Metals and other ligands balance carbon fixation and photorespiration in chloroplasts.

The behavior of many plant enzymes depends on the metals and other ligands to which they are bound. A previous study demonstrated that tobacco Rubisco binds almost equally to magnesium and manganese and rapidly exchanges one metal for the other. The present study characterizes the kinetics of Rubisco and the plastidial malic enzyme when bound to either metal. When Rubisco purified from five C3 species was bound to magnesium rather than manganese, the specificity for CO2 over O2, (Sc/o) increased by 25% and the ratio of the maximum velocities of carboxylation / oxygenation (Vcmax/Vomax) increased by 39%. For the recombinant plastidial malic enzyme, the forward reaction (malate decarboxylation) was 30% slower and the reverse reaction (pyruvate carboxylation) was three times faster when bound to manganese rather than magnesium. Adding 6-phosphoglycerate and NADP+ inhibited carboxylation and oxygenation when Rubisco was bound to magnesium and stimulated oxygenation when it was bound to manganese. Conditions that favored RuBP oxygenation stimulated Rubisco to convert as much as 15% of the total RuBP consumed into pyruvate. These results are consistent with a stromal biochemical pathway in which (1) Rubisco when associated with manganese converts a substantial amount of RuBP into pyruvate, (2) malic enzyme when associated with manganese carboxylates a substantial portion of this pyruvate into malate, and (3) chloroplasts export additional malate into the cytoplasm where it generates NADH for assimilating nitrate into amino acids. Thus, plants may regulate the activities of magnesium and manganese in leaves to balance organic carbon and organic nitrogen as atmospheric CO2 fluctuates.

Structural insights into the disulfide isomerase and chaperone activity of TrbB of the F plasmid type IV secretion system.

Bacteria have evolved elaborate mechanisms to thrive in stressful environments. F-like plasmids in gram-negative bacteria encode for a multi-protein Type IV Secretion System (T4SSF) that is functional for bacterial proliferation and adaptation through the process of conjugation. The periplasmic protein TrbB is believed to have a stabilizing chaperone role in the T4SSF assembly, with TrbB exhibiting disulfide isomerase (DI) activity. In the current report, we demonstrate that the deletion of the disordered N-terminus of TrbBWT, resulting in a truncation construct TrbB37-161, does not affect its catalytic in vitro activity compared to the wild-type protein (p = 0.76). Residues W37-K161, which include the active thioredoxin motif, are sufficient for DI activity. The N-terminus of TrbBWT is disordered as indicated by a structural model of GST-TrbBWT based on ColabFold-AlphaFold2 and Small Angle X-Ray Scattering data and 1H-15N Heteronuclear Single Quantum Correlation (HSQC) spectroscopy of the untagged protein. This disordered region likely contributes to the protein's dynamicity; removal of this region results in a more stable protein based on 1H-15N HSQC and Circular Dichroism Spectroscopies. Lastly, size exclusion chromatography analysis of TrbBWT in the presence of TraW, a T4SSF assembly protein predicted to interact with TrbBWT, does not support the inference of a stable complex forming in vitro. This work advances our understanding of TrbB's structure and function, explores the role of structural disorder in protein dynamics in the context of a T4SSF accessory protein, and highlights the importance of redox-assisted protein folding in the T4SSF.

Optimizing Archaeal Lipid Biosynthesis in Escherichia coli.

Membrane lipid chemistry is remarkably different in archaea compared with bacteria and eukaryotes. In the evolutionary context, this is also termed the lipid divide and is reflected by distinct biosynthetic pathways. Contemporary organisms have almost without exception only one type of membrane lipid. During early membrane evolution, mixed membrane stages likely occurred, and it was hypothesized that the instability of such mixtures was the driving force for the lipid divide. To examine the compatibility between archaeal and bacterial lipids, the bacterium Escherichia coli has been engineered to contain both types of lipids with varying success. Only limited production of archaeal lipid archaetidylethanolamine was achieved. Here, we substantially increased its production in E. coli by overexpression of an archaeal phosphatidylserine synthase needed for ethanolamine headgroup attachment. Furthermore, we introduced a synthetic isoprenoid utilization pathway to increase the supply of isopentenyl-diphosphate and dimethylallyl diphosphate. This improved archaeal lipid production substantially. The archaeal phospholipids also served as a substrate for the E. coli cardiolipin synthase, resulting in archaeal and novel hybrid archaeal/bacterial cardiolipin species not seen in living organisms before. Growth of the E. coli strain with the mixed membrane shows an enhanced sensitivity to the inhibitor of fatty acid biosynthesis, cerulenin, indicating a critical dependence of the engineered E. coli strain on its native phospholipids.

Indication and efficacy of injection laryngoplasty for laryngeal clefts - A retrospective cohort study.

PURPOSE: Laryngeal cleft (LC) is an anatomical defect of the larynx, where there is a gap (or cleft) between the arytenoids. Milder types can be treated with injection laryngoplasty (IL), involving injection with a filler, resulting in a decreased depth of the cleft and thereby reducing tracheal penetration or aspiration. The effect, however, is temporary. The aim of this study was to investigate the possible indications and the efficacy of IL for LC. METHODS: Patients who underwent IL for LC between March 2018 and June 2023 were retrospectively evaluated. The following parameters were studied: incidence of LC symptoms and objective swallowing evaluations before and after IL, the duration of possible symptom improvement, complications, and the number of subsequent suture repairs. RESULTS: Eighty-five patients were included. Before IL, 81 (96 %) patients had symptoms of aspiration during feeding, compared to 41 (54 %) patients after IL (p ≤ 0.001). In 42 (49 %) patients, temporary symptom relief occurred, in 22 (26 %) patients symptoms persisted, in 16 (19 %) patients symptoms decreased permanently. Mild complications such as cough and desaturations in the direct postoperative period occurred. CONCLUSION: This study shows a statistically significant decrease in the number of parents/caretakers reporting swallowing symptoms after injection laryngoplasty, and a decrease in the average percentage of parents/caretakers reporting various other symptoms. Based on our results, injection laryngoplasty can be recommended as a diagnostic tool in the treatment of laryngeal cleft. Furthermore, it can be used as bridge therapy (i.e. until patients outgrow their symptoms, or until suture repair).

Gunshot injuries in Central Europe - Epidemiology and outcome in Germany, Switzerland and Austria - an analysis based on the TraumaRegister DGU®.

BACKGROUND: Firearms are of special interest in trauma research due to high lethality and criminal value. Strong correlation between guns per capita and fire-arm related deaths has been shown. Most of existing literature regarding gun-shot fatalities are from the U.S. and data for Central Europe is lacking. Thus, the aim of this study was to assess the legal frameworks regarding gun-ownership in Germany (DE), Austria (A) and Switzerland (CH), and to retrospectively analyze data from the TraumaRegister DGU® regarding the epidemiology, injury severity, intention and outcome of gunshot-related deaths in these countries. METHODS: All patients from TR-DGU who sustained a gunshot injury in the time period from 1st January 2009 to 31st December 2019 were considered for analysis. Only cases admitted to level 1 or 2 trauma center in Germany, Switzerland, or Austria were included. Predicted mortality was calculated using the RISC-II. Further, the legal framework for firearm posession were explored. RESULTS: The legal frameworks do not differ significantly between the countries. However, only ex-military men from Switzerland are allowed to keep their automatic (military) weapon at home. We assessed 1312 gunshot fatalities (DE 1,099, A 111, CH 102) of which most were due to suspected suicide (A 72.1 %, CH 64.7 %, and DE 56.6 %, p = 0.003). Act of suspected violent crime or accidental gunshots were rare in all three countries. Amongst all gunshot fatalities, Austria showed the highest mean age (57.6 years), followed by DE (53.4 years) and CH (49.4 years; p < 0.01). Gunshot fatalities amongst all assessed countries due to suspected suicide showed a peak at the age of 60 years and above, whereas suspected violent crime delicts with gunshots were mainly seen in younger age groups. The highest mortality was found in suspected suicide cases, showing a mortality of 82.1 % (predicted 65.2 %) in Switzerland, 75.3 % (predicted 65.8 %) in Austria and 63.7 % (predicted 56.2 %) in Germany. CONCLUSION: Gunshot wounds are still rare in central Europe, but gunshot-related suicide rates are high. Gun ownership laws may have an impact on gunshot wounds due to suspected suicide. Injury patterns differ compared to countries where a high incidence of gun ownership is seen.

Adenovirus small E1A directs activation of Alu transcription at YAP/TEAD- and AP-1-bound enhancers through interactions with the EP400 chromatin remodeler.

Alu retrotransposons, which form the largest family of mobile DNA elements in the human genome, have recently come to attention as a potential source of regulatory novelties, most notably by participating in enhancer function. Even though Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, their expression has long been known to increase in response to various types of stress, including viral infection. Here we show that, in primary human fibroblasts, adenovirus small e1a triggered derepression of hundreds of individual Alus by promoting TFIIIB recruitment by Alu-bound TFIIIC. Epigenome profiling revealed an e1a-induced decrease of H3K27 acetylation and increase of H3K4 monomethylation at derepressed Alus, making them resemble poised enhancers. The enhancer nature of e1a-targeted Alus was confirmed by the enrichment, in their upstream regions, of the EP300/CBP acetyltransferase, EP400 chromatin remodeler and YAP1 and FOS transcription factors. The physical interaction of e1a with EP400 was critical for Alu derepression, which was abrogated upon EP400 ablation. Our data suggest that e1a targets a subset of enhancer Alus whose transcriptional activation, which requires EP400 and is mediated by the e1a-EP400 interaction, may participate in the manipulation of enhancer activity by adenoviruses.

Comparative effects of calcitriol and calcimimetic on bone health in renal insufficiency.

Calcitriol and calcimimetics are used to treat hyperparathyroidism secondary to chronic kidney disease (CKD). Calcitriol administration and the subsequent increase in serum calcium concentration decrease parathyroid hormone (PTH) levels, which should reduce bone remodeling. We have previously reported that, when maintaining a given concentration of PTH, the addition of calcimimetics is associated with an increased bone cell activity. Whether calcitriol administration affects bone cell activity while PTH is maintained constant should be evaluated in an animal model of renal osteodystrophy. The aim of the present study was to compare in CKD PTH-clamped rats the bone effects of calcitriol and calcimimetic administration. The results show that the administration of calcitriol and calcimimetic at doses that induced a similar reduction in PTH secretion produced dissimilar effects on osteoblast activity in 5/6 nephrectomized (Nx) rats with secondary hyperparathyroidism and in Nx rats with clamped PTH. Remarkably, in both rat models, the administration of calcitriol decreased osteoblastic activity, whereas calcimimetic increased bone cell activity. In vitro, calcitriol supplementation inhibited nuclear translocation of ß-catenin and reduced proliferation, osteogenesis, and mineralization in mesenchymal stem cells differentiated into osteoblasts. In conclusion, besides the action of calcitriol and calcimimetics at parathyroid level, these treatments have specific effects on bone cells that are independent of the PTH level.

SARS-CoV-2 Delta variant remains viable in environmental biofilms found in meat packaging plants.

To determine why SARS-CoV-2 appears to thrive specifically well in meat packaging plants, we used SARS-CoV-2 Delta variant and meat packaging plant drain samples to develop mixed-species biofilms on materials commonly found within meat packaging plants (stainless steel (SS), PVC, and ceramic tile). Our data provides evidence that SARS-CoV-2 Delta variant remained viable on all the surfaces tested with and without an environmental biofilm after the virus was inoculated with the biofilm for 5 days at 7°C. We observed that SARS-CoV-2 Delta variant was able to remain infectious with each of the environmental biofilms by conducting plaque assay and qPCR experiments, however, we detected a significant reduction in viability post-exposure to Plant B biofilm on SS, PVC, and on ceramic tile chips, and to Plant C biofilm on SS and PVC chips. The numbers of viable SARS-CoV-2 Delta viral particles was 1.81-4.57-fold high than the viral inoculum incubated with the Plant B and Plant C environmental biofilm on SS, and PVC chips. We did not detect a significant difference in viability when SARS-CoV-2 Delta variant was incubated with the biofilm obtained from Plant A on any of the materials tested and SARS-CoV-2 Delta variant had higher plaque numbers when inoculated with Plant C biofilm on tile chips, with a 2.75-fold difference compared to SARS-CoV-2 Delta variant on tile chips by itself. In addition, we detected an increase in the biofilm biovolume in response to SARS-CoV-2 Delta variant which is also a concern for food safety due to the potential for foodborne pathogens to respond likewise when they come into contact with the virus. These results indicate a complex virus-environmental biofilm interaction which correlates to the different bacteria found in each biofilm. Our results also indicate that there is the potential for biofilms to protect SARS-CoV-2 from disinfecting agents and remaining prevalent in meat packaging plants.

Androgen aggravates aortic aneurysms via suppression of PD-1 in mice.

Androgen has long been recognized for its pivotal role in the sexual dimorphism of cardiovascular diseases, including aortic aneurysms (AAs), a devastating vascular disease with a higher prevalence and fatality rate in men than in women. However, the mechanism by which androgen mediates AAs is largely unknown. Here, we found that male, not female, mice developed AAs when exposed to aldosterone and high salt (Aldo-salt). We revealed that androgen and androgen receptors (ARs) were crucial for this sexually dimorphic response to Aldo-salt. We identified programmed cell death protein 1 (PD-1), an immune checkpoint, as a key link between androgen and AAs. Furthermore, we demonstrated that administration of anti-PD-1 Ab and adoptive PD-1-deficient T cell transfer reinstated Aldo-salt-induced AAs in orchiectomized mice and that genetic deletion of PD-1 exacerbated AAs induced by a high-fat diet and angiotensin II (Ang II) in nonorchiectomized mice. Mechanistically, we discovered that the AR bound to the PD-1 promoter to suppress the expression of PD-1 in the spleen. Thus, our study unveils a mechanism by which androgen aggravates AAs by suppressing PD-1 expression in T cells. Moreover, our study suggests that some patients with cancer might benefit from screenings for AAs during immune checkpoint therapy.

SecYEG-mediated translocation in a model synthetic cell.

Giant unilamellar vesicles (GUVs) provide a powerful model compartment for synthetic cells. However, a key challenge is the incorporation of membrane proteins that allow for transport, energy transduction, compartment growth and division. Here, we have successfully incorporated the membrane protein complex SecYEG-the key bacterial translocase that is essential for the incorporation of newly synthesized membrane proteins-in GUVs. Our method consists of fusion of small unilamellar vesicles containing reconstituted SecYEG into GUVs, thereby forming SecGUVs. These are suitable for large-scale experiments while maintaining a high protein:lipid ratio. We demonstrate that incorporation of SecYEG into GUVs does not inhibit its translocation efficiency. Robust membrane protein functionalized proteo-GUVs are promising and flexible compartments for use in the formation and growth of synthetic cells.

Early-onset Colorectal Cancer Patients Do Not Require Shorter Intervals for Post-surgical Surveillance Colonoscopy.

BACKGROUND & AIMS: Early-onset colorectal cancer (EO-CRC), diagnosed before age 50, is rising in incidence worldwide. Although post-surgical colonoscopy surveillance strategies exist, appropriate intervals in EO-CRC remain elusive, as long-term surveillance outcomes remain scant. We sought to compare findings of surveillance colonoscopies of EO-CRC with patients with average onset colorectal cancer (AO-CRC) to help define surveillance outcomes in these groups. METHODS: Single-institution retrospective chart review identified EO-CRC and AO-CRC patients with colonoscopy and no evidence of disease. Surveillance intervals and time to development of advanced neoplasia (CRC and advanced polyps [adenoma/sessile serrated]) were examined. For each group, 3 serial surveillance colonoscopies were evaluated. Statistical analyses were performed utilizing log-ranked Kaplan-Meier method and Cox proportional hazards. RESULTS: A total of 1259 patients with CRC were identified, with 612 and 647 patients in the EO-CRC and AO-CRC groups, respectively. Compared with patients with AO-CRC, patients with EO-CRC had a 29% decreased risk of developing advanced neoplasia from time of initial surgery to first surveillance colonoscopy (hazard ratio, 0.71; 95% confidence interval, 0.52-1.0). Average follow-up time from surgical resection to first surveillance colonoscopy was 12.6 months for both cohorts. Overall surveillance findings differed between cohorts (P = .003), and patients with EO-CRC were found to have less advanced neoplasia compared with their counterparts with AO-CRC (12.4% vs 16.0%, respectively). Subsequent colonoscopies found that, while patients with EO-CRC returned for follow-up surveillance colonoscopy earlier than patients with AO-CRC, the EO-CRC cohort did not have more advanced neoplasia nor non-advanced adenomas. CONCLUSIONS: Patients with EO-CRC do not have an increased risk of advanced neoplasia compared with patients with AO-CRC and therefore do not require more frequent colonoscopy surveillance than current guidelines recommend.

Broadband, High-Reflectivity Dielectric Mirrors at Wafer Scale: Combining Photonic Crystal and Metasurface Architectures for Advanced Lightsails.

Highly ambitious initiatives aspire to propel a miniature spacecraft to a neighboring star within a human generation, leveraging the radiation pressure of lasers for propulsion. One major challenge for this enormous feat is to build a meter-scale, ultralow mass lightsail with broadband reflectivity. In this work, we present the design and fabrication of a lightsail composed of two distinct dielectric layers with photonic crystal/metasurface structure covering a 4" wafer. We achieved broadband reflection of >70% spanning over the full Doppler-shifted laser wavelength range during spacecraft acceleration with a low total mass in the range of a few grams when scaled up to meter size. Furthermore, we find new paths to reliably fabricate these subwavelength structures over macroscopic areas and then systematically characterize their optical performance, confirming their suitability for future lightsail applications. Our innovative device and precise nanofabrication approaches represent a significant leap toward interstellar exploration.

Flicker and reading speed: Effects on individuals with visual sensitivity.

Flicker and patterns of stripes in the modern environment can evoke visual illusions, discomfort migraine, and seizures. We measured reading speed while striped and less striped texts were illuminated with LED lights. In Experiment 1, the lights flickered at 60 Hz and 120 Hz compared to 60 kHz (perceived as steady light). In Experiment 2, the lights flickered at 60 Hz or 600 Hz (at which frequency the phantom array is most visible), and were compared to continuous light. Two types of text were used: one containing words with high horizontal autocorrelation (striped) and another containing words with low autocorrelation (less striped). We measured the number of illusions participants saw in the Pattern Glare (PG) Test. Overall, reading speed was slowest during the 60 Hz and 600 Hz flicker and was slower when reading the high autocorrelation text. Interestingly, the low PG group showed greater effects of flicker on reading speed than the high PG group, which tended to be slower overall. In addition, reading speed in the high PG group was reduced when the autocorrelation of the text was high. These findings suggest that uncomfortable visual environments reduce reading efficiency, the more so in individuals who are visually sensitive.

Distinct genomic and immunologic tumor evolution in germline TP53-driven breast cancers.

Pathogenic germline TP53 alterations cause Li-Fraumeni Syndrome (LFS), and breast cancer is the most common cancer in LFS females. We performed first of its kind multimodal analysis of LFS breast cancer (LFS-BC) compared to sporadic premenopausal BC. Nearly all LFS-BC underwent biallelic loss of TP53 with no recurrent oncogenic variants except ERBB2 (HER2) amplification. Compared to sporadic BC, in situ and invasive LFS-BC exhibited a high burden of short amplified aneuploid segments (SAAS). Pro-apoptotic p53 target genes BAX and TP53I3 failed to be up-regulated in LFS-BC as was seen in sporadic BC compared to normal breast tissue. LFS-BC had lower CD8+ T-cell infiltration compared to sporadic BC yet higher levels of proliferating cytotoxic T-cells. Within LFS-BC, progression from in situ to invasive BC was marked by an increase in chromosomal instability with a decrease in proliferating cytotoxic T-cells. Our study uncovers critical events in mutant p53-driven tumorigenesis in breast tissue.

No trauma-related diagnosis in emergency trauma room whole-body computer tomography of patients with inconspicuous primary survey.

PURPOSE: Whole-body computer tomographic examinations (WBCT) are essential in diagnosing the severely injured. The structured clinical evaluation in the emergency trauma room, according to ATLS® and guidelines, helps to indicate the correct radiological imaging to avoid overtriage and undertriage. This retrospective, single-center study aimed to evaluate the value of WBCT in patients with an inconspicuous primary survey and whether there is any evidence for this investigation in this group of patients. METHODS: This retrospective, single-center study was conducted with patients admitted to a maximum-care hospital and supraregional trauma center in Germany and part of the TraumaNetwork DGU® in southwest Germany between January 2012 and November 2017. Hospital files were used for evaluation, and WBCT was carried out using a 32-row MSCT device from Siemens Healthineers, Volume Zoom, Erlangen, Germany. For evaluation, non-parametric procedures such as the chi-square test, U test, Fisher test, and Wilcoxon rank sum test were used to test for significance (p < 0.05). RESULTS: From 3976 patients treated with WBCT, 120 patients (3.02%) showed an inconspicuous primary survey. This examination did not reveal any trauma sequelae in any of this group. Additionally, 198 patients (4.98%) showed minor clinical symptoms in the primary survey, but no morphological trauma sequence could be diagnosed in WBCT diagnostics. Three hundred forty-two patients were not admitted as inpatients after WBCT and discharged to further outpatient treatment because there were no objectifiable reasons for inpatient treatment. Four hundred fifteen patients did not receive WBCT for, e.g., isolated extremity trauma, child, pregnancy, or death. CONCLUSION: Not one of the clinically asymptomatic patients had an imageable injury after WBCT diagnostics in this study. WBCT should only be performed in severely injured patients after clinical assessment regardless of "trauma mechanism." According to guidelines and ATLS®, the clinical examination seems to be a safe and reliable method for reasonable and responsible decision-making regarding the realization of WBCT with all well-known risk factors.

Ictal-Interictal Continuum in the Pediatric Intensive Care Unit.

BACKGROUND: The ictal-interictal continuum (IIC) consists of several electroencephalogram (EEG) patterns that are common in critically ill adults. Studies focused on the IIC are limited in critically ill children and have focused primarily on associations with electrographic seizures (ESs). We report the incidence of the IIC in the pediatric intensive care unit (PICU). We then compare IIC patterns to rhythmic and periodic patterns (RPP) not meeting IIC criteria looking for associations with acute cerebral abnormalities, ES, and in-hospital mortality. METHODS: This was a retrospective review of prospectively collected data for patients admitted to the PICU at Children's National Hospital from July 2021 to January 2023 with continuous EEG. We excluded patients with known epilepsy and cerebral injury prior to presentation. All patients were screened for RPP. The American Clinical Neurophysiology Society standardized Critical Care EEG terminology for the IIC was applied to each RPP. Associations between IIC and RPP not meeting IIC criteria, with clinical and EEG variables, were calculated using odds ratios (ORs). RESULTS: Of 201 patients, 21% (42/201) had RPP and 12% (24/201) met IIC criteria. Among patients with an IIC pattern, the median age was 3.4 years (interquartile range (IQR) 0.6-12 years). Sixty-seven percent (16/24) of patients met a single IIC criterion, whereas the remainder met two criteria. ESs were identified in 83% (20/24) of patients and cerebral injury was identified in 96% (23/24) of patients with IIC patterns. When comparing patients with IIC patterns with those with RPP not qualifying as an IIC pattern, both patterns were associated with acute cerebral abnormalities (IIC OR 26 [95% confidence interval {CI} 3.4-197], p = 0.0016 vs. RPP OR 3.5 [95% CI 1.1-11], p = 0.03), however, only the IIC was associated with ES (OR 121 [95% CI 33-451], p < 0.0001) versus RPP (OR 1.3 [0.4-5], p = 0.7). CONCLUSIONS: Rhythmic and periodic patterns and subsequently the IIC are commonly seen in the PICU and carry a high association with cerebral injury. Additionally, the IIC, seen in more than 10% of critically ill children, is associated with ES. The independent impact of RPP and IIC patterns on secondary brain injury and need for treatment of these patterns independent of ES requires further study.

Maltose accumulation-induced cell death in Saccharomyces cerevisiae.

Pretreatment of lignocellulose yields a complex sugar mixture that potentially can be converted into bioethanol and other chemicals by engineered yeast. One approach to overcome competition between sugars for uptake and metabolism is the use of a consortium of specialist strains capable of efficient conversion of single sugars. Here, we show that maltose inhibits cell growth of a xylose-fermenting specialist strain IMX730.1 that is unable to utilize glucose because of the deletion of all hexokinase genes. The growth inhibition cannot be attributed to a competition between maltose and xylose for uptake. The inhibition is enhanced in a strain lacking maltase enzymes (dMalX2) and completely eliminated when all maltose transporters are deleted. High-level accumulation of maltose in the dMalX2 strain is accompanied by a hypotonic-like transcriptional response, while cells are rescued from maltose-induced cell death by the inclusion of an extracellular osmolyte such as sorbitol. These data suggest that maltose-induced cell death is due to high levels of maltose uptake causing hypotonic-like stress conditions and can be prevented through engineering of the maltose transporters. Transporter engineering should be included in the development of stable microbial consortia for the efficient conversion of lignocellulosic feedstocks.

Synthetic Vesicles for Sustainable Energy Recycling and Delivery of Building Blocks for Lipid Biosynthesis†.

ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.