An electroolfactogram study of odor response patterns from the mouse olfactory epithelium with reference to receptor zones and odor sorptiveness.

Olfactory sensory neuron (OSN) responses to odors, measured at the population level, tend to be spatially heterogeneous in the vertebrates that have been studied. These response patterns vary between odors but are similar across subjects for a given stimulus. However, few species have been studied making functional interpretation of these patterns problematic. One proximate explanation for the spatial heterogeneity of odor responses comes from evidence that olfactory receptor (OR) genes in rodents are expressed in OSN populations that are spatially restricted to a few zones in the olfactory epithelium (OE). A long-standing functional explanation for response anisotropy in the OE posits that it is the signature of a supplementary mechanism for quality coding, based on the sorptive properties of odor molecules. These theories are difficult to assess because most mapping studies have utilized few odors, provided little replication, or involved but a single species (rat). In fact, to our knowledge, a detailed olfactory response "map" has not been reported for mouse, the species used in most studies of gene localization. Here we report the results of a study of mouse OE response patterns using the electroolfactogram (EOG). We focused on the medial aspect of olfactory turbinates that are accessible in the midsagittal section. This limited approach still allowed us to test predictions derived from the zonal distribution of OSN types and the sorption hypothesis. In 3 separate experiments, 290 mice were used to record EOGs from a set of standard locations along each of 4 endoturbinates utilizing 11 different odors resulting in over 4,400 separate recordings. Our results confirmed a marked spatial heterogeneity in odor responses that varied with odor, as seen in other species. However, no discontinuities were found in the odor-specific response patterns across the OE as might have been predicted given the existence of classical receptor zones nor did we find clear support for the hypothesis that OE response patterns, presumably a reflection of OSN distribution, have been shaped through natural selection by the relative sorptive properties of odors. We propose that receptor zones may be an epiphenomenon of a contingent evolutionary process. In this formulation, constraints on developmental programs for distributing OSN classes within the OE may be minimally related to the odor ligands of specific class members. Further, we propose that odor sorptiveness, which appears to be correlated with the inherent response patterns in the OE of larger species, may be of minimal effect in mice owing to scaling issues.

CTC-5: A novel digital pathology approach to characterise circulating tumour cell biodiversity.

Metastatic progression and tumor evolution complicates the clinical management of cancer patients. Circulating tumor cell (CTC) characterization is a growing discipline that aims to elucidate tumor metastasis and evolution processes. CTCs offer the clinical potential to monitor cancer patients for therapy response, disease relapse, and screen 'at risk' groups for the onset of malignancy. However, such clinical utility is currently limited to breast, prostate, and colorectal cancer patients. Further understanding of the basic CTC biology of other malignancies is required to progress them towards clinical utility. Unfortunately, such basic clinical research is often limited by restrictive characterization methods and high-cost barrier to entry for CTC isolation and imaging infrastructure. As experimental clinical results on applications of CTC are accumulating, it is becoming clear that a two-tier system of CTC isolation and characterization is required. The first tier is to facilitate basic research into CTC characterization. This basic research then informs a second tier specialised in clinical prognostic and diagnostic testing. This study presented in this manuscript describes the development and application of a low-cost, CTC isolation and characterization pipeline; CTC-5. This approach uses an established 'isolation by size' approach (ScreenCell Cyto) and combines histochemical morphology stains and multiparametric immunofluorescence on the same isolated CTCs. This enables capture and characterization of CTCs independent of biomarker-based pre-selection and accommodates both single CTCs and clusters of CTCs. Additionally, the developed open-source software is provided to facilitate the synchronization of microscopy data from multiple sources (https://github.com/CTC5/). This enables high parameter histochemical and immunofluorescent analysis of CTCs with existing microscopy infrastructure without investment in CTC specific imaging hardware. Our approach confirmed by the number of successful tests represents a potential major advance towards highly accessible low-cost technology aiming at the basic research tier of CTC isolation and characterization. The biomarker independent approach facilitates closing the gap between malignancies with poorly, and well-defined CTC phenotypes. As is currently the case for some of the most commonly occurring breast, prostate and colorectal cancers, such advances will ultimately benefit the patient, as early detection of relapse or onset of malignancy strongly correlates with their prognosis.

Early origins of heart disease: low birth weight and determinants of cardiomyocyte endowment.

1. World-wide epidemiological and experimental animal studies demonstrate that adversity in fetal life, resulting in intrauterine growth restriction, programmes the offspring for a greater susceptibility to ischaemic heart disease and heart failure in adult life. 2. After cardiogenesis, cardiomyocyte endowment is determined by a range of hormones and signalling pathways that regulate cardiomyocyte proliferation, apoptosis and the timing of multinucleation/terminal differentiation. 3. The small fetus may have reduced cardiomyocyte endowment owing to the impact of a suboptimal intrauterine environment on the signalling pathways that regulate cardiomyocyte proliferation, apoptosis and the timing of terminal differentiation.

Gulf Stream Deflection by a Bottom Feature off Charleston, South Carolina.

A topographic feature on the continental slope off Charleston at 32 degrees N persistently deflects the Gulf Stream seaward, with the inshore surface thermal front deflected east or south of east in 27 of the 39 cases examined. Meanders often form downstream of the deflection, suggesting that the "Charleston bump" induces Gulf Stream fluctuations.

The endosomal network.

Endocytosis is the process by which extracellular molecules are captured by the cell surface membrane and then taken up into the cell. Once inside the cell, the internalized material is delivered to its final destination via a complex system of organelles, termed the endosomal network. These heterogeneous structures play a key role in the delivery of extracellular and cellular material towards the lysosome for macromolecular degradation. The internalization of mannose-6-phosphate receptors at the cell surface, and their subsequent delivery to the late endosome, is the basis of enzyme replacement therapy in patients with lysosomal storage diseases (LSDs). This review describes the characteristics of the endosomal network and discusses how disturbances in vesicular trafficking or intracellular signaling may be important in the pathology of LSDs.

Enzyme replacement therapy in a feline model of Maroteaux-Lamy syndrome.

We report studies that suggest enzyme replacement therapy will result in a significant reduction in disease progression and tissue pathology in patients with Maroteaux-Lamy syndrome (Mucopolysaccharidosis type VI, MPS VI). A feline model for MPS VI was used to evaluate tissue distribution and clinical efficacy of three forms of recombinant human N-acetylgalactosamine-4-sulfatase (rh4S, EC 3.1.6.1). Intravenously administered rh4S was rapidly cleared from circulation. The majority of rh4S was distributed to liver, but was also detected in most other tissues. Tissue half-life was approximately 2-4 d. Three MPS VI cats given regular intravenous infusions of rh4S for up to 20 mo showed variable reduction of storage vacuoles in Kupffer cells and connective tissues, however cartilage chondrocytes remained vacuolated. Vertebral bone mineral volume was improved in two MPS VI cats in which therapy was initiated before skeletal maturity, and increased bone volume appeared to correlate with earlier age of onset of therapy. One cat showed greater mobility in response to therapy.

Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and Is required for late endosome-lysosome fusion.

Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.

Imaging and lipidomics methods for lipid analysis in metabolic and cardiovascular disease.

Cardiometabolic diseases exhibit changes in lipid biology, which is important as lipids have critical roles in membrane architecture, signalling, hormone synthesis, homoeostasis and metabolism. However, Developmental Origins of Health and Disease studies of cardiometabolic disease rarely include analysis of lipids. This short review highlights some examples of lipid pathology and then explores the technology available for analysing lipids, focussing on the need to develop imaging modalities for intracellular lipids. Analytical methods for studying interactions between the complex endocrine and intracellular signalling pathways that regulate lipid metabolism have been critical in expanding our understanding of how cardiometabolic diseases develop in association with obesity and dietary factors. Biochemical methods can be used to generate detailed lipid profiles to establish links between lifestyle factors and metabolic signalling pathways and determine how changes in specific lipid subtypes in plasma and homogenized tissue are associated with disease progression. New imaging modalities enable the specific visualization of intracellular lipid traffic and distribution in situ. These techniques provide a dynamic picture of the interactions between lipid storage, mobilization and signalling, which operate during normal cell function and are altered in many important diseases. The development of methods for imaging intracellular lipids can provide a dynamic real-time picture of how lipids are involved in complex signalling and other cell biology pathways; and how they ultimately regulate metabolic function/homoeostasis during early development. Some imaging modalities have the potential to be adapted for in vivo applications, and may enable the direct visualization of progression of pathogenesis of cardiometabolic disease after poor growth in early life.

Intracellular distribution and stability of a luminescent rhenium(i) tricarbonyl tetrazolato complex using epifluorescence microscopy in conjunction with X-ray fluorescence imaging.

Optical epifluorescence microscopy was used in conjunction with X-ray fluorescence imaging to monitor the stability and intracellular distribution of the luminescent rhenium(i) complex fac-[Re(CO)3(phen)L], where phen = 1,10-phenathroline and L = 5-(4-iodophenyl)tetrazolato, in 22Rv1 cells. The rhenium complex showed no signs of ancillary ligand dissociation, a conclusion based on data obtained via X-ray fluorescence imaging aligning iodine and rhenium distributions. A diffuse reticular localisation was detected for the complex in the nuclear/perinuclear region of cells, by either optical or X-ray fluorescence imaging techniques. X-ray fluorescence also showed that the rhenium complex disrupted the homeostasis of some biologically relevant elements, such as chlorine, potassium and zinc.

Unprecedented staining of polar lipids by a luminescent rhenium complex revealed by FTIR microspectroscopy in adipocytes.

Fourier transform infrared (FTIR) microspectroscopy and confocal imaging have been used to demonstrate that the neutral rhenium(i) tricarbonyl 1,10-phenanthroline complex bound to 4-cyanophenyltetrazolate as the ancillary ligand is able to localise in regions with high concentrations of polar lipids such as phosphatidylethanolamine (PE), sphingomyelin, sphingosphine and lysophosphatidic acid (LPA) in mammalian adipocytes.

A membrane protein primarily associated with the lysosomal compartment.

A monoclonal antibody designated MBR 39 has been generated against a membrane associated protein found selectively on lysosomes. MBR 39 reacts with the cytosolic face of the lysosome and was used to develop an organelle binding assay which reacted with high density organelles characteristic of lysosomes. These organelles contained lysosomal enzyme markers which included the integral membrane protein acetyl-CoA:alpha-glucosaminide N-acetyltransferase and the soluble lysosomal enzyme markers acid phosphatase (mature form), beta-hexosaminidase, arylsulfatase, and alpha-L-iduronidase. Under conditions which disrupt lysosomes the release of the latter soluble lysosomal enzymes was demonstrated from MBR 39 bound organelles. Immunoblots of MBR 39 with purified fibroblast lysosomal membrane, demonstrated reactivity with polypeptides of molecular mass 63 kDa (major species) and 73 kDa (minor species).

Immune response to enzyme replacement therapy: clinical signs of hypersensitivity reactions and altered enzyme distribution in a high titre rat model.

Immune responses to enzyme replacement therapy (ERT) have been reported and can result in a hypersensitivity/anaphylactic reaction during or immediately after enzyme infusion. We have investigated the infusion of the lysosomal enzyme N-acetylgalactosamine 4-sulphatase (4-sulphatase) into immunized, high titre rats as a model of immune response to ERT. To simulate ERT, high and low titre rats were infused with different doses of radiolabelled recombinant human 4-sulphatase (3H-rh4S). There was evidence of altered targeting, inactivation and degradation of 4-sulphatase in high titre (titre 1024000) compared to low titre (titre 64) rats. There was more 4-sulphatase enzyme activity detected in 5 mg/kg high titre rats when compared to 1 mg/kg high titre rats, suggesting that the antibodies could be saturable in vivo. However, the rats treated with 5 mg/kg 3H-rh4S all had clinical signs of hypersensitivity reactions to 4-sulphatase infusion. There were no apparent signs of adverse reactions in either the high titre 1 mg/kg rats or the low titre rats (1, 5 mg/kg). The high titre 5 mg/kg rats also had changes in 3H-rh4S distribution, with lower levels delivered to the liver and a marked increase in the level remaining in plasma, when compared to either 1 mg/kg high titre rats or low titre rats (1, 5 mg/kg).

Enzyme replacement therapy in Mucopolysaccharidosis VI: evidence for immune responses and altered efficacy of treatment in animal models.

Enzyme replacement therapy (ERT) can potentially result in an immunological response to the introduced protein. The immunological response by Mucopolysaccharidosis type VI (MPS VI) cats to recombinant human N-acetylgalactosamine 4-sulfatase (rh4S) ERT has been investigated. Plasma antibody titres to rh4S were detected in untreated MPS VI and normal control cats, but the antibody titres to rh4S were higher in ERT treated MPS VI cats. The reactivity by cats to rh4S did not appear to be just due to species cross reactivity, as plasma antibodies from normal control, MPS VI and MPS VI ERT cats reacted equally with feline and human 4-sulfatase. Normal control and MPS VI human plasma also had antibody titres to rh4S. Plasma antibodies to rh4S, from an ERT treated cat, could be temporarily removed from circulation by enzyme infusion, confirming specificity for rh4S and indicating a possible window for ERT in the absence of antibody. In enzyme distribution studies with 3H-rh4S, evidence of altered targeting, and enzyme inactivation and degradation were observed in high compared to low titre rats. In high titre rats, the observed loss of 3H-label from vacuolar organelles of the liver may represent either degradation of antibody bound 3H-rh4S for reutilisation within the liver, or antigen presentation. The development of high titre antibody may have a detrimental effect on the efficacy of ERT.

Genotype-phenotype correlations in mucopolysaccharidosis type I using enzyme kinetics, immunoquantification and in vitro turnover studies.

Fibroblasts from 16 patients with known alpha-L-iduronidase gene mutations and different clinical phenotypes of mucopolysaccharidosis type I (MPS I) were investigated in order to establish genotype/phenotype correlations. Enzyme kinetic studies were performed using the specific alpha-L-iduronidase substrate iduronosyl anhydro[1-3H]mannitol-6-sulfate. Specific residual enzyme activities were estimated using the kinetic parameters and an immunoquantification assay which determines levels of alpha-L-iduronidase protein. Cells were cultured in the presence of [35S]sulfate and the in vivo degradation of accumulated labelled glycosaminoglycans measured after different chase times. Residual enzyme activity and different amounts of residual enzyme protein were present in extracts from 9 of 16 cell lines covering a wide spectrum of clinical severity. Catalytic capacity, calculated as the product of kcat/Km and ng iduronidase protein per mg cell protein, was shown in most cases to be directly related to the severity of clinical phenotype, with up to 7% of normal values for patients with the attenuated form of MPS I (Scheie) and less than 0.13% for severely affected patients (Hurler) In vitro turnover studies allowed further refinement of correlations between genotype and phenotype. Scheie disease compared to Hurler disease patients were shown to accumulate smaller amounts of glycosaminoglycans that were also turned over faster. A combination of turnover and residual enzyme data established a correlation between the genotype, the biochemical phenotype and the clinical course of this lysosomal storage disorder.

Atg9 is required for intraluminal vesicles in amphisomes and autolysosomes.

Autophagy is an intracellular recycling and degradation process, which is important for energy metabolism, lipid metabolism, physiological stress response and organism development. During Drosophila development, autophagy is up-regulated in fat body and midgut cells, to control metabolic function and to enable tissue remodelling. Atg9 is the only transmembrane protein involved in the core autophagy machinery and is thought to have a role in autophagosome formation. During Drosophila development, Atg9 co-located with Atg8 autophagosomes, Rab11 endosomes and Lamp1 endosomes-lysosomes. RNAi silencing of Atg9 reduced both the number and the size of autophagosomes during development and caused morphological changes to amphisomes/autolysosomes. In control cells there was compartmentalised acidification corresponding to intraluminal Rab11/Lamp-1 vesicles, but in Atg9 depleted cells there were no intraluminal vesicles and the acidification was not compartmentalised. We concluded that Atg9 is required to form intraluminal vesicles and for localised acidification within amphisomes/autolysosomes, and consequently when depleted, reduced the capacity to degrade and remodel gut tissue during development.

Identification and molecular characterization of alpha-L-iduronidase mutations present in mucopolysaccharidosis type I patients undergoing enzyme replacement therapy.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a deficiency of alpha-L-iduronidase (IDUA). Mutations in the gene are responsible for the enzyme deficiency, which leads to the intralysosomal storage of the partially degraded glycosaminoglycans dermatan sulfate and heparan sulfate. Molecular characterization of MPS I patients has resulted in the identification of over 70 distinct mutations in the IDUA gene. The high degree of molecular heterogeneity reflects the wide clinical variability observed in MPS I patients. Six novel mutations, c.1087C>T (p.R363C), c.1804T>A (p.F602I), c.793G>C, c.712T>A (p.L238Q), c.1727+2T>A, and c.1269C>G (p.S423R), in a total of 14 different mutations, and 13 different polymorphic changes, including the novel c.246C>G (p.H82Q), were identified in a cohort of 10 MPS I patients enrolled in a clinical trial of enzyme-replacement therapy. Five novel amino acid substitutions and c.236C>T (p.A79V) were engineered into the wild-type IDUA cDNA and expressed. A p.G265R read-through mutation, arising from the c.793G>C splice mutation, was also expressed. Each mutation reduced IDUA protein and activity levels to varying degrees with the processing of many of the mutant forms also affected by IDUA. The varied properties of the expressed mutant forms of IDUA reflect the broad range of biochemical and clinical phenotypes of the 10 patients in this study. IDUA kinetic data derived from each patient's cultured fibroblasts, in combination with genotype data, was used to predict disease severity. Finally, residual IDUA protein concentration in cultured fibroblasts showed a weak correlation to the degree of immune response to enzyme-replacement therapy in each patient.

Mutational analysis of mucopolysaccharidosis type VI patients undergoing a trial of enzyme replacement therapy.

Mucopolysaccharidosis type VI (MPS VI), or Maroteaux-Lamy syndrome, is a lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (ARSB). Seven MPS VI patients were chosen for the initial clinical trial of enzyme replacement therapy. Direct sequencing of genomic DNA from these patients was used to identify ARSB mutations. Each individual exon of the ARSB gene was amplified by PCR and subsequently sequenced. Nine substitutions (c.289C>T [p.Q97X], c.629A>G [p.Y210C], c.707T>C [p.L236P], c.936G>T [p.W312C], c.944G>A [p.R315Q], c.962T>C [p.L321P], c.979C>T [p.R327X], c.1151G>A [p.S384N], and c.1450A>G [p.R484G]), two deletions (c.356_358delTAC [p.Y86del] and c.427delG), and one intronic mutation (c.1336+2T>G) were identified. A total of 7 out of the 12 mutations identified were novel (p.Y86del, p.Q97X, p.W312C, p.R327X, c.427delG, p.R484G, and c.1336+2T>G). Two of these novel mutations (p.Y86del and p.W312C) were expressed in Chinese hamster ovary cells and analyzed for residual ARSB activity and mutant ARSB protein. The two common polymorphisms c.1072G>A [p.V358M] and c.1126G>A [p.V376M] were identified among the patients, along with the silent mutation c.1191A>G. Cultured fibroblast ARSB mutant protein and residual activity were determined for each patient, and, together with genotype information, were used to predict the expected clinical severity of each MPS VI patient.

Protein processing: a role in the pathophysiology of genetic disease.

Genetic diseases associated with an enzyme deficiency frequently have reduced intracellular levels of the mutant protein, despite apparently normal levels of message and protein synthesis. It has been suggested that the endoplasmic reticulum (ER) can recognise mutant protein as incorrectly folded and invoke 'quality control' processes which cause the retention and degradation of this protein. This process may occur, even for mutations which do not abrogate protein activity, contributing directly to pathophysiology. Genetic diseases associated with defects in ER and Golgi processing proteins have also been reported and generally result in impaired processing of multiple protein products. In this review the role of the ER and Golgi in the pathogenesis of genetic diseases relating to the vacuolar network are discussed.

An improved method for the purification of IgG monoclonal antibodies from culture supernatants.

A method for the purification of mouse monoclonal antibodies from hybridoma culture supernatants is described. The protocol involves the use of a combination of three previously described methods for the concentration and purification of monoclonal antibodies. Firstly, hybridomas were grown in a Diacult dialysis system (Inter Med Laboratory, Denmark) to yield milligram quantities of monoclonal antibody in a culture supernatant. Monoclonal antibodies were then purified from the culture supernatant by precipitation with polyethylene glycol 6000 (PEG 6000) and finally reprecipitated using an ammonium sulphate procedure. The PEG 6000 treatment caused a density change in the ammonium sulphate immunoglobulin precipitate, and resulted in the formation of a pellicle which contained pure mouse monoclonal antibody. The protocol removed contaminating bovine serum immunoglobulin as well as other serum and cellular protein from the monoclonal antibody preparations.

Design and synthesis of 2-methyl-2-[4-(2-[5-methyl-2-aryloxazol-4-yl]ethoxy)phenoxy]propionic acids: a new class of dual PPARalpha/gamma agonists.

Propionic acid derivative 8, which was designed and synthesized based on putative pharmacophores of known PPARgamma- and PPARalpha-selective compounds, exhibits potent dual PPARalpha/gamma agonist activity as demonstrated by in vitro binding and dose overlap in the newly introduced EOB mouse model for glucose lowering and lipid/cholesterol homeostasis.