Liposomes and nanotechnology in drug development: focus on neurological targets

Neurological diseases represent a medical, social, and economic problem of paramount importance in developed countries. Although their etiology is generally known, developing therapeutic interventions for the central nervous system is challenging due to the impermeability of the blood-brain barrier. Thus, the fight against neurological diseases usually struggles "at the gates" of the brain. Flooding the bloodstream with drugs, where only a minor fraction reaches its target therapeutic site, is an inefficient, expensive, and dangerous procedure, because of the risk of side effects at nontargeted sites. Currently, advances in the field of nanotechnology have enabled development of a generation of multifunctional molecular platforms that are capable of transporting drugs across the blood-brain barrier, targeting specific cell types or functional states within the brain, releasing drugs in a controlled manner, and enabling visualization of processes in vivo using conventional imaging systems. The marriage between drug delivery and molecular imaging disciplines has resulted in a relatively new discipline, known as theranostics, which represents the basis of the concept of personalized medicine. In this study, we review the concepts of the blood-brain barrier and the strategies used to traverse/bypass it, the role of nanotechnology in theranostics, the wide range of nanoparticles (with emphasis on liposomes) that can be used as stealth drug carriers, imaging probes and targeting devices for the treatment of neurological diseases, and the targets and targeting strategies envisaged in the treatment of different types of brain pathology

Study of protein expresion [sic] in peri-infarct tissue after cerebral ischemia

In this work, we report our study of protein expression in rat peri-infarct tissue, 48 h after the induction of permanent focal cerebral ischemia. Two proteomic approaches, gel electrophoresis with mass spectrometry and combined fractional diagonal chromatography (COFRADIC), were performed using tissue samples from the periphery of the induced cerebral ischemic lesions, using tissue from the contra-lateral hemisphere as a control. Several protein spots (3408) were identified by gel electrophoresis, and 11 showed significant differences in expression between peri-infarct and contralateral tissues (at least 3-fold, p < 0.05). Using COFRADIC, 5412 proteins were identified, with 72 showing a difference in expression. Apart from blood-related proteins (such as serum albumin), both techniques showed that the 70 kDa family of heat shock proteins were highly expressed in the peri-infarct tissue. Further studies by 1D and 2D western blotting and immunohistochemistry revealed that only one member of this family (the inducible form, HSP72 or HSP70i) is specifically expressed by the peri-infarct tissue, while the majority of this family (the constitutive form, HSC70 or HSP70c) is expressed in the whole brain. Our data support that HSP72 is a suitable biomarker of peri-infarct tissue in the ischemic brain

Genetic Diversity of Castanea sativa Mill. Accessions from the Tuscan-Emilian Apennines and Emilia Romagna Region (Italy)

This work investigated the genetic diversity of 134 Castanea sativa Mill. accessions present in the Italian region of Emilia-Romagna. Samples were taken from three collection fields (Granaglione, Zocca and Paloneta) in the Tuscan-Emilian Apennines. The accessions were analyzed by using 16 microsatellite markers (SSR). Genetic distances among accessions, calculated through the DICE coefficient, were used to construct an UPGMA cluster analysis. One major genotype (named “Marroni”) was identified across the three investigated collection fields; this variety corresponds to a sweet chestnut cultivar that has been propagated and widely diffused in the Emilia-Romagna region. Other genotypes were represented by different varieties of Italian chestnuts. The results of this study will be used to define and share guidelines for the characterization and varietal certification of the chestnut varieties in the Emilia-Romagna regionField and laboratory work were developed as the contribution of the regional project PSR ‘BIODIVERSAMENTE CASTAGNO’ and of the National Academy of Agriculture (ANA), in particular for supporting the analyses on the Granaglione’s samples. The Parco Didattico Sperimentale del Castagno di Granaglione and the Collection of Zocca host the main varieties of the Tuscan-Emilian Apennines. Sara Alessandri’s fellowship was funded by the Agricultural, Environmental, and Food Science and Technology (STAAA) PhD program offered by the Department of Agriculture and Food Sciences (DISTAL, University of Bologna)S

Polarized light microscopy guarantees the use of autochthonous wheat in the production of flour for the Protected Geographical Indication ‘Galician Bread’

‘Galician Bread’ is a traditional baked product and a national benchmark that has recently been granted the European mark of Protected Geographical Indication (PGI), which requires at least 25% of the flour to be produced from autochthonous varieties such as ‘Caaveiro’. The objective of this work was to find a method that guarantees the presence and the percentage of ‘Caaveiro’ wheat in blended flours by using microscopy techniques. Using optical microscopy, including bright-field and polarizing microscopy, autochthonous and foreign flours were analyzed and compared. ‘Caaveiro’ starch presented a different birefringence pattern (associated with a higher amount of amylose) with respect to other cultivars used to produce flours, a feature used to make a computation of the two starch granule types in the mixtures of ‘Caaveiro’ with foreign flours. Repetitions with different mixture percentages allowed us to develop a mathematical model to estimate the percentage of ‘Caaveiro’ flour present in the mixture. Firstly, the most effective method for preparing samples was determined by ensuring the homogeneity of the samples and, subsequently, a validation was carried out with blind samples. Starch birefringence properties allowed the detection of ‘Caaveiro’ wheat flour in mixtures with foreign/Castilian wheat flours and to determine the percentages used in the flour mixtures applying a calibration line (R2 = 0.9577). Deviations were due to the difficulty in obtaining precise mixtures of the blended flours, as happened with other Simple Sequence Repeat (SSR)-based methods used in the same samples. This is a novel method for detecting contraventions/infractions of the percentage of ‘Caaveiro’ used in wheat flours, which is simple, effective and inexpensiveThis research was supported by the Spanish Ministry of Science and Innovation, Proyectos de Generación de Conocimiento 2021-2023 (PID2021-123905OB-I00). We would like to thank Da Cunha Group for the samples and their support of the ‘Cátedra do Pan e do Cereal’. Nerea Fernández-Canto is grateful to Xunta de Galicia for her predoctoral research fellowship (ED481A-2019/263)S

Corrigendum

Twenty microsatellites (SSRs) reveal two main origins ofvariability in grapevine cultivars from Northwestern SpainVitis 49 (2), 55-62 (2010

Evaluación del tiempo de espera óptimo para la colocación de implantes dentales tras elevaciones de seno maxilar (ESM) con un injerto compuesto por hueso autólogo y biomaterial

Objetivo: Evaluar el tiempo de espera óptimo para la colocación de implantes dentales tras elevaciones de seno maxilar (ES) con un injerto compuesto con la misma cantidad de hueso autólogo que de biomaterial, a través de biopsias humanas, comparando el hueso regenerado obtenido a los 4-5 meses con el obtenido a los 6-8 meses, teniendo el hueso nativo como referencia. Material y Métodos: Fueron analizadas un total de 26 biopsias de 11 pacientes tras ES. Se crearon dos grupos dependiendo del momento de la colocación del implante: grupo t1 a los 4-5 meses (n=13) y grupo t2 a los 6-8 meses (n=13). Fue analizado por microtomografía computarizada (MicroCT) para cada biopsia el mismo volumen para el hueso injertado que para el nativo. Resultados: Se encontraron diferencias estadísticamente significativas en la densidad mineral ósea (BMD), fracción de volumen óseo y separación trabecular (Tb.Sp) entre el hueso nativo e injertado en los grupos t1 y t2, mostrando valores más altos en el hueso injertado excepto para la variable Tb.Sp. que sucedió a la inversa. El descenso de la Tb.Sp en el hueso injertado de los dos grupos puede explicarse por el aumento sigificativo del grosor trabecular en el grupo t2 y por el aumento del número trabecular en el grupo t1, comparándolos con el hueso nativo respectivamente. No se encontró ninguna diferencia estadísticamente significativa entre los dos grupos de hueso injertado cuando se compararon los parámetros morfométricos y de BMD. Conclusiones: Un injerto compuesto por 50% hueso autólogo y 50% biomaterial muestra que no hay diferencias en la microestructura 3D ni en la BMD entre 4-5 o 6-8 meses de espera de cicatrización ósea. Por ello este tiempo de cicatrización se puede acortar a 4 meses con la seguridad de un área injertada con hueso maduroPurpose: To evaluate the ideal implant time insertion in human bone biopsies after maxillary sinus floor elevation (MSFE) with a composite graft consisting of an equal amount of biomaterial and autologous bone, by comparing the bone regeneration obtained 4-5 months after surgery with the obtained after 6-8 months, and having the adjacent native bone as reference. Materials and Methods: A total of 26 biopsies of 11 patients after MSFE were analyzed. Two groups were created depending on the time of implant insertion: t1 group at 4-5 months (n=13) and t2 group at 6-8 months (n=13). The same volume of grafted bone and native bone were analyzed for each biopsy by micro-computed tomography (microCT). Results: Statistically significant differences were found in bone mineral density, bone volume fraction and trabecular separation (Tb. Sp) between native and grafted bone in the t1 and t2 groups, showing grafted bone higher values except for the variable Tb.Sp, which were lower in the grafted bone compared to native bone. The decrease in Tb.Sp in the grafted bone for t1 and t2 groups can be explained by the significant increase in trabecular thickness in t2 group and the trabecular number in t1 group, compared to native bone respectively. Comparing the morphometric parameters and the BMD of the grafted bone between the t1 and t2 groups, no significant differences were found. Conclusions: A composite graft composed of 50% autologous bone and 50% biomaterial shows no differences in 3D microstructure and BMD between 4-5 months and 6-8 months of healing time. Thus, this time can be shortened to 4 months with the security of a grafted area of mature bone

Interleukin-10 facilitates the selection of patients for systemic thrombolysis

Background: Clinical-Diffusion mismatch (CDM; NIHSS score ≥8 & DWI lesion volume ≤25 mL) and Perfusion-Diffusion mismatch (PDM; difference >20% between initial DWI and MTT lesion volumes) have been proposed as surrogates for ischemic brains that are at risk of infarction. However, their utility to improve the selection of patients for thrombolytic treatment remains controversial. Our aim was to identify molecular biomarkers that can be used with neuroimaging to facilitate the selection of ischemic stroke patients for systemic thrombolysis. Methods: We prospectively studied 595 patients with ischemic stroke within 12 h of the stroke onset. A total of 184 patients received thrombolytic treatment according to the SITS-MOST criteria. DWI and MTT volumes were measured at admission. The main outcome variable was good functional outcome at 3 months (modified Rankin scale <3). Serum levels of glutamate (Glu), IL-10, TNF-α, IL-6, NSE, and active MMP-9 also were determined at admission. Results: Patients treated with t-PA who presented with PDM had higher IL-10 levels at admission (p < 0.0001). In contrast, patients with CDM had higher levels of IL-10 (p < 0.0001) as well as Glu and TNF-α (all p < 0.05) and lower levels of NSE and active MMP-9 (all p < 0.0001). IL-10 ≥ 30 pg/mL predicts good functional outcome at 3 months with a specificity of 88% and a sensitibity of 86%. IL-10 levels ≥30 pg/mL independently in both patients with PDM (OR, 18.9) and CDM (OR, 7.5), after an adjustment for covariates. Conclusions: Serum levels of IL-10 facilitate the selection of ischemic stroke patients with CDM and PDM for systemic thrombolysis

In Vivo Theranostics at the Peri-Infarct Region in Cerebral Ischemia

The use of theranostics in neurosciences has been rare to date because of the limitations imposed on the free delivery of substances to the brain by the blood-brain barrier. Here we report the development of a theranostic system for the treatment of stroke, a leading cause of death and disability in developed countries. We first performed a series of proteomic, immunoblotting and immunohistological studies to characterize the expression of molecular biomarkers for the so-called peri-infarct tissue, a key region of the brain for stroke treatment. We confirmed that the HSP72 protein is a suitable biomarker for the peri-infarct region, as it is selectively expressed by at-risk tissue for up to 7 days following cerebral ischemia. We also describe the development of anti-HSP72 vectorized stealth immunoliposomes containing imaging probes to make them traceable by conventional imaging techniques (fluorescence and MRI) that were used to encapsulate a therapeutic agent (citicoline) for the treatment of cerebral ischemia. We tested the molecular recognition capabilities of these nano-platforms in vitro together with their diagnostic and therapeutic properties in vivo, in an animal model of cerebral ischemia. Using MRI, we found that 80% of vectorized liposomes were located on the periphery of the ischemic lesion, and animals treated with citicoline encapsulated on these liposomes presented lesion volumes up to 30% smaller than animals treated with free (non-encapsulated) drugs. Our results show the potential of nanotechnology for the development of effective tools for the treatment of neurological diseases

Corrigendum

Twenty microsatellites (SSRs) reveal two main origins ofvariability in grapevine cultivars from Northwestern SpainVitis 49 (2), 55-62 (2010