Digital Technology Applications in the Management of Adverse Drug Reactions: Bibliometric Analysis.

Adverse drug reactions continue to be not only one of the most urgent problems in clinical medicine, but also a social problem. The aim of this study was a bibliometric analysis of the use of digital technologies to prevent adverse drug reactions and an overview of their main applications to improve the safety of pharmacotherapy. The search was conducted using the Web of Science database for the period 1991-2023. A positive trend in publications in the field of using digital technologies in the management of adverse drug reactions was revealed. A total of 72% of all relevant publications come from the following countries: the USA, China, England, India, and Germany. Among the organizations most active in the field of drug side effect management using digital technologies, American and Chinese universities dominate. Visualization of publication keywords using VOSviewer software 1.6.18 revealed four clusters: "preclinical studies", "clinical trials", "pharmacovigilance", and "reduction of adverse drug reactions in order to improve the patient's quality of life". Molecular design technologies, virtual models for toxicity modeling, data integration, and drug repurposing are among the key digital tools used in the preclinical research phase. Integrating the application of machine learning algorithms for data analysis, monitoring of electronic databases of spontaneous messages, electronic medical records, scientific databases, social networks, and analysis of digital device data into clinical trials and pharmacovigilance systems, can significantly improve the efficiency and safety of drug development, implementation, and monitoring processes. The result of combining all these technologies is a huge synergistic provision of up-to-date and valuable information to healthcare professionals, patients, and health authorities.

Investigation of Mutated in Colorectal Cancer (MCC) Gene Family Evolution History Indicates a Putative Role in Th17/Treg Differentiation.

The MCC family of genes plays a role in colorectal cancer development through various immunological pathways, including the Th17/Treg axis. We have previously shown that MCC1 but not MCC2 plays a role in Treg differentiation. Our understanding of the genetic divergence patterns and evolutionary history of the MCC family in relation to its function, in general, and the Th17/Treg axis, in particular, remains incomplete. In this investigation, we explored 12 species' genomes to study the phylogenetic origin, structure, and functional specificity of this family. In vertebrates, both MCC1 and MCC2 homologs have been discovered, while invertebrates have a single MCC homolog. We found MCC homologs as early as Cnidarians and Trichoplax, suggesting that the MCC family first appeared 741 million years ago (Ma), whereas MCC divergence into the MCC1 and MCC2 families occurred at 540 Ma. In general, we did not detect significant positive selection regulating MCC evolution. Our investigation, based on MCC1 structural similarity, suggests that they may play a role in the evolutionary changes in Tregs' emergence towards complexity, including the ability to utilize calcium for differentiation through the use of the EFH calcium-binding domain. We also found that the motif NPSTGE was highly conserved in MCC1, but not in MCC2. The NPSTGE motif binds KEAP1 with high affinity, suggesting an Nrf2-mediated function for MCC1. In the case of MCC2, we found that the "modifier of rudimentary" motif is highly conserved. This motif contributes to the regulation of alternative splicing. Overall, our study sheds light on how the evolution of the MCC family is connected to its function in regulating the Th17/Treg axis.

Factors regulating the differences in frequency of infiltration of Th17 and Treg of the blood-brain barrier.

Controlling CD4+ immune cell infiltration of the brain is a leading aim in designing therapeutic strategies for a range of neuropathological disorders such as multiple sclerosis, Alzheimer's disease, and depression. CD4+ T cells are a highly heterogeneous and reprogrammable family, which includes various distinctive cell types such as Th17, Th1, and Treg cells. Interestingly Th17 and Treg cells share a related transcriptomic profile, where the TGFß-SMADS pathway plays a fundamental role in regulating the differentiation of both of these cell types. However, Th17 could be highly pathogenic and was shown to promote inflammation in various neuropathological disorders. Conversely, Treg is anti-inflammatory and is known to inhibit Th17. It could be noticed that Th17 frequencies of infiltration of the blood-brain barrier in various neurological disorders are significantly upregulated. However, Treg infiltration numbers are significantly low. The reasons behind these contradicting observations are still unknown. In this perspective, we propose that the difference in the T-cell receptor repertoire diversity, diapedesis pathways, chemokine expression, and mechanical properties of these two cell types could be contributing to answering this intriguing question.

Mouse CCL9 Chemokine Acts as Tumor Suppressor in a Murine Model of Colon Cancer.

Colorectal cancer is the third most frequently diagnosed cancer in the world. Despite extensive studies and apparent progress in modern strategies for disease control, the treatment options are still not sufficient and effective, mostly due to frequently encountered resistance to immunotherapy of colon cancer patients in common clinical practice. In our study, we aimed to uncover the CCL9 chemokine action employing the murine model of colon cancer to seek new, potential molecular targets that could be promising in the development of colon cancer therapy. Mouse CT26.CL25 colon cancer cell line was used for introducing lentivirus-mediated CCL9 overexpression. The blank control cell line contained an empty vector, while the cell line marked as CCL9+ carried the CCL9-overexpressing vector. Next, cancer cells with empty vector (control) or CCL9-overexpressing cells were injected subcutaneously, and the growing tumors were measured within 2 weeks. Surprisingly, CCL9 contributed to a decline in tumor growth in vivo but had no effect on CT26.CL25 cell proliferation or migration in vitro. Microarray analysis of the collected tumor tissues revealed upregulation of the immune system-related genes in the CCL9 group. Obtained results suggest that CCL9 reveals its anti-proliferative functions by interplay with host immune cells and mediators that were absent in the isolated, in vitro system. Under specific study conditions, we determined unknown features of the murine CCL9 that have so far bee reported to be predominantly pro-oncogenic.

The Journey of Cancer Cells to the Brain: Challenges and Opportunities.

Cancer metastases into the brain constitute one of the most severe, but not uncommon, manifestations of cancer progression. Several factors control how cancer cells interact with the brain to establish metastasis. These factors include mediators of signaling pathways participating in migration, infiltration of the blood-brain barrier, interaction with host cells (e.g., neurons, astrocytes), and the immune system. Development of novel therapies offers a glimpse of hope for increasing the diminutive life expectancy currently forecasted for patients suffering from brain metastasis. However, applying these treatment strategies has not been sufficiently effective. Therefore, there is a need for a better understanding of the metastasis process to uncover novel therapeutic targets. In this review, we follow the journey of various cancer cells from their primary location through the diverse processes that they undergo to colonize the brain. These processes include EMT, intravasation, extravasation, and infiltration of the blood-brain barrier, ending up with colonization and angiogenesis. In each phase, we focus on the pathways engaging molecules that potentially could be drug target candidates.

Investigation of the Molecular Evolution of Treg Suppression Mechanisms Indicates a Convergent Origin.

Regulatory T cell (Treg) suppression of conventional T cells is a central mechanism that ensures immune system homeostasis. The exact time point of Treg emergence is still disputed. Furthermore, the time of Treg-mediated suppression mechanisms' emergence has not been identified. It is not yet known whether Treg suppression mechanisms diverged from a single pathway or converged from several sources. We investigated the evolutionary history of Treg suppression pathways using various phylogenetic analysis tools. To ensure the conservation of function for investigated proteins, we augmented our study using nonhomology-based methods to predict protein functions among various investigated species and mined the literature for experimental evidence of functional convergence. Our results indicate that a minority of Treg suppressor mechanisms could be homologs of ancient conserved pathways. For example, CD73, an enzymatic pathway known to play an essential role in invertebrates, is highly conserved between invertebrates and vertebrates, with no evidence of positive selection (w = 0.48, p-value < 0.00001). Our findings indicate that Tregs utilize homologs of proteins that diverged in early vertebrates. However, our findings do not exclude the possibility of a more evolutionary pattern following the duplication degeneration−complementation (DDC) model. Ancestral sequence reconstruction showed that Treg suppression mechanism proteins do not belong to one family; rather, their emergence seems to follow a convergent evolutionary pattern.

Adera2.0: A Drug Repurposing Workflow for Neuroimmunological Investigations Using Neural Networks.

Drug repurposing in the context of neuroimmunological (NI) investigations is still in its primary stages. Drug repurposing is an important method that bypasses lengthy drug discovery procedures and focuses on discovering new usages for known medications. Neuroimmunological diseases, such as Alzheimer's, Parkinson's, multiple sclerosis, and depression, include various pathologies that result from the interaction between the central nervous system and the immune system. However, the repurposing of NI medications is hindered by the vast amount of information that needs mining. We previously presented Adera1.0, which was capable of text mining PubMed for answering query-based questions. However, Adera1.0 was not able to automatically identify chemical compounds within relevant sentences. To challenge the need for repurposing known medications for neuroimmunological diseases, we built a deep neural network named Adera2.0 to perform drug repurposing. The workflow uses three deep learning networks. The first network is an encoder and its main task is to embed text into matrices. The second network uses a mean squared error (MSE) loss function to predict answers in the form of embedded matrices. The third network, which constitutes the main novelty in our updated workflow, also uses a MSE loss function. Its main usage is to extract compound names from relevant sentences resulting from the previous network. To optimize the network function, we compared eight different designs. We found that a deep neural network consisting of an RNN neural network and a leaky ReLU could achieve 0.0001 loss and 67% sensitivity. Additionally, we validated Adera2.0's ability to predict NI drug usage against the DRUG Repurposing Hub database. These results establish the ability of Adera2.0 to repurpose drug candidates that can shorten the development of the drug cycle. The workflow could be download online.

An Update on the Evolutionary History of Bregs.

The relationship between the evolutionary history and the differentiation of Bregs is still not clear. Bregs were demonstrated to possess a regulatory effect on B cells. Various subsets of Bregs have been identified including T2-MZP, MZ, B10, IL10-producing plasma cells, IL10 producing plasmablasts, immature IL10 producing B cells, TIM1, and Br1. It is known that B cells have evolved during fish emergence. However, the origin of Bregs is still not known. Three main models have been previously proposed to describe the origin of Bregs, the first known as single-single (SS) suggests that each type of Bregs subpopulation has emerged from a single pre-Breg type. The second model (single-multi) (SM) assumes that a single Bregs gave rise to multiple types of Bregs that in turn differentiated to other Breg subpopulations. In the third model (multi-multi) (MM), it is hypothesized that Bregs arise from the nearest B cell phenotype. The link between the differentiation of cells and the evolution of novel types of cells is known to follow one of three evolutionary patterns (i.e., homology, convergence, or concerted evolution). Another aspect that controls differentiation and evolution processes is the principle of optimization of energy, which suggests that an organism will always use the choice that requires less energy expenditure for survival. In this review, we investigate the evolution of Breg subsets. We studied the feasibility of Breg origination models based on evolution and energy constraints. In conclusion, our review indicates that Bregs are likely to have evolved under a combination of SM-MM models. This combination ensured successful survival in harsh conditions by following the least costly differentiation pathway, as well as adapting to changing environmental conditions.

RORγt-Expressing Pathogenic CD4+ T Cells Cause Brain Inflammation during Chronic Colitis.

Neurobehavioral disorders and brain abnormalities have been extensively reported in both Crohn's disease and ulcerative colitis patients. However, the mechanism causing neuropathological disorders in inflammatory bowel disease patients remains unknown. Studies have linked the Th17 subset of CD4+ T cells to brain diseases associated with neuroinflammation and cognitive impairment, including multiple sclerosis, ischemic brain injury, and Alzheimer's disease. To better understand how CD4+ T lymphocytes contribute to brain pathology in chronic intestinal inflammation, we investigated the development of brain inflammation in the T cell transfer model of chronic colitis. Our findings demonstrate that CD4+ T cells infiltrate the brain of colitic Rag1 -/- mice in proportional levels to colitis severity. Colitic mice developed hypothalamic astrogliosis that correlated with neurobehavioral disorders. Moreover, the brain-infiltrating CD4+ T cells expressed Th17 cell transcription factor retinoic acid-related orphan receptor γt (RORγt) and displayed a pathogenic Th17 cellular phenotype similar to colonic Th17 cells. Adoptive transfer of RORγt-deficient naive CD4+ T cells failed to cause brain inflammation and neurobehavioral disorders in Rag1 -/- recipients, with significantly less brain infiltration of CD4+ T cells. The finding is mirrored in chronic dextran sulfate sodium-induced colitis in Rorcfl/fl Cd4-Cre mice that showed lower frequency of brain-infiltrating CD4+ T cells and astrogliosis despite onset of significantly more severe colitis compared with wild-type mice. These findings suggest that pathogenic RORγt+CD4+ T cells that aggravate colitis migrate preferentially into the brain, contributing to brain inflammation and neurobehavioral disorders, thereby linking colitis severity to neuroinflammation.

Trends of Anger and Physical Aggression in Russian Women During COVID-19 Lockdown.

The effect of social lockdown during the COVID-19 outbreak on female aggressiveness is not well known. The strict measures of lockdown have resulted in millions of people, worldwide, confined to their homes during the pandemic. However, the consequence of lockdown strategies on females' psychological status including aggressiveness has not yet been investigated. We conducted a cross-sectional study on 31 Russian females' homemakers who are participants in an online fitness platform to investigate the immediate anxiety, depression, and aggression experienced under strict lockdown measures. The participants were surveyed using the hospital anxiety depression scale (HADS) and the Buss-Perry Aggression Questionnaire. We used descriptive and statistical methods to investigate the prevalence of these emotions among two age groups (20-35 and 36-65 years). We found that moderate anxiety prevalence was 77.4% in the entire group while mild depression was 54.8%. Interestingly, the whole sample showed a high level of angriness (p = 0.0002) and physical aggression (p = 0.019). These two emotions seem to be more prevalent than other negative emotions such as hostility, verbal aggression. This relationship was not dependent on age. Overall, there is a significant worsening in female aggression that could lead to higher chances of female victimization and being subjected to partner violence. Future policies designing lockdown strategies should consider this effect on active female homemakers. Due to the small size of our cohort, our results are only indicative of data trends. Larger studies are still needed to confirm the current findings.

Investigation of Evolutionary History and Origin of the Tre1 Family Suggests a Role in Regulating Hemocytes Cells Infiltration of the Blood-Brain Barrier.

Understanding the evolutionary relationship between immune cells and the blood-brain barrier (BBB) is important to devise therapeutic strategies. In vertebrates, immune cells follow either a paracellular or a transcellular pathway to infiltrate the BBB. In Drosophila, glial cells form the BBB that regulates the access of hemocytes to the brain. However, it is still not known which diapedesis route hemocytes cells follow. In vertebrates, paracellular migration is dependent on PECAM1, while transcellular migration is dependent on the expression of CAV1. Interestingly Drosophila genome lacks both genes. Tre1 family (Tre1, moody, and Dmel_CG4313) play a diverse role in regulating transepithelial migration in Drosophila. However, its evolutionary history and origin are not yet known. We performed phylogenetic analysis, together with HH search, positive selection, and ancestral reconstruction to investigate the Tre1 family. We found that Tre1 exists in Mollusca, Arthropoda, Ambulacraria, and Scalidophora. moody is shown to be a more ancient protein and it has existed since Cnidaria emergence and has a homolog (e.g., GPCR84) in mammals. The third family member (Dmel_CG4313) seems to only exist in insects. The origin of the family seems to be related to the rhodopsin-like family and in particular family α. We found that opsin is the nearest receptor to have a common ancestor with the Tre1 family that has diverged in sponges. We investigated the positive selection of the Tre1 family using PAML. Tre1 seems to have evolved under negative selection, whereas moody has evolved during positive selection. The sites that we found under positive selection are likely to play a role in the speciation of function in the case of moody. We have identified an SH3 motif, in Tre1 and, moody and Dmel_CG4313. SH3 is known to play a fundamental role in regulating actin movement in a Rho-dependent manner in PECAM1. Our results suggest that the Tre1 family could be playing an important role in paracellular diapedesis in Drosophila.

RORγt Promotes Foxp3 Expression by Antagonizing the Effector Program in Colonic Regulatory T Cells.

RORγt is the master transcription factor for the Th17 cells. Paradoxically, in the intestine, RORγt is coexpressed in peripherally induced regulatory T cells (pTregs) together with Foxp3, the master transcription factor for Tregs. Unexpectedly, by an unknown mechanism, colonic RORγt+ Tregs show an enhanced suppressor function and prevent intestinal inflammation more efficiently than RORγt-nonexpressing pTregs. Although studies have elucidated the function of RORγt in Th17 cells, how RORγt regulates pTreg function is not understood. In our attempt to understand the role of RORγt in controlling Treg function, we discovered a RORγt-driven pathway that modulates the regulatory (suppressor) function of colonic Tregs. We found that RORγt plays an essential role in maintaining Foxp3 expression. RORγt-deficient Tregs failed to sustain Foxp3 expression with concomitant upregulation of T-bet and IFN-γ expressions. During colitis induced by adoptive transfer of CD45RBhi cells in Rag1 -/- mice, RORγt-deficient colonic Tregs transitioned to a Th1-like effector phenotype and lost their suppressor function, leading to severe colitis with significant mortality. Accordingly, Foxp3-expressing, RORγt-deficient Tregs showed impaired therapeutic efficacy in ameliorating colitis that is not due to their reduced survival. Moreover, using the Treg-specific RORγt and T-bet double-deficient gene knockout mouse, we demonstrate that deletion of T-bet from RORγt-deficient Tregs restored Foxp3 expression and suppression function as well as prevented onset of severe colitis. Mechanistically, our study suggests that RORγt-mediated repression of T-bet is critical to regulating the immunosuppressive function of colonic Tregs during the inflammatory condition.

Interleukins and Interleukin Receptors Evolutionary History and Origin in Relation to CD4+ T Cell Evolution.

Understanding the evolution of interleukins and interleukin receptors is essential to control the function of CD4+ T cells in various pathologies. Numerous aspects of CD4+ T cells' presence are controlled by interleukins including differentiation, proliferation, and plasticity. CD4+ T cells have emerged during the divergence of jawed vertebrates. However, little is known about the evolution of interleukins and their origin. We traced the evolution of interleukins and their receptors from Placozoa to primates. We performed phylogenetic analysis, ancestral reconstruction, HH search, and positive selection analysis. Our results indicated that various interleukins' emergence predated CD4+ T cells divergence. IL14 was the most ancient interleukin with homologs in fungi. Invertebrates also expressed various interleukins such as IL41 and IL16. Several interleukin receptors also appeared before CD4+ T cells divergence. Interestingly IL17RA and IL17RD, which are known to play a fundamental role in Th17 CD4+ T cells first appeared in mollusks. Furthermore, our investigations showed that there is not any single gene family that could be the parent group of interleukins. We postulate that several groups have diverged from older existing cytokines such as IL4 from TGFß, IL10 from IFN, and IL28 from BCAM. Interleukin receptors were less divergent than interleukins. We found that IL1R, IL7R might have diverged from a common invertebrate protein that contained TIR domains, conversely, IL2R, IL4R and IL6R might have emerged from a common invertebrate ancestor that possessed a fibronectin domain. IL8R seems to be a GPCR that belongs to the rhodopsin-like family and it has diverged from the Somatostatin group. Interestingly, several interleukins that are known to perform a critical function for CD4+ T cells such as IL6, IL17, and IL1B have gained new functions and evolved under positive selection. Overall evolution of interleukin receptors was not under significant positive selection. Interestingly, eight interleukin families appeared in lampreys, however, only two of them (IL17B, IL17E) evolved under positive selection. This observation indicates that although lampreys have a unique adaptive immune system that lacks CD4+ T cells, they could be utilizing interleukins in homologous mode to that of the vertebrates' immune system. Overall our study highlights the evolutionary heterogeneity within the interleukins and their receptor superfamilies and thus does not support the theory that interleukins evolved solely in jawed vertebrates to support T cell function. Conversely, some of the members are likely to play conserved functions in the innate immune system.

Paracellular and Transcellular Leukocytes Diapedesis Are Divergent but Interconnected Evolutionary Events.

Infiltration of the endothelial layer of the blood-brain barrier by leukocytes plays a critical role in health and disease. When passing through the endothelial layer during the diapedesis process lymphocytes can either follow a paracellular route or a transcellular one. There is a debate whether these two processes constitute one mechanism, or they form two evolutionary distinct migration pathways. We used artificial intelligence, phylogenetic analysis, HH search, ancestor sequence reconstruction to investigate further this intriguing question. We found that the two systems share several ancient components, such as RhoA protein that plays a critical role in controlling actin movement in both mechanisms. However, some of the key components differ between these two transmigration processes. CAV1 genes emerged during Trichoplax adhaerens, and it was only reported in transcellular process. Paracellular process is dependent on PECAM1. PECAM1 emerged from FASL5 during Zebrafish divergence. Lastly, both systems employ late divergent genes such as ICAM1 and VECAM1. Taken together, our results suggest that these two systems constitute two different mechanical sensing mechanisms of immune cell infiltrations of the brain, yet these two systems are connected. We postulate that the mechanical properties of the cellular polarity is the main driving force determining the migration pathway. Our analysis indicates that both systems coevolved with immune cells, evolving to a higher level of complexity in association with the evolution of the immune system.

What has single-cell RNA sequencing revealed about microglial neuroimmunology?

The use of single-cell RNA sequencing (scRNA-seq) in microglial research is increasing rapidly. The basic workflow of this approach consists of isolating single cells, followed by sequencing. scRNA-seq is capable of examining microglial heterogeneity on a cellular level. However, the results gained from applying this technique suffer from discrepancies due to differences between applied methods characteristics such as the number of cells sequenced and the depth of sequencing. This review aims to shed more light on the recent developments that happened in this field and how they are related to the methods used. To do that, we track the progress and limitations of various scRNA-seq methods currently available. The review then summarizes the current knowledge gained using scRNA-seq in the field of microglia, including novel subpopulations associated with function and development under homeostasis as well during several pathological conditions such as Alzheimer, lipopolysaccharide response, and HIV in relation to the methods employed. Our review points out that despite major developments found using this technique, current scRNA-seq methods suffer from high cost, low yields, and nonstandardization of generated data. Additional development of scRNA-seq methods will raise our awareness of microglia's heterogeneity and plasticity under healthy and pathological conditions.

Drugs Modulating CD4+ T Cells Blood-Brain Barrier Interaction in Alzheimer's Disease.

The effect of Alzheimer's disease (AD) medications on CD4+ T cells homing has not been thoroughly investigated. CD4+ T cells could both exacerbate and reduce AD symptoms based on their infiltrating subpopulations. Proinflammatory subpopulations such as Th1 and Th17 constitute a major source of proinflammatory cytokines that reduce endothelial integrity and stimulate astrocytes, resulting in the production of amyloid ß. Anti-inflammatory subpopulations such as Th2 and Tregs reduce inflammation and regulate the function of Th1 and Th17. Recently, pathogenic Th17 has been shown to have a superior infiltrating capacity compared to other major CD4+ T cell subpopulations. Alzheimer's drugs such as donepezil (Aricept), rivastigmine (Exelon), galantamine (Razadyne), and memantine (Namenda) are known to play an important part in regulating the mechanisms of the neurotransmitters. However, little is known about the effect of these drugs on CD4+ T cell subpopulations' infiltration of the brain during AD. In this review, we focus on understanding the influence of AD drugs on CD4+ T cell subpopulation interactions with the BBB in AD. While current AD therapies improve endothelial integrity and reduce astrocytes activations, they vary according to their influence on various CD4+ T cell subpopulations. Donepezil reduces the numbers of Th1 but not Th2, Rivastigmine inhibits Th1 and Th17 but not Th2, and memantine reduces Th1 but not Treg. However, none of the current AD drugs is specifically designed to target the dysregulated balance in the Th17/Treg axis. Future drug design approaches should specifically consider inhibiting CD4+ Th17 to improve AD prognosis.

Repurposing Zileuton as a Depression Drug Using an AI and In Vitro Approach.

Repurposing drugs to target M1 macrophages inflammatory response in depression constitutes a bright alternative for commonly used antidepressants. Depression is a significant type of mood disorder, where patients suffer from pathological disturbances associated with a proinflammatory M1 macrophage phenotype. Presently, the most commonly used antidepressants such as Zoloft and Citalopram can reduce inflammation, but suffer from dangerous side effects without offering specificity toward macrophages. We employed a new strategy for drug repurposing based on the integration of RNA-seq analysis and text mining using deep neural networks. Our system employs a Google semantic AI universal encoder to compute sentences embedding. Sentences similarity is calculated using a sorting function to identify drug compounds. Then sentence relevance is computed using a custom-built convolution differential network. Our system highlighted the NRF2 pathway as a critical drug target to reprogram M1 macrophage response toward an anti-inflammatory profile (M2). Using our approach, we were also able to predict that lipoxygenase inhibitor drug zileuton could modulate NRF2 pathway in vitro. Taken together, our results indicate that reorienting zileuton usage to modulate M1 macrophages could be a novel and safer therapeutic option for treating depression.

Molecular Evolution and Functional Divergence of the IgLON Family.

IgLON family is a subgroup of cell adhesion molecules which is known to have diverse roles in neuronal development. IgLONs are characterized by possessing 3 Ig-like C2 domains, which play a part in mediating various cellular interactions. Recently, IgLONs have been shown to be expressed at the blood-brain barrier (BBB). However, our understanding of the genetic divergence patterns and evolutionary rates of these proteins in relation to their functions, in general, and at the BBB, in particular, remains inadequate. In this study, 12 species were explored to shed more light on the phylogenetic origins, structure, functional specificity, and divergence of this family. A total of 40 IgLON genes were identified from vertebrates and invertebrates. The absence of IgLON family genes in Hydra vulgaris and Nematostella vectensis but not in Drosophila melanogaster suggests that this family appeared during the time of divergence of Arthropoda 455 Mya. In general, IgLON genes have been subject to strong positive selection in vertebrates. Our study, based on IgLONs' structural similarity, suggests that they may play a role in the evolutionary changes in the brain anatomy towards complexity including regulating neural growth and BBB permeability. IgLONs' functions seem to be performed through complex interactions on the level of motifs as well as single residues. We identified several IgLON motifs that could be influencing cellular migration and proliferation as well as BBB integrity through interactions with SH3 or integrin. Our motif analysis also revealed that NEGR1 might be involved in MAPK pathway as a form of a signal transmitting receptor through its motif (KKVRVVVNF). We found several residues that were both positively selected and with highly functional specificity. We also located functional divergent residues that could act as drug targets to regulate BBB permeability. Furthermore, we identified several putative metalloproteinase cleavage sites that support the ectodomain shedding hypothesis of the IgLONs. In conclusion, our results present a bridge between IgLONs' molecular evolution and their functions.

The landscape of human mutually exclusive splicing.

Mutually exclusive splicing of exons is a mechanism of functional gene and protein diversification with pivotal roles in organismal development and diseases such as Timothy syndrome, cardiomyopathy and cancer in humans. In order to obtain a first genomewide estimate of the extent and biological role of mutually exclusive splicing in humans, we predicted and subsequently validated mutually exclusive exons (MXEs) using 515 publically available RNA-Seq datasets. Here, we provide evidence for the expression of over 855 MXEs, 42% of which represent novel exons, increasing the annotated human mutually exclusive exome more than fivefold. The data provide strong evidence for the existence of large and multi-cluster MXEs in higher vertebrates and offer new insights into MXE evolution. More than 82% of the MXE clusters are conserved in mammals, and five clusters have homologous clusters in Drosophila Finally, MXEs are significantly enriched in pathogenic mutations and their spatio-temporal expression might predict human disease pathology.