Molecular Genetics and Genomics最新文献

{"title":"Expression, prognostic value and function of LMBR1L in gastric cancer.","authors":"Song Liang, Chaosheng Zhang, Chuanjun Wang, Yongkang Liang, Xueyi Feng, Chuansi Wang, Pin Jiang, Shenwei Wu, Xiuming Lu","doi":"10.1007/s00438-026-02420-4","DOIUrl":"10.1007/s00438-026-02420-4","url":null,"abstract":"<p><p>Gastric cancer remains a leading cause of cancer-related death worldwide, and effective prognostic biomarkers are urgently needed to guide clinical management. This study aimed to investigate the role of LMBR1L in gastric cancer. The expression of LMBR1L protein was detected by immunohistochemistry, Western blot and other methods. An LMBR1L knockdown HGC-27 cell line was constructed, and a xenograft experiment in nude mice was conducted to verify its effect on tumor growth in vivo. In addition, a nomogram model was constructed based on the expression level of LMBR1L and other clinical indicators to evaluate its predictive value for postoperative survival of gastric cancer patients. LMBR1L was highly expressed in gastric cancer. Patients with high expression of LMBR1L had lower postoperative overall survival (OS) and disease-free survival (DFS) rates than those with low expression. LASSO regression and Cox multivariate analysis showed that the expression level of LMBR1L was an independent risk factor for the prognosis of gastric cancer patients. The nomogram model constructed based on LMBR1L and other clinical indicators had a good predictive value for postoperative survival of gastric cancer patients, with areas under the ROC curve (AUC)of 0.642, 0.691 and 0.690 for 1-, 3- and 5-year survival rates, respectively. The xenograft experiment in nude mice indicated that knockdown of LMBR1L could significantly inhibit the development of gastric cancer in vivo. LMBR1L is highly expressed in gastric cancer and is closely related to poor prognosis of patients. Its regulatory effects on the biological behavior of gastric cancer in vitro and in vivo further confirm its important value as a prognostic biomarker and potential therapeutic target for gastric cancer, providing new insights into the molecular mechanisms of gastric cancer progression.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":"301 1","pages":""},"PeriodicalIF":2.2,"publicationDate":"2026-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148345723","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"HIPK3 silencing promotes M2 macrophage polarization through NF-κB signaling pathway to enhance oral cancer progression.","authors":"Rongxia Zhang, Yongle Qiu, Wenjing Wang, Si Chen, Liru Zhang, Chencong Li","doi":"10.1007/s00438-026-02476-2","DOIUrl":"10.1007/s00438-026-02476-2","url":null,"abstract":"<p><p>The tumor microenvironment (TME) is crucial for the progression of oral cancer. A key feature of the TME is the dynamic plasticity of tumor-associated macrophages (TAMs), which can polarize into either pro-inflammatory M1 or anti-inflammatory M2 phenotypes. This study aimed to investigate the role of homeodomain-interacting protein kinase 3 (HIPK3) in regulating macrophage polarization and its consequent impact on the behavior of oral squamous cell carcinoma (OSCC) cell line CAL-27. Following transfection of M0 with si-HIPK3 to downregulate HIPK3 expression, we assessed macrophage polarization markers via ELISA. Protein levels of iNOS and Arg1 were further evaluated by Western blot. We subsequently examined the effect of HIPK3-silenced macrophage polarization on the behavior of OSCC cells using functional assays. Finally, the molecular mechanism through which HIPK3 silencing regulates macrophage polarization was investigated. HIPK3 knockdown induced a significant shift from anti-tumor M1 polarization, characterized by decreased iNOS and TNF-α expression, to a tumor-promoting M2 phenotype, as evidenced by elevated levels of Arg1 and CD163. Functional assays further demonstrated that HIPK3 silencing markedly enhanced the proliferation, migration, and invasion capabilities of CAL-27 cells. Mechanistically, HIPK3 likely facilitates macrophage M2 polarization by inhibiting the NF-κB signaling pathway. In conclusion, inhibition of HIPK3 promotes M2 macrophage polarization, which in turn enhances the proliferation and migration of OSCC cells, ultimately contributing to pro-tumorigenic effects.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":"301 1","pages":""},"PeriodicalIF":2.2,"publicationDate":"2026-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148345821","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genetic-epigenetic interactions (meQTLs) in orofacial clefts etiology.","authors":"A L Petrin, L A Machado-Paula, H L Keen, L Hovey, B Doolittle, L Dunlay, W Awotoye, X J Xie, E Zeng, A Butali, M L Marazita, J C Murray, L M Moreno-Uribe","doi":"10.1007/s00438-026-02474-4","DOIUrl":"10.1007/s00438-026-02474-4","url":null,"abstract":"<p><p>Understanding how genetic variants influence disease risk through molecular mechanisms remains a central challenge in complex disease genetics. Nonsyndromic orofacial clefts (OFCs) exemplify this challenge, with most risk loci residing in non-coding regions. We hypothesized that common genetic variants influence OFC risk by modulating DNA methylation at regulatory elements through methylation quantitative trait loci (meQTLs). We analyzed 10 OFC-associated SNPs against genome-wide DNA methylation profiles in 409 cases and 456 controls, identifying 23 potential meQTLs. Findings were validated using 358 cleft-discordant sibling pairs with MethyLight assays. We performed formal mediation analysis, genotype-tissue interaction and cross-referenced with the mQTL Database to assess developmental timing. Nine meQTLs were validated, including rs987525 (8q24)-cg16561172 (MYC) (P = 9.6 × 10⁻⁶), which mapped to a mesendoderm-active enhancer upstream of MYC. Genotype × tissue interaction confirmed tissue-specificity (P = 1.00 × 10^- 3), with stronger effects in oral-derived tissue (saliva). Additional validated SNP-CpG associations involved MAFB-PLCG1, NOG-PPM1E, FOXE1-FRZB, and SPRY2-LGR4. While effect sizes correlated between tissues (r = 0.81), formal mediation analysis indicated individual CpG sites do not fully mediate SNP-phenotype relationships, suggesting coordinated epigenetic mechanisms. Most associations showed peak effects during childhood, while 8q24 showed unique adult-specific patterns. We identified genetic variants influencing methylation at craniofacial regulatory elements, and provided a mechanistic link for a major risk locus, 8q24, with tissue-specific effects in saliva. While individual CpG sites did not fully mediate the genetic risk, our findings identified specific regulatory regions where coordinated epigenetic changes may contribute to OFC susceptibility.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":"301 1","pages":""},"PeriodicalIF":2.2,"publicationDate":"2026-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13290791/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148302447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DDX39B drives the m6A modification of LDHA to promote trophoblast proliferation.","authors":"Cheng Li, Wenjun Zhou, Yuqin Shen, Jing Zhang, Yanqiong Jiang, Ruiman Li","doi":"10.1007/s00438-026-02435-x","DOIUrl":"10.1007/s00438-026-02435-x","url":null,"abstract":"<p><p>Preeclampsia (PE), a hypertensive disorder unique to pregnancy, is linked to impaired trophoblast function. DEAD-box helicase 39B (DDX39B) plays key roles in embryonic development. This study investigated its role in regulating trophoblast biology during PE progression. We conducted functional assays using CCK-8, clone formation, EdU, Transwell, Wound healing and TUNEL in the HTR-8/SVneo trophoblast cells. The interaction between Wilms tumor 1-associating protein (WTAP) and DDX39B was analyzed by Co-IP assay. RIP assay or RNA pull down were used to assess the association between the ELAV-like RNA-binding protein 1 (ELAVL1)/WTAP and L-lactate dehydrogenase A (LDHA) mRNA. Additionally, MeRIP assay was employed to evaluate m6A levels on LDHA transcripts. Overexpression of DDX39B promoted the proliferation and migration of trophoblast cells and suppressed cells apoptosis, while DDX39B knockdown had the opposite result. In addition, WTAP knockdown reversed the promoting effects of DDX39B overexpression on trophoblast proliferation and migration. Mechanistically, DDX39B promoted post-translational stabilization of WTAP by directly interacting with WTAP protein. WTAP enhanced the m6A methylation of LDHA mRNA by recruiting ELAVL1. As expected, LDHA knockdown abrogated the pro-proliferative and anti-apoptotic effects of WTAP overexpression on trophoblasts. Our findings established a novel DDX39B/WTAP/m6A/LDHA regulatory axis, wherein DDX39B acted as an RNA-binding protein to stabilize WTAP, enhancing LDHA expression and promoting trophoblast proliferation, migration, and survival. Dysregulation of this pathway might contribute to PE pathogenesis, offering new avenues for targeted therapies.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":"301 1","pages":""},"PeriodicalIF":2.2,"publicationDate":"2026-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148278111","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single-cell transcriptomics identifies key immune-suppressive cells and their driver genes in the bladder cancer microenvironment with prognostic implications.","authors":"Weihang Song, Guangye Han, Zhenhui Li, Junling Hu, Kuo Ma","doi":"10.1007/s00438-026-02472-6","DOIUrl":"10.1007/s00438-026-02472-6","url":null,"abstract":"<p><p>The immunosuppressive tumor microenvironment (TME) serves as a central driver of bladder cancer (BCa) progression and prognosis. While its significance is widely acknowledged, the key cellular subsets that mediate this immunosuppressive state and their core regulatory genes remain incompletely understood. Key immunosuppressive cellular subsets and their signature genes were systematically identified using single-cell RNA sequencing (scRNA-seq) data from BCa samples. A prognostic risk model was then constructed via univariate and multivariate Cox regression analyses, based on bulk RNA-seq datasets and the identified signature genes. The role of SUSD2 was further validated in vitro using qRT-PCR, Western blot, immunofluorescence, proliferation, and invasion assays. Compared with adjacent normal tissues, BCa tissues showed significant enrichment of stromal cells (e.g., epithelial cells, fibroblasts). Among these stromal populations, the proportion of myofibroblast-like cancer-associated fibroblasts (myCAFs) was significantly increased in BCa tissues, and high myCAF infiltration was closely associated with poor patient prognosis. Pseudotime trajectory analysis confirmed that fibroblast differentiation in BCa shifts toward a terminal state (State 3), which is predominantly composed of myCAFs. A prognostic model established using myCAF-related signature genes (TMEM74B, ABCC9, FCMR, ALG9, SUSD2, and ETV7) exhibited stable predictive performance in both training and validation cohorts, with SUSD2 identified as a risk-related gene. In vitro experiments revealed that SUSD2 knockdown inhibited myCAF activation and extracellular matrix secretion, thereby attenuating its promotional effects on BCa cell proliferation and invasion. The TGF-β receptor inhibitor SB-431542could reverse the facilitative effects of SUSD2 overexpression on tumor cell proliferation and migration. Our findings identify myCAFs as a core regulatory cellular subset and SUSD2 as a key molecule within the immunosuppressive TME of BCa. Additionally, SUSD2 may trigger the activation of the TGF-β/Smad signaling cascade to induce myCAF activation, thereby accelerating BCa progression. These results provide novel potential targets and a theoretical basis for prognosis assessment and TME-targeted therapy in BCa.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":"301 1","pages":""},"PeriodicalIF":2.2,"publicationDate":"2026-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148278098","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genotype-dependent metabolic and inflammatory regulation at the ANRIL locus in human primary macrophages.","authors":"Cemre Buse Kirsan-Kesici, Aybike Sena Ozuynuk-Ertugrul, Evrim Komurcu-Bayrak, Neslihan Coban","doi":"10.1007/s00438-026-02454-8","DOIUrl":"10.1007/s00438-026-02454-8","url":null,"abstract":"<p><p>The long non-coding RNA ANRIL and several single-nucleotide polymorphisms have been strongly implicated in genetic susceptibility to coronary artery disease (CAD). However, how these variants influence gene expression remains incompletely understood. This study aims to investigate genotype-dependent differences in linear and circular ANRIL isoform expression and selected CAD- and diabetes-related genes in monocyte-derived macrophages from healthy adult males carrying rs1333049, rs10757274, and rs564398 variants. Peripheral blood monocytes from healthy male donors with specific ANRIL genotypes were isolated and differentiated into macrophages using macrophage colony-stimulating factor. Monocyte identity and macrophage differentiation were confirmed by flow cytometry and confocal imaging. Gene expression levels of CDKN2A, CDKN2B, ABCA1, PRKAA1, PPARGC1A, TNFA, TNFAIP3, and circular and linear ANRIL transcripts were quantified by qRT-PCR. Expression differences across genotype groups and genotype-dependent correlations were assessed. The rs10757274/rs1333049 AA/CC genotype was associated with reduced expression of PPARGC1A and ABCA1, accompanied by lower TNFA and higher TNFAIP3 expression. In contrast, carriage of the G allele at rs10757274/rs1333049 was associated with increased expression of CDKN2A and linear ANRIL. The rs564398 CC genotype was associated with reduced PPARGC1A expression and altered PRKAA1 levels. Correlation analyses revealed genotype-specific associations between ANRIL isoforms and selected genes. These findings suggest that ANRIL variants are associated with genotype-dependent differences in macrophage gene expression across pathways of cell cycle regulation, inflammation, lipid metabolism, and energy homeostasis. Although exploratory, the results support the possibility that CAD- and diabetes-associated 9p21.3 variants may partly exert their effects through such transcriptional alterations.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":"301 1","pages":""},"PeriodicalIF":2.2,"publicationDate":"2026-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148278058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nocardia fodinahabitans sp. nov. isolated from underground hard coal mine waters shows biotechnological potential for degradation of aromatic hydrocarbons.","authors":"Julia Marciniak, Trine Sørensen, Agnieszka Nowak, Magdalena Noszczyńska, Jakub Smoliński, Katarzyna Machnik, Karolina Solska, Mateusz Pala, Frederik T Hansen, Teis Søndergaard, Mariola Paściak, Piotr Siupka","doi":"10.1007/s00438-026-02456-6","DOIUrl":"10.1007/s00438-026-02456-6","url":null,"abstract":"<p><p>The aim of the study was to describe and evaluate the biotechnological potential of a novel species of Nocardia isolated from underground coal mine water. Nocardia strains represent important human pathogens, but are also common in soil and water environments. Recently, they have gained attention for their biosynthetic potential and vast metabolic abilities to degrade aromatic compounds, including xenobiotics. In this work, we established the taxonomic status of strain MW-W600-9^T. Phylogenetic analysis based on 16 S rRNA gene and whole genome sequencing revealed close relatedness to Nocardia rhizosphaerihabitans and Nocardia asteroides. This is further supported by phenotyping tests, chemotaxonomic analyses and MALDI-ToF profiling. We therefore propose that strain MW-W600-9^T (= DSM 120649^T =PCM 3565^T) constitutes a new species, Nocardia fodinahabitans sp. nov. The strain has 31 putative biosynthetic gene clusters, 12 showing low or no similarity to the clusters of known compounds, indicating its potential to produce novel bioactive compounds. The strain MW-W600-9^T shows degradation potential of aromatic xenobiotics, tested for haloorganic compounds and bisphenols. The concentration of 4-chlorophenol and bisphenol A was decreasing significantly during the strain culture, and the results suggest co-metabolic removal of iohexol. This work shows the biodegradation ability of aromatic xenobiotic compounds by a new species of Nocardia, highlighting the potential use of the strain in bioremediation applications. It also shows for the first time the degradation of bisphenol by a member of the Nocardia genus, along with potential co-metabolic degradation of environmentally persistent haloorganic compounds.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":"301 1","pages":""},"PeriodicalIF":2.2,"publicationDate":"2026-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13269418/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148258930","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrating HTS and CRISPR/Cas for next-generation nucleic and non-nucleic acid diagnostics.","authors":"Manoj Kumar Mishra, Sagar Pamu, Prathap Madeswara Guptha, Murugesan Vanangamudi, Sathish Kumar Mittapalli, Pragyandip Parthasarathi Dash, Vanrajsinh Thakor, Palakurthi Yanadaiah, Sazal Patyar","doi":"10.1007/s00438-026-02468-2","DOIUrl":"10.1007/s00438-026-02468-2","url":null,"abstract":"<p><p>The synergy between HTS and CRISPR/Cas is changing how genes, biomarkers, and diseases are studied. Very useful due to the ability to scan entire molecular libraries in a single assay and its extreme rapidity. CRISPR/Cas systems, however, are crucial for achieving control and specificity, properties essential for precise genetic editing and targeted detection. HTS could be combined with CRISPR in two ways: HTS would expand the search space, and CRISPR would narrow it. This perspective highlights recent advances in which both platforms have been used together - for example, to find genetic variants and molecular markers in cancer, infectious diseases, and even biosensors to track the environment and metabolism. We discuss technical advances as well as practical issues that make the use of CRISPR/Cas more challenging in the clinical environment, including off-target activity, reproducibility, and the increasing complexity and dimensionality of data.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":"301 1","pages":""},"PeriodicalIF":2.2,"publicationDate":"2026-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148258967","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A modality-aware CRISPR actionability framework for functional prioritization of genome-wide significant type 2 diabetes loci.","authors":"Mehboob Uddin Mohd, Syed Mohd Zakariya Ali Khan","doi":"10.1007/s00438-026-02448-6","DOIUrl":"10.1007/s00438-026-02448-6","url":null,"abstract":"<p><p>Genome-wide association studies (GWAS) have identified numerous loci associated with Type 2 Diabetes (T2D), yet translating statistical signals into experimentally testable hypotheses remains a central challenge in post-GWAS biology. The predominance of non-coding regulatory variants complicates target gene assignment and raises uncertainty regarding optimal clustered regularly interspaced short palindromic repeats (CRISPR) perturbation strategy. Here, we present a structured CRISPR Actionability Framework that integrates genomic context, pancreatic islet enhancer overlap, tissue-specific expression validation, and locus clarity into a quantitative CRISPR Actionability Score (CAS). We applied this framework to ten genome-wide significant T2D loci and assigned modality-aware CRISPR strategies (knockout versus CRISPR interference). CAS values ranged from 4 to 10, enabling tiered prioritization into high, moderate, and lower experimental priority classes. High-priority loci included SLC30A8, TCF7L2, and KCNJ11, which demonstrated strong regulatory or coding evidence combined with islet expression support. By explicitly linking genomic architecture to perturbation modality, this framework provides a transparent and reproducible bridge between statistical genetics and functional genome editing. This approach establishes a scalable template for rational CRISPR target selection in complex disease research.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":"301 1","pages":""},"PeriodicalIF":2.2,"publicationDate":"2026-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148258955","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genomic and molecular dynamics analysis of mefA-encoded macrolide efflux protein from Tn2009 and Tn2010 in Streptococcus pneumoniae.","authors":"Yustinus Maladan, Endah Retnaningrum, Budi Setiadi Daryono, Ratna Fathma Sari, Dodi Safari","doi":"10.1007/s00438-026-02457-5","DOIUrl":"10.1007/s00438-026-02457-5","url":null,"abstract":"<p><p>Antibiotic resistance in Streptococcus pneumoniae remains a major clinical challenge, particularly for macrolides such as erythromycin. This resistance is commonly associated with transposons Tn2009 and Tn2010 carrying the mefA gene, which encodes a macrolide efflux protein. This study investigated the genetic characteristics of mefA from Indonesian clinical isolates and elucidated its efflux mechanism using integrated genomic analysis and molecular dynamics simulations. Whole-genome sequencing confirmed the presence of Tn2009 and Tn2010 in clinical isolates. Multiple sequence alignment showed high conservation of mefA (98-100% identity), indicating strong evolutionary stability within the species. A homology model of the mefA encoded efflux protein was constructed and used for molecular docking, revealing stable erythromycin binding within the efflux channel with multiple favorable binding poses. Molecular dynamics simulations (200 ns) demonstrated structural stability of the protein, with an average root mean square deviation of 0.174 nm. Root mean square fluctuation analysis identified localized flexibility in residues Asn195-Ser199, suggesting a functionally relevant intracellular loop involved in conformational dynamics. Despite stable interactions, erythromycin remained confined within a localized channel region and did not undergo spontaneous translocation under equilibrium conditions. Steered molecular dynamics simulations indicated that ligand transport requires external force to overcome an initial energetic barrier of 553.07 kJ/mol/nm, followed by stepwise displacement through multiple transient binding sites, consistent with a multi-site relay mechanism. Umbrella sampling further revealed a rugged free energy landscape, with a maximum potential of mean force (PMF) of ~ 45 kcal/mol near the channel exit region. The PMF profile, reconstructed using WHAM across ~ 30 windows, showed well-converged overlap and multiple intermediate minima, indicating a stable sampling of the reaction coordinate. Collectively, these findings provide structural and energetic insights into mefA mediated erythromycin resistance in S. pneumoniae. The results support a mechanism involving stable substrate binding, localized conformational flexibility, and substantial energy barriers requiring active transport, highlighting potential targets for efflux inhibition strategies.</p>","PeriodicalId":18816,"journal":{"name":"Molecular Genetics and Genomics","volume":"301 1","pages":""},"PeriodicalIF":2.2,"publicationDate":"2026-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148258952","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}