Biochimica et Biophysica Acta (BBA) - General Subjects

Staphylococcus aureus (S. aureus) is a clinically relevant pathogen capable of adapting its membrane composition in response to environmental stress. In this adaptive process, bacterial carotenoids play a crucial role. Although staphyloxanthin (STX) is the main carotenoid produced by the bacterium, S. aureus also synthesizes other pigmented intermediates that play an unknown role in regulating membrane biophysical properties. In this study, we purified 4,4-diaponeurosporenoic acid (4,4'-DNPA) from S. aureus carotenoid extracts and evaluated its effect on the thermotropic and biophysical properties of representative membrane models. The highly rigid triterpenoid 4,4'-DNPA is one of the last precursors in the biosynthesis of STX and is found in high concentrations in the stationary phase of S. aureus. Phase transition temperatures were determined using infrared spectroscopy, while interfacial hydration and hydrophobic core dynamics were investigated using fluorescence spectroscopy through Laurdan generalized polarization and DPH anisotropy. The results show that 4,4'-DNPA increases the main phase transition temperature of lipid bilayers in a concentration-dependent manner. This is in contrast to STX that decreases the transition temperature. This difference is consistent with the additional fatty acid present in STX that changes its effect on the phase behavior. Furthermore, 4,4'-DNPA reduced the interfacial hydration levels and restricted hydrophobic-core dynamics at higher concentrations, consistent with increased molecular order and stability. 4,4'-DNPA therefore complements STX in increasing membrane order and lipid packing. These findings support the notion that the production of bacterial carotenoids functions as a biophysical regulatory mechanism of lipid packing in S. aureus membranes.

Saturated fatty acids such as stearic acid (SA) can exhibit both antimicrobial and growth-promoting effects on bacteria, depending on their concentration and chemical structure. However, the physical properties of the bacterial cell envelope in response to such molecules remain under-explored compared to their biochemical pathways. In this study, a comprehensive investigation is presented on the interaction of SA with the Gram-positive bacterium, Staphylococcus epider-midis (S. epi). SA alters bacterial growth, reflected in a higher maximum specific growth rate, a shorter lag phase, and an extended exponential phase, consistent with a prebiotic effect. Using fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy, we show that SA incorporation leads to significant fluidization of the lipid membrane, characterized by enhanced lateral diffusion and reduced membrane viscosity. Coarse-grained molecular dynamics (CG-MD) simulations demonstrate spontaneous insertion of SA into the membrane and a significant increase in mean-square displacement after insertion, supporting our experimental observations. Importantly, atomic force microscopy measurements show an increase in cell-envelope stiffness, reflected by a higher Youngs modulus which can be attributed to modulations in the glycan-peptide linkage density based on earlier studies that correlate stiffness changes to peptidoglycan (PG) crosslinking in Gram-positive strains [1]. These results provide direct evidence linking membrane fluidization induced by SA and increased cell wall stiffness due to transport modifications in the membrane mediated PG synthesis pathways to enhance bacterial cell viability.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a cytokine that plays a role in immune modulation. Its expression is associated with a multitude of different effects ranging from harmful, as in diseases such as rheumatoid arthritis and multiple sclerosis, to beneficial, as in the case of mitigation of diabetes type I and neutropenia. However, there is a large gap in knowledge explaining how GM-CSF toggles its structure for such physiological and pathological interactions. Our work describes an ongoing attempt to address this gap by focusing on a clustered histidine triad within -helices near the N-terminus, which prior studies have suggested play a role in binding ligands at an acidic pH. While GM-CSF is known to be highly flexible at a more acidic pH, several properties of its histidine triad remain unclear at the physiological pH at which GM-CSF would encounter its binding partners. We describe an effort to characterize the role of the GM-CSF histidines under physiological pH, specifically to determine if these histidines are key to GM-CSF structural integrity, and whether individual histidine residues modulate binding as they do at a lower pH. Our findings reveal that, while the histidine residues have an impact on GM-CSF structure, flexibility, and stability, they alone do not modulate the affinity for ligands at neutral pH. These data provide an initial explanation for the pleiotropic functions and interactions of GM-CSF within a biophysical context. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/700583v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@a6fffcorg.highwire.dtl.DTLVardef@1f00c30org.highwire.dtl.DTLVardef@b04c50org.highwire.dtl.DTLVardef@6224d9_HPS_FORMAT_FIGEXP M_FIG C_FIG

The MYC associated factor X (MAX) is the heterodimeric partner of the MYC paralogs (MYC, MYCN and MYCL). When deregulated, high level of the MYC paralogs contribute to all aspects of tumorigenesis and tumor growth. MAX can also heterodimerize with the MXD proteins, MNT and MGA. Heterodimerization and sequence specific DNA binding to the E-Box sequences at gene promoters is controlled by their heterodimerization with the MAX b-HLH-LZ. As a heterodimer with MAX, MYC proteins activate genes involved in cell metabolism, growth and proliferation whereas MXD proteins, MNT and MGA repress them. MAX can also bind to the E-Bos sequence as a homodimer. Being devoid of a transactivation domain it can act as an antagonist of the MYC/MAX heterodimers. Variants of MAX have been reported to be linked to cancer. These variants are either not expressed, inactivated or lead to missense mutations. This has led to the notion that MAX may have a tumor suppressor role. Here, we characterize three of those variants with missense mutations in the basic region, i.e. E32K, R35P and R35C. We analyzed their heterodimerization with the b-HLH-LZ of MYC and their DNA binding properties as homo-and heterodimers. The R35C variant b-HLH-LZ was found to have a markedly increased affinity for the b-HLH-LZ of MYC. We also observed that all three b-HLH-LZ variants have a lower affinity as homodimers for the E-Box than the WT. This was shown to lead to a preferential binding of all the heterodimeric b-LHLH-LZ to the E-Box. This effect is exacerbated in the case of the R35C variant. We argue that this preferential binding of MYC as heterodimers with these variants to E-Box sequences could contribute to tumorigenesis. Hence, our results suggest that, mechanistically, the MAX homodimer bound to the E-Box could act as a tumor suppressor. MATERIALS AND METHODSO_ST_ABSMolecular modelingC_ST_ABSThe open source version 1.7.6.0 of Pymol was used for modeling and molecular rendering [1]. The crystal structure of the MAX homodimer bound to the E-Box (1HLO [2]) was used as a template for the generation of the models. The variants were generated using the mutagenesis function in the wizard. The conformation of the K32 side chain was manually set in order to avoid introducing steric clashes with DNA. Protein expression and purificationThe cDNA, coding for the MAX b-HLH-LZ (Max* hereafter, residues 22-103, UniProt entry P61244-1) to which are added the GSGC residues in c-terminal, inserted in the pET3a vector was already available in the laboratory [3] and was used as a template to generate the plasmids with inserts coding for each of the mutants (E32K, R35C and R35P) through quick-change PCR with Q5 DNA polymerase and DpnI from New England Biolabs. The primers used were purchased from IDT DNA, their sequences are listed in Table S1. Sequence for each construct was confirmed by Sanger sequencing at the Plateforme de sequencage SANGER - Centre de recherche du CHU de Quebec - Universite Laval. The primary structure for the basic region of each construct is given in Fig. 2A. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/715400v1_fig2.gif" ALT="Figure 2"> View larger version (41K): org.highwire.dtl.DTLVardef@1b05d5eorg.highwire.dtl.DTLVardef@1c1d692org.highwire.dtl.DTLVardef@ee469dorg.highwire.dtl.DTLVardef@15e0ba4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 2.C_FLOATNO Structure schematics, specific and non-specific interactions dictating specificity and stability of binding of the basic region of MAX to the canonical (CACGTG) E-Box. A. Primary structure for the basic region of MAX and each of the variants. Positions making the most important contacts with the E-box are indicated by black arrows. Positions for the variants studied here are colored according to the Zappo colour scheme, following their physico-chemical properties: red for negative, blue for positive, magenta for proline and yellow for cysteine. B. The side chain (carboxylate) of E32 receives H-Bonds from the CA nucleobases in the leading strand (white carbon atoms). R35 and R36 make a salt bridges with phosphate groups while and the guanidino moiety of R36 makes a specific H-Bond with the nucleobase of the G in the strand of the reverse complement (cyan carbon atoms). C. The R35C mutation removes one non-specific salt-bridge at the interface of the complex. D. The aliphatic portion of the K side chain in the E32K variant is unable to accept the H-Bonds from the CA nucleobases and leads to the stabilisation of the complex and the helical structure of the basic region. E. In addition to removing a salt-bride, the Pro residue in the R35P kinks the path of the basic region, prevents the establishment of the specific H-Bonds mandatory for recognition of the E-Box and leads to unfolding of the helical state. C_FIG The MYC b-HLH-LZ (Myc*), the Max*WT b-HLH-LZ and its variants were expressed and purified as previously described [3,4] After lyophilisation, the b-HLH-LZs were kept at -20{degrees}C and solubilised in Myc buffer (50 mM NaCl, 50 mM NaH2PO4 pH 5.5) for Myc* or PBS for Max* at a final concentration of 1 mM before use. Circular dichroismAll circular dichroism (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Peltier-type thermostat. The instrument was routinely calibrated using an aqueous solution of d-10-(+)-camphorsulfonic acid at 290.5 nm. Samples were prepared as follows: Max* (either WT or a variant) was diluted in 100 {micro}l 2X CD buffer (40 mM KCl, 11.4 mM K2HPO4, 28.6 mM KH2PO4, pH 6.8) and the volume adjusted to 106 {micro}l with PBS. 10 {micro}l TCEP 16 mM were added, and the volume further adjusted to 192 {micro}l with ddH2O before samples were incubated overnight at room temperature. After reduction, Myc* was added and the volume adjusted to 198 {micro}l with Myc buffer (Na2HPO4 0.95 mM, NaH2PO4 49.05 mM, 50 mM NaCl, pH 5.5). The DNA complexes were prepared as follows. After a 10 minutes incubation of the protein samples at room temperature, 0, 1 or 2 {micro}l of 2 mM of specific or non-specific DNA duplexes in 10 mM Tris pH 8.0 were added and the volume adjusted to 200 {micro}l with 10 mM Tris pH 8.0. The strands of the specific probe were: 5-ATT ACC CAC GTG TCC T*AC-3 and 5-GTA GGA CAC GTG GGT* AAT-3 (with the E-box sequence underlined) and the non-specific probe: 5-ATT ACC TCC GGA TCC T*AC-3 and 5-GTA GGA TCC GGA GGT* AAT-3 (Integrated DNA Technologies). Samples were further incubated for 10 minutes at room temperature and transferred to a 1 mm path length quartz cuvette. All spectra were recorded from 250 to 195 nm at 0.1 nm intervals by accumulating 10 spectra at 25 {degrees}C. Thermal denaturations were recorded at 222 nm from 5 to 95 {degrees}C at a heating rate of 1 {degrees}C/min. CD signal for spectra and thermal denaturations was corrected by substracting the signal from corresponding spectra or thermal denaturation either for buffer alone or the appropriate DNA duplex. CD signal was then converted to mean residue ellipticity using the following formula [5]: [{theta}] = {delta} {middle dot} MRW/(10{middle dot}c l) where [{theta}] is the mean residue ellipticity in deg {middle dot} cm2 dmol-1, {delta} is the CD signal in millidegrees, MRW is the mean residue weight, c is the concentration in mg/ml and l is the pathlength in mm. For the heterodimers, the concentration used was the sum of Max* and Myc* and the MRW was determined using a weighted average.

Lipopolysaccharide (LPS), a ubiquitous bacterial component of food, is neutralized by a variety of mechanisms that help to establish a threshold, which when exceeded results in an inflammatory TLR4 mediated response. Notably both CEACAM1 and CD36 affect downstream signaling of TLR4 to LPS. Furthermore, CEACAM1 associates with CD36 in hepatocytes, regulating lipid storage and bile acid (BA) secretion that includes reverse transport of LPS to the intestine. Direct binding of LPS-Ra micelles to soluble CEACAM1 or soluble CD36 was analyzed by surface plasmon resonance (SPR), size exclusion chromatography (SEC) and transmission electron microscopy (TEM). Direct binding of CEACAM1 to CD36 was analyzed by SPR and proximity ligation assays. Molecular models were generated by Alpha Fold and Molecular Dynamics. LPS Binding: SPR binding constants of KD= 1.04 x 10-10 M and KD= 3.38 x 10-10 M were obtained for LPS-Ra micelle binding to sCEACAM1 and sCD36, respectively. On SEC, the molecular sizes of LPS-Ra micelles bound to sCEACAM1 and sCD36 were approximately 500 and 800 kDa, respectively. In addition, LPS binding to both was reduced by sodium cholate and sodium deoxycholate. Alpha Fold predicted a binding site of LPS-Ra to CD36, while Molecular Dynamic studies of an N-domain mutant of CEACAM1, that breaks a conserved salt bridge, revealed the presence of an open form that is predicted to bind LPS. sCEACAM1 to sCD36 Binding: A KD of 5.28 x 10-8 M was obtained for sCEACAM1 binding to immobilized sCD36 by SPR. Antibody-based-proximity ligation demonstrated the association of the ectodomains of CEACAM1 and CD36 on hepatic cells and when co-expressed in HEK cells. In addition, biotin-based proximity ligation demonstrated association of the cytoplasmic domains of CEACAM1 and a CD36-BioID2 fusion protein when co-expressed in HEK cells. Alpha Fold predicted both head-to-head (trans) and side-to-side (cis) binding of the N-domain of CEACAM1 to CD36, from which a membrane model of their cis-interaction could account for the proximity ligation results. Both CEACAM1 and CD36 share a common LPS micelle binding function, as well as binding to each other, and together, may regulate uptake and excretion of micellar LPS.

The association between higher levels of physical activity and lower cancer risk and mortality is well established. However, a causal link is yet to be proven. Recent studies showed a decrease in the proliferation rates of cultured human cancer cells when the human serum employed to stimulate them was conditioned by acute exercise. Here, we tested the hypothesis that serum mediates some of the putative benefits of exercise on cancer through alterations to the growth pattern and susceptibility to chemotherapy agents of cancer cells. To this end, human non-small cell lung cancer (NSCLC) cells were exposed to serum from two cohorts that differed significantly on their levels of physical activity and, accordingly, cardiorespiratory fitness, but were otherwise identical (master athletes and non-exercisers), collected before and after an acute exercise intervention. Serum levels of glucose, lipids, albumin, C-reactive protein and cytokines were determined and the impact of the serum responses to acute and lifelong exercise on the above-mentioned parameters were analyzed. We found that acute exercise decreased the cells proliferation rate, yet shortened the cells lag phase after detachment, whereas lifelong exercise had the opposite effects. Significantly, we showed, for the first time, that lifelong exercise increased susceptibility to a chemotherapy agent (cisplatin), which may contribute to the decreased cancer mortality rates found among those who exercise regularly. Similar to the cellular effects, changes to serum cytokine levels - several of them linked to the senescence-associated secretory phenotype - depended on whether serum was conditioned by acute or by chronic exercise. Key pointsChronic exercise increased the in vitro susceptibility of lung cancer cells to cisplatin. Acute and chronic exercise modulated the in vitro tumorigenic potential of lung cancer cells. Effects were mediated by serological changes produced by exercise. Acute and chronic exercise had distinct impacts on serological cytokine levels.

Atherosclerosis is a condition characterized by plaque growths in arteries, consisting of oxidized LDL (low-density lipoprotein) and localized cell cumulation. By the time of diagnosis for patients with atherosclerosis, the disease has often progressed into advanced stages. Statins are commonly prescribed; however, while these drugs can lower blood cholesterol levels, they cannot regress or stop the plaque growth. Currently, there are no treatments available to prevent the formation of new plaques. Such treatment options would require the identification of proteins that act during disease onset, initiating molecular mechanisms that promote plaque formation. Histone deacetylases (HDACs) and Ten Eleven Translocation (TET) demethylases are two important classes of epigenetic mediators. Some isoforms of these two classes of proteins have been found to transcriptionally regulate cellular inflammation, which may favor plaque formation. These transcriptional regulators seem to function early in the molecular mechanisms that are involved in disease progression. In the present work, we identified a clear role of these epigenetic proteins in foam cell formation. Foam cells have been implicated as part of the early steps which ultimately lead to atherosclerosis. Here we showed that in the presence of OxLDL (oxidized LDL), the protein isoform TET1 has a direct role in foam cell formation, while HDAC2 adopts a more indirect role. Using specific inhibitors of TET1 and HDAC2, we showed the inter-regulated molecular mechanisms between these proteins and how they regulate foam cell formation in vitro. In this study, we found that upon inhibition of TET1 in U937-derived macrophages, and subsequent foam cell formation via OxLDL treatment, a lower percentage of foam cells was observed. However, TET2 inhibition under the same treatment conditions had no effect on the inhibition of foam cell formation.

It has been recognized a long time ago that the hedgehog (Hh) and Wnt signaling pathways have numerous similarities that suggest their common evolutionary origin. Although the Hh and Wnt proteins are unrelated they are similar in as much as they carry lipid modifications that are critical for their interaction with their receptors. In our earlier work we have shown that Wnt inhibitory factor 1 (WIF1), originally identified as a Wnt antagonist also binds to and inhibits the signaling activity of sonic hedgehog (Shh), raising the possibility that the lipid moieties of these unrelated morphogens play a dominant role in their interaction with WIF1. In the present work we have compared the interactions of human WIF1 protein with lipidated and non-lipidated forms of human sonic hedgehog (Shh) using Surface Plasmon Resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. Our studies have shown that human WIF1 protein has significantly higher affinity for lipidated than non-lipidated Shh, indicating that lipid modifications of Hhs are important for interactions with WIF1.

Resveratrol, a naturally occurring polyphenol, exhibits anticancer, anti-inflammatory, and antioxidant properties. However, its molecular mechanisms in pancreatic cancer remain incompletely understood, necessitating integrative approaches such as network pharmacology and molecular docking. Potential resveratrol targets were identified using Swiss Target Prediction, while pancreatic cancer-related genes were retrieved from GeneCards and NCBI Gene databases. Overlapping targets were obtained through Venn analysis, followed by protein- protein interaction network construction, hub gene selection, and enrichment analysis. Molecular docking validated compound-target interactions. A total of 100 predicted resveratrol targets and 1,447 pancreatic cancer- associated genes were screened, yielding 39 overlapping genes. Network analysis identified hub genes including EGFR, SRC, MTOR, PIK3CA, PIK3CB, BCL2, and PTGS2. Gene Ontology enrichment indicated roles in cell proliferation, apoptosis regulation, inflammatory response and metabolic regulation while KEGG pathway analysis highlighted the PI3K-Akt, ErbB, and EGFR inhibitor resistance signaling as being closely associated with pancreatic cancer pathway. Docking analysis revealed strong binding of resveratrol with KRAS (-8.2 kcal/mol), EGFR (-7.9 kcal/mol), and MTOR (-7.7 kcal/mol), stabilized by hydrogen bonding. The interaction with KRAS, although not among the predicted targets. Expression profiling validated upregulation of hub genes in tumor samples. Resveratrol exerts multi-targeted effects in pancreatic cancer by modulating oncogenic pathways, particularly KRAS and PI3K/Akt/mTOR signaling. Its favourable safety profile and robust hub gene interactions highlight its potential as a supplementary therapeutic drug, necessitating further preclinical and clinical confirmation.

Oxidative DNA damage caused by endogenous reactive oxygen species (ROS) is a key driver of mutagenesis, cellular dysfunction, and aging, contributing to diseases like cancer, neurodegeneration, rheumatoid arthritis, cardiovascular disorders, and diabetes. Although more than 20 oxidative base lesions have been identified, ROS-induced DNA-protein crosslinks (DPCs) are poorly characterized. ROS-DPCs are unusually bulky and highly toxic lesions that accumulate in metabolically active tissues with age, but their identities, biological consequences, and repair in living cells have remained elusive. In the present work, we characterized ROS-DPCs in human fibrosarcoma (HT1080) cells treated with hydrogen peroxide (H2O2) and elucidated the mechanisms of their removal. Mass spectrometry-based proteomics has identified over 100 cellular proteins that participated in DPC formation, most of which are involved in DNA metabolism. Our data further reveal that DNA replication and transcription facilitate DPC detection and identify a critical role of the ubiquitin-proteasomal system (UPS), replication-coupled activity of SPRTN metalloprotease, and nucleotide excision repair (NER) in removing ROS-induced DPCs. ROS-DPC formation was blocked by pretreatment with metabolically stable and cell-permeable glutathione (GSH) analog ({Psi}-GSH), suggesting a possible therapeutic strategy for preventing diseases associated with increased ROS levels. KEY POINTSMass spectrometry-based proteomics identified over 100 proteins participating in DNA-protein cross-links in human cells treated with ROS Our work reveals the mechanisms through which living cells recognize and remove ROS-DPCs Our study demonstrates the potential of a glutathione analog to prevent ROS-DPC formation GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/704426v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@15d9c33org.highwire.dtl.DTLVardef@ba0307org.highwire.dtl.DTLVardef@1cd46dorg.highwire.dtl.DTLVardef@be80ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

The KRAS oncogene, central to cellular signaling via MAPK and PI3K-AKT pathways, is a notorious cancer driver frequently activated in pancreatic, colorectal, and lung carcinomas. Regulation of human KRAS oncogene expression is important due to its capital role in cell growth, proliferation, and survival. Misregulation of its expression contributes directly to the development and progression of multiple types of cancer. In previous studies, the role of G-quadruplexes elements in both the promoter and 5 UTR regions have shown to play important roles in KRAS expression, particularly when these G4s elements interact with regulatory protein hnRNPA1. In this study, we reveal that KRAS expression is also modulated at the post-transcriptional level through the formation of RNA G-quadruplexes (rG4s) situated at the 5 untranslated region (5UTR) of the mRNA. Biophysical and binding studies were carried out to probe the interaction. Through isothermal titration calorimetry (ITC), we quantified a strong binding affinity between the UP1 domain of hnRNPA1 and short-nucleotide RNA segments capable of adopting different G-quadruplex fold. The binding interaction is characterized by a favorable Gibbs free energy change in the range of {Delta}G {approx} -32 to -34 kJ/mol, suggesting a specific and energetically favorable association. One-dimensional and two-dimensional 1H-15N HSQC NMR spectroscopy revealed pronounced chemical shift changes in residues of both RNA recognition motifs (RRMs) of UP1, signifying direct contact with the rG4 structure.

The IRE1-XBP1 axis is the most conserved of three major unfolded protein response (UPR) branches that are triggered by the endoplasmic reticulum (ER) stress. Although the transcription factor XBP1 is involved in the development and function of several hematopoietic lineages, the role of XBP1 in the activation of mast cells (MCs) that play key role in allergic response remains largely unknown. Because we have identified salicylaldehyde (SA), which inhibits IRE1 nuclease activity that is essential for production of XBP1, as an inhibitor of MC activation in our previous screening, we investigated the effects of additional IRE1 inhibitors, 3-methyl-6-bromo-salichylaldehyde (MBSA) and KIRA6, targeting nuclease domain and kinase domain, respectively, on MC activation. MBSA and KIRA6 suppressed IgE-dependent degranulation and cytokine release of bone marrow-derived MCs (BMMCs), whereas these inhibitors did not suppress the Ca2+ ionophore- or compound48/80-induced degranulation. Treatment with inhibitors against two other branches of UPR, the PERK and the ATF6 pathways, did not affect IgE-induced activation of BMMCs. Intraperitoneal administration of MBSA or KIRA6 significantly suppressed IgE-induced passive anaphylaxis in mice. Furthermore, to evaluate the effect of XBP1, siRNA-mediated knockdown was performed. It was confirmed that Xbp1 siRNA introduction reduced IgE-dependent degranulation of BMMCs in parallel with the knockdown level of Xbp1 mRNA. Taken together, the IRE1-XBP1 axis plays a significant role in IgE-dependent and MC-mediated allergic response, which is considered to be therapeutic target of allergic diseases.

Polycystic Ovary Syndrome (PCOS) is known as an endocrine and metabolic disorder; however, emerging molecular evidence suggests a far more complex systems-level pathology. In this study, we performed an integrative transcriptomic and pathway-level analysis of endometrial tissue from women with PCOS to gain a deeper understanding of the underlying mechanism facilitating the disorder. The findings of the study highlighted mitochondrial dysfunction, chronic oxidative stress, and multi-layered immune dysregulation, adding some new insight apart from classical hyperandrogenism and insulin resistance. We identified some novel gene disease associations which involve C15orf48, ODF3B PRR15-DT, LINC01176, and LOC105379193. The upstream regulators such as (NFE2L2, TWNK, ALKBH1, BCOR, SMARCA4) involved in processes including mitochondrial genome, redox balance, and chromatin remodeling provided new insights into regulatory mechanisms. The IPA pathway analysis validated the compromised immune recovery with low grade inflammations and mitochondrial dysfunctionality. The observations emphasize on complex associations discarding its PCOS pure endocrine nature through immunometabolic-mitochondrial dysfunctionalities.

Elastic and viscoelastic properties of extracellular matrices (ECM) are known to regulate cellular behavior and mechanosensation differently, with implications for morphogenesis, wound healing, and pathophysiology. Most in vitro cellular processes, including cell migration, are studied on linear-elastic substrates to mimic extracellular matrices. However, most tissues are viscoelastic and display a loss modulus (G) that may be 10-20% of their storage modulus (G) under biophysically relevant conditions. Recent research has shown that cells can distinguish between elastic and viscoelastic ECM, leading to alterations in their cellular morphology, migration rates, and contractility. Here, we present a protocol for creating PAH-based model ECMs that enables the fabrication of viscoelastic substrates with storage moduli similar to those of their elastic counterparts. To explore how G influences epithelial cell mechanobiology, we fabricated tunable viscoelastic model ECMs with G of 3 kPa, 8 kPa, and 12 kPa, and for each, independently tuned G values to approximately 300 Pa, 500 Pa, and 700 Pa, respectively. We found that A549 cells cultured on stiff elastic model ECMs migrated [~]30% slower and formed larger focal adhesions compared to their viscoelastic counterparts. Conversely, A549 cells on intermediate viscoelastic model ECMs exhibited a [~]54% reduction in migration speed, with no significant difference in focal adhesion size relative to their elastic counterparts. These findings highlight the complex interplay between substrate (ECM) elastic and viscoelastic properties in regulating epithelial cell mechanobiology and emphasize the importance of time-dependent matrix mechanics in governing epithelial responses.

The human cathelicidin host defense peptide LL-37 forms complexes with nucleic acids that can have either beneficial or detrimental health effects. We suggest that these differential impacts are directly connected to dsDNA binding by LL-37 and to complex formation between protomers. Here, we show using phage {lambda} DNA that LL-37 binds non-specifically to dsDNA, condensing it, followed by complex formation between LL-37 peptides. We find that complex formation is concentration-dependent, with low LL-37 amounts yielding loosely aggregated DNA structures, while higher LL-37 concentrations lead to well-defined, disc-like structures of about 150 nm in diameter. The condensation of the nucleic acids, which causes a loss of the characteristic B-DNA features, results from interactions of the phosphodiester backbone with protonated amino acid side chains of the peptide at physiological pH, predominantly in A-T rich sequences of the nucleic acid. However, in our studies, electrostatic interactions did not appear to be the driving force for complexation, but rather we found the -helical structure of the peptide with its amphipathic and hydrophobic surfaces to be essential. Further, we show that LL-37 also interacts with nucleic acids from neutrophil extracellular traps (NETs) in a concentration-dependent way, causing a reduction in NET aggregate area, which may offer new biophysical insights into diseases such as systemic lupus erythematosus (SLE), which involve slower-than-normal NET clearance. Our results indicate the key importance of LL-37 expression levels for regulation of the innate immune system for optimal human health, since the relative amounts of expressed LL-37 present to interact with extracellular DNA will determine the extent to which the DNA can be condensed, which in turn will affect the ability of the body to clear the NETs before they can cause inflammatory conditions.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in the TSC1 and TSC2 genes and is characterized by benign hamartoma formation in multiple organs. The TSC1-TSC2 complex regulates mTORC1 signaling in response to cellular growth conditions. This study aims to predict the structural stability and functional effects of non-synonymous single-nucleotide polymorphisms (nsSNPs) in human TSC1 and TSC2 using computational approaches. Twelve computational tools were assessed using receiver operating characteristic (ROC) analysis and applied to identify deleterious nsSNPs. Protein stability was predicted using I-Mutant 2.0 and MUpro, while evolutionary conservation was analyzed with ConSurf. NetPhos 3.1 identified potential PTM sites, and MutPred2.0 evaluated their functional impact. Project HOPE assessed mutation-induced physicochemical changes. Structural models were validated using multiple tools, visualized in ChimeraX 1.9, and further evaluated by molecular dynamics simulation to confirm wild-type and mutant stability. All twelve tools had AUC values above 0.90. A combined in silico analysis identified twelve high-risk nsSNPs in TSC1 and sixteen in TSC2, all reducing protein stability, located in conserved regions, and potentially disrupting phosphorylation sites. MutPred and Project HOPE confirmed their impact on protein function. Functional analysis showed TSC1 and TSC2 affect mTORC1 and PI3K-Akt pathways. RMSF and RMSD analyses revealed that TSC1 variants rs1846545280 (G236E), and rs2132135678 (V234E), and TSC2 variants rs45517223 (S758C), rs2151354925 (T836P), and rs45517365 (R1570W) had the largest structural fluctuations. Substitution with glutamic acid, a negatively charged and bulkier residue, may disrupt local folding of TSC1. Similarly, replacement of arginine with tyrosine at position 1570 may impair Rheb binding at the GAP domain of TSC2. These findings highlight potentially pathogenic nsSNPs in TSC1 and TSC2.

The objective of this research was to characterize the proteome of the Apis mellifera royal pupae to evaluate its potential as a nutraceutical and functional food. Six pupal instars (E1-E6) were analyzed using liquid chromatography, mass spectrometry, and bioinformatics techniques to determine their properties and biological functions. The results showed 15 proteins across the different instars. In E1, the Isoform X2 of the Caf1 protein and the vitellogenin precursor were found, both critical in genetic regulation and nutrient transport. E2 revealed three proteins linked to energy and genetic processes. Proteins identified in E3 were associated with sugar metabolism and cellular structure. E4 presented proteins related to cellular stress and oxidative processes. In E5, three proteins were identified, associated with molecular transport and energy metabolism. Results for instar E6 were inconclusive since the complexity of peptide identification. From a nutraceutical and functional perspective, the identified proteins show significant potential due to their antioxidant activities, metabolic control, and cellular regulation. Noteworthy proteins include aldose reductase for its role in diabetes management, glutamate dehydrogenase for its importance in amino acid metabolism, vitellogenin as a nutrient source and immune system stimulant, and heat shock protein 60 A, with therapeutic potential in cardiovascular diseases.

Polyethylene terephthalate (PET) is a commonly used plastic worldwide and reducing its prevalence is crucial to improving environmental pollution. PETase that degrades PET plastic have received a lot of attention recently. This paper evaluates the ester hydrolysis process under both acidic and basic conditions, and shows that the local environment of the protein active site takes advantage of both. High pH in the protein buffer creates a better nucleophile to attack the ester through a proton shuttle channel in the protein, while local hydrogen bonds to the carbonyl of the ester stabilizes the intermediate/transition state of the hydrolysis reaction. With the understanding at the atomic level, we propose two engineering directions that can potentially improve the reactivity of the PETase: 1) increase the alkaline stability of the protein in general; 2) perturb the local hydrogen bond network to increase the partial charge on the PET carbonyl to be hydrolyzed. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/703441v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@151b69borg.highwire.dtl.DTLVardef@1abb95dorg.highwire.dtl.DTLVardef@116a225org.highwire.dtl.DTLVardef@ef2bb1_HPS_FORMAT_FIGEXP M_FIG C_FIG

We have conducted all atom molecular dynamics simulations of POPC and DPPC lipid bilayers using AMBER Lipid21 force field with eight different water models, including SPC/E, TIP3P, TIP3P-FB, TIP4P-FB, TIP4P-Ew, TIP4P/2005, TIP4P-D, and OPC, to identify the most compatible one without any modification. A number of parameters have been computed in order to understand the structure of the lipid bilayer: Area per lipid, Isothermal compressibility modulus, average Volume per lipid, electron density profile, bilayer thickness, X-ray and neutron scattering form factors, deuterium order parameter, and radial distribution function. The estimated Area per lipid, Isothermal compressibility factor, volume per lipid and bilayer thickness are highly consistent with experimental results for the SPC/E water model, indicating its suitability with the AMBER Lipid21 force field, insted of any modification. The bilayer electron density profiles of both the lipid bilayers demonstrate a little augmentation of water penetration with respect to the membrane surface for TIP4P-D water model. However, the experimental X-ray and neutron scattering form factors are aligning well with the simulated results for all studied water models, and TIP4P-D shows better for X-ray data. The deuterium order parameter for lipid acyl chains value less than 0.25 for all observed water models, depicting their disorderness for both the lipid bilayers. The lateral diffusion and reorientation autocorrelation function of the lipid molecules in both the bilayers are computed to reveal their dynamics across all water models. In comparison to other water models, the simulated trajectories predict better structure and reasonably fair dynamic properties for the SPC/E water model. The TIP4P-Ew water model reproduces the lateral diffusion co-efficient in close agreement with experiment. Reorientational dynamics for both the lipids in the bilayers for eight different water models are observed; the presence of slow and slowest time components corresponds to the lipid axial motion (wobble motion) and Twist/Splay motions. So, in view of the overall performance of the different water models with the AMBER Lipid21 all atom force field in reproducing membrane physical properties, the SPC/E water model appears to be an optimal choice.

NLRP3 inflammasome is a cytosolic multi-protein complex that plays a crucial role in the immune system, responding to various exogenous and endogenous stimuli by triggering protective inflammatory responses. However, aberrant NLRP3 inflammasome activation is implicated in numerous inflammatory diseases. Therefore, the NLRP3 inflammasome is an important pharmacological target for the treatment of multiple diseases. In this context, we screened various US-FDA-approved drugs for NLRP3 inflammasome inhibition. We found that among various drugs, minoxidil hydrochloride (MXL) effectively inhibits NLRP3 inflammasome, evidenced by reduced secretion of IL-1{beta} and IL-18 in J774A.1 cells treated with MXL. The IC50 values of MXL for inhibition of IL-1{beta} and IL-18 were calculated to be 1.2 and 1.06 {micro}M, respectively. MXL was found to prevent ASC oligomerization, thereby inhibiting the NLRP3 inflammasome and leading to CASP1 cleavage. Further investigation revealed that MXL also utilizes AMPK-mediated autophagy to modulate NLRP3 inflammasome activity. Using siAMPK and bafilomycin A1, an end-stage autophagy inhibitor, we elucidated crosstalk between the NLRP3 inflammasome and autophagic pathways, which was modulated by MXL. Furthermore, we demonstrated the efficacy of MXL in two different mouse models of inflammation, involving the NLRP3 inflammasome. MXL at doses of 10 and 20 mg/kg effectively inhibited the activation of NLRP3 inflammasome by monosodium urate in the air pouch model and by ATP in the peritoneal inflammation model, as evidenced by reduced secretion of 1{beta} and IL-18 in the lavage. Our study identifies MXL as a potent NLRP3 inflammasome inhibitor, warranting further investigation as a potential therapeutic agent for inflammatory diseases.