Public perception of generic pharmaceuticals in Malta

Objective: To evaluate the perception of the Maltese general public on generic medicinal products. Method: A self-administered questionnaire was compiled and distributed to a sample of the general public. Data collected from the questionnaires was analysed using SPSS ® version 19. Key findings: Five hundred and forty four questionnaires were completed. Fifty one percent of the respondents did not know the meaning of the term ‘generic medicinal product’ and 47% of the respondents became familiar with the term through the questionnaire. Conclusion: Improved communication amongst patients and healthcare professionals on the correct meaning of generic medicinal products and their medical and financial implications is required.peer-reviewe

Altered hemodynamics and vascular reactivity in a mouse model with severe pericyte deficiency

Pericytes are the mural cells of the microvascular network that are in close contact with underlying endothelial cells. Endothelial-secreted PDGFB leads to recruitment of pericytes to the vessel wall, but this is disrupted in Pdgfb$^{ret/ret}$ mice when the PDGFB retention motif is deleted. This results in severely reduced pericyte coverage on blood vessels. In this study, we investigated vascular abnormalities and hemodynamics in Pdgfb$^{ret/ret}$ mice throughout the cerebrovascular network and in different cortical layers by in vivo two-photon microscopy. We confirmed that Pdgfb$^{ret/ret}$ mice are severely deficient in pericytes throughout the vascular network, with enlarged brain blood vessels and a reduced number of vessel branches. Red blood cell velocity, linear density, and tube hematocrit were reduced in Pdgfb$^{ret/ret}$ mice, which may impair oxygen delivery to the tissue. We also measured intravascular PO$_{2}$ and found that concentrations were higher in cortical Layer 2/3 in Pdgfb$^{ret/ret}$ mice, indicative of reduced blood oxygen extraction. Finally, we found that Pdgfb$^{ret/ret}$ mice had a reduced capacity for vasodilation in response to an acetazolamide challenge during functional MRI imaging. Taken together, these results suggest that severe pericyte deficiency can lead to vascular abnormalities and altered cerebral blood flow, reminiscent of pathologies such as arteriovenous malformations

Single-Cell Analysis of Blood-Brain Barrier Response to Pericyte Loss

Rationale: Pericytes are capillary mural cells playing a role in stabilizing newly formed blood vessels during development and tissue repair. Loss of pericytes has been described in several brain disorders, and genetically induced pericyte deficiency in the brain leads to increased macromolecular leakage across the blood-brain barrier (BBB). However, the molecular details of the endothelial response to pericyte deficiency remain elusive. Objective: To map the transcriptional changes in brain endothelial cells resulting from lack of pericyte contact at single-cell level, and to correlate them with regional heterogeneities in BBB function and vascular phenotype. Methods and Results: We reveal transcriptional, morphological and functional consequences of pericyte absence for brain endothelial cells using a combination of methodologies, including single-cell RNA sequencing, tracer analyses and immunofluorescent detection of protein expression in pericyte-deficient adult Pdgfbret/ret mice. We find that endothelial cells without pericyte contact retain a general BBB-specific gene expression profile, however, they acquire a venous-shifted molecular pattern and become transformed regarding the expression of numerous growth factors and regulatory proteins. Adult Pdgfbret/ret brains display ongoing angiogenic sprouting without concomitant cell proliferation providing unique insights into the endothelial tip cell transcriptome. We also reveal heterogeneous modes of pericyte-deficient BBB impairment, where hotspot leakage sites display arteriolar-shifted identity and pinpoint putative BBB regulators. By testing the causal involvement of some of these using reverse genetics, we uncover a reinforcing role for angiopoietin 2 at the BBB. Conclusions: By elucidating the complexity of endothelial response to pericyte deficiency at cellular resolution, our study provides insight into the importance of brain pericytes for endothelial arterio-venous zonation, angiogenic quiescence and a limited set of BBB functions. The BBB-reinforcing role of ANGPT2 is paradoxical given its wider role as TIE2 receptor antagonist and may suggest a unique and context-dependent function of ANGPT2 in the brain

A molecular atlas of cell types and zonation in the brain vasculature

Cerebrovascular disease is the third most common cause of death in developed countries, but our understanding of the cells that compose the cerebral vasculature is limited. Here, using vascular single-cell transcriptomics, we provide molecular definitions for the principal types of blood vascular and vessel-associated cells in the adult mouse brain. We uncover the transcriptional basis of the gradual phenotypic change (zonation) along the arteriovenous axis and reveal unexpected cell type differences: a seamless continuum for endothelial cells versus a punctuated continuum for mural cells. We also provide insight into pericyte organotypicity and define a population of perivascular fibroblast-like cells that are present on all vessel types except capillaries. Our work illustrates the power of single-cell transcriptomics to decode the higher organizational principles of a tissue and may provide the initial chapter in a molecular encyclopaedia of the mammalian vasculature.Peer reviewe

Modeling the structural implications of an alternatively spliced Exoc3l2, a paralog of the tunneling nanotube-forming M-Sec.

The exocyst is a molecular tether that retains secretory vesicles at the plasma membrane prior to SNARE-mediated docking and fusion. However, individual exocyst complex components (EXOCs) may also function independently of exocyst assembly. Alternative splice variants of EXOC mRNA and paralogs of EXOC genes have been described and several have been attributed functions that may be independent of the exocyst complex. Here we describe a novel splice variant of murine Exoc3l2, which we term Exoc3l2a. We discuss possible functional implications of the resulting domain excision from this isoform of EXOC3L2 based on structural similarities with its paralog M-Sec (EXOC3L3), which is implicated in tunneling nanotube formation. The identification of this Exoc3l2 splice variant expands the potential for subunit diversity within the exocyst and for alternative functionality of this component independently of the exocyst

SWI and phase imaging reveal intracranial calcifications in the P301L mouse model of human tauopathy

OBJECTIVE Brain calcifications are associated with several neurodegenerative diseases. Here, we describe the occurrence of intracranial calcifications as a new phenotype in transgenic P301L mice overexpressing four repeat tau, a model of human tauopathy. MATERIALS AND METHODS Thirty-six P301L mice (Thy1.2) and ten age-matched non-transgenic littermates of different ages were assessed. Gradient echo data were acquired in vivo and ex vivo at 7 T and 9.4 T for susceptibility-weighted imaging (SWI) and phase imaging. In addition, ex vivo micro-computed tomography (μCT) was performed. Histochemistry and immunohistochemistry were used to investigate the nature of the imaging lesions. RESULTS SW images revealed regional hypointensities in the hippocampus, cortex, caudate nucleus, and thalamus of P301L mice, which in corresponding phase images indicated diamagnetic lesions. Concomitantly, µCT detected hyperdense lesions, though fewer lesions were observed compared to MRI. Diamagnetic susceptibility lesions in the hippocampus increased with age. The immunochemical staining of brain sections revealed osteocalcin-positive deposits. Furthermore, intra-neuronal and vessel-associated osteocalcin-containing nodules co-localized with phosphorylated-tau (AT8 and AT100) in the hippocampus, while vascular osteocalcin-containing nodules were detected in the thalamus in the absence of phosphorylated-tau deposition. DISCUSSION SWI and phase imaging sensitively detected intracranial calcifications in the P301L mouse model of human tauopathy

Design of splice site-specific primers for <i>Exoc3l2a</i>.

<p><b>A.</b> A forward (P^sj1) and reverse (P^sj2) primer specific to the alternative splice junction between E6 and E11 of <i>Exoc3l2</i> were designed. PCR of cDNA prepared from RNA isolated from mouse heart (E18.5 and P5) using P^sj1 and P¹¹ or P^4:5 and P^sj2 yielded single products, of predicted sizes. <b>B.</b> Melt curve profiles following RT-PCR of adult mouse heart cDNA (<i>n</i> = 5) with P^sj1 and P¹¹ (left plots) or P^4:5 and P^sj2 (right plots).</p

Detection of an alternative splice variant of murine <i>Exoc3l2</i>.

<p><b>A.</b> Gene map of murine <i>Exoc3l2</i> illustrating its twelve exons, as annotated at the NCBI (XM_006540475.1) and Ensembl genome browsers (ENSMUSG00000011263). <b>B.</b> PCR of cDNA prepared from RNA isolated from mouse liver using the <i>Exoc3l2</i>-specific primers P⁹ and P¹¹, and P^4:5 and P^11. <b>C.</b> PCR products of cDNA, prepared from RNA isolated from mouse organs, using the EXOC3L2 specific primers P^10:11 with P¹¹, and P^4:5 with P¹¹. Note the second bands detected at 250 bp in kidney and heart tissue in samples amplified with P^4:5+11.</p