Arterial Klotho Expression and FGF23 Effects on Vascular Calcification and Function

Recent studies support a role for FGF23 and its co-receptor Klotho in cardiovascular pathology, yet the underlying mechanisms remain largely elusive. Herein, we analyzed the expression of Klotho in mouse arteries and generated a novel mouse model harboring a vascular smooth muscle cell specific deletion of Klotho (Sm22-KL^−/−). Arterial Klotho expression was detected at very low levels with quantitative real-time PCR; Klotho protein levels were undetectable by immunohistochemistry and Western blot. There was no difference in arterial Klotho between Sm22-KL^−/− and wild-type mice, as well as no changes in serum markers of mineral metabolism. Intravenous delivery of FGF23 elicited a rise in renal (0.005; p<0.01) but not arterial Egr-1 expression, a marker of Klotho-dependent FGF23 signaling. Further, the impact of FGF23 on vascular calcification and endothelial response was evaluated in bovine vascular smooth muscle cells (bVSMC) and in a murine ex vivo model of endothelial function, respectively. FGF23 treatment (0.125–2 ng/mL) did not modify calcification in bVSMCs or dilatory, contractile and structural properties in mice arterial specimen ex vivo. Collectively, these results demonstrate that FGF23-Klotho signaling is absent in mouse arteries and that the vascular response was unaffected by FGF23 treatment. Thus, our data do not support Klotho-mediated FGF23 effects in the vasculature although confirmative studies in humans are warranted.     

Structural Mimicry of A-Loop Tyrosine Phosphorylation by a Pathogenic FGF Receptor 3 Mutation

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Production of avian influenza virus vaccine using primary cell cultures generated from host organs

The global availability of a therapeutically effective influenza virus vaccine during a pandemic remains a major challenge for the biopharmaceutical industry. Long production time, coupled with decreased supply of embryonated chicken eggs (ECE), significantly affects the conventional vaccine production. Transformed cell lines have attained regulatory approvals for vaccine production. Based on the fact that the avian influenza virus would infect the cells derived from its natural host, the viral growth characteristics were studied on chicken embryo-derived primary cell cultures. The viral propagation was determined on avian origin primary cell cultures, transformed mammalian cell lines, and in ECE. A comparison was made between these systems by utilizing various cell culture-based assays. In-vitro substrate susceptibility and viral infection characteristics were evaluated by performing hemagglutination assay (HA), 50 % tissue culture infectious dose (TCID₅₀) and monitoring of cytopathic effects (CPE) caused by the virus. The primary cell culture developed from chicken embryos showed stable growth characteristics with no contamination. HA, TCID₅₀, and CPE exhibited that these cell systems were permissive to viral infection, yielding 2–10 times higher viral titer as compared to mammalian cell lines. Though the viral output from the ECE was equivalent to the chicken cell culture, the time period for achieving it was decreased to half. Some of the prerequisites of inactivated influenza virus vaccine production include generation of higher vial titer, independence from exogenous sources, and decrease in the production time lines. Based on the tests, it can be concluded that chicken embryo primary cell culture addresses these issues and can serve as a potential alternative for influenza virus vaccine production.     

Amyloid fibril formation by native and modified bovine β-lactoglobulins proceeds through unfolded form of proteins: a comparative study

The misfolding and extracellular amyloid deposition of specific proteins are associated with a large family of human pathologies, often called protein conformational diseases. Despite the many efforts expended to characterize amyloid formation in vitro, there is no deep knowledge about the environment (in which aggregation occurs) as well as mechanism of this type of protein aggregation. Recently, β-lactoglobulin (β-lg) was driven toward amyloid aggregation under specific extreme conditions. In the present study, citraconylation was employed to neutralize the charges on accessible lysine residues of β-lg and different approaches such as turbidimetry, thermodynamic analysis, extrinsic fluorimetry and theoretical studies have been successfully used to compare the different behaviors of the native and modified proteins. Kinetic analyses of native β-lg aggregation showed a gradual development of turbidity, whereas the modified β-lg displayed an increased propensity toward aggregation. Our results clearly demonstrated that the stability of modified β-lg is markedly reduced, compared to the native one. Using of TANGO and WALTZ algorithms (as well as modelling softwares) which describe aggregation tendencies of different parts of a protein structure, we suggested critical importance of some of the lysine residues in the aggregation process. The results highlighted the critical role of protein stability and elucidated the underlying role of hydrophobic/electrostatic interactions in lactoglobulin-based experimental system.     

Molecular Mechanisms of Fibroblast Growth Factor Signaling in Physiology and Pathology

Fibroblast growth factors (FGFs) signal in a paracrine or endocrine fashion to mediate a myriad of biological activities, ranging from issuing developmental cues, maintaining tissue homeostasis, and regulating metabolic processes. FGFs carry out their diverse functions by binding and dimerizing FGF receptors (FGFRs) in a heparan sulfate (HS) cofactor- or Klotho coreceptor-assisted manner. The accumulated wealth of structural and biophysical data in the past decade has transformed our understanding of the mechanism of FGF signaling in human health and development, and has provided novel concepts in receptor tyrosine kinase (RTK) signaling. Among these contributions are the elucidation of HS-assisted receptor dimerization, delineation of the molecular determinants of ligand–receptor specificity, tyrosine kinase regulation, receptor cis-autoinhibition, and tyrosine trans-autophosphorylation. These structural studies have also revealed how disease-associated mutations highjack the physiological mechanisms of FGFR regulation to contribute to human diseases. In this paper, we will discuss the structurally and biophysically derived mechanisms of FGF signaling, and how the insights gained may guide the development of therapies for treatment of a diverse array of human diseases.Fibroblast growth factor (FGF) signaling fulfills essential roles in metazoan development and metabolism. A wealth of literature has documented the requirement for FGF signaling in multiple processes during embryogenesis, including implantation (Feldman et al. 1995), gastrulation (Sun et al. 1999), somitogenesis (Dubrulle and Pourquie 2004; Wahl et al. 2007; Lee et al. 2009; Naiche et al. 2011; Niwa et al. 2011), body plan formation (Martin 1998; Rodriguez Esteban et al. 1999; Tanaka et al. 2005; Mariani et al. 2008), morphogenesis (Metzger et al. 2008; Makarenkova et al. 2009), and organogenesis (Goldfarb 1996; Kato and Sekine 1999; Sekine et al. 1999; Sun et al. 1999; Colvin et al. 2001; Serls et al. 2005; Vega-Hernandez et al. 2011). Recent clinical and biochemical data have uncovered unexpected roles for FGF signaling in metabolic processes, including phosphate/vitamin D homeostasis (Consortium 2000; Razzaque and Lanske 2007; Nakatani et al. 2009; Gattineni et al. 2011; Kir et al. 2011), cholesterol/bile acid homeostasis (Yu et al. 2000a; Holt et al. 2003), and glucose/lipid metabolism (Fu et al. 2004; Moyers et al. 2007). Highlighting its diverse biology, deranged FGF signaling contributes to many human diseases, such as congenital craniosynostosis and dwarfism syndromes (Naski et al. 1996; Wilkie et al. 2002, 2005), Kallmann syndrome (Dode et al. 2003; Pitteloud et al. 2006a), hearing loss (Tekin et al. 2007, 2008), and renal phosphate wasting disorders (Shimada et al. 2001; White et al. 2001), as well as many acquired forms of cancers (Rand et al. 2005; Pollock et al. 2007; Gartside et al. 2009; di Martino et al. 2012). Endocrine FGFs have also been implicated in the progression of acquired metabolic disorders, including chronic kidney disease (Fliser et al. 2007), obesity (Inagaki et al. 2007; Moyers et al. 2007; Reinehr et al. 2012), and insulin resistance (Fu et al. 2004; Chen et al. 2008b; Chateau et al. 2010; Huang et al. 2011), giving rise to many opportunities for drug discovery in the field of FGF biology (Beenken and Mohammadi 2012).Based on sequence homology and phylogeny, the 18 mammalian FGFs are grouped into six subfamilies (Ornitz and Itoh 2001; Popovici et al. 2005; Itoh and Ornitz 2011). Five of these subfamilies act in a paracrine fashion, namely, the FGF1 subfamily (FGF1 and FGF2), the FGF4 subfamily (FGF4, FGF5, and FGF6), the FGF7 subfamily (FGF3, FGF7, FGF10, and FGF22), the FGF8 subfamily (FGF8, FGF17, and FGF18), and the FGF9 subfamily (FGF9, FGF16, and FGF20). In contrast, the FGF19 subfamily (FGF19, FGF21, and FGF23) signals in an endocrine manner (Beenken and Mohammadi 2012). FGFs exert their pleiotropic effects by binding and activating the FGF receptor (FGFR) subfamily of receptor tyrosine kinases that are coded by four genes (FGFR1, FGFR2, FGFR3, and FGFR4) in mammals (Johnson and Williams 1993; Mohammadi et al. 2005b). The extracellular domain of FGFRs consists of three immunoglobulin (Ig)-like domains (D1, D2, and D3), and the intracellular domain harbors the conserved tyrosine kinase domain flanked by the flexible amino-terminal juxtamembrane linker and carboxy-terminal tail (Lee et al. 1989; Dionne et al. 1991; Givol and Yayon 1992). A unique feature of FGFRs is the presence of a contiguous segment of glutamic and aspartic acids in the D1–D2 linker, termed the acid box (AB). The two-membrane proximal D2 and D3 and the intervening D2–D3 linker are necessary and sufficient for ligand binding/specificity (Dionne et al. 1990; Johnson et al. 1990), whereas D1 and the D1–D2 linker are implicated in receptor autoinhibition (Wang et al. 1995; Roghani and Moscatelli 2007; Kalinina et al. 2012). Alternative splicing and translational initiation further diversify both ligands and receptors. The amino-terminal regions of FGF8 and FGF17 can be differentially spliced to yield FGF8a, FGF8b, FGF8e, FGF8f (Gemel et al. 1996; Blunt et al. 1997), and FGF17a and FGF17b isoforms (Xu et al. 1999), whereas cytosine-thymine-guanine (CTG)-mediated translational initiation gives rise to multiple high molecular weight isoforms of FGF2 and FGF3 (Florkiewicz and Sommer 1989; Prats et al. 1989; Acland et al. 1990). The tissue-specific alternative splicing in D3 of FGFR1, FGFR2, and FGFR3 yields “b” and “c” receptor isoforms which, along with their temporal and spatial expression patterns, is the major regulator of FGF–FGFR specificity/promiscuity (Orr-Urtreger et al. 1993; Ornitz et al. 1996; Zhang et al. 2006). A large body of structural data on FGF–FGFR complexes has begun to reveal the intricate mechanisms by which different FGFs and FGFRs combine selectively to generate quantitatively and qualitatively different intracellular signals, culminating in distinct biological responses. In addition, these structural data have unveiled how pathogenic mutations hijack the normal physiological mechanisms of FGFR regulation to lead to pathogenesis. We will discuss the current state of the structural biology of the FGF–FGFR system, lessons learned from studying the mechanism of action of pathogenic mutations, and how the structural data are beginning to shape and advance the translational research.     

Quality of Medicines Commonly Used in the Treatment of Soil Transmitted Helminths and Giardia in Ethiopia: A Nationwide Survey

Background

The presence of poor quality medicines in the market is a global threat on public health, especially in developing countries. Therefore, we assessed the quality of two commonly used anthelminthic drugs [mebendazole (MEB) and albendazole (ALB)] and one antiprotozoal drug [tinidazole (TNZ)] in Ethiopia.

Methods/Principal Findings

A multilevel stratified random sampling, with as strata the different levels of supply chain system in Ethiopia, geographic areas and government/privately owned medicines outlets, was used to collect the drug samples using mystery shoppers. The three drugs (106 samples) were collected from 38 drug outlets (government/privately owned) in 7 major cities in Ethiopia between January and March 2012. All samples underwent visual and physical inspection for labeling and packaging before physico-chemical quality testing and evaluated based on individual monographs in Pharmacopoeias for identification, assay/content, dosage uniformity, dissolution, disintegration and friability. In addition, quality risk was analyzed using failure mode effect analysis (FMEA) and a risk priority number (RPN) was assigned to each quality attribute. A clinically rationalized desirability function was applied in quantification of the overall quality of each medicine. Overall, 45.3% (48/106) of the tested samples were substandard, i.e. not meeting the pharmacopoeial quality specifications claimed by their manufacturers. Assay was the quality attribute most often out-of-specification, with 29.2% (31/106) failure of the total samples. The highest failure was observed for MEB (19/42, 45.2%), followed by TNZ (10/39, 25.6%) and ALB (2/25, 8.0%). The risk analysis showed that assay (RPN = 512) is the most critical quality attribute, followed by dissolution (RPN = 336). Based on Derringer''s desirability function, samples were classified into excellent (14/106,13%), good (24/106, 23%), acceptable (38/106, 36%%), low (29/106, 27%) and bad (1/106,1%) quality.

Conclusions/Significance

This study evidenced that there is a relatively high prevalence of poor quality MEB, ALB and TNZ in Ethiopia: up to 45% if pharmacopoeial acceptance criteria are used in the traditional, dichotomous approach, and 28% if the new risk-based desirability approach was applied. The study identified assay as the most critical quality attributes. The country of origin was the most significant factor determining poor quality status of the investigated medicines in Ethiopia.     

Comparative study of the population level effects of carbaryl and abamectin on larval ectoparasitoid <Emphasis Type="Italic">Habrobracon hebetor</Emphasis> Say (Hymenoptera: Braconidae)

Habrobracon hebetor Say is one of the most important ectoparasitoids of the larval stage of many invasive agricultural pests such as noctuid and pyralid moths. Lethal and sublethal effects of carbaryl and abamectin were assessed on the ectoparasitoid H. hebetor in laboratory conditions. Dose–response bioassays were carried out on immature and adult stages by using dipping and contact residue methods, respectively. LC₅₀ (lethal concentration for 50%) values of carbaryl and abamectin were 31.19 and 4.16 mg a.i. l⁻¹ on larval stage and 12.6 and 4.42 mg a.i. l⁻¹ on adults, respectively. In order to assess the sublethal effects, adult wasps were exposed to an LC₃₀ of each insecticide and then the demographic parameters of live wasps were studied. The results showed that the intrinsic rate of increase (r _m ), finite rate of increase (λ), net reproductive rate (R₀) and gross reproductive rate (GRR) were significantly affected by the insecticides. The highest and the lowest r _m values were 0.23 and 0.17 in control and carbaryl-treated insects, respectively. The sex ratio of H. hebetor offspring was not significantly affected by either insecticide. The results showed that carbaryl had more adverse effects on population parameters of the parasitoid compared to abamectin. Field studies are needed to determine the total effects of the pesticides on H. hebetor.     

The potential of established turf cover for cleaning oily desert soil using rhizosphere technology

The rhizosphere of two turf cover sorts; Bermuda grass and American grass contained high numbers, 8.1 to 16.8 x 10(6) g(-1) of cultivable oil-utilizing and diazotrophic bacteria belonging predominantly to the genera Agrobacterium, Arthrobacter, Pseudomonas, Gordonia, and Rhodococcus. Those bacteria also grew on a nitrogen-free medium and demonstrated the ability to reduce acetylene to ethylene. These isolates grew on a wide range of n-alkanes (C9 to C40) and aromatic hydrocarbons, as sole sources of carbon. Quantitative determinations revealed that predominant bacteria consumed crude oil and representative aliphatic (n-octadecane) and aromatic (phenanthrene) hydrocarbons efficiently. The fact that those organisms had the combined activities of hydrocarbon-utilization and nitrogen-fixation makes them suitable tools for bioremediating oily desert areas that are normally poor in nitrogenous compounds. Phytoremediation experiments showed that spreading turf cover on oily desert soil inhibited oil volatilization and enhanced oil loss in soil by about 15%. Oil loss was also enhanced in turf free soil samples fertilized with NH4NO3. In conclusion, covering this oil-polluted soil with turf cover minimized atmospheric pollution, increased the numbers of the oil-utilizing/nitrogen-fixing bacteria by about 20 to 46% thus, encouraging oil attenuation.     

Measuring nonhomologous end-joining,homologous recombination and alternative end-joining simultaneously at an endogenous locus in any transfectable human cell

Double strand break (DSB) repair primarily occurs through 3 pathways: non-homologous end-joining (NHEJ), alternative end-joining (Alt-EJ), and homologous recombination (HR). Typical methods to measure pathway usage include integrated cassette reporter assays or visualization of DNA damage induced nuclear foci. It is now well understood that repair of Cas9-induced breaks also involves NHEJ, Alt-EJ, and HR pathways, providing a new format to measure pathway usage. Here, we have developed a simple Cas9-based system with validated repair outcomes that accurately represent each pathway and then converted it to a droplet digital PCR (ddPCR) readout, thus obviating the need for Next Generation Sequencing and bioinformatic analysis with the goal to make Cas9-based system accessible to more laboratories. The assay system has reproduced several important insights. First, absence of the key Alt-EJ factor Pol θ only abrogates ∼50% of total Alt-EJ. Second, single-strand templated repair (SSTR) requires BRCA1 and MRE11 activity, but not BRCA2, establishing that SSTR commonly used in genome editing is not conventional HR. Third, BRCA1 promotes Alt-EJ usage at two-ended DSBs in contrast to BRCA2. This assay can be used in any system, which permits Cas9 delivery and, importantly, allows rapid genotype-to-phenotype correlation in isogenic cell line pairs.