Regulatory Proteins Research Articles

Abstract We113: Intravascular Theranostic Photoactivation Guided By OCT-NIRF Dual-Modal Imaging Facilitates Inflammation Resolution of High-Risk Plaque

Introduction and Hypothesis: Photoactivation eliminates inflammatory cells and promotes healing, paving the way for atherosclerosis intervention. We hypothesized that intravascular macrophage-targeted near-infrared fluorescence (NIRF) emitting photoactivation could facilitate inflammation resolution and stabilize the inflamed high-risk plaques. Method and Results: We newly fabricated a Dectin-1-targeted photoactivatable theranostic agent through the chemical conjugation of the NIRF-emitting photosensitizer chlorin e6 (Ce6) and the Dectin-1 ligand laminarin (LAM-Ce6). By intravascular OCT-NIRF structural-molecular imaging at 0- vs 4 weeks, LAM-Ce6 under light irradiation through a customized intravascular fiber effectively attenuated inflammatory NIRF signals, resulting in replacement of fibrotic composition in the targeted plaques (Figure A, P <0.01). Mechanistically, one hour after the light irradiation, autophagy was strongly augmented with the formation of autophagolysosomes (Figure B, P <0.001) and an increase of CD206-positive cells at 7 days. Furthermore, TUNEL/RAM11- and CD206/MerTK-positive cells were prominently increased at 1 day, suggestive of enhanced efferocytosis in the treated plaques (Figure C, the white arrow: macrophage associated apoptotic cells, P <0.05). In line with inflammation resolution, photoactivation enhanced the cholesterol efflux regulatory protein ABCA1 and induced TGF-β in macrophages, which subsequently stimulated smooth muscle cells and produced collagen within the lesions (Figure D). Conclusions: The macrophage Dectin-1 targetable intravascular photoactivation assisted by OCT-NIRF catheter imaging could ultimately transit the plaque phenotype to collagen-rich fibrotic one by autophagy-mediated inflammation resolution with a fibrotic replacement. This novel targeted photoactivation is expected to offer new opportunity for the catheter-based theranostic strategy in patients with macrophage/lipid-rich inflamed coronary plaques.

Abstract Or101: Phospho-Mimic Mutation in Beta Tubulin Suppresses Arrhythmias and Improves Cardiac Connexin 43 Localization in Duchenne Muscular Dystrophy Mice

Duchenne Muscular Dystrophy (DMD) is a severe X-linked neuromuscular disorder, causing progressive muscle degeneration and premature death due to heart and skeletal muscle dysfunction. It is caused by mutations in the dmd gene, leading to the absence of dystrophin, a crucial protein for muscle integrity. There is no cure for DMD. Its associated dystrophic cardiomyopathy is the primary cause of mortality by age 20. Connexin-43 (Cx43) is a gap junction protein responsible for electrical signaling in the heart. Cx43 localizes at the intercalated disc of the cardiomyocyte (CM), forming an intercellular channel with adjacent CMs; In DMD, Cx43 hemichannel remodels to the lateral side of the cardiomyocyte. Our previous research indicated that phospho-mimic Cx43 carrying serine to glutamic acid substitutions at the triplet regulatory site S325, S328, S330 in mdx hearts suppressed Cx43 lateralization and improved cardiac rhythm. The phospho-mimic Cx43 also reduced pathological hyperdensity and transverse directionality of microtubules in mdx cardiomyocytes. Colchicine (Colch) treatment, known to inhibit microtubule polymerization, also improved cardiac rhythm and Cx43 localization in mdx mice. To phenocopy the effect observed with Colch, we generated a mouse model with a beta tubulin mutation at serine 172, which was replaced by glutamic acid (S172E). We found reduced arrhythmias and improved localization of Cx43 in mdx hearts carrying the S172E phospho-mimic mutant tubulin by electrocardiography and immunofluorescent staining, respectively. Moreover, the proteomics data showed that the level of phospho-Cx43 was also upregulated in mdx hearts with S172E tubulin mutation. Further investigation will explore how this mutation impacts protein regulation and electrophysiology, and elucidate its mechanism of action in microtubule polymerization. These studies investigate a regulatory link between microtubule dynamics and localization of Cx43, potentially suggesting a novel therapeutic target for DMD-associated cardiomyopathy.

Impact of protein and small molecule interactions on kinase conformations.

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

Impact of protein and small molecule interactions on kinase conformations

Protein kinases act as central molecular switches in the control of cellular functions. Alterations in the regulation and function of protein kinases may provoke diseases including cancer. In this study we investigate the conformational states of such disease-associated kinases using the high sensitivity of the kinase conformation (KinCon) reporter system. We first track BRAF kinase activity conformational changes upon melanoma drug binding. Second, we also use the KinCon reporter technology to examine the impact of regulatory protein interactions on LKB1 kinase tumor suppressor functions. Third, we explore the conformational dynamics of RIP kinases in response to TNF pathway activation and small molecule interactions. Finally, we show that CDK4/6 interactions with regulatory proteins alter conformations which remain unaffected in the presence of clinically applied inhibitors. Apart from its predictive value, the KinCon technology helps to identify cellular factors that impact drug efficacies. The understanding of the structural dynamics of full-length protein kinases when interacting with small molecule inhibitors or regulatory proteins is crucial for designing more effective therapeutic strategies.

EPInformer: A Scalable Deep Learning Framework for Gene Expression Prediction by Integrating Promoter-enhancer Sequences with Multimodal Epigenomic Data.

Transcriptional regulation, critical for cellular differentiation and adaptation to environmental changes, involves coordinated interactions among DNA sequences, regulatory proteins, and chromatin architecture. Despite extensive data from consortia like ENCODE, understanding the dynamics of cis-regulatory elements (CREs) in gene expression remains challenging. Deep learning is a powerful tool for learning gene expression and epigenomic signals from DNA sequences, exhibiting superior performance compared to conventional machine learning approaches. However, even the most advanced deep learning-based methods may fall short in capturing the regulatory effects of distal elements such as enhancers, limiting their predictive accuracy. In addition, these methods may require significant resources to train or to adapt to newly generated data. To address these challenges, we present EPInformer, a scalable deep-learning framework for predicting gene expression by integrating promoter-enhancer interactions with their sequences, epigenomic signals, and chromatin contacts. Our model outperforms existing gene expression prediction models in rigorous cross-chromosome validation, accurately recapitulates enhancer-gene interactions validated by CRISPR perturbation experiments, and identifies crucial transcription factor motifs within regulatory sequences. EPInformer is available as open-source software at https://github.com/pinellolab/EPInformer.

Understanding Inherited Bleeding Disorders: Genetic Mutations in Blood Coagulation Factors and Regulatory Proteins

Understanding Inherited Bleeding Disorders: Genetic Mutations in Blood Coagulation Factors and Regulatory Proteins

Potential use of HSP70 as an indicator of heat stress in dairy cows – a review

Heat stress (HS) poses significant challenges to the dairy industry, resulting in reduced milk production, impaired reproductive performance, and compromised animal welfare. Therefore, understanding the molecular mechanisms underlying cellular responses to HS is crucial for developing effective strategies to mitigate its adverse effects. Heat shock protein 70 (HSP70) has emerged as a potential player involved in cellular thermotolerance in dairy cows. This review provides a comprehensive overview of the role of HSP70 as a molecular chaperone in cellular thermotolerance in dairy cows under HS. HSP70 facilitates proper protein folding and prevents the aggregation of denatured proteins. By binding to misfolded proteins, it helps maintain protein homeostasis and prevents the accumulation of damaged proteins during HS. Additionally, HSP70 interacts with various regulatory proteins and signaling pathways, contributing to the cellular adaptive response to HS. The upregulation of HSP70 expression in response to HS is regulated by a complex network involving heat-shock factors (HSFs), heat-shock element-binding proteins, and HSF co-chaperones. Therefore, HSP70 holds the potential to be a useful indicator of tissue stress due to its role in maintaining cellular balance, and as it is released both inside and outside cells in response to stress. Traditional methods of measuring HSP70 in blood samples are labor-intensive, and with the process being potentially stressful for the animals and may subsequently affect the results. Therefore, measuring HSP expression in cow's milk has shown promise as an easy, non-invasive, and accurate way to detect HS in dairy cows. Monitoring HSP70 levels in milk can be applied as a supplementary approach to identify HS or HS resistance of individual cows, selection of suitable animals and to guide targeted management strategies. However, despite the potential advantages of using HSP70 as a biomarker for monitoring HS on dairy cows, challenges remain in standardizing measurement protocols, establishing species-specific reference ranges, addressing inter-individual variations, and determining the specificity of changes in HSP70 due to HS. Future research should focus on developing non-invasive techniques for HSP70 detection, with consideration of climatic conditions, and unravelling the molecular interactions and regulatory networks involving HSP70.

Genome-wide identification and characterization of SNW/SKIP domain-containing proteins in plants.

Sessile organisms, such as plants, developed various ways to sense and respond to external and internal stimuli to maximize their fitness through evolutionary time. Transcripts and protein regulation are, among many, the main mechanisms that plants use to respond to environmental changes. SKIP protein is one such, presenting an SNKW interacting domain, which is highly conserved among eukaryotes, where SKI interacting protein acts in regulating key processes. In the present work, many bioinformatics tools, such as phylogenetic relationships, gene structure, physical-chemical properties, conserved motifs, prediction of regulatory cis-elements, chromosomal localization, and protein-protein interaction network, were used to better understand the genome-wide SNW/SKIP domain-containing proteins. In total, 28 proteins containing the SNW/SKIP domain were identified in different plant species, including plants of agronomic interest. Two main protein clusters were formed in phylogenetic analysis, and gene structure analysis revealed that, in general, the coding region had no introns. Also, expression of these genes is possibly induced by abiotic stress stimuli. Primary structure analysis of the proteins revealed the existence of an evolutionarily conserved functional unit. But physicochemical properties show that proteins containing the SNW/SKIP domain are commonly unstable under in vivo conditions. In addition, the protein network, demonstrated that SKIP homologues could act by modulating plant fitness through gene expression regulation at the transcriptional and post-transcriptional levels. This could be corroborated by the expression number of gene copies of SKIP proteins in many species, highlighting it's crucial role in plant development and tolerance through the course of evolution.

Studies on the Comparative Response of Fibers Obtained from the Pastazzo of Citrus bergamia and Cladodes of Opuntia ficus-indica on In Vitro Model of Neuroinflammation.

Adhering to a healthy diet has a protective effect on human health, including a decrease in inflammatory diseases due to consuming fiber. The purpose of this manuscript was to obtain and compare two extracts based on fiber (BF and IF-C), derived from two plants particularly present in the Mediterranean region: bergamot (Citrus bergamia) and prickly pear (Opuntia ficus-indica). The parts used by these plants have been the "pastazzo" for the bergamot and the cladodes for the prickly pear. In addition to in vitro evaluations, the antioxidant activity was also measured on human neurons under inflammatory conditions. Furthermore, the extracts of interest were examined for their effects on the cell cycle and the regulation of pro-apoptotic proteins, caspase 9 and 3, induced by LPS. The results indicated that both extracts had a protective effect against LPS-induced damage, with BF consistently exhibiting superior functionality compared to IF-C. Moreover, the extracts can reduce inflammation, which is a common process of disease. By exploring this avenue, studying the consumption of dietary fiber could enhance our understanding of its positive effects, but additional experiments are needed to confirm this.

Examination of Akt and GSK3β in BDNF-mediated reductions in BACE1 activity in neuronal cells.

Brain-derived neurotrophic factor (BDNF) content and signaling has been identified as one potential regulator of amyloid precursor protein (APP) processing. Recently published work has demonstrated that BDNF reduces BACE1 activity while also elevating the inhibition of GSK3β in the prefrontal cortex of male C57BL/6J mice. These results provide evidence that BDNF alters APP processing by reducing BACE1 activity, which may act through GSK3β inhibition. The purpose of this study was to further explore the role of GSK3β in BDNF-induced regulation on BACE1 activity. We utilized a cell culture and an invitro activity assay model to pharmacologically target BDNF and GSK3β signaling to confirm its involvement in the BDNF response. Treatment of differentiated SH-SY5Y neuronal cells with 75 ng/mL BDNF resulted in elevated pTrkB content, pAkt content, pGSK3β content, and reduced BACE1 activity. An invitro BACE1 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF reduced BACE1 activity; however, in the presence of TrkB or Akt inhibition, this effect was abolished. An invitro ADAM10 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF did not alter ADAM10 activity. However, inhibiting BDNF signaling reduced ADAM10 activity. Collectively these studies suggest that GSK3β inhibition may be necessary for BDNF-induced reductions in BACE1 activity. These findings will allow for the optimization of future therapeutic strategies by selectively targeting TrkB activation and GSK3β inhibition.

Conformational Dynamics, Energetics, and the Divergent Evolution of Allosteric Regulation: The Case of the Yeast MAPK Family.

Allosteric mechanisms provide finely-tuned control over signalling proteins. Proteins of the same family may share high sequence identity and structural similarity but show distinct traits of allosteric control and evolutionary divergent regulation. Revealing the determinants of such properties may be important to understand the molecular bases of different regulatory pathways. Herein, we investigate whether and how evolutionarily-divergent traits of allosteric regulation in homologous proteins can be decoded in terms of internal dynamics and interaction networks that support functionally oriented conformations. In this framework, we start from the comparative analysis of the dynamics and energetics of the yeast MAP Kinases (MAPKs) Fus3 and Kss1 in their native basins. Importantly, distinctive dynamic and energetic stabilization features emerge, which can be related to the two proteins' differential ability to be phosphorylated and engage with the allosteric activator Ste5. We then expanded our study to other evolutionarily-related MAPKs. We show that the dynamical and energetical traits defining the distinct regulatory profiles of Fus3 and Kss1 can be traced along their evolutionary tree. Overall, our approach is able to reconnect (latent) allostery with the principal elements of protein structural stabilization and dynamics, showing how allosteric regulation was encrypted in MAPKs structure well before Ste5 appearance.

Sitagliptin synergizes 5-fluorouracil efficacy in colon cancer cells through MDR1-mediated flux impairment and down regulation of NFκB2 and p-AKT survival proteins.

5-fluorouracil (5-FU) is an inexpensive treatment for colon cancer; however, its efficacy is limited by chemoresistance. This study investigates the combination therapy approach of 5-FU with Sitagliptin (Sita), a diabetic drug with potential cancer-modulating effects. The combination was evaluated in vitro and in silico, focusing on the effects of Sita and 5-FU on colon cancer cells. The results showed that the addition of Sita significantly decreased the IC50 of 5-FU compared to 5-Fu monotherapy. The study also found that Sita and 5-FU interact synergistically, with a combination index below 1. Sita successfully lowered the 5-FU dosage reduction index, decreasing the expression of MDR1 mRNA and p-AKT and NFκB2 subunits p100/p52 protein. Molecular docking analyses confirmed Sita's antagonistic action on MDR1 and thymidylate synthase proteins. The study concludes that sitagliptin can target MDR1, increase apoptosis, and significantly reduce the expression of p-AKT and NFκB2 cell-survival proteins. These effects sensitize colon cancer cells to 5-FU. Repurposing sitagliptin may enhance the anticancer effects of 5-FU at lower dosages.

Knockout of Hsp70 genes significantly affects locomotion speed and gene expression in leg skeletal muscles of Drosophila melanogaster.

The functions of the heat shock protein 70 (Hsp70) genes were studied using a line of Drosophila melanogaster with a knockout of 6 of these genes out of 13. Namely, the effect of knockout of Hsp70 genes on negative geotaxis climbing (locomotor) speed and the ability to adapt to climbing training (0.5-1.5 h/day, 7 days/wk, 19 days) were examined. Seven- and 23-day-old Hsp70- flies demonstrated a comparable reduction (twofold) in locomotor speed and widespread changes in leg skeletal muscle transcriptome (RNA sequencing) compared with w1118 flies. To identify the functions of genes related to decreased locomotor speed, the overlapped differentially expressed genes at both time points were analyzed: the upregulated genes encoded extracellular proteins, regulators of drug metabolism, and the antioxidant response, whereas downregulated genes encoded regulators of carbohydrate metabolism and transmembrane proteins. In addition, in Hsp70- flies, activation of transcription factors related to disruption of the fibril structure and heat shock response (Hsf) was predicted, using the position weight matrix approach. In control flies, adaptation to chronic exercise training was associated mainly with gene response to a single exercise bout, whereas the predicted transcription factors were related to stress/immune (Hsf, NF-κB, etc.) and early gene response. In contrast, Hsp70- flies demonstrated no adaptation to training as well as a significantly impaired gene response to a single exercise bout. In conclusion, the knockout of Hsp70 genes not only reduced physical performance but also disrupted adaptation to chronic physical training, which is associated with changes in the leg skeletal muscle transcriptome and impaired gene response to a single exercise bout.NEW & NOTEWORTHY Knockout of six heat shock protein 70 (Hsp70) genes in Drosophila melanogaster reduced locomotion (climbing) speed that is associated with genotype-specific differences in leg skeletal muscle gene expression. Disrupted adaptation of Hsp70- flies to chronic exercise training is associated with impaired gene response to a single exercise bout.

New functions of pirin proteins and a 2-ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile.

The understanding of how central metabolism and fermentation pathways regulate antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is still incomplete. Our study reveals that B. fragilis encodes two iron-dependent, redox-sensitive regulatory pirin protein genes, pir1 and pir2. The mRNA expression of these genes increases when exposed to oxygen and during growth in iron-limiting conditions. These proteins, Pir1 and Pir2, influence the production of short-chain fatty acids and modify the susceptibility to metronidazole and amixicile, a new inhibitor of pyruvate: ferredoxin oxidoreductase in anaerobes. We have demonstrated that Pir1 and Pir2 interact directly with this oxidoreductase, as confirmed by two-hybrid system assays. Furthermore, structural analysis using AlphaFold2 predicts that Pir1 and Pir2 interact stably with several central metabolism enzymes, including the 2-ketoglutarate:ferredoxin oxidoreductases Kor1AB and Kor2CDAEBG. We used a series of metabolic mutants and electron transport chain inhibitors to demonstrate the extensive impact of bacterial metabolism on metronidazole and amixicile susceptibility. We also show that amixicile is an effective antimicrobial against B. fragilis in an experimental model of intra-abdominal infection. Our investigation led to the discovery that the kor2AEBG genes are essential for growth and have dual functions, including the formation of 2-ketoglutarate via the reverse TCA cycle. However, the metabolic activity that bypasses the function of Kor2AEBG following the addition of phospholipids or fatty acids remains undefined. Overall, our study provides new insights into the central metabolism of B. fragilis and its regulation by pirin proteins, which could be exploited for the development of new narrow-spectrum antimicrobials in the future.

Dietary carbohydrate to lipid ratio affects growth, reproductive performance and health of female yellow catfish (Pelteobagrus fulvidragrus): a lipidomics analysis

This study aimed to examine the impact of dietary carbohydrate to lipid (CHO/L) ratio on the growth, reproductive, and offspring performance of broodstock yellow catfish, and to elucidate the metabolic differences between mothers and offspring using lipidomics. Five isonitrogenous and isoenergetic diets with varying CHO/L ratios (0.65, 1.44, 2.11, 3.13, and 5.36) were fed to three groups of 35 female broodfish in a pond-cage culture system. After an eight-week feeding trial, the dietary CHO/L ratio had a significant impact on the growth and reproductive performance of female yellow catfish. The weight gain ratio (WGR) and specific growth rate (SGR) in the CHO/L0.65 and CHO/L2.11 groups were significantly higher than those in the CHO/L5.36 group (P < 0.05). The fertilization and hatching rates were the highest when the dietary CHO/L ratio was 0.65 and 2.11, respectively. When the dietary CHO/L ratio was 3.13 and 5.36, the plasma contents of testosterone (T) was significantly lower than those of other groups (P = 0.013), and the plasma vitellogenin (VTG) content was the lowest when the CHO/L ratio was 5.36. The plasma contents of estradiol (E2) significantly decreased with increasing dietary CHO/L ratio (PL = 0.012). Lipidomic analysis revealed that the ovary primarily consisted of five subclasses in terms of lipid composition, namely triglyceride, fatty acyl, sterol, glycerophospholipid, and sphingolipid; however, sphingolipids were not detected in the larvae. The relative expression levels of the ovarian lipid metabolism-related genes sterol regulatory element binding protein 1 (srebp1), acetyl-coa carboxylase (acc), delta (12)-oleate desaturase (fad2), and elongation of very long chain fatty acids protein 5 (elvol5) significantly increased with increasing dietary CHO/L ratio (P < 0.05). The relative expression levels of lipid metabolism-related genes srebp 1, peroxisome proliferator activated receptor α (pparα), carnitine palmitoyl transferase 1 isoform (cpt), adipose triglyceride lipase (atgl), fad2, and elvol5 in offspring larvae were initially increased and then decreased with increasing dietary CHO/L ratios until reaching a maximum at a ratio of 2.11 (P < 0.05). In conclusion, based on the broken-line regression of the dietary CHO/L ratio and egg diameter, the optimal dietary CHO/L ratio was 1.91 for broodfish yellow catfish. A high-CHO/L ratio diet results in increased lipogenesis and hepatic lipid accumulation in maternal organisms, leading to impaired reproductive performance and reduced offspring quality.

Inhibition of the sterol regulatory element binding protein (SREB-P) SREBF-1 overcomes docetaxel resistance in advanced prostate cancer.

Resistance to anti-androgens and chemotherapy (Cx) limits therapeutic options for patients with hormone-sensitive (mHSPC) and hormone-resistant prostate cancer (mCRPC). In this context, upregulation of the glucocorticoid receptor (GR) has been identified as a potential bypass mechanism in mCRPC, and a combination of docetaxel and mifepristone (Doc + RU-486), an inhibitor of GR, re-sensitized docetaxel-resistant cell models to Cx. This study was designed to elucidate the molecular mechanisms responsible for this phenomenon. RNA sequencing (RNA-seq) in docetaxel-resistant PCa cell models after Doc + RU-486 treatment with consecutive functional assays were performed. Expression of selected proteins was verified in prostatic tissue from prostate cancer (PCa) patients with progressive disease. Treatment with Doc + RU-486 significantly reduced cancer cell viability and RNA-Seq. revealed sterol regulatory element of binding transcription factor 1 (SREBF-1), a transcription factor of cholesterol and lipid biosynthesis, as a significantly downregulated target. Functional assays confirmed that SREBF-1 downregulation is partially responsible for this observation. In concordance, SREBF-1 knockdown and pharmacological SREBP inhibition, together with other key enzymes in the cholesterol pathway, showed similar results. Furthermore, SREBF-1 expression is significantly elevated in advanced PCa tissues, demonstrating its potential involvement in tumor progression and emerging therapy resistance. Therefore, specific inhibition of cholesterol and lipid biosynthesis might also target Cx-resistant cancer cells and represent a potential additive future therapeutic option to improve mCRPC therapy.

Next generation triplex-forming PNAs for site-specific genome editing of the F508del CFTR mutation

BackgroundCystic Fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein for which there is no cure. One approach to cure CF is to correct the underlying mutations in the CFTR gene. We have used triplex-forming peptide nucleic acids (PNAs) loaded into biodegradable nanoparticles (NPs) in combination with donor DNAs as reagents for correcting mutations associated with genetic diseases including CF. Previously, we demonstrated that PNAs induce recombination between a donor DNA and the CFTR gene, correcting the F508del CFTR mutation in human cystic fibrosis bronchial epithelial cells (CFBE cells) and in a CF murine model leading to improved CFTR function with low off-target effects, however the level of correction was still below the threshold for therapeutic cure. MethodsHere, we report the use of next generation, chemically modified gamma PNAs (γPNAs) containing a diethylene glycol substitution at the gamma position for enhanced DNA binding. These modified γPNAs yield enhanced gene correction of F508del mutation in human bronchial epithelial cells (CFBE cells) and in primary nasal epithelial cells from CF mice (NECF cells). ResultsTreatment of CFBE cells and NECF cells grown at air-liquid interface (ALI) by NPs containing γtcPNAs and donor DNA resulted in increased CFTR function measured by short circuit current and improved gene editing (up to 32 %) on analysis of genomic DNA. ConclusionsThese findings provide the basis for further development of PNA and NP technology for editing of the CFTR gene.

A Multiplexed SEC-MS Approach to Systematically Study the Interplay Between Protein Assembly-States and Phosphorylation Events.

Protein molecular interactions and post-translational modifications (PTMs), such as phosphorylation, can be co-dependent and reciprocally co-regulate each other. Although this interplay is central for many biological processes, a systematic method to simultaneously study assembly-states and PTMs from the same sample is critically missing. Here, we introduce SEC-MX (Size Exclusion Chromatography fractions MultipleXed), a global quantitative method combining Size Exclusion Chromatography and PTM-enrichment for simultaneous characterization of PTMs and assembly-states. SEC-MX enhances throughput, allows phosphopeptide enrichment, and facilitates quantitative differential comparisons between biological conditions. Applying SEC-MX to HEK293 and HCT116 cells, we generated a proof-of-concept dataset mapping thousands of phosphopeptides and their assembly-states. Our analysis revealed intricate relationships between phosphorylation events and assembly-states and generated testable hypotheses for follow-up studies. Overall, we establish SEC-MX as a valuable tool for exploring protein functions and regulation beyond abundance changes.

Proteomic Profile of Endometrial Cancer: A Scoping Review.

Proteomics can be a robust tool in protein identification and regulation, allowing the discovery of potential biomarkers. In clinical practice, the management of endometrial cancer can be challenging. Thus, identifying promising markers could be beneficial, helping both in diagnosis and prognostic stratification, even predicting the response to therapy. Therefore, this manuscript systematically reviews the existing evidence of the proteomic profile of human endometrial cancer. The literature search was conducted via Medline (through PubMed) and the Web of Science. The inclusion criteria were clinical, in vitro, and in vivo original studies reporting proteomic analysis using all types of samples to map the human endometrial cancer proteome. A total of 55 publications were included in this review. Most of the articles carried out a proteomic analysis on endometrial tissue, serum and plasma samples, which enabled the identification of several potential diagnostic and prognostic biomarkers. In addition, eight articles were analyzed regarding the identified proteins, where three studies showed a strong correlation, sharing forty-five proteins. This analysis also allowed the identification of the 10 most frequently reported proteins in these studies: EGFR, PGRMC1, CSE1L, MYDGF, STMN1, CASP3 ANXA2, YBX1, ANXA1, and MYH11. Proteomics-based approaches pointed out potential diagnostic and prognostic candidates for endometrial cancer. However, there is a lack of studies exploring novel therapeutic targets.

Methyltransferase DNMT3B promotes colorectal cancer cell proliferation by inhibiting PLCG2.

Aberrant DNA methylation patterns in the promoter region of PLCG2 are associated with dysregulated signaling pathways and cellular functions. Its role in colorectal cancer cells is still unknown. In this study, qRT-PCR is used to measure DNMT3B expression in colorectal cancer. Western blot analysis and immunohistochemistry are used to analyze DNMT3B and PLCG2 protein levels in colorectal tissues and cell lines. Cell Counting Kit-8 (CCK-8) and colony formation assays are used to assess the proliferation of colorectal cancer cells. Methylation-specific PCR (MSP) and bisulfite-sequencing PCR (BSP) are used to measure DNA methylation level. Our results show that DNMT3B is overexpressed in colorectal cells in the TCGA datasets according to Kaplan-Meier plots. DNMT3B is significantly overexpressed in tumor tissues compared to that in adjacent nontumor tissues. Western blot analysis results demonstrate high expression of DNMT3B in tumor tissues. Compared to normal colonic epithelial cells, colorectal cancer cell lines exhibit elevated level of PLCG2 methylation. Overexpression of PLCG2 effectively prevents the growth of colorectal cancer xenograft tumors in vivo. PLCG2 is identified as a key downstream regulatory protein of DNMT3B in colorectal cancer. DNMT3B inhibits PLCG2 transcription through methylation of the PLCG2 promoter region. DNMT3B controls colorectal cancer cell proliferation through PLCG2, which is useful for developing therapeutic approaches that target PLCG2 expression for the treatment of colorectal cancer.