3D transesophageal echocardiographic visualization of the pulmonary artery catheter through the tricuspid valve and their position relative to the tricuspid valve leaflets.

BACKGROUND: The tricuspid valve is an atrioventricular valve consisting of three lobes. We used the 3D transesophageal echocardiography to visualize position of the pulmonary artery catheter at the tricuspid valve annulus and examined where the catheter passed through at the level of the tricuspid annulus. METHODS: In this prospective and observational study, we monitored the pressure wave on patients undergoing cardiac surgery with the catheter placement by monitoring the pressure waveform for 8 months. We measured the time required for the catheter to pass through the tricuspid and pulmonary valves, respectively. We acquired the 3D image of the tricuspid valve by transesophageal echocardiography and determined the position of the pulmonary artery catheter at the level of the tricuspid annulus. The data were analyzed by Kruskal-Wallis test followed by Mann-Whitney test with Holm multiple comparisons. P < 0.05 was considered significant. RESULTS: Of the 116 cases, the pulmonary artery catheter passed through the tricuspid valve between antero-posterior leaflets in 78 cases (67.2 %), between septal-posterior leaflets in 25 cases (21.6 %), and between antero-septal leaflets in 2 cases (1.7 %) and the center in 11 cases (9.5 %), respectively. The time required for the catheter to pass through the pulmonary valves was significantly different among the catheter positions at the level of the tricuspid annulus. CONCLUSION: The pulmonary artery catheter location at the level of the tricuspid annulus can be identified using 3D transesophageal echocardiography. The location of the catheter significantly affects the pulmonary artery catheter placement time.

Perceiving humanness across ages: neural correlates and behavioral patterns.

Humanness perception, which attributes fundamental and unique human characteristics to other objects or people, has significant consequences for people's interactions. Notably, the failure to perceive humanness in older adults can lead to prejudice. This study investigates the effect of a target's age on humanness perception in terms of two dimensions: agency (the ability to act and do) and experience (the ability to feel and sense). We also examined brain activity using a magnetic resonance imaging (MRI) scanner in order to understand the underlying neural mechanisms. Healthy university students viewed the facial images of older and younger individuals and judged the humanness of each individual in terms of agency and experience while inside the MRI scanner. The results indicated that older adults were rated higher on experience, and no difference was found in ratings for agency between younger and older face images. Analysis of brain imaging data indicated that positive functional connectivity between the ventral and dorsal regions of the medial prefrontal cortex (mPFC) was greater when judging the humanness of younger faces than older faces. We also found that the negative functional connectivity between the left inferior frontal gyrus and postcentral gyrus was greater when judging the humanness of older faces as compared to that of younger faces. Although the current study did not show distinct brain activities related to humanness perception, it suggests the possibility that different brain connectivities are related to humanness perception regarding targets belonging to different age groups.

Optimization of Zwitterionic Polymers for Cell Cryopreservation.

Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC3 C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DSion ) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC3 C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC3 C)_DSion(100%) /1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect.

Super-enhancer trapping by the nuclear pore via intrinsically disordered regions of proteins in squamous cell carcinoma cells.

Master transcription factors such as TP63 establish super-enhancers (SEs) to drive core transcriptional networks in cancer cells, yet the spatiotemporal regulation of SEs within the nucleus remains unknown. The nuclear pore complex (NPC) may tether SEs to the nuclear pore where RNA export rates are maximal. Here, we report that NUP153, a component of the NPC, anchors SEs to the NPC and enhances TP63 expression by maximizing mRNA export. This anchoring is mediated through protein-protein interaction between the intrinsically disordered regions (IDRs) of NUP153 and the coactivator BRD4. Silencing of NUP153 excludes SEs from the nuclear periphery, decreases TP63 expression, impairs cellular growth, and induces epidermal differentiation of squamous cell carcinoma. Overall, this work reveals the critical roles of NUP153 IDRs in the regulation of SE localization, thus providing insights into a new layer of gene regulation at the epigenomic and spatial level.

Cell-compatible isotonic freezing media enabled by thermo-responsive osmolyte-adsorption/exclusion polymer matrices.

During the long-term storage of cells, it is necessary to inhibit ice crystal formation by adding cryoprotectants. Non-cell-permeable cryoprotectants have high osmotic pressure which dehydrates cells, indirectly suppressing intracellular ice crystal formation. However, the high osmotic pressure and dehydration often damage cells. Emerging polymer-type non-cell-permeable cryoprotectants form matrices surrounding cells. These matrices inhibit the influx of extracellular ice nuclei that trigger intracellular ice crystal formation. However, these polymer-type cryoprotectants also require high osmotic pressure to exert an effective cryoprotecting effect. In this study, we designed a poly(zwitterion) (polyZI) that forms firm matrices around cells based on their high affinity to cell membranes. The polyZI successfully cryopreserved freeze-vulnerable cells under isotonic conditions. These matrices also controlled osmotic pressure by adsorbing and desorbing NaCl depending on the temperature, which is a suitable feature for isotonic cryopreservation. Although cell proliferation was delayed by the cellular matrices, washing with a sucrose solution improved proliferation.

Nanoscopic Elucidation of Spontaneous Self-Assembly of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Open Reading Frame 6 (ORF6) Protein.

Open reading frame 6 (ORF6), the accessory protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that suppresses host type-I interferon signaling, possesses amyloidogenic sequences. ORF6 amyloidogenic peptides self-assemble to produce cytotoxic amyloid fibrils. Currently, the molecular properties of the ORF6 remain elusive. Here, we investigate the structural dynamics of the full-length ORF6 protein in a near-physiological environment using high-speed atomic force microscopy. ORF6 oligomers were ellipsoidal and readily assembled into ORF6 protofilaments in either a circular or a linear pattern. The formation of ORF6 protofilaments was enhanced at higher temperatures or on a lipid substrate. ORF6 filaments were sensitive to aliphatic alcohols, urea, and SDS, indicating that the filaments were predominantly maintained by hydrophobic interactions. In summary, ORF6 self-assembly could be necessary to sequester host factors and causes collateral damage to cells via amyloid aggregates. Nanoscopic imaging unveiled the innate molecular behavior of ORF6 and provides insight into drug repurposing to treat amyloid-related coronavirus disease 2019 complications.

Astrocyte-targeting therapy rescues cognitive impairment caused by neuroinflammation via the Nrf2 pathway.

Neuroinflammation is a common feature of neurodegenerative disorders such as Alzheimer's disease (AD). Neuroinflammation is induced by dysregulated glial activation, and astrocytes, the most abundant glial cells, become reactive upon neuroinflammatory cytokines released from microglia and actively contribute to neuronal loss. Therefore, blocking reactive astrocyte functions is a viable strategy to manage neurodegenerative disorders. However, factors or therapeutics directly regulating astrocyte subtypes remain unexplored. Here, we identified transcription factor NF-E2-related factor 2 (Nrf2) as a therapeutic target in neurotoxic reactive astrocytes upon neuroinflammation. We found that the absence of Nrf2 promoted the activation of reactive astrocytes in the brain tissue samples obtained from AD model 5xFAD mice, whereas enhanced Nrf2 expression blocked the induction of reactive astrocyte gene expression by counteracting NF-κB subunit p65 recruitment. Neuroinflammatory astrocytes robustly up-regulated genes associated with type I interferon and the antigen-presenting pathway, which were suppressed by Nrf2 pathway activation. Moreover, impaired cognitive behaviors observed in AD mice were rescued upon ALGERNON2 treatment, which potentiated the Nrf2 pathway and reduced the induction of neurotoxic reactive astrocytes. Thus, we highlight the potential of astrocyte-targeting therapy by promoting the Nrf2 pathway signaling for neuroinflammation-triggered neurodegeneration.

A rare case of pyosalpinx in adolescent girl with Hirschsprung's disease who underwent transvaginal ultrasound-guided drainage.

BACKGROUND: Hydrosalpinx and pyosalpinx are rare gynecologic problems during adolescence, especially in girls without a history of sexual activity. They are even rarer in women with Hirschsprung's disease (HD). We herein report a case of pyosalpinx in an adolescent girl with HD treated by transvaginal ultrasound-guided drainage. CASE PRESENTATION: The present patient was a 12-year-old girl (weight 83 kg; height 159 cm; body mass index 32.8 kg/m2). She had undergone five laparotomies for long-segment HD by 2 years. Her menarche had occurred at 10 years. She was admitted with lower abdominal and anal pain. Computed tomography (CT), magnetic resonance imaging (MRI), and transvaginal ultrasound showed left pyosalpinx and abdominal abscess. Surgical drainage was necessary; however, she had a history of polysurgery and was severely obese, so laparotomy was considered to carry a high risk. Transvaginal ultrasound was deemed more likely to reach the abscess safely. Therefore, she was treated with transvaginal ultrasound-guided drainage by a gynecologist skilled in the procedure. She was discharged home after 52 days. One year and nine months after discharge, there was no reformation of either the abscess or pyosalpinx. CONCLUSIONS: Adolescent girls with HD are at risk of developing hydrosalpinx. Depending on the defecation status, pyosalpinx may also develop. As a less-invasive surgical treatment, transvaginal ultrasound-guided drainage can avoid laparotomy. Collaboration with a gynecologist is essential for the diagnosis and treatment of this clinical condition. Pediatric surgeons should communicate with gynecologists for such cases beginning around puberty for continuous follow-up.

Nanoscopic Assessment of Anti-SARS-CoV-2 Spike Neutralizing Antibody Using High-Speed AFM.

Anti-spike neutralizing antibodies (S NAbs) have been developed for prevention and treatment against COVID-19. The nanoscopic characterization of the dynamic interaction between spike proteins and S NAbs remains difficult. By using high-speed atomic force microscopy (HS-AFM), we elucidate the molecular property of an S NAb and its interaction with spike proteins. The S NAb appeared as monomers with a Y conformation at low density and formed hexameric oligomers at high density. The dynamic S NAb-spike protein interaction at RBD induces neither RBD opening nor S1 subunit shedding. Furthermore, the interaction was stable at endosomal pH. These findings indicated that the S NAb could have a negligible risk of antibody-dependent enhancement. Dynamic movement of spike proteins on small extracellular vesicles (S sEV) resembled that on SARS-CoV-2. The sensitivity of variant S sEVs to S NAb could be evaluated using HS-AFM. Altogether, we demonstrate a nanoscopic assessment platform for evaluating the binding property of S NAbs.

Spatiotemporal tracking of small extracellular vesicle nanotopology in response to physicochemical stresses revealed by HS-AFM.

Small extracellular vesicles (sEVs) play a crucial role in local and distant cell communication. The intrinsic properties of sEVs make them compatible biomaterials for drug delivery, vaccines, and theranostic nanoparticles. Although sEV proteomics have been robustly studied, a direct instantaneous assessment of sEV structure dynamics remains difficult. Here, we use the high-speed atomic force microscopy (HS-AFM) to evaluate nanotopological changes of sEVs with respect to different physicochemical stresses including thermal stress, pH, and osmotic stress. The sEV structure is severely altered at high-temperature, high-pH, or hypertonic conditions. Surprisingly, the spherical shape of the sEVs is maintained in acidic or hypotonic environments. Real-time observation by HS-AFM imaging reveals an irreversible structural change in the sEVs during transition of pH or osmolarity. HS-AFM imaging provides both qualitative and quantitative data at high spatiotemporal resolution (nanoscopic and millisecond levels). In summary, our study demonstrates the feasibility of HS-AFM for structural characterization and assessment of nanoparticles.

Impact of Catheter Ablation on Brain Microstructure and Blood Flow Alterations for Cognitive Improvements in Patients with Atrial Fibrillation: A Pilot Longitudinal Study.

Atrial fibrillation (AF) predisposes patients to develop cognitive decline and dementia. Clinical and epidemiological data propose that catheter ablation may provide further benefit to improve neurocognitive function in patients with AF, but the underlying mechanism is poorly available. Here, we conducted a pilot prospective study to investigate whether AF ablation can alter regional cerebral blood flow (rCBF) and brain microstructures, using multimodal magnetic resonance imaging (MRI) technique. Eight patients (63 ± 7 years) with persistent AF underwent arterial-spin labeling (ASL) perfusion, 3D T1-structural images and cognitive test batteries before and 6 months after intervention. ASL and structural MR images were spatially normalized, and the rCBF and cortical thickness of different brain areas were compared between pre- and 6-month post-treatment. Cognitive-psychological function was improved, and rCBF was significantly increased in the left posterior cingulate cortex (PCC) (p = 0.013), whereas decreased cortical thickness was found in the left posterior insular cortex (p = 0.023). Given that the PCC is a strategic site in the limbic system, while the insular cortex is known to play an important part in the central autonomic nervous system, our findings extend the hypothesis that autonomic system alterations are an important mechanism explaining the positive effect of AF ablation on cognitive function.

Two cases of hemodynamic improvement by modulation of atrioventricular delay in cardiac operations.

BACKGROUND: Symptomatic sick sinus syndrome is one of the indications for pacemaker implantation, and we have to consider to program the pacemaker to an asynchronous pacing mode during an operation. CASE PRESENTATION: We reported two cases with a pacemaker implanted for sick sinus syndrome undergoing cardiac operation. We changed programming of the pacemaker to an asynchronous pacing mode (DOO) and modulated the programmed atrioventricular delay to avoid ventricular pacing, resulting in better hemodynamic condition. Although we observed premature ventricular contraction, no lethal arrhythmias induced by the R-on-T phenomenon were noted. CONCLUSION: Programming of the pacemaker to an asynchronous pacing mode and modulation of the programmed atrioventricular delay to avoid ventricular pacing may be an option for pacemaker management during an operation.

Inhibitory effects of thiol compounds on theaflavin browning and structural analysis of the causative substances.

Theaflavin, a polyphenol responsible for the bright orange color and various bioactivities of black tea exudates, is susceptible to autoxidation at neutral and mild alkaline pH, changing its color to brown. In the presence of cysteine (Cys), glutathione (GSH), or N-acetyl cysteine (NAC), the browning of theaflavin solution was inhibited concomitantly with time-dependent decreases in the concentrations of both theaflavin and thiol group. The rank order of the decrease was Cys â‰… GSH > NAC, suggesting the relevance of the nucleophilic property of the thiol group to its reaction with theaflavin. LC-MS analysis of the reaction products indicated formation of novel derivatives that were mono- or di-molecular adducts of thiol compounds. We determined the chemical structures of theaflavin-Cys and theaflavin-GSH adducts by NMR and proposed the reaction mechanisms. It was found that the theaflavin-Cys adduct was not a simple adduct, to which a new cyclic structure was added.

Discovery of a Novel Aminocyclopropenone Compound That Inhibits BRD4-Driven Nucleoporin NUP210 Expression and Attenuates Colorectal Cancer Growth.

Epigenetic deregulation plays an essential role in colorectal cancer progression. Bromodomains are epigenetic "readers" of histone acetylation. Bromodomain-containing protein 4 (BRD4) plays a pivotal role in transcriptional regulation and is a feasible drug target in cancer cells. Disease-specific elevation of nucleoporin, a component of the nuclear pore complex (NPC), is a determinant of cancer malignancy, but BRD4-driven changes of NPC composition remain poorly understood. Here, we developed novel aminocyclopropenones and investigated their biological effects on cancer cell growth and BRD4 functions. Among 21 compounds developed here, we identified aminocyclopropenone 1n (ACP-1n) with the strongest inhibitory effects on the growth of the cancer cell line HCT116. ACP-1n blocked BRD4 functions by preventing its phase separation ability both in vitro and in vivo, attenuating the expression levels of BRD4-driven MYC. Notably, ACP-1n significantly reduced the nuclear size with concomitant suppression of the level of the NPC protein nucleoporin NUP210. Furthermore, NUP210 is in a BRD4-dependent manner and silencing of NUP210 was sufficient to decrease nucleus size and cellular growth. In conclusion, our findings highlighted an aminocyclopropenone compound as a novel therapeutic drug blocking BRD4 assembly, thereby preventing BRD4-driven oncogenic functions in cancer cells. This study facilitates the development of the next generation of effective and potent inhibitors of epigenetic bromodomains and extra-terminal (BET) protein family.

Effectiveness and safety of kamikihito, a traditional Japanese medicine, in managing anxiety among female patients with intractable chronic constipation.

BACKGROUND AND PURPOSE: The prevalence of anxiety in patients with chronic constipation is particularly high and these individuals are not necessarily satisfied by normal treatments targeting the gastrointestinal tract. Kamikihito, a traditional Japanese Kampo medicine, has been widely used to date in treating anxiety and neurosis in Japan. We conducted a single-arm, open-label pilot study of female patients with intractable chronic constipation and anxiety who took kamikihito by mouth for 12 weeks. MATERIALS AND METHODS: Validated symptom questionnaires on anxiety and gastrointestinal symptoms [the Profile of Mood States, second edition (POMS2); the State-Trait Anxiety Inventory (STAI); and the Gastrointestinal Symptom Rating Scale (GSRS)] were completed at each study visit. Plasma, salivary, and stool samples were also assessed to evaluate levels of clinical bioactive substances linked to stress and inflammation, oxidative levels, the metabolome profile, and gut microbiota. RESULTS: Twenty-four patients completed this study. Anxiety was significantly reduced at four and 12 weeks (Tension-Anxiety subscale of the POMS2, p = 0.006 and p = 0.039; Trait anxiety score of the STAI, p < 0.001 and p = 0.034), while the total GSRS score was improved at 12 weeks (p = 0.039). Targeted metabolomics in plasma showed significant alterations in some metabolites associated with psychological symptoms, such as O-phosphoethanolamine. No significant differences were found between pre- and posttreatment levels of clinical bioactive substances related to stress and inflammation, oxidative levels, and the gut microbiota in this cohort. No serious adverse events occurred. CONCLUSION: Kamikihito ameliorated psychological and gastrointestinal symptoms in patients with chronic constipation. In parallel with the onset of efficacy, kamikihito modulated some anxiety-related metabolites. Kamikihito was safe and well-tolerated.

NSP9 of SARS-CoV-2 attenuates nuclear transport by hampering nucleoporin 62 dynamics and functions in host cells.

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.

Millisecond dynamic of SARS-CoV-2 spike and its interaction with ACE2 receptor and small extracellular vesicles.

SARS-CoV-2 spike protein (S) binds to human angiotensin-converting enzyme 2 (hACE2), allowing virus to dock on cell membrane follow by viral entry. Here, we use high-speed atomic force microscopy (HS-AFM) for real-time visualization of S, and its interaction with hACE2 and small extracellular vesicles (sEVs). Results show conformational heterogeneity of S, flexibility of S stalk and receptor-binding domain (RBD), and pH/temperature-induced conformational change of S. S in an S-ACE2 complex appears as an all-RBD up conformation. The complex acquires a distinct topology upon acidification. S and S2 subunit demonstrate different membrane docking mechanisms on sEVs. S-hACE2 interaction facilitates S to dock on sEVs, implying the feasibility of ACE2-expressing sEVs for viral neutralization. In contrary, S2 subunit docks on lipid layer and enters sEV using its fusion peptide, mimicking the viral entry scenario. Altogether, our study provides a platform that is suitable for real-time visualization of various entry inhibitors, neutralizing antibodies, and sEV-based decoy in blocking viral entry. Teaser: Comprehensive observation of SARS-CoV-2 spike and its interaction with receptor ACE2 and sEV-based decoy in real time using HS-AFM.

A light-switching pyrene probe to detect phase-separated biomolecules.

Biomolecules may undergo liquid-liquid phase separation (LLPS) to spatiotemporally compartmentalize and regulate diverse biological processes. Because the number of tools to directly probe LLPS is limited (ie. FRAP, FRET, fluorescence microscopy, fluorescence anisotropy, circular dichroism, etc.), the physicochemical traits of phase-separated condensates remain largely elusive. Here, we introduce a light-switching dipyrene probe (Pyr-A) that forms monomers in either hydrophobic or viscous environments, and intramolecular excimers in aqueous solutions. By exploiting their distinct fluorescence emission spectra, we used fluorescent microscopic imaging to study phase-separated condensates formed by in vitro protein droplets and membraneless intracellular organelles (centrosomes). Ratiometric measurement of excimer and monomer fluorescence intensities showed that protein droplets became hydrophobic and viscous as their size increased. Moreover, centrosomes became hydrophobic and viscous during maturation. Our results show that Pyr-A is a valuable tool to characterize LLPS and enhance our understanding of phase separation underlying biological functions.

Vaccine temperature management in Lao People's Democratic Republic: A nationwide cross-sectional study.

OBJECTIVE: The objective of the study was to evaluate the duration and frequency of vaccine exposure to suboptimal temperatures during transit from the central vaccine storage in the capital to health centers in Lao PDR. METHODS: Temperature data loggers traveled from the capital to the health centre storages (146) with the vaccines to monitor the vaccine temperature nationwide. One health centre per district was selected using a simple random sampling method for the first round of temperature monitoring. One health centre was selected from every forty-nine high risk districts monitor the trend of vaccine temperature at the health centre storage and during outreach sessions in several districts. Vaccines and temperature data loggers were transported using the normal vaccination transportation. FINDINGS: Overall, the vaccines were exposed to temperatures >8 °C for an average of 1648 min, equivalent to 9.0% of the observational period, and to temperatures <0 °C for an average of 184 min, equivalent to 1.35% of the study period. The proportion of exposure to temperatures >8 °C was the highest during the transit from the capital to the province. The proportion of exposure to temperatures <0 °C was the highest during storage at district level. Examined by region, vaccines in the northern provinces had higher risk of exposure to temperatures >8 °C; however, the risk of exposure to temperatures <0 °C was scattered nationwide. Moreover, some health centers showed fluctuations in storage temperature. CONCLUSIONS: Challenges associated with cold chain management, and the resulting deterioration of vaccines, might account for outbreaks of vaccine-preventable diseases. The government should examine and invest in suitable technologies and approaches to ensure consistency in cold chain management.