy who was referred for urgent heart transplant evaluation. The diagnosis of bilateral adrenal pheochromocytomas was made, and within 3 weeks of medical therapy, left ventricular systolic dysfunction completely reversed, avoiding the need for heart transplantation. The patient later underwent successful adrenalectomy. Unique features of this case of pheochromocytoma-induced cardiomyopathy include (1) serial norepinephine measurements over 3 weeks documenting the efficacy of medical therapy, (2) unique cutaneous manifestations that resolved with medical therapy, and (3) familial multiple endocrine neoplasia syndrome with medullary carcinoma of the thyroid in three generations of this patient's family.

Cytosolic estrogen receptor concentrations in the lumbosacral spinal cord fluctuate during the estrous cycle.

Estrogen responsive neurons have been anatomically identified with autoradiographic and immunohistochemical techniques and their distribution mapped in the lumbosacral spinal cord of female rats. Such neurons contain estrogen receptors (ERs). The present study was undertaken to: 1) quantify cytosolic estrogen receptor (ER) concentrations in the lumbosacral spinal cord and 2) determine if there is a relationship between cytosolic ER concentrations and fluctuations in serum estradiol (SE2) levels during the estrous cycle. Lumbosacral spinal segments were removed from intact cycling rats during the morning of proestrus, the afternoon of proestrus, and the morning of estrus, metestrus and diestrus. Trunk blood was collected at euthanasia and SE2 levels were determined using radioimmunoassay. Cytosolic ER concentrations were measured using a dextran-charcoal coated tube method. Concentrations of cytosolic ERs were low during estrus and metestrus, increased during diestrus with maximum concentrations during the afternoon of proestrus. These changes in ER concentrations paralleled SE2 levels measured in intact cycling animals; i.e., during estrus SE2 levels were low, but began to rise during metestrus, diestrus, and during the morning of proestrus with a maximum peak increase during the afternoon of proestrus. These data indicate there are fluctuations of cytosolic ER concentrations during the estrous cycle and that these changes coincide with changing SE2 concentrations suggesting that ER content is influenced by SE2.

Cardiovascular complications of cocaine.

Cocaine use is escalating in the United States. Cocaine produces sympathomimetic effects and causes generalized vasoconstriction involving multiple organ systems. Its cardiovascular complications may be life-threatening and include myocardial infarction, myocarditis, cardiomyopathy, arrhythmias, and aortic dissection. Accurate diagnosis and prompt management can be life-saving. This review focuses on the current information available on the cardiovascular complications of cocaine and suggests guidelines regarding their management strategies.

Long-term patency of a Cook stent without anticoagulation.

In recent years intracoronary stenting has been proven to be an effective bail out for acute and threatened closure during and after coronary angioplasty. Initial enthusiasm has been hampered by significant anticoagulation related vascular and bleeding problems. We report a patient with intracoronary stent whose stent remained patent despite lack of any anticoagulation.

Inotropic therapy of the failing myocardium.

The clinical syndrome of congestive heart failure remains a therapeutic dilemma and challenge for the physician in 1992. This is a disease process that appears to be increasing in frequency and continues to carry an unacceptably high mortality rate. For years it has been well recognized that the combination of digoxin, Lasix and vasodilator therapy improved symptoms in these patients and decreased hospitalization, but did not increase survival. It was not until 1986 that the combination of digoxin, Lasix, Isordil, and hydralazine was shown to increase survival. Further significant improvement in quality of life and survival has recently been established in three large clinical trials, and it is now safe to say that the standard of care for symptomatic congestive heart failure in 1992 is digoxin, furosemide, and an ACE inhibitor, with the survival trials favoring the ACE inhibitor enalapril. The IV inotropic drug dobutamine remains the mainstay of pharmacological therapy for the treatment of severely refractory heart failure. Unfortunately, the phosphodiesterase inhibitors--amrinone, milrinone, and enoximone--have demonstrated unacceptable clinical side effects and have been withdrawn from further clinical study. In spite of these promising developments, the mortality and morbidity of congestive heart failure remains unacceptably high, and continued investigation in the new fields of pharmacology and the pathophysiology of congestive heart failure still must be aggressively pursued.

Exogenous oestrogen affects calcium metabolism differently from exogenous testosterone in ovariectomized or orchiectomized rats fed a high fructose diet severely deficient in magnesium.

To investigate interactions between sex hormones, dietary fructose, and a severe magnesium deficiency on calcium metabolism, 10 week old ovariectomized (OVX) female, and orchiectomized (ORX) males rats were studied. The OVX and ORX animals were divided into two groups: one half of the animals in each group was injected with beta-oestradiol-3-benzoate dissolved in sesame oil twice a week; the other half was injected with testosterone cypionate in sesame oil twice a week. All animals were pari-fed a severely magnesium-deficient fructose diet. After a 4 week experimental period, a 24 h urine sample was collected for measurements of cAMP, calcium, magnesium, and phosphorus. Blood was collected for determination of calcium, magnesium, phosphorus, 25-hydroxy- and 1.25-dihydroxycholecalciferol [25(OH)D, 1.25(OH)2D], and parathyroid hormone (PTH). Femurs were used for measurements of bone mineral content (BMC) and density (BMD). Oestrogen treatment produced hypercalcaemia and hypercalciuria, and, further, this was higher in female than in male rats. In contrast, testosterone treatment produced hypocalcaemia and hypocalciuria. Hypocalcaemia in testosterone-treated animals may stimulate secretion of PTH. Testosterone-treated animals had significantly lower BMD than oestrogen-treated animals. High circulating PTH seemed to cause bone loss in the testosterone group. High PTH may stimulate hydroxylation of 25(OH) D to 1.25(OH)2D in the kidneys, and high circulating 1.25(OH)2D would antagonize bone formation. Either endogenous or exogenous oestrogen increased kidney calcification. The study indicates that oestrogen-fructose-magnesium interaction on calcium metabolism was significantly different from that of testosterone.

Bone histomorphometry of ovariectomized or orchiectomized rats fed a moderately magnesium-deficient fructose diet and treated with exogenous oestrogen or testosterone.

To investigate the effects of sex hormones on bone histomorphometry, and bone density (BMD), 10 week old ovariectomized (OVX), orchiectomized (ORX), and sham-operated (SHAM) rats fed a moderately magnesium-deficient fructose diet were studied. One third of the OVX and ORX rats were injected with beta-oestradiol-3-benzoate; another third, testosterone cypionate; and the remaining SHAM rats, vehicle only. After 14 weeks, a 24 h urine sample was collected for measurements of calcium, phosphorus and cyclic AMP (cAMP). Blood was collected for determination of calcium, phosphorus, and parathyroid hormone (PTH). Femurs and tibias were removed and weighed. Femurs were used to measure bone areas, mineral contents (BMC), and BMD. Tibias were used for bone histomorphometry (that is, trabecular numbers, thicknesses, % areas and separations). Oestrogen treatment increased serum and urine calcium significantly in both OVX and ORX rats, whereas testosterone decreased serum and urine calcium significantly. Oestrogen decreased urinary cAMP and PTH in both OVX and ORX rats, whereas testosterone treatment increased them significantly. Oestrogen treatment increased BMD, trabecular numbers, thicknesses, and % areas, and decreased bone separations in both OVX and ORX rats. In contrast, testosterone did not increase these bone indices in either OVX or ORX rats; rather, it increased bone separations by decreasing bone strength. Testosterone treatment improved trabecular histomorphometry slightly in OVX rats. The results of the present study are concordant with our previous findings that exogenous oestrogen treatment can prevent osteoporosis in either OVX or ORX rats, whereas exogenous testosterone cannot.

Keratin and carcinoembryonic antigen (CEA) in human melanoma cells.

Human melanomas are known to contain vimentin intermediate filaments but there has been some dispute about their expression of cytokeratins. The cytoplasm of human M21 melanoma cells maintained in culture reacted with a rabbit anti-keratin antibody and two monoclonal anti-keratin antibodies AE1 and AE2. Cells derived directly from subcutaneous xenografts of M21 melanoma in nude mice, however, failed to express cytokeratins. The presence of keratin filaments in cultured M21 cells was confirmed by electronmicroscopic and immuno-electronmicroscopic examinations of cell extracts. Polyacrylamide gel electrophoresis (PAGE), revealed 46 KD keratin proteins in cultured M21 cells. Small amounts of these low molecular weight keratins were detected by PAGE in M21 melanoma xenografts even though immunofluorescence and immunoperoxidase assays failed to demonstrate keratin at the light microscopic level. Immunofluorescence revealed keratin and carcinoembryonic antigen (hitherto undetected in human melanomas) first on the 9th day of culture of xenograft-derived M21 cells. The appearance of keratin and CEA in M21 melanoma cells in vitro was not affected by inhibition of cellular proliferation or as a result of exposure to methotrexate or adriamycin. However, adriamycin altered the cytoplasmic distribution of keratin.