Best practices and tools for reporting reproducible fluorescence microscopy methods.

Although fluorescence microscopy is ubiquitous in biomedical research, microscopy methods reporting is inconsistent and perhaps undervalued. We emphasize the importance of appropriate microscopy methods reporting and seek to educate researchers about how microscopy metadata impact data interpretation. We provide comprehensive guidelines and resources to enable accurate reporting for the most common fluorescence light microscopy modalities. We aim to improve microscopy reporting, thus improving the quality, rigor and reproducibility of image-based science.

Characterization of Amycolatopsis 75iv2 dye-decolorizing peroxidase on O-glycosides.

Dye-decolorizing peroxidases are heme peroxidases with a broad range of substrate specificity. Their physiological function is still largely unknown, but a role in the depolymerization of plant cell wall polymers has been widely proposed. Here, a new expression system for bacterial dye-decolorizing peroxidases as well as the activity with previously unexplored plant molecules are reported. The dye-decolorizing peroxidase from Amycolatopsis 75iv2 (DyP2) was heterologously produced in the Gram-positive bacterium Streptomyces lividans TK24 in both intracellular and extracellular forms without external heme supplementation. The enzyme was tested on a series of O-glycosides, which are plant secondary metabolites with a phenyl glycosidic linkage. O-glycosides are of great interest, both for studying the compounds themselves and as potential models for studying specific lignin-carbohydrate complexes. The primary DyP reaction products of salicin, arbutin, fraxin, naringin, rutin, and gossypin were oxidatively coupled oligomers. A cleavage of the glycone moiety upon radical polymerization was observed when using arbutin, fraxin, rutin, and gossypin as substrates. The amount of released glucose from arbutin and fraxin reached 23% and 3% of the total substrate, respectively. The proposed mechanism suggests a destabilization of the ether linkage due to the localization of the radical in the para position. In addition, DyP2 was tested on complex lignocellulosic materials such as wheat straw, spruce, willow, and purified water-soluble lignin fractions, but no remarkable changes in the carbohydrate profile were observed, despite obvious oxidative activity. The exact action of DyP2 on such lignin-carbohydrate complexes therefore remains elusive. IMPORTANCE: Peroxidases require correct incorporation of the heme cofactor for activity. Heterologous overproduction of peroxidases often results in an inactive enzyme due to insufficient heme synthesis by the host organism. Therefore, peroxidases are incubated with excess heme during or after purification to reconstitute activity. S. lividans as a production host can produce fully active peroxidases both intracellularly and extracellularly without the need for heme supplementation. This reduces the number of downstream processing steps and is beneficial for more sustainable production of industrially relevant enzymes. Moreover, this research has extended the scope of dye-decolorizing peroxidase applications by studying naturally relevant plant secondary metabolites and analyzing the formed products. A previously overlooked artifact of radical polymerization leading to the release of the glycosyl moiety was revealed, shedding light on the mechanism of DyP peroxidases. The key aspect is the continuous addition, rather than the more common approach of a single addition, of the cosubstrate, hydrogen peroxide. This continuous addition allows the peroxidase to complete a high number of turnovers without self-oxidation.

Methanogenic partner influences cell aggregation and signalling of Syntrophobacterium fumaroxidans.

For several decades, the formation of microbial self-aggregates, known as granules, has been extensively documented in the context of anaerobic digestion. However, current understanding of the underlying microbial-associated mechanisms responsible for this phenomenon remains limited. This study examined morphological and biochemical changes associated with cell aggregation in model co-cultures of the syntrophic propionate oxidizing bacterium Syntrophobacterium fumaroxidans and hydrogenotrophic methanogens, Methanospirillum hungatei or Methanobacterium formicicum. Formerly, we observed that when syntrophs grow for long periods with methanogens, cultures tend to form aggregates visible to the eye. In this study, we maintained syntrophic co-cultures of S. fumaroxidans with either M. hungatei or M. formicicum for a year in a fed-batch growth mode to stimulate aggregation. Millimeter-scale aggregates were observed in both co-cultures within the first 5 months of cultivation. In addition, we detected quorum sensing molecules, specifically N-acyl homoserine lactones, in co-culture supernatants preceding the formation of macro-aggregates (with diameter of more than 20 µm). Comparative transcriptomics revealed higher expression of genes related to signal transduction, polysaccharide secretion and metal transporters in the late-aggregation state co-cultures, compared to the initial ones. This is the first study to report in detail both biochemical and physiological changes associated with the aggregate formation in syntrophic methanogenic co-cultures. KEYPOINTS: • Syntrophic co-cultures formed mm-scale aggregates within 5 months of fed-batch cultivation. • N-acyl homoserine lactones were detected during the formation of aggregates. • Aggregated co-cultures exhibited upregulated expression of adhesins- and polysaccharide-associated genes.

Polyphenol Oxidase Activity on Guaiacyl and Syringyl Lignin Units.

The natural heterogeneity of guaiacyl (G) and syringyl (S) compounds resulting from lignin processing hampers their direct use as plant-based chemicals and materials. Herein, we explore six short polyphenol oxidases (PPOs) from lignocellulose-degrading ascomycetes for their capacity to react with G-type and S-type phenolic compounds. All six PPOs catalyze the ortho-hydroxylation of G-type compounds (guaiacol, vanillic acid, and ferulic acid), forming the corresponding methoxy-ortho-diphenols. Remarkably, a subset of these PPOs is also active towards S-compounds (syringol, syringic acid, and sinapic acid) resulting in identical methoxy-ortho-diphenols. Assays with 18O2 demonstrate that these PPOs in particular catalyze ortho-hydroxylation and ortho-demethoxylation of S-compounds and generate methanol as a co-product. Notably, oxidative (ortho-)demethoxylation of S-compounds is a novel reaction for PPOs, which we propose occurs via a distinct reaction mechanism compared to aryl-O-demethylases. We further show that addition of a reducing agent can steer the PPO reaction to form methoxy-ortho-diphenols from both G- and S-type substrates rather than reactive quinones that lead to unfavorable polymerization. Application of PPOs opens for new routes to reduce the heterogeneity and methoxylation degree of mixtures of G and S lignin-derived compounds.

Detecting Sources of Immune Activation and Viral Rebound in HIV Infection.

Anti-retroviral therapy (ART) generally suppresses HIV replication to undetectable levels in peripheral blood, but immune activation associated with increased morbidity and mortality is sustained during ART, and infection rebounds when treatment is interrupted. To identify drivers of immune activation and potential sources of viral rebound, we modified RNAscope in situ hybridization to visualize HIV-producing cells as a standard against which to compare the following assays of potential sources of immune activation and virus rebound following treatment interruption: (i) envelope detection by induced transcription-based sequencing (EDITS) assay; (ii) HIV-Flow; (iii) Flow-FISH assays that can scan tissues and cell suspensions to detect rare cells expressing env mRNA, gag mRNA/Gag protein and p24; and (iv) an ultrasensitive immunoassay that detects p24 in cell/tissue lysates at subfemtomolar levels. We show that the sensitivities of these assays are sufficient to detect one rare HIV-producing/env mRNA+/p24+ cell in one million uninfected cells. These high-throughput technologies provide contemporary tools to detect and characterize rare cells producing virus and viral antigens as potential sources of immune activation and viral rebound. IMPORTANCE Anti-retroviral therapy (ART) has greatly improved the quality and length of life for people living with HIV, but immune activation does not normalize during ART, and persistent immune activation has been linked to increased morbidity and mortality. We report a comparison of assays of two potential sources of immune activation during ART: rare cells producing HIV and the virus' major viral protein, p24, benchmarked on a cell model of active and latent infections and a method to visualize HIV-producing cells. We show that assays of HIV envelope mRNA (EDITS assay), gag mRNA, and p24 (Flow-FISH, HIV-Flow. and ultrasensitive p24 immunoassay) detect HIV-producing cells and p24 at sensitivities of one infected cell in a million uninfected cells, thereby providing validated tools to explore sources of immune activation during ART in the lymphoid and other tissue reservoirs.

Decreased mechanotransduction prevents nuclear collapse in a Caenorhabditis elegans laminopathy.

The function of the nucleus depends on the integrity of the nuclear lamina, an intermediate filament network associated with the linker of nucleoskeleton and cytoskeleton (LINC) complex. The LINC complex spans the nuclear envelope and mediates nuclear mechanotransduction, the process by which mechanical signals and forces are transmitted across the nuclear envelope. In turn, the AAA+ ATPase torsinA is thought to regulate force transmission from the cytoskeleton to the nucleus. In humans, mutations affecting nuclear envelope-associated proteins cause laminopathies, including progeria, myopathy, and dystonia, though the extent to which endogenous mechanical stresses contribute to these pathologies is unclear. Here, we use the Caenorhabditis elegans germline as a model to investigate mechanisms that maintain nuclear integrity as germ cell nuclei progress through meiotic development and migrate for gametogenesis-processes that require LINC complex function. We report that decreasing the function of the C. elegans torsinA homolog, OOC-5, rescues the sterility and premature aging caused by a null mutation in the single worm lamin homolog. We show that decreasing OOC-5/torsinA activity prevents nuclear collapse in lamin mutants by disrupting the function of the LINC complex. At a mechanistic level, OOC-5/torsinA promotes the assembly or maintenance of the lamin-associated LINC complex and this activity is also important for interphase nuclear pore complex insertion into growing germline nuclei. These results demonstrate that LINC complex-transmitted forces damage nuclei with a compromised nuclear lamina. Thus, the torsinA-LINC complex nexus might comprise a therapeutic target for certain laminopathies by preventing damage from endogenous cellular forces.

The impact of entrepreneurship research on other academic fields.

The remarkable ascent of entrepreneurship witnessed as a scientific field over the last 4 decades has been made possible by entrepreneurship's ability to absorb theories, paradigms, and methods from other fields such as economics, psychology, sociology, geography, and even biology. The respectability of entrepreneurship as an academic discipline is now evidenced by many other fields starting to borrow from the entrepreneurship view. In the present paper, seven examples are given from this "pay back" development. These examples were first presented during a seminar at the Erasmus Entrepreneurship Event called what has the entrepreneurship view to offer to other academic fields? This article elaborates on the core ideas of these presentations and focuses on the overarching question of how entrepreneurship research impacts the development of other academic fields. We found that entrepreneurship research questions the core assumptions of other academic fields and provides new insights into the antecedents, mechanisms, and consequences of their respective core phenomena. Moreover, entrepreneurship research helps to legitimize other academic fields both practically and academically.

Entrepreneurship research questions the core assumptions of other academic fields and legitimizes them both practically and academically. Since the 1980s, entrepreneurship research has seen tremendous growth and development, establishing itself as an academic field. Entrepreneurship is also taught extensively in leading business schools around the world. Indeed, few business schools do not address entrepreneurship in their curriculum. This represents a sea change: although entrepreneurs and new ventures had a remarkable impact on society, academia barely noticed it in the 1980s. Simply put: economics and business students rarely, if ever, encountered any mention of entrepreneurship during their studies. While entrepreneurship research has now developed its own methodological toolbox, it has extensively borrowed perspectives, theories, and methods from other fields. In the 2020s, we now find that entrepreneurship scholars are sharing its toolbox with other academic fields, questioning the core assumptions of other academic fields and providing new insights into the antecedents, mechanisms, and consequences of their respective core phenomena. Moreover, entrepreneurship research helps to legitimize other academic fields both practically and academically. Hence, entrepreneurship research now plays not just an important role in entrepreneurship education, practice, and policy but also throughout many other research fields.

Optogenetic-induced sympathetic neuromodulation of brown adipose tissue thermogenesis.

The brown adipose tissue (BAT) is a thermogenic organ that plays a major role in energy balance, obesity, and diabetes due to the potent glucose and lipid clearance that fuels its thermogenesis, which is largely mediated via sympathetic nervous system activation. However, thus far there has been little experimental validation of the hypothesis that selective neuromodulation of the sympathetic nerves innervating the BAT is sufficient to elicit thermogenesis in mice. We generated mice expressing blue light-activated channelrhodopsin-2 (ChR2) in the sympathetic nerves innervating the BAT using two different strategies: injecting the BAT of C57Bl/6J mice with AAV6-hSyn-ChR2 (H134R)-EYFP; crossbreeding tyrosine hydroxylase-Cre mice with floxed-stop ChR2-EYFP mice. The nerves in the BAT expressing ChR2 were selectively stimulated with a blue LED light positioned underneath the fat pad of anesthetized mice, while the BAT and core temperatures were simultaneously recorded. Using immunohistochemistry we confirmed the selective expression of EYFP in TH positive nerves fibers. In addition, local optogenetic stimulation of the sympathetic nerves induced significant increase in the BAT temperature followed by an increase in core temperature in mice expressing ChR2, but not in the respective controls. The BAT activation was also paralleled by increased levels of pre-UCP1 transcript. Our results demonstrate that local optogenetic stimulation of the sympathetic nerves is sufficient to elicit BAT and core thermogenesis, thus suggesting that peripheral neuromodulation has the potential to be exploited as an alternative to pharmacotherapies to elicit organ activation and thus ameliorate type 2 diabetes and/or obesity.

The effect of Clip-tone® and its smartphone application on optimisation of metered-dose inhalers inhalation technique.

BACKGROUNDS: Although metered-dose inhalers (pMDI) therapy is convenient and widely prescribed, its use usually results in repetitive inhalation technique errors. One of the most repetitive errors is inhaling too fast through the pMDI. The present study aimed to evaluate the effect of Clip-tone® along with smartphone visual feedback application on the subject's inhalation time. METHODS: Two hundred subjects were included in the study. They were randomised into four groups. Group 1 received only verbal counselling; group 2 received verbal counselling with resistance (a modified Clip-tone® that does not produce whistle attached to their pMDI); group 3 received verbal counselling plus whistle (as audio feedback) from ordinary Clip-tone® and group 4 received verbal counselling plus audio feedback (whistle) from Clip-tone® and visual feedback (smartphone application). Inhalation time through the pMDI for each subject was recorded three times and inter and intra-subjects variations were calculated. RESULTS: Verbal counselling plus audio feedback and verbal counselling plus audio and visual feedbacks groups had 45/50 (90%) and 37/50 (74%) subjects respectively, having correct inhalation flow (inhaling at between 3 to 7 seconds). Verbal counselling plus audio feedback and verbal counselling plus audio and visual feedbacks groups' inter and intra-subjects variations were lower than that of verbal counselling and verbal counselling with resistance groups which had 28/50 (52%) and 20/50 (40%) subjects respectively, with inhalation time between 3 and 7 seconds. CONCLUSIONS: Providing audio feedback by the Clip-tone® along with smartphone visual feedback application maintained the deep and slow inhalation through pMDI much better compared to verbal counselling only. We recommend the patients to take all their inhaled doses using pMDI attached to a training device like Clip-tone® along with a smartphone visual feedback application for optimisation of the aerosol delivery from the pMDI.

Microalgal swimming signatures and neutral lipids production across growth phases.

Microalgae have been shown as a potential bioresource for food, biofuel, and pharmaceutical products. During the growth phases with corresponding environmental conditions, microalgae accumulate different amounts of various metabolites. We quantified the neutral lipids accumulation and analyzed the swimming signatures (speed and trajectories) of the motile green alga, Dunaliella primolecta, during the lag-exponential-stationary growth cycle at different nutrient concentrations. We discovered significant changes in the neutral lipid content and swimming signatures of microalgae across growth phases. The timing of the maximum swimming speed coincided with the maximum neutral lipid content and both maxima occurred under nutrient stress at the stationary growth phase. Furthermore, the swimming trajectories suggested statistically significant changes in swimming modes at the stationary growth phase when the maximum intracellular neutral lipid content was observed. Our results provide the potential exploitation of microalgal swimming signatures as possible indicators of the cultivation conditions and the timing of microalgal harvest to maximize the lipid yield for biofuel production. The findings can also be implemented to explore the production of food and antibiotics from other microalgal metabolites with low energy costs.

Patient-directed self-management of pain (PaDSMaP) compared to treatment as usual following total knee replacement; a randomised controlled trial.

BACKGROUND: Self-administration of medicines by patients whilst in hospital is being increasingly promoted despite little evidence to show the risks and benefits. Pain control after total knee replacement (TKR) is known to be poor. The aim of the study was to determine if patients operated on with a TKR who self-medicate their oral analgesics in the immediate post-operative period have better pain control than those who receive their pain control by nurse-led drug rounds (Treatment as Usual (TAU)). METHODS: A prospective, parallel design, open-label, randomised controlled trial comparing pain control in patient-directed self-management of pain (PaDSMaP) with nurse control of oral analgesia (TAU) after a TKR. Between July 2011 and March 2013, 144 self-medicating adults were recruited at a secondary care teaching hospital in the UK. TAU patients (n = 71) were given medications by a nurse after their TKR. PaDSMaP patients (n = 73) took oral medications for analgesia and co-morbidities after two 20 min training sessions reinforced with four booklets. Primary outcome was pain (100 mm visual analogue scale (VAS)) at 3 days following TKR surgery or at discharge (whichever came soonest). Seven patients did not undergo surgery for reasons unrelated to the study and were excluded from the intention-to-treat (ITT) analysis. RESULTS: ITT analysis did not detect any significant differences between the two groups' pain scores. A per protocol (but underpowered) analysis of the 60% of patients able to self-medicate found reduced pain compared to the TAU group at day 3/discharge, (VAS -9.9 mm, 95% CI -18.7, - 1.1). One patient in the self-medicating group over-medicated but suffered no harm. CONCLUSION: Self-medicating patients did not have better (lower) pain scores compared to the nurse-managed patients following TKR. This cohort of patients were elderly with multiple co-morbidities and may not be the ideal target group for self-medication. TRIAL REGISTRATION: ISRCTN10868989 . Registered 22 March 2012, retrospectively registered.

Low-Dose Intravenous Immunoglobulin Treatment for Long-Standing Complex Regional Pain Syndrome: A Randomized Trial.

BACKGROUND: Two small trials suggest that low-dose intravenous immunoglobulin (IVIg) may improve the symptoms of complex regional pain syndrome (CRPS), a rare posttraumatic pain condition. OBJECTIVE: To confirm the efficacy of low-dose IVIg compared with placebo in reducing pain during a 6-week period in adult patients who had CRPS from 1 to 5 years. DESIGN: 1:1 parallel, randomized, placebo-controlled, multicenter trial for 6 weeks, with an optional 6-week open extension. Patients were randomly assigned to 1 of 2 study groups between 27 August 2013 and 28 October 2015; the last patient completed follow-up on 21 March 2016. Patients, providers, researchers, and outcome assessors were blinded to treatment assignment. (ISRCTN42179756). SETTING: 7 secondary and tertiary care pain management centers in the United Kingdom. PARTICIPANTS: 111 patients with moderate or severe CRPS of 1 to 5 years' duration. INTERVENTION: IVIg, 0.5 g/kg of body weight, or visually indistinguishable placebo of 0.1% albumin in saline on days 1 and 22 after randomization. MEASUREMENTS: The primary outcome was 24-hour average pain intensity, measured daily between days 6 and 42, on an 11-point (0- to 10-point) rating scale. Secondary outcomes were pain interference and quality of life. RESULTS: The primary analysis sample consisted of 108 eligible patients, 103 of whom had outcome data. Mean (average) pain scores were 6.9 points (SD, 1.5) for placebo and 7.2 points (SD, 1.3) for IVIg. The adjusted difference in means was 0.27 (95% CI, -0.25 to 0.80; P = 0.30), which excluded the prespecified, clinically important difference of -1.2. No statistically significant differences in secondary outcomes were found between the groups. In the open extension, 12 of the 67 patients (18%) who received 2 IVIg infusions had pain reduction of at least 2 points compared with their baseline score. Two patients in the blinded phase (1 in the placebo and 1 in the IVIg group) and 4 in the open IVIg phase had serious events. LIMITATIONS: Results do not apply to patients who have had CRPS for less than 1 year or more than 5 years and do not extend to full-dose treatment (for example, 2 g/kg). The study was inadequately powered to detect subgroup effects. CONCLUSION: Low-dose immunoglobulin treatment for 6 weeks was not effective in relieving pain in patients with moderate to severe CRPS of 1 to 5 years' duration. PRIMARY FUNDING SOURCE: Medical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Program, Pain Relief Foundation, and Biotest United Kingdom.

Mastery of pMDI technique, asthma control and quality-of-life of children with asthma: A randomized controlled study comparing two inhaler technique training approaches.

OBJECTIVE: Verbal counselling (VC) is the clinical standard for training patients on correct inhaler use. Patients fail to recall their VC with time. Ethical approval was obtained to compare the pressurized metered dose inhaler (pMDI) VC with Trainhaler (TH), a novel pMDI inhalation flow and technique training device, in children with asthma. METHODS: At visit 1, 7-17 year-old children with a pMDI hand-lung coordination problem including a fast peak inhalation flow (PIF) through pMDI >60 L/min were randomized into either VC group that received verbal pMDI training; or into TH group that were trained on- and given TH to practice at home. Whereas, children with correct pMDI use formed the control group (CT). Overall pMDI technique, PIF through inhaler, asthma control (AC) and quality of life (QoL) were evaluated. Participants were re-evaluated 6-8 weeks later (visit 2). RESULTS: Of 105 enrolled children; 76 completed the study (VC = 21, TH = 25 and CT = 30). VC decreased non-significantly (p > 0.05) the mean PIF from 104.0 L/min at visit 1 to 84.8 at visit 2. Whilst, the TH did significantly (p < 0.05) reduce the PIF from 113.5 to 71.4 L/min. The two approaches similarly and significantly (p < 0.05) improved the inhaler technique, AC and QoL scores. CONCLUSIONS: The TH improved the inhalation flow through the pMDI close to the ideal needed for adequate lung deposition. Both methods equally enhanced the children's mastery of pMDI use. This was reflected on better AC and QoL. Accessibility to TH might help maintaining the good inhaler use and decreasing regular VC.

A randomized study of the safety and efficacy of fondaparinux versus placebo in the prevention of venous thromboembolism after coronary artery bypass graft surgery.

UNLABELLED: Venous thromboembolism (VTE) after coronary artery bypass graft (CABG) surgery may increase the postoperative morbidity and mortality. Therefore, we examined the current postoperative need for prophylactic antithrombotic therapy after CABG surgery. METHODS: This randomized, placebo-controlled, double-blind study was designed to compare the safety and efficacy of fondaparinux versus placebo in the prevention of VTE after CABG surgery. Between March 2010 and January 2013, 78 patients free from preoperative deep vein thrombosis (DVT) were enrolled, of whom 37 were randomly assigned to placebo and 41 to treatment with fondaparinux. The primary study end point was a composite, up to day 11, of (a) cumulative incidence of all VTE events, defined as symptomatic and asymptomatic DVT, and fatal and nonfatal pulmonary embolisms (efficacy end point), and (b) cumulative incidence of major hemorrhages (safety end point). RESULTS: A single asymptomatic DVT of a lower extremity was detected by duplex ultrasound at the time of discharge from the hospital in the placebo-treated group, and a single major postoperative hemorrhage occurred in the fondaparinux-treated group. CONCLUSIONS: The incidence of postprocedural asymptomatic DVT in this sample of patients undergoing CABG surgery was low. The overall incidence of DVT in the control and investigational treatment groups was similar. Our results showed no benefit of prophylactic postoperative fondaparinux in this population. These findings are congruent with other published studies and provide additional support for recent recommendations not to routinely use anticoagulant prophylaxis after cardiac surgery.

Distribution of topical ocular nepafenac and its active metabolite amfenac to the posterior segment of the eye.

Nepafenac ophthalmic suspensions, 0.1% (NEVANAC(®)) and 0.3% (ILEVRO™), are topical nonsteroidal anti-inflammatory drug (NSAID) products approved in the United States, Europe and various other countries to treat pain and inflammation associated with cataract surgery. NEVANAC is also approved in Europe for the reduction in the risk of postoperative macular edema (ME) associated with cataract surgery in diabetic patients. The efficacy against ME suggests that topical administration leads to distribution of nepafenac or its active metabolite amfenac to the posterior segment of the eye. This article evaluates the ocular distribution of nepafenac and amfenac and the extent of local delivery to the posterior segment of the eye, following topical ocular instillation in animal models. Nepafenac ophthalmic suspension was instilled unilaterally in New Zealand White rabbits as either a single dose (0.1%; one drop) or as multiple doses (0.3%, one drop, once-daily for 4 days, or 0.1% one drop, three-times daily for 3 days and one morning dose on day 4). Nepafenac (0.3%) was also instilled unilaterally in cynomolgus monkeys as multiple doses (one drop, three-times daily for 7 days). Nepafenac and amfenac concentrations in harvested ocular tissues were measured using high-performance liquid chromatography/mass spectrometry. Locally-distributed compound concentrations were determined as the difference in levels between dosed and undosed eyes. In single-dosed rabbit eyes, peak concentrations of locally-distributed nepafenac and amfenac showed a trend of sclera > choroid > retina. Nepafenac peak levels in sub-samples posterior to the eye equator and inclusive of the posterior pole (E-PP) were 55.1, 4.03 and 2.72 nM, respectively, at 0.25 or 0.50 h, with corresponding amfenac peak levels of 41.9, 3.10 and 0.705 nM at 1 or 4 h. By comparison, peak levels in sclera, choroid and retina sub-samples in a band between the ora serrata and the equator (OS-E) were 13- to 40-fold (nepafenac) or 11- to 23-fold (amfenac) higher, indicating an anterior-to-posterior directional concentration gradient. In multiple-dosed rabbit eyes, with 0.3% nepafenac instilled once-daily or 0.1% nepafenac instilled three-times daily, cumulative 24-h locally-distributed levels of nepafenac in E-PP retina were similar between these groups, whereas exposure to amfenac once-daily dosing nepafenac 0.3% was 51% of that achieved with three-times daily dosing of 0.1%. In single-dosed monkey eyes, concentration gradients showed similar directionality as observed in rabbit eyes. Peak concentrations of locally-distributed nepafenac were 1580, 386, 292 and 13.8 nM in E-PP sclera, choroid and retina, vitreous humor, respectively, at 1 or 2 h after drug instillation. Corresponding amfenac concentrations were 21.3, 11.8, 2.58 and 2.82 nM, observed 1 or 2 h post-instillation. The data indicate that topically administered nepafenac and its metabolite amfenac reach pharmacologically relevant concentrations in the posterior eye segment (choroid and retina) via local distribution, following an anterior-to-posterior concentration gradient. The proposed pathway involves a choroidal/suprachoroidal or periocular route, along with an inward movement of drug through the sclera, choroid and retina, with negligible vitreal compartment involvement. Sustained high nepafenac concentrations in posterior segment tissues may be a reservoir for hydrolysis to amfenac.

Involvement of a Hydrophobic Pocket and Helix†11 in Determining the Modes of Action of Prenylated Flavonoids and Isoflavonoids in the Human Estrogen Receptor.

Six prenylated (iso)flavonoids were purified from a licorice root extract and subjected to competition experiments with six commercially available (iso)flavonoids. The agonistic and antagonistic activities of these compounds towards both hERα (human estrogen receptor alpha) and hERß were determined. Differences in the modes of action (agonist or antagonist) were observed for the various compounds tested. In general, each compound had the same mode of action towards both ERs. In silico modeling was performed in order to study the differences in estrogenicity observed between the compounds. It is suggested that prenyl chains fit into a hydrophobic pocket present in the hER, resulting in an increased agonistic activity. In addition, it was shown that an increase in length (≈1.7 Å) of pyran prenylated isoflavonoids resulted in an antagonistic mode of action. This might be caused by collision of the pyran ring with helix 11 in the ligand binding cavity of the hER.