Metabolic flux optimization of iterative pathways through orthogonal gene expression control: Application to the ß-oxidation reversal.

Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse ß-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in E. coli, which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements.

Blood transfusion reactions and risk of acute kidney injury and major adverse kidney events.

BACKGROUND: Blood transfusion reactions may have a negative impact on organ function. It is unknown whether this association holds true for acute kidney injury (AKI). Therefore, we conducted a cohort study to assess the association between transfusion reactions and the incidence of AKI and major adverse kidney events. METHODS: In this retrospective cohort study, we included patients who received transfusion of blood products during hospitalization at the Hospital Civil of Guadalajara. We analyzed them according to the development of transfusion reactions, and the aim was to assess the association between transfusion reactions and AKI during long-term follow-up. RESULTS: From 2017 to 2021, 81,635 patients received a blood product transfusion, and 516 were included in our study. The most common transfusion was red blood cell packaging (50.4%), fresh frozen plasma (28.7%) and platelets (20.9%); of the 516 patients, 129 (25%) had transfusion reactions. Patients who had transfusion reactions were older and had more comorbidities. The most common type of transfusion reaction was allergic reaction (70.5%), followed by febrile nonhemolytic reaction (11.6%) and anaphylactoid reaction (8.5%). Most cases were considered mild. Acute kidney injury was more prevalent among those who had transfusion reactions (14.7%) than among those who did not (7.8%), p = < 0.01; those with AKI had a higher frequency of diabetes, vasopressors, and insulin use. Transfusion reactions were independently associated with the development of AKI (RR 2.1, p = < 0.02). Major adverse kidney events were more common in those with transfusion reactions. The mortality rate was similar between subgroups. CONCLUSION: In our retrospective cohort of patients who received blood product transfusions, 25% experienced transfusion reactions, and this event was associated with a twofold increase in the probability of developing AKI and some of the major adverse kidney events during long follow-up.

Effectiveness and Safety of Teriflunomide in Relapsing-Remitting Multiple Sclerosis and Improvements in Quality of Life: Results from the Real-World TERICARE Study.

INTRODUCTION: Teriflunomide is a once-daily oral immunomodulator approved for relapsing forms of multiple sclerosis (MS) or relapsing-remitting multiple sclerosis (RRMS; depending on the local label), based on extensive evidence from clinical trials and a real-world setting on efficacy, tolerability and patient-reported benefits. The TERICARE study assessed the impact of teriflunomide treatment over 2 years on health-related quality of life (HRQoL) and some of the most common and disabling symptoms of MS, such as fatigue and depression. METHODS: This prospective observational study in Spain included RRMS patients treated with teriflunomide for ≤ 4 weeks. The following patient-reported outcomes (PROs) were collected at baseline and every 6 months for 2 years: the 29-item Multiple Sclerosis Impact Scale version 2 (MSIS-29), the 21-item Modified Fatigue Impact Scale (MFIS-21), the Beck Depression Inventory (BDI-II), the Short Form (SF)-Qualiveen and the Treatment Satisfaction Questionnaire for Medication v1.4 (TSQM). Annualised relapse rate (ARR), disability progression according to the Expanded Disability Status Scale (EDSS), and no evidence of disease activity (NEDA-3) were also assessed. RESULTS: A total of 325 patients were analysed. Patients had a mean (SD) age of 43.2 years (10.4), a mean baseline EDSS score of 1.75 (1.5), a mean number of relapses in the past 2 years of 1.5 (0.7), and 64% had received prior disease-modifying therapy (DMT). Patients showed significant improvements in the psychological domain of MSIS-29 from 35.9 (26.6) at baseline to 29.4 (25.5) at 18 months (p = 0.004) and 29.0 (24.6) at 24 months (p = 0.002). Levels of fatigue and depression were also reduced. After 2 years of treatment with teriflunomide, ARR was reduced to 0.17 (95% CI 0.14-0.21) from the baseline of 0.42 (95% CI 0.38-0.48), representing a 60.1% reduction. Mean EDSS scores remained stable during the study, and 79.9% of patients showed no disability progression. 54.7% of patients achieved NEDA-3 in the first 12 months, which increased to 61.4% during months 12-24. Patients reported increased satisfaction with treatment over the course of the study, regardless of whether they were DMT naive or not. CONCLUSION: Teriflunomide improves psychological aspects of HRQoL and maintains low levels of fatigue and depression. Treatment with teriflunomide over 2 years is effective in reducing ARR and disability progression.

Inhibition of pepper huasteco yellow veins virus by foliar application of ZnO nanoparticles in Capsicum annuum L.

The Pepper huasteco yellow vein virus (PHYVV) is an endemic geminivirus in Mexico causing partial or total losses in the pepper crop since the damage caused by the virus has not been fully controlled. In this work, we evaluated the effect of ZnO NPs (0, 50, 100, 150, and 200 mM) as a preventive (72 h before) and curative (72 h after) treatment of PHYVV infection in two jalapeño pepper varieties. In this study, we observed a decrease in symptoms, and it could be caused by an induction of the defense system in pepper plants and a direct action on PHYVV by foliar application of ZnO NPs. Our findings suggest that ZnO NP application significantly decreased the viral titer for both varieties at 200 mM by 15.11-fold. However, this effect was different depending on the timing of application and the variety of pepper. The greatest decrease in the viral titer in the preventive treatment in both varieties was at the concentration of 200 mM (1781.17 and 274.5 times, respectively). For curative treatment in cv. Don Pancho at the concentration of 200 mM (333.33 times) and cv. Don Benito at 100 mM (43.10 folds). compared to control. Furthermore, virus mobility was generally restricted for both varieties at 100 mM (15.13-fold) compared to the control. The results possibly delineated that ZnO NPs increased plant resistance possibly by increasing POD (2.08 and 0.25 times) and SOD (0.998 and 1.38) in cv. Don Pancho and cv. Don Benito, respectively. On the other hand, in cv. Don Pancho and cv. Don Benito presented a decrease in CAT (0.61 and 0.058) and PAL (0.78 and 0.77), respectively. Taken together, we provide the first evidence to demonstrate the effect of ZnO NPs on viral symptoms depending on the plan-virus-ZnO NP interaction.

Probiotics in septic acute kidney injury, a double blind, randomized control trial.

INTRODUCTION: During acute kidney injury (AKI) due to sepsis, the intestinal microbiota changes to dysbiosis, which affects the kidney function recovery (KFR) and amplifies the injury. Therefore, the administration of probiotics could improve dysbiosis and thereby increase the probability of KFR. METHODS: In this double-blind clinical trial, patients with AKI associated with sepsis were randomized (1:1) to receive probiotics or placebo for 7 consecutive days, with the objectives of evaluate the effect on KFR, mortality, kidney replacement therapy (KRT), urea, urine volume, serum electrolytes and adverse events at day 7. RESULTS: From February 2019 to March 2022, a total of 92 patients were randomized, 48 to the Probiotic and 44 to Placebo group. When comparing with placebo, those in the Probiotics did not observe a higher KFR (HR 0.93, 0.52-1.68, p = 0.81), nor was there a benefit in mortality at 6 months (95% CI 0.32-1.04, p = 0.06). With probiotics, urea values decreased significantly, an event not observed with placebo (from 154 to 80 mg/dl, p = 0.04 and from 130 to 109 mg/dl, p = 0.09, respectively). Urinary volume, need for KRT, electrolyte abnormalities, and adverse events were similar between groups. (ClinicalTrial.gov NCT03877081) (registered 03/15/2019). CONCLUSION: In AKI related to sepsis, probiotics for 7 consecutive days did not increase the probability of KFR, nor did other variables related to clinical improvement, although they were safe.

Methemoglobinemia in Hemodialysis Patients due to Acute Chlorine Intoxication: A Case Series Calling Attention on an Old Problem.

INTRODUCTION: Hemodialysis uses municipal water that must be strictly purified and sterilized to be used for that procedure. Large amounts of decontaminants are often used, such as chlorine, and if these compounds are not subsequently removed they can be transferred to the blood of patients causing complications including methemoglobinemia. METHODS: In this case series study, dialysis patients in one unit were evaluated. We reviewed clinical characteristics and laboratory findings obtained on the day when the water supply was disinfected with chlorine, with the aim to quantify methemoglobin concentrations. Our objective was to characterize the clinical presentation and management of patients who presented with methemoglobinemia on a specific index day. We also reviewed reported cases in the literature regarding this underreported complication. RESULTS: Eight patients who presented with chlorine intoxication were evaluated. The methemoglobin concentrations were between 1.3% and 7.9% (reference value 0-1%). We believe this to be caused by water containing 0.78 mg/L of total chlorine. Seven patients presented with cyanosis, 4 with dizziness, 6 with dark brown blood, 4 with dyspnea, and 4 with headache and hemolytic anemia. Subjects were treated with supplemental oxygen, methylene blue, intravenous vitamin C, blood transfusions, and increased doses of erythropoietin. No patient died, and all continued with their usual hemodialysis sessions. CONCLUSION: Acute chlorine intoxication transferred by the water used during hemodialysis sessions can present with methemoglobinemia accompanied by cyanosis, oxygen desaturation, and hemolytic anemia. Chlorine levels should be carefully monitored in the water used for hemodialysis treatment.

Systolic Blood Pressure and the Risk of Kidney Replacement Therapy and Mortality in Patients with Chronic Kidney Disease Stages 4-5.

INTRODUCTION: In patients with chronic kidney disease stages 4 and 5 (CKD stages 4-5) without dialysis and arterial hypertension, it is unknown if the values of systolic blood pressure (SBP) considered in control (<120 mm Hg) are associated with kidney replacement therapy (KRT) and mortality. METHODS: In this retrospective cohort study, hypertensive CKD stages 4-5 patients attending the Renal Health Clinic at the Hospital Civil de Guadalajara were enrolled. We divided them into those that achieved SBP <120 mm Hg (controlled group) and those who did not (>120 mm Hg), the uncontrolled group. Our primary objective was to analyze the association between the controlled group and KRT; the secondary objective was the mortality risk and if there were subgroups of patients that achieved more benefit. Data were analyzed using Stata software, version 15.1. RESULTS: During 2017-2022, a total of 275 hypertensive CKD stages 4-5 patients met the inclusion criteria for the analysis: 62 in the controlled group and 213 in the uncontrolled group; mean age 61 years; 49.82% were male; SBP was significantly lower in the controlled group (111 mm Hg) compared to the uncontrolled group (140 mm Hg); eGFR was similar between groups (20.41 mL/min/1.73 m2). There was a tendency to increase the mortality risk in the uncontrolled group (HR 6.47 [0.78-53.27]; p = 0.082) and an association by the Kaplan-Meir analysis (Log-rank p = 0.043). The subgroup analysis for risk of KRT in the controlled group revealed that patients ≥61 years had a lower risk of KRT (HR 0.87 [95% CI, 0-76-0.99]; p = 0.03, p of interaction = 0.005), but no differences were found in the subgroup analysis for mortality. In a follow-up of 1.34 years, no association was found in the risk of KRT according to the controlled or uncontrolled groups in a multivariate Cox analysis. CONCLUSION: In a retrospective cohort of patients with CKD stages 4-5 and hypertension, SBP >120 mm Hg was not associated with risk of KRT but could be associated with the risk of death. Clinical trials are required in this group of patients to demonstrate the impact of reaching the SBP goals recommended by the KDIGO guidelines.

Reduction of ethanol content in wine with an improved combination of yeast strains and process conditions.

One interesting strategy to address the increasing alcohol content of wines, associated with climate change, is to reduce the ethanol yield during fermentation. Within this strategy, the approach that would allow the clearest reduction in alcohol content is the respiration of part of the grape sugars by yeasts. Non-Saccharomyces species can be used for this purpose but suffer from a limited ability to dominate the process and complete fermentation. In turn, Saccharomyces cerevisiae shows a high production of acetic acid under the growth conditions required for respiration. Previously proposed procedures used combinations of non-Saccharomyces and S. cerevisiae starters, or a strain of S. cerevisiae (PR1018), with unique metabolic properties. In both cases, precise management of oxygen availability was required to overcome the acetic acid problem. In this work, we have developed a laboratory scale process to take advantage of the properties of PR1018 and a strain of Metschnikowia pulcherrima. This process is more robust than the previous ones and does not rely on strict control of oxygenation or even the use of this particular strain of S. cerevisiae. Aeration can be interrupted instantly without impairing the volatile acidity. Under the selected conditions, an ethanol reduction of around 3% (v/v) was obtained compared to the standard fermentation control.

Engineering Escherichia coli for selective 1-decanol production using the reverse ß-oxidation (rBOX) pathway.

1-Decanol has great value in the pharmaceutical and fragrance industries and plays an important role in the chemical industry. In this study, we engineered Escherichia coli to selectively synthesize 1-decanol by using enzymes of the core reverse ß-oxidation (rBOX) pathway and termination module with overlapping chain-length specificity. Through screening for acyl-CoA reductase termination enzymes and proper regulation of rBOX pathway expression, a 1-decanol titer of 1.4 g/L was achieved. Further improvements were realized by engineering pyruvate dissimilation to ensure the generation of NADH through pyruvate dehydrogenase (PDH) and reducing byproduct synthesis via a tailored YigI thioesterase knockout, increasing 1-decanol titer to 1.9 g/L. The engineered strain produced about 4.4 g/L 1-decanol with a yield of 0.21 g/g in 36 h in a bi-phasic fermentation that used a dodecane overlay to increase 1-decanol transport and reduce its toxicity. Adjustment of pathway expression (varying inducer concentration) and cell growth (oxygen availability) enabled 1-decanol production at 6.1 g/L (0.26 g/g yield) and 10.05 g/L (0.2 g/g yield) using rich medium in shake flasks and bioreactor, respectively. Remarkably, the use of minimal medium resulted in 1-decanol production with 100% specificity at 2.8 g/L (0.14 g/g yield) and a per cell mass yield higher than rich medium. These 1-decanol titers, yields and purity are at least 10-fold higher than others reported to date and the engineered strain shows great potential for industrial production. Taken together, our findings suggest that using rBOX pathway and termination enzymes of proper chain-length specificity in combination with optimal chassis engineering should be an effective approach for the selective production of alcohols.

Antioxidant and drought-acclimation responses in UV-B-exposed transgenic Nicotiana tabacum displaying constitutive overproduction of H2O2.

Hydrogen peroxide (H2O2) is an important molecule that regulates antioxidant responses that are crucial for plant stress resistance. Exposure to low levels of ultraviolet-B radiation (UV-B, 280-315 nm) can also activate antioxidant defenses and acclimation responses. However, how H2O2 and UV-B interact to promote stress acclimation remains poorly understood. In this work, a transgenic model of Nicotiana tabacum cv Xanthi nc, with elevated Mn-superoxide dismutase (Mn-SOD) activity, was used to study the interaction between the constitutive overproduction of H2O2 and a 14-day UV-B treatment (1.75 kJ m-2 d-1 biologically effective UV-B). Subsequently, these plants were subjected to a 7-day moderate drought treatment to evaluate the impact on drought resistance of H2O2- and UV-dependent stimulation of the plants' antioxidant system. The UV-B treatment enhanced H2O2 levels and altered the antioxidant status by increasing the epidermal flavonol index, Trolox Equivalent Antioxidant Capacity, and catalase, peroxidase and phenylalanine ammonia lyase activities in the leaves. UV-B also retarded growth and suppressed acclimation responses in highly H2O2-overproducing transgenic plants. Plants not exposed to UV-B had a higher drought resistance in the form of higher relative water content of leaves. Our data associate the interaction between Mn-SOD transgene overexpression and the UV-B treatment with a stress response. Finally, we propose a hormetic biphasic drought resistance response curve as a function of leaf H2O2 content in N. tabacum cv Xanthi.

Management of Antracnosis with Electrochemically Activated Salt Solutions (EASSs) on Bean Culture.

Common bean (Phaseolus vulgaris L.) is an important crop for food security and for national economics for several countries worldwide. One of the most important factors of risk in common bean production is the fungal disease anthracnose caused by Colletotrichum lindemuthianum, which, in some cases, causes complete yield losses; this kind of plant disease is usually managed through the application of chemical products such as fungicides that are commonly not accepted by society. This rejection is based on the relationship of pesticides with health damage and environmental contamination. In order to help in solving these drawbacks, the present work proposes the use of electrochemically activated salt solutions (EASSs) as a safer pathogen control agent in crops, due to it having shown an elicitor and biostimulant effect on plants. With this background, this manuscript presents in vitro results of the evaluation of the inhibitory effect for multiple bean pathogens and in vivo results of EASS in the common bean-Colletotrichum pathosystem by evaluation of the infection severity and defense activation, such as secondary metabolite production and antioxidant activity. EASS presence in growth media had a strong inhibitory effect at the beginning of experiments for some of the evaluated fungi. EASSs showed an effect against the development of the disease when applied in specific doses to prevent distress in plants.

Directed evolution of Saccharomyces cerevisiae for low volatile acidity during winemaking under aerobic conditions.

The use of yeast respiratory metabolism has been proposed as a promising approach to solve the problem of increasing ethanol content in wine, which is largely due to climate change. The use of S. cerevisiae for this purpose is mostly hampered by acetic acid overproduction generated under the necessary aerobic conditions. However, it was previously shown that a reg1 mutant, alleviated for carbon catabolite repression (CCR), showed low acetic acid production under aerobic conditions. In this work directed evolution of three wine yeast strains was performed to recover CCR-alleviated strains, expecting they will also be improved concerning volatile acidity. This was done by subculturing strains on galactose, in the presence of 2-deoxyglucose for around 140 generations. As expected, all evolved yeast populations released less acetic acid than their parental strains in grape juice, under aerobic conditions. Single clones were isolated from the evolved populations, either directly or after one cycle of aerobic fermentation. Only some clones from one of three original strains showed lower acetic acid production than their parental strain. Most clones isolated from EC1118 showed slower growth. However, even the most promising clones failed to reduce acetic acid production under aerobic conditions in bioreactors. Therefore, despite the concept of selecting low acetic acid producers by using 2-deoxyglucose as selective agent was found to be correct, especially at the population level, the recovery of strains with potential industrial utility by this experimental approach remains a challenge.

Plant hormesis: Revising of the concepts of biostimulation, elicitation and their application in a sustainable agricultural production.

Current research in basic and applied knowledge of plant science has aimed to unravel the role of the interaction between environmental factors and the genome in the physiology of plants to confer the ability to overcome challenges in a climate change scenario. Evidence shows that factors causing environmental stress (stressors), whether of biological, chemical, or physical origin, induce eustressing or distressing effects in plants depending on the dose. The latter suggests the induction of the "hormesis" phenomenon. Sustainable crop production requires a better understanding of hormesis, its basic concepts, and the input variables to make its management feasible. This implies that acknowledging hormesis in plant research could allow specifying beneficial effects to effectively manage environmental stressors according to cultivation goals. Several factors have been useful in this regard, which at low doses show beneficial eustressing effects (biostimulant/elicitor), while at higher doses, they show distressing toxic effects. These insights highlight biostimulants/elicitors as tools to be included in integrated crop management strategies for reaching sustainability in plant science and agricultural studies. In addition, compelling evidence on the inheritance of elicited traits in plants unfolds the possibility of implementing stressors as a tool in plant breeding.

Sex differences in Cardiorenal Syndrome: Insights from CARDIOREN Registry.

PURPOSE OF THE WORK: Although sex-specific differences in heart failure (HF) or kidney disease (KD) have been analyzed separately, the predominant cardiorenal phenotype by sex has not been described. This study aims to explore the sex-related differences in cardiorenal syndrome (CRS) in a contemporary cohort of outpatients with HF. FINDINGS: An analysis of the Cardiorenal Spanish registry (CARDIOREN) was performed. CARDIOREN Registry is a prospective multicenter observational registry including 1107 chronic ambulatory HF patients (37% females) from 13 Spanish HF clinics. Estimated Glomerular Filtration Rate (eGFR) < 60 ml/min/1.73 m2 was present in 59.1% of the overall HF population, being this prevalence higher in the female population (63.2% vs. 56.6%, p = 0.032, median age: 81 years old, IQR:74-86). Among those with kidney dysfunction, women displayed higher odds of showing HF with preserved ejection fraction (HFpEF) (odds ratio [OR] = 4.07; confidence interval [CI] 95%: 2.65-6.25, p < 0.001), prior valvular heart disease (OR = 1.76; CI 95%:1.13-2.75, p = 0.014), anemia (OR: 2.02; CI 95%:1.30-3.14, p = 0.002), more advanced kidney disease (OR for CKD stage 3: 1.81; CI 95%:1.04-3.13, p = 0.034; OR for CKD stage 4: 2.49, CI 95%:1.31-4.70, p = 0.004) and clinical features of congestion (OR:1.51; CI 95%: 1.02-2.25, p = 0.039). On the contrary, males with cardiorenal disease showed higher odds of presenting HF with reduced ejection fraction (HFrEF) (OR:3.13; CI 95%: 1.90-5.16, p < 0.005), ischemic cardiomyopathy (OR:2.17; CI 95%: 1.31-3.61, p = 0.003), hypertension (OR = 2.11; CI 95%:1.18-3.78, p = 0.009), atrial fibrillation (OR:1.71; CI 95%: 1.06-2.75, p = 0.025), and hyperkalemia (OR:2.43, CI 95%: 1.31-4.50, p = 0.005). In this contemporary registry of chronic ambulatory HF patients, we observed sex-related differences in patients with combined heart and kidney disease. The emerging cardiorenal phenotype characterized by advanced CKD, congestion, and HFpEF was predominantly observed in women, whereas HFrEF, ischemic etiology, hypertension, hyperkalemia, and atrial fibrillation were more frequently observed in men.

Characterization of the Key Compounds of Bell Pepper by Spectrophotometry and Gas Chromatography on the Effects of Induced Stress on the Concentration of Secondary Metabolite.

Sweet peppers are consumed worldwide, and traditional uses have sparked interest in their applications as dietary antioxidants, which can be enhanced in plants using elicitors. These are endowed with phytochemicals with potential health benefits such as antioxidants, bioavailability, and bioaccessibility. The trend in metabolomics shows us chemical fingerprints linking metabolomics, innovative analytical form, and bioinformatics tools. The objective was to evaluate the impact of multiple stress interactions, elicitor concentrations, and electrical conductivity on the concentration of secondary metabolites to relate their response to metabolic pathways through the foliar application of a cocktail of said elicitors in pepper crops under greenhouse conditions. The extracts were analyzed by spectrophotometry and gas chromatography, and it was shown that the PCA analysis identified phenolic compounds and low molecular weight metabolites, confirming this as a metabolomic fingerprint in the hierarchical analysis. These compounds were also integrated by simultaneous gene and metabolite simulants to obtain effect information on different metabolic pathways. Showing changes in metabolite levels at T6 (36 mM H2O2 and 3.6 dS/m) and T7 (0.1 mM SA and 3.6 dS/m) but showing statistically significant changes at T5 (3.6 dS/m) and T8 (0.1 mM SA, 36 mM H2O2, and 3.6 dS/m) compared to T1 (32 dS/m) or control. Six pathways changed significantly (p < 0.05) in stress-induced treatments: aminoacyl t-RNA and valine-leucine-isoleucine biosynthesis, and alanine-aspartate-glutamate metabolism, glycoxylate-dicarboxylate cycle, arginine-proline, and citrate. This research provided a complete profile for the characterization of metabolomic fingerprint of bell pepper under multiple stress conditions.

Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo.

Formate can be envisioned at the core of a carbon-neutral bioeconomy, where it is produced from CO2 by (electro-)chemical means and converted into value-added products by enzymatic cascades or engineered microbes. A key step in expanding synthetic formate assimilation is its thermodynamically challenging reduction to formaldehyde. Here, we develop a two-enzyme route in which formate is activated to formyl phosphate and subsequently reduced to formaldehyde. Exploiting the promiscuity of acetate kinase and N-acetyl-γ-glutamyl phosphate reductase, we demonstrate this phosphate (Pi)-based route in vitro and in vivo. We further engineer a formyl phosphate reductase variant with improved formyl phosphate conversion in vivo by suppressing cross-talk with native metabolism and interface the Pi route with a recently developed formaldehyde assimilation pathway to enable C2 compound formation from formate as the sole carbon source in Escherichia coli. The Pi route therefore offers a potent tool in expanding the landscape of synthetic formate assimilation.

Function and dynamics of the intrinsically disordered carboxyl terminus of ß2 adrenergic receptor.

Advances in structural biology have provided important mechanistic insights into signaling by the transmembrane core of G-protein coupled receptors (GPCRs); however, much less is known about intrinsically disordered regions such as the carboxyl terminus (CT), which is highly flexible and not visible in GPCR structures. The ß2 adrenergic receptor's (ß2AR) 71 amino acid CT is a substrate for GPCR kinases and binds ß-arrestins to regulate signaling. Here we show that the ß2AR CT directly inhibits basal and agonist-stimulated signaling in cell lines lacking ß-arrestins. Combining single-molecule fluorescence resonance energy transfer (FRET), NMR spectroscopy, and molecular dynamics simulations, we reveal that the negatively charged ß2AR-CT serves as an autoinhibitory factor via interacting with the positively charged cytoplasmic surface of the receptor to limit access to G-proteins. The stability of this interaction is influenced by agonists and allosteric modulators, emphasizing that the CT plays important role in allosterically regulating GPCR activation.

Danon Disease Presenting as Eosinophilic Myocarditis: Key Role of Endomyocardial Biopsy.

A 21-year-old woman with a history of atopy, peripheral eosinophilia, Wolf-Parkinson-White syndrome, and 5 episodes of myocarditis was diagnosed with eosinophilic myocarditis. Despite adequate immunosuppressive treatment and resolution of the myocarditis episode, the patient developed dilated cardiomyopathy and presented with worsening of her functional class. Finally, genetic testing unveiled an additional diagnosis: Danon disease. (Level of Difficulty: Advanced.).