A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart.

The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.

The Costimulatory Receptor OX40 Inhibits Interleukin-17 Expression through Activation of Repressive Chromatin Remodeling Pathways.

T helper 17 (Th17) cells are prominently featured in multiple autoimmune diseases, but the regulatory mechanisms that control Th17 cell responses are poorly defined. Here we found that stimulation of OX40 triggered a robust chromatin remodeling response and produced a "closed" chromatin structure at interleukin-17 (IL-17) locus to inhibit Th17 cell function. OX40 activated the NF-κB family member RelB, and RelB recruited the histone methyltransferases G9a and SETDB1 to the Il17 locus to deposit "repressive" chromatin marks at H3K9 sites, and consequently repressing IL-17 expression. Unlike its transcriptional activities, RelB acted independently of both p52 and p50 in the suppression of IL-17. In an experimental autoimmune encephalomyelitis (EAE) disease model, we found that OX40 stimulation inhibited IL-17 and reduced EAE. Conversely, RelB-deficient CD4(+) T cells showed enhanced IL-17 induction and exacerbated the disease. Our data uncover a mechanism in the control of Th17 cells that might have important clinic implications.

Evaluation of a novel lysis-based sample processing method to optimize Vibrio vulnificus detecting by loop-mediated isothermal amplification assay.

BACKGROUND: Vibrio vulnificus exists as one of the most serious foodborne pathogens for humans, and rapid and sensitive detection methods are needed to control its infections. As an emerging method, The Loop-Mediated Isothermal Amplification (LAMP) assay has been applied to the early detection of various foodborne pathogens due to its high efficiency, but sample preprocessing still prolongs the complete detection. To optimize the detection process, our study established a novel sample preprocessing method that was more efficient compared to common methods. RESULT: Using V. vulnificus as the detecting pathogen, the water-lysis-based detecting LAMP method shortened the preprocessing time to ≤ 1 min with 100% LAMP specificity; the detection limits of the LAMP assay were decreased to 1.20 × 102 CFU/mL and 1.47 × 103 CFU/g in pure culture and in oyster, respectively. Furthermore, the 100% LAMP specificity and high sensitivity of the water-lysis method were also obtained on detecting V. parahaemolyticus, V. alginolyticus, and P. mirabilis, revealing its excellent LAMP adaption with improvement in sensitivity and efficiency. CONCLUSION: Our study provided a novel LAMP preprocessing method that was more efficient compared to common methods and possessed the practical potential for LAMP application in the future.

Pharmacokinetics of nirmatrelvir/ritonavir and the drug-drug interaction with calcineurin inhibitor in renal transplant recipients.

OBJECTIVES: To describe the pharmacokinetic (PK) characteristics of nirmatrelvir/ritonavir in renal transplant recipients and explore the potential factors that related to the PK variance of nirmatrelvir/ritonavir and its interaction with calcineurin inhibitor (CNI). METHODS: Renal transplant recipients treated with CNI and nirmatrelvir/ritonavir were prospectively enrolled. Steady-state plasma concentrations of nirmatrelvir/ritonavir were determined by high-performance liquid chromatography-tandem mass spectrometry, and the PK parameters were calculated using non-compartmental analysis. Spearman correlation analysis was used for exploring influencing factors. RESULTS: A total of eight recipients were enrolled; for nirmatrelvir and ritonavir, AUC/dose was 0.24179 ± 0.14495 and 0.06196 ± 0.03767 µg·h·mL-1·mg-1. Red blood cell (RBC), hematocrit (Ht), hemoglobins (Hb), and creatinine clearance (Ccr) were negatively correlated with AUC/dose of nirmatrelvir, while Ccr, CYP3A5 genotype, and CYP3A4 genotype were related to the AUC/dose of ritonavir. Ccr was negatively correlated with the C0/dose of tacrolimus (TAC) after termination of nirmatrelvir/ritonavir (rs = -0.943, p = 0.008). CONCLUSIONS: The PK characteristics of nirmatrelvir/ritonavir vary greatly among renal transplant recipients. Factors including Ccr and CYP3A5 genotype were related to the in vivo exposure of nirmatrelvir/ritonavir. During the whole process before and after nirmatrelvir/ritonavir therapy, it is recommended to adjust the CNI basing on renal function to avoid CNI toxicity exposure.

Value of original and modified pathological scoring systems for prognostic prediction in paraffin-embedded donor kidney core biopsy.

BACKGROUND: No study has validated, compared and adapted scoring systems for prognosis prediction based on donor kidney core biopsy (CB), with less glomeruli than wedge biopsy. METHODS: A total of 185 donor kidney CB specimens were reviewed using seven scoring systems. The association between the total score, item scores, score-based grading, and allograft prognosis was investigated. In specimens with less than ten glomeruli (88/185, 47.6%), scoring systems were modified by adjusting weights of the item scores. RESULTS: The Maryland aggregate pathology index (MAPI) score-based grading and periglomerular fibrosis (PGF) associated with delayed graft function (DGF) (Grade: OR = 1.59, p < 0.001; PGF: OR = 1.06, p = 0.006). Total score, score-based grading and chronic lesion score in scoring systems associated with one-year and 3-year eGFR after transplantation. Total-score-based models had similar predictive capacities for eGFR in all scoring systems, except MAPI and Ugarte. Score of glomerulosclerosis (GS), interstitial fibrosis (IF), tubular atrophy (TA), and arteriolar hyalinosis (AH) had good eGFR predictive capacities. In specimens with less than ten glomeruli, modified scoring systems had better eGFR predictive capacities than original scoring systems. CONCLUSIONS: Scoring systems could predict allograft prognosis in paraffin-embedded CB with ten more glomeruli. A simple and pragmatic scoring system should include GS, IF, TA and AH, with weights assigned based on predictive capacity for prognosis. Replacing GS scores with tubulointerstitial scores could significantly improve the predictive capacity of eGFR. The conclusion should be further validated in frozen section.

Clinical practice consensus for the diagnosis and management of melanoma in Taiwan.

Melanoma is rare in Taiwan. Asian melanoma is distinct from Western melanoma because acral and mucosal melanoma accounts for the majority of melanoma cases, leading to distinct tumor behaviors and genetic profiling. With consideration of the clinical guidelines in Western countries, Taiwanese experts developed a local clinical practice consensus guideline. This consensus includes diagnosis, staging, and surgical and systemic treatment, based only on clinical evidence, local epidemiology, and available resources evaluated by experts in Taiwan. This consensus emphasizes the importance of surgical management, particularly for sentinel lymph node biopsies. In addition, molecular testing for BRAF is mandatory for patients before systemic treatment. Furthermore, immunotherapy and targeted therapy are prioritized for systemic treatment. This consensus aimed to assist clinicians in Taiwan in diagnosing and treating patients according to available evidence.

Fracture Toughness Characteristics of Stacked Bilayer Graphene via Far-Field Displacement Measurements.

Mechanical properties of graphene, e.g., strength, modulus, and fracture toughness are extremely sensitive to flaws. Here the fracture properties of stacked bilayer graphene sheets (SBLG) are reported, obtained via stacking two individually grown graphene sheets. The SBLG is presented here as a building block for flaw-resilient nanomaterials. The fracture properties of freestanding SBLG sheets, suspended on transmission electron microscope (TEM) grids, are characterized by stretching the TEM grid inside an scanning electron microscope (SEM) chamber and monitoring the local displacements in real-time. The fracture toughness is measured and expressed as a function of the critical displacement required to propagate existing cracks in the experiment via computational models. This approach decouples force and displacements measurements, and utilizes the known elastic modulus along with the known displacement boundary conditions at the onset of crack growth to estimate the far field force and stress. This strategy represents a breakthrough in nanoscale fracture mechanics for statistical analysis and high throughput experimens on multiple samples at a time. Results demonstrate that the SBLG is markedly tougher than as-grown single or multilayer graphene, with a mode I fracture toughness of ≈28.06 ± 7.5 MPa m $\sqrt m $ . The mechanisms leading to a higher toughness of SBLG are also analyzed and discussed.

Genotype and phenotype analysis and transplantation strategy in children with kidney failure caused by NPHP.

BACKGROUND: Nephronophthisis-related ciliopathies (NPHP-RC) have strong genotype and phenotype heterogeneity, and the transplantation strategy of Boichis syndrome is still controversial. Our purpose was to examine associations of genotype and phenotype in children with NPHP-RC and analyze the transplantation strategies of different phenotypes. METHODS: The records of children with NPHP treated at our center from 01/2018 to 03/2021 were retrospectively reviewed. Inclusion criteria were a diagnosis of NPHP, received kidney transplantation, and received whole exome sequencing (WES) or nephropathy gene panel testing. RESULTS: Twenty-nine children with NPHP were included. Nine children (31%) had NPHP1 mutations, and all presented with isolated nephropathy. Eighteen of 20 patients with non-NPHP1 mutations had compound heterozygous mutations, and 70% had extrarenal phenotype. Age at disease presentation (11.2 ± 1.94 years) and the development of kidney failure (12.4 ± 2.70 years) were later in children with NPHP1 mutations than those with non-NPHP1 mutations (5.2 ± 2.83 years and 5.7 ± 2.92 years, respectively). Four of six children with NPHP3 mutations were diagnosed with Boichis syndrome due to liver fibrosis. Isolated kidney transplantation resulted in good outcomes for patients with mild or moderate liver fibrosis without portal hypertension, while cholestasis was common postoperatively and could be resolved with ursodeoxycholic acid. CONCLUSIONS: NPHP1 mutations are the most common in children with NPHP, and the phenotype of NPHP1 mutation is significantly different from that of non-NPHP1 mutation. For NPHP patients with mild to moderate liver fibrosis without portal hypertension, timely treatment of cholestasis could prevent the rapid progression of liver function damage after isolated kidney transplantation. A higher resolution version of the Graphical abstract is available as Supplementary information.

Prognostic prediction of subjective cognitive decline in major depressive disorder based on immune biomarkers: a prospective observational study.

OBJECTIVE: Subjective cognitive decline (SCD) is highlighted in patients with major depressive disorder (MDD), which impairs objective cognitive performance and worsens the clinical outcomes. Immune dysregulation is supposed to be the potential mechanism of cognitive impairment. However, the peripheral immune biomarkers in patients troubled with MDD and SCD are not conventionally described. METHODS: A prospective-observational study was conducted for 8 weeks. Subjective cognitive function was measured using the Chinese version of the 20-item perceived deficits questionnaire-depression (PDQ-D) and depression symptoms were evaluated with Hamilton Depression Rating Scale-17 (HDRS-17). Luminex assays were used to measure 48 immune cytokines in plasma at baseline. Integrating these results and clinicopathological features, a logistic regression model was used to develop a prognostic prediction. RESULTS: Totally, 114 patients were enrolled in this study. Among the patients who completed follow-up, 56% (N = 50) had residual subjective cognitive decline, and 44% (N = 50) did not. The plasma levels of FGF basic, INF-γ, IL-1ß, MCP-1, M-CSF and SCF were increased and the levels of IL-9, RANTES and PDGF-BB were decreased in the SCD group. Additionally, Basic FGF, IFN-γ, IL-1ß, and SCF were positively correlated and IL-9, RANTES, and PDGF-BB were negatively correlated with the PDQ-D scores after treatment. Notably, combinations of cytokines (SCF and PDGF-BB) and PDQ-D scores at baseline showed good performance (The area under the receiver operating characteristic curve = 0.818) in the prediction of subjective cognitive decline. CONCLUSION: A prognostic model based on protein concentrations of SCF, PDGF-BB, and scores of PDQ-D showed considerable accuracy in predicting residual subjective cognitive decline in depression.

RENAL nephrometry scoring system in bilateral Wilms tumor: predictive application.

AIM: This study aims to explore the application of RENAL nephrometry scoring system in bilateral Wilms tumor (BWT). METHODS: A retrospective review of patients with BWT from January 2010 to June 2022 was performed. Each kidney unit of the BWT was evaluated independently and scored according to RENAL nephrometry scoring system by 2 blinded reviewers, and reviewers were blinded to what surgery the patients ultimately had. Discrepancies were evaluated by a third reviewer to reach a consensus. Tumor anatomical characteristics were summarized and compared. RESULTS: 29 patients with 53 kidney units were included in the study. 53 kidney units included 12 (22.6%) low-complexity, 9 (17.0%) intermediate-complexity, and 32 (60.4%) high-complexity. 2 kidney units (3.8%) had tumor thrombus, and 14 (26.4%) had multiple lesions. A total of 42 kidney units (79.2%) underwent initial nephron-sparing surgery (NSS) and 11 (20.8%) underwent radical nephrectomy. Less complexity tumors were observed in the NSS group. Of the 42 kidney units undergoing initial NSS, 26 were performed in vivo and 16 ex vivo via autotransplantation. The latter group featured a higher complexity. During follow-up, 22 patients survived and 7 died, no statistically significant tumor complexity was observed between the two groups. CONCLUSIONS: The anatomical characteristics of BWT are complex. Despite this study did not indicate that the complexity correlates with prognosis, low-complexity tumors were candidates for NSS, and kidney autotransplantation provided a feasible procedure for high-complexity tumors. A refined system is required due to multiple lesions and tumor thrombus.

Using Both Plasma and Urine Donor-Derived Cell-Free DNA to Identify Various Renal Allograft Injuries.

BACKGROUND: This study was designed to investigate the association between donor-derived cell-free DNA (dd-cfDNA) and renal allograft injuries. METHODS: This single-center study enrolled 113 adult kidney transplant recipients with kidney biopsies. Plasma and urine dd-cfDNA was detected by target region capture sequencing. RESULTS: Plasma dd-cfDNA fraction was increased in multiple types of injuries, but most significantly in antibody-mediated rejection. Plasma dd-cfDNA fraction in isolated antibody-mediated rejection (1.94%, IQR: 1.15%, 2.33%) was higher than in T cell-mediated rejection (0.55%, IQR: 0.50%, 0.73%, P = 0.002) and negative biopsies (0.58%, IQR: 0.42%, 0.78%, P < 0.001), but lower than in mixed rejection (2.49%, IQR: 1.16%, 4.90%, P = 0.342). Increased urine dd-cfDNA concentration was associated with several types of injury, but most significantly with BK polyomavirus-associated nephropathy. Urine dd-cfDNA concentration in BK polyomavirus-associated nephropathy (12.22 ng/mL, IQR: 6.53 ng/mL, 31.66 ng/mL) was respectively higher than that in T cell-mediated rejection (5.24 ng/mL, IQR: 3.22 ng/mL, 6.99 ng/mL, P = 0.001), borderline change (3.93 ng/mL, IQR: 2.45 ng/mL, 6.30 ng/mL, P < 0.001), and negative biopsies (3.09 ng/mL, IQR: 1.94 ng/mL, 5.05 ng/mL, P < 0.001). Plasma dd-cfDNA fraction was positively associated with glomerulitis (r = 0.365, P < 0.001) and peri-tubular capillaritis (r = 0.344, P < 0.001), while urine dd-cfDNA concentration correlated with tubulitis (r = 0.302, P = 0.002). CONCLUSIONS: Both plasma and urine dd-cfDNA are sensitive markers for renal allograft injuries. The interpretation of a specific disease by dd-cfDNA should be combined with other clinical indicators.

Examining the Effect of a Chitosan Biopolymer on Alkali-Activated Inorganic Material for Aqueous Pb(II) and Zn(II) Sorption.

Biopolymers and alkali-activated materials have attracted a great deal of attention as adsorbents for the removal of heavy metal contaminants from aqueous solutions. Both materials are sustainable and feature unique properties, but biopolymers are relatively more expensive or difficult to prepare and exhibit low mechanical and surface properties, a narrow pH range, and thermal stability. In this study, hybrid adsorbents were prepared from both types of material, by alkali activation of low-cost fly ash precursors accompanied by incorporation of 0-2%mass chitosan biopolymer. Two types of alkaline activating solutions, NaOH and Na2SiO3, were employed to generate two sets of hybrid adsorbents with varying chitosan contents. The effect of the chitosan dosage on the aqueous Pb(II) and Zn(II) sorption efficiency was also investigated. The adsorbents exhibited 98-100% removal efficiencies for both metals, but the sorption of Zn(II) was generally higher than that of Pb(II). The addition of 0.1-2.0%mass chitosan resulted in very little improvement in the overall efficiency of the adsorbents. In contrast, 0.05%mass chitosan led to a decrease in the sorption efficiency; this was linked to the decrease in the adsorbents' ζ potential. The Na2SiO3-activated materials featured larger BET surface areas and better overall sorption performance, while the NaOH-activated materials showed the worst Pb(II) sorption performance and hence more noticeable improvement upon addition of chitosan. Mechanistic investigation shows that the sorption process follows second-order kinetics and is a chemisorption-driven process.

Tris(4'-Nitrobiphenyl)amine─An Octupolar Chromophore with High Two-Photon Absorption Cross-Section and Its Application for Uncaging of Calcium Ions in the Near-Infrared Region.

Compounds with high two-photon absorption (2PA) performance in the near-infrared region have attracted great attention because of their application in the material and biological science. In this study, we have developed a simple and novel octupolar chromophore, tris(4'-nitrobiphenyl)amine 1, with three nitro peripheral groups attached to a triphenylamine core via biphenyl linkers. A mono-branched analogue 2 has also been prepared to investigate the effects of octupolar and dipolar systems on photophysical and 2PA behaviors. Compound 1, despite having a much simpler structure than the previous three-branched scaffolds, exhibits comparable σ2 values, reaching 1330 GM at 730 nm and 900 GM at 820 nm in toluene. Combined with an outstanding σ2/MW ratio (2.2 GM g-1 mol) and a high fluorescence quantum yield (0.51), 1 displays potential as a promising two-photon (2P) probe for bioimaging. Subsequently, the ethylene glycol tetraacetic acid-substituted derivatives featuring octupolar (3 and 5) or dipolar (4 and 6) character have been synthesized and their one-photon (1P) and 2P photochemical reactions have been examined. Finally, 1P- and 2P-triggered uncaging of Ca2+ from these calcium chelators has been confirmed.

Extracellular vesicles engineered with valency-controlled DNA nanostructures deliver CRISPR/Cas9 system for gene therapy.

Extracellular vesicles (EVs) hold great promise for transporting CRISPR-Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.

A new scoring system facilitating diagnosis of oral squamous malignancy on biopsy specimens.

BACKGROUND: Morphological evaluation of oral mucosal biopsy is sometimes inconclusive, which may delay the diagnosis and treatment of oral squamous malignancy. Immunohistochemical biomarkers denoting oral squamous malignancy would be clinically helpful in such scenario. METHODS: We first studied the expression patterns of four potential biomarkers (cytokeratin 13, cytokeratin 17, Ki-67 and laminin 5 gamma 2 chain) in an exploratory cohort containing 54 surgical specimens from confirmed oral squamous malignancies. A pattern score was assigned to each specific expression pattern of these four biomarkers. A total score from each specimen was then calculated by summing up the four pattern scores. A cut-off value of total score denoting oral squamous malignancy was then determined. Another 34 oral squamous malignancies that were misdiagnosed as non-malignant lesions on their pre-treatment biopsies were used as a validation cohort to test the clinical utility of this scoring system. RESULTS: In the exploratory cohort, fifty-two (96%) of the 54 confirmed oral squamous malignancies had a total score of 9 and above. In the validation cohort, thirty-one (91%) of the 34 pre-treatment oral biopsy specimens also had a total score of 9 or above, supporting the feasibility of using this scoring system to predict immediate risk of oral squamous malignancy. CONCLUSIONS: Our four-biomarker "oral squamous malignancy scoring system" provides reliable prediction for immediate risk of oral squamous malignancy on pre-treatment oral biopsies.

Signaling Lymphocytic Activation Molecule Family-7 Alleviates Corneal Inflammation by Promoting M2 Polarization.

BACKGROUND: Signaling lymphocytic activation molecule family-7 (SLAMF7) functions as an immune checkpoint molecule on macrophages in antitumor immunity. However, its role in bacterial infection remains largely unknown. METHODS: Bone marrow-derived macrophages (BMDMs) isolated from wild-type (WT) or SLAMF7 knockout (KO) mice were infected with bacteria or treated with lipopolysaccharide/interferon-γ to investigate the expression and function of SLAMF7 in macrophage polarization. A Pseudomonas aeruginosa keratitis murine model was established to explore the effect of SLAMF7 on P. aeruginosa keratitis using WT vs SLAMF7 KO mice, or recombinant SLAMF7 vs phosphate-buffered saline-treated mice, respectively. RESULTS: SLAMF7 expression was enhanced on M1-polarized or bacterial-infected macrophages, and infiltrating macrophages in P. aeruginosa-infected mouse corneas. SLAMF7 promoted M2 polarization by inducing STAT6 activation. In vivo data showed that SLAMF7 KO aggravated, while treatment with recombinant SLAMF7 alleviated, corneal inflammation and disease severity. In addition, blockage of M2 polarization by Arg-1 inhibitor abrogated the effect of recombinant SLAMF7 in disease progression. CONCLUSIONS: SLAMF7 expression in macrophages was induced upon M1 polarization or bacterial infection and alleviated corneal inflammation and disease progression of P. aeruginosa keratitis by promoting M2 polarization. These findings may provide a potential strategy for the treatment of P. aeruginosa keratitis.

Validated LC-MS/MS method for quantitation of total and free mycophenolic acid concentration and its application to a pharmacokinetic study in pediatric renal transplant recipients.

A simple and sensitive LC-MS/MS method was established to quantify total and free mycophenolic acid (MPA) plasma concentrations during immunosuppressive medication for pediatric renal transplantation. The chromatographic separation was performed with the Hypersil GOLD C18 column, using a mobile phase consisting of 0.1% formic acid in water and acetonitrile (60:40, v/v) at an isocratic flow rate of 0.4 ml/min. An Agilent 6420 triple quadrupole mass spectrometer was operated via a positive electrospray ionization interface using the transitions m/z 321.14 → 206.9 for MPA and m/z 324.15 → 209.9 for MPA-d3 (internal standard). The linearity was 0.1-50 µg/ml for total MPA and 0.0025-0.5 µg/ml for free MPA. The within-run and between-run precisions were all <5% and accuracy was within 96.23-107.63%. The validated method was successfully aspplied to a pharmacokinetic study in 28 pediatric renal recipients. The mean free fraction of MPA in our patients was 0.89% (ranging from 0.62 to 1.25%) and albumin level played a major role in the variability of free fraction of MPA, thus, in pediatric patients with hypoproteinemia, close free drug monitoring and dose adjustments should be considered to prevent toxicity.

Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation.

BACKGROUND: Adenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions. However, the spatiotemporal and diversification properties of RNA editing in the brain are largely unknown. Here, we applied in situ sequencing to distinguish between edited and unedited transcripts in distinct regions of the mouse brain at four developmental stages, and investigate the diversity of the RNA landscape. RESULTS: We analyzed RNA editing at codon-altering sites using in situ sequencing at single-cell resolution, in combination with the detection of individual ADAR enzymes and specific cell type marker transcripts. This approach revealed cell-type-specific regulation of RNA editing of a set of transcripts, and developmental and regional variation in editing levels for many of the targeted sites. We found increasing editing diversity throughout development, which arises through regional- and cell type-specific regulation of ADAR enzymes and target transcripts. CONCLUSIONS: Our single-cell in situ sequencing method has proved useful to study the complex landscape of RNA editing and our results indicate that this complexity arises due to distinct mechanisms of regulating individual RNA editing sites, acting both regionally and in specific cell types.

Prognostic Role of Tumoral PD-L1 and IDO1 Expression, and Intratumoral CD8+ and FoxP3+ Lymphocyte Infiltrates in 132 Primary Cutaneous Merkel Cell Carcinomas.

The association of immune markers and clinicopathologic features and patient outcome has not been extensively studied in Merkel cell carcinoma (MCC). We correlated tumoral PD-L1 and IDO1 expression, and intratumoral CD8+ and FoxP3+ lymphocytes count with clinicopathologic variables, Merkel cell polyomavirus (MCPyV) status, and patient outcomes in a series of 132 MCC. By univariate analyses, tumoral PD-L1 expression >1% and combined tumoral PD-L1 >1% and high intratumoral FoxP3+ lymphocyte count correlated with improved overall survival (OS) (p = 0.016, 0.0072), MCC-specific survival (MSS) (p = 0.019, 0.017), and progression-free survival (PFS) (p = 0.043, 0.004, respectively). High intratumoral CD8+ and FoxP3+ lymphocyte count correlated with longer MSS (p = 0.036) and improved PFS (p = 0.047), respectively. Ulceration correlated with worse OS and worse MSS. Age, male gender, and higher stage (3 and 4) significantly correlated with worse survival. MCPyV positivity correlated with immune response. By multivariate analyses, only ulceration and age remained as independent predictors of worse OS; gender and stage remained for shorter PFS. Tumoral PD-L1 expression and increased density of intratumoral CD8+ lymphocytes and FoxP+ lymphocytes may represent favorable prognosticators in a subset of MCCs. Tumoral PD-L1 expression correlated with intratumoral CD8+ and FoxP3+ lymphocytes, which is supportive of an adaptive immune response.

Disulfiram Sensitizes a Therapeutic-Resistant Glioblastoma to the TGF-ß Receptor Inhibitor.

Despite neurosurgery following radiation and chemotherapy, residual glioblastoma (GBM) cells develop therapeutic resistance (TR) leading to recurrence. The GBM heterogeneity confers TR. Therefore, an effective strategy must target cancer stem cells (CSCs) and other malignant cancer cells. TGF-ß and mesenchymal transition are the indicators for poor prognoses. The activity of aldehyde dehydrogenases (ALDHs) is a functional CSC marker. However, the interplay between TGF-ß and ALDHs remains unclear. We developed radiation-resistant and radiation-temozolomide-resistant GBM models to investigate the underlying mechanisms conferring TR. Galunisertib is a drug targeting TGF-ß receptors. Disulfiram (DSF) is an anti-alcoholism drug which functions by inhibiting ALDHs. The anti-tumor effects of combining DSF and Galunisertib were evaluated by in vitro cell grow, wound healing, Transwell assays, and in vivo orthotopic GBM model. Mesenchymal-like phenotype was facilitated by TGF-ß in TR GBM. Additionally, TR activated ALDHs. DSF inhibited TR-induced cell migration and tumor sphere formation. However, DSF did not affect the tumor growth in vivo. Spectacularly, DSF sensitized TR GBM to Galunisertib both in vitro and in vivo. ALDH activity positively correlated with TGF-ß-induced mesenchymal properties in TR GBM. CSCs and mesenchymal-like GBM cells targeted together by combining DSF and Galunisertib may be a good therapeutic strategy for recurrent GBM patients.