Synthesis cost-optimal targeted mutant protein libraries.

Protein variant libraries produced by site-directed mutagenesis are a useful tool utilized by protein engineers to explore variants with potentially improved properties, such as activity and stability. These libraries are commonly built by selecting residue positions and alternative beneficial mutations for each position. All possible combinations are then constructed and screened, by incorporating degenerate codons at mutation sites. These degenerate codons often encode additional unwanted amino acids or even STOP codons. Our study aims to take advantage of annealing based recombination of oligonucleotides during synthesis and utilize multiple degenerate codons per mutation site to produce targeted protein libraries devoid of unwanted variants. Toward this goal we created an algorithm to calculate the minimum number of degenerate codons necessary to specify any given amino acid set, and a dynamic programming method that uses this algorithm to optimally partition a DNA target sequence with degeneracies into overlapping oligonucleotides, such that the total cost of synthesis of the target mutant protein library is minimized. Computational experiments show that, for a modest increase in DNA synthesis costs, beneficial variant yields in produced mutant libraries are increased by orders of magnitude, an effect particularly pronounced in large combinatorial libraries.

Parental Hesitancy towards the Established Childhood Vaccination Programmes in the COVID-19 Era: Assessing the Drivers of a Challenging Public Health Concern.

(1) Background: Vaccine hesitancy remains a major public health concern. The reasons behind this attitude are complex and warrant careful consideration, especially in the context of the COVID-19 era. The purpose of this study was to estimate vaccine hesitancy towards the established childhood immunization programmes in a non-random sample of Greek parents and explore possible links with important drivers of this phenomenon. (2) Methods: An online self-administered questionnaire was used from October 2020 to April 2021 to collect socio-demographic, lifestyle, and health status data and evaluate knowledge, views, and attitudes of the Greek population on COVID-19 pandemic-related issues. Parents were further asked to complete the Parent Attitudes about Childhood Vaccines (PACV) questionnaire. (3) Results: A total of 1095 parents participated in the study with a mean age of 50 years (SD 9.5 years). The hesitancy against the established childhood vaccinations was estimated at 8.9% (95% CI, 7.3-10.8%). Married status and higher education and income were negatively correlated with hesitancy, whereas positive correlations were found for stress and depressive symptoms and current smoking. Variables related to proper awareness, sound knowledge, and trust toward authorities regarding the COVID-19 pandemic were strongly associated with being less hesitant against the established childhood vaccination programmes. (4) Conclusion: The estimated parental hesitancy against the established childhood vaccination programmes is worrisome. Variables related to good awareness and knowledge of the COVID-19 pandemic were strongly associated with being less hesitant against childhood vaccinations. Since controversy surrounding COVID-19 vaccinations may decrease parents' confidence in routine childhood vaccinations, appreciating the complex reasons behind vaccine hesitancy may inform public health policies to overcome barriers and increase vaccine acceptance.

The impact of the COVID-19 pandemic on refugees and asylum seekers in Greece: A retrospective analysis of national surveillance data from 2020.

BACKGROUND: Migrants globally, including refugees and asylum seekers, have experienced adverse clinical and socioeconomic impacts of the COVID-19 pandemic. For approximately 56,000 refugees and asylum seekers in Reception and Identification Centers (RICs) and Reception Sites (RS) in Greece, living in severely substandard living conditions, prevention measures have been impossible with limited provision in terms of routine testing, surveillance, and access to healthcare. These migrant populations have experienced prolonged lockdowns and restricted movement since the pandemic began. We aimed to assess the impact of COVID-19 on refugees and asylum seekers in reception facilities in Greece and explore implications for policy and practice. METHODS: A retrospective analysis of policy documents and national surveillance data was conducted to identify COVID-19 outbreaks and estimate incidence among asylum seekers and refugees residing in these camps during the first 9 months of the epidemic in Greece (26th February - 15th November 2020). Incidence proportion (IP) of COVID-19 confirmed cases was calculated for three population groups (refugees and asylum seekers in RICs, refugees and asylum seekers in RSs, and the general population in Greece) during three time periods (first wave, second wave, and overall across the 9-month period). FINDINGS: Twenty-five COVID-19 outbreaks were identified in refugee and asylum seeker reception facilities, with 6 (85.7%) of 7 RICs and 18 (56.3%) of 32 RSs reporting at least one outbreak during the study period. The overall 9-month COVID-19 IP among refugee and asylum seeker populations residing in RSs on the Greek mainland was 1758 cases per 100,000 population; in RICs the incidence was 2052 cases per 100,000 population. Compared to the general population the risk of COVID-19 infection among refugees and asylum seekers in reception facilities was 2.5 to 3 times higher (p-value<0.001). The risk of acquiring COVID-19 infection was higher among refugee and asylum seeker populations in RSs on the Greek mainland (IP ratio: 2.45; 95% CI: 2.25-2.68) but higher still among refugee and asylum seeker populations in RICs in the Greek islands and the land border with Turkey (IP ratio: 2.86; 95% CI: 2.64-3.10), where living conditions are particularly poor. INTERPRETATION: We identified high levels of COVID-19 transmission among refugees and asylum seekers in reception facilities in Greece. The risk of COVID-19 infection among these enclosed population groups has been significantly higher than the general population of Greece, and risk increases as living conditions deteriorate. These data have immediate implications for policy and practice. Strategies are now needed to ensure refugee and asylum seeker populations are included in national response plans to reduce transmission in at-risk groups for COVID-19, alongside inclusion in plans for COVID-19 vaccine roll out.

Increase of breast-feeding in the past decade in Greece, but still low uptake: cross-sectional studies in 2007 and 2017.

OBJECTIVE: To estimate breast-feeding prevalence in Greece in 2007 and 2017, compare breast-feeding indicators and maternity hospital practices between these years, and investigate breast-feeding determinants. DESIGN: Two national cross-sectional studies (2007 and 2017) using systematic cluster sampling of babies with the same sampling design, data collection and analysis methodology. SETTING: Telephone interview with babies' mothers or fathers. PARTICIPANTS: Representative sample of infants who participated in the national neonatal screening programme (n 549 in 2017, n 586 in 2007). RESULTS: We found that breast-feeding indicators were higher in 2017 compared with 10 years before. In 2017, 94 % of mothers initiated breast-feeding. Breast-feeding rates were 80, 56 and 45 % by the end of the 1st, 4th and 6th completed month of age, respectively. At the same ages, 40, 25 and <1 % of babies, respectively, were exclusively breast-feeding. We also found early introduction of solid foods (after the 4th month of age). Maternity hospital practices favouring breast-feeding were more prevalent in 2017, but still suboptimal (63 % experienced rooming-in; 51 % experienced skin-to-skin contact in the first hour after birth; 19 % received free sample of infant formula on discharge). CONCLUSIONS: We observed an increasing trend in all breast-feeding indicators in the past decade in Greece, but breast-feeding rates - particularly rates of exclusive breast-feeding - remain low. Systematic public health initiatives targeted to health professionals and mothers are needed in order to change the prevailing baby feeding 'culture' and successfully implement the WHO recommendations for exclusive breast-feeding during the first 6 months of life.

Imaging Features of Multiple Myeloma Extramedullary Lesions in the Liver with 18F-FDG PET/CT, Contrast-Enhanced CT and MRI.

Ηepatic involvement in multiple myeloma is not common; nevertheless, it is associated with poorer outcome. Heterogeneous features have been described in few published reports so far. We present the imaging findings of PET/CT in comparison to those of MRI for two multiple myeloma (MM) patients, one with a liver lesion suspicious for myeloma metastasis on PET and one with multiple liver lesions suspicious for myeloma metastases on MRΙ. The subsequent ultrasound-guided needle biopsies confirmed the extramedullary spread of the disease in both patients. The first case exhibited a match in both functional imaging modalities (PET and MRI) but a mismatch of intense metabolic activity on 18F-fluorodeoxyglucose (18F-FDG) PET/CT and iso-attenuating liver parenchyma on contrast-enhanced CT. The second case showed a mismatch of signal elevation persistence on diffusion-weighted imaging (DWI) and physiologic 18F-FDG distribution in the liver parenchyma. These cases present different imaging features in MM lesions of the liver using PET/CT and MRI, reflecting the high disease heterogeneity in patients with MM and demonstrating that the use of both PET/CT and MRI may offer complementary information.

Adverse effect of the financial crisis in Greece on perinatal factors.

Background: Starting in 2008 recession affected many European countries and especially Greece. Previous studies have reported increases in low birth weight, preterm birth and stillbirth rates in Greece during early crisis. In our study we used data on births from 1980 to 2014 that allowed us to distinguish recent changes, which could possibly be attributed to the financial crisis, from long term trends, and controlled for maternal age and country of origin as potential confounders. Our study covered a longer period (up to 2014) than what has been studied before and looked separately at the effect of early and established crisis. Methods: We used national vital statistics data from 1980 to 2014. We performed age standardization and calculated age standardized rates and standardized rate ratios (SRRs) for perinatal factors for three time periods (pre-crisis, early crisis and established crisis) for Greek and non-Greek women. Results: We found an increase in low birth weight deliveries independent of maternal age and origin and an increased stillbirth rate in Greek women younger than 25 in early (RR = 1.42 95%CI: 1.12-1.80) and established crisis periods (RR = 1.36 95%CI: 1.07-1.72) compared with pre-crisis. Non-Greek women have also been affected, with their advantage regarding birth outcomes becoming less profound in the established crisis period (low birth weight: established crisis SRR = 0.84 95%CI: 0.82-0.87, pre-crisis SRR = 0.79 95% CI: 0.76- 0.81). Conclusions: The financial crisis has possibly adversely affected perinatal factors in Greece. Our results highlight the need of appropriate public health interventions and family support policies, especially for younger people, unemployed and immigrants.

Codon Context Optimization in Synthetic Gene Design.

Advances in de novo synthesis of DNA and computational gene design methods make possible the customization of genes by direct manipulation of features such as codon bias and mRNA secondary structure. Codon context is another feature significantly affecting mRNA translational efficiency, but existing methods and tools for evaluating and designing novel optimized protein coding sequences utilize untested heuristics and do not provide quantifiable guarantees on design quality. In this study we examine statistical properties of codon context measures in an effort to better understand the phenomenon. We analyze the computational complexity of codon context optimization and design exact and efficient heuristic gene recoding algorithms under reasonable constraint models. We also present a web-based tool for evaluating codon context bias in the appropriate context.

Low vaccination coverage of Greek Roma children amid economic crisis: national survey using stratified cluster sampling.

Background: Research on Roma health is fragmentary as major methodological obstacles often exist. Reliable estimates on vaccination coverage of Roma children at a national level and identification of risk factors for low coverage could play an instrumental role in developing evidence-based policies to promote vaccination in this marginalized population group. Methods: We carried out a national vaccination coverage survey of Roma children. Thirty Roma settlements, stratified by geographical region and settlement type, were included; 7-10 children aged 24-77 months were selected from each settlement using systematic sampling. Information on children's vaccination coverage was collected from multiple sources. In the analysis we applied weights for each stratum, identified through a consensus process. Results: A total of 251 Roma children participated in the study. A vaccination document was presented for the large majority (86%). We found very low vaccination coverage for all vaccines. In 35-39% of children 'minimum vaccination' (DTP3 and IPV2 and MMR1) was administered, while 34-38% had received HepB3 and 31-35% Hib3; no child was vaccinated against tuberculosis in the first year of life. Better living conditions and primary care services close to Roma settlements were associated with higher vaccination indices. Conclusions: Our study showed inadequate vaccination coverage of Roma children in Greece, much lower than that of the non-minority child population. This serious public health challenge should be systematically addressed, or, amid continuing economic recession, the gap may widen. Valid national estimates on important characteristics of the Roma population can contribute to planning inclusion policies.

[Neuroendocrine tumors: Peptide receptors radionuclide therapy (PRRT)].

Neuroendocrine tumors (neuroendocrine tumors-NET) are a heterogeneous group of neoplasms with a common embryological origin and diverse biological behavior, derived from cells of the neuroendocrine system, the system APUD (amine precursor uptake and decarboxylation). They are characterized by overexpression of all five somatostatin receptors (SSTR1-SSTR5), particularly type 2 (SST2). Surgical resection of the tumor is the treatment option, with a possibility of complete remission in patients with limited disease. Somatostatin analogs (octreotide and lanreotide) are the treatment of choice in patients with residual disease, particularly when it comes to NET non-pancreatic origin. Systemic chemotherapy is administered primarily to patients with poorly differentiated carcinomas. PRRT treatment is recommended in case of non-responsiveness of the disease. The ideal candidates for PRRT are patients with unresectable disease of high and intermediate differentiation. Somatostatine analogs radiolabelled with Indium-111 ((111)In), Yttrium-90 ((90)Y), Lutetium-177 ((177)Lu) and Bismuth-213 ((213)Bi), are selectively concentrated in the tumor cells, causing maximum tissue damage to tumors and with fewer effects on healthy tissue and the immune system. In the current review, it was demonstrated that patients with unresectable grade 1 or 2 disease showed increased PFS (progression free survival) and OS (overall survival), while quality of life was improved after PRRT treatment as compared to somatostatin analogs, chemotherapy and other targeted therapies.

Interventions to increase seasonal influenza vaccine coverage in healthcare workers: A systematic review and meta-regression analysis.

Influenza vaccination is recommended for healthcare workers (HCWs), but coverage is often low. We reviewed studies evaluating interventions to increase seasonal influenza vaccination coverage in HCWs, including a meta-regression analysis to quantify the effect of each component. Fourty-six eligible studies were identified. Domains conferring a high risk of bias were identified in most studies. Mandatory vaccination was the most effective intervention component (Risk Ratio of being unvaccinated [RRunvacc] = 0.18, 95% CI: 0.08-0.45), followed by "soft" mandates such as declination statements (RRunvacc = 0.64, 95% CI: 0.45-0.92), increased awareness (RRunvacc = 0.83, 95% CI: 0.71-0.97) and increased access (RRunvacc = 0.88, 95% CI: 0.78-1.00). For incentives the difference was not significant, while for education no effect was observed. Heterogeneity was substantial (τ(2) = 0.083). These results indicate that effective alternatives to mandatory HCWs influenza vaccination do exist, and need to be further explored in future studies.

Computational tools and algorithms for designing customized synthetic genes.

Advances in DNA synthesis have enabled the construction of artificial genes, gene circuits, and genomes of bacterial scale. Freedom in de novo design of synthetic constructs provides significant power in studying the impact of mutations in sequence features, and verifying hypotheses on the functional information that is encoded in nucleic and amino acids. To aid this goal, a large number of software tools of variable sophistication have been implemented, enabling the design of synthetic genes for sequence optimization based on rationally defined properties. The first generation of tools dealt predominantly with singular objectives such as codon usage optimization and unique restriction site incorporation. Recent years have seen the emergence of sequence design tools that aim to evolve sequences toward combinations of objectives. The design of optimal protein-coding sequences adhering to multiple objectives is computationally hard, and most tools rely on heuristics to sample the vast sequence design space. In this review, we study some of the algorithmic issues behind gene optimization and the approaches that different tools have adopted to redesign genes and optimize desired coding features. We utilize test cases to demonstrate the efficiency of each approach, as well as identify their strengths and limitations.

Population seroprevalence study after a West Nile virus lineage 2 epidemic, Greece, 2010.

INTRODUCTION: During summer 2010, 262 human cases including 35 deaths from West Nile virus (WNV) infection were reported from Central Macedonia, Greece. Evidence from mosquitoes, birds and blood donors demonstrated that the epidemic was caused by WNV lineage 2, which until recently was considered of low virulence. We conducted a household seroprevalence study to estimate the spread of infection in the population during the epidemic, ascertain the relationship of infection to clinical disease, and identify risk factors for infection. METHODS: We used a two-stage cluster design to select a random sample of residents aged ≥18 years in the outbreak epicentre. We collected demographic, medical, and risk factor data using standard questionnaires and environmental checklists, and tested serum samples for presence of WNV IgG and IgM antibodies using ELISA. RESULTS: Overall, 723 individuals participated in the study, and 644 blood samples were available. Weighted seropositivity for IgG antibodies was 5.8% (95% CI: 3.8-8.6; n=41). We estimated that about 1 in 130 (1:141 to 1:124) infected individuals developed WNV neuroinvasive disease, and approximately 18% had clinical manifestations attributable to their infection. Risk factors for infection reflected high exposure to mosquitoes; rural residents were particularly at risk (prevalence ratio: 8.2, 95% CI: 1.1-58.7). DISCUSSION: This study adds to the evidence that WNV lineage 2 strains can cause significant illness, demonstrating ratios of infection to clinical disease similar to those found previously for WNV lineage 1.

Rational design of orthogonal libraries of protein coding genes.

Array-based oligonucleotide synthesis technologies provide access to thousands of custom-designed sequence variants at low cost. Large-scale synthesis and high-throughput assays have become valuable experimental tools to study in detail the interplay between sequence and function. We have developed a methodology and corresponding algorithms for the design of diverse protein coding gene libraries, to exploit the potential of multiplex synthesis and help elucidate the effects of codon utilization and other factors in gene expression. Using our algorithm, we have computationally designed gene libraries with hundreds to thousands of orthogonal codon usage variants, uniformly exploring the design space of codon utilization, while demanding only a small fraction of the synthesis cost that would be required if these variants were synthesized independently.

Ecosystem approach to water resources management using the MIKE 11 modeling system in the Strymonas River and Lake Kerkini.

The ability to apply an ecosystem approach to the Strymonas River catchment was investigated using the MIKE 11 modeling system for the simulation of surface water. The Strymonas River catchment is shared mainly between Bulgaria and Greece. The river feeds the artificial Lake Kerkini, a significant wetland ecosystem, and further downstream it outflows to the Gulf of Strymonikos, whose estuary ecosystem is very important for fisheries, biodiversity and tourism. MIKE 11-NAM was used for the simulation of rainfall-runoff process in the Strymonas River catchment and MIKE 11-HD was used to simulate the unsteady flow of the Strymonas River and to apply management rules based on the water level of Lake Kerkini. Two water level management scenarios were investigated. The first scenario referred to the mean daily-observed water level of Lake Kerkini between 1986 and 2006, and the second scenario represented adjustments necessary to fulfill the lake's ecosystem requirements. Under the current water level management practices (Scenario 1), the Strymonas River-Lake Kerkini system has enough water to fulfill its Irrigation Water Requirements (IWR) in normal and wet years while a slight deficit is appeared in dry years; however, both Lake Kerkini and the Strymonas River estuary ecosystems are subject to pressures, since reduction of the forest area has been recorded. Applying the ecosystem approach (Scenario 2), the protection of the riparian forest of Lake Kerkini is achieved while in normal and wet years the IWR are fulfilled and the deficit of the IWR is increased in dry years. Compared to Scenario 1, the pressure of the Strymonas River estuary ecosystem is slightly increased.

Designed reduction of Streptococcus pneumoniae pathogenicity via synthetic changes in virulence factor codon-pair bias.

In this study, we used a previously described method of controlling gene expression with computer-based gene design and de novo DNA synthesis to attenuate the virulence of Streptococcus pneumoniae. We produced 2 S. pneumoniae serotype 3 (SP3) strains in which the pneumolysin gene (ply) was recoded with underrepresented codon pairs while retaining its amino acid sequence and determined their ply expression and pneumolysin production in vitro and their virulence in a mouse pulmonary infection model. Expression of ply and production of pneumolysin of the recoded SP3 strains were decreased, and the recoded SP3 strains were less virulent in mice than the wild-type SP3 strain or a Δply SP3 strain. Further studies showed that the least virulent recoded strain induced a markedly reduced inflammatory response in the lungs compared with the wild-type or Δply strain. These findings suggest that reducing pneumococcal virulence gene expression by altering codon-pair bias could hold promise for rational design of live-attenuated pneumococcal vaccines.

Live attenuated influenza virus vaccines by computer-aided rational design.

Despite existing vaccines and enormous efforts in biomedical research, influenza annually claims 250,000-500,000 lives worldwide, motivating the search for new, more effective vaccines that can be rapidly designed and easily produced. We applied the previously described synthetic attenuated virus engineering (SAVE) approach to influenza virus strain A/PR/8/34 to rationally design live attenuated influenza virus vaccine candidates through genome-scale changes in codon-pair bias. As attenuation is based on many hundreds of nucleotide changes across the viral genome, reversion of the attenuated variant to a virulent form is unlikely. Immunization of mice by a single intranasal exposure to codon pair-deoptimized virus conferred protection against subsequent challenge with wild-type (WT) influenza virus. The method can be applied rapidly to any emerging influenza virus in its entirety, an advantage that is especially relevant when dealing with seasonal epidemics and pandemic threats, such as H5N1- or 2009-H1N1 influenza.

Improved algorithms for approximate string matching (extended abstract).

BACKGROUND: The problem of approximate string matching is important in many different areas such as computational biology, text processing and pattern recognition. A great effort has been made to design efficient algorithms addressing several variants of the problem, including comparison of two strings, approximate pattern identification in a string or calculation of the longest common subsequence that two strings share. RESULTS: We designed an output sensitive algorithm solving the edit distance problem between two strings of lengths n and m respectively in time O((s - |n - m|).min(m, n, s) + m + n) and linear space, where s is the edit distance between the two strings. This worst-case time bound sets the quadratic factor of the algorithm independent of the longest string length and improves existing theoretical bounds for this problem. The implementation of our algorithm also excels in practice, especially in cases where the two strings compared differ significantly in length. CONCLUSION: We have provided the design, analysis and implementation of a new algorithm for calculating the edit distance of two strings with both theoretical and practical implications. Source code of our algorithm is available online.

Virus attenuation by genome-scale changes in codon pair bias.

As a result of the redundancy of the genetic code, adjacent pairs of amino acids can be encoded by as many as 36 different pairs of synonymous codons. A species-specific "codon pair bias" provides that some synonymous codon pairs are used more or less frequently than statistically predicted. We synthesized de novo large DNA molecules using hundreds of over-or underrepresented synonymous codon pairs to encode the poliovirus capsid protein. Underrepresented codon pairs caused decreased rates of protein translation, and polioviruses containing such amino acid-independent changes were attenuated in mice. Polioviruses thus customized were used to immunize mice and provided protective immunity after challenge. This "death by a thousand cuts" strategy could be generally applicable to attenuating many kinds of viruses.

Elevated atmospheric CO2 affects soil microbial diversity associated with trembling aspen.

The effects of elevated atmospheric CO(2) (560 p.p.m.) and subsequent plant responses on the soil microbial community composition associated with trembling aspen was assessed through the classification of 6996 complete ribosomal DNA sequences amplified from the Rhinelander WI free-air CO(2) and O(3) enrichment (FACE) experiments microbial community metagenome. This in-depth comparative analysis provides an unprecedented, detailed and deep branching profile of population changes incurred as a response to this environmental perturbation. Total bacterial and eukaryotic abundance does not change; however, an increase in heterotrophic decomposers and ectomycorrhizal fungi is observed. Nitrate reducers of the domain bacteria and archaea, of the phylum Crenarchaea, potentially implicated in ammonium oxidation, significantly decreased with elevated CO(2). These changes in soil biota are evidence for altered interactions between trembling aspen and the microorganisms in its surrounding soil, and support the theory that greater plant detritus production under elevated CO(2) significantly alters soil microbial community composition.