Dietary supplements in retinal diseases, glaucoma, and other ocular conditions

Environmental pollution, inadequate eating habits and unhealthy lifestyles have led to a tremendous increase in ocular diseases worldwide. Given the costly treatments that are currently available for the most common and threatening eye diseases (such as cataract, dry eye disorder, or diabetic retinopathy), curing these diseases or preventing refractive errors by taking nutraceuticals and natural compounds that are present in our daily diet is a very valuable intervention. The eyes are the most important part of our visual system and require micronutrients such as vitamins, carotenoids, trace metals, and omega-3 fatty acids in order to function properly and to protect themselves against light-induced and age-mediated degenerative disorders. The Mediterranean Diet (MedDiet) has been in the limelight since the 1980s because of the several health benefits it provides, including eye health. MedDiet is characterized by the consumption of small amounts of red meat, while emphasizing the intake of fish, eggs, nuts, legumes, citrus fruits, green vegetables, olives and their derivatives, especially olive oil, and dairy products in a proportionate manner, in order to achieve the maximum health benefits. The antioxidant, anti-inflammatory, and neuroprotective properties of these foods – both when used as an ingredient in the dietary regime or as a source of nutritional supplements – have shown promising results in the management of chronic degenerative ocular diseases, both in animal models and in human subjects. In this chapter, we will focus on the importance of MedDiet and natural compounds for the visual system and its role in slowing down age-related ocular degeneration

Impaired Ca2+ sensitivity of a novel GCAP1 variant causes cone dystrophy and leads to abnormal synaptic transmission between photoreceptors and bipolar cells

Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca2+/cGMP negative feedback. While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+. As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1-cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers' accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified

The role of 5-arylalkylamino- and 5-piperazino- moieties on the 7-aminopyrazolo[4,3-d]pyrimidine core in affecting adenosine A1 and A2A receptor affinity and selectivity profiles

New 7-amino-2-phenylpyrazolo[4,3-d]pyrimidine derivatives, substituted at the 5-position with aryl(alkyl)amino- and 4-substituted-piperazin-1-yl- moieties, were synthesized with the aim of targeting human (h) adenosine A1 and/or A2A receptor subtypes. On the whole, the novel derivatives 1-24 shared scarce or no affinities for the off-target hA2B and hA3 ARs. The 5-(4-hydroxyphenethylamino)- derivative 12 showed both good affinity (Ki = 150 nM) and the best selectivity for the hA2A AR while the 5-benzylamino-substituted 5 displayed the best combined hA2A (Ki = 123 nM) and A1 AR affinity (Ki = 25 nM). The 5-phenethylamino moiety (compound 6) achieved nanomolar affinity (Ki = 11 nM) and good selectivity for the hA1 AR. The 5-(N4-substituted-piperazin-1-yl) derivatives 15-24 bind the hA1 AR subtype with affinities falling in the high nanomolar range. A structure-based molecular modeling study was conducted to rationalize the experimental binding data from a molecular point of view using both molecular docking studies and Interaction Energy Fingerprints (IEFs) analysis.[Formula: see text]

Bilateral Symmetry of Visual Function Loss in Cone-Rod Dystrophies.

PURPOSE: To investigate bilateral symmetry of visual impairment in cone-rod dystrophy (CRD) patients and understand the feasibility of clinical trial designs treating one eye and using the untreated eye as an internal control. METHODS: This was a retrospective study of visual function loss measures in 436 CRD patients followed at the Ophthalmology Department of the Catholic University in Rome. Clinical measures considered were best-corrected visual acuity, focal macular cone electroretinogram (fERG), and Ganzfeld cone-mediated and rod-mediated electroretinograms. Interocular agreement in each of these clinical indexes was assessed by t- and Wilcoxon tests for paired samples, structural (Deming) regression analysis, and intraclass correlation. Baseline and follow-up measures were analyzed. A separate analysis was performed on the subset of 61 CRD patients carrying likely disease-causing mutations in the ABCA4 gene. RESULTS: Statistical tests show a very high degree of bilateral symmetry in the extent and progression of visual impairment in the fellow eyes of CRD patients. CONCLUSIONS: These data contribute to a better understanding of CRDs and support the feasibility of clinical trial designs involving unilateral eye treatment with the use of fellow eye as internal control

Towards a Long-Read Sequencing Approach for the Molecular Diagnosis of RPGRORF15 Genetic Variants

Sequencing of the low-complexity ORF15 exon of RPGR, a gene correlated with retinitis pigmentosa and cone dystrophy, is difficult to achieve with NGS and Sanger sequencing. False results could lead to the inaccurate annotation of genetic variants in dbSNP and ClinVar databases, tools on which HGMD and Ensembl rely, finally resulting in incorrect genetic variants interpretation. This paper aims to propose PacBio sequencing as a feasible method to correctly detect genetic variants in low-complexity regions, such as the ORF15 exon of RPGR, and interpret their pathogenicity by structural studies. Biological samples from 75 patients affected by retinitis pigmentosa or cone dystrophy were analyzed with NGS and repeated with PacBio. The results showed that NGS has a low coverage of the ORF15 region, while PacBio was able to sequence the region of interest and detect eight genetic variants, of which four are likely pathogenic. Furthermore, molecular modeling and dynamics of the RPGR Glu-Gly repeats binding to TTLL5 allowed for the structural evaluation of the variants, providing a way to predict their pathogenicity. Therefore, we propose PacBio sequencing as a standard procedure in diagnostic research for sequencing low-complexity regions such as RPGRORF15, aiding in the correct annotation of genetic variants in online databases

Ethical considerations regarding animal experimentation

Animal experimentation is widely used around the world for the identification of the root causes of various diseases in humans and animals and for exploring treatment options. Among the several animal species, rats, mice and purpose-bred birds comprise almost 90% of the animals that are used for research purpose. However, growing awareness of the sentience of animals and their experience of pain and suffering has led to strong opposition to animal research among many scientists and the general public. In addition, the usefulness of extrapolating animal data to humans has been questioned. This has led to Ethical Committees’ adoption of the ‘four Rs’ principles (Reduction, Refinement, Replacement and Responsibility) as a guide when making decisions regarding animal experimentation. Some of the essential considerations for humane animal experimentation are presented in this review along with the requirement for investigator training. Due to the ethical issues surrounding the use of animals in experimentation, their use is declining in those research areas where alternative in vitro or in silico methods are available. However, so far it has not been possible to dispense with experimental animals completely and further research is needed to provide a road map to robust alternatives before their use can be fully discontinued

Methodology for clinical research

A clinical research requires a systematic approach with diligent planning, execution and sampling in order to obtain reliable and validated results, as well as an understanding of each research methodology is essential for researchers. Indeed, selecting an inappropriate study type, an error that cannot be corrected after the beginning of a study, results in flawed methodology. The results of clinical research studies enhance the repertoire of knowledge regarding a disease pathogenicity, an existing or newly discovered medication, surgical or diagnostic procedure or medical device. Medical research can be divided into primary and secondary research, where primary research involves conducting studies and collecting raw data, which is then analysed and evaluated in secondary research. The successful deployment of clinical research methodology depends upon several factors. These include the type of study, the objectives, the population, study design, methodology/techniques and the sampling and statistical procedures used. Among the different types of clinical studies, we can recognize descriptive or analytical studies, which can be further categorized in observational and experimental. Finally, also pre-clinical studies are of outmost importance, representing the steppingstone of clinical trials. It is therefore important to understand the types of method for clinical research. Thus, this review focused on various aspects of the methodology and describes the crucial steps of the conceptual and executive stages