Optimized multilocus sequence analysis for laboratory identification of pathogens of ixodid tick-borne borreliosis

Introduction. The most common etiological agents of ixodid tick-borne borreliosis (ITBB) in Russia are Borrelia garinii, B. afzelii, B. bavariensis. Multilocus sequence typing and multilocus sequence analysis (MLSA) have been used in recent studies for Borrelia species identification. The results of using the MLSA scheme for identification of pathogens causing erythemic forms of ITBB have been presented earlier. The purpose of the study was to explore the possibility of MLSA optimization for laboratory identification of ITBB pathogens. Objectives: comparative analysis of nucleotide sequences of 6 conserved genes (rrs, hbb, fla, groEL, recA, ospA) and the rrfA-rrlB intergenic spacer, which are recommended by the MLSA protocol; identification of the minimum set of genes, the concatenated sequences of which are essential for species identification of Borrelia isolates. Materials and methods. The sequences of the above loci of 23 reference isolates collected from patients with ITBB and assigned, using MLSA, to B. bavariensis were compared with the sequences of similar genes of other Borrelia species available in international databases. The UPGMA method was used to build and analyze dendrograms based on the obtained data. Results. The sequences of ospA gene loci of reference species demonstrated the greatest difference (not less than 8.5%) from the sequences of the above gene in other analyzed species of Borrelia; approximately similar species-related differences (not less than 6.7%) were demonstrated by the comparison of recA gene sequences. The sequences of the identified variants of these two genes in B. bavariensis differed from the sequences of the similar genes in the most closely related species B. garinii. The dendrogram of the concatenated nucleotide sequences of recA and ospA genes demonstrated that it was totally consistent with the results of identification of the isolates based on the MLSA protocol. Conclusion. The optimized approach to MLSA of the B. burgdorferi sensu lato group suggests that species identification should be based on the concatenated analysis of loci of only two genes (recA and ospA) out of 7 loci recommended by the MLSA protocol

Green Tea Polyphenols Rescue of Brain Defects Induced by Overexpression of DYRK1A

Individuals with partial HSA21 trisomies and mice with partial MMU16 trisomies containing an extra copy of the DYRK1A gene present various alterations in brain morphogenesis. They present also learning impairments modeling those encountered in Down syndrome. Previous MRI and histological analyses of a transgenic mice generated using a human YAC construct that contains five genes including DYRK1A reveal that DYRK1A is involved, during development, in the control of brain volume and cell density of specific brain regions. Gene dosage correction induces a rescue of the brain volume alterations. DYRK1A is also involved in the control of synaptic plasticity and memory consolidation. Increased gene dosage results in brain morphogenesis defects, low BDNF levels and mnemonic deficits in these mice. Epigallocatechin gallate (EGCG) — a member of a natural polyphenols family, found in great amount in green tea leaves — is a specific and safe DYRK1A inhibitor. We maintained control and transgenic mice overexpressing DYRK1A on two different polyphenol-based diets, from gestation to adulthood. The major features of the transgenic phenotype were rescued in these mice

C6 Peptide-Based Multiplex Phosphorescence Analysis (PHOSPHAN) for Serologic Confirmation of Lyme Borreliosis

<div><p>Background</p><p>A single-tier immunoassay using the C6 peptide of VlsE (C6) from <i>Borrelia burgdorferi</i> sensu stricto (<i>Bb)</i> has been proposed as a potential alternative to conventional two-tier testing for the serologic diagnosis of Lyme disease in the United States and Europe.</p><p>Objective</p><p>To evaluate the performance of C6 peptide based multiplex Phosphorescence Analysis (PHOSPHAN) for the serologic confirmation of Lyme borreliosis (LB) in Russian patients.</p><p>Methods</p><p>Serum samples (<i>n</i> = 351) were collected from 146 patients with erythema migrans (EM); samples from 131 of these patients were taken several times prior to treatment and at different stages of recovery. The control group consisted of 197 healthy blood donors and 31 patients with other diseases, all from the same highly endemic region of Russia. All samples were analyzed by PHOSPHAN for IgM and IgG to <i>Bb</i> C6, recombinant OspC and VlsE proteins, and C6 peptides from <i>B</i>. <i>garinii</i> and <i>B</i>. <i>afzelii</i>.</p><p>Results</p><p>IgM and IgG to <i>Bb</i> C6 were identified in 43 and 95 out of 131 patients (32.8 and 72.5%, respectively); seroconversion of IgM antibodies was observed in about half of the patients (51.2%), and of IgG antibodies, in almost all of them (88.4%). Additional detection of OspC-IgM and VlsE-IgM or IgG to C6 from <i>B</i>. <i>garinii</i> or <i>B</i>. <i>afzelii</i> did not contribute significantly to the overall sensitivity of the multiplex immunoassay.</p><p>Conclusions</p><p>The multiplex phosphorescence immunoassay is a promising method for simultaneously revealing the spectrum of antibodies to several <i>Borrelia</i> antigens. Detection of IgM and IgG to <i>Bb</i> C6 in the sera of EM patients provides effective serologic confirmation of LB and, with high probability, indicates an active infection process.</p></div

Lyme Index values (LIV) for sera from LB patients with erythema migrans (N = 351) and healthy blood donors (N = 197).

<p>(A) PHOSPHAN variants M₁–M₃ and (B) PHOSPHAN variants G₁–G₃ and C6 ELISA (C6). Blood donors, *; line with arrowhead, threshold level (LIV = 1). Immunoassay codes: M₁, <i>Bb</i> C6 IgM; M₂, OspC IgM; M₃, VlsE IgM; G₁, <i>Bb</i> C6 IgG; G₂, <i>Bg</i> C6 IgG; G₃, <i>Ba</i> C6 IgG; C6, Immunetics C6 Lyme ELISA kit. (<i>Bb</i>) <i>B</i>.<i>burgdorferi</i>, (<i>Bg</i>) <i>B</i>.<i>garinii</i>, (<i>Ba</i>) <i>B</i>.<i>afzelii</i>. Legend: Transparent square, Mean; Spotted rectangle, ± Standard Error; Dotted rectangle, Non-Outlier Range; Transparent circle, Outliers; Black square, Extremes.</p

Sensitivity of PHOSPHAN and C6 ELISA tests for serum IgM and IgG antibody responses to <i>B</i>. <i>burgdorferi</i> C6 in samples from EM patients.

<p>(A) Sensitivity of PHOSPHAN variants M₁, G₁, M₁G₁ and C6 ELISA (C6) at the baseline prior to treatment (N = 146). (B) Sensitivity of PHOSPHAN variants M₁ and G₁, and (C) PHOSPHAN variant M₁G₁ and C6 ELISA in tests of serum samples taken at the baseline (n = 146) and on days 8–14 (<i>n</i> = 75), 15–30 (n = 48), and 31–66 (<i>n</i> = 82) after disease onset. Values that are statistically significant (Fisher's exact test, <i>p</i> < 0.05) are denoted by brackets or asterisks. Immunoassay codes: M₁, <i>Bb</i> C6 IgM; G₁, <i>Bb</i> C6 IgG; M₁G₁, <i>Bb</i> C6 IgM+IgG; C6, Immunetics C6 Lyme ELISA kit. (<i>Bb</i>) <i>B</i>.<i>burgdorferi</i>. Legend: (B) Transparent square, M1; Black diamond, G1. (C) Black square, M1G1; Transparent circle, C6.</p

Sensitivity of PHOSPHAN tests for serum IgG antibody responses to <i>B</i>. <i>burgdorferi</i> C6, <i>B</i>. <i>garinii</i> C6, and <i>B</i>. <i>afzelii</i> C6 in samples from EM patients.

<p>(A) Total sensitivity of PHOSPHAN variants G₁, G₂, G₃, and G_1–3 at the baseline prior to treatment (N = 146). (B) Sensitivity of PHOSPHAN variants G₁–G₃ in tests of samples taken at the baseline (<i>n</i> = 146) and on days 8–14 (<i>n</i> = 75), 15–30 (n = 48), and 31–66 (<i>n</i> = 82) after disease onset. Values that are statistically significant (Fisher's exact test, <i>p</i> < 0.05) are denoted by brackets or asterisks. Immunoassay codes: G₁, <i>Bb</i> C6 IgG; G₂, <i>Bg</i> C6 IgG; G₃, <i>Ba</i> C6 IgG; G_1–3, any of the three antigens <i>Bb</i> C6, <i>Bg</i> C6, or <i>Ba</i> C6. (<i>Bb</i>) <i>B</i>.<i>burgdorferi</i>, (<i>Bg</i>) <i>B</i>.<i>garinii</i>, (<i>Ba</i>) <i>B</i>.<i>afzelii</i>. Legend: (B) Transparent square, G1; Black diamond, G2; Black triangle, G3.</p