Keratoconus (KTCN) is a degenerative disorder of the eye characterized by the conical shape and thinning of the cornea. The abnormal structure of KTCN-affected cornea results in loss of visual acuity. While many studies examine how environmental factors influence disease development, finding the genetic triggers has been a major emphasis of KTCN research. This paper focuses on genomic strategies that were implemented for finding candidate genes, including linkage and association studies, and presents different approaches of mutation screening. The advantages and limitations of particular tools are discussed based on literature and personal experience. Since etiology underlying KTCN is complex, numerous findings indicating heterogeneity of genetic factors involved KTCN etiology are presented. Keratoconus (KTCN) is a degenerating, usually bilateral disorder of the eye characterized by progressive stromal thinning, which results in the conical shape of the cornea. These structural changes in the corneal layers induce optical aberrations, leading to a loss of visual acuity due to distorted blurred vision, which is caused by irregular astigmatism, and high myopia  and contain numerous candidate genes to be further evaluated. Mutation screening of all putative genes from these regions requires substantial additional effort; thus, post-linkage analysis is often narrowed to the sequencing of a few functional genes localized in the genetic interval between markers with the maximum LOD. However, functional and positional candidate genes often do not contain variants related to the disease phenotype.COL8A1  gene, localized at 3p14-q13 KTCN is a good example for that , STK24 (serine/threonine kinase 24), and DOCK9 (dedicator of cytokinesis 9) genes , for which the maximum LOD score (4.1 in multipoint parametric linkage and 3.2 for multipoint non-parametric linkage) has been obtained. Interestingly, molecular screening of FARP1 has revealed no pathogenic variants in the affected members of the KTCN-014 family  and SLC4A11 (solute carrier family 4 member 11) at 2q13-q14.3 and 20p13-p12.2  has revealed variants that were observed significantly more frequently in family members with KTCN  promoter gene, which may be involved in inflammatory processes in KTCN , is used to identify the common sequence variation that contributes to a disease risk. This type of study is performed in case–control cohorts with hundreds or thousands individuals and allows for genome-wide analyses due to microarray-based SNP genotyping Manolio .HGF (hepatocyte growth factor) gene, which was previously reported as a high myopia gene in a Han Chinese population  and KTCN in a Caucasian cohort from the USA  gene, encoding an enzyme responsible for collagen cross-linking in different tissues, including the cornea  in patients may lead to false positive associations. Moreover, significant loci often explain only a small proportion of the phenotypic variation, suggesting that rare variants may underlie the genetic causes of the disease. As GWAS recognizes only common risk alleles, the locus associated with GWAS signals may be far larger than assumed. Thus, a good solution for revealing rare variants would be to extend the sequencing region to at least a few additional megabases around the GWAS signal  and SOD1 (superoxide dismutase isoenzyme 1), the first two genes proposed as significant for KTCN development. The gene VSX1 is mapped to chromosome 20p11.2 and encodes a vertebrate paired-like homeodomain transcription factor. It is expressed in vitro and in vivo in human keratocytes during their differentiation into myofibroblasts in response to wound healing , CTSH (cathepsin H), CRABP1 (cellular retinoic acid binding protein 1), and RASGRF1 (RAS protein-specific guanine nucleotide-releasing factor 1), has revealed no significant variants in KTCN patients , IPO5 (c.2377-132A>C), and STK24 (c.1053+29G>C) formed a disease-related haplotype that was carried by all affected individuals in the Ecuadorian KTCN family  is another candidate gene screened in KTCN patients. The pathogenic mutations in ZNF469 were originally reported in patients with brittle cornea syndrome, a condition characterized by an extremely thin cornea that tends to rupture , which is abnormal in a wide variety of corneal diseases, including KTCN or corneal dystrophies  in 20 Indian KTCN patients has revealed 84 variants, including two novel frameshift variants  was heteroplasmic, whereas the remaining nine were homoplasmic  in different compartments of human body, including cornea, which cause a toxic effect on a number of cellular components, such as proteins, nucleic acids, and membrane phospholipids. ROS may be induced by UV light, and since the cornea is constantly exposed to the UV radiation, it is particularly vulnerable to the oxidative stress and damage from ROS . Current NGS technologies offer different methods for template preparation, sequencing, visualizing, genome alignment, and assembly. Still, each NGS strategy allows for direct and complete DNA sequencing in a high-throughput manner, as well as the production of an enormous volume of low-cost reads per instrument run Metzker . Since 2miR-184 gene in a family with severe KTCN combined with the early onset anterior polar cataract . WES is a promising tool that enables selective sequencing of nearly all protein-coding sequences. Unlike the gold standard—exon-by-exon analysis by conventional Sanger sequencing—WES is time- and cost-effective and more suitable for the precise determination of rare and unique coding sequence variants in an individual exome. The literature data provide many examples of the efficacy of WES in variants identification in both rare and common complex disorders, including ocular diseases. Rare coding sequence variants have been found in patients with the sporadic and the familial form of retinitis pigmentosa ; in members of family with congenital cataracts and corneal abnormalities including KTCN, and in Greek sporadic KTCN patients  in search of de novo mutations is quite simple, looking for hereditable mutations in large families with a complex disease, such as KTCN, could be problematic and requires additional Sanger-segregation analyses. Despite these limitations, WES is a promising tool to study genetic causes of many disorders. The increasing interest in new molecular techniques and easier access to the necessary equipment leads us to believe that this approach will be more frequently implemented in KTCN research in the near future.As we indicated above, WES allows for analysis of the coding region of genes only. Since many DNA variants associated with diseases lie outside the coding regions of genomic DNA, WGS, which allows for the characterization of entire genomes of any size, has become a promising and more appropriate solution for research looking for the unidentified genetic causes of diseases. However, while the newest machines can process whole human genomes for only $1000 each, WGS is not routinely used in research laboratories. An expanded computational infrastructure is required in processing and interpreting data sets of full genomes. Literature data showed that WGS may be successfully implemented in the identification of pathogenic DNA variants in genetically heterogeneous diseases, including autism spectrum disorder and the autosomal recessive retinitis pigmentosa  promoter. It was shown that overexpression of TIMP3 might induce apoptosis in corneal stromal cell cultures leading to KTCN (Matthews et al. TIMP3 were not identified suggesting that alteration of TIMP3 expression might be due to epigenetic factors (De Bonis et al. TIMP3 (De Bonis et al. Methylation status in KTCN was studied in One of the issues limiting KTCN studies is a problem with the collection of the appropriate tissue for analyses. While blood samples are usually not difficult to collect from all studied individuals, the collection of whole corneal tissues or their fragment is limited to patients with corneal transplantation. Moreover, it is not possible to obtain the healthy cornea from living donors for comparative analyses. Therefore, the corneal tissues are often derived from non-KTCN individuals with other corneal events, who were referred for corneal transplantation.A good model for KTCN research could be cell lines derived from different parts of human corneas, i.e., corneal epithelial or endothelial cell lines, and corneal fibroblasts. However, the establishment and cultivation of cell lines are difficult and often fail. In addition, corneal cell lines as other cell lines derived from different cell types are not well characterized, have a limited life span, and may not reflect the properties of in vivo cells.There is sufficient data to suggest that KTCN has a strong genetic component. Based on genetic studies, it has become clear that KTCN etiology might involve more than one causing factor and the traditional molecular techniques may not be sufficient for identifying all elements involved in KTCN. The rapid advances of high-throughput molecular technologies have led to the development of innovative approaches allowing relationship analyses between disease phenotype and variations in the human genome. With increased using of next-generation sequencing technologies, it may be possible to identify genetic variants underlying the KTCN etiology.


The central control of energy balance involves a highly regulated neuronal network within the hypothalamus and the dorsal vagal complex. In these structures, pro-opiomelanocortin (POMC) neurons are known to reduce meal size and to increase energy expenditure. In addition, leptin, a peripheral signal that relays information regarding body fat content, modulates the activity of melanocortin pathway neurons including POMC-, Agouti-related peptide (AgRP)/Neuropeptide Y (NPY)-, melanocortin receptors (MC3R and MC4R)-expressing neurons. MicroRNAs (miRNAs) are short non-coding RNAs of 22–26 nucleotides that post-transcriptionally interfere with target gene expression by binding to their mRNAs. Evidence has demonstrated that miRNAs play important roles in the central regulation of energy balance. In this context, different studies identified miRNAs including miR-200 family, miR-103, or miR-488 that could target the genes of melanocortin pathway. More precisely, these different miRNAs can modulate energy homeostasis by affecting leptin transduction pathway in the POMC, or AgRP/NPY neurons. This article reviews the role of identified miRNAs in the modulation of melanocortin pathway in the context of energy homeostasis. Overweight and obesity are significant risk factors for various chronic diseases, including cancer, heart diseases, and type 2 diabetes. In 2014, World Health Organization estimated that more than 1.9 billion adults were overweight. Of these over 600 million were obese. Dramatically, 41 million children under the age of 5 were overweight or obese in 2014. With such a high and expanding prevalence, and considering the associated diseases, obesity has an important economic impact on health care systems. For instance, the global medical costs related to obesity were estimated to reach up to 147 billion dollars per year in the USA  continuously follows modifications in metabolic parameters  or hormones  and elicits adaptive responses like food intake regulation or autonomic nervous system modulation of glucose homeostasis and energy expenditure Figure . More paOne important goal of present research is to identify the molecular mechanism and the intracellular mediators allowing these POMC and NPY/AgRP neurons to respond to energy status variations. Then, it appears crucial to increase our knowledge of the mechanisms controlling the melanocortin system activity, particularly by the discovery of new signaling pathways involved in the control of POMC and AgRP genes expression by leptin. Such pathways should provide beneficial pharmacological targets, and lead to the development of new generation drugs that can safely and effectively treat overweight and obesity linked to leptin resistance. In this context, it has been recently discovered new mechanisms involved in the control of the expression of the melanocortin pathway's genes. In particular, epigenetic mechanisms, including DNA methylation, the modifications of histones, and specific microRNAs (miRNAs) expression, have been proposed to mediate the expression of the melanocortin system , the mammalian target of rapamycin (mTOR), and SIRT1 deacetylase in the hypothalamus, are essential for leptin sensing and then energy homeostasis. More precisely, inactivation of AMPK in POMC neurons induced obesity while SIRT1 in POMC neurons is required for adaptations against diet-induced obesity  of message encoding RNAs. The miRNAs are small non-coding RNA molecules of 21 to 26 nucleotides that regulate gene expression Bartel, . They weAs indicated before, miRNAs are important for neurodevelopment but also neurotransmission or synaptic plasticity  has been observed in the hypothalamus of anx/anx mice  and the differential regulation of RISC genes need to be clarified.As mentioned above, hypothalamus and DVC are important for the detection of circulating nutrients and hormones and in turn, these neuronal structures modulate the pancreas, liver, and adipose tissue physiology through efferent pathways. The function of miRNAs in the hypothalamus and DVC has not been clearly addressed. However, as in the other organs involved in energy homeostasis, miRNAs undoubtedly play a key role in hypothalamus and DVC neurons, and particularly in the function of melanocortin pathway. In accordance with this point, it has been shown that in the anorexia mouse model, ob/ob mice (Schneeberger et al., A large number of miRNAs are expressed in the brain, and deletion of Dicer, a specific enzyme involved in miRNA maturation, in specific brain structures or neuronal cell type can lead to behavioral defect and neurodegeneration (Schaefer et al., It has been established that each tissue exhibit a specific profile of miRNA expression (Babak et al., ob/ob mice (Crépin et al., ob/ob mice compared to the control animals (Crépin et al., ob/ob mice as compared to control animals by real time PCR (Crépin et al., ob/ob mice decreased after leptin treatment (Crépin et al., ob/ob mice can down-regulate Insulin receptor substrate-2 and leptin receptor hypothalamic expression that are involved in the insulin and leptin pathways (Crépin et al., The functions of hypothalamic miRNAs are highly investigated. In particular, potential impact of leptin on hypothalamic miRNAs expression profile begins to be clarified. Recently, the group of Dr Taouis performed a large-scale expression analysis using Taqman Low Density Arrays methodology to analyse 524 rodent mature miRNAs on the hypothalamus of in situ hybridization, we demonstrated that these three miRNAs are present in the POMC neurons of the ARC (Derghal et al., ob/ob and db/db mice models (Derghal et al., ob/ob mice suggesting a role of leptin in the expression of mir-383, mir-384-3p, and mir-488 (Derghal et al., Regarding the functions of miRNAs in the POMC neurons, it has been demonstrated that specific deletion of Dicer in POMC-expressing cells leads to obesity and diabetes which is associated with loss of POMC neurons in the ARC (Schneeberger et al., As indicated above, a large number of miRNAs are involved in the central regulation of energy homeostasis. We postulate that miRNAs are energy sensors involved in the hypothalamic control of systemic energy balance Figure . MoreoveLM and AD wrote the manuscript. MD and JT helped with manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Neurodegenerative diseases are rarely caused by a mutation in a single gene but rather influenced by a combination of genetic, epigenetic and environmental factors. Emerging high-throughput technologies such as RNA sequencing have been instrumental in deciphering the molecular landscape of neurodegenerative diseases, however, the interpretation of such large amounts of data remains a challenge. Network biology has become a powerful platform to integrate multiple omics data to comprehensively explore the molecular networks in the context of health and disease. In this review article, we highlight recent advances in network biology approaches with an emphasis in brain-networks that have provided insights into the molecular mechanisms leading to the most prevalent neurodegenerative diseases including Alzheimer’s (AD), Parkinson’s (PD) and Huntington’s diseases (HD). We discuss how integrative approaches using multi-omics data from different tissues have been valuable for identifying biomarkers and therapeutic targets. In addition, we discuss the challenges the field of network medicine faces toward the translation of network-based findings into clinically actionable tools for personalized medicine applications. Neurodegenerative diseases are usually sporadic in nature and commonly influenced by a wide range of genetic, epigenetic and environmental factors. With the advent of new high-throughput technologies such as RNA sequencing, it has become essential to develop methods beyond the classical pathway analysis to systematically interpret large amounts of data in the context of health and disease. Despite the progress of high-throughput genomic studies the precise pathogenic mechanisms leading to the most prevalent neurodegenerative diseases remain elusive. To this end, the applications of network biology have been successful to provide biological insight and to decipher the molecular underpinnings of neurodegenerative diseases. Network biology is based on the premise that complex diseases, like neurodegenerative diseases, are frequently caused by alterations in many genes comprising multiple biological pathways. A network consists of nodes and edges that may represent genes, proteins, miRNAs, noncoding RNAs, drugs, or diseases connected through a wide range of interactions including, but not limited to physical, genetic, co-expression and colocalization. An example of network analysis of Alzheimer’s disease (AD) that identifies central hubs is shown in Figure Seminal work in network biology including the construction of the human disease network , presenilin 1 (PSEN1) and presenilin 2 (PSEN2) are documented to cause early onset AD, which accounts for approximately 2% of the cases with symptoms appearing before the age of 65  and brain derived neurotrophic factor (BDNF) interacting with the retinoic acid receptor related orphan receptor  are increasingly being used to find highly co-expressed gene modules associated with a particular biological pathway or a clinical trait of interest  and NFT scores, both widely utilized indicators of disease severity in AD  in intraneuronal cytoplasmic inclusion known as Lewy bodies and the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Dopamine restorative drugs and deep brain stimulation are current therapies to treat patients, however, these treatments only alleviate motor symptoms but do not impact disease progression. Mutations in the SNCA with an extended 3′ untranslated region, termed aSynL, which influenced SNCA accumulation  toxicity , the Parkinson’s disease database (ParkDB) and the Comparative Toxicogenomics Database (CTD), identified alvespimycin (17-DMAG) as a candidate neuroprotective agent for PD  is one of the most common dominantly inherited neurodegenerative disorders. The symptoms include some motor symptoms, such as chorea and dystonia, as well as non-motor symptoms, including psychological changes, and cognitive decline leading to dementia  tail in the huntingtin gene  genes expression were dysregulated in four of the modules, indicating that regulatory factors of these genes, such as Rest, Ctcf and Rad21, could be involved in HTT toxicity in mice , inhibitor of DNA binding 2 (ID2), inhibitor of DNA binding 4 (ID4), paired-like homeodomain transcription factor 2 (PITX2), thrombospondin 1 (THBS1), and left-right determination factor 2  studies from cerebellum and frontal cortex of AD patients, GWAS from AD and PD and PPI networks indicated that some PD variants  were associated with the expression of In addition to the studies focused on AD and PD, recent network-based approaches have been applied to understand the molecular networks shared among other neurodegenerative diseases. One study focused on the dorsolateral prefrontal cortex (DLPFC) which is commonly affected in both AD and HD to construct coexpression networks using genome wide expression data from 600 postmorterm DLPFC tissues from AD, HD, and non-dementia controls. Differential coexpression analysis revealed a subnetwork of 242 genes enriched in pathways related to neuron differentiation, apoptosis, gap junction trafficking, and cellular metabolic processes  reversed the gene network changes elicited by fructose (Meng et al., Nutrition is also recognized as an important component in the development and treatment of neurodegenerative diseases (Seidl et al., In addition to a nutrient-rich diet, both physical exercise and cognitive training promote healthy aging Kraft, . It has JAS and VB wrote the first draft of the manuscript. JAS, VB and JAP edited and reviewed the final draft of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


On May 25, 2016, a detainee at a U.S. Immigration and Customs Enforcement (ICE) detention center in Arizona who had been hospitalized with fever and a generalized maculopapular rash was confirmed to have measles by real-time polymerase chain reaction (rPCR). A second case of measles in a staff member was confirmed by rPCR the next day. The privately operated, city-contracted facility housed 1,425 detainees, and employed 510 staff members, including 95 federal ICE staff and 415 contract staff of four distinct employers. Outbreak control measures consisted of administration of measles-mumps-rubella (MMR) vaccine to 1,424 detainees housed at the facility during May 29–31 and isolation of the detainee patient and any additional detainee patients identified during their remaining infectious period (until 4 days after rash onset). Recommendations were made by federal, state, and local public health partners to exclude staff members with measles-compatible symptoms as well as exposed staff members without presumptive evidence of immunity to measles.*Epidemiologic investigations by local and state health departments and CDC identified 31 total cases of measles in 22 detainees and nine staff members, with rash onsets occurring May 6–June 26 . InitialReports of illness from personnel who had developed measles might have prompted other staff members to get vaccinated; by August 4, a total of 445 (87%) staff members were considered to have evidence of immunity, including 119 (23%) with documentation of receipt of 2 MMR doses before the start of the outbreak, 307 (60%) who had received 1 previous MMR dose and received a second dose during the outbreak, and 19 (4%) with serologic evidence of immunity. Although recommendations to exclude infectious staff members and nonimmune staff members suspected to have been exposed were made as soon as the outbreak was recognized, slow compliance with vaccination recommendations and incomplete implementation of exclusion recommendations, and restrictions on enforcing them, might have prolonged this outbreak.Outbreak response is expensive and resource-intensive (Recommendations for implementing measles control policies for detention and correctional facilities, similar to those recommended in health care facilities, could be considered. If permissible, contractual and interagency agreements could include similar provisions, such as requiring MMR vaccination for staff members who work in detention facilities and do not have documented evidence of immunity.


Saccharomyces, allowing in depth studies on chromosomal numerical aberrations. Many wild type strains naturally develop chromosomal rearrangements while adapting to different environmental conditions. Translocations, in particular, are valuable not only because they naturally drive species evolution, but because they might allow the artificial generation of new strains that can be optimized for industrial purposes. In this area, several methodologies to artificially trigger chromosomal translocations have been conceived in the past years, such as the chromosomal fragmentation vector (CFV) technique, the Cre-loxP procedure, the FLP/FRT recombination method and, recently, the bridge - induced translocation (BIT) system. An overview of the methodologies to generate chromosomal translocations in yeast will be presented and discussed considering advantages and drawbacks of each technology, focusing in particular on the recent BIT system. Translocants are important for clinical studies because translocated yeast cells resemble cancer cells from morphological and physiological points of view and because the translocation event ensues in a transcriptional de-regulation with a subsequent multi-factorial genetic adaptation to new, selective environmental conditions. The phenomenon of post-translocational adaptation (PTA) is discussed, providing some new unpublished data and proposing the hypothesis that translocations may drive evolution through adaptive genetic selection.In this review we will focus on chromosomal translocations (either spontaneous or induced) in budding yeast. Indeed, very few organisms tolerate so well aneuploidy like  Saccharomyces cerevisiae for its genomic plasticity, aneuploidy tolerance and finest recombination machinery is the ideal model organism to study chromosome changes selectively established during environmental adaptation. The majority of these genomic variations are limited to single nucleotide polymorphisms, intra-chromosomal gene copy number duplication, and difference in sub-telomeric lengths. However, recent studies on industrial strains of S. cerevisae used for biofuel and wine production, suggested that massive alterations in gene transcription were associated with adaptive evolution during long-term fermentations. Genomic analysis of these new strains revealed that spontaneous gross chromosomal rearrangements and in particular chromosomal translocations were responsible for this transcriptional shift and were due to adaptation to sulfites 2Candida glabrata, which shows high-occurrence of spontaneous chromosome translocation in mitosis S. cerevisiae strains following 100-500 generations of growth in glucose-limited chemostats Adaptive evolution emerges through random genomic mutations that usually reduce and rarely improve the fitness of an organism. FRT Cre-loxP12. In most of the cases, the initial double strand break (DSB) is generated thanks to the I-SceI or the HO endonuclease. More recently, the BIT system was developed, which allows the formation of chromosomal translocations without pre-engineering of the genome. All these GCRs  - providing methodologies are based on homology or micro-homology recombination and will be analyzed in details in the next paragraphs. Non-homologous end joining - based techniques and physical/chemical approaches will not be surveyed in this review.In order to understand the control of genome stability and adaptive evolution and to have valid tools to manipulate the yeast genome, several biological methods have been developed. Ploidy maintenance was altered through many systems such as the FLP-FRT and the Cre/loxP techniques.The site-specific recombination systems most commonly used in yeast as well as in higher eukaryotes are the FLP/FRTFRT, Flippase Recognition Target, with an in vivo efficiency of 100% FRT system has been widely utilized in many higher eukaryotes such as plants, Caenorhabditis, mice, somatic cell lines and in embryonic stem cells 14151617Saccharomyces, the recombination efficiency of FLP allowed the generation of a large number of applications such as a marker recycling system for multiple gene disruption specific targeted integration of kanamycin resistance DNA) without a resident selective marker FRT system was used specifically to induce translocations via homologous recombination in mitosis in DrosophilaP. bergheiSaccharomyces, it is possible to obtain a translocation exploiting two FRTs sites cloned within two different chromosomes and expressing FLP transiently (under an inducible promoter) in a [cir0] (= lacking the 2µ plasmid) diploid strain. It is well know that a single mutation in the core region of the FRT abolishes the self-recombination with the wild type FRT in [cir+] strains and that the identity between the core regions of two FRT sites is necessary, but not sufficient for recombination in [cir0] strains FRT system can be used in multiple rounds of gene deletions, leaving scars that cannot recombine again between them also in [cir+] strains 20FRT system if compared with other enzyme-dependent technologies that allow the construction of many different couples of identical recombination sites recombining between them, but not among them The first is based on 2µ plasmid, a circular DNA molecule of 6,318 bp present in about 40-60 copies per haploid cell in most yeast strains. Recombination between two 599-bp DNA inverted repeats of the plasmid, mediated by its own encoded FLP-recombinase, generates the inversion of a segment of the DNA sequence of the plasmid, leading to two isoforms, A and B, that are found in equal concentrations in yeast cells. No cellular function has been so far elucidated for 2µ and defining its unknown role will be one of the great challenges for the yeast scientists in the next years. However, this selfish plasmid provides one of the most efficient recombination system ever discovered: the FLP/CRE gene was firstly expressed in tobacco cells lox sites were present in direct or inverted orientation respectively. The Cre/lox site-specific recombination system from bacteriophage P1 belongs to the tyrosine integrase family, it promotes the circularization of the bacteriophage after bacterial infection, and maintains the phage genome as unit-copy plasmid by resolving dimeric plasmids during bacterial division. Surprisingly, the minimal nucleotides length required for recombination of the lox (locus of crossing-over), 34 bp, is exactly the same as the one of the FRT . Cre and FLP share some other features, but their comparison leads also to outline substantial differences between them. Cre/lox has been extensively used to direct DNA rearrangements in heterologous organisms also thanks to its natural thermo-stability at 37°C. The optimal temperature of action of the wild type recombinase FLP is of course lower (30°C), since it naturally functions in Saccharomyces. The development of a thermo-stable mutant of FLP, with four amino acids substitutions FRT, both recombinases binding the two halves of the target cooperatively, while FLP has the highest excision efficiency and the highest recombinase-mediated cassette exchange rate loxP has been extensively applied for industrial gene targeting Saccharomycessensu stricto yeasts lox system, could have positive fitness and their position could be fixed by natural selection lox sites (pre-existing pseudo-recognition sites) exist naturally in the Saccharomyces genome and that they can recombine among them even if with low frequency (( 1 x 10-7) loxP sites is an aspect to consider when choosing this targeting system. In fact, promiscuity of different lox sites can affect genome stability and intensive studies to generate lox mutants have been made so far trying to minimalize the problem 34Few years after the discovery of FLP, another site-directed recombination system was explored se lower °C, sinceRS system from Zygosaccharomyces rouxiiAcetinetobacterStreptomyces phage phiC31 system RS, because it operates efficiently in Saccharomyces [cir+] strains and independently from the host recombination system. pRS1 is a plasmid similar to 2µ that can replicate in S.cerevisiae as well as in its native host Z. rouxii. Reciprocal recombination between two non-homologous chromosomes were easily obtained with this method Other site-specific recombination systems have been exploited in the past years to produce translocants: the Gin-gix system from bacteriophage Mu Streptomyces, tested in the fission yeast Saccharomyces, is currently under evaluation for translocation studies in higher eukaryotes; unfortunately, the unspecific translocation rate, due to integration within cryptic sites, may reach 15%, independently from the cell line used Among the other systems, the phiC31 from In conclusion, recombinase-dependent chromosomal rearrangements are widely utilized but need implementation to avoid spurious integrations and consequent secondary rearrangements among the target sequences in sequential rounds of transformations. Moreover, their utilization requires a time-consuming previous engineering of the host strain and, in few cases, the co-presence of additional factors. Unlike endogenous HR, enzyme-mediated recombination efficiency drops with increasing distance between recombination sites. Sometimes the expression level of the recombinases is difficult to be modulated and it reaches toxic levels. Natural hotspots widespread in the yeast genome may heavily affect the outcome of the targeted rearrangement, interfering with the specific recombination event. The strict sequence requirements imposed by site-specific recombinases have limited their applications to organisms that contain artificially-introduced recombination sites or natural pseudo-recognition sites. To address this limitation, laborious works of directed evolution have been used to alter the sequence specificity towards naturally occurring DNA sequences Saccharomyces have contemporarily been studied and improved. These enzyme-independent site-specific methodologies will be described in details in the next paragraphs.For all these drawbacks, targeted systems based merely on the endogenous homologous recombination machinery of homologous recombination system (HRS). The frequency of a homologous DNA targeting in Saccharomyces is considerably higher than an illegitimate integration and varies between 10-4 and 10-647Saccharomyces senses exogenous free ends as endogenous chromosomal fragment ends that result after a DSB, attempting a repair by homologous recombination (HR). The observation that free ends are highly recombinogenic in Saccharomyces allowed easy DNA-mediated manipulation of the yeast genome and its re-shaping.The yeast genome is relatively easy to manipulate thanks to its proficient S. cerevisiae resulted in target homologous recombination events with consequent alteration in the strain’s karyotype. The CFVs contain URA3 as selectable marker, SUP11 for visual monitoring of the chromosomal fragment stability based on the red/white Roman effect, sequences from the Y’ sub-telomeric repeat to generate a new yeast telomere  and they carry a unique DNA segment of the yeast genome (= size of fragmentation: 1 - 5 Kb). The CVF is linearized, transformed into yeast and stable colonies are selected on -URA. The in vivo result is a chromosome fragment with a centromere, telomeres, and genomic sequences from the site of fragmentation chosen as target to the telomere. It was demonstrated that the chromosome fragment formation results from a replicative repair event, named “break-copy duplication” Almost twenty years ago, few authors created an interesting methodology to restructure the yeast chromosomes -11). These endonuclease-based systems have been largely utilized to study homologous recombination In many recent works, the original DSB to generate free ends and provoke the repair demand is artificially produced by the cleavage of well-known homing endonucleases. The most used are the yeast-made I-SceI Chromosomal translocations have been triggered via HRS thanks to these endonucleases functioning in yeast and in higher eukaryotes 5354loxp and FLP/FRT systems, a previous engineering of the strain is necessary to introduce the endonuclease recognition sites and to induce their transcription. The regulatory network of inducible promoters in yeast  is extremely complex and differs significantly among the strains due to evolution and adaptive genome rewiring GAL system, such as the repressor Gal80 and the binding sites of the activator Gal4, often show spontaneous mutations S. cerevisiae. However, they are usually less inducible than galactose promoters, few of them are dependent on toxic inducers (i.e. copper) and give a background expression that is higher under non-repressing and non-inducing conditions . In industrial fermentation processes, the ADH2-related system is frequently used because its expression is turned on in stationary phase, when biomass is high, and does not require any specific inducer 57ADH2 is several hundred-fold repressed in presence of glucose, and a de-repression mechanism with a trans-acting positive transcriptional effector is needed.A further bottleneck of these methodologies is that, as in the Cre/For all these reasons, in the last years, more naïve and portable systems that would work on any wild type yeast strain were deeply investigated, trying to avoid artifacts and addition of exogenous effectors. As a result, BIT technology was invented and will be discussed in the next paragraphs.More than ten years ago, we were studying the possibility to set up a simple system to detect mitotic recombination hotspots in the yeast genome. The idea was based on the observation that a sort of consensus came out aligning several known hotspot sequences (the majority of them meiosis-related). Using degenerated sequences named KARMA (from a short degeneration motif present within the consensus DNA sequence) we built by PCR a linear DNA cassette carrying a selectable marker and used it to transform yeast diploid cells. Unexpectedly, the KARMA cassette did not integrate in specific loci of the genome revealing hotspots, but led to aneuploidies. In fact, yeast cells preferred to consider the two free ends of the linear cassette as independent sequences, suitable for independent integration events. The result was chromosomal translocations with consequent ploidy variations. dur3), another for chromosome XV (locus adh1) and kanamycin in between generated a non-reciprocal chromosomal translocation 8 cells with 10 µg of linear DNA, the average frequency of the translocation, among all the transformants recovered, varied from 2 to 10%. The fluctuation in the frequency depends on the strain used and on the intrinsic recombinogenicity of the targeted genomic locus. Stable translocation-derived recombinants are generated among an ensemble of genomic rearrangements, comprising: i) intrachromosomal deletion, ii) ectopic integration (with a preference for the repeated rDNA region of chromosome XII), iii) aspecific translocations promoted by DNA micro-homology and, iv) recombination with the endogenous 2μ plasmid. In more than 30% of all transformants, only one of the two free ends with 40 nt of homology is correctly integrated via HRS at its homologous genomic site while the remaining majority show ectopic integrations. The results were also confirmed in trisomic strains suggesting that the phenomenon is not restricted to diploid cells We decided to verify the hypothesis that the cell sensed two different homologies toward two different chromosomes as two independent events. The new DNA cassette, carrying a homology of 40 nt for chromosome VIII  level could explain the appearance of cells with elongated buds, possibly arrested at the G2/M transition phase 6061Furthermore, it suggests that the translocant cells have adapted to the checkpoint response to the initial DNA damage generated by BIT chromosome translocation. In fact, besides the DSBs, necessary for the homologous DNA integration, BIT produces extensive genomic damages that, thanks to the signal transduction pathway of the damage checkpoint system, triggers an impairment-induced G2/M arrest until completion of the repair. However, after the translocation event, the cells do not remain permanently arrested, but resume progression through the cell cycle S. cerevisiae. At least eight different pathways were exploited to heal the broken chromosome and allow survival. Endo-reduplication of both chromosomes was possible, leading to strains trisomic for chromosome XVI, tetraploid for chromosome IX, or more complex rearrangements whose outcome was a partial duplication of one of the two chromosomes through a template switching - like mechanism The concept that a heterogeneous phenotypic population may arise from a single translocation event was analyzed in details The ploidy condition and its related phenotype are unique for each translocant as unique is its transcription pattern We speculated whether the same deregulation could happen when performing BIT between homologous chromosomes in diploid cells. The next paragraph will focus on this topic.ad hoc without genetically engineering the strains, simulating the situation of in vivo linear DNA - induced GCRs. We wondered if this system could be applied to bridge together two homologs in a diploid yeast cell. Bridging together two homologous chromosomes VIII of S. cerevisiae, it was found i) that the specificity and the efficiency of gene targeting is decreased with the increasing of the distance between the two homologies within the genome, ii) that the occurrence of targeted BIT between homologous chromosomes is lower than between heterologous, and iii) that nearly - reciprocal translocants are formed with the loss of genetic material that could provoke the loss of heterozygosity (LOH) The main advantage of BIT is that specific translocations can be generated The phenotypical and transcriptional aberrations of the translocant between homologues are negligible if compared with those of non-reciprocal translocants between different chromosomes. Moreover, the quantitative analysis of the expression of several genes, located within 80 Kb of chromosome VIII around the translocation point, did not detect any significant increase in expression with the exception of multidrug resistance genes. The nearly - reciprocal translocation is probably due to a migration of the holliday junction followed by a recession of the homology with consequent LOH  although it was never demonstrated to be exactly the same phenomenon The directionality of migration of the holliday junction after BIT between homologs can be strongly affected by the action of Rdh54 RAD54 triggers secondary rearrangements between long-terminal repeats (LTRs) located far from the breaks, producing an array of further multiple translocations. A schematic summary of the multiple rearrangements deduced from twelve different hybridizations is represented in Fig. 3A. In the case of a BIT translocation between chromosome VIII and XV, we verified that two almost identical LTRs (one on chromosome VIII and the other on chromosome XII) were recombining after the initial BIT event in translocants over-expressing RAD54 and that these further rearrangements might correlate with an abnormal phenotype and poor cell viability . Rearrangements between LTRs as well as abnormal phenotypes were present in all the translocants over-expressing RAD54 and resulted stable once formed RAD54 in break-distal rearrangements between repeats, which are landmark traits of tumor cells. Many chromatin remodelers and in particular the SWI2/SNF2 family of ATPase-dependent DNA translocases, have been correlated to ploidy maintenance RAD54 is found over-expressed up to 5-fold in prostatic cancer cells characterized by recurrent non-reciprocal translocations One of the most interesting observations is on the role of Rad54 in chromosomal translocations. Its absence, as predictable, eliminates the possibility of translocation through the HRS. On the contrary, the over-expression of Candida spp. and Cryptococcus neoformans72C. albicans associated with point mutations in the ERG11 gene has been well documented LIG4 does not contributes to adaptive chromosomal translocations C. albicans C. glabrata is responsible for an increased fitness and drug resistance Saccharomyces and in Candida show pseudo-hypha formation 71PRK1 serine/threonine protein kinase gene. Other genes belonging to the ABC transporters group, such as PDR1, PDR3 and VMR1, are sometimes over-transcribed in budding yeast carrying translocations between heterologous In several yeasts morphological changes that correspond to acquired drug resistance, can be associated to chromosomal translocations 70Saccharomyces and Candida the HRS-dependent chromosomal translocations might result in morphological alterations and adaptive mutagenesis. On the contrary, the non homologous end joining pathway does not appear to be involved in adaptations mediated by ploidy variation Therefore, it seems that in both Saccharomyces. For example, from a biotechnological point of view, yeast strains adapted to cold temperatures during wine fermentations are needed by the industry because the wine aroma is improved at low temperatures. Recent results indicate that these adapted yeasts show proteomic changes due to a greater activation of some metabolic routes such as glutathione biosynthesis and sulfur assimilation 79Aneuploid strains are generally characterized by strong genomic instability that may result from imbalances in particular genes and/or from proteotoxic stress This hypothesis is supported by our recent unpublished observations on BIT translocants that may acquire some complex phenotypic traits because of genes over-transcription following genetic selection . Adaptation might be also due to antagonistic pleiotropy, the ability of a specific gene to perform differently in different phenotypic conditions. Antagonistic pleiotropy was initially proposed as an explanation for aging and could be seen as adaptive evolution when the same genes are responsible for vitality and reproduction in cellular earlier life but a detriment at cellular later life It was already known that Ty-dependent recombination might have a beneficial effect in the long term, restructuring the yeast genome architecture As conclusive remarks, chromosomal translocations can become a useful model to mimic chromosome reassortments catalyzed by aneuploidy in cancer cells and to optimize biotechnological products through the selection of beneficial properties of new strains. These studies may result in important industrial outcomes providing novel chemotherapy adjuvants and unique foodstuff manufacturing processes on the market.astra) through adaptation and difficulties (aspera). In one word, through some sort of trial and error, they have evolved.New strains are the final upshot of a genome remodeling due to a translocation. The strains coming from the same translocation event are all different because different is the answer to a destabilizing event in terms of transcriptional genetic alteration and pathway choice to heal the broken chromosome. The resulting strains are usually impaired and rarely improved in fitness, having in this last case the chance to survive, progress and develop. They have reached a novel stable advantageous condition (


EGR3) of IEGs family was robustly repressed in postmortem prefrontal cortex of BD patients. As a central transcription factor, EGR3 regulates an array of target genes that mediate critical neurobiological processes such as synaptic plasticity, memory and cognition. Considering that EGR3 expression is induced by brain-derived neurotrophic factor (BDNF) that has been consistently related to BD pathophysiology, we suggest a link between BDNF and EGR3 and their potential role in BD. A growing body of data from our group and others has shown that peripheral BDNF levels are reduced during mood episodes and also with illness progression. In this same vein, BDNF has been proposed as an important growth factor in the impaired cellular resilience related to BD. Taken together with the fact that EGR3 regulates the expression of the neurotrophin receptor p75NTR and may also indirectly induce BDNF expression, here we propose a feed-forward gene regulatory network involving EGR3 and BDNF and its potential role in BD.Bipolar disorder (BD) is a severe psychiatric illness with a consistent genetic influence, involving complex interactions between numerous genes and environmental factors. Immediate early genes (IEGs) are activated in the brain in response to environmental stimuli, such as stress. The potential to translate environmental stimuli into long-term changes in brain has led to increased interest in a potential role for these genes influencing risk for psychiatric disorders. Our recent finding using network-based approach has shown that the regulatory unit of early growth response gene 3 ( Bipolar disorder (BD) is a chronic and potentially severe and disabling mental illness that affects between 1% and 3% of the population worldwide . Single nucleotide polymorphisms (SNPs) in EGR3 are associated with schizophrenia  in the promoter region of the gene. However, the findings regarding association between the val66met polymorphism and BD are divergent (Sklar et al., Several studies have been performed to elucidate the mechanisms involved in the presumed reduction in BDNF levels in BD. Some of them have studied polymorphisms in the The apparent decrease in BDNF levels seen in patients would be expected to result in disruption of the intracellular signaling cascades that are normally regulated by BDNF, such as PLC/PKC, PI3K/Akt and Ras/Erk pathways, interfering with processes regulated by this neurotrophin, such as neuronal differentiation and survival, and synaptic plasticity. In fact, BD has been associated with changes in factors involved in neuroplasticity and resilience pathways, including alterations in apoptotic factors, synaptic markers, neurotrophic and inflammatory factors and oxidative stress markers, as well as in processes related to circadian rhythm, neuronal development and calcium metabolism (Kim H. W. et al., Neuropathological findings in the postmortem brains of BD patients demonstrate the types of abnormalities in neuroplasticity one would expect to see from a deficit in neurotrophic factors. For example, morphometric studies show that patients have enlarged third and lateral ventricles, decreased volume of the orbital and medial prefrontal cortices, ventral striatum and mesotemporal cortex and increased volume of the amygdala compared to controls (Strakowski et al., These findings support the “allostatic load” hypothesis that we have previously described (Kapczinski et al., This perspective article presents an accumulation of findings indicating that changes in BDNF are a consistent feature of BD, and may contribute to the pathophysiology of this mental illness. Here we summarize the potential molecular links between BDNF and the IEG transcription factor EGR3, two molecules that may each play a critical role in the impaired cellular resilience related to BD (Manji et al., As we have discussed, BDNF is reported to be altered in BD in both peripheral and central tissue, and it is possible that blood BDNF levels correlate positively with brain BDNF levels (Klein et al., EGR3 have deficits in hippocampal LTD and exhibit dysfunction in NMDAR subclasses (NR1/NR2B; Gallitano-Mendel et al., Alternatively, or in addition, EGR3 may indirectly induce BDNF expression via regulation of NMDAR. In mice, EGR3 has been shown to be required for the function of NR2B-containing NMDARs (Gallitano-Mendel et al., p75NTR, Gao et al., in vitro after treatment with BDNF (Young et al., In addition, EGR3 also regulates the expression of NGFR (Taking this into account, we also suggest that EGR3 repression seen in BD patients could be responsible for the reduced BDNF levels associated to this illness. It is most likely to think in a feedback network than in a cause-and-effect relationship considering that EGR3 is responsive to BDNF and regulates NMDAR, which transcription is also mediated by BDNF through activation of the TrKB receptor and in turn induce BDNF synthesis (Kim et al., EGR3 thus emerges as a potential central player responsible for some of changes observed in BD such as a reduced neurotrophic support. Taking into consideration that EGR3 translates environmental events into neural long-term alterations, the possible disturbance in molecular pathways involving EGR3 could result in an impaired response and adaptation to stress.Altogether, the findings discussed in this article suggest a potential regulatory pathway that possibly is disrupted in BD. For many years, evidence has pointed to alterations in neurotrophic factors in BD suggesting these changes could contribute to an impaired neuroplasticity and resilience. However, the mechanisms underlying this impairment remain unknown. The identified network focused on EGR3 might be beneficial since they could provide a dynamic and fast response to neural activity and thus a sustained adaptation through regulation of an entire regulatory gene network.We suggest experimental approaches to test the hypothesis regarding a potential feed-forward mechanism involving BDNF and EGR3 and its role in BD, which might contribute to understanding its pathophysiology. Moreover, as a central transcription factor of a gene network that regulates crucial neurobiological processes, EGR3 may be a promising pharmacological target. Modulation of IEGs as With this perspective, we propose that a reduction in EGR3 in BD could contribute to alterations in a neurotrophin cascade in this disorder, which includes reduced BDNF levels. A feedback-loop reinforcing this dysfunctional pathway could, in turn, impair neuroplasticity and resilience Figure . This prAll authors provided substantial contributions to the work. BP, FKa and FKl participated in the article design and outline. BP wrote the first draft. ALG revised it critically for relevant scientific content. After a few revisions and editing by all authors, the article was submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Understanding etiology of autoimmune diseases has been a great challenge for designing drugs and vaccines. The pathophysiology of many autoimmune diseases may be attributed to molecular mimicry provoked by microbes. Molecular mimicry hypothesizes that a sequence homology between foreign and self-peptides leads to cross-activation of autoreactive T cells. Different microbial proteins are implicated in various autoimmune diseases, including multiple sclerosis, human type 1 diabetes, primary biliary cirrhosis and rheumatoid arthritis. It may be imperative to identify the microbial epitopes that initiate the activation of autoreactive T cells. Consequently, in the present study, we employed immunoinformatics tools to delineate homologous antigenic regions between microbes and human proteins at not only the sequence level but at the structural level too. Interestingly, many cross-reactive MHC class II binding epitopes were detected from an array of microbes. Further, these peptides possess a potential to skew immune response toward Th1-like patterns. The present study divulges many microbial target proteins, their putative MHC-binding epitopes, and predicted structures to establish the fact that both sequence and structure are two important aspects for understanding the relationship between molecular mimicry and autoimmune diseases. Such findings may enable us in designing potential immunotherapies to tolerize autoreactive T cells. Therefore, it is very important to understand the mechanisms involved in the breakdown of tolerance in autoreactive T cells and pathophysiology of the autoimmune diseases. Our immune system maintains a finely tuned balance that discriminates between the self and non-self antigens. During thymic education, the majority of the autoreactive T cells are deleted, yet a tiny population escapes and are considered to be responsible for provoking autoimmune diseases. Another well-balanced mechanism known as peripheral tolerance ensures the suppression of remaining autoreactive T cells. Despite the fact that this well-established system operates in our body to eliminate or tolerize autoreactive T and B cells, these cells tend to get activated and inflict debilitating autoimmune diseases   is the well-established proteomics server that allows browsing through several databases . Next, we identified and analyzed the homologous microbial peptide sequences that are similar to human protein sequences obtained from UNIPROT database. A total of 216 well-characterized peptides of different microbial origin were selected from the database. Further, these sequences were grouped together on the basis of their frequency of occurrence and were provided with unique peptide ID  to each allele. The peptides were classified based on predicted IC50 values as strong binders (IC50 ≤ 50), weak binders (50 ≤ IC50 ≤ 500) and non-binders (IC50 ≥ 500) ; Six DQ alleles , and 14 DR alleles  were selected for this study  is commonly used as a metric for measuring the structural similarity of two protein models. In the present study, TM-score was used for establishing the quantitative assessment of the peptides structural similarity based on the RMSD values  followed by DRB1*03:01 (41.7%) and DRB1*04:01 (41.6%) worldwide  and molecular docking (EpiDOCK) approaches have been applied for the selected peptides. Concurring results across these different predicting algorithms confirmed not only the promiscuity but also the high MHC class II binding affinity of microbial peptides  that had multiple child nodes, as found in different or same proteins expressed by varied class of microbes Figure . We nextWe identified many MHC class II promiscuous binders from an array of microbial antigens like transketolase, epidermal growth factors, CDA peptide synthetase I, tRNA/rRNA methyltransferase, DNA topoisomerase I, dihydrolipoyllysine-residue acetyltransferase etc. These antigens are associated with insulin-dependent diabetes mellitus, primary biliary cirrhosis, sarcoidosis, type-2 autoimmune hepatitis etc. Some of these antigens exhibited high affinity binding with MHC class II molecules. These findings may signify that the microbial antigens expressing putative autoreactive T cell epitopes exhibit both sequence and structural similarity that may be involved in the progression of autoimmune diseases.Cytokines like IL-12 and IL-4 differentiate naïve CD4 T cells into Th1 and Th2 cells, respectively. In addition, costimulatory molecules, PRRs, peptides interaction with MHC molecules can influence the release of cytokines and the generation of Th1 and Th2 cells  an occurrence of a large number of microbial epitopes, which exhibit sequence homology with human proteins that are possibly involved in the triggering of autoimmune responses; (ii) a considerable number of such peptides show promiscuous binding to several MHC class II molecules, which have high relative risks of autoimmune diseases; (iii) microbial components exhibiting cross-reactivity with human peptides show high-affinity binding to MHC class II molecules; (iv) not only sequence alone but the structure of peptides was found to be equally important in ascertaining the molecular mimicry; (v) cross reactive epitopes could differentially activate Th1 and Th2 cells.Molecular mimicry is based on the fact that T cell and B cell antigenic determinants present in the pathogen may have its counterpart in the host that may possibly trigger autoimmunity  that are perceived by the immune system as danger signals through pattern recognition receptors (PRRs) (Mogensen, in silico tools have been successfully used to study molecular mimicry in various diseases like tuberculosis, rheumatoid arthritis, multiple sclerosis and ulcerative colitis (Kovvali and Das, *01:01, DRB1*04:01 and DRB1*04:04 HLA alleles are known to be associated with rheumatoid arthritis, haemolytic transfusion reactions, psoriasis vulgaris, recurrent respiratory papillomatosis and rheumatoid vasculitis autoimmune diseases (Carcassi et al., In the past, In past, mimicry at the level of the sequence of the microbial peptides has been shown to be associated with autoimmune diseases. However, at a cellular level, this mistaken identity actually occurs due to the structural similarity in the MHC class II binding microbial and human peptides. Taking this into account, we have integrated the concept of the involvement of not only sequence but also the structure of the peptide in contributing in the molecular mimicry. Our current findings suggest that both sequence and structure of the peptides are important in contributing in the molecular mimicry.in-silico tools are available to identify MHC class II-binding peptides and possible release of cytokines IFN-γ and IL-4 (Dhanda et al., in-silico prediction. The distinct production of cytokines by T cells may be due to the engagement of bacterial PAMPs with PRRs expressed on host APCs during antigen presentation to T cells. It has been reported earlier that the activation and differentiation of Th1 and Th2 cells can occur through the recognition of PAMPs of the microbes by PRRs of the host immune cells (Iwasaki and Medzhitov, The cells of the immune system viz. Th17 cells, Th1 cells, Th2 cells, and B cells mainly contribute to the pathogenesis of the autoimmune diseases (Crane and Forrester, Overall, the study implies that immunoinformatics tools are cost effective, less time consuming, convenient and efficient in identifying microbial peptides that share sequence and structure homology with human peptides. Subsequently, responsible for activating autoreactive T cells and perpetuating autoimmune diseases. Further, identifying the sequence and analyzing the structure of microbial determinants can help in designing potential drug and vaccine targets.in silico procedures were conducted by SP, DC, SN, JK, and BS. The data analysis and manuscript were written by SP, DC, and JA.In this study, the concept designing and the work was done by JA, SP, and DC. The The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The use of ionic liquids (ILs) as organocatalysts is reviewed for transesterification reactions, specifically for the conversion of nontoxic compounds such as dialkyl carbonates to both linear mono-transesterification products or alkylene carbonates. An introductory survey compares pros and cons of classic catalysts based on both acidic and basic systems, to ionic liquids. Then, innovative green syntheses of task-specific ILs and their representative applications are introduced to detail the efficiency and highly selective outcome of ILs-catalyzed transesterification reactions. A mechanistic hypothesis is discussed by the concept of cooperative catalysis based on the dual (electrophilic/nucleophilic) activation of reactants. The recycling of the ILs is therefore imperative not only to limit their release to the environment, but also for economic reasons. One strategy to cope with the recycling issue is based on the immobilization of ionic liquids onto solid supports. In the specific field of transesterification reactions, supported ionic liquid (SILs) catalysts are achieved by the dispersion of liquid organic salts on highly porous materials, amongst which montmorillonite clays, modified silica, and polystyrene-based solids are the most frequently used [CF3SO3]) has been chosen as a model organocatalyst to explore the kinetics of the transesterification of methyl acetate with ethanol . Th. ThBasic2 catalyst is synthesized starting from butyl-imidazole and DMC  and 5 mol % BMIM-2-COMore recently, Yuxuan Yi et al. described the transesterification of glycerol with DMC in the presence of four catalytic ionic liquids such as 1-methyl-3-butylimidazolium imidazolium ([BMIM]Im), 1-methyl-3-allylimidazolium imidazolium ([AMIM]Im), 1-methyl-3-butylimidazolium hydroxide ([BMIM]OH), and 1-methyl-3-allylimidazolium hydroxide ([AMIM]OH) . The higAccording to the authors, the result was due to a higher basicity of the imidazolium anion with respect to the hydroxy group, and to the poorer steric hindrance of the AMIM cation with respect to the BMIM cation, the latter exerting less effective interactions with the corresponding (imidazolium) anion. Regardless of its nature, the cation might also activate DMC towards nucleophilic addition: a cooperative nucleophilic–electrophilic mechanism could therefore operate  63]..63].2  with an alcohol ROH and CO2  64]..2 , conversion and selectivity (towards glycerol carbonate) were 96% and 82%, respectively, after 30 min reaction time. Glycidol  was the major byproduct. In a further study by the same group, the formation of glycidol from glycerol carbonate was examined in the presence of the ionic liquid DABCO–DMC obtained in situ by reacting DABCO and DMC  65]..65].DABCO by itself is more basic than the DABCO–DMC IL as indicated by its pH in aqueous solution. Nonetheless the DABCO–DMC IL promoted higher glycerol conversion (77% vs 19% after 10 minutes), and, most importantly, higher GD selectivity (63% compared to 45% after 30 minutes) under the same reaction conditions. In order to explain such a behavior, a cooperative mechanism for the ionic liquid catalysis was invoked, whereby the electrophilic nitrogen atom aids in activating the carbonyl moiety .It must be noted that in these two examples the selectivity of the reaction could be tuned just by changing the catalyst precursor: by switching from the DBU-based IL to the DABCO–DMC IL, the selectivity changed from 82% for glycerol carbonate, to 83% for glycidol.In another publication, Gade et al. also achieved a high selectivity toward GD in a one-pot reaction starting from glycerol and DMC . Using tThis implied that both, the presence of a basic (anionic) center and an electrophilic (cationic) center in the ionic liquid were involved in the reaction. It was therefore proposed that an interaction of the quaternary ammonium center with the carbonyl oxygen of glycerol carbonate (GlyC) could weaken the C=O bond .It is worth mentioning that glycidol was previously obtained at much higher temperatures (170–200 °C).An acyclic organic carbonate that has recently received attention from the synthetic lubricant market is dipentyl carbonate (DPC). An environmentally friendly process for its synthesis has been recently proposed by the transesterification of DMC with 1-pentanol in the presence of 2 mol % of 1-butyl-3-methylimidazolium hydroxide ([BMIM]OH) as a basic ionic liquid catalyst . At the n-butanol has been accomplished using tetraethylammonium-based amino acid ionic liquids ([N2222][AA]) as homogeneous catalysts  product. Quantum-mechanical calculations indicated that the catalyst synergistically activated both BuOH and DMC.A similar transesterification reaction of dimethyl carbonate with atalysts  68]. [HSO4] and choline chloride zinc chloride ([ChCl][ZnCl2]). The ionic liquid increases the catalytic activity of dibutyltin oxide fourfold probably by forming a highly active tin species where the anion of the ionic liquid acts as a ligand. The developed protocol was further studied for various substituted phenols, proving that electron-donating groups (EDG) at the para position enhance the substrate conversion, while electron-withdrawing groups (EWG) provide the aryl methyl carbonate with a very low conversion. Any substituents in the ortho position led to lower conversions due to an increase of the steric hindrance. liquids . The autIonic liquid catalyzed transesterification for dimethyl carbonate production: Ionic liquid-based catalysts brought about a number of improvements for the synthesis of DMC. As mentioned above, the synthesis of DMC through CO2 insertion into an epoxide and the subsequent transesterification of the formed cyclic carbonate with methanol represent a valid alternative for the industrial production of DMC [4DABCO]OH), that achieved 90% conversion, 81% DMC yield and 82% EC yield under optimized conditions . The catalyst reusability was tested in four successive runs, in which the conversion decreased from 90 to 88% and the DMC yield from 81 to 79%, thereby proving the high stability of the investigated IL and the greenness of the process.n of DMC . Althougn of DMC . In thei2 and methanol was proposed by Li et al., using a series of quaternary ammonium ILs in reactions carried out in an autoclave at 150 °C, and under CO2 pressure (2 MPa) I as the catalyst. The reusability of the catalyst was further studied in eight subsequent reactions. Wang et al. investigated the dependence of the catalytic activity on the structure of IL cations and anions for the synthesis of DMC through the transesterification of EC with methanol [MeOCO2]) and its anion metathesis analogues  acetate and phenolate salt derivatives could be obtained from [P8881][CH3OCO2] through a chlorine-free metathesis with acetic acid and phenol , as opposed to the outcome using both solid bases and zeolites, that generate large amounts of CO2.These results highlight the excellent activity and selectivity of these catalytic systems (conversion >99% and yield >90%) with respect to conventional organic and inorganic bases. In addition, the reactions proceed without decarboxylation even at high temperatures  was active for the synthesis of cellulose dialkyl carbonates which find applications as intermediates, supports for the delivery of therapeutics, imaging agents and packaging films and coatings [Recently, phosphonium salts have been reported as transesterification catalysts of light organic carbonates (dimethyl and diethyl carbonate) with complex polyalcohols, such as cellulose. In particular, trioctylphosphonium acetate ([Pcoatings .The literature survey illustrated in this review highlights three main facts. Firstly, organocatalysis by ionic liquids can be an efficient tool for base and even acid-catalyzed transesterification reactions in place of traditional inorganic or solid acids and bases. Advantages in this case are mainly the recovery and recyclability of the catalyst system and the improved selectivity that is often achievable. Secondly, the here presented reactions have a common mechanistic feature based on the cooperative nucleophilic–electrophilic catalysis by the ionic liquid. This type of ambiphilic catalysis is characterized by the nucleophile and the electrophile both being activated respectively by the anion and by the cation of the ionic liquid. Thirdly, organic carbonates – used as feedstocks or produced by transesterification – are valuable synthetic targets in view of the development of new greener solvents, additives, reagents, and in general of chemical products with improved safety and chemical properties.


Saccharomyces cerevisiae have provided evidence that age-related changes in some aspects of mitochondrial functionality can create certain molecular signals. These signals can then define the rate of cellular aging by altering unidirectional and bidirectional communications between mitochondria and other organelles. Several aspects of mitochondrial functionality are known to impact the replicative and/or chronological modes of yeast aging. They include mitochondrial electron transport, membrane potential, reactive oxygen species, and protein synthesis and proteostasis, as well as mitochondrial synthesis of iron-sulfur clusters, amino acids, and NADPH. Our recent findings have revealed that the composition of mitochondrial membrane lipids is one of the key aspects of mitochondrial functionality affecting yeast chronological aging. We demonstrated that exogenously added lithocholic bile acid can delay chronological aging in yeast because it elicits specific changes in mitochondrial membrane lipids. These changes allow mitochondria to operate as signaling platforms that delay yeast chronological aging by orchestrating an institution and maintenance of a distinct cellular pattern. In this review, we discuss molecular and cellular mechanisms underlying the essential role of mitochondrial membrane lipids in yeast chronological aging.The functional state of mitochondria is vital to cellular and organismal aging in eukaryotes across phyla. Studies in the yeast  Mitochondria are indispensable for organismal physiology and health in all eukaryotes –9. The eSaccharomyces cerevisiae have uncovered several mechanisms underlying the essential role of mitochondria in the replicative and chronological modes of aging in this yeast , 5150, 5Because LCA increases the relative concentrations of PA, which is known for the ability to decrease mitochondrial number by promoting fusion of small mitochondria –150, we LCA lessens the relative concentrations of the nonbilayer forming classes of phospholipids known to increase the extent of membrane curving for the IMM (see above); LCA also rises the relative concentrations of the bilayer forming classes of phospholipids shown to decrease the extent of membrane curving for the IMM (as described above). We therefore expected that LCA may increase the proportion of mitochondrial cristae detached from the IMM and having flat bilayer conformation and may also increase the abundance of these detached from the IMM cristae within the mitochondrial matrix. In support of these expectations, yeast cultured in the presence of LCA exhibited the following major morphological changes in the IMM and mitochondrial cristae: (1) many cristae were disconnected from the IMM and accumulated within the mitochondrial matrix in flat bilayer conformation and (2) the ratio of “total length of cristae/total length of the OMM” was significantly increased  50, 51], 5150, 50rho ; (2) S1 ; (3) general TCA cycle dysfunction; (4) kgd1Δ, kgd2Δ, or lpd1Δ ; (5) yme1Δ mdl1Δ  protease Yme1 and the mitochondrial ABC (ATP-binding cassette) transporter Mdl1, both involved in peptide export from mitochondria); and (6) afo1Δ   . The OS subunit)   but also numerous proteins in different cellular locations outside of mitochondria . TherefoΨ) and allows to sustain ΔΨ at a critical threshold; (4) in chronologically “old” yeast, it increases the concentrations of mitochondrial proteins involved in the synthesis and assembly of iron-sulfur clusters, inorganic cofactors of many mitochondrial, nuclear, and cytosolic proteins playing essential roles in vital cellular processes; and (5) in chronologically “young” and “old” yeast, it increases the concentrations of mitochondrial and nonmitochondrial proteins known to be upregulated in response to a simultaneous lack of the mitochondrial i-AAA protease Yme1 and the mitochondrial ABC-transporter Mdl1 involved in peptide export from mitochondria  anchondria  chondria  Our findings provide evidence that LCA delays yeast chronological aging by eliciting a remodeling of mitochondrial lipidome to cause specific changes in the relative concentrations of different classes of membrane lipids . This LCS. cerevisiae allows to establish an aging-delaying cellular pattern is reminiscent of the mechanism in which the UPRmt-driven remodeling of mitochondrial lipidome in the nematode C. elegans triggers a cascade of events that institute an aging-delaying cellular pattern [S. cerevisiae [C. elegans [D. melanogaster [Of note, the proposed mechanism here for how the LCA-dependent remodeling of mitochondrial lipidome in the yeast  pattern . Moreoverevisiae , 62–70,  elegans –95, 98, nogaster , 100, manogaster , 101–109nogaster , 111.