Living myofibroblast-silicon composites for probing electrical coupling in cardiac systems.

Traditional bioelectronics, primarily comprised of nonliving synthetic materials, lack cellular behaviors such as adaptability and motility. This shortcoming results in mechanically invasive devices and nonnatural signal transduction across cells and tissues. Moreover, resolving heterocellular electrical communication in vivo is extremely limited due to the invasiveness of traditional interconnected electrical probes. In this paper, we present a cell-silicon hybrid that integrates native cellular behavior (e.g., gap junction formation and biosignal processing) with nongenetically enabled photosensitivity. This hybrid configuration allows interconnect-free cellular modulation with subcellular spatial resolution for bioelectric studies. Specifically, we hybridize cardiac myofibroblasts with silicon nanowires and use these engineered hybrids to synchronize the electrical activity of cardiomyocytes, studying heterocellular bioelectric coupling in vitro. Thereafter, we inject the engineered myofibroblasts into heart tissues and show their ability to seamlessly integrate into contractile tissues in vivo. Finally, we apply local photostimulation with high cell specificity to tackle a long-standing debate regarding the existence of myofibroblast-cardiomyocyte electrical coupling in vivo.

Silicon Nanowires for Intracellular Optical Interrogation with Subcellular Resolution.

Current techniques for intracellular electrical interrogation are limited by substrate-bound devices, technically demanding methods, or insufficient spatial resolution. In this work, we use freestanding silicon nanowires to achieve photoelectric stimulation in myofibroblasts with subcellular resolution. We demonstrate that myofibroblasts spontaneously internalize silicon nanowires and subsequently remain viable and capable of mitosis. We then show that stimulation of silicon nanowires at separate intracellular locations results in local calcium fluxes in subcellular regions. Moreover, nanowire-myofibroblast hybrids electrically couple with cardiomyocytes in coculture, and photostimulation of the nanowires increases the spontaneous activation rate in coupled cardiomyocytes. Finally, we demonstrate that this methodology can be extended to the interrogation of signaling in neuron-glia interactions using nanowire-containing oligodendrocytes.

[Enhancement of Pesticide Peak Response in GC-MS in the Presence of Multiple Co-Existing Reference Pesticides].

We carried out a collaborative study in six laboratories to confirm the universality of the enhancing effect of co-existing reference pesticides on the GC-MS peak response to a target pesticide (malathion, procymidone, or flucythrinate). First, we confirmed the response enhancement of the target pesticides with increasing numbers of co-existing reference pesticides in solution. Then, using diluted green soybean matrix, we analyzed the target pesticides with two types of matrix-matched calibration, containing the target pesticides or 166 other pesticides. In both cases, the response-enhancing effect of co-existing pesticides was confirmed in all laboratories. The enhancement was reduced by addition of green soybean matrix to the sample and calibration solutions. Our results show that it is necessary to estimate the peak response-enhancing effect of co-existing pesticides in the calibration solution to obtain accurate results with GC-MS determination. The enhancing effect could be reduced by addition of food matrix to the sample and calibration solutions.

Application of proteotyping Strain Solution™ ver. 2 software and theoretically calculated mass database in MALDI-TOF MS typing of Salmonella serotype.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based microbial identification is a popular analytical method. Strain Solution proteotyping software available for MALDI-TOF MS has great potential for the precise and detailed discrimination of microorganisms at serotype- or strain-level, beyond the conventional mass fingerprinting approaches. Here, we constructed a theoretically calculated mass database of Salmonella enterica subspecies enterica consisting of 12 biomarker proteins: ribosomal proteins S8, L15, L17, L21, L25, and S7, Mn-cofactor-containing superoxide dismutase (SodA), peptidyl-prolyl cis-trans isomerase C (PPIase C), and protein Gns, and uncharacterized proteins YibT, YaiA, and YciF, that can allow serotyping of Salmonella. Strain Solution ver. 2 software with the novel database constructed in this study demonstrated that 109 strains (94%), including the major outbreak-associated serotypes, Enteritidis, Typhimurium, and Infantis, could be correctly identified from others by colony-directed MALDI-TOF MS using 116 strains belonging to 23 kinds of typed and untyped serotypes of S. enterica from culture collections, patients, and foods. We conclude that Strain Solution ver. 2 software integrated with the accurate mass database will be useful for the bacterial proteotyping by MALDI-TOF MS-based microbial classification in the clinical and food safety fields.

Enhancing immunogenic responses through CDK4/6 and HIF2α inhibition in Merkel cell carcinoma.

Approximately 50% of Merkel cell carcinoma (MCC) patients facing this highly aggressive skin cancer initially respond positively to PD-1-based immunotherapy. Nevertheless, the recurrence of MCC post-immunotherapy emphasizes the pressing need for more effective treatments. Recent research has highlighted Cyclin-dependent kinases 4 and 6 (CDK4/6) as pivotal cell cycle regulators gaining prominence in cancer studies. This study reveals that the CDK4/6 inhibitor, palbociclib can enhance PD-L1 gene transcription and surface expression in MCC cells by activating HIF2α. Inhibiting HIF2α with TC-S7009 effectively counteracts palbociclib-induced PD-L1 transcription and significantly intensifies cell death in MCC. Simultaneously, co-targeting CDK4/6 and HIF2α boosts ROS levels while suppressing SLC7A11, a key regulator of cellular redox balance, promoting ferroptosis- a form of immunogenic cell death linked to iron. Considering the rising importance of immunogenic cell death in immunotherapy, this strategy holds promise for improving future MCC treatments, markedly increasing immunogenic cell death various across various MCC cell lines, thus advancing cancer immunotherapy.

SRC inhibition enables formation of a growth suppressive MAGI1-PP2A complex in isocitrate dehydrogenase-mutant cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive bile duct malignancy that frequently exhibits isocitrate dehydrogenase (IDH1/IDH2) mutations. Mutant IDH (IDHm) ICC is dependent on SRC kinase for growth and survival and is hypersensitive to inhibition by dasatinib, but the molecular mechanism underlying this sensitivity is unclear. We found that dasatinib reduced p70 S6 kinase (S6K) and ribosomal protein S6 (S6), leading to substantial reductions in cell size and de novo protein synthesis. Using an unbiased phosphoproteomic screen, we identified membrane-associated guanylate kinase, WW, and PDZ domain containing 1 (MAGI1) as an SRC substrate in IDHm ICC. Biochemical and functional assays further showed that SRC inhibits a latent tumor-suppressing function of the MAGI1-protein phosphatase 2A (PP2A) complex to activate S6K/S6 signaling in IDHm ICC. Inhibiting SRC led to activation and increased access of PP2A to dephosphorylate S6K, resulting in cell death. Evidence from patient tissue and cell line models revealed that both intrinsic and extrinsic resistance to dasatinib is due to increased phospho-S6 (pS6). To block pS6, we paired dasatinib with the S6K/AKT inhibitor M2698, which led to a marked reduction in pS6 in IDHm ICC cell lines and patient-derived organoids in vitro and substantial growth inhibition in ICC patient-derived xenografts in vivo. Together, these results elucidated the mechanism of action of dasatinib in IDHm ICC, revealed a signaling complex regulating S6K phosphorylation independent of mTOR, suggested markers for dasatinib sensitivity, and described a combination therapy for IDHm ICC that may be actionable in the clinic.

Micelle-enabled self-assembly of porous and monolithic carbon membranes for bioelectronic interfaces.

Real-world bioelectronics applications, including drug delivery systems, biosensing and electrical modulation of tissues and organs, largely require biointerfaces at the macroscopic level. However, traditional macroscale bioelectronic electrodes usually exhibit invasive or power-inefficient architectures, inability to form uniform and subcellular interfaces, or faradaic reactions at electrode surfaces. Here, we develop a micelle-enabled self-assembly approach for a binder-free and carbon-based monolithic device, aimed at large-scale bioelectronic interfaces. The device incorporates a multi-scale porous material architecture, an interdigitated microelectrode layout and a supercapacitor-like performance. In cell training processes, we use the device to modulate the contraction rate of primary cardiomyocytes at the subcellular level to target frequency in vitro. We also achieve capacitive control of the electrophysiology in isolated hearts, retinal tissues and sciatic nerves, as well as bioelectronic cardiac sensing. Our results support the exploration of device platforms already used in energy research to identify new opportunities in bioelectronics.

Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) Can Precisely Discriminate the Lineages of Listeria monocytogenes and Species of Listeria.

The genetic lineages of Listeria monocytogenes and other species of the genus Listeria are correlated with pathogenesis in humans. Although matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a prevailing tool for rapid and reliable microbial identification, the precise discrimination of Listeria species and lineages remains a crucial issue in clinical settings and for food safety. In this study, we constructed an accurate and reliable MS database to discriminate the lineages of L. monocytogenes and the species of Listeria (L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, L. grayi, and L. rocourtiae) based on the S10-spc-alpha operon gene encoded ribosomal protein mass spectrum (S10-GERMS) proteotyping method, which relies on both genetic information (genomics) and observed MS peaks in MALDI-TOF MS (proteomics). The specific set of eight biomarkers (ribosomal proteins L24, L6, L18, L15, S11, S9, L31 type B, and S16) yielded characteristic MS patterns for the lineages of L. monocytogenes and the different species of Listeria, and led to the construction of a MS database that was successful in discriminating between these organisms in MALDI-TOF MS fingerprinting analysis followed by advanced proteotyping software Strain Solution analysis. We also confirmed the constructed database on the proteotyping software Strain Solution by using 23 Listeria strains collected from natural sources.

Development of a Functional Biohybrid Implant Formed from Periodontal Tissue Utilizing Bioengineering Technology.

Current osseointegrated dental implants have been widely used for the rehabilitation of tooth loss. Although dental implants are considered an available treatment in the paradigm shift from traditional dental therapies, such as fixed dental bridges and removable dentures, the fundamental problems must be overcome before their clinical use in young patients who are still undergoing jawbone growth. Here, we show a novel bioengineering method for a functional biohybrid implant that is combined with adult-derived periodontal tissue and attached with bone tissue as a substitute for cementum. This biohybrid implant was successfully engrafted using the bioengineered periodontal ligament, and it restored physiological function, such as orthodontic movement through bone remodeling and appropriate responsiveness to noxious stimuli. Thus, this study represents the functional biohybrid implant's potential for clinical use as a next-generation dental implant using adult-derived tissues.

Assessing the performance of novel software Strain Solution on automated discrimination of Escherichia coli serotypes and their mixtures using matrix-assisted laser desorption ionization-time of flight mass spectrometry.

O157, O26, and O111 are the most important O serogroups of enterohemorrhagic Escherichia coli worldwide. Recently we reported a strategy for discriminating these serotypes from the others using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) based on the S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum (S10-GERMS) method. To realize the fully automated identification of microorganisms at species- or serotype-level with the concept of S10-GERMS method, novel software named Strain Solution for MALDI-TOF MS was developed. In this study, the Strain Solution was evaluated with a total of 45 E. coli isolates including O26, O91, O103, O111, O115, O121, O128, O145, O157, O159, and untyped serotypes. The Strain Solution could accurately discriminate 92% (11/12) of O157 strains, 100% (13/13) of O26 and O111 strains from the others with three biomarkers in an automated manner. In addition, this software could identify 2 different E. coli strains (K-12 as a non-O157 representative and O157) in mixed samples. The results suggest that Strain Solution will be useful for species- or serotype-level classification of microorganisms in the fields of food safety and diagnostics.

Functional tooth restoration utilising split germs through re-regionalisation of the tooth-forming field.

The tooth is an ectodermal organ that arises from a tooth germ under the regulation of reciprocal epithelial-mesenchymal interactions. Tooth morphogenesis occurs in the tooth-forming field as a result of reaction-diffusion waves of specific gene expression patterns. Here, we developed a novel mechanical ligation method for splitting tooth germs to artificially regulate the molecules that control tooth morphology. The split tooth germs successfully developed into multiple correct teeth through the re-regionalisation of the tooth-forming field, which is regulated by reaction-diffusion waves in response to mechanical force. Furthermore, split teeth erupted into the oral cavity and restored physiological tooth function, including mastication, periodontal ligament function and responsiveness to noxious stimuli. Thus, this study presents a novel tooth regenerative technology based on split tooth germs and the re-regionalisation of the tooth-forming field by artificial mechanical force.

Discrimination of Escherichia coli O157, O26 and O111 from other serovars by MALDI-TOF MS based on the S10-GERMS method.

Enterohemorrhagic Escherichia coli (EHEC), causes a potentially life-threatening infection in humans worldwide. Serovar O157:H7, and to a lesser extent serovars O26 and O111, are the most commonly reported EHEC serovars responsible for a large number of outbreaks. We have established a rapid discrimination method for E. coli serovars O157, O26 and O111 from other E. coli serovars, based on the pattern matching of mass spectrometry (MS) differences and the presence/absence of biomarker proteins detected in matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS). Three biomarkers, ribosomal proteins S15 and L25, and acid stress chaperone HdeB, with MS m/z peaks at 10138.6/10166.6, 10676.4/10694.4 and 9066.2, respectively, were identified as effective biomarkers for O157 discrimination. To distinguish serovars O26 and O111 from the others, DNA-binding protein H-NS, with an MS peak at m/z 15409.4/15425.4 was identified. Sequence analysis of the O157 biomarkers revealed that amino acid changes: Q80R in S15, M50I in L25 and one mutation within the start codon ATG to ATA in the encoded HdeB protein, contributed to the specific peak pattern in O157. We demonstrated semi-automated pattern matching using these biomarkers and successfully discriminated total 57 O157 strains, 20 O26 strains and 6 O111 strains with 100% reliability by conventional MALDI-TOF MS analysis, regardless of the sample conditions. Our simple strategy, based on the S10-spc-alpha operon gene-encoded ribosomal protein mass spectrum (S10-GERMS) method, therefore allows for the rapid and reliable detection of this pathogen and may prove to be an invaluable tool both clinically and in the food industry.

[Efficacy of patient skin dose reduction by a compensating filter through of irradiation field overlaps on the area during percutaneous coronary intervention].

Our study was involved with entrance surface dose reduction and irradiation field by the filter use of PCI, and insertion in place of an effective compensating filter to maximize entrance surface dose reduction, which we verified. The radiation dosimetry put a 6cc ion chamber on the back side of the thorax phantom, and changed the filter of the four corners (a: upper left, b: upper right, c: lower right, d: lower left) of the monitor confirmed with fluoroscopy [(0) no filter, (1) one filter, (2) two filters]. The angle of C arm was assumed to be eight directions and 0 degrees adopted by this hospital. It was compared with a corrective rate of which one was no filter. Next, the presence of filter and irradiation field overlaps on the area in monitor in the angle of C arm was verified by this hospital's classic example. As for corrective rate, (1) becomes 0.41 and (2) become 0.25 at fluoroscopy, (1) becomes 0.26 and (2) become 0.16 at exposure. Irradiation field overlaps on the area (+) compensating filter (-) was many with d of RAO/CAU, a of RAO and c of CAU at left CAG, c of LAO at right CAG, b of LAO/CRA (left CAG), b of CRA (right CAG) and a and d of RAO (right CAG) at both CAG. Irradiation field overlaps on the area (+) compensating filter (+) was many with b of CRA at left CAG, a of LAO/CRA at right CAG, b of CRA (left CAG) and b of RAO (right CAG) at both CAG. When the compensating filter is used the entrance surface dose reduction effect was great. If automatic exposure control protects the part of irradiation field overlaps on the area in the range without operating excessively, the radiological risk can be reduced, and it is conceivable as useful clinical setting.

Effect of aerobic leg exercise training on subcutaneous adipose tissue of thigh in young Japanese women.

A one-legged training model was adopted to assess the influence of moderate-intensity exercise training on subcutaneous adipose tissue, especially focusing on the trained limb. Eight young sedentary Japanese women (ages 21-23 yr) participated in a 12-week training program. The leg was assigned randomly to trained or untrained limb. Each subject performed a supervised 60-min one-legged cycle ergometer training session three times a week. The exercise intensity was set at approximately 40% of peak VO(2) ("moderate" intensity, i.e., below the estimated lactate threshold), which was determined before training. Each subject performed a one-legged incremental cycle exercise test until exhaustion to determine the peak VO(2) of each leg (the trained and untrained legs being investigated separately). The areas of subcutaneous fat and the remaining nonfat tissues of the thigh were evaluated by our novel visualized measuring system based on ultrasonography. The fat cross-sectional areas of the trained and untrained thigh were not different after training (trained: 68.6+/-17.8 vs. untrained: 68.3+/-18.5 cm(2)). The non-fat (muscle and bone) area was also similar between the trained and untrained limb. In addition, there was no systematic influence of the training on the total and lean body mass. After training, the duration time of the one-legged cycle incremental exercise test by the trained leg was significantly improved unilaterally (trained: 1049+/-122 vs. untrained: 930+/-109 s, p<0.05) without any difference of peak VO(2). The results indicate that moderate one-legged aerobic training did not induce any compositional change in the trained thigh; rather, what did improve were certain peripheral factor alone relating to endurance.