Identification of Reference Gene for Quantitative Gene Expression in Early-Term and Late-Term Cultured Canine Fibroblasts Derived from Ear Skin.

Fibroblasts are cells that reside within the fibrous or loose connective tissues of most mammalian organs. For research purposes, fibroblasts are often subjected to long-term culture under defined conditions, during which their properties can significantly change. It is essential to understand and document these changes to obtain reliable outcomes. For the quantification of specific gene expressions, the most reliable and widely used technique is quantitative real-time polymerase chain reaction (qRT-PCR). Here, we assessed the impact of a reference gene's stability on a qRT-PCR analysis of long-term cultured canine skin fibroblasts. After successfully isolating the fibroblasts from canine skin tissues, they were cultured and evaluated for proliferation and ß-galactosidase activity at different passage numbers. With extended culture, the fibroblasts showed a long doubling time and elevated ß-galactosidase activity. Using three widely used algorithms, geNorm, Normfinder, and Bestkeeper, we identified HPRT1, YWHAZ, and GUSB as the most stable reference genes for both early- and late-passage fibroblasts. Conventional reference genes such as GAPDH were found to be less stable than those genes. The normalization of Vimentin by the stable genes showed statistical differences, whereas normalization by an unstable gene did not. Collectively, this study indicates that using stable reference genes is essential for accurately and reliably measuring gene expression in both early- and late-passage fibroblasts. These findings provide valuable insights into internal controls for gene expression studies and are expected to be utilized for analyzing gene expression patterns in molecular biology research.

Histological and Molecular Biological Changes in Canine Skin Following Acute Radiation Therapy-Induced Skin Injury.

Radiation therapy is a crucial cancer treatment, but it can damage healthy tissues, leading to side effects like skin injuries and molecular alterations. This study aimed to elucidate histological and molecular changes in canine skin post-radiation therapy (post-RT) over nine weeks, focusing on inflammation, stem cell activity, angiogenesis, keratinocyte regeneration, and apoptosis. Four male beagles received a cumulative radiation dose of 48 Gy, followed by clinical observations, histological examinations, and an RT-qPCR analysis of skin biopsies. Histological changes correlated with clinical recovery from inflammation. A post-RT analysis revealed a notable decrease in the mRNA levels of Oct4, Sox2, and Nanog from weeks 1 to 9. VEGF 188 levels initially saw a slight increase at week 1, but they had significantly declined by week 9. Both mRNA and protein levels of COX-2 and Keratin 10 significantly decreased over the 9 weeks following RT, although COX-2 expression surged in the first 2 weeks, and Keratin 10 levels increased at weeks 4 to 5 compared to normal skin. Apoptosis peaked at 2 weeks and diminished, nearing normal by 9 weeks. These findings offer insights into the mechanisms of radiation-induced skin injury and provide guidance for managing side effects in canine radiation therapy.

Optical Methods for Optimizing Fluorescence Imaging Field of View and Image Quality in Surgical Guidance Procedures.

Cancer surgery is aimed at complete tumor resection and accurate lymph node detection. However, numerous blood vessels are distributed within the tumor, and the colors of the tumor, blood vessels, and lymph nodes are similar, making observations with the naked eye difficult. Therefore, tumors, blood vessels, and lymph nodes can be monitored via color classification using an operating microscope to induce fluorescence emission. However, as the beam width of the LED required to induce fluorescence emission is narrow and the power loss of the beam is significant at a certain working distance, there are limitations to inducing fluorescence emission, and light reflection occurs in the observation image, obstructing the view of the observation area. Therefore, the removal of reflected light is essential to avoid missing the diagnosis of the lesion under observation. This paper proposes the use of a beam mirror and polarizing filter to increase the beam width and beam intensity. The refraction and reflection effects of the beam were utilized using the beam mirror, and the rotation angle of the polarizing filter was adjusted to remove light reflection. Consequently, the minimum beam power using the beam mirror was 10.9 mW, the beam width was doubled to 40.2°, and more than 98% of light reflection was removed at 90° and 270°. With light reflection effectively eliminated, clear observation of lesions is possible. This method is expected to be used effectively in surgical, procedural, and diagnostic departments.

Increasing the Beam Width and Intensity with Refraction Power Effect Using a Combination of Beam Mirrors and Concave Mirrors for Surgical-Fluorescence-Emission-Guided Cancer Monitoring Method.

The primary goal during cancer removal surgery is to completely excise the malignant tumor. Because the color of the tumor and surrounding tissues is very similar, it is difficult to observe with the naked eye, posing a risk of damaging surrounding blood vessels during the tumor removal process. Therefore, fluorescence emission is induced using a fluorescent contrast agent, and color classification is monitored through camera imaging. LEDs must be irradiated to generate the fluorescent emission electromotive force. However, the power and beam width of the LED are insufficient to generate this force effectively, so the beam width and intensity must be increased to irradiate the entire lesion. Additionally, there should be no shaded areas in the beam irradiation range. This paper proposes a method to enhance the beam width and intensity while eliminating shadow areas. A total reflection beam mirror was used to increase beam width and intensity. However, when the beam width increased, a shadow area appeared at the edge, limiting irradiation of the entire lesion. To compensate for this shadow area, a concave lens was combined with the beam mirror, resulting in an increase in beam width and intensity by more than 1.42 times and 18.6 times, respectively. Consequently, the beam width reached 111.8°, and the beam power was 13.6 mW. The proposed method is expected to be useful for observing tumors through the induction of fluorescence emission during cancer removal surgery or for pathological examination in the pathology department.

The Long-Term Outcomes and Risk Factors of Complications After Fontan Surgery: From the Korean Fontan Registry (KFR).

BACKGROUND AND OBJECTIVES: This study aimed to analyze the outcomes of Fontan surgery in the Republic of Korea, as there were only a few studies from Asian countries. METHODS: The medical records of 1,732 patients who underwent Fontan surgery in 10 cardiac centers were reviewed. RESULTS: Among them, 1,040 (58.8%) were men. The mean age at Fontan surgery was 4.3±4.2 years, and 395 (22.8%) patients presented with heterotaxy syndrome. According to the types of Fontan surgery, 157 patients underwent atriopulmonary (AP) type; 303, lateral tunnel (LT) type; and 1,266, extracardiac conduit (ECC) type. The overall survival rates were 91.7%, 87.1%, and 74.4% at 10, 20, and 30 years, respectively. The risk factors of early mortality were male, heterotaxy syndrome, AP-type Fontan surgery, high mean pulmonary artery pressure (mPAP) in pre-Fontan cardiac catheterization, and early Fontan surgery year. The risk factors of late mortality were heterotaxy syndrome, genetic disorder, significant atrioventricular valve regurgitation (AVVR) before Fontan surgery, high mPAP in pre-Fontan cardiac catheterization, and no fenestration. CONCLUSIONS: In Asian population with a high incidence of heterotaxy syndrome, the heterotaxy syndrome was identified as the poor prognostic factors for Fontan surgery. The preoperative low mPAP and less AVVR are associated with better early and long-term outcomes of Fontan surgery.

Structure and function of the human apoptotic scramblase Xkr4.

Phosphatidylserine externalization on the surface of dying cells is a key signal for their recognition and clearance by macrophages and is mediated by members of the X-Kell related (Xkr) protein family. Defective Xkr-mediated scrambling impairs clearance, leading to inflammation. It was proposed that activation of the Xkr4 apoptotic scramblase requires caspase cleavage, followed by dimerization and ligand binding. Here, using a combination of biochemical approaches we show that purified monomeric, full-length human Xkr4 (hXkr4) scrambles lipids. CryoEM imaging shows that hXkr4 adopts a novel conformation, where three conserved acidic residues create an electronegative surface embedded in the membrane. Molecular dynamics simulations show this conformation induces membrane thinning, which could promote scrambling. Thinning is ablated or reduced in conditions where scrambling is abolished or reduced. Our work provides insights into the molecular mechanisms of hXkr4 scrambling and suggests the ability to thin membranes might be a general property of active scramblases.

Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.

Histo-pillar strip for optimal histogel block construction and biomarker analysis in 3D-lung cancer patient-derived organoids.

This study proposed an optimized histogel construction method for histological analysis by applying lung cancer patient-derived organoids (PDOs) to the developed histo-pillar strip. Previously, there is the cultured PDOs damage problem during the histogel construction due to forced detachment of the Matrigel spots from the 96-well plate bottom. To address this issue, we cultured PDO on the proposed Histo-pillar strips and then immersed them in 4% paraformaldehyde fixation solution to self-isolate PDO without damage. The 4µl patient-derived cell (PDC)/Matrigel mixtures were dispensed on the surface of a U-shaped histo-pillar strip, and the PDCs were aggregated by gravity and cultured into PDOs. Cultured PDOs were self-detached by simply immersing them in a paraformaldehyde fixing solution without physical processing, showing about two times higher cell recovery rate than conventional method. In addition, we proposed a method for embedding PDOs under conditions where the histogel temperature was maintained such that the histogel did not harden, thereby improving the problem of damaging the histogel block in the conventional sandwich histogel construction method. We performed histological and genotyping analyses using tumor tissues and PDOs from two patients with lung adenocarcinoma. Therefore, the PDO culture and improved histogel block construction method using the histo-pillar strip proposed in this study can be employed as useful tools for the histological analysis of a limited number of PDCs.

U-Shape Pillar Strip for 3D Cell-Lumped Organoid Model (3D-COM) Mimicking Lung Cancer Hypoxia Conditions in High-Throughput Screening (HTS).

Hypoxia is a representative tumor characteristic associated with malignant progression in clinical patients. Engineered in vitro models have led to significant advances in cancer research, allowing for the investigation of cells in physiological environments and the study of disease mechanisms and processes with enhanced relevance. In this study, we propose a U-shape pillar strip for a 3D cell-lumped organoid model (3D-COM) to study the effects of hypoxia on lung cancer in a high-throughput manner. We developed a U-pillar strip that facilitates the aggregation of PDCs mixed with an extracellular matrix to make the 3D-COM in 384-plate array form. The response to three hypoxia-activated prodrugs was higher in the 3D-COM than in the 2D culture model. The protein expression of hypoxia-inducible factor 1 alpha (HIF-1α) and HIF-2α, which are markers of hypoxia, was also higher in the 3D-COM than in the 2D culture. The results show that 3D-COM better recapitulated the hypoxic conditions of lung cancer tumors than the 2D culture. Therefore, the U-shape pillar strip for 3D-COM is a good tool to study the effects of hypoxia on lung cancer in a high-throughput manner, which can efficiently develop new drugs targeting hypoxic tumors.

Normal Fermi Surface in the Nodal Superconductor CeCoIn_{5} Revealed via Thermal Conductivity.

The thermal conductivity of heavy-fermion superconductor CeCoIn_{5} was measured with a magnetic field rotating in the tetragonal a-b plane, with the heat current in the antinodal direction, J|| [100]. We observe a sharp resonance in thermal conductivity for the magnetic field at an angle Θ≈12°, measured from the heat current direction [100]. This resonance corresponds to the reported resonance at an angle Θ^{'}≈33° from the direction of the heat current applied along the nodal direction, J||[110]. Both resonances, therefore, occur when the magnetic field is applied in the same crystallographic orientation in the two experiments, regardless of the direction of the heat current, proving conclusively that these resonances are due to the structure of the Fermi surface of CeCoIn_{5}. We argue that the uncondensed Landau quasiparticles, emerging with field, are responsible for the observed resonance. We support our experimental results with density-functional-theory model calculations of the density of states in a rotating magnetic field. Our calculations, using a model Fermi surface of CeCoIn_{5}, reveal several sharp peaks as a function of the field direction. Our study demonstrates that the thermal-conductivity measurement in rotating magnetic field can probe the normal parts of the Fermi surface deep inside the superconducting state.

E proteins control the development of NKγδT cells through their invariant T cell receptor.

T cell receptor (TCR) signaling regulates important developmental transitions, partly through induction of the E protein antagonist, Id3. Although normal γδ T cell development depends on Id3, Id3 deficiency produces different phenotypes in distinct γδ T cell subsets. Here, we show that Id3 deficiency impairs development of the Vγ3+ subset, while markedly enhancing development of NKγδT cells expressing the invariant Vγ1Vδ6.3 TCR. These effects result from Id3 regulating both the generation of the Vγ1Vδ6.3 TCR and its capacity to support development. Indeed, the Trav15 segment, which encodes the Vδ6.3 TCR subunit, is directly bound by E proteins that control its expression. Once expressed, the Vγ1Vδ6.3 TCR specifies the innate-like NKγδT cell fate, even in progenitors beyond the normally permissive perinatal window, and this is enhanced by Id3-deficiency. These data indicate that the paradoxical behavior of NKγδT cells in Id3-deficient mice is determined by its stereotypic Vγ1Vδ6.3 TCR complex.

A Method to Reduce Tension Differences in Pull/Push Manipulation for a Robot in Fluorescence Emission-Guided Surgical Microscopy.

MOTIVATION: A fluorescence emission-guided microscope used to monitor the outcome of cancer removal surgery is highly effective when employing a manipulator to motorize and switch the observation direction. It is necessary to minimize the alignment of looper tension between the stands for pull/push to change the direction of the manipulator and reduce the error rate caused by tension differences. This paper presents a method to minimize the error rate of looper tension between the stands. METHODS: \The looper is inserted between the stands of the manipulator to minimize the difference in tension and make the stress on the pull and push of the looper constant. The constant stress allows the manipulator to move stably in left/right, up/down, and left/right movements, which will be effective for full-camera observation and close-up shots of the end effector. RESULTS: Reducing the tolerance for differences in the manipulator's looper tension (angle and tension) is crucial. When the input value of the looper tension angle is 50°, the output should closely match 50°. Consequently, the measured response has a tolerance of ±49.98%, resulting in an error rate of .02% (1/50th level). CONCLUSION: A method is proposed to minimize the error rate of the manipulator's looper tension in a robot-based fluorescence emission-guided microscope used to observe the status of cancer surgery. As a result, a stable manipulator with a minimal error rate can achieve a 3.986x magnification for close-up observation by switching between high and low orientations.

Estimating entanglement entropy via variational quantum circuits with classical neural networks.

Entropy plays a crucial role in both physics and information science, encompassing classical and quantum domains. In this paper, we present the quantum neural entropy estimator (QNEE), an approach that combines classical neural network (NN) with variational quantum circuits to estimate the von Neumann and Rényi entropies of a quantum state. QNEE provides accurate estimates of entropy while also yielding the eigenvalues and eigenstates of the input density matrix. Leveraging the capabilities of classical NN, QNEE can classify different phases of quantum systems that accompany the changes of entanglement entropy. Our numerical simulation demonstrates the effectiveness of QNEE by applying it to the 1D XXZ Heisenberg model. In particular, QNEE exhibits high sensitivity in estimating entanglement entropy near the phase transition point. We expect that QNEE will serve as a valuable tool for quantum entropy estimation and phase classification.

The long-term effects of the fenestration in patients with extracardiac Fontan circulation-a multicenter Korean cohort study based on national Fontan registry.

Introduction: The long-term effects of fenestration in patients with Fontan circulation remain unclear. We aim to evaluate the fenestration impact on early and late outcomes in patients with extracardiac Fontan (ECF) using a propensity score matching analysis. Methods: We performed an extensive retrospective multicenter clinical data review of the Korean Fontan registry and included 1,233 patients with surgical ECF (779 fenestrated, 454 non-fenestrated). Demographics, baseline, and follow-up data were collected and comprehensively analyzed. Patients were divided into two groups according to the baseline presence or absence of surgical fenestration. Subsequently, patients were sub-divided according to the fenestration status at the last follow-up. Propensity-score matching was performed to account for collected data between the 2 groups using a multistep approach. The primary outcomes were survival and freedom from Fontan failure (FFF). We also looked at postoperative hemodynamics, cardiopulmonary exercise test results, oxygen saturations, and functional status. Results: After propensity-score matching (454 matched pairs), there was no difference in survival or FFF between the 2 groups. However, ECF patients with baseline fenestration had significantly lower oxygen saturation (p = 0.001) and lower functional status (p < 0.001). Patients with fenestration had significantly longer bypass times, higher postoperative central venous pressure, higher postoperative left atrial pressure, and less prolonged pleural effusion in the early postoperative period. The propensity score matching according to the fenestration status at the last follow-up (148 matched pairs) showed that patients with a persistent fenestration had significantly lower oxygen saturation levels (p < 0.001). However there were no intergroup differences in the functional status, survival and FFF. Conclusions: Our results showed no long-term benefits of the Fenestration in terms of survival and FFF. Patients with persistent fenestration showed oxygen desaturation but no difference in exercise intolerance was shown between the 2 groups.

Decreased incidence of Kawasaki disease in South Korea during the SARS-CoV-2 pandemic.

Purpose: Analyzing Kawasaki disease epidemiology during the SARS-CoV-2 pandemic in South Korea using 2012-2020 National Health Insurance Service data. Methods: The incidence of Kawasaki disease for 2012-2020 was investigated to identify changes in incidence after the start of the pandemic. National Health Insurance Service data from the Republic of Korea were used. Kawasaki disease was defined based on the International Statistical Classification of Diseases and Related Health Problems, the Tenth Revision diagnostic code (M30.3), and the intravenous immunoglobulin prescription code. Prescription history was collected for the following medications: intravenous immunoglobulin, aspirin, corticosteroids, tumor necrosis factor-α antagonist, clopidogrel, and anticoagulation drugs. Results: The Kawasaki disease incidence per 100,000 individuals younger than 5 years was 238.9, 230.0, and 141.2 in 2018, 2019, and 2020, respectively. Regarding the incidence from 2012 to 2020, it was the highest in 2018 and decreased to 141.2 (p < 0.001) in 2020, after the start of the pandemic. In 2020, 28.3% of all patients with KD were infants, a percentage significantly higher than that of the previous year (p < 0.001). There was biphasic seasonality in the monthly Kawasaki disease incidence. The Kawasaki disease incidence was the highest in winter followed by that in early summer. Conclusion: After the start of the pandemic, the Kawasaki disease incidence decreased, and the percentage of patients with Kawasaki disease aged <1 year increased. These findings provide support for the hypothesis suggesting an infectious trigger in Kawasaki disease.

Gadolinium-Based Magnetic Resonance Theranostic Agent with Gallic Acid as an Anti-Neuroinflammatory and Antioxidant Agent.

Studies in the field have actively pursued the incorporation of diverse biological functionalities into gadolinium-based contrast agents, aiming at the amalgamation of MRI imaging and therapeutic capabilities. In this research, we present the development of Gd-Ga, an anti-neuroinflammatory MR contrast agent strategically designed to target inflammatory mediators for comprehensive imaging diagnosis and targeted lesion treatment. Gd-Ga is a gadolinium complex composed of 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A) conjugated with gallic acid (3,4,5-trihydroxybenzoic acid). Upon intravenous administration in LPS-induced mouse models, Gd-Ga demonstrated a remarkable three-fold increase in signal-to-noise (SNR) variation compared to Gd-DOTA, particularly evident in both the cortex and hippocampus 30 min post-MR monitoring. In-depth investigations, both in vitro and in vivo, into the anti-neuroinflammatory properties of Gd-Ga revealed significantly reduced protein expression levels of pro-inflammatory mediators compared to the LPS group. The alignment between in silico predictions and phantom studies indicates that Gd-Ga acts as an anti-neuroinflammatory agent by directly binding to MD2. Additionally, the robust antioxidant activity of Gd-Ga was confirmed by its effective scavenging of NO and ROS. Our collective findings emphasize the immense potential of this theranostic complex, where a polyphenol serves as an anti-inflammatory drug, presenting an exceptionally efficient platform for the diagnosis and treatment of neuroinflammation.

Development of an Artificial Soft Solid Gel Using Gelatin Material for High-Quality Ultrasound Diagnosis.

For ultrasound diagnosis, a gel is applied to the skin. Ultrasound gel serves to block air exposure and match impedance between the skin and the probe, enhancing imaging efficiency. However, if use of the ultrasound gel exceeds a certain period of time, it may dry out and be exposed to air, causing impedance mismatch and reducing imaging resolution. In such cases, the use of a soft, solid gel proves advantageous, as it can be employed for an extended period without succumbing to the drying phenomenon and can be reused after disinfection. Its soft consistency ensures excellent skin adhesion. Our soft solid gel demonstrated approximately 1.2 times better performance than water, silicone, and traditional ultrasound gels. When comparing the dimensions of grayscale, dead zone, vertical, and horizontal regions, the measurements for the traditional ultrasound gel were 93.79 mm, 45.32 mm, 103.13 mm, 83.86 mm, and 83.86 mm, respectively. In contrast, the proposed soft solid gel exhibited dimensions of 105.64 mm, 34.48 mm, 141.1 mm, and 102.8 mm.

Structural basis of pH-dependent activation in a CLC transporter.

CLCs are dimeric chloride channels and anion/proton exchangers that regulate processes such as muscle contraction and endo-lysosome acidification. Common gating controls their activity; its closure simultaneously silences both protomers, and its opening allows them to independently transport ions. Mutations affecting common gating in human CLCs cause dominant genetic disorders. The structural rearrangements underlying common gating are unknown. Here, using single-particle cryo-electron microscopy, we show that the prototypical Escherichia coli CLC-ec1 undergoes large-scale rearrangements in activating conditions. The slow, pH-dependent remodeling of the dimer interface leads to the concerted opening of the intracellular H+ pathways and is required for transport. The more frequent formation of short water wires in the open H+ pathway enables Cl- pore openings. Mutations at disease-causing sites favor CLC-ec1 activation and accelerate common gate opening in the human CLC-7 exchanger. We suggest that the pH activation mechanism of CLC-ec1 is related to the common gating of CLC-7.

Ideal chest compression site for cardiopulmonary resuscitation in fontan circulation patients with dextrocardia.

BACKGROUND: We aimed to identify the ideal chest compression site for cardiopulmonary resuscitation (CPR) in patients with a single ventricle with dextrocardia corrected by Fontan surgery. METHODS: The most recent stored chest computed tomography images of all patients with a single ventricle who underwent Fontan surgery were retrospectively analysed. We reported that the ideal chest compression site is the largest part of the compressed single ventricle. To identify the ideal chest compression site, we measured the distance from the midline of the sternum to the point of the maximum sagittal area of the single ventricle as a deviation and calculated the area fraction of the compressed structures. RESULTS: 58 patients (67.2% male) were analysed. The mean right deviation from the midline of the sternum to the ideal compression site was similar to the mean sternum width (32.85 ± 15.61 vs. 31.05 ± 6.75 mm). When chest compression was performed at the ideal site, the area fraction of the single ventricle significantly increased by 7%, which was greater than that of conventional compression (0.15 ± 0.10 vs. 0.22 ± 0.11, P < 0.05). CONCLUSIONS: When performing CPR on a patient with Fontan circulation with dextrocardia, right-sided chest compression may be better than the conventional location.