Glaucoma is a group of diverse diseases characterized by cupping of the optic nerve head due to the loss of retinal ganglion cells. It is the most common cause of irreversible blindness throughout the word; therefore, its timely diagnosis and early detection through an ophthalmological examination are very important. We, herein, present the information on the epidemiology, pathophysiology, clinical diagnosis, and treatment of glaucoma. We also emphasize the investigations of the last decades that have allowed identifying numerous genes and susceptible genetic factors. We have also described in detail the genes whose mutations cause or contribute to the development of the disease.

Over the last decade, gene set analysis has become the first choice for gaining insights into underlying complex biology of diseases through gene expression and gene association studies. It also reduces the complexity of statistical analysis and enhances the explanatory power of the obtained results. Although gene set analysis approaches are extensively used in gene expression and genome wide association data analysis, the statistical structure and steps common to these approaches have not yet been comprehensively discussed, which limits their utility. In this article, we provide a comprehensive overview, statistical structure and steps of gene set analysis approaches used for microarrays, RNA-sequencing and genome wide association data analysis. Further, we also classify the gene set analysis approaches and tools by the type of genomic study, null hypothesis, sampling model and nature of the test statistic, etc. Rather than reviewing the gene set analysis approaches individually, we provide the generation-wise evolution of such approaches for microarrays, RNA-sequencing and genome wide association studies and discuss their relative merits and limitations. Here, we identify the key biological and statistical challenges in current gene set analysis, which will be addressed by statisticians and biologists collectively in order to develop the next generation of gene set analysis approaches. Further, this study will serve as a catalog and provide guidelines to genome researchers and experimental biologists for choosing the proper gene set analysis approach based on several factors.

Extracellular vesicles (EVs) have numerous potential applications in the field of healthcare and diagnostics, and research into their biological functions is rapidly increasing. Mainly because of their small size and heterogeneity, there are significant challenges associated with their analysis and despite overt evidence of the potential of EVs in clinical diagnostic practice, guidelines for analytical procedures have not yet been properly established. Here, we present an overview of the main methods for studying the properties of EVs based on the principles of fluorescence. Setting aside the isolation, purification and physicochemical characterization strategies which answer questions about the size, surface charge and stability of EVs (reviewed elsewhere), we focus on available optical tools that enable the direct analysis of phenotype and mechanisms of interaction with tissues. In brief, the topics on which we elaborate range from the most popular approaches such as nanoparticle tracking analysis and flow cytometry, to less commonly used techniques such as fluorescence depolarization and microarrays as well as emerging areas such as fast fluorescence lifetime imaging microscopy (FLIM). We highlight that understanding the strengths and limitations of each method is essential for choosing the most appropriate combination of analytical tools. Finally, future directions of this rapidly developing area of medical diagnostics are discussed.

BACKGROUND: Microgravity (μG) negatively influences bone metabolism by affecting normal osteoblast and osteoclast function. μG effects on bone metabolism has been an extensive field of study in recent years, due to the challenges presented by space flight.
METHODS: We systematically reviewed research data from genomic studies performed in real or simulat-ed μG, on osteoblast and osteoclast cells. Our search yielded 50 studies, of which 39 concerned cells of the osteoblast family and 11 osteoclast precursors.
RESULTS: Osteoblastic cells under μG show a decreased differentiation phenotype, proved by diminished expression levels of Alkaline Phosphatase (ALP) and Osteocalcin (OCN) but no apoptosis. Receptor Activator of NF-κB Ligand (RANKL)/ Osteoprotegerine (OPG) ratio is elevated in favor of RANKL in a time-dependent manner, and further RANKL production is caused by upregulation of Interleukin-6 (IL-6) and the inflammation pathway. Extracellular signals and changes in the gravitational environment are perceived by mechanosensitive proteins of the cytoskeleton and converted to intracellular signals through the Mitogen Activated Protein Kinase pathway (MAPK). This is followed by changes in the ex-pression of nuclear transcription factors of the Activator Protein-1 (AP-1) family and in turn of the NF-κB, thus affecting osteoblast differentiation, cell cycle, proliferation and maturation. Pre-osteoclastic cells show increased expression of the marker proteins such as Tryptophan Regulated Attenuation Protein (TRAP), cathepsin K, Matrix Metalloproteinase-9 (MMP-9) under μG conditions and become sensitized to RANKL.
CONCLUSIONS: Suppressing the expression of fusion genes such as syncytine-A which acts independently of RANKL, could be possible future therapeutic targets for microgravity side effects.

An accurate etiological classification is key to optimize secondary prevention after ischemic stroke, but the cause remains undetermined in one third of patients. Several studies pointed out the usefulness of circulating gene expression markers to discriminate cardioembolic (CE) strokes, mainly due to atrial fibrillation (AF), while only exploring them in small cohorts. A systematic review of studies analyzing high-throughput gene expression in blood samples to discriminate CE strokes was performed. Significantly dysregulated genes were considered as candidates, and a selection of them was validated by RT-qPCR in 100 patients with defined CE or atherothrombotic (LAA) stroke etiology. Longitudinal performance was evaluated in 12 patients at three time points. Their usefulness as biomarkers for AF was tested in 120 cryptogenic strokes and 100 individuals at high-risk for stroke. Three published studies plus three unpublished datasets were considered for candidate selection. Sixty-seven genes were found dysregulated in CE strokes. CREM, PELI1, and ZAK were verified to be up-regulated in CE vs LAA (p = 0.010, p = 0.003, p < 0.001, respectively), without changes in their expression within the first 24 h after stroke onset. The combined up-regulation of these three biomarkers increased the probability of suffering from CE stroke by 23-fold. In cryptogenic strokes with subsequent AF detection, PELI1 and CREM showed overexpression (p = 0.017, p = 0.059, respectively), whereas in high-risk asymptomatic populations, all three genes showed potential to detect AF (p = 0.007, p = 0.007, p = 0.015). The proved discriminatory capacity of these gene expression markers to detect cardioembolism even in cryptogenic strokes and asymptomatic high-risk populations might bring up their use as biomarkers.

Many scientific articles have been published that use gene-expression-based technologies to discriminate a trait of interest, typically a disease subgroup, within a patient population. However, few gene-expression-based signatures have at present reached the market and become a financially and clinically successful product. The technological, scientific and medical challenges, the regulatory environment and the financial considerations are all essential parts of the development process. Here we discuss the scientific aspects of successfully developing a gene-expression-based signature and review the global strategy of six products that made it to the market. We also present a point-to-point guide that should help researchers to successfully develop genomic signatures, thus paving the way towards personalized medicine.

Begomoviruses are a group of icosahedral single stranded DNA viruses exclusively transmitted by the sweet potato whitefly Bemisia tabaci in a persistent, circulative manner. In this mode of transmission, begomoviruses are acquired by their insect vector as intact virions from the plant phloem, move along the food canal, foregut and esophagus and reach the midgut where they are absorbed into the hemolymph via the filter chamber. The filter chamber is the site where most of the ingested food is filtered, and the first site where the majority of begomoviruses appear to be translocated into the hemolymph via unknown proteins or receptors. Transport from the filter chamber to the hemolymph is aided by a Heat Shock Protein 70. Virus particles not translocated across the filter chamber circulate in the midgut loop but it is not known whether absorption into the hemolymph occurs along this loop. Localization studies have confirmed that begomoviruses are not associated with the hindgut and absorption of virions in this organ is unlikely. In the hemolymph, virions have been shown to interact with a GroEL chaperone produced by the whitefly\'s endosymbiontic bacteria for ensuring their safe journey to the salivary glands. Virions penetrate the primary salivary glands via unknown proteins or receptors and are transported and secreted outside the whitefly to the plant with salivary secretions. Several recent studies have demonstrated the implications of insect and endosymbiont proteins such as the heat shock protein 70 and the bacterial GroEL protein, in the transmission of begomoviruses by B. tabaci. Additional studies attempting to identify other proteins that aid or interact with begomoviruses along their circulation pathway in the whitefly are reviewed in this paper.

The mycotoxin Ochratoxin A (OTA) is a potent renal carcinogen in male rats. Transcriptomic studies on OTA (4 in vitro, 6 in vivo, 2 in vitro/in vivo) have been reviewed. The aim of 6 of them was mainly mechanistic whereas the rest had mostly predictive (1) or evaluation (5) purposes. An overall tendency towards gene expression downregulation was observed, probably as a result of protein synthesis inhibition. DNA damage response genes were not deregulated in most of the studies. Genes involved in acute renal injury, cell survival and cell proliferation were upregulated in several in vivo studies. Apoptosis genes were deregulated in vitro but less affected in vivo; activation of several MAPKs has been observed. Many genes related to oxidative stress or involved in cell-to-cell interaction pathways (Wnt) or cytoskeleton structure appeared to be deregulated either in vitro or in vivo. Regucalcin was highly downregulated in vivo and other calcium homeostasis genes were significantly deregulated in vitro. Genes related to OTA transport (OATs) and metabolism (CYPs) appeared downregulated in vivo. Overall, the mechanism of action of OTA remains unclear, however transcriptomic data have contributed to new mechanistic hypothesis generation and to in vitro-in vivo comparison.

BACKGROUND: Genetic testing of preimplantation embryos has been used for preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Microarray technology is being introduced in both these contexts, and whole genome sequencing of blastomeres is also expeted to become possible soon. The amount of extra information such tests will yield may prove to be beneficial for embryo selection, will also raise various ethical issues. We present an overview of the developments and an agenda-setting exploration of the ethical issues.
METHODS: The paper is a joint endeavour by the presenters at an explorative \'campus meeting\' organized by the European Society of Human Reproduction and Embryology in cooperation with the department of Health, Ethics & Society of the Maastricht University (The Netherlands).
RESULTS: The increasing amount and detail of information that new screening techniques such as microarrays and whole genome sequencing offer does not automatically coincide with an increasing understanding of the prospects of an embryo. From a technical point of view, the future of comprehensive embryo testing may go together with developments in preconception carrier screening. From an ethical point of view, the increasing complexity and amount of information yielded by comprehensive testing techniques will lead to challenges to the principle of reproductive autonomy and the right of the child to an open future, and may imply a possible larger responsibility of the clinician towards the welfare of the future child. Combinations of preconception carrier testing and embryo testing may solve some of these ethical questions but could introduce others.
CONCLUSIONS: As comprehensive testing techniques are entering the IVF clinic, there is a need for a thorough rethinking of traditional ethical paradigms regarding medically assisted reproduction.