Expression Pattern Of Isoforms Research Articles

Alternative splicing and its regulation in the malaria vector Anopheles gambiae

IntroductionAlternative splicing (AS) is a highly conserved mechanism that allows for the expansion of the coding capacity of the genome, through modifications of the way that multiple isoforms are expressed or used to generate different phenotypes. Despite its importance in physiology and disease, genome-wide studies of AS are lacking in most insects, including mosquitoes. Even for model organisms, chromatin associated processes involved in the regulation AS are poorly known.MethodsIn this study, we investigated AS in the mosquito Anopheles gambiae in the context of tissue-specific gene expression and mosquito responses to a Plasmodium falciparum infection, as well as the relationship between patterns of differential isoform expression and usage with chromatin accessibility changes. For this, we combined RNA-seq and ATAC-seq data from A. gambiae midguts and salivary glands, infected and non-infected.ResultsWe report differences between tissues in the expression of 392 isoforms and in the use of 247 isoforms. Secondly, we find a clear and significant association between chromatin accessibility states and tissue-specific patterns of AS. The analysis of differential accessible regions located at splicing sites led to the identification of several motifs resembling the binding sites of Drosophila transcription factors. Finally, the genome-wide analysis of tissue-dependent enhancer activity revealed that approximately 20% of A. gambiae transcriptional enhancers annotate to a differentially expressed or used isoform, and that their activation status is linked to AS differences between tissues.ConclusionThis research elucidates the role of AS in mosquito vector gene expression and identifies regulatory regions potentially involved in AS regulation, which could be important in the development of novel strategies for vector control.

Expression of neuronal NO synthase α- and β-isoforms in skeletal muscle of mice.

Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and β-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-μ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/μ, α/non-μ, β/μ and β/non-μ. Furthermore, immunoblotting suggests that the transition between nNOS α- and β-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS β-isoforms that form homodimers or heterodimers with non-nNOS proteins.

Expression of PDLIM5 Spliceosomes and Regulatory Functions on Myogenesis in Pigs

Expression of PDLIM5 Spliceosomes and Regulatory Functions on Myogenesis in Pigs

The Expression and Secretion Profile of TRAP5 Isoforms in Gaucher Disease.

Gaucher disease (GD) is caused by glucocerebrosidase (GCase) enzyme deficiency, leading to glycosylceramide (Gb-1) and glucosylsphingosine (Lyso-Gb-1) accumulation. The pathological hallmark for GD is an accumulation of large macrophages called Gaucher cells (GCs) in the liver, spleen, and bone marrow, which are associated with chronic organ enlargement, bone manifestations, and inflammation. Tartrate-resistant acid phosphatase type 5 (TRAP5 protein, ACP5 gene) has long been a nonspecific biomarker of macrophage/GCs activation; however, the discovery of two isoforms of TRAP5 has expanded its significance. The discovery of TRAP5's two isoforms revealed that it is more than just a biomarker of macrophage activity. While TRAP5a is highly expressed in macrophages, TRAP5b is secreted by osteoclasts. Recently, we have shown that the elevation of TRAP5b in plasma is associated with osteoporosis in GD. However, the role of TRAP isoforms in GD and how the accumulation of Gb-1 and Lyso-Gb-1 affects TRAP expression is unknown. 39 patients with GD were categorized into cohorts based on bone mineral density (BMD). TRAP5a and TRAP5b plasma levels were quantified by ELISA. ACP5 mRNA was estimated using RT-PCR. An increase in TRAP5b was associated with reduced BMD and correlated with Lyso-Gb-1 and immune activator chemokine ligand 18 (CCL18). In contrast, the elevation of TRAP5a correlated with chitotriosidase activity in GD. Lyso-Gb-1 and plasma seemed to influence the expression of ACP5 in macrophages. As an early indicator of BMD alteration, measurement of circulating TRAP5b is a valuable tool for assessing osteopenia-osteoporosis in GD, while TRAP5a serves as a biomarker of macrophage activation in GD. Understanding the distinct expression pattern of TRAP5 isoforms offers valuable insight into both bone disease and the broader implications for immune system activation in GD.

Pax6 isoforms shape eye development: Insights from developmental stages and organoid models

Pax6 is a critical transcription factor involved in the development of the central nervous system. However, in humans, mutations in Pax6 predominantly result in iris deficiency rather than neurological phenotypes. This may be attributed to the distinct functions of Pax6 isoforms, Pax6a and Pax6b. In this study, we investigated the spatial and temporal expression patterns of Pax6 isoforms during different stages of mouse eye development. We observed a strong correlation between Pax6a expression and the neuroretina gene Sox2, while Pax6b showed a high correlation with iris-component genes, including the mesenchymal gene Foxc1. During early patterning from E10.5, Pax6b was expressed in the hinge of the optic cup and neighboring mesenchymal cells, whereas Pax6a was absent in these regions. At E14.5, both Pax6a and Pax6b were expressed in the future iris and ciliary body, coinciding with the integration of mesenchymal cells and Mitf-positive cells in the outer region. From E18.5, Pax6 isoforms exhibited distinct expression patterns as lineage genes became more restricted. To further validate these findings, we utilized ESC-derived eye organoids, which recapitulated the temporal and spatial expression patterns of lineage genes and Pax6 isoforms. Additionally, we found that the spatial expression patterns of Foxc1 and Mitf were impaired in Pax6b-mutant ESC-derived eye organoids. This in vitro eye organoids model suggested the involvement of Pax6b-positive local mesodermal cells in iris development. These results provide valuable insights into the regulatory roles of Pax6 isoforms during iris and neuroretina development and highlight the potential of ESC-derived eye organoids as a tool for studying normal and pathological eye development.

Expression landscape of cancer-FOXP3 and its prognostic value in pancreatic adenocarcinoma

FOXP3, a key identifier of Treg, has also been identified in tumor cells, which is referred to as cancer-FOXP3 (c-FOXP3). Human c-FOXP3 undergoes multiple alternative splicing events, generating several isoforms, like c-FOXP3FL and c-FOXP3Δ3. Previous research on c-FOXP3 often ignore its cellular source (immune or tumor cells) and isoform expression patterns, which may obscure our understanding of its clinical significance. Our immunohistochemistry investigations which conducted across 18 tumors using validated c-FOXP3 antibodies revealed distinct expression landscapes for c-FOXP3 and its variants, with the majority of tumors exhibited a predominantly expression of c-FOXP3Δ3. In pancreatic ductal adenocarcinoma (PDAC), we further discovered a potential link between nuclear c-FOXP3Δ3 in tumor cells and poor prognosis. Overexpression of c-FOXP3Δ3 in tumor cells was associated with metastasis. This work elucidates the expression pattern of c-FOXP3 in pan-cancer and indicates its potential as a prognostic biomarker in clinical settings, offering new perspectives for its clinical application.

Combinatorial expression of γ-protocadherins regulates synaptic connectivity in the mouse neocortex.

In the process of synaptic formation, neurons must not only adhere to specific principles when selecting synaptic partners but also possess mechanisms to avoid undesirable connections. Yet, the strategies employed to prevent unwarranted associations have remained largely unknown. In our study, we have identified the pivotal role of combinatorial clustered protocadherin gamma (γ-PCDH) expression in orchestrating synaptic connectivity in the mouse neocortex. Through 5' end single-cell sequencing, we unveiled the intricate combinatorial expression patterns of γ-PCDH variable isoforms within neocortical neurons. Furthermore, our whole-cell patch-clamp recordings demonstrated that as the similarity in this combinatorial pattern among neurons increased, their synaptic connectivity decreased. Our findings elucidate a sophisticated molecular mechanism governing the construction of neural networks in the mouse neocortex.

Combinatorial expression of γ-protocadherins regulates synaptic connectivity in the mouse neocortex

In the process of synaptic formation, neurons must not only adhere to specific principles when selecting synaptic partners but also possess mechanisms to avoid undesirable connections. Yet, the strategies employed to prevent unwarranted associations have remained largely unknown. In our study, we have identified the pivotal role of combinatorial clustered protocadherin gamma (γ-PCDH) expression in orchestrating synaptic connectivity in the mouse neocortex. Through 5' end single-cell sequencing, we unveiled the intricate combinatorial expression patterns of γ-PCDH variable isoforms within neocortical neurons. Furthermore, our whole-cell patch-clamp recordings demonstrated that as the similarity in this combinatorial pattern among neurons increased, their synaptic connectivity decreased. Our findings elucidate a sophisticated molecular mechanism governing the construction of neural networks in the mouse neocortex.

Small-Molecule U2 Auxiliary Factor Homology Motif (UHM) Domain Inhibitors Cause Splicing Pattern Changes in U2AF1 Mutant Leukemia Cells and Induce Sub-G1 Cell Cycle Arrest

Mutations in factors involved in the spliceosomal pathway have emerged to be an important contributor to the development of myeloid neoplasms including MDS and secondary AML (sAML). A set of frequently mutated genes encoding RNA splicing factors including SF3B1, U2AF1, SRSF2, and ZRSR2 or haploinsufficiency of LUC7L2 have beenreported, but pharmacological intervention targeting these genes remains limited and under-developed. Combination of mutations of these splicing factors are rarely found in patients indicating that multiple defects in a spliceosome pathway may be deleterious. Heterozygous mutation in U2AF1 found in MDS leads to the acquisition of the neomorphic mutant U2AF1 (U2AF1 wt) that relies on wild-type U2AF1 (U2AF1 wt) to survive and proliferate. To support our hypothesis, we undertook a discovery campaign to identify small-molecule compounds targeting U2AF1. By recognizing the challenge of discovering compounds selectively targeting the mutant zinc-finger domains in U2AF1 found clinically, we devised a tractable intervention strategy to inhibit the UHM domain of U2AF1 (U2AF1-UHM). U2AF1-UHM binds with U2AF2 to form a protein complex (U2AF1/U2AF2) that recognizes the 3' splice site in mRNA processing. U2AF1-UHM inhibitors may decrease the binding between U2AF1 and U2AF2 and abrogate U2AF1/U2AF2 functions to change expression patterns of protein isoforms. To identify hit compounds for development, we performed screening of ~3000 fragment molecules against U2AF1-UHM using the thermal shift assay. Two of the top hits were derivatives of the same chemical scaffold. We verified that both hits inhibited the binding of U2AF1-UHM and U2AF2-ULM (U2AF ligand motifs) in our Homogenous Time-Resolved Fluorescence (HTRF) assay. We next evaluated the top hit, SF-1-8, in our K562-U2AF1 S34F mutant cell line and obtained an IC50 at the micromolar range. In contrast, SF-1-8 had no activity in K562-U2AF1 wt cell line and bone marrow cells obtained from healthy individuals. Based on SF-1-8, we performed chemical modifications to develop structure-activity relationship (SAR) of SF-1-8 and obtained SF-1-50 that was two-fold more potent than SF-1-8. To assess the selectivity of SF-1-8 and analogs to other UHM containing proteins, we further determined the IC50 values of our SF-1-8 analogs to RBM39-UHM, SPF45-UHM, and PUF60-UHM to construct the selectivity profiles of our compounds. The selectivity profiles of SF-1-8 and SF-1-50 showed they were selectively more effective to U2AF1-UHM than RBM39-UHM, SPF45-UHM, and PUF60-UHM. We then studied pathways impacted by these inhibitors in K562-U2AF1 S34F cells by performing RNA-seq in K562-U2AF1 S34F cells treated with SF-1-8 at 5 uM and control. We found that 36 and 63 genes were significantly (p <0.002) up- and down-regulated respectively by SF-1-8 in K562-U2AF1 S34F cells. Downregulated genes included CBL, CBLL1, and a subset of collagen genes ( COL1, COL3, COL5) and upregulated genes included ATF3 and BCL2. When analyzing the transcript changes affected by SF-1-8, we identified substantial protein isoform changes in genes involved with proteosome, endocytosis, apoptosis, extracellular matrix/cell adhesion, histones, and stress response. Primary effects from changes of protein isoform patterns ameliorated the restoration of the trans-Golgi network ( ERGIC3, COPB2), secretory pathway ( RUSC1, AP4E1) and impairment of clathrin mediated endocytosis and endosome-lysosome transport ( CBL, PICALM, VAMP7, ASAP1). NKM-1 cell line was previously characterized to carry U2AF1 mutation. We also found SF-1-50 caused accumulation of sub-G1 cells in NKM-1 cells in a dose-dependent manner. In summary, SF-1-8 caused disruption of extra- and intracellular protein transport in K562-U2AF1 S34F cells and represented a new class of small-molecule inhibitors to target U2AF1-UHM. Further optimization of this class of compounds will allow us to develop effective chemical probes for study in the U2af1 murine models and assess the potential of U2AF1 as a therapeutic target in MDS and sAML.

P199 - Utilization of Tropomyosin isoform expression pattern in development of a urine-based assay for detection of urothelial bladder cancer

P199 - Utilization of Tropomyosin isoform expression pattern in development of a urine-based assay for detection of urothelial bladder cancer

Expression pattern of AGPATs isoforms indicate different functions during the triacylglyceride synthesis in Chinese mitten crab, Eriocheir sinensis

The 1-acylglycerol-3-phosphate acyltransferase (AGPAT) acts as a crucial enzyme in the process of triacylglycerol (TAG) synthesis, enabling the acylation of lysophosphatidic acid (LPA) into phosphatidic acid (PA). In order to decode the distinctive roles of AGPAT isoforms in the TAG production pathway, three AGPAT isoforms were detected for the first time in the Chinese mitten crab Eriocheir sinensis (Es-agpat2, Es-agpat3, and Es-agpat4). The mRNA levels of Es-agpat2 and Es-agpat4 demonstrated a conspicuous presence in the hepatopancreas, with subsequent high levels in the heart, muscle, and thoracic ganglion. On the other hand, the thoracic ganglion exhibited abundant levels of Es-agpat3, while other tissues recorded relatively low expression levels. Observing the molting cycle of E. sinensis, the hepatopancreas showed minimum expression levels of Es-agpat2 and Es-agpat4 at stage A/B. A peak at stage C was noted, which was then followed by a gradual drop until stage E. For the ovarian development cycle, stage II witnessed the maximum expression level of Es-agpat2 and Es-agpat4, succeeded by a sharp fall in stage III. After this, there was an increasing trend from stage III up to stage V. Expression of Es-agpat3 in the hepatopancreas was consistently lower than Es-agpat2 and Es-agpat4 during either the molting or ovarian development. However, in terms of ovarian expression, Es-agpat3 outperformed Es-agpat2 and Es-agpat4. It exhibited a steep increase in expression, peaking at stage II and subsequently diminishing. In situ hybridization (ISH) revealed that in stages II and IV hepatopancreas, Es-agpat4-mRNA was primarily located in fibrillar cells (F cell) and resorptive cells (R cell), with no signal from Es-agpat3. During stage II of ovarian development, both Es-agpat3-mRNA and Es-agpat4-mRNA were located in the cytoplasm of previtellogenic oocyte (PRO) and endogenous vitellogenic oocyte (EN), with no expression at stage IV. Additionally, the silencing of Es-agpat2 and Es-agpat4 caused a downward trend in the expression levels of all subsequent genes in the E. sinensis TAG synthesis pathway. To sum up, these findings suggest that the three Es-agpats may have unique functions in TAG synthesis during either the molting process or ovarian maturation of E. sinensis.

Expression of OCT4 isoforms is reduced in primary colorectal cancer.

Colorectal cancer (CRC) is one of the most common types of cancer worldwide. The carcinogenesis of CRC is indeed complex, and there are many different mechanisms and pathways that contribute to the development of malignancy and the progression from primary to metastatic tumors. The OCT4A, encoded by the POU5F1 gene, is a transcription factor responsible for the phenotype of stem cells, maintaining pluripotency and regulation of differentiation. The POU5F1 gene is made up of five exons that can create numerous isoforms through alternative promoter or alternative splicing. In addition to OCT4A, other isoforms called OCT4B are also translated into protein; however, their role in cells has been unclear. The aim of our work was to investigate the expression patterns of OCT4 isoforms in primary and metastatic CRC, providing us with useful information about their role in the development and progression of CRC. Surgical specimens from a total of 78 patients were collected and isolated from primary tumors (n = 47) and metastases (n = 31). The relative gene expression of OCT4 isoforms was investigated using the RT-qPCR method together with the TaqMan probes for particular OCT4 isoforms. Our results suggest significantly downregulated expression of the OCT4A and OCT4Bs isoforms in both primary (p = 0.0002 and p < 0.0001, respectively) and metastatic tumors (p = 0.0006 and p = 0.00051, respectively) when compared with the control samples. We also observed a correlation between reduced expression of all OCT4 isoforms and both primary and left-sided tumors (p = 0.001 and p = 0.030, respectively). On the other hand, the expression of all OCT4 isoforms was significantly upregulated in metastases compared with primary tumors (p < 0.0001). Unlike previous reports, we found out that the expression of OCT4A, OCT4Bs, and all OCT4 isoforms was significantly reduced in primary tumors and metastases compared with control samples. On the other hand, we supposed that the expression rate of all OCT4 isoforms may be related to the cancer type and side, as well as to liver metastases. However, further studies are required to investigate the detailed expression patterns and significance of individual OCT4 isoforms in carcinogenesis.

Top-down proteomics of myosin light chain isoforms define chamber-specific expression in the human heart

Myosin functions as the “molecular motor” of the sarcomere and generates the contractile force necessary for cardiac muscle contraction. Myosin light chains 1 and 2 (MLC-1 and -2) play important functional roles in regulating the structure of the hexameric myosin molecule. Each of these light chains has an ‘atrial’ and ‘ventricular’ isoform, so called because they are believed to exhibit chamber-restricted expression in the heart. However, recently the chamber-specific expression of MLC isoforms in the human heart has been questioned. Herein, we analyzed the expression of MLC-1 and -2 atrial and ventricular isoforms in each of the four cardiac chambers in adult non-failing donor hearts using top-down mass spectrometry (MS)-based proteomics. Strikingly, we detected an isoform thought to be ventricular, MLC-2v (gene: MYL2), in the atria and confirmed the protein sequence using tandem MS (MS/MS). For the first time, a putative deamidation post-translation modification (PTM) located on MLC-2v in atrial tissue was localized to amino acid N13. MLC-1v (MYL3) and MLC-2a (MYL7) were the only MLC isoforms exhibiting chamber-restricted expression patterns across all donor hearts. Importantly, our results unambiguously show that MLC-1v, not MLC-2v, is ventricle-specific in adult human hearts. Moreover, we found elevated MLC-2 phosphorylation in male hearts compared to female hearts across each cardiac chamber. Overall, top-down proteomics allowed an unbiased analysis of MLC isoform expression throughout the human heart, uncovering previously unexpected isoform expression patterns and PTMs.

ARPC5 isoforms and their regulation by calcium-calmodulin-N-WASP drive distinct Arp2/3-dependent actin remodeling events in CD4 T cells.

CD4 T cell activation induces nuclear and cytoplasmic actin polymerization via the Arp2/3 complex to activate cytokine expression and strengthen T cell receptor (TCR) signaling. Actin polymerization dynamics and filament morphology differ between nucleus and cytoplasm. However, it is unclear how the Arp2/3 complex mediates distinct nuclear and cytoplasmic actin polymerization in response to a common stimulus. In humans, the ARP3, ARPC1, and ARPC5 subunits of the Arp2/3 complex exist as two different isoforms, resulting in complexes with different properties. Here, we show that the Arp2/3 subunit isoforms ARPC5 and ARPC5L play a central role in coordinating distinct actin polymerization events in CD4 T cells. While ARPC5L is heterogeneously expressed in individual CD4 T cells, it specifically drives nuclear actin polymerization upon T cell activation. In contrast, ARPC5 is evenly expressed in CD4 T cell populations and is required for cytoplasmic actin dynamics. Interestingly, nuclear actin polymerization triggered by a different stimulus, DNA replication stress, specifically requires ARPC5 but not ARPC5L. TCR signaling but not DNA replication stress induces nuclear actin polymerization via nuclear calcium-calmodulin signaling and N-WASP. Diversity in the molecular properties and individual expression patterns of ARPC5 subunit isoforms thus tailors Arp2/3-mediated actin polymerization to different physiological stimuli.

In Vitro Investigation of the Interaction of Avian Metapneumovirus and Newcastle Disease Virus with Turkey Respiratory and Reproductive Tissue.

In poultry, several respiratory viral infections lead to a drop in egg production associated with high economic losses. While the virus-host interactions at the respiratory epithelium are well studied, less is known about these interactions in the oviduct. To investigate possible differences between virus infections at these epithelial structures, we compared the interactions of two important poultry viruses on turkey organ cultures. Two members of the order Mononegavirales, the Avian Metapneumovirus (AMPV) and the Newcastle disease virus (NDV), were selected to conduct the in vitro experiments since these viruses can infect both the trachea and oviduct. In addition, we used different strains of these viruses, a subtype A and a subtype B strain for AMPV and the NDV strains Komarow and Herts'33, to detect possible differences not only between the tissues but also between different viral strains. Turkey tracheal and oviduct organ cultures (TOC and OOC) were prepared to investigate viral replication, antigen localisation, lesion development, and the expression pattern of interferon-λ and importin-α isoforms. All viruses replicated more efficiently in the oviduct than in the tracheal epithelium (p < 0.05). In addition, we observed higher expression levels of both, IFN-λ and importin-α in OOCs compared to TOCs. Our results indicated strain-dependent differences, with the AMPV-B- and Herts'33 strains being more virulent in organ cultures than the AMPV-A- and Komarow strains, based on the higher viral genome loads, more severe histological lesions, and higher upregulation of IFN-λ. Overall, our findings reveal tissue- and virus strain-dependent differences, which may have consequences for disease development in the host tissue and, subsequently, possible treatment strategies.

Switching ubiquitous and muscle-specific isoforms of mitochondrial respiratory complex IV in skeletal muscle fine-tunes complex IV activity.

Respiratory complex IV (CIV, cytochrome c oxidase) is the terminal enzyme of the mitochondrial electron transport chain. Some CIV subunits have two or more isoforms, which are ubiquitously expressed or are expressed in specific tissues like the lung, muscle, and testis. Among the tissue-specific CIV isoforms, the muscle-specific isoforms are expressed in adult cardiac and skeletal muscles. To date, the physiological and biochemical association between the muscle-specific CIV isoforms and aerobic respiration in muscles remains unclear. In this study, we profiled the CIV organization and expression pattern of muscle-specific CIV isoforms in different mouse muscle tissues. We found extensive CIV-containing supramolecular organization in murine musculature at advanced developmental stages, while a switch in the expression from ubiquitous to muscle-specific isoforms of CIV was also detected. Such a switch was confirmed during the in vitro differentiation of mouse C2C12 myoblasts. Unexpectedly, a CIV expression decrease was observed during C2C12 differentiation, which was probably due to a small increase in the expression of muscle-specific isoforms coupled with a dramatic decrease in the ubiquitous isoforms. We also found that the enzymatic activity of CIV containing the muscle-specific isoform COX6A2 was higher than that with COX6A1 in engineered HEK293T cells. Overall, our results indicate that switching the expression from ubiquitous to muscle-specific CIV isoforms is indispensable for optimized oxidative phosphorylation in mature skeletal muscles. We also note that the in vitro C2C12 differentiation model is not suitable for the study of muscular aerobic respiration due to insufficient expression of muscle-specific CIV isoforms.

Immunohistochemical Expression Analysis of Caldesmon Isoforms in Colorectal Carcinoma Reveals Interesting Correlations with Tumor Characteristics.

Colorectal cancer is a notorious disease, with almost half of the patients succumbing to the disease. The prevalence and incidence rates of colorectal cancer are increasing in many parts of the world, highlighting the need to discover new biomarkers for diagnosis and therapy. Caldesmon (CaD), an actin-binding protein that plays a significant role in controlling cell motility, has emerged as a promising biomarker. The CALD1 gene encodes CaD as multiple transcripts that mainly encode two protein isoforms: High-molecular-weight (h-CaD), expressed in smooth muscle, and low-molecular-weight (l-CaD), expressed in nonsmooth muscle cells. Most studies have suggested an oncogenic role of CaD in colorectal cancer, but the exact subcellular localization of the two CaD isoforms in tumor cells and stroma have not been clarified yet. Here, we analyzed tissue samples from 262 colorectal cancer patients by immunohistochemistry analysis using specific antibodies for l-CaD and h-CaD. The results showed elevated cytoplasmic expression levels of l-Cad in 187/262 (71.4%) cases. l-Cad was expressed at low levels in the normal colon mucosa and was also consistently expressed in the cancer-associated stroma of all cases, suggesting that it could play a role in modulating the tumor microenvironment. l-CaD expression in cancer cells was associated with preinvasive stages of cancer. Survival analysis indicated that patients with high l-CaD expression in tumor cells could respond poorly to selective chemotherapeutic 5FU, but not combination chemotherapy. h-CaD was expressed in colonic and vascular smooth muscle cells as expected and to a lesser extent in the tumor-associated stroma, but it was not expressed in the cancer cells or normal colon mucosal epithelial cells. Collectively, these data clarify how the expression patterns of CaD isoforms in colorectal cancer can have applications in the management of colorectal cancer patients.

New Insights into the Diverse Functions of the NR2F Nuclear Orphan Receptor Family.

Following gene expansion during evolution, today's phylogenetic tree of the NR2F family of nuclear orphan receptors in mammals is represented by three different isoforms: NR2F1, NR2F2, and NR2F6. Structural analysis of the NR2F family members has revealed that NR2F1 and NR2F2 are closely related and grouped together apart from NR2F6, which is more divergent in its biochemical characteristics. In this review, we highlight current knowledge on the cellular functions of NR2F family members. NR2F family members have been reported to be causally involved in carcinogenesis. Mechanistically, NR2F proteins are localized in the nucleus, where they bind to target DNA enhancer sequences and have been implicated in the regulation of de novo gene transcription, though this is not sufficiently understood. Based on apparently divergent and non-uniform expression patterns of the NR2F isoforms in different tissues and cell types, non-redundant functions of the individual family members appear to exist. Notably, NR2F2 appears to be more closely related functionally to NR2F6 than NR2F1. Along these lines, NR2F2 and NR2F6 have been reported to be involved in cellular neoplasia. Furthermore, enhanced expression of NR2F isoforms has been established as prognostic biomarkers in various cancer entities. Therefore, it is tempting to speculate that NR2F isoforms represent innovative targets for therapeutic intervention in defined types of cancer. Thus, NR2F family nuclear receptors can be viewed as gatekeepers balancing cell type-specific regulation of proliferation and the suppression of terminal differentiation in health and disease.

Malignant DFFB isoform switching promotes leukemia survival in relapse pediatric T-cell acute lymphoblastic leukemia.

With modern treatment most children with acute lymphoblastic leukemia (ALL) survive without relapse. However, for children who relapse the prognosis is still poor, especially in children with T-cell phenotype (T-ALL) and remains the major cause of death. The exact mechanism of relapse is currently not known. While contribution of RNA processing alteration has been linked to other hematological malignancies, its contribution in pediatric T-ALL may provide new insights. Almost all human genes express more than one alternative splice isoform. Thus, gene modulation producing a diverse repertoire of the transcriptome and proteome have become a significant molecular marker of cancer and a potential therapeutic vulnerability. To study this, we performed RNA-sequencing analysis on patient-derived samples followed by splice isoform-specific PCR. We uncovered a distinct RNA splice isoform expression pattern characteristic for relapse samples compared to the leukemia samples from the time of diagnosis. We also identified deregulated splicing and apoptosis pathways specific for relapse T-ALL. Moreover, patients with T-ALL displayed pro-survival splice isoform switching favoring pro-survival isoforms compared to normal healthy stem cells. Cumulatively, pro-survival isoform switching and DFFB isoform regulation of SOX2 and MYCN may play a role in T-ALL proliferation and survival, thus serving as a potential therapeutic option.

Selective PDE4 subtype inhibition provides new opportunities to intervene in neuroinflammatory versus myelin damaging hallmarks of multiple sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by focal inflammatory lesions and prominent demyelination. Even though the currently available therapies are effective in treating the initial stages of disease, they are unable to halt or reverse disease progression into the chronic progressive stage. Thus far, no repair-inducing treatments are available for progressive MS patients. Hence, there is an urgent need for the development of new therapeutic strategies either targeting the destructive immunological demyelination or boosting endogenous repair mechanisms. Using in vitro, ex vivo, and in vivo models, we demonstrate that selective inhibition of phosphodiesterase 4 (PDE4), a family of enzymes that hydrolyzes and inactivates cyclic adenosine monophosphate (cAMP), reduces inflammation and promotes myelin repair. More specifically, we segregated the myelination-promoting and anti-inflammatory effects into a PDE4D- and PDE4B-dependent process respectively. We show that inhibition of PDE4D boosts oligodendrocyte progenitor cells (OPC) differentiation and enhances (re)myelination of both murine OPCs and human iPSC-derived OPCs. In addition, PDE4D inhibition promotes in vivo remyelination in the cuprizone model, which is accompanied by improved spatial memory and reduced visual evoked potential latency times. We further identified that PDE4B-specific inhibition exerts anti-inflammatory effects since it lowers in vitro monocytic nitric oxide (NO) production and improves in vivo neurological scores during the early phase of experimental autoimmune encephalomyelitis (EAE). In contrast to the pan PDE4 inhibitor roflumilast, the therapeutic dose of both the PDE4B-specific inhibitor A33 and the PDE4D-specific inhibitor Gebr32a did not trigger emesis-like side effects in rodents. Finally, we report distinct PDE4D isoform expression patterns in human area postrema neurons and human oligodendroglia lineage cells. Using the CRISPR-Cas9 system, we confirmed that pde4d1/2 and pde4d6 are the key targets to induce OPC differentiation. Collectively, these data demonstrate that gene specific PDE4 inhibitors have potential as novel therapeutic agents for targeting the distinct disease processes of MS.