RESUMO
Many community- and hospital-acquired bacterial infections are caused by antibiotic-resistant pathogens. Methicillin-resistant Staphylococcus aureus (MRSA) predisposes humans to invasive infections that are difficult to eradicate. We designed a closed-loop gene network programming mammalian cells to autonomously detect and eliminate bacterial infections. The genetic circuit contains human Toll-like receptors as the bacterial sensor and a synthetic promoter driving reversible and adjustable expression of lysostaphin, a bacteriolytic enzyme highly lethal to S. aureus. Immunomimetic designer cells harboring this genetic circuit exhibited fast and robust sense-and-destroy kinetics against live staphylococci. When tested in a foreign-body infection model in mice, microencapsulated cell implants prevented planktonic MRSA infection and reduced MRSA biofilm formation by 91%. Notably, this system achieved a 100% cure rate of acute MRSA infections, whereas conventional vancomycin treatment failed. These results suggest that immunomimetic designer cells could offer a therapeutic approach for early detection, prevention, and cure of pathogenic infections in the post-antibiotic era.
Assuntos
Biomimética/métodos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Infecções Estafilocócicas/prevenção & controle , Fosfatase Alcalina/sangue , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Feminino , Células HEK293 , Humanos , Receptores de Lipopolissacarídeos/genética , Lisostafina/metabolismo , Lisostafina/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/genética , Plasmídeos/metabolismo , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Receptor 1 Toll-Like/genética , Receptor 2 Toll-Like/genética , Receptor 6 Toll-Like/genética , Fator de Transcrição AP-1/metabolismoRESUMO
Retinitis pigmentosa (RP) is a Mendelian disease characterized by gradual loss of vision, due to the progressive degeneration of retinal cells. Genetically, it is highly heterogeneous, with pathogenic variants identified in more than 100 genes so far. Following a large-scale sequencing screening, we identified five individuals (four families) with recessive and non-syndromic RP, carrying as well bi-allelic DNA changes in COQ8B, a gene involved in the biosynthesis of coenzyme Q10. Specifically, we detected compound heterozygous assortments of five disease-causing variants (c.187C>T [p.Arg63Trp], c.566G>A [p.Trp189Ter], c.1156G>A [p.Asp386Asn], c.1324G>A [p.Val442Met], and c.1560G>A [p.Trp520Ter]), all segregating with disease according to a recessive pattern of inheritance. Cell-based analysis of recombinant proteins deriving from these genotypes, performed by target engagement assays, showed in all cases a significant decrease in ligand-protein interaction compared to the wild type. Our results indicate that variants in COQ8B lead to recessive non-syndromic RP, possibly by impairing the biosynthesis of coenzyme Q10, a key component of oxidative phosphorylation in the mitochondria.
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In the framework of the UK 100 000 Genomes Project, we investigated the genetic origin of a previously undescribed recessive dermatological condition, which we named LIPHAK (LTV1-associated Inflammatory Poikiloderma with Hair abnormalities and Acral Keratoses), in four affected individuals from two UK families of Pakistani and Indian origins, respectively. Our analysis showed that only one gene, LTV1, carried rare biallelic variants that were shared in all affected individuals, and specifically they bore the NM_032860.5:c.503A > G, p.(Asn168Ser) change, found homozygously in all of them. In addition, high-resolution homozygosity mapping revealed the presence of a small 652-kb stretch on chromosome 6, encompassing LTV1, that was haploidentical and common to all affected individuals. The c.503A > G variant was predicted by in silico tools to affect the correct splicing of LTV1's exon 5. Minigene-driven splicing assays in HEK293T cells and in a skin sample from one of the patients confirmed that this variant was indeed responsible for the creation of a new donor splice site, resulting in aberrant splicing and in a premature termination codon in exon 6 of this gene. LTV1 encodes one of the ribosome biogenesis factors that promote the assembly of the small (40S) ribosomal subunit. In yeast, defects in LTV1 alter the export of nascent ribosomal subunits to the cytoplasm; however, the role of this gene in human pathology is unknown to date. Our data suggest that LIPHAK could be a previously unrecognized ribosomopathy.
Assuntos
Doenças do Cabelo , Ribossomos , Dermatopatias , Humanos , Doenças do Cabelo/genética , Células HEK293 , Mutação , Ribossomos/genética , Dermatopatias/genética , SíndromeRESUMO
The two-component phosphorylation network is of critical importance for bacterial growth and physiology. Here, we address plasticity and interconnection of distinct signal transduction pathways within this network. In Caulobacter crescentus antagonistic activities of the PleC phosphatase and DivJ kinase localized at opposite cell poles control the phosphorylation state and subcellular localization of the cell fate determinator protein DivK. We show that DivK functions as an allosteric regulator that switches PleC from a phosphatase into an autokinase state and thereby mediates a cyclic di-GMP-dependent morphogenetic program. Through allosteric activation of the DivJ autokinase, DivK also stimulates its own phosphorylation and polar localization. These data suggest that DivK is the central effector of an integrated circuit that operates via spatially organized feedback loops to control asymmetry and cell fate determination in C. crescentus. Thus, single domain response regulators can facilitate crosstalk, feedback control, and long-range communication among members of the two-component network.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/citologia , Caulobacter crescentus/metabolismo , Proteínas Quinases/metabolismo , Regulação Alostérica , Proteínas de Bactérias/genética , Caulobacter crescentus/enzimologia , Caulobacter crescentus/genética , Histidina Quinase , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Fosfotransferases/metabolismo , Transdução de SinaisRESUMO
INTRODUCTION: Retinitis pigmentosa (RP) is a rare degenerative retinal disease caused by mutations in approximately seventy genes. Currently, despite the availability of large-scale DNA sequencing technologies, â¼30-40% of patients still cannot be diagnosed at the molecular level. In this study, we investigated a novel intronic deletion of PDE6B, encoding the beta subunit of phosphodiesterase 6 in association with recessive RP. METHODS: Three unrelated consanguineous families were recruited from the northwestern part of Pakistan. Whole exome sequencing was performed for the proband of each family, and the data were analyzed according to an in-house computer pipeline. Relevant DNA variants in all available members of these families were assessed through Sanger sequencing. A minigene-based splicing assay was also performed. RESULTS: The clinical phenotype for all patients was compatible with rod cone degeneration, with the onset during childhood. Whole exome sequencing revealed a homozygous 18 bp intronic deletion (NM_000283.3:c.1921-20_1921-3del) in PDE6B, which co-segregated with disease in 10 affected individuals. In vitro splicing tests showed that this deletion causes aberrant RNA splicing of the gene, leading to the in-frame deletion of 6 codons and, likely, to disease. CONCLUSION: Our findings further expand the mutational spectrum of the PDE6B gene.
Assuntos
Retinose Pigmentar , Humanos , Análise Mutacional de DNA , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/genética , Mutação , Splicing de RNA , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6/genética , Linhagem , Proteínas do Olho/genéticaRESUMO
Conjunctival melanoma (CJM) is a rare but potentially lethal and highly-recurrent cancer of the eye. Similar to cutaneous melanoma (CM), it originates from melanocytes. Unlike CM, however, CJM is relatively poorly characterized from a genomic point of view. To fill this knowledge gap and gain insight into the genomic nature of CJM, we performed whole-exome (WES) or whole-genome sequencing (WGS) of tumor-normal tissue pairs in 14 affected individuals, as well as RNA sequencing in a subset of 11 tumor tissues. Our results show that, similarly to CM, CJM is also characterized by a very high mutation load, composed of approximately 500 somatic mutations in exonic regions. This, as well as the presence of a UV light-induced mutational signature, are clear signs of the role of sunlight in CJM tumorigenesis. In addition, the genomic classification of CM proposed by TCGA seems to be well-applicable to CJM, with the presence of four typical subclasses defined on the basis of the most frequently mutated genes: BRAF, NF1, RAS, and triple wild-type. In line with these results, transcriptomic analyses revealed similarities with CM as well, namely the presence of a transcriptomic subtype enriched for immune genes and a subtype enriched for genes associated with keratins and epithelial functions. Finally, in seven tumors we detected somatic mutations in ACSS3, a possible new candidate oncogene. Transfected conjunctival melanoma cells overexpressing mutant ACSS3 showed higher proliferative activity, supporting the direct involvement of this gene in the tumorigenesis of CJM. Altogether, our results provide the first unbiased and complete genomic and transcriptomic classification of CJM.
Assuntos
Neoplasias da Túnica Conjuntiva/genética , Variações do Número de Cópias de DNA , Melanoma/genética , Mutação , Transcriptoma , Linhagem Celular Tumoral , Neoplasias da Túnica Conjuntiva/metabolismo , Feminino , Humanos , Masculino , Melanoma/metabolismo , Pessoa de Meia-Idade , Neurofibromina 1/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas ras/genéticaRESUMO
Synthetic biology advances the rational engineering of mammalian cells to achieve cell-based therapy goals. Synthetic gene networks have nearly reached the complexity of digital electronic circuits and enable single cells to perform programmable arithmetic calculations or to provide dynamic remote control of transgenes through electromagnetic waves. We designed a synthetic multilayered gaseous-fragrance-programmable analog-to-digital converter (ADC) allowing for remote control of digital gene expression with 2-bit AND-, OR- and NOR-gate logic in synchronized cell consortia. The ADC consists of multiple sampling-and-quantization modules sensing analog gaseous fragrance inputs; a gas-to-liquid transducer converting fragrance intensity into diffusible cell-to-cell signaling compounds; a digitization unit with a genetic amplifier circuit to improve the signal-to-noise ratio; and recombinase-based digital expression switches enabling 2-bit processing of logic gates. Synthetic ADCs that can remotely control cellular activities with digital precision may enable the development of novel biosensors and may provide bioelectronic interfaces synchronizing analog metabolic pathways with digital electronics.
Assuntos
Conversão Análogo-Digital , Odorantes/análise , Biologia Sintética/métodos , Células Cultivadas , Células HEK293 , Humanos , Perfumes/análiseRESUMO
Graves' disease is an autoimmune disorder that causes hyperthyroidism because of autoantibodies that bind to the thyroid-stimulating hormone receptor (TSHR) on the thyroid gland, triggering thyroid hormone release. The physiological control of thyroid hormone homeostasis by the feedback loops involving the hypothalamus-pituitary-thyroid axis is disrupted by these stimulating autoantibodies. To reset the endogenous thyrotrophic feedback control, we designed a synthetic mammalian gene circuit that maintains thyroid hormone homeostasis by monitoring thyroid hormone levels and coordinating the expression of a thyroid-stimulating hormone receptor antagonist (TSHAntag), which competitively inhibits the binding of thyroid-stimulating hormone or the human autoantibody to TSHR. This synthetic control device consists of a synthetic thyroid-sensing receptor (TSR), a yeast Gal4 protein/human thyroid receptor-α fusion, which reversibly triggers expression of the TSHAntag gene from TSR-dependent promoters. In hyperthyroid mice, this synthetic circuit sensed pathological thyroid hormone levels and restored the thyrotrophic feedback control of the hypothalamus-pituitary-thyroid axis to euthyroid hormone levels. Therapeutic plug and play gene circuits that restore physiological feedback control in metabolic disorders foster advanced gene- and cell-based therapies.
Assuntos
Modelos Animais de Doenças , Redes Reguladoras de Genes , Genes Sintéticos , Doença de Graves/genética , Hipófise/fisiopatologia , Glândula Tireoide/fisiopatologia , Animais , Células Cultivadas , Retroalimentação , Doença de Graves/fisiopatologia , Humanos , Camundongos , Hormônios Tireóideos/sangueRESUMO
The author group of above-mentioned review paper was incorrectly published in the online article.
RESUMO
Advances at the interface between the biological sciences and engineering are giving rise to emerging research fields such as synthetic biology. Harnessing the potential of synthetic biology requires timely and adequate translation into clinical practice. However, the translational research enterprise is currently facing fundamental obstacles that slow down the transition of scientific discoveries from the laboratory to the patient bedside. These obstacles including scarce financial resources and deficiency of organizational and logistic settings are widely discussed as primary impediments to translational research. In addition, a number of socio-ethical considerations inherent in translational research need to be addressed. As the translational capacity of synthetic biology is tightly linked to its social acceptance and ethical approval, ethical limitations may-together with financial and organizational problems-be co-determinants of suboptimal translation. Therefore, an early assessment of such limitations will contribute to proactively favor successful translation and prevent the promising potential of synthetic biology from remaining under-expressed. Through the discussion of two case-specific inventions in synthetic biology and their associated ethical implications, we illustrate the socio-ethical challenges ahead in the process of implementing synthetic biology into clinical practice. Since reducing the translational lag is essential for delivering the benefits of basic biomedical research to society at large and promoting global health, we advocate a moral obligation to accelerating translational research: the "translational imperative."
Assuntos
Invenções/ética , Biologia Sintética/ética , Pesquisa Translacional Biomédica/ética , Bioética , HumanosRESUMO
Second messengers control a wide range of important cellular functions in eukaryotes and prokaryotes. Here we show that cyclic di-GMP, a global bacterial second messenger, promotes cell cycle progression in Caulobacter crescentus by mediating the specific degradation of the replication initiation inhibitor CtrA. During the G1-to-S-phase transition, both CtrA and its cognate protease ClpXP dynamically localize to the old cell pole, where CtrA is rapidly degraded. Sequestration of CtrA to the cell pole depends on PopA, a newly identified cyclic di-GMP effector protein. PopA itself localizes to the cell pole and directs CtrA to this subcellular site via the direct interaction with a mediator protein, RcdA. We present evidence that c-di-GMP regulates CtrA degradation during the cell cycle by controlling the dynamic sequestration of the PopA recruitment factor to the cell pole. Furthermore, we show that cell cycle timing of CtrA degradation relies on converging pathways responsible for substrate and protease localization to the old cell pole. This is the first report that links cyclic di-GMP to protein dynamics and cell cycle control in bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Ciclo Celular/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Proteínas de Ligação a DNA/metabolismo , Ligação Proteica , Transporte Proteico/fisiologia , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Synthetic biology has significantly advanced the design of genetic devices that can reprogram cellular activities and provide novel treatment strategies for future gene- and cell-based therapies. However, many metabolic disorders are functionally linked while developing distinct diseases that are difficult to treat using a classic one-drug-one-disease intervention scheme. For example, hypertension, hyperglycemia, obesity, and dyslipidemia are interdependent pathologies that are collectively known as the metabolic syndrome, the prime epidemic of the 21st century. We have designed a unique therapeutic strategy in which the clinically licensed antihypertensive drug guanabenz (Wytensin) activates a synthetic signal cascade that stimulates the secretion of metabolically active peptides GLP-1 and leptin. Therefore, the signal transduction of a chimeric trace-amine-associated receptor 1 (cTAAR1) was functionally rewired via cAMP and cAMP-dependent phosphokinase A (PKA)-mediated activation of the cAMP-response element binding protein (CREB1) to transcription of synthetic promoters containing CREB1-specific cAMP response elements. Based on this designer signaling cascade, it was possible to use guanabenz to dose-dependently control expression of GLP-1-Fc(mIgG)-Leptin, a bifunctional therapeutic peptide hormone that combines the glucagon-like peptide 1 (GLP-1) and leptin via an IgG-Fc linker. In mice developing symptoms of the metabolic syndrome, this three-in-one treatment strategy was able to simultaneously attenuate hypertension and hyperglycemia as well as obesity and dyslipidemia. Using a clinically licensed drug to coordinate expression of therapeutic transgenes combines drug- and gene-based therapies for coordinated treatment of functionally related metabolic disorders.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Guanabenzo/farmacologia , Leptina/metabolismo , Síndrome Metabólica/tratamento farmacológico , Modelos Moleculares , Transdução de Sinais/efeitos dos fármacos , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Desenho de Fármacos , Camundongos , Receptores Acoplados a Proteínas G/metabolismo , Transgenes/genéticaRESUMO
Cyclic guanosine monophosphate (cGMP) is a universal second messenger that is synthesized from guanosine triphosphate (GTP) by guanylyl cyclases (GCs) and hydrolyzed into guanosine monophosphate (GMP) by phosphodiesterases (PDEs). Small-molecule drugs that induce high cGMP levels in specialized tissues by boosting GC activity or inhibiting PDE activity have become the predominant treatment strategy for a wide range of medical conditions, including congestive heart failure, pulmonary hypertension, atherosclerosis-based claudication and erectile dysfunction. By fusing the cGMP receptor protein (CRP) of Rhodospirillum centenum to the Herpes simplex-derived transactivation domain VP16, we created a novel synthetic mammalian cGMP-sensing transcription factor (GTA) that activates synthetic promoters (PGTA) containing newly identified GTA-specific operator sites in a concentration-dependent manner. In cell lines expressing endogenous natriuretic peptide receptor A (NPR-A) (HeLa), GTA/PGTA-driven transgene expression was induced by B-type natriuretic peptide (BNP; Nesiritide(®)) in a concentration-dependent manner, which activated NPR-A׳s intracellular GC domain and triggered a corresponding cGMP surge. Ectopic expression of NPR-A in NPR-A-negative cell lines (HEK-293T) produced high cGMP levels and mediated maximum GTA/PGTA-driven transgene expression, which was suppressed by co-expression of PDEs (PDE-3A, PDE-5A and PDE-9A) and was re-triggered by the corresponding PDE inhibitor drugs (Pletal(®), Perfan(®), Primacor(®) (PDE-3A), Viagra(®), Levitra(®), Cialis(®) (PDE-5A) and BAY73-6691 (PDE-9A)). Mice implanted with microencapsulated designer cells co-expressing the GTA/PGTA device with NPR-A and PDE-5A showed control of blood SEAP levels through administration of sildenafil (Viagra(®)). Designer cells engineered for PDE inhibitor-modulated transgene expression may provide a cell-based PDE-targeting drug discovery platform and enable drug-adjusted gene- and cell-based therapies.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte , GMP Cíclico/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Rhodospirillum centenum/genética , Citrato de Sildenafila/farmacologia , Animais , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , CamundongosRESUMO
Synthetic biology has significantly advanced the design of synthetic control devices, gene circuits and networks that can reprogram mammalian cells in a trigger-inducible manner. Prokaryotic helix-turn-helix motifs have become the standard resource to design synthetic mammalian transcription factors that tune chimeric promoters in a small molecule-responsive manner. We have identified a family of Actinomycetes transcriptional repressor proteins showing a tandem TetR-family signature and have used a synthetic biology-inspired approach to reveal the potential control dynamics of these bi-partite regulators. Daisy-chain assembly of well-characterized prokaryotic repressor proteins such as TetR, ScbR, TtgR or VanR and fusion to either the Herpes simplex transactivation domain VP16 or the Krueppel-associated box domain (KRAB) of the human kox-1 gene resulted in synthetic bi- and even tri-partite mammalian transcription factors that could reversibly program their individual chimeric or hybrid promoters for trigger-adjustable transgene expression using tetracycline (TET), γ-butyrolactones, phloretin and vanillic acid. Detailed characterization of the bi-partite ScbR-TetR-VP16 (ST-TA) transcription factor revealed independent control of TET- and γ-butyrolactone-responsive promoters at high and double-pole double-throw (DPDT) relay switch qualities at low intracellular concentrations. Similar to electromagnetically operated mechanical DPDT relay switches that control two electric circuits by a fully isolated low-power signal, TET programs ST-TA to progressively switch from TetR-specific promoter-driven expression of transgene one to ScbR-specific promoter-driven transcription of transgene two while ST-TA flips back to exclusive transgene 1 expression in the absence of the trigger antibiotic. We suggest that natural repressors and activators with tandem TetR-family signatures may also provide independent as well as DPDT-mediated control of two sets of transgenes in bacteria, and that their synthetic transcription-factor analogs may enable the design of compact therapeutic gene circuits for gene and cell-based therapies.
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Regulação da Expressão Gênica , Proteínas Repressoras/química , Transativadores/química , Actinobacteria/genética , Motivos de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Células HEK293 , Proteína Vmw65 do Vírus do Herpes Simples/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Regiões Promotoras Genéticas , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Repressoras/classificação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Biologia Sintética/métodos , Transativadores/genética , Transativadores/metabolismo , TransgenesRESUMO
Precise spatiotemporal control of physiological processes by optogenetic devices inspired by synthetic biology may provide novel treatment opportunities for gene- and cell-based therapies. An erectile optogenetic stimulator (EROS), a synthetic designer guanylate cyclase producing a blue-light-inducible surge of the second messenger cyclic guanosine monophosphate (cGMP) in mammalian cells, enabled blue-light-dependent penile erection associated with occasional ejaculation after illumination of EROS-transfected corpus cavernosum in male rats. Photostimulated short-circuiting of complex psychological, neural, vascular, and endocrine factors to stimulate penile erection in the absence of sexual arousal may foster novel advances in the treatment of erectile dysfunction.
Assuntos
Luz , Optogenética/métodos , Ereção Peniana/efeitos dos fármacos , Animais , GMP Cíclico/biossíntese , GMP Cíclico/metabolismo , Disfunção Erétil/tratamento farmacológico , Guanilato Ciclase/metabolismo , Masculino , RatosRESUMO
WhiB-like (Wbl) proteins are well known for their diverse roles in actinobacterial morphogenesis, cell division, virulence, primary and secondary metabolism, and intrinsic antibiotic resistance. Gene disruption experiments showed that three different Actinobacteria (Mycobacterium smegmatis, Streptomyces lividans, and Rhodococcus jostii) each exhibited a different whiB7-dependent resistance profile. Heterologous expression of whiB7 genes showed these resistance profiles reflected the host's repertoire of endogenous whiB7-dependent genes. Transcriptional activation of two resistance genes in the whiB7 regulon, tap (a multidrug transporter) and erm(37) (a ribosomal methyltransferase), required interaction of WhiB7 with their promoters. Furthermore, heterologous expression of tap genes isolated from Mycobacterium species demonstrated that divergencies in drug specificity of homologous structural proteins contribute to the variation of WhiB7-dependent drug resistance. WhiB7 has a specific tryptophan/glycine-rich region and four conserved cysteine residues; it also has a peptide sequence (AT-hook) at its C terminus that binds AT-rich DNA sequence motifs upstream of the promoters it activates. Targeted mutagenesis showed that these motifs were required to provide antibiotic resistance in vivo. Anaerobically purified WhiB7 from S. lividans was dimeric and contained 2.1 ± 0.3 and 2.2 ± 0.3 mol of iron and sulfur, respectively, per protomer (consistent with the presence of a 2Fe-2S cluster). However, the properties of the dimer's absorption spectrum were most consistent with the presence of an oxygen-labile 4Fe-4S cluster, suggesting 50% occupancy. These data provide the first insights into WhiB7 iron-sulfur clusters as they exist in vivo, a major unresolved issue in studies of Wbl proteins.
Assuntos
Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Proteínas Ferro-Enxofre/genética , Mycobacterium smegmatis/metabolismo , Rhodococcus/metabolismo , Streptomyces lividans/metabolismo , Fatores de Transcrição/genética , Actinobacteria , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Proteínas Ferro-Enxofre/metabolismo , Metiltransferases/metabolismo , Mutagênese , Mycobacterium smegmatis/genética , Rhodococcus/genética , Especificidade da Espécie , Streptomyces lividans/genética , Fatores de Transcrição/isolamento & purificação , Fatores de Transcrição/metabolismo , Ativação Transcricional/genéticaRESUMO
BACKGROUND: Current first-line disease-modifying therapies (DMT) for multiple sclerosis (MS) patients are injectable or oral treatments. The Optogenerapy consortium is developing a novel bioelectronic cell-based implant for controlled release of beta-interferon (IFNß1a) protein into the body. The current study estimated the potential cost effectiveness of the Optogenerapy implant (hereafter: Optoferon) compared with injectable IFNß1a (Avonex). METHODS: A Markov model simulating the costs and effects of Optoferon compared with injectable 30 mg IFNß1a over a 9-year time horizon from a Dutch societal perspective. Costs were reported in 2019 Euros and discounted at a 4% annual rate; health effects were discounted at a 1.5% annual rate. The cohort consisted of 35-year-old, relapsing-remitting MS patients with mild disability. The device is implanted in a daycare setting, and is replaced every 3 years. In the base-case analysis, we assumed equal input parameters for Optoferon and Avonex regarding disability progression, health effects, adverse event probabilities, and acquisition costs. We assumed reduced annual relapse rates and withdrawal rates for Optoferon compared with Avonex. Sensitivity, scenario, value of information, and headroom analysis were performed. RESULTS: Optoferon was the dominant strategy with cost reductions (- 26,966) and health gains (0.45 quality-adjusted life-years gained). A main driver of cost differences are the acquisition costs of Optoferon being 2.5 times less than the costs of Avonex. The incremental cost-effectiveness ratio was most sensitive to variations in the annual acquisition costs of Avonex, the annual withdrawal rate of Avonex and Optoferon, and the disability progression of Avonex. CONCLUSION: Innovative technology such as the Optoferon implant may be a cost-effective therapy for patients with MS. The novel implantable mode of therapeutic protein administration has the potential to become a new mode of treatment administration for MS patients and in other disease areas. However, trials are needed to establish safety and effectiveness.
Assuntos
Esclerose Múltipla Recidivante-Remitente , Esclerose Múltipla , Adulto , Análise Custo-Benefício , Humanos , Interferon beta-1a , Interferon beta , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológicoRESUMO
PURPOSE: The purpose of this study was to investigate the clinical and genetic features of a man and his daughter with posterior polymorphous corneal dystrophy (PPCD), referred to our clinic for Descemet membrane endothelial keratoplasty. No other known relatives were affected. METHODS: Ophthalmic examination and histology, including electron microscopy, were performed. Genetic testing was conducted by means of whole exome sequencing, and variant analysis was achieved by using an internal in silico pipeline. Molecular tests included a dual-luciferase assay. RESULTS: Slowly progressive blurred vision was reported from childhood by the daughter. The father's symptoms started at age 55. Best-corrected visual acuity was reduced in both patients (0.2-0.4). Slit-lamp examination in both patients revealed bilateral corneal clouding with gray endothelial lesions; other family members had no ophthalmological signs. Descemet membrane endothelial keratoplasty was performed uneventfully in both patients. Histology showed thickened Descemet membrane and abnormal endothelium resembling epithelial-like cells. Both patients carried the OVOL2 5' untranslated region NM_021220.4.c.-61G>A variant in the heterozygous state. This change was associated with increased promoter activity and was not present in the unaffected members of the family. CONCLUSIONS: The 5' untranslated region mutation c.-61G>A in OVOL2 has been previously found in 1 individual with PPCD1 and reported as a variant of unknown significance because of insufficient evidence supporting its pathogenicity. Identification of the second family with 2 individuals affected by PPCD1 carrying this change, together with functional data, provides further proofs that it is disease-causing.
Assuntos
Regiões 5' não Traduzidas/genética , Distrofias Hereditárias da Córnea/genética , Endotélio Corneano/ultraestrutura , Mutação , Fatores de Transcrição/genética , Adulto , Idoso , Distrofias Hereditárias da Córnea/diagnóstico , Distrofias Hereditárias da Córnea/metabolismo , Análise Mutacional de DNA , Endotélio Corneano/patologia , Feminino , Humanos , Masculino , Microscopia Eletrônica , Linhagem , Regiões Promotoras Genéticas , Microscopia com Lâmpada de Fenda , Dedos de ZincoRESUMO
Wireless powered optogenetic cell-based implant provides a strategy to deliver subcutaneously therapeutic proteins. Immortalize Human Mesenchymal Stem Cells (hMSC-TERT) expressing the bacteriophytochrome diguanylate cyclase (DGCL) were validated for optogenetic controlled interferon-ß delivery (Optoferon cells) in a bioelectronic cell-based implant. Optoferon cells transcriptomic profiling was used to elaborate an in-silico model of the recombinant interferon-ß production. Wireless optoelectronic device integration was developed using additive manufacturing and injection molding. Implant cell-based optoelectronic interface manufacturing was established to integrate industrial flexible compact low-resistance screen-printed Near Field Communication (NFC) coil antenna. Optogenetic cell-based implant biocompatibility, and device performances were evaluated in the Experimental Autoimmune Encephalomyelitis (EAE) mouse model of multiple sclerosis.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Camundongos , Animais , Humanos , Esclerose Múltipla/terapia , Encefalomielite Autoimune Experimental/terapia , Interferon beta/genética , Interferon beta/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Camundongos Endogâmicos C57BLRESUMO
Improving a drug delivery system is critical to treat central nervous system disorders. Here we studied an innovative approach based on implantation of a wireless-powered cell-based device in mice. This device, coupling biologic material and electronics, is the first of its kind. The advantage of this technology is its ability to control the secretion of a therapeutic molecule and to switch the classical permanent delivery to activation on demand. In diseases with relapsing-remitting phases such as multiple sclerosis, such activation could be selectively achieved in relapsing phases. However, the safety (tolerance to biomaterials and surgical procedure) of such a clinical device needs to be verified. Therefore, the development of tools to assess the biocompatibility of the system in animal models is an essential step. We present the development of this new therapeutic approach, the challenges we encountered during the different steps of its development (such as cell loading in the chamber, surgery protocol for subcutaneous implantation of the device) and the tools we used to evaluate cell viability and biocompatibility of the device.