RESUMO
BACKGROUND: A variety of bacterial and fungal co-infections may be attributed to the coronavirus disease 2019 (COVID-19), particularly in people who already have a medical condition such diabetes mellitus or those who received large dosages of steroids. CASE REPORT: We described a 52-year-old diabetic man who was receiving high doses of dexamethasone and antibiotics while receiving ambulatory care for COVID-19 pneumonia. His anterior rhinoscopy revealed a necrotic scab, and a sample confirmed Mucor spp. He underwent surgery and was given amphotericin as a result of the severity of the condition, palpebral ptosis, and right ocular palsy he was experiencing. The patien Ìs progression was satisfactory. CONCLUSIONS: pre-existing diabetes mellitus, previous steroid and antimicrobial use, as well as SARS-CoV-2 infection are some of the risk factors associated with Mucor spp. infection. Prompt detection of mucormycosis is important in the management of these affected patients.
ANTECEDENTES: A la enfermedad por coronavirus (COVID-19) se le han atribuido diversas coinfecciones bacterianas y fúngicas, especialmente en sujetos con enfermedades preexistentes (diabetes mellitus) o en quienes han recibido altas dosis de corticosteroides. REPORTE DE CASO: Paciente masculino de 52 años, con antecedente de diabetes mellitus, quien recibió altas dosis de dexametasona y antibióticos mientras recibía atención ambulatoria por neumonía secundaria a COVID-19. La rinoscopia anterior reveló una costra necrótica, y una muestra de exudado confirmó la coexistencia de Mucor spp. Debido a la complicación del cuadro clínico, ptosis palpebral y parálisis ocular derecha, se le administró anfotericina B y fue intervenido quirúrgicamente. La evolución del paciente fue satisfactoria. CONCLUSIONES: La diabetes mellitus preexistente, el consumo de corticosteroides y antimicrobianos, además de la infección por SARS-CoV-2 son factores de riesgo asociados con la infección por Mucor spp. Es importante la detección oportuna de mucormicosis en el tratamiento de estos pacientes.
Assuntos
COVID-19 , Mucormicose , Masculino , Humanos , Pessoa de Meia-Idade , Mucormicose/complicações , Mucormicose/diagnóstico , COVID-19/complicações , SARS-CoV-2 , NarizRESUMO
Mayaro virus (MAYV) is an emerging arbovirus with an increasing circulation across the Americas. In the present study, we evaluated the potential antiviral activity of the following natural compounds against MAYV and other arboviruses: Sanguinarine, (R)-Shikonin, Fisetin, Honokiol, Tanshinone IIA, and α-Mangostin. Sanguinarine and Shikonin showed significant cytotoxicity, whereas Fisetin, Honokiol, Tanshinone IIA, and α-Mangostin were well tolerated in all the cell lines tested. Honokiol and α-Mangostin treatment protected Vero-E6 cells against MAYV-induced damage and resulted in a dose-dependent reduction in viral progeny yields for each of the MAYV strains and human cell lines assessed. These compounds also reduced MAYV viral RNA replication in HeLa cells. In addition, Honokiol and α-Mangostin disrupted MAYV infection at different stages of the virus life cycle. Moreover, Honokiol and α-Mangostin decreased Una, Chikungunya, and Zika viral titers and downmodulated the expression of E1 and nsP1 viral proteins from MAYV, Una, and Chikungunya. Finally, in Honokiol- and α-Mangostin-treated HeLa cells, we observed an upregulation in the expression of type I interferon and specific interferon-stimulated genes, including IFNα, IFNß, MxA, ISG15, OAS2, MDA-5, TNFα, and IL-1ß, which may promote an antiviral cellular state. Our results indicate that Honokiol and α-Mangostin present potential broad-spectrum activity against different arboviruses through different mechanisms.
Assuntos
Alphavirus , Arbovírus , Febre de Chikungunya , Infecção por Zika virus , Zika virus , Humanos , Células HeLa , Alphavirus/genética , Replicação Viral , Antivirais/farmacologiaRESUMO
Animals detect changes in both their environment and their internal state and modify their behavior accordingly. Yet, it remains largely to be clarified how information of environment and internal state is integrated and how such integrated information modifies behavior. Well-fed C. elegans migrates to past cultivation temperature on a thermal gradient, which is disrupted when animals are starved. We recently reported that the neuronal activities synchronize between a thermosensory neuron AFD and an interneuron AIY, which is directly downstream of AFD, in well-fed animals, while this synchrony is disrupted in starved animals. However, it remained to be determined whether the disruption of the synchrony is derived from modulation of the transmitter release from AFD or from the modification of reception or signal transduction in AIY. By performing forward genetics on a transition of thermotaxis behavior along starvation, we revealed that OLA-1, an Obg-like ATPase, functions in AFD to promote disruption of AFD-AIY synchrony and behavioral transition. Our results suggest that the information of hunger is delivered to the AFD thermosensory neuron and gates transmitter release from AFD to disrupt thermotaxis, thereby shedding light onto a mechanism for the integration of environmental and internal state to modulate behavior.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Adenosina Trifosfatases/genética , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Fome , Células Receptoras Sensoriais , TemperaturaRESUMO
Mayaro virus (MAYV) manipulates cell machinery to successfully replicate. Thus, identifying host proteins implicated in MAYV replication represents an opportunity to discover potential antiviral targets. PIM kinases are enzymes that regulate essential cell functions and also appear to be critical factors in the replication of certain viruses. In this study we explored the consequences of PIM kinase inhibition in the replication of MAYV and other arboviruses. Cytopathic effects or viral titers in samples from MAYV-, Chikungunya-, Una- or Zika-infected cells treated with PIM kinase inhibitors were evaluated using an inverted microscope or plaque-forming assays. The expression of viral proteins E1 and nsP1 in MAYV-infected cells was assessed using an immunofluorescence confocal microscope or Western blot. Our results revealed that PIM kinase inhibition partially prevented MAYV-induced cell damage and also promoted a decrease in viral titers for MAYV, UNAV and ZIKV. The inhibitory effect of PIM kinase blocking was observed for each of the MAYV strains tested and also occurred as late as 8 h post infection (hpi). Finally, PIM kinase inhibition suppressed the expression of MAYV E1 and nsP1 proteins. Taken together, these findings suggest that PIM kinases could represent an antiviral target for MAYV and other arboviruses.
Assuntos
Alphavirus/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Linhagem Celular , Vírus Chikungunya/efeitos dos fármacos , Humanos , Zika virus/efeitos dos fármacosRESUMO
Classical Rett syndrome (RTT) is a neurodevelopmental disorder where most of cases carry MECP2 mutations. Atypical RTT variants involve mutations in CDKL5 and FOXG1. However, a subset of RTT patients remains that do not carry any mutation in the described genes. Whole exome sequencing was carried out in a cohort of 21 female probands with clinical features overlapping with those of RTT, but without mutations in the customarily studied genes. Candidates were functionally validated by assessing the appearance of a neurological phenotype in Caenorhabditis elegans upon disruption of the corresponding ortholog gene. We detected pathogenic variants that accounted for the RTT-like phenotype in 14 (66.6 %) patients. Five patients were carriers of mutations in genes already known to be associated with other syndromic neurodevelopmental disorders. We determined that the other patients harbored mutations in genes that have not previously been linked to RTT or other neurodevelopmental syndromes, such as the ankyrin repeat containing protein ANKRD31 or the neuronal acetylcholine receptor subunit alpha-5 (CHRNA5). Furthermore, worm assays demonstrated that mutations in the studied candidate genes caused locomotion defects. Our findings indicate that mutations in a variety of genes contribute to the development of RTT-like phenotypes.
Assuntos
Proteínas de Transporte/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas do Tecido Nervoso/genética , Receptores Nicotínicos/genética , Síndrome de Rett/genética , Adolescente , Adulto , Animais , Caenorhabditis elegans/genética , Proteínas de Ciclo Celular , Criança , Pré-Escolar , Análise Mutacional de DNA , Exoma/genética , Feminino , Fatores de Transcrição Forkhead/genética , Variação Genética , Humanos , Proteína 2 de Ligação a Metil-CpG/genética , Mutação , Proteínas Serina-Treonina Quinases/genética , Síndrome de Rett/fisiopatologiaRESUMO
Objetivo: Comparar los efectos hemodinámicos y el consumo de remifentanilo entre perfusión manual versus infusión controlada por objetivo (TCI) sitio-efecto en anestesia general balanceada para pacientes sometidos a colecistectomía laparoscópica. Material y métodos: Estudio observacional donde se formaron 2 grupos, modo manual y modo TCI sitioûefecto, en un periodo de 4 meses en el Hospital Nacional Arzobispo Loayza. Se establecieron recomendaciones para que la única diferencia sea el modo de perfusión y posteriormente se hizo un emparejamiento por edad. Se tomaron la totalidad de los datos de la hoja de monitoreo de anestesia y una ficha elaborada por el investigador. La recolección estuvo a cargo de los médicos asistentes y residentes de anestesiología del hospital. Resultados: Se analizaron 50 casos por grupo; las características demográficas y valores hemodinámicos basales fueron similares en ambos grupos. La frecuencia cardiaca fue similar en ambos grupos; la presión arterial media tuvo valores ligeramente mayores en el grupo TCI en ciertos tiempos. El consumo de remifentanilo fue menor en el grupo TCI de 0.80 ± 0.41 mg versus 1.08 ± 0.77 mg en el grupo manual (p = 0.0031). Conclusiones: El consumo de remifentanilo fue significativamente menor en el grupo TCI con efectos hemodinámicos similares frente al grupo manual en la población estudiada.
Objective: To compare the hemodynamic effects and remifentanil consumption between manual infusion versus target controlled infusion (TCI) site effect on patients under general anesthesia for undergoing laparoscopic cholecystectomy. Material and methods: Observational study. Two groups were formed, manual mode and TCI siteeffect over a period of 4 months at the Hospital Nacional Arzobispo Loayza. Recommendations were established so that the only difference was the infusion mode and then a match by age was made was made. The data were taken from the anesthesia records and put into a sheet prepared by the investigator. The collection was made by attending physicians and anesthesiology residents of the hospital. Results: We studied 50 patients per group, the demographic and baseline hemodynamic values were similar in both groups. Heart rate was similar in both groups; mean arterial pressure had slightly higher values in the TCI group at certain times. Remifentanil consumption was lower in the TCI group 0.80 ± 0.41 mg versus 1.08 ± 0.77 mg in the manual group (p = 0.0031). Conclusions: Remifentanil consumption was significantly lower in the TCI group with similar hemodynamic effects in the manual group in the population studied.
Assuntos
Humanos , Masculino , Adolescente , Adulto , Feminino , Adulto Jovem , Pessoa de Meia-Idade , Analgésicos Opioides , Bombas de Infusão , Colecistectomia Laparoscópica , Hemodinâmica , Estudos Observacionais como AssuntoRESUMO
Objetivos Los objetivos de este estudio fueron conocer el grado de control del asma bronquial en niños entre 6 y 14 años atendidos en un centro de Atención Primaria. Método Estudio descriptivo transversal que incluyó a niños de entre 6 y 14 años diagnosticados de asma bronquial, atendidos por un equipo de Atención Primaria del área metropolitana de Barcelona. La determinación del control del asma bronquial se realizó mediante el test de control del asma (TCA). Se consideró que tenían un buen control del asma los niños que obtenían una puntuación en el TCA superior a 20. Resultados Se encuestaron un total de 46 pacientes (45% niñas y 55% niños), con una edad media de 8,9 (DE 2,8) años. La prevalencia del asma mal controlada fue del 41,3%. Un 36% de los niños tenían antecedentes familiares de asma y un 43% de los niños refirieron tener dificultades para realizar actividad física. Conclusiones El alto porcentaje de mal control del asma bronquial en niños plantea la necesidad de revisar la efectividad de los protocolos de intervención existentes para mejorar su control (AU)
Objectives: This study aimed to assess the level of asthma control in children aged 6 to 14 years old in a Primary Care Centre. Methodology: A cross-sectional descriptive study was conducted on asthmatic children aged 6 to14 years old followed up in a Primary Health Care Centre in a metropolitan area of Barcelona. The Asthma Control Test (ACT) was the tool used to measure asthma control. Asthma was considered to be well controlled with an ACT score >20. Demographic and disease specific data were also collected. Results: A total of 46 patients (45% girls, 55% boys) completed the ACT. Their mean age was 8,9 years (SD 2.8). The prevalence of poorly controlled asthma was 41.3%. A family history of asthma was found in 36% of the children, and 43% of them declared to have activity limitations. Conclusions: The high percentage of poorly controlled asthma in children raises the need for reviewing the effectiveness in the processes to improve asthma control in these patients (AU)
Assuntos
Humanos , Masculino , Feminino , Criança , Adolescente , Atenção Primária à Saúde/métodos , Asma/epidemiologia , Estudos Transversais , Avaliação de Resultado de Ações PreventivasRESUMO
OBJECTIVES: This study aimed to assess the level of asthma control in children aged 6 to 14 years old in a Primary Care Centre. METHODOLOGY: A cross-sectional descriptive study was conducted on asthmatic children aged 6 to 14 years old followed up in a Primary Health Care Centre in a metropolitan area of Barcelona. The Asthma Control Test (ACT) was the tool used to measure asthma control. Asthma was considered to be well controlled with an ACT score >20. Demographic and disease specific data were also collected. RESULTS: A total of 46 patients (45% girls, 55% boys) completed the ACT. Their mean age was 8,9 years (SD 2.8). The prevalence of poorly controlled asthma was 41.3%. A family history of asthma was found in 36% of the children, and 43% of them declared to have activity limitations. CONCLUSIONS: The high percentage of poorly controlled asthma in children raises the need for reviewing the effectiveness in the processes to improve asthma control in these patients.
Assuntos
Asma/prevenção & controle , Adolescente , Criança , Estudos Transversais , Feminino , Instalações de Saúde , Humanos , Masculino , Atenção Primária à SaúdeRESUMO
The number of alternative sigma factor molecules per bacterial cell determines either stochasticity or evenness of transcription of cognate promoters. An approach for examining the abundance of sigmas in any sample of bacterial origin is explained here which relies on the production of a recombinant highly specific, high-affinity single-chain variable Fv domain (scFv) targeted towards unique protein sites of the factor. Purposely, a super-binder scFv recognizing a distinct epitope of the less abundant sigma σ(54) of Pseudomonas putida (also known as σ(N)) was obtained and its properties examined in detail. To this end, an scFv library was generated from mRNA extracted from lymphocytes of mice immunized with the purified σ(54) protein of this bacterium. The library was displayed on a phage system and subjected to various rounds of panning with purified σ(54) for capturing extreme binders. The resulting high-affinity anti-σ(54) phage antibody (Phab) clone named C2 strongly attached a small region located between positions 172 and 183 of the primary amino acid sequence of σ(54) that overlaps its core RNA polymerase-binding region. The purified scFv-C2 detected minute amounts of σ(54) in whole cell protein extracts not only of P. putida but also Escherichia coli cells and putatively in other bacteria as well. The affinity constant of the purified antibody was measured by surface plasmon resonance (SPR) and found to have a K(D) (k(off)/k(on)) in the range of 2×10(-9)M. The considerable affinity and specificity of this recombinant antibody makes it a tool of choice for quantitative studies on gene expression of σ(54)-dependent promoters in P. putida and other Gram-negative bacteria.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Pseudomonas putida/química , Pseudomonas putida/genética , RNA Polimerase Sigma 54/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Anticorpos de Cadeia Única/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Mapeamento de Epitopos , Escherichia coli , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação Proteica , Pseudomonas putida/imunologia , RNA Polimerase Sigma 54/química , RNA Polimerase Sigma 54/imunologia , RNA Polimerase Sigma 54/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/metabolismoRESUMO
Horizontal transfer of antibiotic resistance genes carried by conjugative plasmids poses a serious health problem. As conjugative relaxases are transported to recipient cells during bacterial conjugation, we investigated whether blocking relaxase activity in the recipient cell might inhibit conjugation. For that purpose, we used an intrabody approach generating a single-chain Fv antibody library against the relaxase TrwC of conjugative plasmid R388. Recombinant single-chain Fv antibodies were engineered for cytoplasmic expression in Escherichia coli cells and either selected in vitro for their specific binding to TrwC, or in vivo by their ability to interfere with conjugation using a high-throughput mating assay. Several intrabody clones were identified showing specific inhibition against R388 conjugation upon cytoplasmic expression in the recipient cell. The epitope recognized by one of these intrabodies was mapped to a region of TrwC containing Tyr-26 and involved in the conjugative DNA-processing termination reaction. These findings demonstrate that the transferred relaxase plays an important role in the recipient cell and open a new approach to identify specific inhibitors of bacterial conjugation.
Assuntos
Anticorpos Antibacterianos/imunologia , Conjugação Genética , DNA Nucleotidiltransferases/antagonistas & inibidores , DNA Nucleotidiltransferases/imunologia , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/imunologia , Escherichia coli/enzimologia , Anticorpos Antibacterianos/genética , DNA Nucleotidiltransferases/metabolismo , Mapeamento de Epitopos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Região Variável de Imunoglobulina/imunologia , Modelos MolecularesRESUMO
The identification of single-chain antibodies (scFvs) that interfere in vivo with the building of the complex that activate the prokaryotic, sigma54-dependent promoter Pu of the catabolic TOL plasmid pWW0 is reported. To this end, a phage M13 library of scFvs was raised against the cognate prokaryotic enhancer-binding activator, XylR. The scFv pool was then expressed intracellularly in a reporter Pu-lacZ strain of Escherichia coli designed to permit formation of intramolecular disulphide bonds in cytoplasmic proteins. This strain allowed the assembly of functional scFvs and the direct testing of their activity on the Pu promoter in vivo. Specifically, genetic screening for lacZ-minus colonies yielded a number of scFvs able to downregulate transcriptional output in live cells. Two antibody clones were purified and shown to inhibit the activity of the same promoter in vitro as well. These scFvs targeted the DNA-binding domain of XylR and its ATP binding site respectively. This work provides a proof of principle that mimetic regulatory factors can be derived from an antibody repertoire that specifically interact with given transcriptional activators. As assembly of initiation complexes is stimulated or inhibited by regulatory proteins we argue that anti-XylR scFvs operate as bona fide transcriptional inhibitors of the Pu promoter.
Assuntos
Anticorpos/metabolismo , Região Variável de Imunoglobulina , Plasmídeos/genética , Regiões Promotoras Genéticas , RNA Polimerase Sigma 54/metabolismo , Sequência de Aminoácidos , Anticorpos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Fenótipo , Plasmídeos/metabolismo , Ligação Proteica , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In this study we investigate the effect of thioredoxin (Trx1) protein fusions in the production, oxidation, and folding of single chain Fv (scFv) antibodies in the cytoplasm of Escherichia coli. We analyze the expression levels, solubility, disulfide-bond formation, and antigen-binding properties of Trx1-scFv fusions in E. coli wild-type cells and isogenic strains carrying mutations in the glutathione oxidoreductase (gor) and/or thioredoxin reductase (trxB) genes. We compare the Trx1-scFv fusions with other reported systems for production of scFv in the cytoplasm of E. coli, including protein fusions to the maltose-binding protein. In addition, we analyze the effect of co-expressing a signal-sequence-less derivative of the periplasmic chaperone and disulfide-bond isomerase DsbC (DeltassDsbC), which has been shown to act as a chaperone for scFvs in the cytoplasm. The results reported here demonstrate that Trx1 fusions produce the highest expression level and induce the correct folding of scFvs even in the absence of DeltassDsbC in the cytoplasm of E. coli trxB gor cells. The disulfide bridges of Trx1-scFv fusions were formed correctly in E. coli trxB gor cells, but not in trxB single mutants. Antigen-binding assays showed that Trx1 has only a minor influence in the affinity of the scFv, indicating that Trx1-scFv fusions can be used without removal of the Trx1 moiety. In addition, we proved that a Trx1"AGPA" variant, having its catalytic cysteine residues mutated to alanine, was fully capable of assisting the folding of the fused scFvs. Taken together, our data indicate that the Trx1 moiety acts largely as an intramolecular protein chaperone, not as a disulfide bond catalyst, inducing the correct folding of scFvs in the cytoplasm of E. coli trxB gor cells.
Assuntos
Escherichia coli/metabolismo , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tiorredoxinas/metabolismo , Citoplasma/metabolismo , Dissulfetos/metabolismo , Escherichia coli/citologia , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Oxirredução , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Tiorredoxinas/química , Tiorredoxinas/genéticaRESUMO
The cellular levels of the alternative sigma factor sigma(54) of Pseudomonas putida have been examined in a variety of growth stages and culture conditions with a single-chain Fv antibody tailored for detection of scarce proteins. The levels of sigma(54) were also monitored in P. putida strains with knockout mutations in ptsO or ptsN, known to be required for the C-source control of the sigma(54)-dependent Pu promoter of the TOL plasmid. Our results show that approximately 80 +/- 26 molecules of sigma(54) exist per cell. Unlike that in relatives of Pseudomonas (e.g., Caulobacter), where fluctuations of sigma(54) determine adaptation and differentiation when cells face starvation, sigma(54) in P. putida remains unexpectedly constant at different growth stages, in nitrogen starvation and C-source repression conditions, and in the ptsO and ptsN mutant strains analyzed. The number of sigma(54) molecules per cell in P. putida is barely above the predicted number of sigma(54)-dependent promoters. These figures impose a framework on the mechanism by which Pu (and other sigma(54)-dependent systems) may become amenable to physiological control.
Assuntos
Proteínas de Ligação a DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Pseudomonas putida/crescimento & desenvolvimento , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meios de Cultura , Mutação , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Plasmídeos , Pseudomonas putida/fisiologia , RNA Polimerase Sigma 54 , Transcrição GênicaRESUMO
Production of intracellular antibodies in Escherichia coli has been thought unlikely owing to an inability to form stable disulfide bonds in the cytoplasm, a necessary step in the folding of most immunoglobulin (Ig) domains. This work investigates whether E. coli strains carrying mutations in the major intracellular disulfide bond-reduction systems (i.e. the thioredoxin and the glutathione/glutaredoxin pathways) allow the oxidation and folding of single chain variable fragment (scFv) antibodies in the cytoplasm. The effect of the co-expression of disulfide bond chaperones in these cells was also examined. An scFv that recognizes the alternative sigma factor sigma(54) was used as a model to investigate disulfide bond formation and the folding of Ig domains in E. coli. The results demonstrate that functional intrabodies, with oxidized disulfide bonds in their Ig domains, are produced efficiently in E. coli cells carrying mutations in the glutathione oxidoreductase (gor) and the thioredoxin reductase (trxB) genes and co-expressing a signal-sequence-less derivative of the disulfide-bond isomerase DsbC ((Delta)ssDsbC). We obtained evidence indicating that (Delta)ssDsbC acts as a chaperone promoting the correct folding and oxidation of scFvs.
