Tuning capsid formation dynamics in recombinant adeno-associated virus producing synthetic cell lines to enhance full particle productivity.

Recombinant adeno-associated virus (rAAV) is widely used as an in vivo delivery vector for gene therapy. It is used in a very large dose, and the large quantities required for broad applications present manufacturing challenges. We have developed a synthetic biology platform of constructing cell lines integrated with essential viral genes which can be induced to produce rAAV without plasmid transfection or virus transduction. Through iterative design-construct-characterization cycles, we have showcased the potential of this synthetic cell production system. Systems characterization of the dynamics of viral transcripts and proteins as well as virus assembly and packaging revealed that the expression level and balance of viral genome and capsid protein are keys to not only the productivity but also the full particle content, an important product quality attribute. Boosting cap gene expression by sequential transfection and integration of multiple copies of the cap gene elevated the rAAV titer to levels on a par with traditional plasmid transfection and virus infection. However, overexpression of the cap gene shifted the balance and kinetics of the genome and capsid. We independently tuned the dynamics of genome amplification and capsid protein synthesis by modulating the induction concentration as well as the time profile, and significantly enhanced full particle content while maintaining a high productivity. This strategy of constructing an inducible stable producer cell line is readily adaptable to rAAV vectors of different serotypes and payloads. It can greatly facilitate scalable production of gene therapy vectors.

An unusual uterine papillary serous carcinoma with post therapy disseminating metastasis presenting as primary renal malignancy: a case report.

Introduction: Uterine papillary serous carcinoma (UPSC) is a highly aggressive endometrial carcinoma that often presents as a high-stage disease. UPSC has a high propensity for metastasis and recurrence, even with little or no myometrial invasion. It usually metastasizes to the pelvis, retroperitoneal lymph nodes, upper abdomen, or peritoneum. However, renal metastasis of UPSC is extremely rare. Case presentation: The authors reported a unique UPSC case in a 75-year-old unmarried woman. Twenty years ago, she had a history of right breast cancer and underwent a modified radical mastectomy. Three years ago, she was diagnosed with endometrial carcinoma, and six courses of chemotherapy and radiotherapy were administered. Computed tomography and retrograde pyelography revealed a right renal pelvic tumor, and a right nephroureterectomy was performed. Renal metastatic UPSC was diagnosed. The patient was administered adjuvant chemotherapy. Clinical discussion: Metastatic UPSCs initially presenting at distant sites are uncommon manifestations. This tumor should be differentially diagnosed in patients presenting with metastatic high-grade serous papillary carcinoma of unknown primary origin. Conclusion: Diagnosing metastatic renal UPSC, based on preoperative and imaging examinations, is often challenging. Thus, a review of the past history, histopathology, and immunohistochemical evaluation plays a crucial and valuable role in the definite and differential diagnosis of this tumor type.

A novel computer-assisted tool for 3D imaging of programmed death-ligand 1 expression in immunofluorescence-stained and optically cleared breast cancer specimens.

BACKGROUND: Programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) are the two most common immune checkpoints targeted in triple-negative breast cancer (BC). Refining patient selection for immunotherapy is non-trivial and finding an appropriate digital pathology framework for spatial analysis of theranostic biomarkers for PD-1/PD-L1 inhibitors remains an unmet clinical need. METHODS: We describe a novel computer-assisted tool for three-dimensional (3D) imaging of PD-L1 expression in immunofluorescence-stained and optically cleared BC specimens (n = 20). The proposed 3D framework appeared to be feasible and showed a high overall agreement with traditional, clinical-grade two-dimensional (2D) staining techniques. Additionally, the results obtained for automated immune cell detection and analysis of PD-L1 expression were satisfactory. RESULTS: The spatial distribution of PD-L1 expression was heterogeneous across various BC tissue layers in the 3D space. Notably, there were six cases (30%) wherein PD-L1 expression levels along different layers crossed the 1% threshold for admitting patients to PD-1/PD-L1 inhibitors. The average PD-L1 expression in 3D space was different from that of traditional immunohistochemistry (IHC) in eight cases (40%). Pending further standardization and optimization, we expect that our technology will become a valuable addition for assessing PD-L1 expression in patients with BC. CONCLUSION: Via a single round of immunofluorescence imaging, our approach may provide a considerable improvement in patient stratification for cancer immunotherapy as compared with standard techniques.

Enhancing the production of recombinant adeno-associated virus in synthetic cell lines through systematic characterization.

Recombinant adeno-associated virus (rAAV) is among the most commonly used in vivo gene delivery vehicles and has seen a number of successes in clinical application. Current manufacturing processes of rAAV employ multiple plasmid transfection or rely on virus infection and face challenges in scale-up. A synthetic biology approach was taken to generate stable cell lines with integrated genetic modules, which produced rAAV upon induction albeit at a low productivity. To identify potential factors that restrained the productivity, we systematically characterized virus production kinetics through targeted quantitative proteomics and various physical assays of viral components. We demonstrated that reducing the excessive expression of gene of interest by its conditional expression greatly increased the productivity of these synthetic cell lines. Further enhancement was gained by optimizing induction profiles and alleviating proteasomal degradation of viral capsid protein by the addition of proteasome inhibitors. Altogether, these enhancements brought the productivity close to traditional multiple plasmid transfection. The rAAV produced had comparable full particle contents as those produced by conventional transient plasmid transfection. The present work exemplified the versatility of our synthetic biology-based viral vector production platform and its potential for plasmid- and virus-free rAAV manufacturing.

Decoding the prognostic significance of integrator complex subunit 9 (INTS9) in glioma: links to TP53 mutations, E2F signaling, and inflammatory microenvironments.

INTRODUCTION: Gliomas, a type of brain neoplasm, are prevalent and often fatal. Molecular diagnostics have improved understanding, but treatment options are limited. This study investigates the role of INTS9 in processing small nuclear RNA (snRNA), which is crucial to generating mature messenger RNA (mRNA). We aim to employ advanced bioinformatics analyses with large-scale databases and conduct functional experiments to elucidate its potential role in glioma therapeutics. MATERIALS AND METHODS: We collected genomic, proteomic, and Whole-Exon-Sequencing data from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) for bioinformatic analyses. Then, we validated INTS9 protein expression through immunohistochemistry and assessed its correlation with P53 and KI67 protein expression. Gene Set Enrichment Analysis (GSEA) was performed to identify altered signaling pathways, and functional experiments were conducted on three cell lines treated with siINTS9. Then, we also investigate the impacts of tumor heterogeneity on INTS9 expression by integrating single-cell sequencing, 12-cell state prediction, and CIBERSORT analyses. Finally, we also observed longitudinal changes in INTS9 using the Glioma Longitudinal Analysis (GLASS) dataset. RESULTS: Our findings showed increased INTS9 levels in tumor tissue compared to non-neoplastic components, correlating with high tumor grading and proliferation index. TP53 mutation was the most notable factor associated with upregulated INTS9, along with other potential contributors, such as combined chromosome 7 gain/10 loss, TERT promoter mutation, and increased Tumor Mutational Burden (TMB). In GSEA analyses, we also linked INTS9 with enhanced cell proliferation and inflammation signaling. Downregulating INTS9 impacted cellular proliferation and cell cycle regulation during the function validation. In the context of the 12 cell states, INTS9 correlated with tumor-stem and tumor-proliferative-stem cells. CIBERSORT analyses revealed increased INTS9 associated with increased macrophage M0 and M2 but depletion of monocytes. Longitudinally, we also noticed that the INTS9 expression declined during recurrence in IDH wildtype. CONCLUSION: This study assessed the role of INTS9 protein in glioma development and its potential as a therapeutic target. Results indicated elevated INTS9 levels were linked to increased proliferation capacity, higher tumor grading, and poorer prognosis, potentially resulting from TP53 mutations. This research highlights the potential of INTS9 as a promising target for glioma treatment.

Apoptotic Cell-Derived CD14(+) Microparticles Promote the Phagocytic Activity of Neutrophilic Precursor Cells in the Phagocytosis of Apoptotic Cells.

Membranous CD14 is crucial in the phagocytic activity of neutrophils. However, the role of CD14(+) microparticles (MPs) derived from apoptotic neutrophils (apo-MP) during the phagocytic process is not clear. All trans-retinoic acid (ATRA) induces acute promyelocytic leukemic NB4 cells along granulocytic differentiation. In this study, we investigated the role of CD14(+)apo-MP in the cell-cell interaction during the phagocytic process of apoptotic cells by viable ATRA-NB4 cells. We firstly demonstrate that CD14 expression and phagocytic activity of NB4 cells were upregulated simultaneously after ATRA treatment in a time-dependent manner, and both were significantly enhanced via concurrent lipopolysaccharide treatment. The phagocytic activity of ATRA-NB4 cells and lipopolysaccharide-treated ATRA-NB4 cells were both significantly attenuated by pre-treating cells with an antibody specific to either CD14 or TLR4. Further flow cytometric analysis demonstrates that apoptotic ATRA-NB4 cells release CD14(+)apo-MP in an idarubicin dosage-dependent manner. Both CD14 expression and the phagocytic activity of viable ATRA-NB4 cells were significantly enhanced after incubation with apo-MP harvested from apoptotic ATRA-NB4 cells, and the apo-MP-enhanced phagocytic activity was significantly attenuated by pre-treating apo-MP with an anti-CD14 antibody before incubation with viable cells. We conclude that CD14(+)apo-MP derived from apoptotic ATRA-NB4 cells promotes the phagocytic activity of viable ATRA-NB4 cells in engulfing apoptotic cells.

Unusually aggressive primary testicular diffuse large B-cell lymphoma initially presenting as systemic disseminating metastases in older adult men: a case report.

Primary testicular lymphoma (PTL) accounts for 1-2% of all nonHodgkin lymphomas (NHL), 4% of extranodal nonHodgkin lymphomas, and ~9% of testicular malignancies. A rare subtype of PTL is primary testicular diffuse large B-cell lymphoma (PT-DLBCL), which may initially present as disseminating metastasis in older adult males and has a poor prognosis. Case presentation: Herein, the authors describe the case of a 64-year-old man with the chief complaint of a painless unilateral scrotal mass. Computed tomography scans of the abdomen and a pelvic examination demonstrated a left testicular tumor with multiple lymphadenopathies partially aggregated in the para-aortic area and disseminated to multiple soft tissues and organs. Subsequently, the patient underwent a left radical orchiectomy. Pathological and immunohistochemical examinations confirmed the diagnosis of left PT-DLBCL with systemic disseminating metastases. Clinical discussion: PTL often aggressively spreads to other extranodal organs, such as the contralateral testis, central nervous system, lung, pleura, Waldeyer's ring, and soft tissues. In men over 60 years of age, PT-DLBCL is the most common testicular malignancy. However, extensive systemic metastasis as the initial presentation is extremely rare. PT-DLBCL has a dismal prognosis and requires radical orchiectomy followed by multimodal therapy and central nervous system prophylaxis or systemic intervention to improve survival. Conclusion: The diagnosis of PT-DLBCL through preoperative and imaging examinations is often challenging. Thus, histopathology and immunohistochemical markers play a crucial and valuable role in the definite diagnosis and differential diagnosis of PTLs.

Panoramic Tissue Examination That Integrates 3-Dimensional Pathology Imaging and Gene Mutation: Potential Utility in Non-Small Cell Lung Cancer.

Novel therapeutics have significantly improved the survival and quality of life of patients with malignancies in this century. Versatile precision diagnostic data were used to formulate personalized therapeutic strategies for patients. However, the cost of extensive information depends on the consumption of the specimen, raising the challenges of effective specimen utilization, particularly in small biopsies. In this study, we proposed a tissue-processing cascaded protocol that obtains 3-dimensional (3D) protein expression spatial distribution and mutation analysis from an identical specimen. In order to reuse the thick section tissue evaluated after the 3D pathology technique, we designed a novel high-flatness agarose-embedded method that could improve tissue utilization rate by 1.52 fold, whereas it reduced the tissue-processing time by 80% compared with the traditional paraffin-embedding method. In animal studies, we demonstrated that the protocol would not affect the results of DNA mutation analysis. Furthermore, we explored the utility of this approach in non-small cell lung cancer because it is a compelling application for this innovation. We used 35 cases including 7 cases of biopsy specimens of non-small cell lung cancer to simulate future clinical application. The cascaded protocol consumed 150-µm thickness of formalin-fixed, paraffin-embedded specimens, providing 3D histologic and immunohistochemical information approximately 38 times that of the current paraffin-embedding protocol, and 3 rounds of DNA mutation analysis, offering both essential guidance for routine diagnostic evaluation and advanced information for precision medicine. Our designed integrated workflow provides an alternative way for pathological examination and paves the way for multidimensional tumor tissue assessment.

Incidental finding of multiple diffuse large B-cell lymphomas masquerading as jejunojejunal intussusception with unexplained pleural effusion: a case report.

Primary non-Hodgkin's lymphoma of the gastrointestinal (GI) tract is rare. It is aggressive and necessitates early diagnosis and management. Simultaneous primary GI lymphomas are unusual with rarely reported cases. Case presentation: This novel case report describes an 84-year-old man with multiple primary diffuse large B-cell lymphomas (DLBCLs) of the jejunum with disseminating pleural and multiple regional lymph nodes involvement presenting as intestinal obstruction and segments of jejunojejunal intussusception. The patient underwent surgical intervention and adjuvant chemotherapy. Unfortunately, the patient suffered from multiple organ failure and died 4 months after surgery. Clinical discussion: Obstruction and perforation are rare and life-threatening complications of GI lymphoma. Multiple DLBCLs of the jejunum are rare. Moreover, primary GI-DLBCL that initially presents with pleural effusion or with intestinal perforation is uncommon. This report aims to remind clinicians that lymphoma should be considered when assessing the cause of unexplained pleural effusion, especially when the available examination data cannot be confirmed by clinical manifestations. Conclusion: Through this case report, the authors learn that clinical manifestations, morphological characteristics, immunophenotypes, and molecular biological characteristics are vastly different and important. This poses the biggest challenge before surgery and should not be ignored.

Ancient ubiquitous protein 1 (AUP1) is a prognostic biomarker connected with TP53 mutation and the inflamed microenvironments in glioma.

INTRODUCTION: Glioblastoma (GBM) is the most common and lethal brain tumor. The current treatment is surgical removal combined with radiotherapy and chemotherapy, Temozolomide (TMZ). However, tumors tend to develop TMZ resistance which leads to therapeutic failure. Ancient ubiquitous protein 1 (AUP1) is a protein associated with lipid metabolism, which is widely expressed on the surface of ER and Lipid droplets, involved in the degradation of misfolded proteins through autophagy. It has recently been described as a prognostic marker in renal tumors. Here, we aim to use sophisticated bioinformatics and experimental validation to characterize the AUP1's role in glioma. MATERIAL AND METHODS: We collected the mRNA, proteomics, and Whole-Exon-Sequencing from The Cancer Genome Atlas (TCGA) for bioinformatics analyses. The analyses included the expression difference, Kaplan-Meier-survival, COX-survival, and correlation to the clinical factors (tumor mutation burden, microsatellite instability, and driven mutant genes). Next, we validated the AUP1 protein expression using immunohistochemical staining on the 78 clinical cases and correlated them with P53 and KI67. Then, we applied GSEA analyses to identify the altered signalings and set functional experiments (including Western Blot, qPCR, BrdU, migration, cell-cycle, and RNAseq) on cell lines when supplemented with small interfering RNA targeting the AUP1 gene (siAUP1) for further validation. We integrated the single-cell sequencing and CIBERSORT analyses at the Chinese Glioma Genome Atlas (CGGA) and Glioma Longitudinal AnalySiS (GLASS) dataset to rationale the role of AUP1 in glioma. RESULTS: Firstly, the AUP1 is a prognostic marker, increased in the tumor component, and correlated with tumor grade in both transcriptomes and protein levels. Secondly, we found higher AUP1 associated with TP53 status, Tumor mutation burden, and increased proliferation. In the function validation, downregulated AUP1 expression merely impacted the U87MG cells' proliferation instead of altering the lipophagy activity. From the single-cell sequencing and CIBERSORT analyses at CGGA and GLASS data, we understood the AUP1 expression was affected by the tumor proliferation, stromal, and inflammation compositions, particularly the myeloid and T cells. In the longitudinal data, the AUP1 significantly dropped in the recurrent IDH wildtype astrocytoma, which might result from increased AUP1-cold components, including oligodendrocytes, endothelial cells, and pericytes. CONCLUSION: According to the literature, AUP1 regulates lipophagy by stabilizing the ubiquitination of lipid droplets. However, we found no direct link between AUP1 suppression and altered autophagy activity in the functional validation. Instead, we noticed AUP1 expression associated with tumor proliferation and inflammatory status, contributed by myeloid cells and T cells. In addition, the TP53 mutations seem to play an important role here and initiate inflamed microenvironments. At the same time, EGFR amplification and Chromosome 7 gain combined 10 loss are associated with increased tumor growth related to AUP1 levels. This study taught us that AUP1 is a poorer predictive biomarker associated with tumor proliferation and could report inflamed status, potentially impacting the clinical application.

Deep-learning based breast cancer detection for cross-staining histopathology images.

Hematoxylin and eosin (H&E) staining is the gold standard for tissue characterization in routine pathological diagnoses. However, these visible light dyes do not exclusively label the nuclei and cytoplasm, making clear-cut segmentation of staining signals challenging. Currently, fluorescent staining technology is much more common in clinical research for analyzing tissue morphology and protein distribution owing to its advantages of channel independence, multiplex labeling, and the possibility of enabling 3D tissue labeling. Although both H&E and fluorescent dyes can stain the nucleus and cytoplasm for representative tissue morphology, color variation between these two staining technologies makes cross-analysis difficult, especially with computer-assisted artificial intelligence (AI) algorithms. In this study, we applied color normalization and nucleus extraction methods to overcome the variation between staining technologies. We also developed an available workflow for using an H&E-stained segmentation AI model in the analysis of fluorescent nucleic acid staining images in breast cancer tumor recognition, resulting in 89.6% and 80.5% accuracy in recognizing specific tumor features in H&E- and fluorescent-stained pathological images, respectively. The results show that the cross-staining inference maintained the same precision level as the proposed workflow, providing an opportunity for an expansion of the application of current pathology AI models.

Dual-frequency ultrasound enhances functional neuron differentiation from neural stem cells by modulating Ca2+ dynamics and the ERK1/2 signaling pathway.

Our previous study demonstrated that ultrasound is able to promote differentiation on neural stem cells (NSCs), and dual-frequency ultrasound promotes this effect due to enhanced acoustic cavitation compared with single-frequency ultrasound. However, the underlying biological reasons have not been well disclosed. The purpose of this study was to investigate the underlying bioeffects, mechanisms and signaling pathways of dual-frequency ultrasound on NSC differentiation. The morphology, neurite outgrowth, and differentiation percentages were investigated under various dual-frequency simulation parameters with exposure periods varying from 5 to 15 min. Morphological observations identified that dual-frequency ultrasound stimulation promoted ultrasound dose-dependent neurite outgrowth. In particular, cells exposed for 10 min/2 days showed optimal neurite outgrowth and neuron differentiation percentages. In addition, live cell calcium images showed that dual-frequency ultrasound enhanced the internal calcium content of the cells, and calcium ions entering cells from the extracellular environment could be observed. Dual frequency ultrasound exposure enhanced extracellular calcium influx and upregulated extracellular signal-regulated kinases 1/2 (ERK1/2) expression. Observations from immunostaining and protein expression examinations also identified that dual-frequency ultrasound promoted brain-derived neurotrophic factor (BDNF) secretion from astrocytes derived from NSCs. In summary, evidence supports that dual-frequency ultrasound effectively enhances functional neuron differentiation via calcium channel regulation via the downstream ERK1/2 pathway and promotes BDNF secretion to serve as feedback to cascade neuron differentiation. The results may provide an alternative for cell-based therapy in brain injury.

A domain knowledge enhanced yield based deep learning classifier identifies perineural invasion in oral cavity squamous cell carcinoma.

Background: Perineural invasion (PNI), a form of local invasion defined as the ability of cancer cells to invade in, around, and through nerves, has a negative prognostic impact in oral cavity squamous cell carcinoma (OCSCC). Unfortunately, the diagnosis of PNI suffers from a significant degree of intra- and interobserver variability. The aim of this pilot study was to develop a deep learning-based human-enhanced tool, termed domain knowledge enhanced yield (Domain-KEY) algorithm, for identifying PNI in digital slides. Methods: Hematoxylin and eosin (H&E)-stained whole-slide images (WSIs, n = 85) were obtained from 80 patients with OCSCC. The model structure consisted of two parts to simulate human decision-making skills in diagnostic pathology. To this aim, two semantic segmentation models were constructed (i.e., identification of nerve fibers followed by the diagnosis of PNI). The inferred results were subsequently subjected to post-processing of generated decision rules for diagnostic labeling. Ten H&E-stained WSIs not previously used in the study were read and labeled by the Domain-KEY algorithm. Thereafter, labeling correctness was visually inspected by two independent pathologists. Results: The Domain-KEY algorithm was found to outperform the ResnetV2_50 classifier for the detection of PNI (diagnostic accuracy: 89.01% and 61.94%, respectively). On analyzing WSIs, the algorithm achieved a mean diagnostic accuracy as high as 97.50% versus traditional pathology. The observed accuracy in a validation dataset of 25 WSIs obtained from seven patients with oropharyngeal (cancer of the tongue base, n = 1; tonsil cancer, n = 1; soft palate cancer, n = 1) and hypopharyngeal (cancer of posterior wall, n = 2; pyriform sinus cancer, n = 2) malignancies was 96%. Notably, the algorithm was successfully applied in the analysis of WSIs to shorten the time required to reach a diagnosis. The addition of the hybrid intelligence model decreased the mean time required to reach a diagnosis by 15.0% and 23.7% for the first and second pathologists, respectively. On analyzing digital slides, the tool was effective in supporting human diagnostic thinking. Conclusions: The Domain-KEY algorithm successfully mimicked human decision-making skills and supported expert pathologists in the routine diagnosis of PNI.

Rose Virome Analysis and Identification of a Novel Ilarvirus in Taiwan.

Rose (Rosa spp.), especially R. hybrida, is one of the most popular ornamental plants in the world and the third largest cut flower crop in Taiwan. Rose mosaic disease (RMD), showing mosaic, line patterns and ringspots on leaves, is a common rose disease caused by the complex infection of various viruses. Due to pests and diseases, the rose planting area in Taiwan has been decreasing since 2008; however, no rose virus disease has been reported in the past five decades. In the spring of 2020, rose samples showing RMD-like symptoms were observed at an organic farm in Chiayi, central Taiwan. The virome in the farm was analyzed by RNA-seq. Rose genomic sequences were filtered from the obtained reads. The remaining reads were de novo assembled to generate 294 contigs, 50 of which were annotated as viral sequences corresponding to 10 viruses. Through reverse transcription-polymerase chain reaction validation, a total of seven viruses were detected, including six known rose viruses, namely apple mosaic virus, prunus necrotic ringspot virus, rose partitivirus, apple stem grooving virus, rose spring dwarf-associated virus and rose cryptic virus 1, and a novel ilarvirus. After completing the whole genome sequencing and sequence analysis, the unknown ilarvirus was demonstrated as a putative new species, tentatively named rose ilarvirus 2. This is the first report of the rose virus disease in Taiwan.

Rapid Histological Assessment of Prostate Specimens in the Three-dimensional Space by Hydrophilic Tissue Clearing and Confocal Microscopy.

Microscopic examination of biopsied and resected prostatic specimens is the mainstay in the diagnosis of prostate cancer. However, conventional analysis of hematoxylin and eosin (H&E)-stained tissue is time-consuming and offers limited two-dimensional (2D) information. In the current study, we devised a method-termed Prostate Rapid Optical examination for cancer STATus (proSTAT)-for rapid screening of prostate cancer using high-resolution 2D and three-dimensional (3D) confocal images obtained after hydrophilic tissue clearing of 100-µm-thick tissue slices. The results of the proSTAT method were compared with those of traditional H&E stains for the analysis of cores (n=15) obtained from radical prostatectomy specimens (n=5). Gland lumen formation, consistent with Gleason pattern 3, was evident following tracking of multiple optical imaging sections. In addition, 3D rendering allowed visualizing a tubular network of interconnecting branches. Rapid 3D fluorescent labeling of tumor protein p63 accurately distinguished prostate adenocarcinoma from normal tissue and benign lesions. Compared with conventional stains, the 3D spatial and molecular information extracted from proSTAT may significantly increase the amount of available data for pathological assessment of prostate specimens. Our approach is amenable to automation and-subject to independent validation-can find a wide spectrum of clinical and research applications.

Effects of sinoaortic denervation on hemodynamic perturbations of prolonged paradoxical sleep deprivation and rapid cold stress in rats.

BACKGROUND: Sleep disturbances and aversive cold stress (CS) are cardiovascular risk factors. This study investigates how homeostatic control autonomic baroreflex influences the hemodynamic perturbations evoked by paradoxical sleep deprivation (PSD) and CS. METHODS: Conscious adult male rats were randomly divided into four groups (Sham/CON [control], Sham/PSD, sinoaortic denervation [SAD]/CON, and SAD/PSD). Spectral analysis and SAD were employed to evaluate the effects of a 72-hr PSD with 10-min CS on blood pressure variability and heart rate variability (BPV and HRV) at total power (TP) and three frequency power densities, very-low-frequency (VLF), low frequency (LF), and high frequency (HF). RESULTS: Key findings showed: (I) Compared with the control sham surgery (Sham/CON), in the natural baseline (PreCS) trial, SAD surgery (SAD/CON) causes high systolic blood pressure (SBP), heart rate (HR), increases LFBPV (low-frequency power of BPV), LF/HFHRV (the ratio LF/HF of HRV), and TPBPV (the total power of BPV), but decreases HFHRV (high-frequency power of HRV) and VLFHRV (very-low-frequency power of HRV) than the Sham/CON does. In the CS trial, SAD/CON increases the CS-induced pressor, increases the CS-elicited spectral density, LF/HFHRV, but decreases HFBPV than the Sham/CON does. (II) Compared with SAD/CON and Sham/PSD (PSD under sham surgery), in both PreCS and CS trials, SAD/PSD (PSD under SAD) causes high SBP and HR than both SAD/CON and Sham/PSD their SBP and HR. In PreCS, SAD-PSD also changes the spectral density, including increasing Sham-PSD's LFBPV, LF/HFHRV, VLFBPV, and TPBPV but decreasing Sham-PSD's VLFHRV and TPHRV. However, in CS, SAD-PSD changes the CS-elicited spectral density, including increasing Sham-PSD's VLFBPV, LF/HFHRV, and TPHRV but decreasing Sham-PSD's HFBPV and LFBPV. CONCLUSION: The results suggest baroreflex combined with other reflex pathways, such as inhibitory renorenal reflex, modulates the vascular and cardiorespiratory responses to PSD under PreCS and subsequent CS trials.

Single-cell transcriptomics links malignant T cells to the tumor immune landscape in cutaneous T cell lymphoma.

Cutaneous T cell lymphoma (CTCL) represents a heterogeneous group of non-Hodgkin lymphoma distinguished by the presence of clonal malignant T cells. The heterogeneity of malignant T cells and the complex tumor microenvironment remain poorly characterized. With single-cell RNA analysis and bulk whole-exome sequencing on 19 skin lesions from 15 CTCL patients, we decipher the intra-tumor and inter-lesion diversity of CTCL patients and propose a multi-step tumor evolution model. We further establish a subtyping scheme based on the molecular features of malignant T cells and their pro-tumorigenic microenvironments: the TCyEM group, demonstrating a cytotoxic effector memory T cell phenotype, shows more M2 macrophages infiltration, while the TCM group, featured by a central memory T cell phenotype and adverse patient outcome, is infiltrated by highly exhausted CD8+ reactive T cells, B cells and Tregs with suppressive activities. Our results establish a solid basis for understanding the nature of CTCL and pave the way for future precision medicine for CTCL patients.

Computer-assisted three-dimensional quantitation of programmed death-ligand 1 in non-small cell lung cancer using tissue clearing technology.

Immune checkpoint blockade therapy has revolutionized non-small cell lung cancer treatment. However, not all patients respond to this therapy. Assessing the tumor expression of immune checkpoint molecules, including programmed death-ligand 1 (PD-L1), is the current standard in predicting treatment response. However, the correlation between PD-L1 expression and anti-PD-1/PD-L1 treatment response is not perfect. This is partly caused by tumor heterogeneity and the common practice of assessing PD-L1 expression based on limited biopsy material. To overcome this problem, we developed a novel method that can make formalin-fixed, paraffin-embedded tissue translucent, allowing three-dimensional (3D) imaging. Our protocol can process tissues up to 150 µm in thickness, allowing anti-PD-L1 staining of the entire tissue and producing high resolution 3D images. Compared to a traditional 4 µm section, our 3D image provides 30 times more coverage of the specimen, assessing PD-L1 expression of approximately 10 times more cells. We further developed a computer-assisted PD-L1 quantitation method to analyze these images, and we found marked variation of PD-L1 expression in 3D. In 5 of 33 needle-biopsy-sized specimens (15.2%), the PD-L1 tumor proportion score (TPS) varied by greater than 10% at different depth levels. In 14 cases (42.4%), the TPS at different depth levels fell into different categories (< 1%, 1-49%, or ≥ 50%), which can potentially influence treatment decisions. Importantly, our technology permits recovery of the processed tissue for subsequent analysis, including histology examination, immunohistochemistry, and mutation analysis. In conclusion, our novel method has the potential to increase the accuracy of tumor PD-L1 expression assessment and enable precise deployment of cancer immunotherapy.

Removal of Isopropanol by synergistic non-thermal plasma and photocatalyst.

The dielectric barrier discharge (DBD) of non-thermal plasmas was combined with a self-made photocatalyst to remove isopropanol (IPA). Synthesis conditions for the novel photocatalyst, including calcination temperature and copper loading, were varied before photocatalysis to obtain at the optimal reaction efficiency. The effects of initial IPA concentration, oxygen content, and catalyst dosage were also observed. Finally, catalyst reusability was analyzed. X-ray photoelectron spectroscopy fitting revealed Ti, Cu, C, and O peaks in the synthesized catalyst. After a 60-min reaction with 100% oxygen as the carrying gas, nearly 100% of the IPA was converted. Overall, the optimal IPA conversion efficiency and acetone and carbon dioxide selectivity were achieved when the photocatalyst was synthesized at a calcination temperature of 550 °C and copper loading of 1.8%, along with a 100% oxygen carrying gas and a 3-mm discharge gap.

PEG10 amplification at 7q21.3 potentiates large-cell transformation in cutaneous T-cell lymphoma.

Mycosis fungoides (MF), the most common form of cutaneous T-cell lymphoma, undergo large-cell transformation (LCT) in the late stage, manifesting aggressive behavior, resistance to treatments, and poor prognosis, but the mechanisms involved remain unclear. To identify the molecular driver of LCT, we collected tumor samples from 133 MF patients and performed whole-transcriptome sequencing on 49 advanced-stage MF patients, followed by integrated copy number inference and genomic hybridization. Tumors with LCT showed unique transcriptional programs and enriched expressions of genes at chr7q. Paternally expressed gene 10 (PEG10), an imprinted gene at 7q21.3, was ectopically expressed in malignant T cells from LCT, driven by 7q21.3 amplification. Mechanistically, aberrant PEG10 expression increased cell size, promoted cell proliferation, and conferred treatment resistance by a PEG10/KLF2/NF-κB axis in in vitro and in vivo models. Pharmacologically targeting PEG10 reversed the phenotypes of proliferation and treatment resistance in LCT. Our findings reveal new molecular mechanisms underlying LCT and suggest that PEG10 inhibition may serve as a promising therapeutic approach in late-stage aggressive T-cell lymphoma.