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In recent years, biotechnological conversion of the alternative carbon source acetate has attracted much attention. So far, acetate has been mainly used for microbial production of bioproducts with bulk applications. In this study, we aimed to investigate the potential of acetate as carbon source for heterologous protein production using the acetate‐utilizing platform organism Corynebacterium glutamicum. For this purpose, expression of model protein eYFP with the promoter systems T7lac and tac was characterized during growth of C. glutamicum on acetate as sole carbon source. The results indicated a 3.3‐fold higher fluorescence level for acetate‐based eYFP production with T7 expression strain MB001(DE3) pMKEx2‐eyfp compared to MB001 pEKEx2‐eyfp. Interestingly, comparative eyfp expression studies on acetate or glucose revealed an up to 83% higher biomass‐specific production for T7 RNAP‐dependent eYFP production using acetate as carbon source. Furthermore, high‐level protein accumulation on acetate was demonstrated for the first time in a high cell density cultivation process with pH‐coupled online feeding control, resulting in a final protein titer of 2.7 g/L and product yield of 4 g per 100 g cell dry weight. This study presents a first proof of concept for efficient microbial upgrading of potentially low‐cost acetate into high‐value bioproducts, such as recombinant proteins. In this study, we aimed to investigate the potential of acetate as alternative carbon source for heterologous protein production using the acetate‐utilizing platform organism Corynebacterium glutamicum. For this purpose, expression of model protein eYFP was characterized during batch and fed‐batch cultures of C. glutamicum T7 expression system on acetate as sole carbon source. This study presents a first proof of concept for efficient microbial upgrading of potentially low‐cost acetate into high‐value bioproducts, such as recombinant proteins.

Background Itaconic acid is a promising platform chemical for a bio-based polymer industry. Today, itaconic acid is biotechnologically produced with Aspergillus terreus at industrial scale from sugars. The production of fuels but also of chemicals from food substrates is a dilemma since future processes should rely on carbon sources which do not compete for food or feed. Therefore, the production of chemicals from alternative substrates such as acetate is desirable to develop novel value chains in the bioeconomy. Results In this study, Corynebacterium glutamicum ATCC 13032 was engineered to efficiently produce itaconic acid from the non-food substrate acetate. Therefore, we rewired the central carbon and nitrogen metabolism by inactivating the transcriptional regulator RamB, reducing the activity of isocitrate dehydrogenase, deletion of the gdh gene encoding glutamate dehydrogenase and overexpression of cis- aconitate decarboxylase (CAD) from A. terreus optimized for expression in C. glutamicum. The final strain C. glutamicum Δ ramB Δ gdh IDH R453C  (pEKEx2- malE cad opt ) produced 3.43 ± 0.59 g itaconic acid L −1 with a product yield of 81 ± 9 mmol mol −1 during small-scale cultivations in nitrogen-limited minimal medium containing acetate as sole carbon and energy source. Lowering the cultivation temperature from 30 °C to 25 °C improved CAD activity and further increased the titer and product yield to 5.01 ± 0.67 g L −1 and 116 ± 15 mmol mol −1 , respectively. The latter corresponds to 35% of the theoretical maximum and so far represents the highest product yield for acetate-based itaconic acid production. Further, the optimized strain C. glutamicum Δ ramB Δ gdh IDH R453C  (pEKEx2- malE cad opt ), produced 3.38 ± 0.28 g itaconic acid L −1 at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis. Conclusion As shown in this study, acetate represents a suitable non-food carbon source for itaconic acid production with C. glutamicum . Tailoring the central carbon and nitrogen metabolism enabled the efficient production of itaconic acid from acetate and therefore this study offers useful design principles to genetically engineer C. glutamicum for other products from acetate.

Currently, most biotechnological products are based on microbial conversion of carbohydrate substrates that are predominantly generated from sugar- or starch-containing plants. However, direct competitive uses of these feedstocks in the food and feed industry represent a dilemma, so using alternative carbon sources has become increasingly important in industrial biotechnology. A promising alternative carbon source that may be generated in substantial amounts from lignocellulosic biomass and C1 gases is acetate. This review discusses the underexploited potential of acetate to become a next-generation platform substrate in future industrial biotechnology and summarizes alternative sources and routes for acetate production. Furthermore, biotechnological aspects of microbial acetate utilization and the state of the art of biotechnological acetate conversion into value-added bioproducts are highlighted. The search for alternative carbon sources in industrial biotechnology is driven by the competing use of commonly used sugar-based substrates in the food and feed industry.Acetate represents a highly attractive, alternative microbial carbon source for industrial biotechnology.The most interesting routes to alternatively generate acetate comprise the depolymerization of lignocellulosic materials and the Wood-Ljungdahl pathway of acetogenic bacteria to produce acetate as the main product via gas fermentation, microbial electrosynthesis, or microbial photosynthesis.Acetate and acetate-containing streams have emerged as promising carbon sources for microorganisms to produce a variety of value-added bioproducts, such as platform chemicals (e.g., succinic acid), microbial lipids, bioplastics (e.g., polyhydroxyalkanoates), and biosurfactants (e.g., rhamnolipids).

Glioblastoma presents characteristically with an exuberant, poorly functional vasculature that causes malperfusion, hypoxia and necrosis. Despite limited clinical efficacy, anti-angiogenesis resulting in vascular normalization remains a promising therapeutic approach. Yet, fundamental questions concerning anti-angiogenic therapy remain unanswered, partly due to the scale and resolution gap between microscopy and clinical imaging and a lack of quantitative data readouts. To what extend does treatment lead to vessel regression or vessel normalization and does it ameliorate or aggravate hypoxia? Clearly, a better understanding of the underlying mechanisms would greatly benefit the development of desperately needed improved treatment regimens. Here, using orthotopic transplantation of Gli36 cells, a widely used murine glioma model, we present a mesoscopic approach based on light sheet fluorescence microscopic imaging of wholemount stained tumors. Deep learning-based segmentation followed by automated feature extraction allowed quantitative analyses of the entire tumor vasculature and oxygenation statuses. Unexpectedly in this model, the response to both cytotoxic and anti-angiogenic therapy was dominated by vessel normalization with little evidence for vessel regression. Equally surprising, only cytotoxic therapy resulted in a significant alleviation of hypoxia. Taken together, we provide and evaluate a quantitative workflow that addresses some of the most urgent mechanistic questions in anti-angiogenic therapy.

Objectives To investigate whether gravitational valves reduce the risk of overdrainage complications compared with programmable valves in ventriculoperitoneal (VP) shunt surgery for idiopathic normal pressure hydrocephalus (iNPH). Background Patients with iNPH may benefit from VP shunting but are prone to overdrainage complications during posture changes. Gravitational valves with tantalum balls are considered to reduce the risk of overdrainage but their clinical effectiveness is unclear. Methods We conducted a pragmatic, randomised, multicentre trial comparing gravitational with non-gravitational programmable valves in patients with iNPH eligible for VP shunting. The primary endpoint was any clinical or radiological sign (headache, nausea, vomiting, subdural effusion or slit ventricle) of overdrainage 6 months after randomisation. We also assessed disease specific instruments (Black and Kiefer Scale) and Physical and Mental Component Scores of the Short Form 12 (SF-12) generic health questionnaire. Results We enrolled 145 patients (mean (SD) age 71.9 (6.9) years), 137 of whom were available for endpoint analysis. After 6 months, 29 patients in the standard and five patients in the gravitational shunt group developed overdrainage (risk difference −36%, 95% CI −49% to −23%; p<0.001). This difference exceeded predetermined stopping rules and resulted in premature discontinuation of patient recruitment. Disease specific outcome scales did not differ between the groups although there was a significant advantage of the gravitational device in the SF-12 Mental Component Scores at the 6 and 12 month visits. Conclusions Implanting a gravitational rather than another type of valve will avoid one additional overdrainage complication in about every third patient undergoing VP shunting for iNPH.

Abstract Background Brain biopsy of intracranial lesions is often necessary to determine specific therapy. The cost of the currently used stereotactic rigid frame and optical tracking systems for brain biopsy in dogs is often prohibitive or accuracy is not sufficient for all types of lesion. Objectives To evaluate the application accuracy of an inexpensive magnetic resonance imaging-based personalized, 3D printed brain biopsy device. Animals Twenty-two dog heads from cadavers were separated into 2 groups according to body weight (<15 kg, >20 kg). Methods Experimental study. Two target points in each cadaver head were used (target point 1: caudate nucleus, target point 2: piriform lobe). Comparison between groups was performed using the independent Student's t test or the nonparametric Mann-Whitney U Test. Results The total median target point deviation was 0.83 mm (range 0.09-2.76 mm). The separate median target point deviations for target points 1 and 2 in all dogs were 0.57 mm (range: 0.09-1.25 mm) and 0.85 mm (range: 0.14-2.76 mm), respectively. Conclusion and Clinical Importance This magnetic resonance imaging-based 3D printed stereotactic brain biopsy device achieved an application accuracy that was better than the accuracy of most brain biopsy systems that are currently used in veterinary medicine. The device can be applied to every size and shape of skull and allows precise positioning of brain biopsy needles in dogs.

Shigella flexneri proliferate in infected human epithelial cells at exceptionally high rates. This vigorous growth has important consequences for rapid progression to life-threatening bloody diarrhea, but the underlying metabolic mechanisms remain poorly understood. Here, we used metabolomics, proteomics, and genetic experiments to determine host and Shigella metabolism during infection in a cell culture model. The data suggest that infected host cells maintain largely normal fluxes through glycolytic pathways, but the entire output of these pathways is captured by Shigella , most likely in the form of pyruvate. This striking strategy provides Shigella with an abundant favorable energy source, while preserving host cell ATP generation, energy charge maintenance, and survival, despite ongoing vigorous exploitation. Shigella uses a simple three-step pathway to metabolize pyruvate at high rates with acetate as an excreted waste product. The crucial role of this pathway for Shigella intracellular growth suggests targets for antimicrobial chemotherapy of this devastating disease.

There is an increased risk of death due to drug toxicity after release from incarceration. The purpose of this study was to describe the timing, rate and circumstances of drug toxicity deaths following release from incarceration. This information can be used to help design potential preventive interventions. We reviewed coroner's files to identify deaths in adults in Ontario between 2006 and 2013 caused by drug toxicity (n = 6,978) and these records were matched with provincial correctional records to identify individuals who died within one year of being released from incarceration (n = 702). Twenty percent (n = 137) of the 702 deaths occurred within one week of release. The majority (77%, n = 538) of deaths after release involved one or more opioids. Of the deaths involving opioids, intervention by another person may have been possible in 318 cases. Between 2006 and 2013 in Ontario, one in ten drug toxicity deaths in adults occurred within one year of release from provincial incarceration. These findings may help to inform the implemention and assessment of interventions aimed at reducing drug toxicity deaths following release from incarceration.

A challenge for metabolomics analysis is ensuring that sample preparation does not alter the cells' metabolic state. This protocol describes LC/MS analysis of core metabolites from adherent mammalian cells after rapid wash and quenching steps. Metabolomics has emerged as a powerful tool for addressing biological questions. Liquid chromatography coupled with mass spectrometry (LC-MS) is widely used for metabolic characterization, including targeted and untargeted approaches. Despite recent innovations, a crucial aspect of this technique is the sample preparation for accurate data analyses. In this protocol, we present a robust and adaptable workflow for metabolic analyses of mammalian cells from adherent cell cultures, which is particularly suited for qualitative and quantitative central metabolite characterization by LC-MS. Each sample consists of 600,000 mammalian cells grown on cover glasses, allowing for fast and complete transfer of the cells for metabolite extraction or medium exchange, e.g., for labeling experiments. The sampling procedure includes a fast and efficient washing step in liquid flow in water, which reduces cross-contamination and matrix effects while minimizing perturbation of the metabolic steady state of the cells; it is followed by quenching cell metabolism. The latter is achieved by using a −20 °C cold methanol acetonitrile mixture acidified with formic acid, followed by freeze drying, metabolite extraction and LC-MS. The protocol requires 2 s for cell sampling until quenching, and the entire protocol takes a total of 1.5 h per sample when the provided nanoscale LC-MS method is applied.

Research with the evaluative priming paradigm has shown that affective evaluation processes reliably influence cognition and behavior, even when triggered outside awareness. However, the precise mechanisms underlying such subliminal evaluative priming effects, response activation vs semantic processing, are matter of a debate. In this study, we determined the relative contribution of semantic processing and response activation to masked evaluative priming with pictures and words. To this end, we investigated the modulation of masked pictorial vs verbal priming by previously activated perceptual vs semantic task sets and assessed the electrophysiological correlates of priming using event-related potential (ERP) recordings. Behavioral and electrophysiological effects showed a differential modulation of pictorial and verbal subliminal priming by previously activated task sets: Pictorial priming was only observed during the perceptual but not during the semantic task set. Verbal priming, in contrast, was found when either task set was activated. Furthermore, only verbal priming was associated with a modulation of the N400 ERP component, an index of semantic processing, whereas a priming-related modulation of earlier ERPs, indexing visuo-motor S-R activation, was found for both picture and words. The results thus demonstrate that different neuro-cognitive processes contribute to unconscious evaluative priming depending on the stimulus format.