RESUMO
Speciation leads to adaptive changes in organ cellular physiology and creates challenges for studying rare cell-type functions that diverge between humans and mice. Rare cystic fibrosis transmembrane conductance regulator (CFTR)-rich pulmonary ionocytes exist throughout the cartilaginous airways of humans1,2, but limited presence and divergent biology in the proximal trachea of mice has prevented the use of traditional transgenic models to elucidate ionocyte functions in the airway. Here we describe the creation and use of conditional genetic ferret models to dissect pulmonary ionocyte biology and function by enabling ionocyte lineage tracing (FOXI1-CreERT2::ROSA-TG), ionocyte ablation (FOXI1-KO) and ionocyte-specific deletion of CFTR (FOXI1-CreERT2::CFTRL/L). By comparing these models with cystic fibrosis ferrets3,4, we demonstrate that ionocytes control airway surface liquid absorption, secretion, pH and mucus viscosity-leading to reduced airway surface liquid volume and impaired mucociliary clearance in cystic fibrosis, FOXI1-KO and FOXI1-CreERT2::CFTRL/L ferrets. These processes are regulated by CFTR-dependent ionocyte transport of Cl- and HCO3-. Single-cell transcriptomics and in vivo lineage tracing revealed three subtypes of pulmonary ionocytes and a FOXI1-lineage common rare cell progenitor for ionocytes, tuft cells and neuroendocrine cells during airway development. Thus, rare pulmonary ionocytes perform critical CFTR-dependent functions in the proximal airway that are hallmark features of cystic fibrosis airway disease. These studies provide a road map for using conditional genetics in the first non-rodent mammal to address gene function, cell biology and disease processes that have greater evolutionary conservation between humans and ferrets.
Assuntos
Fibrose Cística , Modelos Animais de Doenças , Furões , Pulmão , Transgenes , Animais , Humanos , Animais Geneticamente Modificados , Linhagem da Célula , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Furões/genética , Furões/fisiologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Pulmão/patologia , Traqueia/citologia , Transgenes/genéticaRESUMO
Using dissolved inorganic carbon (DIC) as a major carbon source, as autotrophs do, is complicated by the bedeviling nature of this substance. Autotrophs using the Calvin-Benson-Bassham cycle (CBB) are known to make use of a toolkit comprised of DIC transporters and carbonic anhydrase enzymes (CA) to facilitate DIC fixation. This minireview provides a brief overview of the current understanding of how toolkit function facilitates DIC fixation in Cyanobacteria and some Proteobacteria using the CBB and continues with a survey of the DIC toolkit gene presence in organisms using different versions of the CBB and other autotrophic pathways (reductive citric acid cycle, Wood-Ljungdahl pathway, hydroxypropionate bicycle, hydroxypropionate-hydroxybutyrate cycle, and dicarboxylate-hydroxybutyrate cycle). The potential function of toolkit gene products in these organisms is discussed in terms of CO2 and HCO3- supply from the environment and demand by the autotrophic pathway. The presence of DIC toolkit genes in autotrophic organisms beyond those using the CBB suggests the relevance of DIC metabolism to these organisms and provides a basis for better engineering of these organisms for industrial and agricultural purposes.
Assuntos
Archaea , Bactérias , Archaea/genética , Archaea/metabolismo , Bactérias/genética , Bactérias/metabolismo , Processos Autotróficos/genética , Carbono/metabolismo , Hidroxibutiratos/metabolismo , Dióxido de Carbono/metabolismo , Ciclo do Carbono/genéticaRESUMO
Fluorine magnetic resonance imaging (19 F MRI) has emerged as an attractive alternative to conventional 1 H MRI due to enhanced specificity deriving from negligible background signal in this modality. We report a dual nanoparticle conjugate (DNC) platform as an aptamer-based sensor for use in 19 F MRI. DNC consists of core-shell nanoparticles with a liquid perfluorocarbon core and a mesoporous silica shell (19 F-MSNs), which give a robust 19 F MR signal, and superparamagnetic iron oxide nanoparticles (SPIONs) as magnetic quenchers. Due to the strong magnetic quenching effects of SPIONs, this platform is uniquely sensitive and functions with a low concentration of SPIONs (4 equivalents) relative to 19 F-MSNs. The probe functions as a "turn-on" sensor using target-induced dissociation of DNA aptamers. The thrombin binding aptamer was incorporated as a proof-of-concept (DNCThr ), and we demonstrate a significant increase in 19 F MR signal intensity when DNCThr is incubated with human α-thrombin. This proof-of-concept probe is highly versatile and can be adapted to sense ATP and kanamycin as well. Importantly, DNCThr generates a robust 19 F MRI "hot-spot" signal in response to thrombin in live mice, establishing this platform as a practical, versatile, and biologically relevant molecular imaging probe.
Assuntos
Nanopartículas , Trombina , Humanos , Animais , Camundongos , Imageamento por Ressonância Magnética/métodos , Nanopartículas/química , Nanopartículas Magnéticas de Óxido de Ferro , Dióxido de Silício/químicaRESUMO
Two sulphur-oxidizing, chemolithoautotrophic aerobes were isolated from the chemocline of an anchialine sinkhole located within the Weeki Wachee River of Florida. Gram-stain-negative cells of both strains were motile, chemotactic rods. Phylogenetic analysis of the 16S rRNA gene and predicted amino acid sequences of ribosomal proteins, average nucleotide identities, and alignment fractions suggest the strains HH1T and HH3T represent novel species belonging to the genus Thiomicrorhabdus. The genome G+C fraction of HH1T is 47.8 mol% with a genome length of 2.61 Mb, whereas HH3T has a G+C fraction of 52.4 mol% and 2.49 Mb genome length. Major fatty acids of the two strains included C16â:â1, C18â:â1 and C16â:â0, with the addition of C10:0 3-OH in HH1T and C12â:â0 in HH3T. Chemolithoautotrophic growth of both strains was supported by elemental sulphur, sulphide, tetrathionate, and thiosulphate, and HH1T was also able to use molecular hydrogen. Neither strain was capable of heterotrophic growth or use of nitrate as a terminal electron acceptor. Strain HH1T grew from pH 6.5 to 8.5, with an optimum of pH 7.4, whereas strain HH3T grew from pH 6 to 8 with an optimum of pH 7.5. Growth was observed between 15-35 °C with optima of 32.8 °C for HH1T and 32 °C for HH3T. HH1T grew in media with [NaCl] 80-689 mM, with an optimum of 400 mM, while HH3T grew at 80-517 mM, with an optimum of 80 mM. The name Thiomicrorhabdus heinhorstiae sp. nov. is proposed, and the type strain is HH1T (=DSM 111584T=ATCC TSD-240T). The name Thiomicrorhabdus cannonii sp. nov is proposed, and the type strain is HH3T (=DSM 111593T=ATCC TSD-241T).
Assuntos
DNA Bacteriano , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Florida , Hospitais , Oxirredução , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Enxofre/metabolismoRESUMO
There are limited prospective data on lenalidomide, subcutaneous bortezomib, and dexamethasone (RsqVd) in transplant-eligible/transplant-ineligible patients with newly diagnosed multiple myeloma. Reliable biomarkers for efficacy and toxicity are required to better tailor therapy. Two parallel studies were conducted by Cancer Trials Ireland (CTI; NCT02219178) and the Dana-Farber Cancer Institute (DFCI; NCT02441686). Patients received four 21-day cycles of RsqVd and could then receive either another 4 cycles of RsqVd or undergo autologous stem cell transplant. Postinduction/posttransplant, patients received lenalidomide maintenance, with bortezomib included for high-risk patients. The primary endpoint was overall response rate (ORR) after 4 cycles of RsqVd. Eighty-eight patients were enrolled and 84 treated across the two studies; median age was 64.7 (CTI study) and 60.0 years (DFCI study), and 59% and 57% had stage II-III disease. Pooled ORR after 4 cycles in evaluable patients was 93.5%, including 48.1% complete or very good partial responses (CTI study: 91.9%, 59.5%; DFCI study: 95.0%, 37.5%), and in the all-treated population was 85.7% (44.0%). Patients received a median of 4 (CTI study) and 8 (DFCI study) RsqVd cycles; 60% and 31% of patients (CTI study) and 33% and 51% of patients (DFCI study) underwent transplant or received further RsqVd induction, respectively. The most common toxicity was peripheral neuropathy (pooled: 68%, 7% grade 3-4; CTI study: 57%, 7%; DFCI study: 79%, 7%). Proteomics analyses indicated elevated kallikrein-6 in good versus poor responders, decreased midkine in good responders, and elevated macrophage inflammatory protein 1-alpha in patients who stopped treatment from neurotoxicity, suggesting predictive biomarkers warranting further investigation.
Assuntos
Mieloma Múltiplo , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Bortezomib/efeitos adversos , Dexametasona/efeitos adversos , Humanos , Quimioterapia de Indução , Lenalidomida/efeitos adversos , Pessoa de Meia-Idade , Mieloma Múltiplo/terapia , Estudos ProspectivosRESUMO
The purpose of this article is to highlight and provide solutions for the complexities of the communication disorder of cluttering. The article includes a 12-part case study of a school-aged child who is referred for an evaluation due to decreased speech intelligibility. The case is woven throughout the article to illustrate all aspects of client management: initial referral, evaluation and differential diagnosis, treatment, and discharge. The case reflects the challenges of increasing client awareness and the importance of advocating for client's needs throughout the process. This article provides background on myths about cluttering and current research findings to debunk these myths. Additionally, methods for evaluation, differential diagnosis, and treatment of cluttering are presented. An indirect approach to increasing client awareness and family and client education is included. The overall focus is to help the clinician better understand how to evaluate, treat, discharge, and advocate for clients with cluttering in ways that meet clients where they are.
Assuntos
Distúrbios da Fala , Inteligibilidade da Fala , Criança , Diagnóstico Diferencial , Humanos , Distúrbios da Fala/diagnósticoRESUMO
In nature, concentrations of dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) can be low, and autotrophic organisms adapt with a variety of mechanisms to elevate intracellular DIC concentrations to enhance CO2 fixation. Such mechanisms have been well studied in Cyanobacteria, but much remains to be learned about their activity in other phyla. Novel multisubunit membrane-spanning complexes capable of elevating intracellular DIC were recently described in three species of bacteria. Homologs of these complexes are distributed among 17 phyla in Bacteria and Archaea and are predicted to consist of one, two, or three subunits. To determine whether DIC accumulation is a shared feature of these diverse complexes, seven of them, representative of organisms from four phyla, from a variety of habitats, and with three different subunit configurations, were chosen for study. A high-CO2-requiring, carbonic anhydrase-deficient (ΔyadF ΔcynT) strain of Escherichia coli Lemo21(DE3), which could be rescued via elevated intracellular DIC concentrations, was created for heterologous expression and characterization of the complexes. Expression of all seven complexes rescued the ability of E. coli Lemo21(DE3) ΔyadF ΔcynT to grow under low-CO2 conditions, and six of the seven generated measurably elevated intracellular DIC concentrations when their expression was induced. For complexes consisting of two or three subunits, all subunits were necessary for DIC accumulation. Isotopic disequilibrium experiments clarified that CO2 was the substrate for these complexes. In addition, the presence of an ionophore prevented the accumulation of intracellular DIC, suggesting that these complexes may couple proton potential to DIC accumulation. IMPORTANCE To facilitate the synthesis of biomass from CO2, autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multisubunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, raising the question of whether these complexes share this capability across the 17 phyla of Bacteria and Archaea where they are found. This study shows that DIC accumulation is a trait shared by complexes with various subunit structures, from organisms with diverse physiologies and taxonomies, suggesting that this trait is universal among them. Successful expression in E. coli suggests the possibility of their expression in engineered organisms synthesizing compounds of industrial importance from CO2.
Assuntos
Processos Autotróficos/fisiologia , Bactérias/classificação , Bactérias/metabolismo , Carbono/metabolismo , Bactérias/genética , Proteínas de Bactérias , Dióxido de Carbono/metabolismo , Cromatografia Líquida , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Concentração de Íons de Hidrogênio , Espectrometria de Massas em TandemRESUMO
Genome and proteome data predict the presence of both the reductive citric acid cycle (rCAC; also called the reductive tricarboxylic acid cycle) and the Calvin-Benson-Bassham cycle (CBB) in "Candidatus Endoriftia persephonae," the autotrophic sulfur-oxidizing bacterial endosymbiont from the giant hydrothermal vent tubeworm Riftia pachyptila. We tested whether these cycles were differentially induced by sulfide supply, since the synthesis of biosynthetic intermediates by the rCAC is less energetically expensive than that by the CBB. R. pachyptila was incubated under in situ conditions in high-pressure aquaria under low (28 to 40 µmol · h-1) or high (180 to 276 µmol · h-1) rates of sulfide supply. Symbiont-bearing trophosome samples excised from R. pachyptila maintained under the two conditions were capable of similar rates of CO2 fixation. Activities of the rCAC enzyme ATP-dependent citrate lyase (ACL) and the CBB enzyme 1,3-bisphosphate carboxylase/oxygenase (RubisCO) did not differ between the two conditions, although transcript abundances for ATP-dependent citrate lyase were 4- to 5-fold higher under low-sulfide conditions. δ13C values of internal dissolved inorganic carbon (DIC) pools were varied and did not correlate with sulfide supply rate. In samples taken from freshly collected R. pachyptila, δ13C values of lipids fell between those collected for organisms using either the rCAC or the CBB exclusively. These observations are consistent with cooccurring activities of the rCAC and the CBB in this symbiosis. IMPORTANCE Previous to this study, the activities of the rCAC and CBB in R. pachyptila had largely been inferred from "omics" studies of R. pachyptila without direct assessment of in situ conditions prior to collection. In this study, R. pachyptila was maintained and monitored in high-pressure aquaria prior to measuring its CO2 fixation parameters. Results suggest that ranges in sulfide concentrations similar to those experienced in situ do not exert a strong influence on the relative activities of the rCAC and the CBB. This observation highlights the importance of further study of this symbiosis and other organisms with multiple CO2-fixing pathways, which recent genomics and biochemical studies suggest are likely to be more prevalent than anticipated.
Assuntos
Gammaproteobacteria/fisiologia , Poliquetos/microbiologia , Simbiose , Animais , Processos Autotróficos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico , Gammaproteobacteria/classificação , Gammaproteobacteria/genética , Gammaproteobacteria/isolamento & purificação , Fontes Hidrotermais/microbiologia , Fontes Hidrotermais/parasitologia , Fotossíntese , Poliquetos/fisiologia , Sulfetos/metabolismo , Enxofre/metabolismoRESUMO
RubisCO, the CO2 fixing enzyme of the Calvin-Benson-Bassham (CBB) cycle, is responsible for the majority of carbon fixation on Earth. RubisCO fixes 12 CO2 faster than 13 CO2 resulting in 13 C-depleted biomass, enabling the use of δ13 C values to trace CBB activity in contemporary and ancient environments. Enzymatic fractionation is expressed as an ε value, and is routinely used in modelling, for example, the global carbon cycle and climate change, and for interpreting trophic interactions. Although values for spinach RubisCO (ε = ~29) have routinely been used in such efforts, there are five different forms of RubisCO utilized by diverse photolithoautotrophs and chemolithoautotrophs and ε values, now known for four forms (IA, B, D and II), vary substantially with ε = 11 to 27. Given the importance of ε values in δ13 C evaluation, we measured enzymatic fractionation of the fifth form, form IC RubisCO, which is found widely in aquatic and terrestrial environments. Values were determined for two model organisms, the 'Proteobacteria' Ralstonia eutropha (ε = 19.0) and Rhodobacter sphaeroides (ε = 22.4). It is apparent from these measurements that all RubisCO forms measured to date discriminate less than commonly assumed based on spinach, and that enzyme ε values must be considered when interpreting and modelling variability of δ13 C values in nature.
Assuntos
Proteínas de Bactérias/química , Cupriavidus necator/enzimologia , Rhodobacter sphaeroides/enzimologia , Ribulose-Bifosfato Carboxilase/química , Proteínas de Bactérias/metabolismo , Ciclo do Carbono , Isótopos de Carbono/química , Cupriavidus necator/química , Cupriavidus necator/isolamento & purificação , Ecossistema , Fotossíntese , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/isolamento & purificação , Ribulose-Bifosfato Carboxilase/metabolismo , Microbiologia do Solo , Microbiologia da ÁguaRESUMO
Use of hydrogen gas (H2) as an electron donor is common among free-living chemolithotrophic microorganisms. Given the presence of this dissolved gas at deep-sea hydrothermal vents, it has been suggested that it may also be a major electron donor for the free-living and symbiotic chemolithoautotrophic bacteria that are the primary producers at these sites. Giant Riftia pachyptila siboglinid tubeworms and their symbiotic bacteria ("Candidatus Endoriftia persephone") dominate many vents in the Eastern Pacific, and their use of sulfide as a major electron donor has been documented. Genes encoding hydrogenase are present in the "Ca Endoriftia persephone" genome, and proteome data suggest that these genes are expressed. In this study, high-pressure respirometry of intact R. pachyptila and incubations of trophosome homogenate were used to determine whether this symbiotic association could also use H2 as a major electron donor. Measured rates of H2 uptake by intact R. pachyptila in high-pressure respirometers were similar to rates measured in the absence of tubeworms. Oxygen uptake rates in the presence of H2 were always markedly lower than those measured in the presence of sulfide, as was the incorporation of 13C-labeled dissolved inorganic carbon. Carbon fixation by trophosome homogenate was not stimulated by H2, nor was hydrogenase activity detectable in these samples. Though genes encoding [NiFe] group 1e and [NiFe] group 3b hydrogenases are present in the genome and transcribed, it does not appear that H2 is a major electron donor for this system, and it may instead play a role in intracellular redox homeostasis.IMPORTANCE Despite the presence of hydrogenase genes, transcripts, and proteins in the "Ca Endoriftia persephone" genome, transcriptome, and proteome, it does not appear that R. pachyptila can use H2 as a major electron donor. For many uncultivable microorganisms, omic analyses are the basis for inferences about their activities in situ However, as is apparent from the study reported here, there are dangers in extrapolating from omics data to function, and it is essential, whenever possible, to verify functions predicted from omics data with physiological and biochemical measurements.
Assuntos
Crescimento Quimioautotrófico/fisiologia , Gammaproteobacteria/metabolismo , Hidrogênio/metabolismo , Fontes Hidrotermais , Poliquetos/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Genes Bacterianos , Genoma Bacteriano , Interações entre Hospedeiro e Microrganismos/fisiologia , Hidrogenase/genética , Hidrogenase/metabolismo , Fontes Hidrotermais/química , Fontes Hidrotermais/microbiologia , Poliquetos/metabolismo , Substâncias Redutoras/metabolismo , SimbioseRESUMO
Members of the genera Hydrogenovibrio, Thiomicrospira, and Thiomicrorhabdus fix carbon at hydrothermal vents, coastal sediments, hypersaline lakes, and other sulfidic habitats. The genome sequences of these ubiquitous and prolific chemolithoautotrophs suggest a surprising diversity of mechanisms for the uptake and fixation of dissolved inorganic carbon (DIC); these mechanisms are verified here. Carboxysomes are apparent in the transmission electron micrographs of most of these organisms but are lacking in Thiomicrorhabdus sp. strain Milos-T2 and Thiomicrorhabdus arctica, and the inability of Thiomicrorhabdus sp. strain Milos-T2 to grow under low-DIC conditions is consistent with the absence of carboxysome loci in its genome. For the remaining organisms, genes encoding potential DIC transporters from four evolutionarily distinct families (Tcr_0853 and Tcr_0854, Chr, SbtA, and SulP) are located downstream of carboxysome loci. Transporter genes collocated with carboxysome loci, as well as some homologs located elsewhere on the chromosomes, had elevated transcript levels under low-DIC conditions, as assayed by reverse transcription-quantitative PCR (qRT-PCR). DIC uptake was measureable via silicone oil centrifugation when a representative of each of the four types of transporter was expressed in Escherichia coli The expression of these genes in the carbonic anhydrase-deficient E. coli strain EDCM636 enabled it to grow under low-DIC conditions, a result consistent with DIC transport by these proteins. The results from this study expand the range of DIC transporters within the SbtA and SulP transporter families, verify DIC uptake by transporters encoded by Tcr_0853 and Tcr_0854 and their homologs, and introduce DIC as a potential substrate for transporters from the Chr family.IMPORTANCE Autotrophic organisms take up and fix DIC, introducing carbon into the biological portion of the global carbon cycle. The mechanisms for DIC uptake and fixation by autotrophic Bacteria and Archaea are likely to be diverse but have been well characterized only for "Cyanobacteria" Based on genome sequences, members of the genera Hydrogenovibrio, Thiomicrospira, and Thiomicrorhabdus have a variety of mechanisms for DIC uptake and fixation. We verified that most of these organisms are capable of growing under low-DIC conditions, when they upregulate carboxysome loci and transporter genes collocated with these loci on their chromosomes. When these genes, which fall into four evolutionarily independent families of transporters, are expressed in E. coli, DIC transport is detected. This expansion in known DIC transporters across four families, from organisms from a variety of environments, provides insight into the ecophysiology of autotrophs, as well as a toolkit for engineering microorganisms for carbon-neutral biochemistries of industrial importance.
Assuntos
Dióxido de Carbono/metabolismo , Piscirickettsiaceae/isolamento & purificação , Piscirickettsiaceae/metabolismo , Sulfetos/metabolismo , Processos Autotróficos , Ciclo do Carbono , Dióxido de Carbono/análise , Ecossistema , Fontes Hidrotermais/química , Fontes Hidrotermais/microbiologia , Filogenia , Piscirickettsiaceae/classificação , Piscirickettsiaceae/genéticaRESUMO
Chemolithoautotrophic bacteria from the genera Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira are common, sometimes dominant, isolates from sulfidic habitats including hydrothermal vents, soda and salt lakes and marine sediments. Their genome sequences confirm their membership in a deeply branching clade of the Gammaproteobacteria. Several adaptations to heterogeneous habitats are apparent. Their genomes include large numbers of genes for sensing and responding to their environment (EAL- and GGDEF-domain proteins and methyl-accepting chemotaxis proteins) despite their small sizes (2.1-3.1 Mbp). An array of sulfur-oxidizing complexes are encoded, likely to facilitate these organisms' use of multiple forms of reduced sulfur as electron donors. Hydrogenase genes are present in some taxa, including group 1d and 2b hydrogenases in Hydrogenovibrio marinus and H. thermophilus MA2-6, acquired via horizontal gene transfer. In addition to high-affinity cbb3 cytochrome c oxidase, some also encode cytochrome bd-type quinol oxidase or ba3 -type cytochrome c oxidase, which could facilitate growth under different oxygen tensions, or maintain redox balance. Carboxysome operons are present in most, with genes downstream encoding transporters from four evolutionarily distinct families, which may act with the carboxysomes to form CO2 concentrating mechanisms. These adaptations to habitat variability likely contribute to the cosmopolitan distribution of these organisms.
Assuntos
Crescimento Quimioautotrófico , Genoma Bacteriano , Piscirickettsiaceae/genética , Ecossistema , Hidrogenase/genética , Filogenia , Piscirickettsiaceae/classificação , Piscirickettsiaceae/enzimologia , Piscirickettsiaceae/metabolismo , Enxofre/metabolismoRESUMO
Thiothrix is the type genus of the Thiotrichaceae in the Thiotrichales of the Gammaproteobacteria, comprising nine species of sulfur-oxidising filamentous bacteria, which are variously autotrophic, heterotrophic or have mixed metabolic modes. Within the genus, four species show 16S rRNA gene identities lower the Yarza threshold for the rank of genus (94.5â%) - Thiothrix disciformis, Thiothrix flexilis, Thiothrix defluvii and Thiothrix eikelboomii - as they show no affiliation to extant genera, a polyphasic study was undertaken including biochemical, physiological and genomic properties and phylogeny based on the 16S rRNA gene (rrs), recombination protein A (RecA), polynucleotide nucleotidyltransferase (Pnp), translation initiation factor IF-2 (InfB), glyceraldehyde-3-phosphate dehydrogenase (GapA), glutaminyl-tRNA synthetase (GlnS), elongation factor EF-G (FusA) and concatamers of 53 ribosomal proteins encoded by rps, rpl and rpm operons, all of which support the reclassification of these species. We thus propose Thiolinea gen. nov. and Thiofilum gen. nov. for which the type species are Thiolinea disciformis gen. nov., comb. nov. and Thiofilum flexile gen. nov., comb. nov. We also propose that these genera are each circumscribed into novel families Thiolinaceae fam. nov. and Thiofilaceae fam. nov., and that Leucothrix and Cocleimonas are circumscribed into Leucotrichaceaefam. nov. and provide emended descriptions of Thiothrix and Thiotrichaceae.
Assuntos
Filogenia , Thiothrix/classificação , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Genes Bacterianos , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
The synergistic activity of algae and prokaryotic microorganisms can be used to improve the efficiency of biological wastewater treatment, particularly with regards to nitrogen removal. For example, algae can provide oxygen through photosynthesis needed for aerobic degradation of organic carbon and nitrification and harvested algal-prokaryotic biomass can be used to produce high value chemicals or biogas. Algal-prokaryotic consortia have been used to treat wastewater in different types of reactors, including waste stabilization ponds, high rate algal ponds and closed photobioreactors. This review addresses the current literature and identifies research gaps related to the following topics: 1) the complex interactions between algae and prokaryotes in wastewater treatment; 2) advances in bioreactor technologies that can achieve high nitrogen removal efficiencies in small reactor volumes, such as algal-prokaryotic biofilm reactors and enhanced algal-prokaryotic treatment systems (EAPS); 3) molecular tools that have expanded our understanding of the activities of algal and prokaryotic communities in wastewater treatment processes.
Assuntos
Reatores Biológicos , Nitrogênio/química , Águas Residuárias , Biomassa , Nitrificação , Fotobiorreatores , Eliminação de Resíduos LíquidosRESUMO
Many autotrophic microorganisms are likely to adapt to scarcity in dissolved inorganic carbon (DIC; CO2 + HCO3- + CO32-) with CO2 concentrating mechanisms (CCM) that actively transport DIC across the cell membrane to facilitate carbon fixation. Surprisingly, DIC transport has been well studied among cyanobacteria and microalgae only. The deep-sea vent gammaproteobacterial chemolithoautotroph Thiomicrospira crunogena has a low-DIC inducible CCM, though the mechanism for uptake is unclear, as homologs to cyanobacterial transporters are absent. To identify the components of this CCM, proteomes of T. crunogena cultivated under low- and high-DIC conditions were compared. Fourteen proteins, including those comprising carboxysomes, were at least 4-fold more abundant under low-DIC conditions. One of these proteins was encoded by Tcr_0854; strains carrying mutated copies of this gene, as well as the adjacent Tcr_0853, required elevated DIC for growth. Strains carrying mutated copies of Tcr_0853 and Tcr_0854 overexpressed carboxysomes and had diminished ability to accumulate intracellular DIC. Based on reverse transcription (RT)-PCR, Tcr_0853 and Tcr_0854 were cotranscribed and upregulated under low-DIC conditions. The Tcr_0853-encoded protein was predicted to have 13 transmembrane helices. Given the mutant phenotypes described above, Tcr_0853 and Tcr_0854 may encode a two-subunit DIC transporter that belongs to a previously undescribed transporter family, though it is widespread among autotrophs from multiple phyla.IMPORTANCE DIC uptake and fixation by autotrophs are the primary input of inorganic carbon into the biosphere. The mechanism for dissolved inorganic carbon uptake has been characterized only for cyanobacteria despite the importance of DIC uptake by autotrophic microorganisms from many phyla among the Bacteria and Archaea In this work, proteins necessary for dissolved inorganic carbon utilization in the deep-sea vent chemolithoautotroph T. crunogena were identified, and two of these may be able to form a novel transporter. Homologs of these proteins are present in 14 phyla in Bacteria and also in one phylum of Archaea, the Euryarchaeota Many organisms carrying these homologs are autotrophs, suggesting a role in facilitating dissolved inorganic carbon uptake and fixation well beyond the genus Thiomicrospira.
Assuntos
Dióxido de Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Fontes Hidrotermais/microbiologia , Piscirickettsiaceae/metabolismo , Carbono/metabolismo , Mutação , Filogenia , Piscirickettsiaceae/genética , ProteomaRESUMO
The genus Thiomicrorhabdus (Tmr) in the Piskirickettsiaceae in the Thiotrichales of the Gammaproteobacteria contains four species of sulfur-oxidising obligate chemolithoautotroph with validly published names, all previously classified as Thiomicrospira (Tms) species. Here we demonstrate that Thiomicrospira hydrogeniphila, a recently published hydrogen-utilising chemolithoautotroph closely related to Thiomicrorhabdus frisia (type species of Thiomicrorhabdus) should be classified as a member of the genus Thiomicrorhabdus and not Thiomicrospira, as Thiomicrorhabdus hydrogeniphila comb. nov., on the basis of comparative physiology and morphology as well as 16S rRNA (rrs) gene identity of Tms. hydrogeniphila MAS2T being closer to that of Tmr. frisia JB-A2T (99.1â%) than to Tms. pelophila DSM 1534T (90.5â%) or Hydrogenovibrio marinus MH-110T (94.1â%), and on the basis of the topology of 16S rRNA gene maximum likelihood trees, which clearly place Tms. hydrogeniphila within the genus Thiomicrorhabdus. It was also noted that thiosulfate-grown Thiomicrorhabdus spp. can be distinguished from Thiomicrospira spp. or Hydrogenovibrio spp. on the basis of the 3 dominant fatty acids (C16â:â1, C18â:â1 and C16â:â0), and from other Thiomicrorhabdus spp. on the basis of the fourth dominant fatty acid, which varies between the species of this genus - which could provide a useful diagnostic method. We provide an emended description of Thiomicrorhabdus (Boden R, Scott KM, Williams J, Russel S, Antonen K et al.Int J Syst Evol Microbiol 2017;67:1140-1151) to take into account the properties of Thiomicrorhabdus hydrogeniphila comb. nov.
Assuntos
Filogenia , Piscirickettsiaceae/classificação , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , Hidrogênio/metabolismo , Oxirredução , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Enxofre/metabolismo , Tiossulfatos/metabolismoRESUMO
Thiomicrospira(Tms) species are small sulfur-oxidizing chemolithoautotrophic members of the Gammaproteobacteria. Whilst the type species Tms. pelophila and closely related Tms. thyasirae exhibit canonical spiral morphology under sub-optimal growth conditions, most species are vibrios or rods. The 16S rRNA gene diversity is vast, with identities as low as 91.6â% for Tms. pelophila versus Tms. frisia, for example. Thiomicrospira was examined with closely related genera Hydrogenovibrio and Thioalkalimicrobium and, to rationalize organisms on the basis of the 16S rRNA gene phylogeny, physiology and morphology, we reclassify Tms. kuenenii, Tms. crunogena, Tms. thermophila and Tms. halophila to Hydrogenovibrio kuenenii comb. nov., H. crunogenus corrig. comb. nov., H. thermophilus corrig. comb. nov. and H. halophilus corrig. comb. nov. We reclassify Tms. frisia, Tms. arctica, Tms. psychrophila and Tms. chilensis to Thiomicrorhabdus (Tmr) gen. nov., as Tmr. frisia comb. nov., Tmr. arctica comb. nov., Tmr. psychrophila comb. nov. and Tmr. chilensis comb. nov. - the type species of Thiomicrorhabdus is Tmr. frisia. We demonstrate that Thioalkalimicrobium species fall within the genus Thiomicrospira sensu stricto, thus reclassifying them as Tms. aerophila corrig. comb. nov., Tms. microaerophila corrig. comb. nov., Tms. cyclica corrig. comb. nov. and Tms. sibirica corrig. comb. nov. We provide emended descriptions of the genera Thiomicrospira and Hydrogenovibrio and of Tms. thyasirae.
Assuntos
Filogenia , Piscirickettsiaceae/classificação , Técnicas de Tipagem Bacteriana , DNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Enxofre , Bactérias Redutoras de Enxofre/classificaçãoRESUMO
The gammaproteobacterium Thiomicrospira crunogena XCL-2 is an aerobic sulfur-oxidizing hydrothermal vent chemolithoautotroph that has a CO2 concentrating mechanism (CCM), which generates intracellular dissolved inorganic carbon (DIC) concentrations much higher than extracellular, thereby providing substrate for carbon fixation at sufficient rate. This CCM presumably requires at least one active DIC transporter to generate the elevated intracellular concentrations of DIC measured in this organism. In this study, the half-saturation constant (K CO2) for purified carboxysomal RubisCO was measured (276 ± 18 µM) which was much greater than the K CO2 of whole cells (1.03 µM), highlighting the degree to which the CCM facilitates CO2 fixation under low CO2 conditions. To clarify the bioenergetics powering active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis (DCCD) or proton potential (CCCP). Incubations with each of these inhibitors resulted in diminished intracellular ATP, DIC, and fixed carbon, despite an absence of an inhibitory effect on proton potential in the DCCD-incubated cells. Electron transport complexes NADH dehydrogenase and the bc 1 complex were found to be insensitive to DCCD, suggesting that ATP synthase was the primary target of DCCD. Given the correlation of DIC uptake to the intracellular ATP concentration, the ABC transporter genes were targeted by qRT-PCR, but were not upregulated under low-DIC conditions. As the T. crunogena genome does not include orthologs of any genes encoding known DIC uptake systems, these data suggest that a novel, yet to be identified, ATP- and proton potential-dependent DIC transporter is active in this bacterium. This transporter serves to facilitate growth by T. crunogena and other Thiomicrospiras in the many habitats where they are found.
Assuntos
Ciclo do Carbono/fisiologia , Carbono/metabolismo , Piscirickettsiaceae/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Bacteriana da Expressão Gênica , Piscirickettsiaceae/enzimologia , Piscirickettsiaceae/genéticaRESUMO
BACKGROUND: Multiple myeloma is a malignancy of plasma cells accounting for approximately 1% of cancers and 12% of haematological malignancies. The first-in-class proteasome inhibitor, bortezomib, is commonly used to treat newly diagnosed as well as relapsed/refractory myeloma, either as single agent or combined with other therapies. OBJECTIVES: We conducted a systematic review and meta-analysis to assess the effects of bortezomib on overall survival (OS), progression-free survival (PFS), response rate (RR), health-related quality of life (HRQoL), adverse events (AEs) and treatment-related death (TRD). SEARCH METHODS: We searched MEDLINE, the Cochrane Central Register of Controlled Trials and EMBASE (till 27 January 2016) as well as conference proceedings and clinical trial registries for randomised controlled trials (RCTs). SELECTION CRITERIA: We included randomised controlled trials (RCTs) that compared i) bortezomib versus no bortezomib with the same background therapy in each arm; ii) bortezomib versus no bortezomib with different background therapy in each arm or compared to other agent(s) and iii) bortezomib dose comparisons and comparisons of different treatment administrations and schedules. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted outcomes data and assessed risk of bias. We extracted hazard ratios (HR) and their confidence intervals for OS and PFS and odds ratios (OR) for response rates, AEs and TRD. We contacted trial authors to provide summary statistics if missing. We estimated Logrank statistics which were not available. We extracted HRQoL data, where available. MAIN RESULTS: We screened a total of 3667 records, identifying 16 relevant RCTs involving 5626 patients and included 12 trials in the meta-analyses. All trials were randomised and open-label studies. Two trials were published in abstract form and therefore we were unable to assess potential risk of bias in full.There is moderate-quality evidence that bortezomib prolongs OS (four studies, 1586 patients; Peto OR 0.77, 95% CI 0.65 to 0.92) and PFS (five studies, 1855 patients; Peto OR 0.65, 95% CI 0.57 to 0.74) from analysing trials of bortezomib versus no bortezomib with the same background therapy in each arm.There is high-quality evidence that bortezomib prolongs OS (five studies, 2532 patients; Peto OR 0.76, 95% CI 0.67 to 0.88) but low-quality evidence for PFS (four studies, 2489 patients; Peto OR 0.67, 95% CI 0.61 to 0.75) from analysing trials of bortezomib versus no bortezomib with different background therapy in each arm or compared to other agent(s).Four trials (N = 716) examined different doses, methods of administrations and treatment schedules and were reviewed qualitatively only.We identified four trials in the meta-analysis that measured time to progression (TTP) and were able to extract and analyse PFS data for three of the studies, while in the case of one study, we included TTP data as PFS data were not available. We therefore did not analyse TTP separately in this review.Patients treated with bortezomib have increased risk of thrombocytopenia, neutropenia, gastro-intestinal toxicities, peripheral neuropathy, infection and fatigue with the quality of evidence highly variable. There is high-quality evidence for increased risk of cardiac disorders from analysing trials of bortezomib versus no bortezomib with different background therapy in each arm or versus other agents. The risk of TRD in either comparison group analysed is uncertain due to the low quality of the evidence.Only four trials analysed HRQoL and the data could not be meta-analysed.Subgroup analyses by disease setting revealed improvements in all outcomes, whereas for therapy setting, an improved benefit for bortezomib was observed in all outcomes and subgroups except for OS following consolidation therapy. AUTHORS' CONCLUSIONS: This meta-analysis found that myeloma patients receiving bortezomib benefited in terms of OS, PFS and response rate compared to those who did not receive bortezomib. This benefit was observed in trials of bortezomib versus no bortezomib with the same background therapy and in trials of bortezomib versus no bortezomib with different background therapy in each arm or compared to other agent(s). Further evaluation of newer proteasome inhibitors is required to ascertain whether these agents offer an improved risk-benefit profile, while more studies of HRQoL are also required.
Assuntos
Antineoplásicos/uso terapêutico , Bortezomib/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Humanos , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Biocatalytic CO2 sequestration to reduce greenhouse-gas emissions from industrial processes is an active area of research. Carbonic anhydrases (CAs) are attractive enzymes for this process. However, the most active CAs display limited thermal and pH stability, making them less than ideal. As a result, there is an ongoing effort to engineer and/or find a thermostable CA to fulfill these needs. Here, the kinetic and thermal characterization is presented of an α-CA recently discovered in the mesophilic hydrothermal vent-isolate extremophile Thiomicrospira crunogena XCL-2 (TcruCA), which has a significantly higher thermostability compared with human CA II (melting temperature of 71.9°C versus 59.5°C, respectively) but with a tenfold decrease in the catalytic efficiency. The X-ray crystallographic structure of the dimeric TcruCA shows that it has a highly conserved yet compact structure compared with other α-CAs. In addition, TcruCA contains an intramolecular disulfide bond that stabilizes the enzyme. These features are thought to contribute significantly to the thermostability and pH stability of the enzyme and may be exploited to engineer α-CAs for applications in industrial CO2 sequestration.