Metabolic Engineering of Saccharomyces cerevisiae for Fermentative Production of Heme.

Heme is a key ingredient required to mimic the color and flavor of meat in plant-based alternatives. This study aimed to develop a yeast-based microbial cell factory for efficient and sustainable production of heme. To this end, first, Hem12p (uroporphyrinogen decarboxylase) was identified as the rate-limiting enzyme in the heme biosynthetic pathway present in Saccharomyces cerevisiae D452-2. Next, we investigated the effects of disruption of the genes involved in the competition for heme biosynthesis precursors, transcriptional repression, and heme degradation (HMX1) on heme production efficiency. Of the knock-out strains constructed in this study, only the HMX1-deficient strain produced heme at a higher concentration than the background strain without gene disruption. In addition, overexpression of PUG1 encoding a plasma membrane transporter involved in protoporphyrin IX (the precursor to heme biosynthesis) uptake led to a significant increase in intracellular heme concentration. As a result, among the various engineered strains constructed in this study, the ΔHMX1/H3&12 + PUG1 strain, the HMX1-deficient strain overexpressing HEM3, HEM12, and PUG1, produced the highest concentration of heme (4.6 mg/L) in batch fermentation, which was 3.9-fold higher than that produced by the wild-type D452-2 strain. In a glucose-limited fed-batch fermentation, the ΔHMX1/H3&12 + PUG1 strain produced 28 mg/L heme in 66 h.

Atomic-Scale Scanning of Domain Network in the Ferroelectric HfO2 Thin Film.

Ferroelectric HfO2-based thin films have attracted much interest in the utilization of ferroelectricity at the nanoscale for next-generation electronic devices. However, the structural origin and stabilization mechanism of the ferroelectric phase are not understood because the film is typically nanocrystalline with active yet stochastic ferroelectric domains. Here, electron microscopy is used to map the in-plane domain network structures of epitaxially grown ferroelectric Y:HfO2 films in atomic resolution. The ferroelectricity is confirmed in free-standing Y:HfO2 films, allowing for investigating the structural origin for their ferroelectricity by 4D-STEM, high-resolution STEM, and iDPC-STEM. At the grain boundaries of <111>-oriented Pca21 orthorhombic grains, a high-symmetry mixed-(R3m, Pnm21) phase is induced, exhibiting enhanced polarization due to in-plane compressive strain. Nanoscale Pca21 orthorhombic grains and their grain boundaries with mixed-(R3m, Pnm21) phases of higher symmetry cooperatively determine the ferroelectricity of the Y:HfO2 film. It is also found that such ferroelectric domain networks emerge when the film thickness is beyond a finite value. Furthermore, in-plane mapping of oxygen positions overlaid on ferroelectric domains discloses that polarization is suppressed at vertical domain walls, while it is active when domains are aligned horizontally with subangstrom domain walls. In addition, randomly distributed 180° charged domain walls are confined by spacer layers.

Flexible low-profile external ventricular drain catheter for real-time brain monitoring.

External ventricular drainage is one of the most common neurosurgical procedures in the world for acute hydrocephalus, which must be performed carefully by a neurosurgeon. Although various neuromonitoring external ventricular drain (EVD) catheters have been utilized, they still suffer from rigidity and bulkiness to mitigate post-EVD placement trauma. Here, we introduce a flexible and low-profile smart EVD catheter using a class of technologies with sensitive electrical materials, seamless integration, and flexible mechanics, which serves as a highly soft and minimally invasive device to monitor electrical brain signals. This device reliably captures biopotentials in real time while exhibiting remarkable flexibility and reliability. The seamless integration of its sensory system promises a minimally invasive EVD placement on brain tissue. This work validates the device's distinct characteristics and performances through in vitro experiments and computational analysis. Collectively, this device's exceptional patient- and user-friendly attributes highlight its potential as one of the most practical EVD catheters.

Value of circulating neutrophil elastase for detecting recurrence of differentiated thyroid cancer.

Objective: The inflammatory microenvironment has been implicated in differentiated thyroid cancer (DTC). Inflammatory stimuli induce the release of components of neutrophils into extracellular space, leading to formation of neutrophil extracellular trap (NET), which can stimulate growth and progression of cancer. Generation of activated factor XII and thrombin is also involved in cancer progression. This study attempted to determine whether the level of circulating markers of NET, activated factor XII, and endogenous thrombin potential may be useful for detecting the recurrence of DTC. Methods: A total of 122 patients with DTC were recruited during the postoperative follow-up period. Measurement of the levels of circulating markers of NET (neutrophil elastase, histone-DNA complex, cell-free dsDNA), activated factor XII, and endogenous thrombin potential was performed. Results: A significantly elevated level of neutrophil elastase was detected in patients with recurrence (n = 12) compared to those without recurrence (n = 110), while significant elevation of the levels of other markers was not observed. The value for area under the curve (0.717, P = 0.018) of neutrophil elastase for detecting recurrence of DTC was superior to that (0.661, P = 0.051) of serum thyroglobulin. An elevated level of neutrophil elastase was significantly associated with recurrence of DTC independent of serum thyroglobulin. Conclusions: Because an elevated level of neutrophil elastase was detected in patients with recurrence of DTC and showed a significant association with recurrence of DTC, it can be proposed as a novel biomarker for use in detecting recurrence of DTC along with other tests.

Transformer-based Deep Neural Network for Breast Cancer Classification on Digital Breast Tomosynthesis Images.

Purpose: To develop an efficient deep neural network model that incorporates context from neighboring image sections to detect breast cancer on digital breast tomosynthesis (DBT) images. Materials and Methods: The authors adopted a transformer architecture that analyzes neighboring sections of the DBT stack. The proposed method was compared with two baselines: an architecture based on three-dimensional (3D) convolutions and a two-dimensional model that analyzes each section individually. The models were trained with 5174 four-view DBT studies, validated with 1000 four-view DBT studies, and tested on 655 four-view DBT studies, which were retrospectively collected from nine institutions in the United States through an external entity. Methods were compared using area under the receiver operating characteristic curve (AUC), sensitivity at a fixed specificity, and specificity at a fixed sensitivity. Results: On the test set of 655 DBT studies, both 3D models showed higher classification performance than did the per-section baseline model. The proposed transformer-based model showed a significant increase in AUC (0.88 vs 0.91, P = .002), sensitivity (81.0% vs 87.7%, P = .006), and specificity (80.5% vs 86.4%, P < .001) at clinically relevant operating points when compared with the single-DBT-section baseline. The transformer-based model used only 25% of the number of floating-point operations per second used by the 3D convolution model while demonstrating similar classification performance. Conclusion: A transformer-based deep neural network using data from neighboring sections improved breast cancer classification performance compared with a per-section baseline model and was more efficient than a model using 3D convolutions.Keywords: Breast, Tomosynthesis, Diagnosis, Supervised Learning, Convolutional Neural Network (CNN), Digital Breast Tomosynthesis, Breast Cancer, Deep Neural Networks, Transformers Supplemental material is available for this article. © RSNA, 2023.

Epigallocatechin-3-Gallate Attenuates Myocardial Dysfunction via Inhibition of Endothelial-to-Mesenchymal Transition.

Cardiac tissue damage following ischemia leads to cardiomyocyte apoptosis and myocardial fibrosis. Epigallocatechin-3-gallate (EGCG), an active polyphenol flavonoid or catechin, exerts bioactivity in tissues with various diseases and protects ischemic myocardium; however, its association with the endothelial-to-mesenchymal transition (EndMT) is unknown. Human umbilical vein endothelial cells (HUVECs) pretreated with transforming growth factor ß2 (TGF-ß2) and interleukin 1ß (IL-1ß) were treated with EGCG to verify cellular function. In addition, EGCG is involved in RhoA GTPase transmission, resulting in reduced cell mobility, oxidative stress, and inflammation-related factors. A mouse myocardial infarction (MI) model was used to confirm the association between EGCG and EndMT in vivo. In the EGCG-treated group, ischemic tissue was regenerated by regulating proteins involved in the EndMT process, and cardioprotection was induced by positively regulating apoptosis and fibrosis of cardiomyocytes. Furthermore, EGCG can reactivate myocardial function due to EndMT inhibition. In summary, our findings confirm that EGCG is an impact activator controlling the cardiac EndMT process derived from ischemic conditions and suggest that supplementation with EGCG may be beneficial in the prevention of cardiovascular disease.

Efficient production of glutathione in Saccharomyces cerevisiae via a synthetic isozyme system.

Glutathione, a tripeptide consisting of cysteine, glutamic acid, and glycine, has multiple beneficial effects on human health. Previous studies have focused on producing glutathione in Saccharomyces cerevisiae by overexpressing γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2), which are the rate-limiting enzymes involved in the glutathione biosynthetic pathway. However, the production yield and titer of glutathione remain low due to the feedback inhibition on GSH1. To overcome this limitation, a synthetic isozyme system consisting of a novel bifunctional enzyme (GshF) from Gram-positive bacteria possessing both GSH1 and GSH2 activities, in addition to GSH1/GSH2, was introduced into S. cerevisiae, as GshF is insensitive to feedback inhibition. Given the HSP60 chaperonin system mismatch between bacteria and S. cerevisiae, co-expression of Group-I HSP60 chaperonins (GroEL and GroES) from Escherichia coli was required for functional expression of GshF. Among various strains constructed in this study, the SKSC222 strain capable of synthesizing glutathione with the synthetic isozyme system produced 240 mg L-1 glutathione with glutathione content and yield of 4.3% and 25.6 mgglutathione /gglucose , respectively. These values were 6.6-, 4.9-, and 4.3-fold higher than the corresponding values of the wild-type strain. In a glucose-limited fed-batch fermentation, the SKSC222 strain produced 2.0 g L-1 glutathione in 67 h. Therefore, this study highlights the benefits of the synthetic isozyme system in enhancing the production titer and yield of value-added chemicals by engineered strains of S. cerevisiae.

Laboratory Perspective of Concurrent Acute Myeloid Leukemia and COVID-19: a Case Report and Review of Literature.

BACKGROUND: Whether the diagnosis and treatment of coronavirus disease 2019 (COVID-19) affect the clinical course of patients with hematologic malignancies has been of interest during the COVID-19 pandemic. METHODS: We describe a 47-year-old female who was concurrently diagnosed with COVID-19 and acute myeloid leukemia (AML). RESULTS: She developed COVID-19 pneumonia which required treatment with remdesivir and dexamethasone, and induction therapy for t-AML was delayed. After her COVID-19 was resolved and isolation ended, follow-up bone marrow examination showed decreased leukemic burden. CONCLUSIONS: This case describes possible effects of COVID-19 treatment on the clinical course of patients with AML from a laboratory perspective.

Reversibly Controlled Ternary Polar States and Ferroelectric Bias Promoted by Boosting Square-Tensile-Strain.

Interaction between dipoles often emerges intriguing physical phenomena, such as exchange bias in the magnetic heterostructures and magnetoelectric effect in multiferroics, which lead to advances in multifunctional heterostructures. However, the defect-dipole tends to be considered the undesired to deteriorate the electronic functionality. Here, deterministic switching between the ferroelectric and the pinched states by exploiting a new substrate of cubic perovskite, BaZrO3 is reported, which boosts the square-tensile-strain to BaTiO3 and promotes four-variants in-plane spontaneous polarization with oxygen vacancy creation. First-principles calculations propose a complex of an oxygen vacancy and two Ti3+ ions coins a charge-neutral defect-dipole. Cooperative control of the defect-dipole and the spontaneous polarization reveals ternary in-plane polar states characterized by biased/pinched hysteresis loops. Furthermore, it is experimentally demonstrated that three electrically controlled polar-ordering states lead to switchable and nonvolatile dielectric states for application of nondestructive electro-dielectric memory. This discovery opens a new route to develop functional materials via manipulating defect-dipoles and offers a novel platform to advance heteroepitaxy beyond the prevalent perovskite substrates.

Simultaneous production of 2'-fucosyllactose and difucosyllactose by engineered Escherichia coli with high secretion efficiency.

BACKGROUND AND AIM: Difucosyllactose (Di-FL) has strong antimicrobial activity against various pathogens, including group B Streptococcus, identified as the leading cause of neonatal sepsis. In this study, we sought to develop Escherichia coli as a microbial cell factory for efficiently producing Di-FL as well as 2'-fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, by utilizing the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway. MAIN METHODS AND MAJOR RESULTS: The biosynthetic pathway for producing fucosylated oligosaccharides via the salvage pathway requires two enzymes, l-fucokinase/GDP-l-fucose phosphorylase (FKP) from Bacteroides fragilis and α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori. To decrease the intracellular accumulation of 2'-FL while increasing substrate accessibility to FKP and FucT2, we evaluated whether extracellular secretion of FKP and FucT2 would enhance the production of fucosylated oligosaccharides. Among various engineered strains constructed in this study, the ΔLFAR-YA/FF+P-PLA2 strain expressing phospholipase A2 (PLA2 ) from Streptomyces violaceoruber, whose native signal peptide was replaced with the PelB signal peptide (P-PLA2 ), could secrete both FKP and FucT2 into the culture medium. Notably, it was observed that FKP and FucT2 present in the extracellular fraction could catalyze the synthesis of Di-FL from lactose and fucose. As a result, a batch fermentation with the ΔLFAR-YA/FF+P-PLA2 strain resulted in the production of 1.22 ± 0.01 g L-1 Di-FL and 0.47 ± 0.01 g L-1 2'-FL, whereas the control strain could only produce 0.65 ± 0.01 g L-1 2'-FL. CONCLUSIONS AND IMPLICATIONS: This study highlights the benefits of extracellular secretion of enzymes to improve biotransformation efficiency, as the transport of substrates and/or products across the cell membrane is limited.

Conformable microneedle pH sensors via the integration of two different siloxane polymers for mapping peripheral artery disease.

Flexible microneedles are important tools that allow access to the inside of biological tissue from the outside without surgery. However, it had been hard to realize microneedle sensor arrays on flexible substrates because of the difficulty of attaining a needle with a high Young's modulus for a selected area on a thin or soft substrate. In this work, we developed a microneedle sensor on a hybrid substrate based on high Young's modulus epoxy siloxane for the microneedles and low Young's modulus polydimethylsiloxane for the conformable substrate. Polyaniline was deposited on the microneedle for pH sensing. The mechanical durability of the device was assessed by insertion into pig skin 1000 times. Last, the flexible microneedle pH sensors showed their utility for monitoring pH distribution in rats in a peripheral artery diseases model.

Functional expression and extracellular secretion of Clostridium thermocellum Cel48S cellulase in Escherichia coli via the signal recognition particle-dependent translocation pathway.

As the only glycoside hydrolase family 48 member in Clostridium thermocellum, the exoglucanase Cel48S plays a crucial role in the extremely high activity of the cellulosome against crystalline cellulose. Although the importance of Cel48S in the hydrolysis of crystalline cellulose has been widely accepted, an efficient production system has not yet been established because Cel48S is usually expressed in Escherichia coli within inactive inclusion bodies. For unstable proteins like Cel48S, translocation across the inner membrane can be more advantageous than cytoplasmic production due to the presence of folding modulators in the periplasm and the absence of cytoplasmic proteases. In this study, we evaluated whether the production of Cel48S in the periplasmic space of E. coli could enhance its functional expression. To do so, we attached the PelB signal peptide, which mediates post-translational secretion, to the N-terminal end of Cel48S (P-Cel48S). The PelB signal peptide allowed catalytically active Cel48S to be successfully produced in the culture medium. In addition, we investigated the role of an alternative co-translational pathway on the extracellular production of Cel48S, finding that co-translational secretion yielded a specific activity of recombinant Cel48S of 135.1 ± 10.0 U/mg cell in the culture medium, which was 2.2 times higher than that associated with P-Cel48S expression. Therefore, we believe that our approach has potential applications for the cost-effective conversion of lignocellulosic biomass and the industrial production of other unstable proteins.

Development of an Onboard Robotic Platform for Embedded Programming Education.

Robotics has been used as an attractive tool in diverse educational fields. A variety of robotic platforms have contributed to teaching practical embedded programming to engineering students at universities. However, most platforms only support content with a low level of programming skills and are unlikely to support a high level of embedded programming. This low association negatively affects students, such as incomprehension, decreased participation, dissatisfaction with course quality, etc. Therefore, this paper proposed a new robotic platform with relevant curricula to improve their effectiveness. The developed platform provided practical content used in mechatronics classes and the capability to operate a robot with a high level of embedded programming. To verify the effectiveness of the proposed platform, participants (undergraduates) examined course evaluations for educational programs based on the developed platform compared with the previous year's class evaluation. The results showed that the proposed platform positively affects students' intellectual ability (performance) and satisfaction in programming education.

Enhanced ferroelectric switching speed of Si-doped HfO2 thin film tailored by oxygen deficiency.

Investigations concerning oxygen deficiency will increase our understanding of those factors that govern the overall material properties. Various studies have examined the relationship between oxygen deficiency and the phase transformation from a nonpolar phase to a polar phase in HfO2 thin films. However, there are few reports on the effects of oxygen deficiencies on the switching dynamics of the ferroelectric phase itself. Herein, we report the oxygen- deficiency induced enhancement of ferroelectric switching properties of Si-doped HfO2 thin films. By controlling the annealing conditions, we controlled the oxygen deficiency concentration in the ferroelectric orthorhombic HfO2 phase. Rapid high-temperature (800 °C) annealing of the HfO2 film accelerated the characteristic switching speed compared to low-temperature (600 °C) annealing. Scanning transmission electron microscopy and electron energy-loss spectroscopy (EELS) revealed that thermal annealing increased oxygen deficiencies, and first-principles calculations demonstrated a reduction of the energy barrier of the polarization flip with increased oxygen deficiency. A Monte Carlo simulation for the variation in the energy barrier of the polarization flipping confirmed the increase of characteristic switching speed.

On Training Neural Network Decoders of Rate Compatible Polar Codes via Transfer Learning.

Neural network decoders (NNDs) for rate-compatible polar codes are studied in this paper. We consider a family of rate-compatible polar codes which are constructed from a single polar coding sequence as defined by 5G new radios. We propose a transfer learning technique for training multiple NNDs of the rate-compatible polar codes utilizing their inclusion property. The trained NND for a low rate code is taken as the initial state of NND training for the next smallest rate code. The proposed method provides quicker training as compared to separate learning of the NNDs according to numerical results. We additionally show that an underfitting problem of NND training due to low model complexity can be solved by transfer learning techniques.

Scale-free ferroelectricity induced by flat phonon bands in HfO2.

Discovery of robust yet reversibly switchable electric dipoles at reduced dimensions is critical to the advancement of nanoelectronics devices. Energy bands flat in momentum space generate robust localized states that are activated independently of each other. We determined that flat bands exist and induce robust yet independently switchable dipoles that exhibit a distinct ferroelectricity in hafnium dioxide (HfO2). Flat polar phonon bands in HfO2 cause extreme localization of electric dipoles within its irreducible half-unit cell widths (~3 angstroms). Contrary to conventional ferroelectrics with spread dipoles, those intrinsically localized dipoles are stable against extrinsic effects such as domain walls, surface exposure, and even miniaturization down to the angstrom scale. Moreover, the subnanometer-scale dipoles are individually switchable without creating any domain-wall energy cost. This offers unexpected opportunities for ultimately dense unit cell-by-unit cell ferroelectric switching devices that are directly integrable into silicon technology.

Facile Ferroelectric Phase Transition Driven by Si Doping in HfO2.

The recently discovered ferroelectricity in thin-film orthorhombic HfO2, which can be directly integrated into complementary metal-oxide semiconductor technology, has become an important research target. However, the use of orthorhombic HfO2 in practical devices has been limited by undesirable mixing with the monoclinic phase, which is nonpolar and thus degrades the ferroelectric properties. Here, we demonstrate that a Si dopant significantly stabilizes the ferroelectric phase because of its unique bonding characteristics, particularly its intrinsic tendency to form strong covalent bonds with O, thereby weakening the phase boundary to stabilize the ferroelectric orthorhombic phase over the nonpolar monoclinic phase, relatively. On the basis of our theoretical predictions, we conducted transmission electron microscopy measurements and confirmed that Si substitution doping indeed induced monoclinic structural components into the orthorhombic phase, which is a strong indication of the weakened phase boundary and subsequent facilitation of the ferroelectric transition. This work thus provides an atomic-scale picture for understanding the unique role of Si in promoting the ferroelectric phase and the dopant dependence on the wake-up effect in HfO2, offering a substantial advancement toward integrating ferroelectrics into practical devices.

Engineering of a Microbial Cell Factory for the Extracellular Production of Catalytically Active Phospholipase A2 of Streptomyces violaceoruber.

Phospholipase A2 (PLA2) from Streptomyces violaceoruber is a lipolytic enzyme used in a wide range of industrial applications including production of lysolecithins and enzymatic degumming of edible oils. We have therefore investigated expression and secretion of PLA2 in two workhorse microbes, Pichia pastoris and Escherichia coli. The PLA2 was produced to an activity of 0.517 ± 0.012 U/ml in the culture broth of the recombinant P. pastoris. On the other hand, recombinant E. coli BL21 star (DE3), overexpressing the authentic PLA2 (P-PLA2), showed activity of 17.0 ± 1.3 U/ml in the intracellular fraction and 21.7 ± 0.7 U/ml in the culture broth. The extracellular PLA2 activity obtained with the recombinant E. coli system was 3.2-fold higher than the corresponding value reached in a previous study, which employed recombinant E. coli BL21 (DE3) overexpressing codon-optimized PLA2. Finally, we observed that the extracellular PLA2 from the recombinant E. coli P-PLA2 culture was able to hydrolyze 31.1 g/l of crude soybean lecithin, an industrial substrate, to a conversion yield of approximately 95%. The newly developed E. coli-based PLA2 expression system led to extracellular production of PLA2 to a productivity of 678 U/l·h, corresponding to 157-fold higher than that obtained with the P. pastoris-based system. This study will contribute to the extracellular production of a catalytically active PLA2.

Stable Subloop Behavior in Ferroelectric Si-Doped HfO2.

The recent demand for analogue devices for neuromorphic applications requires modulation of multiple nonvolatile states. Ferroelectricity with multiple polarization states enables neuromorphic applications with various architectures. However, deterministic control of ferroelectric polarization states with conventional ferroelectric materials has been met with accessibility issues. Here, we report unprecedented stable accessibility with robust stability of multiple polarization states in ferroelectric HfO2. Through the combination of conventional voltage measurements, hysteresis temperature dependence analysis, piezoelectric force microscopy, first-principles calculations, and Monte Carlo simulations, we suggest that the unprecedented stability of intermediate states in ferroelectric HfO2 is due to the small critical volume size for nucleation and the large activation energy for ferroelectric dipole flipping. This work demonstrates the potential of ferroelectric HfO2 for analogue device applications enabling neuromorphic computing.

Noncontact RF Vital Sign Sensor for Continuous Monitoring of Driver Status.

In this paper, a radio frequency vital sign sensor based on double voltage-controlled oscillators (VCOs) combined with a switchable phase-locked loop (PLL) is proposed for a noncontact remote vital sign sensing system. Our sensing system primarily detects the periodic movements of the human lungs and the hearts via the impedance variation of the resonator. With a change in impedance, both the VCO oscillation frequency and the PLL feedback voltage also change. Thus, by tracking the feedback voltage of the PLL, breath and heart rate signals can be acquired simultaneously. However, as the distance between the body and the sensor varies, there are certain points with minimal sensitivity, making it is quite difficult to detect vital signs. These points, called impedance null points, periodically occur at distances proportional to the wavelength. To overcome the impedance null point problem, two resonators operating at different frequencies, 2.40 and 2.76 GHz, are employed as receiving components. In an experiment to investigate the sensing performance as a function of distance, the measurement distance was accurately controlled by a linear actuator. Furthermore, to evaluate the sensing performance in a real environment, experiments were carried out with a male and a female subject in a static vehicle. To demonstrate the real-time vital sign monitoring capability, spectrograms were utilized, and the accuracy was assessed relative to reference sensors. Based on the results, it is demonstrated that the proposed remote sensor can reliably detect vital signs in a real vehicle environment.