Oral Health in 12- and 15-Year-Old Children in Serbia: A National Pathfinder Study.

The aim of the paper is to present the oral health profile of 12- and 15-year-old schoolchildren in Serbia. Basic Methods for Oral Health Surveys of the WHO were implemented to record: Decayed, Missing, and Filled Teeth/Surfaces Index (DMFT/DMFS), gingival bleeding, enamel fluorosis and other structural anomalies, dental erosion, dental trauma, and oral mucosal lesions. In addition, Silness and Löe plaque index and orthodontic status were assessed. A total of 36% of 12-year-olds and 22% of 15-year-olds in Serbia were caries-free. The mean DMFT was 2.32 ± 2.69 for 12-year-olds and 4.09 ± 3.81 for 15-year-olds. DMFT was made up largely by the decayed component. Gingival bleeding was present in 26% of examined 12-year-old and 18% of 15-year-old children. Dental plaque was observed in 63% of both 12- and 15-year-olds. Fluorosis, structural anomalies, dental erosion, dental trauma, and oral mucosal lesion were rarely detected. Low prevalence of malocclusions was found. Oral disease is still a common public health problem among schoolchildren in Serbia. A significant increase in the prevalence of caries disease between 12- and 15-year-old groups implies that preventive care for adolescents requires special attention. Corrective actions and reforms to the current school-based oral health prevention program are needed to further improve oral health in Serbian children

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A longstanding design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method that performs rigid three-body fusion of homo-oligomer and spacer building blocks to generate user-defined architectures, while at the same time significantly increasing the number of geometric solutions over typical symmetric fusion. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies from a set of designed homo-dimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from DARPins (designed ankyrin repeat proteins), anchored on one end by α-helical fusion and on the other by a designed homo-dimer interface, and we explored their use for cryo-EM structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects, small scaffold size, and the low-order symmetry of these dihedral scaffolds, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins

Software Algorithms for Design of Symmetric Protein Complexes Applied to Cryo-Electron Microscopy Scaffolds and Antibody Nanoparticles

Thesis (Ph.D.)--University of Washington, 2020Innovation in the symmetric assembly and protein material design space has the potential to – eventually – reinvent medicine and nanotechnology. One leading strategy for design of symmetric assemblies uses genetic fusion of protein homo-oligomer subunits via α-helical linkers. For the nearly two decades since its inception in 2001, this method has been applied as it was originally formulated. In this dissertation, I present the development and application of a tripartite fusion method that builds on this work and addresses two of its principal limitations: linker flexibility and a dearth of geometric solutions. The method is applied to investigate two proof-of-principle design concepts: 1) target capture and structure determination on symmetric cryo-EM scaffolds 2) antibody display and integration into nanoparticles. Designs are characterized by native mass spectrometry, small angle X-ray scattering, and electron microscopy. The experimental results in both of these areas showcase the viability and promise of this design strategy for further use

Determination of Fluorine Ions Penetration Degree Into Enamel by Empa.

Exogen of different preparations was applied to completely healthy teeth of children up to 11 years. A month after application of fluorine, teeth were extracted and prepared by standard methods for investigation by electron microprobe ARL, type SEMQ. The results obtained showed that the highest degree of fluorine ions migration was stimulated by organic fluorite' (amin fluoride). Very poor results were obtained by NaF, while the values obtained by 'fluor-protector' ranges between these two preparates. All up to date investigations showed that protective characteristics of fluorine against caries formation were reached only in case when enamel surface contained fluorine concentration of 10**3, mu g. Thus, our results proved that the first condition for good prophylaxy is obtained by application of aminofluoride and then by 'fluor-protector'

Predicting severity and intrahospital mortality in CovID-19: The place and role of oxidative stress

SARS-CoV-2 virus causes infection which led to a global pandemic in 2020 with the development of severe acute respiratory syndrome. Therefore, this study was aimed at examining its possible role in predicting severity and intrahospital mortality of COVID-19, alongside with other laboratory and biochemical procedures, clinical signs, symptoms, and comorbidity. This study, approved by the Ethical Committee of Clinical Center Kragujevac, was designed as an observational prospective cross-sectional clinical study which was conducted on 127 patients with diagnosed respiratory COVID-19 viral infection from April to August 2020. The primary goals were to determine the predictors of COVID-19 severity and to determine the predictors of the negative outcome of COVID-19 infection. All patients were divided into three categories: patients with a mild form, moderate form, and severe form of COVID-19 infection. All biochemical and laboratory procedures were done on the first day of the hospital admission. Respiratory (p < 0:001) and heart (p = 0:002) rates at admission were significantly higher in patients with a severe form of COVID-19. From all observed hematological and inflammatory markers, only white blood cell count (9:43 ± 4:62, p = 0:001) and LDH (643:13 ± 313:3, p = 0:002) were significantly higher in the severe COVID-19 group. We have observed that in the severe form of SARS-CoV-2, the levels of superoxide anion radicals were substantially higher than those in two other groups (11:3 ± 5:66, p < 0:001) and the nitric oxide level was significantly lower in patients with the severe disease (2:66 ± 0:45, p < 0:001). Using a linear regression model, TA, anosmia, ageusia, O2-, and the duration at the ICU are estimated as predictors of severity of SARS-CoV-2 disease. The presence of dyspnea and a higher heart rate were confirmed as predictors of a negative, fatal outcome. Results from our study show that presence of hypertension, anosmia, and ageusia, as well as the duration of ICU stay, and serum levels of O2- are predictors of COVID-19 severity, while the presence of dyspnea and an increased heart rate on admission were predictors of COVID-19 mortality

Predicting Severity and Intrahospital Mortality in COVID-19: The Place and Role of Oxidative Stress

SARS-CoV-2 virus causes infection which led to a global pandemic in 2020 with the development of severe acute respiratory syndrome. Therefore, this study was aimed at examining its possible role in predicting severity and intrahospital mortality of COVID-19, alongside with other laboratory and biochemical procedures, clinical signs, symptoms, and comorbidity. This study, approved by the Ethical Committee of Clinical Center Kragujevac, was designed as an observational prospective cross-sectional clinical study which was conducted on 127 patients with diagnosed respiratory COVID-19 viral infection from April to August 2020. The primary goals were to determine the predictors of COVID-19 severity and to determine the predictors of the negative outcome of COVID-19 infection. All patients were divided into three categories: patients with a mild form, moderate form, and severe form of COVID-19 infection. All biochemical and laboratory procedures were done on the first day of the hospital admission. Respiratory (p<0.001) and heart (p=0.002) rates at admission were significantly higher in patients with a severe form of COVID-19. From all observed hematological and inflammatory markers, only white blood cell count (9.43±4.62, p=0.001) and LDH (643.13±313.3, p=0.002) were significantly higher in the severe COVID-19 group. We have observed that in the severe form of SARS-CoV-2, the levels of superoxide anion radicals were substantially higher than those in two other groups (11.3±5.66, p<0.001) and the nitric oxide level was significantly lower in patients with the severe disease (2.66±0.45, p<0.001). Using a linear regression model, TA, anosmia, ageusia, O2−, and the duration at the ICU are estimated as predictors of severity of SARS-CoV-2 disease. The presence of dyspnea and a higher heart rate were confirmed as predictors of a negative, fatal outcome. Results from our study show that presence of hypertension, anosmia, and ageusia, as well as the duration of ICU stay, and serum levels of O2− are predictors of COVID-19 severity, while the presence of dyspnea and an increased heart rate on admission were predictors of COVID-19 mortality

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method for rigidly fusing homo-oligomer and spacer building blocks to generate user-defined architectures that generates far more geometric solutions than previous approaches. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies using a set of designed homodimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small-angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from designed ankyrin repeat proteins (DARPins), held in place on one end by α-helical fusion and on the other by a designed homodimer interface, and we explored their use for cryogenic electron microscopy (cryo-EM) structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects and small scaffold size, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins

Nature versus design: the conformational propensities of D-amino acids and the importance of side chain chirality

NoD-amino acids are useful building blocks for de novo peptide design and they play a role in aging-related diseases associated with gradual protein racemization. For amino acids with achiral side chains, one should be able to presume that the conformational propensities of L- and D-amino acids are a reflection of one another due to the straightforward geometric inversion at the Cα atom. However, this presumption does not account for the directionality of the backbone dipole and the inverted propensities have never been definitively confirmed in this context. Furthermore, there is little known of how alternative side chain chirality affects the backbone conformations of isoleucine and threonine. Using a GGXGG host-guest pentapeptide system, we have completed exhaustive sampling of the conformational propensities of the D-amino acids, including D-allo-isoleucine and D-allo-threonine, using atomistic molecular dynamics simulations. Comparison of these simulations with the same systems hosting the cognate L-amino acids verifies that the intrinsic backbone conformational propensities of the D-amino acids are the inverse of their cognate L-enantiomers. Where amino acids have a chiral center in their side chain (Thr, Ile) the β-configuration affects the backbone sampling, which in turn can confer different biological properties.NI

New Dynamic Rotamer Libraries: Data-Driven Analysis of Side-Chain Conformational Propensities

NoMost rotamer libraries are generated from subsets of the PDB and do not fully represent the conformational scope of protein side chains. Previous attempts to rectify this sparse coverage of conformational space have involved application of weighting and smoothing functions. We resolve these limitations by using physics-based molecular dynamics simulations to determine more accurate frequencies of rotameric states. This work forms part of our Dynameomics initiative and uses a set of 807 proteins selected to represent 97% of known autonomous protein folds, thereby eliminating the bias toward common topologies found within the PDB. Our Dynameomics derived rotamer libraries encompass 4.8 × 10(9) rotamers, sampled from at least 51,000 occurrences of each of 93,642 residues. Here, we provide a backbone-dependent rotamer library, based on secondary structure ϕ/ψ regions, and an update to our 2011 backbone-independent library that addresses the doubling of our dataset since its original publication.NI