RESUMEN

Recently, it was reported that a testicular organ culture system (TOCS) using polydimethylsiloxane (PDMS) chips with excellent oxygen permeability and biocompatibility, called the PDMS-chip ceiling (PC) method, enables improved spermatogenesis efficiency. We investigated whether this PC method is useful for detecting impaired spermatogenesis caused by busulfan (Bu), a typical testicular toxicant. In this study, testicular tissue fragments from Acro3-EGFP mice, which express the green fluorescent protein (GFP) and reflect the progression of spermatogenesis, were subjected to the PC method. When treated with Bu, cultured tissues shrank in volume, and their GFP-expressing area decreased or disappeared. Histological examination confirmed the regression of spermatogenesis. In addition, immunohistochemical examination revealed that spermatogonia, including spermatogonial stem cells (SSCs), were the primary targets of Bu toxicity. Time-course analysis demonstrated that the recovery of spermatogenesis, dependent on Bu concentration, correlated closely with the severity of damage to these target cells. These results suggest that the PC method is a useful approach for detecting spermatogenesis impairment accurately through faithful recapitulation of spermatogenesis in vivo.

Asunto(s)

Busulfano , Técnicas de Cultivo de Órganos , Espermatogénesis , Testículo , Animales , Masculino , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacos , Testículo/citología , Técnicas de Cultivo de Órganos/métodos , Ratones , Busulfano/farmacología , Espermatogonias/efectos de los fármacos , Espermatogonias/citología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética

RESUMEN

Genetically modified organisms are commonly used in disease research and agriculture but the precise genomic alterations underlying transgenic mutations are often unknown. The position and characteristics of transgenes, including the number of independent insertions, influences the expression of both transgenic and wild-type sequences. We used long-read, Oxford Nanopore Technologies (ONT) to sequence and assemble two transgenic strains of Caenorhabditis elegans commonly used in the research of neurodegenerative diseases: BY250 (pPdat-1::GFP) and UA44 (GFP and human α-synuclein), a model for Parkinson's research. After scaffolding to the reference, the final assembled sequences were ∼102 Mb with N50s of 17.9 Mb and 18.0 Mb, respectively, and L90s of six contiguous sequences, representing chromosome-level assemblies. Each of the assembled sequences contained more than 99.2% of the Nematoda BUSCO genes found in the C. elegans reference and 99.5% of the annotated C. elegans reference protein-coding genes. We identified the locations of the transgene insertions and confirmed that all transgene sequences were inserted in intergenic regions, leaving the organismal gene content intact. The transgenic C. elegans genomes presented here will be a valuable resource for Parkinson's research as well as other neurodegenerative diseases. Our work demonstrates that long-read sequencing is a fast, cost-effective way to assemble genome sequences and characterize mutant lines and strains.

Asunto(s)

Animales Modificados Genéticamente , Caenorhabditis elegans , Secuenciación de Nanoporos , Transgenes , Caenorhabditis elegans/genética , Animales , Transgenes/genética , Animales Modificados Genéticamente/genética , Secuenciación de Nanoporos/métodos , alfa-Sinucleína/genética , Genoma de los Helmintos , Mutagénesis Insercional , Humanos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo

RESUMEN

BACKGROUND: Fetal microchimerism occurs in the mother after a pregnancy. To investigate the role of fetal microchimerism cells (FMCs) in rheumatoid arthritis, we analyzed the population of fetal cells in pregnant experimental arthritis mice. METHODS: We used EGFP+ fetuses, which were mated with either healthy female mice or CIA mice, and male C57BL/6J-Tg (Pgk1-EGFP)03Narl mice, to detect the population of FMCs in maternal circulation. The disease progression was determined by measuring the clinical score and histological stains during pregnancy. The fetal cells have been analyzed if expressing EGFP, CD45, and Scal by flow cytometry. We also detected the expression of CD14+ IL-10+ cells in vivo and in vitro. RESULTS: Our data showed that the pregnancy ameliorated the arthritis progression of CIA mice. The IHC stains showed the CD45 -Sca-1+ EGFP+ FMCs were expressed in the bone marrow and peripheral blood mononuclear cells (PBMC) at 14 gestation days. However, Treg and Tc cell populations showed no significant change in the bone marrow. The data showed the H2Kb + fetal cells induced CD14+ IL10+ cell populations increased in the bone marrow in vitro and in vivo. CONCLUSION: Our investigations demonstrated that the FMCs protected the CIA mice from cartilage damage and triggered an immunosuppressive response in them by increasing the number of CD14+ IL10+ cells. In conclusion, the FMCs could potentially exhibit protective properties within the context of inflammatory arthritis that arises during pregnancy.

Asunto(s)

Artritis Experimental , Quimerismo , Progresión de la Enfermedad , Interleucina-10 , Receptores de Lipopolisacáridos , Ratones Endogámicos C57BL , Animales , Femenino , Embarazo , Interleucina-10/metabolismo , Masculino , Receptores de Lipopolisacáridos/metabolismo , Artritis Experimental/inmunología , Artritis Experimental/patología , Células Cultivadas , Ratones Endogámicos DBA , Ratones Transgénicos , Artritis Reumatoide/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Edad Gestacional , Intercambio Materno-Fetal , Fenotipo , Antígenos Comunes de Leucocito

RESUMEN

Near-infrared (NIR) probes are highly sought after as fluorescent tags for multicolor cellular and in vivo imaging. Here we develop small NIR fluorescent nanobodies, termed NIR-FbLAG16 and NIR-FbLAG30, enabling background-free visualization of various GFP-derived probes and biosensors. We also design a red-shifted variant, NIR-Fb(718), to simultaneously target several antigens within the NIR spectral range. Leveraging the antigen-stabilizing property of the developed NIR-Fbs, we then create two modular systems for precise control of gene expression in GFP-labeled cells. Applying the NIR-Fbs in vivo, we target cells expressing GFP and the calcium biosensor GCaMP6 in the somatosensory cortex of transgenic mice. Simultaneously tracking calcium activity and the reference signal from NIR-FbLAGs bound to GCaMP6 enables ratiometric deep-brain in vivo imaging. Altogether, NIR-FbLAGs present a promising approach for imaging and manipulating various processes in live cells and behaving animals expressing GFP-based probes.

Asunto(s)

Técnicas Biosensibles , Proteínas Fluorescentes Verdes , Ratones Transgénicos , Anticuerpos de Dominio Único , Animales , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Técnicas Biosensibles/métodos , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/química , Ratones , Humanos , Colorantes Fluorescentes/química , Calcio/metabolismo , Espectroscopía Infrarroja Corta/métodos , Corteza Somatosensorial/metabolismo , Corteza Somatosensorial/diagnóstico por imagen , Células HEK293

RESUMEN

Peritonitis is a common and life-threatening inflammatory disease. Myeloid cells are elevated in the peripheral blood and contribute to peritonitis, but their circulating dynamics are not clear. In vivo flow cytometry (IVFC) is a noninvasive technique for monitoring the dynamics of circulating cells in live animals. It has been extensively used to detect circulating tumor cells, but rarely for monitoring immune cells. Here, we describe a method adapting an intravital microscope for IVFC so that we can monitor LysM-EGFP-labeled circulating myeloid cells in a tumor necrosis factor (TNF) α-induced peritonitis mouse model. Using this IVFC method, we quantified the blood flow velocity and cell concentration in circulation. We observed a significant increase in LysM-EGFP+ cells in circulation after TNFα intraperitoneal (i.p.) injection, which reached a plateau in ~20 min. Conventional cytometry analysis showed that most LysM-EGFP+ cells were neutrophils. Increasing blood neutrophils were accompanied by neutrophil recruitment to the peritoneal cavity and neutrophil emigration from the bone marrow. We then monitored neutrophil CD64 expression in vivo and found a significant increase in TNFα-induced peritonitis. We also found that CD18 blockade doubled the circulating neutrophil number in TNFα-induced peritonitis, suggesting that CD18 is critical for neutrophil recruitment in peritonitis. Overall, we demonstrate that IVFC techniques are useful for studying the circulating dynamics of immune cells during inflammatory diseases.

Asunto(s)

Citometría de Flujo , Células Mieloides , Peritonitis , Factor de Necrosis Tumoral alfa , Animales , Peritonitis/inducido químicamente , Peritonitis/sangre , Citometría de Flujo/métodos , Ratones , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/sangre , Células Mieloides/metabolismo , Neutrófilos/metabolismo , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética

RESUMEN

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional ß-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of ß-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as ß-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal ß-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.

Asunto(s)

Diferenciación Celular , Genes Reporteros , Células Secretoras de Insulina , Islotes Pancreáticos , Células Madre Pluripotentes , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Línea Celular , Insulina/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética

RESUMEN

Cells crucially rely on the interactions of biomolecules at their plasma membrane to maintain homeostasis. Yet, a methodology to systematically quantify biomolecular organisation, measuring diffusion dynamics and oligomerisation, represents an unmet need. Here, we introduce the brightness-transit statistics (BTS) method based on fluorescence fluctuation spectroscopy and combine information from brightness and transit times to elucidate biomolecular diffusion and oligomerisation in both cell-free in vitro and in vitro systems incorporating living cells. We validate our approach in silico with computer simulations and experimentally using oligomerisation of EGFP tethered to supported lipid bilayers. We apply our pipeline to study the oligomerisation of CD40 ectodomain in vitro and endogenous CD40 on primary B cells. While we find a potential for CD40 to oligomerize in a concentration or ligand depended manner, we do not observe mobile oligomers on B cells. The BTS method combines sensitive analysis, quantification, and intuitive visualisation of dynamic biomolecular organisation.

Asunto(s)

Membrana Celular , Proteínas Fluorescentes Verdes , Membrana Dobles de Lípidos , Membrana Celular/metabolismo , Membrana Celular/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Humanos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/química , Difusión , Espectrometría de Fluorescencia/métodos , Linfocitos B/metabolismo , Simulación por Computador , Multimerización de Proteína , Animales

RESUMEN

We describe a binary expression aleatory mosaic (BEAM) system, which relies on DNA delivery by transfection or viral transduction along with nested recombinase activity to generate two genetically distinct, non-overlapping populations of cells for comparative analysis. Control cells labeled with red fluorescent protein (RFP) can be directly compared with experimental cells manipulated by genetic gain or loss of function and labeled with GFP. Importantly, BEAM incorporates recombinase-dependent signal amplification and delayed reporter expression to enable sharper delineation of control and experimental cells and to improve reliability relative to existing methods. We applied BEAM to a variety of known phenotypes to illustrate its advantages for identifying temporally or spatially aberrant phenotypes, for revealing changes in cell proliferation or death, and for controlling for procedural variability. In addition, we used BEAM to test the cortical protomap hypothesis at the individual radial unit level, revealing that area identity is cell autonomously specified in adjacent radial units.

Asunto(s)

Recombinasas , Animales , Recombinasas/metabolismo , Recombinasas/genética , Mosaicismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Expresión Génica/genética , Proteína Fluorescente Roja , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Humanos

RESUMEN

Dynamic properties are essential for microtubule (MT) physiology. Current techniques for in vivo imaging of MTs present intrinsic limitations in elucidating the isotype-specific nuances of tubulins, which contribute to their versatile functions. Harnessing the power of the AlphaFold2 pipeline, we engineered a strategy for the minimally invasive fluorescence labeling of endogenous tubulin isotypes or those harboring missense mutations. We demonstrated that a specifically designed 16-amino acid linker, coupled with sfGFP11 from the split-sfGFP system and integration into the H1-S2 loop of tubulin, facilitated tubulin labeling without compromising MT dynamics, embryonic development, or ciliogenesis in Caenorhabditis elegans. Extending this technique to human cells and murine oocytes, we visualized MTs with the minimal background fluorescence and a pathogenic tubulin isoform with fidelity. The utility of our approach across biological contexts and species set an additional paradigm for studying tubulin dynamics and functional specificity, with implications for understanding tubulin-related diseases known as tubulinopathies.

Asunto(s)

Caenorhabditis elegans , Proteínas Fluorescentes Verdes , Microtúbulos , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Humanos , Microtúbulos/metabolismo , Ratones , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Ingeniería de Proteínas/métodos , Oocitos/metabolismo

RESUMEN

EXPRESSION OF CONCERN: J.-Z. Yu, J. Kuret, and M. M. Rasenick, "Transient Expression of Fluorescent Tau Proteins Promotes Process Formation in PC12 Cells: Contributions of the Tau C-terminus to This Process," Journal of Neuroscience Research 67, no. 5 (2002): 625-633, https://doi.org/10.1002/jnr.10152. This Expression of Concern for the above article published online on 16 January 2002, in Wiley Online Library (wileyonlinelibrary.com), has been published by agreement between the journal Editors-in-Chief, Cristina A. Ghiani and J. Paula Warrington; and Wiley Periodicals LLC. The Expression of Concern has been agreed following concerns raised regarding suspected duplication between the two images, Tau23-GFP (72 hours) presented in Figure 4a and Tau 24 (174-383)-GFP (24 hours) presented in Figure 5a. The authors acknowledge the duplication but due to the length of time that has elapsed since the study was conducted and published, they were unable to provide an explanation or the original data. The journal has decided to issue an Expression of Concern to alert the readers.

Asunto(s)

Proteínas tau , Proteínas tau/metabolismo , Proteínas tau/genética , Células PC12 , Animales , Ratas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Neuritas/metabolismo

RESUMEN

BACKGROUND: Selection markers are useful in genetic modification of yeast Pichia pastoris. However, the leakage of the promoter caused undesired expression of selection markers especially those toxic proteins like MazF, halting the cell growth and hampering the genetic manipulation in procaryotic system. In this study, a new counter-selectable marker-based strategy has been established for seamless modification with high efficiency and low toxicity. RESULTS: At first, the leaky expression of the enhanced green fluorescent protein (EGFP) as a reporter gene under the control of six inducible promoters of P. pastoris was investigated in two hosts Escherichia coli and P. pastoris, respectively. The results demonstrated that the DAS1 and FDH1 promoters (PDAS1 and PFDH1) had the highest leakage expression activities in procaryotes and eukaryotes, and the DAS2 promoter (PDAS2) was inducible with medium strength but low leakage expression activity, all of which were selected for further investigation. Next, Mirabilis antiviral proteins (MAPs) c21873-1, c21873-1T (truncated form of c21873-1) and c23467 were mined as the new counter-selectable markers, and hygromycin B (Hyg B) resistance gene was used as the positive-selectable marker, respectively. Then, modular plasmids with MAP-target gene-Hyg B cassettes were constructed and used to transform into P. pastoris cells after linearization, and the target genes were integrated into its genome at the BmT1 locus through single-crossover homologous recombination (HR). After counter-selection induced by methanol medium, the markers c21873-1 and c21873-1T were recycled efficiently. But c23467 failed to be recycled due to its toxic effect on the P. pastoris cells. At last, the counter-selectable marker c21873-1 under the tightly regulated PDAS2 enabled the encoding genes of reporter EGFP and tested proteins to be integrated into the target locus and expressed successfully. CONCLUSIONS: We have developed MAP c21873-1 as a novel counter-selectable marker which could perform efficient gene knock-in by site-directed HR. Upon counter-selection, the marker could be recycled for repeated use, and no undesirable sequences were introduced except for the target gene. This unmarked genetic modification strategy may be extended to other genetic modification including but not limited to gene knock-out and site-directed mutagenesis in future.

Asunto(s)

Regiones Promotoras Genéticas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Marcadores Genéticos , Saccharomycetales/genética , Saccharomycetales/metabolismo

RESUMEN

Selective autophagy of protein aggregates, called aggrephagy, is vital for maintaining cellular homeostasis. Classically, studying aggrephagy has been challenging due to the infrequent occurrence of autophagic events and the lack of control over the specificity and timing of protein aggregation. We previously reported two variants of a PIM (particles induced by multimerization) assay that enable the formation of chemically induced, fluorescently labeled protein aggregates in cells. PIMs are recognized by the selective autophagy machinery and are subsequently degraded in the lysosome. By making use of pH-sensitive fluorescent proteins, such as GFP or mKeima, the PIM assay allows for direct visualization of aggregate clearance in cells. Here, we describe a protocol for the use of the PIM assay to study aggrephagy in live and fixed cells.

Asunto(s)

Autofagia , Agregado de Proteínas , Humanos , Multimerización de Proteína , Lisosomas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética

RESUMEN

Virus-like particles (VLPs) have untapped potential for packaging and delivery of macromolecular cargo. To be a broadly useful platform, there needs to be a strategy for attaching macromolecules to the inside or the outside of the VLP with minimal modification of the platform or cargo. Here, we repurpose antiviral compounds that bind to hepatitis B virus (HBV) capsids to create a chemical tag to noncovalently attach cargo to the VLP. Our tag consists of a capsid assembly modulator, HAP13, connected to a linker terminating in maleimide. Our cargo is a green fluorescent protein (GFP) with a single addressable cysteine, a feature that can be engineered in many proteins. The HAP-GFP construct maintained HAP's intrinsic ability to bind HBV capsids and accelerate assembly. We investigated the capacity of HAP-GFP to coassemble with HBV capsid protein and bind to preassembled capsids. HAP-GFP binding was concentration-dependent, sensitive to capsid stability, and dependent on linker length. Long linkers had the greatest activity to bind capsids, while short linkers impeded assembly and damaged intact capsids. In coassembly reactions, >20 HAP-GFP molecules were presented on the outside and inside of the capsid, concentrating the cargo by more than 100-fold compared to bulk solution. We also tested an HAP-GFP with a cleavable linker so that external GFP molecules could be removed, resulting in exclusive internal packaging. These results demonstrate a generalizable strategy for attaching cargo to a VLP, supporting development of HBV as a modular VLP platform.

Asunto(s)

Cápside , Proteínas Fluorescentes Verdes , Virus de la Hepatitis B , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/química , Cápside/química , Cápside/metabolismo , Ensamble de Virus , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Virión/metabolismo , Virión/química , Propiedades de Superficie

RESUMEN

BmNPV is a pathogen that infects silkworms exclusively. Although the interaction between BmNPV and the silkworm has been widely noticed and studied, its specific mechanism has still not been elucidated. In this study, we investigated whether BmNPV infection induces the onset of host cell autophagy to enhance viral replication. We observed a significant increase in double- or single-membrane vesicles and an accumulation of enhanced green fluorescent protein eGFP-ATG8 spots in virus-infected cells 72 h after BmNPV infection, accompanied by a conversion of ATG8 to ATG8-PE. In addition, we observed changes in the mitochondrial morphology of BmN cells after BmNPV infection by transmission electron microscopy. By detecting the mitochondrial membrane potential, we found that BmNPV infection resulted in the decrease of mitochondrial membrane potential, and that eGFP-ATG8 was able to co-localise with mitochondria after virus infection of the cells. Moreover, the use of drugs to regulate the occurrence of autophagy affects the replication of cellular BmNPV. Our data demonstrates that BmNPV infection induces host cell autophagy and leads to cellular mitochondrial damage, which in turn may lead to mitochondrial autophagy, and that BmNPV-induced host autophagy promotes its replication in cells. These findings will provide clues for further understanding of host-virus interactions.

Asunto(s)

Autofagia , Bombyx , Mitocondrias , Nucleopoliedrovirus , Replicación Viral , Animales , Nucleopoliedrovirus/fisiología , Bombyx/virología , Mitocondrias/metabolismo , Mitocondrias/virología , Línea Celular , Potencial de la Membrana Mitocondrial , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Interacciones Huésped-Patógeno

RESUMEN

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications.

Asunto(s)

Genes Reporteros , Replicación de ARN , ARN Viral , Replicón , SARS-CoV-2 , Humanos , Adenosina Monofosfato/análogos & derivados , Adenosina Monofosfato/farmacología , Alanina/análogos & derivados , Cromosomas Artificiales Bacterianos/genética , COVID-19/virología , COVID-19/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Regiones Promotoras Genéticas , Replicón/genética , Replicación de ARN/genética , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Transfección , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

The advancement of effective nasal mucoadhesive delivery faces challenges due to rapid mucociliary clearance (MCC). Conventional studies have employed mucoadhesive materials, mainly forming spherical nanoparticles, but these offer limited adhesion to the nasal mucosa. This study hypothesizes that a 2D nanoscale structure utilizing adhesive polyphenols can provide a superior strategy for countering MCC, aligning with the planar mucosal layers. We explore the use of tannic acid (TA), a polyphenolic molecule known for its adhesive properties and ability to form complexes with biomolecules. Our study introduces an unprecedented 2D nanopatch, assembled through the interaction of TA with green fluorescent protein (GFP), and cell-penetrating peptide (CPP). This 2D nanopatch demonstrates robust adhesion to nasal mucosa and significantly enhances immunoglobulin A secretions, suggesting its potential for enhancing nasal vaccine delivery. The promise of a polyphenol-enabled adhesive 2D nanopatch signifies a pivotal shift from conventional spherical nanoparticles, opening new pathways for delivery strategies through respiratory mucoadhesion.

Asunto(s)

Mucosa Nasal , Polifenoles , Taninos , Taninos/química , Polifenoles/química , Polifenoles/administración & dosificación , Mucosa Nasal/metabolismo , Mucosa Nasal/inmunología , Animales , Nanopartículas/química , Humanos , Péptidos de Penetración Celular/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/química , Adhesivos/química , Depuración Mucociliar/efectos de los fármacos , Inmunoglobulina A , Ratones

RESUMEN

Galectin-8 is a small soluble lectin with two carbohydrate recognition domains (CRDs). N- and C-terminal CRDs of Gal-8 differ in their specificity for glycan ligands. Here, we wanted to find out whether oligomerization of individual CRDs of galectin-8 affects its biological activity. Using green fluorescent protein polygons (GFPp) as an oligomerization scaffold, we generated intrinsically fluorescent CRDs with altered valency. We show that oligomers of C-CRD are characterized by significant cell surface affinity. Furthermore, the multivalency of the resulting variants has an impact on cellular activities such as cell signaling, heparin binding and proliferation. Our data indicates that tunable valence is a useful tool for modifying the biological activity of CRDs of galectins.

Asunto(s)

Galectinas , Galectinas/metabolismo , Galectinas/química , Humanos , Ligandos , Unión Proteica , Ingeniería de Proteínas/métodos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/química , Multimerización de Proteína , Proliferación Celular , Heparina/química , Heparina/metabolismo

RESUMEN

We generated a human induced pluripotent stem cell (hiPSC) line (CMCi014-A-78) expressing a GFP reporter in the 3'-UTR region of the KLOTHO locus using CRISPR/Cas9-mediated homologous recombination to screen for candidates regulating KLOTHO. The established cell line exhibits a normal karyotype, typical stem cell morphology, expression of pluripotency markers, and the ability to differentiate into the three germ layers. Consequently, this hiPSC line could serve as a valuable resource for screening KLOTHO regulators in hiPSC-derived target cells or organoids.

Asunto(s)

Regiones no Traducidas 3' , Glucuronidasa , Proteínas Fluorescentes Verdes , Células Madre Pluripotentes Inducidas , Proteínas Klotho , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Glucuronidasa/metabolismo , Glucuronidasa/genética , Línea Celular , Sistemas CRISPR-Cas , Genes Reporteros , Diferenciación Celular , Técnicas de Sustitución del Gen/métodos , Sitios Genéticos

RESUMEN

E-cadherin, a transmembrane protein, is essential for maintaining the integrity and structure of human pluripotent stem cells (hPSCs) by facilitating strong cell-cell adhesion and communication, which is crucial for their colony formation and pluripotency. Here, we used the CRISPR/Cas9 system to introduce the enhanced green fluorescent protein (EGFP)-tagged CDH1 into the AAVS1 locus, a safe harbour site, of human induced pluripotent stem cells (hiPSCs). The engineered cell line, KSCBi002-A-3, expressed functional CDH1-EGFP fusion protein, exhibited normal cell morphology, maintained a normal karyotype, and retained pluripotent state.

Asunto(s)

Cadherinas , Proteínas Fluorescentes Verdes , Células Madre Pluripotentes Inducidas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Cadherinas/metabolismo , Cadherinas/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Fluorescentes Verdes/genética , Línea Celular , Sistemas CRISPR-Cas , Antígenos CD/metabolismo , Antígenos CD/genética , Diferenciación Celular

RESUMEN

Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.

Asunto(s)

Inteínas , Proteínas de Unión a Maltosa , Rec A Recombinasas , beta-Galactosidasa , Inteínas/genética , beta-Galactosidasa/genética , beta-Galactosidasa/química , beta-Galactosidasa/aislamiento & purificación , beta-Galactosidasa/metabolismo , Proteínas de Unión a Maltosa/genética , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , Rec A Recombinasas/genética , Rec A Recombinasas/química , Rec A Recombinasas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Elastina/química , Elastina/genética , Elastina/aislamiento & purificación , Precipitación Química , Escherichia coli/genética , Escherichia coli/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/química