Directed evolution of highly sensitive and stringent choline-induced gene expression controllers.

Gene expression controllers are useful tools for microbial production of recombinant proteins and valued bio-based chemicals. Despite its usefulness, they have rarely been applied to the practical industrial bioprocess, due to the lack of systems that meets the three requirements: low cost, safety, and tight control, to the inducer molecules. Previously, we have developed the high-spec gene induction system controlled by safe and cheap inducer choline. However, the system requires relatively high concentration (~100 mM) of choline to fully induce the gene under control. In this work, we attempted to drastically improve the sensitivity of this induction system to further reduce the induction costs. To this end, we devised a simple circuit which couple gene induction system with positive-feedback loop (P-loop) of choline importer protein BetT. After the tuning of translation level of BetT (strength of the P-loop) and deletion of endogenous betI (noise sources), highly active yet stringent control of gene expression was achieved using about 100 times less amount of inducer molecules. The choline induction system developed in this study has the lowest basal expression, the lowest choline needed to be activated, and the highest amplitude of induction as the highest available promoter such as those known as PT5 system. With this system, one can tightly control the expression level of genes of interest with negligible cost for inducer molecule, which has been the bottleneck for the application to the large-scale industrial processes.

Performance of plasma Aß42/40, measured using a fully automated immunoassay, across a broad patient population in identifying amyloid status.

BACKGROUND: Plasma biomarkers have emerged as promising screening tools for Alzheimer's disease (AD) because of their potential to detect amyloid ß (Aß) accumulation in the brain. One such candidate is the plasma Aß42/40 ratio (Aß42/40). Unlike previous research that used traditional immunoassay, recent studies that measured plasma Aß42/40 using fully automated platforms reported promising results. However, its utility should be confirmed using a broader patient population, focusing on the potential for early detection. METHODS: We recruited 174 participants, including healthy controls (HC) and patients with clinical diagnoses of AD, frontotemporal lobar degeneration, dementia with Lewy bodies/Parkinson's disease, mild cognitive impairment (MCI), and others, from a university memory clinic. We examined the performance of plasma Aß42/40, measured using the fully automated high-sensitivity chemiluminescence enzyme (HISCL) immunoassay, in detecting amyloid-positron emission tomography (PET)-derived Aß pathology. We also compared its performance with that of Simoa-based plasma phosphorylated tau at residue 181 (p-tau181), glial fibrillary acidic protein (GFAP), and neurofilament light (NfL). RESULTS: Using the best cut-off derived from the Youden Index, plasma Aß42/40 yielded an area under the receiver operating characteristic curve (AUC) of 0.949 in distinguishing visually assessed 18F-Florbetaben amyloid PET positivity. The plasma Aß42/40 had a significantly superior AUC than p-tau181, GFAP, and NfL in the 167 participants with measurements for all four biomarkers. Next, we analyzed 99 participants, including only the HC and those with MCI, and discovered that plasma Aß42/40 outperformed the other plasma biomarkers, suggesting its ability to detect early amyloid accumulation. Using the Centiloid scale (CL), Spearman's rank correlation coefficient between plasma Aß42/40 and CL was -0.767. Among the 15 participants falling within the CL values indicative of potential future amyloid accumulation (CL between 13.5 and 35.7), plasma Aß42/40 categorized 61.5% (8/13) as Aß-positive, whereas visual assessment of amyloid PET identified 20% (3/15) as positive. CONCLUSION: Plasma Aß42/40 measured using the fully automated HISCL platform showed excellent performance in identifying Aß accumulation in the brain in a well-characterized cohort. This equipment may be useful for screening amyloid pathology because it has the potential to detect early amyloid pathology and is readily applied in clinical settings.

Surface-limited reactions for spatial control of kinesin-microtubule motility assays using indirect irradiation of an electron beam.

Gliding of microtubules (MTs) on kinesins has been applied to lab-on-a-chip devices, which enable autonomous transportation and detection of biomolecules in the field of bioengineering. For rapid fabrication and evaluation of the kinesin-MT based devices, optical control techniques have been developed for control of kinesin activity and density; however, use of caged molecules lacks spatial controllability for long-term experiments, and direct irradiations of UV light onto kinesin-coated surfaces are inherently damaging to MTs due to their depth limit since the heights of the kinesin-MT systems are at the tens of a nanometer scale. Considering surface electric fields in electrolytic solutions are shielded at the nanometer scale due to Debye shielding, in this study, we show that fine spatial control of kinesin density and activity is enabled using surface-limited electrochemical reactions induced by indirect irradiations of an electron beam (EB). An EB is indirectly irradiated onto the kinesins through a 100-nm-thick silicon nitride membrane, and the electrons scattered in the membrane can cause localized electrochemical effects to the kinesins. We show that these localized electrochemical effects cause both ablation of kinesins and motility control of kinesin activity by changing the EB acceleration voltage. In particular, the latter is achieved without complete ablation of MTs, though the MTs are indirectly irradiated by the EB. As a demonstration of on-demand control of gliding MTs, we show the accumulation of the MTs on a target area by scanning the EB. The proposed accumulation technique will lead to rapid prototyping of microdevices based on MT-kinesin motility assay systems.

Sequential Learning on sEMGs in Short- and Long-term Situations via Self-training Semi-supervised Support Vector Machine.

The purpose of this study it to assess the effect of sequential learning of self-training support vector machine (ST-S3VM) on short- and long-term surface electromyogram (sEMG) datasets. A machine learning-based supervised classi-fier is enabling stable, complex, and high-performance motion control. Unlabeled sEMG measurements are easy by the devel-opment of wearable sensing technology. Thus, semi-supervised learning methods are attracted attention to utilize unlabeled sEMG data for supervised classifier with a small amount of labeled data. To evaluate robustness of ST-S3VM in realistic conditions, two public datasets which respectively contain a short- and long-term dataset were used. We compared the performance of ST-S3VM with four-kinds of SVM classifiers. In both short- and long-term situations, ST combined classifiers (ST-SVM and ST-S3VM) showed higher performances than the methods without ST (SVM and S3VM). In some cases, ST-S3VM had the best performance, but in other cases, ST-SVM had better performance than ST-S3VM. In order to make better use of unlabeled data, we will develop ST-S3VM to reduce the impact of harmful unlabeled data.

Time-domain Mixup Source Data Augmentation of sEMGs for Motion Recognition towards Efficient Style Transfer Mapping.

Motion recognition based on surface electromyogram (sEMG) recorded from the forearm is attracting attention for its applicability because it easily integrates with wearable devices and has a high signal-to-noise ratio. Inter-subject variability and inadequate data availability are common problems encountered in classifiers. Transfer learning (TL) techniques can reduce the inter-subject variability; however, when the amount of data recorded from each source subject is small, the TL-combined classifier is prone to overfitting problems. In this study, we tested the accuracy of motion recognition with and without TL when the source dataset was increased up to 10 times with a time-domain data augmentation method called mixup. The performance was evaluated using an 8-class sEMG dataset containing wearable sensing data from 25 subjects. We found that mixup improved the performance of TL-combined classifiers (support vector machine and 4-layered fully connected feedforward neural network). In future work, we plan to investigate the relationship between the amount of data and sEMG-based motion recognition by comparing multiple sEMG datasets and multiple data augmentation methods.

Brain-controlled cycling system for rehabilitation following paraplegia with delay-time prediction.

Objective.Robotic rehabilitation systems have been investigated to assist with motor dysfunction recovery in patients with lower-extremity paralysis caused by central nervous system lesions. These systems are intended to provide appropriate sensory feedback associated with locomotion. Appropriate feedback is thought to cause synchronous neuron firing, resulting in the recovery of function.Approach.In this study, we designed and evaluated an ergometric cycling wheelchair, with a brain-machine interface (BMI), that can force the legs to move by including normal stepping speeds and quick responses. Experiments were conducted in five healthy subjects and one patient with spinal cord injury (SCI), who experienced the complete paralysis of the lower limbs. Event-related desynchronization in theßband (18-28 Hz) was used to detect lower-limb motor images.Main results.An ergometer-based BMI system was able to safely and easily force patients to perform leg movements, at a rate of approximately 1.6 s/step (19 rpm), with an online accuracy rate of 73.1% for the SCI participant. Mean detection time from the cue to pedaling onset was 0.83±0.31 s.Significance.This system can easily and safely maintain a normal walking speed during the experiment and be designed to accommodate the expected delay between the intentional onset and physical movement, to achieve rehabilitation effects for each participant. Similar BMI systems, implemented with rehabilitation systems, may be applicable to a wide range of patients.

Analysis and Usage: Subject-to-subject Linear Domain Adaptation in sEMG Classification.

Before the operation of a biosignal-based application, long-duration calibration is required to adjust the pre-trained classifier to a new user data (target data). For reducing such time-consuming step, linear domain adaptation (DA) transfer learning approaches, which transfer pooled data (source data) related to the target data, are highlighted. In the last decade, they have been applied to surface electromyogram (sEMG) data with the implicit assumption that sEMG data are linear. However, sEMGs typically have non-linear characteristics, and due to the discrepancy between the assumption and actual characteristics, linear DA approaches would cause a negative transfer. This study investigated how the correlation between the source and target data affects an 8-class forearm movement classification after applying linear DA approaches. As a result, we found significant positive correlations between the classification accuracy and the source-target correlation. Additionally, the source-target correlation depended on the motion class. Therefore, our results suggest that we should choose a non-linear DA approach when the source-target correlation among subjects or motion classes is low.

Motor Imagery Classification of Finger Motions Using Multiclass CSP.

Electroencephalogram (EEG) data during motor imagery tasks regarding small-scale physical dynamics such as finger motions have low discriminability because capturing the spatial difference of the motions is difficult. We assumed that more discriminative features can be captured if spatial filters maximize the independence of each class data. This study constructed spatial filters named multiclass common spatial pattern (CSP), which maximize an approximation of mutual in-formation of extracted components and class labels, and applied them to a five-class motor-imagery dataset containing finger motion tasks. By applying multiclass CSP, the classification accuracies were improved (Mean SD: 40.6 ± 10.1%) compared with classical CSP (21.8 ± 2.5%) and no spatial filtering case (38.7±10.0%). In addition, we visualized learned spatial filters to assess the trend of discriminative features of finger motions. For these results, it was clear that multiclass CSP captured task-specific spatial maps for each finger motion and outperformed multiclass motor-imagery classification performance about 2% even when the tasks are small-scale physical dynamics.

Independent Low-Rank Matrix Analysis-Based Automatic Artifact Reduction Technique Applied to Three BCI Paradigms.

Electroencephalogram (EEG)-based brain-computer interfaces (BCIs) can potentially enable people to non-invasively and directly communicate with others using brain activities. Artifacts generated from body activities (e.g., eyeblinks and teeth clenches) often contaminate EEGs and make EEG-based classification/identification hard. Although independent component analysis (ICA) is the gold-standard technique for attenuating the effects of such contamination, the estimated independent components are still mixed with artifactual and neuronal information because ICA relies only on the independence assumption. The same problem occurs when using independent vector analysis (IVA), an extended ICA method. To solve this problem, we designed an independent low-rank matrix analysis (ILRMA)-based automatic artifact reduction technique that clearly models sources from observations under the independence assumption and a low-rank nature in the frequency domain. For automatic artifact reduction, we combined the signal separation technique with an independent component classifier for EEGs named ICLabel. To assess the comparative efficiency of the proposed method, the discriminabilities of artifact-reduced EEGs using ICA, IVA, and ILRMA were determined using an open-access EEG dataset named OpenBMI, which contains EEG data obtained through three BCI paradigms [motor-imagery (MI), event-related potential (ERP), and steady-state visual evoked potential (SSVEP)]. BCI performances were obtained using these three paradigms after applying artifact reduction techniques, and the results suggested that our proposed method has the potential to achieve higher discriminability than ICA and IVA for BCIs. In addition, artifact reduction using the ILRMA approach clearly improved (by over 70%) the averaged BCI performances using artifact-reduced data sufficiently for most needs of the BCI community. The extension of ICA families to supervised separation that leaves the discriminative ability would further improve the usability of BCIs for real-life environments in which artifacts frequently contaminate EEGs.

Cobalt chloride, a chemical hypoxia-mimicking agent, suppresses myoblast differentiation by downregulating myogenin expression.

Cobalt chloride can create hypoxia-like state in vitro (referred to as chemical hypoxia). Several studies have suggested that chemical hypoxia may cause deleterious effects on myogenesis. The intrinsic underlying mechanisms of myoblast differentiation, however, are not fully understood. Here, we show that cobalt chloride strongly suppresses myoblast differentiation in a dose-dependent manner. The impaired myoblast differentiation is accompanied by downregulation of myogenic regulatory factor myogenin. Under chemical hypoxia, myogenin stability is decreased at mRNA and protein levels. A muscle-specific E3 ubiquitin ligase MAFbx, which can target myogenin protein for proteasomal degradation, is upregulated along with changes in Akt/Foxo and AMPK/Foxo signaling pathways. A proteasome inhibitor completely prevents cobalt chloride-mediated decrease in myogenin protein. These results suggest that cobalt chloride might modulate myogenin expression at post-transcriptional and post-translational levels, resulting in the failure of the myoblasts to differentiate into myotubes.

Engineered topographical structure to control spatial cell density using cell migration.

Control of the spatial distribution of various cell types is required to construct functional tissues. Here, we report a simple topographical structure changed the spatial cell density. A concave curved boundary was designed, which allowed the spatial descent moving of cells and the change in spatial distributions of co-cultured cells. We utilized the difference in cell motility between myoblast cells (C2C12) and neuronal cells (PC12) to demonstrate the feasibility of spontaneous change in spatial cell density. Without the curved boundaries, high motility cells (C2C12) did not migrate to the adjacent area, which resulted in a slight temporal change (< 15%) in the spatial cell distribution. In contrast, with the curved boundaries, the cell density of the high motility cells in the groove to those cells on the ridge showed an increase exceeding 45%. On the other hand, the temporal change in the spatial cell distribution of low motility cells (PC12) was below 15% with or without the curved boundaries. In addition, as groove width increased, both cells displayed more initially gathering in groove. Importantly, these cell-type dependent results were also maintained under co-culture conditions. Our results suggest that designing topographical interfaces changes spatial cell density without any manipulation and is useful for multi-cellular constructs.

Pause of the target gliding microtuble on the virtual cathode.

We report the transient response of gliding microtubules on a virtual cathode. In vivo activities, microtubule-kinesin systems are known to act as motor proteins with respect to cell motility cytokinesis and cellular transport by hydrolyzing ATP molecules. With development of in vitro assays, motor proteins have been attracting much attention as a key component for highly efficient nano-transportation systems. The molecular functions based on structural states are affected by changing the ionic condition of the molecular functions and by changing the electrical field in solution because of electrical charges of the molecules. The virtual cathode, which was generated on the SiN display surface by a low energy electron beam, locally induced electrochemical reactions and electric field around the targeted molecules on the display surface, and then the gliding motions of the targeted microtubules were regulated. In this study, we demonstrated that the virtual cathode display temporally stops a selected gliding microtubule by only applying the virtual cathode to the microtubule. The pause mode of the microtubule was easily canceled by simply turning the virtual cathode off, and then the gliding motion was restarted.

Pinpoint Delivery of Molecules by Using Electron Beam Addressing Virtual Cathode Display.

Electroporation, a physical transfection method to introduce genomic molecules in selective living cells, could be implemented by microelectrode devices. A local electric field generated by a finer electrode can induces cytomembrane poration in the electrode vicinity. To employ fine, high-speed scanning electrodes, we developed a fine virtual cathode pattern, which was generated on a cell adhesive surface of 100-nm-thick SiN membrane by inverted-electron beam lithography. The SiN membrane works as both a vacuum barrier and the display screen of the virtual cathode. The kinetic energy of the incident primary electrons to the SiN membrane was completely blocked, whereas negative charges and leaking electric current appeared on the surface of the dielectric SiN membrane within a region of 100 nm. Locally controlled transmembrane molecular delivery was demonstrated on adhered C2C12 myoblast cells in a culturing medium with fluorescent dye propidium iodide (PI). Increasing fluorescence of pre-diluted PI indicated local poration and transmembrane inflow at the virtual cathode position, as well as intracellular diffusion. The transmembrane inflows depended on beam duration time and acceleration voltage. At the post-molecular delivery, a slight decrease in intracellular PI fluorescence intensity indicates membrane recovery from the poration. Cell viability was confirmed by time-lapse cell imaging of post-exposure cell migration.

Pharmacological targeting of HSP90 with 17-AAG induces apoptosis of myogenic cells through activation of the intrinsic pathway.

We have shown that pharmacological inhibition of HSP90 ATPase activity induces apoptosis of myoblasts during their differentiation. However, the signaling pathways remain not fully characterized. We report that pharmacological targeting of HSP90 with 17-AAG activates the intrinsic pathway including caspase-dependent and caspase-independent pathways. 17-AAG induces the typical apoptotic phenotypes including PARP cleavage, chromatin condensation, and nuclear fragmentation with mitochondrial release of cytochrome c, Smac/DIABLO, procaspase-9 processing, and caspase-3 activation. AIF and EndoG redistribute from the mitochondria into the cytosol and are partially translocated to the nucleus in 17-AAG-treated cells. These results suggest that caspase-dependent and caspase-independent pathways should be considered in apoptosis of myogenic cells induced by inhibition of HSP90 ATPase activity.

Dynamic electromechanical control of biomolecules using a nano virtual cathode display.

The dynamic electromechanical control of spatial structures of biomolecules in aqueous solutions was demonstrated using a nano virtual cathode display. By generating a focused electric field around the biomolecules using an electron beam (EB), the molecules' spatiotemporal responses to the electrical stimuli, such as globule transition of DNA random coils and deformation of planar lipid bilayers and vesicles, were successfully observed. The proposed system may be applied to high resolution and high degree-of-freedom manipulations to measure the mechanical and structural properties of bio-nanomaterials.

Directed cell migration in co-cultures by topographic curvature for heterogeneous tissue engineering.

Placing cells in the proper position is important for tissue engineering. Previous works addressed this subject in the way of controlling cell migration by micro- or nano-patterning the substrates. However, the problem of changing spatial cell density freely under co-culture conditions is remaining. To solve this problem, in this work, we report that C2C12 spatial cell density changes by the patterning geometric boundary of the topographical structures. In 48 h after seeding cells, at the linear boundary (ridge-groove) structures, C2C12 Groove/Ridge ratio was under 0.70 both under monoculture conditions and under co-culture conditions. In contrast, at the combining the linear boundary and the round boundary (ridge-groove + hole) structures, the ratio was over 0.89 under both culture conditions. This our finding will provide a new device which enables to manipulate spatial cell density under co-culture conditions for heterogeneous tissue engineering.

Temporal relation between neural activity and neurite pruning on a numerical model and a microchannel device with micro electrode array.

Synapse elimination and neurite pruning are essential processes for the formation of neuronal circuits. These regressive events depend on neural activity and occur in the early postnatal days known as the critical period, but what makes this temporal specificity is not well understood. One possibility is that the neural activities during the developmentally regulated shift of action of GABA inhibitory transmission lead to the critical period. Moreover, it has been reported that the shifting action of the inhibitory transmission on immature neurons overlaps with synapse elimination and neurite pruning and that increased inhibitory transmission by drug treatment could induce temporal shift of the critical period. However, the relationship among these phenomena remains unclear because it is difficult to experimentally show how the developmental shift of inhibitory transmission influences neural activities and whether the activities promote synapse elimination and neurite pruning. In this study, we modeled synapse elimination in neuronal circuits using the modified Izhikevich's model with functional shifting of GABAergic transmission. The simulation results show that synaptic pruning within a specified period like the critical period is spontaneously generated as a function of the developmentally shifting inhibitory transmission and that the specific firing rate and increasing synchronization of neural circuits are seen at the initial stage of the critical period. This temporal relationship was experimentally supported by an in vitro primary culture of rat cortical neurons in a microchannel on a multi-electrode array (MEA). The firing rate decreased remarkably between the 18-25 days in vitro (DIV), and following these changes in the firing rate, the neurite density was slightly reduced. Our simulation and experimental results suggest that decreasing neural activity due to developing inhibitory synaptic transmission could induce synapse elimination and neurite pruning at particular time such as the critical period. Additionally, these findings indicate that we can estimate the maturity level of inhibitory transmission and the critical period by measuring the firing rate and the degree of synchronization in engineered neural networks.

Identification of a VapBC toxin-antitoxin system in a thermophilic bacterium Thermus thermophilus HB27.

There are 12 putative toxin-antitoxin (TA) loci in the Thermus thermophilus HB27 genome, including four VapBC and three HicBA families. Expression of these seven putative toxin genes in Escherichia coli demonstrated that one putative VapC toxin TTC0125 and two putative HicA toxins, TTC1395 and TTC1705, inhibited cell growth, and co-expression with cognate antitoxin genes rescued growth, indicating that these genes function as TA loci. In vitro analysis with the purified TTC0125 and total RNA/mRNA from E. coli and T. thermophilus showed that TTC0125 has RNase activity to rRNA and mRNA; this activity was inhibited by the addition of the purified TTC0126. Translation inhibition assays showed that TTC0125 inhibited protein synthesis by degrading mRNA but not by inactivating ribosomes. Amino acid substitutions of 14 predicted catalytic and conserved residues in VapC toxins to Ala or Asp in TTC0125 indicated that nine residues are important for its in vivo toxin activity and in vitro RNase activity. These data demonstrate that TTC0125-TTC0126 functions as a VapBC TA module and causes growth inhibition by degrading free RNA. This is the first study to identify the function of TA systems in T. thermophilus.

Assessment of associations between ischaemic attacks in patients with type 2 diabetes mellitus and air concentrations of particulate matter <2.5 µm.

OBJECTIVE: To investigate the relationship between air concentrations of fine particulate matter <2.5 µm in diameter (PM2.5) and ischaemic attacks in high-risk elderly patients (aged ≥60 years). METHODS: This retrospective data analysis study extracted clinical data from an electronic medical chart system and environmental air quality data from the Ministry of the Environment Atmospheric Environmental Regional Observation System. Patients were stratified into four groups according to whether or not they had type 2 diabetes mellitus (T2DM) and/or an ischaemic attack (i.e. cerebral infarctions and myocardial infarctions) during the 2-year study period. Analysis of the combined clinical and environmental data produced the odds ratio for the likelihood of experiencing an ischaemic attack. RESULTS: Clinical data were collected from 94 647 patients. On days when the air temperature was ≥25℃ and PM2.5 concentration was ≥20µg/m(3), an ischaemic attack was more likely to occur in the T2DM group than in the group without T2DM. An ischaemic attack was more likely to occur 2 days after an increase in the PM2.5 concentration compared with the same day as and 3-6 days after the increase. CONCLUSION: The results of this study confirmed an association between PM2.5 and ischaemic attacks in high-risk patients.

Effects of ageing on expression of the muscle-specific E3 ubiquitin ligases and Akt-dependent regulation of Foxo transcription factors in skeletal muscle.

Controversy exists as to whether the muscle-specific E3 ubiquitin ligases MAFbx and MuRF1 are transcriptionally upregulated in the process of sarcopenia. In the present study, we investigated the effects of ageing on mRNA/protein expression of muscle-specific E3 ubiquitin ligases and Akt/Foxo signalling in gastrocnemius muscles of female mice. Old mice exhibited a typical sarcopenic phenotype, characterized by loss of muscle mass and strength, decreased amount of myofibrillar proteins, incidence of aberrant muscle fibres, and genetic signature to sarcopenia. Activation levels of Akt were lower in adult and old mice than in young mice. Consequently, Akt-mediated phosphorylation levels of Foxo1 and Foxo3 proteins were decreased. Nuclear levels of Foxo1 and Foxo3 proteins showed an overall increasing trend in old mice. MAFbx mRNA expression was decreased in old mice relative to adult mice, whereas MuRF1 mRNA expression was less affected by ageing. At the protein level, MAFbx was less affected by ageing, whereas MuRF1 was increased in old mice relative to adult mice, with ubiquitin-protein conjugates being increased with ageing. In conclusion, we provided evidence for no mRNA upregulation of muscle-specific E3 ubiquitin ligases and disconnection between their expression and Akt/Foxo signalling in sarcopenic mice. Their different responsiveness to ageing may reflect different roles in sarcopenia.