The impact of XPC gene single nucleotide polymorphism rs2228001 on head and neck cancer patients' response to radiotherapy treatment.

Background: Head and neck squamous carcinoma (HNSC) is the sixth most common neoplasm, with a 40-50% overall survival rate. HNSC standard treatment depends on tumor size, metastasis or human papillomavirus (HPV) status including surgery, chemotherapy, and radiotherapy. The last two may lead to defects in the tumor microenvironment and cancer cell biology as disorders in DNA damage repair systems. Here, we evaluate the correlation between single nucleotide polymorphism (SNP) rs2228001 in the XPC gene with the early and late adverse effects of radiotherapy, determine the distribution of the SNP and post-treatment follow-up in HNSC patients. Materials and methods: Head and neck cancer tissues and clinical data were obtained from 79 patients. The SNP of the XPC gene (rs2228001) was evaluated with polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP). The chi-square test was used to determine the correlation between mutation and adverse effects occurrence. Results/Conclusion: Single nucleotide polymorphism rs2228001 in the XPC gene is correlated with the early adverse effect of skin reaction and the late adverse effect of elevated C-reactive protein (CRP) levels in the HNSC patients.

ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs.

In addition to the canonical ISGF3 and non-canonical STAT2/IRF9 complexes, evidence is emerging of the role of their unphosphorylated counterparts in IFN-dependent and -independent ISG transcription. To better understand the relation between ISGF3 and U-ISGF3 and STAT2/IRF9 and U-STAT2/IRF9 in IFN-I-stimulated transcriptional responses, we performed RNA-Seq and ChIP-Seq, in combination with phosphorylation inhibition and antiviral experiments. First, we identified a group of ISRE-containing ISGs that were commonly regulated in IFNα-treated WT and STAT1-KO cells. Thus, in 2fTGH and Huh7.5 WT cells, early and long-term IFNα-inducible transcription and antiviral activity relied on the DNA recruitment of the ISGF3 components STAT1, STAT2 and IRF9 in a phosphorylation- and time-dependent manner. Likewise, in ST2-U3C and Huh-STAT1KO cells lacking STAT1, delayed IFN responses correlated with DNA binding of phosphorylated STAT2/IRF9 but not U-STAT2/IRF9. In addition, comparative experiments in U3C (STAT1-KO) cells overexpressing all the ISGF3 components (ST1-ST2-IRF9-U3C) revealed U-ISGF3 (and possibly U-STAT2/IRF9) chromatin interactions to correlate with phosphorylation-independent ISG transcription and antiviral activity. Together, our data point to the dominant role of the canonical ISGF3 and non-canonical STAT2/IRF9, without a shift to U-ISGF3 or U-STAT2/IRF9, in the regulation of early and prolonged ISG expression and viral protection. At the same time, they suggest the threshold-dependent role of U-ISFG3, and potentially U-STAT2/IRF9, in the regulation of constitutive and possibly long-term IFNα-dependent responses.

STAT2/IRF9 directs a prolonged ISGF3-like transcriptional response and antiviral activity in the absence of STAT1.

Evidence is accumulating for the existence of a signal transducer and activator of transcription 2 (STAT2)/interferon regulatory factor 9 (IRF9)-dependent, STAT1-independent interferon alpha (IFNα) signalling pathway. However, no detailed insight exists into the genome-wide transcriptional regulation and the biological implications of STAT2/IRF9-dependent IFNα signalling as compared with interferon-stimulated gene factor 3 (ISGF3). In STAT1-defeicient U3C cells stably overexpressing human STAT2 (hST2-U3C) and STAT1-deficient murine embryonic fibroblast cells stably overexpressing mouse STAT2 (mST2-MS1KO) we observed that the IFNα-induced expression of 2'-5'-oligoadenylate synthase 2 (OAS2) and interferon-induced protein with tetratricopeptide repeats 1 (Ifit1) correlated with the kinetics of STAT2 phosphorylation, and the presence of a STAT2/IRF9 complex requiring STAT2 phosphorylation and the STAT2 transactivation domain. Subsequent microarray analysis of IFNα-treated wild-type (WT) and STAT1 KO cells overexpressing STAT2 extended our observations and identified ∼120 known antiviral ISRE-containing interferon-stimulated genes (ISGs) commonly up-regulated by STAT2/IRF9 and ISGF3. The STAT2/IRF9-directed expression profile of these IFN-stimulated genes (ISGs) was prolonged as compared with the early and transient response mediated by ISGF3. In addition, we identified a group of 'STAT2/IRF9-specific' ISGs, whose response to IFNα was ISGF3-independent. Finally, STAT2/IRF9 was able to trigger an antiviral response upon encephalomyocarditis virus (EMCV) and vesicular stomatitis Indiana virus (VSV). Our results further prove that IFNα-activated STAT2/IRF9 induces a prolonged ISGF3-like transcriptome and generates an antiviral response in the absence of STAT1. Moreover, the existence of 'STAT2/IRF9-specific' target genes predicts a novel role of STAT2 in IFNα signalling.

Erratum: Structural, topological, and functional characterization of transmembrane proteins TMEM213, 207, 116, 72 and 30B provides a potential link to ccRCC etiology.

[This corrects the article on p. 1863 in vol. 13, PMID: 37293153.].

The modulatory effect of melatonin on genotoxicity of irinotecan in healthy human lymphocytes and cancer cells.

Drug-target interactions can be modified by adding modulatory agents to increase treatment efficacy and clinical outcome. Combination chemotherapy has become increasingly important because drugs acting synergistically can achieve therapeutic effects at substantially lower doses and with a limited spectrum of side effects. Irinotecan, known as one of the camptothecin analogs, has shown a broad spectrum of antitumor activity against various malignancies. In this study, we evaluated the effect of melatonin on the genotoxic activity of irinotecan in healthy human lymphocytes and a lung cancer cell line (A549) and a colorectal adenocarcinoma cell line (HT29) in vitro. Irinotecan, as a single agent, was shown to induce DNA damage in all types of analyzed cells. The combination of melatonin at concentrations of 50 µM with increasing doses of irinotecan (7.5, 15, 30, and 60 µM) resulted in an increase in the amount of DNA damage in A549 and HT29 cancer cells, but was not effective in inducing DNA damage in healthy human lymphocytes. Analysis of the efficacy of DNA repair, performed after 60 and 120 minutes of postincubation, showed the gradual decrease of DNA percentage in comet tails during repair postincubation in all experimental samples. Our results indicate that melatonin can modulate the genotoxic activity of irinotecan and DNA repair efficacy in human cancer cells in vitro. These findings may be supportive for the optimization of therapeutic efficacy in irinotecan treatment.

Structural, topological, and functional characterization of transmembrane proteins TMEM213, 207, 116, 72 and 30B provides a potential link to ccRCC etiology.

Due to their involvement in the development of various cancers Transmembrane Proteins (TMEMs) are the focus of many recent studies. Previously we reported TMEM de-regulation in clear cell Renal Cell Carcinoma (ccRCC) with TMEM213, 207, 116, 72 and 30B being among the most downregulated on mRNA level. TMEM down-regulation was also more pronounced in advanced ccRCC tumors and was potentially linked to clinical parameters such as: metastasis (TMEM72 and 116), Fuhrman grade (TMEM30B) and overall survival (TMEM30B). To further investigate these findings, first, we set off to prove experimentally that selected TMEMs are indeed membrane-bound as predicted in silico, we verified the presence of signaling peptides on their N-termini, orientation of TMEMs within the membrane and validated their predicted cellular localization. To investigate the potential role of selected TMEMs in cellular processes overexpression studies in HEK293 and HK-2 cell lines were carried out. Additionally, we tested TMEM isoform expression in ccRCC tumors, identified mutations in TMEM genes and examined chromosomal aberrations in their loci. We confirmed the membrane-bound status of all selected TMEMs, assigned TMEM213, and 207 to early endosomes, TMEM72 to early endosomes and plasma membrane, TMEM116 and 30B to the endoplasmic reticulum. The N-terminus of TMEM213 was found to be exposed to the cytoplasm, the C-terminus of TMEM207, 116 and 72 were directed toward the cytoplasm, and both termini of TMEM30B faced the cytoplasm. Interestingly, TMEM mutations and chromosomal aberrations were infrequent in ccRCC tumors, yet we identified potentially damaging mutations in TMEM213 and TMEM30B and found deletions in the TMEM30B locus in nearly 30% of the tumors. Overexpression studies suggested selected TMEMs may take part in carcinogenesis processes such as cell adhesion, regulation of epithelial cell proliferation, and regulation of adaptive immune response, which could indicate a link to the development and progression of ccRCC.

A novel type of biochar from chitinous Hermetia illucens waste with a built-in stimulating effect on plants and soil arthropods.

The breeding of insects generates waste in the form of insect excrement and feed residues. In addition, a specific chitinous waste in the form of insect larvae and pupae exuvia is also left. Recent research tries to manage it, e.g., by producing chitin and chitosan, which are value-added products. The circular economy approach requires testing new, non-standard management methods that can develop products with unique properties. To date, the possibility of biochar production from chitinous waste derived from insects has not been evaluated. Here we show that the puparia of Hermetia illucens are suitable for biochar production, which in turn exhibits original characteristics. We found that the biochars have a high nitrogen level, which is rarely achievable in materials of natural origin without artificial doping. This study presents a detailed chemical and physical characterization of the biochars. Moreover, ecotoxicological analysis has revealed the biochars' stimulation effect on plant root growth and the reproduction of the soil invertebrate Folsomia candida, as well as the lack of a toxic effect on its mortality. This predisposes these novel materials with already built-in stimulating properties to be used in agronomy, for example as a carriers for fertilizers or beneficial bacteria.

Dysregulated Interferon Response and Immune Hyperactivation in Severe COVID-19: Targeting STATs as a Novel Therapeutic Strategy.

A disease outbreak in December 2019, caused by a novel coronavirus SARS-CoV-2, was named COVID-19. SARS-CoV-2 infects cells from the upper and lower respiratory tract system and is transmitted by inhalation or contact with infected droplets. Common clinical symptoms include fatigue, fever, and cough, but also shortness of breath and lung abnormalities. Still, some 5% of SARS-CoV-2 infections progress to severe pneumonia and acute respiratory distress syndrome (ARDS), with pulmonary edema, acute kidney injury, and/or multiple organ failure as important consequences, which can lead to death. The innate immune system recognizes viral RNAs and triggers the expression of interferons (IFN). IFNs activate anti-viral effectors and components of the adaptive immune system by activating members of the STAT and IRF families that induce the expression of IFN-stimulated genes (ISG)s. Among other coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV, common strategies have been identified to antagonize IFN signaling. This typically coincides with hyperactive inflammatory host responses known as the "cytokine storm" that mediate severe lung damage. Likewise, SARS-CoV-2 infection combines a dysregulated IFN response with excessive production of inflammatory cytokines in the lungs. This excessive inflammatory response in the lungs is associated with the local recruitment of immune cells that create a pathogenic inflammatory loop. Together, it causes severe lung pathology, including ARDS, as well as damage to other vulnerable organs, like the heart, spleen, lymph nodes, and kidney, as well as the brain. This can rapidly progress to multiple organ exhaustion and correlates with a poor prognosis in COVID-19 patients. In this review, we focus on the crucial role of different types of IFN that underlies the progression of SARS-CoV-2 infection and leads to immune cell hyper-activation in the lungs, exuberant systemic inflammation, and multiple organ damage. Consequently, to protect from systemic inflammation, it will be critical to interfere with signaling cascades activated by IFNs and other inflammatory cytokines. Targeting members of the STAT family could therefore be proposed as a novel therapeutic strategy in patients with severe COVID-19.

The unique role of STAT2 in constitutive and IFN-induced transcription and antiviral responses.

In the canonical pathway of IFN-I-mediated signaling, phosphorylation of STAT1 and STAT2 leads to heterodimerization and interaction with IRF9. This complex, also known as IFN-stimulated gene factor 3 (ISGF3), then translocates into the nucleus and binds the IFN-I-stimulated response element (ISRE) leading to the activation of transcription of over 300 interferon stimulated genes (ISGs). In addition, STAT1 homodimers [known as γ-activated factor (GAF)] are formed and translocate to the nucleus, where they target genes containing the γ-activated sequence (GAS). The primary function of ISGF3 is to mediate a rapid and robust IFN-I activated response by regulating transient transcription of antiviral ISGs. This requires the quick assembly of ISGF3 from its pre-existing components STAT1, STAT2 and IRF9 and transport to the nucleus to bind ISRE-containing ISGs. The exact events that take place in formation, nuclear translocation and DNA-binding of active ISGF3 are still not clear. Over the years many studies have provided evidence for the existence of a multitude of alternative STAT2-containing (ISRE or GAS-binding) complexes involved in IFN-I signaling, emphasizing the importance of STAT2 in the regulation of specific IFN-I-induced transcriptional programs, independent of its involvement in the classical ISGF3 complex. This review describes the unique role of STAT2 in differential complex formation of unphosphorylated and phosphorylated ISGF3 components that direct constitutive and IFN-I-stimulated transcriptional responses. In addition, we highlight the existence of a STAT1-independent IFN-I signaling pathway, where STAT2/IRF9 can potentially substitute for the role of ISGF3 and offer a back-up response against viral infection.

STAT1-dependent signal integration between IFNγ and TLR4 in vascular cells reflect pro-atherogenic responses in human atherosclerosis.

Signal integration between IFNγ and TLRs in immune cells has been associated with the host defense against pathogens and injury, with a predominant role of STAT1. We hypothesize that STAT1-dependent transcriptional changes in vascular cells involved in cross-talk between IFNγ and TLR4, reflect pro-atherogenic responses in human atherosclerosis. Genome-wide investigation identified a set of STAT1-dependent genes that were synergistically affected by interactions between IFNγ and TLR4 in VSMCs. These included the chemokines Cxcl9, Ccl12, Ccl8, Ccrl2, Cxcl10 and Ccl5, adhesion molecules Cd40, Cd74, and antiviral and antibacterial genes Rsad2, Mx1, Oasl1, Gbp5, Nos2, Batf2 and Tnfrsf11a. Among the amplified genes was also Irf8, of which Ccl5 was subsequently identified as a new pro-inflammatory target in VSMCs and ECs. Promoter analysis predicted transcriptional cooperation between STAT1, IRF1, IRF8 and NFκB, with the novel role of IRF8 providing an additional layer to the overall complexity. The synergistic interactions between IFNγ and TLR4 also resulted in increased T-cell migration and impaired aortic contractility in a STAT1-dependent manner. Expression of the chemokines CXCL9 and CXCL10 correlated with STAT1 phosphorylation in vascular cells in plaques from human carotid arteries. Moreover, using data mining of human plaque transcriptomes, expression of a selection of these STAT1-dependent pro-atherogenic genes was found to be increased in coronary artery disease (CAD) and carotid atherosclerosis. Our study provides evidence to suggest that in ECs and VSMCs STAT1 orchestrates a platform for cross-talk between IFNγ and TLR4, and identifies a STAT1-dependent gene signature that reflects a pro-atherogenic state in human atherosclerosis.