Gene expression differentiation in the reproductive tissues of Drosophila willistoni subspecies and their hybrids.

Early lineage diversification is central to understand what mutational events drive species divergence. Particularly, gene misregulation in interspecific hybrids can inform about what genes and pathways underlie hybrid dysfunction. In Drosophila hybrids, how regulatory evolution impacts different reproductive tissues remains understudied. Here, we generate a new genome assembly and annotation in Drosophila willistoni and analyse the patterns of transcriptome divergence between two allopatrically evolved D. willistoni subspecies, their male sterile and female fertile hybrid progeny across testis, male accessory gland, and ovary. Patterns of transcriptome divergence and modes of regulatory evolution were tissue-specific. Despite no indication for cell-type differences in hybrid testis, this tissue exhibited the largest magnitude of expression differentiation between subspecies and between parentals and hybrids. No evidence for anomalous dosage compensation in hybrid male tissues was detected nor was a differential role for the neo- and the ancestral arms of the D. willistoni X chromosome. Compared to the autosomes, the X chromosome appeared enriched for transgressively expressed genes in testis despite being the least differentiated in expression between subspecies. Evidence for fine genome clustering of transgressively expressed genes suggests a role of chromatin structure on hybrid gene misregulation. Lastly, transgressively expressed genes in the testis of the sterile male progeny were enriched for GO terms not typically associated with sperm function, instead hinting at anomalous development of the reproductive tissue. Our thorough tissue-level portrait of transcriptome differentiation between recently diverged D. willistoni subspecies and their hybrids provides a more nuanced view of early regulatory changes during speciation.

Approaches to Psychosocial Care Within Tertiary Diabetes Care Centres in Canada: An Environmental Scan.

BACKGROUND: Patients with diabetes facing psychosocial challenges often struggle with diabetes self-management, and thereby are at risk of complications arising from hyperglycemia and hypoglycemia. Despite the Diabetes Canada Clinical Practice Guidelines' recommendations for diabetes centres to offer psychosocial services for the screening and support of patients with psychosocial barriers, the extent to which these recommendations have been implemented in centres across Canada is unknown. METHODS: Tertiary adult diabetes centres affiliated with academic institutions were invited to participate in our quantitative survey (n=40). At each centre, a structured telephone interview was administered to a manager, as well as a social worker and psychologist (if employed at the centre), to inquire regarding the psychosocial services offered. RESULTS: Thirty diabetes centres (75%) completed at least one questionnaire. Participating diabetes centres varied considerably in capacity, employing from 3 to more than 50 diabetes educators and from 0 to 20 endocrinologists. Approximately 80% of centres reported providing patients with access to psychosocial services, but only about 50% had a dedicated, internal psychosocial team. Stratified results showed centres that are the main centres affiliated with academic teaching hospitals were more likely to have embedded psychosocial services and to make referrals for psychosocial concerns when compared with satellite centres. The majority of managers reported that their staffing and funding model provided inadequate psychosocial support and that increased funding for psychosocial care was either somewhat of a priority or a high priority. CONCLUSIONS: Diabetes centres across Canada struggle to offer adequate psychosocial services; greater funding and training for staff is required to uphold the Diabetes Canada Clinical Practice Guidelines.

Multiscale analysis of the randomization limits of the chromosomal gene organization between Lepidoptera and Diptera.

How chromosome gene organization and gene content evolve among distantly related and structurally malleable genomes remains unresolved. This is particularly the case when considering different insect orders. We have compared the highly contiguous genome assemblies of the lepidopteran Danaus plexippus and the dipteran Drosophila melanogaster, which shared a common ancestor around 290 Ma. The gene content of 23 out of 30 D. plexippus chromosomes was significantly associated with one or two of the six chromosomal elements of the Drosophila genome, denoting common ancestry. Despite the phylogenetic distance, 9.6% of the 1-to-1 orthologues still reside within the same ancestral genome neighbourhood. Furthermore, the comparison D. plexippus-Bombyx mori indicated that the rates of chromosome repatterning are lower in Lepidoptera than in Diptera, although still within the same order of magnitude. Concordantly, 14 developmental gene clusters showed a higher tendency to retain full or partial clustering in D. plexippus, further supporting that the physical association between the SuperHox and NK clusters existed in the ancestral bilaterian. Our results illuminate the scope and limits of the evolution of the gene organization and content of the ancestral chromosomes to the Lepidoptera and Diptera while helping reconstruct portions of the genome in their most recent common ancestor.

A de novo transcriptional atlas in Danaus plexippus reveals variability in dosage compensation across tissues.

A detailed knowledge of gene function in the monarch butterfly is still lacking. Here we generate a genome assembly from a Mexican nonmigratory population and used RNA-seq data from 14 biological samples for gene annotation and to construct an atlas portraying the breadth of gene expression during most of the monarch life cycle. Two thirds of the genes show expression changes, with long noncoding RNAs being particularly finely regulated during adulthood, and male-biased expression being four times more common than female-biased. The two portions of the monarch heterochromosome Z, one ancestral to the Lepidoptera and the other resulting from a chromosomal fusion, display distinct association with sex-biased expression, reflecting sample-dependent incompleteness or absence of dosage compensation in the ancestral but not the novel portion of the Z. This study presents extended genomic and transcriptomic resources that will facilitate a better understanding of the monarch's adaptation to a changing environment.

Poloxamer 188-Coated Ammonium Methacrylate Copolymer Nanocarriers Enhance Loperamide Permeability across Pgp-Expressing Epithelia.

Loperamide is a µ-opioid agonist with poor gastrointestinal absorption, mainly because of its modest aqueous solubility and being a P-glycoprotein (Pgp) efflux substrate. Nevertheless, studies associated with therapeutic effects strongly suggest that loperamide holds potential pharmacological advantages over traditional µ-opioid agonists commonly used for analgesia. Thus, in this Communication, we assessed in MDCK-hMDR1 cell lines the effects over loperamide uptake and efflux ratio, when loaded into Eudragit RS (ERS) nanocarriers coated with poloxamer 188 (P188). ERS was chosen for enhancing loperamide aqueous dispersibility and P188 as a potential negative Pgp modulator. In uptake assays, it was observed that Pgp limited the accumulation of loperamide into cells and that preincubation with P188, but not coincubation, led to increasing loperamide uptake at a similar extent of Pgp pharmacological inhibition. On the other hand, the efflux ratio displayed no alterations when Pgp was pharmacologically inhibited, whereas ERS/P188 nanocarriers effectively enhanced loperamide uptake and absorptive transepithelial transport. The latter suggests that loperamide transport across cells is significantly influenced by the presence of the unstirred water layer (UWL), which could hinder the visualization of Pgp-efflux effects during transport assays. Thus, results in this work highlight that formulating loperamide into this nanocarrier enhances its uptake and transport permeability.

Metformin Transport in Native MDCK-Wt and MDCK-II Monolayers Unveils Functional Inter-Strains Differences Influencing Drug Permeability.

PURPOSE: MDCK cells are commonly used to assess drug permeability, but the existence of various strains merits a comparative functional study. Since metformin absorption is largely mediated by transporters and paracellular diffusion, we used it to functionally compare MDCK-wt and MDCK-II. METHODS: Uptake, bidirectional transport and efflux experiments were performed using different buffers, pH, and a panel of transporter inhibitors. Relative contributions to total transport in both strains were estimated. RESULTS: Metformin uptake into MDCK-wt was linear but saturable in MDCK-II. Uptake into MDCK-wt or -II was promoted at pH 5.4 or 8.4, respectively. Quinidine and cimetidine similarly inhibited uptake in both strains. Lopinavir (PMAT specific) at pH 5.4 or pyrimethamine (MATE specific) at pH 8.4 differentially inhibited MDCK-wt or -II, respectively. Transport at pH 7.4 was absorptive regardless of strains, but secretory (MDCK-II) or absorptive (MDCK-wt) at pH 5.4. Efflux was largely basolateral in both strains. While paracellular permeability was similar between strains, total transport was dominated by transporters in MDCK-II or paracellular diffusion in MDCK-wt. CONCLUSIONS: Metformin transport revealed functional differences between MDCK strains. Apical uptake was governed by MATE in MDCK-II or PMAT in MDCK-wt, such that metformin transport was either secretory or absorptive, respectively.

"Development of Fixed Dose Combination Products" Workshop Report: Considerations of Gastrointestinal Physiology and Overall Development Strategy.

The gastrointestinal (GI) tract is one of the most popular and used routes of drug product administration due to the convenience for better patient compliance and reduced costs to the patient compared to other routes. However, its complex nature poses a great challenge for formulation scientists when developing more complex dosage forms such as those combining two or more drugs. Fixed dose combination (FDC) products are two or more single active ingredients combined in a single dosage form. This formulation strategy represents a novel formulation which is as safe and effective compared to every mono-product separately. A complex drug product, to be dosed through a complex route, requires judicious considerations for formulation development. Additionally, it represents a challenge from a regulatory perspective at the time of demonstrating bioequivalence (BE) for generic versions of such drug products. This report gives the reader a summary of a 2-day short course that took place on the third and fourth of November at the Annual Association of Pharmaceutical Scientists (AAPS) meeting in 2018 at Washington, D.C. This manuscript will offer a comprehensive view of the most influential aspects of the GI physiology on the absorption of drugs and current techniques to help understand the fate of orally ingested drug products in the complex environment represented by the GI tract. Through case studies on FDC product development and regulatory issues, this manuscript will provide a great opportunity for readers to explore avenues for successfully developing FDC products and their generic versions.

Structural requirements of the human sodium-dependent bile acid transporter (hASBT): role of 3- and 7-OH moieties on binding and translocation of bile acids.

Bile acids (BAs) are the end products of cholesterol metabolism. One of the critical steps in their biosynthesis involves the isomerization of the 3ß-hydroxyl (-OH) group on the cholestane ring to the common 3α-configuration on BAs. BAs are actively recaptured from the small intestine by the human Apical Sodium-dependent Bile Acid Transporter (hASBT) with high affinity and capacity. Previous studies have suggested that no particular hydroxyl group on BAs is critical for binding or transport by hASBT, even though 3ß-hydroxylated BAs were not examined. The aim of this study was to elucidate the role of the 3α-OH group on BAs binding and translocation by hASBT. Ten 3ß-hydroxylated BAs (Iso-bile acids, iBAs) were synthesized, characterized, and subjected to hASBT inhibition and uptake studies. hASBT inhibition and uptake kinetics of iBAs were compared to that of native 3α-OH BAs. Glycine conjugates of native and isomeric BAs were subjected to molecular dynamics simulations to identify topological descriptors related to binding and translocation by hASBT. Iso-BAs bound to hASBT with lower affinity and exhibited reduced translocation than their respective 3α-epimers. Kinetic data suggests that, in contrast to native BAs where hASBT binding is the rate-limiting step, iBAs transport was rate-limited by translocation and not binding. Remarkably, 7-dehydroxylated iBAs were not hASBT substrates, highlighting the critical role of 7-OH group on BA translocation by hASBT, especially for iBAs. Conformational analysis of gly-iBAs and native BAs identified topological features for optimal binding as: concave steroidal nucleus, 3-OH "on-" or below-steroidal plane, 7-OH below-plane, and 12-OH moiety toward-plane. Our results emphasize the relevance of the 3α-OH group on BAs for proper hASBT binding and transport and revealed the critical role of 7-OH group on BA translocation, particularly in the absence of a 3α-OH group. Results have implications for BA prodrug design.

Putative irreversible inhibitors of the human sodium-dependent bile acid transporter (hASBT; SLC10A2) support the role of transmembrane domain 7 in substrate binding/translocation.

PURPOSE: To explore the involvement of transmembrane domain (TM) 7 of the human apical sodium-dependent bile acid transporter (hASBT) on bile acid (BA) binding/translocation, using two electrophilic BA derivatives as molecular probes. METHODS: Two electrophilic derivatives of chenodeoxycholic acid (CDCA) were designed, synthesized and evaluated for their ability to inactivate hASBT, and the human organic cation/carnitine transporter (hOCTN2) as a control (i.e. a non-BA transporting model). The ability of electrophilic derivatives to interact with hASBT was evaluated by 2-aminoethyl-methanethiosulfonate (MTSEA)-biotin labeling of thiol groups in TM7 cysteine mutants. RESULTS: Unlike native BAs, the electrophilic CDCA derivatives specifically inactivated hASBT, but not hOCTN2, and inhibited hASBT in a time- and concentration-dependent fashion. Preincubation of hASBT Cys-mutants in the exofacial half of TM7 with reactive electrophilic probes blocked transporter biotinylation by MTSEA-biotin, similar to 2-(trimethylammonium)ethyl-methanethiosulfonate (MTSET) blocking. This blocking pattern differed from that produced by native BAs, which exposed exofacial TM7 residues, thereby increasing staining. CONCLUSION: Kinetic and biochemical data indicate these novel electrophilic BAs are potent and specific irreversible inhibitors of hASBT and offer new evidence about the role of TM7 in binding/translocation of bile acids.

Inhibition requirements of the human apical sodium-dependent bile acid transporter (hASBT) using aminopiperidine conjugates of glutamyl-bile acids.

PURPOSE: Synthesize aminopiperidine conjugates of glutamyl-bile acids (glu-BAs) and develop a hASBT inhibition model using the conformationally sampled pharmacophore (CSP) approach. METHODS: glu-BAs aminopiperidine conjugates were synthesized. hASBT inhibition was measured as K(i). A CSP-SAR model was built using structural and physico-chemical descriptors and evaluated via cross-validation. RESULTS: Twenty-nine aminopiperidine conjugates were synthesized. All inhibited hASBT, with K(i) ranging from 0.95 to 31.8 muM. Amidation of the piperidine nitrogen slightly decreased activity, while replacement by a carbon increased potency. Esterification of the glutamic acid linker had a minor impact, suggesting that a negative charge around C-24 is not required for binding. Three quantitative CSP-SAR models were developed. The best model (r (2) = 0.813, Q (2) = 0.726) included two descriptors: angle between 7-OH, alpha-substituent and centroid of rings B and C, and electrostatic contribution to the solvation free-energy. The model successfully distinguished between compounds with K(i) < 16muM and K(i) > 16muM. Models indicated that hydrophobicity, alpha substituent orientation, and partially compacted side chain conformation promote inhibitory potency. Qualitative CSP-SAR analysis indicated that the presence of an internal salt bridge, resulting in a locked conformation of the side chain, yielded weaker inhibitors. CONCLUSIONS: Aminopiperidine conjugates of glu-BAs were potent hASBT inhibitors. A predictive and robust CSP-SAR model was developed.

Method to screen substrates of apical sodium-dependent bile acid transporter.

Human apical sodium-dependent bile acid transporter (hASBT) is a potential prodrug target under study. Development of prodrugs that target hASBT may yield compounds with low solubility and/or susceptibility to hydrolysis. A transport uptake method is needed that increases compound solubility and avoids NaOH for cell lysis during postexperimental cell sample preparation. The overall goal was to develop an assay method to screen for hASBT uptake of novel compounds. The first objective was to determine the maximum cosolvent concentrations that are compatible with an hASBT active transport assay. The second objective was to develop a NaOH-free cell lysis method to process cell samples from these uptake studies. The following cosolvents were studied: dimethylacetamide (DMAC), dimethylformamide (DMF), dimethylsulfoxide (DMSO), ethanol, methanol, polyethylene glycol-400, propylene glycol, and dioxane. Initial studies included taurocholate flux studies across hASBT-Madin-Darby canine kidney monolayers using up to 10% cosolvent, as well as cytotoxicity studies. The effect of selected cosolvent concentrations on the hASBT Michaelis-Menten kinetic parameters was evaluated. Additionally, two acetonitrile-based cell lysis methods that do not use NaOH were evaluated in terms of percent sample recovery and hASBT kinetic parameters. Results showed that the maximum permissible cosolvent concentrations for hASBT uptake studies, without compromising assay results or causing cytotoxicity, are 1% DMAC, 1% DMF, 2.5% DMSO, 2.5% methanol, and 2.5% ethanol. Additionally, both NaOH-free, acetonitrile-based cell lysis methods provided similar recovery and hASBT results, compared to NaOH method. Hence, an assay method was developed to screen for active transport, allowing for cosolvents that can solubilize compounds and avoid NaOH sample treatment, which can otherwise degrade compound.