Zebrafish Model as a Screen to Prevent Cyst Inflation in Autosomal Dominant Polycystic Kidney Disease.

In autosomal dominant polycystic kidney disease (ADPKD), kidney cyst growth requires the recruitment of CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel that is defective in cystic fibrosis. We have been studying cyst inflation using the zebrafish Kupffer's vesicle (KV) as model system because we previously demonstrated that knocking down polycystin 2 (PC2) induced a CFTR-mediated enlargement of the organ. We have now quantified the PC2 knockdown by showing that it causes a 73% reduction in the number of KV cilia expressing PC2. According to the literature, this is an essential event in kidney cystogenesis in ADPKD mice. Additionally, we demonstrated that the PC2 knockdown leads to a significant accumulation of CFTR-GFP at the apical region of the KV cells. Furthermore, we determined that KV enlargement is rescued by the injection of Xenopus pkd2 mRNA and by 100 µM tolvaptan treatment, the unique and approved pharmacologic approach for ADPKD management. We expected vasopressin V2 receptor antagonist to lower the cAMP levels of KV-lining cells and, thus, to inactivate CFTR. These findings further support the use of the KV as an in vivo model for screening compounds that may prevent cyst enlargement in this ciliopathy, through CFTR inhibition.

Role of the sugar moiety on the opioid receptor binding and conformation of a series of enkephalin neoglycopeptides.

Glycosylation by simple sugars is a drug discovery alternative that has been explored with varying success for enhancing the potency and bioavailability of opioid peptides. Long ago we described two O-glycosides having either ß-Glucose and ß-Galactose of (d-Met2, Pro5)-enkephalinamide showing one of the highest antinociceptive activities known. Here, we report the resynthesis of these two analogs and the preparation of three novel neoglycopeptide derivatives (α-Mannose, ß-Lactose and ß-Cellobiose). Binding studies to cloned zebrafish opioid receptors showed very small differences of affinity between the parent compound and the five glycopeptides thus suggesting that the nature of the carbohydrate moiety plays a minor role in determining the binding mode. Indeed, NMR conformational studies, combined with molecular mechanics calculations, indicated that all glycopeptides present the same major conformation either in solution or membrane-like environment. The evidences provided here highlight the relevance for in vivo activity of the conjugating bond between the peptide and sugar moieties in opioid glycopeptides.

Enhanced colonic delivery of ciclosporin A self-emulsifying drug delivery system encapsulated in coated minispheres.

OBJECTIVES: Investigate the potential of coated minispheres (SmPill®) to enhance localized Ciclosporin A (CsA) delivery to the colon. METHODS: CsA self-emulsifying drug delivery systems (SEDDS) were encapsulated into SmPill® minispheres. Varying degrees of coating thickness (low, medium and high) were applied using ethylcellulose and pectin (E:P) polymers. In vitro CsA release was evaluated in simulated gastric and intestinal media. Bioavailability of CsA in vivo following oral administration to pigs of SmPill® minispheres was compared to Neoral® po and Sandimmun® iv in a pig model. CsA concentrations in blood and intestinal tissue were determined by HPLC-UV. RESULTS: In vitro CsA release from coated minispheres decreased with increasing coating thickness. A linear relationship was observed between in vitro CsA release and in vivo bioavailability (r(2) = 0.98). CsA concentrations in the proximal, transverse and distal colon were significantly higher following administration of SmPill®, compared to Neoral® po and Sandimmun® iv (p < 0.05). Analysis of transverse colon tissue subsections also revealed significantly higher CsA concentrations in the mucosa and submucosa using SmPill® minispheres (p < 0.05). CONCLUSIONS: Modulating E:P coating thickness controls release of CsA from SmPill® minispheres. Coated minispheres limited CsA release in the small intestine and enhanced delivery and uptake in the colon. These findings demonstrate clinical advantages of an oral coated minisphere-enabled CsA formulation in the treatment of inflammatory conditions of the large intestine.

Improving Heat Transfer at the Bottom of Vials for Consistent Freeze Drying with Unidirectional Structured Ice.

The quality of lyophilized products is dependent of the ice structure formed during the freezing step. Herein, we evaluate the importance of the air gap at the bottom of lyophilization vials for consistent nucleation, ice structure, and cake appearance. The bottom of lyophilization vials was modified by attaching a rectified aluminum disc with an adhesive material. Freezing was studied for normal and converted vials, with different volumes of solution, varying initial solution temperature (from 5°C to 20°C) and shelf temperature (from -20°C to -40°C). The impact of the air gap on the overall heat transfer was interpreted with the assistance of a computational fluid dynamics model. Converted vials caused nucleation at the bottom and decreased the nucleation time up to one order of magnitude. The formation of ice crystals unidirectionally structured from bottom to top lead to a honeycomb-structured cake after lyophilization of a solution with 4% mannitol. The primary drying time was reduced by approximately 35%. Converted vials that were frozen radially instead of bottom-up showed similar improvements compared with normal vials but very poor cake quality. Overall, the curvature of the bottom of glass vials presents a considerable threat to consistency by delaying nucleation and causing radial ice growth. Rectifying the vials bottom with an adhesive material revealed to be a relatively simple alternative to overcome this inconsistency.

Optimized Proteomic Mass Spectrometry Characterization of Recombinant Human µ-Opioid Receptor Functionally Expressed in Pichia pastoris Cell Lines.

Human µ-opioid receptor (hMOR) is a class-A G-protein-coupled receptor (GPCR), a prime therapeutic target for the management of moderate and severe pain. A chimeric form of the receptor has been cocrystallized with an opioid antagonist and resolved by X-ray diffraction; however, further direct structural analysis is still required to identify the active form of the receptor to facilitate the rational design of hMOR-selective agonist and antagonists with therapeutic potential. Toward this goal and in spite of the intrinsic difficulties posed by the highly hydrophobic transmembrane motives of hMOR, we have comprehensively characterized by mass spectrometry (MS) analysis the primary sequence of the functional hMOR. Recombinant hMOR was overexpressed as a C-terminal c-myc and 6-his tagged protein using an optimized expression procedure in Pichia pastoris cells. After membrane solubilization and metal-affinity chromatography purification, a procedure was devised to enhance the concentration of the receptor. Subsequent combinations of in-solution and in-gel digestions using either trypsin, chymotrypsin, or proteinase K, followed by matrix-assisted laser desorption ionization time-of-flight MS or nanoliquid chromatography coupled with tandem MS analyses afforded an overall sequence coverage of up to >80%, a level of description first attained for an opioid receptor and one of the six such high-coverage MS-based analyses of any GPCR.

Influence of polar side chains modifications on the dual enkephalinase inhibitory activity and conformation of human opiorphin, a pain perception related peptide.

The dual inhibitory action of the pain related peptide opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH) against neutral endopeptidase (NEP) and aminopeptidase N (AP-N) was further investigated by a SAR study involving minor modifications on the polar side chains of Arg residues and glycosylation with monosaccharides at Ser. None of them exerted dual or individual inhibitory potency superior than opiorphin. However, the correlations deduced offer further proof for the key role of these residues upon the binding and bioactive conformational stabilization of opiorphin. NMR conformational studies on the glycopeptides suggest that they are still very flexible compounds that may attain their respective bioactive conformations.

Intramuscular degloving injuries to the rectus femoris: findings at MRI.

OBJECTIVE: The purpose of this study was to describe the MRI characteristics of intramuscular degloving injuries to the rectus femoris whereby the inner bipennate component of the indirect head is dissociated from its superficial unipennate component. CONCLUSION: Injuries to the rectus femoris muscle are common in athletes, especially European football (soccer) players. The complex muscle-within-a-muscle anatomic configuration of the rectus femoris makes it susceptible to various types of injuries.

Singlet oxygen in antimicrobial photodynamic therapy: photosensitizer-dependent production and decay in E. coli.

Several families of photosensitizers are currently being scrutinized for antimicrobial photodynamic therapy applications. Differences in physical and photochemical properties can lead to different localization patterns as well as differences in singlet oxygen production and decay when the photosensitizers are taken up by bacterial cells. We have examined the production and fate of singlet oxygen in Escherichia coli upon photosensitization with three structurally-different cationic photosensitizers, namely New Methylene Blue N (NMB), a member of the phenothiazine family, ACS268, a hydrophobic porphyrin with a single cationic alkyl chain, and zinc(II)-tetramethyltetrapyridinoporphyrazinium salt, a phthalocyanine-like photosensitizer with four positive charges on the macrocycle core. The kinetics of singlet oxygen production and decay indicate different localization for the three photosensitizers, whereby NMB appears to localize in an aqueous-like microenvironment, whereas ACS268 localizes in an oxygen-shielded site, highly reactive towards singlet oxygen. The tetracationic zinc(II) tetrapyridinoporphyrazine is extensively aggregated in the bacteria and fails to produce any detectable singlet oxygen.

Lyophilization cycle design for highly concentrated protein formulations supported by micro freeze-dryer and heat flux sensor.

High-concentration protein formulations (HCPFs) represent a common strategy and freeze-drying can mitigate the stability challenges of HCPFs. In general, an in-depth characterization of the lyophilization process is essential to not impair the product quality by inappropriate process parameters. The aim of this study was to create a primary drying design space for lyophilized HCPFs by utilizing the heat flux sensor (HFS) integrated in a MicroFD with a minimum number of cycles and product vials. All the necessary data to obtain the design space were determined starting from only two lyophilization cycles, each holding 19 vials. The vial heat transfer coefficient (Kv) was determined by the HFS and compared to gravimetric values. The results indicate a consistant offset between the HFS and the gravimetry based values for annealed samples with higher protein content. This work highlights a possibility of integrating new technologies, the HFS and the MicroFD to generate a design space for lyophilization of HCPFs, which enables to implement a QbD approach at minimal material and time investment.

Design of freeze-drying cycles: The determination of heat transfer coefficient by using heat flux sensor and MicroFD.

The complexity of biopharmaceuticals requires often the freeze-drying as stabilizing process. Inadequate parameters in the primary drying phase can impair product quality, besides, increasing time and costs. Therefore, the process requires a thorough characterization and with this purpose, heat flux sensor (HFS) and miniaturized freeze-dryers conceived to emulate larger equipment, were recently introduced. Our study investigates, for the first time, the use of HFS and miniaturized freeze-dryer (MicroFD) in combination to obtain the heat transfer coefficient (Kv) for two formulation types and freezing protocols. First, as the MicroFD presents the possibility to set the temperature of vial surrounding (LyoSIM), it was determined which set-up was representative for a lab-scale freeze drying process. Additionally, the HFS-based results were compared with the data obtained by the most accurate, but time-consuming and invasive gravimetric method. Second, the role of atypical heat transfer was evaluated for HFS and gravimetric methodology with gold-coated and un-coated vials. Obtained results revealed the HFS and the MicroFD can be used in combination to obtain Kv real-time with much less effort that gravimetrically, to study different vial scenarios, and to design lyophilization processes with a limited amount of material and experiments.

Burnout and Self-Perceived Stress in Workers in Essential Services after the Impact of Hurricanes Irma and Maria.

OBJECTIVE: To examine the levels of burnout and self-perceived stress in workers at the Puerto Rico Electric Power Authority, who experienced major disasters: Hurricanes Irma and Maria. METHODS: A quantitative descriptive cross-sectional study with a non-probabilistic convenience sample was conducted in workers at the Puerto Rico Electric Power Authority. A structured questionnaire was administered to a sample of 163 eligible participants, aged 21 years and older, who participated voluntarily. Using employees of the electric company, the study examined the relationships between burnout and several characteristics (years of employment, existing health conditions, and coping strategies) both before and after Hurricanes Irma and Maria. Burnout was assessed with Gil-Monte's Spanish Burnout Inventory, and self-perceived stress was assessed with the 14-item Perceived Stress Scale. RESULTS: Before the hurricanes, 16.6% of the workers reported high levels of burnout syndrome, while, after the hurricanes, the proportion increased to one-fifth (20.9%). Prior to the 2 hurricanes, more than one-fourth (23.4%) of the sample reported being extremely stressed; after the hurricanes, that proportion increased to 55%. Factors such as years of employment, counseling, and self-perceived stress showed significant statistical associations (P < .05) with burnout. CONCLUSION: From a public health standpoint, priority should be given to this population, thereby preventing burnout and any other negative effects of the aftermath (i.e., the lengthy response, recovery, and reconstruction) of these kinds of major disaster.

Antigenic diversity among Portuguese clinical isolates of Helicobacter pylori.

BACKGROUND: The human gastroduodenal pathogen, Helicobacter pylori, is characterized by an unusual extent of genetic heterogeneity. This dictates differences in the antigenic pattern of strains resulting in heterogeneous human humoral immune responses. Here, we examined the antigenic variability among a group of 10 strains isolated from Portuguese patients differing in age, gender, and H. pylori-associated gastric diseases. MATERIAL AND METHODS: Immunoassays were performed on two-dimensional electrophoresis gels obtained for the proteome of each strain, using a commercial pool of antibodies produced in rabbit, against the whole cell lysate of an Australian H. pylori strain. Relevant proteins were identified by mass spectrometry. RESULTS: Immunoproteomes of the Portuguese strains showed no correlation between the number of antigenic proteins or the antigenic profile, and the disease to which each strain was associated. The Heat shock protein B was the unique immunoreactive protein common to all of them. Additionally, seven proteins were found to be antigenic in at least 80% of strains: enoyl-(acyl-carrier-protein) reductase (NADH); Catalase; Flagellin A; 2 isoforms of alkyl hydroperoxide reductase; succinyl-CoA transferase subunit B; and an unidentified protein. These proteins were present in the proteome of all tested strains, suggesting that differences in their antigenicity are related to antigenic variance. CONCLUSIONS: This study showed evidence of the variability of antigenic pattern among H. pylori strains. We believe that this fact contributes to the failure of anti-H. pylori vaccines and the low accuracy of serological tests based on a low number of proteins or antigens of only one strain.

Synthesis, biological evaluation and structural characterization of novel glycopeptide analogues of nociceptin N/OFQ.

To examine if the biological activity of the N/OFQ peptide, which is the native ligand of the pain-related and viable drug target NOP receptor, could be modulated by glycosylation and if such effects could be conformationally related, we have synthesized three N/OFQ glycopeptide analogues, namely: [Thr(5)-O-α-D-GalNAc-N/OFQ] (glycopeptide 1), [Ser(10)-O-α-D-GalNAc]-N/OFQ (glycopeptide 2) and [Ser(10)-O-ß-D-GlcNAc]-N/OFQ] (glycopeptide 3). They were tested for biological activity in competition binding assays using the zebrafish animal model in which glycopeptide 2 exhibited a slightly improved binding affinity, whereas glycopeptide 1 showed a remarkably reduced binding affinity compared to the parent compound and glycopeptide 3. The structural analysis of these glycopeptides and the parent N/OFQ peptide by NMR and circular dichroism indicated that their aqueous solutions are mainly populated by random coil conformers. However, in membrane mimic environments a certain proportion of the molecules of all these peptides exist as α-helix structures. Interestingly, under these experimental conditions, glycopeptide 1 (glycosylated at Thr-5) exhibited a population of folded hairpin-like geometries. From these facts it is tempting to speculate that nociceptin analogues showing linear helical structures are more complementary and thus interact more efficiently with the native NOP receptor than folded structures, since glycopeptide 1 showed a significantly reduced binding affinity for the NOP receptor.

Pkd2 Affects Cilia Length and Impacts LR Flow Dynamics and Dand5.

The left-right (LR) field recognizes the importance of the mechanism involving the calcium permeable channel Polycystin-2. However, whether the early LR symmetry breaking mechanism is exclusively via Polycystin-2 has not been tested. For that purpose, we need to be able to isolate the effects of decreasing the levels of Pkd2 protein from any eventual effects on flow dynamics. Here we demonstrate that curly-up (cup) homozygous mutants have abnormal flow dynamics. In addition, we performed one cell stage Pkd2 knockdowns and LR organizer specific Pkd2 knockdowns and observed that both techniques resulted in shorter cilia length and abnormal flow dynamics. We conclude that Pkd2 reduction leads to LR defects that cannot be assigned exclusively to its putative role in mediating mechanosensation because indirectly, by modifying cell shape or decreasing cilia length, Pkd2 deficit affects LR flow dynamics.

Detecting chromosomal interactions in Capture Hi-C data with CHiCAGO and companion tools.

Capture Hi-C is widely used to obtain high-resolution profiles of chromosomal interactions involving, at least on one end, regions of interest such as gene promoters. Signal detection in Capture Hi-C data is challenging and cannot be adequately accomplished with tools developed for other chromosome conformation capture methods, including standard Hi-C. Capture Hi-C Analysis of Genomic Organization (CHiCAGO) is a computational pipeline developed specifically for Capture Hi-C analysis. It implements a statistical model accounting for biological and technical background components, as well as bespoke normalization and multiple testing procedures for this data type. Here we provide a step-by-step guide to the CHiCAGO workflow that is aimed at users with basic experience of the command line and R. We also describe more advanced strategies for tuning the key parameters for custom experiments and provide guidance on data preprocessing and downstream analysis using companion tools. In a typical experiment, CHiCAGO takes ~2-3 h to run, although pre- and postprocessing steps may take much longer.

CiliarMove: new software for evaluating ciliary beat frequency helps find novel mutations by a Portuguese multidisciplinary team on primary ciliary dyskinesia.

Evaluation of ciliary beat frequency (CBF) performed by high-speed videomicroscopy analysis (HVMA) is one of the techniques required for the correct diagnosis of primary ciliary dyskinesia (PCD). Currently, due to lack of open-source software, this technique is widely performed by visually counting the ciliary beatings per a given time-window. Our aim was to generate open-source, fast and intuitive software for evaluating CBF, validated in Portuguese PCD patients and healthy volunteers. Nasal brushings collected from 17 adult healthy volunteers and 34 PCD-referred subjects were recorded using HVMA. Evaluation of CBF was compared by two different methodologies: the new semi-automated computer software CiliarMove and the manual observation method using slow-motion movies. Clinical history, nasal nitric oxide and transmission electron microscopy were performed for diagnosis of PCD in the patient group. Genetic analysis was performed in a subset (n=8) of suspected PCD patients. The correlation coefficient between the two methods was R2=0.9895. The interval of CBF values obtained from the healthy control group (n=17) was 6.18-9.17 Hz at 25°C. In the PCD-excluded group (n=16), CBF ranged from 6.84 to 10.93 Hz and in the PCD group (n=18), CBF ranged from 0 to 14.30 Hz. We offer an automated open-source programme named CiliarMove, validated by the manual observation method in a healthy volunteer control group, a PCD-excluded group and a PCD-confirmed group. In our hands, comparisons between CBF intervals alone could discern between healthy and PCD groups in 78% of the cases.

Heat flux sensor to create a design space for freeze-drying development.

Freeze-drying methodology requires an in-depth understanding and characterization for optimal processing of biopharmaceuticals. Particularly the primary drying phase, the longest and most expensive stage of the process, is of interest for optimization. The currently used process analytical technology (PAT) tools give highly valuable insights but come with limitations. Our study describes, for the first time, the application of a heat flux sensor (HFS) to build a primary drying design space and predict the process evolution. First, the heat transfer coefficient (Kv) generated by HFS and by the most accurate, but time-consuming and invasive, gravimetric method were compared. Second, the applicability to generate a design space was tested and verified. Obtained results revealed a good agreement of the values generated from this new and fast HFS compared to the gravimetric determination. Additionally, residual moisture assessed by Karl-Fischer titration and frequency modulated spectroscopy (FMS) support the quality of the obtained predictions. Thus, the HFS approach can substantially accelerate evaluation, development and transfer of a freeze-drying cycle.

Evaluation of Sucrose Laurate as an Intestinal Permeation Enhancer for Macromolecules: Ex Vivo and In Vivo Studies.

Oral delivery of macromolecules requires permeation enhancers (PEs) adaptable to formulation. Sucrose laurate (SL) (D1216), a food grade surfactant, was assessed in Caco-2 monolayers, isolated rat intestinal tissue mucosae, and rat intestinal instillations. Accordingly, 1 mM SL increased the apparent permeability coefficient (Papp) of [14C]-mannitol and reduced transepithelial electrical resistance (TEER) across monolayers. It altered expression of the tight junction protein, ZO-1, increased plasma membrane potential, and decreased mitochondrial membrane potential in Caco-2 cells. The concentrations that increased flux were of the same order as those that induced cytotoxicity. In rat colonic tissue mucosae, the same patterns emerged in respect to the concentration-dependent increases in paracellular marker fluxes and TEER reductions with 5 mM being the key concentration. While the histology revealed some perturbation, ion transport capacity was retained. In rat jejunal and colonic instillations, 50 and 100 mM SL co-administered with insulin induced blood glucose reductions and achieved relative bioavailability values of 2.4% and 8.9%, respectively, on a par with the gold standard PE, sodium caprate (C10). The histology of the intestinal loops revealed little damage. In conclusion, SL is a candidate PE with high potential for emulsion-based systems. The primary action is plasma membrane perturbation, leading to tight junction openings and a predominant paracellular flux.

Alpha-galactosylceramide enhances mucosal immunity to oral whole-cell cholera vaccines.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae) that results in 3-4 million cases globally with 100,000-150,000 deaths reported annually. Mostly confined to developing nations, current strategies to control the spread of cholera include the provision of safe drinking water and improved sanitation and hygiene, ideally in conjunction with oral vaccination. However, difficulties associated with the costs and logistics of these strategies have hampered their widespread implementation. Specific challenges pertaining to oral cholera vaccines (OCVs) include a lack of safe and effective adjuvants to further enhance gut immune responses, the complex and costly multicomponent vaccine manufacturing, limitations of conventional liquid formulation and the lack of an integrated delivery platform. Herein we describe the use of the orally active adjuvant α-Galactosylceramide (α-GalCer) to strongly enhance intestinal bacterium- and toxin-specific IgA responses to the OCV, Dukoral® in C57BL/6 and BALB/c mice. We further demonstrate the mucosal immunogenicity of a novel multi-antigen, single-component whole-cell killed V. cholerae strain and the enhancement of its immunogenicity by adding α-GalCer. Finally, we report that combining these components and recombinant cholera toxin B subunit in the SmPill® minisphere delivery system induced strong intestinal and systemic antigen-specific antibody responses.

An oral alpha-galactosylceramide adjuvanted Helicobacter pylori vaccine induces protective IL-1R- and IL-17R-dependent Th1 responses.

Helicobacter pylori causes chronic gastric infection that can lead to peptic ulcers and is an identified risk factor for gastric cancer development. Although much effort has been put into the development of a Helicobacter pylori vaccine over the last three decades, none has yet reached clinical application. Specific challenges pertaining to effective H. pylori vaccine development include the lack of proven vaccine-effective antigens and safe mucosal adjuvants to enhance local immune responses as well as the lack of accepted correlates of protection. Herein, we demonstrate that prophylactic intragastric immunisation with a whole-cell killed H. pylori antigen administered together with the non-toxic oral adjuvant α-galactosylceramide (α-GalCer) induced effective immune protection against H. pylori infection in mice, which was of similar magnitude as when using the "gold standard" cholera toxin as adjuvant. We further describe that this α-GalCer-adjuvanted vaccine formulation elicited strong intestinal and systemic Th1 responses as well as significant antigen-specific mucosal and systemic antibody responses. Finally, we report that the protective intestinal Th1 responses induced by α-GalCer are dependent on CD1d, IL-1R as well as IL-17R signalling. In summary, our results show that α-GalCer is a promising adjuvant for inclusion in an oral vaccine against H. pylori infection.