Highly Durable, Stretchable Multielectrode Array for Electro-mechanical Co-stimulation of Cells.

Electro-mechanical co-stimulation of cells can be a useful cue for tissue engineering. However, reliable co-stimulation platforms still have limitations due to low durability of the components and difficulty in optimizing the stimulation parameters. Although various electro-mechanical co-simulation systems have been explored, integrating materials and components with high durability is still limited. To tackle this problem, we designed an electro-mechanical co-stimulation system that facilitates uniaxial cyclic stretching, electrical stimulation, and optical monitoring. This system utilizes a robust and autoclavable stretchable multielectrode array housed within a compact mini-incubator. To illustrate its effectiveness, we conducted experiments that highlighted how electro-mechanical co-stimulation using this system can enhance the maturation of cardiomyocytes derived from human induced pluripotent stem cells. The results showed great potential of our co-stimulation platform as an effective tool for tissue engineering.

Guidelines for Packaging, Transport, and Storage of Source Cells for Organoids.

This paper presents guidelines for the systematic management of packaging, storage, transportation, and traceability of source cells used for organoid research. Given the important role of source cells in organoid studies, it is important to ensure the preservation of their quality and integrity throughout transportation and distribution processes. The proposed guidelines, therefore, call for a cohesive strategy through these stages to minimize the risks of contamination, deterioration, and loss-threats that significantly compromise the safety, efficacy, and efficiency of source cells. Central to these guidelines is the quality control measures that include roles and responsibilities across the entire supply chain, with recommendations specific to packaging materials, transportation facilities, and storage management. Furthermore, the need for an integrated management system is emphasized, spanning from source cell collection to the final application. This system is crucial for maintaining the traceability and accountability of source cells, facilitating the sharing, distribution, and utilization on a global scale, and supporting to advance organoid research and development.

Contrast-enhanced ultrasound for the diagnosis of acute pyelonephritis in pediatric patients with urinary tract infection: A feasibility study.

PURPOSE: This study aimed to assess the feasibility of contrast-enhanced ultrasound (CEUS) for the diagnosis of acute pyelonephritis (APN) in pediatric patients with febrile urinary tract infection (UTI). MATERIALS AND METHODS: Between March 2019 and January 2021, study participants with suspected UTI were assessed for APN using ultrasound. Parenchymal echogenicity changes, renal pelvis dilatation, and the presence of a focal suspected lesion were assessed using conventional grayscale ultrasound. The presence and location of a decreased perfusion area were evaluated using color Doppler ultrasound (CDUS) and CEUS. Agreement between each ultrasound examination and a 99mTc‒dimercaptosuccinic acid (DMSA) scan was assessed using the κ value, and the most visible period of the lesion was evaluated using CEUS. RESULTS: This study enrolled 21 participants (median age, 8.0 months; range, 2.0-61.0 months) with isolated urinary tract pathogens. Five increased parenchymal echotextures (11.9%) and 14 renal pelvic dilatations (33.3%) were confirmed, but no focal lesions were detected on the grayscale images. CDUS and CEUS showed decreased local perfusion suggestive of APN in two and five kidneys, respectively. DMSA scan showed substantial agreement with CEUS findings (κ = 0.80, P = 0.010), but other grayscale and CDUS findings did not agree with DMSA scan results (P > 0.05). All lesions were best observed in the late parenchymal phase on CEUS. CONCLUSION: CEUS can reveal renal perfusion defects in pediatric patients with suspected APN without radiation exposure or sedation; therefore, CEUS may be a feasible and valuable diagnostic technique.

Coordinated and Distinct Roles of Peptidoglycan Carboxypeptidases DacC and DacA in Cell Growth and Shape Maintenance under Stress Conditions.

Peptidoglycan (PG) is an essential bacterial architecture pivotal for shape maintenance and adaptation to osmotic stress. Although PG synthesis and modification are tightly regulated under harsh environmental stresses, few related mechanisms have been investigated. In this study, we aimed to investigate the coordinated and distinct roles of the PG dd-carboxypeptidases (DD-CPases) DacC and DacA in cell growth under alkaline and salt stresses and shape maintenance in Escherichia coli. We found that DacC is an alkaline DD-CPase, the enzyme activity and protein stability of which are significantly enhanced under alkaline stress. Both DacC and DacA were required for bacterial growth under alkaline stress, whereas only DacA was required for growth under salt stress. Under normal growth conditions, only DacA was necessary for cell shape maintenance, while under alkaline stress conditions, both DacA and DacC were necessary for cell shape maintenance, but their roles were distinct. Notably, all of these roles of DacC and DacA were independent of ld-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and the outer membrane lipoprotein Lpp. Instead, DacC and DacA interacted with penicillin-binding proteins (PBPs)-dd-transpeptidases-mostly in a C-terminal domain-dependent manner, and these interactions were necessary for most of their roles. Collectively, our results demonstrate the coordinated and distinct novel roles of DD-CPases in bacterial growth and shape maintenance under stress conditions and provide novel insights into the cellular functions of DD-CPases associated with PBPs. IMPORTANCE Most bacteria have a peptidoglycan architecture for cell shape maintenance and protection against osmotic challenges. Peptidoglycan dd-carboxypeptidases control the amount of pentapeptide substrates, which are used in the formation of 4-3 cross-links by the peptidoglycan synthetic dd-transpeptidases, penicillin-binding proteins (PBPs). Seven dd-carboxypeptidases exist in Escherichia coli, but the physiological significance of their redundancy and their roles in peptidoglycan synthesis are poorly understood. Here, we showed that DacC is an alkaline dd-carboxypeptidase for which both protein stability and enzyme activity are significantly enhanced at high pH. Strikingly, dd-carboxypeptidases DacC and DacA physically interacted with PBPs, and these interactions were necessary for cell shape maintenance as well as growth under alkaline and salt stresses. Thus, cooperation between dd-carboxypeptidases and PBPs may allow E. coli to overcome various stresses and to maintain cell shape.

Divergent Effects of Peptidoglycan Carboxypeptidase DacA on Intrinsic ß-Lactam and Vancomycin Resistance.

Vancomycin and ß-lactams are clinically important antibiotics that inhibit the formation of peptidoglycan cross-links, but their binding targets are different. The binding target of vancomycin is d-alanine-d-alanine (d-Ala-d-Ala), whereas that of ß-lactam is penicillin-binding proteins (PBPs). In this study, we revealed the divergent effects of peptidoglycan (PG) carboxypeptidase DacA on vancomycin and ß-lactam resistance in Escherichia coli and Bacillus subtilis. The deletion of DacA induced sensitivity to most ß-lactams, whereas it induced strong resistance toward vancomycin. Notably, both phenotypes did not have a strong association with ld-transpeptidases, which are necessary for the formation of PG 3-3 cross-links and covalent bonds between PG and an Lpp outer membrane (OM) lipoprotein. Vancomycin resistance was induced by an increased amount of decoy d-Ala-d-Ala residues within PG, whereas ß-lactam sensitivity was associated with physical interactions between DacA and PBPs. The presence of an OM permeability barrier strongly strengthened vancomycin resistance, but it significantly weakened ß-lactam sensitivity. Collectively, our results revealed two distinct functions of DacA, which involved inverse modulation of bacterial resistance to clinically important antibiotics, ß-lactams and vancomycin, and presented evidence for a link between DacA and PBPs. IMPORTANCE Bacterial PG hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and stress adaptation. Of all the PG hydrolases, the role of PG carboxypeptidases is poorly understood, especially regarding their impacts on antibiotic resistance. We have revealed two distinct functions of PG carboxypeptidase DacA with respect to antibiotic resistance. The deletion of DacA led to sensitivity to most ß-lactams, while it caused strong resistance to vancomycin. Our study provides novel insights into the roles of PG carboxypeptidases in the regulation of antibiotic resistance and a potential clue for the development of a drug to improve the clinical efficacy of ß-lactam antibiotics.

Distinct Amino Acid Availability-Dependent Regulatory Mechanisms of MepS and MepM Levels in Escherichia coli.

Peptidoglycan (PG) hydrolases play important roles in various aspects of bacterial physiology, including cytokinesis, PG synthesis, quality control of PG, PG recycling, and antibiotic resistance. However, the regulatory mechanisms of their expression are poorly understood. In this study, we have uncovered novel regulatory mechanisms of the protein levels of the synthetically lethal PG endopeptidases MepS and MepM, which are involved in PG synthesis. A mutant defective for both MepS and MepM was lethal in an amino acid-rich medium, whereas it exhibited almost normal growth in a minimal medium, suggesting the expendability of MepS and MepM in a minimal medium. Protein levels of MepS and MepM dramatically decreased in the minimal medium. Although MepM was revealed as a substrate of Prc, a periplasmic protease involved in the proteolysis of MepS, only the decrease in the MepS level in the minimal medium was affected by the prc depletion. Phenotypic and biochemical analyses showed that the presence of aromatic amino acids in the medium induced the accumulation of MepS, but not MepM, while the presence of glutamate increased the level of MepM, but not MepS. Together, these results demonstrate that the protein levels of the two major PG endopeptidases are regulated in an amino acid availability-dependent manner, but their molecular mechanisms and signaling are significantly distinct.

The inner membrane protein LapB is required for adaptation to cold stress in an LpxC-independent manner.

The inner membrane protein lipopolysaccharide assembly protein B (LapB) is an adaptor protein that activates the proteolysis of LpxC by an essential inner membrane metalloprotease, FtsH, leading to a decrease in the level of lipopolysaccharide in the membrane. In this study, we revealed the mechanism by which the essential inner membrane protein YejM regulates LapB and analyzed the role of the transmembrane domain of LapB in Escherichia coli. The transmembrane domain of YejM genetically and physically interacted with LapB and inhibited its function, which led to the accumulation of LpxC. The transmembrane domain of LapB was indispensable for both its physical interaction with YejM and its regulation of LpxC proteolysis. Notably, we found that the lapB mutant exhibited strong cold sensitivity and this phenotype was not associated with increased accumulation of LpxC. The transmembrane domain of LapB was also required for its role in adaptation to cold stress. Taken together, these results showed that LapB plays an important role in both the regulation of LpxC level, which is controlled by its interaction with the transmembrane domain of YejM, and adaptation to cold stress, which is independent of LpxC.

Genomic profile of metastatic breast cancer patient-derived xenografts established using percutaneous biopsy.

BACKGROUND: Metastatic breast cancer (mBC) is a complex and life-threatening disease and although it is difficult to cure, patients can benefit from sequential anticancer treatment, including endocrine therapy, targeted therapy and cytotoxic chemotherapy. The patient-derived xenograft (PDX) model is suggested as a practical tool to predict the clinical outcome of this disease as well as to screen novel drugs. This study aimed to establish PDX models in Korean patients and analyze their genomic profiles and utility for translational research. METHODS: Percutaneous core needle biopsy or punch biopsy samples were used for xenotransplantation. Whole exome sequencing and transcriptome analysis were performed to assess the genomic and RNA expression profiles, respectively. Copy number variation and mutational burden were analyzed and compared with other metastatic breast cancer genomic results. Mutational signatures were also analyzed. The antitumor effect of an ATR inhibitor was tested in the relevant PDX model. RESULTS: Of the 151 cases studied, 40 (26%) PDX models were established. Notably, the take rate of all subtypes, including the hormone receptor-positive (HR +) subtype, exceeded 20%. The PDX model had genomic fidelity and copy number variation that represented the pattern of its donor sample. TP53, PIK3CA, ESR1, and GATA3 mutations were frequently found in our samples, with TP53 being the most frequently mutated, and the somatic mutations in these genes strengthened their frequency in the PDX model. The ESR1 mutation, CCND1 amplification, and the APOBEC signature were significant features in our HR + HER2- PDX model. Fulvestrant in combination with palbociclib showed a partial response to the relevant patient's tumor harboring the ESR1 mutation, and CCND1 amplification was found in the PDX model. AZD6738, an ATR inhibitor, delayed tumor growth in a relevant PDX model. CONCLUSIONS: Our PDX model was established using core needle biopsy samples from primary and metastatic tissues. Genomic profiles of the samples reflected their original tissue characteristics and could be used for the interpretation of clinical outcomes.

A Class I Histone Deacetylase Inhibitor Attenuates Insulin Resistance and Inflammation in Palmitate-Treated C2C12 Myotubes and Muscle of HF/HFr Diet Mice.

Histone deacetylase (HDAC) inhibitors, which regulate gene expression by inhibiting the deacetylation of histones and nonhistone proteins, have been shown to exert a wide array of biological effects; these include anti-cancer, anti-obesity, and anti-diabetes effects, as well as cardiovascular-protective activity. However, the effects of class I HDAC inhibition on lipotoxicity in C2C12 myotubes and skeletal muscle tissue remain poorly understood. In this study, we investigated the molecular mechanism underlying the protective effect of class I HDAC inhibition under lipotoxic conditions, i.e., in palmitate (PA)-treated C2C12 myotubes and skeletal muscle tissue in high fat (HF)/high fructose (HFr) diet mice. PA treatment of C2C12 myotubes increased HDAC3 protein expression and impaired mitochondrial oxidation, resulting in increased mitochondrial ROS generation and an accumulation of intracellular triglycerides (TG). Prolonged exposure led to increased inflammatory cytokine expression and insulin resistance. In contrast, MS-275, a class I HDAC inhibitor, dramatically attenuated lipotoxicity, preventing PA-induced insulin resistance and inflammatory cytokine expression. Similar beneficial effects were also seen following HDAC3 knockdown. In addition, MS-275 increased the mRNA expression of peroxisome proliferator activator receptor γ-coactivator 1α (PGC1α) and mitochondrial transcription factor A (TFAM), which serve as transcriptional coactivators in the context of mitochondrial metabolism and biogenesis, and restored expression of peroxisome proliferator-activated receptor alpha (PPARα), medium-chain acyl-coenzyme A dehydrogenase (MCAD), enoyl-CoA hydratase, and 3-hydroxyacyl CoA dehydrogenase (EHHADH). In vivo, treatment of HF/HFr-fed mice with MS-275 ameliorated hyperglycemia, insulin resistance, stress signals, and TNF-α expression in skeletal muscle. Taken together, these results suggest that HDAC3 inhibition rather than HDAC1/2 inhibition by MS-275 protects against lipotoxicity in C2C12 myotubes and skeletal muscle, and may be effective for the treatment of obesity and insulin resistance.

Genetic Evidence for Distinct Functions of Peptidoglycan Endopeptidases in Escherichia coli.

Peptidoglycan (PG) is an essential component of the bacterial exoskeleton that plays a pivotal role in the maintenance of cell shape and resistance to cell lysis under high turgor pressures. The synthesis and degradation of PG must be tightly regulated during bacterial cell elongation and division. Unlike enzymes involved in PG synthesis, PG hydrolases show high redundancy in many bacteria including Escherichia coli. In this study, we showed that PG endopeptidases have distinct roles in cell growth and division. Phenotypic analysis of mutants lacking one of seven PG endopeptidases identified a MepM-specific phenotype, salt sensitivity, and a MepS-specific phenotype, EDTA sensitivity. Complementation test in each phenotype showed that the phenotype of the mepM mutant was restored only by MepM, whereas the phenotype of the mepS mutant was restored by MepS or by overexpression of MepH, PbpG, or MepM. These distinct phenotypes depend on both the specific localizations and specific domains of MepM and MepS. Finally, using the identified phenotypes, we revealed that MepM and MepH were genetically associated with both penicillin-binding protein 1a (PBP1a) and PBP1b, whereas MepS and PbpG were genetically associated with only PBP1b. Notably, a defect in PBP1a or PBP1b phenocopied the mepM mutant, suggesting the importance of MepM on PG synthesis. Therefore, our results indicate that each PG endopeptidase plays a distinct role in cell growth and division, depending on its distinct domains and cellular localizations.

A wearable lab-on-a-patch platform with stretchable nanostructured biosensor for non-invasive immunodetection of biomarker in sweat.

Conformable, wearable biosensor-integrated systems are a promising approach to non-invasive and quantitative on-body detection of biomarkers in body fluids. However, realizing such a system has been slowed by the difficulty of fabricating a soft affinity-based biosensor patch capable of precise on-body fluid handling with minimal wearer intervention and a simple measurement protocol. Herein, we demonstrate a conformable, wearable lab-on-a-patch (LOP) platform composed of a stretchable, label-free, impedimetric biosensor and a stretchable microfluidic device for on-body detection of the hormone biomarker, cortisol. The all-in-one, stretchable microfluidic device can precisely collect and deliver sweat for cortisol quantitation and offers one-touch operation of reagent delivery for simultaneous electrochemical signal generation and washing. Three-dimensional nanostructuring of the Au working electrode enables the high sensitivity required to detect the pM-levels of cortisol in sweat. Our integrated LOP detected sweat cortisol quantitatively and accurately during exercise. This LOP will open a new horizon for non-invasive, highly sensitive, and quantitative on-body immunodetection for wearable personal diagnostics.

GLP­1 improves palmitate­induced insulin resistance in human skeletal muscle via SIRT1 activity.

The present study investigated whether glucagon like peptide­1 (GLP­1) improves glucose uptake through glucose transporter type 4 (GLUT4), mediated by the activation of sirtuin 1 (SIRT1), in skeletal muscle cells with palmitate induced­insulin resistance. The levels of glucose uptake, GLUT4, protein kinase A (PKA), and cyclic adenosine monophosphate (cAMP) were determined in human skeletal muscle myotubes (HSMMs) exposed to palmitate and GLP­1. Then, to determine whether PKA/cAMP were downstream signals of GLP­1, a PKA inhibitor was used. To determine whether SIRT­1 contributes to GLP­1 action in HSMMs with palmitate­induced insulin resistance, the levels of peroxisome proliferator­activated receptor γ coactivator 1α (PGC1α) deacetylation and SIRT­1 activity were assessed using a SIRT1 inhibitor and small interfering RNA (siRNA). The phosphorylation levels of protein kinase B (Akt) and insulin receptor substrate 1 (IRS­1) as insulin signaling pathways, were assessed in GLP­1­treated HSMMs exposed to palmitate. The influence of SIRT1 on the GLP­1­induced activation of insulin signaling pathway was determined using a SIRT1 inhibitor. GLP­1 restored the palmitate­induced reductions in the levels of glucose uptake, GLUT4 mRNA, GLUT4 promoter activity, and GLUT4 protein in HSMMs. PKA and cAMP, as GLP­1 downstream signals, played a role in this process. GLP­1 increased the deacetylation levels of PGC1α, and stimulated SIRT1 in HSMMs. Moreover, the SIRT1 inhibitor and siRNA of SIRT1 suppressed the effect of GLP­1 on GLUT4 expression in HSMMs exposed to palmitate. The SIRT1 inhibitor also prevented the GLP­1­induced phosphorylation of IRS­1 and Akt in palmitate­treated HSMMs. The present findings suggest that in palmitate­induced insulin­resistant HSMM, GLP­1 activates SIRT1 through the PKA/cAMP pathway, which in turn enhances glucose uptake through GLUT4 and the insulin signaling pathway.

Fully Stretchable Capillary Microfluidics-Integrated Nanoporous Gold Electrochemical Sensor for Wearable Continuous Glucose Monitoring.

Biosensor systems for wearable continuous monitoring are desired to be developed into conformal patch platforms. However, developing such patches is very challenging owing to the difficulty of imparting materials and components with both high stretchability and high performance. Herein, we report a fully stretchable microfluidics-integrated glucose sensor patch comprised of an omnidirectionally stretchable nanoporous gold (NPG) electrochemical biosensor and a stretchable passive microfluidic device. A highly electrocatalytic NPG electrode was formed on a stress-absorbing 3D micropatterned polydimethylsiloxane (PDMS) substrate to confer mechanical stretchability, high sensitivity, and durability in non-enzymatic glucose detection. A thin, stretchable, and tough microfluidic device was made by embedding stretchable cotton fabric as a capillary into a thin polyurethane nanofiber-reinforced PDMS channel, enabling collection and passive, accurate delivery of sweat from skin to the electrode surface, with excellent replacement capability. The integrated glucose sensor patch demonstrated excellent ability to continuously and accurately monitor the sweat glucose level.

Neurite Guidance on Laser-Scribed Reduced Graphene Oxide.

This paper describes a one-step, chemical-free method to generate micropatterned in vitro neuronal networks on chemically unmodified reduced graphene oxide. The suggested method relies on infrared-based photothermal reduction of graphene oxide, which concurrently leads to the formation of submicrometer-scale surface roughness that promotes neuronal adhesion and guides neurite outgrowth. A commercially available laser source (LightScribe DVD drive) controlled by a computer software can be used to reduce graphene oxide (GO), and its repetitive scribing to a GO film brings about gradual increase and decrease in electrical conductivity and neurite guiding ability of the scribed regions, respectively. Our results also indicate that the observed adhesion-promoting and neurite guiding effect originate from the contrast in surface nanotopography, but not that in conductivity. This method is readily applicable to diverse graphene-based biomedical devices.

An Omnidirectionally Stretchable Photodetector Based on Organic-Inorganic Heterojunctions.

Omnidirectionally stretchable photodetectors are limited by difficulties in designing material and fabrication processes that enable stretchability in multiaxial directions. Here, we propose a new approach involving an organic-inorganic p-n heterojunction photodetector comprised of free-standing ZnO nanorods grown on a poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate transport layer coated on a three-dimensional micropatterned stretchable substrate containing bumps and valleys. This structure allows for efficient absorption of stretching strain. This approach allows the device to accommodate large tensile strain in all of the directions. The device behaves as a photogated p-n heterojunction photodetector in which current modulation was obtained by sensing the mechanisms that rely on photovoltage and photogating effects. The device exhibits a high photoresponse to UV light and reliable electrical performance under applied stretching in uniaxial and omniaxial directions. Furthermore, the device can be easily and conformally attached to a human wrist. This allowed us to investigate the response of the device to UV light during human activity.

A Solution-Processable, Omnidirectionally Stretchable, and High-Pressure-Sensitive Piezoresistive Device.

The development of omnidirectionally stretchable pressure sensors with high performance without stretching-induced interference has been hampered by many challenges. Herein, an omnidirectionally stretchable piezoresistive pressure-sensing device is demonstrated by combining an omniaxially stretchable substrate with a 3D micropattern array and solution-printing of electrode and piezoresistive materials. A unique substrate structural design and materials mean that devices that are highly sensitive are rendered, with a stable out-of-plane pressure response to both static (sensitivity of 0.5 kPa-1 and limit of detection of 28 Pa) and dynamic pressures and the minimized in-plane stretching responsiveness (a small strain gauge factor of 0.17), achieved through efficient strain absorption of the electrode and sensing materials. The device can detect human-body tremors, as well as measure the relative elastic properties of human skin. The omnidirectionally stretchable pressure sensor with a high pressure sensitivity and minimal stretch-responsiveness yields great potential to skin-attachable wearable electronics, human-machine interfaces, and soft robotics applications.

Mogul-Patterned Elastomeric Substrate for Stretchable Electronics.

A mogul-patterned stretchable substrate with multidirectional stretchability and minimal fracture of layers under high stretching is fabricated by double photolithography and soft lithography. Au layers and a reduced graphene oxide chemiresistor on a mogul-patterned poly(dimethylsiloxane) substrate are stable and durable under various stretching conditions. The newly designed mogul-patterned stretchable substrate shows great promise for stretchable electronics.

Engineering Optical and Electronic Properties of WS2 by Varying the Number of Layers.

The optical constants, bandgaps, and band alignments of mono-, bi-, and trilayer WS2 were experimentally measured, and an extraordinarily high dependency on the number of layers was revealed. The refractive indices and extinction coefficients were extracted from the optical-contrast oscillation for various thicknesses of SiO2 on a Si substrate. The bandgaps of the few-layer WS2 were both optically and electrically measured, indicating high exciton-binding energies. The Schottky-barrier heights (SBHs) with Au/Cr contact were also extracted, depending on the number of layers (1-28). From an engineering viewpoint, the bandgap can be modulated from 3.49 to 2.71 eV with additional layers. The SBH can also be reduced from 0.37 eV for a monolayer to 0.17 eV for 28 layers. The technique of engineering materials' properties by modulating the number of layers opens pathways uniquely adaptable to transition-metal dichalcogenides.