Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis.

Syphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides.

Impact of oppositely charged shell and cores on interaction of core-shell colloids with differently charged proteins as a route for tuning of the colloids cytotoxicity.

The present work represents interactions between the core-shell nanoparticles and different proteins, exemplified by lysozyme (LSZ), pepsin, bovine serum albumin (BSA), thioredoxin (TRX) and yellow fluorescent protein (YFP). The core-shell morphology derives from the non-covalent deposition of polyethyleneimine (PEI) onto nanoprecipitated luminescent complex (AuCl)2L (L is cyclic PNNP ligand). Analysis of the data obtained by DLS, CD spectroscopy, luminescence derived from both (AuCl)2L and YFP reveal the electrostatically driven interaction of negatively charged proteins with the shell of PEI-(AuCl)2L. The fluorescence of YFP enables to reveal the inclusion of the protein molecules into the shell. The lack of any luminescent response of PEI-(AuCl)2L on TRX conforms its electrostatically driven interactions with the shell which, in turn, excludes a binding of the exposed thiol moieties with (AuCl)2L. The negatively charged surface of pepsin provides the greatest recharging of the PEI-based shell versus the other proteins, which is followed by the enhanced luminescence of (AuCl)2L. The significant effect of PEI-(AuCl)2L on the CD spectra of LSZ followed by the decreased intensity of (AuCl)2L-based luminescence points to specific interaction mode of PEI-(AuCl)2L with LSZ. The flow cytometry and fluorescent microscopy measurements revealed efficient internalization of PEI-(AuCl)2L into the Wi-38 cell samples resulting in the efficient staining of all cell organelles. The concentration dependent cytotoxicity of PEI-(AuCl)2L is detectably enhanced by LSZ, which is correlated with its interaction mode with the nanoparticles.

Isoforms of Base Excision Repair Enzymes Produced by Alternative Splicing.

Transcripts of many enzymes involved in base excision repair (BER) undergo extensive alternative splicing, but functions of the corresponding alternative splice variants remain largely unexplored. In this review, we cover the studies describing the common alternatively spliced isoforms and disease-associated variants of DNA glycosylases, AP-endonuclease 1, and DNA polymerase beta. We also discuss the roles of alternative splicing in the regulation of their expression, catalytic activities, and intracellular transport.

Interfacial uploading of luminescent hexamolybdenum cluster units onto amino-decorated silica nanoparticles as new design of nanomaterial for cellular imaging and photodynamic therapy.

The present work introduces a facile synthetic route to embed phosphorescent K2[{Mo6I8}I6] and (nBu4N)2[{Mo6I8}(CH3COO)6] clusters (C) onto silica-water interface of amino-decorated silica nanoparticles (SNs, 60 ±â€¯6 nm). The assembled C-SNs gain in the luminescence intensity, which remains stable within three months after their assembly. High uptake capacity of the clusters (8700 of K2[{Mo6I8}I6] and 6500 of (nBu4N)2[{Mo6I8}(CH3COO)6] per the each nanoparticle) derives from ionic self-assembly and coordination bonds between the cluster complexes and ionic (amino- and siloxy-) groups at the silica surface. The coordination via amino- or siloxy-groups restricts aquation and hydrolysis of the embedded clusters, in comparison with the parent K2[{Mo6I8}I6] and (nBu4N)2[{Mo6I8}(CH3COO)6. High potential of the assembled nanoparticles in the ROS generation was revealed by EPR measurements facilitated by spin trapping. The high positive charge and convenient colloid stability of the assembled C-SNs hybrids are the prerequisite for their efficient cellular uptake, which is exemplified in the work by MCF-7 cell line. The measured dark and photoinduced cytotoxicity of the C-SNs hybrids reveals significant photodynamic therapy effect on the MCF-7 cancer cell line versus the normal cells. This effect is entirely due to the embedded clusters and is dependent on the chemical composition of the cluster.

Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol.

Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6-6.5, low salt concentrations and 3 mM Mg2+ or 0.3-3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37ºC. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

ATPase-deficient mitochondrial inner membrane protein ATAD3A disturbs mitochondrial dynamics in dominant hereditary spastic paraplegia.

De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.

Syphilis epidemiology in 1994-2013, molecular epidemiological strain typing and determination of macrolide resistance in Treponema pallidum in 2013-2014 in Tuva Republic, Russia.

The incidence of syphilis in the Tuva Republic (geographical centre of Asia), Russia has been exceedingly high historically. No detailed examinations and no molecular investigations of Treponema pallidum strains transmitted in the Tuva Republic, or in general, in Russia, were published internationally. We examined the syphilis epidemiology in 1994-2013, and the molecular epidemiology and macrolide resistance in T. pallidum strains in 2013-2014 in the Tuva Republic. Among 95 mainly primary or secondary syphilis patients, the arp, tpr, tp0548 and 23S rRNA genes in 85 polA gene-positive genital ulcer specimens were characterized. The syphilis incidence in Tuva Republic peaked in 1998 (1562), however declined to 177 in 2013. Among the 70 (82%) completely genotyped specimens, six molecular strain types were found. Strain type 14d/f accounted for 91%, but also 14c/f, 14d/g, 14b/f, 14i/f, 9d/f, and 4d/f were identified. Two (2.4%) specimens contained the 23S rRNA A2058G macrolide resistance mutation. This is the first internationally published typing study regarding T. pallidum in Russia, performed in the Tuva Republic with the highest syphilis incidence in Russia. The two molecular strain types 4d/f and 9d/f have previously been described only in Eastern and Northern China and for the first time, macrolide-resistant syphilis was described in Russia.

Russian gonococcal antimicrobial susceptibility programme (RU-GASP)--resistance in Neisseria gonorrhoeae during 2009-2012 and NG-MAST genotypes in 2011 and 2012.

BACKGROUND: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major concern worldwide and gonococcal AMR surveillance globally is imperative for public health purposes. In Eastern Europe, gonococcal AMR surveillance is exceedingly rare. However, in 2004 the Russian gonococcal antimicrobial susceptibility programme (RU-GASP) was initiated. The aims of this study were to describe the prevalence and trends of gonococcal AMR from 2009 to 2012, and molecular epidemiological genotypes in 2011 and 2012 in Russia. METHODS: Gonococcal isolates from 12-46 surveillance sites distributed across Russia, obtained in 2009 (n = 1200), 2010 (n = 407), 2011 (n = 423), and 2012 (n = 106), were examined for antimicrobial susceptibility using agar dilution method. Gonococcal isolates from 2011 and 2012 were investigated with N. gonorrhoeae multi-antigen sequence typing (NG-MAST). RESULTS: During 2009-2012, the proportions of gonococcal isolates resistant to ciprofloxacin, penicillin G, azithromycin and spectinomycin ranged from 25.5% to 44.4%, 9.6% to 13.2%, 2.3% to 17.0% and 0.9% to 11.6%, respectively. Overall, the resistance level to penicillin G was stable, the resistance level to ciprofloxacin was decreasing, however, the level of resistance to azithromycin increased. All isolates were susceptible to ceftriaxone using the US CLSI breakpoints. However, using the European breakpoints 58 (2.7%) of the isolates were resistant to ceftriaxone. Interestingly, this proportion was decreasing, i.e. from 4.8% in 2009 to 0% in 2012. CONCLUSIONS: In Russia, the diversified gonococcal population showed a high resistance to ciprofloxacin, penicillin G and azithromycin. In general, the MICs of ceftriaxone were relatively high, however, they were decreasing from 2009 to 2012. Ceftriaxone should be the first-line for empiric antimicrobial monotherapy of gonorrhoea in Russia. It is essential to further strengthen the surveillance of gonococcal AMR (ideally also gonorrhoea treatment failures) in Russia.

Cloning, sequencing, expression, and characterization of thermostability of oligopeptidase B from Serratia proteamaculans, a novel psychrophilic protease.

Protease from Serratia proteamaculans (PSP) is the first known psychrophilic oligopeptidase B. The gene of S. proteamaculans 94 oligopeptidase B was cloned, sequenced and expressed in Escherichia coli. The unfolding of PSP molecule following heat treatment at 37°C by measuring fluorescence spectra was examined in parallel with the residual activity determination. The effect of PSP thermostabilization by glycerol at 37-50 °Ð¡ was revealed. Calcium ions and buffer solution of low molarity cause the opposite effect - the acceleration of PSP inactivation at 37°C. The thermal stability of PSP molecule in the presence of 0-100mM CaCl2 was also investigated by means of high-sensitivity differential scanning calorimetry. The artificial reconstruction of the natural complex PSP-chaperonin from S. рroteamaculans was carried out: the stable complex (1:1) of chaperonin E. сoli GroEL with active recombinant enzyme PSP was obtained. It was shown that complex formation with chaperonin promotes PSP thermostability at 37°C.