{"title":"An artifact of recombinatorial cloning challenges established beliefs of plasmid cotransformation, selection, and maintenance.","authors":"Courtney L Geer, J Michael Charette","doi":"10.1139/bcb-2025-0096","DOIUrl":"10.1139/bcb-2025-0096","url":null,"abstract":"<p><p>Gateway cloning is an easy, efficient, accurate, and versatile cloning strategy. During Expression clone validation, we sometimes see an additional band co-migrating with the pDONR (Entry) backbone. We show that this \"mystery\" band is not an artifact of aberrant recombination but instead originates from a cotransformation event, where more than one different plasmid is transformed into a single <i>Escherichia coli</i> cell simultaneously and in the absence of antibiotic selection. We find that the unselected pDONR Entry plasmid is cotransformed into <i>E. coli</i> with the desired Expression vector in 9%-29% of colonies and is maintained without antibiotic selection, despite plasmid incompatibility. We propose an easy strategy to screen for and eliminate cotransformants. Our results challenge accepted beliefs of bacterial plasmid transformation, selection, and maintenance and comprise the first documented occurrence of cotransformation in Gateway cloning.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":"1-13"},"PeriodicalIF":2.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144156069","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}
Genwang Wang, Di Liu, Junzhi Leng, Dong Jin, Qi Wang, Hao Wang, Yang Bu, Feng Wang, Yongfeng Hui
{"title":"TMCO1 regulates energy metabolism and mitochondrial function of hepatocellular carcinoma cells through TOMM20, affecting the growth of subcutaneous graft tumors and infiltration of CAFs.","authors":"Genwang Wang, Di Liu, Junzhi Leng, Dong Jin, Qi Wang, Hao Wang, Yang Bu, Feng Wang, Yongfeng Hui","doi":"10.1139/bcb-2024-0091","DOIUrl":"10.1139/bcb-2024-0091","url":null,"abstract":"<p><p>This study mainly shows the role of endoplasmic reticulum transmembrane and coiled coil domains 1 (TMCO1) in the regulatory mechanism of hepatocellular carcinoma (HCC). Invasion and migration capacity were detected by Transwell and wound healing after TMCO1 and TOMM20 overexpression and knockdown, and mitochondrial function was detected through reactive oxygen species (ROS), mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), and ATP production. A model of subcutaneous tumor formation in nude mice was established to detect the effect of TMCO1 on tumor formation. The results showed that overexpression of TMCO1 significantly promoted HCC cell metastasis, promoted cell proliferation and ATP production, inhibited cell apoptosis, mPTP opening and ROS production, mediated the increase of MMP level and cytoskeletal remodeling. However, knocking down TMCO1 can have the opposite effect. More importantly, knocking down TOMM20 can block the regulation effect of TMCO1, and TOMM20 overexpression can alleviate the inhibitory effect of knocking down TMCO1 on the development of liver cancer cells. In animal models, knockdown of TMCO1 expression significantly inhibited the growth of subcutaneous implant tumors. This suggests that TMCO1 may be a potential and valuable therapeutic target for liver cancer.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":"1-15"},"PeriodicalIF":2.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142680701","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}
Berta Buey, Eva Latorre, Marta Castro, Marta Sofía Valero, Miguel Ángel Plaza, María Pilar Arruebo, Inés Abad, Ana Rodríguez-Largo, Lourdes Sánchez, José Emilio Mesonero
{"title":"Neuroprotective effects of whey and buttermilk-based formulas on a DSS-induced colitis murine model.","authors":"Berta Buey, Eva Latorre, Marta Castro, Marta Sofía Valero, Miguel Ángel Plaza, María Pilar Arruebo, Inés Abad, Ana Rodríguez-Largo, Lourdes Sánchez, José Emilio Mesonero","doi":"10.1139/bcb-2024-0153","DOIUrl":"10.1139/bcb-2024-0153","url":null,"abstract":"<p><p>Inflammatory bowel disease is a gut-brain axis disorder that comprises chronic inflammatory conditions affecting the gastrointestinal tract, where alterations in the mood of patients are common. Gut-brain axis is a bidirectional communication that link gut and brain. The close association between inflammatory bowel disease and neuroinflammation has far-reaching implications, as is increasingly recognized as a contributing factor to neuropsychiatric and neurodegenerative diseases. The increasing prevalence and high economic cost, together with the loss of life quality of people suffering from these diseases, point to the need to find alternatives to alleviate them. Exploring new therapeutic avenues prompts us to consider the potential benefits of milk fractions, taking advantage of the use of dairy by-products, such as whey and buttermilk. This study examines the impact of cow's whey- and buttermilk-based formulas supplemented with bovine lactoferrin and milk fat globule membrane on the expression of cytokines, as well as on the components of immune and serotonergic system of the brain in a murine model of dextran sodium sulfate-induced colitis. Our results show the potential of these dairy by-products, especially whey, as functional foods in ameliorating neuroinflammation and safeguarding the central nervous system function amid the neurological complications induced or concomitant with intestinal inflammatory processes.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":"1-11"},"PeriodicalIF":2.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142364213","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}
Amjad R Khan, Rabia Javed, Tariq Sadad, Saeed Ali Bahaj, Gabriel Avelino Sampedro, Mideth Abisado
{"title":"Early pigment spot segmentation and classification from iris cellular image analysis with explainable deep learning and multiclass support vector machine.","authors":"Amjad R Khan, Rabia Javed, Tariq Sadad, Saeed Ali Bahaj, Gabriel Avelino Sampedro, Mideth Abisado","doi":"10.1139/bcb-2023-0183","DOIUrl":"10.1139/bcb-2023-0183","url":null,"abstract":"<p><p>Globally, retinal disorders impact thousands of individuals. Early diagnosis and treatment of these anomalies might halt their development and prevent many people from developing preventable blindness. Iris spot segmentation is critical due to acquiring iris cellular images that suffer from the off-angle iris, noise, and specular reflection. Most currently used iris segmentation techniques are based on edge data and noncellular images. The size of the pigment patches on the surface of the iris increases with eye syndrome. In addition, iris images taken in uncooperative settings frequently have negative noise, making it difficult to segment them precisely. The traditional diagnosis processes are costly and time consuming since they require highly qualified personnel and have strict environments. This paper presents an explainable deep learning model integrated with a multiclass support vector machine to analyze iris cellular images for early pigment spot segmentation and classification. Three benchmark datasets MILE, UPOL, and Eyes SUB were used in the experiments to test the proposed methodology. The experimental results are compared on standard metrics, demonstrating that the proposed model outperformed the methods reported in the literature regarding classification errors. Additionally, it is observed that the proposed parameters are highly effective in locating the micro pigment spots on the iris surfaces.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":"1-10"},"PeriodicalIF":2.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71420299","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":"The role of the polybromo-associated BAF complex in development.","authors":"JinYoung Park, Jacob G Kirkland","doi":"10.1139/bcb-2024-0224","DOIUrl":"10.1139/bcb-2024-0224","url":null,"abstract":"<p><p>Chromatin is dynamically regulated during development, where structural changes affect the transcription of genes required to promote different cell types. One of the chromatin regulatory factors responsible for transcriptional regulation during development is the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, an ATP-dependent chromatin remodeling factor conserved throughout eukaryotes. The catalytic subunit of this complex, BRG1, is shared in all three SWI/SNF complexes subfamilies and is essential for developing most cell lineages. Interestingly, many human developmental diseases have correlative or causative mutations in different SWI/SNF subunits. Many polybromo-associated BAF (pBAF) complex-specific subunit genetic alterations result in developmental failures in tissue-specific ways. This observation suggests that the pBAF complex plays a vital role in development and differentiation, and studying the pBAF complex may provide an opportunity to better understand gene regulation during development. In this mini-view, we will focus on the functions of pBAF-specific subunits and their influence on the development of various cell and tissue types by regulating developmental gene expression.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":"1-8"},"PeriodicalIF":2.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11752563/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142614211","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":"A novel mouse model of pulmonary fibrosis: twice-repeated oropharyngeal bleomycin administration mimicking human pathology.","authors":"Jingyu Wang, Fengqing Zhu, Yuxuan Liu, Renru Luo, Zixuan Fan, Wanqin Dai, Shuquan Wei, Chuwen Lin","doi":"10.1139/bcb-2024-0221","DOIUrl":"10.1139/bcb-2024-0221","url":null,"abstract":"<p><p>Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible lung disease with high mortality and limited treatment options. While single-dose bleomycin-induced models are commonly used to investigate the pathogenesis of IPF, they fail to adequately replicate the complex pathological features in human patients, thereby hindering comprehensive investigations. Previous studies utilizing repetitive bleomycin injections have demonstrated a closer resemblance to human IPF pathology; however, the time- and resource-intensive nature of this approach presents significant drawbacks. Here, we propose a novel methodology involving twice-repeated oropharyngeal administration of bleomycin in mice, which closely mirrors the pathological manifestations observed in IPF patients. This model exhibited the honeycomb-like cyst formation, fibroblastic foci, bronchiolization of alveolar epithelium, emergence of metaplastic alveolar KRT5<sup>+</sup> basal cells, and sustainability of these fibrotic phenotypes, thereby providing a robust model for IPF. Our findings establish a more efficient and translatable preclinical platform for investigating IPF pathogenesis and exploring potential therapeutic strategies.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":"103 ","pages":"1-7"},"PeriodicalIF":2.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143057889","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}
Xiangming Gao, Li Liu, Xueqin Liu, Yanru Shen, Yang Fan
{"title":"Study on the mechanism of USP7 promoting endometriosis by regulating DNMT1 deubiquitination level.","authors":"Xiangming Gao, Li Liu, Xueqin Liu, Yanru Shen, Yang Fan","doi":"10.1139/bcb-2024-0301","DOIUrl":"10.1139/bcb-2024-0301","url":null,"abstract":"<p><p>Endometriosis is characterized by aberrant epigenetic regulation, and our study reveals that both Ubiquitin Specific Protease 7 (USP7) and DNA methyltransferase 1 (DNMT1) are significantly overexpressed in endometriosis tissues. Functional analyses in ectopic endometrial stromal cells (EESCs) indicate that elevated USP7 levels markedly enhance cellular proliferation, migration, and invasion, whereas silencing DNMT1 mitigates these oncogenic properties. Notably, USP7 and DNMT1 co-localize within the nucleus and interact directly, with USP7 modulating DNMT1 stability by attenuating its ubiquitination. The reduction in DNMT1 protein levels following USP7 silencing was reversed by proteasome inhibition, underscoring the pivotal role of USP7-mediated deubiquitination in maintaining DNMT1 stability. Furthermore, treatment with the USP7 inhibitor FT-671 significantly reduced DNMT1 protein expression while leaving its mRNA levels unaffected, and FT-671 effectively suppressed EESCs proliferation, migration, and invasion. Collectively, these findings suggest that USP7 contributes to the pathogenesis of endometriosis by sustaining DNMT1 expression and promoting aberrant cellular behaviors, thereby representing a promising therapeutic target for this disease.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":"1-14"},"PeriodicalIF":2.4,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144109562","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":"Quercetin alleviates the progression of chronic rhinosinusitis by affecting nasal mucosal epithelial remodeling, inflammation, and Treg/Th17 imbalance.","authors":"Xiaopeng Qu, Pengyu Tao, Jiajia Dong, Lingzhao Meng","doi":"10.1139/bcb-2025-0049","DOIUrl":"10.1139/bcb-2025-0049","url":null,"abstract":"<p><p>Nasal mucosal epithelial tissue remodeling and persist inflammation are related to the development of chronic rhinosinusitis (CRS). Quercetin possesses multiple biological properties in several inflammatory diseases. However, its roles in CRS remain unclear. In this study, <i>Serumstaphylococcus aureus enterotoxin B</i> (SEB) increased inflammatory response in human nasal epithelial cells (hNECs), which was reversed by quercetin. Moreover, quercetin inhibited SEB-evoked epithelial-mesenchymal transition (EMT) in hNECs by increasing EMT marker E-cadherin and decreasing N-cadherin expression. Concomitantly, SEB-induced increases in transcripts and release of MMP-9 were reduced by quercetin. Mechanistically, quercetin inhibited SEB-induced activation of the TLR2-NF-kB axis in hNECs. Moreover, restoring TLR2 signaling reversed quercetin-mediated inhibition of SEB-induced inflammation, EMT and MMP-9 expression. In vivo, quercetin attenuated histopathological changes of nasal mucosal tissues in <i>Staphylococcus aureus</i>-constructed CRS mice. Concomitantly, quercetin alleviated inflammatory response and nasal mucosal remodeling by suppressing EMT and MMP-9 levels. Additionally, quercetin ameliorated imbalance of Treg/Th17 proportions. Notably, quercetin suppressed activation of the TLR2-NF-kB axis, while restoring this signaling reversed quercetin-mediated protection against CRS. Thus, quercetin may attenuate pathological progression of CRS by inhibiting nasal mucosal tissue remodeling, inflammation and Treg/Th17 imbalance, which may be associated with inhibition of TLR2-NF-kB axis, supporting a promising therapeutic agent for CRS.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":"1-11"},"PeriodicalIF":2.1,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144551836","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 reliable Western blot workflow with improved dynamic range for the detection of myelin proteins in murine brain.","authors":"Victor P Liu, Shaheer Lakhani, Kendra L Furber","doi":"10.1139/bcb-2025-0093","DOIUrl":"10.1139/bcb-2025-0093","url":null,"abstract":"<p><p>Myelin is a highly structured multilamellar sheath produced by oligodendrocytes, which insulates neuronal axons to facilitate neurotransmission. Maturation of oligodendrocytes in cortical regions of the developing murine brain occurs postnatally and corresponds to the marked upregulation of myelin-specific genes. Western blotting is a conventional technique used to study protein expression but historically has only been considered semiquantitative. This study aims to optimize a Western blot workflow for the quantification of myelin proteins in murine brain, including the examination of the following parameters: sample preparation, electrophoretic transfer conditions, detection parameters, data normalization, and linear dynamic range. As a proof of principle, the optimized protocol was employed to characterize both the absolute and relative expression of myelin oligodendrocyte glycoprotein (MOG) throughout neurodevelopment. A dynamic loading paradigm, which varied total protein load across different age groups to ensure antigen detection remained in the linear dynamic range of the assay, showed a greater relative increase in expression when compared to standard loading paradigm. This approach resulted in comparable MOG expression profiles from both absolute and relative quantification. The optimized Western blot workflow will facilitate protein quantification and will improve data reproducibility when investigating the molecular mechanisms of myelination in development, aging, and disease.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":"1-13"},"PeriodicalIF":2.1,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144289382","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}