Indication of West Nile Virus (WNV) Lineage 2 Overwintering among Wild Birds in the Regions of Peloponnese and Western Greece.

West Nile virus (WNV), a zoonotic mosquito-borne virus, has recently caused human outbreaks in Europe, including Greece. Its transmission cycle in nature includes wild birds as amplifying hosts and ornithophilic mosquito vectors. The aim of this study was to assess WNV circulation among wild birds from two regions of Greece, Peloponnese and Western Greece, during 2022. To this end, a total of 511 birds belonging to 37 different species were sampled and molecularly screened. WNV RNA was detected from February to November in a total of 71 wild birds of nine species originating from both investigated regions. The first eight positive samples were sequenced on a part of NS3 and, according to the phylogenetic analysis, they belonged to evolutionary lineage 2 and presented similarity to previous outbreak-causing Greek strains (Argolis 2017, Macedonia 2010 and 2012). It was more likely to identify a PCR positive bird as the population density and the distance from water sources decreased. The present report provides evidence of WNV occurrence in both Peloponnese and Western Greece during 2022 and underlines its possible overwintering, highlighting the need for avian species surveillance to be conducted annually and throughout the year. Magpies are proposed as sentinels for WNV monitoring.

Genetic Analysis and Status of Brown Bear Sub-Populations in Three National Parks of Greece Functioning as Strongholds for the Species' Conservation.

In order to optimize the appropriate conservation actions for the brown bear (Ursus arctos L.) population in Greece, we estimated the census (Nc) and effective (Ne) population size as well as the genetic status of brown bear sub-populations in three National Parks (NP): Prespa (MBPNP), Pindos (PINDNP), and Rhodopi (RMNP). The Prespa and Pindos sub-populations are located in western Greece and the Rhodopi population is located in eastern Greece. We extracted DNA from 472 hair samples and amplified through PCR 10 microsatellite loci. In total, 257 of 472 samples (54.5%) were genotyped for 6-10 microsatellite loci. Genetic analysis revealed that the Ne was 35, 118, and 61 individuals in MBPNP, PINDNP, and RMNP, respectively, while high levels of inbreeding were found in Prespa and Rhodopi but not in Pindos. Moreover, analysis of genetic structure showed that the Pindos population is genetically distinct, whereas Prespa and Rhodopi show mutual overlaps. Finally, we found a notable gene flow from Prespa to Rhodopi (10.19%) and from Rhodopi to Prespa (14.96%). Therefore, targeted actions for the conservation of the bears that live in the abovementioned areas must be undertaken, in order to ensure the species' viability and to preserve the corridors that allow connectivity between the bear sub-populations in Greece.

Maternal serum pregnancy-associated plasma protein-A concentration at 11-14 weeks of gestation and preeclampsia risk of women with common congenital anatomic uterine abnormalities.

To evaluate maternal serum pregnancy-associated plasma protein-A (PAPP-A) levels at 11-14 weeks of gestation and preeclampsia risk in women with common congenital anatomic uterine abnormalities (AUAs). First trimester screening markers were compared between 12 AUA pregnancies, 60 age matched controls and 12 cases of early preeclampsia. PAPP-A level and birth weight were significantly lower in AUA compared to control and early preeclampsia group (p<.001). Preeclampsia was absent in the AUAs pregnancies group. Birth weight were similar in AUA group when we compared AUA and control group regarding weeks of gestation at delivery and lower but not significantly, when we compared AUA and early preeclampsia group. Our findings suggest that AUA pregnancies are associated with low first trimester maternal serum PAPP-A concentrations not predictive of susceptibility to preeclampsia.Impact statementWhat is already known on this subject? During first trimester screening for preeclampsia based on maternal pregnancy-associated plasma protein A (PAPP-A) levels, various parameters are used, such as the somatometric characteristics of pregnant woman, single or multiple pregnancy, smoking status, family history, diabetes, hypertension and measurement of blood pressure and uterine artery Dopplers.What do the results of this study add? Our pioneer study revealed that there is drastic difference in PAPP-A concentration in women with common anatomic uterine abnormalities (AUAs), in comparison with their age matched control women with normal uterus.What are the implications of these findings for clinical practice and further research? Based on our results, uterine anatomical deviations, is another factor which must be taken in account for preeclampsia risk calculation and further clinical consultation and follow up in those pregnancies. Lower PAPP-A levels in AUA cases is a weak predictor of susceptibility to preeclampsia and could be associated to smaller placental size rather than poor placentation and in future research the calculation of the uterine cavity functional dimension may lead to a more accurate clinical assessment.

Renin-Angiotensin System Single Nucleotide Polymorphisms Are Associated with Bladder Cancer Risk.

The renin-angiotensin system (RAS), besides being a major regulator of blood pressure, is also involved in tumor angiogenesis. Emerging evidence suggests a correlation between the use of pharmacologic RAS inhibitors and a delay in urothelial bladder cancer (BC) progression. However, it is unknown whether RAS gene variants may predispose to the development of BC. This study examined the association of RAS single nucleotide polymorphisms (SNPs) including AT1R rs5186, AT2R rs11091046, REN rs12750834, ANG rs4762, and ANG rs699 with the risk of developing non-invasive BC. Peripheral blood samples from 73 patients with T1 urothelial BC (66 men, seven women) and an equal number of healthy subjects (control group) were collected. The TT genotype of the REN rs12750834 SNP (OR: 2.8 [1.3-6.05], p = 0.008) and to a lesser extent the presence of the T allele (OR: 2.3 [1.2-4.48], p = 0.01) conferred a higher risk of BC. The highest risk for BC within SNP carriers of the RAS system was associated with the presence of the CC genotype (OR: 17.6 [7.5-41.35], p < 0.001) and C allele (OR: 17.7 [8.8-35.9], p < 0.001) of the ANG rs699 SNP. The presence of the AT2R rs11091046 SNP, particularly the AA genotype, was associated with a protective effect against developing BC (OR: 0.268 [0.126-057], p < 0.001). In conclusion, these results support the clinical utility of RAS gene SNPs AT2R rs11091046, REN rs12750834, and ANG rs699 in the genetic cancer risk assessment of patients and families with BC.

Dysregulation of Caveolin-1 Phosphorylation and Nuclear Translocation Is Associated with Senescence Onset.

We recently reported that the inability of osteoarthritic (OA) chondrocytes to repair oxidative stress (OS) induced DNA damage is linked to Cav-1 overexpression/improper localization. We speculated that the senescent status of OA cells was responsible for this Cav-1 dysregulation. Here, to further investigate this hypothesis, we used Wharton Jelly derived mesenchymal stem cells (WJ-MSCs) and investigated Cav-1 function as cells reached replicative senescence or upon stress induced senescence (SIPS). We showed that Cav-1 is upregulated, phosphorylated and translocated to the nucleus in young WJ-MSCs upon acute exogenous OS, and that it returns back to basal/nonphosphorylated levels and exports the nucleus in the recovery phase. However, as cells reach senescence, this regulation is lost. OS did not induce any Cav-1-mediated response, which is concomitant with the inability of older cells to restore DNA damage. Furthermore, downregulation of Cav-1 resulted in persistent OS-induced DNA damage and subsequent onset of senescence. We also report that the establishment of senescence is mediated by autophagy stimulation, since downregulation of autophagy key molecule Atg5, simultaneously with Cav-1 downregulation, was found to inhibit SIPS. Basically, we propose that Cav-1 involvement in DNA damage response can lead to senescence, either because the damage is extensive or because Cav-1 is absent/unable to perform its homeostatic role.

Development of a Multiplex Bead Assay for Simultaneous Serodiagnosis of Antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis in Wild Boar.

The aim of this study was to evaluate the diagnostic performance of a multiplex bead assay for the simultaneous detection of antibodies against Mycobacterium bovis, Brucella suis, and Trichinella spiralis. Sera from Eurasian wild boar of known serological status for TB (64 seropositive, 106 seronegative), Brucella (30 seropositive, 39 seronegative), and Trichinella (21 seropositive, 97 seronegative) were used for the development and evaluation of the assay. Magnetic beads coated with recombinant MPB83 antigen (TB), a whole-cell B. suis 1330 antigen, and an E/S T. spiralis antigen were used for the detection of specific antibodies using Bio-Rad Bio-Plex technology. The sensitivities (Se) and specificities (Sp) of the multiplex assay were, for M. bovis, 0.98 and 0.86; for B. suis, 1.00 and 0.97; and for T. spiralis, 0.90 and 0.99 (Se and Sp, respectively). The results show the diagnostic potential of this assay for the simultaneous detection of antibodies against M. bovis, B. suis, and T. spiralis in wild boar.

Poultry and Wild Birds as a Reservoir of CMY-2 Producing Escherichia coli: The First Large-Scale Study in Greece.

Resistance mediated by ß-lactamases is a globally spread menace. The aim of the present study was to determine the occurrence of Escherichia coli producing plasmid-encoded AmpC ß-lactamases (pAmpC) in animals. Fecal samples from chickens (n = 159), cattle (n = 104), pigs (n = 214), and various wild bird species (n = 168), collected from different Greek regions during 2018-2020, were screened for the presence of pAmpC-encoding genes. Thirteen E. coli displaying resistance to third-generation cephalosporins and a positive AmpC confirmation test were detected. blaCMY-2 was the sole pAmpC gene identified in 12 chickens' and 1 wild bird (Eurasian magpie) isolates and was in all cases linked to an upstream ISEcp1-like element. The isolates were classified into five different sequence types: ST131, ST117, ST155, ST429, and ST1415. Four chickens' stains were assigned to ST131, while five chickens' strains and the one from the Eurasian magpie belonged to ST117. Seven pAmpC isolates co-harbored genes conferring resistance to tetracyclines (tetM, tetB, tetC, tetD), 3 carried sulfonamide resistance genes (sulI and sulII), and 10 displayed mutations in the quinolone resistance-determining regions of gyrA (S83L+D87N) and parC (S80I+E84V). This report provides evidence of pAmpC dissemination, describing for the first time the presence of CMY-2 in chickens and wild birds from Greece.

Association of maternal Toll-like receptor-4 alleles with susceptibility to early-onset preeclampsia in central Greece.

INTRODUCTION: Altered maternal inflammatory responses may play a role in the development of hypertensive disorders of pregnancy like preeclampsia, its more severe early-onset form and intrauterine growth restriction. We evaluated the relation of common allelic variants of Toll-like receptor 4 (TLR4), known to impair the inflammatory response, with the susceptibility to early-onset preeclampsia in Central Greece. METHODS: We compared the occurrence of TLR4 (Asp299Gly and Thr399Ile) alleles in heterozygous (A/G, C/T) and homozygous (G/G, T/T) states in 84 women with a history of early-onset preeclampsia and 94 age matched controls with a history of only uneventful pregnancies, by direct sequencing. RESULTS: Heterozygous TLR4 allelic variants were more common in women with a history of early-onset preeclampsia than in controls (GA for Asp299Gly: 14.3% vs 6.4% (AA), p = 0.053 & CT for Thr399Ile: 16.7% vs. 6.4% (CC), p = 0.019) and a stronger association was obtained when homozygous allelic carriers were also included (GA/GG for Asp299Gly: 16.7% vs. 6.4% (AA), p = 0.03 & TC/TT for Thr399Ile: 19.0% vs. 6.4% (CC), p = 0.01). DISCUSSION: We recorded association between common TLR4 gene variants and early-onset preeclampsia. Our findings support the involvement of maternal innate immune system in severe hypertensive disorders of pregnancy and point to the potential value of maternal TLR4 polymorphisms as predictors-risk factors of susceptibility to early-onset preeclampsia in Central Greece.

Impact of renin-angiotensin-aldosterone system polymorphisms on myocardial perfusion: Correlations with myocardial single photon emission computed tomography-derived parameters.

BACKGROUND: Renin-angiotensin-aldosterone system (RAAS) has an important role in atherosclerosis. We investigated the effects of six RAAS gene polymorphisms on myocardial perfusion. METHODS AND RESULTS: We examined 810 patients with known or suspected coronary artery disease (CAD) using stress-rest myocardial single-photon emission computed tomography. Summed stress score (SSS), summed rest score (SRS), summed difference score (SDS), transient ischemic dilation (TID), and lung/heart ratio (LHR) were recorded. The following gene polymorphisms were investigated: angiotensin-converting enzyme (ACE) insertion/deletion (I/D), angiotensinogen (AGT) M235T and T174M, angiotensin II type 1 receptor (AT1R) A1166C, renin (REN) C5312T, and angiotensin II type 2 receptor (AT2R) C3123A. The heterozygotes or homozygotes on ACE D allele were 7.54 times more likely to have abnormal SSS, while the AGT (T174M) heterozygotes were 5.19 times more likely to have abnormal SSS. The homozygotes of ACE D had significantly higher values on TID and LHR, while the AGT (T174M) heterozygotes had higher values on TID. The AT1R heterozygotes had greater odds for having SSS ≥ 3. The patients carried AT1R homozygosity of C allele had significantly higher values on TID, while heterozygotes of AT1R had significantly higher values on LHR. CONCLUSIONS: Among the polymorphisms investigated, ACE D allele had the strongest association with abnormal myocardial perfusion.

Human Papilloma Virus (HPV) and Fertilization: A Mini Review.

Human papilloma virus (HPV) is one of the most prevalent viral sexually transmitted diseases. The ability of HPV to induce malignancy in the anogenital tract and stomato-pharyngeal cavity is well documented. Moreover, HPV infection may also affect reproductive health and fertility. Although, the impact of HPV on female fertility has not been thoroughly studied it has been found also to have an impact on semen parameters. Relative information can be obtained from studies investigating the relationship between HPV and pregnancy success. Furthermore, there is an ongoing debate whether HPV alters the efficacy of assisted reproductive technologies. An association between HPV and assisted reproductive technologies (ART) programs has been reported. Nevertheless, due to conflicting data and the small number of existing studies further research is required. It remains to be clarified whether HPV detection and genotyping could be included in the diagnostic procedures in couples undergoing in vitro fertilization (IVF)/intrauterine insemination (IUI) treatments. Vaccination of both genders against HPV can reduce the prevalence of HPV infection and eliminate its implications on human fertility. The aim of the present mini-review is to reiterate the association between HPV and human fertility through a systematic literature review.

Pregnancy prediction by free sperm DNA and sperm DNA fragmentation in semen specimens of IVF/ICSI-ET patients.

The purpose of this study was to evaluate the predictive value of free sperm plasma DNA (f-spDNA) and sperm DNA fragmentation (SDF), in semen specimens from men undergoing in vitro fertilization/intracytoplasmic sperm injection-embryo transfer (IVF/ICSI-ET) treatments. Fifty-five semen samples were evaluated during 55 consecutive IVF/ICSI-ET cycles. F-spDNA was determined by conventional quantitative real-time PCR-Sybr green detection approach, while evaluation of sperm DNA damage was performed using the sperm chromatin dispersion (SCD) assay. While f-spDNA only correlated with total sperm count, SDF correlated with many semen parameters (including sperm concentration, total sperm count and the per cent of non-progressive sperm). Neither SDF nor the proportion of sperm with small or no halos correlated with f-spDNA. Interestingly, smoking status correlated with f-spDNA but not with SDF. Although these two factors seem to interact for the prediction of pregnancy, receiver-operating characteristics (ROC) analysis revealed that SDF had a stronger predictive value (AUC = 0.7, p < 0.05) than f-spDNA (AUC = 0.6, p > 0.05). SDF and f-spDNA may not be associated together but they interact at a significant level in order to exert their actions on pregnancy outcome. Among the two markers, SDF appears to have stronger and significantly predictive value for pregnancy success.

Sequence variations in the FII, FV, F13A1, FGB and PAI-1 genes are associated with differences in myocardial perfusion.

AIMS: Coronary artery disease (CAD) is a significant cause of morbidity and mortality in modern societies. The association between genetic markers and CAD is still poorly understood. In this study, we evaluated the effect of five genetic variants: Factor V Leiden (FV:c.1691G>A) (rs6025), Factor II prothrombin (FII:c.20210G>A; rs1799963), plasminogen activator inhibitor 1 (PAI-1) -675(4G/5G; SERPINE1:g.4329_4330insG; rs34857375), ß-fibrinogen -455G>A (FGB:c.4577G>A; rs1800790) and Factor XIII (F13A1:c.103G>T; rs5985) on myocardial perfusion. MATERIALS & METHODS: We examined 523 patients using exercise-rest myocardial perfusion single photon emission computed tomography, where the summed stress score (SSS), summed rest score and summed difference score (SDS) indexes, were calculated. In order to examine the independent prognostic ability of genotype on SSS and SDS, multiple linear regression models were used. RESULTS: It was found that Factor V Leiden, Factor XIII, ß-fibrinogen and PAI-1 genotypes were independent prognostic predictors of SSS and SDS with Factor XIII exhibiting the strongest association. Moreover, Factor II prothrombin proved an independent prognostic predictor of SSS. CONCLUSION: Our study provides the first evidence of an association between these polymorphisms and myocardial perfusion, suggesting that the process of coronary artery disease and also patients' prognosis, may be modified by the FV:c.1691G>A, FII:c.20210G>A, PAI-1 -675 (4G/5G), ß-fibrinogen FGB:c.4577G>A and F13A1:c.103G>T genotypes.

Sequence-based genotyping HPV L1 DNA and RNA transcripts in clinical specimens.

We developed a direct sequence-based genotyping method to detect single and multiple HPV L1 DNA and RNA types in genital and dermatological specimens. Our method couples PCR amplification of a highly conserved HPV L1 segment using a broad spectrum-generic primer cocktail mix with automated sequencing of amplified PCR products, followed by GenBank sorting of sequencing data. We genotyped 5 skin and 30 cervical HPV DNA-positive specimens using this method and established its first experimentally derived working cutoff value with the aid of commercial hybridization-based techniques. We suggest that sequence-based genotyping of appropriately amplified DNA and RNA products may serve as a primary HPV detection method in dermatological specimens. It can be applied as an all-purpose genotyping method for rare HPV types not detectable by commercial hybridization-based techniques and for sorting multiple HPV infections by order of prevalence.

Epigenetic regulation of hTERT promoter in hepatocellular carcinomas.

Although hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide, the molecular pathogenesis of the disease has not been elucidated. Several studies have shown that telomerase activity and hTERT expression are increased in HCCs. In the present study we tried to elucidate hTERT transcriptional and epigenetic regulatory mechanisms in HCC. hTERT expression was tested by real-time PCR and DNA methylation status was assessed by MethyLight and DNA bisulfite sequencing analyses in 106 tissues (64 with HCC and 42 without liver disorders) and also in 7 hepatocarcinoma cell lines (HepG2, HepG3B2, C3A, SNU-182, SNU-398, SBU-449 and SNU-475). hTERT expression levels were inversely correlated with DNA methylation levels in HCC and normal tissues (r=-0.859). hTERT expression was found to be regulated by DNA methylation and histone H3-K9 modifications, affecting the ability of c-myc binding in E-box 1 site in hTERT promoter. Additionally, c-myc siRNA liposomal down-regulation inhibited significantly hTERT expression (p<0.05). Thus, we propose that hTERT is regulated by a combination of epigenetic mechanisms (DNA methylation and histone modifications) and by the transcription factor c-myc in HCC.

Influence of interleukin 1alpha (IL-1alpha), IL-4, and IL-6 polymorphisms on genetic susceptibility to chronic osteomyelitis.

The association between cytokine gene polymorphisms and chronic osteomyelitis was investigated in order to determine whether genetic variability in cytokine genes predisposes to osteomyelitis susceptibility. Significant genotypic and allelic associations were observed between interleukin 1alpha (IL-1alpha) -889-C/T, IL-4 -1098-G/T and -590-C/T, and IL-6 -174-G/C polymorphisms and osteomyelitis in the Greek population, pointing towards their potential involvement in osteomyelitis pathogenesis.

Association of KLOTHO gene polymorphisms with knee osteoarthritis in Greek population.

Based on the fact that klotho-deficient mice exhibit multiple aging phenotypes, including osteopenia and subchondral sclerosis of joints and on the recent observation that KLOTHO gene plays an important role in calcium/phosphate homeostasis, we explored the possibility whether human KLOTHO gene polymorphisms are associated with osteoarthritis (OA). A total of 752 individuals participated in the study. The knee OA group consisted of 369 patients; 298 women (mean age 65.9 +/- 8.2; range 40-92 years) and 71 men (mean age 65.7 +/- 9.1; range 30-82 years). The control population consisted of 383 subjects; 231 women (mean age 65.8 +/- 8.4; range 35-90 years) and 152 men (mean age 61.5 +/- 9.3; range 28-87 years). Four SNPs--G395A in the promoter region, G1110C in exon 2, C1818T and C2298T in exon 4--were genotyped. A significant genotypic and allelic association was observed in SNP C2998T and knee OA, while genotype GA of SNP G395A was significantly associated (p = 0.039) with knee OA in females only. For the first time, an association was observed between SNPs G395A and C2998T of the KLOTHO gene and knee osteoarthritis implicating KLOTHO in OA pathogenesis.

On the mechanism of phenotypic conversion of human cervical adenocarcinoma HeLa cells surviving infection by influenza B virus: potential implications for biological management of adenocarcinomas.

We infected HeLa cells with low (10(-9) units), medium (10(-6) units), and high (10(-2) units) influenza B titers and compared the resulting human papilloma virus (HPV), retinoic acid receptor alpha subunit (RARalpha) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA content of surviving infected hosts with that of their uninfected precursors by semi-quantitative reverse transcriptase/polymerase chain reaction amplification (RT/PCR). This comparison revealed a moderate and drastic dependence of HPV and RARalpha mRNA content, respectively, but a complete independence of GAPDH mRNA expression on viral titer. A mechanism of adoptive replacement of tolerable cellular with viral gene expression was proposed to explain these findings. We conclude that the reported ability of influenza B viruses to specifically target and eliminate the cervical adenocarcinoma HeLa cell line studied may find practical applications in biological cancer management.

The growth differentiation factor 5 (GDF5) core promoter polymorphism is not associated with knee osteoarthritis in the Greek population.

Genetic factors have been shown to play an important role in the etiology of osteoarthritis (OA). A functional single nucleotide polymorphism (SNP) +104T/C; rs143383 in the 5' UTR of the GDF5 gene was recently associated with susceptibility to osteoarthritis in the Japanese and Chinese population. Our objective was to assess whether this SNP was also associated with knee OA in a Greek Caucasian population sample. The +104T/C SNP was genotyped in a total of 519 case-control cohort; 251 patients with idiopathic knee OA and 268 controls were used. No significant differences were found in genotype or allele frequencies of the +104T/C SNP of GDF5 gene between cases and controls (p < 0.05). Also, no significant differences in allelic and genotypic frequencies were found when the individuals were stratified by sex. Our data implied that the +104T/C; rs143383 GDF5 core promoter polymorphism is not a risk factor for OA etiology in Greek Caucasians. Our study highlights the heterogeneous nature of OA genetic susceptibility.