Toxin bioportides: exploring toxin biological activity and multifunctionality

Toxins have been shown to have many biological functions and to constitute a rich source of drugs and biotechnological tools. We focus on toxins that not only have a specific activity, but also contain residues responsible for transmembrane penetration, which can be considered bioportides—a class of cell-penetrating peptides that are also intrinsically bioactive. Bioportides are potential tools in pharmacology and biotechnology as they help deliver substances and nanoparticles to intracellular targets. Bioportides characterized so far are peptides derived from human proteins, such as cytochrome c (CYCS), calcitonin receptor (camptide), and endothelial nitric oxide synthase (nosangiotide). However, toxins are usually disregarded as potential bioportides. In this review, we discuss the inclusion of some toxins and molecules derived thereof as a new class of bioportides based on structure activity relationship, minimization, and biological activity studies. The comparative analysis of the amino acid residue composition of toxin-derived bioportides and their short molecular variants is an innovative analytical strategy which allows us to understand natural toxin multifunctionality in vivo and plan novel pharmacological and biotechnological products. Furthermore, we discuss how many bioportide toxins have a rigid structure with amphiphilic properties important for both cell penetration and bioactivity.     

Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood–brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000? was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.     

On glioblastoma and the search for a cure: where do we stand?

Although brain tumours have been documented and recorded since the nineteenth century, 2016 marked 90 years since Percival Bailey and Harvey Cushing coined the term “glioblastoma multiforme”. Since that time, although extensive developments in diagnosis and treatment have been made, relatively little improvement on prognosis has been achieved. The resilience of GBM thus makes treating this tumour one of the biggest challenges currently faced by neuro-oncology. Aggressive and robust development, coupled with difficulties of complete resection, drug delivery and therapeutic resistance to treatment are some of the main issues that this nemesis presents today. Current treatments are far from satisfactory with poor prognosis, and focus on palliative management rather than curative intervention. However, therapeutic research leading to developments in novel treatment stratagems show promise in combating this disease. Here we present a review on GBM, looking at the history and advances which have shaped neurosurgery over the last century that cumulate to the present day management of GBM, while also exploring future perspectives in treatment options that could lead to new treatments on the road to a cure.     

Skeletal muscle secretome in Duchenne muscular dystrophy: a pivotal anti-inflammatory role of adiponectin

Background

Persistent inflammation exacerbates the progression of Duchenne muscular dystrophy (DMD). The hormone, adiponectin (ApN), which is decreased in the metabolic syndrome, exhibits anti-inflammatory properties on skeletal muscle and alleviates the dystrophic phenotype of mdx mice. Here, we investigate whether ApN retains its anti-inflammatory action in myotubes obtained from DMD patients. We unravel the underlying mechanisms by studying the secretome and the early events of ApN.

Methods

Primary cultures of myotubes from DMD and control patients were treated or not by ApN after an inflammatory challenge. Myokines secreted in medium were identified by cytokine antibody-arrays and ELISAs. The early events of ApN signaling were assessed by abrogating selected genes.

Results

ApN retained its anti-inflammatory properties in both dystrophic and control myotubes. Profiling of secretory products revealed that ApN downregulated the secretion of two pro-inflammatory factors (TNFα and IL-17A), one soluble receptor (sTNFRII), and one chemokine (CCL28) in DMD myotubes, while upregulating IL-6 that exerts some anti-inflammatory effects. These changes were explained by pretranslational mechanisms. Earlier events of the ApN cascade involved AdipoR1, the main receptor for muscle, and the AMPK-SIRT1-PGC-1α axis leading, besides alteration of the myokine profile, to the upregulation of utrophin A (a dystrophin analog).

Conclusion

ApN retains its beneficial properties in dystrophic muscles by activating the AdipoR1-AMPK-SIRT1-PGC-1α pathway, thereby inducing a shift in the secretion of downstream myokines toward a less inflammatory profile while upregulating utrophin. ApN, the early events of the cascade and downstream myokines may be therapeutic targets for the management of DMD.

     

Germline-specific dgcr8 knockout in zebrafish using a BACK approach

Zebrafish is an important model to study developmental biology and human diseases. However, an effective approach to achieve spatial and temporal gene knockout in zebrafish has not been well established. In this study, we have developed a new approach, namely bacterial artificial chromosome-rescue-based knockout (BACK), to achieve conditional gene knockout in zebrafish using the Cre/loxP system. We have successfully deleted the DiGeorge syndrome critical region gene 8 (dgcr8) in zebrafish germ line and demonstrated that the maternal-zygotic dgcr8 (MZdgcr8) embryos exhibit MZdicer-like phenotypes with morphological defects which could be rescued by miR-430, indicating that canonical microRNAs play critical role in early development. Our findings establish that Cre/loxP-mediated tissue-specific gene knockout could be achieved using this BACK strategy and that canonical microRNAs play important roles in early embryonic development in zebrafish.     

IFT54 regulates IFT20 stability but is not essential for tubulin transport during ciliogenesis

Intraflagellar transport (IFT) is required for ciliogenesis by ferrying ciliary components using IFT complexes as cargo adaptors. IFT54 is a component of the IFT-B complex and is also associated with cytoplasmic microtubules (MTs). Loss of IFT54 impairs cilia assembly as well as cytoplasmic MT dynamics. The N-terminal calponin homology (CH) domain of IFT54 interacts with tubulins/MTs and has been proposed to transport tubulin during ciliogenesis, whereas the C-terminal coiled-coil (CC) domain binds IFT20. However, the precise function of these domains in vivo is not well understood. We showed that in Chlamydomonas, loss of IFT54 completely blocks ciliogenesis but does not affect spindle formation and proper cell cycle progression, even though IFT54 interacts with mitotic MTs. Interestingly, IFT54 lacking the CH domain allows proper flagellar assembly. The CH domain is required for the association of IFT54 with the axoneme but not with mitotic MTs, and also regulates the flagellar import of IFT54 but not IFT81 and IFT46. The C-terminal CC domain is essential for IFT54 to bind IFT20, and for its recruitment to the basal body and incorporation into IFT complexes. Complete loss of IFT54 or the CC domain destabilizes IFT20. ift54 mutant cells expressing the CC domain alone rescue the stability of IFT20 and form stunted flagella with accumulation of both IFT-A component IFT43 and IFT-B component IFT46, indicating that IFT54 also functions in IFT turn-around at the flagellar tip.     

Human heart disease: lessons from human pluripotent stem cell-derived cardiomyocytes

Technical advances in generating and phenotyping cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are now driving their wider acceptance as in vitro models to understand human heart disease and discover therapeutic targets that may lead to new compounds for clinical use. Current literature clearly shows that hPSC-CMs recapitulate many molecular, cellular, and functional aspects of human heart pathophysiology and their responses to cardioactive drugs. Here, we provide a comprehensive overview of hPSC-CMs models that have been described to date and highlight their most recent and remarkable contributions to research on cardiovascular diseases and disorders with cardiac traits. We conclude discussing immediate challenges, limitations, and emerging solutions.     

The emerging link between O-GlcNAcylation and neurological disorders

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is involved in the regulation of many cellular cascades and neurological diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and stroke. In the brain, the expression of O-GlcNAcylation is notably heightened, as is that of O-linked N-acetylglucosaminyltransferase (OGT) and β-N-acetylglucosaminidase (OGA), the presence of which is prominent in many regions of neurological importance. Most importantly, O-GlcNAcylation is believed to contribute to the normal functioning of neurons; conversely, its dysregulation participates in the pathogenesis of neurological disorders. In neurodegenerative diseases, O-GlcNAcylation of the brain’s key proteins, such as tau and amyloid-β, interacts with their phosphorylation, thereby triggering the formation of neurofibrillary tangles and amyloid plaques. An increase of O-GlcNAcylation by pharmacological intervention prevents neuronal loss. Additionally, O-GlcNAcylation is stress sensitive, and its elevation is cytoprotective. Increased O-GlcNAcylation ameliorated brain damage in victims of both trauma-hemorrhage and stroke. In this review, we summarize the current understanding of O-GlcNAcylation’s physiological and pathological roles in the nervous system and provide a foundation for development of a therapeutic strategy for neurological disorders.     

Regulation of centriolar satellite integrity and its physiology

Centriolar satellites comprise cytoplasmic granules that are located around the centrosome. Their molecular identification was first reported more than a quarter of a century ago. These particles are not static in the cell but instead constantly move around the centrosome. Over the last decade, significant advances in their molecular compositions and biological functions have been achieved due to comprehensive proteomics and genomics, super-resolution microscopy analyses and elegant genetic manipulations. Centriolar satellites play pivotal roles in centrosome assembly and primary cilium formation through the delivery of centriolar/centrosomal components from the cytoplasm to the centrosome. Their importance is further underscored by the fact that mutations in genes encoding satellite components and regulators lead to various human disorders such as ciliopathies. Moreover, the most recent findings highlight dynamic structural remodelling in response to internal and external cues and unexpected positive feedback control that is exerted from the centrosome for centriolar satellite integrity.     

Vascular smooth muscle cells in Marfan syndrome aneurysm: the broken bricks in the aortic wall

Marfan syndrome (MFS) is a connective tissue disorder with multiple organ manifestations. The genetic cause of this syndrome is the mutation of the FBN1 gene, encoding the extracellular matrix (ECM) protein fibrillin-1. This genetic alteration leads to the degeneration of microfibril structures and ECM integrity in the tunica media of the aorta. Indeed, thoracic aortic aneurysm and dissection represent the leading cause of death in MFS patients. To date, the most effective treatment option for this pathology is the surgical substitution of the damaged aorta. To highlight novel therapeutic targets, we review the molecular mechanisms related to MFS etiology in vascular smooth muscle cells, the foremost cellular type involved in MFS pathogenesis.