Doramectin Induces Apoptosis in B16 Melanoma Cells.

INTRODUCTION/OBJECTIVE: Metastatic melanoma resists current pharmacological regimens that act through apoptosis. This indicates that therapies acting via non-apoptotic cell-death pathways could be pursued. Doramectin has shown promising results in another cancer of neural crest origin, neuroblastoma, through the inhibition of growth via autophagy. Our research hypothesis is that doramectin induces autophagy in B16F10 melanoma cells. METHODS: Cells were treated with doramectin (15 uM) or a combination of both doramectin and a cell-death inhibitor, compared to untreated control cells (media), and then analyzed with MTT analysis. Likewise, MDC analysis was completed to detect autophagy involvement with doramectin treatment. Flow cytometry and TUNEL Assay were conducted to observe cell death-related effects. RESULTS: MTT analysis of doramectin-treated cells displayed a decrease in cell growth compared to control. Apoptotic morphology was prominent in melanoma cells treated with doramectin. Increased autophagy was not detected by fluorometric microscopic analysis. Flow cytometry analysis of doramectin-treated cells showed apoptosis as a major mode of cell death with some necrosis. CONCLUSION: Doramectin induces a novel cell-death mechanism in melanoma compared to other forms of cancer and should be studied as an effective anti-cancer agent for melanoma treatment.

Shikonin Causes Non-apoptotic Cell Death in B16F10 Melanoma.

BACKGROUND: Melanoma treatment is highly resistant to current chemotherapeutic agents. Due to its resistance towards apoptotic cell death, non-apoptotic cell death pathways are sought after. OBJECTIVE: We investigated a Chinese herbal medicine, shikonin, and its effect on B16F10 melanoma cells in vitro. METHODS: Cell growth of B16F10 melanoma cells treated with shikonin was analyzed using an MTT assay. Shikonin was combined with necrostatin, an inhibitor of necroptosis; caspase inhibitor; 3-methyladenine, an inhibitor of autophagy; or N-acetyl cysteine, an inhibitor of reactive oxygen species. Flow cytometry was used to assess types of cell death resulting from treatment with shikonin. Cell proliferation was also analyzed utilizing a BrdU labeling assay. Monodansylcadaverine staining was performed on live cells to gauge levels of autophagy. Western blot analysis was conducted to identify specific protein markers of necroptosis including CHOP, RIP1, and pRIP1. MitoTracker staining was utilized to identify differences in mitochondrial density in cells treated with shikonin. RESULTS: Analysis of MTT assays revealed a large decrease in cellular growth with increasing shikonin concentrations. The MTT assays with necrostatin, 3-methyladenine, and N-acetyl cysteine involvement, suggested that necroptosis, autophagy, and reactive oxygen species are a part of shikonin's mechanism of action. Cellular proliferation with shikonin treatment was also decreased. Western blotting confirmed that shikonin-treated melanoma cells increase levels of stress-related proteins, e.g., CHOP, RIP, pRIP. CONCLUSION: Our findings suggest that mainly necroptosis is induced by the shikonin treatment of B16F10 melanoma cells. Induction of ROS production and autophagy are also involved.

Does lower extremity fracture fixation technique influence neurologic outcomes in patients with traumatic brain injury? The EAST Brain vs. Bone multicenter trial.

OBJECTIVE: This study aimed to determine whether lower extremity fracture fixation technique and timing (≤24 vs. >24 hours) impact neurologic outcomes in TBI patients. METHODS: A prospective observational study was conducted across 30 trauma centers. Inclusion criteria were age 18 years and older, head Abbreviated Injury Scale (AIS) score of >2, and a diaphyseal femur or tibia fracture requiring external fixation (Ex-Fix), intramedullary nailing (IMN), or open reduction and internal fixation (ORIF). The analysis was conducted using analysis of variamce, Kruskal-Wallis, and multivariable regression models. Neurologic outcomes were measured by discharge Ranchos Los Amigos Revised Scale (RLAS-R). RESULTS: Of the 520 patients enrolled, 358 underwent Ex-Fix, IMN, or ORIF as definitive management. Head AIS was similar among cohorts. The Ex-Fix group experienced more severe lower extremity injuries (AIS score, 4-5) compared with the IMN group (16% vs. 3%, p = 0.01) but not the ORIF group (16% vs. 6%, p = 0.1). Time to operative intervention varied between the cohorts with the longest time to intervention for the IMN group (median hours: Ex-Fix, 15 [8-24] vs. ORIF, 26 [12-85] vs. IMN, 31 [12-70]; p < 0.001). The discharge RLAS-R score distribution was similar across the groups. After adjusting for confounders, neither method nor timing of lower extremity fixation influenced the discharge RLAS-R. Instead, increasing age and head AIS score were associated with a lower discharge RLAS-R score (odds ratio [OR], 1.02; 95% confidence interval [CI], 1.002-1.03 and OR, 2.37; 95% CI, 1.75-3.22), and a higher Glasgow Coma Scale motor score on admission (OR, 0.84; 95% CI, 0.73-0.97) was associated with higher RLAS-R score at discharge. CONCLUSION: Neurologic outcomes in TBI are impacted by severity of the head injury and not the fracture fixation technique or timing. Therefore, the strategy of definitive fixation of lower extremity fractures should be dictated by patient physiology and the anatomy of the injured extremity and not by the concern for worsening neurologic outcomes in TBI patients. LEVEL OF EVIDENCE: Prognostic and Epidemiological; Level III.

Medical Students' Diagnostic Accuracy and Treatment Plans.

Physician-mentored patient rounds (PMPR) were used to assess diagnostic accuracy and treatment plans of preclinical medical students. During 4 PMPR sessions, students gathered patient history, observed a physical exam, analyzed diagnostic tests, and developed treatment plans for a patient with chronic obstructive pulmonary disease. Of 470 students, 99.4% correctly diagnosed the patient. Nearly 78% prescribed long-acting beta-agonists or long-acting muscarinic antagonists. Most included appropriate pharmacologic treatments. Only 47% included smoking cessation in their treatment.

Physician-Mentored Patient Rounds to Observe and Assess Entrustable Professional Activities 1 and 2 in Preclinical Medical Students.

CONTEXT: Thirteen entrustable professional activities (EPAs) for entering residency were created to aid medical educators as they prepare preclinical students for their residency and to assess student readiness for residency. The A.T. Still University Kirksville College of Osteopathic Medicine (ATSU-KCOM) developed a program called physician-mentored patient rounds (PMPR), which focuses on EPA 1 and EPA 2. OBJECTIVE: To determine whether PMPRs could be used to assess expected behaviors of EPA 1 (gather a history and perform a physical examination) and EPA 2 (prioritize a differential diagnosis after a clinical encounter). METHODS: The PMPR sessions at ATSU-KCOM take place over several weeks (30-minute sessions per week), during which students gather a patient's history (sessions 1 and 2), observe a physical examination by the physician mentor (session 2), analyze diagnostic test results (session 3), and formulate a treatment plan (session 4). The PMPRs in this study used a real patient with confirmed chronic obstructive pulmonary disease (COPD). This study did not include the session-4 treatment plan. Between sessions, students completed an assignment to further demonstrate their behaviors as expected in the EPAs. Student responses were analyzed and summarized for physician feedback in the subsequent PMPR session. Students' diagnostic accuracy was measured at the conclusion of each session. RESULTS: A total of 516 students were included in the study. The PMPR weekly attendance was high (453-475). Although history gathering in the large-group setting was disorderly, diagnostic accuracy over the 3-session period improved. After history taking, 411 students (86.5%) included COPD in the differential diagnosis. A smaller number, 235 students (49.5%), listed COPD as the most likely diagnosis. After the physical examination, 439 included COPD in the differential diagnosis, and 385 listed COPD as the most likely diagnosis. After analysis of diagnostic test results, 468 students listed COPD as the most likely diagnosis. CONCLUSION: Physician-mentored patient rounds seem to be an effective means to assess preclinical students' expected behaviors as described in EPA 1 and EPA 2.

The immediate effect of individual manipulation techniques on pulmonary function measures in persons with chronic obstructive pulmonary disease.

BACKGROUND: The use of manipulation has long been advocated in the treatment of chronic obstructive pulmonary disease (COPD), but few randomized controlled clinical trials have measured the effect of manipulation on pulmonary function. In addition, the effects of individual manipulative techniques on the pulmonary system are poorly understood. Therefore, the purpose of this study was to determine the immediate effects of four osteopathic techniques on pulmonary function measures in persons with COPD relative to a minimal-touch control protocol. METHODS: Persons with COPD aged 50 and over were recruited for the study. Subjects received five, single-technique treatment sessions: minimal-touch control, thoracic lymphatic pump (TLP) with activation, TLP without activation, rib raising, and myofascial release. There was a 4-week washout period between sessions. Protocols were given in random order until all five techniques had been administered. Pulmonary function measures were obtained at baseline and 30-minutes posttreatment. For the actual pulmonary function measures and percent predicted values, Wilcoxon signed rank tests were used to test within-technique changes from baseline. For the percent change from baseline, Friedman tests were used to test for between-technique differences. RESULTS: Twenty-five subjects were enrolled in the study. All four tested osteopathic techniques were associated with adverse posttreatment changes in pulmonary function measures; however, different techniques changed different measures. TLP with activation increased posttreatment residual volume compared to baseline, while TLP without activation did not. Side effects were mild, mostly posttreatment chest wall soreness. Surprisingly, the majority of subjects believed they could breathe better after receiving osteopathic manipulation. CONCLUSION: In persons with COPD, TLP with activation, TLP without activation, rib raising, and myofascial release mildly worsened pulmonary function measures immediately posttreatment relative to baseline measurements. The activation component of the TLP technique appears to increase posttreatment residual volume. Despite adverse changes in pulmonary function measures, persons with COPD subjectively reported they benefited from osteopathic manipulation.

Microbiological Safety of Blue and Cheddar Cheeses Containing Naturally Modified Milk Fat.

Milk containing naturally modified fat was obtained by feeding lactating dairy cows a Control diet and two experimental diets containing either extruded soybeans or sunflower seeds. Milk from cows fed the experimental diets contained higher levels of both long chain (C18-C18:2) and unsaturated fatty acids than the milk from cows fed the Control diet. Each milk was pasteurized, standardized to 3.6% milk fat, and inoculated with Listeria monocytogenes (strains Scott A and V7), Salmonella typhimurium and Salmonella senftenberg , before manufacturing into Blue or stirred-curd Cheddar cheeses. Populations of L. monocytogenes and Salmonella spp. were monitored during manufacture and aging using Oxford and Xylose Lysine Desoxycholate agars, respectively. During the manufacture of Blue and Cheddar cheese, and during the aging of Blue cheese, behavior of Salmonella spp. and L. monocytogenes in the experimental cheese was similar to the Control cheese. During aging of Cheddar cheese, the rate and extent of decline of Salmonella spp. and L. monocytogenes varied among the cheeses. Declines correlated with the accumulation of specific fatty acids, namely C12, C14, C18:1 and C18:2. These fatty acids were also found to be inhibitory to S. typhimurium and L. monocytogenes when incorporated into tryptic soy agar plates at 37°C. Therefore, the natural fat modification of Blue and Cheddar cheeses enhanced the safety of these cheeses.

Alteration of the Fatty Acid Content of Milk Fat.

Milk fatty acid composition can be influenced by several factors, many of which are interactive. These include stage of lactation, seasonal variation, low milk fat syndrome, feeding, genetic variation, and changes in the energy status of the cow due to administration of bovine somatotropin. Utilization of feeding, genetic variation, and bovine somatotropin should produce a milk fat lower in saturated and higher in unsaturated fatty acids. This may be beneficial to consumers, as many health professionals are recommending diets lower in saturated fatty acids. Giving consumers the option of purchasing low saturated fatty acid dairy products may also assist in alleviating the current milk fat surplus in the dairy industry.

Influence of Milk Fat Higher in Unsaturated Fatty Acids on the Accuracy of Milk Fat Analyses by the Mid-Infrared Spectroscopic Method.

An experiment was conducted to investigate the reliability of milk fat measurement by the mid-infrared spectroscopic method when analyzing milk fat containing greater than normal amounts of unsaturated fatty acids. Sixteen mid-lactation Holstein cows were divided into four treatments including a control (C), control with bovine somatotropin (C+), bovine somatotropin and added dietary fat from sunflower seeds (Sun+), or bovine somatotropin and added dietary fat from safflower seeds (Saff+). Milks were sampled weekly for 16 weeks (n=256). Unsaturated fatty acid percentages in milk fat were 25.0, 28.4, 39.6, and 37.9 for C, C+, Sun+, and Saff+ treatments, respectively. Milk fat percentages measured by the Mojonnier fat extraction and mid-infrared spectroscopic methods were 2.99, 2.97; 3.06, 3.01; 2.73, 2.56; and 2.86, 2.74 for C, C+, Sun+, and Saff+ treatments, respectively. Results indicate the mid-infrared spectroscopic method underestimates the fat content in milk which is higher in unsaturated fatty acids. Dairy producers feeding diets with added fat from unsaturated fat sources may be underpaid for milk fat content when the milk is analyzed by the mid-infrared spectroscopic method. A possible remedy for this problem may be to have milk plants calibrate the mid-infrared spectroscopic instrument with milk samples containing higher than normal amounts of unsaturated fatty acids in milk fat.

Composition of Grade A and Manufacturing Grade Herd Milks in South Dakota.

Biweekly Grade A and manufacturing grade herd milk samples were collected from April 1, 1985, to March 31, 1986, from 203 herds in the Sioux Falls, SD, area and were analyzed to compare composition. The average herd milk composition was 3.70% fat, 3.24% protein, 4.80% lactose, 0.63% ash, 8.67% solids-not-fat (SNF), and 12.37% total solids (TS). Grade A milk had higher % lactose (4.83 and 4.76), % SNF (8.70 and 8.61), and % TS (12.41 and 12.30) than manufacturing grade milk. These compositional differences between grades were consistent throughout the year. Grade A milk has more value in the production of dried dairy products (nonfat dry milk and whey powders) and ice cream than manufacturing grade milk due to its higher lactose and SNF content. New milk pricing plans for protein and other constituents should consider compositional differences (lactose, SNF and TS) that exist between grade A and manufacturing grade herd milk before implementing them.

Use of the Osmometer for Quality Control of Ice Cream Mix 1.

Fourteen brands of commercial vanilla ice cream, purchased at three different times, were analyzed for freezing point, firmness, fat, solids-not-fat, total solids, and net weight. Mean freezing points ranged from -2.65 ± .04 to -3.24 ± .19°C, which indicated ice cream composition and mix formulations varied. Values for mean penetrometer readings ranged from 120 ± 6.0 to 193 ± 34.9 mm; fat, 9.3 ± .23 to 16.8 ± .20%; solids-not-fat, 25.2 ± .17 to 29.6 ± .64%; total solids, 35.5 ± .25 to 42.0 ± .26%; and net weights, 1.026 ± .045 to 1.756 ± .017 kg/1.89 liter. The freezing point osmometer has potential use as a rapid quality control instrument to measure the freezing point of ice cream mix and detect when improper quantities of soluble mix ingredients have been incorporated into the mix. More testing of mix formulations will improve product uniformity and quality and assure compliance with regulatory standards.