Predicting potentially pathogenic effects of hRPE65 missense mutations: a computational strategy based on molecular dynamics simulations

The human retinal pigment epithelium-specific 65-kDa protein (hRPE65) plays a crucial role within the retinoid visual cycle and several mutations affecting either its expression level or its enzymatic function are associated with inherited retinal diseases such as Retinitis Pigmentosa. The gene therapy product voretigene neparvovec (Luxturna) has been recently approved for treating hereditary retinal dystrophies; however, the treatment is currently accessible only to patients presenting confirmed biallelic mutations that severely impair hRPE65 function, and many reported hRPE65 missense mutations lack sufficient evidences for proving their pathogenicity. In this context, we developed a computational approach aimed at evaluating the potential pathogenic effect of hRPE65 missense variants located on the dimerisation domain of the protein. The protocol evaluates how mutations may affect folding and conformation stability of this protein region, potentially helping clinicians to evaluate the eligibility for gene therapy of patients diagnosed with this type of hRPE65 variant of uncertain significance

Optimal low-intensity pulsed ultrasound stimulation for promoting anti-inflammatory effects in macrophages

: In this paper, we stimulated M1-like macrophages (obtained from U937 cells) with low-intensity pulsed ultrasound (LIPUS) to lower pro-inflammatory cytokine production. A systematic screening of different frequencies, intensities, duty cycles, and exposure times was performed. The optimal stimulation conditions leading to a marked decrease in the release of inflammatory cytokines were determined to be 38 kHz, 250 mW/cm2, 20%, and 90 min, respectively. Using these parameters, we verified that up to 72 h LIPUS did not affect cell viability, resulting in an increase in metabolic activity and in a reduction of reactive oxygen species (ROS) production. Moreover, we found that two mechanosensitive ion channels (PIEZO1 and TRPV1) were involved in the LIPUS-mediated cytokine release modulation. We also assessed the role of the nuclear factor κB (NF-κB) signaling pathway and observed an enhancement of actin polymerization. Finally, transcriptomic data suggested that the bioeffects of LIPUS treatment occur through the modulation of p38 MAPK signaling pathway

Studio della proteina redox MICAL in vitro : caratterizzazione della sua funzione nel controllo della vitalita e motilita cellulare.

Il principale obiettivo di questa Tesi consiste nello studio della funzione della proteina MICAL in vitro (cellule immortalizzate in coltura). La nostra ipotesi è che MICAL grazie alla sua struttura, alla capacità di legare ossigeno, al controllo sull’assetto del citoscheletro [dati di letteratura], e al fatto di essere espressa in cellule endoteliali [nostri dati non pubblicati], possa modificare l’assetto locale del citoscheletro delle CE in relazione alla concentrazione locale di ossigeno, e, in questo modo dirigere la motilità orientata da ipossia delle CE durante l’angiogenesi. La funzione di MICAL potrebbe essere deregolata nella neo-angiogenesi patologica

Microgravity inhibits autophagy in human capillary endothelial cells in space flight

Microgravity and space radiation (SR) are the two environmental factors that most affect human crews in space flight (SF). The endothelium is highly sensitive to gravitational unloading and several health problems reported by astronauts derive from endothelial dysfunction and impaired homeostasis. Recently, we found that space-flown, endothelial cells show cell softening, the presence of stress granules, reduced motility, profound cytoskeletal reorganization, an increased number of primary cilia, mitochondrial senescence, activation of DNA repair mechanisms, changes of chromosome territories, telomere shortening and increased apoptosis. The transcriptomic study showed activation of oxidative stress, inflammation and DNA damage repair pathways. In general, pathways for metabolism and a pro-proliferative phenotype are activated by microgravity and downregulated by SR. SR upregulates pathways for endothelial activation (hypoxia, cytokines, inflammation), DNA repair and apoptosis, promoting macroautophagy/autophagy flux and an ageing-like phenotype, which instead are downregulated by microgravity. Microgravity and SR exert opposite effects on the MTORC1 gene pathway: SR inhibits the pathway (with consequent enhancement of autophagy), while microgravity strongly stimulates MTORC1 (with consequent inhibition of autophagy). The sum of both contributions results in the net effect of autophagy inhibition in space-flown cells. Microgravity and SR should be considered separately to tailor effective countermeasures to protect astronauts’ health. Potentiation of autophagy is worthy of further investigation as a possible physiological countermeasure to SF-induced cell stress

HCN1 channels: A versatile tool for signal processing by primary sensory neurons

Most primary sensory neurons (PSNs) generate a slowly-activating inward current in response to membrane hyperpolarization (Ih) and express HCN1 along with additional isoforms coding for hyperpolarization-activated channels (HCN). Changes in HCN expression may affect the excitability and firing patterns of PSNs, but retinal and inner ear PSNs do not fire action potentials, suggesting HCN channel roles may extend beyond excitability and cell firing control. In patients taking Ih blockers, photopsia triggered in response to abrupt changes in luminance correlates with impaired visual signal processing via parallel rod and cone pathways. Furthermore, in a mouse model of inherited retinal degeneration, HCN blockers or Hcn1 genetic ablation may worsen photoreceptors' demise. PSN's use of HCN channels to adjust either their firing rate or process signals generated by sensory transduction in non-spiking PSNs indicates HCN1 channels as a versatile tool with a novel role in sensory processing beyond firing control

Human Pathophysiological Adaptations to the Space Environment

Space is an extreme environment for the human body, where during long-term missions microgravity and high radiation levels represent major threats to crew health. Intriguingly, space flight (SF) imposes on the body of highly selected, well-trained, and healthy individuals (astronauts and cosmonauts) pathophysiological adaptive changes akin to an accelerated aging process and to some diseases. Such effects, becoming manifest over a time span of weeks (i.e., cardiovascular deconditioning) to months (i.e., loss of bone density and muscle atrophy) of exposure to weightlessness, can be reduced through proper countermeasures during SF and in due time are mostly reversible after landing. Based on these considerations, it is increasingly accepted that SF might provide a mechanistic insight into certain pathophysiological processes, a concept of interest to pre-nosological medicine. In this article, we will review the main stress factors encountered in space and their impact on the human body and will also discuss the possible lessons learned with space exploration in reference to human health on Earth. In fact, this is a productive, cross-fertilized, endeavor in which studies performed on Earth yield countermeasures for protection of space crew health, and space research is translated into health measures for Earth-bound population

Inducible Pluripotent Stem Cells to Model and Treat Inherited Degenerative Diseases of the Outer Retina: 3D-Organoids Limitations and Bioengineering Solutions

Inherited retinal degenerations (IRD) affecting either photoreceptors or pigment epithelial cells cause progressive visual loss and severe disability, up to complete blindness. Retinal organoids (ROs) technologies opened up the development of human inducible pluripotent stem cells (hiPSC) for disease modeling and replacement therapies. However, hiPSC-derived ROs applications to IRD presently display limited maturation and functionality, with most photoreceptors lacking well-developed outer segments (OS) and light responsiveness comparable to their adult retinal counterparts. In this review, we address for the first time the microenvironment where OS mature, i.e., the subretinal space (SRS), and discuss SRS role in photoreceptors metabolic reprogramming required for OS generation. We also address bioengineering issues to improve culture systems proficiency to promote OS maturation in hiPSC-derived ROs. This issue is crucial, as satisfying the demanding metabolic needs of photoreceptors may unleash hiPSC-derived ROs full potential for disease modeling, drug development, and replacement therapies

Human pathophysiological adaptations to the space environment

Space is an extreme environment for the human body, where during long-term missions microgravity and high radiation levels represent major threats to crew health. Intriguingly, space flight (SF) imposes on the body of highly selected, well-trained, and healthy individuals (astronauts and cosmonauts) pathophysiological adaptive changes akin to an accelerated aging process and to some diseases. Such effects, becoming manifest over a time span of weeks (i.e., cardiovascular deconditioning) to months (i.e., loss of bone density and muscle atrophy) of exposure to weightlessness, can be reduced through proper countermeasures during SF and in due time are mostly reversible after landing. Based on these considerations, it is increasingly accepted that SF might provide a mechanistic insight into certain pathophysiological processes, a concept of interest to pre-nosological medicine. In this article, we will review the main stress factors encountered in space and their impact on the human body and will also discuss the possible lessons learned with space exploration in reference to human health on Earth. In fact, this is a productive, cross-fertilized, endeavor in which studies performed on Earth yield countermeasures for protection of space crew health, and space research is translated into health measures for Earth-bound population

Hypergravity activates a pro-angiogenic homeostatic response by human capillary endothelial cells

Capillary endothelial cells are responsible for homeostatic responses to organismic and environmental stimulations. When malfunctioning, they may cause disease. Exposure to microgravity is known to have negative effects on astronauts’ physiology, the endothelium being a particularly sensitive organ. Microgravity-related dysfunctions are striking similar to the consequences of sedentary life, bed rest, and ageing on Earth. Among different countermeasures implemented to minimize the effects of microgravity, a promising one is artificial gravity. We examined the effects of hypergravity on human microvascular endothelial cells of dermal capillary origin (HMEC-1) treated at 4 g for 15 min, and at 20 g for 15 min, 3 and 6 h. We evaluated cell morphology, gene expression and 2D motility and function. We found a profound rearrangement of the cytoskeleton network, dose-dependent increase of Focal Adhesion kinase (FAK) phosphorylation and Yes-associated protein 1 (YAP1) expression, suggesting cell stiffening and increased proneness to motility. Transcriptome analysis showed expression changes of genes associated with cardiovascular homeostasis, nitric oxide production, angiogenesis, and inflammation. Hypergravity-treated cells also showed significantly improved motility and function (2D migration and tube formation). These results, expanding our knowledge about the homeostatic response of capillary endothelial cells, show that adaptation to hypergravity has opposite effect compared to microgravity on the same cell type