A Protocol for Preparing Mucoadhesive Emulsion Microgels and Assessing Their Mucoadhesion Properties In Vitro.

Intravesical instillation is an efficient therapeutic technique based on targeted administration of a drug directly into the lesion for the treatment of bladder diseases. This is an alternative to traditional systemic administration of drugs. However, this technique requires repeated procedures, which can lead to even greater inflammation and infection of the urethra. To date, novel systems that allow prolonged drug retention in the bladder cavity are actively being developed. We recently reported a targeted drug delivery system based on the mucoadhesive emulsion microgels consisting of the natural component whey protein isolate. Such micron-sized carriers possess high loading capacity, a prolonged drug release profile, and efficient mucoadhesive properties to the bladder urothelium. As a continuation of this work, we present a protocol for the synthesis of mucoadhesive emulsion microgels. Detailed procedures for preparing precursor solutions as well as studying the physico-chemical parameters of microgels (including loading capacity and drug release rate) and the mucoadhesive properties using the model of porcine bladder urothelium are discussed. Precautionary measures and nuances that are worth paying attention to during each experimental stage are given as well. Key features • The protocol for the synthesis of mucoadhesive emulsion microgels based on whey protein isolate is presented. The experimental conditions of emulsion microgels synthesis are discussed. • Methods for studying the physico-chemical properties of mucoadhesive emulsion microgels (size of emulsion microgels particles, loading capacity, release kinetics) are described. • The method for assessing mucoadhesive properties of emulsion microgels is demonstrated using the porcine bladder tissue model ex vivo.

Mucoadhesive Emulsion Microgels for Intravesical Drug Delivery: Preparation, Retention at Urothelium, and Biodistribution Study.

The intravesical instillation procedure is a proven method in modern urology for the treatment of bladder diseases. However, the low therapeutic efficiency and painfulness of the instillation procedure are significant limitations of this method. In the present study, we propose an approach to solving this problem by using microsized mucoadhesive macromolecular carriers based on whey protein isolate with the possibility of prolonged release of drugs as a drug delivery system. The optimal water-to-oil ratio (1:3) and whey protein isolate concentration (5%) were determined to obtain emulsion microgels with sufficient loading efficiency and mucoadhesive properties. The droplet diameter of emulsion microgels varies from 2.2 to 3.8 µm. The drug release kinetics from the emulsion microgels was evaluated. The release of the model dye in saline and artificial urine in vitro was observed for 96 h and reached up to 70% of loaded cargo for samples. The effect of emulsion microgels on the morphology and viability of two cell lines was observed: L929 mouse fibroblasts (normal adherent cells) and THP-1 human monocytes (cancer suspension cells). Developed emulsion microgels (5%, 1:3 and 1:5) showed sufficient mucoadhesion to a porcine bladder urothelium ex vivo. The biodistribution of emulsion microgels (5%, 1:3 and 1:5) in mice (n = 3) after intravesical (instillation) and systemic (intravenous) administration was assessed in vivo and ex vivo using near-infrared fluorescence live imaging for real time. It was demonstrated that intravesical instillation allows approximately 10 times more efficient accumulation of emulsion microgels in the mice urinary bladder in vivo 1 h after injection compared to systemic injection. The retention of the emulsion of mucoadhesive microgels in bladders after the intravesical instillation was observed for 24 h.

The Effect of Cooling Rate on Crystallographic Features of Phase Transformations in Zr-2.5Nb.

Zirconium (Zr) alloys are utilized as structural components for the cores of nuclear reactors due to the excellent combination of their mechanical properties and corrosion resistance under intense neutron irradiation conditions in water. The characteristics of microstructures formed during heat treatments play a crucial role in obtaining the operational performance of parts made from Zr alloys. This study investigates the morphological features of (α + ß)-microstructures in the Zr-2.5Nb alloy, as well as the crystallographic relationships between α- and ß-phases. These relationships are induced by the ß→α(α″) displacive transformation that occurs during water quenching (WQ) and the diffusion-eutectoid transformation that takes place during furnace cooling (FC). To conduct this analysis, samples solution treated at 920 °C were examined using EBSD and TEM. The experimental distribution of α/ß-misorientations for both cooling regimes deviates from the Burgers orientation relationship (BOR) at a discrete set of angles close to 0, 29, 35, and 43°. The experimental α/ß-misorientation spectra are confirmed by crystallographic calculations for the ß→α→ß-transformation path based on the BOR. Similar spectra of misorientation angle distribution in α-phase and between α and ß phases in Zr-2.5Nb after WQ and FC point to similar transformation mechanisms and the significant role of shear and shuffle in ß→α-transformation.

Glass Film Formation on GOES Surface during High-Temperature Annealing: The Mechanism with Amorphous Phase Formation.

Ceramic insulation coating (glass film) is an important constituent of grain-oriented electrical steel (GOES) designed for use in transformers. Within the scope of this study, the glass film was obtained by means of interaction between the surface of GOES containing 0.5 wt. % Cu and a heat-resistant MgO coating during annealing up to 1100 °C in the 75%H2 + 25%N2 atmosphere. The structure of glass film was analyzed using X-ray diffraction, glow-discharge optical emission spectroscopy, scanning probe microscopy, scanning electron microscopy, differential scanning calorimetry and thermodynamic calculations. After annealing, the glass film contained the following phases: crystalline (MgFe)2SiO4 and amorphous Fe-based solid solutions. The multi-stage mechanism of the glass film formation on GOES surface during high-temperature annealing was determined.

Crystallographic Features of Phase Transformations during the Continuous Cooling of a Ti6Al4V Alloy from the Single-Phase ß-Region.

Crystallographic relationships between α- and ß-phases resulting from phase transformations, which took place during the continuous water quenching (WQ), air cooling (AC) and furnace cooling (FC) of a Ti6Al4V plates solution treated at 1065 °C, were investigated by methods of electron backscatter diffraction (EBSD) and transmission electron microscopy (TEM). WQ, AC and FC resulted in typical martensite, basket-weave and parallel-plate Widmanstatten structures, respectively. The experimental distribution of α/ß-misorientations deviated from BOR at set discrete angles close to 22, 30, 35 and 43°. The experimental spectra of angles were confirmed by theoretical calculations of the possible misorientations between the α and ß phases through the ßo→α→ßII -transformation path based on Burgers orientation relationship (BOR). Joint analysis of the experimental data and theoretical calculations revealed that the secondary ßII-phase was precipitated according to the sequence ßo→α→ßII during continuous cooling from the single-phase ß-region. Similar spectra for α/ß-phase misorientations for all investigated cooling rates acknowledged the similar transformation mechanisms and dominant shear component of the phase transformations.

Tensile Deformation and Fracture Behavior of API-5L X70 Line Pipe Steel.

Thermo-mechanical controlled processing (TMCP) is employed to obtain the required level of mechanical properties of contemporary HSLA steel plates utilized for gas and oil pipeline production. The strength and crack resistance of pipeline steels are mainly determined by its microstructure and crystallographic texture. In this study, the influence of the structural and textural states of industrially produced API-5L X70-X80 pipeline steels on tensile mechanical properties was analyzed. TMCP routes with different hot rolling temperatures and cooling rates were employed. The texture of steel was assessed using the Taylor factor, which was calculated based on electron backscatter diffraction (EBSD). The decrease in rolling temperature resulted in the sharper texture characterized by {001} planes banding (cleavage planes in the bcc lattice) parallel to rolling direction. The tensile deformation behavior at the stage of necking was determined by the crystallographic and morphological texture of the material and demonstrated significant anisotropy. Rupture of all investigated samples was accompanied by the development of splitting on the fracture surface. The splitting was localized in the rolling plane similar to the splitting in standard Charpy tests of pipeline steels.

Is there an advantageous arrangement of aromatic residues in proteins? Statistical analysis of aromatic interactions in globular proteins.

The aim of this study was to evaluate the favorability of different conformations of aromatic residues in proteins by analysing the occurrence of particular conformations. The clustering of protein structures from the Protein Data Bank (PDB) was performed. Conformations of interacting aromatic residues were analyzed for 511 282 pairs in 35 493 protein structures sharing less than 50% identity. Pairs with a parallel arrangement of aromatic residues made up 6.2% of all possible ones, which was twice as much as expected. Pairs with a perpendicular arrangement of aromatic residues made up 25%. We demonstrate that the most favorable arrangement was at an angle of 60° between the interacting aromatic residues. Among all possible aromatic pairs, the His-His pair was twice as frequent as expected, and the His-Phe pair was less frequent than expected. A server (CARP - Contacts of Aromatic Residues in Proteins) has been created for calculating essential structural features of interacting aromatic residues in proteins: http://bioproteom.protres.ru/arom_q_prog/.

Texturing and Phase Evolution in Ti-6Al-4V: Effect of Electron Beam Melting Process, Powder Re-Using, and HIP Treatment.

The research demonstrates microstructural changes and development of specific texture in Ti-6Al-4V specimens produced by electron beam melting (EBM) under different conditions. The effect of two factors, namely, raw material (powder) recycling and hot isostatic pressing (HIP), on the EBM produced samples structure and properties, has been explored. The as-printed and treated samples were investigated using electron backscattered diffraction (EBSD) analysis. Modification of mechanical properties after the EBM and HIP are explained by the EBSD data on microstructural phenomena and phase transformations. The work is devoted to assessing the possibility of reusing the residual titanium alloy powder for the manufacture of titanium components by the combination of EBM and HIP methods.

Myosin Binding Protein-C Forms Amyloid-Like Aggregates In Vitro.

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5-10 min) formation of large (>2 µm) aggregates. sMyBP-C oligomers formed both at the initial 5-10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7-10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-ß quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S-S bonds providing for general stability.

Disordered Residues and Patterns in the Protein Data Bank.

We created a new library of disordered patterns and disordered residues in the Protein Data Bank (PDB). To obtain such datasets, we clustered the PDB and obtained the groups of chains with different identities and marked disordered residues. We elaborated a new procedure for finding disordered patterns and created a new version of the library. This library includes three sets of patterns: unique patterns, patterns consisting of two kinds of amino acids, and homo-repeats. Using this database, the user can: (1) find homologues in the entire Protein Data Bank; (2) perform a statistical analysis of disordered residues in protein structures; (3) search for disordered patterns and homo-repeats; (4) search for disordered regions in different chains of the same protein; (5) download clusters of protein chains with different identity from our database and library of disordered patterns; and (6) observe 3D structure interactively using MView. A new library of disordered patterns will help improve the accuracy of predictions for residues that will be structured or unstructured in a given region.

Repair of cystocele and apical genital prolapse using 6-strap mesh implant.

OBJECTIVE: To assess the outcomes of surgical repair of anterior apical prolapse using the 6-strap mesh implant. STUDY DESIGN: The prospective study included 100 patients with genitourinary prolapse. We used advanced 6-strap mesh implant. The results were assessed at 1 (n = 100) and 12 (n = 93) months after surgery. Maximum follow-up was over 4 years. The anatomical outcomes according to the Pelvic Organ Prolapse Quantification system and intraoperative and postoperative complications were assessed. Stage II and higher prolapse was considered to be a recurrence. The quality of life and sexual function were assessed using Pelvic Organ Prolapse Distress Inventory 20, Pelvic Floor Impact Questionnaire 7, and Pelvic Organ Prolapse/Incontinence Sexual Questionnaire 12. RESULTS: Median age was 57 years (34-78 years (95% confidence interval)). All patients had stage III cystocele. The anterior vaginal wall descent in all the patients was associated with uterine descent: 37 (37%), stage II; 60 (60%), stage III; in 3 (3%), stage IV. In eight cases, postoperative de novo stress urinary incontinence developed. The quality of life improved in 93 (93%) women as judged by the Pelvic Floor Distress Inventory 20 data and in 87 (87%) women, according to the Pelvic Floor Impact Questionnaire 7 data. The desirable anatomical result (⩽stage I according to the Pelvic Organ Prolapse Quantification system) was achieved in 97 (97%) patients. With the exception of mesh fragment excision due to erosion (grade 3a), all the complications were classified as grade I according to the Clavien-Dindo classification. CONCLUSION: Genitourinary prolapse repair using 6-strap mesh is efficacious and relatively safe. The method demonstrates good anatomical results in relation to both anterior and apical prolapses with relatively short-term complications.

Repeats in S1 Proteins: Flexibility and Tendency for Intrinsic Disorder.

An important feature of ribosomal S1 proteins is multiple copies of structural domains in bacteria, the number of which changes in a strictly limited range from one to six. For S1 proteins, little is known about the contribution of flexible regions to protein domain function. We exhaustively studied a tendency for intrinsic disorder and flexibility within and between structural domains for all available UniProt S1 sequences. Using charge-hydrophobicity plot cumulative distribution function (CH-CDF) analysis we classified 53% of S1 proteins as ordered proteins; the remaining proteins were related to molten globule state. S1 proteins are characterized by an equal ratio of regions connecting the secondary structure within and between structural domains, which indicates a similar organization of separate S1 domains and multi-domain S1 proteins. According to the FoldUnfold and IsUnstruct programs, in the multi-domain proteins, relatively short flexible or disordered regions are predominant. The lowest percentage of flexibility is in the central parts of multi-domain proteins. Our results suggest that the ratio of flexibility in the separate domains is related to their roles in the activity and functionality of S1: a more stable and compact central part in the multi-domain proteins is vital for RNA interaction, terminals domains are important for other functions.

Is there codon usage bias for poly-Q stretches in the human proteome?

We have analyzed codon usage for poly-Q stretches of different lengths for the human proteome. First, we have obtained that all long poly-Q stretches in Protein Data Bank (PDB) belong to the disordered regions. Second, we have found the bias for codon usage for glutamine homo-repeats in the human proteome. In the cases when the same codon is used for poly-Q stretches only CAG triplets are found. Similar results are obtained for human proteins with glutamine homo-repeats associated with diseases. Moreover, for proteins associated with diseases (from the HraDis database), the fraction of proteins for which the same codon is used for glutamine homo-repeats is less (22%) than for proteins from the human proteome (26%). We have demonstrated for poly-Q stretches in the human proteome that in some cases (28) the splicing sites correspond to the homo-repeats and in 11 cases, these sites appear at the C -terminal part of the homo-repeats with statistical significance 10 -8 .

Extracorporeal ureter handling during laparoscopic pyeloplasty: tips and tricks for beginners.

INTRODUCTION: Laparoscopic preparation of the ureter is a challenging part of upper urinary tract reconstruction, due to limited depth perception provided by the camera and lack of wristed motion of most laparoscopic instruments needed for adequate spatulation and scar tissue removal. One solution has been to perform the more difficult portions of the surgery in an extracorporeal manner. A hybrid intracorporeal-extracorporeal approach to upper tract ureteral reconstruction facilitates ureteral preparation at the stage of mastering the technique. MATERIAL AND METHODS: This retrospective study included 100 patients with primary ureteropelvic junction obstruction, who underwent laparoscopic pyeloplasty from 2014 to 2017. The patients were stratified into 2 groups: those who underwent conventional laparoscopic surgery and those who were managed with the hybrid approach. For the hybrid approach, externalizing the ureter to skin level required additional mobilization of the upper urinary tract. RESULTS: A total of 47 patients underwent conventional laparoscopic pyeloplasty and 53 - hybrid surgery. The maximum body mass index was 32. The hybrid approach was 8.5 minutes shorter compared to the conventional approach (p <0.001). No complications higher than Clavien-Dindo IIIb (n = 2) were observed (in both groups). Complete success (the resolution of pain and/or hydronephrosis) was observed in 92.5% in the hybrid group and in 95.7% in the conventional treatment group. CONCLUSIONS: Hybrid pyeloplasty may be considered safe and effective. It has the advantage of making the surgery less challenging and time-consuming while offering improved precision. The advantages of the technique are particularly apparent during training. This technique can be recommended in the learning process of the surgeon.

Proteome Evolution of Deep-Sea Hydrothermal Vent Alvinellid Polychaetes Supports the Ancestry of Thermophily and Subsequent Adaptation to Cold in Some Lineages.

Temperature, perhaps more than any other environmental factor, is likely to influence the evolution of all organisms. It is also a very interesting factor to understand how genomes are shaped by selection over evolutionary timescales, as it potentially affects the whole genome. Among thermophilic prokaryotes, temperature affects both codon usage and protein composition to increase the stability of the transcriptional/translational machinery, and the resulting proteins need to be functional at high temperatures. Among eukaryotes less is known about genome evolution, and the tube-dwelling worms of the family Alvinellidae represent an excellent opportunity to test hypotheses about the emergence of thermophily in ectothermic metazoans. The Alvinellidae are a group of worms that experience varying thermal regimes, presumably having evolved into these niches over evolutionary times. Here we analyzed 423 putative orthologous loci derived from 6 alvinellid species including the thermophilic Alvinella pompejana and Paralvinella sulfincola. This comparative approach allowed us to assess amino acid composition, codon usage, divergence, direction of residue changes and the strength of selection along the alvinellid phylogeny, and to design a new eukaryotic thermophilic criterion based on significant differences in the residue composition of proteins. Contrary to expectations, the alvinellid ancestor of all present-day species seems to have been thermophilic, a trait subsequently maintained by purifying selection in lineages that still inhabit higher temperature environments. In contrast, lineages currently living in colder habitats likely evolved under selective relaxation, with some degree of positive selection for low-temperature adaptation at the protein level.

How Common Is Disorder? Occurrence of Disordered Residues in Four Domains of Life.

Disordered regions play important roles in protein adaptation to challenging environmental conditions. Flexible and disordered residues have the highest propensities to alter the protein packing. Therefore, identification of disordered/flexible regions is important for structural and functional analysis of proteins. We used the IsUnstruct program to predict the ordered or disordered status of residues in 122 proteomes, including 97 eukaryotic and 25 large bacterial proteomes larger than 2,500,000 residues. We found that bacterial and eukaryotic proteomes contain comparable fraction of disordered residues, which was 0.31 in the bacterial and 0.38 in the eukaryotic proteomes. Additional analysis of the total of 1540 bacterial proteomes of various sizes yielded a smaller fraction of disordered residues, which was only 0.26. Together, the results showed that the larger is the size of the proteome, the larger is the fraction of the disordered residues. A continuous dependence of the fraction of disordered residues on the size of the proteome is observed for four domains of life: Eukaryota, Bacteria, Archaea, and Viruses. Furthermore, our analysis of 122 proteomes showed that the fraction of disordered residues increased with increasing the length of homo-repeats for polar, charged, and small residues, and decreased for hydrophobic residues. The maximal fraction of disordered residues was obtained for proteins containing lysine and arginine homo-repeats. The minimal fraction was found in valine and leucine homo-repeats. For 15-residue long homo-repeats these values were 0.2 (for Val and Leu) and 0.7 (for Lys and Arg).

HRaP: database of occurrence of HomoRepeats and patterns in proteomes.

We focus our attention on multiple repeats of one amino acid (homorepeats) and create a new database (named HRaP, at http://bioinfo.protres.ru/hrap/) of occurrence of homorepeats and disordered patterns in different proteomes. HRaP is aimed at understanding the amino acid tandem repeat function in different proteomes. Therefore, the database includes 122 proteomes, 97 eukaryotic and 25 bacterial ones that can be divided into 9 kingdoms and 5 phyla of bacteria. The database includes 1,449,561 protein sequences and 771,786 sequences of proteins with GO annotations. We have determined homorepeats and patterns that are associated with some function. Through our web server, the user can do the following: (i) search for proteins with the given homorepeat in 122 proteomes, including GO annotation for these proteins; (ii) search for proteins with the given disordered pattern from the library of disordered patterns constructed on the clustered Protein Data Bank in 122 proteomes, including GO annotations for these proteins; (iii) analyze lengths of homorepeats in different proteomes; (iv) investigate disordered regions in the chosen proteins in 122 proteomes; (v) study the coupling of different homorepeats in one protein; (vi) determine longest runs for each amino acid inside each proteome; and (vii) download the full list of proteins with the given length of a homorepeat.

A novel web server predicts amino acid residue protection against hydrogen-deuterium exchange.

MOTIVATION: To clarify the relationship between structural elements and polypeptide chain mobility, a set of statistical analyses of structures is necessary. Because at present proteins with determined spatial structures are much less numerous than those with amino acid sequence known, it is important to be able to predict the extent of proton protection from hydrogen-deuterium (HD) exchange basing solely on the protein primary structure. RESULTS: Here we present a novel web server aimed to predict the degree of amino acid residue protection against HD exchange solely from the primary structure of the protein chain under study. On the basis of the amino acid sequence, the presented server offers the following three possibilities (predictors) for user's choice. First, prediction of the number of contacts occurring in this protein, which is shown to be helpful in estimating the number of protons protected against HD exchange (sensitivity 0.71). Second, probability of H-bonding in this protein, which is useful for finding the number of unprotected protons (specificity 0.71). The last is the use of an artificial predictor. Also, we report on mass spectrometry analysis of HD exchange that has been first applied to free amino acids. Its results showed a good agreement with theoretical data (number of protons) for 10 globular proteins (correlation coefficient 0.73). We pioneered in compiling two datasets of experimental HD exchange data for 35 proteins. AVAILABILITY: The H-Protection server is available for users at http://bioinfo.protres.ru/ogp/ SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.