Dr. Farkas-Pool, Carlos

The identification of new proteins that regulate the function of one of the main cellular phosphatases, protein phosphatase 1 (PP1), is essential to find possible pharmacological targets to alter phosphatase function in various cellular processes, including the initiation and development of multiple diseases. IIIG9 is a regulatory subunit of PP1 initially identified in highly polarized ciliated cells. In addition to its ciliary location in ependymal cells, we recently showed that IIIG9 has extraciliary functions that regulate the integrity of adherens junctions. In this review, we perform a detailed analysis of the expression, localization, and function of IIIG9 in adult and developing normal brains. In addition, we provide a 3D model of IIIG9 protein structure for the first time, verifying that the classic structural and conformational characteristics of the PP1 regulatory subunits are maintained. Our review is especially focused on finding evidence linking IIIG9 dysfunction with the course of some pathologies, such as ciliopathies, drug dependence, diseases based on neurological development, and the development of specific high-malignancy and -frequency brain tumors in the pediatric population. Finally, we propose that IIIG9 is a relevant regulator of PP1 function in physiological and pathological processes in the CNS.

Background The significant role of embryonic cerebrospinal fluid (eCSF) in the initial stages of brain development has been thoroughly studied. This fluid contains crucial molecules for proper brain development such as members of the Wnt and FGF families, apolipoproteins, and retinol binding protein. Nevertheless, the source of these molecules remains uncertain since they are present before the formation of the choroid plexus, which is conventionally known as the primary producer of cerebrospinal fluid. The subcommissural organ (SCO) is a highly conserved gland located in the diencephalon and is one of the earliest differentiating brain structures. The SCO secretes molecules into the eCSF, prior to the differentiation of the choroid plexus, playing a pivotal role in the homeostasis and dynamics of this fluid. One of the key molecules secreted by the SCO is SCO-spondin, a protein involved in maintenance of the normal ventricle size, straight spinal axis, neurogenesis, and axonal guidance. Furthermore, SCO secretes transthyretin and basic fibroblast growth factor 2, while other identified molecules in the eCSF could potentially be secreted by the SCO. Additionally, various transcription factors have been identified in the SCO. However, the precise mechanisms involved in the early SCO development are not fully understood. Results To uncover key molecular players and signaling pathways involved in the role of the SCO during brain development, we conducted a transcriptomic analysis comparing the embryonic chick SCO at HH23 and HH30 stages (4 and 7 days respectively). Additionally, a public transcriptomic data from HH30 entire chick brain was used to compare expression levels between SCO and whole brain transcriptome. These analyses revealed that, at both stages, the SCO differentially expresses several members of bone morphogenic proteins, Wnt and fibroblast growth factors families, diverse proteins involved in axonal guidance, neurogenic and differentiative molecules, cell receptors and transcription factors. The secretory pathway is particularly upregulated at stage HH30 while the proliferative pathway is increased at stage HH23. Conclusión The results suggest that the SCO has the capacity to secrete several morphogenic molecules to the eCSF prior to the development of other structures, such as the choroid plexus.

Degenerative Cervical Myelopathy (DCM) is the most common cause of spinal cord impairment in elderly populations. It describes a spectrum of disorders that cause progressive spinal cord compression, neurological impairment, loss of bladder and bowel functions, and gastrointestinal dysfunction. The gut microbiota has been recognized as an environmental factor that can modulate both the function of the central nervous system and the immune response through the microbiota-gut-brain axis. Changes in gut microbiota composition or microbiota-producing factors have been linked to the progression and development of several pathologies. However, little is known about the potential role of the gut microbiota in the pathobiology of DCM. Here, DCM was induced in C57BL/6 mice by implanting an aromatic polyether material underneath the C5-6 laminae. The extent of DCM-induced changes in microbiota composition was assessed by 16S rRNA sequencing of the fecal samples. The immune cell composition was assessed using flow cytometry. To date, several bacterial members have been identified using BLAST against the largest collection of metagenome-derived genomes from the mouse gut. In both, female and males DCM caused gut dysbiosis compared to the sham group. However, dysbiosis was more pronounced in males than in females, and several bacterial members of the families Lachnospiraceae and Muribaculaceae were significantly altered in the DCM group. These changes were also associated with altered microbe-derived metabolic changes in propionate-, butyrate-, and lactate-producing bacterial members. Our results demonstrate that DCM causes dynamic changes over time in the gut microbiota, reducing the abundance of butyrate-producing bacteria, and lactate-producing bacteria to a lesser extent. Genome-scale metabolic modeling using gapseq successfully identified pyruvate-to-butanoate and pyruvate-to-propionate reactions involving genes such as Buk and ACH1, respectively. These results provide a better understanding of the sex-specific molecular effects of changes in the gut microbiota on DCM pathobiology.

The establishment and maintenance of nucleosome-free regions (NFRs) are prominent processes within chromatin dynamics. Transcription factors, ATP-dependent chromatin remodeling complexes (CRCs) and DNA sequences are the main factors involved. In Saccharomyces cerevisiae, CRCs such as RSC contribute to chromatin opening at NFRs, while other complexes, including ISW1a, contribute to NFR shrinking. Regarding DNA sequences, growing evidence points to poly(dA:dT) tracts as playing a direct role in active processes involved in nucleosome positioning dynamics. Intriguingly, poly(dA:dT)-tract-containing NFRs span asymmetrically relative to the location of the tract by a currently unknown mechanism. In order to obtain insight into the role of poly(dA:dT) tracts in nucleosome remodeling, we performed a systematic analysis of their influence on the activity of ISW1a and RSC complexes. Our results show that poly(dA:dT) tracts differentially affect the activity of these CRCs. Moreover, we found differences between the effects exerted by the two alternative tract orientations. Remarkably, tract-containing linker DNA is taken as exit DNA for nucleosome sliding catalyzed by RSC. Our findings show that defined DNA sequences, when present in linker DNA, can dictate in which direction a remodeling complex has to slide nucleosomes and shed light into the mechanisms underlying asymmetrical chromatin opening around poly(dA:dT) tracts.

Background: The advancement of hybrid sequencing technologies is increasingly expanding genome assemblies that are often annotated using hybrid sequencing transcriptomics, leading to improved genome characterization and the identification of novel genes and isoforms in a wide variety of organisms. Results: We developed an easy-to-use genome-guided transcriptome annotation pipeline that uses assembled transcripts from hybrid sequencing data as input and distinguishes between coding and long non-coding RNAs by integration of several bioinformatic approaches, including gene reconciliation with previous annotations in GTF format. We demonstrated the efficiency of this approach by correctly assembling and annotating all exons from the chicken SCO-spondin gene (containing more than 105 exons), including the identification of missing genes in the chicken reference annotations by homology assignments. Conclusions: Our method helps to improve the current transcriptome annotation of the chicken brain. Our pipeline, implemented on Anaconda/Nextflow and Docker is an easy-to-use package that can be applied to a broad range of species, tissues, and research areas helping to improve and reconcile current annotations. The code and datasets are publicly available at https://github.com/cfark as/annotate_my_genomes

Spalt-like proteins are Zinc finger transcription factors from Caenorhabditis elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family’s most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.

Acute myeloid leukemia (AML) is a diverse malignancy originating from myeloid progenitor cells, with significant genetic and clinical variability. Modern classification systems like those from the World Health Organization (WHO) and European LeukemiaNet use immunophenotyping, molecular genetics, and clinical features to categorize AML subtypes. This classification highlights crucial genetic markers such as FLT3, NPM1 mutations, and MLL-AF9 fusion, which are essential for prognosis and directing targeted therapies. The MLL-AF9 fusion protein is often linked with therapy-resistant AML, highlighting the risk of relapse due to standard chemotherapeutic regimes. In this sense, factors like the ZEB, SNAI, and TWIST gene families, known for their roles in epithelial–mesenchymal transition (EMT) and cancer metastasis, also regulate hematopoiesis and may serve as effective therapeutic targets in AML. These genes contribute to cell proliferation, differentiation, and extramedullary hematopoiesis, suggesting new possibilities for treatment. Advancing our understanding of the molecular mechanisms that promote AML, especially how the bone marrow microenvironment affects invasion and drug resistance, is crucial. This comprehensive insight into the molecular and environmental interactions in AML emphasizes the need for ongoing research and more effective treatments.

Degenerative cervical myelopathy (DCM) describes a spectrum of disorders that cause progressive and chronic cervical spinal cord compression. The clinical presentation can be complex and can include locomotor impairment, hand and upper extremity dysfunction, pain, loss of bladder and bowel function, as well as gastrointestinal dysfunction. Once diagnosed, surgical decompression is the recommended treatment for DCM patients with moderate to severe impairment. Our body is composed of a large community of microorganisms, known as the microbiota. Traumatic and nontraumatic spinal cord injuries (SCIs) can induce changes in the gut microbiota and gut microbiota derived metabolites. These changes have been reported as important disease-modifying factors after injury. However, whether gut dysbiosis is associated with functional neurological recovery after surgical decompression has not been examined to date. Here, DCM was induced in C57BL/6 mice by implanting an aromatic polyether material underneath the C5–6 laminae. The extent of gut dysbiosis was assessed by gas chromatography and 16S rRNA sequencing from fecal samples before and after decompression. Neuromotor activity was assessed using the Catwalk test. Our results show that DCM pre- and post- surgical decompression is associated with gut dysbiosis, without altering short chain fatty acids (SCFAs) levels. Significant differences in Clostridia, Verrumicrobiae, Lachnospiracea, Firmicutes, Bacteroidales, and Clostridiaceae were observed between the DCM group (before decompression) and after surgical decompression (2 and 5 weeks). The changes in gut microbiota composition correlated with locomotor features of the Catwalk. For example, a longer duration of ground contact and dysfunctional swing in the forelimbs, were positively correlated with gut dysbiosis. These results show for the first time an association between gut dysbiosis and locomotor deterioration after delayed surgical decompression. Thus, providing a better understanding of the extent of changes in microbiota composition in the setting of DCM pre- and postsurgical decompression.

Colorectal cancer (CRC) is one of the most common malignant neoplasms and the second leading cause of death from tumors worldwide. Therefore, there is a great need to study new therapeutical strategies, such as effective immunotherapies against these malignancies. Unfortunately, many CRC patients do not respond to current standard immunotherapies, making it necessary to search for adjuvant treatments. Histone deacetylase 6 (HDAC6) is involved in several processes, including immune response and tumor progression. Specifically, it has been observed that HDAC6 is required to activate the Signal Transducer and Activator of Transcription 3 (STAT3), a transcription factor involved in immunogenicity, by activating different genes in these pathways, such as PD-L1. Over-expression of immunosuppressive pathways in cancer cells deregulates T-cell activation. Therefore, we focused on the pharmacological inhibition of HDAC6 in CRC cells because of its potential as an adjuvant to avoid immunotolerance in immunotherapy. We investigated whether HDAC6 inhibitors (HDAC6is), such as Nexturastat A (NextA), affected STAT3 activation in CRC cells. First, we found that NextA is less cytotoxic than the non-selective HDACis panobinostat. Then, NextA modified STAT3 and decreased the mRNA and protein expression levels of PD-L1. Importantly, transcriptomic analysis showed that NextA treatment affected the expression of critical genes involved in immunomodulatory pathways in CRC malignancies. These results suggest that treatments with NextA reduce the functionality of STAT3 in CRC cells, impacting the expression of immunomodulatory genes involved in the inflammatory and immune responses. Therefore, targeting HDAC6 may represent an interesting adjuvant strategy in combination with immunotherapy.