Ratiometric gibberellin biosensors for the analysis of signaling dynamics and metabolism in plant protoplasts.

Gibberellins (GAs) are major regulators of developmental and growth processes in plants. Using the degradation-based signaling mechanism of GAs, we have built transcriptional regulator (DELLA)-based, genetically encoded ratiometric biosensors as proxies for hormone quantification at high temporal resolution and sensitivity that allow dynamic, rapid and simple analysis in a plant cell system, i.e. Arabidopsis protoplasts. These ratiometric biosensors incorporate a DELLA protein as a degradation target fused to a firefly luciferase connected via a 2A peptide to a renilla luciferase as a co-expressed normalization element. We have implemented these biosensors for all five Arabidopsis DELLA proteins, GA-INSENSITIVE, GAI; REPRESSOR-of-ga1-3, RGA; RGA-like1, RGL1; RGL2 and RGL3, by applying a modular design. The sensors are highly sensitive (in the low pm range), specific and dynamic. As a proof of concept, we have tested the applicability in three domains: the study of substrate specificity and activity of putative GA-oxidases, the characterization of GA transporters, and the use as a discrimination platform coupled to a GA agonists' chemical screening. This work demonstrates the development of a genetically encoded quantitative biosensor complementary to existing tools that allow the visualization of GA in planta.

bHLH heterodimer complex variations regulate cell proliferation activity in the meristems of Arabidopsis thaliana.

Root, shoot, and lateral meristems are the main regions of cell proliferation in plants. It has been proposed that meristems might have evolved dedicated transcriptional networks to balance cell proliferation. Here, we show that basic helix-loop-helix (bHLH) transcription factor heterodimers formed by members of the TARGET OF MONOPTEROS5 (TMO5) and LONESOME HIGHWAY (LHW) subclades are general regulators of cell proliferation in all meristems. Yet, genetics and expression analyses suggest specific functions of these transcription factors in distinct meristems, possibly due to their expression domains determining heterodimer complex variations within meristems, and to a certain extent to the absence of some of them in a given meristem. Target gene specificity analysis for heterodimer complexes focusing on the LONELY GUY gene targets further suggests differences in transcriptional responses through heterodimer diversification that could allow a common bHLH heterodimer complex module to contribute to cell proliferation control in multiple meristems.

Genetically Encoded Biosensors for the Quantitative Analysis of Auxin Dynamics in Plant Cells.

Plants, as sessile organisms, possess complex and intertwined signaling networks to react and adapt their behavior toward different internal and external stimuli. Due to this high level of complexity, the implementation of quantitative molecular tools in planta remains challenging. Synthetic biology as an ever-growing interdisciplinary field applies basic engineering principles in life sciences. A plethora of synthetic switches, circuits, and even higher order networks has been implemented in different organisms, such as bacteria and mammalian cells, and facilitates the study of signaling and metabolic pathways. However, the application of such tools in plants lags behind, and thus only a few genetically encoded biosensors and switches have been engineered toward the quantitative investigation of plant signaling. Here, we present a protocol for the quantitative analysis of auxin signaling in Arabidopsis thaliana protoplasts. We implemented genetically encoded, ratiometric, degradation-based luminescent biosensors and applied them for studying auxin perception dynamics. For this, we utilized three different Aux/IAAs as sensor modules and analyzed their degradation behavior in response to auxin. Our experimental approach requires simple hardware and experimental reagents and can thus be implemented in every plant-related or cell culture laboratory. The system allows for the analysis of auxin perception and signaling aspects on various levels and can be easily expanded to other hormones, as for example strigolactones. In addition, the modular sensor design enables the implementation of sensor modules in a straightforward and time-saving approach.

Interdisciplinary patient-centred poststroke care in Follow-up After Stroke, Screening and Treatment (FASST) clinic model: a proof-of-concept pilot study.

Background: Although secondary stroke prevention is important, the optimal outpatient model that improves risk factor control and decreases post-stroke complications effectively has not been established. We created Follow-up After Stroke, Screening and Treatment (FASST), an interdisciplinary clinic involving stroke physicians and pharmacists to address poststroke complications and secondary stroke prevention systemically. We present our approach to assess its proof-of-concept in our pilot study. Methods: We included the patients attending FASST clinic after their hospital discharge. We used validated survey screens to assess for complications: depression, anxiety, sleep disorders, cognitive impairment, disability, social support, quality of life and functional status. Data were collected including risk factors, complication screening results and outcome scores. Clinical pharmacists assessed risk factor control and health-related behaviours for modification. Results: Of the 25 patients enrolled in the interdisciplinary clinic, all had comorbid hyperlipidaemia and hypertension, and 44% had diabetes mellitus. About one-third needed medication changes for risk factor control. On screening, 16% of patients were found to have depression, 12% had anxiety and 20% had sleep apnoea. These patients were either managed in the clinic or were referred to relevant subspeciality clinics. The status of risk factor control was assessed in all patients, and 32% had medications adjustments. Conclusion: Our preliminary data found that FASST clinic model is feasible and potentially useful. It represents an integrated approach to post-stroke care, with pharmacist collaboration to improve risk factor control, while assessing for poststroke complications. Further study is needed to improve health outcomes through integrated poststroke care.

Phase II, Double-Blind, Vehicle-Controlled Study to Determine the Cantharidin Dose Regimen, Efficacy, Safety, and Tolerability of VP-102 in Subjects with External Genital Warts.

BACKGROUND: External genital warts are caused by various subtypes of the human papilloma virus and spread through direct skin-to-skin contact. Approximately 1% of the US population have external genital warts. Although cantharidin has been used to treat external genital warts for decades, there are no US Food and Drug Administration-approved cantharidin products and no reliable or controlled sources of cantharidin available. VP-102 is a drug-device combination product containing cantharidin (0.7% w/v) in a single-use shelf-stable applicator. OBJECTIVE: The objective of this randomized, double-blind, vehicle-controlled, phase II clinical trial was to determine the optimal regimen for the treatment, safety, and efficacy of VP-102 in external genital warts. METHODS: The study was conducted in two parts. Part A was dose finding and Part B was performed following the completion of Part A for a safety and efficacy evaluation. Following completion of Part A, 6-h and 24-h VP-102 regimens under occlusion were selected to be evaluated in Part B. RESULTS: Pooled results from Part B and Part A of the 6-h and 24-h VP-102 treatment regimens showed that 36.7% and 33.3% of participants achieved complete clearance of all treatable external genital warts at the end of treatment vs 4.2% (p < 0.0048) and 0% (p < 0.0075) with the vehicle. Adverse events experienced by the VP-102-treated participants were consistent with the pharmacodynamic action of cantharidin as a vesicant and were primarily mild to moderate in severity. The most common adverse events included application-site vesicles, pain, and erythema. No participants discontinued the study because of adverse events and no serious adverse events were deemed treatment related. CONCLUSIONS: The adverse event profile and efficacy of VP-102 under occlusion demonstrated in this study support the conclusion that a 6-h or up to 24-h exposure regimen represents an acceptable risk:benefit profile and justifies the conduct of a larger vehicle-controlled phase III study in external genital warts. CLINICAL TRIAL REGISTRATION: NCT03981822, actual study start date: 25 June, 2019; actual primary completion date: 21 May, 2020; actual study completion date: 8 July, 2020.

Blue Light-Operated CRISPR/Cas13b-Mediated mRNA Knockdown (Lockdown).

The introduction of optogenetics into cell biology has furnished systems to control gene expression at the transcriptional and protein stability level, with a high degree of spatial, temporal, and dynamic light-regulation capabilities. Strategies to downregulate RNA currently rely on RNA interference and CRISPR/Cas-related methods. However, these approaches lack the key characteristics and advantages provided by optical control. "Lockdown" introduces optical control of RNA levels utilizing a blue light-dependent switch to induce expression of CRISPR/Cas13b, which mediates sequence-specific mRNA knockdown. Combining Lockdown with optogenetic tools to repress gene-expression and induce protein destabilization with blue light yields efficient triple-controlled downregulation of target proteins. Implementing Lockdown to degrade endogenous mRNA levels of the cyclin-dependent kinase 1 (hCdk1) leads to blue light-induced G2/M cell cycle arrest and inhibition of cell growth in mammalian cells.

A Protoplast-Based Bioassay to Quantify Strigolactone Activity in Arabidopsis Using StrigoQuant.

Understanding the biological background of strigolactone (SL) structural diversity and the SL signaling pathway at molecular level requires quantitative and sensitive tools that precisely determine SL dynamics. Such biosensors may be also very helpful in screening for SL analogs and mimics with defined biological functions.Recently, the genetically encoded, ratiometric sensor StrigoQuant was developed and allowed the quantification of the activity of a wide concentration range of SLs. StrigoQuant can be used for studies on the biosynthesis, function and signal transduction of this hormone class.Here, we provide a comprehensive protocol for establishing the use of StrigoQuant in Arabidopsis protoplasts. We first describe the generation and transformation of the protoplasts with StrigoQuant and detail the application of the synthetic SL analogue GR24. We then show the recording of the luminescence signal and how the obtained data are processed and used to assess/determine SL perception.

Treatment of Hepatitis C Virus and Long-Term Effect on Glycemic Control.

BACKGROUND: Chronic hepatitis C virus (HCV) infection has been linked to worsening glycemic control in patients with diabetes due to insulin resistance. Studies have shown that treating HCV improves glycemic control in this patient population. Most studies assess glycemic control until the patient's sustained viral response at 12 weeks (SVR12). OBJECTIVES: To (a) assess the sustainability of glycemic control after the SVR12 date and (b) determine the change in diabetic medication use over time. METHODS: This was a retrospective chart review of patients treated at an academic medical center's hepatology clinic from 2014 through 2017. Patients were eligible for review if they were treated for hepatitis C and had type 2 diabetes mellitus (DM) or pre-DM, defined by a hemoglobin A1c (A1c) > 5.7%, at baseline. Data were collected from the EPIC database available to Temple University Hospital. Results were analyzed using a linear mixed model and descriptive statistics. RESULTS: Of the 1,073 patients screened, 310 met the eligibility criteria. Most patients achieved SVR12 (87.8%). A statistically significant decrease in A1c from baseline to treatment completion with direct-acting antivirals (DAAs) and until current reading was seen (P < 0.05). Overall, A1c was reduced in patients treated for HCV with DAA by -0.27% (95% CI = -0.479% to -0.055%, P = 0.014) from baseline to current reading. No statistically significant difference existed in A1c at time of SVR12 to current reading (difference in A1c = 0.07%, 95% CI = -0.26% to 0.4%, P = 0.67), indicating that the reduction in A1c achieved by treating HCV can be sustained over time. Insulin dose also decreased from baseline to current values. CONCLUSIONS: Overall, patients with diabetes successfully treated for the hepatitis C virus may be able to maintain improved glycemic control past SVR12. This could lead to less antidiabetic medication use and decreased insulin requirements for this patient population. DISCLOSURES: This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. The authors report no conflicts of interest.

Pharmacist Comfort and Awareness of HIV and HCV Point-of-Care Testing in Community Settings.

OBJECTIVE: To address the public health concern of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) transmission, various intrapersonal and organizational factors were identified to explore opportunities for pharmacists as part of a HIV/HCV prevention strategy. The awareness and comfort of pharmacists practicing in independent pharmacies and student pharmacists on providing HIV and HCV point-of-care (POC) tests in an urban setting were investigated. METHOD: Surveys were anonymously completed by pharmacists practicing in independent pharmacies within a 2-mile radius of our institution and student pharmacists attending our institution. Surveys were administered over a period of 10 weeks. Data were analyzed using descriptive statistics. RESULTS: A total of 119 pharmacy students and 23 practicing licensed pharmacists completed the survey. Only 21.7% of pharmacists were aware that HIV and HCV screenings are available as POC tests. Pharmacists were more likely to feel comfortable administering other POC tests and not HIV or HCV POC tests. Student pharmacists felt more comfortable than pharmacists in administering HIV and HCV POC tests and had a higher perceived level of comfort of their ability to administer these tests as licensed pharmacists. CONCLUSIONS: In this urban setting, awareness and comfort in pharmacist-provided HIV and HCV POC testing is low, however, with proper training and education, pharmacists in these community pharmacy practice settings can expand HIV and HCV screening opportunities for the community.

Hepatitis C Treatment Differences in Elderly Patients: Single-Center Retrospective Study.

Background: Clinical studies evaluating direct-acting antivirals (DAAs) for hepatitis C virus (HCV) treatment show sustained virological response at 12 weeks (SVR12) rates >90%. However, there are few elderly patients included in these studies; thus, generalizability of high success rates to patients >70 years old cannot be assumed. Objective: To identify treatment differences between elderly and nonelderly patients. Methods: This is a retrospective cohort study of all patients who were treated with DAAs between June 2014 and September 2016 at our institution. Patients were divided into 2 groups: elderly, age ≥70 years at the time of initiation of DAAs, and nonelderly, <70 years. The primary outcome was achievement of SVR12. Results: Among the 551 patients, 60 with age range 70 to 86 years comprised the elderly group. SVR12 rates were significantly lower in the elderly population, especially in those with liver cirrhosis. SVR12 was achieved in 81% of the elderly group as compared with 95% in the nonelderly group. Among cirrhotic patients, 69.4% in the elderly group, and 94.1% in the nonelderly group achieved SVR12. Binary logistic regression modeling showed age >70 years to be the strongest predictor of treatment failure (odds ratio = 3.4), along with diagnosis of cirrhosis (odds ratio = 2.4), when corrected for gender, race, prior treatment experience, genotype, and presence of hepatocellular carcinoma. Conclusion and Relevance: Lower SVR12 was seen in elderly cirrhotic patients (69.4%), who are at higher risk of complications related to advanced liver disease and untreated HCV infection, highlighting the need to treat patients before cirrhosis develops.

Synthetic Switches and Regulatory Circuits in Plants.

Synthetic biology is an established but ever-growing interdisciplinary field of research currently revolutionizing biomedicine studies and the biotech industry. The engineering of synthetic circuitry in bacterial, yeast, and animal systems prompted considerable advances for the understanding and manipulation of genetic and metabolic networks; however, their implementation in the plant field lags behind. Here, we review theoretical-experimental approaches to the engineering of synthetic chemical- and light-regulated (optogenetic) switches for the targeted interrogation and control of cellular processes, including existing applications in the plant field. We highlight the strategies for the modular assembly of genetic parts into synthetic circuits of different complexity, ranging from Boolean logic gates and oscillatory devices up to semi- and fully synthetic open- and closed-loop molecular and cellular circuits. Finally, we explore potential applications of these approaches for the engineering of novel functionalities in plants, including understanding complex signaling networks, improving crop productivity, and the production of biopharmaceuticals.

Influential Factors of Successful Hepatitis C Treatment in Elderly Patients.

BACKGROUND: Chronic Hepatitis C virus (HCV) is an infection associated with an increased risk of cirrhosis, hepatocellular carcinoma (HCC), and morbidity and mortality. Treating HCV poses challenges in the elderly population due to the lack of evidence and complexity of patients. OBJECTIVE: This study aims to evaluate factors that influence HCV treatment success in elderly patients, especially those over age of 70, such as pill burden and comorbidities, in addition to drug interactions and adverse effects. METHODS: This was a retrospective chart review of patients treated at our urban academic institution from 2014-2016. RESULTS: Sixty-two patients over the age of 70 were included in this study. The sustained virologic response rate 12 weeks after the completion of treatment (SVR12) was 79%. In a multi-variate analysis, cirrhosis, age closer to 70, and longer duration of treatment were statistically significantly more likely to lead to treatment failure. Though not statistically significant, other factors that may negatively influence achievement of SVR12 were cognitive impairment, cardiovascular disease, multi-tablet HCV regimen, time to initiation of HCV treatment > 90 days, and prior treatment experience. Pill burden of other prescribed medications did not impact SVR12. Adverse events and drug interactions were common in the population. CONCLUSIONS: Overall SVR12 rate in the elderly population was lower than that reported in the literature. Factors associated with lower treatment success, especially cirrhosis, should be considered when treating an elderly population. Further data is needed on the impact of other factors on SVR12 attainment in an elderly patient population.

Pharmacists' Impact on Secondary Stroke Prevention.

BACKGROUND: Patients admitted to our institution with a cerebrovascular accident (stroke) or transient ischemic attack (TIA) are referred to the pharmacist-run stroke prevention clinic (SPC) for medication and risk factor management. OBJECTIVE: The objective was to determine if patients receiving care from the SPC have better outcomes than patients who received usual care. METHODS: This was a retrospective chart review of patients referred to the SPC. At the time of stroke/TIA, before initial visit, and after last SPC visit, risk factor data was collected. Hospital readmissions were reviewed for secondary stroke/TIA, myocardial infarction (MI), and new or incidental peripheral artery disease (PAD). For patients that did not attend SPC visits, data was used as a control. RESULTS: Patients referred to the SPC from October 2012 to December 2014 were reviewed. 455 records were reviewed. The primary composite end point of readmission for stroke/TIA, myocardial infarction, and new or incidental PAD was statistically significantly lower in the SPC group than the control group (P = .013). All surrogate markers, including blood pressure, Low Density Lipoprotein, Hemoglobin A1c, and smoking status, improved in the SPC group. CONCLUSION: Pharmacists can play a role in reducing risk factors for secondary stroke/TIA and prevent future hospital admissions.

Eight-Week Outcomes of Ledipasvir/Sofosbuvir in Noncirrhotic Treatment-Naive Patients with Hepatitis C: Analysis of Pharmacy-Based Data.

BACKGROUND: Clinical trials have demonstrated that 8 weeks of ledipasvir and sofosbuvir (LDV/SOF) achieved high rates of sustained virologic response at 12 weeks (SVR12) in patients with hepatitis C viral (HCV) genotype 1 infection. The effectiveness of this combination was noted to be robust in treatment-naive noncirrhotic patients and in patients with an HCV viral load of < 6 million IU/mL before treatment. Generalizability of these results to community clinical practice, however, was advised with caution due to the variability of staging methods, fluctuating nature of viral loads, lack of prospective trials, and real-life confirmation. OBJECTIVE: To evaluate the efficacy, defined as SVR12, of LDV/SOF in a real-world setting. METHODS: Patients met inclusion criteria if given 8 weeks of LDV/SOF by a specialty pharmacy from October 2014 to October 2015 and if SVR12 was assessed after therapy completion. Clinical outcomes data were obtained from the pharmacy database. RESULTS: Of the 6,391 prescriptions of LDV/SOF received by the pharmacy, 3,648 (57%) were covered by insurance, and among them, only 511 (14%) were for an 8-week regimen. SVR12 data were available for 380 (74%) patients who completed an 8-week regimen. 230 different prescribers wrote prescriptions, and 57 different insurance plans approved the 8-week regimen. The majority (74%) of patients were followed by gastroenterology clinics. The 380 patients included in the analysis were all treatment-naive HCV genotype 1 patients. Overall, SVR12 was achieved in 97% of patients, while 10 patients relapsed. The SVR12 rates were lower (93%) in patients with stage 3 fibrosis, particularly in African Americans (29 of 35: 83%). CONCLUSIONS: Outcomes were favorable for the 8-week use of LDV/SOF in a noncontrolled real-world setting in treatment-naive noncirrhotic patients with a baseline viral load < 6 million IU/mL. Use of this approach in African Americans with evidence of advanced fibrosis should be avoided. DISCLOSURES: No outside funding supported this study. Lott is an employee of Diplomat Pharmacy. Andres and Qureshi have nothing to disclose. Study concept and design were contributed by Lott and Qureshi, who also collected the data. All authors contributed equally to data interpretation and manuscript preparation. Andres revised the manuscript, along with Lott and Qureshi. This work was presented in part as a poster at the 2016 International Liver Conference; Barcelona, Spain; April 13-17, 2016.

An Unexpected Interaction between Sofosbuvir/Ledipasvir and Atorvastatin and Colchicine Causing Rhabdomyolysis in a Patient with Impaired Renal Function.

Hepatitis C virus (HCV) infection affects roughly 170 million people worldwide. Sofosbuvir/Ledipasvir (Sof/Led) is a new once daily direct acting antiviral combination pill that was approved in October 2014 for use in patients with HCV genotype 1 infection. Coadministration of Sof/Led is studied only with rosuvastatin which shows significantly increased level of drug and is associated with increased risk of myopathy, including rhabdomyolysis. There is no mention of such HMG-CoA reductase inhibitor interaction as a class, as pravastatin did not have any clinically significant interaction with Sof/Led. Other myotoxic drugs, including colchicine are not studied. We present a case of a serious drug interaction between Sof/Led and atorvastatin, in the background of CKD and colchicine use.

Treatment Considerations for Unique Patient Populations With HCV Genotype 1 Infection.

OBJECTIVE: To review the literature for the treatment of hepatitis C virus (HCV) genotype 1 in certain populations of patients that require further considerations before therapy initiation. DATA SOURCES: A systematic electronic literature search using the MEDLINE database was performed using the search terms hepatitis C, chronic hepatitis C, drug therapy, end stage liver disease, liver transplantation, HIV, hepatitis B, African Americans, renal insufficiency, obesity, pregnancy, and pediatrics. STUDY SELECTION AND DATA EXTRACTION: English language studies from January 1985 to March 2015 were considered. Additional references were identified from ongoing trials obtained from clinicaltrials.gov, conference proceedings, online databases, and citations in relevant review articles. DATA SELECTION: Direct-acting antivirals are first-line recommendations for the treatment of HCV genotype 1 infection, and these include combinations of sofosbuvir, simeprevir, ledipasvir/sofosbuvir, ombitasvir/paritaprevir/ritonavir plus dasabuvir, and ribarvirin. Historical and clinical data focusing on the treatment of HCV with these agents in the following populations were selected: decompensated cirrhosis, post-liver transplant, HIV, African Americans, obesity, hepatitis B coinfection, renal impairment, pregnancy, and pediatrics. CONCLUSION: Depending on the population studied, clinicians must consider differences in efficacy outcomes, potential drug interactions, and adverse effects that patients may experience.

A practical guide to concentrated insulin for pharmacists.

PURPOSE: Insulin improves glycemic control in several ways, for example, by stimulating glucose uptake in the muscle and inhibiting hepatic glucose production. It has other mechanisms of action for correcting the abnormal metabolism of proteins, fats, and carbohydrates. The formulation of concentrated insulin (U-500) is a higher potency of insulin than the U-100 regular formulation. It is indicated for children and adults with type 1 and type 2 diabetes who have not achieved adequate glycemic control with exercise and proper dietary habits. However, the unique characteristics of concentrated insulin require that a patient be educated on its use. This article provides a practical guide for pharmacists on the use of concentrated insulin in both inpatient and outpatient settings and highlights specific concerns and management strategies. CONCLUSION: Concentrated insulin works in the same mechanism as U-100 insulin formulations for treating type 1 and type 2 diabetes. Pharmacists are knowledgeable about managing the disease and can identify patients who will benefit with treatment of concentrated insulin. They can provide recommendations to prevent and resolve situations, such as dosing errors, which arise in patients on concentrated insulin and can educate patients and health care professionals on dosing conversions and titration.

A Possible Case of Gabapentin-Induced Mild Hyperglycemia.

Objective: A possible case of gabapentin-induced mild hyperglycemia is reported. Case Summary: A 63-year-old Caucasian gentleman was treated in a pharmacist-run pharmacotherapy clinic for type 2 diabetes management. His comorbidities included type 2 diabetes mellitus, hypertension, dyslipidemia, coronary artery disease, peripheral vascular disease, Barrett's esophagus, chronic back pain, and obesity. His medications for these comorbidities were continued throughout his care. The patient had no known drug allergies. After 3 months of pharmacist-managed diabetes management, the patient's glucose levels had improved and were within the desired range for a majority of the readings. The patient was consulted to the pain clinic for uncontrolled pain and initiated and titrated on gabapentin from 300 mg per day to 600 to 900 mg 3 times per day. Two weeks later, the patient's glucose levels increased to levels of 150 to 165 mg/dL. The patient's increasing blood glucose values were treated with NPH insulin, which was titrated for several months until he was switched to insulin glargine. Gabapentin was not discontinued since it provided adequate pain control. Discussion: An objective causality assessment revealed that the adverse effect was possible. No other new medications were introduced, current medications were not adjusted, and no alternate causes could be found for increased glucose values. Conclusion: A 63-year-old Caucasian gentleman with type 2 diabetes mellitus developed a possible case of gabapentin-induced mild hyperglycemia after receiving gabapentin for several months with a dose titration. Gabapentin could be considered as a cause for otherwise unexplained hyperglycemia in a patient.