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OBJECTIVES: Hearing aid processing in realistic listening environments is difficult to study effectively. Often the environment is unpredictable or unknown, such as in wearable aid trials with subjective report by the wearer. Some laboratory experiments create listening environments to exert tight experimental control, but those environments are often limited by physical space, a small number of sound sources, or room absorptive properties. Simulation techniques bridge this gap by providing greater experimental control over listening environments, effectively bringing aspects of the real-world into the laboratory. This project used simulation to study the effects of wide-dynamic range compression (WDRC) and digital noise reduction (DNR) on speech intelligibility in a reverberant environment with six spatialized competing talkers. The primary objective of this study was to determine the efficacy of WDRC and DNR in a complex listening environment using virtual auditory space techniques. DESIGN: Participants of greatest interest were listeners with hearing impairment. A group of listeners with clinically normal hearing was included to assess the effects of the simulation absent the complex effects of hearing loss. Virtual auditory space techniques were used to simulate a small restaurant listening environment with two different reverberation times (0.8 and 1.8 sec) in a range of signal to noise ratios (SNRs) (-8.5 to 11.5 dB SNR). Six spatialized competing talkers were included to further enhance realism. A hearing aid simulation was used to examine the degree to which speech intelligibility was affected by slow and fast WDRC in conjunction with the presence or absence of DNR. The WDRC and DNR settings were chosen to be reasonable estimates of hearing aids currently available to consumers. RESULTS: A WDRC × DNR × Hearing Status interaction was observed, such that DNR was beneficial for speech intelligibility when combined with fast WDRC speeds, but DNR was detrimental to speech intelligibility when WDRC speeds were slow. The pattern of the WDRC × DNR interaction was observed for both listener groups. Significant main effects of reverberation time and SNR were observed, indicating better performance with lower reverberation times and more positive SNR. CONCLUSIONS: DNR reduced low-amplitude noise before WDRC-amplified the low-intensity portions of the signal, negating one potential downside of fast WDRC and leading to an improvement in speech intelligibility in this simulation. These data suggest that, in some real-world environments that include both reverberation and noise, older listeners with hearing impairment may find speech to be more intelligible if DNR is activated when the hearing aid has fast compression time constants. Additional research is needed to determine the appropriate DNR strength and to confirm results in wearable hearing aids and a wider range of listening environments.
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Auxiliares de Audição , Perda Auditiva Neurossensorial , Percepção da Fala , Humanos , Perda Auditiva Neurossensorial/reabilitação , Razão Sinal-Ruído , Restaurantes , RuídoRESUMO
Consumers can influence ecological patterns and processes through their trophic roles and contributions to the flow of energy through ecosystems. However, the diet and associated trophic roles of consumers commonly change during ontogeny. Despite the prevalence of ontogenetic variation in trophic roles of most animals, we lack an understanding of whether they change consistently across local populations and broad geographic gradients. We examined how the diet and trophic position of a generalist marine predator varied with ontogeny across seven broadly separated locations (~ 750 km). We observed a high degree of heterogeneity in prey consumed without evidence of spatial structuring in this variability. However, compound-specific isotope analysis of amino acids revealed remarkably consistent patterns of increasing trophic position through ontogeny across local populations, suggesting that the roles of this generalist predator scaled with its body size across space. Given the high degree of diet heterogeneity we observed, this finding suggests that even though the dietary patterns differed, the underlying food web architecture transcended variation in prey species across locations for this generalist consumer. Our research addresses a gap in empirical field work regarding the interplay between stage-structured populations and food webs, and suggests ontogenetic changes in trophic position can be consistent in generalist consumers.
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Ecossistema , Cadeia Alimentar , Animais , Estado Nutricional , Dieta , Tamanho CorporalRESUMO
BACKGROUND: To investigate mechanisms of injury and recovery in neonatal encephalopathy (NE), we performed targeted metabolomic analysis of plasma using liquid chromatography with tandem mass spectrometry (LC/MS/MS) from healthy term neonates or neonates with NE. METHODS: Plasma samples from the NE (n = 45, day of life 0-1) or healthy neonatal (n = 30, ≥36 weeks gestation) cohorts had LC/MS/MS metabolomic profiling with a 193-plex targeted metabolite assay covering >366 metabolic pathways. Metabolite levels were compared to 2-year neurodevelopmental outcomes measured by the Bayley Scales of Infant and Toddler Development III (Bayley-III). RESULTS: Out of 193 metabolites, 57 met the pre-defined quality control criteria for analysis. Significant (after false discovery rate correction) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways included aminoacyl-tRNA biosynthesis, arginine biosynthesis, and metabolism of multiple amino acids. Significant disease pathways included seizures. In regression models, histidine and C6 sugar amine were significantly associated with cognitive, motor, and language and betaine with cognitive and motor Bayley-III composite scores. The addition of histidine, C6 sugar amine, and betaine to a Sarnat score-based clinical regression model significantly improved model performance (Akaike information criterion and adjusted r2) for Bayley-III cognitive, motor, and language scores. CONCLUSIONS: Plasma metabolites may help to predict neurological outcomes in neonatal brain injury and enhance current clinical predictors. IMPACT: Plasma metabolites may help to predict neurological outcomes in NE and supplement current clinical predictors. Current metabolomics research is limited in terms of clinical application and association with long-term outcomes. Our study presents novel associations of plasma metabolites from the first 24 h of life and 2-year neurodevelopmental outcomes for infants with NE. Our metabolomics discovery provides insight into possible disease mechanisms and methods to rescue and/or supplement metabolic pathways involved in NE. Our metabolomics discovery of metabolic pathway supplementations and/or rescue mechanisms may serve as adjunctive therapies for NE.
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Lesões Encefálicas , Doenças do Recém-Nascido , Arginina , Betaína , Histidina , Humanos , Lactente , Recém-Nascido , Metabolômica , RNA de Transferência , Açúcares , Espectrometria de Massas em TandemRESUMO
OBJECTIVES: Previous research has shown that the association between hearing aid-processed speech recognition and individual working memory ability becomes stronger in more challenging conditions (e.g., higher background noise levels) and with stronger hearing aid processing (e.g., fast-acting wide dynamic range compression, WDRC). To date, studies have assumed omnidirectional microphone settings and collocated speech and noise conditions to study such relationships. Such conditions fail to recognize that most hearing aids are fit with directional processing that may improve the signal to noise ratio (SNR) and speech recognition in spatially separated speech and noise conditions. Here, we considered the possibility that directional processing may reduce the signal distortion arising from fast-acting WDRC and in turn influence the relationship between working memory ability and speech recognition with WDRC processing. The combined effects of hearing aid processing (WDRC and directionality) and SNR were quantified using a signal modification metric (cepstral correlation), which measures temporal envelope changes in the processed signal with respect to a linearly amplified reference. It was hypothesized that there will be a weaker association between working memory ability and speech recognition for hearing aid processing conditions that result in overall less signal modification (i.e., fewer changes to the processed envelope). DESIGN: Twenty-three individuals with bilateral, mild to moderately severe sensorineural hearing loss participated in the study. Participants were fit with a commercially available hearing aid, and signal processing was varied in two dimensions: (1) Directionality (omnidirectional [OMNI] versus fixed-directional [DIR]), and (2) WDRC speed (fast-acting [FAST] versus slow-acting [SLOW]). Sentence recognition in spatially separated multi-talker babble was measured across a range of SNRs: 0 dB, 5 dB, 10 dB, and quiet. Cumulative signal modification was measured with individualized hearing aid settings, for all experimental conditions. A linear mixed-effects model was used to determine the relationship between speech recognition, working memory ability, and cumulative signal modification. RESULTS: Signal modification results showed a complex relationship between directionality and WDRC speed, which varied by SNR. At 0 and 5 dB SNRs, signal modification was lower for SLOW than FAST regardless of directionality. However, at 10 dB SNR and in the DIR listening condition, there was no signal modification difference between FAST and SLOW. Consistent with previous studies, the association of speech recognition in noise with working memory ability depended on the level of signal modification. Contrary to the hypothesis above, however, there was a significant association of speech recognition with working memory only at lower levels of signal modification, and speech recognition increased at a faster rate for individuals with better working memory as signal modification decreased with DIR and SLOW. CONCLUSIONS: This research suggests that working memory ability remains a significant predictor of speech recognition when WDRC and directionality are applied. Our findings revealed that directional processing can reduce the detrimental effect of fast-acting WDRC on speech cues at higher SNRs, which affects speech recognition ability. Contrary to some previous research, this study showed that individuals with better working memory ability benefitted more from a decrease in signal modification than individuals with poorer working memory ability.
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Auxiliares de Audição , Perda Auditiva Neurossensorial , Percepção da Fala , Perda Auditiva Bilateral , Humanos , Memória de Curto Prazo , FalaRESUMO
A recent goal of synthetic biology has been to identify new chassis that provide benefits lacking in model organisms. Vibrio natriegens is a marine Gram-negative bacterium which is an emergent synthetic biology chassis with inherent benefits: An extremely fast growth rate, genetic tractability, and the ability to grow on a variety of carbon sources ("feedstock flexibility"). Given these inherent benefits, we sought to determine its potential to heterologously produce natural products, and chose beta-carotene and violacein as test cases. For beta-carotene production, we expressed the beta-carotene biosynthetic pathway from the sister marine bacterium Vibrio campbellii, as well as the mevalonate biosynthetic pathway from the Gram-positive bacterium Lactobacillus acidophilus to improve precursor abundance. Violacein was produced by expressing a biosynthetic gene cluster derived from Chromobacterium violaceum. Not only was V. natriegens able to heterologously produce these compounds in rich media, illustrating its promise as a new chassis for small molecule drug production, but it also did so in minimal media using a variety of feedstocks. The ability for V. natriegens to produce natural products with multiple industrially-relevant feedstocks argues for continued investigations into the production of more complex natural products in this chassis.
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Produtos Biológicos/metabolismo , Indóis/metabolismo , Vibrio/metabolismo , beta Caroteno/biossíntese , Vias Biossintéticas , Chromobacterium/genética , Família Multigênica , Biologia Sintética , Vibrio/genéticaRESUMO
Streptococcus pneumoniae coinfection is a major cause of influenza-associated mortality; however, the mechanisms underlying pathogenesis or protection remain unclear. Using a clinically relevant mouse model, we identify immune-mediated damage early during coinfection as a new mechanism causing susceptibility. Coinfected CCR2(-/-) mice lacking monocytes and monocyte-derived cells control bacterial invasion better, show reduced epithelial damage and are overall more resistant than wild-type controls. In influenza-infected wild-type lungs, monocytes and monocyte-derived cells are the major cell populations expressing the apoptosis-inducing ligand TRAIL. Accordingly, anti-TRAIL treatment reduces bacterial load and protects against coinfection if administered during viral infection, but not following bacterial exposure. Post-influenza bacterial outgrowth induces a strong proinflammatory cytokine response and massive inflammatory cell infiltrate. Depletion of neutrophils or blockade of TNF-α facilitate bacterial outgrowth, leading to increased mortality, demonstrating that these factors aid bacterial control. We conclude that inflammatory monocytes recruited early, during the viral phase of coinfection, induce TRAIL-mediated lung damage, which facilitates bacterial invasion, while TNF-α and neutrophil responses help control subsequent bacterial outgrowth. We thus identify novel determinants of protection versus pathology in influenza-Streptococcus pneumoniae coinfection.
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Coinfecção/imunologia , Vírus da Influenza A Subtipo H3N2 , Monócitos/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/patogenicidade , Animais , Coinfecção/fisiopatologia , Citocinas/imunologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/fisiopatologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Infecções por Orthomyxoviridae/complicações , Infecções Pneumocócicas/complicações , Receptores CCR2/deficiência , Receptores CCR2/genética , Streptococcus pneumoniae/crescimento & desenvolvimento , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Despite the fact that actinomycetes harbor the genetic potential to produce terpenes, terpenoid natural products tend to be a rare occurrence in fermentation broths. Here we report two new halimane-type diterpenoids, micromonohalimanes A (1) and B (2), that were isolated from a Micromonospora sp. cultivated from the marine ascidian Symplegma brakenhielmi. This is the first report of the halimane-type diterpenoids from Micromonospora. The structures were determined using spectroscopic methods including X-ray crystallography to establish the absolute configuration. Micromonohalimane B demonstrated moderate antibacterial activity against methicillin-resistant Staphylococcus aureus.
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Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Diterpenos/isolamento & purificação , Diterpenos/farmacologia , Micromonospora/química , Animais , Antibacterianos/química , Cristalografia por Raios X , Diterpenos/química , Florida , Biologia Marinha , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Conformação Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Urocordados/microbiologiaRESUMO
With respect to bacterial natural products, a significant outcome of the genomic era was that the biosynthetic potential in many microorganisms surpassed the number of compounds isolated under standard laboratory growth conditions, particularly among certain members in the phylum Actinobacteria. Our group, as well as others, investigated interspecies interactions, via co-culture, as a technique to coax bacteria to produce novel natural products. While co-culture provides new opportunities, challenges exist and questions surrounding these methods remain unanswered. In marine bacteria, for example, how prevalent are interspecies interactions and how commonly do interactions result in novel natural products? In an attempt to begin to answer basic questions surrounding co-culture of marine microorganisms, we have tested both antibiotic activity-based and LC/MS-based methods to evaluate Micromonosporaceae secondary metabolite production in co-culture. Overall, our investigation of 65 Micromonosporaceae led to the identification of 12 Micromonosporaceae across three genera that produced unique metabolites in co-culture. Our results suggest that interspecies interactions were prevalent between marine Micromonosporaceae and marine mycolic acid-containing bacteria. Furthermore, our approach highlights a sensitive and rapid method for investigating interspecies interactions in search of novel antibiotics, secondary metabolites, and genes.
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Antibacterianos/isolamento & purificação , Produtos Biológicos/isolamento & purificação , Técnicas de Cocultura , Micromonosporaceae/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Micromonosporaceae/classificação , Metabolismo Secundário , Especificidade da EspécieRESUMO
Two novel trialkyl-substituted aromatic acids, solwaric acids A and B, were isolated from a marine Solwaraspora sp. cultivated from the ascidian Trididemnum orbiculatum. Solwaric acids A and B were isotopically labeled with U-¹³C glucose, and analysis of a ¹³C-¹³C COSY allowed for unambiguous determination of the location of the phenyl methyl group. The two novel compounds demonstrated antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA).
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Ácidos/farmacologia , Antibacterianos/farmacologia , Urocordados/química , Ácidos/química , Ácidos/isolamento & purificação , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacosRESUMO
The objective of this project was to establish cutoff scores on the tinnitus subscale of the Tinnitus and Hearing Survey (THS) using a large sample of United States service members (SM) with the end goal of guiding clinical referrals for tinnitus evaluation. A total of 4,589 SM undergoing annual audiometric surveillance were prospectively recruited to complete the THS tinnitus subscale (THS-T). A subset of 1,304 participants also completed the Tinnitus Functional Index (TFI). The original 5-point response scale of the THS (THS-T16) was modified to an 11-point scale (THS-T40) for some participants, to align with the response scale of the TFI. Age, sex, hearing loss, and self-reported tinnitus bother were also recorded. The THS-T was relatively insensitive to hearing, but self-reported bothersome tinnitus was significantly associated with the THS-T40 score. Receiver operating characteristic analysis was used to determine cutoff scores on the THS-T that aligned with recommended cutoff values for clinical intervention on the TFI. A cutoff of 9 on the THS-T40 aligns with a TFI cutoff of 25, indicating a patient may need intervention for tinnitus. A cutoff of 15 aligns with a TFI cutoff of 50, indicating that more aggressive intervention for tinnitus is warranted. The THS-T is a viable tool to identify patients with tinnitus complaints warranting clinical evaluation for use by hearing conservation programs and primary care clinics. The THS-T40 cutoff scores of 9 and 15 provide clinical reference points to guide referrals to audiology.
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Zumbido , Humanos , Zumbido/diagnóstico , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Adulto , Estudos Prospectivos , Estados Unidos , Audição , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Curva ROC , Inquéritos e Questionários , Autorrelato , Audiometria/métodosRESUMO
Low- (or mild-) gain hearing aids (LGHAs) are increasingly considered for individuals with normal peripheral hearing but significant self-reported hearing difficulties (SHDs). This study assesses the benefits of LGHAs as a management option for individuals with normal hearing thresholds (NHTs) and SHDs, comparing LGHA use and benefit to individuals with non-significant hearing difficulties (NHDs) and those with peripheral hearing loss. Questionnaires addressing hearing aid usage, benefit, hearing difficulties, and tinnitus were administered to 186 individuals who self-identified as hearing aid users in a sample of 6652 service members who were receiving their annual hearing tests. Participants were divided into SHD and NHD groups based on the normative cutoff of the Tinnitus and Hearing Survey-Hearing Subscale (THS-H), and into hearing impairment (HI) and NHT based on their audiometric air-conduction thresholds. Individuals with SHDs and NHTs reported higher LGHA usage and benefit than individuals with NHDs and NHTs. Comparable use and benefit were noted between groups with SHDs regardless of peripheral hearing loss status. The findings support LGHAs as a suitable management option for individuals with NHTs and SHDs, as indicated by hearing aid use and benefit. Quantifying the level of perceived auditory processing deficits (i.e., SHDs), notably with the THS-H, enhances sensitivity in identifying those who may benefit the most from this treatment option.
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Synthetic biology is touted as the next industrial revolution as it promises access to greener biocatalytic syntheses to replace many industrial organic chemistries. Here, it is shown to what synthetic biology can offer in the form of multienzyme cascades for the synthesis of the most basic of new materials-chemicals, including especially designer chemical products and their analogs. Since achieving this is predicated on dramatically expanding the chemical space that enzymes access, such chemistry will probably be undertaken in cell-free or minimalist formats to overcome the inherent toxicity of non-natural substrates to living cells. Laying out relevant aspects that need to be considered in the design of multi-enzymatic cascades for these purposes is begun. Representative multienzymatic cascades are critically reviewed, which have been specifically developed for the synthesis of compounds that have either been made only by traditional organic synthesis along with those cascades utilized for novel compound syntheses. Lastly, an overview of strategies that look toward exploiting bio/nanomaterials for accessing channeling and other nanoscale materials phenomena in vitro to direct novel enzymatic biosynthesis and improve catalytic efficiency is provided. Finally, a perspective on what is needed for this field to develop in the short and long term is presented.
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Nanoestruturas , Biocatálise , CatáliseRESUMO
Evolution has gifted enzymes with the ability to synthesize an abundance of small molecules with incredible control over efficiency and selectivity. Central to an enzyme's role is the ability to selectively catalyze reactions in the milieu of chemicals within a cell. However, for chemists it is often desirable to extend the substrate scope of reactions to produce analogue(s) of a desired product and therefore some degree of enzyme promiscuity is often desired. Herein, we examine this dichotomy in the context of the violacein biosynthetic pathway. Importantly, we chose to interrogate this pathway with tryptophan analogues in vitro, to mitigate possible interference from cellular components and endogenous tryptophan. A total of nine tryptophan analogues were screened for by analyzing the substrate promiscuity of the initial enzyme, VioA, and compared to the substrate tryptophan. These results suggested that for VioA, substitutions at either the 2- or 4-position of tryptophan were not viable. The seven analogues that showed successful substrate conversion by VioA were then applied to the five enzyme cascade (VioABEDC) for the production of violacein, where l-tryptophan and 6-fluoro-l-tryptophan were the only substrates which were successfully converted to the corresponding violacein derivative(s). However, many of the other tryptophan analogues did convert to various substituted intermediaries. Overall, our results show substrate promiscuity with the initial enzyme, VioA, but much less for the full pathway. This work demonstrates the complexity involved when attempting to analyze substrate analogues within multienzymatic cascades, where each enzyme involved within the cascade possesses its own inherent promiscuity, which must be compatible with the remaining enzymes in the cascade for successful formation of a desired product.
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Total syntheses of putative (±)-trichodermatides B and C are described. These efficient syntheses feature the oxa-[3 + 3] annulation strategy, leading to B and C along with their respective C2-epimers. However, these synthetic samples are spectroscopically very different from the natural products. DFT calculations of C13 chemical shifts are conducted and the predicted values are in good agreement with those of synthetic samples, thereby questioning in the accuracy of structural assignments of trichodermatides B and C.
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Hearing difficulties are frequently reported by patients in audiology clinics, including patients with normal audiometric thresholds. However, because all individuals experience some difficulty understanding speech in noisy environments, it can be difficult to assess hearing complaints objectively across patients. Normative values help address this issue by providing an objective cutoff score for determining what is or is not clinically significant. The goal of this study was to establish normative values for the four-item hearing subscale of the Tinnitus and Hearing Survey (THS-H). Respondents completing the THS-H rate the level of difficulty understanding speech in the situations most commonly reported as being difficult: in the presence of noise, on TV or in movies, soft voices and group conversations. In this study, 22,583 US Service Members (SMs) completed the THS-H using an 11-point scale ranging from 0 (not a problem) to 10 (a very big problem). Responses to the four items were summed to produce values between 0 and 40. The distribution of the final scores was analyzed based on severity of hearing loss, age, and sex. Only 5% of SMs with clinically normal hearing scored above 27, so this score was selected as a cutoff for "clinically significant hearing problems." Due to its ease of administration and interpretation, the THS-H could be a useful tool for identifying patients with subjective hearing difficulty warranting audiological evaluation and management.
Assuntos
Surdez , Perda Auditiva , Percepção da Fala , Zumbido , Humanos , Zumbido/diagnóstico , Zumbido/etiologia , Audição/fisiologia , Testes Auditivos , Perda Auditiva/diagnóstico , Perda Auditiva/complicações , Inquéritos e QuestionáriosRESUMO
The use of biological systems in manufacturing and medical applications has seen a dramatic rise in recent years as scientists and engineers have gained a greater understanding of both the strengths and limitations of biological systems. Biomanufacturing, or the use of biology for the production of biomolecules, chemical precursors, and others, is one particular area on the rise as enzymatic systems have been shown to be highly advantageous in limiting the need for harsh chemical processes and the formation of toxic products. Unfortunately, biological production of some products can be limited due to their toxic nature or reduced reaction efficiency due to competing metabolic pathways. In nature, microbes often secrete enzymes directly into the environment or encapsulate them within membrane vesicles to allow catalysis to occur outside the cell for the purpose of environmental conditioning, nutrient acquisition, or community interactions. Of particular interest to biotechnology applications, researchers have shown that membrane vesicle encapsulation often confers improved stability, solvent tolerance, and other benefits that are highly conducive to industrial manufacturing practices. While still an emerging field, this review will provide an introduction to biocatalysis and bacterial membrane vesicles, highlight the use of vesicles in catalytic processes in nature, describe successes of engineering vesicle/enzyme systems for biocatalysis, and end with a perspective on future directions, using selected examples to illustrate these systems' potential as an enabling tool for biotechnology and biomanufacturing.
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Enzyme activity can be many times enhanced in configurations where they are displayed on a nanoparticle (NP) and this same format sometimes even provides access to channeling phenomena within multienzyme cascades. Here, we demonstrate that such enhancement phenomena can be expanded to enzymatic cofactor recycling along with the coupled enzymatic processes that they are associated with. We begin by showing that the efficiency of glucose driven reduction of nicotinamide adenine dinucleotide (NAD+ â NADH) by glucose dehydrogenase (GDH) is enhanced ca. 5-fold when the enzyme is displayed on nanocrystalline semiconductor quantum dots (QDs) which are utilized as prototypical NP materials in our experimental assays. Coupling this enzymatic step with NADH-dependent lactate dehydrogenase (LDH) conversion of lactate to pyruvate also increases the latter's rate by a similar amount when both enzymes were jointly incorporated into self-assembled QD-based nanoclusters. Detailed agarose gel mobility assays and transmission electron microscopy imaging studies confirm that both tetrameric enzymes assemble to and crosslink the QDs into structured nanoclusters via their multiple-pendant terminal (His)6 sequences. Unexpectedly, control experiments utilizing blocking peptides to prevent enzyme-crosslinking of QDs resulted in even further enhancement of individual enzyme on-QD kinetic activity. This activity was also probed revealing that 200-fold excess peptide/QD addition enhanced individual GDH and LDH on-QD kcat a further 2- and 1.5×, respectively, above that seen just by QD display to a maximum of â¼10-fold GDH enhancement. The potential implications for how these enzyme kinetics-enhancing phenomena can be applied to single and multi-enzyme cascaded reactions in the context of cofactor recycling and cell-free synthetic biology are discussed.
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Nanopartículas , Pontos Quânticos , NAD/química , Cinética , Nanopartículas/química , Pontos Quânticos/química , L-Lactato Desidrogenase/metabolismo , Peptídeos/químicaRESUMO
All Gram-negative bacteria are believed to produce outer membrane vesicles (OMVs), proteoliposomes shed from the outermost membrane. We previously separately engineered E. coli to produce and package two organophosphate (OP) hydrolyzing enzymes, phosphotriesterase (PTE) and diisopropylfluorophosphatase (DFPase), into secreted OMVs. From this work, we realized a need to thoroughly compare multiple packaging strategies to elicit design rules for this process, focused on (1) membrane anchors or periplasm-directing proteins (herein "anchors/directors") and (2) the linkers connecting these to the cargo enzyme; both may affect enzyme cargo activity. Herein, we assessed six anchors/directors to load PTE and DFPase into OMVs: four membrane anchors, namely, lipopeptide Lpp', SlyB, SLP, and OmpA, and two periplasm-directing proteins, namely, maltose-binding protein (MBP) and BtuF. To test the effect of linker length and rigidity, four different linkers were compared using the anchor Lpp'. Our results showed that PTE and DFPase were packaged with most anchors/directors to different degrees. For the Lpp' anchor, increased packaging and activity corresponded to increased linker length. Our findings demonstrate that the selection of anchors/directors and linkers can greatly influence the packaging and bioactivity of enzymes loaded into OMVs, and these findings have the potential to be utilized for packaging other enzymes into OMVs.
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Access to efficient enzymatic channeling is desired for improving all manner of designer biocatalysis. We demonstrate that enzymes constituting a multistep cascade can self-assemble with nanoparticle scaffolds into nanoclusters that access substrate channeling and improve catalytic flux by orders of magnitude. Utilizing saccharification and glycolytic enzymes with quantum dots (QDs) as a model system, nanoclustered-cascades incorporating from 4 to 10 enzymatic steps are prototyped. Along with confirming channeling using classical experiments, its efficiency is enhanced several fold more by optimizing enzymatic stoichiometry with numerical simulations, switching from spherical QDs to 2-D planar nanoplatelets, and by ordering the enzyme assembly. Detailed analyses characterize assembly formation and clarify structure-function properties. For extended cascades with unfavorable kinetics, channeled activity is maintained by splitting at a critical step, purifying end-product from the upstream sub-cascade, and feeding it as a concentrated substrate to the downstream sub-cascade. Generalized applicability is verified by extending to assemblies incorporating other hard and soft nanoparticles. Such self-assembled biocatalytic nanoclusters offer many benefits towards enabling minimalist cell-free synthetic biology.
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Nanopartículas , Pontos Quânticos , Nanopartículas/química , Pontos Quânticos/química , Biocatálise , Catálise , CinéticaRESUMO
Inefficient cellular delivery limits the landscape of macromolecular drugs. Boronic acids readily form boronate esters with the 1,2- and 1,3-diols of saccharides, such as those that coat the surface of mammalian cells. Here pendant boronic acids are shown to enhance the cytosolic delivery of a protein toxin. Thus, boronates are a noncationic carrier that can deliver a polar macromolecule into mammalian cells.