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SAGA (Spt-Ada-Gcn5-acetyltransferase) is a 19-subunit complex that stimulates transcription via two chromatin-modifying enzymatic modules and by delivering the TATA box binding protein (TBP) to nucleate the pre-initiation complex on DNA, a pivotal event in the expression of protein-encoding genes1. Here we present the structure of yeast SAGA with bound TBP. The core of the complex is resolved at 3.5 Å resolution (0.143 Fourier shell correlation). The structure reveals the intricate network of interactions that coordinate the different functional domains of SAGA and resolves an octamer of histone-fold domains at the core of SAGA. This deformed octamer deviates considerably from the symmetrical analogue in the nucleosome and is precisely tuned to establish a peripheral site for TBP, where steric hindrance represses binding of spurious DNA. Complementary biochemical analysis points to a mechanism for TBP delivery and release from SAGA that requires transcription factor IIA and whose efficiency correlates with the affinity of DNA to TBP. We provide the foundations for understanding the specific delivery of TBP to gene promoters and the multiple roles of SAGA in regulating gene expression.
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Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Pichia , Regiones Promotoras Genéticas/genética , Proteína de Unión a TATA-Box/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Sitios de Unión , ADN de Hongos/química , ADN de Hongos/metabolismo , Regulación Fúngica de la Expresión Génica , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Histonas/química , Histonas/metabolismo , Modelos Moleculares , Pichia/química , Pichia/genética , Unión Proteica , Conformación Proteica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores Asociados con la Proteína de Unión a TATA/química , Factores Asociados con la Proteína de Unión a TATA/metabolismo , Proteína de Unión a TATA-Box/química , Factor de Transcripción TFIIA/química , Factor de Transcripción TFIIA/metabolismo , Factor de Transcripción TFIID/química , Factor de Transcripción TFIID/metabolismoRESUMEN
The Pat1 protein is a central player of eukaryotic mRNA decay that has also been implicated in translational control. It is commonly considered a central platform responsible for the recruitment of several RNA decay factors. We demonstrate here that a yeast-specific C-terminal region from Pat1 interacts with several short motifs, named helical leucine-rich motifs (HLMs), spread in the long C-terminal region of yeast Dcp2 decapping enzyme. Structures of Pat1-HLM complexes reveal the basis for HLM recognition by Pat1. We also identify a HLM present in yeast Xrn1, the main 5'-3' exonuclease involved in mRNA decay. We show further that the ability of yeast Pat1 to bind HLMs is required for efficient growth and normal mRNA decay. Overall, our analyses indicate that yeast Pat1 uses a single binding surface to successively recruit several mRNA decay factors and show that interaction between those factors is highly polymorphic between species.
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Endorribonucleasas/metabolismo , Exorribonucleasas/metabolismo , Proteínas Fúngicas/metabolismo , ARN Mensajero/metabolismo , Levaduras/metabolismo , Unión Proteica/fisiología , Dominios Proteicos/fisiología , Estabilidad del ARN/fisiología , Proteínas de Unión al ARN/metabolismoRESUMEN
The positive transcription elongation factor (P-TEFb) is required for the transcription of most genes by RNA polymerase II. Hexim proteins associated with 7SK RNA bind to P-TEFb and reversibly inhibit its activity. P-TEFb comprises the Cdk9 cyclin-dependent kinase and a cyclin T. Hexim proteins have been shown to bind the cyclin T subunit of P-TEFb. How this binding leads to inhibition of the kinase activity of Cdk9 has remained elusive, however. Using a photoreactive amino acid incorporated into proteins, we show that in live cells, cell extracts, and in vitro reconstituted complexes, Hexim1 cross-links and thus contacts Cdk9. Notably, replacement of a phenylalanine, F208, belonging to an evolutionary conserved Hexim1 peptide (202PYNTTQFLM210) known as the "PYNT" sequence, cross-links a peptide within the activation segment that controls access to the Cdk9 catalytic cleft. Reciprocally, Hexim1 is cross-linked by a photoreactive amino acid replacing Cdk9 W193, a tryptophan within this activation segment. These findings provide evidence of a direct interaction between Cdk9 and its inhibitor, Hexim1. Based on similarities with Cdk2 3D structure, the Cdk9 peptide cross-linked by Hexim1 corresponds to the substrate binding-site. Accordingly, the Hexim1 PYNT sequence is proposed to interfere with substrate binding to Cdk9 and thereby to inhibit its kinase activity.
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Colon Transverso , Neoplasias del Colon , Laparoscopía , Colectomía , Colon Transverso/patología , Colon Transverso/cirugía , Neoplasias del Colon/patología , Neoplasias del Colon/cirugía , Humanos , Escisión del Ganglio Linfático , Ganglios Linfáticos/patología , Ganglios Linfáticos/cirugíaRESUMEN
In the yeast Saccharomyces cerevisiae, the Edc3 protein was previously reported to participate in the auto-regulatory feedback loop controlling the level of the RPS28B messenger RNA (mRNA). We show here that Edc3 binds directly and tightly to the globular core of Rps28 ribosomal protein. This binding occurs through a motif that is present exclusively in Edc3 proteins from yeast belonging to the Saccharomycetaceae phylum. Functional analyses indicate that the ability of Edc3 to interact with Rps28 is not required for its general function and for its role in the regulation of the YRA1 pre-mRNA decay. In contrast, this interaction appears to be exclusively required for the auto-regulatory mechanism controlling the RPS28B mRNA decay. These observations suggest a plausible model for the evolutionary appearance of a Rps28 binding motif in Edc3.
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Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Retroalimentación Fisiológica , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
This paper provides an extensive examination of a sizable dataset of English tweets focusing on nine widely recognized cryptocurrencies, specifically Cardano, Binance, Bitcoin, Dogecoin, Ethereum, Fantom, Matic, Shiba, and Ripple. Our goal was to conduct a psycholinguistic and emotional analysis of social media content associated with these cryptocurrencies. Such analysis can enable researchers and experts dealing with cryptocurrencies to make more informed decisions. Our work involved comparing linguistic characteristics across the diverse digital coins, shedding light on the distinctive linguistic patterns emerging in each coin's community. To achieve this, we utilized advanced text analysis techniques. Additionally, this work unveiled an understanding of the interplay between these digital assets. By examining which coin pairs are mentioned together most frequently in the dataset, we established co-mentions among different cryptocurrencies. To ensure the reliability of our findings, we initially gathered a total of 832,559 tweets from X. These tweets underwent a rigorous preprocessing stage, resulting in a refined dataset of 115,899 tweets that were used for our analysis. Overall, our research offers valuable perception into the linguistic nuances of various digital coins' online communities and provides a deeper understanding of their interactions in the cryptocurrency space.
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Cancer is unquestionably a global healthcare challenge, spurring the exporation of novel treatment approaches. In recent years, nanomaterials have garnered significant interest with the greatest hopes for targeted nanoformulations due to their cell-specific delivery, improved therapeutic efficacy, and reduced systemic toxicity for the organism. The problem of successful clinical translation of nanoparticles may be related to the fact that most in vitro tests are performed at pH values of normal cells and tissues, ranging from 7.2 to 7.4. The extracellular pH values of tumors are characterized by a shift to a more acidic region in the range of 5.6-7.0 and represent a crucial target for enhancing nanoparticle delivery to cancer cells. Here we show the method of non-active protein incorporation into the surface of HER2-targeted nanoparticles to achieve optimal cellular uptake within the pH range of the tumor microenvironment. The method efficacy was confirmed in vitro and in vivo showing the maximum binding of nanoparticles to cells at a pH value 6.4. Namely, fluorescent magnetic nanoparticles, modified with HER2-recognising affibody ZHER2:342, with proven specificity in terms of HER2 recognition (with 62-fold higher cellular uptake compared to control nanoparticles) were designed for targeting cancer cells at slightly acidic pH values. The stabilizing protein, namely, bovine serum albumin, one of the major blood components with widespread availability and biocompatibility, was used for the decoration of the nanoparticle surface to alter the pH response of the targeting magnetic conjugates. The optimally designed nanoparticles showed a bell-shaped dependency of interaction with cancer cells in the pH range of 5.6-8.0 with maximum cellular uptake at pH value 6.4 close to that of the tumor microenvironment. In vivo experiments revealed that after i.v. administration, BSA-decorated nanoparticles exhibited 2 times higher accumulation in tumors compared to magnetic nanoparticles modified with affibody only. Thus, we demonstrated a valid method for enhancing the specificity of targeted nanoparticle delivery to cancer cells without changing the functional components of nanoparticles.
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Mutations in human mitochondrial DNA are often associated with incurable human neuromuscular diseases. Among these mutations, an important number have been identified in tRNA genes, including 29 in the gene MT-TL1 coding for the tRNA(Leu(UUR)). The m.3243A>G mutation was described as the major cause of the MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). This mutation was reported to reduce tRNA(Leu(UUR)) aminoacylation and modification of its anti-codon wobble position, which results in a defective mitochondrial protein synthesis and reduced activities of respiratory chain complexes. In the present study, we have tested whether the mitochondrial targeting of recombinant tRNAs bearing the identity elements for human mitochondrial leucyl-tRNA synthetase can rescue the phenotype caused by MELAS mutation in human transmitochondrial cybrid cells. We demonstrate that nuclear expression and mitochondrial targeting of specifically designed transgenic tRNAs results in an improvement of mitochondrial translation, increased levels of mitochondrial DNA-encoded respiratory complexes subunits, and significant rescue of respiration. These findings prove the possibility to direct tRNAs with changed aminoacylation specificities into mitochondria, thus extending the potential therapeutic strategy of allotopic expression to address mitochondrial disorders.
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Genes Mitocondriales , Síndrome MELAS/genética , Mutación Puntual , ARN de Transferencia de Leucina/genética , Aminoacilación , Secuencia de Bases , Línea Celular , Respiración de la Célula , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Transporte de ARN , ARN de Transferencia de Leucina/química , ARN de Transferencia de Leucina/metabolismo , ARN de Transferencia de Lisina/química , ARN de Transferencia de Lisina/metabolismoRESUMEN
Photothermal therapy is one of the most promising and rapidly developing fields in modern oncology due to its high efficiency, localized action, and minimal invasiveness. Polymeric nanoparticles (NPs) incorporating low molecular-weight photothermal dyes are capable of delivering therapeutic agents to the tumor site, releasing them in a controlled manner, and providing tumor treatment under external light irradiation. The nanoparticle synthesis components are critically important factors that influence the therapeutically significant characteristics of polymeric NPs. Here, we show the impact of stabilizers and solvents used for synthesis on the properties of PLGA NPs for photothermal therapy. We synthesized PLGA nanocarriers using the microemulsion method and varied the nature of the solvent and the concentration of the stabilizer-namely, chitosan oligosaccharide lactate. A phthalocyanine-based photosensitizer, which absorbs light in the NIR window, was encapsulated in the PLGA NPs. When mQ water was used as a solvent and chitosan oligosaccharide lactate was used at a concentration of 1 g/L, the PLGA NPs exhibited highly promising photothermal properties. The final composite of the nanocarriers demonstrated photoinduced cytotoxicity against EMT6/P cells under NIR laser irradiation in vitro and was suitable for bioimaging.
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Targeted nanoparticles of different origins are considered as new-generation diagnostic and therapeutic tools. However, there are no targeted drug formulations within the composition of nanoparticles approved by the FDA for use in the clinic, which is associated with the insufficient effectiveness of the developed candidates, the difficulties of their biotechnological production, and inadequate batch-to-batch reproducibility. Targeted protein self-assembling nanoparticles circumvent this problem since proteins are encoded in DNA and the final protein product is produced in only one possible way. We believe that the combination of the endless biomedical potential of protein carriers as nanoparticles and the standardized protein purification protocols will make significant progress in "magic bullet" creation possible, bringing modern biomedicine to a new level. In this review, we are focused on the currently existing platforms for targeted self-assembling protein nanoparticles based on transferrin, lactoferrin, casein, lumazine synthase, albumin, ferritin, and encapsulin proteins, as well as on proteins from magnetosomes and virus-like particles. The applications of these self-assembling proteins for targeted delivery in vitro and in vivo are thoroughly discussed, including bioimaging applications and different therapeutic approaches, such as chemotherapy, gene delivery, and photodynamic and photothermal therapy. A critical assessment of these protein platforms' efficacy in biomedicine is provided and possible problems associated with their further development are described.
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Loss of H2A-H2B histone dimers is a hallmark of actively transcribed genes, but how the cellular machinery functions in the context of noncanonical nucleosomal particles remains largely elusive. In this work, we report the structural mechanism for adenosine 5'-triphosphate-dependent chromatin remodeling of hexasomes by the INO80 complex. We show how INO80 recognizes noncanonical DNA and histone features of hexasomes that emerge from the loss of H2A-H2B. A large structural rearrangement switches the catalytic core of INO80 into a distinct, spin-rotated mode of remodeling while its nuclear actin module remains tethered to long stretches of unwrapped linker DNA. Direct sensing of an exposed H3-H4 histone interface activates INO80, independently of the H2A-H2B acidic patch. Our findings reveal how the loss of H2A-H2B grants remodelers access to a different, yet unexplored layer of energy-driven chromatin regulation.
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Chaetomium , Ensamble y Desensamble de Cromatina , Cromatina , Histonas , Nucleosomas , Cromatina/química , ADN/química , Histonas/química , Nucleosomas/química , Microscopía por Crioelectrón , Chaetomium/química , Chaetomium/ultraestructuraRESUMEN
In the yeast Saccharomyces cerevisiae, nuclear DNA-encoded is partially imported into mitochondria. We previously found that the synthetic transcripts of yeast tRNA(Lys) and a number of their mutant versions could be specifically internalized by isolated yeast and human mitochondria. The mitochondrial targeting of tRNA(Lys) in yeast was shown to depend on the cytosolic precursor of mitochondrial lysyl-tRNA synthetase and the glycolytic enzyme enolase. Here we applied the approach of in vitro selection (SELEX) to broaden the spectrum of importable tRNA-derived molecules. We found that RNAs selected for their import into isolated yeast mitochondria have lost the potential to acquire a classical tRNA-shape. Analysis of conformational rearrangements in the importable RNAs by in-gel fluorescence resonance energy transfer (FRET) approach permitted us to suggest that protein factor binding and subsequent import require formation of an alternative structure, different from a classic L-form tRNA model. We show that in the complex with targeting protein factor, enolase 2, tRK1 adopts a particular conformation characterized by bringing together the 3'-end and the TPsiC loop. This is a first evidence for implication of RNA secondary structure rearrangement in the mechanism of mitochondrial import selectivity. Based on these data, a set of small RNA molecules with significantly improved efficiency of import into yeast and human mitochondria was constructed, opening the possibility of creating a new mitochondrial vector system able to target therapeutic oligoribonucleotides into deficient human mitochondria.
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Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Mitocondrias/metabolismo , Aptámeros de Nucleótidos/química , Secuencia de Bases , Transporte Biológico Activo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Técnicas In Vitro , Lisina-ARNt Ligasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Fosfopiruvato Hidratasa/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Técnica SELEX de Producción de Aptámeros , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
Single-stranded breaks (SSBs) are the most frequent DNA lesions threatening genomic integrity. A highly kinked DNA structure in complex with human PARP-1 domains led to the proposal that SSB sensing in Eukaryotes relies on dynamics of both the broken DNA double helix and PARP-1's multi-domain organization. Here, we directly probe this process at the single-molecule level. Quantitative smFRET and structural ensemble calculations reveal how PARP-1's N-terminal zinc fingers convert DNA SSBs from a largely unperturbed conformation, via an intermediate state into the highly kinked DNA conformation. Our data suggest an induced fit mechanism via a multi-domain assembly cascade that drives SSB sensing and stimulates an interplay with the scaffold protein XRCC1 orchestrating subsequent DNA repair events. Interestingly, a clinically used PARP-1 inhibitor Niraparib shifts the equilibrium towards the unkinked DNA conformation, whereas the inhibitor EB47 stabilizes the kinked state.
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Roturas del ADN de Cadena Simple , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/metabolismo , Reparación del ADN , Daño del ADN , ADN/metabolismoRESUMEN
This paper presents studies on the processing of enrichment tailings as a component of a raw mixture in order to obtain cement clinker, with simultaneous distillation of zinc. Thermodynamic studies were carried out in the temperature range of 600-1600 °C using the software application "HSC Chemistry 6" developed by the metallurgical company Outokumpu (Finland). As a result of the conducted studies, we found that zinc contributes to the intensification of mineral formation of cement clinker. In particular, it was found that the formation of belite is possible in the temperature range from 990.7 to 1500 °C with Gibbs energy values of -0.01 and -323.8 kJ (which is better than the standard process by -11.4 kJ), respectively; the formation of alite is possible in the temperature range from 982.9 to 1500 °C with Gibbs energy values of -0.05 and -402.1 kJ (better than the standard process by -11.4 kJ), respectively; the formation of tricalcium aluminate is thermodynamically possible in the temperature range from 600 °C at ΔGTo = -893.8 kJ to 1500 °C at ΔGTo = -1899.3 kJ (better than the standard process by -1570.1 kJ), respectively; and the formation of four calcium aluminoferrite is possible in the temperature range from 600 °C at ΔGTo = -898.9 kJ to 1500 °C at ΔGTo = -1959.3 kJ (better than the standard process by -1570.2 kJ), respectively, with simultaneous distillation of zinc into a gaseous state for its further capture.
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This paper presents studies on the possibility of utilization of technogenic waste from the metallurgical industry by the method of complex processing in order to reduce the anthropogenic load on the environment of the region with the example of the zinc silicate-magnetite-carbon system. The selected sample of clinker dump from welting was subjected to chemical and scanning electron microscopic analyses and thermodynamic modeling. Thermodynamic studies were carried out in the temperature range 1600-2200 K and pressure p = 0.1 MPa, modeling the process of electric melting of clinker from welting in an arc furnace using the software application Astra 4 developed at the Bauman Moscow State Technical University (Moscow, Russian Federation). As a result of the thermodynamic modeling, the optimal temperature range was established, which was 1800-1900 K. Thermodynamic studies established that it is possible to drive away zinc from the system under study by 99-100% in the entire temperature range under study. The maximum degree of silicon extraction (αSi) in the alloy is up to 69.44% at T = 1900 K, and the degree of iron extraction (αFe) in the alloy is up to 99.996%. In particular, it was determined and proved that clinker waste from welting can act as a secondary technogenic raw material when it is processed as a mono mixture to produce iron silicides with a silicon content of 18 to 28%.
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The paper studies the properties of brass workpieces for antifriction rings under severe plastic deformation by high-pressure torsion. The evolution of microstructure and mechanical properties of deformed workpieces after six cycles of deformation by high-pressure torsion at 500 °C have been studied. All metallographic studies were performed using modern methods: transmission electron microscopy (TEM) and analysis electron back scatter diffraction patterns (EBSD). The deformation resulted in an ultrafine grained structure with a large number of large-angle boundaries. The strength properties of brass increased compared to the initial state almost by three times, the microhardness also increases by three times, i.e., increased from 820 MPa in the initial state to 2115 MPa after deformation. In this case, the greatest increase in strength properties occurs in the first two cycles of deformation.
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During mining, only 4-8% is converted to final products, and the rest is accumulated in landfills. There is a lack of research on the study of various patterns and mechanisms of the formation of cement clinker minerals during the simultaneous distillation of zinc. This paper presents studies of thermodynamic stimulation of environmental and population protection by utilization of technogenic enrichment waste as secondary raw materials for clinker production and zinc extraction. In particular, a comparison of the Gibbs energy (ΔG) of clinker formation under standard chemical equations and under non-standard chemical equations is given. According to the results of the study, using thermodynamic simulation, the temperature intervals of mineral formation, the dependence of the Gibbs energy on temperature (ΔGT°), and the approximation equations were found; it was established that the presence of zinc ferrite contributes to the intensification of the formation of clinker minerals and the extraction of Zn to gas.
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Nanoparticle-based chemotherapy is considered to be an effective approach to cancer diagnostics and therapy in modern biomedicine. However, efficient tumor targeting remains a great challenge due to the lack of specificity, selectivity, and high dosage of chemotherapeutic drugs required. A two-step targeted drug delivery strategy (DDS), involving cancer cell pre-targeting, first with a first nontoxic module and subsequent targeting with a second complementary toxic module, is a solution for decreasing doses for administration and lowering systemic toxicity. To prove two-step DDS efficiency, we performed a direct comparison of one-step and two-step DDS based on chemotherapy loaded PLGA nanoparticles and barnase*barstar interface. Namely, we developed and thoroughly characterized the two-step targeting strategy of HER2-overexpressing cancer cells. The first targeting block consists of anti-HER2 scaffold polypeptide DARPin9_29 fused with barstar. Barstar exhibits an extremely effective binding to ribonuclease barnase with Kaff = 1014 M-1, thus making the barnase*barstar protein pair one of the strongest known protein*protein complexes. A therapeutic PLGA-based nanocarrier coupled to barnase was used as a second targeting block. The PLGA nanoparticles were loaded with diagnostic dye, Nile Blue, and a chemotherapeutic drug, doxorubicin. We showed that the two-step DDS increases the performance of chemotherapy-loaded nanocarriers: IC50 of doxorubicin delivered via two-step DDS was more than 100 times lower than that for one-step DDS: IC50 = 43 ± 3 nM for two-step DDS vs. IC50 = 4972 ± 1965 nM for one-step DDS. The obtained results demonstrate the significant efficiency of two-step DDS over the classical one-step one. We believe that the obtained data will significantly change the direction of research in developing targeted anti-cancer drugs and promote the creation of new generation cancer treatment strategies.
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The extreme aggressiveness and lethality of many cancer types appeal to the problem of the development of new-generation treatment strategies based on smart materials with a mechanism of action that differs from standard treatment approaches. The targeted delivery of nanoparticles to specific cancer cell receptors is believed to be such a strategy; however, there are no targeted nano-drugs that have successfully completed clinical trials to date. To meet the challenge, we designed an alternative way to eliminate tumors in vivo. Here, we show for the first time that the targeting of lectin-equipped polymer nanoparticles to the glycosylation profile of cancer cells, followed by photodynamic therapy (PDT), is a promising strategy for the treatment of aggressive tumors. We synthesized polymer nanoparticles loaded with magnetite and a PDT agent, IR775 dye (mPLGA/IR775). The magnetite incorporation into the PLGA particle structure allows for the quantitative tracking of their accumulation in different organs and the performing of magnetic-assisted delivery, while IR775 makes fluorescent in vivo bioimaging as well as light-induced PDT possible, thus realizing the theranostics concept. To equip PLGA nanoparticles with targeting modality, the particles were conjugated with lectins of different origins, and the flow cytometry screening revealed that the most effective candidate for breast cancer cell labeling is ConA, a lectin from Canavalia ensiformis. In vivo experiments showed that after i.v. administration, mPLGA/IR775-ConA nanoparticles efficiently accumulated in the allograft tumors under the external magnetic field; produced a bright fluorescent signal for in vivo bioimaging; and led to 100% tumor growth inhibition after the single session of PDT, even for large solid tumors of more than 200 mm3 in BALB/c mice. The obtained results indicate that the mPLGA/IR775 nanostructure has great potential to become a highly effective oncotheranostic agent.
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The Baculovirus/insect cell expression system is a powerful technology for reconstitution of eukaryotic macromolecular assemblies. Most multigene expression platforms rely on Tn7-mediated transposition for transferring the expression cassette into the baculoviral genome. This allows a rigorous characterization of recombinant bacmids but involves multiple steps, a limitation when many constructs are to be tested. For parallel expression screening and potential high throughput applications, we have established an open source multigene-expression toolbox exploiting homologous recombination, thus reducing the recombinant baculovirus generation to a single-step procedure and shortening the time from cloning to protein production to 2 weeks. The HR-bac toolbox is composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. They contain single or dual expression cassettes bearing different affinity tags and their design facilitates the mix and match utilization of expression units from Multibac constructs. The overall cost of virus generation with HR-bac toolbox is relatively low as the preparation of linearized baculoviral DNA only requires standard reagents. Various multiprotein assemblies (nuclear hormone receptor heterodimers, the P-TEFb or the ternary CAK kinase complex associated with the XPD TFIIH subunit) are used as model systems to validate the toolbox presented.