Pub Date : 2024-06-18DOI: 10.1016/j.bbrep.2024.101750
Yuanxia Zheng , Yi Zhang , Xuegang Li , Liangwei Liu
The widely used ET recombination requires an ssDNA product degraded by Rac phage protein E588 from dsDNA for strand invasion. However, proof of the ssDNA product is still elusive. The study provided three levels of proof sequentially. The probable ssDNAs degraded by E588 from the fluorescent plus-, minus-, or double-stranded dsDNA pET28a-xylanase exhibited a half fluorescence intensity of the corresponding dsDNAs, equivalent to the E588 degradation nucleotides half that of the total nucleotides degraded from the corresponding dsDNA. The ssDNA product degraded by E588 from the fluorescent minus-stranded dsDNA was confirmed by gradient gel-electrophoresis and two nuclease degradation reactions. Degraded by E588 from the dsDNA pET28a-xylanase that had a phosphorothioated plus-stranded 5′-terminus, the plus-stranded ssDNA product was separated via gel electrophoresis and recovered via a DNAclean kit. The recovered ssDNA product was proven to have intact 5′- and 3′-ends by DNA sequencing analysis. This study provides a solid foundation for the mechanism of ssDNA invasion.
{"title":"Proof of ssDNA degraded from dsDNA for ET recombination","authors":"Yuanxia Zheng , Yi Zhang , Xuegang Li , Liangwei Liu","doi":"10.1016/j.bbrep.2024.101750","DOIUrl":"https://doi.org/10.1016/j.bbrep.2024.101750","url":null,"abstract":"<div><p>The widely used ET recombination requires an ssDNA product degraded by Rac phage protein E588 from dsDNA for strand invasion. However, proof of the ssDNA product is still elusive. The study provided three levels of proof sequentially. The probable ssDNAs degraded by E588 from the fluorescent plus-, minus-, or double-stranded dsDNA pET28a-xylanase exhibited a half fluorescence intensity of the corresponding dsDNAs, equivalent to the E588 degradation nucleotides half that of the total nucleotides degraded from the corresponding dsDNA. The ssDNA product degraded by E588 from the fluorescent minus-stranded dsDNA was confirmed by gradient gel-electrophoresis and two nuclease degradation reactions. Degraded by E588 from the dsDNA pET28a-xylanase that had a phosphorothioated plus-stranded 5′-terminus, the plus-stranded ssDNA product was separated via gel electrophoresis and recovered via a DNAclean kit. The recovered ssDNA product was proven to have intact 5′- and 3′-ends by DNA sequencing analysis. This study provides a solid foundation for the mechanism of ssDNA invasion.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-06-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001146/pdfft?md5=e12edba587563f22a95b54dc75132163&pid=1-s2.0-S2405580824001146-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141423855","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-16DOI: 10.1016/j.bbrep.2024.101756
Megan M. Aoki , Anna B. Kisiala , Scott C. Farrow , Craig R. Brunetti , Robert J. Huber , R.J. Neil Emery
Lonely guy (LOG) proteins are phosphoribohydrolases (PRHs) that are key cytokinin (CK)-activating enzymes in plant and non-plant CK-producing organisms. During CK biosynthesis, LOGs catalyze the conversion of precursor CK-nucleotides (CK-NTs) to biologically active free base forms. LOG/PRH activity has been detected in bacteria, archaea, algae, and fungi. However, in these organisms, the LOG/PRH activity for CK-NTs and non-CK-NTs (e.g., adenine-NTs) has not been assessed simultaneously, which leaves limited knowledge about the substrate specificity of LOGs. Thus, we performed bioinformatic analyses and a biochemical characterization of a LOG ortholog from Dictyostelium discoideum, a soil-dwelling amoeba, which produces CKs during unicellular growth and multicellular development. We show that DdLog exhibits LOG/PRH activity on two CK-NTs, N6-isopentenyladenosine-5′-monophosphate (iPMP) and N6-benzyladenosine-5′-monophosphate (BAMP), and on adenosine 5′-monophosphate (AMP) but not on 3′, 5′-cyclic adenosine-monophosphate (cAMP). Additionally, there were higher turnover rates for CK-NTs over AMP. Together, these findings confirm that DdLog acts as a CK-activating enzyme; however, in contrast to plant LOGs, it maintains a wider specificity for other substrates (e.g., AMP) reflecting it has maintained its original, non-CK related role even after diversifying into a CK-activating enzyme.
孤独者(LOG)蛋白是一种磷酸核糖水解酶(PRHs),是植物和非植物细胞分裂素(CK)产生生物体中的关键细胞分裂素(CK)活化酶。在 CK 生物合成过程中,LOG 催化前体 CK 核苷酸(CK-NTs)转化为具有生物活性的游离碱基形式。在细菌、古菌、藻类和真菌中都检测到了 LOG/PRH 活性。然而,在这些生物体中,CK-NT 和非 CK-NT(如腺嘌呤-NT)的 LOG/PRH 活性尚未同时得到评估,因此对 LOG 的底物特异性了解有限。因此,我们对来自盘状竹荪的 LOG 直向同源物进行了生物信息学分析和生化鉴定,盘状竹荪是一种生活在土壤中的变形虫,在单细胞生长和多细胞发育过程中会产生 CKs。我们发现,DdLog 对两种 CK-NTs(N6-异戊烯基腺苷-5′-单磷酸(iPMP)和 N6-苄基腺苷-5′-单磷酸(BAMP))以及腺苷-5′-单磷酸(AMP)具有 LOG/PRH 活性,但对 3′,5′-环腺苷-单磷酸(cAMP)不具有 LOG/PRH 活性。此外,CK-NTs 的周转率高于 AMP。这些发现共同证实了 DdLog 起着 CK 激活酶的作用;然而,与植物 LOG 不同的是,它对其他底物(如 AMP)保持着更广泛的特异性,这反映出它即使在多样化为 CK 激活酶后,仍保持着原来与 CK 无关的作用。
{"title":"Biochemical characterization of a unique cytokinin and nucleotide phosphoribohydrolase Lonely Guy protein from Dictyostelium discoideum","authors":"Megan M. Aoki , Anna B. Kisiala , Scott C. Farrow , Craig R. Brunetti , Robert J. Huber , R.J. Neil Emery","doi":"10.1016/j.bbrep.2024.101756","DOIUrl":"https://doi.org/10.1016/j.bbrep.2024.101756","url":null,"abstract":"<div><p>Lonely guy (LOG) proteins are phosphoribohydrolases (PRHs) that are key cytokinin (CK)-activating enzymes in plant and non-plant CK-producing organisms. During CK biosynthesis, LOGs catalyze the conversion of precursor CK-nucleotides (CK-NTs) to biologically active free base forms. LOG/PRH activity has been detected in bacteria, archaea, algae, and fungi. However, in these organisms, the LOG/PRH activity for CK-NTs and non-CK-NTs (e.g., adenine-NTs) has not been assessed simultaneously, which leaves limited knowledge about the substrate specificity of LOGs. Thus, we performed bioinformatic analyses and a biochemical characterization of a LOG ortholog from <em>Dictyostelium discoideum</em>, a soil-dwelling amoeba, which produces CKs during unicellular growth and multicellular development. We show that <em>Dd</em>Log exhibits LOG/PRH activity on two CK-NTs, <em>N</em><sup><em>6</em></sup>-isopentenyladenosine-5′-monophosphate (iPMP) and <em>N</em><sup><em>6</em></sup>-benzyladenosine-5′-monophosphate (BAMP), and on adenosine 5′-monophosphate (AMP) but not on 3′, 5′-cyclic adenosine-monophosphate (cAMP). Additionally, there were higher turnover rates for CK-NTs over AMP. Together, these findings confirm that <em>Dd</em>Log acts as a CK-activating enzyme; however, in contrast to plant LOGs, it maintains a wider specificity for other substrates (e.g., AMP) reflecting it has maintained its original, non-CK related role even after diversifying into a CK-activating enzyme.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001201/pdfft?md5=c689c8c6b1e4fc62ca4820540feb3ed3&pid=1-s2.0-S2405580824001201-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141333168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Although antimicrobial peptides are considered one of the most promising alternatives to conventional antibiotics given the alarming increase in bacterial multidrug resistance, many aspects of their mechanism of action remain unclear, in particular the emergence and role of collective phenomena such as the spontaneous formation of nano-sized unstructured objects (clusters) and their effects on the biodynamics. We study this process using two novel peptides from the mucus of the garden snail Cornu aspersum as an example to reveal its dynamics and bioactivity implications through coordinated in silico and in vitro techniques — molecular dynamics simulations, UV–Vis and fluorescence spectroscopy, and antibacterial activity tests against two representative bacterial strains — one gram-negative (Escherichia coli 3458) and one gram-positive (Bacillus subtilis). The results obtained confirm the impact of the aggregation processes of the peptides on their biological activity and provide insight into possible synergies in their action.
{"title":"In silico and physico-chemical characterization of cluster formation dynamics in peptide solutions","authors":"Dimitar Kaynarov , Karina Marinova , Rossitsa Marinova , Peicho Petkov , Lyudmila Velkova , Aleksandar Dolashki , Petar Petrov , Leandar Litov , Elena Lilkova , Pavlina Dolashka , Nevena Ilieva","doi":"10.1016/j.bbrep.2024.101753","DOIUrl":"https://doi.org/10.1016/j.bbrep.2024.101753","url":null,"abstract":"<div><p>Although antimicrobial peptides are considered one of the most promising alternatives to conventional antibiotics given the alarming increase in bacterial multidrug resistance, many aspects of their mechanism of action remain unclear, in particular the emergence and role of collective phenomena such as the spontaneous formation of nano-sized unstructured objects (clusters) and their effects on the biodynamics. We study this process using two novel peptides from the mucus of the garden snail <em>Cornu aspersum</em> as an example to reveal its dynamics and bioactivity implications through coordinated in silico and in vitro techniques — molecular dynamics simulations, UV–Vis and fluorescence spectroscopy, and antibacterial activity tests against two representative bacterial strains — one gram-negative (<em>Escherichia coli</em> 3458) and one gram-positive (<em>Bacillus subtilis</em>). The results obtained confirm the impact of the aggregation processes of the peptides on their biological activity and provide insight into possible synergies in their action.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001171/pdfft?md5=64d70db8a1811f8bb84c23c6a7f41c46&pid=1-s2.0-S2405580824001171-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141325321","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-13DOI: 10.1016/j.bbrep.2024.101755
Diana Luísa Almeida-Nunes , Mariana Nunes , Hugo Osório , Verónica Ferreira , Cláudia Lobo , Paula Monteiro , Miguel Henriques Abreu , Carla Bartosch , Ricardo Silvestre , Ricardo Jorge Dinis-Oliveira , Sara Ricardo
Ovarian cancer (OC) patients develop ascites, an accumulation of ascitic fluid in the peritoneal cavity anda sign of tumour dissemination within the peritoneal cavity. This body fluid is under-researched, mainly regarding the ascites formed during tumour progression that have no diagnostic value and, therefore, are discarded. We performed a discovery proteomics study to identify new biomarkers in the ascites supernatant of OC patients. In this preliminary study, we analyzed a small amount of OC ascites to highlight the importance of not discarding such biological material during treatment, which could be valuable for OC management. Our findings reveal that OC malignant ascitic fluid (MAF) displays a proliferative environment that promotes the growth of OC cells that shift the metabolic pathway using alternative sources of nutrients, such as the cholesterol pathway. Also, OC ascites drained from patients during treatment showed an immunosuppressive environment, with up-regulation of proteins from the signaling pathways of IL-4 and IL-13 and down-regulation from the MHC-II. This preliminary study pinpointed a new protein (Transmembrane Protein 132A) in the OC context that deserves to be better explored in a more extensive cohort of patients’ samples. The proteomic profile of MAF from OC patients provides a unique insight into the metabolic kinetics of cancer cells during disease progression, and this information can be used to develop more effective treatment strategies.
{"title":"Ovarian cancer ascites proteomic profile reflects metabolic changes during disease progression","authors":"Diana Luísa Almeida-Nunes , Mariana Nunes , Hugo Osório , Verónica Ferreira , Cláudia Lobo , Paula Monteiro , Miguel Henriques Abreu , Carla Bartosch , Ricardo Silvestre , Ricardo Jorge Dinis-Oliveira , Sara Ricardo","doi":"10.1016/j.bbrep.2024.101755","DOIUrl":"https://doi.org/10.1016/j.bbrep.2024.101755","url":null,"abstract":"<div><p>Ovarian cancer (OC) patients develop ascites, an accumulation of ascitic fluid in the peritoneal cavity anda sign of tumour dissemination within the peritoneal cavity. This body fluid is under-researched, mainly regarding the ascites formed during tumour progression that have no diagnostic value and, therefore, are discarded. We performed a discovery proteomics study to identify new biomarkers in the ascites supernatant of OC patients. In this preliminary study, we analyzed a small amount of OC ascites to highlight the importance of not discarding such biological material during treatment, which could be valuable for OC management. Our findings reveal that OC malignant ascitic fluid (MAF) displays a proliferative environment that promotes the growth of OC cells that shift the metabolic pathway using alternative sources of nutrients, such as the cholesterol pathway. Also, OC ascites drained from patients during treatment showed an immunosuppressive environment, with up-regulation of proteins from the signaling pathways of IL-4 and IL-13 and down-regulation from the MHC-II. This preliminary study pinpointed a new protein (Transmembrane Protein 132A) in the OC context that deserves to be better explored in a more extensive cohort of patients’ samples. The proteomic profile of MAF from OC patients provides a unique insight into the metabolic kinetics of cancer cells during disease progression, and this information can be used to develop more effective treatment strategies.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001195/pdfft?md5=d2e6a95d1726d359ee927f1270f1f59d&pid=1-s2.0-S2405580824001195-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141325320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-13DOI: 10.1016/j.bbrep.2024.101739
Xiangxiong Deng , Su Zhang , Quan Qing , Pengfei Wang , Haiyang Ma , Qinghua Ma , Weixiang Zhao , Hanjing Tang , Min Lu
Mesenchymal stem cells (MSCs) have tremendous potential in cell therapy and regenerative medicine. The placenta-derived MSCs (PMSCs) are becoming favorable sources as they are ethically preferable and rich in MSCs. Although several subgroups of PMSCs have been identified from human term placenta, optimal sources for specific clinical applications remain to be elucidated. This study aimed to isolate MSCs from various components of the placenta, and compare their biological characteristics, including morphology, proliferation, immunophenotype, differentiation potential, growth factor and cytokine secretion, and immunomodulatory properties. Finally, four distinct groups of PMSCs were isolated from the placenta: amniotic membrane-derived MSCs (AM-MSCs), chorionic membrane-derived MSCs (CM-MSCs), chorionic plate-derived MSCs (CP-MSCs), and chorionic villi-derived MSCs (CV-MSCs). The results showed that CV-MSCs had good proliferation ability, and were easier to induce osteogenic and chondrogenic differentiation; CP-MSCs exhibited the strongest inhibitory effect on the proliferation of activated T cells, secreted high levels of EGF and IL-6, and could well differentiate into osteoblasts, adipocytes, and chondroblasts; AM-MSCs showed good growth dynamics in the early generations, were able to grow at high density, and tended to induce differentiation into osteogenic and neural lineages. These findings may provide novel evidence for the selection of seed cells in clinical application.
{"title":"Distinct biological characteristics of mesenchymal stem cells separated from different components of human placenta","authors":"Xiangxiong Deng , Su Zhang , Quan Qing , Pengfei Wang , Haiyang Ma , Qinghua Ma , Weixiang Zhao , Hanjing Tang , Min Lu","doi":"10.1016/j.bbrep.2024.101739","DOIUrl":"https://doi.org/10.1016/j.bbrep.2024.101739","url":null,"abstract":"<div><p>Mesenchymal stem cells (MSCs) have tremendous potential in cell therapy and regenerative medicine. The placenta-derived MSCs (PMSCs) are becoming favorable sources as they are ethically preferable and rich in MSCs. Although several subgroups of PMSCs have been identified from human term placenta, optimal sources for specific clinical applications remain to be elucidated. This study aimed to isolate MSCs from various components of the placenta, and compare their biological characteristics, including morphology, proliferation, immunophenotype, differentiation potential, growth factor and cytokine secretion, and immunomodulatory properties. Finally, four distinct groups of PMSCs were isolated from the placenta: amniotic membrane-derived MSCs (AM-MSCs), chorionic membrane-derived MSCs (CM-MSCs), chorionic plate-derived MSCs (CP-MSCs), and chorionic villi-derived MSCs (CV-MSCs). The results showed that CV-MSCs had good proliferation ability, and were easier to induce osteogenic and chondrogenic differentiation; CP-MSCs exhibited the strongest inhibitory effect on the proliferation of activated T cells, secreted high levels of EGF and IL-6, and could well differentiate into osteoblasts, adipocytes, and chondroblasts; AM-MSCs showed good growth dynamics in the early generations, were able to grow at high density, and tended to induce differentiation into osteogenic and neural lineages. These findings may provide novel evidence for the selection of seed cells in clinical application.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001031/pdfft?md5=9b06b6b2d4479ca61b7129e336b9d897&pid=1-s2.0-S2405580824001031-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141325319","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The oldest human coronavirus that started pandemics is severe acute respiratory syndrome virus (SARS-CoV). While SARS-CoV was eradicated, its new version, SARS-CoV2, caused the global pandemic of COVID-19. Evidence highlights the harmful events orchestrated by these viruses are mediated by Spike (S)P protein. Experimental epitopes of the S protein which were overlapping and ancestral between SARS-CoV and SARS-CoV-2 were obtained from the immune epitopes database (IEDB). The epitopes were then assembled in combination with a 50 S ribosomal protein L7/L12 adjuvant, a Mycobacterium tuberculosis-derived element and mediator of dendritic cells (DCs) and toll-like receptor 4 (TLR4). The immunogenic sequence was modeled by the GalaxyWeb server. After the improvement and validation of the protein structure, the physico-chemical properties and immune simulation were performed. To investigate the interaction with TLR3/4, Molecular Dynamics Simulation (MDS) was used. By merging the 17 B- and T-lymphocyte (HTL/CTL) epitopes, the vaccine sequence was created. Also, the Ramachandran plot presented that most of the residues were located in the most favorable and allowed areas. Moreover, SnapGene was successful in cloning the DNA sequence linked to our vaccine in the intended plasmid. A sequence was inserted between the XhoI and SacI position of the pET-28a (+) vector, and simulating the agarose gel revealed the existence of the inserted gene in the cloned plasmid with SARS vaccine (SARSV) construct, which has a 6565 bp in length overall. In terms of cytokines/IgG response, immunological simulation revealed a strong immune response. The stabilized vaccine showed strong interactions with TLR3/4, according to Molecular Dynamics Simulation (MDS) analysis. The present ancestral vaccine targets common sequences which seem to be valuable targets even for the new variant SARS-CoV-2.
{"title":"Development of an ancestral DC and TLR4-inducing multi-epitope peptide vaccine against the spike protein of SARS-CoV and SARS-CoV-2 using the advanced immunoinformatics approaches","authors":"Cena Aram , Parsa Alijanizadeh , Kiarash Saleki , Leila Karami","doi":"10.1016/j.bbrep.2024.101745","DOIUrl":"https://doi.org/10.1016/j.bbrep.2024.101745","url":null,"abstract":"<div><p>The oldest human coronavirus that started pandemics is severe acute respiratory syndrome virus (SARS-CoV). While SARS-CoV was eradicated, its new version, SARS-CoV2, caused the global pandemic of COVID-19. Evidence highlights the harmful events orchestrated by these viruses are mediated by Spike (S)P protein. Experimental epitopes of the S protein which were overlapping and ancestral between SARS-CoV and SARS-CoV-2 were obtained from the immune epitopes database (IEDB). The epitopes were then assembled in combination with a 50 S ribosomal protein L7/L12 adjuvant, a <em>Mycobacterium tuberculosis</em>-derived element and mediator of dendritic cells (DCs) and toll-like receptor 4 (TLR4). The immunogenic sequence was modeled by the GalaxyWeb server. After the improvement and validation of the protein structure, the physico-chemical properties and immune simulation were performed. To investigate the interaction with TLR3/4, Molecular Dynamics Simulation (MDS) was used. By merging the 17 B- and T-lymphocyte (HTL/CTL) epitopes, the vaccine sequence was created. Also, the Ramachandran plot presented that most of the residues were located in the most favorable and allowed areas. Moreover, SnapGene was successful in cloning the DNA sequence linked to our vaccine in the intended plasmid. A sequence was inserted between the <em>Xho</em>I and <em>Sac</em>I position of the pET-28a (+) vector, and simulating the agarose gel revealed the existence of the inserted gene in the cloned plasmid with SARS vaccine (SARSV) construct, which has a 6565 bp in length overall. In terms of cytokines/IgG response, immunological simulation revealed a strong immune response. The stabilized vaccine showed strong interactions with TLR3/4, according to Molecular Dynamics Simulation (MDS) analysis. The present ancestral vaccine targets common sequences which seem to be valuable targets even for the new variant SARS-CoV-2.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-06-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001092/pdfft?md5=615fc3278f0abb7d49b792a68ac21b86&pid=1-s2.0-S2405580824001092-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141314656","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-08DOI: 10.1016/j.bbrep.2024.101748
Md. Mazharul Islam Chowdhury , Nafisa Kabir , Rezwana Ahmed , Kazushige Yokota , Randy Mullins , Hasan Mahmud Reza
Prostacyclin or prostaglandin I2 (PGI2), a metabolite of arachidonic cyclooxygenase pathway, has been demonstrated as an effector of adipocyte differentiation. However, due to its instability in biological fluid, it is difficult to evaluate the role of PGI2 in regulating adipocyte differentiation in different stages in culture. Therefore, this study aimed to establish a simple and rapid method for the production of monoclonal antibody against 6-Keto PGF1α, a stable PGI2 metabolite, and its quantification to determine the role of PGI2 in culture medium. Eight-week-old female BALB/c mice were immunized with the hapten of 6-Keto PGF1α and BSA for several weeks until a higher antibody titer (absorbance value > 0.9 at 1000-times dilution) against 6-Keto PGF1α was found. Then, fusion of antibody-producing spleen lymphocytes with SP-2 myeloma cells and thymocytes was performed and cultured in HAT-medium supplemented with hypoxanthine, aminopterin, and thymine. Specific antibody-producing cells (M2-A4-B8-D10) against 6-Keto PGF1α were identified and separated. A standard ELISA calibration curve was developed with 100% reactivity for 6-Keto-PGF 1 α ranging from 0.26 pg to 6.44 ng corresponding to 90% and 10% of the maximum binding capacity for the immobilized antigen respectively. This method can easily be applied to monitor PGI2 regulation in different stages of cultured adipocytes to reveal the regulatory roles of PGI2 in maintaining homeostasis and adipocyte differentiation.
{"title":"Generation of monoclonal antibody against 6-Keto PGF1α and development of ELISA for its quantification in culture medium","authors":"Md. Mazharul Islam Chowdhury , Nafisa Kabir , Rezwana Ahmed , Kazushige Yokota , Randy Mullins , Hasan Mahmud Reza","doi":"10.1016/j.bbrep.2024.101748","DOIUrl":"https://doi.org/10.1016/j.bbrep.2024.101748","url":null,"abstract":"<div><p>Prostacyclin or prostaglandin I<sub>2</sub> (PGI<sub>2</sub>), a metabolite of arachidonic cyclooxygenase pathway, has been demonstrated as an effector of adipocyte differentiation. However, due to its instability in biological fluid, it is difficult to evaluate the role of PGI<sub>2</sub> in regulating adipocyte differentiation in different stages in culture. Therefore, this study aimed to establish a simple and rapid method for the production of monoclonal antibody against 6-Keto PGF<sub>1</sub>α, a stable PGI<sub>2</sub> metabolite, and its quantification to determine the role of PGI<sub>2</sub> in culture medium. Eight-week-old female BALB/c mice were immunized with the hapten of 6-Keto PGF<sub>1</sub>α and BSA for several weeks until a higher antibody titer (absorbance value > 0.9 at 1000-times dilution) against 6-Keto PGF<sub>1</sub>α was found. Then, fusion of antibody-producing spleen lymphocytes with SP-2 myeloma cells and thymocytes was performed and cultured in HAT-medium supplemented with hypoxanthine, aminopterin, and thymine. Specific antibody-producing cells (M2-A4-B8-D10) against 6-Keto PGF<sub>1</sub>α were identified and separated. A standard ELISA calibration curve was developed with 100% reactivity for 6-Keto-PGF 1 α ranging from 0.26 pg to 6.44 ng corresponding to 90% and 10% of the maximum binding capacity for the immobilized antigen respectively. This method can easily be applied to monitor PGI<sub>2</sub> regulation in different stages of cultured adipocytes to reveal the regulatory roles of PGI<sub>2</sub> in maintaining homeostasis and adipocyte differentiation.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001122/pdfft?md5=13a159d6eb5def427e7325a56862fb03&pid=1-s2.0-S2405580824001122-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141290600","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Zika virus represents the primary cause of infection during pregnancy and can lead to various neurological disorders such as microcephaly and Guillain-Barré syndrome affecting both children and adults. This infection is also associated with urological and nephrological problems. So far, evidence of mosquito-borne Zika virus infection has been reported in a total of 89 countries and territories. However, surveillance efforts primarily concentrate on outbreaks that this virus can cause, yet the measures implemented are typically limited. Currently, there are no specific treatments or vaccines designed for the prevention or treatment of Zika virus infection or its associated disease. The development of effective therapeutic agents presents an urgent need. Importantly, an alternative for advancing the discovery of new molecules could be dermaseptins, a family of antimicrobial peptides known for their potential antiviral properties. In this study, we carried out the synthesis of dermaseptins and their analogs and subsequently assessed the bioactivity tests against Zika virus (ZIKV PF13) of dermaseptins B2 and S4 and their derivatives. The cytotoxicity of these peptides was investigated on HMC3 cell line and HeLa cells by CellTiter-Glo® Luminescent Cell Viability Assay. Thereafter, we evaluated the antiviral activity caused by the action of our dermaseptins on the viral envelope using the Fluorescence Activated Cell Sorting (FACS). The cytotoxicity of our molecules was concentration-dependent at microgram concentrations Expect for dermaseptin B2 and its derivative which present no toxicity against HeLa and HMC3 cell lines. It was observed that all tested analogs from S4 family exhibited antiviral activity with low concentrations ranging from 3 to 12.5 μg/ml , unlike the native B2 and its derivative which increased the infectivity. Pre-incubating of dermaseptins with ZIKV PF13 before infection revealed that these derivatives inhibit the initial stages of virus infection. In summary, these results suggest that dermaseptins could serve as novel lead structures for the development of potent antiviral agents against Zika virus infections.
{"title":"Evaluation of the antiviral activity of new dermaseptin analogs against Zika virus","authors":"Houda Haddad , Frédéric Tangy , Ines Ouahchi , Wissal Sahtout , Bouraoui Ouni , Amira Zaïri","doi":"10.1016/j.bbrep.2024.101747","DOIUrl":"https://doi.org/10.1016/j.bbrep.2024.101747","url":null,"abstract":"<div><p>Zika virus represents the primary cause of infection during pregnancy and can lead to various neurological disorders such as microcephaly and Guillain-Barré syndrome affecting both children and adults. This infection is also associated with urological and nephrological problems. So far, evidence of mosquito-borne Zika virus infection has been reported in a total of 89 countries and territories. However, surveillance efforts primarily concentrate on outbreaks that this virus can cause, yet the measures implemented are typically limited. Currently, there are no specific treatments or vaccines designed for the prevention or treatment of Zika virus infection or its associated disease. The development of effective therapeutic agents presents an urgent need. Importantly, an alternative for advancing the discovery of new molecules could be dermaseptins, a family of antimicrobial peptides known for their potential antiviral properties. In this study, we carried out the synthesis of dermaseptins and their analogs and subsequently assessed the bioactivity tests against Zika virus (ZIKV PF13) of dermaseptins B2 and S4 and their derivatives. The cytotoxicity of these peptides was investigated on HMC3 cell line and HeLa cells by CellTiter-Glo® Luminescent Cell Viability Assay. Thereafter, we evaluated the antiviral activity caused by the action of our dermaseptins on the viral envelope using the Fluorescence Activated Cell Sorting (FACS). The cytotoxicity of our molecules was concentration-dependent at microgram concentrations Expect for dermaseptin B2 and its derivative which present no toxicity against HeLa and HMC3 cell lines. It was observed that all tested analogs from S4 family exhibited antiviral activity with low concentrations ranging from 3 to 12.5 μg/ml , unlike the native B2 and its derivative which increased the infectivity. Pre-incubating of dermaseptins with ZIKV PF13 before infection revealed that these derivatives inhibit the initial stages of virus infection. In summary, these results suggest that dermaseptins could serve as novel lead structures for the development of potent antiviral agents against Zika virus infections.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001110/pdfft?md5=96d64c14aa77a2845ab92539c5a92ac7&pid=1-s2.0-S2405580824001110-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141289449","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-06-07DOI: 10.1016/j.bbrep.2024.101749
Sara MT. Persson , Anna Casselbrant , Aiham Alarai , Erik Elebring , Lars Fändriks , Ville Wallenius
Background
Roux-en-Y gastric bypass (RYGB) is an effective treatment for obesity, resulting in long-term weight loss and rapid remission of type 2 diabetes mellitus. Improved glucagon-like peptide 1 (GLP-1) levels is one factor that contributes to the positive effects. Prior to RYGB, GLP-1 response is blunted which can be attributed to intestinal ketogenesis. Intestinal produced ketone bodies inhibit GLP-1 secretion in enteroendocrine cells via an unidentified G-protein coupled receptors (GPCRs). A possible class of GPCRs through which ketone bodies may reach are the free fatty acid receptors (FFARs) located at the basolateral membrane of enteroendocrine cells.
Aim
To evaluate FFAR3 expression in enteroendocrine cells of the small intestine under different circumstances, such as diet and bariatric surgery, as well as explore the link between ketone bodies and GLP-1 secretion.
Materials and methods
FFAR3 and enteroendocrine cell expression was analyzed using Western blot and immunohistochemistry in biopsies from healthy volunteers, obese patients undergoing RYGB and mice. GLUTag cells were used to study GLP-1 secretion and FFAR3 signaling pathways.
Results
The expression of FFAR3 is markedly influenced by diet, especially high fat diet, which increased FFAR3 protein expression. Lack of substrate such as free fatty acids in the alimentary limb after RYGB, downregulate FFAR3 expression. The number of enteroendocrine cells was affected by diet in the normal weight individuals but not in the subjects with obesity. In GLUTag cells, we show that the ketone bodies exert its blocking effect on GLP-1 secretion via the FFAR3, and the Gαi/o signaling pathway.
Conclusion
Our findings that ketone bodies via FFAR3 inhibits GLP-1 secretion bring important insight into the pathophysiology of T2D. This highlights the role of FFAR3 as a possible target for future anti-diabetic drugs and treatments.
{"title":"Role of FFAR3 in ketone body regulated glucagon-like peptide 1 secretion","authors":"Sara MT. Persson , Anna Casselbrant , Aiham Alarai , Erik Elebring , Lars Fändriks , Ville Wallenius","doi":"10.1016/j.bbrep.2024.101749","DOIUrl":"https://doi.org/10.1016/j.bbrep.2024.101749","url":null,"abstract":"<div><h3>Background</h3><p>Roux-en-Y gastric bypass (RYGB) is an effective treatment for obesity, resulting in long-term weight loss and rapid remission of type 2 diabetes mellitus. Improved glucagon-like peptide 1 (GLP-1) levels is one factor that contributes to the positive effects. Prior to RYGB, GLP-1 response is blunted which can be attributed to intestinal ketogenesis. Intestinal produced ketone bodies inhibit GLP-1 secretion in enteroendocrine cells via an unidentified G-protein coupled receptors (GPCRs). A possible class of GPCRs through which ketone bodies may reach are the free fatty acid receptors (FFARs) located at the basolateral membrane of enteroendocrine cells.</p></div><div><h3>Aim</h3><p>To evaluate FFAR3 expression in enteroendocrine cells of the small intestine under different circumstances, such as diet and bariatric surgery, as well as explore the link between ketone bodies and GLP-1 secretion.</p></div><div><h3>Materials and methods</h3><p>FFAR3 and enteroendocrine cell expression was analyzed using Western blot and immunohistochemistry in biopsies from healthy volunteers, obese patients undergoing RYGB and mice. GLUTag cells were used to study GLP-1 secretion and FFAR3 signaling pathways.</p></div><div><h3>Results</h3><p>The expression of FFAR3 is markedly influenced by diet, especially high fat diet, which increased FFAR3 protein expression. Lack of substrate such as free fatty acids in the alimentary limb after RYGB, downregulate FFAR3 expression. The number of enteroendocrine cells was affected by diet in the normal weight individuals but not in the subjects with obesity. In GLUTag cells, we show that the ketone bodies exert its blocking effect on GLP-1 secretion via the FFAR3, and the Gα<sub>i/o</sub> signaling pathway.</p></div><div><h3>Conclusion</h3><p>Our findings that ketone bodies via FFAR3 inhibits GLP-1 secretion bring important insight into the pathophysiology of T2D. This highlights the role of FFAR3 as a possible target for future anti-diabetic drugs and treatments.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001134/pdfft?md5=56c7dbabc00bad665cfb069aa310d2c2&pid=1-s2.0-S2405580824001134-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141289448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Dermal fibroblasts play a crucial role in skin structure and function by producing hyaluronic acid. Piceatannol (PIC), a polyphenol abundant in passion fruit seeds, has been reported to activate sirtuin 1 (SIRT1). Clinical trials have demonstrated that PIC intake improves skin moisture and maintains skin elasticity, yet the underlying mechanism remains unclear. This study aimed to investigate the effects of PIC on hyaluronic acid biosynthesis and the involvement of SIRT1 in this process. Human dermal fibroblast Hs68 cells were stimulated with PIC, and the expression levels of HAS2 and HYAL2, key enzymes in hyaluronic acid biosynthesis, as well as SIRT1 expression, were assessed using quantitative real-time PCR. Additionally, the role of SIRT1 in the hyaluronic acid biosynthesis pathway during PIC stimulation was examined using a SIRT1 inhibitor. The results demonstrated that PIC increased HAS2 expression while decreasing HYAL2 expression in human dermal fibroblasts. Furthermore, PIC enhanced SIRT1 expression, and pre-treatment with a SIRT1 inhibitor mitigated PIC-induced upregulation of HAS2, suggesting that PIC promotes hyaluronic acid synthesis by inducing SIRT1. These findings suggest that PIC could serve as a beneficial food ingredient, enhancing skin structure and function by promoting hyaluronic acid biosynthesis via SIRT1 induction.
{"title":"Piceatannol enhances hyaluronic acid synthesis through SIRT1-Mediated HAS2 upregulation in human dermal fibroblasts","authors":"Mizuki Yoshihara, Shinpei Kawakami, Yuko Matsui, Toshihiro Kawama","doi":"10.1016/j.bbrep.2024.101746","DOIUrl":"https://doi.org/10.1016/j.bbrep.2024.101746","url":null,"abstract":"<div><p>Dermal fibroblasts play a crucial role in skin structure and function by producing hyaluronic acid. Piceatannol (PIC), a polyphenol abundant in passion fruit seeds, has been reported to activate sirtuin 1 (SIRT1). Clinical trials have demonstrated that PIC intake improves skin moisture and maintains skin elasticity, yet the underlying mechanism remains unclear. This study aimed to investigate the effects of PIC on hyaluronic acid biosynthesis and the involvement of SIRT1 in this process. Human dermal fibroblast Hs68 cells were stimulated with PIC, and the expression levels of <em>HAS2</em> and <em>HYAL2</em>, key enzymes in hyaluronic acid biosynthesis, as well as <em>SIRT1</em> expression, were assessed using quantitative real-time PCR. Additionally, the role of <em>SIRT1</em> in the hyaluronic acid biosynthesis pathway during PIC stimulation was examined using a SIRT1 inhibitor. The results demonstrated that PIC increased <em>HAS2</em> expression while decreasing <em>HYAL2</em> expression in human dermal fibroblasts. Furthermore, PIC enhanced <em>SIRT1</em> expression, and pre-treatment with a SIRT1 inhibitor mitigated PIC-induced upregulation of <em>HAS2</em>, suggesting that PIC promotes hyaluronic acid synthesis by inducing <em>SIRT1</em>. These findings suggest that PIC could serve as a beneficial food ingredient, enhancing skin structure and function by promoting hyaluronic acid biosynthesis via <em>SIRT1</em> induction.</p></div>","PeriodicalId":8771,"journal":{"name":"Biochemistry and Biophysics Reports","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2405580824001109/pdfft?md5=881ca0a0c853ba5a10efe8acb335904d&pid=1-s2.0-S2405580824001109-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141242364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}