Non-canonical deubiquitination of OTUB1 induces IFNγ-mediated cell cycle arrest via regulation of p27 stability.

The deubiquitinase OTUB1, implicated as a potential oncogene in various tumors, lacks clarity in its regulatory mechanism in tumor progression. Our study investigated the effects and underlying mechanisms of OTUB1 on the breast cancer cell cycle and proliferation in IFNγ stimulation. Loss of OTUB1 abrogated IFNγ-induced cell cycle arrest by regulating p27 protein expression, whereas OTUB1 overexpression significantly enhanced p27 expression even without IFNγ treatment. Tyr26 phosphorylation residue of OTUB1 directly bound to p27, modulating its post-translational expression. Furthermore, we identified crucial lysine residues (K134, K153, and K163) for p27 ubiquitination. Src downregulation reduced OTUB1 and p27 expression, suggesting that IFNγ-induced cell cycle arrest is mediated by the Src-OTUB1-p27 signaling pathway. Our findings highlight the pivotal role of OTUB1 in IFNγ-induced p27 expression and cell cycle arrest, offering therapeutic implications.

Development of a Mature B Lymphocyte Probe through Gating-Oriented Live-Cell Distinction (GOLD) and Selective Imaging of Topical Spleen.

B lymphocytes play a pivotal role in the adaptive immune system by facilitating antibody production. Young B cell progenitors originate in the bone marrow and migrate to the spleen for antigen-dependent maturation, leading to the development of diverse B cell subtypes. Thus, tracking B cell trajectories through cell type distinction is essential for an appropriate checkpoint assessment. Despite its significance, monitoring specific B cell subclasses in live states has been hindered by a lack of suitable molecular tools. In this study, we introduce CDoB as the first mature B cell-selective probe, enabling real-time discrimination of three classified stages in B-cell development: progenitor, transitional, and mature B cells, through a single analysis using CyTOF. The selective mechanism of CDoB, elucidated as gating-oriented live-cell distinction (GOLD), targets SLC25A16, identified through systematic screening of SLC-CRISPRa and CRISPRi libraries. CDoB selectively brightens mature B cells in the mitochondrial area using SLC25A16 as the main gate, and the staining intensity correlates positively with the expression level of SLC25A16 along the B cell maturation continuum. In spleen tissues, CDoB demonstrates selective marking in mature B cell areas in live tissue status, representing the first performance achieved by a small-molecule fluorescent probe.

Targeting Heme Oxygenase 2 (HO2) with TiNIR, a Theragnostic Approach for Managing Metastatic Non-Small Cell Lung Cancer.

Despite notable advancements in cancer therapeutics, metastasis remains a primary obstacle impeding a successful prognosis. Our prior study has identified heme oxygenase 2 (HO2) as a promising therapeutic biomarker for the aggressive subsets within tumor. This study aims to systematically evaluate HO2 as a therapeutic target of cancer, with a specific emphasis on its efficacy in addressing cancer metastasis. Through targeted inhibition of HO2 by TiNIR (tumor-initiating cell probe with near infrared), we observed a marked increase in reactive oxygen species. This, in turn, orchestrated the modulation of AKT and cJUN activation, culminating in a substantial attenuation of both proliferation and migration within a metastatic cancer cell model. Furthermore, in a mouse model, clear inhibition of cancer metastasis was unequivocally demonstrated with an HO2 inhibitor administration. These findings underscore the therapeutic promise of targeting HO2 as a strategic intervention to impede cancer metastasis, enhancing the effectiveness of cancer treatments.

Additive roles of antiferromagnetically coupled elements in the magnetic proximity effect in the GdFeCo/Pt system.

Interfacial magnetic interactions between different elements are the origin of various spin-transport phenomena in multi-elemental magnetic systems. We investigate the coupling between the magnetic moments of the rare-earth, transition-metal, and heavy-metal elements across the interface in a GdFeCo/Pt thin film, an archetype system to investigate ferrimagnetic spintronics. The Pt magnetic moments induced by the antiferromagnetically aligned FeCo and Gd moments are measured using element-resolved x-ray measurements. It is revealed that the proximity-induced Pt magnetic moments are always aligned parallel to the FeCo magnetic moments, even below the ferrimagnetic compensation temperature where FeCo has a smaller moment than Gd. This is understood by a theoretical model showing distinct effects of the rare-earth Gd 4f and transition-metal FeCo 3d magnetic moments on the Pt electronic states. In particular, the Gd and FeCo work in-phase to align the Pt moment in the same direction, despite their antiferromagnetic configuration. The unexpected additive roles of the two antiferromagnetically coupled elements exemplify the importance of detailed interactions among the constituent elements in understanding magnetic and spintronic properties of thin film systems.

Characterization of Ralstonia insidiosa C1 isolated from Alpine regions: Capability in polyhydroxyalkanoates degradation and production.

This study ventures into the exploration of potential poly-3-hydroxybutyrate (PHB) degradation in alpine environments. PHB-degrading bacteria were identified in both campus soil, representing a residential area, and Mt. Kurodake soil, an alpine region in Hokkaido, Japan. Next-generation sequencing analysis indicated that the campus soil exhibited higher microbial diversity, while Ralstonia insidiosa C1, isolated from Mt. Kurodake soil, displayed the highest proficiency in PHB degradation. R. insidiosa C1 efficiently degraded up to 3% (w/v) of PHB and various films composed of other biopolymers at 14 °C. This bacterium synthesized homopolymers using substrates such as 3-hydroxybutyric acid, sugars, and acetic acid, while also produced copolymers using a mixture of fatty acids. The analysis results confirmed that the biopolymer synthesized by strain C1 using glucose was PHB, with physical properties comparable to commercial products. The unique capabilities of R. insidiosa C1, encompassing both the production and degradation of bioplastics, highlight its potential to establish a novel material circulation model.

Dynamic Assessment of Modified Quick Sequential Organ Failure Assessment in Acutely Deteriorated Patients with Chronic Liver Disease.

Background/Aims: Quick sequential organ failure assessment (qSOFA) has been suggested to identify those who have poor outcomes in patients with suspected infection. We aimed to evaluate the ability of the modified qSOFA (m-qSOFA) to identify high-risk patients in acutely deteriorated patients with chronic liver disease (CLD), especially acute-on-chronic liver failure (ACLF). Methods: We used the data of both Korean Acute-on-Chronic Liver Failure (KACLiF) and Asian Pacific Association for the Study of the Liver ACLF Research Consortium (AARC) cohorts. qSOFA was modified by replacing the Glasgow Coma Scale with hepatic encephalopathy, and m-qSOFA≥2 was considered high. Results: Patients with high m-qSOFA had a significantly lower 1-month transplant-free survival (TFS) in both cohorts and higher organ failure development in KACLiF than patients with low m-qSOFA (Ps<0.05). Subgroup analysis by ACLF showed that patients with high m-qSOFA had lower TFS than patients with low m-qSOFA. m-qSOFA was an independent prognostic factor (hazard ratios (HR)=2.604, 95% confidence interval (CI) 1.353-5.013, P=0.004 in KACLiF and HR=1.904, 95% CI 1.484-2.442, P<0.001 in AARC). The patients with low m-qSOFA at baseline but high m-qSOFA on the 7th day had a significantly lower 1-month TFS than the patients with high m-qSOFA at baseline but low m-qSOFA on the 7th day (52.6% vs. 89.4%, P<0.001 in KACLiF and 26.9% vs. 61.5%, P<0.001 in AARC). Conclusion: Baseline and dynamic changes in m-qSOFA were useful to identify patients with a high risk of organ failure development and short-term mortality among CLD patients with acute deterioration.

MIM-CyCIF: masked imaging modeling for enhancing cyclic immunofluorescence (CyCIF) with panel reduction and imputation.

Cyclic Immunofluorescence (CyCIF) can quantify multiple biomarkers, but panel capacity is limited by technical challenges. We propose a computational panel reduction approach that can impute the information content from 25 markers using only 9 markers, learning co-expression and morphological patterns while concurrently increasing speed and panel content and decreasing cost. We demonstrate strong correlations in predictions and generalizability across breast and colorectal cancer, illustrating applicability of our approach to diverse tissue types.

Correlation of handgrip strength with quality of life-adjusted pulmonary function in adults.

BACKGROUND: Handgrip strength (HGS) is acknowledged as a key indicator of overall physical fitness and is associated with various health outcomes. OBJECTIVES: This research investigates the correlation between HGS and quality of life (QoL), focusing on its relation to pulmonary function in the general adult population. METHOD: The study involved 19,402 participants aged 40 and above, spanning from 2014 to 2019, who underwent pulmonary function and HGS tests. Participants were categorized based on lung function, and regression analyses were employed to examine the relationship between HGS and QoL, with adjustments made for lung function. RESULTS: The average age of the cohort was 58.2 years, comprising 44.6% males and 41.2% smokers. Out of the 18,708 participants who completed the European Quality of Life Scale-Five Dimensions (EQ-5D-3L) assessment, higher severity levels in mobility, self-care, usual activities, pain or discomfort, and anxiety or depression were linked to lower HGS in both sexes. Additionally, among the 3,723 participants who completed the Health-related Quality of Life Instrument with 8 Items (HINT-8) assessment, higher severity levels in pain, work, and depression were associated with lower HGS in men. In women, higher severity levels in climbing stairs, pain, vitality, and work correlated with lower HGS. CONCLUSIONS: As problems indicated by EQ-5D worsened, there was a consistent decrease in handgrip strength (HGS) across both genders. The HINT-8 assessment further revealed that increased severity in pain and work-related issues led to reduced HGS in both men and women. This study highlights the relationship between HGS and Quality of Life (QoL), taking lung function into consideration, and underscores the importance of HGS as a potential marker of physical health and fitness.

Detection of sulfur mustard simulant by trisaryl phosphoric triamide-based resin using a quartz crystal microbalance sensor.

Chemical warfare agents (CWAs) pose a persistent threat to human safety, and bis(2-chloroethyl) sulfide, or sulfur mustard (SM) is one of the most dangerous substances and is able to cause serious harm. Detecting SM gas is vital, but current methods have high-temperature requirements and limited selectivity, mainly because of the lack of CWA receptor development, and this makes them challenging to use. To address this issue, we present a trisaryl phosphoric triamide-based resin receptor that preferentially interacts with a SM simulant 2-chloroethyl ethyl sulfide (2-CEES) through dipole interactions. The receptor was synthesized through a facile process using an amine and a triethyl phosphate and the properties of its coating were enhanced using epoxy chemistry. The receptor's superior triamide structure was evaluated using a quartz crystal microbalance and reactivity was confirmed by observing the variations in reactivity according to the number of phosphoramides. The receptor showed better reactivity to 2-CEES vapor than to the known poly(epichlorohydrin) and showed selectivity to other volatile organic compounds. Moreover, its durability was evident even 30 days post-coating. The applicability of this receptor extends to array sensors, sound acoustic wave sensors, and chemo-resistive and chemo-capacitive sensors, and it promises advances in chemical warfare agent detection.

Ultra high content analyses of circulating and tumor associated hybrid cells reveal phenotypic heterogeneity.

Persistently high, worldwide mortality from cancer highlights the unresolved challenges of disease surveillance and detection that impact survival. Development of a non-invasive, blood-based biomarker would transform survival from cancer. We demonstrate the functionality of ultra-high content analyses of a newly identified population of tumor cells that are hybrids between neoplastic and immune cells in patient matched tumor and peripheral blood specimens. Using oligonucleotide conjugated antibodies (Ab-oligo) permitting cyclic immunofluorescence (cyCIF), we present analyses of phenotypes among tumor and peripheral blood hybrid cells. Interestingly, the majority of circulating hybrid cell (CHC) subpopulations were not identified in tumor-associated hybrids. These results highlight the efficacy of ultra-high content phenotypic analyses using Ab-oligo based cyCIF applied to both tumor and peripheral blood specimens. The combination of a multiplex phenotypic profiling platform that is gentle enough to analyze blood to detect and evaluate disseminated tumor cells represents a novel approach to exploring novel tumor biology and potential utility for developing the population as a blood-based biomarker in cancer.

Novel Fluorescent Strategy for Discriminating T and B Lymphocytes Using Transport System.

Fluorescent bioprobes are invaluable tools for visualizing live cells and deciphering complex biological processes by targeting intracellular biomarkers without disrupting cellular functions. In addition to protein-binding concepts, fluorescent probes utilize various mechanisms, including membrane, metabolism, and gating-oriented strategies. This study introduces a novel fluorescent mechanism distinct from existing ways. Here, we developed a B cell selective probe, CDrB, with unique transport mechanisms. Through SLC-CRISPRa screening, we identified two transporters, SLCO1B3 and SLC25A41, by sorting out populations exhibiting higher and lower fluorescence intensities, respectively, demonstrating contrasting activities. We confirmed that SLCO1B3, with comparable expression levels in T and B cells, facilitates the transport of CDrB into cells, while SLC25A41, overexpressed in T lymphocytes, actively exports CDrB. This observation suggests that SLC25A41 plays a crucial role in discriminating between T and B lymphocytes. Furthermore, it reveals the potential for the reversible localization of SLC25A41 to demonstrate its distinct activity. This study is the first report to unveil a novel strategy of SLC by exporting the probe. We anticipate that this research will open up new avenues for developing fluorescent probes.

Discovery of Live Cell Selective Fluorescent Probes and Elucidation of Their Mechanisms: Case Study of B Cell Selective Probe CDgB.

The development of small-molecule fluorescent probes for specific immune cell identification offers an economical alternative to expensive antibodies. Moreover, it enables the identification of live target cells and provides insights into the distinct properties of cells, leveraging their specific staining mechanisms. This chapter presents a comprehensive elucidation of the methodology employed for screening fluorescent compounds using flow cytometry measurements. A novel analytical approach is proposed to distinguish a fluorescent compound with a specific carbon length for B lymphocytes, involving an assessment of the staining index and the predominant ratio of immune cells. Moreover, a protocol is presented for investigating the staining mechanisms of these probes by employing cell mimicking models such as small unilamellar vesicles (SUVs).

Quantitative multiplex immunohistochemistry reveals inter-patient lymphovascular and immune heterogeneity in primary cutaneous melanoma.

Introduction: Quantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma. Methods: We established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas. Results: Here we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates. Discussion: We describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for metastasis or immune infiltration. This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

Quantitative image analysis pipeline for detecting circulating hybrid cells in immunofluorescence images with human-level accuracy.

Circulating hybrid cells (CHCs) are a newly discovered, tumor-derived cell population found in the peripheral blood of cancer patients and are thought to contribute to tumor metastasis. However, identifying CHCs by immunofluorescence (IF) imaging of patient peripheral blood mononuclear cells (PBMCs) is a time-consuming and subjective process that currently relies on manual annotation by laboratory technicians. Additionally, while IF is relatively easy to apply to tissue sections, its application to PBMC smears presents challenges due to the presence of biological and technical artifacts. To address these challenges, we present a robust image analysis pipeline to automate the detection and analysis of CHCs in IF images. The pipeline incorporates quality control to optimize specimen preparation protocols and remove unwanted artifacts, leverages a ß-variational autoencoder (VAE) to learn meaningful latent representations of single-cell images, and employs a support vector machine (SVM) classifier to achieve human-level CHC detection. We created a rigorously labeled IF CHC data set including nine patients and two disease sites with the assistance of 10 annotators to evaluate the pipeline. We examined annotator variation and bias in CHC detection and provided guidelines to optimize the accuracy of CHC annotation. We found that all annotators agreed on CHC identification for only 65% of the cells in the data set and had a tendency to underestimate CHC counts for regions of interest (ROIs) containing relatively large amounts of cells (>50,000) when using the conventional enumeration method. On the other hand, our proposed approach is unbiased to ROI size. The SVM classifier trained on the ß-VAE embeddings achieved an F1 score of 0.80, matching the average performance of human annotators. Our pipeline enables researchers to explore the role of CHCs in cancer progression and assess their potential as a clinical biomarker for metastasis. Further, we demonstrate that the pipeline can identify discrete cellular phenotypes among PBMCs, highlighting its utility beyond CHCs.

The Effect of 3D-Printed Sequential Dual Drug-Releasing Patch on the Capsule Formation Around the Silicone Implant in a Rat Model.

BACKGROUND: Implant-based breast reconstruction is associated with increased risk of early infection and late-stage capsular contracture. OBJECTIVES: We evaluated the feasibility of a dual drug-releasing patch that enabled the controlled delivery of antibiotics and immunosuppressants in a temporally and spatially appropriate manner to the implant site. METHODS: The efficacy of a dual drug-releasing patch, which was 3D-printed using tissue-derived biomaterial ink, was evaluated in rats with silicone implants. The groups included implant only (n = 10); implant plus bacterial inoculation (n = 14); implant, bacterial inoculation, and patch loaded with gentamycin placed on the ventral side of the implant (n = 10), and implant, bacterial inoculation, and patch loaded with gentamycin and triamcinolone acetonide (n = 9). Histologic and immunohistochemical analyses were 8 weeks after implantation. RESULTS: The two drugs were sequentially released from the dual drug-releasing patch and exhibited different release profiles. Compared to the animals with bacterial inoculation, those with the antibiotic-only and the dual drug-releasing patch exhibited thinner capsules, lower myofibroblast activity and inflammation, indicating better tissue integration and less foreign body response. These effects were more pronounced with the dual drug-releasing patch than with the antibiotic-only patch. CONCLUSIONS: The 3D-printed dual drug-releasing patch effectively reduced inflammation and capsule formation in a rat model of silicone breast reconstruction. The beneficial effect of the dual drug-releasing patch was better than that of the antibiotic-only patch, indicating its therapeutic potential as a novel approach to prevent capsular contracture while reducing concerns of systemic side effects.

Stereotypic T cell receptor clonotypes in the thymus and peripheral blood of Myasthenia gravis patients.

Myasthenia Gravis (MG) patients with anti-acetylcholine receptor (AChR) antibodies frequently show hyperplastic thymi with ectopic germinal centers, where autoreactive B cells proliferate with the aid of T cells. In this study, thymus and peripheral blood (PB) samples were collected from ten AChR antibody-positive MG patients. T cell receptor (TCR) repertoires were analyzed using next-generation sequencing (NGS), and compared with that of an age and sex matched control group generated from a public database. Certain V genes and VJ gene recombination pairs were significantly upregulated in the TCR chains of αß-T cells in the PB of MG patients compared to the control group. Furthermore, the TCR chains found in the thymi of MG patients had a weighted distribution to longer CDR3 lengths when compared to the PB of MG patients, and the TCR beta chains (TRB) in the MG group's PB showed increased clonality encoded by one upregulated V gene. When TRB sequences were sub-divided into groups based on their CDR3 lengths, certain groups showed decreased clonality in the MG group's PB compared to the control group's PB. Finally, we demonstrated that stereotypic MG patient-specific TCR clonotypes co-exist in both the PB and thymi at a much higher frequency than that of the clonotypes confined to the PB. These results strongly suggest the existence of a biased T cell-mediated immune response in MG patients, as observed in other autoimmune diseases.

Computed Tomography Radiomics for Preoperative Prediction of Spread Through Air Spaces in the Early Stage of Surgically Resected Lung Adenocarcinomas.

PURPOSE: To assess the added value of radiomics models from preoperative chest CT in predicting the presence of spread through air spaces (STAS) in the early stage of surgically resected lung adenocarcinomas using multiple validation datasets. MATERIALS AND METHODS: This retrospective study included 550 early-stage surgically resected lung adenocarcinomas in 521 patients, classified into training, test, internal validation, and temporal validation sets (n=211, 90, 91, and 158, respectively). Radiomics features were extracted from the segmented tumors on preoperative chest CT, and a radiomics score (Rad-score) was calculated to predict the presence of STAS. Diagnostic performance of the conventional model and the combined model, based on a combination of conventional and radiomics features, for the diagnosis of the presence of STAS were compared using the area under the curve (AUC) of the receiver operating characteristic curve. RESULTS: Rad-score was significantly higher in the STAS-positive group compared to the STAS-negative group in the training, test, internal, and temporal validation sets. The performance of the combined model was significantly higher than that of the conventional model in the training set {AUC: 0.784 [95% confidence interval (CI): 0.722-0.846] vs. AUC: 0.815 (95% CI: 0.759-0.872), p=0.042}. In the temporal validation set, the combined model showed a significantly higher AUC than that of the conventional model (p=0.001). The combined model showed a higher AUC than the conventional model in the test and internal validation sets, albeit with no statistical significance. CONCLUSION: A quantitative CT radiomics model can assist in the non-invasive prediction of the presence of STAS in the early stage of lung adenocarcinomas.

Aerococcus kribbianus sp. nov., a facultatively anaerobic bacterium isolated from pig faeces.

Two cocci-shaped, facultatively anaerobic, Gram-positive bacteria isolated from the faeces of a pig were designated as strains YH-aer221T and YH-aer222. Analysis of the 16S rRNA gene sequences revealed that the isolates were most closely related to Aerococcus suis JCM 18035T with 96.6 % similarity. The multi-locus sequence tree revealed that the isolates formed a sub-cluster adjacent to A. suis JCM 18035T. The average nucleotide identity values for the isolates and their most closely related strains were 71.8 and 71.7 %, respectively; and the digital DNA-DNA hybridization values for the isolates and their most closely related strains were 25.6 and 25.5 %, respectively. The main fatty acids were C18 : 1ω9c, C16 : 0 and C18 : 0. The cell wall contained the meso-diaminopimelic acid-based peptidoglycan. The two isolates shared the same metabolic pathways. Isolates YH-aer221T and YH-aer222 harboured the same CRISPR array with 33 and 46 spacers, respectively. Single-genome vs. metagenome analysis showed that the genomes of the isolates were not found in the available metagenome database. Given their chemotaxonomic, phenotypic and phylogenetic properties, YH-aer221T (= KCTC 25571T=JCM 35699T) and YH-aer222 (=KCTC 25573=JCM 35700) represent a novel taxon. The name Aerococcus kribbianus sp. nov. is proposed.

Gait quality in prosthesis users is reflected by force-based metrics when learning to walk on a new research-grade powered prosthesis.

Introduction: Powered prosthetic feet require customized tuning to ensure comfort and long-term success for the user, but tuning in both clinical and research settings is subjective, time intensive, and the standard for tuning can vary depending on the patient's and the prosthetist's experience levels. Methods: Therefore, we studied eight different metrics of gait quality associated with use of a research-grade powered prosthetic foot in seven individuals with transtibial amputation during treadmill walking. We compared clinically tuned and untuned conditions with the goal of identifying performance-based metrics capable of distinguishing between good (as determined by a clinician) from poor gait quality. Results: Differences between the tuned and untuned conditions were reflected in ankle power, both the vertical and anterior-posterior impulse symmetry indices, limb-force alignment, and positive ankle work, with improvements seen in all metrics during use of the tuned prosthesis. Discussion: Notably, all of these metrics relate to the timing of force generation during walking which is information not directly accessible to a prosthetist during a typical tuning process. This work indicates that relevant, real-time biomechanical data provided to the prosthetist through the future provision of wearable sensors may enhance and improve future clinical tuning procedures associated with powered prostheses as well as their long-term outcomes.