Gene expression involves a series of consequential processes, beginning with mRNA synthesis and culminating in translation. Traditionally studied as a linear sequence of events, recent findings challenge this perspective, revealing coupling mechanisms that coordinate key steps of gene expression, even when spatially and temporally distant. In this review, we focus on translation, the final stage of gene expression, and examine its coupling with key stages of mRNA metabolism: synthesis, processing, export, and decay. For each of these processes, we provide an overview of known instances of coupling with translation. Furthermore, we discuss the role of high-throughput technologies in uncovering these intricate interactions on a genome-wide scale. Finally, we highlight key challenges and propose future directions to advance our understanding of how coupling mechanisms orchestrate robust and adaptable gene expression programs.

Naive T cell activation requires both TCR and CD28 signals. The CARMIL2 cytosolic protein enables CD28-dependent activation of the NF-κB transcription factor via its ability to link CD28 to the CARD11 adaptor protein. Here, we developed mice expressing a mutation named Carmil2QE and mimicking a mutation found in human T cell malignancies. Naive T cells from Carmil2QE mice contained preformed CARMIL2QE-CARD11 complexes in numbers comparable to those assembling in wild-type T cells after CD28 engagement. Such ready-made CARMIL2QE-CARD11 complexes also formed in CD28-deficient mice where they unexpectedly induced most of the functions that normally result from CD28 engagement in a manner that remains antigen-dependent. In turn, tumor-specific T cells expressing Carmil2QE do not require CD28 engagement and thereby escape to both PD-1 and CTLA-4 inhibition. In conclusion, we uncovered the overarching role played by CARMIL2-CARD11 signals among those triggered by CD28 and exploited them to induce potent solid tumor-specific T cell responses in the absence of CD28 ligands and immune checkpoint inhibitors.

Infections caused by carbapenem-resistant Acinetobacter baumannii (A. baumannii; CRAb) are associated with high patient morbidity and mortality. The serious threat for human health imposed by CRAb was recently underscored by identification of close-to-untouchable carbapenem- and tetracycline-resistant isolates. Since outer membrane vesicles (OMVs) of Gram-negative bacteria may contribute to antimicrobial resistance, our present study was aimed at investigating OMVs produced by the first two carbapenem- and tetracycline-resistant A. baumannii isolates in Europe. These isolates, denoted CRAb1 and CRAb2, contain large, nearly identical plasmids that specify multiple resistances. Both isolates produce OMVs that were analyzed by differential light scattering, transmission electron microscopy and proteomics. By comparison with OMVs from the plasmid-free non-carbapenem-resistant A. baumannii isolate Ab1, which is an isogenic ancestor of the CRAb1 isolate, we show that plasmid carriage by the CRAb1 and CRAb2 isolates leads to an increased OMV size that is accompanied by increased diversity of the OMV proteome. Our analyses show that OMVs from CRAb1 and CRAb2 are major reservoirs of proteins involved in antimicrobial resistance, including the plasmid-encoded carbapenemases New Delhi metallo-β-lactamase-1 (NDM-1), and carbapenem-hydrolyzing oxacillinase OXA-97 (OXA-97). Here we report that these OMV-borne carbapenemases hydrolyze imipenem and protect otherwise carbapenem-sensitive A. baumannii and Escherichia coli (E. coli) isolates against this antibiotic. In conclusion, our findings demonstrate that OMVs from highly drug-resistant CRAb confer protection against last-resort antibiotics to non-resistant bacterial pathogens.

Cancer cells frequently undergo energy metabolic stress induced by the increased dynamics of nutrient supply. Hepatocyte nuclear factor 4A (HNF4A) is a master transcription factor (TF) in hepatocytes that regulates metabolism and differentiation. However, the mechanism underlying how HNF4A functions in cancer progression remains unclear due to conflicting results observed in numerous studies. To address the roles of HNF4A in hepatocellular carcinoma (HCC), we investigated the regulatory functions of HNF4A in HCC cells under different glucose supply conditions. We found that HNF4A exhibited tumor-suppressive effects on the proliferation and migration of HCC cells in glucose-sufficient conditions and tumor-promotive effects on HCC cells in glucose-insufficient conditions. Further investigation revealed that this diverse function of HNF4A was dependent upon the AMPK pathway activity. Similarly, the prognosis predicted by HNF4A was also correlated with whether the AMPKa expression levels were low or high in clinical HCC patients. Multiomics approaches consisting of proteomics and ChIP-seq revealed that key HNF4A target genes, including NEDD4 and RPS6KA2, are involved in the diverse function of HNF4A in HCC in response to the AMPK activity status. Specifically, HNF4A could bind to the promoter region of NEDD4 and RPS6KA2, and upregulating their expression. Our study has demonstrated the relationship between and synergism of AMPK and HNF4A in the progression of HCC under diverse nutrient conditions.

The field of quantitative biology (q-bio) seeks to provide precise and testable explanations for observed biological phenomena by applying mathematical and computational methods. The central goals of q-bio are to (1) systematically propose quantitative hypotheses in the form of mathematical models, (2) demonstrate that these models faithfully capture a specific essence of a biological process, and (3) correctly forecast the dynamics of the process in new, and previously untested circumstances. Achieving these goals depends on accurate analysis and incorporating informative experimental data to constrain the set of potential mathematical representations. In this introductory tutorial, we provide an overview of the state of the field and introduce some of the computational methods most commonly used in q-bio. In particular, we examine experimental techniques in single-cell imaging, computational tools to process images and extract quantitative data, various mechanistic modeling approaches used to reproduce these quantitative data, and techniques for data-driven model inference and model-driven experiment design. All topics are presented in the context of additional online resources, including open-source Python notebooks and open-ended practice problems that comprise the technical content of the annual Undergraduate Quantitative Biology Summer School (UQ-Bio).

We report here transport of full-length epidermal growth factor receptor (EGFR), Insulin Receptor, 7-pass transmembrane receptor Smoothened, and 13-pass Sodium-iodide symporter to extracellular vesicles (EVs) for structural and functional studies. Mass spectrometry confirmed the transported proteins are the most abundant in EV membranes, and the presence of many receptor-interacting proteins in EVs demonstrates their utility for characterizing membrane protein interactomes. Cryo-electron tomography of EGFR-containing EVs reveals that EGFR forms clusters in both the presence and absence of EGF with a ~3 nm gap between the inner membrane and cytoplasmic density. EGFR extracellular region (ECR) dimers do not form regular arrays in these clusters. Subtomogram averaging of the 150 kDa EGF-bound EGFR ECR dimer yielded a 15 Å map into which the crystal structure of the ligand-bound EGFR ECR dimer fits well. These findings refine our understanding of EGFR activation, clustering, and signaling and establish EVs as a versatile platform for structural and functional characterization of human membrane proteins in cell-derived membranes.

The efficacy of oocyte cryopreservation still requires enhancement. Current understanding of cryodamage is largely limited to the organelle level, and the impact of freezing on oocyte gene expression remains unclear.

In this study, we employed an innovative dual-omics approach to assess the transcriptional and translational profiles of mouse metaphase II (MII) oocytes during the initial 4 h post-thaw.

Our mapping of the translational recovery in vitrified mouse oocytes post-thaw revealed a critical 2-h window that is optimal for recovery. We confirmed the mitochondrial damage associated with vitrification and identified the activation of autophagy and proteasomal degradation during this period. Additionally, our analysis indicates that vitrified oocytes have another repair response to counteract cryoinjury, involving spindle remodeling and membrane recycling.

These findings can guide future efforts to improve oocyte vitrification outcomes by improving repair processes and not focusing solely on the damage processes.

Kidney ischemia-reperfusion (I/R) is associated with endothelial injury. Administration of miRNA (miR)-486-5p protects against rat kidney I/R injury, with localisation to capillary endothelial cells, although it inhibits I/R-induced endothelial nitric oxide synthase (eNOS) protein expression. Here, we studied the effect of miR-486-5p on eNOS and endothelial cell function and determined its mRNA targets. Human umbilical vein endothelial cells (HUVECs) were transfected with the miR-486-5p mimic and assayed for proliferation, migration and network formation. Biotinylated miR-486-5p was transfected for pulldown of bound mRNA, followed by RNA sequencing. miR-486-5p markedly decreased eNOS mRNA and protein in HUVECs (p < 0.001) and decreased eNOS protein in human pulmonary microvascular endothelial cells (p < 0.05), although eNOS was not a direct target of miR-486-5p. miR-486-5p inhibited angiogenesis, which was rescued with eNOS plasmid transfection. RNA sequencing of biotinylated miR-486-5p pulldown RNA revealed highly significant enrichment in predicted targets FOXO1, FOXP1, TNFSF4, MAML3 and CELSR3, and in the non-predicted target SPCS2. RT-qPCR validated these transcripts as inhibited by miR-486-5p. While silencing of FOXO1 had no impact on eNOS protein, MAML3 silencing inhibited eNOS levels. miR-486-5p inhibits angiogenesis in endothelial cells via eNOS down-regulation, which involves selective targeting of MAML3. These data support a novel pathway regulating endothelial cell function.

Mycobacterium tuberculosis, the pathogen responsible for human tuberculosis, responds to iron limitation by increasing the production of extracellular vesicles. This study examined the protein composition of induced M. tuberculosis extracellular membrane vesicles using chromatography coupled with mass spectrometry. The results revealed that vesicles contain key pathogenicity factors, including proteins that enhance bacterial survival, immune evasion, and inflammation. These findings deepen our understanding of the potential role of extracellular vesicles in M. tuberculosis-host interactions. The data can also aid in identifying new biomarkers of infection and developing vesicle-based, culture-independent TB diagnostic platforms.

Recent advancements in single-cell technologies have enabled comprehensive characterization of cellular states through transcriptomic, epigenomic, and proteomic profiling at single-cell resolution. These technologies have significantly deepened our understanding of cell functions and disease mechanisms from various omics perspectives. As these technologies evolve rapidly and data resources expand, there is a growing need for computational methods that can integrate information from different modalities to facilitate joint analysis of single-cell multi-omics data. However, integrating single-cell omics datasets presents unique challenges due to varied feature correlations and technology-specific limitations. To address these challenges, we introduce scMODAL, a deep learning framework tailored for single-cell multi-omics data alignment using feature links. scMODAL integrates datasets with limited known positively correlated features, leveraging neural networks and generative adversarial networks to align cell embeddings and preserve feature topology. Our experiments demonstrate scMODAL's effectiveness in removing unwanted variation, preserving biological information, and accurately identifying cell subpopulations across diverse datasets. scMODAL not only advances integration tasks but also supports downstream analyses such as feature imputation and feature relationship inference, offering a robust solution for advancing single-cell multi-omics research.

Palaeoproteomics leverages the persistence, diversity, and biological import of ancient proteins to explore the past, and answer fundamental questions about phylogeny, environment, diet, and disease. These insights are largely gleaned from hard tissues like bone and teeth, as well-established protocols exist for extracting ancient proteins from mineralised tissues. No such method, however, exists for the soft tissues, which are underexplored in palaeoproteomics given permission for destructive analysis routinely depends on a proven methodology. Considering less than one-tenth of all human proteins are expressed in bone, compared to three-quarters in the internal organs, the amount of biological information presently inaccessible is substantial. We address this omission with an optimised LC-FAIMS-MS/MS workflow yielding the largest, most diverse palaeoproteome yet described. Using archaeological human brains, we test ten protocols with varied chemistries and find that urea lysis effectively disrupts preserved membrane regions to expose low-abundant, intracellular analytes. Further, we show that ion mobility spectrometry improves unique protein identification by as much as 40%, and represents a means of "cleaning" dirty archaeological samples. Our methodology will be useful for improving protein recovery from a range of ancient tissues and depositional environments.

Stem cells differ from other somatic cells in that they possess self-renewal and differentiation potential, which endows them with unique characteristics, and have great therapeutic potential. Studies have shown that the self-renewal and differentiation potential of stem cells is regulated by ribosomes during protein synthesis. In this review, we discuss the translation regulation mechanisms and ribosome biogenesis in stem cells. Protein translation levels and ribosome biogenesis change dynamically during the development and differentiation of stem cells, and hierarchical translational regulation promotes stem cell differentiation. We also demonstrate that mitochondrial protein translation plays an important role in the regulation of stem cell fate. Ribosomes not only mediate the self-renewal and differentiation of stem cells through protein synthesis. They are also a key target for stem cell therapy. Understanding the mechanism of ribosome regulation in stem cells will allow better control of stem cells for their application.

Eukaryotic translation initiation typically involves recruitment of the 43S ribosomal pre-initiation complex (PIC) to the 5'-end of the mRNA to form the 48S PIC, followed by scanning in search of a start codon in a favorable nucleotide complex. The start codon is recognized through base-pairing with the anticodon of the initiator Met-tRNAi. The stringency of start codon selection controls the probability of initiation from a start codon in a suboptimal nucleotide context. Met-tRNAi itself is recruited to the 43S PIC by the eukaryotic translation initiation factor 2 (eIF2), in the form of the eIF2-GTP•Met-tRNAi ternary complex (TC). GTP hydrolysis by eIF2, promoted by its GTPase-activating protein eIF5, leads to the release of eIF2-GDP from the PIC. Recycling of eIF2-GDP to TC is promoted by the guanine nucleotide exchange factor eIF2B. Its inhibition by a number of stress factors triggers the integrated stress response (ISR). This review describes the recent advances in elucidating the interactions of eIF2 and its partners, with an emphasis on the timing and dynamics of their binding to, and release from the PIC. Special attention is given to the regulation of the stringency of start codon selection and the ISR. The discussion is mostly limited to translation initiation in mammals and budding yeast.

mRNA therapy is a promising approach in oncology, offering innovative applications such as tumor vaccines, protein replacement therapy, cell therapy, and gene therapy. However, challenges such as mRNA stability and delivery efficiency must be addressed. Advances in delivery system technologies are crucial for precise mRNA delivery, enhancing treatment safety and efficacy. The development of delivery systems requires accurate organ or cell targeting, intelligent release mechanisms, and optimized administration routes. This review outlines the applications of mRNA therapy in oncology, as well as the utilization of nonviral vectors, encompassing organic, inorganic, and biomimetic systems. It further elucidates the strategies for passive and active vector targeting and examines recent advances in the realm of stimuli-responsive delivery systems that are sensitive to pH and ultrasound. Additionally, the review addresses the development of noninvasive mRNA delivery systems designed for oral and pulmonary administration. The current challenges and emerging trends of mRNA therapy are discussed, and the potential strategies to mitigate these issues are emphasized.

Trisomy of chromosome 21, the cause of Down syndrome (DS), is the most commonly occurring genetic cause of Alzheimer's disease (AD). Here, we compare the frontal cortex proteome of people with Down syndrome-Alzheimer's disease (DSAD) to demographically matched cases of early onset AD and healthy ageing controls. We find dysregulation of the proteome, beyond proteins encoded by chromosome 21, including an increase in the abundance of the key AD-associated protein, APOE, in people with DSAD compared to matched cases of AD. To understand the cell types that may contribute to changes in protein abundance, we undertook a matched single-nuclei RNA-sequencing study, which demonstrated that APOE expression was elevated in subtypes of astrocytes, endothelial cells, and pericytes in DSAD. We further investigate how trisomy 21 may cause increased APOE. Increased abundance of APOE may impact the development of, or response to, AD pathology in the brain of people with DSAD, altering disease mechanisms with clinical implications. Overall, these data highlight that trisomy 21 alters both the transcriptome and proteome of people with DS in the context of AD, and that these differences should be considered when selecting therapeutic strategies for this vulnerable group of individuals who have high risk of early onset dementia.

Background/Objectives: The recent COVID-19 pandemic caused by SARS-CoV-2 infection has highlighted the need for protocols for rapid development of efficient screening methods to search for the optimal mRNA vaccine structures against mutable viral agents. The unmatched success of mRNA vaccines by Pfizer and Moderna encoding the spike protein of SARS-CoV-2 confirms the potential of lipid nanoparticles for mRNA delivery for an accelerated development of new vaccines. The efficacy of vaccination and the production cost of mRNA-based vaccines largely depend on the composition of mRNA components, since the synthesis of an immunogenic protein requires precise and efficient translation in vivo. The composition of 5' and 3' UTR combinations of mRNA has a strong impact on the translation efficiency. The major objective of this study was to increase the probability of producing the immunogenic protein encoded by vaccine mRNA. For this purpose, we proposed to find a new combination of natural UTRs and, in parallel with that, to design and test the system for in vivo selection of translationally active UTRs. Methods: By using Ribo-Seq analysis, sets of candidate short UTRs were generated. These UTRs were tested both in cell cultures and in mice for effective production of secreted nanoluciferase (NLuc) and the S protein of SARS-CoV-2. A combination of the most effective UTRs was used to generate a prototype of an mRNA vaccine capable of inducing neutralizing antibodies against coronavirus. Results: The usefulness of the selected UTRs for vaccine development was tested by implicating the full-length coding sequence of SARS-CoV-2 S protein to produce the main immunogen. As a result, the system for functional screening of UTRs was created by using the NLuc gene. Conclusions: The proposed approach allows non-invasive quantitative assessment of the translational activity of UTRs in the blood serum of mice. By using the full-length sequence of SARS-CoV-2 S protein as a prototype, we demonstrated that the combination of UTRs selected using our luciferase-based reporter assay induces IgG titers and neutralization rates comparable to those obtained by using UTRs from commercial S-protein-based mRNA vaccines.

The regulation of RNA polymerase II (RNAPII) activity requires orchestrated responses among genomic regulatory sequences and an expansive set of proteins and protein complexes. Despite intense study over five decades, mechanistic insights continue to emerge. Within the past 10 years, live-cell imaging and single-cell transcriptomics experiments have yielded new information about enhancer-promoter communication, transcription factor dynamics, and the kinetics of RNAPII transcription activation. These insights have established RNAPII re-initiation and bursting as a common regulatory phenomenon with widespread implications for gene regulation in health and disease. Here, we summarize regulatory strategies that help control RNAPII bursting in eukaryotic cells, which is defined as short periods of active transcription followed by longer periods of inactivity. We focus on RNAPII re-initiation (i.e., a "burst" of two or more polymerases that initiate from the same promoter), with an emphasis on molecular mechanisms, open questions, and controversies surrounding this distinct regulatory stage.

In a human cell, DNA is packed with histones, RNA, and chromatin-associated proteins, forming a cohesive gel. At any given moment, only a subset of the proteome has physical access to the DNA and organizes its structure, transcription, replication, repair, and other essential molecular functions. We have developed a "zero-distance" photo-crosslinking approach to quantify proteins in direct contact with DNA in living cells. Collecting DNA interactomes from human breast cancer cells, we present an atlas of over one thousand proteins with physical access to DNA and hundreds of peptide-nucleotide crosslinks pinpointing protein-DNA interfaces with single-amino-acid resolution. Quantitative comparisons of DNA interactomes from differentially treated cells recapitulate the recruitment of key transcription factors as well as DNA repair proteins and uncover fast-acting restrictors of chromatin accessibility on a timescale of minutes. This opens a direct way to explore genomic regulation in a hypothesis-free manner, applicable to many organisms and systems.

N7-methylguanosine (m7G) modification of transfer RNA (tRNA) is essential for the biological functions of tRNAs and has been found to play a regulatory role in a variety of human cancers. However, the biological function of METTL1-mediated m7G tRNA modification in papillary thyroid cancer (PTC) is unclear. Here, we found that METTL1 is significantly upregulated in PTC tissues compared to normal control tissues and is associated with poor PTC prognosis. Functional analysis confirmed that METTL1 promotes the proliferation and metastasis of PTC cells in a manner dependent on its tRNA methyltransferase activity. Mechanistically, METTL1 knockdown leads to a decrease in the abundance of certain m7G-modified tRNAs, which suppresses the m7G tRNA modification-mediated codon-specific translation of TNF-α. Furthermore, exogenous supplementation with TNF-α partially reversed the decrease in the proliferation and metastasis of PTC cells induced by METTL1 deletion. Positive correlations between METTL1, WDR4, and TNF-α expression, which affect the proliferation and metastasis of PTC, were confirmed via analysis of microarrays containing PTC tissues. These results demonstrate the oncogenic role of METTL1-mediated m7G tRNA modification in regulating codon-specific translation efficiency in PTC and suggest that targeting METTL1 may be a promising therapeutic approach for overcoming PTC progression by inhibiting PTC cell proliferation and metastasis.

Identifying detectable peptides, known as flyers, is key in mass spectrometry-based proteomics. Peptide detectability is strongly related to peptide sequences and their resulting physicochemical properties. Moreover, the high variability in MS data challenges the development of a generic model for detectability prediction, underlining the need for customizable tools. We present Pfly, a deep learning model developed to predict peptide detectability based solely on peptide sequence. Pfly is a versatile and reliable state-of-the-art tool, offering high performance, accessibility, and easy customizability for end-users. This adaptability allows researchers to tailor Pfly to specific experimental conditions, improving accuracy and expanding applicability across various research fields. Pfly is an encoder-decoder with an attention mechanism, classifying peptides as flyers or non-flyers, and providing both binary and categorical probabilities for four distinct classes defined in this study. The model was initially trained on a synthetic peptide library and subsequently fine-tuned with a biological dataset to mitigate bias toward synthesizability, improving predictive capacity and outperforming state-of-the-art predictors in benchmark comparisons across different human and cross-species datasets. The study further investigates the influence of protein abundance and rescoring, illustrating the negative impact on peptide identification due to misclassification. Pfly has been integrated into the DLOmix framework and is accessible on GitHub at https://github.com/wilhelm-lab/dlomix.

Translational regulation plays a pivotal role in cardiac gene expression, influencing protein synthesis in response to physiological and pathological stimuli. Although transcription regulates gene expression, translation ultimately determines protein levels, making it a crucial research focus. In cardiomyocytes, disruptions in this process contribute to various cardiac diseases, including hypertrophy, fibrosis, dilated cardiomyopathy, ischemic heart disease, and diabetic cardiomyopathy. Emerging evidence highlights the significance of translational regulation across multiple cardiac cell types, such as cardiomyocytes and fibroblasts, and its role in disease progression. During cardiac remodeling, transcriptomic changes are often modest, suggesting that post-transcriptional mechanisms, particularly translation, play a dominant role in cellular adaptation. This review discusses key methodologies for studying translational regulation and novel mechanisms of translational regulation related to different cardiac pathologies and highlights relevant therapeutic avenues for targeting these pathways.

Oilseed rape (Brassica napus) seeds are of major economic and nutritional value since they are rich in both oil and proteins, which accumulate predominantly in the embryonic cotyledons during the filling period. Developmental phases such as embryogenesis, seed filling, and maturation have been associated with specific changes in the transcriptional landscape and are controlled by interactions of regulatory components, particularly transcription factors and cis-regulatory elements. However, the global changes on the protein level remain largely elusive. Here, we investigated the dynamics of seed development by an integrative analysis of the seed transcriptome and proteome. Plants of the winter-type cultivar Express 617 were grown under controlled, field-like conditions in the IPK PhenoSphere, and developing seeds were collected for temporally and spatially resolved multi-omics analyses. The dataset covers five stages, from pre-storage to seed maturation, and includes spatial information on four dissected organs/tissues. It provides comprehensive insights into differentiation and developmental processes of the Brassica napus seed and may serve as starting point to select potentially important genes for detailed functional investigations.

The Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation in acute myeloid leukaemia (AML) is associated with adverse clinical outcomes, including poor prognosis, high relapse rates and reduced responses to conventional treatment regimens. While venetoclax (VEN) monotherapy has shown limited efficacy in FLT3-ITD AML due to intrinsic resistance mechanisms, this study demonstrates that ciclosporin A (CsA) synergistically enhances VEN's anti-leukaemic activity. CsA significantly suppresses cell proliferation, induces mitochondrial apoptosis and impairs mitochondrial bioenergetics in FLT3-ITD AML cells. Mechanistically, CsA enhances the effects of VEN through the downregulation of NFATC1, a critical regulator of the PI3K/AKT/mTOR signalling pathway. This suppression of NFATC1 leads to the coordinated downregulation of the anti-apoptotic proteins BCL-2 and MCL-1, thereby overcoming resistance and reinstating therapeutic susceptibility in FLT3-ITD AML.

Extracellular vesicles (EVs) are potential non-invasive diagnostic, prognostic and therapeutic tools. Additionally, they are important contributors to tumorigenesis. Glycosylation has been found to modulate the composition of the EV proteome. Increased amounts of β1,6-branched N-glycans, synthesized by N-acetylglucosaminyltransferase V (GnT-V), are most commonly observed in melanoma and are associated with decreased cell adhesion and increased metastasis. The opposite effect is caused by the addition of bisecting GlcNAc by N-acetylglucosaminyltransferase III (GnT-III). To date, the impact of these enzymes on EV cargo in melanoma remains unexplored. Flow cytometry was used to study the surface glycosylation of genetic variants of WM266-4 melanoma cells with induced overexpression of GnT-III or GnT-V encoding genes (MGAT3 or MGAT5) and EVs released by these cells. LC-MS/MS proteomics was applied to analyze the effect of altered glycosylation on the proteome of released EVs, followed by detailed bioinformatic analysis. Flow cytometry analysis revealed dynamic changes in the surface glycosylation of EVs derived from melanoma cells overexpressing MGAT3 or MGAT5. Induced overexpression of MGAT3 or MGAT5 also caused significant changes in the proteome of EVs. The proteomic analysis identified a total of 1770 microvesicular and 704 exosomal proteins that play different roles in melanoma progression, including those with established diagnostic/prognostic potential and those closely associated with melanoma onset. Proteomic profiling of EVs derived from cells overexpressing MGAT3 and MGAT5 revealed functional changes in EV protein content driven by glycosylation modifications. The study presented a potential multifaced application of melanoma-derived EVs for diagnostic and prognostic purposes.

Single-cell proteomics (SCPs) has advanced significantly, yet it remains largely unidimensional, focusing primarily on protein abundances. In this study, we employed a pulsed stable isotope labeling by amino acids in cell culture (pSILAC) approach to simultaneously analyze protein abundance and turnover in single cells (SC-pSILAC). Using a state-of-the-art SCP workflow, we demonstrated that two SILAC labels are detectable from ∼4,000 proteins in single HeLa cells recapitulating known biology. We performed a large-scale time-series SC-pSILAC analysis of undirected differentiation of human induced pluripotent stem cells (iPSCs) encompassing 6 sampling times over 2 months and analyzed >1,000 cells. Protein turnover dynamics highlighted differentiation-specific co-regulation of protein complexes with core histone turnover, discriminating dividing and non-dividing cells. Lastly, correlating cell diameter with the abundance of individual proteins showed that histones and some cell-cycle proteins do not scale with cell size. The SC-pSILAC method provides a multidimensional view of protein dynamics in single-cell biology.

Amylase trypsin inhibitors (ATIs) potentially play a role in irritable bowel syndrome (IBS) and non-celiac wheat sensitivity (NCWS). These cereal-derived inhibitors are suspected to bind to the TLR4-MD2-CD14 complex and trigger intestinal pro-inflammatory responses, but confirmation through more extensive cell line studies is required. In this study, an amylase trypsin inhibitors enriched fraction (AEF) was prepared and characterized. Then, AEF binding potential to TLR4-MD2-CD14 was investigated using the human TLR4 reporter cell line HEK-BlueTM. The method took into account the presence of lipopolysaccharides (LPS) using Polymyxin B (PMB) to block LPS binding to TLR4. Proteinase K was also used to hydrolyze AEF proteins and eliminate their induced response. The cell line experiments showed that PMB treatment of AEF reduced the binding signal by 92 %. Complete hydrolysis of the protein by Proteinase K doubled the TLR4 activation signal and might indicate that protein-LPS complexation reduced LPS's ability to activate the TLR4-receptor. These finding underline the need for future work to consider the non-protein part in cell assays, especially the LPS bias. Altogether, these results indicated that LPS activates TLR4-MD2-CD14 and challenges ATIs' intestinal inflammation capacity contributing in irritable bowel syndrome and non-celiac wheat sensitivity.

Maintaining the proper balance between granulosa cells (Gc) proliferation and apoptosis is crucial for folliculogenesis and female fertility. Our previous study showed expression of omentin-1 (intelectin-1, ITLN1) in the porcine ovarian follicles; however, its impact on Gc functions remains unknown. Therefore, this study aimed to determine the in vitro effects of ITLN1 on Gc proliferation and apoptosis in Large White (LW) and Meishan (MS) pigs. These breeds were chosen due to their distinct reproductive characteristics: MS pigs are known for maintaining a higher number of follicles during the follicular phase and exhibiting greater estradiol synthesis compared to LW pigs. Porcine Gc were incubated with ITLN1 (10-100 ng/mL) for 24-72 h, and the viability/proliferation (alamarBlue/BrdU assays), cell cycle progression (flow cytometry) and the gene and protein expression of proliferation/apoptotic markers (PCNA, cyclins A1, B2, D1, E1, caspases-3, -9, BCL-2, BAX, FAS, FADD, XIAP) (real-time PCR, western blotting) were assessed. Next, the effect of ITLN1 on the phosphorylation of several kinases (AKT, AMPK, ERK1/2, STAT3, PKA) and the gene and protein expression of the insulin receptor (INSR) were studied (real-time PCR, western blotting). Then, using pharmacological inhibitors of ERK1/2 (PD98059, 5 μM), AKT (LY294002, 10 μM) and INSR (1 μM), treated alone or with ITLN1 (S961, 50 ng/mL), we analyzed its involvement in the effects of ITLN1 on Gc proliferation/apoptosis. We demonstrated that ITLN1 had a mitogenic effect on Gc by enhancing cell cycle progression and modulating the levels of PCNA, cyclins and apoptotic factors via ERK1/2, AKT, and INSR, suggesting that ITLN1 is a newly identified regulator in ovarian folliculogenesis, regardless of the fatness degree of pigs.

Lysine-specific demethylase 1 (LSD1) is a histone demethylase and regulator of differentiation, including in cancer. A neuronal-specific isoform of LSD1-LSD1+8a-has been shown to play a key role in promoting neuronal differentiation in the developing brain. We previously determined that LSD1+8a transcripts were detected in an aggressive subtype of prostate cancer harboring a neuronal program-neuroendocrine prostate cancer (NEPC)-but not in prostate adenocarcinomas harboring a glandular program. However, the number of samples examined was limited.

Using a large collection of prostate cancer patient cell lines and patient-derived xenografts (PDXs), we measured LSD1+8a using quantitative polymerase chain reaction (qPCR), RNA in situ hybridization (RNA-ISH), and protein detection methods. We then validated our findings using an independent cohort of patient tumor samples.

LSD1+8a mRNA expression was detected in every NEPC cell line and PDX examined by qPCR and RNA-ISH but in none of the prostate adenocarcinomas. We validated the RNA-ISH results in patient tumors, confirming that LSD1+8a was expressed in all NEPC tumors but in none of the adenocarcinomas. Finally, we generated a rabbit monoclonal antibody specific to LSD1+8a protein and confirmed its specificity using normal neuronal tissue samples. However, LSD1+8a protein was not detectable in NEPC tumors-likely due to the substantially lower levels of LSD1+8a mRNA in NEPC tumors vs. normal neuronal tissues.

Measuring LSD1+8a mRNA is a sensitive and specific method for the diagnosis of NEPC, which is often challenging.

Glecirasib potently and selectively inhibits KRAS G12C and reduces ERK and AKT phosphorylation in KRAS G12C-mutant cancer cells, further inducing cell-cycle arrest and apoptosis. Glecirasib monotherapy leads to tumor regression in KRAS G12C-mutant animal models and shows synergistic effects with cetuximab or JAB-3312 (sitneprotafib).

Huntington's disease (HD) is a debilitating neurodegenerative disorder affecting an individual's cognitive and motor abilities. HD is caused by a mutation in the huntingtin gene producing a toxic polyglutamine-expanded protein (mHTT) and leading to degeneration in the striatum and cortex. Yet, the molecular signatures that underlie tissue-specific vulnerabilities remain unclear. Here, we investigate this aspect by leveraging multi-epitope protein interaction assays, subcellular fractionation, thermal proteome profiling, and genetic modifier assays. The use of human cell, mouse, and fly models afforded capture of distinct subcellular pools of epitope-enriched and tissue-dependent interactions linked to dysregulated cellular pathways and disease relevance. We established an HTT association with nearly all subunits of the transcriptional regulatory Mediator complex (20/26), with preferential enrichment of MED15 in the tail domain. Using HD and KO models, we find HTT modulates the subcellular localization and assembly of the Mediator. We demonstrated striatal enriched and functional interactions with regulators of calcium homeostasis and chromatin remodeling, whose disease relevance was supported by HD fly genetic modifiers assays. Altogether, we offer insights into tissue- and localization-dependent (m)HTT functions and pathobiology.

Do the levels of adipokines (adiponectin, apelin, chemerin and vaspin) in plasma, hypothalamus, ovaries and periovarian adipose tissue differ during polycystic ovarian syndrome (PCOS)?

The PCOS was induced in rats by oral administration of non-steroidal aromatase inhibitor letrozole. To determine the plasma levels of adiponectin, apelin, chemerin and vaspin enzyme-linked immunosorbent assays were carried out. To assess the expression (gene and protein) and immunolocalization of these adipokines and their receptors, namely Adipor1 and Adipor2 for adiponectin, Aplnr for apelin, Ccrl2, Cmklr1 and Gpr1 for chemerin and Grp78 for vaspin in the hypothalamus, real-time polymerase chain reaction, and Western blot and immunohistochemistry were used to analyse ovaries and periovarian adipose tissue respectively.

In PCOS, the plasma level of adiponectin decreased (P = 0.0003), whereas apelin, chemerin and vaspin increased (P ≤ 0.0479). Moreover, PCOS modulates the expression of adipokines and their receptors in the hypothalamus, ovaries and periovarian adipose tissue compared with healthy rats (P ≤ 0.487).

A strong relationship was found between PCOS and adipokines, which suggests that adipokines may be a biomarker of PCOS.

Chinese hamster ovary (CHO) cells are major expression platforms for the transient production of recombinant therapeutic proteins (RTPs). Most improvement strategies have focused on promoting transcriptional expression in CHO cells. However, methods for promoting the yield of RTPs through translational regulation remain unclear. In this study, we investigated characteristics of the 5'-untranslated region (UTR) that influence recombinant protein expression in CHO cells and identified sequences that have positive effects on protein expression using ribosome sequencing. Some elements and characteristics of 5'-UTR differentially affected the translation of the main open reading frame and increased recombinant protein expression by 1.5-fold in CHO cells. The findings may help relieve the bottleneck of the yield of RTPs on translation enhancement.

Changes in gene expression are a key driver of phenotypic evolution, leading to a persistent interest in the evolution of transcriptomes. Traditionally, gene expression is modeled as a continuous trait, leaving qualitative transitions largely unexplored. In this paper, we detail the development of new Bayesian inference techniques to study the evolutionary turnover of organ-specific transcriptomes, which we define as instances where orthologous genes gain or lose expression in a particular organ. To test these techniques, we analyze the transcriptomes of 2 male reproductive organs, testes and accessory glands, across 11 species of the Drosophila melanogaster species group. We first discretize gene expression states by estimating the probability that each gene is expressed in each organ and species. We then define a phylogenetic model of correlated transcriptome evolution in 2 or more organs and fit it to the expression state data. Inferences under this model imply that many genes have gained and lost expression in each organ, and that the 2 organs experienced accelerated transcriptome turnover on different branches of the Drosophila phylogeny.

Collagen XII (COL12A1) is a type of FACIT collagen that plays an important role in the extracellular matrix structuring, participating in the regulation of collagen fiber size, and serves as a link between different components of the extracellular matrix. However, it is still unclear whether exogenous administration of collagen XII has a direct regulatory effect. In this study, we successfully produced recombinant human XII-type collagen (rh12C) through genetic engineering approach, which is composed of different functional domains. A Pichia pastoris host cell strain was constructed based on the intracellular translation regulatory mechanism of collagen, achieving a maximum yield of 4.89 g/L. After purification and structural characterization of the protein, its potential biological efficacy was evaluated through in vitro cell experiments.