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Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.
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SIR2-HerA, a bacterial two-protein anti-phage defense system, induces bacterial death by depleting NAD+ upon phage infection. Biochemical reconstitution of SIR2, HerA, and the SIR2-HerA complex reveals a dynamic assembly process. Unlike other ATPases, HerA can form various oligomers, ranging from dimers to nonamers. When assembled with SIR2, HerA forms a hexamer and converts SIR2 from a nuclease to an NAD+ hydrolase, representing an unexpected regulatory mechanism mediated by protein assembly. Furthermore, high concentrations of ATP can inhibit NAD+ hydrolysis by the SIR2-HerA complex. Cryo-EM structures of the SIR2-HerA complex reveal a giant supramolecular assembly up to 1 MDa, with SIR2 as a dodecamer and HerA as a hexamer, crucial for anti-phage defense. Unexpectedly, the HerA hexamer resembles a spiral staircase and exhibits helicase activities toward dual-forked DNA. Together, we reveal the supramolecular assembly of SIR2-HerA as a unique mechanism for switching enzymatic activities and bolstering anti-phage defense strategies.
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Proteínas de Escherichia coli , Escherichia coli , Sirtuinas , Fagos T , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , NAD , Sirtuinas/metabolismo , Escherichia coli/enzimología , Escherichia coli/virología , Proteínas de Escherichia coli/metabolismoRESUMEN
In response to the persistent exposure to phage infection, bacteria have evolved diverse antiviral defense mechanisms. In this study, we report a bacterial two-component defense system consisting of a Sir2 NADase and a HerA helicase. Cryo-electron microscopy reveals that Sir2 and HerA assemble into a â¼1 MDa supramolecular octadecamer. Unexpectedly, this complex exhibits various enzymatic activities, including ATPase, NADase, helicase, and nuclease, which work together in a sophisticated manner to fulfill the antiphage function. Therefore, we name this defense system "Nezha" after a divine warrior in Chinese mythology who employs multiple weapons to defeat enemies. Our findings demonstrate that Nezha could sense phage infections, self-activate to arrest cell growth, eliminate phage genomes, and subsequently deactivate to allow for cell recovery. Collectively, Nezha represents a paradigm of sophisticated and multifaceted strategies bacteria use to defend against viral infections.
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Caudovirales , Escherichia coli , Adenosina Trifosfatasas , Microscopía por Crioelectrón , ADN Helicasas , NAD+ Nucleosidasa , Escherichia coli/enzimología , Escherichia coli/virologíaRESUMEN
Introduction: The ability to modulate and enhance the anti-tumor immune responses is critical in developing novel therapies in cancer. The Tumor Necrosis Factor (TNF) Receptor Super Family (TNFRSF) are potentially excellent targets for modulation which result in specific anti-tumor immune responses. CD40 is a member of the TNFRSF and several clinical therapies are under development. CD40 signaling plays a pivotal role in regulating the immune system from B cell responses to myeloid cell driven activation of T cells. The CD40 signaling axis is well characterized and here we compare next generation HERA-Ligands to conventional monoclonal antibody based immune modulation for the treatment of cancer. Methods & results: HERA-CD40L is a novel molecule that targets CD40 mediated signal transduction and demonstrates a clear mode of action in generating an activated receptor complex via recruitment of TRAFs, cIAP1, and HOIP, leading to TRAF2 phosphorylation and ultimately resulting in the enhanced activation of key inflammatory/survival pathway and transcription factors such asNFkB, AKT, p38, ERK1/2, JNK, and STAT1 in dendritic cells. Furthermore, HERA-CD40L demonstrated a strong modulation of the tumor microenvironment (TME) via the increase in intratumoral CD8+ T cells and the functional switch from pro-tumor macrophages (TAMs) to anti-tumor macrophages that together results in a significant reduction of tumor growth in a CT26 mouse model. Furthermore, radiotherapy which may have an immunosuppressive modulation of the TME, was shown to have an immunostimulatory effect in combination with HERA-CD40L. Radiotherapy in combination with HERA-CD40L treatment resulted in an increase in detected intratumoral CD4+/8+ T cells compared to RT alone and, additionally, the repolarization of TAMs was also observed, resulting in an inhibition of tumor growth in a TRAMP-C1 mouse model. Discussion: Taken together, HERA-CD40L resulted in activating signal transduction mechanisms in dendritic cells, resulting in an increase in intratumoral T cells and manipulation of the TME to be pro-inflammatory, repolarizing M2 macrophages to M1, enhancing tumor control.
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Ligando de CD40 , Neoplasias , Animales , Ratones , Antígenos CD40 , Células Presentadoras de Antígenos , Macrófagos , Neoplasias/radioterapia , Modelos Animales de Enfermedad , Microambiente TumoralRESUMEN
BACKGROUND: The nuclease NurA and the ATPase/translocase HerA play a vital role in repair of double-strand breaks (DSB) during the homologous recombination in archaea. A NurA-HerA complex is known to mediate DSB DNA end resection, leading to formation of a free 3' end used to search for the homologous sequence. Despite the structures of individual archaeal types of NurA and HerA having been reported, there is limited information regarding the molecular mechanisms underlying this process. Some bacteria also possess homologs of NurA and HerA; however, the bacterial type of this complex, as well as the detailed mechanisms underlying the joining of NurA-HerA in DSB DNA end resection, remains unclear. RESULTS: We report for the first time the crystal structures of Deinococcus radiodurans HerA (drHerA) in the nucleotide-free and ADP-binding modes. A D. radiodurans NurA-HerA complex structure was constructed according to a low-resolution cryo-electron microscopy map. We performed site-directed mutagenesis to map the drNurA-HerA interaction sites, suggesting that their interaction is mainly mediated by ionic links, in contrast to previously characterized archaeal NurA-HerA interactions. The key residues responsible for the DNA translocation activity, DNA unwinding activity, and catalytic activities of the drNurA-HerA complex were identified. A HerA/FtsK-specific translocation-related motif (TR motif) that guarantees the processivity of double-stranded DNA (dsDNA) translocation was identified. Moreover, a mechanism for the translocation-regulated resection of the 5' tail of broken dsDNA and the corresponding generation of a recombinogenic 3' single-stranded DNA tail by the drNurA-HerA complex was elucidated. CONCLUSIONS: Our work provides new insights into the mechanism underlying bacterial NurA-HerA-mediated DSB DNA end resection, and the way this complex digests the 5' tail of a DNA duplex and provides long 3' free end for strand invasion in the bacterial homologous recombination process.
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Adenosina Trifosfatasas , Bacterias , Microscopía por Crioelectrón , Adenosina Trifosfatasas/genética , Bacterias/genética , Reparación del ADN , ADN , ADN Bacteriano , Archaea/genéticaAsunto(s)
Planificación en Desastres , Contramedidas Médicas , Humanos , Europa (Continente) , Salud PúblicaRESUMEN
RNA helicases catalyze the ATP-dependent destabilization of RNA duplexes. DEAD-box helicases share a helicase core that mediates ATP binding and hydrolysis, RNA binding and unwinding. Most members of this family contain domains flanking the core that can confer RNA substrate specificity and guide the helicase to a specific RNA. However, the in vivo RNA substrates of most helicases are currently not defined. The DEAD-box helicase Hera from Thermus thermophilus contains a helicase core, followed by a dimerization domain and an RNA binding domain that folds into an RNA recognition motif (RRM). The RRM mediates high affinity binding to an RNA hairpin, and an adjacent duplex is then unwound by the helicase core. Hera is a cold-shock protein, and has been suggested to act as an RNA chaperone under cold-shock conditions. Using crosslinking immunoprecipitation of Hera/RNA complexes and sequencing, we show that Hera binds to a large fraction of T. thermophilus RNAs under normal-growth and cold-shock conditions without a strong sequence preference, in agreement with a structure-specific recognition of RNAs and a general function in RNA metabolism. Under cold-shock conditions, Hera is recruited to RNAs with high propensities to form stable secondary structures. We show that selected RNAs identified, including a set of tRNAs, bind to Hera in vitro, and activate the Hera helicase core. Gene ontology analysis reveals an enrichment of genes related to translation, including mRNAs of ribosomal proteins, tRNAs, tRNA ligases, and tRNA-modifying enzymes, consistent with a key role of Hera in ribosome and tRNA metabolism.
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ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Thermus thermophilus/crecimiento & desarrollo , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Respuesta al Choque por Frío , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , Estructura Secundaria de Proteína , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Thermus thermophilus/enzimología , Thermus thermophilus/genéticaRESUMEN
Viruses of archaea represent one of the most enigmatic parts of the virosphere. Most of the characterized archaeal viruses infect extremophilic hosts and display remarkable diversity of virion morphotypes, many of which have never been observed among bacteriophages or viruses of eukaryotes. However, recent environmental studies have shown that archaeal viruses are widespread also in moderate ecosystems, where they play an important ecological role by influencing the turnover of microbial communities, with a global impact on the carbon and nitrogen cycles. In this review, we summarize recent advances in understanding the molecular details of virion organization and assembly of archaeal viruses. We start by briefly introducing the 20 officially recognized families of archaeal viruses and then outline the similarities and differences of archaeal virus assembly with the morphogenesis pathways used by bacterial and eukaryotic viruses, and discuss the evolutionary implications of these observations. Generally, the assembly of the icosahedral archaeal viruses closely follows the mechanisms employed by evolutionarily related bacterial and eukaryotic viruses with the HK97 fold and double jelly-roll major capsid proteins, emphasizing the overall conservation of these pathways over billions of years of evolution. By contrast, archaea-specific viruses employ unique virion assembly mechanisms. We also highlight some of the molecular adaptations underlying the stability of archaeal viruses in extreme environments. Despite considerable progress during the past few years, the archaeal virosphere continues to represent one of the least studied parts of the global virome, with many molecular features awaiting to be discovered and characterized.
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Archaea/virología , Virus de Archaea/química , Virus de Archaea/fisiología , Ensamble de Virus , Virus de Archaea/clasificación , Virus de Archaea/genética , Bacterias/virología , Libros , Proteínas de la Cápside , Ecosistema , Genoma Viral , Filogenia , Proteínas Virales/genéticaRESUMEN
TNF Receptor Superfamily (TNF-R-SF) signaling is a structurally well-defined event that requires proper receptor clustering and trimerization. While the TNF-SF ligands naturally exist as trivalent functional units, the receptors are usually separated on the cell surface. Critically, receptor assembly into functional trimeric signaling complexes occurs through binding of the natural ligand unit. TNF-R-SF members, including CD40, have been key immunotherapeutic targets for over 20 years. CD40, expressed by antigen-presenting cells, endothelial cells, and many tumors, plays a fundamental role in connecting innate and adaptive immunity. The multiple investigated strategies to induce CD40 signaling can be broadly grouped into antibody-based or CD40L-based approaches. Currently, seven different antibodies and one CD40L-based hexavalent fusion protein are in active clinical trials. In this review, we describe the biology and structural properties of CD40, requirements for agonistic signal transduction through CD40 and summarize current attempts to exploit the CD40 signaling pathway for the treatment of cancer.
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Células Endoteliales , Neoplasias , Antígenos CD40 , Ligando de CD40 , Humanos , Neoplasias/terapia , Receptores del Factor de Necrosis Tumoral , Transducción de SeñalRESUMEN
Abstract Introduction: After post-septoplasty nasal packing removal, a certain proportion of nasal secretion occurs, leading to local and sometimes systemic infections. Objective: The aim was to determine if standardized dry ivy leaf extract application after nasal packing removal influences the reduction of nasal secretion and diminish the occurrence of local infections. Methods: The study included 70 post-septoplasty patients (divided into two equal groups) whose nasal packing was removed on the third day after the procedure. Group I was treated with standardized dry ivy leaf extract syrup along with regular nasal irrigation for the five days after the nasal packing removal whereas the Group II had only nasal lavage. On the sixth day after nasal packing removal, the quantity of nasal secretion was determined using a visual analog scale and nasal endoscopic examination. Results: The group treated with standardized dry ivy leaf extract syrup had significantly lesser nasal secretion both by subjective patients' assessment (p < 0.001) and by nasal endoscopic examination (p = 0.003). The post-surgical follow up examination on the sixth day after nasal packing removal showed no development of local infection in the Group I, while in the Group II a local infection was evident in five patients (14.29%) and antibiotic therapy was required. Conclusion: The use of the standardized dry ivy leaf extract after nasal packing removal significantly lowers the proportion of nasal secretion.
Resumo Introdução: Após a remoção do tampão nasal pós-septoplastia, ocorre produção de secreção nasal, predispondo infecções locais e, por vezes, sistêmicas. Objetivo: O objetivo foi determinar se a aplicação do extrato padronizado de folhas de hera seca após a remoção do tampão nasal influencia a redução da secreção nasal e diminui a ocorrência de infecções locais. Método: O estudo incluiu 70 pacientes pós-septoplastia (divididos em dois grupos iguais) cujo tampão nasal foi retirado no terceiro dia após o procedimento. O grupo I foi tratado com xarope padronizado de extrato de folha seca de hera juntamente com irrigação nasal regular por cinco dias após a remoção do tamponamento nasal, enquanto ao grupo II foi recomendado apenas lavagem nasal. No sexto dia após a remoção do tampão nasal, a quantidade de secreção nasal foi determinada pela escala EVA (escala visual analógica) e pelo exame endoscópico nasal. Resultados: O grupo tratado com xarope de extrato seco de folhas de hera apresentou secreção nasal significativamente menor tanto pela avaliação subjetiva dos pacientes (p < 0,001) quanto pelo exame endoscópico nasal (p = 0,003). O exame de acompanhamento pós-cirúrgico no sexto dia após a remoção do tampão nasal não mostrou desenvolvimento de infecção local nos pacientes do grupo I, enquanto que no grupo II, cinco apresentaram sinais de infecção local (14,29%) com necessidade de antibioticoterapia. Conclusão: O uso do extrato padronizado de folhas secas de hera após a remoção do tampão nasal reduz significativamente a produção de secreção nasal.
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Humanos , Masculino , Femenino , Adolescente , Adulto , Persona de Mediana Edad , Adulto Joven , Cuidados Posoperatorios/métodos , Rinoplastia/métodos , Extractos Vegetales/uso terapéutico , Hedera/química , Tabique Nasal/cirugía , Epistaxis/prevención & control , Nariz/microbiología , Hojas de la Planta/química , Hemorragia Posoperatoria/prevención & control , Fitoterapia , Antibacterianos/uso terapéuticoRESUMEN
BACKGROUND: Glucocorticoid-induced TNFR-related protein (TNFRSF18, GITR, CD357), expressed by T cells, and its ligand (TNFSF18, GITRL), expressed by myeloid populations, provide co-stimulatory signals that boost T cell activity. Due to the important role that GITR plays in regulating immune functions, agonistic stimulation of GITR is a promising therapeutic concept. Multiple strategies to induce GITR signaling have been investigated. The limited clinical efficacy of antibody-based GITR agonists results from structural and functional characteristics of antibodies that are unsuitable for stimulating the well-defined trimeric members of the TNFRSF. METHODS: To overcome limitations of antibody-based TNFRSF agonists, we have developed HERA-GITRL, a fully human hexavalent TNF receptor agonist (HERA) targeting GITR and mimicking the natural signaling concept. HERA-GITRL is composed of a trivalent but single-chain GITRL-receptor-binding-domain (scGITRL-RBD) unit fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. A specific mouse surrogate, mmHERA-GITRL, was also generated to examine in vivo activity in respective mouse tumor models. RESULTS: For functional characterization of HERA-GITRL in vitro, human immune cells were isolated from healthy-donor blood and stimulated with anti-CD3 antibody in the presence of HERA-GITRL. Consistently, HERA-GITRL increased the activity of T cells, including proliferation and differentiation, even in the presence of regulatory T cells. In line with these findings, mmHERA-GITRL enhanced antigen-specific clonal expansion of both CD4+ (OT-II) and CD8+ (OT-I) T cells in vivo while having no effect on non-specific T cells. In addition, mmHERA-GITRL showed single-agent anti-tumor activity in two subcutaneous syngeneic colon cancer models (CT26wt and MC38-CEA). Importantly, this activity is independent of its FcγR-binding functionality, as both mmHERA-GITRL with a functional Fc- and a silenced Fc-domain showed similar tumor growth inhibition. Finally, in a direct in vitro comparison to a bivalent clinical benchmark anti-GITR antibody and a trivalent GITRL, only the hexavalent HERA-GITRL showed full biological activity independent of additional crosslinking. CONCLUSION: In this manuscript, we describe the development of HERA-GITRL, a true GITR agonist with a clearly defined mechanism of action. By clustering six receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional FcγR-mediated crosslinking.
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Receptores del Factor de Necrosis Tumoral/agonistas , Anticuerpos de Cadena Única/administración & dosificación , Linfocitos T Reguladores/inmunología , Factores de Necrosis Tumoral/química , Animales , Línea Celular Tumoral , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Activación de Linfocitos , Macaca fascicularis , Ratones , Proteínas Recombinantes de Fusión/inmunología , Transducción de Señal , Anticuerpos de Cadena Única/inmunología , Factores de Necrosis Tumoral/metabolismoRESUMEN
INTRODUCTION: After post-septoplasty nasal packing removal, a certain proportion of nasal secretion occurs, leading to local and sometimes systemic infections. OBJECTIVE: The aim was to determine if standardized dry ivy leaf extract application after nasal packing removal influences the reduction of nasal secretion and diminish the occurrence of local infections. METHODS: The study included 70 post-septoplasty patients (divided into two equal groups) whose nasal packing was removed on the third day after the procedure. Group I was treated with standardized dry ivy leaf extract syrup along with regular nasal irrigation for the five days after the nasal packing removal whereas the Group II had only nasal lavage. On the sixth day after nasal packing removal, the quantity of nasal secretion was determined using a visual analog scale and nasal endoscopic examination. RESULTS: The group treated with standardized dry ivy leaf extract syrup had significantly lesser nasal secretion both by subjective patients' assessment (p<0.001) and by nasal endoscopic examination (p=0.003). The post-surgical follow up examination on the sixth day after nasal packing removal showed no development of local infection in the Group I, while in the Group II a local infection was evident in five patients (14.29%) and antibiotic therapy was required. CONCLUSION: The use of the standardized dry ivy leaf extract after nasal packing removal significantly lowers the proportion of nasal secretion.
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Hedera/química , Tabique Nasal/cirugía , Extractos Vegetales/uso terapéutico , Cuidados Posoperatorios/métodos , Rinoplastia/métodos , Adolescente , Adulto , Antibacterianos/uso terapéutico , Epistaxis/prevención & control , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nariz/microbiología , Fitoterapia , Hojas de la Planta/química , Hemorragia Posoperatoria/prevención & control , Adulto JovenRESUMEN
The original approach of the Hemispheric Encoding/Retrieval Asymmetry model (HERA) was aimed at the operations of encoding and retrieving episodic memories. However, whether HERA presumptions can apply to different types of stimuli (e.g., words and pictures) continues to be a matter of debate. Therefore, in order to analyse the effects of brain pre-activation on subsequent memory, HERA was tested through a hand-clenching paradigm using four types of stimuli: words, fractal images, silhouettes of common objects, and pseudowords. Results revealed that only the memory of words and pseudowords was enhanced by hand-clenching pre-activation, according to HERA predictions. Since the cognitive processes underlying recognition of verbal stimuli are considered to follow a cognitive route involving grapheme-morpheme conversion, it could be hypothesized that hand-clenching pre-activation might be associated with a selective pre-activation of the brain circuits participating in that pathway. Hence, the present work broadens possible interpretations behind the effects of hand-clenching on memory, based on the process engaged and the type of stimulus to be remembered.
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Mapeo Encefálico/psicología , Lateralidad Funcional/fisiología , Memoria Episódica , Reconocimiento en Psicología/fisiología , Adolescente , Adulto , Análisis de Varianza , Cerebro/fisiología , Cognición , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Recuerdo Mental/fisiología , Tomografía de Emisión de Positrones , Adulto JovenRESUMEN
Tumor necrosis factor receptor superfamily member 7 (TNFRSF7, CD27), expressed primarily by T cells, and its ligand CD27L (TNFSF7, CD70) provide co-stimulatory signals that boost T cell activation, differentiation, and survival. Agonistic stimulation of CD27 is therefore a promising therapeutic concept in immuno-oncology intended to boost and sustain T cell driven anti-tumor responses. Endogenous TNFSF/TNFRSF-based signal transmission is a structurally well-defined event that takes place during cell-to-cell-based contacts. It is well-established that the trimeric-trivalent TNFSF-receptor binding domain (TNFSF-RBD) exposed by the conducting cell and the resulting multi-trimer-based receptor clustering on the receiving cell are essential for agonistic signaling. Therefore, we have developed HERA-CD27L, a novel hexavalent TNF receptor agonist (HERA) targeting CD27 and mimicking the natural signaling concept. HERA-CD27L is composed of a trivalent but single-chain CD27L-receptor-binding-domain (scCD27L-RBD) fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. The hexavalent agonist significantly boosted antigen-specific T cell responses while having no effect on non-specific T cells and was superior over stabilized recombinant trivalent CD27L. In addition, HERA-CD27L demonstrated potent single-agent anti-tumor efficacy in two different syngeneic tumor models, MC38-CEA and CT26wt. Furthermore, the combination of HERA-CD27L and an anti-PD-1 antibody showed additive anti-tumor effects highlighting the importance of both T cell activation and checkpoint inhibition in anti-tumor immunity. In this manuscript, we describe the development of HERA-CD27L, a true CD27 agonist with a clearly defined forward-signaling mechanism of action.
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In view of the rising cost pressure and an increasing number of drug shortages, switches between generic drug preparations have become a daily routine in hospitals. To ensure consistently high treatment quality and best possible patient safety, the equivalence of the new and the previous drug preparation must be ensured before any change in the purchase of pharmaceutical products takes place. So far, no easily usable, transparent and standardized instrument for this kind of comparison between generic drug products has been available. A group of pharmaceutical experts has developed the drug HTA (health technology assessment) model "HERA" (HTA Evaluation of geneRic phArmaceutical products) through a multi-step process. The instrument is designed to perform both a qualitative and economic comparison of equivalent drug preparations ("aut idem" substitution) before switching products. The economic evaluation does not only consider unit prices and consumption quantity, but also the processing costs associated with a product change process. The qualitative comparison is based on the evaluation of 34 quality criteria belonging to six evaluation fields (e.g., approval status, practical handling, packaging design). The objective evaluation of the quality criteria is complemented by an assessment of special features of the individual hospital for complex drug switches, including the feedback of the physicians utilizing the drug preparation. Thus potentially problematic switches of pharmaceutical products can be avoided at the best possible rate, contributing to the improvement of patient safety. The novel drug HTA model HERA is a tool used in clinical practice that can add to an increase in quality, therapeutic safety and transparency of drug use while simultaneously contributing to the economic optimization of drug procurement in hospitals. Combining these two is essential for hospitals facing the tension between rising cost pressure and at the same time increasing demands on quality and transparency, triggered by, amongst others, current legislation (Hospital Structures Act, anti-corruption legislation).
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Medicamentos Genéricos/normas , Seguridad del Paciente , Análisis Costo-Beneficio , Medicamentos Genéricos/economía , Alemania , Hospitales , Humanos , Evaluación de la Tecnología BiomédicaRESUMEN
Intense gene flux between prokaryotes result in high percentage of archaeal genes in the genome of the thermophilic bacteria Thermus spp. Among these archaeal genes a homolog to the Sulfolobus spp. HerA protein appears in all of the Thermus spp. strains so far sequenced (HepA). The role of HepA in Thermus thermophilus HB27 has been analyzed using deletion mutants, and its structure resolved at low resolution by electron microscopy. Recombinant HepA shows DNA-dependent ATPase activity and its structure revealed a double ring, conically-shaped hexamer with an upper diameter of 150 Å and a bottom module of 95 Å. A central pore was detected in the structure that ranges from 13 Å at one extreme, to 30 Å at the other. Mutants lacking HepA show defective natural competence and DNA donation capability in a conjugation-like process termed "transjugation", and also high sensitivity to UV and dramatic sensitivity to high temperatures. These data support that acquisition of an ancestral archaeal HerA has been fundamental for the adaptation of Thermus spp. to high temperatures.
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Homologous recombination (HR) generates error-free repair products, which plays an important role in double strand break repair and replication fork rescue processes. DNA end resection, the critical step in HR, is usually performed by a series of nuclease/helicase. RecJ was identified as a 5'-3' exonuclease involved in bacterial DNA end resection. Typical RecJ possesses a conserved DHH domain, a DHHA1 domain, and an oligonucleotide/oligosaccharide-binding (OB) fold. However, RecJs from Deinococcus-Thermus phylum, such as Deinococcus radiodurans RecJ (DrRecJ), possess an extra C-terminal domain (CTD), of which the function has not been characterized. Here, we showed that a CTD-deletion of DrRecJ (DrRecJΔC) could not restore drrecJ mutant growth and mitomycin C (MMC)-sensitive phenotypes, indicating that this domain is essential for DrRecJ in vivo. DrRecJΔC displayed reduced DNA nuclease activity and DNA binding ability. Direct interaction was identified between DrRecJ-CTD and DrHerA, which stimulates DrRecJ nuclease activity by enhancing its DNA binding affinity. Moreover, DrNurA nuclease, another partner of DrHerA, inhibited the stimulation of DrHerA on DrRecJ nuclease activity by interaction with DrHerA. Opposing growth and MMC-resistance phenotypes between the recJ and nurA mutants were observed. A novel modulation mechanism among DrRecJ, DrHerA, and DrNurA was also suggested.
RESUMEN
DNA double-strand breaks can be repaired by homologous recombination, during which the DNA ends are long-range resected by helicase-nuclease systems to generate 3' single strand tails. In archaea, this requires the Mre11-Rad50 complex and the ATP-dependent helicase-nuclease complex HerA-NurA. We report the cryo-EM structure of Sulfolobus solfataricus HerA-NurA at 7.4Å resolution and present the pseudo-atomic model of the complex. HerA forms an ASCE hexamer that tightly interacts with a NurA dimer, with each NurA protomer binding three adjacent HerA HAS domains. Entry to NurA's nuclease active sites requires dsDNA to pass through a 23Å wide channel in the HerA hexamer. The structure suggests that HerA is a dsDNA translocase that feeds DNA into the NurA nuclease sites.
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Roturas del ADN de Doble Cadena , ADN Helicasas/química , ADN Helicasas/metabolismo , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Adenosina Trifosfato/metabolismo , Dominio Catalítico , ADN/genética , ADN/metabolismo , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Sulfolobus solfataricus/enzimologíaRESUMEN
The capacity for semantic memory-the ability to acquire and store knowledge of the world-is highly developed in the human brain. In particular, semantic memory assimilated through an auditory route may be a uniquely human capacity. One method of obtaining neurobiological insight into memory mechanisms is through the study of experts. In this work, we study a group of Hindu Vedic priests, whose religious training requires the memorization of vast tracts of scriptural texts through an oral tradition, recalled spontaneously during a lifetime of subsequent spiritual practice. We demonstrate focal increases of cortical thickness in regions of the left prefrontal lobe and right temporal lobe in Vedic priests, in comparison to a group of matched controls. The findings are relevant to current hypotheses regarding cognitive processes underlying storage and recall of long-term declarative memory.
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BACKGROUND: Trastuzumab treatment improves survival of HER2-positive primary breast cancer. HER2 staining intensity varies widely in HER2-positive tumours. PATIENTS AND METHODS: We investigated whether differences in immunohistochemical (IHC) staining intensity for HER2 in HER2-positive tumors (IHC 3+ or FISH ratio ≥2.0) was associated with prognosis or benefit from trastuzumab treatment in patients randomized to 1 year or no trastuzumab in the HERceptin Adjuvant (HERA) trial. Median follow-up was 2 years. The nested case-control analysis, included 425 patients (cases) with a disease-free survival (DFS) event and two matched controls (no DFS event) per case. Tissue sections stained for HER2 were assessed for HER2 staining intensity by image analysis. RESULTS: HER2 staining intensity varied widely and correlated with HER2 gene copy number (Spearman, r = 0.498, P < 0.001) or less closely with HER2/CEP17 FISH ratio (r = 0.396, P < 0.001). We found no significant difference in DFS in the observation arm according to staining intensity (odds ratio [OR] change per 10 unit change in intensity: 1.015, 95% confidence interval [CI] 0.930-1.108) and no impact of staining intensity on benefit derived from 1-year trastuzumab (OR: 1.017, 95% CI 0.925-1.120). CONCLUSIONS: Variability in HER2 staining in HER2-positive tumours has no role in clinical management with adjuvant trastuzumab. HERA TRIAL NO: NCT00045032.