Development of Wafer-Type Plasma Monitoring Sensor with Automated Robot Arm Transfer Capability for Two-Dimensional In Situ Processing Plasma Diagnosis.

In this work, we propose our newly developed wafer-type plasma monitoring sensor based on a floating-type double probe method that can be useful for two-dimensional (2D) in situ plasma diagnosis within a semiconductor processing chamber. A key achievement of this work is the first realization of an ultra-thin plasma monitoring sensor with a system thickness of ~1.4 mm, which supports a fully automated robot arm transfer capability for in situ plasma diagnosis. To the best of our knowledge, it is the thinnest accomplishment among all wafer-type plasma monitoring sensors. Our proposed sensor is assembled with two Si wafers and SiO2-based probes; accordingly, it makes it possible to monitor the actual dynamics of processing plasmas under electrostatic chucking (ESC) conditions. Also, it allows for the prevention of chamber contamination issues after continuously exposing the radio frequency (RF) to various processing gases. Using a test-bed chamber, we successfully demonstrated the feasibility and system performance of the proposed sensor, including robot arm transfer capability, vacuum and thermal stress durability, and data integrity and reproducibility. Consequently, compared with the conventional plasma diagnostic tools, we expect that our proposed sensor will be highly beneficial for tool-to-tool matching (TTTM) and/or for studying various plasma-related items by more accurately providing the parameters of processing plasmas, further saving both time and manpower resources required for preventive maintenance (PM) routines as well.

Mobile genetic element-based gene editing and genome engineering: Recent advances and applications.

Genome engineering has revolutionized several scientific fields, ranging from biochemistry and fundamental research to therapeutic uses and crop development. Diverse engineering toolkits have been developed and used to effectively modify the genome sequences of organisms. However, there is a lack of extensive reviews on genome engineering technologies based on mobile genetic elements (MGEs), which induce genetic diversity within host cells by changing their locations in the genome. This review provides a comprehensive update on the versatility of MGEs as powerful genome engineering tools that offers efficient solutions to challenges associated with genome engineering. MGEs, including DNA transposons, retrotransposons, retrons, and CRISPR-associated transposons, offer various advantages, such as a broad host range, genome-wide mutagenesis, efficient large-size DNA integration, multiplexing capabilities, and in situ single-stranded DNA generation. We focused on the components, mechanisms, and features of each MGE-based tool to highlight their cellular applications. Finally, we discussed the current challenges of MGE-based genome engineering and provided insights into the evolving landscape of this transformative technology. In conclusion, the combination of genome engineering with MGE demonstrates remarkable potential for addressing various challenges and advancing the field of genetic manipulation, and promises to revolutionize our ability to engineer and understand the genomes of diverse organisms.

Novel evaluation of upper-limb motor performance after stroke based on normal reaching movement model.

BACKGROUND: Upper-limb rehabilitation robots provide repetitive reaching movement training to post-stroke patients. Beyond a pre-determined set of movements, a robot-aided training protocol requires optimization to account for the individuals' unique motor characteristics. Therefore, an objective evaluation method should consider the pre-stroke motor performance of the affected arm to compare one's performance relative to normalcy. However, no study has attempted to evaluate performance based on an individual's normal performance. Herein, we present a novel method for evaluating upper limb motor performance after a stroke based on a normal reaching movement model. METHODS: To represent the normal reaching performance of individuals, we opted for three candidate models: (1) Fitts' law for the speed-accuracy relationship, (2) the Almanji model for the mouse-pointing task of cerebral palsy, and (3) our proposed model. We first obtained the kinematic data of healthy (n = 12) and post-stroke (n = 7) subjects with a robot to validate the model and evaluation method and conducted a pilot study with a group of post-stroke patients (n = 12) in a clinical setting. Using the models obtained from the reaching performance of the less-affected arm, we predicted the patients' normal reaching performance to set the standard for evaluating the affected arm. RESULTS: We verified that the proposed normal reaching model identifies the reaching of all healthy (n = 12) and less-affected arm (n = 19; 16 of them showed an R2 > 0.7) but did not identify erroneous reaching of the affected arm. Furthermore, our evaluation method intuitively and visually demonstrated the unique motor characteristics of the affected arms. CONCLUSIONS: The proposed method can be used to evaluate an individual's reaching characteristics based on an individuals normal reaching model. It has the potential to provide individualized training by prioritizing a set of reaching movements.

Relationship between Coma Recovery Scale-Revised and the Thalamocortical Tract of Ascending Reticular Activating System in Hypoxic-Ischemic Brain Injury: A Pilot Study.

BACKGROUND: This pilot study examined the relationship between the Coma Recovery Scale-Revised (CRS-R) and the five subparts of the thalamocortical tract in chronic patients with hypoxic-ischemic brain injury by diffusion tensor tractography (DTT). METHODS: Seventeen consecutive chronic patients with hypoxic-ischemic brain injury were recruited. The consciousness state was evaluated using CRS-R. The five subparts of the thalamocortical tract (the prefrontal cortex, the premotor cortex, the primary motor cortex, the primary somatosensory cortex, and the posterior parietal cortex) were reconstructed using DTT. Fractional anisotropy and the tract volume of each subpart of the thalamocortical tract were estimated. RESULTS: The CRS-R score showed a moderate positive correlation with the tract volume of the prefrontal cortex part of the thalamocortical tract (p < 0.05). In addition, the tract volume of the prefrontal cortex component of the thalamocortical tract could explain the variability in the CRS-R score (p < 0.05). CONCLUSION: The prefrontal cortex part was closely related to the CRS-R score in chronic patients with hypoxic-ischemic brain injury. In addition, the change in the remaining number of neural fibers of the prefrontal cortex part appeared to be related to the change in conscious state.

Riboswitch-guided chalcone synthase engineering and metabolic flux optimization for enhanced production of flavonoids.

Flavonoids are a group of secondary metabolites from plants that have received attention as high value-added pharmacological substances. Recently, a robust and efficient bioprocess using recombinant microbes has emerged as a promising approach to supply flavonoids. In the flavonoid biosynthetic pathway, the rate of chalcone synthesis, the first committed step, is a major bottleneck. However, chalcone synthase (CHS) engineering was difficult because of high-level conservation and the absence of effective screening tools, which are limited to overexpression or homolog-based combinatorial strategies. Furthermore, it is necessary to precisely regulate the metabolic flux for the optimum availability of malonyl-CoA, a substrate of chalcone synthesis. In this study, we engineered CHS and optimized malonyl-CoA availability to establish a platform strain for naringenin production, a key molecular scaffold for various flavonoids. First, we engineered CHS through synthetic riboswitch-based high-throughput screening of rationally designed mutant libraries. Consequently, the catalytic efficiency (kcat/Km) of the optimized CHS enzyme was 62% higher than that of the wild-type enzyme. In addition to CHS engineering, we designed genetic circuits using transcriptional repressors to fine-tune the malonyl-CoA availability. The best mutant with synergistic effects of the engineered CHS and the optimized genetic circuit produced 98.71 mg/L naringenin (12.57 mg naringenin/g glycerol), which is the highest naringenin concentration and yield from glycerol in similar culture conditions reported to date, a 2.5-fold increase compared to the parental strain. Overall, this study provides an effective strategy for efficient production of flavonoids.

An EEG-based asynchronous MI-BCI system to reduce false positives with a small number of channels for neurorehabilitation: A pilot study.

Many studies have used motor imagery-based brain-computer interface (MI-BCI) systems for stroke rehabilitation to induce brain plasticity. However, they mainly focused on detecting motor imagery but did not consider the effect of false positive (FP) detection. The FP could be a threat to patients with stroke as it can induce wrong-directed brain plasticity that would result in adverse effects. In this study, we proposed a rehabilitative MI-BCI system that focuses on rejecting the FP. To this end, we first identified numerous electroencephalogram (EEG) signals as the causes of the FP, and based on the characteristics of the signals, we designed a novel two-phase classifier using a small number of EEG channels, including the source of the FP. Through experiments with eight healthy participants and nine patients with stroke, our proposed MI-BCI system showed 71.76% selectivity and 13.70% FP rate by using only four EEG channels in the patient group with stroke. Moreover, our system can compensate for day-to-day variations for prolonged session intervals by recalibration. The results suggest that our proposed system, a practical approach for the clinical setting, could improve the therapeutic effect of MI-BCI by reducing the adverse effect of the FP.

Parallel screening and cheminformatics modeling of flavonoid activated aptasensors.

A parallel screening of 27 different flavonoids and chalcones was conducted using 6 artificial naringenin-activated riboswitches (M1, M2, M3, O, L and H). A quantitative structure-property relationship approach was applied to understand the physicochemical properties of the flavonoid structures resulting in specificity differences relied on the fluorescence intensity of a green fluorescent protein reporter. Robust models of riboswitches M1, M2 and O that had good predictive power were constructed with descriptors selected for their high correlation. Increased electronegativity and hydrophilicity of the flavonoids structures were identified as two properties that increased binding affinity to RNA riboswitches. Hydroxyl groups at the C-3' and C-4' positions of the flavonoid molecule were strictly required for ligand-activation with riboswitches M1 and M2. Riboswitches O and L preferred multi-hydroxylated flavones as ligands. Substitutions on the A ring of the flavonoid molecule were not important in the molecular recognition process. O-glycosylated derivatives were not recognized by any of the riboswitches, presumably due to steric hindrances. Despite the challenges of detecting RNA conformational change after ligand binding, the resulting models elucidate important physicochemical features in the ligands for conformational structural studies of artificial aptamer complexes and for design of ligands having higher binding specificity.

Role of Diffusion Tensor Imaging in the Diagnosis of Traumatic Axonal Injury in Individual Patients with a Concussion or Mild Traumatic Brain Injury: A Mini-Review.

Present review paper aims to understand role of diffusion tensor imaging (DTI) and diffusion tensor tractography (DTT) in diagnosis of traumatic axonal injury (TAI), induced by head trauma, in individual patients with a concussion or mild traumatic brain injury (mTBI). Precise information on presence and severity of TAI in brain is necessary for determining appropriate therapeutic strategies. Several hundred DTI-based studies have reported TAI in concussion or mTBI. Majority of these DTI-based studies have been performed in a group of patients, whereas case studies that have reported TAI in individual patients with a concussion or mTBI are fewer. Summary of these DTI-based studies for individual patients is as follows: DTI can be used as a non-invasive tool for determining presence and severity of TAI in individual patients with concussion or mTBI. However, for diagnosis of TAI in an individual patient, several conditions are required to be met: no past history of head trauma, presence of possible conditions for TAI occurrence during head trauma, development of new clinical features after head trauma, and DTI observed abnormality of a neural structure that coincides with a newly developed clinical feature. However, further studies for a more precise diagnosis of TAI in individual patients should be encouraged.

Bioengineered Co-culture of organoids to recapitulate host-microbe interactions.

The recent spike in the instances of complex physiological host-microbe interactions has raised the demand for developing in vitro models that recapitulate the microbial microenvironment in the human body. Organoids are steadily emerging as an in vitro culture system that closely mimics the structural, functional, and genetic features of complex human organs, particularly for better understanding host-microbe interactions. Recent advances in organoid culture technology have become new avenues for assessing the pathogenesis of symbiotic interactions, pathogen-induced infectious diseases, and various other diseases. The co-cultures of organoids with microbes have shown great promise in simulating host-microbe interactions with a high level of complexity for further advancement in related fields. In this review, we provide an overview of bioengineering approaches for microbe-co-cultured organoids. Latest developments in the applications of microbe-co-cultured organoids to study human physiology and pathophysiology are also highlighted. Further, an outlook on future research on bioengineered organoid co-cultures for various applications is presented.

Development of Synthetic Riboswitches to Guide the Evolution of Metabolite Production in Microorganisms.

The untranslated region (UTR) of prokaryotic mRNA contains riboswitches, which are gene regulating modules. Riboswitches can be used as biosensors to regulate the expression of a gene or an operon depending on the intracellular level of a target molecule and consequently modulate the cellular responses. In evolutionary engineering, riboswitch-based biosensors have been widely applied for high-throughput screening or selection of target phenotypes. Evolutionary approaches can overcome the limitations of rational approaches in metabolic engineering. Previous studies have reported synthetic riboswitches equipped with novel aptamers and marker genes based on a deep understanding of the operation mechanism of the riboswitch. Here, we introduce the development process of novel synthetic riboswitches for applications in metabolic engineering.

Role of Diffusion Tensor Imaging in Diagnosis and Estimation of Shunt Effect for Hydrocephalus in Stroke Patients: A Narrative Review.

Hydrocephalus is a dilatation of the brain ventricular system by the accumulation of cerebrospinal fluid within the ventricle caused by impaired cerebrospinal fluid circulation or clearance. A diagnosis of hydrocephalus at the chronic stage of stroke has been mainly made by clinical features and radiologic findings on brain computed tomography and magnetic resonance imaging. On the other hand, it could not determine the effect of hydrocephalus or shunt effect on the periventricular neural structures. By contrast, these effects on the periventricular neural structures can be estimated using diffusion tensor imaging (DTI). This article reviewed 10 DTI-based studies related to the diagnosis and estimation of the shunt effect for hydrocephalus in stroke patients. These studies suggest that DTI could be a useful diagnostic and estimation tool of the shunt effect for hydrocephalus in stroke patients. In particular, some studies suggested that fractional anisotropy value in the periventricular white matter could be a diagnostic biomarker for hydrocephalus. As a result, the role of DTI in diagnosing and estimating the shunt effect for hydrocephalus in stroke patients appears to be promising. However, the number of studies and patients of all reviewed studies were limited (10 studies including a total of 58 stroke patients with heterogenous brain pathologies).

Entropy Could Quantify Brain Activation Induced by Mechanical Impedance-Restrained Active Arm Motion: A Functional NIRS Study.

Brain activation has been used to understand brain-level events associated with cognitive tasks or physical tasks. As a quantitative measure for brain activation, we propose entropy in place of signal amplitude and beta value, which are widely used, but sometimes criticized for their limitations and shortcomings as such measures. To investigate the relevance of our proposition, we provided 22 subjects with physical stimuli through elbow extension-flexion motions by using our exoskeleton robot, measured brain activation in terms of entropy, signal amplitude, and beta value; and compared entropy with the other two. The results show that entropy is superior, in that its change appeared in limited, well established, motor areas, while signal amplitude and beta value changes appeared in a widespread fashion, contradicting the modularity theory. Entropy can predict increase in brain activation with task duration, while the other two cannot. When stimuli shifted from the rest state to the task state, entropy exhibited a similar increase as the other two did. Although entropy showed only a part of the phenomenon induced by task strength, it showed superiority by showing a decrease in brain activation that the other two did not show. Moreover, entropy was capable of identifying the physiologically important location.

Relationship between Dizziness and the Core Vestibular Projection Injury in Patients with Mild Traumatic Brain Injury.

Some studies have reported that a core vestibular projection (CVP) injury is associated with dizziness following a brain injury using diffusion tensor tractography (DTT). On the other hand, there has been no DTT study on dizziness caused by a CVP injury in patients with mild traumatic brain injury (TBI). In this study, DTT was used to examine the relationship between dizziness and CVP injury in patients with mild TBI. Forty-three patients with mild TBI and twenty-nine normal subjects were recruited. The patients were classified into two groups based on the dizziness score: group A, patients with a dizziness score less than 2 on the sub-item score for dizziness in the Rivermead Post-concussion Symptoms Questionnaire; group B, patients with a dizziness score above 2. The tract volume (TV) in group B was significantly lower than group A and the control group (p < 0.05). By contrast, the TV in group A was similar to the control group (p > 0.05). Regarding the correlation, the dizziness score of all patients showed a strong negative correlation with the TV of the CVP (r = -0.711, p < 0.05). DTT revealed the CVP injury in patients with dizziness after mild TBI. In addition, the severity of dizziness of these patients was closely related to the injury severity of the CVP.

Multi-level rebalancing of the naringenin pathway using riboswitch-guided high-throughput screening.

Recombinant microbes have emerged as promising alternatives to natural sources of naringenin-a key molecular scaffold for flavonoids. In recombinant strains, expression levels of the pathway genes should be optimized at both transcription and the translation stages to precisely allocate cellular resources and maximize metabolite production. However, the optimization of the expression levels of naringenin generally relies on evaluating a small number of variants from libraries constructed by varying transcription efficiency only. In this study, we introduce a systematic strategy for the multi-level optimization of biosynthetic pathways. We constructed a multi-level combinatorial library covering both transcription and translation stages using synthetic T7 promoter variants and computationally designed 5'-untranslated regions. Furthermore, we identified improved strains through high-throughput screening based on a synthetic naringenin riboswitch. The most-optimized strain obtained using this approach exhibited a 3-fold increase in naringenin production, compared with the parental strain in which only the transcription efficiency was modulated. Furthermore, in a fed-batch bioreactor, the optimized strain produced 260.2 mg/L naringenin, which is the highest concentration reported to date using glycerol and p-coumaric acid as substrates. Collectively, this work provides an efficient strategy for the expression optimization of the biosynthetic pathways.

Signal amplification and optimization of riboswitch-based hybrid inputs by modular and titratable toehold switches.

BACKGROUND: Synthetic biological circuits are widely utilized to control microbial cell functions. Natural and synthetic riboswitches are attractive sensor modules for use in synthetic biology applications. However, tuning the fold-change of riboswitch circuits is challenging because a deep understanding of the riboswitch mechanism and screening of mutant libraries is generally required. Therefore, novel molecular parts and strategies for straightforward tuning of the fold-change of riboswitch circuits are needed. RESULTS: In this study, we devised a toehold switch-based modulator approach that combines a hybrid input construct consisting of a riboswitch and transcriptional repressor and de-novo-designed riboregulators named toehold switches. First, the introduction of a pair of toehold switches and triggers as a downstream signal-processing module to the hybrid input for coenzyme B12 resulted in a functional riboswitch circuit. Next, several optimization strategies that focused on balancing the expression levels of the RNA components greatly improved the fold-change from 260- to 887-fold depending on the promoter and host strain. Further characterizations confirmed low leakiness and high orthogonality of five toehold switch pairs, indicating the broad applicability of this strategy to riboswitch tuning. CONCLUSIONS: The toehold switch-based modulator substantially improved the fold-change compared to the previous sensors with only the hybrid input construct. The programmable RNA-RNA interactions amenable to in silico design and optimization can facilitate further development of RNA-based genetic modulators for flexible tuning of riboswitch circuitry and synthetic biosensors.

Process of Obtaining Social Consensus and 3-Year Functional Outcomes of the First Hand Allotransplantation in Korea.

BACKGROUND: On February 2, 2017, the surgical team of ten board-certified hand specialists of W Hospital in Korea successfully performed the nation's first hand transplantation at Yeungnam University Medical Center (YUMC). This paper reports on the legal, financial, and cultural hurdles that were overcome to open the way for hand transplantation and its functional outcomes at 36 months after the operation. METHODS: W Hospital formed a memorandum of understanding with Daegu city and YUMC to comply with government regulations regarding hand transplantation. Campaigns were initiated in the media to increase public awareness and understanding. With the city's financial and legal support and the university's medical cooperation, a surgical team performed a left distal forearm hand transplantation from a brain-dead 48-year-old man to a 35-year-old left-handed man. RESULTS: With this successful allotransplantation, the Korean Act on Organ Transplantation has now been amended to include hand transplantation. Korean national health insurance has also begun covering hand transplantation. Functional outcome at 36 months after the operation showed satisfactory progress in both motor and sensory functions. The disabilities of the arm, shoulder, and hand score were 23. The final Hand Transplantation Score was 90 points. Functional brain magnetic resonance imaging shows significant cortical reorganization of the corticospinal tract, and reinnervation of intrinsic muscle is observed. CONCLUSIONS: Hand transplantation at the distal forearm shows very satisfactory outcomes in functional, aesthetical, and psychological aspects. Legal and financial barriers against hand transplantation have long been the most burdensome issues. Despite this momentous success, there have been no other clinical applications of vascularized composite allotransplantation due to the limited acceptance by Korean doctors and people. Further public education campaigns for vascularized composite allotransplantation are needed to increase awareness and acceptance.

Sensitive fluorescence detection of SARS-CoV-2 RNA in clinical samples via one-pot isothermal ligation and transcription.

The control of viral outbreaks requires nucleic acid diagnostic tests that are sensitive, simple and fast. Here, we report a highly sensitive and specific one-pot assay for the fluorescence-based detection of RNA from pathogens. The assay, which can be performed within 30-50 min of incubation time and can reach a limit of detection of 0.1-attomolar RNA concentration, relies on a sustained isothermal reaction cascade producing an RNA aptamer that binds to a fluorogenic dye. The RNA aptamer is transcribed by the T7 RNA polymerase from the ligation product of a promoter DNA probe and a reporter DNA probe that hybridize with the target single-stranded RNA sequence via the SplintR ligase (a Chlorella virus DNA ligase). In 40 nasopharyngeal SARS-CoV-2 samples, the assay reached positive and negative predictive values of 95 and 100%, respectively. We also show that the assay can rapidly detect a range of viral and bacterial RNAs.

Artificial Caprolactam-Specific Riboswitch as an Intracellular Metabolite Sensor.

Caprolactam is a monomer used for the synthesis of nylon-6, and a recombinant microbial strain for biobased production of nylon-6 was recently developed. An intracellular biosensor for caprolactam can facilitate high-throughput metabolic engineering of recombinant microbial strains. Because of the mixed production of caprolactam and valerolactam in the recombinant strain, a caprolactam biosensor should be highly specific for caprolactam. However, a highly specific caprolactam sensor has not been reported. Here, we developed an artificial riboswitch that specifically responds to caprolactam. This riboswitch was prepared using a coupled in vitro- in vivo selection strategy with a heterogeneous pool of RNA aptamers obtained from in vitro selection to construct a riboswitch library used in in vivo selection. The caprolactam riboswitch successfully discriminated caprolactam from valerolactam. Moreover, the riboswitch was activated by 3.36-fold in the presence of 50 mM caprolactam. This riboswitch enabled caprolactam-dependent control of cell growth, which will be useful for improving caprolactam production and is a valuable tool for metabolic engineering.

Tools and systems for evolutionary engineering of biomolecules and microorganisms.

Evolutionary approaches have been providing solutions to various bioengineering challenges in an efficient manner. In addition to traditional adaptive laboratory evolution and directed evolution, recent advances in synthetic biology and fluidic systems have opened a new era of evolutionary engineering. Synthetic genetic circuits have been created to control mutagenesis and enable screening of various phenotypes, particularly metabolite production. Fluidic systems can be used for high-throughput screening and multiplexed continuous cultivation of microorganisms. Moreover, continuous directed evolution has been achieved by combining all the steps of evolutionary engineering. Overall, modern tools and systems for evolutionary engineering can be used to establish the artificial equivalent to natural evolution for various research applications.

Multi-level engineering of Baeyer-Villiger monooxygenase-based Escherichia coli biocatalysts for the production of C9 chemicals from oleic acid.

Whole-cell biotransformation is one of the promising alternative approaches to microbial fermentation for producing high-value chemicals. Baeyer-Villiger monooxygenase (BVMO)-based Escherichia coli biocatalysts have been engineered to produce industrially relevant C9 chemicals, such as n-nonanoic acid and 9-hydroxynonanoic acid, from a renewable long-chain fatty acid. The key enzyme in the biotransformation pathway (i.e., BVMO from Pseudomonans putida KT2440) was first engineered, using structure modeling-based design, to improve oxidative and thermal stabilities. Using a stable and tunable plasmid (STAPL) system, E. coli host cells were engineered to have increased plasmid stability and homogeneity of the recombinant E. coli population, as well as to optimize the level of BVMO expression. Multi-level engineering of the key enzyme in host cells, allowed recombinant E. coli expressing a fatty acid double-bond hydratase, a long-chain secondary alcohol dehydrogenase, and the engineered BVMO from P. putida KT2440 (i.e., E6BVMO_C302L/M340L), to ultimately produce C9 chemicals (i.e., n-nonanoic acid and 9-hydroxynonanoic acid) from oleic acid, with a yield of up to 6 mmoL/g dry cells. This yield was 2.4-fold greater than the yield in the control strain before engineering. Therefore, this study will contribute to the development of improved processes for the biosynthesis of industrially relevant medium chain fatty acids via whole-cell biocatalysis.