{"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":"https://doi.org/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 semi-quantitative. This study aims to optimize a western blot workflow for 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":""},"PeriodicalIF":2.4,"publicationDate":"2025-06-13","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}
{"title":"Bovine lactoferrin suppresses the proliferation of endometriotic stromal cells via the PI3K/Akt/mTOR pathway.","authors":"Akiko Nakamura, Yuji Tanaka, Shunichiro Tsuji, Tsukuru Amano, Akie Takebayashi, Akimasa Takahashi, Ayako Inatomi, Tetsuro Hanada, Takashi Murakami","doi":"10.1139/bcb-2025-0014","DOIUrl":"https://doi.org/10.1139/bcb-2025-0014","url":null,"abstract":"<p><p>The most common medical therapy for endometriosis suppresses ovulation, which is a barrier for patients planning pregnancy. To address this issue, we focused on the cell proliferation-suppressing effects of lactoferrin, which reportedly in various malignant tumours. Despite being a benign disease, endometriotic cells have similar characteristics to malignant tumours, which may be involved in its onset and progression. Endometriotic and endometrial stromal cells were obtained from patients with endometriosis. After culture with 1 mg/mL of bovine lactoferrin, cell proliferation was significantly suppressed in endometriotic stromal cells compared to controls, but this remained unchanged in endometrial stromal cells. Bovine lactoferrin also significantly increased the number of endometriotic stromal cells in the G0/G1 phase and significantly decreased those in the S phase, and suppressed the protein expression of phosphorylated-AKT, phosphorylated-mTOR, phosphorylated-S6K, and cyclin D1. Bovine lactoferrin inhibits the transition from the G1 to the S phase by suppressing the PI3K/Akt/mTOR pathway and reducing the synthesis of cyclin D1, thereby arresting the cell cycle at the G1 phase. Bovine lactoferrin suppressed the proliferation of endometriotic stromal cells without suppressing the proliferation of endometrial stromal cells. Lactoferrin, which allows for pregnancy and lactation during administration, has potential as a novel therapeutic candidate for endometriosis.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144257224","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}
Ming Xue, Hailing Ding, Yongxin Han, Yuhua Wei, Xuming Wang, Xiaohan Wang, Xiangqian Kong
{"title":"MCPIP1 deficiency alleviates abdominal aortic aneurysm formation by inhibiting MAPK signaling.","authors":"Ming Xue, Hailing Ding, Yongxin Han, Yuhua Wei, Xuming Wang, Xiaohan Wang, Xiangqian Kong","doi":"10.1139/bcb-2024-0260","DOIUrl":"https://doi.org/10.1139/bcb-2024-0260","url":null,"abstract":"<p><p>Abdominal aortic aneurysm (AAA) is a chronic and severe aortic disease. Our previous studies have indicated that monocyte chemotactic protein-induced protein-1 (MCPIP1) is involved in AAA. However, the exact effect of MCPIP1 on angiotensin II (Ang II)-induced AAA formation is currently unknown. MCPIP1 deficiency reduced AAA formation in Ang II-induced mice. Less collagen and elastin degradation were observed in MCPIP1 deficient mice treated with Ang II. Ang II decreased αSMA and SM22α levels in aortas and VSMCs, whereas MCPIP1 deficiency reduced this decrease. MCPIP1 deficiency also attenuated Ang II-induced expression of MAPK signaling-associated proteins in aortas and VSMCs. Silencing MCPIP1 decreased proliferation and migration in Ang II-induced VSMCs. Furthermore, inactivation of ERK1/2 with PD98059 reduced Ang II-induced proliferation and migration of VSMCs. Dual luciferase and chromatin immunoprecipitation assay results confirmed that MCPIP1 was transcriptionally regulated by KLF4. KLF4 knockdown reversed the facilitating effect of Ang II on MCPIP1 expression. In conclusion, our findings suggest that MCPIP1 promotes Ang II-induced VSMCs phenotypic switching via the MAPK signaling pathway.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-06-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144246217","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":"Subacute exposure to aluminium chloride induces cytotoxicity and oxidative stress in rat erythrocytes: A dose-dependent study.","authors":"Farha Shahabuddin, Samina Naseem, Farah Khan","doi":"10.1139/bcb-2024-0236","DOIUrl":"https://doi.org/10.1139/bcb-2024-0236","url":null,"abstract":"<p><p>Aluminium (Al) toxicity has attracted considerable interest due to its bioavailability, environmental persistence, and adverse health effects. The present study investigates the effect of Al on rat erythrocytes under in vivo conditions. Rats were administered 0 (control), 25 (Al 1), 35 (Al 2), 45 (Al 3), and 55 (Al 4) mg/kg b.wt of AlCl3 orally for 30 days. Hemolysates were prepared from different experimental groups and assayed for various biochemical parameters. AlCl₃ administration significantly increased protein oxidation and lipid peroxidation, while decreasing total sulfhydryl and glutathione levels in rat erythrocytes. Methemoglobin level was increased and methemoglobin reductase activity was decreased upon AlCl3 treatment. Prolonged AlCl3 exposure inhibited the activities of antioxidant enzymes, and lowered the cells' antioxidant power. It also caused an increase in H2O2 and NO levels showing generation of oxidative and nitrosative stress. AlCl3 intoxication adversely affected the membrane-bound and metabolic enzyme activities. Alterations in all the biochemical parameters were found in an AlCl3 dose-dependent manner. Scanning electron microscopy showed gross morphological changes in erythrocytes, from discocytes to acanthocytes and echinocytes, further supporting the damaging effect of aluminium. The aluminium-induced oxidative stress seems to be the key mechanism of damage to the cellular components that could lead to red cell senescence.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-05-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144186429","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}
T Deandre Chevannes, Antony D St-Jacques, Matthew Eric Loewen, Michele C Loewen
{"title":"A combinatorial multi-site directed mutagenesis solution for improved thermal stability of Lactobacillus plantarum tannase.","authors":"T Deandre Chevannes, Antony D St-Jacques, Matthew Eric Loewen, Michele C Loewen","doi":"10.1139/bcb-2025-0134","DOIUrl":"https://doi.org/10.1139/bcb-2025-0134","url":null,"abstract":"<p><p>This study used a modified flapless (FLT) version of tannase from Lactobacillus plantarum, (LpTan) to explore the effects of 'stacking' site mutations predicted by Protein Repair One Stop Shop (PROSS) to increase stability. Four different LpTan structural-state models (including apo, substrate- and product- bound as well as FLT) were comparatively applied, yielding 143 predicted mutations. Of these, 8 mutations (including Q63T, A65D, A184Y, A257D, V276Y, T321G, G421D and G439D (FLT numbering)) were selected to stack, based on conservation of the prediction across all four structural states. Combinatorial screening of the arising 256-member library yielded a selection of possible hits, of which four were further characterized. Variant P6H7 contained 7 of the 8 mutations (excluding V276Y) and showed the highest significant kcat, 17 % higher than FLT and 30 % higher than LpTan, and a 4.5 °C increase in Tm. Variant P8E5 with 6 of 8 mutations (excluding A257D and G439D), yielded a 6.5 °C increase in Tm compared to FLT. The two other variants showed more moderate increases, albeit still greater than FLT or LpTan. Overall, the ability to design thermal stabilized versions of a tannase is emphasized. Putative mechanisms underlying the stabilization imparted by the highlighted variations are discussed.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-05-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144180443","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":"Ophiopogonin D protects against cerebral ischemia-reperfusion injury in rats by inhibiting STAT3 phosphorylation.","authors":"Zheng Tang, Chunmei Liu","doi":"10.1139/bcb-2024-0328","DOIUrl":"https://doi.org/10.1139/bcb-2024-0328","url":null,"abstract":"<p><p>Our aim is to explore the protective effect of ophiopogonin D (OPD) on cerebral ischemia/reperfusion injury (CIRI) and its relevant molecular mechanisms. OPD effect on brain injury of CIRI rats was evaluated using 2,3,5-triphenyltetrazolium chloride staining, neurological deficit score, brain water content, hematoxylin and eosin staining, Nissl staining and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling staining. Immunofluorescence, immunohistochemistry, qRT-PCR, western blot and relative kits were used for detecting inflammatory factors, oxidative stress related factors, and mRNA and protein levels. The Cell Counting Kit-8 assay and flow cytometry were applied for assessing the viability and apoptosis of oxygen-glucose deprivation)/reoxygenation (OGD/R)-induced PC12 cells. OPD ameliorated brain injury via inhibiting neurons apoptosis, oxidative stress and inflammatory response in cerebral infarction rats. In addition, we found that OPD attenuated the apoptosis, oxidative stress and inflammatory response in OGD/R-induced PC12 cells. Both in CIRI rats and OGD/R-induced PC12 cells, OPD was demonstrated to inhibit signal transducer and activator of transcription 3 (STAT3) phosphorylation. Moreover, Colivelin TFA (a STAT3 activator) reversed OPD effect on the apoptosis, oxidative stress and inflammatory response in OGD/R-induced PC12 cells. Our findings demonstrated that OPD could protect against CIRI in rats by inhibiting STAT3 phosphorylation.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144156078","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":"An artifact of recombinatorial cloning challenges established beliefs of plasmid cotransformation, selection, and maintenance.","authors":"Courtney L Geer, Michael Charette","doi":"10.1139/bcb-2025-0096","DOIUrl":"https://doi.org/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 E. coli cell simultaneously and in the absence of antibiotic selection. We find that the unselected pDONR Entry plasmid is cotransformed into E. coli 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":""},"PeriodicalIF":2.4,"publicationDate":"2025-05-27","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}
Sherri L Christian, Valerie Booth, Scott Harding, Amy M Todd, Mark D Berry
{"title":"Equity in Action: A Four-Year journey towards Gender Parity and Racial Diversity in Biochemistry Hiring.","authors":"Sherri L Christian, Valerie Booth, Scott Harding, Amy M Todd, Mark D Berry","doi":"10.1139/bcb-2025-0114","DOIUrl":"https://doi.org/10.1139/bcb-2025-0114","url":null,"abstract":"<p><p>Recruitment of faculty members in academic departments shapes the department for decades in research and teaching arenas. A diverse department is beneficial for all students as representation of underrepresented minority groups in the professoriate can inspire a greater diversity of students to pursue higher levels of education or research-focused careers. Increased diversity benefits research directly as diverse teams have been shown to have better ideas and outcomes. In 2020, our department had lower gender diversity than expected based on the pool of qualified personnel in Canada. Therefore, we altered our hiring process, primarily by redacting applications, for recruitment into entry-level tenure-track faculty positions. This resulted in the increased hiring of women (17% to 80%) with no substantial change in hiring of racially diverse individuals (50% to 40%). Overall, combined with retirements, the percentage of women faculty in the department went from 25% to 50% and the percentage of racialized faculty went from 38% to 44%. Thus, our intervention was successful in increasing the diversity of our department within a short timeframe. Our experience could provide other departments with a template for making substantive change, even in the absence of internal expertise in the area.</p>","PeriodicalId":8775,"journal":{"name":"Biochemistry and Cell Biology","volume":" ","pages":""},"PeriodicalIF":2.4,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144109558","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":"https://doi.org/10.1139/bcb-2024-0301","url":null,"abstract":"<p><p>Endometriosis is characterized by aberrant epigenetic regulation, and our study reveals that both USP7 and 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":""},"PeriodicalIF":2.4,"publicationDate":"2025-05-20","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}
Marie-Frédérique Gauthier, Giada Ostinelli, Mélissa Pelletier, André Tchernof
{"title":"Origin of dedifferentiated adipocyte-derived cells (DFAT) during ceiling culture in an Adiponectin Cre-Recombinase mouse model.","authors":"Marie-Frédérique Gauthier, Giada Ostinelli, Mélissa Pelletier, André Tchernof","doi":"10.1139/bcb-2024-0140","DOIUrl":"10.1139/bcb-2024-0140","url":null,"abstract":"<p><p>DFAT cells represent an attractive source of stem cells in tissue engineering and in the potential treatment of several clinical conditions. Our objective was to determine whether DFAT cells originate from mature adipocytes and address whether contamination from the stromal vascular fraction (SVF) could be as a source for these cells. A murine adiponectin-creERT;mT/mG model was used with the excision of the cassette induced by tamoxifen injection for the cells expressing adiponectin (adipoq). This model allows distinguishing of mature adipocytes (green fluorescence) from other SVF cell types (red fluorescence) based on the fluorescent protein expressed. Mature adipocytes and SVF cells were isolated from adipose tissues by collagenase digestion. Ceiling cultures were imaged by time-lapse microscopy. Confocal microscopy was used to follow cells over 21 days. Time-lapse microscopy experiments showed liposecretion occurring in mature adipocytes displaying green fluorescence. Confocal imaging allowed the identification of a heterogeneous cell population expressing green but also red fluorescence after 21 days of culture. Asymmetrical division of mature adipocytes was not observed. In conclusion, liposecretion of mature adipocytes is a phenomenon that can be observed in vitro and DFAT cells do originate from mature adipocytes<i>.</i> However, the population of DFAT cells is heterogenous.</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":"142543388","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}