Multi-omic based production strain improvement (MOBpsi) for bio-manufacturing of toxic chemicals.

Robust systematic approaches for the metabolic engineering of cell factories remain elusive. The available models for predicting phenotypical responses and mechanisms are incomplete, particularly within the context of compound toxicity that can be a significant impediment to achieving high yields of a target product. This study describes a Multi-Omic Based Production Strain Improvement (MOBpsi) strategy that is distinguished by integrated time-resolved systems analyses of fed-batch fermentations. As a case study, MOBpsi was applied to improve the performance of an Escherichia coli cell factory producing the commodity chemical styrene. Styrene can be bio-manufactured from phenylalanine via an engineered pathway comprised of the enzymes phenylalanine ammonia lyase and ferulic acid decarboxylase. The toxicity, hydrophobicity, and volatility of styrene combine to make bio-production challenging. Previous attempts to create styrene tolerant E. coli strains by targeted genetic interventions have met with modest success. Application of MOBpsi identified new potential targets for improving performance, resulting in two host strains (E. coli NST74ΔaaeA and NST74ΔaaeA cpxPo) with increased styrene production. The best performing re-engineered chassis, NST74ΔaaeA cpxPo, produced ∼3 × more styrene and exhibited increased viability in fed-batch fermentations. Thus, this case study demonstrates the utility of MOBpsi as a systematic tool for improving the bio-manufacturing of toxic chemicals.

Letrozole-Induced Polycystic Ovary Syndrome Attenuates Cystathionine-ß Synthase mRNA and Protein Abundance in the Ovaries of Female Sprague Dawley Rats.

BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine disorder that affects 10% of reproductive-aged women and leads to hyperandrogenism, anovulation, and infertility. PCOS has been associated with elevated serum homocysteine as well as altered methylation status; however, characterization of one-carbon metabolism (OCM) in PCOS remains incomplete. OBJECTIVES: The aim of our research was to assess OCM in a letrozole-induced Sprague Dawley rat model of PCOS. METHODS: Five-week-old female rats (n = 36) were randomly assigned to letrozole [0.9 mg/kg body weight (BW)] treatment or vehicle (carboxymethylcellulose) control that was administered via subcutaneously implanted slow-release pellets every 30 d. For both treatment groups, 12 rats were randomly assigned to be euthanized during proestrus at one of the following time points: 8, 16, or 24 wk of age. Daily BW was measured and estrous cyclicity was monitored during the last 30 d of the experimental period. Ovaries were collected to assess mRNA and protein abundance of OCM enzymes. RESULTS: Letrozole-induced rats exhibited 1.9-fold higher cumulative BW gain compared with control rats across all age groups (P < 0.0001). Letrozole reduced the time spent at proestrus (P = 0.0001) and increased time in metestrus (P < 0.0001) of the estrous cycle. Cystathionine ß-synthase (Cbs) mRNA abundance was reduced in the letrozole-induced rats at 16 (59%; P < 0.05) and 24 (77%; P < 0.01) wk of age. In addition, CBS protein abundance was 32% lower in 8-wk-old letrozole-induced rats (P = 0.02). Interestingly, betaine-homocysteine S-methyltransferase mRNA abundance increased as a function of age in letrozole-induced rats (P = 0.03). CONCLUSION: These data demonstrate that letrozole-induced PCOS Sprague Dawley rats temporally decrease the ovarian abundance of Cbs mRNA and protein in the early stages of PCOS.

From formulation to in vivo model: A comprehensive study of a synergistic relationship between vancomycin, carvacrol, and cuminaldehyde against Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VRE) has become endemic in healthcare settings, reducing treatment options for enterococcal infections. New antimicrobials for VRE infections are a high priority, but the development of novel antibiotics is time-consuming and expensive. Essential oils (EOs) synergistically enhance the activity of some existing antibiotics, suggesting that EO-antibiotic combinations could resensitise resistant bacteria and maintain the antibiotic repertoire. The mechanism of resensitisation of bacteria to antibiotics by EOs is relatively understudied. Here, the synergistic interactions between carvacrol (1.98 mM) and cuminaldehyde (4.20 mM) were shown to reestablish susceptibility to vancomycin (0.031 mg/L) in VRE, resulting in bactericidal activity (4.73 log10 CFU/ml reduction). Gene expression profiling, coupled with ß-galactosidase leakage and salt tolerance assays, suggested that cell envelope damage contributes to the synergistic bactericidal effect against VRE. The EO-vancomycin combination was also shown to kill clinical isolates of VRE (2.33-5.25 log10 CFU/ml reduction), and stable resistance did not appear to develop even after multiple passages. The in vivo efficacy of the EO-vancomycin combination was tested in a Galleria mellonella larvae assay; however, no antimicrobial action was observed, indicating that further drug development is required for the EO-vancomycin combination to be clinically useful for treatment of VRE infections.

Dietary Whole Egg Reduces Body Weight Gain in a Dose-Dependent Manner in Zucker Diabetic Fatty Rats.

BACKGROUND: We previously reported that a whole-egg-based diet attenuated weight gain in rats with type 2 diabetes (T2D) and more effectively maintained vitamin D status than an equivalent amount of supplemental cholecalciferol. OBJECTIVES: The objective of this study was to determine the lowest dose of whole egg effective at maintaining vitamin D homeostasis and attenuating the obese phenotype in T2D rats. METHODS: Zucker diabetic fatty (ZDF) rats (n = 40; age 6 wk; prediabetic) and their lean controls (n = 40; age 6 wk) were randomly assigned to a diet containing 20% casein (CAS) or 20%, 10%, 5%, or 2.5% protein from whole egg (20% EGG, 10% EGG, 5% EGG, and 2.5% EGG, respectively). All diets contained 20% total protein (wt:wt). All rats received their respective diets for 8 wk, at a stage of growth and development that translates to adolescence in humans, until 14 wk of age, a point at which ZDF rats exhibit overt T2D. Weight gain was measured 5 d/wk, and circulating 25-hydroxyvitamin D [25(OH)D] was measured by ELISA. Mean values were compared by 2-factor ANOVA. RESULTS: The 20% EGG diet maintained serum 25(OH)D at 30 nmol/L in ZDF rats, whereas the 10%, 5%, and 2.5% EGG diets did not prevent insufficiency, resulting in mean serum 25(OH)D concentrations of 24 nmol/L in ZDF rats. Body weight gain was reduced by 29% (P < 0.001) and 31% (P < 0.001) in ZDF rats consuming 20% and 10% EGG diets, respectively, and by 16% (P = 0.004) and 12% (P = 0.030) in ZDF rats consuming 5% and 2.5% EGG diets, respectively, compared with CAS. CONCLUSIONS: Whole-egg-based diets exerted a dose-dependent response with respect to attenuating weight gain. These data could support dietary recommendations aimed at body weight management in individuals predisposed to obesity and T2D.

When the larger objective matters more: support workers' epistemic and deontic authority over adult service-users.

We report on how support workers sometimes over-ride the wishes of people living with cognitive impairments. This can happen when they are both involved in some project (such as an institutionally-managed game, a physical journey, an educational activity and so on). The support worker might use their deontic authority (to propose, decide or announce future actions) to do things that advance the over-arching project, in spite of proposals for what are cast as diversions from the person with impairments. They might also use their epistemic authority (their greater knowledge or cognitive capacity) to trump their clients' choices and preferences in subordinate projects. Not orienting to suggested courses of actions is generally interactionally dispreferred and troublesome, but, although the providers do sometimes orient to their actions as balking their clients' wishes, they usually do not, and encounter little resistance. We discuss how people with disabilities may resist or palliate such loss of control, and the dilemmas that support staff face in carrying out their duties.

Efficient bio-production of citramalate using an engineered Escherichia coli strain.

Citramalic acid is a central intermediate in a combined biocatalytic and chemocatalytic route to produce bio-based methylmethacrylate, the monomer used to manufacture Perspex and other high performance materials. We developed an engineered E. coli strain and a fed-batch bioprocess to produce citramalate at concentrations in excess of 80 g l-1 in only 65 h. This exceptional efficiency was achieved by designing the production strain and the fermentation system to operate synergistically. Thus, a single gene encoding a mesophilic variant of citramalate synthase from Methanococcus jannaschii, CimA3.7, was expressed in E. coli to convert acetyl-CoA and pyruvate to citramalate, and the ldhA and pflB genes were deleted. By using a bioprocess with a continuous, growth-limiting feed of glucose, these simple interventions diverted substrate flux directly from central metabolism towards formation of citramalate, without problematic accumulation of acetate. Furthermore, the nutritional requirements of the production strain could be satisfied through the use of a mineral salts medium supplemented only with glucose (172 g l-1 in total) and 1.4 g l-1 yeast extract. Using this system, citramalate accumulated to 82±1.5 g l-1, with a productivity of 1.85 g l-1 h-1 and a conversion efficiency of 0.48 gcitramalate g-1glucose. The new bioprocess forms a practical first step for integrated bio- and chemocatalytic production of methylmethacrylate.

Applying asymptotic methods to synthetic biology: Modelling the reaction kinetics of the mevalonate pathway.

The mevalonate pathway is normally found in eukaryotes, and allows for the production of isoprenoids, a useful class of organic compounds. This pathway has been successfully introduced to Escherichia coli, enabling a biosynthetic production route for many isoprenoids. In this paper, we develop and solve a mathematical model for the concentration of metabolites in the mevalonate pathway over time, accounting for the loss of acetyl-CoA to other metabolic pathways. Additionally, we successfully test our theoretical predictions experimentally by introducing part of the pathway into Cupriavidus necator. In our model, we exploit the natural separation of time scales as well as of metabolite concentrations to make significant asymptotic progress in understanding the system. We confirm that our asymptotic results agree well with numerical simulations, the former enabling us to predict the most important reactions to increase isopentenyl diphosphate production whilst minimizing the levels of HMG-CoA, which inhibits cell growth. Thus, our mathematical model allows us to recommend the upregulation of certain combinations of enzymes to improve production through the mevalonate pathway.

Family history and TOMM40 '523 interactive associations with memory in middle-aged and Alzheimer's disease cohorts.

INTRODUCTION: Family history (FH) of Alzheimer's disease (AD) affects mitochondrial function and may modulate effects of translocase of the outer mitochondrial membrane 40 kDa (TOMM40) rs10524523 ('523) poly-T length on memory decline. METHODS: For 912 nonapolipoprotein ε4 middle-aged adults and 365 aged adults across the AD spectrum, linear mixed models gauged FH and TOMM40 '523 interactions on memory and global cognition between baseline and up to 10 years later. A cerebrospinal fluid mitochondrial function biomarker was also assessed. RESULTS: For FH negative participants, gene-dose preservation of memory and global cognition was seen for "very long" versus "short" carriers. For FH positive, an opposite gene-dose decline was seen for very long versus short carriers. Maternal FH was a stronger predictor in aged, but not middle-aged, participants. Similar gene-dose effects were seen for the mitochondrial biomarker aspartate aminotransferase. DISCUSSION: These results may clarify conflicting findings on TOMM40 poly-T length and AD-related decline.

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging.

Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

Remembering visual and linguistic common ground in shared history.

Successful communication requires speakers and listeners to refer to information in their common ground. Shared history is one of the bases for common ground, as information from a communicative episode in the past can be referred to in future communication. However, to draw upon shared history, communicative partners need to have an accurate memory record that they can refer to. The memory mechanism for shared history is poorly understood. The current study investigated the ways in which memory for shared history is prioritised. Two experiments presented a referential communication task followed by a surprise recognition memory task, with the former task serving as an episode of shared history. Experiment 1 revealed superior memory for information that was both seen in the communicators' common ground and referred to, followed by information that was seen but not referred to, and finally by information privileged to the participants. Experiment 2 provided a replication of Experiment 1 and further demonstrated that these co-presence effects are not dependent on the presence of a speaker with a different perspective to the participant.

Overexpression of CREB in the nucleus accumbens shell increases cocaine reinforcement in self-administering rats.

Chronic exposure to addictive drugs enhances cAMP response element binding protein (CREB)-regulated gene expression in nucleus accumbens (NAc), and these effects are thought to reduce the positive hedonic effects of passive cocaine administration. Here, we used viral-mediated gene transfer to produce short- and long-term regulation of CREB activity in NAc shell of rats engaging in volitional cocaine self-administration. Increasing CREB expression in NAc shell markedly enhanced cocaine reinforcement of self-administration behavior, as indicated by leftward (long-term) and upward (short-term) shifts in fixed ratio dose-response curves. CREB also increased the effort exerted by rats to obtain cocaine on more demanding progressive ratio schedules, an effect highly correlated with viral-induced modulation of BDNF protein in the NAc shell. CREB enhanced cocaine reinforcement when expressed either throughout acquisition of self-administration or when expression was limited to postacquisition tests, indicating a direct effect of CREB independent of reinforcement-related learning. Downregulating endogenous CREB in NAc shell by expressing a short hairpin RNA reduced cocaine reinforcement in similar tests, while overexpression of a dominant-negative CREB(S133A) mutant had no significant effect on cocaine self-administration. Finally, increasing CREB expression after withdrawal from self-administration enhanced cocaine-primed relapse, while reducing CREB levels facilitated extinction of cocaine seeking, but neither altered relapse induced by cocaine cues or footshock stress. Together, these findings indicate that CREB activity in NAc shell increases the motivation for cocaine during active self-administration or after withdrawal from cocaine. Our results also highlight that volitional and passive drug administration can lead to substantially different behavioral outcomes.

Exploiting Violet-Blue Light to Kill Campylobacter jejuni: Analysis of Global Responses, Modeling of Transcription Factor Activities, and Identification of Protein Targets.

Campylobacter jejuni is a microaerophilic foodborne zoonotic pathogen of worldwide concern as the leading cause of bacterial gastroenteritis. Many strains are increasingly antibiotic resistant and new methods of control are required to reduce food-chain contamination. One possibility is photodynamic inactivation (PDI) using violet-blue (VB) light, to which C. jejuni is highly susceptible. Here, we show that flavin and protoporphyrin IX are major endogenous photosensitizers and that exposure of cells to VB light increases intracellular reactive oxygen species (ROS) to high levels, as indicated by a dichlorodihydrofluorescein reporter. Unusually for an oxygen-respiring bacterium, C. jejuni employs several ROS-sensitive iron-sulfur cluster enzymes in central metabolic pathways; we show that VB light causes rapid inactivation of both pyruvate and 2-oxoglutarate oxidoreductases, thus interrupting the citric acid cycle. Cells exposed to VB light also lose heme from c-type cytochromes, restricting electron transport, likely due to irreversible oxidation of heme-ligating cysteine residues. Evaluation of global gene expression changes by RNAseq and probabilistic modeling showed a two-stage protein damage/oxidative stress response to VB light, driven by specific regulators, including HspR, PerR, Fur, and RacR. Deletion mutant analysis showed that superoxide dismutase and the cytochrome CccA were particularly important for VB light survival and that abolishing repression of chaperones and oxidative stress resistance genes by HcrA, HspR, or PerR increased tolerance to VB light. Our results explain the high innate sensitivity of C. jejuni to VB light and provide new insights that may be helpful in exploiting PDI for novel food-chain interventions to control this pathogen. IMPORTANCE Campylobacteriosis caused by C. jejuni is one of the most widespread zoonotic enteric diseases worldwide and represents an enormous human health and economic burden, compounded by the emergence of antibiotic-resistant strains. New interventions are urgently needed to reduce food-chain contamination. Although UV light is well known to be bactericidal, it is highly mutagenic and problematic for continuous exposure in food production facilities; in contrast, narrow spectrum violet-blue (VB) light is much safer. We confirmed that C. jejuni is highly susceptible to VB light and then identified some of the global regulatory networks involved in responding to photo-oxidative damage. The identification of damaged cellular components underpins efforts to develop commercial applications of VB light-based technologies.

Antibiotic prescribing in UK out-of-hours primary care services: a realist-informed scoping review of training and guidelines for healthcare professionals.

BACKGROUND: Antibiotic overuse has contributed to antimicrobial resistance, which is a global public health problem. In the UK, despite the fall in rates of antibiotic prescription since 2013, prescribing levels remain high in comparison with other European countries. Prescribing in out-of-hours (OOH) care provides unique challenges for prudent prescribing, for which professionals may not be prepared. AIM: To explore the guidance available to professionals on prescribing antibiotics for common infections in OOH primary care within the UK, with a focus on training resources, guidelines, and clinical recommendations. DESIGN & SETTING: A realist-informed scoping review of peer-reviewed articles and grey literature. METHOD: The review focused on antibiotic prescribing OOH (for example, clinical guidelines and training videos). General prescribing guidance was searched whenever OOH-focused resources were unavailable. Electronic databases and websites of national agencies and professional societies were searched following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standards. Findings were organised according to realist review components, that is, mechanisms, contexts, and outcomes. RESULTS: In total, 46 clinical guidelines and eight training resources were identified. Clinical guidelines targeted adults and children, and included recommendations on prescription strategy, spectrum of the antibiotic prescribed, communication with patients, treatment duration, and decision-making processes. No clinical guidelines or training resources focusing specifically on OOH were found. CONCLUSION: The results highlight a lack of knowledge about whether existing resources address the challenges faced by OOH antibiotic prescribers. Further research is needed to explore the training needs of OOH health professionals, and whether further OOH-focused resources need to be developed given the rates of antibiotic prescribing in this setting.

A microbubble-sparged yeast propagation-fermentation process for bioethanol production.

BACKGROUND: Industrial biotechnology will play an increasing role in creating a more sustainable global economy. For conventional aerobic bioprocesses supplying O2 can account for 15% of total production costs. Microbubbles (MBs) are micron-sized bubbles that are widely used in industry and medical imaging. Using a fluidic oscillator to generate energy-efficient MBs has the potential to decrease the costs associated with aeration. However, little is understood about the effect of MBs on microbial physiology. To address this gap, a laboratory-scale MB-based Saccharomyces cerevisiae Ethanol Red propagation-fermentation bioethanol process was developed and analysed. RESULTS: Aeration with MBs increased O2 transfer to the propagation cultures. Titres and yields of bioethanol in subsequent anaerobic fermentations were comparable for MB-propagated and conventional, regular bubble (RB)-propagated yeast. However, transcript profiling showed significant changes in gene expression in the MB-propagated yeast compared to those propagated using RB. These changes included up-regulation of genes required for ergosterol biosynthesis. Ergosterol contributes to ethanol tolerance, and so the performance of MB-propagated yeast in fed-batch fermentations sparged with 1% O2 as either RBs or MBs were tested. The MB-sparged yeast retained higher levels of ergosteryl esters during the fermentation phase, but this did not result in enhanced viability or ethanol production compared to ungassed or RB-sparged fermentations. CONCLUSIONS: The performance of yeast propagated using energy-efficient MB technology in bioethanol fermentations is comparable to that of those propagated conventionally. This should underpin the future development of MB-based commercial yeast propagation.

Multimodal Multiplexed Immunoimaging with Nanostars to Detect Multiple Immunomarkers and Monitor Response to Immunotherapies.

The overexpression of immunomarker programmed cell death protein 1 (PD-1) and engagement of PD-1 to its ligand, PD-L1, are involved in the functional impairment of cluster of differentiation 8+ (CD8+) T cells, contributing to cancer progression. However, heterogeneities in PD-L1 expression and variabilities in biopsy-based assays render current approaches inaccurate in predicting PD-L1 status. Therefore, PD-L1 screening alone is not predictive of patient response to treatment, which motivates us to simultaneously detect multiple immunomarkers engaged in immune modulation. Here, we have developed multimodal probes, immunoactive gold nanostars (IGNs), that accurately detect PD-L1+ tumor cells and CD8+ T cells simultaneously in vivo, surpassing the limitations of current immunoimaging techniques. IGNs integrate the whole-body imaging of positron emission tomography with high sensitivity and multiplexing of Raman spectroscopy, enabling the dynamic tracking of both immunomarkers. IGNs also monitor response to immunotherapies in mice treated with combinatorial PD-L1 and CD137 agonists and distinguish responders from those nonresponsive to treatment. Our results showed a multifunctional nanoscale probe with capabilities that cannot be achieved with either modality alone, allowing multiplexed immunologic tumor profiling critical for predicting early response to immunotherapies.

Whole Egg Consumption Impairs Insulin Sensitivity in a Rat Model of Obesity and Type 2 Diabetes.

BACKGROUND: The literature regarding the relation between egg consumption and type 2 diabetes (T2D) is inconsistent and there is limited evidence pertaining to the impact of egg consumption on measures of insulin sensitivity. OBJECTIVES: The aim of this study was to investigate the effect of dietary whole egg on metabolic biomarkers of insulin resistance in T2D rats. METHODS: Male Zucker diabetic fatty (ZDF) rats (n = 12; 6 wk of age) and age-matched lean controls (n = 12) were randomly assigned to be fed a casein- or whole egg-based diet. At week 5 of dietary treatment, an insulin tolerance test (ITT) was performed on all rats and blood glucose was measured by glucometer. After 7 wk of dietary treatment, rats were anesthetized and whole blood was collected via a tail vein bleed. Following sedation, the extensor digitorum longus muscle was removed before and after an intraperitoneal insulin injection, and insulin signaling in skeletal muscle was analyzed by Western blot. Serum glucose and insulin were analyzed by ELISA for calculation of the homeostatic model assessment of insulin resistance (HOMA-IR). RESULTS: Mean ITT blood glucose over the course of 60 min was 32% higher in ZDF rats fed the whole egg-based diet than in ZDF rats fed the casein-based diet. Furthermore, whole egg consumption increased fasting blood glucose by 35% in ZDF rats. Insulin-stimulated phosphorylation of key proteins in the insulin signaling pathway did not differ in skeletal muscle of ZDF rats fed casein- and whole egg-based diets. In lean rats, no differences were observed in insulin tolerance, HOMA-IR and skeletal muscle insulin signaling, regardless of experimental dietary treatment. CONCLUSIONS: These data suggest that whole body insulin sensitivity may be impaired by whole egg consumption in T2D rats, although no changes were observed in skeletal muscle insulin signaling that could explain this finding.

Proteomic Profiling, Transcription Factor Modeling, and Genomics of Evolved Tolerant Strains Elucidate Mechanisms of Vanillin Toxicity in Escherichia coli.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an economically important flavor compound that can be made in bacterial cell factories, but toxicity is a major problem for cells producing this aromatic aldehyde. Using (i) a global proteomic analysis supported by multiple physiological experiments, mutant analyses, and inferred transcription factor modeling and (ii) adaptive laboratory evolution (ALE) of vanillin tolerance combined with genome-wide analysis of the underlying mutations, mechanisms of vanillin toxicity in Escherichia coli have been elucidated. We identified 147 proteins that exhibited a significant change in abundance in response to vanillin, giving the first detailed insight into the cellular response to this aldehyde. Vanillin caused accumulation of reactive oxygen species invoking adaptations coordinated by a MarA, OxyR, and SoxS regulatory network and increased RpoS/DksA-dependent gene expression. Differential fumarase C upregulation was found to prevent oxidative damage to FumA and FumB during growth with vanillin. Surprisingly, vanillin-dependent reduction pf copper (II) to copper (I) led to upregulation of the copA gene and growth in the presence of vanillin was shown to be hypersensitive to inhibition by copper ions. AcrD and AaeAB were identified as potential vanillin efflux systems. Vanillin-tolerant strains isolated by ALE had distinct nonsynonymous single nucleotide polymorphisms (SNPs) in gltA that led to increased citrate synthase activity. Strain-specific mutations in cpdA, rob, and marC were also present. One strain had a large (∼10-kb) deletion that included the marRAB region. Our data provide new understanding of bacterial vanillin toxicity and identify novel gene targets for future engineering of vanillin-tolerant strains of E. coli IMPORTANCE A particular problem for the biotechnological production of many of the valuable chemicals that we are now able to manufacture in bacterial cells is that these products often poison the cells producing them. Solutions to improve product yields or alleviate such toxicity using the techniques of modern molecular biology first require a detailed understanding of the mechanisms of product toxicity. Here we have studied the economically important flavor compound vanillin, an aromatic aldehyde that exerts significant toxic effects on bacterial cells. We used high-resolution protein abundance analysis as a starting point to determine which proteins are upregulated and which are downregulated by growth with vanillin, followed by gene expression and mutant studies to understand the mechanism of the response. In a second approach, we evolved bacterial strains with higher vanillin tolerance. Their genome sequences have yielded novel insights into vanillin tolerance that are complementary to the proteomics data set.

Systems Analyses Reveal the Resilience of Escherichia coli Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate.

Productivity of bacterial cell factories is frequently compromised by stresses imposed by recombinant protein synthesis and carbon-to-product conversion, but little is known about these bioprocesses at a systems level. Production of the unnatural metabolite citramalate in Escherichia coli requires the expression of a single gene coding for citramalate synthase. Multiomic analyses of a fermentation producing 25 g liter-1 citramalate were undertaken to uncover the reasons for its productivity. Metabolite, transcript, protein, and lipid profiles of high-cell-density, fed-batch fermentations of E. coli expressing either citramalate synthase or an inactivated enzyme were similar. Both fermentations showed downregulation of flagellar genes and upregulation of chaperones IbpA and IbpB, indicating that these responses were due to recombinant protein synthesis and not citramalate production. Citramalate production did not perturb metabolite pools, except for an increased intracellular pyruvate pool. Gene expression changes in response to citramalate were limited; none of the general stress response regulons were activated. Modeling of transcription factor activities suggested that citramalate invoked a GadW-mediated acid response, and changes in GadY and RprA regulatory small RNA (sRNA) expression supported this. Although changes in membrane lipid composition were observed, none were unique to citramalate production. This systems analysis of the citramalate fermentation shows that E. coli has capacity to readily adjust to the redirection of resources toward recombinant protein and citramalate production, suggesting that it is an excellent chassis choice for manufacturing organic acids.IMPORTANCE Citramalate is an attractive biotechnology target because it is a precursor of methylmethacrylate, which is used to manufacture Perspex and other high-value products. Engineered E. coli strains are able to produce high titers of citramalate, despite having to express a foreign enzyme and tolerate the presence of a nonnative biochemical. A systems analysis of the citramalate fermentation was undertaken to uncover the reasons underpinning its productivity. This showed that E. coli readily adjusts to the redirection of metabolic resources toward recombinant protein and citramalate production and suggests that E. coli is an excellent chassis for manufacturing similar small, polar, foreign molecules.

Diagnosis of immunomarkers in vivo via multiplexed surface enhanced Raman spectroscopy with gold nanostars.

In this work, we demonstrate the targeted diagnosis of immunomarker programmed death ligand 1 (PD-L1) and simultaneous detection of epidermal growth factor receptor (EGFR) in breast cancer tumors in vivo using gold nanostars (AuNS) with multiplexed surface enhanced Raman spectroscopy (SERS). Real-time longitudinal tracking with SERS demonstrated maximum accumulation of AuNS occurred 6 h post intravenous (IV) delivery, enabling detection of both biomarkers simultaneously. Raman signal correlating to both PD-L1 and EGFR decreased by ∼30% in control tumors where receptors were pre-blocked prior to AuNS delivery, indicating both the sensitivity and specificity of SERS in distinguishing tumors with different levels of PD-L1 and EGFR expression. Our in vivo study was combined with the first demonstration of ex vivo SERS spatial maps of whole tumor lesions that provided both a qualitative and quantitative assessment of biomarker status with near cellular-level resolution. High resolution SERS maps also provided an overview of AuNS distribution in tumors which correlated well with the vascular density. Mass spectrometry showed AuNS accumulation in tumor and liver, and clearance via spleen, and electron microscopy revealed AuNS were endocytosed in tumors, Kupffer cells in the liver, and macrophages in the spleen. This study demonstrates that SERS-based diagnosis mediated by AuNS provides an accurate measure of multiple biomarkers both in vivo and ex vivo, which will ultimately enable a clinically-translatable platform for patient-tailored immunotherapies and combination treatments.