DNA and histone synthesis are coupled and ongoing replication is required to maintain histone gene expression. Here, we expose S phase-arrested cells to the kinase inhibitors caffeine and LY294002. This uncouples DNA replication from histone messenger RNA (mRNA) abundance, altering the efficiency of replication stress-induced histone mRNA down-regulation. Interference with caffeine-sensitive checkpoint kinases ataxia telangiectasia and Rad3 related (ATR)/ataxia telangiectasia mutated (ATM) does not affect histone mRNA down- regulation, which indicates that ATR/ATM alone cannot account for such coupling. LY294002 potentiates caffeine's ability to uncouple histone mRNA stabilization from replication only in cells containing functional DNA-activated protein kinase (DNA-PK), which indicates that DNA-PK is the target of LY294002. DNA-PK is activated during replication stress and DNA-PK signaling is enhanced when ATR/ATM signaling is abrogated. Histone mRNA decay does not require Chk1/Chk2. Replication stress induces phosphorylation of UPF1 but not hairpin[corrected]-binding protein/stem-loop binding protein at S/TQ sites, which are preferred substrate recognition motifs of phosphatidylinositol 3-kinase-like kinases, which indicates that histone mRNA stability may be directly controlled by ATR/ATM- and DNA-PK-mediated phosphorylation of UPF1.

Tumor cells might resist therapy with ionizing radiation (IR) by non-homologous end-joining (NHEJ) of IR-induced double-strand breaks. One of the key players in NHEJ is DNA-dependent protein kinase (DNA-PK). The catalytic subunit of DNA-PK, i.e. DNA-PKcs, can be inhibited with the small-molecule inhibitor NU7026. In the current study, the in vitro potential of NU7026 to radiosensitize neuroblastoma cells was investigated. DNA-PKcs is encoded by the PRKDC (protein kinase, DNA-activated, catalytic polypeptide) gene. We showed that PRKDC levels were enhanced in neuroblastoma patients and correlated with a more advanced tumor stage and poor prognosis, making DNA-PKcs an interesting target for radiosensitization of neuroblastoma tumors. Optimal dose finding for combination treatment with NU7026 and IR was performed using NGP cells. One hour pre-treatment with 10 μM NU7026 synergistically sensitized NGP cells to 0.63 Gy IR. Radiosensitizing effects of NU7026 increased in time, with maximum effects observed from 96 h after IR-exposure on. Combined treatment of NGP cells with 10 μM NU7026 and 0.63 Gy IR resulted in apoptosis, while no apoptotic response was observed for either of the therapies alone. Inhibition of IR-induced DNA-PK activation by NU7026 confirmed the capability of NGP cells to, at least partially, resist IR by NHEJ. NU7026 also synergistically radiosensitized other neuroblastoma cell lines, while no synergistic effect was observed for low DNA-PKcs-expressing non-cancerous fibroblasts. Results obtained for NU7026 were confirmed by PRKDC knockdown in NGP cells. Taken together, the current study shows that DNA-PKcs is a promising target for neuroblastoma radiosensitization.

Recognition of cytosolic DNA initiates a series of innate immune responses by inducing IFN-I production and subsequent triggering JAK1-STAT1 signaling which plays critical roles in the pathogenesis of infection, inflammation and autoimmune diseases through promoting B cell activation and antibody responses. The stimulator of interferon genes protein (STING) has been demonstrated to be a critical hub of type I IFN induction in cytosolic DNA-sensing pathways. However, it still remains unknown whether cytosolic DNA can directly activate the JAK1-STAT1 signaling or not. And the role of STING is also unclear in this response. In the present study, we found that dsDNA directly triggered the JAK1-STAT1 signaling by inducing phosphorylation of the Lyn kinase. Moreover, this response is not dependent on type I IFN receptors. Interestingly, STING could inhibit dsDNA-triggered activation of JAK1-STAT1 signaling by inducing SHP-1 and SHP-2 phosphorylation. In addition, compared with normal B cells, the expression of STING was significantly lower and the phosphorylation level of JAK1 was significantly higher in B cells from MRL/lpr lupus-prone mice, highlighting the close association between STING low-expression and JAK1-STAT1 signaling activation in B cells in autoimmune diseases. Our data provide a molecular insight into the novel role of STING in dsDNA-mediated inflammatory disorders.

We have established monoclonal antibodies (MoAbs) that are directed against hepatitis C virus (HCV)-expressing cells. They showed enhanced tumorigenicity after passage in culture for more than 44 days (RzM6-44d cells). To address the mechanism underlying this phenomenon, we characterized the MoAbs, and found that one of the clones recognized a molecule that was down-regulated in the RzM6-44d cells. This molecule was purified and identified as the 70-kDa thyroid autoantigen Ku70. Moreover, expression of the full-length HCV genome or HCV-core protein sequence in WRL68 human embryonic liver cells reduced the level of Ku70 protein, enhanced the ubiquitination of Ku70, and decreased the activity of DNA-activated protein kinase (DNA-PK). Therefore, it appears that the HCV-core protein facilitates the degradation of Ku70 and reduces DNA-PK activity in noncancerous liver cells.

Here we tested anti-tumor activity of KU-0060648 in preclinical hepatocellular carcinoma (HCC) models. Our results demonstrated that KU-0060648 was anti-proliferative and pro-apoptotic in established (HepG2, Huh-7 and KYN-2 lines) and primary human HCC cells, but was non-cytotoxic to non-cancerous HL-7702 hepatocytes. DNA-PKcs (DNA-activated protein kinase catalytic subunit) is an important but not exclusive target of KU-0060648. DNA-PKcs knockdown or dominant negative mutation inhibited HCC cell proliferation. On the other hand, overexpression of wild-type DNA-PKcs enhanced HepG2 cell proliferation. Importantly, KU-0060648 was still cytotoxic to DNA-PKcs-silenced or -mutated HepG2 cells, although its activity in these cells was relatively weak. Further studies showed that KU-0060648 inhibited PI3K-AKT-mTOR activation, independent of DNA-PKcs. Introduction of constitutively-active AKT1 (CA-AKT1) restored AKT-mTOR activation after KU-0060648 treatment in HepG2 cells, and alleviated subsequent cytotoxicity. In vivo, intraperitoneal (i.p.) injection of KU-0060648 significantly inhibited HepG2 xenograft growth in nude mice. AKT-mTOR activation was also inhibited in xenografted tumors. Finally, we showed that DNA-PKcs expression was significantly upregulated in human HCC tissues. Yet miRNA-101, an anti-DNA-PKcs miRNA, was downregulated. Over-expression of miR-101 in HepG2 cells inhibited DNA-PKcs expression and cell proliferation. Together, these results indicate that KU-0060648 inhibits HCC cells through DNA-PKcs-dependent and -independent mechanisms.

Chronic low levels of survival motor neuron (SMN) protein cause spinal muscular atrophy (SMA). SMN is ubiquitously expressed, but the mechanisms underlying predominant neuron degeneration in SMA are poorly understood. We report that chronic low levels of SMN cause Senataxin (SETX)-deficiency, which results in increased RNA-DNA hybrids (R-loops) and DNA double-strand breaks (DSBs), and deficiency of DNA-activated protein kinase-catalytic subunit (DNA-PKcs), which impairs DSB repair. Consequently, DNA damage accumulates in patient cells, SMA mice neurons and patient spinal cord tissues. In dividing cells, DSBs are repaired by homologous recombination (HR) and non-homologous end joining (NHEJ) pathways, but neurons predominantly use NHEJ, which relies on DNA-PKcs activity. In SMA dividing cells, HR repairs DSBs and supports cellular proliferation. In SMA neurons, DNA-PKcs-deficiency causes defects in NHEJ-mediated repair leading to DNA damage accumulation and neurodegeneration. Restoration of SMN levels rescues SETX and DNA-PKcs deficiencies and DSB accumulation in SMA neurons and patient cells. Moreover, SETX overexpression in SMA neurons reduces R-loops and DNA damage, and rescues neurodegeneration. Our findings identify combined deficiency of SETX and DNA-PKcs stemming downstream of SMN as an underlying cause of DSBs accumulation, genomic instability and neurodegeneration in SMA and suggest SETX as a potential therapeutic target for SMA.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a key member of the phosphatidylinositol-3 kinase-like (PIKK) family of protein kinases with critical roles in DNA-double strand break repair, transcription, metastasis, mitosis, RNA processing, and innate and adaptive immunity. The absence of DNA-PKcs from many model organisms has led to the assumption that DNA-PKcs is a vertebrate-specific PIKK. Here, we find that DNA-PKcs is widely distributed in invertebrates, fungi, plants, and protists, and that threonines 2609, 2638, and 2647 of the ABCDE cluster of phosphorylation sites are highly conserved amongst most Eukaryotes. Furthermore, we identify highly conserved amino acid sequence motifs and domains that are characteristic of DNA-PKcs relative to other PIKKs. These include residues in the Forehead domain and a novel motif we have termed YRPD, located in an α helix C-terminal to the ABCDE phosphorylation site loop. Combining sequence with biochemistry plus structural data on human DNA-PKcs unveils conserved sequence and conformational features with functional insights and implications. The defined generally progressive DNA-PKcs sequence diversification uncovers conserved functionality supported by Evolutionary Trace analysis, suggesting that for many organisms both functional sites and evolutionary pressures remain identical due to fundamental cell biology. The mining of cancer genomic data and germline mutations causing human inherited disease reveal that robust DNA-PKcs activity in tumors is detrimental to patient survival, whereas germline mutations compromising function are linked to severe immunodeficiency and neuronal degeneration. We anticipate that these collective results will enable ongoing DNA-PKcs functional analyses with biological and medical implications.

In addition to silencing specific genes, small interfering RNA (siRNA) transfection is also associated with the non-specific induction of inflammatory cytokines and type I interferon. Those so-called "off-target" effects have considerable implications for the interpretation of in vitro studies and clinical application of siRNA. The present study attempted to develop a better understanding of the mechanism involved in these off target effects. Synthesized siRNA significantly enhances DNA-mediated interferon lambda-1 response (IFN-λ1/IL-29), a newly characterized antiviral interferon in non-immune or primary immune cells. This enhancement was most pronounced by double-stranded siRNA with at least a 2-nucleotide overhang at one 3' terminus in a dose-dependent manner, while the presence of DNA was indispensable. A pull-down assay using biotinylated siRNA- or DNA-conjugated beads indicated that retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) were involved in the sensing of siRNA and DNA, respectively. Co-immunoprecipitation analysis further revealed that RIG-I and IFI16 formed a complex via siRNA, and the dissociation of IFI16 from this complex in the presence of DNA activated the downstream STING-TBK1-IRF3 (stimulator of interferon genes - tank-binding kinase 1 - interferon regulatory factor 3) pathway, shedding light on a new physiological signalling pathway to activate innate immunity. Collectively, these findings may provide rational information for siRNA-induced innate immunity, with important implications for developing siRNA-based reagents to control human diseases.

Colorectal cancer is the third most common type of cancer and the fourth leading cause of cancer-associated mortality worldwide. Serine/arginine-rich splicing factor 1 (SRSF1) is a well-characterized oncogenic factor that promotes tumorigenesis by controlling a number of alternative splicing events. However, there is limited network analysis, from a global aspect, to study the effect of SRSF1 on colorectal cancer. In the present study, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of available gene regulation data from The Cancer Genome Atlas database revealed the enriched functions and signaling pathways of SRSF1. Subsequently, Oncomine analysis was performed, which demonstrated that SRSF1 was upregulated in a number of types of colon cancer. From overlapping the analysis of 2,678 SRSF1-related genes and 3,625 colorectal cancer genes in GeneCards, 468 genes were identified as SRSF1-related colorectal cancer genes. The GO results revealed that these overlapped genes were primarily enriched in metabolic processes, response to DNA damage, regulation of the cell cycle and a number of additional biological processes. KEGG pathway analysis revealed that SRSF1-related colorectal cancer genes were associated with the cell cycle, deregulated signaling pathways associated with cancer progression and colorectal cancer signaling pathways. In addition, the Search Tool for the Retrieval of Interacting Genes/Proteins database and Cytoscape analysis demonstrated that 468 SRSF1-related colorectal cancer genes exhibit potential interaction networks in which these genes were enriched in DNA metabolic processes, cell cycle regulation and regulation of apoptosis. The results of the present study suggested that SRSF1 exhibited an increased degree of interaction with key molecules, including NUF2 NDC80 kinetochore complex component, kinesin family member 2C, structural maintenance of chromosomes 3, ATM serine/threonine kinase, BRCA1 DNA repair associated, protein kinase DNA-activated catalytic polypeptide, heat shock protein 90 alpha family class A member 1, ras homolog family member A, and phosphatase and tensin homolog. Collectively, the bioinformatics analysis of the present study indicated that SRSF1 may have key functions in the progression and development of colorectal cancer.

Chronic fetal hypoxia is a common complication observed in human pregnancy, impacting pregnancies across global contexts. Exposure to chronic intrauterine hypoxia has major short- and long-term consequences for offspring health. However, the impact of chronic gestational hypoxia on female reproductive system development is unknown. We aimed to understand the impact of exposure to chronic fetal hypoxia on the developing female reproductive system. Wistar rat dams underwent normoxia (21%) or hypoxia (13%) during pregnancy. Postnatally, all female offspring were maintained in normoxic conditions into early adulthood. Female rats exposed to chronic gestational hypoxia (13%) during their intrauterine development had decreased ovarian primordial follicular reserve compared to controls (P < 0.05). Adult females who had been exposed to chronic fetal hypoxia had significantly reduced somatic ovarian telomere length (P < 0.05) and reduced ovarian protein expression of KU70, a critical component of the DNA-activated protein kinase repair complex (P < 0.01). Gene expression of NADPH oxidase 2-mediated oxidative stress markers was increased (P < 0.05). Exposure to chronic hypoxia during fetal development leads to accelerated aging of the somatic ovary and decreased ovarian reserve in adulthood. Ovarian aging is highly sensitive to gestational hypoxia, with implications for future fertility in next-generation offspring of high-risk pregnancies.-Aiken, C. E., Tarry-Adkins, J. L., Spiroski, A.-M., Nuzzo, A. M., Ashmore, T. J., Rolfo, A., Sutherland, M. J., Camm, E. J., Giussani, D. A., Ozanne, S. E. Chronic gestational hypoxia accelerates ovarian aging and lowers ovarian reserve in next-generation adult rats.

Hepatitis B virus infection remains a major health problem worldwide due to its high risk of liver failure and hepatocellular carcinoma. Covalently closed circular DNA (cccDNA), which is present as an individual minichromosome, serves as the template for transcription of all viral RNAs and pla ays critical role in viral persistence. Therefore, there is an urgent need to gain broader insight into the transcription regulation of cccDNA. Here, we combined a modified Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with an engineered ascorbate peroxidase 2 (APEX2) to identify cccDNA associated proteins systematically in living cells. By functional screening, we verified that protein kinase, DNA-activated, catalytic subunit (PRKDC) was an effective activator of HBV cccDNA transcription in HBV-infected HepG2-NTCP cells and primary human hepatocytes. Mechanismly, PRKDC interacted with POLR2A and POLR2B, the two largest subunits of RNA polymerase II (Pol II) and recruited Pol II to HBV cccDNA minichromosome in a kinase-dependent manner. PRKDC knockdown or inhibitor treatment significantly decreased the enrichment of POLR2A and POLR2B on cccDNA, as well as reducing the levels of cccDNA associated Pol II Ser5 and Ser2 phosphorylation, which eventually inhibited the HBV cccDNA activity. Collectively, these findings give us new insights into cccDNA transcription regulation, thus providing new potential targets for HBV treatment in patients.

Prostate cancer is still one of the most common malignancies in men all around the world. The mechanism of how prostate cancer initiates and develops is still not clear. Here in this study, we show that tumor suppressor ZBTB38 could suppress the migration and proliferation of prostate cancer cells. We find lower ZBTB38 expression in prostate cancer tissues, which also strongly predicts a poorer prognosis of prostate cancer. ZBTB38 binds DKK1 (Dickkopf WNT signaling pathway inhibitor 1) locus and promotes DKK1 expression in prostate cancer cell lines. Consistently, reduction of DKK1 expression significantly restores ZBTB38-mediated suppression of migration and proliferation of prostate cancer cell lines. Mechanistically, we find that ZBTB38 primarily binds the promoters of target genes, and differentially regulates the expression of 1818 genes. We also identify PRKDC (protein kinase, DNA-activated, catalytic subunit) as a ZBTB38-interacting protein that could repress the function of ZBTB38 in suppressing migration and proliferation of prostate cancer cells. Taken together, our results indicate that ZBTB38 could repress cell migration and proliferation in prostate cancer via promoting DKK1 expression, and also provide evidence supporting ZBTB38 as a potential prognosis marker for prostate cancer.

S-nitrosylation of proteins by nitric oxide is a major mode of signalling in cells. S-nitrosylation can mediate the regulation of a range of proteins, including prominent nuclear proteins, such as HDAC2 (ref. 2) and PARP1 (ref. 3). The high reactivity of the nitric oxide group with protein thiols, but the selective nature of nitrosylation within the cell, implies the existence of targeting mechanisms. Specificity of nitric oxide signalling is often achieved by the binding of nitric oxide synthase (NOS) to target proteins, either directly or through scaffolding proteins such as PSD-95 (ref. 5) and CAPON. As the three principal isoforms of NOS--neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS)--are primarily non-nuclear, the mechanisms by which nuclear proteins are selectively nitrosylated have been elusive. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is physiologically nitrosylated at its Cys 150 residue. Nitrosylated GAPDH (SNO-GAPDH) binds to Siah1, which possesses a nuclear localization signal, and is transported to the nucleus. Here, we show that SNO-GAPDH physiologically transnitrosylates nuclear proteins, including the deacetylating enzyme sirtuin-1 (SIRT1), histone deacetylase-2 (HDAC2) and DNA-activated protein kinase (DNA-PK). Our findings reveal a novel mechanism for targeted nitrosylation of nuclear proteins and suggest that protein-protein transfer of nitric oxide groups may be a general mechanism in cellular signal transduction.

MicroRNAs (miRNAs) have been suggested to repress transcription via binding the 3'-untranslated regions of mRNAs. However, the involvement and details of miRNA-mediated epigenetic regulation, particularly in targeting genomic DNA and mediating epigenetic regulation, remain largely uninvestigated. In the present study, transcription factor CCAAT/enhancer binding protein delta (CEBPD) was responsive to the anticancer drug bortezomib, a clinical and highly selective drug for leukemia treatment, and contributed to bortezomib-induced cell death. Interestingly, following the identification of CEBPD-induced miRNAs, we found that miR-744, miR-3154 and miR-3162 could target CpG islands in the 5'-flanking region of the CEBPD gene. We previously demonstrated that the Yin Yang 1 (YY1)/polycomb group (PcG) protein/DNA methyltransferase (DNMT) complex is important for CCAAT/enhancer binding protein delta (CEBPD) gene inactivation; we further found that Argonaute 2 (Ago2) interacts with YY1 and binds to the CEBPD promoter. The miRNA/Ago2/YY1/PcG group protein/DNMT complex linked the inactivation of CEBPD and genes adjacent to its 5'-flanking region, including protein kinase DNA-activated catalytic polypeptide (PRKDC), minichromosome maintenance-deficient 4 (MCM4) and ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), upon bortezomib treatment. Moreover, we revealed that miRNA binding is necessary for YY1/PcG group protein/DNMT complex-mediated epigenetic gene silencing and is associated with bortezomib-induced methylation on genomic DNA. The present study successfully characterized the interactions of the miRNA/Ago2/YY1/PcG group protein/DNMT complex and provided new insights for miRNA-mediated epigenetic regulation in bortezomib-induced leukemic cell arrest and cell death.

Little is known about the function of Keratin 80 (KRT80), an epithelial keratin, in cancer. This study investigated the role of KRT80 in the prognosis of colorectal carcinoma (CRC) and the underlying mechanisms involved in CRC migration and invasion. We analyzed the expression of KRT80 using The Cancer Genome Atlas and Oncomine databases. Higher expression of KRT80 was found to be significantly associated with multiple pathological parameters, lower disease-free survival, and overall survival in CRC patients. Also, KRT80 was an independent prognostic indicator for CRC. Furthermore, altered KRT80 expression impacted migration and invasion of CRC cells, as well as the expression of epithelial-mesenchymal transition (EMT)-related markers and cell morphology via the AKT pathway. Inhibiting the expression of AKT could reverse these phenomena. Liquid Chromatograph Mass Spectrometer/Mass Spectromete, Co-immunoprecipitation, and laser scanning confocal microscopy techniques showed that KRT80 could interact with protein kinase, DNA-activated, catalytic polypeptide (PRKDC). Suppressing PRKDC could inhibit the expression of AKT and EMT, as well as the migration and invasion of CRC cells. Taken together, these results demonstrated that KRT80 was an independent prognostic biomarker for CRC and promoted CRC migration and invasion by interacting with PRKDC via activation of the AKT pathway.

Predictive biomarkers of response to immune checkpoint inhibitors (ICI) help to identify cancer patients who will benefit from immunotherapy. Protein kinase, DNA-activated, catalytic subunit (PRKDC) is an important gene for DNA double-strand break (DSB) repair and central T-cell tolerance. We aimed to investigate the association between PRKDC mutations and tumor mutation burden (TMB), tumor microenvironment (TME), and response to ICI. Whole-exome sequencing data of 4023 solid tumor samples from the Cancer Genome Atlas (TCGA) and panel-based sequencing data of 3877 solid tumor samples from Geneplus-Beijing, China, were used to analyze the TMB. The mRNA expression data of 3541 solid tumor samples from TCGA were used to explore the effect of PRKDC mutations on the TME. Four ICI-treated cohorts were analyzed for verifying the correlation between PRKDC mutations and the response to ICI. In both the TCGA and Geneplus datasets, we found that the TMB in PRKDC mutation samples was significantly higher than in PRKDC wild-type samples (P < 0.05 and P < 0.0001, respectively). Further, TCGA datasets showed that PRKDC mutation samples were associated with a significantly increased expression of CD8+ T cells, NK cells, immune checkpoint, chemokines, etc. compared to PRKDC wild-type samples (P < 0.05). In ICI-treated cohorts, we also found the PRKDC mutations were associated with increased survival (median PFS, not reached vs. 6.8 months, HR, 0.2893; 95% CI, 0.1255-0.6672; P = 0.0650, Hellmann cohort; median OS, 1184 days vs. 250 days, HR, 0.5126; 95% CI, 0.2715-0.9679; P = 0.1020, Allen cohort), and the increase was significant in multivariate analysis (HR, 0.361; 95% CI, 0.155-0.841; P = 0.018, Allen cohort; HR, 0.240 95% CI, 0.058-0.998; P = 0.050, Hellmann cohort). In summary, we found that PRKDC mutation often appeared to co-exist with deficiency in some other DNA damage repair mechanism and is nonetheless one of the important factors associated with increased TMB, inflamed TME, and better response to ICI.

Prostaglandin (PG) A2, one of cyclopentenone PGs, is known to induce activation of apoptosis in various cancer cells. Although PGA2 has been reported to cause activation of apoptosis by altering the expression of apoptosis-related genes, the role of p53, one of the most critical pro-apoptotic genes, on PGA2-induced apoptosis has not been clarified yet. To address this issue, we compared the apoptosis in HCT116 p53 null cells (HCT116 p53-/-) to that in HCT116 cells containing the wild type p53 gene. Cell death induced by PGA2 was associated with phosphorylation of histone H2A variant H2AX (H2AX), activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase 1 in HCT116 cells. Induction of apoptosis in PGA2-treated cells was almost completely prevented by pretreatment with a pan-caspase inhibitor, z-VAD-Fmk, or an inhibitor of protein synthesis, cycloheximide. While PGA2 induced apoptosis in HCT116 cells, phosphorylation of p53 and transcriptional induction of p53-target genes such as p21WAF1, PUMA, BAX, NOXA, and DR5 occurred. Besides, pretreatment of pifithrin-α (PFT-α), a chemical inhibitor of p53's transcriptional activity, interfered with the induction of apoptosis in PGA2-treated HCT116 cells. Pretreatment of NU7441, a small molecule inhibitor of DNA-activated protein kinase (DNA-PK) suppressed PGA2-induced phosphorylation of p53 and apoptosis as well. Moreover, among target genes of p53, knockdown of DR5 expression by RNA interference, suppressed PGA2-induced apoptosis. In the meanwhile, in HCT116 p53-/- cells, PGA2 induced apoptosis in delayed time points and with less potency. Delayed apoptosis by PGA2 in HCT116 p53-/- cells was also associated with phosphorylation of H2AX but was not inhibited by either PFT- or NU7441. Collectively, these results suggest the following. PGA2 may induce p53-dependent apoptosis in which DNA-PK activates p53, and DR5, a transcriptional target of p53, plays a pivotal role in HCT116 cells. In contrast to apoptosis in HCT116 cells, PGA2 may induce apoptosis in a fashion of less potency, which is independent of p53 and DNA-PK in HCT116 p53-/- cells.

The human skin is a significant barrier for protection against pathogen transmission. Rodent models used to investigate human-specific pathogens that target the skin are generated by introducing human skin grafts to immunocompromised rodent strains. Infection-induced immunopathogenesis has been separately studied in humanized rodent models developed with human lymphoid tissue and hematopoietic stem cell transplants. Successful co-engraftment of human skin, autologous lymphoid tissues, and autologous immune cells in a rodent model has not yet been achieved, though it could provide a means of studying the human immune response to infection in the human skin. Here, we introduce the human Skin and Immune System (hSIS)-humanized NOD-scid IL2Rγnull (NSG) mouse and Sprague-Dawley-Rag2tm2hera Il2rγtm1hera (SRG) rat models, co-engrafted with human full-thickness fetal skin, autologous fetal lymphoid tissues, and autologous fetal liver-derived hematopoietic stem cells. hSIS-humanized rodents demonstrate the development of human full-thickness skin, along with autologous lymphoid tissues, and autologous immune cells. These models also support human skin infection following intradermal inoculation with community-associated methicillin-resistant Staphylococcus aureus. The co-engraftment of these human skin and immune system components into a single humanized rodent model could provide a platform for studying human skin infections.

Bladder cancer (BLCA) is the fourth common cancer among males in the United States, which is also the fourth leading cause of cancer-related death in old males. BLCA has a high recurrence rate, with over 50% of patients which has at least one recurrence within five years. Due to the complexity of the molecular mechanisms and heterogeneous cancer feature, BLCA clinicians find it hard to make an efficient management decision as they lack reliable assessment of mortality risk. Meanwhile, there is currently no screening suitable prognostic signature or method recommended for early detection, which is significantly important to early-stage detection and prognosis. In this study, a novel model, named the risk-weighted sparse regression (RWSR) model, is constructed to identify a robust signature for patients of early-stage BLCA. The 17-gene signature is generated and then validated as an independent prognostic factor in BLCA cohorts from GSE13507 and TCGA_BLCA datasets. Meanwhile, a risk score model is developed and validated among the 17-gene signature. The risk score is also considered an independent factor for prognosis prediction, which is confirmed through prognosis analysis. The Kaplan-Meier with the log-rank test is used to assess survival difference. Furthermore, the predictive capacity of the signature is proved through stratification analysis. Finally, an effective patient classification is completed by a combination of the 17-gene signature and stage information, which is for better survival prediction and treatment decisions. Besides, 11 genes in the signature, such as coiled-coil domain containing 73 (CCDC73) and protein kinase, DNA-activated, and catalytic subunit (PRKDC), are proved to be prognosis marker genes or strongly associated with prognosis and progress of other types of cancer in published literature already. As a result, this paper would more accurately predict a patient's prognosis and improve surveillance in the clinical setting, which may provide a quantitative and reliable decision-making basis for the treatment plan.

The CRISPR/Cas systems in prokaryotes such as bacteria and archaea are the adaptive immune system to prevent infection from viruses, phages, or other foreign substances. When viruses or phages first invade the bacteria, Cas proteins recognize and cut the DNA from viruses or phages into short fragments that will be integrated into the CRISPR array. Once bacteria are invaded again, the modified CRISPR and Cas proteins react quickly to cut DNA at the specified target location, protecting the host. Due to its high efficiency, versatility, and simplicity, the CRISPR/Cas system has become one of the most popular gene editing technologies. In this review, we briefly introduce the CRISPR/Cas systems, focus on several delivery methods including physical delivery, viral vector delivery, and non-viral vector delivery, and the applications of disease therapy. Finally, some problems in CRISPR/Cas9 technology have been proposed, such as the off-target effects, the efficiency of DNA repair mechanisms, and delivery of CRISPR/Cas system safely and efficiently to the target location.