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Villins are versatile, multifunctional actin regulatory proteins. They promote actin stabilization and remodeling mainly via their actin bundling and Ca 2+ -dependent severing activities, respectively. Arabidopsis subclass II and III villins normally coexist in cells, but the biological significance of their coexistence remains unknown. Here we demonstrate that subclass II villin binds to Ca 2+ with high affinity and exhibits strong severing but weak bundling activity compared to subclass III villin. Subclass II villin plays a dominant role in promoting actin remodeling, which requires its Ca 2+ -dependent severing activity. Subclass II villin is also strictly required for physiological processes including oriented organ growth and stress tolerance. By comparison, subclass III villin binds to Ca 2+ with low affinity and exhibits weak severing but strong bundling activity, and acts as the major player in controlling actin stabilization and organization. Thus, we demonstrate that multifunctional villin isovariants have diverged biochemically to ensure exquisite control of the actin cytoskeleton to meet different cellular needs in plants. This study provides new insights into the role of villins in fine-tuning actin dynamics and plant development., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, MDA5 and LGP2, recognize viral RNA to mount an antiviral interferon (IFN) response RLRs share three different protein domains: C-terminal domain, DExD/H box RNA helicase domain, and an N-terminal domain with two tandem repeats (CARDs). LGP2 lacks tandem CARD and is not able to induce an IFN response. However, LGP2 positively enhances MDA5 and negatively regulates RIG-I signaling. In this study, we determined the LGP2 alternative transcripts in humans to further comprehend the mechanism of its regulation, their evolutionary origin, and the isoforms functionallity. The results showed new eight alternative transcripts in the samples tested. The presence of these transcripts demonstrated that the main mechanisms for the regulation of LGP2 expression are both by insertion of introns and by the loss of exons. The phylogenetic analysis of the comparison between sequences from exon 1 to exon 3 of humans and those previously described in non-human primates showed three well-differentiated groups (lineages) originating from gorillas, suggesting that the transspecies evolution has been maintained for 10 million years. The corresponding protein models (isoforms) were also established, obtaining four isoforms: one complete and three others lacking the C-terminal domain or this domain and the partial or total He2 Helicase domain, which would compromise the functionality of LGP2. In conclusion, this is the first study that elucidate the large genomic organization and complex transcriptional regulation of human LGP2, its pattern of sequence generation, and a mode of evolutionary inheritance across species., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In Brief: Protein phosphatase 1 catalytic subunit gamma isoform 2 (PP1γ2) is a unique phosphatase expressed only in mammalian testes and sperm cells. The PP1γ2 isoform is indispensable for sperm motility and fertility and cannot be replaced by the PP1γ1 isoform for these functions., Abstract: The serine-threonine phosphatase has four paralogs - PP1α, PP1β, PP1γ1 and PP1γ2 - encoded by three genes, Ppp1ca, Ppp1cb and Ppp1cc. Protein phosphatase PP1γ2, one of two isoforms of the gene Ppp1cc, is expressed in spermatogenic cells in the testes and sperm, while PP1γ1 is found in somatic cells. The two PP1γ isoforms, formed by alternate splicing that occurs only in mammals, are identical except at their C-termini. Global or testis-specific knockout of Ppp1cc in mice results in male infertility due to disrupted spermiation and mid-to-late spermiogenesis. Transgenic expression of PP1γ2, driven by a testis-specific promoter in differentiating spermatogenic cells, rescues spermatogenesis and fertility in the Ppp1cc-null mice. Why PP1γ2 is essential and present only in mammalian sperm is a mystery. We have generated a knock-in mouse where the Ppp1cc gene is edited to express only PP1γ1. Spermatogenesis was normal in knock-in mice. Testis-expressed PP1γ1 in the knock-in mice and PP1γ2 in the wild-type mice were incorporated in equal amounts into sperm. Sperm bearing PP1γ1 have reduced flagellar beat amplitude and motility, and male mice were severely sub-fertile. Although in the wild-type mice, PP1γ2 is present in both the head and tail, in the knock-in mice, PP1γ1 is absent in sperm heads, leading to an altered intra-sperm protein phosphatase landscape. Phosphoproteomic analysis of sperm proteins suggested a plausible molecular basis for compromised PP1γ1 functions: it identified GSK3α, a known substrate of PP1, to be dysregulated in knock-in sperm. This study provides a preliminary explanation for the isoform-specific requirement of PP1γ2 for male fertility.

Eukaryotic cells typically express multiple tubulin isoforms that form the microtubule cytoskeleton. A new study of the evolution and functional diversification of pools of tubulin isoforms suggests that these proteins are part of a co-evolving network that includes the extensive microtubule interactome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Death-Associated Protein Kinase 1 (DAPK1) family members are calcium/calmodulin-regulated serine/threonine kinases implicated in cell death, normal development, and human diseases. However, the regulation of DAPK1 expression in cancer remains unclear., Methods: We examined the expression and functional impact of a DAPK1 splice variant, DAPK1-215, in multiple cancer cell lines. DAPK1 and DAPK1-215 expression levels were quantified by qRT-PCR and Western blotting. Cell migration, invasion, and proliferation assays were conducted in vitro, and a zebrafish model was employed to evaluate metastatic potential. RNA pull-down and CLIP-seq analyses were performed to identify potential RNA-binding proteins. Finally, clinical liver cancer specimens were analyzed to assess the prognostic relevance of DAPK1-215 and DAPK1 mRNA levels., Results: DAPK1-215 downregulated DAPK1 expression in liver, kidney, and gastric cancer cells by reducing DAPK1 mRNA stability. DAPK1-215 promoted migratory and invasive capabilities in liver and kidney cancer cells, but inhibited these processes in gastric cancer cells, without affecting cell proliferation. Mechanistically, DEAD-Box Helicase 3 X-Linked (DDX3X) stabilized both DAPK1-215 and DAPK1 mRNAs, suggesting that DAPK1-215 may act by competing for DDX3X binding to modulate DAPK1 mRNA stability. Importantly, high levels of DAPK1-215 correlated inversely with DAPK1 mRNA in liver cancer specimens and predicted poor prognosis, whereas high DAPK1 expression predicted improved patient outcomes., Conclusions: Our findings unveil DAPK1-215 as a molecular brake on DAPK1 expression, influencing cancer cell migration and invasion in a context-dependent manner. These results highlight the potential of DAPK1-215 as an important regulator of malignant progression and as a prognostic marker in liver cancer., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Patient consent for publication: Not applicable. Competing interests: The authors declare no conflict of interest for this article., (© 2025. The Author(s).)

The quick-to-court (qtc) gene is expressed in both males and females but affects only the mating behavior of males, probably due to the different composition of isoforms between the sexes. We tested this hypothesis and examined the sex-specific expression of qtc transcripts in the tissues of male and female Oregon-R flies. It was found that some qtc transcripts, such as qtc-RM and qtc-RN, are testis-specific, while others like qtc-RH are found in ovaries but absent in testes. No sex-specific transcripts were identified in the brain, suggesting further investigation into specific brain structures may be needed. There is likely a complex regulation of qtc gene expression, which is potentially influenced by various factors in different tissues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The innate immune response serves as the primary defense against viral infections, with the recognition of viral nucleic acids by pattern recognition receptors (PRRs) initiating antiviral responses. Mitochondrial antiviral-signaling protein (MAVS) acts as a pivotal adaptor protein in the RIG-I pathway. Alternative splicing further diversifies MAVS isoforms. In this study, we identified and characterized a novel rat MAVS variant (MAVS500) with a twenty-one-nucleotide deletion, resulting in a protein seven amino acids shorter than the wild-type (WT) rat MAVS. The MAVS500 was cloned from the rat bladder cancer cell line, NBT-II, using specific primers, and subsequently sequenced. MAVS500 was overexpressed in HEK293T and NBT-II cells and then analyzed using Western Blotting and fluorescence microscopy. MAVS500 overexpression increased downstream signaling proteins, NFκβ and pNFκβ, compared to WT rat MAVS in both human and rat cell lines. Structural analysis revealed a high similarity between MAVS500 and WT rat MAVS. The seven-amino-acid deletion in MAVS500 induces significant conformational rearrangements, reducing helical turns and altering structural dynamics, which may impact its interactions with downstream signaling molecules in the innate immune pathway. The identification of MAVS500 enhances our understanding of MAVS regulation and its role in the innate immune response, providing valuable insights into alternative splicing as a mechanism for diversifying protein function.

Background: Identification of global transcriptional events is crucial for genome annotation, as accurate annotation enhances the efficiency and comparability of genomic information across species. However, the annotation of transcripts in the cucumber genome remains to be improved, and many transcriptional events have not been well studied., Results: We collected 1,904 high-quality public cucumber transcriptome samples from the National Center for Biotechnology Information (NCBI) to identify and annotate transcript isoforms in the cucumber genome. Over 44.26 billion Q30 clean reads were mapped to the cucumber genome with an average mapping rate of 92.75%. Transcriptome assembly identified 151,453 transcripts spanning 20,442 loci. Among these, 12.7% of transcripts exactly matched annotated genes in the cucumber reference genome. More than 80% of the transcripts were classified as novel isoforms. Approximately 96.6% of these isoforms originated from known gene loci, while around 3.3% were derived from novel gene loci. Coding potential prediction identified 4,543 long non-coding RNAs (lncRNAs) across 3,376 loci. Building on these results, we identified tissue-specific transcripts in 10 tissues. Among that, 1,655 annotated genes and 4,214 predicted transcripts were considered as tissue-specific. The root exhibited the highest number of tissue-specific transcripts, followed by shoot apex. Subsequent selective pressure analysis revealed that tissue-specific regions experienced stronger directional selection compared to non-specific regions., Conclusions: By analyzing thousands of published transcriptome data, we identified abundant transcriptional events and tissue-specific transcripts in cucumbers. This study presented here adds the great value to the public data and offers insights for further exploration of a more comprehensive tissue regulatory network in cucumber., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Mutations in RAS and PI3Kα are major drivers of human cancer. Their interaction plays a crucial role in activating PI3Kα and amplifying the PI3K-AKT-mTOR pathway. Disrupting RAS-PI3Kα interaction enhances survival in lung and skin cancer models and reduces tumor growth and angiogenesis, although the structural details of this interaction remain unclear. Here, we present structures of KRAS, RRAS2, and MRAS bound to the catalytic subunit (p110α) of PI3Kα, elucidating the interaction interfaces and local conformational changes upon complex formation. Structural and mutational analyses highlighted key residues in RAS and PI3Kα impacting binding affinity and revealed isoform-specific differences at the interaction interface in RAS and PI3K isoforms, providing a rationale for their differential affinities. Notably, in the RAS-p110α complex structures, RAS interaction with p110α is limited to the RAS-binding domain and does not involve the kinase domain. This study underscores the pivotal role of the RAS-PI3Kα interaction in PI3Kα activation and provides a blueprint for designing PI3Kα isoform-specific inhibitors to disrupt this interaction., Competing Interests: Competing interests: All authors, except F.M., have no competing interests. F.M. is a consultant for Ideaya Biosciences, Kura Oncology, Leidos Biomedical Research, Pfizer, Daiichi Sankyo, Amgen, PMV Pharma, OPNA-IO, and Quanta Therapeutics, has received research grants from Boehringer-Ingelheim, and is a consultant for and cofounder of BridgeBio Pharma., (© 2025. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Biallelic pathogenic variants in the gene encoding nebulin (NEB) are a known cause of congenital myopathy. We present two brothers with congenital myopathy and compound heterozygous variants (NC_000002.12:g.151692086G>T; NM_001271208.2: c.2079C>A; p.(Cys693Ter) and NC_000002.12:g.151533439T>C; NM_001271208.2:c.21522+3A>G) in NEB. Transcriptomic sequencing on affected individual muscles revealed that the extended splice variant c.21522+3A>G causes exon 144 skipping. Nebulin isoforms containing exon 144 are known to be mutually exclusive with isoforms containing exon 143, and these isoforms are differentially expressed during development and in adult skeletal muscles. Affected individuals' MRI patterns of muscle involvement were compared with the known pattern of relative abundance of these two isoforms in muscle. We propose that the pattern of muscle involvement in these affected individuals better fits the distribution of exon 144-containing isoforms in muscle than with previously published MRI findings in NEB-related disease due to other variants. Our report introduces disease pathogenesis and manifestation as a result of alteration of isoform distributions in muscle., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Alternative splicing (AS) is a mechanism that generates translational diversity within a genome. Equally important is the dynamic adaptability of the splicing machinery, which can give preference to one isoform over others encoded by a single gene. These isoform preferences change in response to the cell's state and function. Particularly significant is the impact of physiological alternative splicing in T lymphocytes, where specific isoforms can enhance or reduce the cells' reactivity to stimuli. This process makes splicing isoforms defining features of cell states, exemplified by CD45 splice isoforms, which characterize the transition from naïve to memory states. Two developments have accelerated the use of AS dynamics for therapeutic interventions: advancements in long-read RNA sequencing and progress in nucleic acid chemical modifications. Improved oligonucleotide stability has enabled their use in directing splicing to specific sites or modifying sequences to enhance or silence particular splicing events. This review highlights immune regulatory splicing patterns with potential significance for enhancing anticancer immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Tzaban, Stern, Zisman, Eisenberg, Klein, Frankenburg and Lotem.)

The transcription factor p63 is expressed in many different isoforms as a result of differential promoter use and splicing. Some of these isoforms have very specific physiological functions in the development and maintenance of epithelial tissues and surveillance of genetic integrity in oocytes. The ASPP family of proteins is involved in modulating the transcriptional activity of the p53 protein family members, including p63. In particular, iASPP plays an important role in the development and differentiation of epithelial tissues. Here we characterize the interaction of iASPP with p63 and show that it binds to the linker region between the DNA binding domain and the oligomerization domain. We further demonstrate that this binding site is removed in a splice variant of p63 where a stretch of five amino acids is replaced with a single alanine residue. This stretch contains a degenerate class II SH3 domain binding motif that is responsible for interaction with iASPP, as well as two positively charged amino acids. Moreover, the concomitant loss of the charged amino acids in the alternatively spliced version decreases the affinity of p63 to its cognate DNA element two- to threefold. mRNAs encoding full-length p63, as well as its alternatively spliced version, are present in all tissues that we investigated, albeit in differing ratios. We speculate that, through the formation of hetero-complexes of both isoforms, the affinity to DNA, as well as the interaction with iASPP, can be fine-tuned in a tissue-specific manner., Competing Interests: Competing interests: The authors declare no competing interests. Ethical statement: All methods were performed in accordance with the relevant guidelines and regulations. As this is an in vitro study without human patients or animals involved no ethics committee approval was required. The human cDNA from ten different tissues was purchased directly from the company Zyagen without any connection to identifiable human patients., (© 2025. The Author(s).)

Alternative splicing is a crucial cellular process in eukaryotes, enabling the generation of multiple protein isoforms with diverse functions from a single gene. To better understand the impact of alternative splicing on protein structures, protein-protein interaction and human diseases, we developed ASpdb (https://biodataai.uth.edu/ASpdb/), a comprehensive database integrating experimentally determined structures and AlphaFold 2-predicted models for human protein isoforms. ASpdb includes over 3400 canonical isoforms, each represented by both experimentally resolved and predicted structures, and >7200 alternative isoforms with AlphaFold 2 predictions. In addition to detailed splicing events, 3D structures, sequence variations and functional annotations, ASpdb uniquely offers comparative analyses and visualization of structural alterations among isoforms. This resource is invaluable for advancing research in alternative splicing, structural biology and disease mechanisms., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Dissecting the mechanisms underlying heat tolerance is important for understanding how plants acclimate to heat stress. Here, we identify a heat-responsive gene in Arabidopsis thaliana, RNA-DIRECTED DNA METHYLATION 16 (RDM16), which encodes a pre-mRNA splicing factor. Knockout mutants of RDM16 are hypersensitive to heat stress, which is associated with impaired splicing of the mRNAs of 18 out of 20 HEAT SHOCK TRANSCRIPTION FACTOR (HSF) genes. RDM16 forms condensates upon exposure to heat. The arginine residues in intrinsically disordered region 1 (IDR1) of RDM16 are responsible for RDM16 condensation and its function in heat stress tolerance. Notably, RDM16 produces two alternatively spliced transcripts designated RDM16-LONG (RDL) and RDM16-SHORT (RDS). RDS also forms condensates and can promote RDL condensation to improve heat tolerance. Our findings provide insight into the cooperative condensation of the two RDM16 isoforms encoded by RDM16 splice variants in enhancing heat tolerance in Arabidopsis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

This review examines the giant elastic protein titin and its critical roles in heart function, both in health and disease, as discovered since its identification nearly 50 years ago. Encoded by the TTN (titin gene), titin has emerged as a major disease locus for cardiac disorders. Functionally, titin acts as a third myofilament type, connecting sarcomeric Z-disks and M-bands, and regulating myocardial passive stiffness and stretch sensing. Its I-band segment, which includes the N2B element and the PEVK (proline, glutamate, valine, and lysine-rich regions), serves as a viscoelastic spring, adjusting sarcomere length and force in response to cardiac stretch. The review details how alternative splicing of titin pre-mRNA produces different isoforms that greatly impact passive tension and cardiac function, under physiological and pathological conditions. Key posttranslational modifications, especially phosphorylation, play crucial roles in adjusting titin's stiffness, allowing for rapid adaptation to changing hemodynamic demands. Abnormal titin modifications and dysregulation of isoforms are linked to cardiac diseases such as heart failure with preserved ejection fraction, where increased stiffness impairs diastolic function. In addition, the review discusses the importance of the A-band region of titin in setting thick filament length and enhancing Ca² + sensitivity, contributing to the Frank-Starling Mechanism of the heart. TTN truncating variants are frequently associated with dilated cardiomyopathy, and the review outlines potential disease mechanisms, including haploinsufficiency, sarcomere disarray, and altered thick filament regulation. Variants in TTN have also been linked to conditions such as peripartum cardiomyopathy and chemotherapy-induced cardiomyopathy. Therapeutic avenues are explored, including targeting splicing factors such as RBM20 (RNA binding motif protein 20) to adjust isoform ratios or using engineered heart tissues to study disease mechanisms. Advances in genetic engineering, including CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), offer promise for modifying TTN to treat titin-related cardiomyopathies. This comprehensive review highlights titin's structural, mechanical, and signaling roles in heart function and the impact of TTN mutations on cardiac diseases., Competing Interests: None.

Papillary thyroid cancer (PTC) is often characterized by indolent behavior, small tumors with slow cell proliferation and a tendency to metastasize to cervical lymph node simultaneously, and the molecular mechanisms underlying that remain poorly understood. In this study, FN1 was the hottest gene of PTC and distinctive expression in PTC cells. FN1 deficiency severely inhibited the p53 signaling pathway, especially cyclin proteins, resulting in increased cell growth but hampered invasion. The alternatively splicing EDB region of FN1 was exclusively expressed in tumors, which impacted integrin β1 (ITGB1) bonding FN1 and its secretion process, resulting in completely distinct roles of two isoforms that FN1 including and skipping EDB domain. The isoform EDB(-)FN1 intracellularly inhibited tumor proliferation by upregulating p21 expression, whereas extracellular EDB(+)FN1 promoted lymph node metastasis via the VEGF signaling pathway in vitro and in vivo. Moreover, the alternative splicing EDB region of FN1 was modulated by p53-targeted protein ZMAT3 which activated cell migration and lymphoangiogenesis. Collectively, combined with p53-induced proteins, FN1 played both anti- and pro-cancer roles owing to EDB domain alternative splicing. FN1 is a potential determinant behind the characteristic behavior of PTC, which may contribute to a deeper understanding of the peculiarity of PTC and provide a promising target for regional lymph node metastasis., Competing Interests: Declarations. Ethics approval and consent to participate: Ethical approval was provided by the Committees for Ethical Review of Research involving Human Subjects and Animal experiments at Xiangya Hospital, Central South University (No. 2019030140). Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The molecular mechanisms underlying cell migration remain incompletely understood. Here, we show that knock-out cells for NHSL3, the most recently identified member of the Nance-Horan Syndrome family, are more persistent than parental cells in single cell migration, but that, in wound healing, follower cells are impaired in their ability to follow leader cells. The NHSL3 locus encodes several isoforms. We identify the partner repertoire of each isoform using proteomics and predict direct partners and their binding sites using an AlphaFold2-based pipeline. Rescue with specific isoforms, and lack of rescue when relevant binding sites are mutated, establish that the interaction of a long isoform with MENA/VASP proteins is critical at cell-cell junctions for collective migration, while the interaction of a short one with 14-3-3θ in lamellipodia is critical for single cell migration. Taken together, these results demonstrate that NHSL3 regulates single and collective cell migration through distinct mechanisms., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Adenosine to inosine deaminases acting on RNA (ADARs) enzymes are found in all metazoa. Their sequence and protein organization is conserved but also shows distinct differences. Moreover, the number of ADAR genes differs between organisms, ranging from one in flies to three in mammals. The distinct isoforms of ADARs and their specific roles determine the complexity of A-to-I RNA editing, its regulation and the versatility of these enzymes. Understanding the different isoform-specific functions and targets will provide a deeper understanding of the diverse biological processes influenced by ADARs, either through ADAR editing of dsRNAs or the interaction with RNAs and proteins. The detailed identification and assigning of isoform-specific targets is a crucial step towards our understanding of functional differences amongst ADAR isoforms and will help us to understand their individual implications for health and disease. This chapter delves into unique characteristics and functional implications of ADAR isoforms. We describe the ectopic overexpression in editing free cells and the use of RNA immunoprecipitation coupled with sequencing to determine isoform-specific interactions with RNAs and their editing sites. Additionally, we discuss new challenges in editing detection by different ADARs in the context of other modifications and provide ideas for potentially better methods to determine the "true editome"., (Copyright © 2025. Published by Elsevier Inc.)

Brain-specific angiogenesis inhibitor 1 (BAI1) belongs to the adhesion G-protein-coupled receptors, which exhibit large multi-domain extracellular N termini that mediate cell-cell and cell-matrix interactions. To explore the existence of BAI1 isoforms, we queried genomic datasets for markers of active chromatin and new transcript variants in the ADGRB1 (adhesion G-protein-coupled receptor B1) gene. Two major types of mRNAs were identified in human/mouse brain, those with a start codon in exon 2 encoding a full-length protein of a predicted size of 173.5/173.3 kDa and shorter transcripts starting from alternative exons at the intron 17/exon 18 boundary with new or exon 19 start codons, predicting two shorter isoforms of 76.9/76.4 and 70.8/70.5 kDa, respectively. Immunoblots on wild-type and Adgrb1 exon 2-deleted mice, reverse transcription PCR, and promoter-luciferase reporter assay confirmed that the shorter isoforms originate from an alternative promoter in intron 17. The shorter BAI1 isoforms lack most of the N terminus and are very close in structure to the truncated BAI1 isoform generated through GPS processing from the full-length receptor. The cleaved BAI1 isoform has a 19 amino acid extracellular stalk that may serve as a receptor agonist, while the alternative transcripts generate BAI1 isoforms with extracellular N termini of 5 or 60 amino acids. Further studies are warranted to compare the functions of these isoforms and examine the distinct roles they play in different tissues and cell types., Competing Interests: Declarations. Ethical Approval: All animal procedures were performed under Institutional Animal Care and Use Committee (IACUC) approval. The human tissue was obtained with Institutional Review Board for Human Use (IRB) approval. Consent to Participate: Not applicable. Consent to Publication: Not applicable. Competing interests: EGVM is co-founder, Chief Scientific Officer, and shareholder of OncoSpherix, Inc. (not related to the current study). Other authors declare no competing interest., (© 2024. The Author(s).)

The lack of targeted therapy for triple-negative breast cancer (TNBC) is among the mainsprings of its poor prognosis. This study aimed to elucidate the role of the androgen receptor (AR) and its splice variant 7 (ARv7) in TNBC patients. Further, the molecular impact of their blockers, Enzalutamide and EPI-001, on the TNBC cell line MDA-MB-231 was investigated. Thereby, immunohistochemical expression of AR/ARv7 was assessed for TNBC Egyptian patients. Moreover, bioinformatics analysis of AR/ARv7 RNA status was carried out on TNBC patients from The Cancer Genome Atlas Breast Carcinoma project (TCGA-BRCA). Data from both groups was correlated with patients' clinicopathological features. Besides, scratch wound healing assay and ELISA were employed to assess the effect of AR/ARv7 blockers on several metastasis markers in MDA-MB-231 cell line. In the Egyptian-TNBC patients, AR expression was associated with worse 7-year DFS (40.6 ± 18.6 %). In addition, ARv7 showed cytoplasmic and nuclear patterns, and both cytoplasmic and nuclear ARv7 + patients demonstrated a worse 7-year DFS (22.7 ± 17.7 % and 20 ± 17.9 %) and overall survival (63.6 ± 14.5 % and 40 ± 21.8 %). Importantly, 80 % of the nuclear ARv7 + patients developed distant metastasis. The data of the TCGA-TNBC patients showed a tendency for poor outcomes in the high ARv7-expressing patients. Molecularly, in MDA-MB-231, both inhibitors modulated metastasis and epithelial to mesenchymal transition (EMT) markers ROCK1, ROCK2, c-Myc, E-cadherin and N-cadherin, with EPI-001 downregulating NF-ĸB level as well. We concluded that ARv7 indicated poor prognosis in the studied cohorts and that blocking of AR/ARv7 abated metastasis and key regulators of EMT in MDA-MB-231, at least in part by targeting ROCK/NF-ĸB/c-Myc axis., Competing Interests: Declaration of interest The authors declare no conflict of interest exists., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Calcifying organisms are suffering from negative impacts induced by climate change, such as CO 2 -induced acidification, which may impair external calcified structures. Freshwater mollusks have the potential to suffer more from CO 2 -induced acidification than marine calcifiers due to the lower buffering capacity of many freshwater systems. One of the most important enzymes contributing to the biomineralization reaction is carbonic anhydrase (CA), which catalyzes the reversible conversion of CO 2 to bicarbonate, the major carbon source of the calcareous structure in calcifiers. In this study we characterized two α-CA isoforms (LsCA1 and LsCA4) from the freshwater snail Lymnaea stagnalis using a combination of gene sequencing, gene expression, phylogenetic analysis and biochemical assays. Both CA isoforms demonstrated high expression levels in the mantle tissue, the major site for biomineralization. Furthermore, expression of LsCA4 during development parallels shell formation. The primary protein structure analysis, active site configuration and the catalytic activity of LsCA4 together suggest that the LsCA4 is embedded in the apical and basolateral membranes of mantle cells; while LsCA1 is proposed to be cytosolic and might play an important role in acid-base regulation. These findings of LsCA isoforms form a strong basis for a more detailed physiological understanding of the effects of elevated CO 2 on calcification in freshwater mollusks., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Gliblastoma is a malignant brain tumor; despite available treatment modalities, the tumor reoccurrence rate persist in the currently prescribed Temozolomide chemotherapy. Study aimed to study the inquisitive role of RNA binding splice factor protein hnRNPA1 in promoting glioma resistance against Temozolomide drug and therapeutic insights., Methods: In this study two non-expressing O 6 -methylguanine-DNA methyltransferase (MGMT) glioma cell lines U87MG & LN229. U87MG cells were grown in Temozolomide from 50μM upto 400μM & LN229 cells grown upto 200μM, till then both these cells acquired Temozolomide resistance. Both of these cells were grown & maintained continously in its highest dose of Temozolomide (TMZ). Splice factor protein SF2/ASF1 was functionally correlated with abundance of hnRNPA1 protein in Temozolomide (TMZ) resistant cells using its specific siRNA transfection approach, in detrmining SF2/ASF1 mediated hnRNPA1 splicing and Temozolomide resistant reversal., Results: U87MG TMZ resistance, results an increase in the expression of pre mRNA-splicing factor SF2/ASF1, Heterogeneous Ribonucleoprotein A1 (hnRNPA1) and O 6 -methylguanine-DNA methyltransferase (MGMT) protein. MGMT expression was not observed in LN229 TMZ resistant cells. Further, mRNA sequencing of hnRNPA1 confirmed the exclusive abundance of its higher isoform in TMZ- resistant cells along with increase in SF2/ASF1 expression. Knocking down of SF2/ASF1 using its specific siRNA reverted the higher isoform of hnRNPA1 isoform Var2 to its lower isoform hnRNPA1 Var1 in U87 TMZ resistant cells, reveals hnRNPA1 alternative higher isoform abundance is SF2/ASF1 splice factor dependent. Additionally, selective knock down of hnRNPA1 higher isoform Var2 in TMZ resistant U87MG & LN229 promotes apoptosis, was further specfically enhanced on Wortmannin (PI3Kinase inhibitor) treatment., Conclusion: Targeting higher isoform Var2 of hnRNPA1 specifically induces chemosensitization in MGMT expressed Temozolomide resistant U87MG as well as in MGMT non-expressed LN229 TMZ resistant cells., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Ethical approval: Protocols for the handling of human tissues and cells were approved by the Ethics committee of Dr. B.R Ambedkar Center for Biomedical Research (No.F.50–2/Eth.Com/ACBR/16/2379) and Rajiv Gandhi Cancer Institute and Research Centre (Res/SCM/17/2016/59). Consent for publication: Obtained procedural consent from the human ethical committee., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Actin is one of the most widespread and most conserved proteins. At the same time, six actin isoforms are known, encoded by different genes. These isoforms differ slightly in amino acid sequence and have similar structures, but differ in localization and functioning. During functioning, actin interacts with a large number of proteins, which are combined according to this feature into a pool of so-called actin-binding proteins. The question arises whether and how the proteins interacting with different actin isoforms differ. Since the pool of actin-binding proteins includes hundreds of proteins, it was logical to use bioinformatics analysis to solve the questions. In this work, it is shown that the functionality of the α-, β-, and γ-actin interactomes differ significantly, but their structural characteristics are close., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Alternative splicing (AS), a crucial mechanism in post-transcriptional regulation, has been implicated in diverse cancer processes. Several splicing variants of solute carrier (SLC) transporters reportedly play pivotal roles in tumorigenesis and tumor development. However, an in-depth analysis of AS landscapes of SLCs in colon adenocarcinoma (COAD) is lacking. Herein, we analyzed data from The Cancer Genome Atlas and identified 1215 AS events across 243 SLC genes, including 109 differentially expressed AS (DEAS) events involving 62 SLC genes in COAD. Differentially spliced SLCs were enriched in biological processes, including transmembrane transporter activity, transporter activity, ferroptosis, and choline metabolism. In patients with COAD, tumor tissues exhibited higher expression of longer mitochondrial carrier SLC25A16 isoforms than adjacent normal tissues, consistent with bioinformatics analysis. Protein-coding sequences and transmembrane helices of survival-related DEAS were predicted, revealing that shifts in splicing sites altered the number and structure of their transmembrane proteins. We developed a prognostic risk model based on the screened 6-SLC-AS (SLC7A6_RI_37208 (SLC7A6-RI), SLC11A2_AP_21724, SLC2A8_ES_87631, SLC35B1_AA_42317, SLC39A11_AD_43204, and SLC7A8_AP_26712). Knockdown of the intronic region of SLC7A6-RI isoform enhanced colon cancer cell proliferation. In vivo, knockdown of the intronic region of SLC7A6-RI isoform enhanced tumor growth in colon cancer. Mechanistically, si-SLC7A6-RI isoform exerted oncogenic effects by activating the PI3K-Akt-mTOR signaling pathway and promoting cell proliferation, evidenced by increased expression of key regulators Phosphorylated Mammalian Target of Rapamycin (p-mTOR) and a cell proliferation marker Proliferating Cell Nuclear Antigen (PCNA) using western blotting. Our study elucidated SLC-AS in COAD, highlighting its potential as a prognostic and therapeutic target and emphasizing the suppressive influence of SLC7A6-RI in colon cancer progression., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)