Journal of Biological Engineering最新文献

{"title":"Chitosan biomineralized with ions-doped nano-hydroxyapatite tunes osteoblasts metabolism and DNA damage.","authors":"Franco Furlani, Matilde Clarissa Malfatti, Alfredo Rondinella, Elisabetta Campodoni, Monica Sandri, Lorenzo Fedrizzi, Gianluca Tell","doi":"10.1186/s13036-024-00458-9","DOIUrl":"10.1186/s13036-024-00458-9","url":null,"abstract":"<p><p>Hydroxyapatite (HA) is a bioceramic material widely used as a bone biomimetic substitute and can be synthesized by biomineralization, according to which HA nanoparticles are formed on a polymer template. Nevertheless, little is known about the effect of ion doping and biomineralization on cell metabolism, oxidative stress, and DNA damage. In the present contribution, we report on synthesizing and characterizing biomineralized chitosan as a polymer template with HA nanoparticles doped with magnesium (MgHA) and iron ions (FeHA). The physical-chemical and morphological characterization confirmed the successful synthesis of low crystalline ions-doped HA nanoparticles on the chitosan template, whereas the biochemical activity of the resulting nanoparticles towards human osteoblasts-like cells (MG63 and HOBIT) was investigated considering their effect on cell metabolism, proliferation, colony formation, redox status, and DNA damage extent. Data obtained suggest that particles enhance cell metabolism but partially limit cell proliferation. The redox status of cells was measured suggesting a slight increase in Reactive Oxygen Species production with chitosan biomineralized with iron-doped HA, whereas no effect with magnesium-doped HA and no effect of all formulations on the oxidation level of Peroxiredoxin. On the other hand, DNA damage was investigated by COMET assay, and expression and foci γH2AX. These latter tests indicated that HA-based nanoparticles promote DNA damage which is enhanced by chitosan thus suggesting that chitosan favors the nanoparticles' internalization by cells and modulates their biological activity. The potential DNA damage should be considered - and potentially exploited for instance in anticancer treatment - when HA-based particles are used to devise biomaterials.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"60"},"PeriodicalIF":5.7,"publicationDate":"2024-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11515322/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142500937","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":"Phytoconstituents of Withania somnifera (L.) Dunal (Ashwagandha) unveiled potential cerebroside sulfotransferase inhibitors: insight through virtual screening, molecular dynamics, toxicity, and reverse pharmacophore analysis.","authors":"Nivedita Singh, Anil Kumar Singh","doi":"10.1186/s13036-024-00456-x","DOIUrl":"10.1186/s13036-024-00456-x","url":null,"abstract":"<p><p>Cerebroside sulfotransferase (CST) is considered as therapeutic target for substrate reduction therapy (SRT) for metachromatic leukodystrophy (MLD). The present study evaluates the therapeutic potential of 57 phytoconstituents of Withania somnifera against CST. Using binding score cutoff ≤-7.0 kcal/mol, top 10 compounds were screened and after ADME and toxicity-based screening, Withasomidienone, 2,4-methylene-cholesterol, and 2,3-Didehydrosomnifericin were identified as safe and potent drug candidates for CST inhibition. Key substrate binding site residues involved in interaction were LYS82, LYS85, SER89, TYR176, PHE170, PHE177. Four steroidal Lactone-based withanolide backbone of these compounds played a critical role in stabilizing their position in the active site pocket. 100 ns molecular dynamics simulation and subsequent trajectory analysis through structural deviation and compactness, principal components, free energy landscape and correlation matrix confirmed the stability of CST-2,3-Didehydrosomnifericin complex throughout the simulation and therefore is considered as the most potent drug candidate for CST inhibition and Withasomidienone as the second most potent drug candidate. The reverse pharmacophore analysis further confirmed the specificity of these two compounds towards CST as no major cross targets were identified. Thus, identified compounds in this study strongly present their candidature for oral drug and provide route for further development of more specific CST inhibitors.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"59"},"PeriodicalIF":5.7,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11515467/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142500938","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":"Microglia morphological response to mesenchymal stromal cell extracellular vesicles demonstrates EV therapeutic potential for modulating neuroinflammation.","authors":"Kanupriya R Daga, Andrew M Larey, Maria G Morfin, Kailin Chen, Sara Bitarafan, Jana M Carpenter, Hannah M Hynds, Kelly M Hines, Levi B Wood, Ross A Marklein","doi":"10.1186/s13036-024-00449-w","DOIUrl":"https://doi.org/10.1186/s13036-024-00449-w","url":null,"abstract":"<p><strong>Background: </strong>Mesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation- relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs.</p><p><strong>Results: </strong>Using an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-α /IFN-γ stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism.</p><p><strong>Conclusion: </strong>This study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphometric approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"58"},"PeriodicalIF":5.7,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11488223/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142466148","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":"Construction of bispecific antibodies by specific pairing between the heavy chain and the light chain using removable SpyCatcher/SnoopCatcher units.","authors":"Jyunna Yoshida, Yuki Kato, Ai Isogawa, Yoshikazu Tanaka, Izumi Kumagai, Ryutaro Asano, Takeshi Nakanishi, Koki Makabe","doi":"10.1186/s13036-024-00454-z","DOIUrl":"https://doi.org/10.1186/s13036-024-00454-z","url":null,"abstract":"<p><p>During the production of bispecific antibodies (bsAbs), nonspecific pairing results in low yields of target bsAb molecules, an issue known as the \"mispairing problem.\" Several antibody engineering techniques have been developed to overcome mispairing issues. Here, we introduce \"bsAb by external pairing and excision\" (BAPE), a novel chain pairing method that induces specific chain pairing by fusing external SpyCatcher/Tag and SnoopCatcher/Tag units. These tags are then removed via protease cleavage. In this study, we applied this method to force the correct pairings of heavy and light chains while the heavy-chain pairing was achieved by the Knobs-into-Holes mutation. We then confirmed the formation of interchain bridges with covalent isopeptide bonds. Both anti-CD3/anti-Her2 and anti-CD3/anti-EGFR bsAbs displayed satisfactory target binding activities and in vitro cell-killing activity with activated T-cells.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"57"},"PeriodicalIF":5.7,"publicationDate":"2024-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11476941/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142466147","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":"Enhancing fatty acid and omega-3 production in Schizochytrium sp. using developed safe-harboring expression system.","authors":"Ae Jin Ryu, Won-Sub Shin, Sunghoon Jang, Yejin Lin, Yejee Park, Yujung Choi, Ji Young Kim, Nam Kyu Kang","doi":"10.1186/s13036-024-00447-y","DOIUrl":"10.1186/s13036-024-00447-y","url":null,"abstract":"<p><strong>Background: </strong>Schizochytrium, a group of eukaryotic marine protists, is an oleaginous strain, making it a highly promising candidate for the production of lipid-derived products such as biofuels and omega-3 fatty acids. However, the insufficient advancement of genetic engineering tools has hindered further advancements. Therefore, the development and application of genetic engineering tools for lipid enhancement are crucial for industrial production.</p><p><strong>Results: </strong>Transgene expression in Schizochytrium often encounters challenges such as instability due to positional effects. To overcome this, we developed a safe-harbor transgene expression system. Initially, the sfGFP gene was integrated randomly, and high-expressing transformants were identified using fluorescence-activated cell sorting. Notably, HRsite 2, located approximately 3.2 kb upstream of cytochrome c, demonstrated enhanced sfGFP expression and homologous recombination efficiency. We then introduced the 3-ketoacyl-ACP reductase (KR) gene at HRsite 2, resulting in improved lipid and docosahexaenoic acid (DHA) production. Transformants with KR at HRsite 2 exhibited stable growth, increased glucose utilization, and a higher lipid content compared to those with randomly integrated transgenes. Notably, these transformants showed a 25% increase in DHA content compared to the wild-type strain.</p><p><strong>Conclusion: </strong>This study successfully established a robust homologous recombination system in Schizochytrium sp. by identifying a reliable safe harbor site for gene integration. The targeted expression of the KR gene at this site not only enhanced DHA production but also maintained growth and glucose consumption rates, validating the efficacy of the safe-harbor approach. This advancement in synthetic biology and metabolic engineering paves the way for more efficient biotechnological applications in Schizochytrium sp.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"56"},"PeriodicalIF":5.7,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11468124/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400356","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":"Correction: Follicular reconstruction and neo-oogenesis in xenotransplantation of human ovarian isolated cells derived from chemotherapy-induced POF patients.","authors":"Sara Khaleghi, Farideh Eivazkhani, Somayeh Tavana, Ashraf Moini, Marefat Ghaffari Novin, Petkov Stoyan, Hamid Nazarian, Rouhollah Fathi","doi":"10.1186/s13036-024-00452-1","DOIUrl":"https://doi.org/10.1186/s13036-024-00452-1","url":null,"abstract":"","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"55"},"PeriodicalIF":5.7,"publicationDate":"2024-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11459889/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142390786","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":"Avoiding overflow metabolite formation in Komagataella phaffii fermentations to enhance recombinant protein production.","authors":"Thomas Steimann, Judith Wegmann, Monica I Espinosa, Lars Mathias Blank, Jochen Büchs, Marcel Mann, Jørgen Barsett Magnus","doi":"10.1186/s13036-024-00453-0","DOIUrl":"10.1186/s13036-024-00453-0","url":null,"abstract":"<p><strong>Background: </strong>Komagataella phaffii (K. phaffii), formerly known as Pichia pastoris, is a widely utilized yeast for recombinant protein production. However, due to the formation of overflow metabolites, carbon yields may be reduced and product recovery becomes challenging. This study investigates the impact of oxygen availability, different glucose concentrations and feeding strategies on overflow metabolite formation and recombinant protein production in K. phaffii.</p><p><strong>Results: </strong>High glucose concentrations in batch fermentation, as applied in literature, lead to substantial ethanol accumulation, adversely affecting biomass yield and product formation. Increasing dissolved oxygen setpoints does not significantly reduce ethanol formation, indicating that glucose surplus, rather than oxygen availability, drives overflow metabolism. Decreasing the initial glucose concentration to 5 g/L and adapting the feeding strategy of the fed-batch phase, effectively mitigates overflow metabolite formation, improving biomass yield by up to 9% and product concentration by 40% without increasing process time.</p><p><strong>Conclusions: </strong>These findings underscore the importance of a suitable glucose-feeding strategy in K. phaffii fermentation processes and highlight the detrimental effects of overflow metabolites on productivity. By optimizing carbon source utilization, it is possible to enhance fermentation efficiency and recombinant protein production with K. phaffii.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"54"},"PeriodicalIF":5.7,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11448000/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142371944","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 allele-specific PCR with CRISPR-Cas13a for sensitive KRAS mutation detection in pancreatic cancer.","authors":"Samuel Amintas, Grégoire Cullot, Mehdi Boubaddi, Julie Rébillard, Laura Karembe, Béatrice Turcq, Valérie Prouzet-Mauléon, Aurélie Bedel, François Moreau-Gaudry, David Cappellen, Sandrine Dabernat","doi":"10.1186/s13036-024-00450-3","DOIUrl":"10.1186/s13036-024-00450-3","url":null,"abstract":"<p><strong>Background: </strong>The clustered regulatory interspaced short palindromic repeats (CRISPR)-Cas13a system has strong potential for highly sensitive detection of exogenous sequences. The detection of KRAS^G12 point mutations with low allele frequencies may prove powerful for the formal diagnosis of pancreatic ductal adenocarcinoma (PDAC).</p><p><strong>Results: </strong>We implemented preamplification of KRAS alleles (wild-type and mutant) to reveal the presence of mutant KRAS with CRISPR-Cas13a. The discrimination of KRAS^G12D from KRAS^WT was poor for the generic KRAS preamplification templates and depended on the crRNA design, the secondary structure of the target templates, and the nature of the mismatches between the guide and the templates. To improve the specificity, we used an allele-specific PCR preamplification method called CASPER (Cas13a Allele-Specific PCR Enzyme Recognition). CASPER enabled specific and sensitive detection of KRAS^G12D with low DNA input. CASPER detected KRAS mutations in DNA extracted from patients' pancreatic ultrasound-guided fine-needle aspiration fluid.</p><p><strong>Conclusion: </strong>CASPER is easy to implement and is a versatile and reliable method that is virtually adaptable to any point mutation.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"53"},"PeriodicalIF":5.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11445877/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142361553","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":"Probing the orthogonality and robustness of the mammalian RNA-binding protein Musashi-1 in Escherichia coli.","authors":"Roswitha Dolcemascolo, Raúl Ruiz, Sara Baldanta, Lucas Goiriz, María Heras-Hernández, Roser Montagud-Martínez, Guillermo Rodrigo","doi":"10.1186/s13036-024-00448-x","DOIUrl":"10.1186/s13036-024-00448-x","url":null,"abstract":"<p><p>RNA recognition motifs (RRMs) are widespread RNA-binding protein domains in eukaryotes, which represent promising synthetic biology tools due to their compact structure and efficient activity. Yet, their use in prokaryotes is limited and their functionality poorly characterized. Recently, we repurposed a mammalian Musashi protein containing two RRMs as a translation regulator in Escherichia coli. Here, employing high-throughput RNA sequencing, we explored the impact of Musashi expression on the transcriptomic and translatomic profiles of E. coli, revealing certain metabolic interference, induction of post-transcriptional regulatory processes, and spurious protein-RNA interactions. Engineered Musashi protein mutants displayed compromised regulatory activity, emphasizing the importance of both RRMs for specific and sensitive RNA binding. We found that a mutation known to impede allosteric regulation led to similar translation control activity. Evolutionary experiments disclosed a loss of function of the synthetic circuit in about 40 generations, with the gene coding for the Musashi protein showing a stability comparable to other heterologous genes. Overall, this work expands our understanding of RRMs for post-transcriptional regulation in prokaryotes and highlight their potential for biotechnological and biomedical applications.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"52"},"PeriodicalIF":5.7,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11443895/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347184","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":"Editorial Expression of Concern: Recent advances in nanomaterial-based biosensor for periodontitis detection.","authors":"Mohammad Hosseini Hooshiar, Masoud Amiri Moghaddam, Mohammad Kiarashi, Athraa Y Al-Hijazi, Abbas Fadel Hussein, Hareth A Alrikabi, Sara Salari, Samar Esmaelian, Hassan Mesgari, Saman Yasamineh","doi":"10.1186/s13036-024-00451-2","DOIUrl":"https://doi.org/10.1186/s13036-024-00451-2","url":null,"abstract":"","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"51"},"PeriodicalIF":5.7,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11438296/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142347183","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}