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Journal of Visualized Experiments : JoVE Sep 2023Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites,...
Solute carriers (SLCs) are membrane transporters that import and export a range of endogenous and exogenous substrates, including ions, nutrients, metabolites, neurotransmitters, and pharmaceuticals. Despite having emerged as attractive therapeutic targets and markers of disease, this group of proteins is still relatively underdrugged by current pharmaceuticals. Drug discovery projects for these transporters are impeded by limited structural, functional, and physiological knowledge, ultimately due to the difficulties in the expression and purification of this class of membrane-embedded proteins. Here, we demonstrate methods to obtain high-purity, milligram quantities of human SLC transporter proteins using codon-optimized gene sequences. In conjunction with a systematic exploration of construct design and high-throughput expression, these protocols ensure the preservation of the structural integrity and biochemical activity of the target proteins. We also highlight critical steps in the eukaryotic cell expression, affinity purification, and size-exclusion chromatography of these proteins. Ultimately, this workflow yields pure, functionally active, and stable protein preparations suitable for high-resolution structure determination, transport studies, small-molecule engagement assays, and high-throughput in vitro screening.
Topics: Humans; Membrane Transport Proteins; Solute Carrier Proteins; Drug Discovery; High-Throughput Screening Assays; Membrane Proteins; Pharmaceutical Preparations
PubMed: 37843272
DOI: 10.3791/65878 -
The American Journal of Drug and... Nov 2023There is currently no format-independent method to determine delta-9-tetrahydrocannabinol (THC) in milligrams for self-report studies. Validate self-report method for...
There is currently no format-independent method to determine delta-9-tetrahydrocannabinol (THC) in milligrams for self-report studies. Validate self-report method for quantifying mg THC from commercially available cannabis products using product labeling, which includes both net weight and product potency. 53 adult cannabis users (24 M, 29F), 21-39 years of age ( = 28.38, SD = 4.15), were instructed to report daily use via a weekly survey for two consecutive weeks, provide product label photographs, abstain from use for 24 h, submit a urine sample and complete the Cannabis Use Disorder Identification Test - Revised (CUDIT-R) and the Marijuana Craving Questionnaire - Short Form (MCQ-SF). Milligrams of THC were determined by multiplying quantity of product used by its THC concentration. Urine was analyzed for the urine metabolite 11-nor-carboxy-THC (THC-COOH) via liquid chromatography mass spectroscopy. THC and THC-COOH values were log10 transformed prior to correlational analyses. Median daily THC consumption was 102.53 mg ( = 203.68, SD = 268.13). Thirty-three (62%) of the 53 participants reported using two or more formats over the 2-week period. There was a significant positive correlation between log10 THC-COOH and log10 THC mg (r(41) = .59, < .001), log10 THC mg and MCQ-SF score (r(41) = .59, < .001), and log10 THC mg dose and CUDIT-R score, (r(41) = .39, = .010). Our label-based methodology provides consumption information across all modalities of cannabis use in standard units that can be combined across products for calculation of dose. It is a viable and valid method for quantifying mg of THC consumed and can be utilized in any region where cannabis is legal, and labeling is regulated.
Topics: Adult; Humans; Cannabis; Dronabinol; Self Report; Gas Chromatography-Mass Spectrometry; Hallucinogens; Cannabinoid Receptor Agonists; Substance Abuse Detection
PubMed: 37506343
DOI: 10.1080/00952990.2023.2232525 -
MMWR. Morbidity and Mortality Weekly... Feb 2024In 2021, an 8-mg intranasal naloxone product was approved by the Food and Drug Administration; however, no studies have examined outcomes among persons who receive the... (Comparative Study)
Comparative Study
Comparison of Administration of 8-Milligram and 4-Milligram Intranasal Naloxone by Law Enforcement During Response to Suspected Opioid Overdose - New York, March 2022-August 2023.
In 2021, an 8-mg intranasal naloxone product was approved by the Food and Drug Administration; however, no studies have examined outcomes among persons who receive the 8-mg naloxone product and those who receive the usual 4-mg product. During March 2022-August 2023, New York State Department of Health (NYSDOH) supplied some New York State Police (NYSP) troops with 8-mg intranasal naloxone; other troops continued to receive 4-mg intranasal naloxone to treat suspected opioid overdose. NYSP submitted detailed reports to NYSDOH when naloxone was administered. No significant differences were observed in survival, mean number of naloxone doses administered, prevalence of most postnaloxone signs and symptoms, postnaloxone anger or combativeness, or hospital transport refusal among 4-mg and 8-mg intranasal naloxone recipients; however, persons who received the 8-mg intranasal naloxone product had 2.51 times the risk for opioid withdrawal signs and symptoms, including vomiting, than did those who received the 4-mg intranasal naloxone product (95% CI = 1.51-4.18). This initial study suggests no benefits to law enforcement administration of higher-dose naloxone were identified; more research is needed to guide public health agencies in considering whether 8-mg intranasal naloxone confers additional benefits for community organizations.
Topics: Humans; Analgesics, Opioid; Drug Overdose; Law Enforcement; Naloxone; Narcotic Antagonists; New York; Opiate Overdose
PubMed: 38329911
DOI: 10.15585/mmwr.mm7305a4 -
Journal of Ovarian Research Jan 2024Women have polycystic ovarian syndrome (PCOS) at higher rates than any other endocrine condition with an average incidence rate of 6 to 8%. Acne is an immune mediate... (Clinical Trial)
Clinical Trial
BACKGROUND
Women have polycystic ovarian syndrome (PCOS) at higher rates than any other endocrine condition with an average incidence rate of 6 to 8%. Acne is an immune mediate common condition frequently affecting adolescents and adults and is often associated with PCOS. The objective of the study was to assess the impact of oral isotretinoin on ovarian functions of acne patients suffering from PCOS. Forty women with a clinical diagnosis of acne as well as PCOS participated in this prospective clinical trial. Participants were given oral doses of isotretinoin ranging from 0.5 to 1 milligram per kilogram (mg/kg), for a total of 120 to 150 mg/kg. To establish baseline values of hormone levels, on days 2-5 of the menstrual cycle, venous blood samples were obtained. Moreover, global acne grading system (GAGS), follicle count, and bilateral ovarian volumes were evaluated both before and after isotretinoin treatment.
RESULTS
A significant reduction in global acne score from pre-treatment levels to post-treatment levels was observed (11.58 ± 5.857 vs. 1.65 ± 1.369). Ovarian volume was significantly reduced from 10.26 ± 1.539 before treatment to 8.74 ± 1.436 after treatment on the right side (P < 0.001) and from 11.08 ± 1.564 before treatment to 9.36 ± 1.479 after treatment on the left side (P < 0.001). A significant reduction in free testosterone level and hirsutism scores were observed after treatment (P < 0.001; P < 0.01 respectively.
CONCLUSION
Isotretinoin may exert beneficial effects in hyperandrogenic women with PCOS and needs to be further evaluated by large multicentre controlled trials.
Topics: Adult; Adolescent; Humans; Female; Polycystic Ovary Syndrome; Isotretinoin; Prospective Studies; Acne Vulgaris
PubMed: 38245761
DOI: 10.1186/s13048-024-01347-x -
The American Surgeon Dec 2023The opioid overdose epidemic remains one of the leading focuses of the United States' public health agenda. Current literature has suggested that many surgical...
The opioid overdose epidemic remains one of the leading focuses of the United States' public health agenda. Current literature has suggested that many surgical procedures are associated with an increased risk of chronic opioid use in the post-operative period of opioid-naïve patients. We aimed to assess whether providing feedback on the average morphine milligram equivalents (MMED) and opioid utilization by selected post-operative patients would impact the provider opioid prescribing patterns. An opioid stewardship educational intervention provided didactic and email feedback to general surgeons about their prescribing patterns and summary feedback on opioid usage among post-operative patients from the pre-intervention period. We used descriptive statistics, Chi Square, Fisher's Exact test, Wilcoxon Rank Sum, two sample t test, and Spearman's rho to analyze the data gathered. A total of 5142 patients with an average age of 43.9 years were included in the study period. Women accounted for 3096 (60.2%) and 2046 (39.8%) were men. The surgeries during the study period included 1928 (37.5%) appendectomies and 3214 (62.5%) cholecystectomies. The predominant surgical approach was laparoscopic 5028 (97.8%). In both groups, the total MMED and total number of pills prescribed decreased significantly after the intervention was implemented. There were no refill prescriptions nor 30-day readmissions among those discharged with an opioid prescription in either study phase. An intervention that provided general surgeons with feedback about their post-operative prescription patterns and data on post-operative opioid utilization by patients decreased prescribed MMED.
Topics: Male; Humans; Female; Adult; Analgesics, Opioid; Feedback; Pain, Postoperative; Practice Patterns, Physicians'; Opioid-Related Disorders
PubMed: 36418926
DOI: 10.1177/00031348221129511 -
Diseases of the Colon and Rectum Aug 2023In the United States, 37% of all opioids are prescribed in the surgical setting, many of which report initial exposure in the postoperative period.
BACKGROUND
In the United States, 37% of all opioids are prescribed in the surgical setting, many of which report initial exposure in the postoperative period.
OBJECTIVE
This study aimed to assess the impact of a narcotic-sparing enhanced recovery after surgery protocol on postoperative narcotic use by patients and to assess its impact on the narcotic-prescribing practices of physicians.
DESIGN
Data regarding consecutive narcotic-naïve patients who underwent a surgical procedure from January 2013 to August 2017 were retrospectively reviewed.
SETTINGS
Patients were divided into 2 cohorts: preimplementation (2013-2015) and postimplementation (2015-2017) of the enhanced recovery after surgery protocol.
PATIENTS
This study included patients who underwent elective inpatient abdominal colorectal surgery at the University of Florida Health.
MAIN OUTCOME MEASURES
The primary outcome measure was 30-day postoperative narcotic use (inpatient and outpatient). Other outcomes measured included pain scores, time to diet institution, length of hospital stay, cost of hospitalization, and postoperative complications.
RESULTS
Baseline characteristics were similar between the preprotocol group (n = 537) and postprotocol group (n = 790). Protocol implementation was associated with a decrease in the total 30-day postoperative narcotic amount used by patients (2481 vs 31 morphine milligram equivalents; p = 0.05), inpatient patient-controlled analgesia use (63% vs 0.5%; p < 0.00001; dosage 1254 vs 5 morphine milligram equivalents), inpatient on-demand oral narcotic use (90% vs 32%; p = 0.001; dosage 47 vs 5 morphine milligram equivalents), and outpatient narcotic amount used (46 vs 6 morphine milligram equivalents; p = 0.001). Average pain scores were similar.
LIMITATIONS
Retrospective nature of the study and possible underestimation of pre- and postoperative narcotic use.
CONCLUSIONS
Implementation of a narcotic-sparing enhanced recovery after surgery protocol was associated with a decrease in both inpatient and 30-day outpatient postoperative narcotic use. Variation in resident physician prescribing practices suggests the need for ongoing education to accompany these protocols. See Video Abstract at http://links.lww.com/DCR/B936 .
EL IMPACTO DE UN PROTOCOLO DE RECUPERACIN MEJORADO CON AHORRO DE NARCTICOS EN EL USO POSTOPERATORIO DE NARCTICOS DESPUS DE UNA COLECTOMA
ANTECEDENTES:En los Estados Unidos, el 37 % de todos los opioides se prescriben en el entorno quirúrgico. Entre los adictos a los narcóticos, muchos reportan una exposición inicial en el período posoperatorio.OBJETIVO:Nuestro objetivo fue evaluar el impacto de un protocolo de recuperación mejorada después de la cirugía que ahorra narcóticos en el uso de narcóticos postoperatorios por parte de los pacientes y evaluar su impacto en las prácticas de prescripción de narcóticos de los médicos.DISEÑO:Se revisaron retrospectivamente los datos de pacientes consecutivos sin tratamiento previo con narcóticos que se sometieron a un procedimiento quirúrgico colorrectal abdominal electivo para pacientes hospitalizados desde enero de 2013 hasta agosto de 2017.AJUSTE:Los pacientes se dividieron en 2 cohortes: antes de la implementación (2013-2015) y después de la implementación (2015-2017) del protocolo de recuperación mejorada después de la cirugía.PACIENTES:Pacientes de cirugía colorrectal abdominal electiva para pacientes internados en University of Florida Health.MEDIDAS DE RESULTADO PRINCIPALES:La medida de resultado primaria fue el uso de narcóticos postoperatorios de 30 días (pacientes hospitalizados y ambulatorios). Otros resultados medidos incluyeron puntuaciones de dolor, tiempo hasta la institución de la dieta, duración de la estancia hospitalaria, costo de la hospitalización y complicaciones postoperatorias.RESULTADOS:Las características iniciales fueron similares entre los grupos antes (n = 537) y después del protocolo (n = 790). La implementación del protocolo se asoció con una disminución en la cantidad total de narcóticos postoperatorios de 30 días utilizada por los pacientes (2481 mg frente a 31 mg de equivalentes de morfina, p = 0,05), uso de analgesia controlada por pacientes hospitalizados (63 % frente a 0,5 %, p < 0,00001; dosis 1254 mg frente a 5 mg), uso de narcóticos orales a demanda en pacientes hospitalizados (90 % frente a 32 %, p = 0,001; dosis de 47 mg frente a 5 mg) y cantidad de narcóticos utilizados en pacientes ambulatorios (46 mg frente a 6 mg, p = 0,001). Las puntuaciones medias de dolor fueron similares.LIMITACIONES:La naturaleza retrospectiva del estudio y la posible sub estimación del uso de narcóticos antes y después de la operación fueron limitaciones de los hallazgos del estudio.CONCLUSIÓN:La implementación de un protocolo de recuperación mejorada después de la cirugía que ahorra narcóticos se asoció con una disminución en el uso de narcóticos en el postoperatorio de pacientes hospitalizados y ambulatorios de 30 días. La variación en las prácticas de prescripción de los médicos residentes sugiere la necesidad de una educación continua que acompañe a estos protocolos. Consulte Video Resumen en http://links.lww.com/DCR/B936 . (Traducción-Dr. Mauricio Santamaria ).
Topics: Humans; United States; Retrospective Studies; Narcotics; Opioid-Related Disorders; Postoperative Complications; Colectomy; Postoperative Period; Pain; Morphine
PubMed: 35316244
DOI: 10.1097/DCR.0000000000002292 -
The American Surgeon May 2024There is evidence that providers often overprescribe opiates in the postoperative period. Despite an ever-growing geriatric population, there is little research...
There is evidence that providers often overprescribe opiates in the postoperative period. Despite an ever-growing geriatric population, there is little research detailing current opiate usage in older patients after trauma. This population presents a unique set of challenges for pain management and prescription drug dependence due to sensitivity, a narrow therapeutic window, and high rates of pre-existing polypharmacy. Assess the use of narcotics in geriatric trauma patients with various injury patterns to establish a reference point for future intervention for reduction in narcotic dependence. We created a database of trauma patients' age ≥65 years admitted to a single level 1 trauma center in the Southeastern United States during the 2019 calendar year. Information gathered included patient factors, injury patterns, operative intervention, pain medications prescribed during hospitalization and at discharge, total and average daily morphine milligram equivalents (MME) inpatient and outpatient, and requests/prescriptions for narcotics at follow-up. In 2019, there were 475 patients aged ≥65 admitted to our level 1 trauma center for acute traumatic injuries. 219 of those patients required operative intervention. Average total inpatient MME for this cohort was 169.0 with average daily MME of 22.89. The average total prescribed MME upon discharge was 79.27. There were 29 patients documented to request narcotic prescription refill at time of clinic follow-up, 27 of whom were prescribed a narcotic medication at follow-up. This dataset establishes a reference point for opiate use in geriatric trauma patients to facilitate further research for mitigation of risk in this population.
PubMed: 38782463
DOI: 10.1177/00031348241256063 -
Orthopedics 2023The aim of this quality improvement initiative was to reduce unnecessary opioid prescribing by sharing data with prescribers on opioid use by patients. In our study,...
The aim of this quality improvement initiative was to reduce unnecessary opioid prescribing by sharing data with prescribers on opioid use by patients. In our study, transition of care clinicians performed follow-up phone calls to select postoperative orthopedic patients to determine opioid use. We implemented a standardized postoperative 7-day opioid wean and designed a dashboard to track the information gathered. We calculated descriptive statistics for continuous and categorical variables. In the initial assessment of opioid use by orthopedic patients, the study consisted of 296 patients with a mean age of 64.8±11.4 years, 147 females (49.7%) and 149 males (50.3%), 59.1% joint replacements (hip, knee, shoulder), and 40.9% spine surgeries (lumbar decompression, cervical fusion, hemilaminectomy). Among those prescribed an opioid, 50% received a prescription for 30 pills or less and 52.4% reported taking more than 80% of the opioid pills, while 35.1% reported taking less than 60%. In the prescribing quality improvement assessment, there were a total of 1547 hospitalizations for joint replacement surgeries from June 2018 to June 2020: 774 (50.0%) hips and 773 (50.0%) knees. There was a significant difference in morphine milligram equivalents per day and quantity prescribed when comparing the preintervention period with the postintervention period without significant increases in opioid refill requests or return visit rates. In our study, sharing data around patient opioid use and provider-facing prescribing metrics reduced postoperative opioid prescribing without significantly increasing opioid refill or emergency department return visit rates. [. 2023;46(4):e230-e236.].
Topics: Male; Female; Humans; Middle Aged; Aged; Analgesics, Opioid; Pain, Postoperative; Quality Improvement; Practice Patterns, Physicians'; Arthroplasty, Replacement; Retrospective Studies
PubMed: 36779731
DOI: 10.3928/01477447-20230207-05 -
Journal of Experimental Pharmacology 2024Wiens and Polhill (Loranthaceae) is a parasitic plant native to north eastern Africa and Ethiopia. In Ethiopia, it is traditionally used to treat breast swelling,...
BACKGROUND
Wiens and Polhill (Loranthaceae) is a parasitic plant native to north eastern Africa and Ethiopia. In Ethiopia, it is traditionally used to treat breast swelling, mastitis, morning illnesses and vomiting.
OBJECTIVE
This study aimed to screen the main phytochemical constituents; determine the total amounts of phenolics, flavonoids, and tannins; and evaluate the antimicrobial (against and ) and antioxidant (against DPPH radical and ferric ion) activities of leaves extracts.
METHODS
Powdered leaves were macerated using -hexane, chloroform, ethyl acetate, ethanol, and methanol. All crude extracts were qualitatively screened for phytochemical identification. The total phenolic, flavonoid, and condensed tannin contents of the chloroform, ethanol, and methanol extracts were determined by UV-Vis spectrophotometry. The -hexane, chloroform, and methanol extracts were evaluated for their antimicrobial activity against the aforementioned microbes using agar disc diffusion and broth micro-dilution techniques. Chloroform, ethanol, and methanol extracts were also evaluated for antioxidant activity by DPPH and ferric ion reduction antioxidant power (FRAP) assays.
RESULTS
Methanol (17.56 ± 16%) and ethanol (16.45 ± 19%) showed better extraction efficiency. Flavonoids, polyphenols, tannins, terpenoids, saponins, and sterols were detected in all extracts. The highest total content of phenolics (22.63 ± 0.69 mgGAE/gDCE), flavonoids (5.38 ± 0.52 mgCE/gDCE) and tannins (39.18 ± 38 mg CE/g DCE), as milligram of gallic acid and catechin per gram of dried crude extract, were recorded in the methanolic extract. The methanolic extract also presented best anti -DPPH strength (IC, 4.31 μg/mL) and ferric ion reduction power (absorbance of 0.71) though found weak compared to the ascorbic acid (IC of 0.49 μg/mL and absorbance of 0.93, respectively).
CONCLUSION
All evaluated extracts displayed antifungal activity against both and strains (minimum inhibitory concentration values of 12.5-25 mg/mL), whereas they were found to have negligible activity against all tested bacterial strains. This report provides preliminary information for further phytochemical investigation of to isolate potential antioxidant and antifungal compounds.
PubMed: 38371428
DOI: 10.2147/JEP.S452098 -
The Journal of Biological Chemistry Feb 2024Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers,...
Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers, and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at a quantity, stability, and purity that meets the demand of the planned experiment. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favoring inhibitor factor 1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the glyco-diosgenin to lauryl maltose neopentyl glycol ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.
Topics: Animals; Cattle; Adenosine Triphosphate; Dimerization; Mitochondria, Heart; Mitochondrial Membranes; Mitochondrial Proton-Translocating ATPases; Centrifugation, Density Gradient
PubMed: 38159856
DOI: 10.1016/j.jbc.2023.105603