Enhanced production of 3,4-dihydroxybutyrate from xylose by engineered yeast via xylonate re-assimilation under alkaline condition.

To realize lignocellulose-based bioeconomy, efficient conversion of xylose into valuable chemicals by microbes is necessary. Xylose oxidative pathways that oxidize xylose into xylonate can be more advantageous than conventional xylose assimilation pathways because of fewer reaction steps without loss of carbon and ATP. Moreover, commodity chemicals like 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone can be produced from the intermediates of xylose oxidative pathway. However, successful implementations of xylose oxidative pathway in yeast have been hindered because of the secretion and accumulation of xylonate which is a key intermediate of the pathway, leading to low yield of target product. Here, high-yield production of 3,4-dihydroxybutyrate from xylose by engineered yeast was achieved through genetic and environmental perturbations. Specifically, 3,4-dihydroxybutyrate biosynthetic pathway was established in yeast through deletion of ADH6 and overexpression of yneI. Also, inspired by the mismatch of pH between host strain and key enzyme of XylD, alkaline fermentations (pH ≥ 7.0) were performed to minimize xylonate accumulation. Under the alkaline conditions, xylonate was re-assimilated by engineered yeast and combined product yields of 3,4-dihydroxybutyrate and 3-hydroxybutyrolactone resulted in 0.791 mol/mol-xylose, which is highest compared with previous study. These results shed light on the utility of the xylose oxidative pathway in yeast.

Optimization of 1,2,4-butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance.

1,2,4-Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2-ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2-keto-3-deoxy-xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2-ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.

Improving the functionality of surface-engineered yeast cells by altering the cell wall morphology of the host strain.

The expression of functional proteins on the cell surface using glycosylphosphatidylinositol (GPI)-anchoring technology is a promising approach for constructing yeast cells with special functions. The functionality of surface-engineered yeast strains strongly depends on the amount of functional proteins displayed on their cell surface. On the other hand, since the yeast cell wall space is finite, heterologous protein carrying capacity of the cell wall is limited. Here, we report the effect of CCW12 and CCW14 knockout, which encode major nonenzymatic GPI-anchored cell wall proteins (GPI-CWPs) involved in the cell wall organization, on the heterologous protein carrying capacity of yeast cell wall. Aspergillus aculeatus ß-glucosidase (BGL) was used as a reporter to evaluate the protein carrying capacity in Saccharomyces cerevisiae. No significant difference in the amount of cell wall-associated BGL and cell-surface BGL activity was observed between CCW12 and CCW14 knockout strains and their control strain. In contrast, in the CCW12 and CCW14 co-knockout strains, the amount of cell wall-associated BGL and its activity were approximately 1.4-fold higher than those of the control strain and CCW12 or CCW14 knockout strains. Electron microscopic observation revealed that the total cell wall thickness of the CCW12 and CCW14 co-knockout strains was increased compared to the parental strain, suggesting a potential increase in heterologous protein carrying capacity of the cell wall. These results indicate that the CCW12 and CCW14 co-knockout strains are a promising host for the construction of highly functional recombinant yeast strains using cell-surface display technology. KEY POINTS: • CCW12 and/or CCW14 of a BGL-displaying S. cerevisiae strain were knocked out. • CCW12 and CCW14 co-disruption improved the display efficiency of BGL. • The thickness of the yeast cell wall was increased upon CCW12 and CCW14 knockout.

Production of 1,2,4-butanetriol from xylose by Saccharomyces cerevisiae through Fe metabolic engineering.

1,2,4-Butanetriol can be used to produce energetic plasticizer as well as several pharmaceutical compounds. Although Saccharomyces cerevisiae has some attractive characters such as high robustness for industrial production of useful chemicals by fermentation, 1,2,4-butanetriol production by S. cerevisiae has not been reported. 1,2,4-butanteriotl is produced by an oxidative xylose metabolic pathway completely different from the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways conventionally used for xylose assimilation in S. cerevisiae. In the present study, S. cerevisiae was engineered to produce 1,2,4-butanetriol by overexpression of xylose dehydrogenase (XylB), xylonate dehydratase (XylD), and 2-ketoacid decarboxylase. Further improvement of the recombinant strain was performed by the screening of optimal 2-ketoacid decarboxylase suitable for 1,2,4-butanetriol production and the enhancement of Fe uptake ability to improve the XylD enzymatic activity. Eventually, 1.7 g/L of 1,2,4-butanetriol was produced from 10 g/L xylose with a molar yield of 24.5%. Furthermore, 1.1 g/L of 1,2,4-butanetriol was successfully produced by direct fermentation of rice straw hydrolysate.

Etiology of out-of-hospital cardiac arrest in psychiatric patients: Chart review.

AIM: Although sudden cardiac deaths are more common in psychiatric patients than the general population, data on their causes are very limited. The aim of this study was to investigate initial rhythms and causes of out-of-hospital cardiac arrest (OHCA) in patients with psychiatric disorders. METHODS: We conducted a systematic chart review of patients resuscitated after OHCA and hospitalized in the Tertiary Emergency Medical Center of Tokyo Metropolitan Bokutoh Hospital in Japan between January 2010 and December 2017. The initial rhythms and causes of OHCA were compared between psychiatric patients and non-psychiatric patients. Parameters of interest were compared using chi-squared test, Fisher's exact test, or the Mann-Whitney U-test, as appropriate. RESULTS: A total of 49 psychiatric and 600 non-psychiatric patients were eligible for this study. Fatal but shockable arrhythmias (i.e. ventricular fibrillation and ventricular tachycardia) were less frequently observed as initial rhythms in patients with psychiatric disorders than the others (22.4% vs 49.7%, P < 0.001). Cardiac origin was less common as the cause of OHCA (26.5% vs 58.5%, P < 0.01), while airway obstruction and pulmonary embolism were more frequent in psychiatric versus non-psychiatric patients (24.5% vs 6.5%, P < 0.01; and 12.2% vs 1.5%, P < 0.01, respectively). The results were similar when psychiatric patients were compared with sex- and age-matched controls selected from the non-psychiatric patient group. CONCLUSION: Although fatal arrhythmias may be less common, non-cardiac causes such as pulmonary embolism and airway obstruction need to be treated with high clinical suspicion in an event of sudden cardiac arrest in psychiatric patients.

Combined Cell Surface Display of ß-d-Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass.

Xylitol is a highly valuable commodity chemical used extensively in the food and pharmaceutical industries. The production of xylitol from d-xylose involves a costly and polluting catalytic hydrogenation process. Biotechnological production from lignocellulosic biomass by micro-organisms like yeasts is a promising option. In this study, xylitol is produced from lignocellulosic biomass by a recombinant strain of Saccharomyces cerevisiae (S. cerevisiae) (YPH499-SsXR-AaBGL) expressing cytosolic xylose reductase (Scheffersomyces stipitis xylose reductase [SsXR]), along with a ß-d-glucosidase (Aspergillus aculeatus ß-glucosidase 1 [AaBGL]) displayed on the cell surface. The simultaneous cofermentation of cellobiose/xylose by this strain leads to an ≈2.5-fold increase in Yxylitol/xylose (=0.54) compared to the use of a glucose/xylose mixture as a substrate. Further improvement in the xylose uptake by the cell is achieved by a broad evaluation of several homologous and heterologous transporters. Homologous maltose transporter (ScMAL11) shows the best performance in xylose transport and is used to generate the strain YPH499-XR-ScMAL11-BGL with a significantly improved xylitol production capacity from cellobiose/xylose coutilization. This report constitutes a promising proof of concept to further scale up the biorefinery industrial production of xylitol from lignocellulose by combining cell surface and metabolic engineering in S. cerevisiae.

Treatment of a patient with acute aortic dissection using extracorporeal cardiopulmonary resuscitation after an out-of-hospital cardiac arrest: a case report.

Case: Circulatory support using veno-arterial extracorporeal membrane oxygenation for aortic disease is conventionally contraindicated. In this case, a 66-year-old man experienced cardiopulmonary arrest caused by acute aortic dissection. When exercising in the gym, he experienced chest discomfort, so the staff immediately called an ambulance. While in the ambulance, he experienced cardiopulmonary arrest. His initial electrocardiogram showed ventricular fibrillation. At the emergency department, we immediately performed extracorporeal cardiopulmonary resuscitation. We suspected acute coronary syndrome, so coronary angiography was carried out. Enlargement of ascending aorta was noted. Whole-body enhanced computed tomography was subsequently performed, leading to a final diagnosis of acute aortic dissection. Outcome: Emergency ascending aorta prosthesis implantation was performed. The patient received intensive care and was discharged on day 49 of hospitalization. His cerebral performance category score was 4 at discharge. Conclusion: This case suggests that veno-arterial extracorporeal membrane oxygenation may be used for patients with aortic dissection presenting with cardiac arrest.

Conventional cardiopulmonary resuscitation-induced refractory cardiac arrest due to latent left ventricular outflow tract obstruction due to a sigmoid septum: a case report.

BACKGROUND: Patients with left ventricular outflow tract obstruction who do not exhibit a dynamic pressure gradient at rest, experience pressure gradient increases of ≥ 30 mmHg only during specific situations; this is called latent left ventricular outflow tract obstruction. It is provoked by increased cardiac contraction and preload and afterload depletion. There are a few reports of patients with it developing cardiac arrest. We present a case of latent left ventricular outflow tract obstruction in which the patient with a sigmoid septum experienced refractory pulseless electrical activity due to conventional advanced cardiac life support. CASE PRESENTATION: A 73-year-old Asian woman on escitalopram and lorazepam was transported to our hospital for chest and back pain with altered consciousness. On arrival, she was in shock and developed pulseless electrical activity. After initiation of conventional cardiopulmonary resuscitation according to adult advanced cardiovascular life support guidelines, she could not regain spontaneous circulation. She was ultimately resuscitated via venoarterial extracorporeal membrane oxygenation initiation. The only abnormal laboratory result at admission was anemia. Her hemodynamic status stabilized after red blood cell transfusion, and venoarterial extracorporeal membrane oxygenation was subsequently terminated. Transthoracic echocardiography showed a sigmoid septum; dobutamine-infused Doppler echocardiography revealed a significant outflow gradient, and continuous monitoring showed Brockenbrough-Braunwald sign, which confirmed a diagnosis of latent left ventricular outflow tract obstruction due to a sigmoid septum. As a result, carvedilol and verapamil were initiated. A follow-up dobutamine-infused Doppler echocardiography showed a reduction of outflow gradient, and she was discharged without any sequelae. Latent left ventricular outflow tract obstruction worsened due to increasing cardiac contraction and the depletion of preload and afterload. Depleted preload occurred due to dehydration and anemia, whereas depleted afterload occurred due to the prescribed drugs, which subsequently caused pulseless electrical activity. Moreover, ß-stimulation from the adrenaline probably enhanced the hypercontractile state and caused refractory pulseless electrical activity in our case. CONCLUSIONS: Patients with latent left ventricular outflow tract obstruction can progress to cardiogenic shock and pulseless electrical activity due to increased cardiac contraction and depletion of preload and afterload. We should consider the patient's underlying conditions that induced pulseless electrical activity.

Neurological outcomes and duration from cardiac arrest to the initiation of extracorporeal membrane oxygenation in patients with out-of-hospital cardiac arrest: a retrospective study.

BACKGROUND: We investigated the relationship between neurological outcomes and duration from cardiac arrest (CA) to the initiation of extracorporeal membrane oxygenation (ECMO) (CA-to-ECMO) in patients with out-of-hospital cardiac arrest (OHCA) treated with extracorporeal cardiopulmonary resuscitation (ECPR) and determined the ideal time at which ECPR should be performed. METHODS: During the time period in which this study was conducted, 3451 patients experienced OHCA. This study finally included 79 patients aged 18 years or older whose OHCA had been witnessed and who underwent ECPR in the emergency room between January 2011 and December 2015. Our primary endpoint was survival to hospital discharge with good neurological outcomes (a cerebral performance category of 1 or 2). RESULTS: Of the 79 patients included, 11 had good neurological outcomes. The median duration from CA-to-ECMO was significantly shorter in the good neurological outcome group (33 min, interquartile range [IQR], 27-50 vs. 46 min, IQR, 42-56: p = 0.03). After controlling for potential confounders, we found that the adjusted odds ratio of CA-to-ECMO time for a good neurological outcome was 0.92 (95% confidence interval: 0.87-0.98, p = 0.007). The area under the receiver operating characteristic curve of CA-to-ECMO for predicting a good neurological outcome was 0.71, and the optimal CA-to-ECMO cutoff time was 40 min. The dynamic probability of survival with good neurological outcomes based on CA-to-ECMO time showed that the survival rate with good neurological outcome decreased abruptly from over 30% to approximately 15% when the CA-to-ECMO time exceeded 40 min. DISCUSSION: In this study, CA-to-ECMO time was significantly shorter among patients with good neurological outcomes, and significantly associated with good neurological outcomes at hospital discharge. In addition, the probability of survival with good neurological outcome decreased when the CA-to-ECMO time exceeded 40 minutes. The indication for ECPR for patients with OHCA should include several factors. However, the duration of CPR before the initiation of ECMO is a key factor and an independent factor for good neurological outcomes in patients with OHCA treated with ECPR. Therefore, the upper limit of CA-to-ECMO time should be inevitably included in the indication for ECPR for patients with OHCA. In the present study, there was a large difference in the rate of survival to hospital discharge with good neurological outcome between the patients with a CA-to-ECMO time within 40 minutes and those whose time was over 40 minutes. Based on the present study, the time limit of the duration of CPR before the initiation of ECMO might be around 40 minutes. We should consider ECPR in patients with OHCA if they are relatively young, have a witness and no terminal disease, and the initiation of ECMO is presumed to be within this time period. CONCLUSIONS: The duration from CA-to-ECMO was significantly associated with good neurological outcomes. The indication for patients with OHCA should include a criterion for the ideal time to initiate ECPR.

A case of circumferential type A aortic dissection with intimal intussusception diagnosed using repeat transthoracic echocardiography examination.

Case: Sometimes it is difficult to diagnose circumferential aortic dissection with enhanced computed tomography alone. A 58-year-old woman presented with sudden-onset chest discomfort and loss of consciousness. Transthoracic echocardiogram showed mild aortic regurgitation. Enhanced computed tomography scans showed no obvious intimal tear or flap at the proximal ascending aorta, but an intimal flap was observed from the aortic arch to both common iliac arteries. Stanford type B dissection was tentatively diagnosed. Repeat detailed transthoracic echocardiography examination showed an intimal tear and flap at the ascending aorta; prolapse into the left ventricle caused severe aortic regurgitation. Type A aortic dissection was definitively diagnosed; emergent operation showed a circumferential intimal tear originating from the ascending aorta. Outcome: The ascending aorta was replaced; aortic regurgitation disappeared. The patient was discharged in a good condition 58 days postoperatively. Conclusion: Dynamic evaluations with transthoracic echocardiography should be carried out to diagnose circumferential aortic dissection.

Chest compression-related fatal internal mammary artery injuries manifesting after venoarterial extracorporeal membrane oxygenation: a case series.

BACKGROUND: Cardiopulmonary resuscitation-related bleeding, especially internal mammary artery injuries, can become life-threatening complications after initiating venoarterial extracorporeal membrane oxygenation owing to the frequent involvement of concomitant anticoagulant treatment, antiplatelet treatment, targeted temperature management, and bleeding coagulopathy. We report the cases of five patients who experienced this complication and discuss their management. CASE PRESENTATION: We retrospectively evaluated five patients with cardiopulmonary resuscitation-related internal mammary artery injuries who were treated between February 2011 and February 2016 at our institution. All five patients were Asian men, aged 56 to 68-years old, who had received concomitant intravenously administered unfractionated heparin (3000 units) with antiplatelet therapy. Four patients received targeted temperature management. The injuries and hematomas were detected using contrast-enhanced computed tomography in all cases. Three patients were treated using transcatheter arterial embolization within 6 hours following cardiopulmonary arrest, and two were resuscitated and received appropriate treatment following early recognition of their injuries. Two patients died of hemorrhagic shock with delayed intervention. Four of the five patients had excessively prolonged activated partial thromboplastin times before their interventions. CONCLUSIONS: Computed tomography should be performed as soon as possible after the return of spontaneous circulation to identify injuries and consider appropriate treatments for patients who have experienced cardiac arrest. Delayed bleeding may develop after treating hypovolemic shock and relieving arterial spasms; therefore, transcatheter arterial embolization should be performed aggressively to prevent delayed bleeding even in the absence of extravasation. This approach may be superior to thoracotomy because it is less invasive, causes less bleeding, and can selectively stop arterial bleeding sooner. A 3000-unit intravenous bolus of unfractionated heparin may be redundant; heparin-free extracorporeal cardiopulmonary resuscitation may be a more appropriate alternative. Unfractionated heparin treatment can commence after the bleeding has stopped.