Amelioration of hepatic steatosis by dietary essential amino acid-induced ubiquitination.

Nonalcoholic fatty liver disease (NAFLD) is a global health concern with no approved drugs. High-protein dietary intervention is currently the most effective treatment. However, its underlying mechanism is unknown. Here, using Drosophila oenocytes, the specialized hepatocyte-like cells, we find that dietary essential amino acids ameliorate hepatic steatosis by inducing polyubiquitination of Plin2, a lipid droplet-stabilizing protein. Leucine and isoleucine, two branched-chain essential amino acids, strongly bind to and activate the E3 ubiquitin ligase Ubr1, targeting Plin2 for degradation. We further show that the amino acid-induced Ubr1 activity is necessary to prevent steatosis in mouse livers and cultured human hepatocytes, providing molecular insight into the anti-NAFLD effects of dietary protein/amino acids. Importantly, split-intein-mediated trans-splicing expression of constitutively active UBR2, an Ubr1 family member, significantly ameliorates obesity-induced and high fat diet-induced hepatic steatosis in mice. Together, our results highlight activation of Ubr1 family proteins as a promising strategy in NAFLD treatment.

Stuxnet fine-tunes Notch dose during development using a functional Polycomb response element.

Evolutionarily conserved Notch signaling is highly sensitive to changes in Notch receptor dose caused by intrinsic and environmental fluctuations. It is well known that epigenetic regulation responds dynamically to genetic, cellular and environmental stresses. However, it is unclear whether the Notch receptor dose is directly regulated at the epigenetic level. Here, by studying the role of the upstream epigenetic regulator Stuxnet (Stx) in Drosophila developmental signaling, we find that Stx promotes Notch receptor mRNA expression by counteracting the activity of Polycomb repressive complex 1 (PRC1). In addition, we provide evidence that Notch is a direct PRC1 target by identifying and validating in vivo the only bona fide Polycomb response element (PRE) among the seven Polycomb group (PcG)-binding sites revealed by DamID-seq and ChIP-seq analysis. Importantly, in situ deletion of this PRE results in increased Notch expression and phenotypes resembling Notch hyperactivation in cell fate specification. These results not only underscore the importance of epigenetic regulation in fine-tuning the Notch activity dose, but also the need to assess the physiological significance of omics-based PcG binding in development.

Epigenetic regulator Stuxnet modulates octopamine effect on sleep through a Stuxnet-Polycomb-Octß2R cascade.

Sleep homeostasis is crucial for sleep regulation. The role of epigenetic regulation in sleep homeostasis is unestablished. Previous studies showed that octopamine is important for sleep homeostasis. However, the regulatory mechanism of octopamine reception in sleep is unknown. In this study, we identify an epigenetic regulatory cascade (Stuxnet-Polycomb-Octß2R) that modulates the octopamine receptor in Drosophila. We demonstrate that stuxnet positively regulates Octß2R through repression of Polycomb in the ellipsoid body of the adult fly brain and that Octß2R is one of the major receptors mediating octopamine function in sleep homeostasis. In response to octopamine, Octß2R transcription is inhibited as a result of stuxnet downregulation. This feedback through the Stuxnet-Polycomb-Octß2R cascade is crucial for sleep homeostasis regulation. This study demonstrates a Stuxnet-Polycomb-Octß2R-mediated epigenetic regulatory mechanism for octopamine reception, thus providing an example of epigenetic regulation of sleep homeostasis.

Protein modifications in Hedgehog signaling: Cross talk and feedback regulation confer divergent Hedgehog signaling activity.

The complexity of the Hedgehog (Hh) signaling cascade has increased over the course of evolution; however, it does not suffice to accommodate the dynamic yet robust requirements of differential Hh signaling activity needed for embryonic development and adult homeostatic maintenance. One solution to solve this dilemma is to apply multiple forms of post-translational modifications (PTMs) to the core Hh signaling components, modulating their abundance, localization, and signaling activity. This review summarizes various forms of protein modifications utilized to regulate Hh signaling, with a special emphasis on crosstalk between different forms of PTMs and their feedback regulation by Hh signaling.

Impact of Surfactants on Polymer Maintained Nifedipine Supersaturation in Aqueous Solution.

PURPOSE: To study the impact of different surfactants on the supersaturation of nifedipine stabilized with HPMC and PVP-VA. METHODS: Different kinds of surfactants, including one cationic surfactant, two anionic surfactants, and three nonionic surfactants, were used to evaluate their impacts on the supersaturation of nifedipine stabilized with HPMC and PVP-VA. Polymer-surfactant interaction was studied by nuclear magnetic resonance (NMR) and fluorescent method. Solubility of nifedipine in solutions containing different amounts of polymers and surfactants was measured. Drug-polymer affinity was evaluated by measuring the percentage of polymer coprecipitated together with the drug from supersaturated solutions. RESULTS: Different polymer-surfactant combinations had different impacts on the supersaturation of nifedipine. Some combinations, such as PVP-VA/SLS and PVP-VA/NaTC under higher surfactant concentrations, showed improved drug supersaturation, due to increased drug solubility or polymer-surfactant synergy; while other combinations, such as HPMC/SLS and HPMC/Tween 20 under lower surfactant concentrations, showed reduced drug supersaturation, which could result from competitive surfactant-polymer or drug-surfactant interaction that disrupted pre-existent drug-polymer interaction. CONCLUSIONS: The ultimate impacts of various surfactants on polymer stabilized nifedipine supersaturation could be attributed to the interplay between different factors, including solubility enhancement of the drug, drug-polymer-surfactant interactions, and polymer-surfactant synergy.

Recognition of early and late stages of bladder cancer using metabolites and machine learning.

INTRODUCTION: Bladder cancer (BCa) is one of the most common and aggressive cancers. It is the sixth most frequently occurring cancer in men and its rate of occurrence increases with age. The current method of BCa diagnosis includes a cystoscopy and biopsy. This process is expensive, unpleasant, and may have severe side effects. Recent growth in the power and accessibility of machine-learning software has allowed for the development of new, non-invasive diagnostic methods whose accuracy and sensitivity are uncompromising to function. OBJECTIVES: The goal of this research was to elucidate the biomarkers including metabolites and corresponding genes for different stages of BCa, show their distinguishing and common features, and create a machine-learning model for classification of stages of BCa. METHODS: Sets of metabolites for early and late stages, as well as common for both stages were analyzed using MetaboAnalyst and Ingenuity® Pathway Analysis (IPA®) software. Machine-learning methods were utilized in the development of a binary classifier for early- and late-stage metabolites of BCa. Metabolites were quantitatively characterized using EDragon 1.0 software. The two modeling methods used are Multilayer Perceptron (MLP) and Stochastic Gradient Descent (SGD) with a logistic regression loss function. RESULTS: We explored metabolic pathways related to early-stage BCa (Galactose metabolism and Starch and sucrose metabolism) and to late-stage BCa (Glycine, serine, and threonine metabolism, Arginine and proline metabolism, Glycerophospholipid metabolism, and Galactose metabolism) as well as those common to both stages pathways. The central metabolite impacting the most cancerogenic genes (AKT, EGFR, MAPK3) in early stage is D-glucose, while late-stage BCa is characterized by significant fold changes in several metabolites: glycerol, choline, 13(S)-hydroxyoctadecadienoic acid, 2'-fucosyllactose. Insulin was also seen to play an important role in late stages of BCa. The best performing model was able to predict metabolite class with an accuracy of 82.54% and the area under precision-recall curve (PRC) of 0.84 on the training set. The same model was applied to three separate sets of metabolites obtained from public sources, one set of the late-stage metabolites and two sets of the early-stage metabolites. The model was better at predicting early-stage metabolites with accuracies of 72% (18/25) and 95% (19/20) on the early sets, and an accuracy of 65.45% (36/55) on the late-stage metabolite set. CONCLUSION: By examining the biomarkers present in the urine samples of BCa patients as compared with normal patients, the biomarkers associated with this cancer can be pinpointed and lead to the elucidation of affected metabolic pathways that are specific to different stages of cancer. Development of machine-learning model including metabolites and their chemical descriptors made it possible to achieve considerable accuracy of prediction of stages of BCa.

Impact of a Single Hydrogen Substitution by Fluorine on the Molecular Interaction and Miscibility between Sorafenib and Polymers.

We aim to understand the potential impact of a modest chemical modification of a drug molecule on the downstream design of its amorphous solid dispersion (ASD) formulation. To this end, we used sorafenib (SOR) and its fluorinated form, regorafenib (REG), as model drugs, to assess the impact of a single hydrogen substitution by fluorine on the molecular interaction and miscibility between drug and PVP or PVP-VA, two commonly used polymers for ASDs. In this study, we observed that the Tg values of PVP or PVP-VA based ASDs of SOR deviated positively from the Gordon-Taylor prediction, which assumes ideal mixing, yet the Tg of REG ASDs deviated negatively from or matched well with the ideal mixing model, suggesting much stronger drug-polymer interactions in SOR ASDs compared with the REG ASDs. Using solution NMR and computational methods, we proved that a six-member-ring formed between the carbonyl groups on the polymers and the uramido hydrogen of SOR or REG, through intermolecular hydrogen bonding. However, steric hindrance resulting from fluorination in REG caused weaker interaction between REG-polymer than SOR-polymer. To further confirm this mechanism, we investigated the molecular interactions of other two uramido-containing model compounds, triclocarban (TCC) and gliclazide (GCZ), with PVP. We found that TCC but not GCZ formed a hexatomic ring with PVP. We concluded that PVP based polymers can easily interact with N, N'-disubstituted urea compounds with a trans-trans structure in the form of hexatomic rings, and the interaction strength of the hexatomic ring largely depended on the chemistry of drug molecules. This study illustrated that even a slight chemical modification on drug molecules could result in substantial difference in drug-polymer interactions, thus significantly impacting polymer selection and pharmaceutical performance of their ASD formulations.

Aggregation of Hydroxypropyl Methylcellulose Acetate Succinate under Its Dissolving pH and the Impact on Drug Supersaturation.

Hydroxypropyl methylcellulose acetate succinate (HPMC-AS) is one of the commonly selected polymers used in amorphous solid dispersions (ASD) with excellent capabilities to maintain aqueous supersaturation of poorly water-soluble drugs and inhibit their crystallization, but the underlying mechanisms remain elusive. In this study, posaconazole was chosen as the model drug to study the supersaturation maintaining and crystallization inhibition capabilities of different types of HPMC-AS under pH 5.5-7.5. We analyzed the HPMC-AS aggregation status in solution using combination of static and dynamic light scattering and observed higher polymer aggregation number when higher grade HPMC-AS or lower pH was used, which correlates well with prolonged drug supersaturation or crystallization inhibition. The amount of HPMC-AS coprecipitated with PSZ, a direct indicator of drug/HPMC-AS affinity, also showed positive correlation with the drug supersaturation and crystallization inhibition in the dissolution process. Therefore, we conclude that the aggregation behavior of HPMC-AS and the drug/HPMC-AS affinity are the key mechanisms that lead to posaconazole supersaturation and crystallization inhibition when HPMC-AS was applied.

Surface Enrichment and Depletion of the Active Ingredient in Spray Dried Amorphous Solid Dispersions.

PURPOSE: To study the effects of physicochemical properties of drug and polymer, as well as the drug-polymer interactions, on the surface composition of SDDs. METHODS: Ethanol solutions containing a model drug (IMC, NMP or FCZ) and a model polymer (PVPK12, PVPK30 or PVP-VA) were spray dried, and the surface composition of SDDs was analyzed by XPS. The surface tensions of pure components and their solutions were measured using Wilhelmy plate and/or calculated using ACD/Labs. NMR and DLS were used to obtain the diffusion coefficients of IMC, NMP, PVPK12 and PVPK30 in solvents. Flory-Huggins interaction parameters for selected drug-polymer pairs were obtained using a melting point depression method. RESULTS: Significant surface enrichment or depletion of the drug was observed in SDDs depending on the particular drug-polymer combination. With PVP as the dispersion polymer, IMC and NMP were surface enriched; whereas FCZ, a hydrophilic drug, was surface depleted. With increasing PVP molecular weight, the surface drug concentration increased, and the effect was greater in the NMP/PVP and FCZ/PVP systems than in the IMC/PVP system where strong drug-polymer interaction existed. Changing the polymer from PVP to PVP-VA reduced the surface concentration of the drug. CONCLUSIONS: The surface concentration of a SDD can be significantly different from the bulk concentration. The main results of this work are consistent with the notion that the relative surface tensions control surface enrichment or depletion. Besides, the relative diffusion rates of the components and the strength of their interactions may also affect the surface composition of the SDDs.

Ubpy controls the stability of the ESCRT-0 subunit Hrs in development.

Ubiquitylated developmental membrane signaling proteins are often internalized for endocytic trafficking, through which endosomal sorting complexes required for transport (ESCRT) act sequentially to deliver internalized cargos to lysosomes. The ESCRT function in endocytic sorting is well established; however, it is not fully understood how the sorting machinery itself is regulated. Here, we show that Ubiquitin isopeptidase Y (Ubpy) plays a conserved role in vivo in the homeostasis of an essential ESCRT-0 complex component Hrs. We find that, in the absence of Drosophila Ubpy, multiple membrane proteins that are essential components of important signaling pathways accumulate in enlarged, aberrant endosomes. We further demonstrate that this phenotype results from endocytic pathway defects. We provide evidence that Ubpy interacts with and deubiquitylates Hrs. In Ubpy-null cells, Hrs becomes ubiquitylated and degraded in lysosomes, thus disrupting the integrity of ESCRT sorting machinery. Lastly, we find that signaling proteins are enriched in enlarged endosomes when Hrs activity is abolished. Together, our data support a model in which Ubpy plays a dual role in both cargo deubiquitylation and the ESCRT-0 stability during development.

Genetic interaction screens identify a role for hedgehog signaling in Drosophila border cell migration.

BACKGROUND: Cell motility is essential for embryonic development and physiological processes such as the immune response, but also contributes to pathological conditions such as tumor progression and inflammation. However, our understanding of the mechanisms underlying migratory processes is incomplete. Drosophila border cells provide a powerful genetic model to identify the roles of genes that contribute to cell migration. RESULTS: Members of the Hedgehog signaling pathway were uncovered in two independent screens for interactions with the small GTPase Rac and the polarity protein Par-1 in border cell migration. Consistent with a role in migration, multiple Hh signaling components were enriched in the migratory border cells. Interference with Hh signaling by several different methods resulted in incomplete cell migration. Moreover, the polarized distribution of E-Cadherin and a marker of tyrosine kinase activity were altered when Hh signaling was disrupted. Conservation of Hh-Rac and Hh-Par-1 signaling was illustrated in the wing, in which Hh-dependent phenotypes were enhanced by loss of Rac or par-1. CONCLUSIONS: We identified a pathway by which Hh signaling connects to Rac and Par-1 in cell migration. These results further highlight the importance of modifier screens in the identification of new genes that function in developmental pathways.

Breast Cancer Method of Detection: 5-Year Outcomes Across a Multisite Health Care Enterprise.

PURPOSE: To investigate the feasibility and accuracy of radiologists categorizing the method of detection (MOD) when performing image-guided breast biopsies. METHODS: This retrospective, observational study was conducted across a health care enterprise that provides breast imaging services at 18 imaging sites across four US states. Radiologists used standardized templates to categorize the MOD, defined as the first test, sign, or symptom that triggered the subsequent workup and recommendation for biopsy. All image-guided breast biopsies since the implementation of the MOD-inclusive standardized template-from October 31, 2017 to July 6, 2023-were extracted. A random sample of biopsy reports was manually reviewed to evaluate the accuracy of MOD categorization. RESULTS: A total of 29,999 biopsies were analyzed. MOD was reported in 29,423 biopsies (98.1%) at a sustained rate that improved over time. The 10 MOD categories in this study included the following: 15,184 mammograms (51.6%); 4,561 MRIs (15.5%); 3,473 ultrasounds (11.8%); 2,382 self-examinations (8.1%); 2,073 tomosynthesis studies (7.0%); 432 clinical examinations (1.5%); 421 molecular breast imaging studies (1.4%); 357 other studies (1.2%); 338 contrast-enhanced digital mammograms (1.1%); and 202 PET studies (0.7%). Original assignments of the MOD agreed with author assignments in 87% of manually reviewed biopsies (n = 100, 95% confidence interval: [80.4%, 93.6%]). CONCLUSIONS: This study demonstrates that US radiologists can consistently and accurately categorize the MOD over an extended time across a health care enterprise.

Stereotactic central/core ablative radiation therapy: results of a phase I study of a novel strategy to treat bulky tumor.

Purpose: Bulky tumor remains as a challenge to surgery, chemotherapy and conventional radiation therapy. Hence, in efforts to overcome this challenge, we designed a novel therapeutic paradigm via strategy of Stereotactic Central/Core Ablative Radiation Therapy (SCART).), which is based on the principles of SBRT (stereotactic body radiation therapy and spatially fractionated radiation therapy (SFRT). We intend to safely deliver an ablative dose to the core of the tumor and with a low dose at tumor edge. The purpose of the phase 1 study was to determine dose-limiting toxicities (DLT)s and the Maximum Tolerated Dose (MTD) of SCART. Methods and materials: We defined a SCART-plan volume inside the tumor, which is proportional to the dimension of tumor. VMAT/Cyberknife technique was adopted. In the current clinical trial; Patients with biopsy proven recurrent or metastatic bulky cancers were enrolled. The five dose levels were 15 Gy X1, 15Gy X3, 18GyX3, 21GyX3 and 24GyX3, while keeping the whole tumor GTV's border dose at 5Gy each fraction. There was no restriction on concurrent systemic chemotherapy agents. Results: 21 patients were enrolled and underwent SCART. All 21 patients have eligible data for study follow-up. Radiotherapy was well tolerated with all treatment completed as scheduled. The dose was escalated for two patients to 24GyX3. No grade 3 or higher toxicity was observed in any of the enrolled patients. The average age of patients was 66 years (range: 14-85) and 13 (62%) patients were male. The median SCART dose was 18Gy (range: 15 - 24). Six out of the 18 patients with data for overall survival (OS) died, and the median time to death was 16.3 months (range: 1 - 25.6). The mean percent change for tumor shrinkage between first visit volumes and post-SCART volumes was 49.5% (SD: 40.89, p-value:0.009). Conclusion: SCART was safely escalated to 24 GyX 3 fractions, which is the maximum Tolerated Dose (MTD) for SCART. This regimen will be used in future phase II trials.

Identification of Ubr1 as an amino acid sensor of steatosis in liver and muscle.

BACKGROUND: Malnutrition is implicated in human metabolic disorders, including hepatic steatosis and myosteatosis. The corresponding nutrient signals and sensors as well as signalling pathways have not yet been well studied. This study aimed to unravel the nutrient-sensing mechanisms in the pathogenesis of steatosis. METHODS: Plin2, a lipid droplet (LD) protein-inhibiting lipolysis, is associated with steatosis in liver and muscle. Taking advantage of the Gal4-UAS system, we used the Drosophila melanogaster wing imaginal disc as an in vivo model to study the regulation of Plin2 proteostasis and LD homeostasis. Drosophila Schneider 2 (S2) cells were used for western blotting, immunoprecipitation assays, amino acid-binding assays and ubiquitination assays to further investigate the regulatory mechanisms of Plin2 in response to nutrient signals. Mouse AML12 hepatocytes, human JHH-7 and SNU-475 hepatoma cells were used for immunofluorescence, western blotting and immunoprecipitation to demonstrate that the mode of Plin2 regulation is evolutionarily conserved. In addition, we purified proteins from HEK293 cells and reconstituted in vitro cell-free systems in amino acid-binding assays, pulldown assays and ubiquitination assays to directly demonstrate the molecular mechanism by which Ubr1 senses amino acids to regulate Plin2 proteostasis. RESULTS: As a lipolysis inhibitor, Plin2 was significantly elevated in liver (P < 0.05) and muscle (P < 0.05) in patients with steatosis. Consistently, we found that the ubiquitin moiety can be conjugated to any Lys residue in Plin2, ensuring robust clearance of Plin2 by protein degradation. We further demonstrated that the E3 ubiquitin ligase Ubr1 targets Plin2 for degradation in an amino acid-dependent manner. Ubr1 uses two canonical substrate-binding pockets, independent of each other, to bind basic and bulky hydrophobic amino acids, respectively. Mechanistically, amino acid binding allosterically activates Ubr1 by alleviating Ubr1's auto-inhibition. In the absence of amino acids, or when the amino acid-binding capacity of Ubr1 is diminished, Ubr1-mediated Plin2 degradation is inactivated, leading to steatosis. CONCLUSIONS: We identified Ubr1 as an amino acid sensor regulating Plin2 proteostasis, bridging the knowledge gap between steatosis and nutrient sensing. Our work may provide new strategies for the prevention and treatment of steatosis.

Dissection of the microRNA Network Regulating Hedgehog Signaling in Drosophila.

The evolutionarily conserved Hedgehog (Hh) signaling plays a critical role in embryogenesis and adult tissue homeostasis. Aberrant Hh signaling often leads to various forms of developmental anomalies and cancer. Since altered microRNA (miRNA) expression is associated with developmental defects and tumorigenesis, it is not surprising that several miRNAs have been found to regulate Hh signaling. However, these miRNAs are mainly identified through small-scale in vivo screening or in vitro assays. As miRNAs preferentially reduce target gene expression via the 3' untranslated region, we analyzed the effect of reduced expression of core components of the Hh signaling cascade on downstream signaling activity, and generated a transgenic Drosophila toolbox of in vivo miRNA sensors for core components of Hh signaling, including hh, patched (ptc), smoothened (smo), costal 2 (cos2), fused (fu), Suppressor of fused (Su(fu)), and cubitus interruptus (ci). With these tools in hand, we performed a genome-wide in vivo miRNA overexpression screen in the developing Drosophila wing imaginal disc. Of the twelve miRNAs identified, seven were not previously reported in the in vivo Hh regulatory network. Moreover, these miRNAs may act as general regulators of Hh signaling, as their overexpression disrupts Hh signaling-mediated cyst stem cell maintenance during spermatogenesis. To identify direct targets of these newly discovered miRNAs, we used the miRNA sensor toolbox to show that miR-10 and miR-958 directly target fu and smo, respectively, while the other five miRNAs act through yet-to-be-identified targets other than the seven core components of Hh signaling described above. Importantly, through loss-of-function analysis, we found that endogenous miR-10 and miR-958 target fu and smo, respectively, whereas deletion of the other five miRNAs leads to altered expression of Hh signaling components, suggesting that these seven newly discovered miRNAs regulate Hh signaling in vivo. Given the powerful effects of these miRNAs on Hh signaling, we believe that identifying their bona fide targets of the other five miRNAs will help reveal important new players in the Hh regulatory network.

Vitrectomy versus Combined Vitrectomy and Scleral Buckle for Repair of Primary Rhegmatogenous Retinal Detachment with Vitreous Hemorrhage.

PURPOSE: To compare pars plana vitrectomy (PPV) with combined PPV and scleral buckle (PPV/SB) for repair of primary rhegmatogenous retinal detachment (RRD) with associated vitreous hemorrhage (VH). DESIGN: Retrospective, observational study. PARTICIPANTS: Patients with RRD and associated VH who underwent PPV or PPV/SB from January 1, 2010, through August 31, 2020, were analyzed. METHOD: We performed a single-institution, retrospective, observational study of 224 eyes with RRD and VH at the time of detachment. We excluded eyes with <6 months of follow-up, a prior history of retinal detachment (RD) repair with vitrectomy or SB, VH that resolved before surgical intervention, and tractional or combined tractional and rhegmatogenous detachments. MAIN OUTCOME MEASURES: Single-surgery anatomic success (SSAS) at 6 months, defined as no recurrent RD requiring surgical intervention. RESULTS: Pars plana vitrectomy and PPV/SB were performed on 138 eyes (62%) and 85 eyes (38%), respectively. The mean age of the PPV and PPV/SB patients was 61.9 and 60.2 years, respectively. Single-surgery anatomic success was achieved in 107 of 138 eyes (77.5%) that underwent PPV and 78 of 85 eyes (91.7%) that underwent PPV/SB. The difference in SSAS between types of treatment was significant (P = 0.006). Mean visual acuity improvement in the PPV/SB group was 0.54 logMAR units greater than that in the PPV group (P = 0.126). The incidence of postoperative proliferative vitreoretinopathy in the PPV/SB group (11.7%) was lower than that in the PPV group (19.5%; P = 0.128). The rate of repeat PPV for non-RD reasons was similar for both the groups (P = 0.437). Final reattachment status was achieved in 137 of the 138 and 84 of the 85 eyes in the PPV and PPV/SB groups, respectively. Final visual acuity improvement was significantly better in eyes with PPV/SB than in eyes with PPV alone (logMAR 2.12 vs. 1.26, respectively; P = 0.011). CONCLUSIONS: In patients with RRD and VH, SSAS was superior in patients treated with PPV/SB compared with those treated with PPV alone. Although not significantly different, the PPV/SB group had better visual outcomes and a lower postoperative proliferative vitreoretinopathy rate.

Competition between two phosphatases fine-tunes Hedgehog signaling.

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. How its signaling activity is fine-tuned in response to fluctuated Hh gradient is less known. Here, we identify protein phosphatase V (PpV), the catalytic subunit of protein phosphatase 6, as a homeostatic regulator of Hh signaling. PpV is genetically upstream of widerborst (wdb), which encodes a regulatory subunit of PP2A that modulates high-level Hh signaling. We show that PpV negatively regulates Wdb stability independent of phosphatase activity of PpV, by competing with the catalytic subunit of PP2A for Wdb association, leading to Wdb ubiquitination and subsequent proteasomal degradation. Thus, regulated Wdb stability, maintained through competition between two closely related phosphatases, ensures graded Hh signaling. Interestingly, PpV expression is regulated by Hh signaling. Therefore, PpV functions as a Hh activity sensor that regulates Wdb-mediated PP2A activity through feedback mechanisms to maintain Hh signaling homeostasis.

A widely diverged locus involved in locomotor adaptation in Heliconius butterflies.

Heliconius butterflies have undergone adaptive radiation and therefore serve as an excellent system for exploring the continuum of speciation and adaptive evolution. However, there is a long-lasting paradox between their convergent mimetic wing patterns and rapid divergence in speciation. Here, we characterize a locus that consistently displays high divergence among Heliconius butterflies and acts as an introgression hotspot. We further show that this locus contains multiple genes related to locomotion and conserved in Lepidoptera. In light of these findings, we consider that locomotion traits may be under selection, and if these are heritable traits that are selected for, then they might act as species barriers.

A high-sensitivity HPLC-ELSD method for HPMC-AS quantification and its application in elucidating the release mechanism of HPMC-AS based amorphous solid dispersions.

Hydroxypropyl methylcellulose acetate succinate (HPMC-AS) is one of the most widely used polymers used in amorphous solid dispersions (ASD) for solubility and bioavailability enhancement of poorly water-soluble drugs. Once released from ASDs, HPMC-AS was often found to be highly effective in maintaining drug supersaturation, and this capability is dependent on the concentration and substitution types of this pH-dependent polymer. Therefore, accurate quantification of different grades of HPMC-AS allows us to better understand the release and supersaturation mechanisms of HPMC-AS based ASDs. Since previously reported analytical methods were unable to quantify HPMC-AS in a complex medium with enough sensitivity, we hereby developed a high-sensitivity HPLC-ELSD (evaporative light scattering detector) method with satisfactory specificity, linearity, accuracy and precision, to quantify HPMC-AS down to 20 µg/mL in dissolution media, with the presence of various commonly used pharmaceutical excipients. With the assistance of this method, we compared the intrinsic dissolution rates (IDR) of both the drug and the polymer of posaconazole ASDs based on different types of HPMC-AS. We observed that: 1) For ASDs that were spray dried and uniformly mixed, drug and polymer released simultaneously into the medium with practically identical IDRs slower than the IDR of pure HPMC-AS; 2) For ASDs that were heterogeneously mixed, IDRs of the drug and polymer were significantly slower or faster than the IDRs of the drug and polymer of the uniform ASDs, respectively. In summary, the high sensitivity HPLC-ELSD method established here can be readily applied to quantify HPMC-AS in various dissolution media, thus helps to reveal the release kinetics and mechanisms of different HPMC-AS based ASDs.

Predictive Modeling of Micellar Solubilization by Single and Mixed Nonionic Surfactants.

Micellar solubilization is an important concept in the delivery of poorly water-soluble drugs. The rational selection of the type and the amount of surfactant to be incorporated in a formulation require comprehensive solubility studies. These studies are time and material demanding, both of which are scarce, especially during late discovery and early development stages. We hypothesized that, if the solubilization mechanism or molecular interaction is similar, the solubilization capacity ratio (a newly defined parameter) is dictated by micellar structures, independent of drugs. We tested this hypothesis by performing solubility studies using 8 commonly used nonionic surfactants and 17 insoluble compounds with diverse characteristics. The results show a striking constant solubilization capacity ratio among the 8 nonionic surfactants, which allow us to develop predictive solubility models for both single and mixed surfactant systems. The vast majority of the predicted solubility values, using our developed models, fall within 2-fold of the experimentally determined values with high correlation coefficients. As expected, systems involving ionic surfactant sodium dodecyl sulfate, used as a negative control, do not follow this trend. Deviations from the model, observed in this study or envisioned, were discussed. In conclusion, we have established predictive models that are capable of predicting solubility in a wide range of nonionic micellar solutions with only 1 experimental measurement. The application of such a model will significantly reduce resource and greatly enhance drug product development efficiency.