An expandable, modular de novo protein platform for precision redox engineering.

The electron-conducting circuitry of life represents an as-yet untapped resource of exquisite, nanoscale biomolecular engineering. Here, we report the characterization and structure of a de novo diheme "maquette" protein, 4D2, which we subsequently use to create an expanded, modular platform for heme protein design. A well-folded monoheme variant was created by computational redesign, which was then utilized for the experimental validation of continuum electrostatic redox potential calculations. This demonstrates how fundamental biophysical properties can be predicted and fine-tuned. 4D2 was then extended into a tetraheme helical bundle, representing a 7 nm molecular wire. Despite a molecular weight of only 24 kDa, electron cryomicroscopy illustrated a remarkable level of detail, indicating the positioning of the secondary structure and the heme cofactors. This robust, expressible, highly thermostable and readily designable modular platform presents a valuable resource for redox protein design and the future construction of artificial electron-conducting circuitry.

Does Vaccinating against Influenza in a Given Epidemic Season Have an Impact on Vaccination in the Next Season: A Follow-Up Study.

To improve the uptake of influenza vaccine in the elderly, it is important to understand the factors that predict vaccination. The study objective was to explain influenza vaccination uptake in the next season (2019/2020) in a sample of primary care clinic patients from Gryfino, Poland, vaccinated in 2018/2019 with the free-of-charge quadrivalent vaccine. A baseline and a follow-up survey assessed respondent intentions to receive a vaccine (2018), then (2020) vaccine uptake and its predictors. Patients (n = 108, 54.6% males, Mage = 66.7 ± 6.7) filled in a researcher-administered questionnaire. A majority (69.3%) intended to get vaccinated in the next season, with 25.9% receipt. Of those willing to be immunized, only 31.9% were vaccinated in the next season; of those whose decision was dependent on reimbursement, none received influenza vaccine; of undecided patients, 23.1% were vaccinated. Multivariable analysis indicated that living with a partner (OR 6.22, p = 0.01), being employed (OR = 4.55, p = 0.05) and past vaccination behavior (OR 4.12; p = 0.04) were predictors of vaccine uptake. The findings show limited follow-through on initial influenza vaccination plans for the nearest season in previously vaccinated elderly patients. Future interventions should additionally focus on unanticipated barriers to vaccination, such as those revealed in this study, to increase vaccination coverage rates.

Vaccination concerns, beliefs and practices among Ukrainian migrants in Poland: a qualitative study.

BACKGROUND: Ukrainians numbering approximately 1.2 million are the largest migrant group in Poland. Data on vaccination coverage among migrants are not collected in EU, including Poland. Therefore, this qualitative study aimed to identify vaccination practices in this migrant group, to explore facilitators and barriers to vaccination and related access to Polish healthcare services. METHODS: In September 2019, a qualitative study of Ukrainian migrants (UMs) living in Szczecin, Poland, and recruited through a snowball sampling method, was conducted. Using a semi-structured topic guide, four focus groups were held with 22 UMs aged 18-45. Participants were asked about their attitudes towards vaccination in general with comparison between services in Poland and Ukraine. Following transcription and translation, a thematic analysis was conducted. RESULTS: Respondents were distrustful of Ukrainian vaccination policy, medical personnel and individual vaccines, however, they often returned to Ukraine for dental and gynaecological appoint-ments. While critical with regards to registering with Polish GPs practices, UMs were confident in health professionals, as well as vaccine delivery. Vaccines were perceived as safer and of better quality than in Ukraine. Difficulties in translating vaccination records were rarely reported, verbal communi-cation was not problematic due to language similarities. All UM parents reported vaccinating their children according to the Polish schedule. However, a significant number of adult UMs have not completed mandatory vaccinations, although they may have obtained false immunization certificates; according to UMs those can be obtained by bribing. Participants reported lower acceptance of the influenza vaccine, mainly due to perceptions around its importance; none had been vaccinated against influenza. None of UMs had heard of the HPV vaccine. UMs experienced challenges in accessing credible online vaccination information in Ukrainian, no official local health authority vaccination material existed either, except for information about measles. CONCLUSIONS: This study pinpointed positive UM attitudes and practices regarding child vaccination in the Polish healthcare system and identified issues for improvement, such as adult vaccination. Health communication should be more tailored within UMs information delivery systems to enable migrants to make informed choices about vaccination. Further research is needed to better assess factors affecting vaccine uptake identified in this study.

School life and influenza immunization: A cross-sectional study on vaccination coverage and influencing determinants among Polish teachers.

OBJECTIVE: The general public influenza vaccination coverage in Poland is one of the lowest in the EU (3.7% in 2017/2018). Teachers have the potential to be a target group for immunization programs against influenza infection, however, there is yet to be a study in Poland, or even in the EU that has assessed influenza vaccine uptake among this group. The study objective was to evaluate influenza vaccination coverage and to assess influencing determinants amongst Polish teachers. METHOD: A cross-sectional questionnaire-based study was conducted among 277 teachers from 9 primary schools of two regional capitals of two Polish regions: Szczecin and Lublin. RESULTS: A quarter of teachers have ever received influenza vaccine; 4.5% reported receiving vaccination in the 2018/2019 season. The main reasons for not being vaccinated were a lack of confidence in its effectiveness (56.9%) and concerns related to adverse effects (30.6%). Forty four percent (43.8%) of teachers believed that they are at risk of influenza infection, only 62.5% indicated vaccination as an effective method of preventing influenza. Previous information about influenza (OR = 15.70), high knowledge level about influenza (OR = 2.56), family physician recommendation (OR = 2.39), belief that influenza vaccination should be mandatory for teachers (OR = 3.29), and having a vaccinated family member (OR = 2.68) were each associated with higher odds of immunization. Willingness to be vaccinated against influenza in the next season was strongly associated with current vaccination status (OR = 7.16). CONCLUSIONS: Influenza vaccination coverage among Polish teachers is alarmingly low; vaccine receipt was associated with teacher attitudes and beliefs. Future interventions related to maximizing vaccination coverage in this group should take advantage of the involvement of family physicians and specifically focus on teachers who have never been vaccinated before. As knowledge about influenza positively influences vaccination decisions, education strategies should focus on reducing knowledge gaps to alter attitudes and increase uptake.

Quadrivalent Influenza Vaccine-Induced Antibody Response and Influencing Determinants in Patients ≥ 55 Years of Age in the 2018/2019 Season.

The effects of immunization with subunit inactivated quadrivalent influenza vaccine (QIV) are not generally well assessed in the elderly Polish population. Therefore, this study evaluated vaccine-induced antibody response and its determinants. METHODS: Consecutive patients ≥ 55 years old, attending a Primary Care Clinic in Gryfino, Poland, received QIV (A/Michigan/ 45/2015(H1N1)pdm09, A/Singapore/INFIMH-16-0019/2016 (H3N2), B/Colorado/06/2017, B/Phuket/ 3073/2013) between October-December 2018. Hemagglutination inhibition assays measured antibody response to vaccine strains from pre/postvaccination serum samples. Geometric mean titer ratio (GMTR), protection rate (PR) and seroconversion rate (SR) were also calculated. RESULTS: For 108 patients (54.6% males, mean age: 66.7 years) the highest GMTR (61.5-fold) was observed for A/H3N2/, then B/Colorado/06/2017 (10.3-fold), A/H1N1/pdm09 (8.4-fold) and B/Phuket/ 3073/2013 (3.0-fold). Most patients had post-vaccination protection for A/H3N2/ and B/Phuket/3073/ 2013 (64.8% and 70.4%, respectively); lower PRs were observed for A/H1N1/pdm09 (41.8%) and B/Colorado/06/ 2017 (57.4%). The SRs for A/H3N2/, A/H1N1/pdm09, B Victoria and B Yamagata were 64.8%, 38.0%, 46.8%, and 48.2%, respectively. Patients who received QIV vaccination in the previous season presented lower (p < 0.001 and p = 0.03, respectively) response to B Victoria and B Yamagata. CONCLUSIONS: QIV was immunogenic against the additional B lineage strain (B Victoria) without significantly compromising the immunogenicity of the other three vaccine strains, therefore, adding a second B lineage strain in QIV could broaden protection against influenza B infection in this age group. As the QIV immunogenicity differed regarding the four antigens, formulation adjustments to increase the antigen concentration of the serotypes that have lower immunogenicity could increase effectiveness. Prior season vaccination was associated with lower antibody response to a new vaccine, although not consistent through the vaccine strains.

Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine.

Cytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l-1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s' role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

Characterization and structure-guided engineering of the novel versatile terpene monooxygenase CYP109Q5 from Chondromyces apiculatus DSM436.

One of the major challenges in chemical synthesis is the selective oxyfunctionalization of non-activated C-H bonds, which can be enabled by biocatalysis using cytochrome P450 monooxygenases. In this study, we report on the characterization of the versatile CYP109Q5 from Chondromyces apiculatus DSM436, which is able to functionalize a wide range of substrates (terpenes, steroids and drugs), including the ring of ß-ionone in non-allylic positions. The crystal structure of CYP109Q5 revealed flexibility within the active site pocket that permitted the accommodation of bulky substrates, and enabled a structure-guided approach to engineering the enzyme. Some variants of CYP109Q5 displayed a switch in selectivity towards the non-allylic positions of ß-ionone, allowing the simultaneous production of 2- and 3-hydroxy-ß-ionone, which are chemically challenging to synthesize and are important precursors for carotenoid synthesis. An efficient whole-cell system finally enabled the production of up to 0.5 g l-1 hydroxylated products of ß-ionone; this system can be applied to product identification in further biotransformations. Overall, CYP109Q5 proved to be highly evolvable and active. The studies in this work demonstrate that, using rational mutagenesis, the highly versatile CYP109Q5 generalist can be progressively evolved to be an industrially valuable specialist for the synthesis of specific products.

Risk Model in Women with Ovarian Cancer Without Mutations.

Ovarian cancer is characterised by the greatest mortality among all tumors of the reproductive tract. This study included 246 patients which consisted of 136 women with ovarian cancer without genetic mutation and 110 women with benign ovarian cysts. We created two mathematical logic models containing positive and negative risk factors of ovarian cancer such as: age at last menstruation cycle, patient age, OC, HRT, smoking, education status, and alcohol consumption. The calculated cut-off point for the first model was 0.5117. Classification determined on the basis of that cut-off point yielded 87.19% of correctly classified cases, of which 91.38% are "case" and 81.61% - "noncase". For the second model the designated cut-off point was set at 0.5149 and the percentage of correctly classified patients was 88.12%, with 92.24% correctly rated as cancer patients and 82.56% of the cases rightly recognised as having no ovarian cancer. Logit is a simple mathematical model that can be a useful tool for identification of patients with increased risk of ovarian cancer.