Single-cell transcriptome sequencing analysis reveals intra-tumor heterogeneity in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor of the digestive system that poses a significant threat to human life and health. It is crucial to thoroughly investigate the mechanisms of esophageal carcinogenesis and identify potential key molecular events in its carcinogenesis. Single-cell transcriptome sequencing is an emerging technology that has gained prominence in recent years for studying molecular mechanisms, which may help to further explore the underlying mechanisms of the ESCC tumor microenvironment in depth. The single-cell dataset was obtained from GSE160269 in the Gene Expression Omnibus database, including 60 tumor samples and four paracancer samples. The single-cell data underwent dimensional reduction clustering analysis to identify clusters and annotate expression profiles. Subcluster analysis was conducted for each cellular taxon. Copy number variation analysis of tumor cell subpopulations was performed to primarily identify malignant cells within them. A proposed chronological analysis was performed to obtain the process of cell differentiation. In addition, cell communication, transcription factor analysis, and tumor pathway analysis were also performed. Relevant risk models and key genes were established by univariate COX regression and LASSO analysis. The key genes obtained from the screen were subjected to appropriate silencing and cellular assays, including CCK-8, 5-ethynyl-2'-deoxyuridine, colony formation, and western blot. Single-cell analysis revealed that normal samples contained a large number of fibroblasts, T cells, and B cells, with fewer other cell types, whereas tumor samples exhibited a relatively balanced distribution of cell types. Subclassification analysis of immune cells, fibroblasts, endothelial cells, and epithelial cells revealed their specific spatial characteristics. The prognostic risk model, we constructed successfully, achieved accurate prognostic stratification for ESCC patients. The screened key gene, UPF3A, was found to be significantly associated with the development of ESCC by cellular assays. This process might be linked to the phosphorylation of ERK and P38. Single-cell transcriptome analysis successfully revealed the distribution of cell types and major expressed factors in ESCC patients, which could facilitate future in-depth studies on the therapeutic mechanisms of ESCC.

Cytisine eye drops for benzalkonium chloride-induced dry eye: safety and efficacy evaluation.

This experiment aimed to investigate the feasibility of cytisine (CYT) in treating eye diseases with ocular topical application. An in vitro cytotoxicity test, a hen's egg test-chorioallantoic membrane (HET-CAM), and a mouse eye tolerance test were used to fully reveal the ocular safety profiles of CYT. For the efficacy evaluations, CYT's effects on cell wound healing, against H2O2-induced oxidative stress damages on cells, and on benzalkonium chloride (BAC)-induced dry eye disease (DED) in mice were evaluated. Results showed that CYT did not show any cytotoxicities at concentrations no higher than 250 µg/ml, while lipoic acid (α-LA) at 250 µg/ml and BAC at 1.25 µg/ml showed significant cytotoxicities within 48 h incubation. The HET-CAM and mouse eye tolerance test confirmed that 0.5% CYT eye drops demonstrated good safety characteristics. Efficacy evaluations showed that CTY significantly promoted cell migration and wound healing. CYT significantly improved cell survival against H2O2-induced oxidative stress damage by reversing the imbalance between the reactive oxygen species (ROS) and antioxidant defense mechanisms. The animal evaluation of the BAC-induced dry eye model revealed that CYT demonstrated a strong treatment effect, including reversing ocular surface damages, recovering corneal sensitivity, and inhibiting neovascularization; HMGB1/NF-κB signaling was involved in this DED treatment by CTY. In conclusion, CYT had strong experimental treatment efficacy against DED with good ocular safety profiles, and it might be a novel and promising drug for DED.

Imatinib@glycymicelles entrapped in hydrogel: preparation, characterization, and therapeutic effect on corneal alkali burn in mice.

Imatinib (IMB) is a type of tyrosine kinase inhibitor with great application potential for inhibiting corneal neovascularization (CNV), but its poor water solubility limits its application in eye disease treatment. In this study, novel IMB@glycymicelles entrapped in hydrogel (called IMB@glycymicelle-hydrogel) were prepared, characterized, and evaluated for their therapeutic effects on corneal alkali burn in mice. Imatinib could be successfully loaded in glycymicelles using glycyrrhizin as a nanocarrier with an optimized weight ratio of IMB:nanocarrier. The apparent solubility of IMB was significantly improved from 61.69 ± 5.55 µg/mL to bare IMB to 359,967.62 ± 20,059.42 µg/mL to IMB@glycymicelles. Then, the IMB@glycymicelles were entrapped in hydrogel fabricated with hydroxypropyl methylcellulose and sodium hyaluronate (HA) to prolong retention time on the ocular surface. Rabbit eye tolerance tests showed that IMB@glycymicelle-hydrogel possessed good ocular safety profiles. In a mouse model of corneal alkali burns, the topical administration of IMB@glycymicelle-hydrogel showed strong efficacy by prompting corneal wound healing, recovering corneal sensitivity, relieving corneal opacities, and inhibiting CNV, and these efficacy evaluation parameters were better than those of the positive drug HA. Overall, these results demonstrated that IMB@glycymicelle-hydrogel may be a promising candidate for the effective treatment of alkali ocular damage.

Integrated multi-omics analyses identify anti-viral host factors and pathways controlling SARS-CoV-2 infection.

Host anti-viral factors are essential for controlling SARS-CoV-2 infection but remain largely unknown due to the biases of previous large-scale studies toward pro-viral host factors. To fill in this knowledge gap, we perform a genome-wide CRISPR dropout screen and integrate analyses of the multi-omics data of the CRISPR screen, genome-wide association studies, single-cell RNA-Seq, and host-virus proteins or protein/RNA interactome. This study uncovers many host factors that are currently underappreciated, including the components of V-ATPases, ESCRT, and N-glycosylation pathways that modulate viral entry and/or replication. The cohesin complex is also identified as an anti-viral pathway, suggesting an important role of three-dimensional chromatin organization in mediating host-viral interaction. Furthermore, we discover another anti-viral regulator KLF5, a transcriptional factor involved in sphingolipid metabolism, which is up-regulated, and harbors genetic variations linked to COVID-19 patients with severe symptoms. Anti-viral effects of three identified candidates (DAZAP2/VTA1/KLF5) are confirmed individually. Molecular characterization of DAZAP2/VTA1/KLF5-knockout cells highlights the involvement of genes related to the coagulation system in determining the severity of COVID-19. Together, our results provide further resources for understanding the host anti-viral network during SARS-CoV-2 infection and may help develop new countermeasure strategies.

Ocular topical application of alpha-glucosyl hesperidin as an active pharmaceutical excipient: in vitro and in vivo experimental evaluation.

Alpha-glucosyl hesperidin (GH) is an aqueous soluble, amphipathic hesperidin derivative with several pharmacological effects, and it is postulated in this manuscript that GH could potentially be utilized as an active pharmaceutical excipient in eyedrops. The ocular safety of GH was evaluated according to in vitro cytotoxicity and in vivo ocular tolerance. The in vivo corneal permeation of coumarin-6 (Cou-6) with or without GH was characterized, and the in vivo inducing corneal wound healing using bisdemethoxycurcumin (BDMC) with or without GH was also evaluated to determine whether GH is an active pharmaceutical excipient in eyedrops. The results demonstrated that as high as 30 mg/ml of GH exhibits high-level in vitro and in vivo safety profiles according to four in vitro and in vivo evaluations. GH improved the corneal permeation of Cou-6 in mice, as well as demonstrated in vitro antioxidant activity. Concerning in vivo activity, a BDMC-GH suspension was shown to be synergistic in promoting corneal wound healing in mice, as well as restoring corneal sensitivity, promoting corneal epithelial wound healing, and restoring the corneal tissue structure without inflammatory cell infiltration. Overall, GH could be a novel and promising active excipient in eyedrops.

Fabrication of resveratrol-loaded soy protein isolate-glycyrrhizin nanocomplex for improving bioavailability via pH-responsive hydrogel properties.

Resveratrol (RES) is a functional polyphenol that suffers from low water solubility and poor bioavailability. A novel RES-loaded soy protein isolate-dipotassium glycyrrhizinate (SPI-DG) nanocomplex (RES@SPI-DG) was designed and evaluated in this study. RES@SPI-DG was prepared using a simple but novel self-assembly ultrasonic-assisted pH-driven method. The interactions between RES and SPI-DG were non-covalent bonds, including hydrophobic interactions, hydrogen bonds, and van der Waals interactions. RES@SPI-DG exhibited high encapsulation efficiency (97.60 ± 0.38 %) and loading capacity (8.74 ± 0.03 %) of RES with a uniform small size (68.39 ± 1.10 nm). RES in RES@SPI-DG was in an amorphous state and demonstrated a 24-h apparent solubility 482.53-fold higher than bare RES. RES@SPI-DG also showed strong in vitro antioxidant properties. The pH-responsive hydrogel character of SPI-DG makes it an effective intestine-targeted delivery system that could retard the release of RES in a simulated stomach and accelerate it in a simulated intestine. In animal experiments, the bioavailability of RES@SPI-DG was 5.17 times higher than that of bare RES, and the biodistribution was also significantly improved. RES@SPI-DG demonstrated a strong hepatoprotective effect against overdose acetaminophen-induced liver injury. The SPI-DG complex might be a promising nano-platform for enhancing the bioavailability and efficacy of hydrophobic polyphenols such as RES.

CRISPR-Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames.

Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR-Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the 'dark' proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.

Leveraging a disulfidptosis-related signature to predict the prognosis and immunotherapy effectiveness of cutaneous melanoma based on machine learning.

BACKGROUND: Disulfidptosis is a recently discovered programmed cell death pathway. However, the exact molecular mechanism of disulfidptosis in cutaneous melanoma remains unclear. METHODS: In this study, clustering analysis was performed using data from public databases to construct a prognostic model, which was subsequently externally validated. The biological functions of the model genes were then investigated through various experimental techniques, including qRT-PCR, Western blotting, CCK-8 assay, wound healing assay, and Transwell assay. RESULTS: We constructed a signature using cutaneous melanoma (CM) data, which accurately predicts the overall survival (OS) of patients. The predictive value of this signature for prognosis and immune therapy response was validated using multiple external datasets. High-risk CM subgroups may exhibit decreased survival rates, alterations in the tumor microenvironment (TME), and increased tumor mutation burden. We initially verified the expression levels of five optimum disulfidptosis-related genes (ODRGs) in normal tissues and CM. The expression levels of these genes were further confirmed in HaCaT cells and three melanoma cell lines using qPCR and protein blotting analysis. HLA-DQA1 emerged as the gene with the highest regression coefficient in our risk model, highlighting its role in CM. Mechanistically, HLA-DQA1 demonstrated the ability to suppress CM cell growth, proliferation, and migration. CONCLUSION: In this study, a novel signature related to disulfidptosis was constructed, which accurately predicts the survival rate and treatment sensitivity of CM patients. Additionally, HLA-DQA1 is expected to be a feasible therapeutic target for effective clinical treatment of CM.

Simple and novel icariin-loaded pro-glycymicelles as a functional food: physicochemical characteristics, in vitro biological activities, and in vivo experimental hyperlipidemia prevention evaluations.

A novel functional food for hyperlipidemia named icariin (ICA) pro-glycymicelles (ICA-PGs) using glycyrrhizin as a phytonanomaterial was easily prepared with improved storage, pH, and salt stabilities. ICA-PGs can easily dissolve in water to self-assemble into a clear glycymicelle solution with high ICA encapsulation efficiency. The ICA in ICA-PGs exhibits significantly increased aqueous solubility, faster in vitro release, and higher bioaccessibility than bare ICA. The ICA-PGs exhibited improved in vitro activities including antioxidant, anti-α-glucosidase, anti-lipase, and anti-cholesterol esterase activities. The ICA-PG also demonstrated improved antioxidant activity in cells. In vivo evaluation confirmed that the ICA-PG demonstrated a significant protective effect against experimental hyperlipidemia in mice, exhibiting decreasing levels of triglycerides (TGs), total cholesterol (TC), and low-density lipoprotein-cholesterol (LDL-C) in the serum, and restoring the hepatic morphology to the normal state. These results indicated that the ICA-PG could improve in vitro/in vivo profiles of ICA, providing a new concept and a promising functional food for hyperlipidemia.

Identification of cuproptosis and immune-related gene prognostic signature in lung adenocarcinoma.

Background: Cuproptosis is a novel form of programmed cell death that differs from other types such as pyroptosis, ferroptosis, and autophagy. It is a promising new target for cancer therapy. Additionally, immune-related genes play a crucial role in cancer progression and patient prognosis. Therefore, our study aimed to create a survival prediction model for lung adenocarcinoma patients based on cuproptosis and immune-related genes. This model can be utilized to enhance personalized treatment for patients. Methods: RNA sequencing (RNA-seq) data of lung adenocarcinoma (LUAD) patients were collected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. The levels of immune cell infiltration in the GSE68465 cohort were determined using gene set variation analysis (GSVA), and immune-related genes (IRGs) were identified using weighted gene coexpression network analysis (WGCNA). Additionally, cuproptosis-related genes (CRGs) were identified using unsupervised clustering. Univariate COX regression analysis and least absolute shrinkage selection operator (LASSO) regression analysis were performed to develop a risk prognostic model for cuproptosis and immune-related genes (CIRGs), which was subsequently validated. Various algorithms were utilized to explore the relationship between risk scores and immune infiltration levels, and model genes were analyzed based on single-cell sequencing. Finally, the expression of signature genes was confirmed through quantitative real-time PCR (qRT-PCR), immunohistochemistry (IHC), and Western blotting (WB). Results: We have identified 5 Oncogenic Driver Genes namely CD79B, PEBP1, PTK2B, STXBP1, and ZNF671, and developed proportional hazards regression models. The results of the study indicate significantly reduced survival rates in both the training and validation sets among the high-risk group. Additionally, the high-risk group displayed lower levels of immune cell infiltration and expression of immune checkpoint compared to the low-risk group.

Multiomics analyses reveal DARS1-AS1/YBX1-controlled posttranscriptional circuits promoting glioblastoma tumorigenesis/radioresistance.

The glioblastoma (GBM) stem cell-like cells (GSCs) are critical for tumorigenesis/therapeutic resistance of GBM. Mounting evidence supports tumor-promoting function of long noncoding RNAs (lncRNAs), but their role in GSCs remains poorly understood. By combining CRISPRi screen with orthogonal multiomics approaches, we identified a lncRNA DARS1-AS1-controlled posttranscriptional circuitry that promoted the malignant properties of GBM cells/GSCs. Depleting DARS1-AS1 inhibited the proliferation of GBM cells/GSCs and self-renewal of GSCs, prolonging survival in orthotopic GBM models. DARS1-AS1 depletion also impaired the homologous recombination (HR)-mediated double-strand break (DSB) repair and enhanced the radiosensitivity of GBM cells/GSCs. Mechanistically, DARS1-AS1 interacted with YBX1 to promote target mRNA binding and stabilization, forming a mixed transcriptional/posttranscriptional feed-forward loop to up-regulate expression of the key regulators of G1-S transition, including E2F1 and CCND1. DARS1-AS1/YBX1 also stabilized the mRNA of FOXM1, a master transcription factor regulating GSC self-renewal and DSB repair. Our findings suggest DARS1-AS1/YBX1 axis as a potential therapeutic target for sensitizing GBM to radiation/HR deficiency-targeted therapy.

Novel bisdemethoxycurcumin@phytomicelle ophthalmic solution: In vitro formulation appraisal and in vivo prompting rapid corneal wound healing evaluations.

A simple and novel phytochemical-based nano-ophthalmic solution was developed for the treatment of eye diseases. This nanoformulation was produced from the mixture of the phytochemicals glycyrrhizin and alpha-glycosyl hesperidin, which serve as the phytonanomaterials that solubilize bisdemethoxycurcumin (BDMC), a promising phytochemical with strong pharmacological activities but with poor water solubility. This novel nanoformulation is a clear solution named as BDMC@phytomicelle ophthalmic solution, which was formulated using a simple preparation process. The BDMC@phytomicelles were characterized by a BDMC encapsulation efficiency of 98.37% ± 2.26%, a small phytomicelle size of 4.06 ± 0.22 nm, and a small polydispersity index of 0.25 ± 0.04. With the optimization of the BDMC@phytomicelles, the apparent solubility of BDMC (i.e., the loading of BDMC in the phytomicelles) in the simulated lacrimal fluid was 3.19 ± 0.02 mg/ml. The BDMC@phytomicelle ophthalmic solution demonstrated a good storage stability. Moreover, it did not cause irritations in rabbit eyes, and it facilitated the excellent corneal permeation of BDMC in mice. The BDMC@phytomicelles demonstrated a marked effect on the in vivo induction of corneal wound healing both in healthy and denervated corneas, as seen in the induction of corneal epithelial wound healing, recovery of corneal sensitivity, and increase in corneal subbasal nerve fiber density. These strong pharmacological activities involve the inhibition of hmgb1 signaling and the induction of VIP signaling. Overall, the BDMC@phytomicelle ophthalmic solution is a novel and promising simple ocular nano-formulation of BDMC with significantly improved in vivo profiles.

Cuproptosis/ferroptosis-related gene signature is correlated with immune infiltration and predict the prognosis for patients with breast cancer.

Background: Breast invasive carcinoma (BRCA) is a malignant tumor with high morbidity and mortality, and the prognosis is still unsatisfactory. Both ferroptosis and cuproptosis are apoptosis-independent cell deaths caused by the imbalance of corresponding metal components in cells and can affect the proliferation rate of cancer cells. The aim in this study was to develop a prognostic model of cuproptosis/ferroptosis-related genes (CFRGs) to predict survival in BRCA patients. Methods: Transcriptomic and clinical data for breast cancer patients were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Cuproptosis and ferroptosis scores were determined for the BRCA samples from the TCGA cohort using Gene Set Variation Analysis (GSVA), followed by weighted gene coexpression network analysis (WGCNA) to screen out the CFRGs. The intersection of the differentially expressed genes grouped by high and low was determined using X-tile. Univariate Cox regression and least absolute shrinkage and selection operator (LASSO) were used in the TGCA cohort to identify the CFRG-related signature. In addition, the relationship between risk scores and immune infiltration levels was investigated using various algorithms, and model genes were analyzed in terms of single-cell sequencing. Finally, the expression of the signature genes was validated with quantitative real-time PCR (qRT‒PCR) and immunohistochemistry (IHC). Results: A total of 5 CFRGs (ANKRD52, HOXC10, KNOP1, SGPP1, TRIM45) were identified and were used to construct proportional hazards regression models. The high-risk groups in the training and validation sets had significantly worse survival rates. Tumor mutational burden (TMB) was positively correlated with the risk score. Conversely, Tumor Immune Dysfunction and Exclusion (TIDE) and tumor purity were inversely associated with risk scores. In addition, the infiltration degree of antitumor immune cells and the expression of immune checkpoints were lower in the high-risk group. In addition, risk scores and mTOR, Hif-1, ErbB, MAPK, PI3K/AKT, TGF-ß and other pathway signals were correlated with progression. Conclusion: We can accurately predict the survival of patients through the constructed CFRG-related prognostic model. In addition, we can also predict patient immunotherapy and immune cell infiltration.

Morphological decomposition in Chinese compound word recognition: Electrophysiological evidence.

The present study examined the effect of both morphological complexity and semantic transparency in Chinese compound word recognition. Using a visual lexical decision task, our electrophysiological results showed that transparent and opaque compounds induced stronger Left Anterior Negativity (LAN) than monomorphemic words. This result suggests that Chinese compounds might be decomposed into their constituent morphemes at the lemma level, whereas monomorphemic words are accessed as a whole-word lemma directly from the form level. In addition, transparent and opaque compounds produced a similar N400 as each other, suggesting that transparency did not show an effect on the involvement of constituent morphemes during access to the whole-word lemma. Two behavioral experiments additionally showed similar patterns to the EEG results. These findings support morphological decomposition for compounds at the lemma level as proposed by the full-parsing model, and no evidence is found to support the role of transparency during Chinese compound word recognition.

CRISPR/Cas9 screen uncovers functional translation of cryptic lncRNA-encoded open reading frames in human cancer.

Emerging evidence suggests that cryptic translation within long noncoding RNAs (lncRNAs) may produce novel proteins with important developmental/physiological functions. However, the role of this cryptic translation in complex diseases (e.g., cancer) remains elusive. Here, we applied an integrative strategy combining ribosome profiling and CRISPR/Cas9 screening with large-scale analysis of molecular/clinical data for breast cancer (BC) and identified estrogen receptor α-positive (ER+) BC dependency on the cryptic ORFs encoded by lncRNA genes that were upregulated in luminal tumors. We confirmed the in vivo tumor-promoting function of an unannotated protein, GATA3-interacting cryptic protein (GT3-INCP) encoded by LINC00992, the expression of which was associated with poor prognosis in luminal tumors. GTE-INCP was upregulated by estrogen/ER and regulated estrogen-dependent cell growth. Mechanistically, GT3-INCP interacted with GATA3, a master transcription factor key to mammary gland development/BC cell proliferation, and coregulated a gene expression program that involved many BC susceptibility/risk genes and impacted estrogen response/cell proliferation. GT3-INCP/GATA3 bound to common cis regulatory elements and upregulated the expression of the tumor-promoting and estrogen-regulated BC susceptibility/risk genes MYB and PDZK1. Our study indicates that cryptic lncRNA-encoded proteins can be an important integrated component of the master transcriptional regulatory network driving aberrant transcription in cancer, and suggests that the "hidden" lncRNA-encoded proteome might be a new space for therapeutic target discovery.

A simple but novel glycymicelle ophthalmic solution based on two approved drugs empagliflozin and glycyrrhizin: in vitro/in vivo experimental evaluation for the treatment of corneal alkali burns.

A simple but novel ophthalmic solution based on two approved drugs was developed to reposition existing drugs to treat new diseases. This nanoformulation was developed using the phytochemical drug glycyrrhizin as an amphiphilic nanocarrier to micellarly solubilize empagliflozin (EMP), an oral drug that is widely used to control high blood glucose but has poor water solubility. This novel nanoformulation, which we designated the EMP@glycymicelle ophthalmic solution, was obtained using a simple preparation process. The resulting solution was a clear solution with an EMP encapsulation efficiency of 97.91 ± 0.50%, a small glycymicelle size of 6.659 ± 0.196 nm, and a narrow polydispersity index of 0.226 ± 0.059. The optimized formulation demonstrated that EMP was soluble in water up to 18 mg ml-1 because of its encapsulation within glycymicelles. The EMP@glycymicelle ophthalmic solution exhibited excellent characteristics, including good storage stability, fast in vitro release profiles, improved in vitro antioxidant activity, and no ocular irritation. Ocular permeation evaluation showed that the EMP@glycymicelle ophthalmic solution had strong ocular permeation of EMP, and it reached the posterior segment of mouse eyes after ocular topical administration. The treatment efficacy evaluation showed that the EMP@glycymicelle ophthalmic solution had a significant effect against corneal alkali burns in mice, prompting corneal wound healing, recovering corneal sensitivity, reducing corneal haze, and relieving corneal NV invasion. The mechanism of inhibiting HMGB1 signaling was involved in this strong treatment effect. These results indicated that the EMP@glycymicelle ophthalmic solution provided a new concept of drug repurposing and a promising ocular system for the nano-delivery of EMP with significantly improved in vivo profiles.

Single-cell CRISPR immune screens reveal immunological roles of tumor intrinsic factors.

Genetic screens are widely exploited to develop novel therapeutic approaches for cancer treatment. With recent advances in single-cell technology, single-cell CRISPR screen (scCRISPR) platforms provide opportunities for target validation and mechanistic studies in a high-throughput manner. Here, we aim to establish scCRISPR platforms which are suitable for immune-related screens involving multiple cell types. We integrated two scCRISPR platforms, namely Perturb-seq and CROP-seq, with both in vitro and in vivo immune screens. By leveraging previously generated resources, we optimized experimental conditions and data analysis pipelines to achieve better consistency between results from high-throughput and individual validations. Furthermore, we evaluated the performance of scCRISPR immune screens in determining underlying mechanisms of tumor intrinsic immune regulation. Our results showed that scCRISPR platforms can simultaneously characterize gene expression profiles and perturbation effects present in individual cells in different immune screen conditions. Results from scCRISPR immune screens also predict transcriptional phenotype associated with clinical responses to cancer immunotherapy. More importantly, scCRISPR screen platforms reveal the interactive relationship between targeting tumor intrinsic factors and T cell-mediated antitumor immune response which cannot be easily assessed by bulk RNA-seq. Collectively, scCRISPR immune screens provide scalable and reliable platforms to elucidate molecular determinants of tumor immune resistance.

Fabrication, characterization of carboxymethyl konjac glucomannan/ovalbumin-naringin nanoparticles with improving in vitro bioaccessibility.

Naringin is potential functional and therapeutic ingredient, has low bioavailability because of poor aqueous solubility. In this study, an ovalbumin (OVA)-carboxymethyl konjac glucomannan (CKGM) nano-delivery system was developed to enhance the bioavailability of naringin. The effects of proportion (OVA: CKGM), pH and naringin concentration were studied on the formation, encapsulation efficiency (EE) and bioaccessibility of OVA/CKGM-Naringin nanoparticles (OVA/CKGM-Naringin NPs). Its morphology and size were viewed by Scanning Electron Microscope (SEM) and Transmission Electron Microscopy (TEM). The cross-linkage between OVA and CKGM was verified by Fourier Transform Infrared Spectroscopy (FTIR) and Fluorescence Intensity analysis. The size of OVA/CKGM-Naringin NPs were 463.83 ± 18.50 nm (Polydispersity Index-PDI, 0.42 ± 0.05). It indicated that 2:1 of OVA: CKGM, pH 3 and 7 mg/mL of naringin concentration were optimized processing parameters of OVA/CKGM-Naringin NPs with EE (97.90 ± 2.97 %) and remarkably improved bioaccessibility (85.01 ± 2.52 %). The OVA/CKGM-Naringin NPs was energy efficiently prepared and verified as an ideal carrier of naringin.

Visual-Auditory Integration and High-Variability Speech Can Facilitate Mandarin Chinese Tone Identification.

PURPOSE: Previous studies have demonstrated that tone identification can be facilitated when auditory tones are integrated with visual information that depicts the pitch contours of the auditory tones (hereafter, visual effect). This study investigates this visual effect in combined visual-auditory integration with high- and low-variability speech and examines whether one's prior tonal-language learning experience shapes the strength of this visual effect. METHOD: Thirty Mandarin-naïve listeners, 25 Mandarin second language learners, and 30 native Mandarin listeners participated in a tone identification task in which participants judged whether an auditory tone was rising or falling in pitch. Moving arrows depicted the pitch contours of the auditory tones. A priming paradigm was used with the target auditory tones primed by four multimodal conditions: no stimuli (A-V-), visual-only stimuli (A-V+), auditory-only stimuli (A+V-), and both auditory and visual stimuli (A+V+). RESULTS: For Mandarin naïve listeners, the visual effect in accuracy produced under the cross-modal integration (A+V+ vs. A+V-) was superior to a unimodal approach (A-V+ vs. A-V-), as evidenced by a higher d prime of A+V+ as opposed to A+V-. However, this was not the case in response time. Additionally, the visual effect in accuracy and response time under the unimodal approach only occurred for high-variability speech, not for low-variability speech. Across the three groups of listeners, we found that the less tonal-language learning experience one had, the stronger the visual effect. CONCLUSION: Our study revealed the visual-auditory advantage and disadvantage of the visual effect and the joint contribution of visual-auditory integration and high-variability speech on facilitating tone perception via the process of speech symbolization and categorization. SUPPLEMENTAL MATERIAL: https://doi.org/10.23641/asha.21357729.

Critical role of lncEPAT in coupling dysregulated EGFR pathway and histone H2A deubiquitination during glioblastoma tumorigenesis.

Histone 2A (H2A) monoubiquitination is a fundamental epigenetics mechanism of gene expression, which plays a critical role in regulating cell fate. However, it is unknown if H2A ubiquitination is involved in EGFR-driven tumorigenesis. In the current study, we have characterized a previously unidentified oncogenic lncRNA (lncEPAT) that mediates the integration of the dysregulated EGFR pathway with H2A deubiquitination in tumorigenesis. LncEPAT was induced by the EGFR pathway, and high-level lncEPAT expression positively correlated with the glioma grade and predicted poor survival of glioma patients. Mass spectrometry analyses revealed that lncEPAT specifically interacted with deubiquitinase USP16. LncEPAT inhibited USP16's recruitment to chromatin, thereby blocking USP16-mediated H2A deubiquitination and repressing target gene expression, including CDKN1A and CLUSTERIN. Depletion of lncEPAT promoted USP16-induced cell cycle arrest and cellular senescence, and then repressed GBM cell tumorigenesis. Thus, the EGFR-lncEPAT-ubH2A coupling represents a previously unidentified mechanism for epigenetic gene regulation and senescence resistance during GBM tumorigenesis.