Biomolecules最新文献

The blastocyst develops a unique metabolism that facilitates the creation of a specialized microenvironment at the site of implantation characterized by high levels of lactate and reduced pH. While historically perceived as a metabolic waste product, lactate serves as a signaling molecule which facilitates the invasion of surrounding tissues by cancers and promotes blood vessel formation during wound healing. However, the role of lactate in reproduction, particularly at the implantation site, is still being considered. Here, we detail the biological significance of the microenvironment created by the blastocyst at implantation, exploring the origin and significance of blastocyst-derived lactate, its functional role at the implantation site and how understanding this mediator of the maternal-fetal dialogue may help to improve implantation in assisted reproduction.

Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer-related deaths globally, with a persistently low five-year survival rate of only 14-17%. High rates of metastasis contribute significantly to the poor prognosis of NSCLC, in which inflammation plays an important role by enhancing tumor growth, angiogenesis, and metastasis. Targeting inflammatory pathways within cancer cells may thus represent a promising strategy for inhibiting NSCLC metastasis. This study evaluated the anti-inflammatory and anti-metastatic properties of morin, a bioactive compound derived from a Thai medicinal herb, focusing on its effects on NLRP3 inflammasome-mediated pathways in an in vitro NSCLC model. The A549 and H1299 cell lines were stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP) to activate the NLRP3 pathway. The inhibition effects exhibited by morin in reducing pro-inflammatory secretion in LPS- and ATP-stimulated NSCLC cells were assessed by ELISA, while wound healing and trans-well invasion assays evaluated its impact on cell migration and invasion. RT-qPCR measurement quantified the expression of inflammatory genes, and zymography and Western blotting were used to examine changes in invasive protein levels, epithelial-to-mesenchymal transition (EMT) markers, and underlying molecular mechanisms. Our findings demonstrated the significant ability of morin to decrease the production of IL-1β, IL-18, and IL-6 in a dose-dependent manner (p < 0.05), as well as suppress NSCLC cell migration and invasion. Morin downregulated invasive proteins (MMP-2, MMP-9, u-PAR, u-PA, MT1-MMP) and EMT markers (fibronectin, N-cadherin, vimentin) (p < 0.01) while also reducing the mRNA levels of NLRP3, IL-1β, IL-18, and IL-6. Mechanistic investigations revealed that morin suppressed NLRP3 inflammasome activity and inactivated MAPK pathways. Specifically, it decreased the expression of NLRP3 and ASC proteins and reduced caspase-1 activity, while reducing the phosphorylation of ERK, JNK, and p38 proteins. Collectively, these findings suggest that morin's inactivation of the NLRP3 inflammasome pathway could offer a novel therapeutic strategy for counteracting pro-tumorigenic inflammation and metastatic progression in NSCLC.

Synthetic cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODNs) are promising candidates for vaccine adjuvants, because they activate immune responses through the Toll-like receptor 9 (TLR9) pathway. However, unmodified CpG ODNs are quickly degraded by serum nucleases, and their negative charge hinders cellular uptake, limiting their clinical application. Our group previously reported that guanine-quadruplex (G4)-forming CpG ODNs exhibit enhanced stability and cellular uptake. G4 structures can form in parallel, anti-parallel, or hybrid topologies, depending on strand orientation, but the effects of these topologies on CpG ODNs have not yet been explored. In this study, we designed three distinct G4 topologies as scaffolds for CpG ODNs. Among the three topology, the parallel G4 CpG ODN demonstrated the highest serum stability and cellular uptake, resulting in the strongest immune response from macrophage cells. Additionally, we investigated the binding affinities of the different G4 topologies to macrophage scavenger receptor-1 and TLR9, both of which are key to immune activation. These findings provide valuable insights into the development of CpG ODN-based vaccine adjuvants.

Myalgic Encephalomyelitis or Chronic Fatigue Syndrome (ME/CFS) is a chronic multisystem disease characterized by severe muscle fatigue, pain, dizziness, and brain fog. The two most common symptoms are post-exertional malaise (PEM) and orthostatic intolerance (OI). ME/CFS patients with OI (ME+OI) suffer from dizziness or faintness due to a sudden drop in blood pressure while maintaining an upright posture. Clinical research has demonstrated that patients with OI display severe cardiovascular abnormalities resulting in reduced effective blood flow in the cerebral blood vessels. However, despite intense investigation, it is not known why the effective cerebral blood flow is reduced in OI patients. Based on our recent findings, we observed that tetrahydrobiopterin (BH4) metabolism was highly dysregulated in ME+OI patients. In the current review article, we attempted to summarize our recent findings on BH4 metabolism to shed light on the molecular mechanisms of OI.

Cellular behavior is strongly influenced by mechanical signals in the surrounding microenvironment, along with external factors such as temperature fluctuations, changes in blood flow, and muscle activity, etc. These factors are key in shaping cellular states and can contribute to the development of various diseases. In the realm of rehabilitation physical therapies, therapeutic exercise and manual treatments, etc., are frequently employed, not just for pain relief but also to support recovery from diverse health conditions. However, the detailed molecular pathways through which these therapies interact with tissues and influence gene expression are not yet fully understood. The identification of YAP has been instrumental in closing this knowledge gap. YAP is known for its capacity to perceive and translate mechanical signals into specific transcriptional programs within cells. This insight has opened up new perspectives on how physical and rehabilitation medicine may exert its beneficial effects. The review investigates the involvement of the Hippo/YAP signaling pathway in various diseases and considers how different rehabilitation techniques leverage this pathway to aid in healing. Additionally, it examines the therapeutic potential of modulating the Hippo/YAP pathway within the context of rehabilitation, while also addressing the challenges and controversies that surround its use in physical and rehabilitation medicine.

The Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is a multidomain protein consisting of two protein-protein interaction domains, the Src homology 2 (SH2) domain, and the proline-rich region (PRR), as well as three phosphoinositide-binding domains, the pleckstrin homology-like (PHL) domain, the 5-phosphatase (5PPase) domain, and the C2 domain. SHIP1 is commonly known for its involvement in the regulation of the PI3K/AKT signaling pathway by dephosphorylation of phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P₃) at the D5 position of the inositol ring. However, the functional role of each domain of SHIP1 for the regulation of its enzymatic activity is not well understood. To determine the contribution of the individual domains to catalytic activity, the full-length protein was compared with truncated constructs lacking one or more domain(s), regarding the substrate turnover (k_cat) and catalytic efficiency (k_cat/K_m) towards ci8-PtdIns(3,4,5)P₃. With this approach, it was possible to verify the allosteric activation of SHIP1 mediated by the C2 domain as described previously, while the PHL domain seemed instead to have a negative effect regarding catalytic efficiency. The full-length SHIP1 clearly displayed the highest turnover and the second-highest catalytic efficiency, showing the role of the SH2 domain and PRR not only in protein-protein interactions but also in catalysis. The SH2 domain increased substrate turnover but negatively affected catalytic efficiency. The linker between the SH2 and the PHL domains decreased the turnover number but positively influenced the catalytic efficiency. The PRR increased both the substrate turnover and the protein's catalytic efficiency. The regression analysis of the Michaelis-Menten graph revealed SHIP1 to be an allosteric enzyme, with the PRR and the linker being the most involved domains in that regard. In summary, our data indicate a complex regulation of the enzymatic activity of SHIP1 by its individual domains. While the C2 domain and PRR at the carboxy-terminus have a positive effect on enzymatic activity, the SH2 and PHL domain at the amino-terminus inhibit catalytic efficiency.

Liver transplantation is the only curative option for end-stage liver disease and is necessary for an increasing number of patients with advanced primary or secondary liver cancer. Many patient groups can benefit from this treatment, however the shortage of liver grafts remains an unsolved problem. Liver bioengineering offers a promising method for expanding the donor pool through the production of acellular scaffolds that can be seeded with recipient cells. Decellularization protocols involve the removal of cells using various chemical, physical, and enzymatic steps to create a collagenous network that provides support for introduced cells and future vascular and biliary beds. However, the removal of the cells causes varying degrees of matrix damage, that can affect cell seeding and future organ performance. The main objective of this review is to present the existing techniques of producing decellularized livers, with an emphasis on the assessment and definition of acellularity. Decellularization agents are discussed, and the standard process of acellular matrix production is evaluated. We also introduce the concept of the stepwise assessment of the matrix during decellularization through decellularization cycles. This method may lead to shorter detergent exposure times and less scaffold damage. The introduction of apoptosis induction in the field of organ engineering may provide a valuable alternative to existing long perfusion protocols, which lead to significant matrix damage. A thorough understanding of the decellularization process and the action of the various factors influencing the final composition of the scaffold is essential to produce a biocompatible matrix, which can be the basis for further studies regarding recellularization and retransplantation.

The methylotrophic yeast Komagataella kurtzmanii belongs to the group of homothallic fungi that are able to spontaneously change their mating type by inversion of chromosomal DNA in the MAT locus region. As a result, natural and genetically engineered cultures of these yeasts typically contain a mixture of sexually dimorphic cells that are prone to self-diploidisation and spore formation accompanied by genetic rearrangements. These characteristics pose a significant challenge to the development of genetically stable producers for industrial use. In the present study, we constructed heterothallic strains of K. kurtzmanii, ensuring a constant mating type by unifying the genetic sequences in the active and silent MAT loci. To obtain such strains, we performed site-directed inactivation of one of the two yeast MAT loci, replacing its sequence with a selective HIS4 gene surrounded by I-SceI meganuclease recognition sites. We then used transient expression of the SCE1 gene, encoding a recombinant I-SceI meganuclease, to induce site-specific cleavage of HIS4, followed by damage repair by homologous recombination in mutant cells. As a result, heterothallic strains designated 'Y-727-2(alpha)' and 'Y-727-9(a)', which correspond to the α and a mating type, respectively, were obtained. The strains demonstrated a loss of the ability to self-diploidize. The results of PCR and whole genome analysis confirmed the identity of the contents of the MAT loci. Analysis of the genomes of the final strains, however, revealed a fusion of chromosome 3 and chromosome 4 in strain Y-727-2(alpha)-1. This finding was subsequently confirmed by pulsed-field gel electrophoresis of yeast chromosomes. However, the ability of the Y-727-2(alpha)-derived producers to efficiently secrete recombinant β-galactosidase was unaffected by this genomic rearrangement.

HtpB, the chaperonin of the bacterial pathogen L. pneumophila, is found in extracellular locations, even the cytoplasm of host cells. Although chaperonins have an essential cytoplasmic function in protein folding, HtpB exits the cytoplasm to perform extracellular virulence-related functions that support L. pneumophila's lifestyle. The mechanism by which HtpB reaches extracellular locations is not currently understood. To address this experimental gap, immunoelectron microscopy, trypsin-accessibility assays, and cell fractionation were used to localize HtpB in various L. pneumophila secretion mutants. Dot/Icm type IV secretion mutants displayed less surface-exposed HtpB and more periplasmic HtpB than parent strains. The analysis of periplasmic extracts and outer membrane vesicles of these mutants, where HtpB co-localized with bona fide periplasmic proteins, confirmed the elevated levels of periplasmic HtpB. Genetic complementation of the mutants recovered parent strain levels of surface-exposed and periplasmic HtpB. The export of GSK-tagged HtpB into the cytoplasm of infected cells was also Dot/Icm-dependent. The translocating role of the Dot/Icm system was not specific for HtpB because GroEL, the chaperonin of Escherichia coli, was found at the cell surface and accumulated in the periplasm of Dot mutants when expressed in L. pneumophila. These findings establish that a functional Dot/Icm system is required for HtpB to reach extracellular locations, but the mechanism by which cytoplasmic HtpB reaches the periplasm remains partially unidentified.

In the present work, we performed calculations of the kinetic isotope effect (KIE) on H/D, ¹⁴N/¹⁵N, ¹⁶O/¹⁸O, and ¹²C/¹³C isotopic substitution in the dissociation of beta-sheet polyglycine dimers of different lengths into two monomer chains. This dissociation reaction, proceeding via breaking of the interchain hydrogen bonds (H-bonds), is considered to be a model of unfolding of the secondary structure of proteins. The calculated strengthening of the interchain hydrogen bonds N-H⋯O=C due to heavy isotope substitution decreases in the row H/D >> ¹⁴N/¹⁵N > ¹⁶O/¹⁸O > ¹²C/¹³C. The KIE for H/D substitution, defined as the ratio of the rate constants k(H)k(D), was calculated with the use of a "completely loose" transition state model. The results of the calculations show that a very high H/D isotope effect can be achieved for proteins even with moderately long chains connected by dozens of interchain H-bonds. The results obtained also indicate that the heavy isotope substitution in the internal (interchain) and external H-bonds, located on the periphery of a dimer, can provide comparable effects on secondary structure stabilization.