Doping control of estra-4,9-diene-3,17-dione in horses.

Estra-4,9-diene-3,17-dione (dienedione) is an anabolic-androgenic steroid (AAS) available on the market as a dietary supplement for bodybuilding. It is prohibited in both human and equine sports due to its potential performance-enhancing effect. With the rare presence of the 4,9-diene configuration in endogenous steroids, dienedione has been considered as a synthetic AAS. Nevertheless, the reoccurring detection of dienedione in entire male horse urine samples led to the investigation of its possible endogenous nature in horses, and its endogenous nature in entire male horses has been recently confirmed and reported by the authors' laboratory. While dienedione is not detected in castrated horses (geldings), it is essential to study its elimination and identify its metabolites for its effective control. To study the elimination and biotransformation of dienedione, administration experiments were performed by giving three castrated horses (geldings) each single oral dose of 1500 mg of dienedione powder for seven consecutive days. The postulated in vivo metabolites included 17-hydroxyestra-4,9-dien-3-one (M1a and M1b), hydroxylated dienedione (M2a, M2b, M3a, M3b, M4, M5) and hydroxylated M1 (M6a, M6b, M7a, M7b, M8a and M8b), formed from hydroxylation and reduction of dienedione. To control the misuse of dienedione in geldings, M3a and M3b are the potential targets that gave the longest detection time, which could be detected for up to 2-5 days in urine and 0.4-4 days in plasma.

Endogenous nature of estra-4,9-diene-3,17-dione in entire male horses.

Estra-4,9-diene-3,17-dione (dienedione) is an anabolic androgenic steroid (AAS) sold as a bodybuilding supplement. It is prohibited in both human and equine sports. With no report of 4,9-diene configuration in endogenous steroids, dienedione has long been considered a synthetic AAS. Nevertheless, the reoccurring detection of dienedione in colt (entire male horse) urine samples lead to the investigation of its possible endogenous nature in horses. This paper describes (i) the detection of naturally occurring dienedione in colts, (ii) the conjugation study of dienedione and (iii) the population study of free and glucuronide-conjugated dienedione in colt urine. Qualitative and quantitative analyses of dienedione content in colt urine were performed, employing liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Qualitative analyses showed that dienedione was endogenous in colt urine and mainly in the form of glucuronide conjugates. Glucuronidation of dienedione was believed to happen at 3-enol leading to dienedione-3-glucuronide. Upon the population study of free and glucuronide-conjugated dienedione in colt urine samples (n = 175), the mean ± SD was determined to be 2.5 ± 3.5 ng/ml. The population data fitted a normal distribution after a fifth root transformation with the exclusion of one outlier by Grubb's test. A possible in-house threshold was proposed at 30 ng/ml of free and glucuronide-conjugated dienedione in colt urine associated with a risk factor of 1 in 14,269 (with a degree of freedom of 173). This is the first report of endogenous dienedione in entire male horses and the approach for controlling its potential misuse by using a threshold is also presented.

Operative Time and 30-Day Outcomes in Bariatric Surgery: Comparison between Robotic and Laparoscopic Approach: 4-Year MBSAQIP Database Analysis.

BACKGROUND: The main criticism of robotic surgery is longer operative time (OT). The aim of this study was to examine the variables that determine OT, the association between OT and 30-day outcomes, and the effect of the robotic approach in bariatric surgery. STUDY DESIGN: MBSAQIP data for 2016 to 2019 were queried. Logistic regression was performed to examine the association between OT and outcomes for each surgical approach while adjusting for patients' characteristics. The results of each fitted logistic regression model were reported as odds ratio and the associated 95% CI. RESULTS: A total of 666,182 patients underwent robotic sleeve gastrectomy (R-SG), laparoscopic sleeve gastrectomy, robotic Roux-en-Y gastric bypass (R-RYGB), laparoscopic Roux-en-Y gastric bypass, robotic duodenal switch (R-DS), and laparoscopic duodenal switch). More patients underwent laparoscopic surgery (89.7%) than robotic surgery (10.3%). OT for robotic cases was longer than for laparoscopic cases (p < 0.0001). Longer OT was associated with increased odds of adverse 30-day outcomes irrespective of the surgical approach. The association between OT and adverse outcomes was stronger in the laparoscopic cohort. There was no significant difference in postoperative outcomes when comparing the laparoscopic and robotic approaches after adjusting for OT, except a lower reoperation rate for R-SG (p = 0.03) and readmission rates in R-RYGB and R-DS (p < 0.01). The variability of OT was higher in the laparoscopic group and was more affected by the first assistant. CONCLUSIONS: The outcomes in robotic bariatric surgery were comparable with the laparoscopic approach despite longer OT. Use of robotic surgery decreased the variability in OT.

Minimally invasive vs open pancreatoduodenectomy on oncological adequacy: a propensity score-matched analysis.

BACKGROUND: The adoption of minimally invasive pancreatoduodenectomy (MIPD) has increased over the last decade. Most of the data on perioperative and oncological outcomes derives from single-center high-volume hospitals. The impact of MIPD on oncological outcomes in a multicenter setting is poorly understood. METHODS: The National Cancer Database was utilized to perform a propensity score matching analysis between MIPD vs open pancreatoduodenectomy (OPD). The primary outcomes were lymphadenectomy ≥ 15 nodes and surgical margins. Secondary outcomes were 90-day mortality, length of stay, and overall survival. RESULTS: A total of 10,246 patients underwent pancreatoduodenectomy for ductal adenocarcinoma between 2010 and 2016. Among these patients, 1739 underwent MIPD. A propensity score matching analysis with a 1:2 ratio showed that the rate of lymphadenectomy ≥ 15 nodes was significantly higher for MIPD compared to OPD, 68.4% vs 62.5% (P < .0001), respectively. There was no statistically significant difference in the rate of positive margins, 90-day mortality, and overall survival. OPD was associated with an increased rate of length of stay > 10 days, 36.6% vs 33% for MIPD (P < .01). Trend analysis for the patients who underwent MIPD revealed that the rate of adequate lymphadenectomy increased during the study period, 73.1% between 2015 and 2016 vs 63.2% between 2010 and 2012 (P < .001). In addition, the rate of conversion to OPD decreased over time, 29.3% between 2010 and 2012 vs 20.2% between 2015 and 2016 (P < .001). CONCLUSION: In this propensity score matching analysis, the MIPD approach was associated with a higher rate of adequate lymphadenectomy and a shorter length of stay compared to OPD. The surgical margins status, 90-day mortality, and overall survival were similar between the groups.

Surgeon Experience Is Associated With Prolonged Air Leak After Robotic-assisted Pulmonary Lobectomy.

BACKGROUND: Prolonged air leak (PAL) (>5 days) after robotic-assisted pulmonary lobectomy is a significant complication. This study aimed to determine patient- and surgeon-related factors that can predict PAL after robotic lobectomy for lung cancer. METHODS: This study was a retrospective review of a single-center experience of robotic-assisted lobectomy for lung cancer. Perioperative variables, including surgeon case experience, patient demographics, diffusion capacity of lung for carbon monoxide, forced expiratory volume in 1 second, body mass index, and smoking status were evaluated. RESULTS: A total of 305 robotic-assisted lobectomies performed by 4 surgeons met inclusion criteria from June 2016 to February 2019. The 30-day postoperative mortality was 1.2%. PAL developed in 27 of 305 (8.8%) patients. Surgeons' robotic experience was grouped by 10-case increments. When adjusted for age and sex, the odds for PAL decreased by 15% for every 10 robotic lobectomies the surgeons performed (odds ratio [OR], 0.85; 95% CI, 0.74-0.99; P = .0384). Logistic regression models showed a linear transition curve at the 50th case. Female sex (OR, 2.62; 95% CI, 1.03-6.69; P = .0314) and younger age (OR, 0.61; 95% CI, 0.41-0.91; P = .0184) were statistically significant risk factors for PAL. Cumulative sum analysis similarly showed a strong association between experience and PAL. Preoperative diffusing capacity of lung for carbon monoxide, forced expiratory volume in 1 second, body mass index, and smoking status were not statistically significant predictive factors. CONCLUSIONS: These results show that surgeon robotic case experience is associated with the rate of postoperative PAL: as the number of robotic lobectomies increases, the rate of PAL significantly decreases. It is imperative to emphasize that a learning curve exists for this approach that directly affects patient outcomes.

Reverse-translational identification of a cerebellar satiation network.

The brain is the seat of body weight homeostasis. However, our inability to control the increasing prevalence of obesity highlights a need to look beyond canonical feeding pathways to broaden our understanding of body weight control1-3. Here we used a reverse-translational approach to identify and anatomically, molecularly and functionally characterize a neural ensemble that promotes satiation. Unbiased, task-based functional magnetic resonance imaging revealed marked differences in cerebellar responses to food in people with a genetic disorder characterized by insatiable appetite. Transcriptomic analyses in mice revealed molecularly and topographically -distinct neurons in the anterior deep cerebellar nuclei (aDCN) that are activated by feeding or nutrient infusion in the gut. Selective activation of aDCN neurons substantially decreased food intake by reducing meal size without compensatory changes to metabolic rate. We found that aDCN activity terminates food intake by increasing striatal dopamine levels and attenuating the phasic dopamine response to subsequent food consumption. Our study defines a conserved satiation centre that may represent a novel therapeutic target for the management of excessive eating, and underscores the utility of a 'bedside-to-bench' approach for the identification of neural circuits that influence behaviour.

Surgery and invasive diagnostic procedures for benign disease are rare in a large low-dose computed tomography lung cancer screening program.

OBJECTIVE: Lung cancer screening with low-dose chest computed tomography improves survival. However, concerns about overdiagnosis and unnecessary interventions persist. We reviewed our lung cancer screening program to determine the rate of surgery and invasive procedures for nonmalignant disease. METHODS: We reviewed all patients undergoing lung cancer screening from January 2012 to June 2017 with follow-up through January 2019. Patients with suspicious findings (Lung CT Screening Reporting and Data System 4) were referred for further evaluation. RESULTS: Of 3280 patients screened, 345 (10.5%) had Lung CT Screening Reporting and Data System 4 findings. A total of 311 patients had complete follow-up, of whom 93 (29.9%) were diagnosed with lung cancer. Eighty-three patients underwent lung surgery (2.5% of screened patients). Forty patients underwent lobectomy (48.2%), 3 patients (3.6%) underwent bilobectomy, and 40 patients (48.2%) underwent sublobar resection. Fourteen patients underwent surgery for benign disease (0.43% of screened patients). Fifty-four patients, 5 with benign disease, had at least 1 invasive diagnostic procedure but never underwent surgery. The incidence of any invasive intervention for nonmalignant disease was 0.95% (31/3280 patients). There were no postprocedural deaths within 60 days. Twenty-five patients (0.76%) underwent stereotactic body radiation therapy; 19 patients (76%) had presumed lung cancer without pretreatment pathologic confirmation. CONCLUSIONS: Surgical resection for benign disease occurred in 0.43% of patients undergoing lung cancer screening. The combined incidence of any invasive diagnostic or therapeutic intervention, including surgical resection, for benign disease was only 0.95%. Periprocedural complications were rare. These results indicate that concern over unnecessary interventions is overstated and should not hinder adoption of lung cancer screening. A multidisciplinary team approach, including thoracic surgeons, is critical to maintain an appropriate rate of interventions in lung cancer screening.

Bitter bottle gourd (Lagenaria siceraria) toxicity.

BACKGROUND: Bottle gourd (Lagenaria siceraria) is an edible plant in the Cucurbitaceae family. When extremely bitter, ingestion of bottle gourd can cause rapid onset diarrhea, vomiting, gastrointestinal bleeding, and hypotension due to release of a substance named cucurbitacin. OBJECTIVE: Our aim was to increase physician awareness of cucurbitacin poisoning in order to facilitate accurate diagnosis and appropriate management. CASE REPORT: Five adult patients presented with nausea, vomiting, and diarrhea within 5 to 25 min of ingesting cooked bitter bottle gourd. One patient developed severe diarrhea, hematemesis, and hypotension requiring hospitalization. All patients improved within a few days with intravenous fluids and proton pump inhibitors. To our knowledge, this is the first reported group of patients with toxicity due to ingestion of bottle gourd in the United States (US). CONCLUSIONS: Physicians should be suspicious of cucurbitacin toxicity in patients who present with symptoms within minutes of ingestion of a plant in the Cucurbitaceae family. Patients should be asked if the plant tasted unusually bitter. The most common symptoms include diarrhea and hematemesis. More than half of patients develop hypotension. There is no known antidote for bottle gourd poisoning; treatment is supportive. Proton pump inhibitors should be given to patients with gastrointestinal mucosal injury.

Polo-like kinase is required for Golgi and bilobe biogenesis in Trypanosoma brucei.

A bilobed structure marked by TbCentrin2 regulates Golgi duplication in the protozoan parasite Trypanosoma brucei. This structure must itself duplicate during the cell cycle for Golgi inheritance to proceed normally. We show here that duplication of the bilobed structure is dependent on the single polo-like kinase (PLK) homologue in T. brucei (TbPLK). Depletion of TbPLK leads to malformed bilobed structures, which is consistent with an inhibition of duplication and an increase in the number of dispersed Golgi structures with associated endoplasmic reticulum exit sites. These data suggest that the bilobe may act as a scaffold for the controlled assembly of the duplicating Golgi.

Laurate Biosensors Image Brain Neurotransmitters In Vivo: Can an Antihypertensive Medication Alter Psychostimulant Behavior?

Neuromolecular Imaging (NMI) with novel biosensors enables the selective detection of neurotransmitters in vivo within seconds, on line and in real time. Biosensors remain in place for continuing studies over a period of months. This biotechnological advance is based on conventional electrochemistry; the biosensors detect neurotransmitters by electron transfer. Simply stated, biosensors adsorb electrons from each neurotransmitter at specific oxidation potentials; the current derived from electron transfer is proportional to neurotransmitter concentration. Selective electron transfer properties of these biosensors permit the imaging of neurotransmitters, metabolites and precursors. The novel BRODERICK PROBE® biosensors we have developed, differ in formulation and detection capabilities from biosensors/electrodes used in conventional electrochemistry/ voltammetry. In these studies, NMI, specifically, the BRODERICK PROBE® laurate biosensor images neurotransmitter signals within mesolimbic neuronal terminals, nucleus accumbens (NAc); dopamine (DA), serotonin (5-HT), homovanillic acid (HVA) and Ltryptophan (L-TP) are selectively imaged. Simultaneously, we use infrared photobeams to monitor open-field movement behaviors on line with NMI in the same animal subjects. The goals are to investigate integrated neurochemical and behavioral effects of cocaine and caffeine alone and co-administered and further, to use ketanserin to decipher receptor profiles for these psychostimulants, alone and co-administered. The rationale for selecting this medication is: ketanserin (a) is an antihypertensive and cocaine and caffeine produce hypertension and (b) acts at 5-HT2A/2C receptors, prevalent in NAc and implicated in hypertension and cocaine addiction. Key findings are: (a) the moderate dose of caffeine simultaneously potentiates cocaine's neurochemical and behavioral responses. (b) ketanserin simultaneously inhibits cocaine-increased DA and 5-HT release in NAc and open-field behaviors and (c) ketanserin inhibits 5-HT release in NAc and open-field behaviors produced by caffeine, but, surprisingly, acts to increase DA release in NAc. Importantly, the latter effect may be a possible adverse effect of the moderate dose of caffeine in hypertensive patients. Thus, an antihypertensive medication is shown here to play a role in inhibiting brain reward possibly via antihypertensive mechanisms at DA and 5-HT receptor subtypes within DA motor neurons. An explanatory note for the results obtained, is the role likely played by the G Protein Receptor Complex (GPRC) family of proteins. Empirical evidence shows that GPRC dimers, heteromers and heterotrimers may cause cross-talk between distinct signalling cascade pathways in the actions of cocaine and caffeine. Ligand-directed functional selectivity, particularly for ketanserin, in addition to GPRCs, may also cause differential responses. The results promise new therapeutic strategies for drug addiction, brain reward and cardiovascular medicine.

Safety belt use by law enforcement officers on reality television: a missed opportunity for injury prevention?

BACKGROUND: Although safety belt usage rates are increasing nationwide, motor vehicle crashes (MVCs) remain a leading cause of death for young people and are emerging as a leading cause for police officers specifically. A content analysis was performed on the television show, COPS, to determine on-air safety belt usage rates. METHODS: A sample of 63 unique episodes of the reality-based television series, COPS, was viewed during a 4-month period (September 1, 2005 to January 1, 2006). Episodes had original airing dates ranging from 1990 to 2004. Safety belt usage status was determined per police officer per driving scene (N = 250). A driving scene represented a continuous trip (start to finish) with a total on-camera time exceeding 5 seconds. Scenes with indeterminate safety belt status were excluded. High-speed driving, officer gender, and officer race were also recorded. RESULTS: Of the 203 scenes included, 77 (38%) demonstrated safety belt usage. High-speed driving scenes had higher safety belt usage rates compared with low-speed (48% versus 29%, p = 0.005). More contemporary episodes (1999 to 2004) had higher safety belt usage rates as well (51% versus 28%, p = 0.001). Officer gender and race revealed no significant differences in safety belt usage rates (p = 0.930 and p = 0.900, respectively). CONCLUSIONS: In this popular, reality-based television series, safety belt usage by police officers is extremely low. These findings suggest the need to increase safety belt usage by police officers, especially those filmed for television.

Suspected DLIS interference in the dimension DGNA digoxin assay method and the clinical application of the revised digoxin target range.

The authors present a case of an elderly female patient with heart failure and renal dysfunction treated with digoxin, where 2 commercial immunoassay methods (DRI, Microgenics, and DGNA, Dade Behring) showed a clinically very significant discrepancy on the same plasma sample, viz. 0.5 and 2.3 nmol/L, respectively. The sample was also referred to a third external laboratory that returned a result of 0.9 nmol/L using mFPIA (AxSYM, Abbott). Subsequent ultrafiltration (30,000 Dalton) on the sample essentially eliminated the difference, suggesting an interference from a large molecular weight compound(s), potentially the well-described digoxin-like immunoreactive substance(s) (DLIS). Although further study is required to verify that the DLIS implicated was indeed the interfering species, it does again highlight the importance of careful method selection in the clinical therapeutic drug monitoring laboratory to ensure that such well-established potential problems do not result in inappropriate dosage reduction with consequent lack of adequate drug exposure and serious clinical sequelae.

Ordered assembly of the duplicating Golgi in Trypanosoma brucei.

The new Golgi in the protozoan parasite Trypanosoma brucei grows near to the old and adjacent to the growing new endoplasmic reticulum exit site. Growth is now shown to be at least a two-stage process, in which a representative matrix marker (GRASP) and enzyme (GntB) are delivered to the site of assembly, followed approximately 10 min later by a COPI component (epsilon-COP) and a trans-Golgi network (TGN) marker (GRIP70). A secretory cargo marker (signal sequence-YFP) appeared early near the new endoplasmic reticulum exit site but did not enter the Golgi until the second stage. Together these data suggest that structural and enzymatic components of the new Golgi stack are laid down first, followed by those needed to move and sort the cargo passing through it.

Golgi duplication in Trypanosoma brucei.

Duplication of the single Golgi apparatus in the protozoan parasite Trypanosoma brucei has been followed by tagging a putative Golgi enzyme and a matrix protein with variants of GFP. Video microscopy shows that the new Golgi appears de novo, near to the old Golgi, about two hours into the cell cycle and grows over a two-hour period until it is the same size as the old Golgi. Duplication of the endoplasmic reticulum (ER) export site follows exactly the same time course. Photobleaching experiments show that the new Golgi is not the exclusive product of the new ER export site. Rather, it is supplied, at least in part, by material directly from the old Golgi. Pharmacological experiments show that the site of the new Golgi and ER export is determined by the location of the new basal body.

Is the light chain subcellular localization an important factor in botulinum toxin duration of action?

Botulinum neurotoxins (BoNT) are therapeutic proteins that are specific, potent, and effective. They are highly specific in binding to motor neurons but do not bind to other non-neuronal cells. These proteins are zinc-dependent endopeptidases that inhibit exocytosis by specific cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor) proteins involved in vesicle docking and fusion. The therapeutic effect of BoNT/A in humans lasts from 3 to 12 months, depending upon the condition treated. Data from animal and cell culture models suggests that the long-lasting duration of inhibition of neurotransmitter release induced by BoNT/A maybe due to the persistence of the endopeptidase activity of the light chain (LC/A) in cells, interactions of the cleaved substrates, and/or the response of the nerve to the temporary disruption of communication with its target tissue. We have analyzed the subcellular localization of the light chains from serotypes A, B, and E and have demonstrated that each light chain displays a distinct distribution within cells. LC/A localizes at the plasma membrane, LC/B is dispersed throughout the cell including the nucleus, and LC/E is mainly cytosolic. Localization is similar in non-neuronal cell lines, suggesting that the signals involved in proper subcellular localization are within the LC sequences and the moiety the light chain interacts with is present in both neuronal and non-neuronal cells.

Plasma membrane localization signals in the light chain of botulinum neurotoxin.

Botulinum neurotoxin (BoNT) is a potent biological substance used to treat neuromuscular and pain disorders. Both BoNT type A and BoNT type E display high-affinity uptake into motor neurons and inhibit exocytosis through cleavage of the synaptosome-associated protein of 25 kDa (SNAP25). The therapeutic effects of BoNT/A last from 3 to 12 months, whereas the effects of BoNT/E last less than 4 weeks. Using confocal microscopy and site-specific mutagenesis, we have determined that the protease domain of BoNT/A light chain (BoNT/A-LC) localizes in a punctate manner to the plasma membrane, colocalizing with the cleaved product, SNAP25(197). In contrast, the short-duration BoNT/E serotype is cytoplasmic. Mutations in the BoNT/A-LC have revealed sequences at the N terminus necessary for plasma membrane localization, and an active dileucine motif in the C terminus that is likely involved in trafficking and interaction with adaptor proteins. These data support sequence-specific signals as determinants of intracellular localization and as a basis for the different durations of action in these two BoNT serotypes.