RNA-sequencing suggests extracellular matrix and vasculature dysregulation could impair neurogenesis in schizophrenia cases with elevated inflammation.

A subgroup of schizophrenia cases with elevated inflammation have reduced neurogenesis markers and increased macrophage density in the human subependymal zone (SEZ; also termed subventricular zone or SVZ) neurogenic niche. Inflammation can impair neurogenesis; however, it is unclear which other pathways are associated with reduced neurogenesis. This research aimed to discover transcriptomic differences between inflammatory subgroups of schizophrenia in the SEZ. Total RNA sequencing was performed on SEZ tissue from schizophrenia cases, designated into low inflammation (n = 13) and high inflammation (n = 14) subgroups, based on cluster analysis of inflammation marker gene expression. 718 genes were differentially expressed in high compared to low inflammation schizophrenia (FDR p < 0.05) and were most significantly over-represented in the pathway 'Hepatic Fibrosis/Hepatic Stellate-Cell Activation'. Genes in this pathway relate to extracellular matrix stability (including ten collagens) and vascular remodelling suggesting increased angiogenesis. Collagen-IV, a key element of the basement membrane and fractones, had elevated gene expression. Immunohistochemistry revealed novel collagen-IV+ fractone bulbs within the human SEZ hypocellular gap. Considering the extracellular matrix's regulatory role in SEZ neurogenesis, fibrosis-related alterations in high inflammation schizophrenia may disrupt neurogenesis. Increased angiogenesis could facilitate immune cell transmigration, potentially explaining elevated macrophages in high inflammation schizophrenia. This discovery-driven analysis sheds light on how inflammation may contribute to schizophrenia neuropathology in the neurogenic niche.

CRISPR-Cas13d screens identify KILR, a breast cancer risk-associated lncRNA that regulates DNA replication and repair.

BACKGROUND: Long noncoding RNAs (lncRNAs) have surpassed the number of protein-coding genes, yet the majority have no known function. We previously discovered 844 lncRNAs that were genetically linked to breast cancer through genome-wide association studies (GWAS). Here, we show that a subset of these lncRNAs alter breast cancer risk by modulating cell proliferation, and provide evidence that a reduced expression on one lncRNA increases breast cancer risk through aberrant DNA replication and repair. METHODS: We performed pooled CRISPR-Cas13d-based knockdown screens in breast cells to identify which of the 844 breast cancer-associated lncRNAs alter cell proliferation. We selected one of the lncRNAs that increased cell proliferation, KILR, for follow-up functional studies. KILR pull-down followed by mass spectrometry was used to identify binding proteins. Knockdown and overexpression studies were performed to assess the mechanism by which KILR regulates proliferation. RESULTS: We show that KILR functions as a tumor suppressor, safeguarding breast cells against uncontrolled proliferation. The half-life of KILR is significantly reduced by the risk haplotype, revealing an alternative mechanism by which variants alter cancer risk. Mechanistically, KILR sequesters RPA1, a subunit of the RPA complex required for DNA replication and repair. Reduced KILR expression promotes breast cancer cell proliferation by increasing the available pool of RPA1 and speed of DNA replication. Conversely, KILR overexpression promotes apoptosis in breast cancer cells, but not normal breast cells. CONCLUSIONS: Our results confirm lncRNAs as mediators of breast cancer risk, emphasize the need to annotate noncoding transcripts in relevant cell types when investigating GWAS variants and provide a scalable platform for mapping phenotypes associated with lncRNAs.

Redefining normal breast cell populations using long noncoding RNAs.

Single-cell RNAseq has allowed unprecedented insight into gene expression across different cell populations in normal tissue and disease states. However, almost all studies rely on annotated gene sets to capture gene expression levels and sequencing reads that do not align to known genes are discarded. Here, we discover thousands of long noncoding RNAs (lncRNAs) expressed in human mammary epithelial cells and analyze their expression in individual cells of the normal breast. We show that lncRNA expression alone can discriminate between luminal and basal cell types and define subpopulations of both compartments. Clustering cells based on lncRNA expression identified additional basal subpopulations, compared to clustering based on annotated gene expression, suggesting that lncRNAs can provide an additional layer of information to better distinguish breast cell subpopulations. In contrast, these breast-specific lncRNAs poorly distinguish brain cell populations, highlighting the need to annotate tissue-specific lncRNAs prior to expression analyses. We also identified a panel of 100 breast lncRNAs that could discern breast cancer subtypes better than protein-coding markers. Overall, our results suggest that lncRNAs are an unexplored resource for new biomarker and therapeutic target discovery in the normal breast and breast cancer subtypes.

An Alzheimer's Disease Patient-Derived Olfactory Stem Cell Model Identifies Gene Expression Changes Associated with Cognition.

An early symptom of Alzheimer's disease (AD) is an impaired sense of smell, for which the molecular basis remains elusive. Here, we generated human olfactory neurosphere-derived (ONS) cells from people with AD and mild cognitive impairment (MCI), and performed global RNA sequencing to determine gene expression changes. ONS cells expressed markers of neuroglial differentiation, providing a unique cellular model to explore changes of early AD-associated pathways. Our transcriptomics data from ONS cells revealed differentially expressed genes (DEGs) associated with cognitive processes in AD cells compared to MCI, or matched healthy controls (HC). A-Kinase Anchoring Protein 6 (AKAP6) was the most significantly altered gene in AD compared to both MCI and HC, and has been linked to cognitive function. The greatest change in gene expression of all DEGs occurred between AD and MCI. Gene pathway analysis revealed defects in multiple cellular processes with aging, intellectual deficiency and alternative splicing being the most significantly dysregulated in AD ONS cells. Our results demonstrate that ONS cells can provide a cellular model for AD that recapitulates disease-associated differences. We have revealed potential novel genes, including AKAP6 that may have a role in AD, particularly MCI to AD transition, and should be further examined.

Identifying gene expression profiles associated with neurogenesis and inflammation in the human subependymal zone from development through aging.

The generation of new neurons within the mammalian forebrain continues throughout life within two main neurogenic niches, the subgranular zone (SGZ) of the hippocampal dentate gyrus, and the subependymal zone (SEZ) lining the lateral ventricles. Though the SEZ is the largest neurogenic niche in the adult human forebrain, our understanding of the mechanisms regulating neurogenesis from development through aging within this region remains limited. This is especially pertinent given that neurogenesis declines dramatically over the postnatal lifespan. Here, we performed transcriptomic profiling on the SEZ from human post-mortem tissue from eight different life-stages ranging from neonates (average age ~ 2 months old) to aged adults (average age ~ 86 years old). We identified transcripts with concomitant profiles across these decades of life and focused on three of the most distinct profiles, namely (1) genes whose expression declined sharply after birth, (2) genes whose expression increased steadily with age, and (3) genes whose expression increased sharply in old age in the SEZ. Critically, these profiles identified neuroinflammation as becoming more prevalent with advancing age within the SEZ and occurring with time courses, one gradual (starting in mid-life) and one sharper (starting in old age).

Reduced adult neurogenesis is associated with increased macrophages in the subependymal zone in schizophrenia.

Neural stem cells in the human subependymal zone (SEZ) generate neuronal progenitor cells that can differentiate and integrate as inhibitory interneurons into cortical and subcortical brain regions; yet the extent of adult neurogenesis remains unexplored in schizophrenia and bipolar disorder. We verified the existence of neurogenesis across the lifespan by chartering transcriptional alterations (2 days-103 years, n = 70) and identifying cells indicative of different stages of neurogenesis in the human SEZ. Expression of most neural stem and neuronal progenitor cell markers decreased during the first postnatal years and remained stable from childhood into ageing. We next discovered reduced neural stem and neuronal progenitor cell marker expression in the adult SEZ in schizophrenia and bipolar disorder compared to controls (n = 29-32 per group). RNA sequencing identified increased expression of the macrophage marker CD163 as the most significant molecular change in schizophrenia. CD163+ macrophages, which were localised along blood vessels and in the parenchyma within 10 µm of neural stem and progenitor cells, had increased density in schizophrenia but not in bipolar disorder. Macrophage marker expression negatively correlated with neuronal progenitor marker expression in schizophrenia but not in controls or bipolar disorder. Reduced neurogenesis and increased macrophage marker expression were also associated with polygenic risk for schizophrenia. Our results support that the human SEZ retains the capacity to generate neuronal progenitor cells throughout life, although this capacity is limited in schizophrenia and bipolar disorder. The increase in macrophages in schizophrenia but not in bipolar disorder indicates that immune cells may impair neurogenesis in the adult SEZ in a disease-specific manner.

Genes with human-specific features are primarily involved with brain, immune and metabolic evolution.

BACKGROUND: Humans have adapted to widespread changes during the past 2 million years in both environmental and lifestyle factors. This is evident in overall body alterations such as average height and brain size. Although we can appreciate the uniqueness of our species in many aspects, molecular variations that drive such changes are far from being fully known and explained. Comparative genomics is able to determine variations in genomic sequence that may provide functional information to better understand species-specific adaptations. A large number of human-specific genomic variations have been reported but no currently available dataset comprises all of these, a problem which contributes to hinder progress in the field. RESULTS: Here we critically update high confidence human-specific genomic variants that mostly associate with protein-coding regions and find 856 related genes. Events that create such human-specificity are mainly gene duplications, the emergence of novel gene regions and sequence and structural alterations. Functional analysis of these human-specific genes identifies adaptations to brain, immune and metabolic systems to be highly involved. We further show that many of these genes may be functionally associated with neural activity and generating the expanded human cortex in dynamic spatial and temporal contexts. CONCLUSIONS: This comprehensive study contributes to the current knowledge by considerably updating the number of human-specific genes following a critical bibliographic survey. Human-specific genes were functionally assessed for the first time to such extent, thus providing unique information. Our results are consistent with environmental changes, such as immune challenges and alterations in diet, as well as neural sophistication, as significant contributors to recent human evolution.

Adar3 Is Involved in Learning and Memory in Mice.

The amount of regulatory RNA encoded in the genome and the extent of RNA editing by the post-transcriptional deamination of adenosine to inosine (A-I) have increased with developmental complexity and may be an important factor in the cognitive evolution of animals. The newest member of the A-I editing family of ADAR proteins, the vertebrate-specific ADAR3, is highly expressed in the brain, but its functional significance is unknown. In vitro studies have suggested that ADAR3 acts as a negative regulator of A-I RNA editing but the scope and underlying mechanisms are also unknown. Meta-analysis of published data indicates that mouse Adar3 expression is highest in the hippocampus, thalamus, amygdala, and olfactory region. Consistent with this, we show that mice lacking exon 3 of Adar3 (which encodes two double stranded RNA binding domains) have increased levels of anxiety and deficits in hippocampus-dependent short- and long-term memory formation. RNA sequencing revealed a dysregulation of genes involved in synaptic function in the hippocampi of Adar3-deficient mice. We also show that ADAR3 transiently translocates from the cytoplasm to the nucleus upon KCl-mediated activation in SH-SY5Y cells. These results indicate that ADAR3 contributes to cognitive processes in mammals.

THC exposure of human iPSC neurons impacts genes associated with neuropsychiatric disorders.

There is a strong association between cannabis use and schizophrenia but the underlying cellular links are poorly understood. Neurons derived from human-induced pluripotent stem cells (hiPSCs) offer a platform for investigating both baseline and dynamic changes in human neural cells. Here, we exposed neurons derived from hiPSCs to Δ9-tetrahydrocannabinol (THC), and identified diagnosis-specific differences not detectable in vehicle-controls. RNA transcriptomic analyses revealed that THC administration, either by acute or chronic exposure, dampened the neuronal transcriptional response following potassium chloride (KCl)-induced neuronal depolarization. THC-treated neurons displayed significant synaptic, mitochondrial, and glutamate signaling alterations that may underlie their failure to activate appropriately; this blunted response resembles effects previously observed in schizophrenia hiPSC- derived neurons. Furthermore, we show a significant alteration in THC-related genes associated with autism and intellectual disability, suggesting shared molecular pathways perturbed in neuropsychiatric disorders that are exacerbated by THC.

Long Non-Coding RNAs in Neuronal Aging.

The expansion of long non-coding RNAs (lncRNAs) in organismal genomes has been associated with the emergence of sophisticated regulatory networks that may have contributed to more complex neuronal processes, such as higher-order cognition. In line with the important roles of lncRNAs in the normal functioning of the human brain, dysregulation of lncRNA expression has been implicated in aging and age-related neurodegenerative disorders. In this paper, we discuss the function and expression of known neuronal-associated lncRNAs, their impact on epigenetic changes, the contribution of transposable elements to lncRNA expression, and the implication of lncRNAs in maintaining the 3D nuclear architecture in neurons. Moreover, we discuss how the complex molecular processes that are orchestrated by lncRNAs in the aged brain may contribute to neuronal pathogenesis by promoting protein aggregation and neurodegeneration. Finally, this review explores the possibility that age-related disturbances of lncRNA expression change the genomic and epigenetic regulatory landscape of neurons, which may affect neuronal processes such as neurogenesis and synaptic plasticity.

Multiple Innovations in Genetic and Epigenetic Mechanisms Cooperate to Underpin Human Brain Evolution.

Our knowledge of how the human brain differs from those of other species in terms of evolutionary adaptations and functionality is limited. Comparative genomics reveal valuable insight, especially the expansion of human-specific noncoding regulatory and repeat-containing regions. Recent studies add to our knowledge of evolving brain function by investigating cellular mechanisms such as protein emergence, extensive sequence editing, retrotransposon activity, dynamic epigenetic modifications, and multiple noncoding RNA functions. These findings present an opportunity to combine newly discovered genetic and epigenetic mechanisms with more established concepts into a more comprehensive picture to better understand the uniquely evolved human brain.

Using Human iPSC-Derived Neurons to Uncover Activity-Dependent Non-Coding RNAs.

Humans are arguably the most complex organisms present on Earth with their ability to imagine, create, and problem solve. As underlying mechanisms enabling these capacities reside in the brain, it is not surprising that the brain has undergone an extraordinary increase in size and complexity within the last few million years. Human induced pluripotent stem cells (hiPSCs) can be differentiated into many cell types that were virtually inaccessible historically, such as neurons. Here, we used hiPSC-derived neurons to investigate the cellular response to activation at the transcript level. Neuronal activation was performed with potassium chloride (KCl) and its effects were assessed by RNA sequencing. Our results revealed the involvement of long non-coding RNAs and human-specific genetic variants in response to neuronal activation and help validate hiPSCs as a valuable resource for the study of human neuronal networks. In summary, we find that genes affected by KCl-triggered activation are implicated in pathways that drive cell proliferation, differentiation, and the emergence of specialized morphological features. Interestingly, non-coding RNAs of various classes are amongst the most highly expressed genes in activated hiPSC-derived neurons, thus suggesting these play crucial roles in neural pathways and may significantly contribute to the unique functioning of the human brain.

Adenine Glycosylase MutY of Corynebacterium pseudotuberculosis presents the antimutator phenotype and evidences of glycosylase/AP lyase activity in vitro.

Corynebacterium pseudotuberculosis is the etiological agent of caseous lymphadenitis, a disease that predominantly affects small ruminants, causing significant economic losses worldwide. As a facultative intracellular pathogen, this bacterium is exposed to an environment rich in reactive oxygen species (ROS) within macrophages. To ensure its genetic stability, C. pseudotuberculosis relies on efficient DNA repair pathways for excision of oxidative damage such as 8-oxoguanine, a highly mutagenic lesion. MutY is an adenine glycosylase involved in adenine excision from 8-oxoG:A mismatches avoiding genome mutation incorporation. The purpose of this study was to characterize MutY protein from C. pseudotuberculosis and determine its involvement with DNA repair. In vivo functional complementation assay employing mutY gene deficient Escherichia coli transformed with CpmutY showed a 13.5-fold reduction in the rate of spontaneous mutation, compared to cells transformed with empty vector. Also, under oxidative stress conditions, CpMutY protein favored the growth of mutY deficient E. coli, relative to the same strain in the absence of CpMutY. To demonstrate the involvement of this enzyme in recognition and excision of 8-oxoguanine lesion, an in vitro assay was performed. CpMutY protein was capable of recognizing and excising 8-oxoG:A but not 8-oxoG:C presenting evidences of glycosylase/AP lyase activity in vitro. In silico structural characterization revealed the presence of preserved motifs related to the MutY activity on DNA repair, such as catalytic residues involved in glycosylase/AP lyase activity and structural DNA-binding elements, such as the HhH motif and the [4Fe-4S] cluster. The three-dimensional structure of CpMutY, generated by comparative modeling, exhibits a catalytic domain very similar to that of E. coli MutY. Taken together, these results indicate that the CpmutY encodes a functional protein homologous to MutY from E. coli and is involved in the prevention of mutations and the repair of oxidative DNA lesions.

Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase.

Trypanosoma cruzi, the etiological agent of Chagas' disease, presents three cellular forms (trypomastigotes, epimastigotes and amastigotes), all of which are submitted to oxidative species in its hosts. However, T. cruzi is able to resist oxidative stress suggesting a high efficiency of its DNA repair machinery.The Base Excision Repair (BER) pathway is one of the main DNA repair mechanisms in other eukaryotes and in T. cruzi as well. DNA glycosylases are enzymes involved in the recognition of oxidative DNA damage and in the removal of oxidized bases, constituting the first step of the BER pathway. Here, we describe the presence and activity of TcNTH1, a nuclear T. cruzi DNA glycosylase. Surprisingly, purified recombinant TcNTH1 does not remove the thymine glycol base, but catalyzes the cleavage of a probe showing an AP site. The same activity was found in epimastigote and trypomastigote homogenates suggesting that the BER pathway is not involved in thymine glycol DNA repair. TcNTH1 DNA-binding properties assayed in silico are in agreement with the absence of a thymine glycol removing function of that parasite enzyme. Over expression of TcNTH1 decrease parasite viability when transfected epimastigotes are submitted to a sustained production of H2O2.Therefore, TcNTH1 is the only known NTH1 orthologous unable to eliminate thymine glycol derivatives but that recognizes and cuts an AP site, most probably by a beta-elimination mechanism. We cannot discard that TcNTH1 presents DNA glycosylase activity on other DNA base lesions. Accordingly, a different DNA repair mechanism should be expected leading to eliminate thymine glycol from oxidized parasite DNA. Furthermore, TcNTH1 may play a role in the AP site recognition and processing.

The Corynebacterium pseudotuberculosis genome contains two formamidopyrimidine-DNA glycosylase enzymes, only one of which recognizes and excises 8-oxoguanine lesion.

The GO-system is a DNA repair mechanism that prevents and corrects oxidative DNA damage. Formamidopyrimidine-DNA glycosylase (FPG/MutM) participates in this system, avoiding the mutagenic effects of 8-oxoguanine lesion into DNA. Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis, is a facultative intracellular microorganism vulnerable to oxidative DNA damage. Since inefficiencies in the DNA damage repair system can lead to death, the characterization of repair genes may provide valuable molecular targets for caseous lymphadenitis therapy. The purposes of this study were to functionally characterize MutM1 and MutM2 proteins from C. pseudotuberculosis in silico, in vivo, and in vitro and to examine their role in the repair of 8-oxoguanine damage. In silico investigation revealed that both proteins have conserved domains typical of DNA glycosylases, such as DNA binding domains and DNA glycosylase/AP lyase catalytic domain. In comparison with the MutM protein of Escherichia coli, however, CpMutM2 was found to lack residues that are essential for recognizing and excising 8-oxoguanine damage. Molecular docking calculations have shown a native-like orientation of 8-oxoguanine at the CpMutM1 active site, while the same is not observed for CpMutM2, which seems to poorly interact with DNA. Surface charge analyses have corroborated this finding. Overexpression of CpMutM1 or CpMutM2 has toxic effects on E. coli strain BH20 (mutM-), as shown by growth curves obtained in the presence of hydrogen peroxide and cell viability assays. This cytotoxicity can be attributed to an imbalance in the repair pathway, resulting from hyperactivity of DNA glycosylases, leading to formation of AP sites and DNA strand breakage at levels that exceed the processing capacity of other enzymes in the BER pathway. In order to demonstrate the involvement of these enzymes in the recognition and excision of 8-oxoguanine lesion, glycosylase activity was evaluated in vitro. Only the CpMutM1 protein was proven to be capable of recognizing and excising 8-oxoguanine. Taken together, these results suggest that although the formamidopyrimidine-DNA glycosylase domain is conserved in both proteins, only one proved to be functional in recognizing and excising 8-oxoguanine lesion.

Evaluation of the Schistosoma mansoni Y-box-binding protein (SMYB1) potential as a vaccine candidate against schistosomiasis.

Schistosomiasis is a neglected tropical disease, and after malaria, is the second most important tropical disease in public health. A vaccine that reduces parasitemia is desirable to achieve mass treatment with a low cost. Although potential antigens have been identified and tested in clinical trials, no effective vaccine against schistosomiasis is available. Y-box-binding proteins (YBPs) regulate gene expression and participate in a variety of cellular processes, including transcriptional and translational regulation, DNA repair, cellular proliferation, drug resistance, and stress responses. The Schistosoma mansoni ortholog of the human YB-1, SMYB1, is expressed in all stages of the parasite life cycle. Although SMYB1 binds to DNA or RNA oligonucleotides, immunohistochemistry assays demonstrated that it is primarily localized in the cytoplasm of parasite cells. In addition, SMYB1 interacts with a protein involved in mRNA processing, suggesting that SMYB1 functions in the turnover, transport, and/or stabilization of RNA molecules during post-transcriptional gene regulation. Here we report the potential of SMYB1 as a vaccine candidate. We demonstrate that recombinant SMYB1 stimulates the production of high levels of specific IgG1 antibodies in a mouse model. The observed levels of specific IgG1 and IgG2a antibodies indicate an actual protection against cercariae challenge. Animals immunized with rSMYB1 exhibited a 26% reduction in adult worm burden and a 28% reduction in eggs retained in the liver. Although proteins from the worm tegument are considered optimal targets for vaccine development, this study demonstrates that unexposed cytoplasmic proteins can reduce the load of intestinal worms and the number of eggs retained in the liver.

Proteomic analysis of Trypanosoma cruzi response to ionizing radiation stress.

Trypanosoma cruzi, the causative agent of Chagas disease, is extremely resistant to ionizing radiation, enduring up to 1.5 kGy of gamma rays. Ionizing radiation can damage the DNA molecule both directly, resulting in double-strand breaks, and indirectly, as a consequence of reactive oxygen species production. After a dose of 500 Gy of gamma rays, the parasite genome is fragmented, but the chromosomal bands are restored within 48 hours. Under such conditions, cell growth arrests for up to 120 hours and the parasites resume normal growth after this period. To better understand the parasite response to ionizing radiation, we analyzed the proteome of irradiated (4, 24, and 96 hours after irradiation) and non-irradiated T. cruzi using two-dimensional differential gel electrophoresis followed by mass spectrometry for protein identification. A total of 543 spots were found to be differentially expressed, from which 215 were identified. These identified protein spots represent different isoforms of only 53 proteins. We observed a tendency for overexpression of proteins with molecular weights below predicted, indicating that these may be processed, yielding shorter polypeptides. The presence of shorter protein isoforms after irradiation suggests the occurrence of post-translational modifications and/or processing in response to gamma radiation stress. Our results also indicate that active translation is essential for the recovery of parasites from ionizing radiation damage. This study therefore reveals the peculiar response of T. cruzi to ionizing radiation, raising questions about how this organism can change its protein expression to survive such a harmful stress.

Nucleotide excision repair in Trypanosoma brucei: specialization of transcription-coupled repair due to multigenic transcription.

Nucleotide excision repair (NER) is a highly conserved genome repair pathway acting on helix distorting DNA lesions. NER is divided into two subpathways: global genome NER (GG-NER), which is responsible for repair throughout genomes, and transcription-coupled NER (TC-NER), which acts on lesions that impede transcription. The extent of the Trypanosoma brucei genome that is transcribed is highly unusual, since most genes are organized in multigene transcription units, each transcribed from a single promoter. Given this transcription organization, we have addressed the importance of NER to T. brucei genome maintenance by performing RNAi against all predicted contributing factors. Our results indicate that TC-NER is the main pathway of NER repair, but only CSB, XPBz and XPG contribute. Moreover, we show that UV lesions are inefficiently repaired in T. brucei, perhaps due to preferential use of RNA polymerase translesion synthesis. RNAi of XPC and DDB was found to be lethal, and we show that these factors act in inter-strand cross-link repair. XPD and XPB appear only to act in transcription, not repair. This work indicates that the predominance of multigenic transcription in T. brucei has resulted in pronounced adaptation of NER relative to the host and may be an attractive drug target.

Comparative modeling of proteins: a method for engaging students' interest in bioinformatics tools.

The huge increase in data being produced in the genomic era has produced a need to incorporate computers into the research process. Sequence generation, its subsequent storage, interpretation, and analysis are now entirely computer-dependent tasks. Universities from all over the world have been challenged to seek a way of encouraging students to incorporate computational and bioinformatics skills since undergraduation in order to understand biological processes. The aim of this article is to report the experience of awakening students' interest in bioinformatics tools during a course focused on comparative modeling of proteins. The authors start by giving a full description of the course environmental context and students' backgrounds. Then they detail each class and present a general overview of the protein modeling protocol. The positive and negative aspects of the course are also reported, and some of the results generated in class and in projects outside the classroom are discussed. In the last section of the article, general perspectives about the course from students' point of view are given. This work can serve as a guide for professors who teach subjects for which bioinformatics tools are useful and for universities that plan to incorporate bioinformatics into the curriculum.