Biochemistry and Biophysics Reports

Ionizing radiation induces DNA damage and oxidative stress; however, the genes and molecular mechanisms involved in bacterial stress responses have not been sufficiently identified. In this study, we used Limnobacter thiooxidans strain CS-K2, which is the closest relative to the bacteria detected in torus room water at the Fukushima Daiichi Nuclear Power Plant according to 16S rRNA gene sequences, and evaluated its response to {gamma}-ray irradiation using integrated transcriptomic and proteomic analyses. We identified three previously uncharacterized genes (LT3105, LT3115, and LT3126) that were strongly induced at the mRNA and protein levels. These genes exhibited low basal expression but were markedly upregulated by {gamma}-ray irradiation. Notably, LT3126 encodes a protein containing VIT (vault protein inter--trypsin) and VWA (von Willebrand factor type A) domains and showed the strongest induction. Overexpression of LT3126 increased survival after 500 Gy irradiation by approximately 200-fold compared with the control bacteria, demonstrating a direct contribution to survival under high-dose stress. Comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, this study identified a novel set of {gamma}-ray-responsive genes and demonstrated a functional role for LT3126 in radiation resistance, providing new insights into molecular adaptation in radiation-associated environments. IMPORTANCEWe identified a novel set of {gamma}-ray-responsive genes (LT3105, LT3115, and LT3126) in the non-model bacterium Limnobacter thiooxidans. These genes are located in relatively close genomic proximity and are coordinately induced upon irradiation, suggesting a shared functional role in stress response. Overexpression of LT3126 increased survival by approximately 200-fold after 500 Gy irradiation compared with the control bacteria, demonstrating a substantial contribution to survival under high-dose stress. These genes were also induced by heat shock and oxidative stress, indicating that their function extends beyond radiation-specific responses to broader environmental stress adaptation. Consistent with this, comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, these findings highlight previously unrecognized molecular strategies that may support bacterial survival in radiation-associated environments.

Temperatures in aquatic ecosystems have been affected by anthropogenic activities such as agricultural and industrial water use, and climate change caused by greenhouse gas emissions. Changes in water temperature directly affect the cellular and organismal physiology of fishes because most fishes are ectotherms. These effects can take different forms, such as increased cellular stress and reactive oxygen species (ROS) production. However, the type and magnitude of response depend on the duration and the frequency of the exposure to elevated water temperature. In this study, we investigated the long-term effects of exposure to daily thermal fluctuations occurring during early-life stages of zebrafish, Danio rerio, on gene expression and CTmax in later developmental stages. To do so, wild-type zebrafish were exposed daily to a + 5{degrees}C fluctuation in temperature from ambient (28 {degrees}C) to 33 {degrees}C over the first 30 days post fertilization (dpf), before being held until 90 dpf at ambient temperature. The fish that experienced daily thermal fluctuation were compared to a control group that was kept at 28 {degrees}C throughout the experiment and sampled at the same timepoints. Samples were collected at 18 (larval), 30, 60 (juvenile), and 90 (adult) dpf to study the expression of heat shock proteins and oxidative stress genes. The thermotolerance of fish was tested using CTmax trials at 60 and 90 dpf. Daily thermal fluctuation over the first 30 dpf led to a significant increase in the expression of hsp47, gstp1a, sod1, and sod2 genes at 60 dpf, and hsp47, hsp90aa1, hsp90ab1, cat, glulb, gstp1a, sod1, and sod2 genes at 90 dpf. The only significant increase detected during the larval stage was glulb at 18 dpf. Fish that experienced thermal fluctuation also had a higher CTmax at 60 dpf, but this increased thermotolerance significantly decreased from 60 to 90 dpf, where it was not different between treatments. Overall, our study demonstrates that early-life thermal stress increased cellular stress responses and thermotolerance in zebrafish into later ontological stages.

Congenital disorders of glycosylation and deglycosylation are rare, serious, and lethal disorders afflicting humans. CDGs and CDDGs result in loss of function enzymes which fail to build or break down oligosaccharides on proteins. This can produce protein aggregates and, in turn, reactive oxygen species that harm the cell eventually leading to autophagy and apoptosis. Because sperm contain high concentrations of polyunsaturated fatty acids, they are especially sensitive to these effects, which is understood as one of the leading factors in human male infertility. Sperm are developed in zebrafish similarly to humans and are useful models to examine human reproductive health, as well as genetic disorders. The combination of these advantages makes the analysis of sperm from zebrafish with heterozygous ALG1 or DPAGT1 CDGs or the NGLY1 CDDG suitable. Analysis of sperm concentration, motility, status, viability, and hypoosmotic swelling demonstrated the effects of these disorders on sperm quality. Results showed a significant decrease in sperm concentration, motility, and hypoosmotic swelling for all mutant zebrafish compared to the wild type. This suggests that CDGs and CDDGs influence the amount of sperm produced, the percentage of sperm cells that are mobile, and the integrity of the plasma membrane.

Ovarian and colorectal cancers have the highest incidence and mortality in the world, after breast cancer. Despite the initial response to Pt-drugs or 5-fluorouracil (5-FU), many cancer cells develop resistance to these drugs. For this reason, new therapeutic strategies represent an important medical need, in particular for drugs that are being studied in combination with methods that promote their entry into the cell. Among these strategies, electrochemotherapy (ECT), the combination of drugs with electroporation (EP), a physical method that uses high-frequency electrical pulses to create pores into which chemotherapy drugs can permeate, is gaining interest. In this study, we have evaluated the effect of ECT on the growth of both ovarian (A2780 and A2780/CP) and colorectal (HCT116) cancer cell lines using platinum derivatives (Cisplatin, Carboplatin and Oxaliplatin), as DNA alkylating agents, and human thymidylate synthase (hTS) inhibitors, both traditional (5FU) and novel TS destabilizers (compounds E3 and E7). To this aim, synergism quotient-like analysis to determine whether electroporation gives an advantage in terms of cytotoxicity was applied to the relative IC20 and IC50 concentrations of each drug. Results showed that two of the three Pt-drugs have greater efficacy when combined with EP. 5-FU and the new TS inhibitors E3 and E7 also take advantage of ECT because EP increases drug uptake into the cell, even in resistant cells. In conclusion, ECT appears to be a viable strategy to obviate the problem of resistance in ovarian and colorectal cancers, to deliver compounds inside cells overcoming uptake limits, especially for the low lipophilic compounds whose cytotoxic efficacy is hampered by the obstacle of biological membranes.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies due to its dense stroma, which limits drug delivery and therapeutic efficacy. Ultrasound (US) mediated strategies using nanobubbles (NBs) offer a promising approach to enhance treatment, yet the biological effects of NB exposure and the timing of US application remain unclear. Here, we investigated how NB exposure with immediate (0h) or delayed (1h) US affects viability, proliferation, metabolism, and stress signaling in PANC-1 and BxPC-3 cells. Immediate US exposure in the presence of extracellular nanobubbles resulted in a greater reduction in cell viability at 24 h compared to delayed US application. Proliferation analysis showed that Ki67 positivity decreased following USNB treatments in both cell lines. Metabolically, NB treatment alone increased cellular activity, whereas combined USNB treatment reduced metabolic activity over time. Seahorse analysis revealed higher basal respiration in PANC-1 cells compared to BxPC-3 cells, consistent with a more glycolytic phenotype, while USNB treatment enhanced glycolytic responses, particularly in PANC-1. Moreover, stress responses were also more pronounced in PANC-1 cells, with HSP70 expression increasing up to 2-fold in NB incubated group and decreasing in USNB groups compared to untreated, whereas BxPC-3 cells exhibited only modest and opposite changes to PANC-1 in HSP70 expression decreasing with NB incubation. Treatment timing critically influenced outcomes, with immediate US producing stronger antiproliferative and cytotoxic effects, highlighting the importance of sequencing in USNB therapeutic strategies. Moreover, NBs alone stimulated metabolic and stress responses that may promote proliferation, whereas NBs combined with US induced stronger stress responses associated with metabolic reprogramming and reduced proliferation.

Zinc is an essential trace element involved in numerous biological processes, including cellular signalling, development, and reproduction. Zinc homeostasis is regulated by zinc transporters, yet the physiological roles of many transporters remain poorly understood in vivo. Here, we investigated the function of the zinc transporter ZIP9 (SLC39A9) using a zebrafish (Danio rerio) knockout model. Elemental imaging using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) revealed altered zinc distribution in zip9-deficient larvae. Synchrotron-based X-ray fluorescence (XRF) imaging further showed reduced zinc levels in the brain region of mutant zebrafish. Consistent with these observations, loss of zip9 was associated with altered expression of key neuroendocrine genes within the hypothalamic-pituitary-gonadal (HPG) axis. Zip9 mutant females exhibited disrupted ovarian follicle development, reduced spawning rates, and decreased egg production. In addition, embryos derived from zip9 mutant parents displayed reduced size, impaired early development, and decreased survival. Together, these findings identify ZIP9 as a regulator of zinc distribution in vivo and suggest that ZIP9-mediated zinc signalling contributes to reproductive regulation in zebrafish.

Glioblastoma (GBM) is the most aggressive primary brain malignancy, characterized by hypoxia-driven proliferation, therapeutic resistance, and poor prognosis. While hypoxia-induced transcriptional changes are well documented, the temporal regulation of cell cycle genes under sustained hypoxia remains unclear. This study profiled transcriptomic alterations in U87MG cells cultured under normoxia and graded hypoxia for one to three days. Differentially expressed genes (DEGs) were identified and analyzed using STRING, Cytoscape, MCODE, and CytoHubba to construct protein-protein interaction (PPI) networks and extract hub genes. Functional enrichment was assessed through DAVID, ClueGO, and KEGG, while prognostic relevance was evaluated using GlioVis and ONCOMINE datasets. qRT-PCR validated expression of selected hub genes. A total of 294 DEGs were identified, forming two main functional modules enriched in cell cycle regulation and chemokine signaling pathways. Eighteen hub genes (KIF20A, CCNB1, AURKA, EGR1, CDCA3, CENPF, CDCA2, ASPM, KIF11, CCL2, CCNA2, DLGAP5, RACGAP1, TPX2, PTGS2, CTGF, and KIFC1) were significantly associated with mitotic processes and GBM progression. Survival analysis demonstrated that 17 of these genes correlated with poor overall survival (p < 0.05). qRT-PCR confirmed that hub gene expression peaked during early hypoxia and declined with prolonged exposure, indicating dynamic regulatory adaptation. These findings identify key hypoxia-responsive genes governing cell cycle progression and highlight their prognostic and therapeutic potential in glioblastoma.

Organisms have spent their life histories exposed to background levels of natural ionizing radiation. To document the role that radiation plays, the deprivation of these natural levels has been studied by incubating organisms in the shielded space of underground laboratories. We report here on two studies (Study I and Study II) using Aedes aegypti for the first time as a model organism incubated 655 meters underground at the Waste Isolation Pilot Plant (WIPP) outside of Carlsbad, New Mexico, U.S.A. Male mosquitos were incubated at the surface exposed to natural background radiation, and were compared to two underground treatments in which incubators were supplemented with radiation sources used to mimic background and these groups were compared to the underground, radiation-deprived treatment. In Study I, the mosquitos incubated underground in the absence of natural radiation had higher levels of mortality compared to those incubated at the surface and PCA plots of the two transcriptomes were clearly differentiated. Study II was conducted the following year and the experiment was narrowed to include only the surface control and underground, radiation-deprived treatment which allowed for four biological replicates. Again, there was a higher level of mortality in the mosquitos grown underground compared mosquitos grown at the surface. Transcriptomes were not as clearly differentiated by PCA analysis and fecundity data were similar between the two groups. Functional analysis of transcriptomic DEGs from two independent studies suggested there are stress responses in radiation deprived mosquitoes. The absence of a secondary stressor in Study II is discussed as an explanation for the transcriptome differences in the two experiments.

Magnetic treatment in tomato seeds (Solanum lycopersicum L.) has been studied as a biotechnological technique to induce a reduction in germination times and enhance plant development. However, the modified cellular mechanisms involved in the reduction of germination times or the improvement of development parameters are not yet clearly established. To explore a possible altered cellular mechanism, the effect of homogeneous static magnetic fields on the structure of the cyclic nucleotide-gated channel 6 (CNGC6), the modification in the organization of POPC lipids in the plasma membrane, and changes in calcium ion mobility were evaluated. For this purpose, coarse-grained molecular dynamics simulations were performed using the Martini 3 model in GROMACS, applying five different magnetic flux densities (0.000, 0.001, 0.010, 0.100, 1.000, and 10.000) T over 1 000 ns. The results showed an anisotropic effect in the longitudinal direction of the protein, which generated heterogeneous behavior among the chains of the homotetramer; this altered the conformation of the CNGC6 channel and modified the pore bottleneck. In contrast, no significant changes were observed in the conformational order of the POPC phospholipid chains. As a preliminary, single-replicate exploratory study, these results suggest that homogeneous static magnetic fields may induce specific structural modifications in the CNGC6 ion channel of Solanum lycopersicum L. without compromising the integrity of the lipid bilayer or the dynamics of ion transport within the analyzed timescale; these preliminary findings provide a molecular-level structural basis for future experimental and computational investigations of magnetic field effects on plant cyclic nucleotide-gated channels.

Chemotherapy treatment of colorectal cancers (CRC) using cisplatin (CDDP) encounters problems of drug resistance by the cancer cells and cytotoxicity to normal cells, highlighting the urgent need for joint therapeutical strategies. Selenium-enriched rapeseed extracts exhibit anti-cancer effects but the bioactive components and mechanisms remain unclear. Here, we applied different solvents to fractionate the extracts from Selenium-enriched rapeseed and found that the water extract (WE) fraction significantly enhanced the cytotoxic effect of CDDP on cancer cells but no damage on normal cells. HPLC-ICP-MS analysis revealed that methylselenocysteine (MSC) and selenocystine (SeCys2) were the main selenium speciation in WE. Through cell biology and integrative multi-omics analysis, we found a synergistic anti-CRC cell effect when combining CDDP with MSC, sulforaphane (SFN), celastrol (Cel), Indole-3-carbinol (I3C), -linolenic acid (ALA) or linoleic acid (LA). We propose that the CDDP-WE combination treatment holds the promise for improving curative efficacy for chemo-refractory CRC patients in the future.

A priori assessment of target proteins' druggability remains an unsolved problem in the field of drug development. The empirical approaches widely used to solve this problem demonstrate low efficiency. In this work, we investigated the factor of hydration of a representative set of 65 evolutionarily and structurally unrelated human enzymes in a water environment. This factor depends only on the structure of the proteins, and not on the physical and chemical properties of any potential ligands. The results show that, unlike the widely used approaches based on calculations of the accessible surface area (ASA), the content of low-entropy water molecules (LEW) in the active sites of human enzymes is systematically higher than that in other areas of their surface, including inactive cavities. Optimal criteria and a step-by-step procedure for identifying protein ligand binding sites are proposed. The proposed approach, based on the calculation of the LEW content in the first hydration layer of potentially interesting target proteins, makes it possible to evaluate their medicinal suitability even before the development of any ligands. The article also presents the results of a comparative analysis of experimental Raman spectroscopy data and the results of molecular dynamics simulations of water hydrogen bonds using three widely used water models (TIP3P, OPC3, and TIP5P) and standard algorithms for calculating hydrogen bond networks.

Accurate evaluation of extracellular matrix (ECM) mineralization at the nano-scale is essential for establishing relevant in vitro bone models. This is particularly important with the development and increased application of three-dimensional (3D) cell models for biological research. Transmission electron microscopy (TEM) allows to perform ultra-structural analysis of cells and ECM organization, but its application in in vitro bone models remains limited, due to the potential alteration or loss of the mineral phase during sample preparation. In this study, we compared two TEM sample preparation methods - the conventional chemical fixation and the anhydrous methods - to evaluate their ability to preserve the mineralized ECM in MC3T3-E1 cells cultured as monolayers and as alginate-encapsulated bone spheroids. Chemical fixation preserved cellular ultra-structure and collagen organization, allowing for detailed assessment of cells and ECM organization. Although mineral deposits were detected and their needle-like morphology assessed, characterization of more immature deposits was partially limited by the effects of uranyl acetate and the overall sample preparation process, which could lead to alteration or loss of less stable mineral phases. The anhydrous preparation method resulted in limited preservation of cellular and ECM morphology and did not allow reliable identification of mineral deposits. When applied to spheroids, the chemical fixation method preserved the 3D architecture, collagen-rich ECM and inner mineral deposits, confirming spheroids as a relevant model for bone studies. Overall, these results highlight the need for optimized sample preparation strategies that preserve both ultra-structure and mineral components for accurate nano-scale characterization of bone mineralization.

BackgroundTriple-negative breast cancer (TNBC) is an aggressive subtype lacking well-defined molecular targets, leaving chemotherapy as the primary treatment despite drug resistance, systemic toxicity, and high recurrence rates. Therefore, the development of effective and less toxic therapeutic agents is essential. This study investigated the anti-cancer potential of gloriosine, a bioactive alkaloid with antiproliferative activity and low toxicity toward normal breast cells. MethodsPotential targets of gloriosine were predicted using SwissTargetPrediction, TargetNet, and PharmMapper, and overlapping genes related to TNBC and glutamine metabolism were selected. Protein-protein interaction networks, Gene Ontology, and KEGG pathway enrichment analyses were performed. Molecular docking evaluated binding affinity, followed by in vitro validation using cell viability, colony formation, and wound healing assays. ROS levels were measured by DCFDA and GSH assays, and ferroptosis was assessed by Western blot and FerroOrange staining in MDA{square}MB{square}231 cells. ResultsA total of 100 potential targets were identified, with 60 overlapping with TNBC and glutamine metabolism-related genes. Key targets included SRC, EGFR, mTOR, and HSP90AA1. Enrichment analyses indicated involvement in cancer progression, metabolic regulation, and resistance pathways, including central carbon metabolism, EGFR inhibitor resistance, and ErbB signaling. Gloriosine showed strong binding affinity toward hub targets. Experimental studies confirmed concentration-dependent inhibition of cell proliferation and migration. Mechanistically, gloriosine suppressed glutamine metabolism via GLS1 downregulation and induced ferroptosis, evidenced by increased ROS, glutathione depletion, GPX4 downregulation, and elevated intracellular iron levels. ConclusionsGloriosine exerts significant anti-cancer effects in TNBC through multi-target modulation and induction of ferroptosis, highlighting its potential as a promising therapeutic candidate. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/725321v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@ce0ebcorg.highwire.dtl.DTLVardef@29603borg.highwire.dtl.DTLVardef@6d0025org.highwire.dtl.DTLVardef@249700_HPS_FORMAT_FIGEXP M_FIG C_FIG Flow chart of the network pharmacological and in vitro study of gloriosine

In many cases, cancer cells develop resistance to chemotherapy and other cancer treatments. Therefore, there is a need for new therapeutic agents using naturally derived compounds that are expected to have low toxicity and fewer side effects. Fucoidan is a sulfated polysaccharide found in brown algae such as kelp and wakame seaweed. Many previous reports have shown that fucoidan exerts anti-bacterial, anti-viral, antioxidant, immunomodulatory effects, and anti-tumor effects. The antitumor and antiviral effects of fucoidan have been reported to vary depending on its origin, as they are influenced by sulfate content and molecular weight. Therefore, it is important to investigate the antitumor effects of various species of fucoidan, but there are few reports on the effects of fucoidan derived from Laminaria japonica on colorectal cancer. In this study, we evaluated the effects of fucoidan from Laminaria japonica on apoptosis in five human colon cancer cells. The apoptotic cell population was significantly increased in fucoidan-treated cells. In addition, the expressions of Bax, Bak, PARP, caspase-8, -9 and -3 were upregulated. The necroptosis-related molecule RIP and MLKL were degraded indicates that necroptosis was not involved in this fucoidan-treated cell death. These results suggest that fucoidan-treated cells showed induction of apoptosis via mitochondrial intrinsic pathway, but not necroptosis via caspase-8. Fucoidan-induced apoptosis may prove useful in the therapeutic protocol of colon cancer.

BackgroundThe delayed effects of radiation exposure on the heart often manifest as cardiac fibrosis and diastolic dysfunction, which can develop years after exposure. However, no FDA-approved serological biomarker is available to assess the risk of individuals for developing radiation-related heart disease (RRHD). ObjectivesSerum pro-N-cadherin (PNC) has shown promise as a marker for predicting the onset of heart failure in the general population. We hypothesize that serum PNC levels will correlate with the risk of RRHD following radiation exposure. MethodsWe examined male non-human primates (NHPs) exposed to total-body irradiation (TBI) and unirradiated controls from the Wake Forest University radiation late effects cohort. NHPs exhibited cardiac fibrosis scores ranging from less severe (F0-1) to more severe (F2-3). Cardiac tissue samples collected at necropsy, median 6.8 years post-irradiation, were stained for PNC by immunohistochemistry. PNC was quantified in longitudinal serum samples collected 2, 1 and 0 years before necropsy. The associations of serum PNC levels with cardiac fibrosis scores and echocardiographic parameters were examined. ResultsHistological examinations showed aberrant localization of PNC in NHPs with cardiac fibrosis. Elevated serum PNC levels significantly correlated with severe cardiac fibrosis (AUC = 0.81, p = 0.006) and echocardiogram parameters of diastolic dysfunction. Cardiac fibrosis was the only measured comorbidity with a significant difference in serum PNC. ConclusionsOur results demonstrate that serum PNC significantly correlates with cardiac fibrosis and diastolic dysfunction in irradiated NHPs. These findings pave the way for future clinical studies to develop serum PNC as a biomarker of RRHD in humans. HIGHLIGHTSO_LIRadiation-related heart disease is an often under-recognized complication of radiation exposure and radiation therapy, which has no FDA-approved biomarkers for assessing risk. C_LIO_LIOur results reveal that serum pro-N-cadherin is a biomarker of cardiac fibrosis and diastolic dysfunction in non-human primates that survived radiation exposure. C_LIO_LIThis study lays the foundation for further research into the development of serum pro-N-cadherin as a biomarker for assessing the risk of radiation-related heart disease in humans. C_LI

To elucidate the early mechanisms underlying the long-term neuroprotective effect of FLASH-RT in the normal brain, spatial transcriptomics (Nanostring) were performed after whole-brain irradiation of C57BL/6J mice with either 1 or 3 fractions of 10 Gy at 5.6x106 Gy/s (1 pulse-FLASH) or at conventional dose-rate 0.1 Gy/s. FLASH -RT induced a distinct transcriptomic signature in the CA3 and DG neurons, with upregulation of genes encoding glutamate receptors, involved in calcium signaling, long-term potentiation and mitochondrial OXPHOS. Early transcriptional upregulation of Gria gene translated into increased AMPAR protein levels at 48h in the DG and CA3 region and sustained higher AMPAR expression at 2 and 4 weeks post-FLASH. These findings support a durable activation of AMPAR. We propose a mechanism to explain FLASH-induced neuroprotection initiated by early calcium influx and subsequent sustained expression of glutamate receptor AMPAR in neurons and/or neural progenitors of the CA3, potentially contributing to long-term cognitive sparing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/725423v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1ae125forg.highwire.dtl.DTLVardef@138357aorg.highwire.dtl.DTLVardef@13f128dorg.highwire.dtl.DTLVardef@1db1cf6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFLASH-RT induces a stronger transcriptional response in the hippocampus than the cortex. C_LIO_LIFLASH-RT induces calcium signaling, LTP and mitochondrial OXPHOS genes. C_LIO_LIEarly AMPAR upregulation leads to sustained protein expression. C_LIO_LIFLASH-RT induces a AMPAR-dependent signaling program in CA3 neurons. C_LI

Members of the transforming growth factor-{beta} (TGF-{beta}) superfamily, including inhibins and activins, are structurally related glycoprotein dimers that regulate reproductive and endocrine signaling. Their high degree of molecular similarity presents challenges for label-free analytical discrimination. To evaluate the ability of Raman spectroscopy to distinguish closely related TGF-{beta} superfamily proteins based on intrinsic vibrational fingerprints. Raman spectra of recombinant human Inhibin -subunit, Inhibin B ({beta}B homodimer), and Activin A ({beta}A--{beta}A) were acquired using confocal Raman microscopy with 532 nm excitation. Spectra were baseline-corrected, area-normalized, and analysed using principal component analysis (PCA). Distinct spectral signatures were observed across the 500--1800 cm-1 region. Differences within the S--S stretching region (500--550 cm-1) were consistent with variations in disulfide-bond environments, with the Inhibin -subunit exhibiting the highest relative intensity in this region. Variations in the amide I band (1600--1700 cm-1) suggested differences in protein secondary structure, while aromatic amino acid vibrations provided additional discriminatory features. PCA revealed clear clustering and separation of all three protein classes based on their Raman fingerprints. Raman spectroscopy enables label-free differentiation of structurally related endocrine glycoproteins and demonstrates potential for the structural characterization and classification of inhibin and activin proteins within the TGF-{beta} superfamily.

The patch-clamp experimental technique is widely used to study the electrical properties of ion channels in biological and artificial lipid membranes. The key to the high quality of the experiments is the manufacturing of glass pipettes that provide highly electrically resistant contact between the edge of the pipette tip and the lipid bilayer. Preparation of the pipettes is particularly challenging for studies of the mitochondrial membranes due to the need for very small pipette tip sizes. Here, we present a robust procedure for producing pipettes suitable for experiments with native mitochondrial membranes. This procedure involves a two-step approach: initial fabrication of relatively large glass micropipettes using a standard micropipette puller, followed by tip refinement using a microforger to achieve smooth glass surface and reduced opening size. Pipette tip diameters and surface structure were examined using field emission - scanning electron microscopy (FE-SEM) imaging to assess the effects of variable parameters on pipette geometry and size. The resulting pipettes were validated in patch-clamp recording of the mitochondrial inner membranes. This approach enables the reproducible production of optimized pipettes for mitochondrial patch-clamp experiments, improving the quality and throughput of electrophysiological recordings of the mitochondrial ion channels.

Cold environments pose substantial metabolic challenges to ectothermic organisms. In amphibians, such as the African clawed frog (Xenopus laevis), exposure to cold temperatures induces pronounced hyperglycemia; however, the molecular mechanisms underlying this response remain unclear. This study investigated the metabolic responses of the liver to cold exposure using transcriptome analysis. Adult frogs were subjected to a temperature of 5{degrees}C for five days, and their liver transcriptome was subsequently analyzed using RNA sequencing. Cold exposure significantly elevated blood glucose levels. Transcriptome analysis revealed extensive alterations in gene expression, including the upregulation of key gluconeogenesis-related genes. Notably, genes involved in FOXO1 signaling exhibited coordinated changes, with increased expression of foxo1 and its regulator prmt1 (arginine methyltransferase) and decreased expression of mdm2 (E3 ubiquitin ligase), suggesting that the phosphorylation of FOXO1 may be suppressed. Consistent with these findings, the expression of gluconeogenic genes (g6pc1 and pck1) was elevated, whereas the glycolytic gene gck was downregulated, indicating a shift towards glucose production. In addition to carbohydrate metabolism, genes involved in lipid and cholesterol metabolism, particularly fatty acid desaturases (scd and fads2), were also upregulated, suggesting that the remodeling of membrane lipid composition may occur under cold conditions. Furthermore, genes related to antioxidant and redox pathways, including those involved in the detoxification of reactive oxygen species and iron sequestration, were induced, indicating enhanced redox regulation. Collectively, these results demonstrate that cold exposure induces coordinated metabolic remodeling in the liver of X. laevis, characterized by enhanced gluconeogenesis, lipid remodeling, and robust redox regulation. SUMMARY STATEMENTCold exposure drives coordinated hepatic metabolic reprogramming in Xenopus laevis, elevating gluconeogenesis, modifying lipid composition, and strengthening antioxidant defenses through integrated transcriptional responses that support survival under a low-temperature environment.

Leukocyte immunoglobulin-like receptor B4 (LILRB4, ILT3) is an inhibitory immune checkpoint expressed on myeloid cells, where it contributes to immunosuppression within the tumor microenvironment. Secretogranin 2 (SCG2) has recently been identified as a functional ligand of LILRB4, yet small molecule modulators of this interaction remain unexplored. Here, we report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to interrogate the LILRB4 (ILT3)-SCG2 interaction. The assay demonstrated robust performance and was validated using a blocking anti-LILRB4 antibody, consistent with orthogonal ELISA measurements. Pilot screening of chemical libraries identified 23 primary hits, of which two compounds, BMS-813160 and PSB-603, showed reproducible, dose-dependent inhibition with TR-FRET IC50 values of 26.7 {+/-} 1.03 {micro}M and 37.2 {+/-} 2.14 {micro}M, respectively. Activity was confirmed by ELISA, supporting the robustness of the assay. This platform enables high-throughput discovery of first-in-class small molecule modulators of the LILRB4-SCG2 immune checkpoint and provides a foundation for targeting myeloid-driven immunosuppression.