HPV16 E6 Oncogene Contributes to Cancer Immune Evasion by Regulating PD-L1 Expression through a miR-143/HIF-1a Pathway.

Human Papillomaviruses have been associated with the occurrence of cervical cancer, the fourth most common cancer that affects women globally, while 70% of cases are caused by infection with the high-risk types HPV16 and HPV18. The integration of these viruses' oncogenes E6 and E7 into the host's genome affects a multitude of cellular functions and alters the expression of molecules. The aim of this study was to investigate how these oncogenes contribute to the expression of immune system control molecules, using cell lines with integrated HPV16 genome, before and after knocking out E6 viral gene using the CRISPR/Cas9 system, delivered with a lentiviral vector. The molecules studied are the T-cell inactivating protein PD-L1, its transcription factor HIF-1a and the latter's negative regulator, miR-143. According to our results, in the E6 knock out (E6KO) cell lines an increased expression of miR-143 was recorded, while a decrease in the expression of HIF-1a and PD-L1 was exhibited. These findings indicate that E6 protein probably plays a significant role in enabling cervical cancer cells to evade the immune system, while we propose a molecular pathway in cervical cancer, where PD-L1's expression is regulated by E6 protein through a miR-143/HIF-1a axis.

Interaction between TCF7L2 rs7903146 Genotype, HbA1c Levels, and the Periodontal Status of Dental Patients.

OBJECTIVE: The aim of the study was to investigate the potential interaction between TCF7L2 rs7903146 genotype, which is implicated for type-2 diabetes mellitus genetic susceptibility, HbA1c levels, and the periodontal status of dental patients. MATERIALS AND METHODS: HbA1c levels, clinical periodontal parameters (probing depth, clinical attachment level, bleeding on probing, and plaque index), and several parameters (such as body mass index [BMI], smoking habits, education level, and age) were recorded in 150 patients who fulfilled the criteria for screening for prediabetes/diabetes of the Centers for Disease Control and Prevention. DNA was extracted and the TCF7L2 single nucleotide polymorphism (SNP) rs7903146 was genotyped in all participants. RESULTS: Thirty-one patients out of 150 tested were found with unknown hyperglycemia (20.7%). Regarding sex, education, parent with diabetes, normal BMI, smoking, age ≥45 years and prior testing for diabetes, no differences were observed between patients displaying HbA1c < 5.7 and ≥ 5.7% (Pearson's Chi-square test, p > 0.05). Regarding periodontal parameters and differences between subgroups (HbA1c levels ≥ 5.7 and HbA1c levels < 5.7), statistically significant differences were observed for probing depth (3.20 ± 0.94 vs. 2.81 ± 0.78 mm), clinical attachment level (3.54 ± 1.20 vs. 3.18 ± 1.06 mm) and bleeding on probing (0.62 ± 0.25 vs. 0.50 ± 0.24%) with hyperglycemic patients exhibiting worse periodontal conditions (Mann-Whitney test p < 0.05). The allelic and genotype frequencies for the transcription factor 7-like 2 (TCF7L2) gene, SNPs 7903146 did not exhibit a significant difference between the HbA1c > 5.7 and HbA1c < 5.7 groups and the periodontitis and nonperiodontitis subgroups respectively (Fisher's exact test >0.05). Statistical Analysis Patient characteristics and their association with prediabetes were tested by Pearson's Chi-square test (asymptotic, two sided). Differences of periodontal parameters between subgroups were tested with the Mann-Whitney U-test. The associations of allele and genotype frequencies in the patient and control groups were analyzed using the Fisher's exact test of independence.The significance level was set at the 0.05 for all tests. CONCLUSION: A statistically significant association between TCF7L2 rs7903146 genotype and periodontal condition or HbA1c levels was not observed in contrast to statistically significant differences of clinical parameters of periodontitis in patients with hyperglycemia.

Diversity, Cyanotoxin Production, and Bioactivities of Cyanobacteria Isolated from Freshwaters of Greece.

Cyanobacteria are a diverse group of photosynthetic Gram-negative bacteria that produce an array of secondary compounds with selective bioactivity against a broad spectrum of organisms and cell lines. In this study, 29 strains isolated from freshwaters in Greece were classified using a polyphasic approach and assigned to Chroococcales, Synechococcales, and Nostocales, representing 11 genera and 17 taxa. There were good agreements between 16S ribosomal RNA (rRNA)-cpcBA-internal genetic spacer (IGS) characterization and morphological features, except for the Jaaginema-Limnothrix group which appears intermixed and needs further elucidation. Methanol extracts of the strains were analyzed for cyanotoxin production and tested against pathogenic bacteria species and several cancer cell lines. We report for the first time a Nostoc oryzae strain isolated from rice fields capable of producing microcystins (MCs) and a Chlorogloeopsis fritschii strain isolated from the plankton of a lake, suggesting that this species may also occur in freshwater temperate habitats. Strains with very high or identical 16S rRNA gene sequences displayed different antibacterial and cytotoxic activities. Extracts from Synechococcus cf. nidulans showed the most potent antibacterial activity against Staphylococcus aureus, whereas Jaaginema sp. strains exhibited potent cytotoxic activities against human colorectal adenocarcinoma and hepatocellular carcinoma cells. Jaaginema Thessaloniki Aristotle University Microalgae and Cyanobacteria (TAU-MAC) 0110 and 0210 strains caused pronounced changes in the actin network and triggered the formation of numerous lipid droplets in hepatocellular carcinoma and green monkey kidney cells, suggesting oxidative stress and/or mitochondrial damage leading to apoptosis.

Superior long-term repopulating capacity of G-CSF+plerixafor-mobilized blood: implications for stem cell gene therapy by studies in the Hbb(th-3) mouse model.

High numbers of genetically modified hematopoietic stem cells (HSCs) equipped with enhanced engrafting potential are required for successful stem cell gene therapy. By using thalassemia as a model, we investigated the functional properties of hematopoietic stem and progenitor cells (HSPCs) from Hbb(th3)/45.2(+) mice after mobilization with G-CSF, plerixafor, or G-CSF+plerixafor and the engraftment kinetics of primed cells after competitive primary and noncompetitive secondary transplantation. G-CSF+plerixafor yielded the highest numbers of HSPCs, while G-CSF+plerixafor-mobilized Hbb(th3)/45.2(+) cells, either unmanipulated or transduced with a reporter vector, achieved faster hematologic reconstitution and higher levels of donor chimerism over all other types of mobilized cells, after competitive transplantation to B6.BoyJ/45.1(+) recipients. The engraftment benefit observed in the G-CSF+plerixafor group was attributed to the more primitive stem cell phenotype of G-CSF+plerixafor-LSK cells, characterized by higher CD150(+)/CD48 expression. Moreover, secondary G-CSF+plerixafor recipients displayed stable or even higher chimerism levels as compared with primary engrafted mice, thus maintaining or further improving engraftment levels over G-CSF- or plerixafor-secondary recipients. Plerixafor-primed cells displayed the lowest competiveness over all other mobilized cells after primary or secondary transplantation, probably because of the higher frequency of more actively proliferating LK cells. Overall, the higher HSC yields, the faster hematological recovery, and the superiority in long-term engraftment indicate G-CSF+plerixafor-mobilized blood as an optimal graft source, not only for thalassemia gene therapy, but also for stem cell gene therapy applications in general.

Mobilization of hematopoietic stem cells in a thalassemic mouse model: implications for human gene therapy of thalassemia.

Granulocyte colony-stimulating factor (G-CSF)-mobilized blood stem cells may become the preferable source of hematopoietic stem cells (HSCs) for gene therapy because of the higher yield of cells compared with conventional bone marrow harvesting. A G-CSF-associated risk of splenic rupture has been recognized in normal donors of HSCs, but limited information is available about the G-CSF effect in the presence of splenomegaly and extramedullary hematopoiesis. We investigated the G-CSF effect in a thalassemic mouse model (HBB(th-3)) as compared with a normal strain (C57BL/6), in terms of safety, mobilization efficacy, and distribution of stem cells among hematopoietic compartments. There was no death or clinical sequelae of splenic rupture in G-CSF-treated animals of either strain; however, hemorrhagic infarcts in the spleen were detected with low frequency in G-CSF-treated HBB(th-3) mice (12.5%). HBB(th-3) mice mobilized less effectively than C57BL/6 mice (Lin(-)Sca-1(+)c-Kit(+) cells/microl of peripheral blood mononuclear cells [PBMCs]: 90 +/- 55 vs. 255 +/- 174, respectively, p = 0.01; CFU-GM/ml PBMCs: 390 +/- 262 vs. 1131 +/- 875, p = 0.01) because of increased splenic trapping of hematopoietic stem and progenitor cells (Lin(-)Sca-1(+)c-Kit(+) cells per spleen (x10(5)): 487 +/- 35 vs. 109 +/- 19.6, p = 0.01; CFU-GM per spleen (x10(2)): 1470 +/- 347 vs. 530 +/- 425, p = 0.0006). Splenectomy restored the mobilization proficiency of thalassemic mice at comparable levels to normal mice and resulted in the development of a hematopoietic compensatory mechanism in the thalassemic liver that protected splenectomized mice from severe anemia. Our data imply that, in view of human gene therapy for thalassemia, either multiple cycles or alternative ways of mobilization may be required for a sufficient yield of transplantable HSCs. In addition, strategies to minimize the risk of G-CSF-induced splenic infarcts should be explored in a clinical setting.

Human ubiquitin specific protease 31 is a deubiquitinating enzyme implicated in activation of nuclear factor-kappaB.

TRAF2 mediates activation of the transcription factors NF-kappaB and AP1 by TNF. A yeast two-hybrid screen of a human cDNA library identified a ubiquitin specific protease homologue (USP31) as a TRAF2-interacting protein. Two cDNAs encoding for USP31 were identified. One cDNA encodes a 1035-amino acid long isoform of USP31 (USP31, long isoform) and the other a 485-amino acid long isoform of USP31 (USP31S1, short isoform). USP31 and USP31S1 share a common amino terminal region with homology to the catalytic region of known deubiquitinating enzymes. Enzymatic assays demonstrated that USP31 but not USP31S1 possess deubiquitinating activity. Furthermore, it was shown that USP31 has a higher activity towards lysine-63-linked as compared to lysine-48-linked polyubiquitin chains. Overexpression of USP31 in HEK 293T cells inhibited TNFalpha, CD40, LMP1, TRAF2, TRAF6 and IKKbeta-mediated NF-kappaB activation, but did not inhibit Smad-mediated transcription activation. In addition, both USP31 isoforms interact with p65/RelA. Our data support a role for USP31 in the regulation of NF-kappaB activation by members of the TNF receptor superfamily.

Identification and characterization of the gene products of open reading frame U86/87 of human herpesvirus 6.

The human herpesvirus 6 (HHV-6) immediate early-A locus (IE-A) locates in the position analogous to the human cytomegalovirus (HCMV) major IE (MIE) locus that is well-known to play critical roles in viral infection. Similarly to HCMV MIE, HHV-6 IE-A consists of two genetic units, IE1 and IE2, corresponding to open reading frames U90-U89 and U90-U86/87, respectively. However, the HHV-6 IE-A locus exhibits limited sequence homology with the HCMV MIE locus. In this study, to characterize HHV-6 IE2 gene products, polyclonal antibodies against four domains of the U86/87 open reading frame were generated by immunization of rabbits with bacterially-expressed proteins. Three polypeptides derived from the U86/87 region with apparent molecular masses of 100, 85 and 55 kD were detected in HHV-6-infected cells 3 days after infection, while IE1 polypeptides with apparent molecular mass greater than 170 kD were detectable as early as 8 h. Mapping of the IE2 gene products with the antibodies suggests differential splicing and alternative translation initiation in the IE2 genetic unit. The IE2 products show a mixed cytoplasmic and nuclear localization pattern. In addition, the 437 amino acid carboxyl-terminus domain bound to a DNA fragment containing the putative IE-A promoter. These results suggest that HHV-6 IE2 plays a critical role in transcriptional regulation and viral growth as does HCMV IE2, although it is likely that HHV-6 IE2 has expression kinetics different from HCMV IE2.