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Structure of lipoprotein lipase in complex with GPIHBP1.
No full textLipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1
Evaluating rectal swab collection method for gut microbiome analysis in the common marmoset (Callithrix jacchus)
No full textThe common marmoset (Callithrix jacchus) is increasingly used as an animal model for biomedical research; however, gastrointestinal diseases causing significant morbidity are endemic in many captive marmoset colonies. Establishing gut microbiome patterns in a marmoset colony may aid in clinical decision-making and model reproducibility. A standardized method of sample collection and storage is essential for proper interpretation of microbiome data. While microbiome studies commonly utilize fecal samples, the goal of this study was to determine whether the microbiome profile from a rectal swab performed on a sedated animal was comparable to the microbiome profile from a fecal sample. During routine physical exams, paired fecal and rectal swab samples were collected from each of 23 marmosets. DNA was extracted from all fecal and rectal swab samples and 16S ribosomal RNA gene sequences were amplified and analyzed. Initial comparison of the relative abundance of bacterial phyla between paired samples had a r2 value of 0.70 with S of 0.08 with no significant differences in α and β diversity metrics between fecal and rectal samples. Initial analysis however, revealed 5 discordant fecal-rectal pairs which corresponded only with the 5 rectal swabs that were classified as free of visible fecal matter during collection. Exclusion of these 5 pairs resulted in an optimized fit of the data as evidenced by a r2 value of 0.91 with S of 0.05. These results demonstrate that rectal swabs are a reliable method for profiling the fecal microbiome in the marmoset since the bacterial composition from a rectal swab with visible fecal contents correlated well with the bacterial composition from a fecal sample from the same marmoset. This study highlights the importance of standardized sample collection methods and exclusion of inappropriate samples
A Review of Pain Assessment Methods in Laboratory Rodents.
No full textEnsuring that laboratory rodent pain is well managed underpins the ethical acceptability of working with these animals in research. Appropriate treatment of pain in laboratory rodents requires accurate assessments of the presence or absence of pain to the extent possible. This can be challenging some situations because laboratory rodents are prey species that may show subtle signs of pain. Although a number of standard algesiometry assays have been used to assess evoked pain responses in rodents for many decades, these methods likely represent an oversimplification of pain assessment and many require animal handling during testing, which can result in stress-induced analgesia. More recent pain assessment methods, such as the use of ethograms, facial grimace scoring, burrowing, and nest-building, focus on evaluating changes in spontaneous behaviors or activities of rodents in their home environments. Many of these assessment methods are time-consuming to conduct. While many of these newer tests show promise for providing a more accurate assessment of pain, most require more study to determine their reliability and sensitivity across a broad range of experimental conditions, as well as between species and strains of animals. Regular observation of laboratory rodents before and after painful procedures with consistent use of 2 or more assessment methods is likely to improve pain detection and lead to improved treatment and care-a primary goal for improving overall animal welfare
The H+-Activated Ovarian Cancer G Protein-Coupled Receptor 1 (OGR1) is responsible for Renal Calcium Loss during Acidosis
No full textABSTRACT
Hypercalciuria is a common feature during metabolic acidosis. However, the mechanisms sensing acidosis and inducing increased urinary calcium excretion during acidosis are still unknown. Here we report that mice deficient for the Ovarian cancer G-protein coupled receptor 1 (OGR1 or Gpr68) did not excrete more calcium during chronic metabolic acidosis. Wild type (OGR1+/+) and OGR1-deficient mice (OGR1-/-) were subjected to standard chow (control) or 0.28 M NH4Cl in water for 1 day (acute metabolic acidosis) or 2 % NH4Cl in food for 7 days (chronic metabolic acidosis). OGR1 mRNA is ubiquitously expressed, including kidneys, and found along the entire nephron. No differences in responding to the acid load were observed in OGR1-/- mice, except for higher plasma [HCO3-] after 1 day. Bone mineral density and resorption activity of osteoclasts were similar between OGR1+/+ and OGR1-/- mice. Plasma PTH and Vitamin D3 levels were indistinguishable. However, the expression levels of key proteins for active transepithelial Ca2+ reabsorption in the distal convoluted tubule, TRPV5 and Calbindin-D28k were increased in OGR1-/- mice under metabolic acidosis. TRPV5 abundance was downregulated in wild type mice during metabolic acidosis but maintained at the same level in the absence of OGR1. OGR1-/- also exhibited higher NHE3 abundance when compared to OGR1+/+ under metabolic acidosis. In conclusion, OGR1 is a pH sensor involved in the hypercalciuria developed during metabolic acidosis and may regulate renal calcium excretion through modulation of proximal tubular NHE3 activity and regulation of the distal tubule TRPV5 calcium channel
A single-cell atlas of the airway epithelium reveals the CFTR-rich pulmonary ionocyte
No full textThe functions of epithelial tissues are dictated by the types, abundance and distribution of the differentiated cells they contain. Attempts to restore tissue function after damage require knowledge of how physiological tasks are distributed among cell types, and how cell states vary between homeostasis, injury–repair and disease. In the conducting airway, a heterogeneous basal cell population gives rise to specialized luminal cells that perform mucociliary clearance1. Here we perform single-cell profiling of human bronchial epithelial cells and mouse tracheal epithelial cells to obtain a comprehensive census of cell types in the conducting airway and their behaviour in homeostasis and regeneration. Our analysis reveals cell states that represent known and novel cell populations, delineates their heterogeneity and identifies distinct differentiation trajectories during homeostasis and tissue repair. Finally, we identified a novel, rare cell type that we call the ‘pulmonary ionocyte’, which co-expresses FOXI1, multiple subunits of the vacuolar-type H+-ATPase (V-ATPase) and CFTR, the gene that is mutated in cystic fibrosis. Using immunofluorescence, modulation of signalling pathways and electrophysiology, we show that Notch signalling is necessary and FOXI1 expression is sufficient to drive the production of the pulmonary ionocyte, and that the pulmonary ionocyte is a major source of CFTR activity in the conducting airway epithelium
Degradation of recombinant proteins of diverse formats by CHO host cell proteases is circumvented via knock-out of CHO matriptase
No full textAn increasing number of biologics are entering the pipeline of pharmaceutical companies. Today, the preferred production host for therapeutic proteins are CHO cells. However one of the major hurdles especially for the production of non-antibody glycoproteins is host cell-related proteolytic degradation which can drastically impact developability and timelines of pipeline projects
N-Butylpyrrolidone as a green solvent for Solid Phase Peptide Synthesis
No full textBy means of a systematic approach, several candidate solvents were tested for their feasibility of replacing the reprotoxic DMF as solvent used in large scale manufacture of peptide by the Solid Phase Peptide Synthesis (SPPS) methodology. N-Butylpyrrolidone (NBP) was found to be a viable green alternative solvent to replace DMF, appropriate to all stages of the SPPS
Discovery of the First Potent, Selective, and Orally Bioavailable Signal Peptide Peptidase-Like 2a (SPPL2a) Inhibitor Displaying Pronounced Immunomodulatory Effects In Vivo
No full textSignal peptide peptidase-like 2a (SPPL2a) is an aspartic intramembrane protease which has recently been shown to play an important role in the development and function of antigen presenting cells such as B lymphocytes and dendritic cells. In this paper, we describe the discovery of the first selective and orally active SPPL2a inhibitor (S)-2-cyclopropyl-N1-((S)-5,11-dioxo-10,11-dihydro-1H,3H,5H-spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1′-cyclopropan]-10-yl)-N4-(5-fluoro-2-methylpyridin-3-yl)succinamide 40 (SPL-707). This compound shows adequate selectivity against the closely related enzymes γ-secretase and SPP and a good pharmacokinetic profile in mouse and rat. Compound 40 significantly inhibited processing of the SPPL2a substrate CD74/p8 fragment in rodents at doses ≤10 mg/kg b.i.d. po. Oral dosing of 40 for 11 days at ≥10 mg/kg b.i.d. recapitulated the phenotype seen in Sppl2a knockout (ko) and ENU mutant mice (reduced number of specific B cells and myeloid dendritic cells). Thus, we believe that SPPL2a represents an interesting and druggable pharmacological target, potentially providing a novel approach for the treatment of autoimmune diseases by targeting B cells and dendritic cells
Verification of a physiologically based pharmacokinetic model of ritonavir to estimate drug–drug interaction potential of CYP3A4 substrates
No full textRitonavir is one of several ketoconazole alternatives used to evaluate strong CYP3A4 inhibition potential in clinical drug–drug interaction (DDI) studies. In this study, four physiologically based pharmacokinetic (PBPK) models of ritonavir as an in vivo time-dependent inhibitor of CYP3A4 were created and verified for oral doses of 20, 50, 100 and 200 mg using the fraction absorbed (Fa) and oral clearance (CLoral) values reported in the literature, because transporter and CYP enzyme reaction phenotyping data were not available. The models were used subsequently to predict and compare the magnitude of the AUC increase in nine reference DDI studies evaluating the effect of ritonavir at steady-state on midazolam (CYP3A4 substrate) exposure. Midazolam AUC and Cmax ratios were predicted within 2-fold of the respective observations in seven studies. Simulations of the hepatic and gut CYP3A4 abundance after multiple oral dosing of ritonavir indicated that a 3-day treatment with ritonavir 100 mg twice daily is sufficient to reach maximal CYP3A4 inhibition and subsequent systemic exposure increase of a CYP3A4 substrate, resulting in the reliable estimation of fm,CYP3A4. The ritonavir model was submitted as part of the new drug application for Kisqali® (ribociclib) and accepted by health authorities
Opportunities and challenges for combining chemo- and biocatalysis
No full textThe last decade has seen a substantial increase of successful examples for the combination of chemo- and biocatalysis for multistep syntheses. This is driven by obvious advantages such as higher yields, decreased costs, environmental benefits and high selectivity. On the downside, efforts must be undertaken to combine the divergent reaction conditions, reagent tolerance, and solvent systems of the 'different worlds of catalysis'. Thanks to progress in enzyme discovery and engineering as well as in the development of milder and more compatible conditions for various chemocatalysts, many limitations could already be overcome. This review highlights the opportunities by prominent examples for combined syntheses, but also the current challenges and emerging solutions keeping in mind the fast progress in transition metal-, organo-, photo- electro-, and biocatalysis