Single-cell RNA sequencing of nc886, a non-coding RNA transcribed by RNA polymerase III, with a primer spike-in strategy.

Single-cell RNA sequencing (scRNA-seq) has emerged as a versatile tool in biology, enabling comprehensive genomic-level characterization of individual cells. Currently, most scRNA-seq methods generate barcoded cDNAs by capturing the polyA tails of mRNAs, which exclude many non-coding RNAs (ncRNAs), especially those transcribed by RNA polymerase III (Pol III). Although previously thought to be expressed constitutively, Pol III-transcribed ncRNAs are expressed variably in healthy and disease states and play important roles therein, necessitating their profiling at the single-cell level. In this study, we developed a measurement protocol for nc886 as a model case and initial step for scRNA-seq for Pol III-transcribed ncRNAs. Specifically, we spiked in an oligo-tagged nc886-specific primer during the polyA tail capture process for the 5'scRNA-seq. We then produced sequencing libraries for standard 5' gene expression and oligo-tagged nc886 separately, to accommodate different cDNA sizes and ensure undisturbed transcriptome analysis. We applied this protocol in three cell lines that express high, low, and zero levels of nc886. Our results show that the identification of oligo tags exhibited limited target specificity, and sequencing reads of nc886 enabled the correction of non-specific priming. These findings suggest that gene-specific primers (GSPs) can be employed to capture RNAs lacking a polyA tail, with subsequent sequence verification ensuring accurate gene expression counting. Moreover, we embarked on an analysis of differentially expressed genes in cell line sub-clusters with differential nc886 expression, demonstrating variations in gene expression phenotypes. Collectively, the primer spike-in strategy allows combined analysis of ncRNAs and gene expression phenotype.

Adenovirus expressing nc886, an anti-interferon and anti-apoptotic non-coding RNA, is an improved gene delivery vector.

Recombinant adenovirus (rAdV) vector is the most promising vehicle to deliver an exogenous gene into target cells and is preferred for gene therapy. Exogenous gene expression from rAdV is often too inefficient to induce phenotypic changes and the amount of administered rAdV must be very high to achieve a therapeutic dose. However, it is often hampered because a high dose of rAdV is likely to induce cytotoxicity by activating immune responses. nc886, a 102-nucleotide non-coding RNA that is transcribed by RNA polymerase III, acts as an immune suppressor and a facilitator of AdV entry into the nucleus. Therefore, in this study, we have constructed an rAdV expressing nc886 (AdV:nc886) to explore whether AdV:nc886 overcomes the aforementioned drawbacks of conventional rAdV vectors. When infected into mouse cell lines and mice, AdV:nc886 expresses a sufficient amount of nc886, which suppresses the induction of interferon-stimulated genes and apoptotic pathways triggered by AdV infection. As a result, AdV:nc886 is less cytotoxic and produces more rAdV-delivered gene products, compared with the parental rAdV vector lacking nc886. In conclusion, this study demonstrates that the nc886-expressing rAdV could become a superior gene delivery vehicle with greater safety and higher efficiency for in vivo gene therapy.

Pharmacokinetics and safety of candidate tocilizumab biosimilar CT-P47 administered by auto-injector or pre-filled syringe: a randomized, open­label, single-dose phase I study.

BACKGROUND: This study compared the pharmacokinetics (PK), immunogenicity, and safety of candidate tocilizumab biosimilar, CT-P47, administered via auto-injector (CT-P47 AI) or pre-filled syringe (CT-P47 PFS), in healthy Asian adults. RESEARCH DESIGN AND METHODS: In this phase I, multicenter, open-label study, participants were randomized 1:1 to receive a single 162 mg/0.9 mL dose of CT-P47 via AI or PFS. Primary endpoints were area under the concentration - time curve from time zero to infinity (AUC0-inf) and maximum serum concentration (Cmax). PK equivalence was determined if 90% confidence intervals (CIs) for the ratios of geometric least-squares means (gLSMs) were within the predefined 80-125% equivalence margin. Secondary PK parameters, immunogenicity, and safety outcomes were also assessed. RESULTS: Of 314 participants randomized (155 CT-P47 AI; 159 CT-P47 PFS), 310 received the study drug (153 CT-P47 AI; 157 CT-P47 PFS). Primary and secondary PK results, immunogenicity and safety were similar between groups. Ninety percent CIs for the ratio of gLSMs were within the predefined equivalence margin for AUC0-inf (85.87-102.94) and Cmax (82.98-98.16). CONCLUSIONS: PK equivalence between CT-P47 AI and CT-P47 PFS was demonstrated in healthy Asian adults, with comparable immunogenicity and safety between the two devices. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05617183.

Tocilizumab is a biologic medicine used to treat inflammatory diseases, such as rheumatoid arthritis. A biosimilar is a drug that is an almost identical copy of an approved original ('reference') biologic medicine; it has identical efficacy and safety to the original medicine but is typically less expensive. CT­P47 is in development as a possible tocilizumab biosimilar.Some patients prefer injections using an auto-injector (AI) rather than a pre-filled syringe (PFS), for reasons including ease of use and convenience. With an AI, medicine is delivered automatically by firmly pressing the device against the skin, whereas, with a PFS, a needle is inserted into the skin and medicine delivered by depressing the plunger. The injection of CT­P47 using a PFS has shown comparable pharmacokinetics (i.e., the uptake, metabolism and excretion of the drug by the body) and safety to tocilizumab. Therefore, if the pharmacokinetics and safety of CT­P47 administered via AI and PFS were shown to be similar, this might expand the choice of administration devices available to patients.In this study, 310 healthy adults received a single injection of CT­P47 via AI or PFS. Blood samples were taken over 43 days to analyze pharmacokinetics. The uptake, metabolism and elimination of CT­P47 by the body was similar when administered by each device, suggesting that CT­P47 can be administered by either AI or PFS.

Vimentin-mediated buffering of internal integrin ß1 pool increases survival of cells from anoikis.

BACKGROUND: The intermediate filament protein vimentin is widely recognized as a molecular marker of epithelial-to-mesenchymal transition. Although vimentin expression is strongly associated with cancer metastatic potential, the exact role of vimentin in cancer metastasis and the underlying mechanism of its pro-metastatic functions remain unclear. RESULTS: This study revealed that vimentin can enhance integrin ß1 surface expression and induce integrin-dependent clustering of cells, shielding them against anoikis cell death. The increased integrin ß1 surface expression in suspended cells was caused by vimentin-mediated protection of the internal integrin ß1 pool against lysosomal degradation. Additionally, cell detachment was found to induce vimentin Ser38 phosphorylation, allowing the translocation of internal integrin ß1 to the plasma membrane. Furthermore, the use of an inhibitor of p21-activated kinase PAK1, one of the kinases responsible for vimentin Ser38 phosphorylation, significantly reduced cancer metastasis in animal models. CONCLUSIONS: These findings suggest that vimentin can act as an integrin buffer, storing internalized integrin ß1 and releasing it when needed. Overall, this study provides insights regarding the strong correlation between vimentin expression and cancer metastasis and a basis for blocking metastasis using this novel therapeutic mechanism.

Phrase boundaries lacking word prosody: An articulatory investigation of Seoul Korean.

This electromagnetic articulography study explores the kinematic profile of Intonational Phrase boundaries in Seoul Korean. Recent findings suggest that the scope of phrase-final lengthening is conditioned by word- and/or phrase-level prominence. However, evidence comes mainly from head-prominence languages, which conflate positions of word prosody with positions of phrasal prominence. Here, we examine phrase-final lengthening in Seoul Korean, an edge-prominence language with no word prosody, with respect to focus location as an index of phrase-level prominence and Accentual Phrase (AP) length as an index of word demarcation. Results show that phrase-final lengthening extends over the phrase-final syllable. The effect is greater the further away that focus occurs. It also interacts with the domains of AP and prosodic word: lengthening is greater in smaller APs, whereas shortening is observed in the initial gesture of the phrase-final word. Additional analyses of kinematic displacement and peak velocity revealed that Korean phrase-final gestures bear the kinematic profile of IP boundaries concurrently to what is typically considered prominence marking. Based on these results, a gestural coordination account is proposed, in which boundary-related events interact systematically with phrase-level prominence as well as lower prosodic levels, and how this proposal relates to the findings in head-prominence languages is discussed.

Neuromuscular blocking agent re-exposure in a retrospective cohort with neuromuscular blocking agent-associated anaphylaxis.

BACKGROUND: Neuromuscular blocking agents (NMBAs) are one of the most common causes of perioperative anaphylaxis. Although skin test positivity may help identify reactive NMBAs, it is unclear whether skin test negativity can guarantee the safety of systemically administered NMBAs. OBJECTIVE: This study aimed to evaluate the real-world safety of alternative NMBAs screened using skin tests in patients with suspected NMBA-induced anaphylaxis. METHODS: A retrospective cohort of suspected NMBA-induced anaphylaxis were recruited among patients at Seoul National University Hospital from June 2009 to May 2021, and their characteristics and outcomes were assessed. RESULTS: A total of 47 cases (0.017%) of suspected anaphylaxis occurred in 282,707 patients who received NMBAs. Cardiovascular manifestations were observed in 95.7%, whereas cutaneous findings were observed in 59.6%. Whereas 83% had a history of undergoing general anesthesia, 17% had no history of NMBA use. In skin tests, the overall positivity to any NMBA was 94.6% (81.1% to culprit NMBAs) and the cross-reactivity was 75.7%, which is related to the chemical structural similarity among NMBAs; the cross-reactivity and chemical structure similarity of rocuronium were 85.3% and 0.814, respectively, with vecuronium; this is in contrast to 50% and 0.015 with cisatracurium and 12.5% and 0.208 with succinylcholine. There were 15 patients who underwent subsequent surgery with a skin test-negative NMBA; whereas 80.0% (12/15) safely completed surgery, 20.0% (3/15) experienced hypotension. CONCLUSION: Similarities in chemical structure may contribute to the cross-reactivity of NMBAs in skin tests. Despite the high negative predictability of skin tests for suspected NMBA-induced anaphylaxis, the potential risk of recurrent anaphylaxis has not been eliminated.

Prosodic Structural Effects on Non-Contrastive Coarticulatory Vowel Nasalization in L2 English by Korean Learners.

This acoustic study explores how Korean learners produce coarticulatory vowel nasalization in English that varies with prosodic structural factors of focus-induced prominence and boundary. N-duration and A1-P0 (degree of V-nasalization) are measured in consonant-vowel-nasal (CVN) and nasal-vowel-consonant (NVC) words in various prosodic structural conditions (phrase-final vs. phrase-medial; focused vs. unfocused). Korean learners show a systematic fine-tuning of the non-contrastive V-nasalization in second language (L2) English in relation to prosodic structure, although it does not pertain to learning new L2 sound categories (i.e., L2 English nasal consonants are directly mapped onto Korean nasal consonants). The prosodic structurally conditioned phonetic detail in English appears to be accessible in most part to Korean learners and was therefore reflected in their production of L2 English. Their L2 production, however, is also found to be constrained by their first language (L1-Korean) to some extent, resulting in some phonetic effects that deviate from both L1 and L2. The results suggest that the seemingly low-level coarticulatory process is indeed under the speaker's control in L2, which reflects interactions of the specificities of the phonetics-prosody interface in L1 and L2. The results are also discussed in terms of their implications for theories of L2 phonetics.

Environment-Sensitive Ectodomain Shedding of Epithin/PRSS14 Increases Metastatic Potential of Breast Cancer Cells by Producing CCL2.

Epithin/PRSS14 is a membrane serine protease that plays a key role in tumor progression. The protease exists on the cell surface until its ectodomain shedding, which releases most of the extracellular domain. Previously, we showed that the remaining portion on the membrane undergoes intramembrane proteolysis, which results in the liberation of the intracellular domain and the intracellular domainmediated gene expression. In this study, we investigated how the intramembrane proteolysis for the nuclear function is initiated. We observed that ectodomain shedding of epithin/PRSS14 in mouse breast cancer 4T1 cells increased depending on environmental conditions and was positively correlated with invasiveness of the cells and their proinvasive cytokine production. We identified selenite as an environmental factor that can induce ectodomain shedding of the protease and increase C-C motif chemokine ligand 2 (CCL2) secretion in an epithin/PRSS14-dependent manner. Additionally, by demonstrating that the expression of the intracellular domain of epithin/PRSS14 is sufficient to induce CCL2 secretion, we established that epithin/PRSS14- dependent shedding and its subsequent intramembrane proteolysis are responsible for the metastatic conversion of 4T1 cells under these conditions. Consequently, we propose that epithin/PRSS14 can act as an environment-sensing receptor that promotes cancer metastasis by liberating the intracellular domain bearing transcriptional activity under conditions promoting ectodomain shedding.

A host non-coding RNA, nc886, plays a pro-viral role by promoting virus trafficking to the nucleus.

Elucidation of the interplay between viruses and host cells is crucial for taming viruses to benefit human health. Cancer therapy using adenovirus, called oncolytic virotherapy, is a promising treatment option but is not robust in all patients. In addition, inefficient replication of human adenovirus in mouse hampered the development of an in vivo model for preclinical evaluation of therapeutically engineered adenovirus. nc886 is a human non-coding RNA that suppresses Protein Kinase R (PKR), an antiviral protein. In this study, we have found that nc886 greatly promotes adenoviral gene expression and replication. Remarkably, the stimulatory effect of nc886 is not dependent on its function to inhibit PKR. Rather, nc886 facilitates the nuclear entry of adenovirus via modulating the kinesin pathway. nc886 is not conserved in mouse and, when xenogeneically expressed in mouse cells, promotes adenovirus replication. Our investigation has discovered a novel mechanism of how a host ncRNA plays a pro-adenoviral role. Given that nc886 expression is silenced in a subset of cancer cells, our study highlights that oncolytic virotherapy might be inefficient in those cells. Furthermore, our findings open future possibilities of harnessing nc886 to improve the efficacy of oncolytic adenovirus and to construct nc886-expressing transgenic mice as an animal model.

SOCS1 counteracts ROS-mediated survival signals and promotes apoptosis by modulating cell cycle to increase radiosensitivity of colorectal cancer cells.

As negative regulators of cytokine signaling pathways, suppressors of cytokine signaling (SOCS) proteins have been reported to possess both pro-tumor and anti-tumor functions. Our recent studies have demonstrated suppressive effects of SOCS1 on epithelial to mesenchymal signaling in colorectal cancer cells in response to fractionated ionizing radiation or oxidative stress. The objective of the present study was to determine the radiosensitizing action of SOCS1 as an anti-tumor mechanism in colorectal cancer cell model. In HCT116 cells exposed to ionizing radiation, SOCS1 over-expression shifted cell cycle arrest from G2/M to G1 and promoted radiation-induced apoptosis in a p53-dependent manner with down-regulation of cyclin B and up-regulation of p21. On the other hand, SOCS1 knock-down resulted in a reduced apoptosis with a decrease in G1 arrest. The regulatory action of SOCS1 on the radiation response was mediated by inhibition of radiation-induced Jak3/STAT3 and Erk activities, thereby blocking G1 to S transition. Radiation-induced early ROS signal was responsible for the activation of Jak3/Erk/STAT3 that led to cell survival response. Our data collectively indicate that SOCS1 can promote radiosensitivity of colorectal cancer cells by counteracting ROS-mediated survival signal, thereby blocking cell cycle progression from G1 to S. The resulting increase in G1 arrest with p53 activation then contributes to the promotion of apoptotic response upon radiation. Thus, induction of SOCS1 expression may increase therapeutic efficacy of radiation in tumors with low SOCS1 levels. [BMB Reports 2022; 55(4): 198-203].

Associations of fasting glucose and glycated hemoglobin with vitamin D levels according to diabetes mellitus status in Korean adults.

OBJECTIVES: According to previous studies, vitamin D deficiency might increase the risk of type 2 diabetes mellitus (DM). However, few studies have examined whether vitamin D continues to affect glucose control after DM diagnosis. Therefore, we examined the association between vitamin D and glucose levels in individuals with and without DM. METHODS: We analyzed data for 32,943 adults aged 19 years and older from the 2008 to 2014 Korea National Health and Nutrition Examination Survey. Patients were classified into 3 groups according to the 25-hydroxyvitamin D concentration. DM was defined as a fasting glucose level ≥126 mg/dL, current use of DM medications or insulin injections, or a self-reported diagnosis of DM by a doctor. RESULTS: In male DM patients, the hemoglobin A1c (HbA1c) level increased significantly as vitamin D levels became severely deficient. In male and postmenopausal female with abnormal HbA1c, those with severe vitamin D deficiency had significantly higher HbA1c levels (p for trend=0.004 and 0.022 for male and postmenopausal female, respectively). Significant differences were found between participants with normal and abnormal HbA1c levels in both male and female. However, regardless of sex or menopausal status, there was no significant association between vitamin D and fasting glucose in any of the fasting glucose subgroups. CONCLUSIONS: Male and female with abnormal HbA1c levels showed markedly elevated blood glucose when they also had vitamin D deficiency. A more distinct difference was observed in the HbA1c subgroups than in the fasting glucose subgroups.

Identification and Functional Characterization of Two Noncoding RNAs Transcribed from Putative Active Enhancers in Hepatocellular Carcinoma.

Enhancers have been conventionally perceived as cis-acting elements that provide binding sites for trans-acting factors. However, recent studies have shown that enhancers are transcribed and that these transcripts, called enhancer RNAs (eRNAs), have a regulatory function. Here, we identified putative eRNAs by profiling and determining the overlap between noncoding RNA expression loci and eRNA-associated histone marks such as H3K27ac and H3K4me1 in hepatocellular carcinoma (HCC) cell lines. Of the 132 HCC-derived noncoding RNAs, 74 overlapped with the eRNA loci defined by the FANTOM consortium, and 65 were located in the proximal regions of genes differentially expressed between normal and tumor tissues in TCGA dataset. Interestingly, knockdown of two selected putative eRNAs, THUMPD3-AS1 and LINC01572, led to downregulation of their target mRNAs and to a reduction in the proliferation and migration of HCC cells. Additionally, the expression of these two noncoding RNAs and target mRNAs was elevated in tumor samples in the TCGA dataset, and high expression was associated with poor survival of patients. Collectively, our study suggests that noncoding RNAs such as THUMPD3-AS1 and LINC01572 (i.e., putative eRNAs) can promote the transcription of genes involved in cell proliferation and differentiation and that the dysregulation of these noncoding RNAs can cause cancers such as HCC.

Nc886, a Novel Suppressor of the Type I Interferon Response Upon Pathogen Intrusion.

Interferons (IFNs) are a crucial component in the innate immune response. Especially the IFN-ß signaling operates in most cell types and plays a key role in the first line of defense upon pathogen intrusion. The induction of IFN-ß should be tightly controlled, because its hyperactivation can lead to tissue damage or autoimmune diseases. Activation of the IFN-ß promoter needs Interferon Regulatory Factor 3 (IRF3), together with Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Activator Protein 1 (AP-1). Here we report that a human noncoding RNA, nc886, is a novel suppressor for the IFN-ß signaling and inflammation. Upon treatment with several pathogen-associated molecular patterns and viruses, nc886 suppresses the activation of IRF3 and also inhibits NF-κB and AP-1 via inhibiting Protein Kinase R (PKR). These events lead to decreased expression of IFN-ß and resultantly IFN-stimulated genes. nc886's role might be to restrict the IFN-ß signaling from hyperactivation. Since nc886 expression is regulated by epigenetic and environmental factors, nc886 might explain why innate immune responses to pathogens are variable depending on biological settings.

Increased Interleukin-11 and Stress-Related Gene Expression in Human Endothelial and Bronchial Epithelial Cells Exposed to Silver Nanoparticles.

This article aimed to identify and distinguish the various responses to silver nanoparticles (NPs) of endothelial and epithelial cells. We also assessed the significantly increased gene expression levels, as shown by microarray analysis. We evaluated the median lethal dose of NPs in each cell line and found that each value was different. We also confirmed the toxicity of 5 nm silver NPs. Meanwhile, cell death was not observed in cells exposed to 100 nm silver NPs at a high concentration. We verified that 5 nm silver NPs affected the variation in gene expression in cells through microarray analysis and observed a noticeable increase in interleukin (IL)-8 and IL-11 gene expression in early stages. This study showed noticeable variation in the expression of oxidative stress-related genes in early stages. Microarray results showed considerable variation in cell death-, apoptosis-, and cell survival-related gene expression. Of note, IL-11 gene expression was particularly increased following the exposure of endothelial and epithelial cells to 5 nm silver NPs. In conclusion, this study demonstrated that intracellular genes specifically responded to silver NPs in respiratory epithelial cells and endothelial cells. Among cytokine genes, IL-11 expression was noticeably increased. Additionally, we confirmed that NP toxicity was affected by NP size and dose.

Topological Adaptation of Transmembrane Domains to the Force-Modulated Lipid Bilayer Is a Basis of Sensing Mechanical Force.

Cells can sense and respond to various mechanical stimuli from their surrounding environment. One of the explanations for mechanosensitivity, a lipid-bilayer model, suggests that a stretch of the membrane induced by mechanical force alters the physical state of the lipid bilayer, driving mechanosensors to assume conformations better matched to the altered membrane. However, mechanosensors of this class are restricted to ion channels. Here, we reveal that integrin αIIbß3, a prototypic adhesion receptor, can be activated by various mechanical stimuli including stretch, shear stress, and osmotic pressure. The force-induced integrin activation was not dependent on its known intracellular activation signaling events and was even observed in reconstituted cell-free liposomes. Instead, these mechanical stimuli were found to alter the lipid embedding of the integrin ß3 transmembrane domain (TMD) and subsequently weaken the αIIb-ß3 TMD interaction, which results in activation of the receptor. Moreover, artificial modulation of the membrane curvature near integrin αIIbß3 can induce its activation in cells as well as in lipid nanodiscs, suggesting that physical deformation of the lipid bilayer, either by mechanical force or curvature, can induce integrin activation. Thus, our results establish the adhesion receptor as a bona fide mechanosensor that directly senses and responds to the force-modulated lipid environment. Furthermore, this study expands the lipid-bilayer model by suggesting that the force-induced topological change of TMDs and subsequent alteration in the TMD interactome is a molecular basis of sensing mechanical force transmitted via the lipid bilayer.

Identification of microRNAs modulated by DNA hypomethylating drugs in extranodal NK/T-cell lymphoma.

To identify epigenetically silenced miRNAs and to investigate their influences on predictive target oncogenes in extranodal natural killer/T-cell lymphoma (NKTCL). Decitabine treatment was performed to evaluate methylated miRNAs in NKTCL cells. The relationship between a given miRNA and its target mRNA was validated using 24 tumor tissues. miR-379, miR-134, miR-20b, miR-376a, miR-654-3p, miR-143, miR-181c, miR-1225-5p, miR-1246, and miR-1275 were epigenetically silenced in SNK6 cells. miR-134, miR-376a, miR-143 and miR-181c significantly affected cellular viability. PDGFRα was regulated by miR-34a and miR-181c. miR-143, miR-20b and miR34a regulated STAT3 expression. miR-20b and miR-143 expression showed inverse correlations with STAT3 mRNA expression in NKTCL tissues. K-RAS was regulated by miR-181c. Downregulation of cell viability by salirasib treatment was identified. miRNAs were downregulated by DNA methylation, and several microRNAs affected the viability of NKTCL cells. miR-34a and miR-181c may be involved in the oncogenic progression of NKTCL through the regulation of PDGFRα, STAT3, and K-RAS.

Programmed cell death ligand-1-mediated enhancement of hexokinase 2 expression is inversely related to T-cell effector gene expression in non-small-cell lung cancer.

BACKGROUND: We investigated the role of PD-L1 in the metabolic reprogramming of non-small cell lung cancer (NSCLC). METHODS: Changes in glycolysis-related molecules and glycolytic activity were evaluated in PD-L1low and PD-L1high NSCLC cells after transfection or knockdown of PD-L1, respectively. Jurkat T-cell activation was assessed after co-culture with NSCLC cells. The association between PD-L1 and immune response-related molecules or glycolysis were analyzed in patients with NSCLC and The Cancer Genome Atlas (TCGA). RESULTS: Transfecting PD-L1 in PD-L1low cells enhanced hexokinase-2 (HK2) expression, lactate production, and extracellular acidification rates, but minimally altered GLUT1 and PKM2 expression and oxygen consumption rates. By contrast, knocking-down PD-L1 in PD-L1high cells decreased HK2 expression and glycolysis by suppressing PI3K/Akt and Erk pathways. Interferon-γ (IFNγ) secretion and activation marker expression was decreased in stimulated Jurkat T-cells when co-cultured with HK2-overexpressing vector-transfected tumor cells rather than empty vector-transfected tumor cells. Immunohistochemistry revealed that PD-L1 expression was positively correlated with HK2 expression in NSCLC (p < 0.001). In TCGA, HK2 exhibited a positive linear association with CD274 (PD-L1) expression (p < 0.001) but an inverse correlation with the expression of CD4, CD8A, and T-cell effector function-related genes in the CD274high rather than CD274low group. Consistently, there were fewer CD8+ T-cells in PD-L1positive/HK2high tumors compared to PD-L1positive/HK2low tumors in squamous cell carcinoma. CONCLUSIONS: PD-L1 enhances glycolysis in NSCLC by upregulating HK2, which might dampen anti-tumor immunity. PD-L1 may contribute to NSCLC oncogenesis by inducing metabolic reprogramming and immune checkpoint.

Mechanism mediated by a noncoding RNA, nc886, in the cytotoxicity of a DNA-reactive compound.

DNA-reactive compounds are harnessed for cancer chemotherapy. Their genotoxic effects are considered to be the main mechanism for the cytotoxicity to date. Because this mechanism preferentially affects actively proliferating cells, it is postulated that the cytotoxicity is specific to cancer cells. Nonetheless, they do harm normal quiescent cells, suggesting that there are other cytotoxic mechanisms to be uncovered. By employing doxorubicin as a representative DNA-reactive compound, we have discovered a cytotoxic mechanism that involves a cellular noncoding RNA (ncRNA) nc886 and protein kinase R (PKR) that is a proapoptotic protein. nc886 is transcribed by RNA polymerase III (Pol III), binds to PKR, and prevents it from aberrant activation in most normal cells. We have shown here that doxorubicin evicts Pol III from DNA and, thereby, shuts down nc886 transcription. Consequently, the instantaneous depletion of nc886 provokes PKR and leads to apoptosis. In a short-pulse treatment of doxorubicin, these events are the main cause of cytotoxicity preceding the DNA damage response in a 3D culture system as well as the monolayer cultures. By identifying nc886 as a molecular signal for PKR to sense doxorubicin, we have provided an explanation for the conundrum why DNA-damaging drugs can be cytotoxic to quiescent cells that have the competent nc886/PKR pathway.

Transdermal Drug Delivery using a Specialized Cavitation Seed for Ultrasound.

GOAL: The Sonophoresis, which utilizes ultrasound for transdermal drug delivery (TDD), can improve the efficiency of drug delivery for a variety of drugs predominantly due to caviation effect. In order to increase the efficacy of sonophoresis, we propose an alternative cavitation seed specialized for sonophoresis, which can be concentrated on the skin surface by gravity adapting perfluorohexane as core. METHODS: An in vitro and in vivo experiments were conducted to assess the effect of the specialized cavitation seed. High performance liquid chromatography was used for in vitro experiments on porcine skin with ferulic acid and an optical imaging system was used for in vivo experiments on rat model with fluorescein isothiocyanate-dextran (FD, 150 kDa), respecitively. RESULTS: The amount of ferulic acid delivered by sonophoresis with the proposed cavitation seed was approximately 1,700 times greater than the amount delivered by diffusion. FD could be delivered to a depth of 500 »m under the skin, and the average total flux in the region of interest was increased 6.4-fold for the group using sonophoresis with the cavitation seed compared to the group using diffusion. CONCLUSION: Conclusively, sonophoresis with the proposed cavitation seed demonstrated significant improvement in TDD and the possibility of macromolecule delivery into the skin. SIGNIFICANCE: This approach has potential to be a main TDD method for variety of applications including medicine and cosmetics.