BMC BiotechnologyPub Date : 2024-08-02DOI: 10.1186/s12896-024-00882-1
Narges Yaghoubi, Amir Gholamzad, Tahere Naji, Mehrdad Gholamzad
{"title":"In vitro evaluation of PLGA loaded hesperidin on colorectal cancer cell lines: an insight into nano delivery system.","authors":"Narges Yaghoubi, Amir Gholamzad, Tahere Naji, Mehrdad Gholamzad","doi":"10.1186/s12896-024-00882-1","DOIUrl":"10.1186/s12896-024-00882-1","url":null,"abstract":"<p><strong>Background: </strong>Colorectal cancer is a common disease worldwide with non-specific symptoms such as blood in the stool, bowel movements, weight loss and fatigue. Chemotherapy drugs can cause side effects such as nausea, vomiting and a weakened immune system. The use of antioxidants such as hesperidin could reduce the side effects, but its low bioavailability is a major problem. In this research, we aimed to explore the drug delivery and efficiency of this antioxidant on the HCT116 colorectal cancer cell line by loading hesperidin into PLGA nanoparticles.</p><p><strong>Materials and methods: </strong>Hesperidin loaded PLGA nanoparticles were produced by single emulsion evaporation method. The physicochemical properties of the synthesized hesperidin-loaded nanoparticles were determined using SEM, AFM, FT-IR, DLS and UV-Vis. Subsequently, the effect of the PLGA loaded hesperidin nanoparticles on the HCT116 cell line after 48 h was investigated by MTT assay at three different concentrations of the nanoparticles.</p><p><strong>Result: </strong>The study showed that 90% of hesperidin were loaded in PLGA nanoparticles by UV-Vis spectrophotometry and FT-IR spectrum. The nanoparticles were found to be spherical and uniform with a hydrodynamic diameter of 76.2 nm in water. The release rate of the drug was about 93% after 144 h. The lowest percentage of cell viability of cancer cells was observed at a concentration of 10 µg/ml of PLGA nanoparticles loaded with hesperidin.</p><p><strong>Conclusion: </strong>The results indicate that PLGA nanoparticles loaded with hesperidin effectively reduce the survival rate of HCT116 colorectal cancer cells. However, further studies are needed to determine the appropriate therapeutic dosage and to conduct animal and clinical studies.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"52"},"PeriodicalIF":3.5,"publicationDate":"2024-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11297711/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141878275","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}
BMC BiotechnologyPub Date : 2024-08-01DOI: 10.1186/s12896-024-00878-x
Ali Es-haghi, Mohammad Sadegh Amiri, Mohammad Ehsan Taghavizadeh Yazdi
{"title":"Ferula latisecta gels for synthesis of zinc/silver binary nanoparticles: antibacterial effects against gram-negative and gram-positive bacteria and physicochemical characteristics","authors":"Ali Es-haghi, Mohammad Sadegh Amiri, Mohammad Ehsan Taghavizadeh Yazdi","doi":"10.1186/s12896-024-00878-x","DOIUrl":"https://doi.org/10.1186/s12896-024-00878-x","url":null,"abstract":"This study explores the potential antibacterial applications of zinc oxide nanoparticles (ZnO NPs) enhanced with silver (Ag) using plant gel (ZnO-AgO NPs). The problem addressed is the increasing prevalence of pathogenic bacteria and the need for new, effective antimicrobial agents. ZnO NPs possess distinctive physicochemical properties that enable them to selectively target bacterial cells. Their small size and high surface area-to-volume ratio allow efficient cellular uptake and interaction with bacterial cells. In this study, the average size of the synthesized ZnO-Ag nanoparticles was 77.1 nm, with a significant standard deviation of 33.7 nm, indicating a wide size distribution. The nanoparticles demonstrated remarkable antibacterial efficacy against gram-negative and gram-positive bacteria, with inhibition zones of 14.33 mm for E. coli and 15.66 mm for B. subtilis at a concentration of 300 µg/ml. Minimum inhibitory concentrations (MIC) were determined to be 100 µg/ml for E. coli and 75 µg/ml for S. saprophyticus. Additionally, ZnO-Ag NPs exhibited excellent biocompatibility, making them appropriate for various pharmacological uses. This study utilizes Ferula latisecta gels, offering a sustainable and eco-friendly approach to nanoparticle synthesis. Incorporating of Ag into ZnO NPs significantly enhances their antimicrobial properties, with the combined results showing great inhibition effects on pathogenic microbes. The findings suggest that ZnO-Ag NPs could be a promising candidate for addressing the challenges posed by drug-resistant bacterial infections and enhancing antimicrobial treatments.","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"87 1","pages":""},"PeriodicalIF":3.5,"publicationDate":"2024-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141868805","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}
BMC BiotechnologyPub Date : 2024-07-19DOI: 10.1186/s12896-024-00869-y
Miguel de la Fuente, Diego Delgado, Maider Beitia, Gabriel Barreda-Gómez, Arantxa Acera, Mikel Sanchez, Elena Vecino
{"title":"Validation of a rapid collagenase activity detection technique based on fluorescent quenched gelatin with synovial fluid samples.","authors":"Miguel de la Fuente, Diego Delgado, Maider Beitia, Gabriel Barreda-Gómez, Arantxa Acera, Mikel Sanchez, Elena Vecino","doi":"10.1186/s12896-024-00869-y","DOIUrl":"10.1186/s12896-024-00869-y","url":null,"abstract":"<p><strong>Background: </strong>Measuring collagenase activity is crucial in the field of joint health and disease management. Collagenases, enzymes responsible for collagen degradation, play a vital role in maintaining the balance between collagen synthesis and breakdown in joints. Dysregulation of collagenase activity leads to joint tissue degradation and diseases such as rheumatoid arthritis and osteoarthritis. The development of methods to measure collagenase activity is essential for diagnosis, disease severity assessment, treatment monitoring, and identification of therapeutic targets.</p><p><strong>Results: </strong>This study aimed to validate a rapid collagenase activity detection technique using synovial fluid samples. Antibody microarray analysis was initially performed to quantify the levels of matrix metalloproteinase-9 (MMP-9), a major collagenase in joints. Subsequently, the developed gelatin-based test utilizing fluorescence measurement was used to determine collagenase activity. There was a significant correlation between the presence of MMP-9 and collagenase activity. In addition, Lower Limit of Detection and Upper Limit of Detection can be preliminary estimated as 8 ng/mL and 48 ng/mL respectively.</p><p><strong>Conclusions: </strong>The developed technique offers a potential point-of-care assessment of collagenase activity, providing real-time information for clinicians and researchers. By accurately quantifying collagenase activity, healthcare professionals can optimize patient care, improve treatment outcomes, and contribute to the understanding and management of joint-related disorders. Further research and validation are necessary to establish the full potential of this rapid collagenase activity detection method in clinical practice.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"50"},"PeriodicalIF":3.5,"publicationDate":"2024-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11264812/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141726834","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}
BMC BiotechnologyPub Date : 2024-07-15DOI: 10.1186/s12896-024-00870-5
Tanveer Majeed, Charles C Lee, William J Orts, Romana Tabassum, Tawaf Ali Shah, Yousef A Bin Jardan, Turki M Dawoud, Mohammed Bourhia
{"title":"Characterization of a thermostable protease from Bacillus subtilis BSP strain.","authors":"Tanveer Majeed, Charles C Lee, William J Orts, Romana Tabassum, Tawaf Ali Shah, Yousef A Bin Jardan, Turki M Dawoud, Mohammed Bourhia","doi":"10.1186/s12896-024-00870-5","DOIUrl":"10.1186/s12896-024-00870-5","url":null,"abstract":"<p><p>This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca<sup>2+</sup> and Fe<sup>2+</sup> increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H<sub>2</sub>O<sub>2</sub>). Characteristics, such as tolerance to high SDS and H<sub>2</sub>O<sub>2</sub> concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"49"},"PeriodicalIF":3.5,"publicationDate":"2024-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11247832/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141619217","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}
BMC BiotechnologyPub Date : 2024-07-09DOI: 10.1186/s12896-024-00875-0
Monalissa Halablab, Lovisa Wallman, Johan Bonde
{"title":"Recombinant human enamelin produced in Escherichia coli promotes mineralization in vitro.","authors":"Monalissa Halablab, Lovisa Wallman, Johan Bonde","doi":"10.1186/s12896-024-00875-0","DOIUrl":"10.1186/s12896-024-00875-0","url":null,"abstract":"<p><strong>Background: </strong>Enamelin is an enamel matrix protein that plays an essential role in the formation of enamel, the most mineralized tissue in the human body. Previous studies using animal models and proteins from natural sources point to a key role of enamelin in promoting mineralization events during enamel formation. However, natural sources of enamelin are scarce and with the current study we therefore aimed to establish a simple microbial production method for recombinant human enamelin to support its use as a mineralization agent.</p><p><strong>Results: </strong>In the study the 32 kDa fragment of human enamelin was successfully expressed in Escherichia coli and could be obtained using immobilized metal ion affinity chromatography purification (IMAC), dialysis, and lyophilization. This workflow resulted in a yield of approximately 10 mg enamelin per liter culture. Optimal conditions for IMAC purification were obtained using Ni<sup>2+</sup> as the metal ion, and when including 30 mM imidazole during binding and washing steps. Furthermore, in vitro mineralization assays demonstrated that the recombinant enamelin could promote calcium phosphate mineralization at a concentration of 0.5 mg/ml.</p><p><strong>Conclusions: </strong>These findings address the scarcity of enamelin by facilitating its accessibility for further investigations into the mechanism of enamel formation and open new avenues for developing enamel-inspired mineralized biomaterials.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"48"},"PeriodicalIF":3.5,"publicationDate":"2024-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11234762/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141562580","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}
BMC BiotechnologyPub Date : 2024-07-08DOI: 10.1186/s12896-024-00874-1
Jaber Hemmati, Mohsen Chiani, Babak Asghari, Ghodratollah Roshanaei, Sara Soleimani Asl, Morvarid Shafiei, Mohammad Reza Arabestani
{"title":"Antibacterial and antibiofilm potentials of vancomycin-loaded niosomal drug delivery system against methicillin-resistant Staphylococcus aureus (MRSA) infections.","authors":"Jaber Hemmati, Mohsen Chiani, Babak Asghari, Ghodratollah Roshanaei, Sara Soleimani Asl, Morvarid Shafiei, Mohammad Reza Arabestani","doi":"10.1186/s12896-024-00874-1","DOIUrl":"10.1186/s12896-024-00874-1","url":null,"abstract":"<p><p>The threat of methicillin-resistant Staphylococcus aureus (MRSA) is increasing worldwide, making it significantly necessary to discover a novel way of dealing with related infections. The quick spread of MRSA isolates among infected individuals has heightened public health concerns and significantly limited treatment options. Vancomycin (VAN) can be applied to treat severe MRSA infections, and the indiscriminate administration of this antimicrobial agent has caused several concerns in medical settings. Owing to several advantageous characteristics, a niosomal drug delivery system may increase the potential of loaded antimicrobial agents. This work aims to examine the antibacterial and anti-biofilm properties of VAN-niosome against MRSA clinical isolates with emphasis on cytotoxicity and stability studies. Furthermore, we aim to suggest an effective approach against MRSA infections by investigating the inhibitory effect of formulated niosome on the expression of the biofilm-associated gene (icaR). The thin-film hydration approach was used to prepare the niosome (Tween 60, Span 60, and cholesterol), and field emission scanning electron microscopy (FE-SEM), an in vitro drug release, dynamic light scattering (DLS), and entrapment efficiency (EE%) were used to investigate the physicochemical properties. The physical stability of VAN-niosome, including hydrodynamic size, polydispersity index (PDI), and EE%, was analyzed for a 30-day storage time at 4 °C and 25 °C. In addition, the human foreskin fibroblast (HFF) cell line was used to evaluate the cytotoxic effect of synthesized niosome. Moreover, minimum inhibitory and bactericidal concentrations (MICs/MBCs) were applied to assess the antibacterial properties of niosomal VAN formulation. Also, the antibiofilm potential of VAN-niosome was investigated by microtiter plate (MTP) and real-time PCR methods. The FE-SEM result revealed that synthesized VAN-niosome had a spherical morphology. The hydrodynamic size and PDI of VAN-niosome reported by the DLS method were 201.2 nm and 0.301, respectively. Also, the surface zeta charge of the prepared niosome was - 35.4 mV, and the EE% ranged between 58.9 and 62.5%. Moreover, in vitro release study revealed a sustained-release profile for synthesized niosomal formulation. Our study showed that VAN-niosome had acceptable stability during a 30-day storage time. Additionally, the VAN-niosome had stronger antibacterial and anti-biofilm properties against MRSA clinical isolates compared with free VAN. In conclusion, the result of our study demonstrated that niosomal VAN could be promising as a successful drug delivery system due to sustained drug release, negligible toxicity, and high encapsulation capacity. Also, the antibacterial and anti-biofilm studies showed the high capacity of VAN-niosome against MRSA clinical isolates. Furthermore, the results of real-time PCR exhibited that VAN-niosome could be proposed as a powerful strategy against MRSA biofilm via down","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"47"},"PeriodicalIF":3.5,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11229259/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141557951","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}
BMC BiotechnologyPub Date : 2024-07-06DOI: 10.1186/s12896-024-00872-3
Sayeda A Abdelhamid, Mostafa M Abo Elsoud, A F El-Baz, Ashraf M Nofal, Heba Y El-Banna
{"title":"Optimisation of indole acetic acid production by Neopestalotiopsis aotearoa endophyte isolated from Thymus vulgaris and its impact on seed germination of Ocimum basilicum.","authors":"Sayeda A Abdelhamid, Mostafa M Abo Elsoud, A F El-Baz, Ashraf M Nofal, Heba Y El-Banna","doi":"10.1186/s12896-024-00872-3","DOIUrl":"10.1186/s12896-024-00872-3","url":null,"abstract":"<p><strong>Background: </strong>Microbial growth during plant tissue culture is a common problem that causes significant losses in the plant micro-propagation system. Most of these endophytic microbes have the ability to propagate through horizontal and vertical transmission. On the one hand, these microbes provide a rich source of several beneficial metabolites.</p><p><strong>Results: </strong>The present study reports on the isolation of fungal species from different in vitro medicinal plants (i.e., Breynia disticha major, Breynia disticha, Duranta plumieri, Thymus vulgaris, Salvia officinalis, Rosmarinus officinalis, and Ocimum basilicum l) cultures. These species were tested for their indole acetic acid (IAA) production capability. The most effective species for IAA production was that isolated from Thymus vulgaris plant (11.16 µg/mL) followed by that isolated from sweet basil plant (8.78 µg/mL). On screening for maximum IAA productivity, medium, \"MOS + tryptophan\" was chosen that gave 18.02 μg/mL. The macroscopic, microscopic examination and the 18S rRNA sequence analysis indicated that the isolate that given code T4 was identified as Neopestalotiopsis aotearoa (T4). The production of IAA by N. aotearoa was statistically modeled using the Box-Behnken design and optimized for maximum level, reaching 63.13 µg/mL. Also, IAA extract was administered to sweet basil seeds in vitro to determine its effect on plant growth traits. All concentrations of IAA extract boosted germination parameters as compared to controls, and 100 ppm of IAA extract exhibited a significant growth promotion effect for all seed germination measurements.</p><p><strong>Conclusions: </strong>The IAA produced from N. aotearoa (T4) demonstrated an essential role in the enhancement of sweet basil (Ocimum basilicum) growth, suggesting that it can be employed to promote the plant development while lowering the deleterious effect of using synthetic compounds in the environment.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"46"},"PeriodicalIF":3.5,"publicationDate":"2024-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11227711/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141544482","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}
BMC BiotechnologyPub Date : 2024-07-05DOI: 10.1186/s12896-024-00873-2
Fouad Qasim Jubair Al-Zayadi, Ali S Shakir, Ahmed Shayaa Kareem, Abdolmajid Ghasemian, Esmaeil Behmard
{"title":"Design of a novel multi-epitope vaccine against Marburg virus using immunoinformatics studies.","authors":"Fouad Qasim Jubair Al-Zayadi, Ali S Shakir, Ahmed Shayaa Kareem, Abdolmajid Ghasemian, Esmaeil Behmard","doi":"10.1186/s12896-024-00873-2","DOIUrl":"10.1186/s12896-024-00873-2","url":null,"abstract":"<p><p>Marburg virus (MARV) is a highly contagious and virulent agent belonging to Filoviridae family. MARV causes severe hemorrhagic fever in humans and non-human primates. Owing to its highly virulent nature, preventive approaches are promising for its control. There is currently no approved drug or vaccine against MARV, and management mainly involves supportive care to treat symptoms and prevent complications. Our aim was to design a novel multi-epitope vaccine (MEV) against MARV using immunoinformatics studies. In this study, various proteins (VP35, VP40 and glycoprotein precursor) were used and potential epitopes were selected. CTL and HTL epitopes covered 79.44% and 70.55% of the global population, respectively. The designed MEV construct was stable and expressed in Escherichia coli (E. coli) host. The physicochemical properties were also acceptable. MARV MEV candidate could predict comprehensive immune responses such as those of humoral and cellular in silico. Additionally, efficient interaction to toll-like receptor 3 (TLR3) and its agonist (β-defensin) was predicted. There is a need for validation of these results using further in vitro and in vivo studies.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"45"},"PeriodicalIF":3.5,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11227231/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141537460","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}
BMC BiotechnologyPub Date : 2024-06-26DOI: 10.1186/s12896-024-00871-4
Olli Huhtinen, Stuart Prince, Urpo Lamminmäki, Rune Salbo, Antti Kulmala
{"title":"Increased stable integration efficiency in CHO cells through enhanced nuclear localization of Bxb1 serine integrase.","authors":"Olli Huhtinen, Stuart Prince, Urpo Lamminmäki, Rune Salbo, Antti Kulmala","doi":"10.1186/s12896-024-00871-4","DOIUrl":"10.1186/s12896-024-00871-4","url":null,"abstract":"<p><strong>Background: </strong>Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.</p><p><strong>Methods: </strong>In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.</p><p><strong>Results: </strong>The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.</p><p><strong>Conclusions: </strong>This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"44"},"PeriodicalIF":3.5,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11210126/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141455036","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}
BMC BiotechnologyPub Date : 2024-06-22DOI: 10.1186/s12896-024-00868-z
Danielly Corrêa-Moreira, Gisela Lara da Costa, Reginaldo Gonçalves de Lima Neto, Tatiana Pinto, Bruna Salomão, Tulio Machado Fumian, Camille Ferreira Mannarino, Tatiana Prado, Marize Pereira Miagostovich, Lívia de Souza Ramos, André Luis Souza Dos Santos, Manoel Marques Evangelista Oliveira
{"title":"Screening of Candida spp. in wastewater in Brazil during COVID-19 pandemic: workflow for monitoring fungal pathogens.","authors":"Danielly Corrêa-Moreira, Gisela Lara da Costa, Reginaldo Gonçalves de Lima Neto, Tatiana Pinto, Bruna Salomão, Tulio Machado Fumian, Camille Ferreira Mannarino, Tatiana Prado, Marize Pereira Miagostovich, Lívia de Souza Ramos, André Luis Souza Dos Santos, Manoel Marques Evangelista Oliveira","doi":"10.1186/s12896-024-00868-z","DOIUrl":"10.1186/s12896-024-00868-z","url":null,"abstract":"<p><p>Fungal diseases are often linked to poverty, which is associated with poor hygiene and sanitation conditions that have been severely worsened by the COVID-19 pandemic. Moreover, COVID-19 patients are treated with Dexamethasone, a corticosteroid that promotes an immunosuppressive profile, making patients more susceptible to opportunistic fungal infections, such as those caused by Candida species. In this study, we analyzed the prevalence of Candida yeasts in wastewater samples collected to track viral genetic material during the COVID-19 pandemic and identified the yeasts using polyphasic taxonomy. Furthermore, we investigated the production of biofilm and hydrolytic enzymes, which are known virulence factors. Our findings revealed that all Candida species could form biofilms and exhibited moderate hydrolytic enzyme activity. We also proposed a workflow for monitoring wastewater using Colony PCR instead of conventional PCR, as this technique is fast, cost-effective, and reliable. This approach enhances the accurate taxonomic identification of yeasts in environmental samples, contributing to environmental monitoring as part of the One Health approach, which preconizes the monitoring of possible emergent pathogenic microorganisms, including fungi.</p>","PeriodicalId":8905,"journal":{"name":"BMC Biotechnology","volume":"24 1","pages":"43"},"PeriodicalIF":3.5,"publicationDate":"2024-06-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11193224/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141440164","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}