Muscle tissue plays a vital role in the maintenance of overall health, contributing to essential body functions such as locomotion, respiration, blood circulation, and metabolic regulation [...].
Muscle tissue plays a vital role in the maintenance of overall health, contributing to essential body functions such as locomotion, respiration, blood circulation, and metabolic regulation [...].
Phage display is widely used in biomedical research. One of the great advantages of phage display is the specificity of the connection of a foreign peptide exposed outside the capsid to the intended target. Secondary detection systems, which are often laborious and costly, are required to identify and quantify the peptide/target interaction. In this study, we generated a novel dual-display phage to facilitate the detection and quantification of the peptide/target interaction. First, we generated a biotin-tagged phage by adding a small biotin-accepting peptide (sBT) to gene-3 of the M13K07 helper phage. Subsequently, we enhanced the M13K07 biotin-tagged phage by incorporating a selective peptide on gene-8, which is then exposed to the phage capsid. The exposed peptide acts as a probe to bind to a selective molecular target, whose interaction can be readily visualized thanks to the biotinylated phage. Our versatile dual-display phage exhibits high flexibility; by swapping the displayed peptide/probe, one can change the phage target while retaining the sBT gene in-frame with the pIII. We expect the generated biotin-tagged dual phages to be used as a multifunctional probe to couple with several streptavidin-biotin-based systems.
The androgen receptor (AR), a member of the nuclear steroid hormone receptor family of transcription factors, plays a crucial role not only in the development of the male phenotype but also in the development and growth of prostate cancer. While AR structure and AR interactions with coregulators and chromatin have been studied in detail, improving our understanding of AR function in gene transcription regulation, the spatio-temporal organization and the role of microscopically discernible AR foci in the nucleus are still underexplored. This review delves into the molecular mechanisms underlying AR foci formation, focusing on liquid-liquid phase separation and its role in spatially organizing ARs and their binding partners within the nucleus at transcription sites, as well as the influence of 3D-genome organization on AR-mediated gene transcription.
Ex vivo fusion confocal microscopy (EVFCM) enables the rapid examination of breast tissue and has the potential to reduce the surgical margins and the necessity for further surgeries. Traditional methods, such as frozen section analysis, are limited by the distortion of tissue and artefacts, leading to false negatives and the need for additional surgeries. This study on observational diagnostic accuracy evaluated the ability of EVFCM to detect breast cancer. A total of 36 breast tissue samples, comprising 20 non-neoplastic and 16 neoplastic cases, were analysed using EVFCM and compared to the results obtained from routine histopathology. A Mohs surgeon experienced in EVFCM (evaluator A) and two breast pathologists unfamiliar with EVFCM (evaluators B and C) performed blinded analyses. EVFCM showed high concordance with the histopathology and the detection of neoplasia, with significant kappa values (p < 0.001). Evaluator A achieved 100% sensitivity and specificity. Evaluators B and C achieved a sensitivity of >87%, a specificity of >94%, positive predictive values of >95%, and negative predictive values of 81% and 94%, respectively. EVFCM therefore offers a promising technique for the assessment of margins in breast-conserving surgery. Its widespread adoption could significantly reduce re-excisions, lower healthcare costs, and improve cosmetic and oncological outcomes.
Background: Humanized mice transplanted with CD34+ hematopoietic cells (HPCs) are broadly used to study human immune responses and infections in vivo and for testing therapies pre-clinically. However, until now, it was not clear whether interactions between the mouse major histocompatibility complexes (MHCs) and/or the human leukocyte antigens (HLAs) were necessary for human T-cell development and immune reactivity.
Methods: We evaluated the long-term (20-week) human hematopoiesis and human T-cell development in NOD Scid Gamma (NSG) mice lacking the expression of MHC class I and II (NSG-DKO). Triplicate experiments were performed with HPCs obtained from three donors, and humanization was confirmed in the reference strain NOD Rag Gamma (NRG). Further, we tested whether humanized NSG-DKO mice would respond to a lentiviral vector (LV) systemic delivery of HLA-A*02:01, HLA-DRB1*04:01, human GM-CSF/IFN-α, and the human cytomegalovirus gB antigen.
Results: Human immune reconstitution was detectable in peripheral blood from 8 to 20 weeks after the transplantation of NSG-DKO. Human single positive CD4+ and CD8+ T-cells were detectable in lymphatic tissues (thymus, bone marrow, and spleen). LV delivery harnessed the detection of lymphocyte subsets in bone marrow (αβ and γδ T-cells and NK cells) and the expression of HLA-DR. Furthermore, RNA sequencing showed that LV delivery increased the expression of different human reactome pathways, such as defense responses to other organisms and viruses.
Conclusions: Human T-cell development and reactivity are independent of the expression of murine MHCs in humanized mice. Therefore, humanized NSG-DKO is a promising new model for studying human immune responses, as it abrogates the xenograft mouse MHC interference.
Heart failure is a significant health issue in developed countries, often stemming from conditions like hypertension, which imposes a pressure overload on the heart. Despite various treatment strategies for heart failure, many lack long-term effectiveness. A critical aspect of cardiac disease is the remodeling of the heart, where compensatory changes in the extracellular matrix exacerbate disease progression. This review explores the processes and changes occurring in the pressure-overloaded heart with respect to the extracellular matrix. It further summarizes current treatment strategies, and then focuses on novel treatment targets for maladaptive cardiac remodeling, derived from transverse aortic constriction-induced pressure overload animal models.
"Bubblegum" acyl-CoA synthetase (ACSBG1) is a pivotal player in lipid metabolism during mouse brain development, facilitating the activation of long-chain fatty acids (LCFA) and their incorporation into lipid species that are crucial for brain function. ACSBG1 converts LCFA into acyl-CoA derivatives, supporting vital metabolic processes. Fruit fly mutants lacking ACSBG1 exhibited neurodegeneration and had elevated levels of very long-chain fatty acids (VLCFA), characteristics of human X-linked adrenoleukodystrophy (XALD). To explore ACSBG1's function and potential as a therapeutic target in XALD, we created an ACSBG1 knockout (Acsbg1-/-) mouse and examined the effects on brain FA metabolism during development. Phenotypically, Acsbg1-/- mice resembled wild type (w.t.) mice. ACSBG1 expression was found mainly in tissue affected pathologically in XALD, namely the brain, adrenal gland and testis. ACSBG1 depletion did not significantly reduce the total ACS enzyme activity in these tissue types. In adult mouse brain, ACSBG1 expression was highest in the cerebellum; the low levels detected during the first week of life dramatically increased thereafter. Unexpectedly, lower, rather than higher, saturated VLCFA levels were found in cerebella from Acsbg1-/- vs. w.t. mice, especially after one week of age. Developmental changes in monounsaturated ω9 FA and polyunsaturated ω3 FA levels also differed between w.t. and Acsbg1-/- mice. ACSBG1 deficiency impacted the developmental expression of several cerebellar FA metabolism enzymes, including those required for the synthesis of ω3 polyunsaturated FA, precursors of bioactive signaling molecules like eicosanoids and docosanoids. These changes in membrane lipid FA composition likely affect membrane fluidity and may thus influence the body's response to inflammation. We conclude that, despite compelling circumstantial evidence, it is unlikely that ACSBG1 directly contributes to the pathology of XALD, decreasing its potential as a therapeutic target. Instead, the effects of ACSBG1 knockout on processes regulated by eicosanoids and/or docosanoids should be further investigated.
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that sense lipophilic molecules and act as transcription factors to regulate target genes. PPARs have been implicated in the regulation of innate immunity, glucose and lipid metabolism, cell proliferation, wound healing, and fibrotic processes. Some synthetic PPAR ligands are promising molecules for the treatment of inflammatory and fibrotic processes in immune-mediated intestinal diseases. Some of these are currently undergoing or have previously undergone clinical trials. Dietary PPAR ligands and changes in microbiota composition could modulate PPARs' activation to reduce inflammatory responses in these immune-mediated diseases, based on animal models and clinical trials. This narrative review aims to summarize the role of PPARs in immune-mediated bowel diseases and their potential therapeutic use.
NRAS belongs to the RAS family of GTPases. In colorectal cancer (CRC), NRAS mutations are rare compared to KRAS, but may lead to worse outcomes. We report the functional characterization of the novel NRAS mutants-G48C, Q43K, and E37K-identified in Filipino young-onset CRC patients. Unlike previously characterized NRAS mutants with no apparent effects on cell proliferation, these mutants enhanced proliferation of both HCT116 and NIH3T3 cells. This was confirmed in 3D spheroid assays to mimic the spatial organization of cells. G48C and E37K showed apoptosis resistance in both cell lines, and Q43K showed resistance in HCT116 cells. All three showed no effect on cellular migration in NIH3T3, but G48C enhanced the migration rate of HCT116 cells. Actin staining of NIH3T3 cells expressing the mutants showed a shrunken cytoplasm and transient structures associated with motility and invasiveness. Docking simulations show that GDP is only able to bind fully within the binding pocket of wild-type NRAS, but not in the mutants. Further, G48C, Q43K, and E37K all have less negative ΔG values, indicating a weaker GDP-binding affinity compared to wild-type NRAS. Taken together, the results suggest that oncogenic readouts of NRAS mutants are codon- and mutation-specific, with potential repercussions on the aggressiveness, resistance, and therapeutic response.
Interleukin-1 Receptor Associated Kinase 1 (IRAK1) is a serine/threonine kinase that plays a critical role as a signaling transducer of the activated Toll-like receptor (TLR)/Interleukin-1 receptor (IL-1R) signaling pathway in both immune cells and cancer cells. Upon hyperphosphorylation by IRAK4, IRAK1 forms a complex with TRAF6, which results in the eventual activation of the NF-κB and MAPK pathways. IRAK1 can translocate to the nucleus where it phosphorylates STAT3 transcription factor, leading to enhanced IL-10 gene expression. In immune cells, activated IRAK1 coordinates innate immunity against pathogens and mediates inflammatory responses. In cancer cells, IRAK1 is frequently activated, and the activation is linked to the progression and therapeutic resistance of various types of cancers. Consequently, IRAK1 is considered a promising cancer drug target and IRAK1 inhibitors have been developed and evaluated preclinically and clinically. This is a comprehensive review that summarizes the roles of IRAK1 in regulating metastasis-related signaling pathways of importance to cancer cell proliferation, cancer stem cells, and dissemination. This review also covers the significance of IRAK1 in mediating cancer resistance to therapy and the underlying molecular mechanisms, including the evasion of apoptosis and maintenance of an inflammatory tumor microenvironment. Finally, we provide timely updates on the development of IRAK1-targeted therapy for human cancers.