Preclinical Development and Characterization of Novel Adeno-Associated Viral Vectors for the Treatment of Lipoprotein Lipase Deficiency.

Lipoprotein lipase deficiency (LPLD) results from mutations within the lipoprotein lipase (LPL) gene that lead to a complete lack of catalytically active LPL protein. Glybera was one of the first adeno-associated virus (AAV) gene replacement therapy to receive European Medicines Agency regulatory approval for the treatment of LPLD. However, Glybera is no longer marketed potentially due to a combination of economical, manufacturing, and vector-related issues. The aim of this study was to develop a more efficacious AAV gene therapy vector for LPLD. Following preclinical biodistribution, efficacy and non-Good Laboratory Practice toxicity studies with novel AAV1 and AAV8-based vectors in mice, we identified AAV8 pVR59. AAV8 pVR59 delivered a codon-optimized, human gain-of-function hLPLS447X transgene driven by a CAG promoter in an AAV8 capsid. AAV8 pVR59 was significantly more efficacious, at 10- to 100-fold lower doses, compared with an AAV1 vector based on Glybera, when delivered intramuscularly or intravenously, respectively, in mice with LPLD. Efficient gene transfer was observed within the injected skeletal muscle and liver following delivery of AAV8 pVR59, with long-term correction of LPLD phenotypes, including normalization of plasma triglycerides and lipid tolerance, for up to 6 months post-treatment. While intramuscular delivery of AAV8 pVR59 was well tolerated, intravenous administration augmented liver pathology. These results highlight the feasibility of developing a superior AAV vector for the treatment of LPLD and provide critical insight for initiating studies in larger animal models. The identification of an AAV gene therapy vector that is more efficacious at lower doses, when paired with recent advances in production and manufacturing technologies, will ultimately translate to increased safety and accessibility for patients.

Interaction of Hoechst 33342 with POPC Membranes at Different pH Values.

Hoechst 33342 (H33342) is a fluorescent probe that is commonly used to stain the DNA of living cells. To do so, it needs to interact with and permeate through cell membranes, despite its high overall charge at physiological pH values. In this work, we address the effect of pH in the association of H33342 with lipid bilayers using a combined experimental and computational approach. The partition of H33342 to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid membranes was experimentally quantified using fluorescence spectroscopy and isothermal titration calorimetry (ITC) measurements. Quantum chemical calculations were performed to select the most stable isomer of H33342 for the overall charges 0, +1, and +2, expected to predominate across the 5 < pH < 10 range. The interaction of these isomers with POPC bilayers was then studied by both unrestrained and umbrella sampling molecular dynamics (MD) simulations. Both experimental results and computational free energy profiles indicate that the partition coefficient of H33342 displays a small variation over a wide pH range, not exceeding one order of magnitude. The enthalpy variation upon partition to the membrane suggests efficient hydrogen bonding between the probe and the lipid, namely, for the protonated +2 form, which was confirmed in the MD simulation studies. The relatively high lipophilicity obtained for the charged species contrasts with the decrease in their general hydrophobicity as estimated from octanol/water partition. This highlights the distinction between lipophilicity and hydrophobicity, as well as the importance of considering the association with lipid bilayers when predicting the affinity for biomembranes.

Incorporating pressure-volume traits into the leaf economics spectrum.

In recent years, attempts have been made in linking pressure-volume parameters and the leaf economics spectrum to expand our knowledge of the interrelationships among leaf traits. We provide theoretical and empirical evidence for the coordination of the turgor loss point and associated traits with net CO2 assimilation (An ) and leaf mass per area (LMA). We measured gas exchange, pressure-volume curves and leaf structure in 45 ferns and angiosperms, and explored the anatomical and chemical basis of the key traits. We propose that the coordination observed between mass-based An , capacitance and the turgor loss point (πtlp ) emerges from their shared link with leaf density (one of the components of LMA) and, specially, leaf saturated water content (LSWC), which in turn relates to cell size and nitrogen and carbon content. Thus, considering the components of LMA and LSWC in ecophysiological studies can provide a broader perspective on leaf structure and function.

Lipid Metabolic Alterations in the ALS-FTD Spectrum of Disorders.

There is an increasing interest in the study of the relation between alterations in systemic lipid metabolism and neurodegenerative disorders, in particular in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). In ALS these alterations are well described and evident not only with the progression of the disease but also years before diagnosis. Still, there are some discrepancies in findings relating to the causal nature of lipid metabolic alterations, partly due to the great clinical heterogeneity in ALS. ALS presentation is within a disorder spectrum with Frontotemporal Dementia (FTD), and many patients present mixed forms of ALS and FTD, thus increasing the variability. Lipid metabolic and other systemic metabolic alterations have not been well studied in FTD, or in ALS-FTD mixed forms, as has been in pure ALS. With the recent development in lipidomics and the integration with other -omics platforms, there is now emerging data that not only facilitates the identification of biomarkers but also enables understanding of the underlying pathological mechanisms. Here, we reviewed the recent literature to compile lipid metabolic alterations in ALS, FTD, and intermediate mixed forms, with a view to appraising key commonalities or differences within the spectrum.

Targeting insulin-like growth factor-1 receptor (IGF1R) for brain delivery of biologics.

The blood-brain barrier (BBB) prevents the majority of drugs from crossing into the brain and reaching neurons. To overcome this challenge, safe and non-invasive technologies targeting receptor-mediated pathways have been developed. In this study, three single-domain antibodies (sdAbs; IGF1R3, IGF1R4, and IGF1R5) targeting the extracellular domain of the human insulin-like growth factor-1 receptor (IGF1R), generated by llama immunization, showed enhanced transmigration across the rat BBB model (SV-ARBEC) in vitro. The rate of brain uptake of these sdAbs fused to mouse Fc (sdAb-mFc) in vivo was estimated using the fluorescent in situ brain perfusion (ISBP) technique followed by optical brain imaging and distribution volume evaluation. Compared to the brains perfused with the negative control A20.1-mFc, the brains perfused with anti-IGF1R sdAbs showed a significant increase of the total fluorescence intensity (~2-fold, p < .01) and the distribution volume (~4-fold, p < .01). The concentration curve for IGF1R4-mFc demonstrated a linear accumulation plateauing at approximately 400 µg (~1 µM), suggesting a saturable mechanism of transport. Capillary depletion and mass spectrometry analyses of brain parenchyma post-ISBP confirmed the IGF1R4-mFc brain uptake with ~25% of the total amount being accumulated in the parenchymal fraction in contrast to undetectable levels of A20.1-mFc after a 5-min perfusion protocol. Systemic administration of IGF1R4-mFc fused with the non-BBB crossing analgesic peptide galanin (2 and 5 mg/kg) induced a dose-dependent suppression of thermal hyperalgesia in the Hargreaves pain model. In conclusion, novel anti-IGF1R sdAbs showed receptor-mediated brain uptake with pharmacologically effective parenchymal delivery of non-permeable neuroactive peptides.

Differentiation of Adipose-Derived Stem Cells into Vascular Smooth Muscle Cells for Tissue Engineering Applications.

Synthetic grafts have been developed for vascular bypass surgery, however, the risks of thrombosis and neointimal hyperplasia still limit their use. Tissue engineering with the use of adipose-derived stem cells (ASCs) has shown promise in addressing these limitations. Here we further characterized and optimized the ASC differentiation into smooth muscle cells (VSMCs) induced by TGF-ß and BMP-4. TGF-ß and BMP-4 induced a time-dependent expression of SMC markers in ASC. Shortening the differentiation period from 7 to 4 days did not impair the functional property of contraction in these cells. Stability of the process was demonstrated by switching cells to regular growth media for up to 14 days. The role of IGFBP7, a downstream effector of TGF-ß, was also examined. Finally, topographic and surface patterning of a substrate is recognized as a powerful tool for regulating cell differentiation. Here we provide evidence that a non-woven PET structure does not affect the differentiation of ASC. Taken together, our results indicate that VSMCs differentiated from ASCs are a suitable candidate to populate a PET-based vascular scaffolds. By employing an autologous source of cells we provide a novel alternative to address major issues that reduces long-term patency of currently vascular grafts.

Predictive Value of Pro-BNP for Heart Failure Readmission after an Acute Coronary Syndrome.

BACKGROUND: N-terminal pro-brain natural peptide (NT-pro-BNP) is a well-established biomarker of tissue congestion and has prognostic value in patients with heart failure (HF). Nonetheless, there is scarce evidence on its predictive capacity for HF re-admission after an acute coronary syndrome (ACS). We performed a prospective, single-center study in all patients discharged after an ACS. HF re-admission was analyzed by competing risk regression, taking all-cause mortality as a competing event. Results are presented as sub-hazard ratios (sHR). Recurrent hospitalizations were tested by negative binomial regression, and results are presented as incidence risk ratio (IRR). RESULTS: Of the 2133 included patients, 528 (24.8%) had HF during the ACS hospitalization, and their pro-BNP levels were higher (3220 pg/mL vs. 684.2 pg/mL; p < 0.001). In-hospital mortality was 2.9%, and pro-BNP was similarly higher in these patients. Increased pro-BNP levels were correlated to increased risk of HF or death during the hospitalization. Over follow-up (median 38 months) 243 (11.7%) patients had at least one hospital readmission for HF and 151 (7.1%) had more than one. Complete revascularization had a preventive effect on HF readmission, whereas several other variables were associated with higher risk. Pro-BNP was independently associated with HF admission (sHR: 1.47) and readmission (IRR: 1.45) at any age. Significant interactions were found for the predictive value of pro-BNP in women, diabetes, renal dysfunction, STEMI and patients without troponin elevation. CONCLUSIONS: In-hospital determination of pro-BNP is an independent predictor of HF readmission after an ACS.

Cell wall thickness and composition are involved in photosynthetic limitation.

The key role of cell walls in setting mesophyll conductance to CO2 (gm) and, consequently, photosynthesis is reviewed. First, the theoretical properties of cell walls that can affect gm are presented. Then, we focus on cell wall thickness (Tcw) reviewing empirical evidence showing that Tcw varies strongly among species and phylogenetic groups in a way that correlates with gm and photosynthesis; that is, the thicker the mesophyll cell walls, the lower the gm and photosynthesis. Potential interplays of gm, Tcw, dehydration tolerance, and hydraulic properties of leaves are also discussed. Dynamic variations of Tcw in response to the environment and their implications in the regulation of photosynthesis are discussed, and recent evidence suggesting an influence of cell wall composition on gm is presented. We then propose a hypothetical mechanism for the influence of cell walls on photosynthesis, combining the effects of thickness and composition, particularly pectins. Finally, we discuss the prospects for using biotechnology for enhancing photosynthesis by altering cell wall-related genes.

In vivo near-infrared fluorescent optical imaging for CNS drug discovery.

INTRODUCTION: In vivo imaging technologies have become integral and essential component of drug discovery, development, and clinical assessment for central nervous system (CNS) diseases. Near-infrared (NIR) fluorescence imaging in the range of 650-950 nm is widely used for pre-clinical in vivo imaging studies. The recent expansion of NIR imaging into the shortwave infrared (SWIR, 1000-1700 nm) window enabled improvements in tissue penetration and resolution required for anatomical, dynamic, and molecular neuroimaging with high potential for clinical translation. AREAS COVERED: This review focuses on the latest progress in near-infrared (NIR)-fluorescent optical imaging modalities with an emphasis on the SWIR window. Advantages and challenges in developing novel organic and inorganic SWIR emitters, with special attention to their toxicology and pharmacology, are discussed. Examples of their application in preclinical imaging of brain function and pathology provide a platform to assess the potential for their clinical translation. EXPERT OPINION: Propelled through concomitant technological advancements in imaging instrumentation, algorithms and new SWIR emitters, SWIR imaging has addressed key barriers to optical imaging modalities used in pre-clinical studies addressing the CNS. Development of biocompatible SWIR emitters and adoption of SWIR into multi-modal imaging modalities promise to rapidly advance optical imaging into translational studies and clinical applications.

Cell wall composition strongly influences mesophyll conductance in gymnosperms.

Cell wall thickness is widely recognized as one of the main determinants of mesophyll conductance to CO2 (gm ). However, little is known about the components that regulate effective CO2 diffusivity in the cell wall (i.e. the ratio between actual porosity and tortuosity, the other two biophysical diffusion properties of cell walls). The aim of this study was to assess, at the interspecific level, potential relationships between cell wall composition, cell wall thickness (Tcw ) and gm . Gymnosperms constitute an ideal group to deepen these relationships, as they present, on average, the thickest cell walls within spermatophytes. We characterized the foliar gas exchange, the morphoanatomical traits related with gm , the leaf fraction constituted by cell walls and three main components of primary cell walls (hemicelluloses, cellulose and pectins) in seven gymnosperm species. We found that, although the relatively low gm of gymnosperms was mainly determined by their elevated Tcw , gm was also strongly correlated with cell wall composition, which presumably sets the final effective CO2 diffusivity. The data presented here suggest that (i) differences in gm are strongly correlated to the pectins to hemicelluloses and cellulose ratio in gymnosperms, and (ii) variations in cell wall composition may modify effective CO2 diffusivity in the cell wall to compensate the negative impact of thickened walls. We speculate that higher relative pectin content allows higher gm because pectins increase cell wall hydrophilicity and CO2 molecules cross the wall dissolved in water.

How do vascular plants perform photosynthesis in extreme environments? An integrative ecophysiological and biochemical story.

In this work, we review the physiological and molecular mechanisms that allow vascular plants to perform photosynthesis in extreme environments, such as deserts, polar and alpine ecosystems. Specifically, we discuss the morpho/anatomical, photochemical and metabolic adaptive processes that enable a positive carbon balance in photosynthetic tissues under extreme temperatures and/or severe water-limiting conditions in C3 species. Nevertheless, only a few studies have described the in situ functioning of photoprotection in plants from extreme environments, given the intrinsic difficulties of fieldwork in remote places. However, they cover a substantial geographical and functional range, which allowed us to describe some general trends. In general, photoprotection relies on the same mechanisms as those operating in the remaining plant species, ranging from enhanced morphological photoprotection to increased scavenging of oxidative products such as reactive oxygen species. Much less information is available about the main physiological and biochemical drivers of photosynthesis: stomatal conductance (gs ), mesophyll conductance (gm ) and carbon fixation, mostly driven by RuBisCO carboxylation. Extreme environments shape adaptations in structures, such as cell wall and membrane composition, the concentration and activation state of Calvin-Benson cycle enzymes, and RuBisCO evolution, optimizing kinetic traits to ensure functionality. Altogether, these species display a combination of rearrangements, from the whole-plant level to the molecular scale, to sustain a positive carbon balance in some of the most hostile environments on Earth.

Interaction of Bile Salts With Lipid Bilayers: An Atomistic Molecular Dynamics Study.

Bile salts (BS) are biosurfactants crucial for emulsification and intestinal absorption of cholesterol and other hydrophobic compounds such as vitamins and fatty acids. Interaction of BS with lipid bilayers is important for understanding their effects on membranes properties. The latter have relevance in passive diffusion processes through intestinal epithelium such as reabsorption of BS, as well as their degree of toxicity to intestinal flora and their potential applications in drug delivery. In this work, we used molecular dynamics simulations to address at the atomic scale the interactions of cholate, deoxycholate, and chenodeoxycholate, as well as their glycine conjugates with POPC bilayers. In this set of BS, variation of three structural aspects was addressed, namely conjugation with glycine, number and position of hydroxyl substituents, and ionization state. From atomistic simulations, the location and orientation of BS inside the bilayer, and their specific interactions with water and host lipid, such as hydrogen bonding and ion-pair formation, were studied in detail. Membrane properties were also investigated to obtain information on the degree of perturbation induced by the different BS. The results are described and related to a recent experimental study (Coreta-Gomes et al., 2015). Differences in macroscopic membrane partition thermodynamics and translocation kinetics are rationalized in terms of the distinct structures and atomic-scale behavior of the bile salt species. In particular, the faster translocation of cholate is explained by its higher degree of local membrane perturbation. On the other hand, the relatively high partition of the polar glycine conjugates is related to the longer and more flexible side chain, which allows simultaneous efficient solvation of the ionized carboxylate and deep insertion of the ring system.

The poor prognosis of low hypodiploidy in adults with B-cell precursor acute lymphoblastic leukaemia is restricted to older adults and elderly patients.

The prognostic significance of low-hypodiploidy has not been extensively evaluated in minimal residual disease (MRD)-oriented protocols for adult acute lymphoblastic leukaemia (ALL). We analysed the outcome of hypodiploid adult ALL patients treated within Programa Español de Tratamientos en Hematología (PETHEMA) protocols. The 5-year cumulative incidence of relapse (CIR) of low-hypodiploid B-cell precursor (BCP)-ALL was significantly higher than that of high-hypodiploids (52% vs. 12%, P = 0.013). Low-hypodiploid BCP-ALL patients aged ≤35 years showed superior survival (71% vs. 21%, P = 0.026) and lower 5-year CIR (17% vs. 66%, P = 0.090) than low-hypodiploids aged >35 years. Older adults and elderly low-hypodiploid BCP-ALL patients show dismal prognosis although achieving an end-induction good MRD response.

Validation of the Spanish-language Cardiff Anomalous Perception Scale.

The Cardiff Anomalous Perceptions Scale (CAPS) is a psychometric measure of hallucinatory experience. It has been widely used in English and used in initial studies in Spanish but a full validation study has not yet been published. We report a validation study of the Spanish-language CAPS, conducted in both Spain and Colombia to cover both European and Latin American Spanish. The Spanish-language version of the CAPS was produced through back translation with slight modifications made for local dialects. In Spain, 329 non-clinical participants completed the CAPS along with 40 patients with psychosis. In Colombia, 190 non-clinical participants completed the CAPS along with 21 patients with psychosis. Participants completed other psychometric scales measuring psychosis-like experience to additionally test convergent and divergent validity. The Spanish-language CAPS was found to have good internal reliability. Test-retest reliability was slightly below the cut-off, although could only be tested in the Spanish non-clinical sample. The scale showed solid construct validity and a principal components analysis broadly replicated previously reported three component factor structures for the CAPS.

Effect of a grapevine-shoot waste extract on red wine aromatic properties.

BACKGROUND: The use of a grapevine-shoot extract (VIN) is being studied as an alternative to sulfur dioxide (SO2 ). VIN stabilizes anthocyanins and preserves polyphenolic compounds, and thus improves chromatic wine properties. In this study, selected aroma compounds (esters, C13 -norisoprenoids, oxidation and vine-shoot-related compounds), sensory analysis and the olfactometric profile were determined in the wines treated with VIN at two concentrations. RESULTS: Treatment with VIN hardly modified the content of esters and oxidation-related compounds in the wines. However, the high ß-damascenone and isoeugenol contents and the increase in astringency at tasting in VIN wines were noteworthy, as were some odorant zones. All these were established as VIN markers after the chemometric data analysis. CONCLUSION: These data revealed that only the lowest dose tested may be recommended as a suitable alternative to SO2 . Although some aromatic properties of these wines may change, these changes are not considered to affect the quality of the wines negatively. These results are useful for wineries, which face having to discover the aroma-related processes in the challenge of producing SO2 -free wines without detriment to their sensory properties. © 2018 Society of Chemical Industry.

IgG subclasses quantitation: Analytical performance of The Binding Site SPAPLUS® human assay and comparison with Siemens BNII® assay.

OBJECTIVES: Accurate evaluation of analyzers is highly recommended before these devices are broadly introduced for routine testing. Concerning quantification of IgG subclasses (IgGSc), standardization has not yet been reached and thus different assays might lead to different results. Here we report the analytical performances of The Binding Site (TBS) SPAPLUS® human IgGSc assay and the concordance with the Siemens BNII® human IgGSc assay. DESIGN AND METHODS: We evaluated precision, LoB, LoD and linearity of TBS SPAPLUS® human IgGSc immunoassay. Quantitation of IgGSc in 53 patients' serum samples was performed in parallel on both analyzers. Results from both assays were compared. RESULTS: Analytical performances of the TBS SPAPLUS® human IgGSc assay are acceptable for routine clinical use. According to the method comparison study, TBS assay measures lower values than Siemens assay for IgG1 and IgG4, whereas for IgG2 and IgG3 TBS provides greater values. All assays present a proportional bias, greater in the case of IgG3 and IgG4 assays. Individual subclass agreement, based on the classification of samples within three categories (low, normal and high) according to assay-specific reference intervals, range from 75% (IgG1) to 92% (IgG2). However, total classification agreement over all four subclasses only account for 55% of samples. CONCLUSION: Results obtained from both assays are not interchangeable. Standardization of IgGSc assay and review of the reference ranges must be accomplished in order to achieve a higher degree of agreement between different methods.

Effect of Acyl Chain Length on the Rate of Phospholipid Flip-Flop and Intermembrane Transfer.

The rate at which phospholipids equilibrate between different membranes and between the non-polar environments in biological fluids is of high importance in the understanding of biomembrane diversity, as well as in the development of liposomes for drug delivery. In this work, we characterize the rate of insertion into and desorption from POPC bilayers for a homologous series of amphiphiles with the fluorescent NBD group attached to phosphoethanolamines of different acyl chain lengths, NBD-diC n -PE with n = 6, 8, 10, and 12. The rate of translocation between bilayer leaflets was also characterized, providing all the relevant parameters for their interaction with lipid bilayers. The results are complemented with data for NBD-diC14-PE obtained from literature (Abreu et al. Biophys J 87:353-365, 2004; Moreno et al. Biophys J 91:873-881, 2006). The rate of translocation between the POPC leaflets is not dependent on the length of the acyl chains, while this affects strongly the rate of desorption from the bilayer. Insertion in the POPC bilayer is not diffusion controlled showing a significant dependence on the acyl chain length and on temperature. The results obtained are compared with those previously reported for NBD-LysoC14-PE (Sampaio et al. Biophys J 88:4064-4071, 2005), and with the homologous series of single chain amphiphiles NBD-C n (Cardoso et al. J Phys Chem B 114:16337-16346, 2010; J Phys Chem B 115:10098-10108, 2011). This allows the establishment of important relations between the rate constants for interaction with the lipid bilayers and the structural properties of the amphiphiles, namely the total surface and the cross-section of their non-polar region.

Effect of microalgae incorporation on physicochemical and textural properties in wheat bread formulation.

The objective of this study was to evaluate the effect of the incorporation of different microalgae on physicochemical and textural properties of bread. Four species of microalgae Isochrysis galbana, Tetraselmis suecica, Scenedesmus almeriensis, and Nannochloropsis gaditana were used in this study. Properties such as water activity, pH, microbiological counts, viscosity, and color were analyzed to determine the effect of microalgae addition on sourdough. The technological quality of breads was analyzed in terms of physicochemical properties, color, texture profile, and porosity. The main effect of microalgae addition was changes in bread color, crust, and crumb that implies an increase of browning and an evolution to more green-yellow tonalities. The textural parameters of breads such as hardness, chewiness, and resilience are not modified by microalgae addition.

Polimiositis secundaria a un linfoma no Hodgkin / Polymyositis secondary to a non-Hodgkins lymphoma

No disponible

Sacroilitis séptica y absceso en el psoas / Septic sacroilitis and psoas abscess

No disponible