Genetic prediction of ICU hospitalization and mortality in COVID-19 patients using artificial neural networks.

There is an unmet need of models for early prediction of morbidity and mortality of Coronavirus disease-19 (COVID-19). We aimed to a) identify complement-related genetic variants associated with the clinical outcomes of ICU hospitalization and death, b) develop an artificial neural network (ANN) predicting these outcomes and c) validate whether complement-related variants are associated with an impaired complement phenotype. We prospectively recruited consecutive adult patients of Caucasian origin, hospitalized due to COVID-19. Through targeted next-generation sequencing, we identified variants in complement factor H/CFH, CFB, CFH-related, CFD, CD55, C3, C5, CFI, CD46, thrombomodulin/THBD, and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS13). Among 381 variants in 133 patients, we identified 5 critical variants associated with severe COVID-19: rs2547438 (C3), rs2250656 (C3), rs1042580 (THBD), rs800292 (CFH) and rs414628 (CFHR1). Using age, gender and presence or absence of each variant, we developed an ANN predicting morbidity and mortality in 89.47% of the examined population. Furthermore, THBD and C3a levels were significantly increased in severe COVID-19 patients and those harbouring relevant variants. Thus, we reveal for the first time an ANN accurately predicting ICU hospitalization and death in COVID-19 patients, based on genetic variants in complement genes, age and gender. Importantly, we confirm that genetic dysregulation is associated with impaired complement phenotype.

Pharmacogenetic analysis of canonical versus noncanonical pathway of NF-kB in Crohn's disease patients under anti-tumor necrosis factor-α treatment.

OBJECTIVES: This study explores the potential of gene polymorphisms in the canonical and noncanonical NF-kB signaling pathway as a prediction biomarker of anti-tumor necrosis factor (TNF)α response in Crohn's patients. MATERIALS AND METHODS: A total of 109 Greek patients with Crohn's disease (CD) were recruited, and the genotype of TLR2 rs3804099, LTA rs909253, TLR4 rs5030728, and MAP3K14/NIK rs7222094 single nucleotide polymorphisms was investigated for association with response to anti-TNFα therapy. Patient's response to therapy was based on the Crohn's Disease Activity Index, depicting the maximum response within 24 months after initiation of treatment. RESULTS: Seventy-three patients (66.7%) were classified as responders while 36 as nonresponders (33.3%). Comparing allelic frequencies between responders and nonresponders, the presence of TLR2 rs3804099 T allele was associated with nonresponse (P = 0.003), even after stratification by anti-TNFα drugs (infliximab: P = 0.032, adalimumab: P = 0.026). No other association was identified for the rest of the polymorphisms under study. Haplotype analysis further enhanced the association of rs3804099 T allele with loss of response, even though the results were NS (P = 0.073). CONCLUSION: Our results suggest that polymorphisms in the canonical NF-kB pathway genes could potentially act as a predictive biomarker of anti-TNFα response in CD.

Towards analyzing the potential of exosomes to deliver microRNA therapeutics.

Exosome selectivity mechanisms underlying exosome-target cell interactions and the specific traits affecting their capability to communicate still remain unclear. Moreover, the capacity of exosomes to efficiently deliver their molecular cargos intracellularly needs precise investigation towards establishing functional exosome-based delivery platforms exploitable in the clinical practice. The current study focuses on: (a) exosome production from normal MRC-5 and Vero cells growing in culture, (b) physicochemical characterization by dynamic light scattering (DLS) and cryo-transmission electron microscopy; (c) cellular uptake studies of rhodamine-labeled exosomes in normal and cancer cells, providing to exosomes either "autologous" or "heterologous" cellular delivery environments; and (d) loading exogenous Alexa Fluor 488-labeled siRNA into exosomes for the assessment of their delivering capacity by immunofluorescence in a panel of recipient cells. The data obtained thus far indicate that MRC-5 and Vero exosomes, indeed exhibit an interesting delivering profile, as promising "bio-shuttles," being pharmacologically exploitable in the context of theranostic applications.

Genetic justification of severe COVID-19 using a rigorous algorithm.

Recent studies suggest excessive complement activation in severe coronavirus disease-19 (COVID-19). The latter shares common characteristics with complement-mediated thrombotic microangiopathy (TMA). We hypothesized that genetic susceptibility would be evident in patients with severe COVID-19 (similar to TMA) and associated with disease severity. We analyzed genetic and clinical data from 97 patients hospitalized for COVID-19. Through targeted next-generation-sequencing we found an ADAMTS13 variant in 49 patients, along with two risk factor variants (C3, 21 patients; CFH,34 patients). 31 (32%) patients had a combination of these, which was independently associated with ICU hospitalization (p = 0.022). Analysis of almost infinite variant combinations showed that patients with rs1042580 in thrombomodulin and without rs800292 in complement factor H did not require ICU hospitalization. We also observed gender differences in ADAMTS13 and complement-related variants. In light of encouraging results by complement inhibitors, our study highlights a patient population that might benefit from early initiation of specific treatment.

The GEnetic Syntax Score: a genetic risk assessment implementation tool grading the complexity of coronary artery disease-rationale and design of the GESS study.

BACKGROUND: Coronary artery disease (CAD) remains one of the leading causes of mortality worldwide and is associated with multiple inherited and environmental risk factors. This study is designed to identify, design, and develop a panel of genetic markers that combined with clinical and angiographic information, will facilitate the creation of a personalized risk prediction algorithm (GEnetic Syntax Score-GESS). GESS score could be a reliable tool for predicting cardiovascular risk for future adverse events and for guiding therapeutic strategies. METHODS: GESS (ClinicalTrials.gov Identifier: NCT03150680) is a prospective, non-interventional clinical study designed to enroll 1080 consecutive patients with no prior history of coronary revascularization procedure, who undergo scheduled or emergency coronary angiography in AHEPA, University General Hospital of Thessaloniki. Next generation sequencing (NGS) technology will be used to genotype specific single-nucleotide polymorphisms (SNPs) across the genome of study participants, which were identified as clinically relevant to CAD after extensive bioinformatic analysis of literature-based SNPs. Enrichment analyses of Gene Ontology-Molecular Function, Reactome Pathways and Disease Ontology terms were also performed to identify the top 15 statistically significant terms and pathways. Furthermore, the SYNTAX score will be calculated for the assessment of CAD severity of all patients based on their angiographic findings. All patients will be followed-up for one-year, in order to record any major adverse cardiovascular events. DISCUSSION: A group of 228 SNPs was identified through bioinformatic and pharmacogenomic analysis to be involved in CAD through a wide range of pathways and was correlated with various laboratory and clinical parameters, along with the patients' response to clopidogrel and statin therapy. The annotation of these SNPs revealed 127 genes being affected by the presence of one or more SNPs. The first patient was enrolled in the study in February 2019 and enrollment is expected to be completed until June 2021. Hence, GESS is the first trial to date aspiring to develop a novel risk prediction algorithm, the GEnetic Syntax Score, able to identify patients at high risk for complex CAD based on their molecular signature profile and ultimately promote pharmacogenomics and precision medicine in routine clinical settings. Trial registration GESS trial registration: ClinicalTrials.gov Number: NCT03150680. Registered 12 May 2017- Prospectively registered, https://clinicaltrials.gov/ct2/show/NCT03150680 .

Awareness, knowledge and trust in the Greek authorities towards COVID-19 pandemic: results from the Epirus Health Study cohort.

BACKGROUND: To assess the level of knowledge and trust in the policy decisions taken regarding the coronavirus disease (COVID-19) pandemic among Epirus Health Study (EHS) participants. METHODS: The EHS is an ongoing and deeply-phenotyped prospective cohort study that has recruited 667 participants in northwest Greece until August 31st, 2020. Level of knowledge on coronavirus (SARS-CoV-2) transmission and COVID-19 severity was labeled as poor, moderate or good. Variables assessing knowledge and beliefs towards the pandemic were summarized overall and by sex, age group (25-39, 40-49, 50-59, ≥60 years) and period of report (before the lifting of lockdown measures in Greece: March 30th to May 3rd, and two post-lockdown time periods: May 4th to June 31st, July 1st to August 31st). A hypothesis generating exposure-wide association analysis was conducted to evaluate the associations between 153 agnostically-selected explanatory variables and participants' knowledge. Correction for multiple comparisons was applied using a false discovery rate (FDR) threshold of 5%. RESULTS: A total of 563 participants (49 years mean age; 60% women) had available information on the standard EHS questionnaire, the clinical and biochemical measurements, and the COVID-19-related questionnaire. Percentages of poor, moderate and good knowledge status regarding COVID-19 were 4.5, 10.0 and 85.6%, respectively. The majority of participants showed absolute or moderate trust in the Greek health authorities for the management of the epidemic (90.1%), as well as in the Greek Government (84.7%) and the official national sources of information (87.4%). Trust in the authorities was weaker in younger participants and those who joined the study after the lifting of lockdown measures (p-value≤0.001). None of the factors examined was associated with participants' level of knowledge after correction for multiple testing. CONCLUSIONS: High level of knowledge about the COVID-19 pandemic and trust in the Greek authorities was observed, possibly due to the plethora of good quality publicly available information and the timely management of the pandemic at its early stages in Greece. Information campaigns for the COVID-19 pandemic should be encouraged even after the lifting of lockdown measures to increase public awareness.

Ceftazidime/avibactam and eravacycline susceptibility of carbapenem-resistant Klebsiella pneumoniae in two Greek tertiary teaching hospitals.

The present study evaluated the carbapenem resistance mechanisms of Klebsiella pneumoniae strains isolated in two Greek tertiary teaching hospitals and their susceptibility to currently used and novel antimicrobial agents.Forty-seven carbapenem resistant K. pneumoniae strains were collected in G. Papanikolaou and Ippokrateio hospital of Thessaloniki between 2016 and 2018. Strain identification and antimicrobial susceptibility was conducted by Vitek 2 system (Biomérieux France). Susceptibility against new antimicrobial agents was examined by disk diffusion method. Polymerase chain reaction (PCR) was used to detect blaKPC, blaVIM, blaNDM and blaOXA-48 genes.The meropenem-EDTA and meropenem-boronic acid synergy test performed on the 24 K. pneumoniae strains demonstrated that 8 (33.3%) yielded positive for metallo-beta-lactamases (MBL) and 16 (66.6%) for K. pneumonia carbapenemases (KPC) production. Colistin demonstrated the highest in vitro activity (87.7%) among the 47 K. pneumoniae strains followed by gentamicin (76.5%) and tigecycline (51%). Among new antibiotics ceftazidime/avibactam showed the highest sensitivity (76.6%) in all strains followed by eravacycline (66.6%). The blaKPC gene was present in 30 strains (63.8%), the blaNDM in 11 (23.4%) and the blaVIM in 6 (12.8%). The blaOXA-48 gene was not detected.Well established antimicrobial agents such as colistin, gentamicin and tigecycline and novel antibiotics like ceftazidime/avibactam and eravacycline can be reliable options for the treatment of invasive infections caused by carbapenem-resistant K. pneumoniae.

First Report of Isolation and Genome Sequence of L. petauri Strain from a Rainbow Trout Lactococcosis Outbreak.

Lactococcosis is a disease encountered in a wide variety of fish species causing mortalities and having great economic impact on farmed fish. In this study, we report for the first time the isolation of a strain of the recently described novel species Lactococcus petauri, from rainbow trout suffering from lactococcosis. The aim of this study was to determine the complete genome sequence of L. petauri strain LG_SAV_20 and to characterize its antimicrobial resistance and virulence. The genome of L. petauri LG_SAV_20 consists of 2,078,949 base pair (bp) with a GC content of 38.05%, 1950 predicted coding sequence (CDS), and 60 RNAs (51 tRNAs, 3 ncRNAs, and 6 rRNAs). Phylogenetic analysis revealed that L. petauri LG_SAV_20 shares most of its genome with L. garvieae strains isolated from rainbow trout. Detection of genes associated with antimicrobial resistance indicated that the isolate possesses the multidrug transporter mdt(A) gene, while using comparative analysis we identified several genes that might be related to bacterial pathogenesis. This genomic information provides new insights into the role of this novel species as an etiological agent of lactococcosis.

Identification of complement-related host genetic risk factors associated with influenza A(H1N1)pdm09 outcome: challenges ahead.

Influenza remains an important threat for human health, despite the extensive study of influenza viruses and the production of effective vaccines. In contrast to virus genetics determinants, host genetic factors with clinical impact remained unexplored until recently. The association between three single nucleotide polymorphisms (SNPs) and influenza outcome in a European population was investigated in the present study. All samples were collected during the influenza A(H1N1)pdm09 post-pandemic period 2010-11 and a sufficient number of severe and fatal cases was included. Host genomic DNA was isolated from pharyngeal samples of 110 patients from northern Greece with severe (n = 59) or mild (n = 51) influenza A(H1N1)pdm09 disease, at baseline, and the genotype of CD55 rs2564978, C1QBP rs3786054 and FCGR2A rs1801274 SNPs was investigated. Our findings suggest a relationship between the two complement-related SNPs, namely, the rare TT genotype of CD55 and the rare AA genotype of C1QBP with increased death risk. No significant differences were observed for FCGR2A genotypes neither with fatality nor disease severity. Additional large-scale genetic association studies are necessary for the identification of reliable host genetic risk factors associated with influenza A(H1N1)pdm09 outcome. Prophylactic intervention of additional high-risk populations, according to their genetic profile, will be a key achievement for the fight against influenza viruses.

An initially unidentified case of urinary tract infection due to Aerococcus urinae.

Aerococcus urinae is a microorganism responsible for urinary tract and blood stream infections which are rarely reported in clinical practice. However, it has been proposed that the infrequency of such reports may be partially due to difficulties related to pathogen identification. We present here a case of an elderly male patient with urinary tract infection where A. urinae was initially not identified by a private microbiology laboratory. Our report highlights the need to consider A. urinae as a causative agent of urinary tract infections because if not identified and properly treated it may lead to endocarditis or septicemia.

Hepatotoxicity assessment of innovative nutritional supplements based on olive-oil formulations enriched with natural antioxidants.

Introduction: This study focuses on the assessment of extra virgin olive-oil and olive fruit-based formulations enriched with natural antioxidants as potential nutritional supplements for alleviating symptoms and long-term consequences of illnesses whose molecular pathophysiology is affected by oxidative stress and inflammation, such as Alzheimer's disease (AD). Methods: Besides evaluating cell viability and proliferation capacity of human hepatocellular carcinoma HepG2 cells exposed to formulations in culture, hepatotoxicity was also considered as an additional safety measure using quantitative real-time PCR on RNA samples isolated from the cell cultures and applying approaches of targeted molecular analysis to uncover potential pathway effects through gene expression profiling. Furthermore, the formulations investigated in this work contrast the addition of natural extract with chemical forms and evaluate the antioxidant delivery mode on cell toxicity. Results: The results indicate minimal cellular toxicity and a significant beneficial impact on metabolic molecular pathways in HepG2 cell cultures, thus paving the way for innovative therapeutic strategies using olive-oil and antioxidants in dietary supplements to minimize the long-term effects of oxidative stress and inflammatory signals in individuals being suffered by disorders like AD. Discussion: Overall, the experimental design and the data obtained support the notion of applying innovative molecular methodologies and research techniques to evidently advance the delivery, as well as the scientific impact and validation of nutritional supplements and dietary products to improve public health and healthcare outcomes.

Vascular access malfunction: towards a more genecentric view.

A well-functioning vascular access is the cornerstone for an optimal hemodialysis treatment and an issue of major importance for the outcome of patients on chronic hemodialysis. Over the last few years reports supporting the aspect that mechanisms involved in vascular access malfunction are genetically controlled have been published. Triggered by two cases reported herein, we present a comprehensive review of the literature on an evolving field of particular significance to patients on hemodialysis.

Single Nucleotide Polymorphisms' Causal Structure Robustness within Coronary Artery Disease Patients.

An ever-growing amount of accumulated data has materialized in several scientific fields, due to recent technological progress. New challenges emerge in exploiting these data and utilizing the valuable available information. Causal models are a powerful tool that can be employed towards this aim, by unveiling the structure of causal relationships between different variables. The causal structure may avail experts to better understand relationships, or even uncover new knowledge. Based on 963 patients with coronary artery disease, the robustness of the causal structure of single nucleotide polymorphisms was assessed, taking into account the value of the Syntax Score, an index that evaluates the complexity of the disease. The causal structure was investigated, both locally and globally, under different levels of intervention, reflected in the number of patients that were randomly excluded from the original datasets corresponding to two categories of the Syntax Score, zero and positive. It is shown that the causal structure of single nucleotide polymorphisms was more robust under milder interventions, whereas in the case of stronger interventions, the impact increased. The local causal structure around the Syntax Score was studied in the case of a positive Syntax Score, and it was found to be resilient, even when the intervention was strong. Consequently, employing causal models in this context may increase the understanding of the biological aspects of coronary artery disease.

Targeted genotyping of COVID-19 patients reveals a signature of complement C3 and factor B coding SNPs associated with severe infection.

We have attempted to explore further the involvement of complement components in the host COVID-19 (Coronavirus disease-19) immune responses by targeted genotyping of COVID-19 adult patients and analysis for missense coding Single Nucleotide Polymorphisms (coding SNPs) of genes encoding Alternative pathway (AP) components. We have identified a small group of common coding SNPs in Survivors and Deceased individuals, present in either relatively similar frequencies (CFH and CFI SNPs) or with stark differences in their relative abundance (C3 and CFB SNPs). In addition, we have identified several sporadic, potentially protective, coding SNPs of C3, CFB, CFD, CFH, CFHR1 and CFI in Survivors. No coding SNPs were detected for CD46 and CD55. Our demographic analysis indicated that the C3 rs1047286 or rs2230199 coding SNPs were present in 60 % of all the Deceased patients (n = 25) (the rs2230199 in 67 % of all Deceased Males) and in 31 % of all the Survivors (n = 105, p = 0.012) (the rs2230199 in 25 % of all Survivor Males). When we analysed these two major study groups using the presence of the C3 rs1047286 or rs2230199 SNPs as potential biomarkers, we noticed the complete absence of the protective CFB rs12614 and rs641153 coding SNPs from Deceased Males compared to Females (p = 0.0023). We propose that in these individuals, C3 carrying the R102G and CFB lacking the R32W or the R32Q amino acid substitutions, may contribute to enhanced association dynamics of the C3bBb AP pre-convertase complex assembly, thus enabling the exploitation of the activation of the Complement Alternative pathway (AP) by SARS-CoV-2.

Robust SARS-COV-2-specific T-cell immune memory persists long-term in immunocompetent individuals post BNT162b2 double shot.

Background: A robust efficiency of mRNA vaccines against coronavirus disease-2019 has been demonstrated, however, the intended long-term protection against SARS-CoV-2 has been challenged by the waning humoral and cellular immunity over time, leading to a third vaccination dose recommendation for immunocompetent individuals, six months after completion of primary mRNA vaccination. Methods: We here measured humoral responses via an immunoassay measuring SARS-CoV-2 neutralizing antibodies and T-cell responses using Elispot for interferon-γ 1- and 8- months post full BNT162b2 vaccination, in 10 health-care professionals. To explore whether the declining abundance of coronavirus-specific T-cells (CoV-2-STs) truly reflects decreased capacity for viral control, rather than the attenuating viral stimulus over time, we modeled ex vivo the T-cellular response upon viral challenge in fully vaccinated immunocompetent individuals, 1- and 8-months post BNT162b2. Findings: Notwithstanding the declining CoV-2-neutralizing antibodies and CoV-2-STs, re-challenged CoV-2-STs, 1- and 8-months post vaccination, presented similar functional characteristics including high cytotoxicity against both the unmutated virus and the delta variant. Interpretation: These findings suggest robust and sustained cellular immune response upon SARS-CοV-2 antigen exposure, 8 months post mRNA vaccination, despite declining CοV-2-STs over time in the presence of an attenuating viral stimulus.

Invariable Ribosome Stoichiometry During Murine Erythroid Differentiation: Implications for Understanding Ribosomopathies.

Heterogeneity of the main ribosomal composition represents an emerging, yet debatable, mechanism of gene expression regulation with a purported role in ribosomopathies, a group of disorders caused by mutations in ribosomal protein genes (RPs). Ribosomopathies, mysteriously relate with tissue-specific symptoms (mainly anemia and cancer predisposition), despite the ubiquitous expression and necessity of the associated RPs. An outstanding question that may shed light into disease pathogenicity and provide potential pharmacological interventions, is whether and how the ribosomal composition is modified during, the highly affected by RP mutations, process of erythroid differentiation. To address this issue, we analyzed ribosome stoichiometry using an established model of erythroid differentiation, through sucrose gradient ultracentrifugation and quantitative proteomics. We found that differentiation associates with an extensive reprogramming of the overall ribosomal levels, characterized by an increase in monosomes and a decrease in polysomes. However, by calculating a stoichiometry score for each independent ribosomal protein, we found that the main ribosomal architecture remained invariable between immature and differentiated cells. In total, none of the 78 Ribosomal Proteins (RPs- 74 core RPs, Rack1, Fau and 2 paralogs) detected was statistically different between the samples. This data was further verified through antibody-mediated quantification of 6 representative RPs. Moreover, bioinformatic analysis of whole cell proteomic data derived out of 4 additional models of erythropoiesis revealed that RPs were co-regulated across these cell types, too. In conclusion, ribosomes maintain an invariant protein stoichiometry during differentiation, thus excluding ribosome heterogeneity from a potential mechanism of toxicity in ribosomopathies and other erythroid disorders.

Immune Response of Adult Sickle Cell Disease Patients after COVID-19 Vaccination: The Experience of a Greek Center.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential weapons to control the spread of the coronavirus disease-19 (COVID-19) pandemic and protect immunocompromised patients. With a greater susceptibility to infection, sickle cell disease (SCD) patients are considered as "high risk" patients during the current COVID-19 pandemic. In our study, we try to determine the immune response of adult SCD patients monitored at our center after the first and second dose of the qualified mRNA vaccines available and correlate them to several disease-specific markers, as well as complement activation. The results demonstrate that the levels of neutralizing antibodies (nAbs) against SARS-CoV-2 were adequate for most patients studied after the second dose and there seemed to be a certain association with complement activation. Further studies are critical to determine the durability of this immune response and the potential benefit of a third dose.

Dissecting miRNA-Gene Networks to Map Clinical Utility Roads of Pharmacogenomics-Guided Therapeutic Decisions in Cardiovascular Precision Medicine.

MicroRNAs (miRNAs) create systems networks and gene-expression circuits through molecular signaling and cell interactions that contribute to health imbalance and the emergence of cardiovascular disorders (CVDs). Because the clinical phenotypes of CVD patients present a diversity in their pathophysiology and heterogeneity at the molecular level, it is essential to establish genomic signatures to delineate multifactorial correlations, and to unveil the variability seen in therapeutic intervention outcomes. The clinically validated miRNA biomarkers, along with the relevant SNPs identified, have to be suitably implemented in the clinical setting in order to enhance patient stratification capacity, to contribute to a better understanding of the underlying pathophysiological mechanisms, to guide the selection of innovative therapeutic schemes, and to identify innovative drugs and delivery systems. In this article, the miRNA-gene networks and the genomic signatures resulting from the SNPs will be analyzed as a method of highlighting specific gene-signaling circuits as sources of molecular knowledge which is relevant to CVDs. In concordance with this concept, and as a case study, the design of the clinical trial GESS (NCT03150680) is referenced. The latter is presented in a manner to provide a direction for the improvement of the implementation of pharmacogenomics and precision cardiovascular medicine trials.