Search

Advances in fluorescence imaging technology have been crucial to the progress of neuroscience. Whether it was specific expression of indicator proteins, detection of neurotransmitters, or miniaturization of fluorescence microscopes, fluorescence imaging has improved upon electrophysiology, the gold standard for monitoring brain activity, and enabled novel methods to sense activity in the brain. Hence, we developed a lightweight and compact implantable CMOS-based lensless Ca2+ imaging device for freely moving transgenic G-CaMP mouse experiments. However, without a lens system, determination of regions of interest (ROI) has proven challenging. Localization of fluorescence activity and separation of signal from noise are difficult. In this study, we report an ROI selection method using a series of adaptive binarizations with a gaussian method and morphological image processing. The parameters for each operation such as the kernel size, sigma and footprint size were optimized. We then validated the utility of the algorithm with simulated data and freely moving nociception experiments using the lensless devices. The device was implanted in the dorsal raphe nucleus to observe pain-related brain activity following a formalin test to stimulate pain. We observed significant increases in fluorescence activity after formalin injection compared to the control group when using the ROI determination algorithm., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Castillo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

[Purpose] No established method for appropriately developing clinical thinking in physical therapy currently exists. This study examined whether clinical thinking can be appropriately developed using a problem-solving model. [Participants and Methods] Physical therapy students were asked to develop clinical thinking in the same two cases: one class using the problem-solving model and the other class using International Classification of Functioning, Disability and Health concepts. Each clinical thought was scored and compared based on consistency. [Results] In both cases, students who used the problem-solving model scored higher. [Conclusion] The hierarchical structure of the problem-solving model clarified the relationship between each element and this was easy to maintain, suggesting that it facilitated appropriate clinical thinking., Competing Interests: The authors declare that there is no conflict of interest., (2024©by the Society of Physical Therapy Science. Published by IPEC Inc.)

[Purpose] A method for evaluating integrated interpretation skills, which is an important educational goal in clinical practice, is yet to be established. This study examined the interrater reliability of a problem-solving model that was developed as a novel assessment method. [Participants and Methods] Three students created problem-solving models using fictitious case data. Ten clinical practice supervisors with at least 3 years of experience were asked to score the models, and the intraclass correlation coefficient (2, 1) was calculated. [Results] The intraclass correlation coefficient was 0.88, which indicates a good result. [Conclusion] Good interrater reliability was obtained using the problem-solving model, which suggests that this model may be used to assess integrated interpretation skills in clinical practice., Competing Interests: The authors declare that there are no conflicts of interest., (2024©by the Society of Physical Therapy Science. Published by IPEC Inc.)

Objective : Current neuronal imaging methods mostly use bulky lenses that either impede animal behavior or prohibit multi-depth imaging. To overcome these limitations, we developed a lightweight lensless biophotonic system for neuronal imaging, enabling compact and simultaneous visualization of multiple brain layers. Approach : Our developed 'CIS-NAIST' device integrates a micro-CMOS image sensor, thin-film fluorescence filter, micro-LEDs, and a needle-shaped flexible printed circuit. With this device, we monitored neuronal calcium dynamics during seizures across the different layers of the hippocampus and employed machine learning techniques for seizure classification and prediction. Main results : The CIS-NAIST device revealed distinct calcium activity patterns across the CA1, molecular interlayer, and dentate gyrus. Our findings indicated an elevated calcium amplitude activity specifically in the dentate gyrus compared to other layers. Then, leveraging the multi-layer data obtained from the device, we successfully classified seizure calcium activity and predicted seizure behavior using Long Short-Term Memory and Hidden Markov models. Significance : Taken together, our 'CIS-NAIST' device offers an effective and minimally invasive method of seizure monitoring that can help elucidate the mechanisms of temporal lobe epilepsy., (Creative Commons Attribution license.)

Infectious and foodborne diseases pose significant global threats, with devastating consequences in low- and middle-income countries. Ozone, derived from atmospheric oxygen, exerts antimicrobial effects against various microorganisms, and degrades fungal toxins, which were initially recognized in the healthcare and food industries. However, highly concentrated ozone gas can be detrimental to human health. In addition, ozonated water is unstable and has a short half-life. Therefore, ultrafine-bubble technology is expected to overcome these issues. Ultrafine bubbles, which are nanoscale entitles that exist in water for considerable durations, have previously demonstrated bactericidal effects against various bacterial species, including antibiotic-resistant strains. This present study investigated the effects of ozone ultrafine bubble water (OUFBW) on various bacterial toxins. This study revealed that OUFBW treatment abolished the toxicity of pneumolysin, a pneumococcal pore-forming toxin, and leukotoxin, a toxin that causes leukocyte injury. Silver staining confirmed the degradation of pneumolysin, leukotoxin, and staphylococcal enterotoxin A, which are potent gastrointestinal toxins, following OUFB treatment. In addition, OUFBW treatment significantly inhibited NF-κB activation by Pam3CSK4, a synthetic triacylated lipopeptide that activates Toll-like receptor 2. Additionally, OUFBW exerted bactericidal activity against Staphylococcus aureus, including an antibiotic-resistant strain, without displaying significant toxicity toward human neutrophils or erythrocytes. These results suggest that OUFBW not only sterilizes bacteria but also degrades bacterial toxins., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Takizawa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In this study, we developed and demonstrated a millimeter-wave electric field imaging system using an electro-optic crystal and a highly sensitive polarization measurement technique using a polarization image sensor, which was fabricated using a 0.35-µm standard CMOS process. The polarization image sensor was equipped with differential amplifiers that amplified the difference between the 0° and 90° pixels. With the amplifier, the signal-to-noise ratio at low incident light levels was improved. Also, an optical modulator and a semiconductor optical amplifier were used to generate an optical local oscillator (LO) signal with a high modulation accuracy and sufficient optical intensity. By combining the amplified LO signal and a highly sensitive polarization imaging system, we successfully performed millimeter-wave electric field imaging with a spatial resolution of 30×60 µm at a rate of 1 FPS, corresponding to 2400 pixels/s.

We developed two methods for three-dimensional (3D) evaluation of spinal alignment in standing position by image matching between biplanar x-ray images and 3D vertebral models. One used a Slot-Scanning 3D x-ray Imager (sterEOS) to obtain biplanar x-ray images, and the other used a conventional x-ray system and a rotating table. The 3D vertebral model was constructed from the CT scan data. The spatial position of the vertebral model was determined by minimizing the contour difference between the projected image of the model and the biplanar x-ray images. Verification experiments were conducted using a torso phantom. The relative positions of the upper vertebrae to the lowest vertebrae of the cervical, thoracic, and lumbar vertebrae were evaluated. The mean, standard deviation, and mean square error of the relative position were less than 1° and 1 mm in all cases for sterEOS. The maximum mean squared errors of the conventional x-ray system and the rotating table were 0.7° and 0.4 mm for the cervical spine, 1.0° and 1.2 mm for the thoracic spine, and 1.1° and 1.2 mm for the lumbar spine. Therefore, both methods could be useful for evaluating the spinal alignment in standing position., Competing Interests: No conflict of interest was reported by the author(s)., (Copyright © 2024 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of PBJ-Associação Porto Biomedical/Porto Biomedical Society.)

Background: The Kerlan-Jobe Orthopedic Clinic (KJOC) questionnaire is a self-reported performance and functional assessment tool with good reliability and validity for overhead athletes with shoulder and elbow injuries. This study aimed to develop a Japanese version of the KJOC (J-KJOC) to clarify its reproducibility and validity for use by Japanese university baseball players., Methods: The J-KJOC was translated according to the guidelines for cross-cultural adaptation. A total of 88 university baseball players completed the J-KJOC and the Quick-Disabilities of the Arm, Shoulder, and Hand (Q-DASH) questionnaires. Thirty players completed the J-KJOC two times after a median interval of two weeks. We assessed the absolute reliability, construct validity, internal consistency, and test-retest reliability., Results: Cronbach's alpha coefficients ranged from 0.88 and the intraclass correlation coefficient for the total score was 0.91. A fixed bias was absent in the J-KJOC scores (mean difference: -2.2, 95% CI: -4.8 to 0.5). Furthermore, the J-KJOC score was correlated with the Q-DASH-disability/symptom (r = -0.60, p<0.01) and Q-DASH-sports/music (r = -0.63, p<0.01) scores but not correlated with the Q-DASH-work score (r = -0.11, p = 0.316)., Conclusions: The J-KJOC questionnaire demonstrated good reproducibility and validity for assessing upper arm performance in Japanese university baseball players. The results of this study support the use of the J-KJOC for Japanese-speaking baseball players. Further research using this instrument on other types of overhead athletes is needed to determine its wider utility in sports medicine applications., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Sasagawa et al.)

We propose and demonstrate a method for equivalent time sampling using image sensors to selectively detect only the target frequency. Shortening the exposure time of the image sensor and using equivalent time sampling allows for the detection of frequency components that are higher than the frame rate. However, the imaging system in our previous work was also sensitive to the frequency component at 1/4 of the frame rate. In this study, we control the phase relationship between the exposure time and observed signal by inserting an additional interval once every four frames to detect the target frequency selectively. With this technique, we conducted electric field imaging based on the electro-optic effect under high noise conditions in the low-frequency band to which the conventional method is sensitive. The results demonstrated that the proposed method improved the signal-to-noise ratio.

Goreisan is a Kampo medicine used to treat headaches associated with climate change. Here, by using an implantable complementary metal-oxide-semiconductor (CMOS) device, we evaluated the effects of Goreisan and loxoprofen on cerebral blood flow (CBF) dynamics associated with barometric pressure fluctuations in freely moving mice. In the vehicle group, decreasing barometric pressure increased CBF that was prevented by Goreisan and loxoprofen. Notably, Goreisan, but not loxoprofen, reduced CBF after returning to atmospheric pressure. These results indicate that, unlike the mechanism of action of antipyretic analgesics, Goreisan normalizes CBF abnormalities associated with barometric pressure fluctuations by actively reducing CBF increase., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest regarding this research and/or publication., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background: The effect of trunk stability and dynamic balance warm-up exercises on physical functional improvement remains unelucidated. This study examined whether exercises could prevent anterior cruciate ligament (ACL) injury and improve trunk muscle activation and dynamic balance in gymnasts., Methods: This comparison study, involving gymnastics practice sessions, included 31 university gymnasts and was conducted in two periods: 1 year of observation followed by 2 years of intervention. Participants performed a trunk and dynamic balance warm-up exercise program during the intervention. The effect of exercise on the incidence of ACL injury was evaluated. In addition, the paired t-test was used to compare the Y-balance distance and the changes in muscle thickness associated with trunk muscle activation at rest and during plank., Results: ACL injury risk during the intervention was significantly lower, with a relative risk of 0.23 (P=0.02, 95% CI: 0.06-0.88). Changes in muscle thickness with activation of the transversus abdominis (P<0.01, mean difference 4.1, 95% CI: 9.97-28.07, Cohen's d=0.52), internal oblique (P<0.01, mean difference 5.2, 95% CI: 9.72-21.55, Cohen's d=0.65), and external oblique (P<0.01, mean difference 5.5, 95% CI: 20.44-39.09, Cohen's d=0.71) muscles were significantly higher during the intervention. The Y-balance distance was also significantly greater in the posterior medial reach (P<0.01, mean difference 3.3, 95% CI: 1.56-6.26, Cohen's d=0.46) during the intervention., Conclusions: Exercise-based warm-up programs may decrease ACL injuries. It can improve physical functions, such as the rate of change in trunk muscle thickness and the posterior medial distance during Y balance.

Background: Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia., Methods: The Actin CreER /R26 VT2/GK3 Rainbow reporter mouse was used for unbiased in vivo survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA-sequencing was used to characterize distinct vessel-forming populations and their interactions., Results: Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells: VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105 high Sca1 low , which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105 low Sca1 high and forms stunted vessels and fat. Interestingly, cotransplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice., Conclusions: These findings suggest that autologous cotransplantation of synergistic VSPCs from nonessential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease., Competing Interests: Disclosures None.

Over the past 2 decades, the prevalence of macrolide-resistant Streptococcus pneumoniae (MRSP) has increased considerably, due to widespread macrolide use. Although macrolide usage has been proposed to be associated with treatment failure in patients with pneumococcal diseases, macrolides may be clinically effective for treating these diseases, regardless of the susceptibility of the causative pneumococci to macrolides. As we previously demonstrated that macrolides downregulate the transcription of various genes in MRSP, including the gene encoding the pore-forming toxin pneumolysin, we hypothesized that macrolides affect the proinflammatory activity of MRSP. Using HEK-Blue cell lines, we found that the supernatants from macrolide-treated MRSP cultures induced decreased NF-κB activation in cells expressing Toll-like receptor 2 and nucleotide-binding oligomerization domain 2 compared to the supernatants from untreated MRSP cells, suggesting that macrolides inhibit the release of these ligands from MRSP. Real-time PCR analysis revealed that macrolides significantly downregulated the transcription of various genes encoding peptidoglycan synthesis-, lipoteichoic acid synthesis-, and lipoprotein synthesis-related molecules in MRSP cells. The silkworm larva plasma assay demonstrated that the peptidoglycan concentrations in the supernatants from macrolide-treated MRSP cultures were significantly lower than those from untreated MRSP cultures. Triton X-114 phase separation revealed that lipoprotein expression decreased in macrolide-treated MRSP cells compared to the lipoprotein expression in untreated MRSP cells. Consequently, macrolides may decrease the expression of bacterial ligands of innate immune receptors, resulting in the decreased proinflammatory activity of MRSP. IMPORTANCE To date, the clinical efficacy of macrolides in pneumococcal disease is assumed to be linked to their ability to inhibit the release of pneumolysin. However, our previous study demonstrated that oral administration of macrolides to mice intratracheally infected with macrolide-resistant Streptococcus pneumoniae resulted in decreased levels of pneumolysin and proinflammatory cytokines in bronchoalveolar lavage fluid samples compared to the levels in samples from untreated infected control mice, without affecting the bacterial load in the fluid. This finding suggests that additional mechanisms by which macrolides negatively regulate proinflammatory cytokine production may be involved in their efficacy in vivo . Furthermore, in this study, we demonstrated that macrolides downregulated the transcription of various proinflammatory-component-related genes in S. pneumoniae, which provides an additional explanation for the clinical benefits of macrolides., Competing Interests: The authors declare no conflict of interest.

In this research, we combined our ultralight micro-imaging device for calcium imaging with microdialysis to simultaneously visualize neural activity in the dorsal raphe nucleus (DRN) and measure serotonin release in the central nucleus of the amygdala (CeA) and the anterior cingulate cortex (ACC). Using this platform, we observed brain activity following nociception induced by formalin injection in the mouse's hind paw. Our device showed that DRN fluorescence intensity increased after formalin injection, and the increase was highly correlated with the elevation in serotonin release in both the CeA and ACC. The increase in calcium fluorescence intensity occurred during the acute and inflammatory phases, which suggests the biphasic response of nociceptive pain. Furthermore, we found that the increase in fluorescence intensity was positively correlated with mouse licking behavior. Lastly, we compared the laterality of pain stimulation and found that DRN fluorescence activity was higher for contralateral stimulation. Microdialysis showed that CeA serotonin concentration increased only after contralateral stimulation, while ACC serotonin release responded bilaterally. In conclusion, our study not only revealed the inter-regional serotonergic connection among the DRN, the CeA, and the ACC, but also demonstrated that our device is feasible for multi-site implantation in conjunction with a microdialysis system, allowing the simultaneous multi-modal observation of different regions in the brain.

Hybrid emission filters, comprising an interference filter and an absorption filter, exhibit high excitation light rejection performance and can act as lensless fluorescent devices. However, it has been challenging to produce them in large batches over a large area. In this study, we propose and demonstrate a method for transferring a Si substrate, on which the hybrid filter is deposited, onto an image sensor by attaching it to the sensor and removing the substrate via plasma etching. Through this method, we can transfer uniform filters onto fine micrometer-sized needle devices and millimeter-sized multisensor chips. Optical evaluation reveals that the hybrid filter emits light in the 500 to 560 nm range, close to the emission region of green fluorescent protein (GFP). Furthermore, by observing the fluorescence emission from the microbeads, a spatial resolution of 12.11 μm is calculated. In vitro experiments confirm that the fabricated device is able to discriminate GFP emission patterns from brain slices.

The macrolide erythromycin (ERM) inhibits excessive neutrophil accumulation and bone resorption in inflammatory tissues. We previously reported that the expression of developmental endothelial locus-1 (DEL-1), an endogenous anti-inflammatory factor induced by ERM, is involved in ERM action. Furthermore, DEL-1 is involved in the induction of bone regeneration. Therefore, in this study, we investigated whether ERM exerts an osteoblastogenic effect by upregulating DEL-1 under inflammatory conditions. We performed in vitro cell-based mechanistic analyses and used a model of Porphyromonas gingivalis lipopolysaccharide (LPS)-induced periodontitis to evaluate how ERM restores osteoblast activity. In vitro, P. gingivalis LPS stimulation suppressed osteoblast differentiation and bone formation. However, ERM treatment combined with P. gingivalis LPS stimulation upregulated osteoblast differentiation-related factors and Del1 , indicating that osteoblast differentiation was restored. Alveolar bone resorption and gene expression were evaluated in a periodontitis model, and the results confirmed that ERM treatment increased DEL-1 expression and suppressed bone loss by increasing the expression of osteoblast-associated factors. In conclusion, ERM restores bone metabolism homeostasis in inflammatory environments possibly via the induction of DEL-1.

Two plasminogen binding proteins were identified from a mouse infected with Streptococcus pneumoniae. The pneumococcal proteins were annotated as ATP-dependent Clp protease ATP-binding subunit (ClpC) and excinuclease ABC subunit C (UvrC) using the isobaric tags for relative and absolute quantification (iTRAQ) method. Recombinants of both proteins showed significant binding to plasminogen and were found to promote plasminogen activation by tissue-type plasminogen activator. In addition, ClpC and UvrC were LytA-dependently released into the culture supernatant and bound to the bacterial surface. These results suggest that S. pneumoniae releases ClpC and UvrC by autolysis and recruits them to the bacterial surface, where they bind to plasminogen and promote its activation, contributing to extracellular matrix degradation and tissue invasion., (© 2022 The Societies and John Wiley & Sons Australia, Ltd.)

Hydroxyapatite (HAp) is a biomaterial used because of its high potential for improving the healing of bone fractures. HAp is typically prepared using synthetic methods. However, synthetic HAp generally does not contain trace elements, which are considered to be important factors in the healing process. Therefore, researchers have focused on developing methods for preparing biomaterials containing trace elements. A novel method for coating calcium phosphate materials with trace elements is proposed in this study. In this method, mineral components with trace elements were obtained from bone samples and coated onto the titanium surface using only electrical stimulation under constant voltage application in phosphate-buffered saline. The resulting material had poor crystallinity, which was similar to apatite structure present in bone tissue, and it contained trace elements. Calcium phosphate depositions were also constructed on the surfaces of bone and vascular specimens under the same conditions. Consequently, this method can potentially be applied as an innovative treatment for implant surfaces and in the preparation of scaffolds for bone tissue engineering., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

A readout device for a dual-functional neural observation system is presented. The authors separately developed the reading operation of an implantable CMOS image sensor and a setup for fast-scan cyclic voltammetry and implemented them together in a microcontroller-based device. The developed imaging readout device with a size of [Formula: see text] can reach the highest reading rate of 160 fps with a 120×268 pixel image sensor. The voltammetry function was verified through an experiment using commercial carbon fiber electrodes in phosphate-buffered saline. When the imaging is sequentially operated with 400 V/s-scan rate voltammetry from -0.4 to 1.3 V, the system can operate at up to 60 fps. With this system, calcium imaging and dopamine recording in a freely behaving mouse can be achieved together in a simpler manner. This study aims to be the basis for the development of an implantable multi-functional sensor.

Recruitment of plasminogen is an important infection strategy of the human pathogen Streptococcus pneumoniae to invade host tissues. In Streptococcus aureus, triosephosphate isomerase (TPI) has been reported to bind plasminogen. In this study, the TPI of S. pneumoniae (TpiA) was identified through proteomic analysis of bronchoalveolar lavage fluid from a murine pneumococcal pneumonia model. The binding kinetics of recombinant pneumococcal TpiA with plasminogen were characterized using surface plasmon resonance (SPR, Biacore), ligand blot analyses, and enzyme-linked immunosorbent assay. Enhanced plasminogen activation and subsequent degradation by plasmin were also shown. Release of TpiA into the culture medium was observed to be dependent on autolysin. These findings suggest that S. pneumoniae releases TpiA via autolysis, which then binds to plasminogen and promotes its activation, thereby contributing to tissue invasion via degradation of the extracellular matrix., (© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Neutrophil elastase (NE) functions as a host defense factor; however, excessive NE activity can potentially destroy human tissues. Although NE activity is positively correlated to gingival crevicular fluid and clinical attachment loss in periodontitis, the underlying mechanisms by which NE aggravates periodontitis remain elusive. In this study, we investigated how NE induces periodontitis severity and whether NE inhibitors were efficacious in periodontitis treatment. In a ligature-induced murine model of periodontitis, neutrophil recruitment, NE activity, and periodontal bone loss were increased in the periodontal tissue. Local administration of an NE inhibitor significantly decreased NE activity in periodontal tissue and attenuated periodontal bone loss. Furthermore, the transcription of proinflammatory cytokines in the gingiva, which was significantly upregulated in the model of periodontitis, was significantly downregulated by NE inhibitor injection. An in vitro study demonstrated that NE cleaved cell adhesion molecules, such as desmoglein 1, occludin, and E-cadherin, and induced exfoliation of the epithelial keratinous layer in three-dimensional human oral epithelial tissue models. The permeability of fluorescein-5-isothiocyanate-dextran or periodontal pathogen was significantly increased by NE treatment in the human gingival epithelial monolayer. These findings suggest that NE induces the disruption of the gingival epithelial barrier and bacterial invasion in periodontal tissues, aggravating periodontitis., (© 2022. The Author(s).)

Background: Coronary artery disease is an incurable, life-threatening disease that was once considered primarily a disorder of lipid deposition. Coronary artery disease is now also characterized by chronic inflammation' notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies., Methods: We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity., Results: In addition to macrophages, we found a high proportion of αβ T cells in the coronary plaques. Most of these T cells lack high expression of CCR7 and L-selectin , indicating that they are primarily antigen-experienced memory cells. Notably, nearly one-third of these cells express the HLA-DRA surface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αβ T cells (CD4

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.

Significance: Intrinsic optical signals (IOS) generated in the cortical tissue as a result of various interacting metabolic processes are used extensively to elucidate the underlying mechanisms that govern neurovascular coupling. However, current IOS measurements still often rely on bulky, tabletop imaging systems, and there remains a dearth of studies in freely moving subjects. Lightweight, miniature head-mounted imaging devices provide unique opportunities for investigating cortical dynamics in small animals under a variety of naturalistic behavioral settings., Aim: The aim of this work was to monitor IOS in the somatosensory cortex of wild-type mice by developing a lightweight, biocompatible imaging device that readily lends itself to animal experiments in freely moving conditions., Approach: Herein we describe a method for realizing long-term IOS imaging in mice using a 0.54-g, compact, CMOS-based, head-mounted imager. The two-part module, consisting of a tethered sensor plate and a base plate, allows facile assembly prior to imaging sessions and disassembly when the sensor is not in use. LEDs integrated into the device were chosen to illuminate the cortical mantle at two different wavelengths in the visible regime (λcenter: 535 and 625 nm) for monitoring volume- and oxygenation state-dependent changes in the IOS, respectively. To test whether the system can detect robust cortical responses, we recorded sensory-evoked IOS from mechanical stimulation of the hindlimbs (HL) of anesthetized mice in both acute and long-term implantation conditions., Results: Cortical IOS recordings in the primary somatosensory cortex hindlimb receptive field (S1HL) of anesthetized mice under green and red LED illumination revealed robust, multiphasic profiles that were time-locked to the mechanical stimulation of the contralateral plantar hindpaw. Similar intrinsic signal profiles observed in S1HL at 40 days postimplantation demonstrated the viability of the approach for long-term imaging. Immunohistochemical analysis showed that the brain tissue did not exhibit appreciable immune response due to the device implantation and operation. A proof-of-principle imaging session in a freely behaving mouse showed minimal locomotor impediment for the animal and also enabled estimation of blood flow speed., Conclusions: We demonstrate the utility of a miniature cortical imaging device for monitoring IOS and related hemodynamic processes in both anesthetized and freely moving mice, cueing potential for applications to some neuroscientific studies of sensation and naturalistic behavior.

Dopamine (DA) is the key regulator of reward behavior. The DA neurons in the ventral tegmental area (VTA) and their projection areas, which include the prefrontal cortex (PFC), nucleus accumbens (NAc), and amygdala, play a primary role in the process of reward-driven behavior induced by the drugs of addiction, including nicotine and alcohol. In our previous study, we developed a novel platform consisting of micro-LED array devices to stimulate a large area of the brain of rats and monkeys with photo-stimulation and a microdialysis probe to estimate the DA release in the PFC. Our results suggested that the platform was able to detect the increased level of dopamine in the PFC in response to the photo-stimulation of both the PFC and VTA. In this study, we used this platform to photo-stimulate the VTA neurons in both ChrimsonR-expressing (non-specific) wild and dopamine transporter (DAT)-Cre (dopamine specific) mice, and measured the dopamine release in the nucleus accumbens shell (NAcShell). We measured the DA release in the NAcShell in response to optogenetic stimulation of the VTA neurons and investigated the effect of GABAergic neurons on dopaminergic neurons by histochemical studies. Comparing the photo-stimulation frequency of 2 Hz with that of 20 Hz, the change in DA concentration at the NAcShell was greater at 20 Hz in both cases. When ChrimsonR was expressed specifically for DA, the release of DA at the NAcShell increased in response to photo-stimulation of the VTA. In contrast, when ChrimsonR was expressed non-specifically, the amount of DA released was almost unchanged upon photo-stimulation. However, for nonspecifically expressed ChrimsonR, intraperitoneal injection of bicuculline, a competitive antagonist at the GABA-binding site of the GABA A receptor, also significantly increased the release of DA at the NAcShell in response to photo-stimulation of the VTA. The results of immunochemical staining confirm that GABAergic neurons in the VTA suppress DA activation, and also indicate that alterations in GABAergic neurons may have serious downstream effects on DA activity, NAcShell release, and neural adaptation of the VTA. This study also confirms that optogenetics technology is crucial to study the relationship between the mesolimbic dopaminergic and GABAergic neurons in a neural-specific manner.

Streptococcus pneumoniae is a causative pathogen of several human infectious diseases including community-acquired pneumonia. Pneumolysin (PLY), a pore-forming toxin, plays an important role in the pathogenesis of pneumococcal pneumonia. In recent years, the use of traditional natural substances for prevention has drawn attention because of the increasing antibacterial drug resistance of S. pneumoniae . According to some studies, green tea exhibits antibacterial and antitoxin activities. The polyphenols, namely the catechins epigallocatechin gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC) are largely responsible for these activities. Although matcha green tea provides more polyphenols than green tea infusions, its relationship with pneumococcal pneumonia remains unclear. In this study, we found that treatment with 20 mg/mL matcha supernatant exhibited significant antibacterial activity against S. pneumoniae regardless of antimicrobial resistance. In addition, the matcha supernatant suppressed PLY-mediated hemolysis and cytolysis by inhibiting PLY oligomerization. Moreover, the matcha supernatant and catechins inhibited PLY-mediated neutrophil death and the release of neutrophil elastase. These findings suggest that matcha green tea reduces the virulence of S. pneumoniae in vitro and may be a promising agent for the treatment of pneumococcal infections.

Significance: Gene expression analysis is an important fundamental area of biomedical research. However, live gene expression imaging has proven challenging due to constraints in conventional optical devices and fluorescent reporters., Aim: Our aim is to develop smaller, more cost-effective, and versatile imaging capabilities compared with conventional devices. Bioluminescence reporter-based gene expression analysis was targeted due to its advantages over fluorescence-based imaging., Approach: We created a small compact imaging system using micro-CMOS image sensors (μCIS). The μCIS model had an improved pixel design and a patterned absorption filter array to detect the low light intensity of bioluminescence., Results: The device demonstrated lower dark current, lower temporal noise, and higher sensitivity compared with previous designs. The filter array enabled us to subtract dark current drift and attain a clearer light signal. These improvements allowed us to measure bioluminescence reporter-based gene expression in living mammalian cells., Conclusion: Using our μCIS system for bioluminescence imaging in the future, the device can be implanted in vivo for simultaneous gene expression imaging, behavioral analysis, and optogenetic modulation.