Bioscience Reports

The Ube2J1 enzyme that mediates the ubiquitination and proteasomal degradation of misfolded proteins at the ER is phosphorylated at serine S184. Following anisomycin treatment of HEK293T cells, we observed an inverse relationship between phosphorylation and dephosphorylation at this site. This suggested a dynamic interchange between the two forms, and we show that S184 is a target for protein phosphatase 2A. The S184-phosphorylated protein is known to exhibit increased sensitivity to proteasomal degradation, and we found that mutation at K186R increased the ratio of S184-phosphorylated to S184-dephosphorylated protein. Although the K186R mutant retained some sensitivity to proteasomal inhibition, our results show that Ube2J1 steady state expression can be exercised at multiple levels, and can involve dynamic phosphorylation and dephosphorylation at S184.

The ubiquitin conjugating enzyme UBE2J1/Ubc6e localizes to the endoplasmic reticulum where it mediates the ubiquitination and proteasomal degradation of terminally misfolded proteins. Although the protein is known to undergo phosphorylation at serine S184, we have considered modification at an additional site and used a bespoke anti-phospho antibody to confirm phosphorylation also at serine residue S266. Despite the well-described role of UBE2J1 in ER associated degradation (ERAD), we found no evidence for regulation at S266 during Unfolded Protein Response (UPR) induction by thapsigargin. Instead, our studies suggest that phosphorylation occurs independently at the S184 and S266 sites, with mutation at one site failing to disrupt basal phosphorylation at the second. We identified several contexts in which these two phosphorylations were differentially regulated. For example, ER localization, which is important for phosphorylation at S184, was not required for modification at S266, and sensitivity to proteasome inhibitors, which is regarded as a distinguishing feature of the S184 phospho-variant, was unaltered by the S266A mutation. Regarding regulation at S266 on the other hand, we found that pharmacological activation of protein kinase A resulted in rapid phosphorylation, with differential use of phospho-specific antibodies confirming that phosphorylation at S184 was unchanged by this treatment. Hormonal stimulation by glucagon resulted in a similar pattern of UBE2J1 phosphorylation, which occurred exclusively at S266 and could be inhibited by H89. The differential regulation demonstrated in these studies extends our understanding of the UBE2J1 enzyme, and may indicate a role in the integration of energy metabolism with environmental stress conditions.

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.

Background: Endometriosis affects approximately 10% of women of reproductive age and is associated with pelvic pain, infertility, and reduced quality of life. Dienogest is widely used for medical management. This study evaluated the effects of dienogest on endometrioma size, serum CA-125 levels, and pelvic pain. Methods: In this retrospective study, medical records of 45 women aged 18-49 years who received oral dienogest (2 mg/day) for at least six months were reviewed. Endometrioma size was assessed by ultrasound, pelvic pain using the Visual Analog Scale (VAS), and serum CA-125 levels from laboratory records. Baseline and six-month values were compared using the Wilcoxon test and correlations were analyzed using Spearman's test. Results: After six months of treatment, significant reductions were observed in endometrioma size and VAS scores (p<0.001) and CA-125 levels (p<0.001) compared with baseline. No significant correlation was found between endometrioma size and VAS scores or CA-125 levels either before or after treatment (p>0.05). A significant negative correlation was identified between patient age and post-treatment endometrioma size (r = -0.320, p<0.05). Conclusion: Six months of dienogest therapy was associated with significant improvements in lesion size, pain, and biochemical markers. Dienogest may represent an effective medical treatment option for symptomatic patients, particularly for those seeking to avoid surgery and preserve ovarian reserve.

Background: Despite significant advancements in obstetric care, the incidence of preeclampsia remains a substantial public health challenge, and effective strategies to prevent the disease progression remain limited, particularly in low-resource settings. N-acetylcysteine (NAC), an antioxidant and glutathione precursor, has demonstrated anti-inflammatory and vasodilatory effects, making it a promising candidate for repurposing. However, robust evidence from well-powered randomized controlled trials is lacking. Objective: This study will evaluate the impact of NAC on the time-to-disease progression in pregnant women with early-onset preeclampsia in Lagos, Nigeria. Methods: This is the study protocol for a proof-of-concept, double-blind, randomized, controlled trial to be conducted between April 2026 to July 2028 at the maternity units of the two teaching hospitals in Lagos, Nigeria. At baseline, n=153 sexually active women aged 18 years or older diagnosed with early-onset preeclampsia at 24 to 34 weeks gestation will be randomised to receive either daily oral tablet containing 600 mg of NAC or a placebo tablet that is matched for appearance and the dosing regimen in addition to standard antenatal care from diagnosis (randomisation) until either 34 weeks gestation or delivery, whichever comes first. The primary endpoint is the time-to-progression (in days) of early-onset preeclampsia to severe disease. The data analysis will be conducted on an intention-to-treat basis. Kaplan-Meier estimates with a Log-rank test will be used to calculate and compare the time-to-disease progression for the treatment groups, while Cox proportional hazard models with a backwards conditional method will be used to compare the primary endpoint between the treatment arms while adjusting for other covariates for precision using hazard ratios (HRs) and 95% confidence intervals (95%CIs). Subgroup analyses will also be performed to assess the differential effects of significant covariates on the impact of NAC on disease progression. Statistical significance will be reported as P<0.05. Discussion: This study will evaluate the efficacy of daily oral NAC compared to placebo in treating pregnant women with early-onset preeclampsia. If proven effective, NAC could offer a safe, affordable, and scalable intervention to reduce the burden of preeclampsia, particularly in resource-constrained settings.

The molecular basis of triple-negative breast cancer (TNBC), a highly aggressive and therapy-resistant subtype of breast cancer, is poorly understood. This study aims to identify key genes and pathways involved in TNBC development and progression using a systems biology approach followed by experimental validation. Here, two transcriptome microarray datasets from the GEO database were analysed using the R package LIMMA to detect differentially expressed genes (DEGs) in TNBC tumors. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analyses using the DAVID database were performed to identify DEGs regulated biological functions and pathways. Further, a protein-protein interaction (PPI) network was constructed using the STRING online database, and the topological properties were determined using MCODE and Cytohubba plug-ins. The expression and the prognostic value of the hub genes were validated using the Cancer Genome Atlas (TCGA) survival analysis. We found 727 DEGs, of which 473 were downregulated and 254 were upregulated in TNBC vs. non-TNBC samples. The GO and KEGG analyses indicated that the DEGs were mainly related to cell adhesion, tumorigenesis, and cellular immunity. The PPI network had shown six hub genes, namely CCND1, CDH1, ESR1, FN1, IL6, and PPARG, as the top key regulators. All these genes were validated by quantitative real-time PCR in the TNBC cell line using non-TNBC cell line as a calibrator, and the obtained results were in accordance with the bioinformatics data. This information may contribute to understanding the various molecular mechanisms that drive the development and progression of TNBC tumors.

BackgroundAlcohol-induced osteonecrosis of the femoral head (AIONFH) is an orthopedic disorder from chronic alcohol abuse, characterized by disrupted femoral head blood supply, osteocyte death and structural collapse. Current hip-preserving therapy is unsatisfactory, and most patients eventually require total hip arthroplasty. Panax Notoginseng Saponins (PNS), the core active component of Panax notoginseng, exerts pro-angiogenic and anti-osteocyte apoptosis effects, but its specific therapeutic mechanism remains unclear. ObjectiveThis study used network pharmacology, molecular dynamics simulation and animal experiments to identify PNSs active components, core targets and key pathways for AIONFH, verify its in vivo efficacy, and provide a scientific basis for clinical application. MethodsPNS active components, their targets and AIONFH-related targets were screened from databases; intersection targets constructed an interaction network, core targets were screened by three machine learning algorithms, with concurrent GO and KEGG analysis. Molecular docking was performed between core targets and PNS components; Gromacs 2022 conducted 100 ns simulation to evaluate complex stability. AIONFH rat models were grouped with 4-week intragastric intervention; pathology, immunofluorescence and PCR were used for detection. Results and DiscussionNetwork pharmacology identified 127 PNS targets and 18 intersections with 672 AIONFH targets. Six core targets (including FGF2, HSD11B1) were screened; KEGG indicated VEGF pathway as key. Ginsenoside Re bound HSD11B1 with the lowest binding energy (-12.4 kcal/mol), and 100 ns simulation confirmed complex stability. Animal experiments showed PNS improved trabecular structure and regulated osteocyte activity. PNS treats AIONFH via multi-component, multi-target mode, core mechanism being osteocyte apoptosis inhibition. Results and DiscussionNetwork pharmacology screening identified 127 potential targets of PNS, and 18 potential intersection targets were obtained by overlapping with 672 AIONFH-related targets. Six core targets including FGF2 and HSD11B1 were screened out by machine learning, and KEGG analysis indicated that the VEGF pathway and other pathways were the key signaling pathways for PNS action. Molecular docking showed that Ginsenoside Re had the lowest binding energy with HSD11B1 (-12.4 kcal/mol), and 100 ns molecular dynamics simulation confirmed the stable conformation of this complex. Animal experiments demonstrated that PNS could improve trabecular bone structure and regulate osteocyte activity. In summary, PNS exerts a therapeutic effect on AIONFH through a multi-component, multi-target and multi-pathway mode, with the core mechanism of inhibiting osteocyte apoptosis.

Parathyroid hormone-related peptide (PTHrP), encoded by PTHLH, has been implicated in tumor progression through its involvement in epithelial-mesenchymal transition (EMT), angiogenesis, and tumor cell migration. Previous experimental studies suggest that PTHrP may promote these processes in colorectal cancer (CRC), partly through the modulation of factors such as secreted protein acidic and rich in cysteine (SPARC) and vascular endothelial growth factor (VEGFA). These events play a key role in the acquisition of an aggressive phenotype in our experimental models. In this study, we performed an integrative in silico analysis of multiple transcriptomic datasets to investigate the potential role of PTHLH in CRC. Differential expression analysis identified a set of consistently dysregulated genes across independent datasets. Functional enrichment and network analyses revealed that PTHLH expression is associated with biological processes related to extracellular matrix remodeling, EMT, and angiogenesis. Correlation analyses showed a positive association between PTHLH and SPARC expression, while network-based approaches suggested a potential functional connection with VEGFA. To assess the clinical relevance of these findings, survival analysis was performed using publicly available datasets. High expression levels of PTHLH, SPARC, and VEGFA were significantly associated with reduced overall survival in patients. Notably, a combined gene signature based on these three factors demonstrated a stronger prognostic effect than individual genes, indicating enhanced predictive value. These findings suggest that PTHrP is associated with molecular pathways involved in tumor progression and, together with SPARC and VEGF, may contribute to a coordinated regulatory axis with prognostic relevance in CRC, warranting further experimental validation.

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.

S-acylation is the addition of fatty acids to cysteine residues to regulate protein function and localization. S-acylation is catalyzed by the ZDHHC (Asp-His-His-Cys) family of protein S-acyltransferases (PATs), which S-acylate protein substrates by first auto-S-acylating the catalytic cysteine of the DHHC active site followed by transfer to the substrate. ZDHHC13 and ZDHHC17 are related ankyrin repeat domain (ANK) PATs that S-acylate multiple neuronal proteins, including huntingtin (HTT), the protein mutated in Huntington disease. However, unlike ZDHHC17 and other human PATs, ZDHHC13 possesses a non-canonical DQHC active site. As the first histidine is essential for auto-S-acylation, it is unclear if ZDHHC13 is catalytically active. Our phylogenetic analysis of eukaryotic ANK-containing PATs shows that ZDHHC13 orthologues are more divergent compared to ZDHHC17. While the ZDHHC17 DHHC is highly conserved, the motif varies among ZDHHC13 orthologues, with some vertebrate lineages containing a serine in place of the catalytic cysteine. Interestingly, we found that the ZDHHC13 S-acylation is lower than that of ZDHHC17, but the ZDHHC13 catalytic cysteine is indeed S-acylated. While expression of wild type (WT) ZDHHC13 in ZDHHC13 deficient HEK293T cells increased S-acylation of a HTT1-588 fragment, surprisingly, expression of catalytically dead DQHS ZDHHC13 was still able to facilitate HTT1-588 S-acylation equally. This suggests the ZDHHC13 catalytic cysteine is not required for S-acylation of target proteins, suggesting ZDHHC13 may coordinate another PAT. Indeed, we identified ZDHHC13 in high-molecular weight complexes. Our results indicate that ZDHHC13 is a likely pseudoenzyme that may function via a non-conventional mechanism reliant on other PATs. This work broadens our understanding of the function of this non-canonical PAT.

{beta}-glucosidase (BglB) from Paenibacillus polymyxa was mutated (G23S, Rosetta/Foldit numbering; G26S, conventional numbering) to assess structural and functional changes. Foldit modeling and prior Design 2 Data (D2D) database results led us to hypothesize that this mutation would increase substrate binding affinity and catalytic efficiency, with a moderate reduction in thermal stability. The mutant protein was expressed, purified, and analyzed using kinetics and thermal stability assays. Relative to the wild-type (WT), G23S exhibited a similar binding affinity (similar Km), an approximately 2-fold increase in turnover number (kcat) and catalytic efficiency (kcat/Km), an almost 14-fold increase in maximum reaction velocity (Vmax) and a slight decrease in thermostability (T50). The results largely support the hypothesis, indicating that changes in residue 23 can enhance catalytic power while minimally compromising stability.

Background Conventionally, infertility has been regarded as primarily a female issue, leading to misconceptions, stigma, and underrepresentation of male infertility in healthcare discussions. This study assessed the knowledge, attitude and perception of Undergraduates towards male infertility in Osun State University. Methods A descriptive cross-sectional design was employed to select 300 undergraduates via multistage sampling. Qualitative data were collected using a focus group discussion guide covering the knowledge, attitude and perception, while quantitative data were collected using a self-administered questionnaire covering socio-demographic characteristics, knowledge, attitude and perception towards male infertility. Qualitative analysis was performed using NVivo software, while IBM SPSS Statistics version 27 was used for the quantitative analysis, with thematic analysis and chi-square tests to determine the association between variables (significance at p < 0.05). Results Respondents were predominantly females (64.0%) with a mean age of 20.99 {+/-} 2.31 years. Overall knowledge was low (47.7%), while more than half had a negative attitude (52.3%). Significant predictors of attitude include faculty (0.049), level (p=0.031), and formal education on male infertility (p=0.007). Conclusion Students demonstrated a poor understanding of male infertility, and their attitudes remain influenced by cultural norms surrounding marriage, masculinity, and gender roles. Hence, the need to foster open dialogues, promote gender-inclusive narratives, and strengthen healthcare support systems.

B-cell leukemia/lymphoma 11B (BCL11B), despite its name, is a key regulator of T-cell development, specification, and T-cell malignancies. BCL11B contains a bipartite DNA binding domain composed of two C2H2 zinc finger arrays: low-affinity ZF2-3 and high affinity ZF4-6. These arrays function as homotypic modules that recognize similar six-nucleotide motifs, TG(O_SCPLOWNC_SCPLOW)CC(O_SCPLOWCC_SCPLOWO_SCPCAP/C_SCPCAPO_SCPLOWTC_SCPLOWO_SCPCAP/C_SCPCAPO_SCPLOWAC_SCPLOW), as seven of the eight DNA base-contacting residues are conserved between them. The most conserved interactions involve GG dinucleotides, contacted by arginine and lysine residues at key base-interacting positions in ZF3 and ZF5. The two ZF arrays are connected by a long [~]300-residue linker that provides flexibility in how the arrays engage DNA, allowing ZF2-3 and ZF4-6 binding to the same or opposite strands with variable orientation, spacing and positioning along the DNA. This extended linker is enriched in serine/threonine, acidic residues (aspartate/glutamate), and structural residues (glycine/proline), providing additional layers of transcriptional regulation possibly through post-translational modification, electrostatic modulation, and/or condensate formation. We also examined six missense mutations in base-interacting residues, that are associated with neurodevelopmental disorders. Substitutions replacing bulky, positively charged arginine or lysine with smaller or hydrophobic residues likely reduce DNA-binding affinity and/or specificity, whereas substitutions between asparagine and lysine may alter base recognition preferences.

The KCNE (KCNE1-6) proteins are single-pass transmembrane auxiliary subunits of the voltage-gated K+ channel KCNQ1. KCNQ1-KCNE complexes have been well studied in jawed vertebrates ranging from zebrafish to humans, but KCNE subunits from earlier-diverging vertebrates remain poorly characterized. Here, we functionally characterize a single KCNE-like gene in lamprey, a jawless vertebrate, and designate it kcne0 as an early-diverging member of the KCNE family. KCNE0 shows moderate amino acid sequence similarity to KCNE1-6 but is not particularly similar to any single isoform. Both kcnq1 and kcne0 transcripts were detected in multiple lamprey organs. When co-expressed with lamprey KCNQ1, KCNE0 produced a constitutively active current, similar to KCNE3. By contrast, KCNE0 modulated KCNQ1 from other species less effectively, suggesting species-specific tuning of KCNQ1-KCNE compatibility. Introducing into KCNE0 an intracellular tetra-leucine motif analogous to that in KCNE4 markedly reduced KCNQ1 current amplitude, conferring a KCNE4-like inhibitory effect. Overall, this work provides a functional reference for comparing KCNE-dependent modulation of KCNQ1 across vertebrates and suggests an underlying compatibility mechanism.

Nucleobindin 1 (NUCB1) is a multifunctional conserved protein located in Golgi luminal, nucleus, extracellular and cytosolic pools. NUCB1 is multidomain protein comprised of a signal peptide, a DNA-binding domain, a leucine zipper and Ca2+ -binding domain. The multiple domains and localization of NUCB1 potentiates its interactions with various partners, such as DNA, Gi3 protein, cyclooxygenase 2, LRP10 and RNA suggests its importance in the regulation of many cellular events. We revealed that NUCB1 contains three RNA-binding regions and able to interact with two RNA fragments. It was suggested possible variants of the participation of NUCB1 in the interaction of the two partially complementary RNAs. The RNA-binding properties of the NUCB1 were also confirmed in vivo experiments.

BackgroundThe ability of TRAIL to specifically induce apoptosis in cancer cells makes it a promising candidate to be an effective chemotherapeutic drug. But resistance to TRAIL treatment is a major obstacle. Finding combinatorial therapies that make resistant tumors more susceptible to TRAIL is an effective preclinical approach. In this work, we investigated the possibility that pre-treatment of paclitaxel may promote apoptosis in TRAIL-resistant breast cancer cells. MethodsIn silico analysis was done to investigate the binding affinity between TRAIL receptors (DR5 and DCR2) and paclitaxel via docking and MD simulation. To check whether any non-lethal dose of paclitaxel can modulate the expression of TRAIL receptors, qPCR was done in paclitaxel treated breast cancer cells. Next, paclitaxel was pre-administered to TRAIL-resistant MCF7 and MDA-MB-453 human breast cancer cells followed by rhTRAIL treatment. Cell viability and survival was evaluated using the MTT assay and colony formation assay, respectively. Immunoblot for caspase-3 was performed to study apoptosis. The expression level changes of DR5 and DCR2 were analyzed post-treatment using qPCR and immunoblot assay. ResultsIn silico analysis showed that paclitaxel can bind with higher stability to DCR2 in comparison to DR5 thereby changing the preference of TRAIL molecules towards DR5. Next, in cell line experiments we observed that administering a non-lethal dose of paclitaxel to MDA-MB-231 and MCF7 breast cancer cells resulted in no significant cell death but led to an increase in DR5 and a decrease in DCR2 expression at both the transcript and protein levels. Furthermore, in TRAIL-resistant MCF7 and MDA-MB-453 cells, pre-treatment with paclitaxel followed by rhTRAIL administration induced significant cell death due to paclitaxel induced increase in DR5 as well as decrease in DCR2 expression at both the transcript and protein levels. Moreover, long term survival of MDA-MB-453 cells was significantly lower when pretreated with paclitaxel and exposed to rhTRAIL compared to control, paclitaxel alone or rhTRAIL alone group. ConclusionThus, our study uncovers a novel therapeutic strategy to overcome TRAIL resistance underscoring the clinical potential of using a non-lethal dose of paclitaxel to modulate TRAIL receptor dynamics. Future research should be aimed at exploring the potentiality of using paclitaxel-based combinatorial approaches in crafting effective TRAIL therapies.

The Na+/K+-ATPase (NKA) regulates ion balance in the kidney and influences cellular processes like proliferation and apoptosis through its signal transduction. The endogenous ligand 20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to inflammation and fibrosis in chronic kidney disease (CKD) and inhibits NKA activity in renal tubules. However, the molecular mechanism of this interaction remains unclear. In this study, we used in-silico approach to investigate the potential interaction between 20-HETE and NKA. Various ligands, including known NKA ligands such as cardiotonic steroids (CTS), 20-HETE, and negative controls, were docked using rigid and Induced Fit Docking to predict the affinity of the ligands toward NKA. Binding free energy calculations with the Prime Molecular mechanics with generalized Born and surface area (Prime MM/GBSA) tools were used to confirm the involvement of key amino acids in ligand-receptor interactions. The docking analyses revealed that 20-HETE exhibited a binding affinity comparable to negative control, with some differences between rigid and induced fit docking. Binding free energy data highlighted key amino acids in the 20-HETE and NKA interaction. Interaction fingerprint and mutations such as Ala330Gly and Val329Ala significantly reduced binding free energy, while Thr804Ala showed a notable decrease, underscoring the potential importance of these amino acids in ligand stabilization. These findings provide computational evidence supporting potential direct interaction between 20-HETE and NKA and identify candidate residues for future experimental validation.

Purine moieties are essential for many functions within the eukaryotic cell, including energy, signaling and nucleic acid synthesis. While purine starvation is known to induce stress resistance in eukaryotic model organism budding yeast Saccharomyces cerevisiae, it remains unclear whether the physiological response is related to disruption of synthesis pathway in particular position or it is uniform across all genetic deficiencies within the de novo adenine biosynthesis pathway. It is also not known how purine starved cells perceive purine shortage - weather they share the same signaling elements with nitrogen starvation or not. MethodsWe characterised physiology of strains with deletions in adenine biosynthesis pathway when cultivated in full or purine deficient and compared to cell physiological parameters when cultivated in nitrogen deficient media. We tested stress tolerance, carbon flux, cell cycle arrest and did transcription profiling (RNA-seq). ResultsOur findings demonstrate that purine starvation-induced stress resistance is significantly modulated by the specific step at which the pathway is interrupted. Transcriptional analysis revealed that purine starvation in many aspects phenocopies nitrogen starvation, particularly - in both starvations strong downregulation of ribosome related genes occurs. In the same time several metabolic features which differ from N- and ade- starvations: pentose phosphate pathway is specifically upregulated within ade4{Delta}-ade2{Delta} and downregulated in N-cells. Notably, the expression of stress-responsive genes such as HSP12, HSP26, and GRE1 varied between mutants, suggesting that the accumulation of pathway intermediates (e.g., AIR in ade2{Delta}) or the absence of downstream precursors (AICAR) alters the perception of starvation especially in the case of ade16{Delta}ade17{Delta} strain. ConclusionsMetabolic and stress-tolerance phenotypes of purine auxotrophs are not merely a result of purine depletion but seems that the response is signalled via the same pathways, like TOR1. The results suggest that strains having mutations within various positions of the purine pathway "perceive" purine limitation a bit differently - especially when we compare the end of the pathway with the other mutants. Different phenotypic outcomes of the occasional purine depletion might give preferences for organisms which have mutations in the beginning rather at the end of the pathway. Besides, our findings might have implications in the design of synthetic pathways and the use of auxotrophic markers in yeast research.

Freshwater algae represent an underexplored source of naturally occurring bioactive metabolites with potential applications in pharmaceutical and biomedical research. This study investigated the phytochemical composition, antioxidant capacity, and preliminary cytotoxic potential of ethanolic and n-hexane extracts of freshwater algal species collected at Jilani Park, Lahore, Pakistan. Algal species were identified morphologically by Dr. Ghazal Yasmeen (Institute of Botany, Punjab University, Lahore). Extracts were analyzed using gas chromatography-mass spectrometry (GC-MS) and qualitative phytochemical screening. Antioxidant activity was evaluated using DPPH radical scavenging, hydrogen peroxide scavenging, and reducing power assays. Cytotoxic potential was assessed using MTT and cell adhesion assays on HeLa and SF767 cell lines as preliminary indicators of bioactivity. GC-MS analysis identified 25 compounds, including sterols, fatty acid esters, terpenoids, phenolic compounds, and volatile metabolites. Phytochemical screening confirmed the presence of flavonoids, phenolics, tannins, and terpenoids in the extracts. Among the tested extracts, the n-hexane fraction demonstrated comparatively higher antioxidant activity across multiple assays. Ethanolic extracts showed moderate reductions in HeLa cell viability, whereas limited effects were observed in SF767 cells. These findings suggest that freshwater algae are promising natural reservoirs of antioxidant metabolites with potential relevance for future isolation and characterization of bioactive compounds for biomedical applications. Further purification and mechanistic studies are required to identify specific active constituents.

BACKGROUNGBisphenol A (BPA) has been linked to hypertension and disturbances in lipid metabolism; however, limited evidence is available regarding its association with hypertensive intracerebral hemorrhage (ICH). METHODSA multicenter, retrospective case-control study was conducted involving 129 participants, including individuals from an ICH group and healthy controls. Standard assays were employed to assess serum thyroid function, lipid profiles, serum fatty acid-binding [x]protein 4 (FABP4), oxidative stress markers, gap junction proteins, Wnt/{beta}-catenin signaling pathway activity, and expression changes of S100A8-mediated inflammatory cytokines involved in gut-brain interactions. Correlation analyses using Pearson and Spearman methods revealed that both BPA exposure and low T3 levels were significantly associated with elevated diastolic blood pressure, altered lipid metabolism, gut microbiota composition, and microglial activation. RESULTSGender-based disparities in lipid metabolism were identified. Changes in {beta}3-adrenergic receptor and neuromodulin-1 expression appear to influence fat regulation and attenuate oxidative stress responses. Subsequently, increased expression of gap junction proteins and activation of the Wnt/{beta}-catenin signaling pathway contribute to metabolic reprogramming and alterations in biochemical kinetics. Gut microbiota analysis demonstrated that, compared to controls, the ICH group exhibited significant dysbiosis and reduced alpha diversity. Further correlation analyses indicated that BPA levels were positively associated with FABP4 and oxidative stress markers, while S100A8 showed a strong dependence on microglial expression. CONCLUSIONThe interplay between lipid metabolism dysfunction and pro-inflammatory cytokines enhances vascular vulnerability. Collectively, BPA exposure, oxidative stress, and microglia-mediated neuroinflammation are significantly associated with an elevated risk of hypertensive ICH. China Clinical Trial Registry registration noticeFrom: China Clinical Trials Registry <chictr@vip.qq.com>+To:guopingwang60a<guopingwang60a@163.com> yunyanshuangfei <yunyanshuangfei@126.com> FUNDINGThis work was supported by the Natural Science Foundation of Shanxi Province (grant no. 201701D121177) Key informationGender-specific differences were observed in lipid metabolism and oxidative stress parameters; BPA exposure was shown to induce lipid metabolic disturbances, promote excessive production of oxidative stress byproducts, and consequently elevate oxidative stress responses; BPA was associated with stress-induced alterations in thyroid hormone function, further exacerbating dysregulation of lipid metabolism and oxidative stress; Fatty acid binding protein 4 (FABP4), a key adipokine implicated in metabolic disorders and adipose tissue inflammation, exhibited a significant positive correlation with serum BPA levels, whereas low levels of triiodothyronine (T3) were negatively correlated with FABP4. These findings suggest that serum FABP4 may serve as a biochemical marker for chronic low-grade adipose tissue inflammation and metabolic dysfunction; Gap junction proteins and the Wnt/{beta}-catenin signaling pathway may contribute to microglial activation and mediate neuroinflammatory responses, nerve injury, and secondary pathological processes in obesity-related cerebral hemorrhage.