Diagnostics of preeclampsia based on Congo red binding to urinary components: Rationales and limitations.

Preeclampsia is a disorder that can occur during pregnancy and is one of the leading causes of death among pregnant women. This disorder occurs after the 20th week of pregnancy and is characterized by arterial hypertension, proteinuria, fetoplacental, and multiple organ dysfunctions. Despite the long history of studying preeclampsia, its etiology and pathogenesis remain poorly understood, and therapy is symptomatic. One of the factors of the disorder is believed to be misfolded proteins that are prone to form amyloid aggregates. The CRD tests, utilizing the binding of the amyloid-specific dye Congo red to urine components, demonstrate high efficiency in diagnosing preeclampsia. However, these tests have also been found to be positive in other disorders with proteinuria, presumably associated with concomitant amyloidosis. To assess the limitations of the CRD tests, we examined urine congophilia and protein components mediating Congo red positivity in patients with proteinuria, including preeclampsia, amyloid and non-amyloid nephropathies. We stained the urine samples and calculated congophilia levels. We also assessed the contribution of large protein aggregates to congophilia values using ultracentrifugation and determined the molecular weights of congophilic urinary proteins using centrifugal concentrators. All proteinuric groups demonstrate positive results in the CRD tests and congophilia levels were more than two times higher compared with the control non-proteinuric groups (p <0.01). There was a strong correlation between urine protein excretion and congophilia in amyloid nephropathy (rs = 0.76), non-amyloid nephropathies (rs = 0.90), and preeclampsia (rs = 0.90). Removal of large aggregates from urine did not affect the congophilia levels. Separation of urine protein fractions revealed congophilic components in the range of 30-100 kDa, including monomeric serum albumin. Our results indicate limitations of CRD tests in preeclampsia diagnostics in women with renal disorders and underscore the need for further research on the mechanisms of Congo red binding with urine components.

Phenotypic and genomic characteristics of oxacillin-susceptible mecA-positive Staphylococcus aureus, rapid selection of high-level resistance to beta-lactams.

The aim of this study is to describe the phenotypic and genetic properties of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) isolates and their beta-lactam resistant derivatives obtained after selection with oxacillin. A collection of hospital- (HA-) and community-acquired (CA-) MRSA was screened for oxacillin susceptibility. Antibiotic susceptibility testing, population analysis profile (PAP), mecA expression analysis, and whole genome sequencing (WGS) were performed for 60 mecA-positive OS-MRSA isolates. Twelve high-level beta-lactam resistant derivatives selected during PAP were also subjected to WGS. OS-MRSA were more prevalent among CA-MRSA (49/205, 24%) than among HA-MRSA (11/575, 2%). OS-MRSA isolates belonged to twelve sequence types (ST), with a predominance of ST22-t223-SCCmec IVc and ST59-t1950-SCCmec V lineages. OS-MRSA were characterized by mecA promoter mutations at - 33 (C→T) or - 7 (G→T/A) along with PBP2a substitutions (S225R or E246G). The basal and oxacillin-induced levels of mecA expression in OS-MRSA isolates were significantly lower than those in control ST8-HA-MRSA isolates. Most of the OS-MRSA isolates were heteroresistant to oxacillin. High-level beta-lactam resistant OS-MRSA derivatives selected with oxacillin carried mutations in mecA auxiliary factors: relA (metabolism of purines), tyrS, cysS (metabolism of tRNAs), aroK, cysE (metabolism of amino acids and glycolysis). Cefoxitin-based tests demonstrated high specificity for OS-MRSA detection. The highest positive predictive values (PPV > 0.95) were observed for broth microdilution, the VITEK® 2 automatic system, and chromogenic media. Susceptibility testing of CA-MRSA requires special attention due to the high prevalence of difficult-to-detect OS-MRSA among them. Mis-prescription of beta-lactams for the treatment of OS-MRSA may lead to selection of high-level resistance and treatment failures.

Adaptive Laboratory Evolution of Staphylococcus aureus Resistance to Vancomycin and Daptomycin: Mutation Patterns and Cross-Resistance.

Vancomycin and daptomycin are first-line drugs for the treatment of complicated methicillin-resistant Staphylococcus aureus (MRSA) infections, including bacteremia. However, their effectiveness is limited not only by their resistance to each antibiotic but also by their associated resistance to both drugs. It is unknown whether novel lipoglycopeptides can overcome this associated resistance. Resistant derivatives from five S. aureus strains were obtained during adaptive laboratory evolution with vancomycin and daptomycin. Both parental and derivative strains were subjected to susceptibility testing, population analysis profiles, measurements of growth rate and autolytic activity, and whole-genome sequencing. Regardless of whether vancomycin or daptomycin was selected, most of the derivatives were characterized by a reduced susceptibility to daptomycin, vancomycin, telavancin, dalbavancin, and oritavancin. Resistance to induced autolysis was observed in all derivatives. Daptomycin resistance was associated with a significant reduction in growth rate. Resistance to vancomycin was mainly associated with mutations in the genes responsible for cell wall biosynthesis, and resistance to daptomycin was associated with mutations in the genes responsible for phospholipid biosynthesis and glycerol metabolism. However, mutations in walK and mprF were detected in derivatives selected for both antibiotics.

Search and Identification of Amyloid Proteins.

Amyloids are fibrillar proteins with a cross-ß structure. Pathological amyloids are associated with the development of a number of incurable diseases, while functional amyloids regulate vital processes. The detection of unknown amyloids in living objects is a difficult task, and therefore the question of the prevalence and biological significance of amyloids remains open. We present a description of two methods, the combination of which makes it possible to find and identify amyloid proteins in the proteome of various organisms. The method of proteomic screening for amyloids allows the detection of the proteins that form SDS-resistant aggregates. SDS resistance is a general feature of amyloid fibrils. Protein aggregates resistant to SDS treatment can be collected by ultracentrifugation and further identified by mass spectrometry. However, in addition to amyloids, SDS-resistant aggregates contain some non-amyloid proteins. To test the amyloid properties of proteins identified by proteomic screening, we developed the method of fibril immunoprecipitation followed by Congo red staining and birefringence analysis. The methods of proteomic screening and immunoprecipitation of fibrillar proteins have been successfully tested and applied for the identification of amyloid proteins in yeast and vertebrates.

Amyloid Properties of the FXR1 Protein Are Conserved in Evolution of Vertebrates.

Functional amyloids are fibrillary proteins with a cross-ß structure that play a structural or regulatory role in pro- and eukaryotes. Previously, we have demonstrated that the RNA-binding FXR1 protein functions in an amyloid form in the rat brain. This RNA-binding protein plays an important role in the regulation of long-term memory, emotions, and cancer. Here, we evaluate the amyloid properties of FXR1 in organisms representing various classes of vertebrates. We show the colocalization of FXR1 with amyloid-specific dyes in the neurons of amphibians, reptiles, and birds. Moreover, FXR1, as with other amyloids, forms detergent-resistant insoluble aggregates in all studied animals. The FXR1 protein isolated by immunoprecipitation from the brains of different vertebrate species forms fibrils, which show yellow-green birefringence after staining with Congo red. Our data indicate that in the evolution of vertebrates, FXR1 acquired amyloid properties at least 365 million years ago. Based on the obtained data, we discuss the possible role of FXR1 amyloid fibrils in the regulation of vital processes in the brain of vertebrates.

RNA-binding protein FXR1 is presented in rat brain in amyloid form.

Amyloids are ß-sheets-rich protein fibrils that cause neurodegenerative and other incurable human diseases affecting millions of people worldwide. However, a number of proteins is functional in the amyloid state in various organisms from bacteria to humans. Using an original proteomic approach, we identified a set of proteins forming amyloid-like aggregates in the brain of young healthy rats. One of them is the FXR1 protein, which is known to regulate memory and emotions. We showed that FXR1 clearly colocalizes in cortical neurons with amyloid-specific dyes Congo-Red, Thioflavines S and T. FXR1 extracted from brain by immunoprecipitation shows yellow-green birefringence after staining with Congo red. This protein forms in brain detergent-resistant amyloid oligomers and insoluble aggregates. RNA molecules that are colocalized with FXR1 in cortical neurons are insensitive to treatment with RNase A. All these data suggest that FXR1 functions in rat brain in amyloid form. The N-terminal amyloid-forming fragment of FXR1 is highly conserved across mammals. We assume that the FXR1 protein may be presented in amyloid form in brain of different species of mammals, including humans.