Aerobiological Monitoring and Metabarcoding of Grass Pollen.

Grass pollen is one of the leading causes of pollinosis, affecting 10-30% of the world's population. The allergenicity of pollen from different Poaceae species is not the same and is estimated from moderate to high. Aerobiological monitoring is a standard method that allows one to track and predict the dynamics of allergen concentration in the air. Poaceae is a stenopalynous family, and thus grass pollen can usually be identified only at the family level with optical microscopy. Molecular methods, in particular the DNA barcoding technique, can be used to conduct a more accurate analysis of aerobiological samples containing the DNA of various plant species. This study aimed to test the possibility of using the ITS1 and ITS2 nuclear loci for determining the presence of grass pollen from air samples via metabarcoding and to compare the analysis results with the results of phenological observations. Based on the high-throughput sequencing data, we analyzed the changes in the composition of aerobiological samples taken in the Moscow and Ryazan regions for three years during the period of active flowering of grasses. Ten genera of the Poaceae family were detected in airborne pollen samples. The representation for most of them for ITS1 and ITS2 barcodes was similar. At the same time, in some samples, the presence of specific genera was characterized by only one sequence: either ITS1 or ITS2. Based on the analysis of the abundance of both barcode reads in the samples, the following order could describe the change with time in the dominant species in the air: Poa, Alopecurus, and Arrhenatherum in early mid-June, Lolium, Bromus, Dactylis, and Briza in mid-late June, Phleum, Elymus in late June to early July, and Calamagrostis in early mid-July. In most samples, the number of taxa found via metabarcoding analysis was higher compared to that in the phenological observations. The semi-quantitative analysis of high-throughput sequencing data well reflects the abundance of only major grass species at the flowering stage.

Clinical Effectiveness of Lurasidone Monotherapy in Patients with Acute Episodes of Schizophrenia and Associated Symptoms of Depression.

AIM: We endeavored to evaluate the efficacy of Lurasidone at doses of 40-160 mg per day on symptoms of schizophrenia associated with symptoms of depression in real clinical practice in a Russian patient population. METHODS: One hundred sixty eight patients aged 18-65 years old, who at the time of the start of the observation were being treated in a hospital or day hospital due to an exacerbation of paranoid schizophrenia accompanied by symptoms of depression, were prescribed lurasidone. Treatment with lurasidone and other concomitant drugs, their prescription, withdrawal, selection, and dose modifications were determined based on the indications for the use of those drugs and the recommended doses in the instructions, clinical need, and patient interests, rather than by the goals of the study. During the observation period, the severity of depressive symptoms according to the Calgary Depression Scale (CDSS) and that of psychotic symptoms according to the Positive and Negative Syndrome Scale (PANSS) were assessed six times (before the start of treatment and then on the 4th, 7th, 14th, 28th, and 42nd days). RESULTS: A statistically significant reduction in the severity of the symptoms was observed with the use of lurasidone in doses ranging from 40 mg to 160 mg per day. The fastest and most significant (p <0.001) reductions in the total PANSS and CDSS scores were observed with lurasidone 120 mg. A somewhat lower efficacy of lurasidone was observed at a dose of 160 mg. The largest reductions in the total PANSS and CDSS scores with lurasidone 120 mg were associated with the highest survival rate and the longest median time from treatment initiation to discontinuation or follow-up. The most commonly reported side effects with lurasidone in this study (nausea, akathisia, tremor and drowsiness) were consistent with the known safety profile of the drug. Adverse events in most cases were assessed as mild, or occasionally moderate. CONCLUSION: A six-week prospective observational study of the real-world clinical effectiveness of lurasidone in doses ranging from 40 mg to 160 mg per day established statistically and clinically significant improvements in both psychotic and depressive symptoms in patients with acute exacerbation of schizophrenia and associated symptoms of depression.

Optimization of in vitro trophoblast assay for real-time impedimetric sensing of trophoblast-erythrocyte interactions in Plasmodium falciparum malaria.

Sequestration of Plasmodium falciparum-infected erythrocytes (IEs) is responsible for the pathophysiology of placental malaria, leading to serious complications such as intrauterine growth restriction and low birth weight. However, it is an experimental challenge to study the biology of human placenta. Conventional cell culture-based in vitro placental models rely on immunostaining techniques and high-magnification microscopy is limited in providing real-time quantitative analysis. Impedimetric sensing in combination with cell culture may offer a useful tool. In this paper, we report that real-time label-free measurement of cellular electrical impedance using xCELLigence technology can be used to quantify the proliferation, syncytial fusion, and long-term response of BeWo cells to IEs cytoadhesion. Specifically, we optimized key experimental parameters of cell seeding density and concentration of forskolin, a compound used to promote cell syncitiation, based on electrical signals and immunostaining results. Prolonged time of infection with IEs that led to cell-cell junction vanishment in BeWo cells and release of inflammatory cytokines were monitored in real time by continuous change in electrical impedance. The results suggest that the impedimetric technique is sensitive and can offer new opportunities for the study of cellular responses of trophoblast cells to IEs. The developed system can provide potentially a high-throughput screening tool of anti-adhesion or anti-inflammatory drugs for placental malaria infections.

Supercoil Levels in E. coli and Salmonella Chromosomes Are Regulated by the C-Terminal 35⁻38 Amino Acids of GyrA.

Prokaryotes have an essential gene-gyrase-that catalyzes negative supercoiling of plasmid and chromosomal DNA. Negative supercoils influence DNA replication, transcription, homologous recombination, site-specific recombination, genetic transposition and sister chromosome segregation. Although E. coli and Salmonella Typhimurium are close relatives with a conserved set of essential genes, E. coli DNA has a supercoil density 15% higher than Salmonella, and E. coli cannot grow at the supercoil density maintained by wild type (WT) Salmonella. E. coli is addicted to high supercoiling levels for efficient chromosomal folding. In vitro experiments were performed with four gyrase isoforms of the tetrameric enzyme (GyrA2:GyrB2). E. coli gyrase was more processive and faster than the Salmonella enzyme, but Salmonella strains with chromosomal swaps of E. coli GyrA lost 40% of the chromosomal supercoil density. Reciprocal experiments in E. coli showed chromosomal dysfunction for strains harboring Salmonella GyrA. One GyrA segment responsible for dis-regulation was uncovered by constructing and testing GyrA chimeras in vivo. The six pinwheel elements and the C-terminal 35⁻38 acidic residues of GyrA controlled WT chromosome-wide supercoiling density in both species. A model of enzyme processivity modulated by competition between DNA and the GyrA acidic tail for access to ß-pinwheel elements is presented.

Nicotinamide inhibits the growth of P. falciparum and enhances the antimalarial effect of artemisinin, chloroquine and pyrimethamine.

Nicotinamide (vitamin B3) - is a water soluble amide derivative of nicotinic acid, which has been used at high doses for a variety of therapeutic applications. However, its antimalarial effect has not been intensively explored. In this work we analysed the effect of nicotinamide alone and in combination with artemisinin, chloroquine andpyrimethamine on the growth of blood stages of P. falciparum. Our results demonstrate that nicotinamide effectively inhibits the growth of blood stage parasites with IC50 of 6.9±0.1mM and 2.2±0.3mM for CS2 and 3G8 strains, respectively. The combination of nicotinamide with artemisinin, chloroquine and pyrimethamine demonstrated synergistic effects at IC 10-90%. Treatment of uninfected red blood cells with high dose of nicotinamide (60mM) did not provoke the significant LDH release, demonstrating its non-toxicity for erythrocytes. Nicotinamide acts below the level of tolerance and reduces the effective concentration of anti-malarial drugs due to synergism. These in vitro results suggest that nicotinamide might be useful not only as a vitamin supplement but also as an enhancer of the anti-parasitic effect of common antimalarial drugs including artemisinin, chloroquine and pyrimethamine.

Anti-malarial effect of semi-synthetic drug amitozyn.

BACKGROUND: Malaria caused by Plasmodium falciparum is the most virulent form of malaria, leading to approximately a half million deaths per year. Chemotherapy continues to be a key approach in malaria prevention and treatment. Due to widespread parasite drug resistance, identification and development of new anti-malarial compounds remains an important task of malarial parasitology. The semi-synthetic drug amitozyn, obtained through alkylation of major celandine (Chelidonium majus) alkaloids with N,N'N'-triethylenethiophosphoramide (ThioTEPA), is a widely used Eastern European folk medicine for the treatment of various tumours. However, its anti-malarial effect has never been studied. METHODS: The anti-malarial effects of amitozyn alone and in combination with chloroquine, pyrimethamine and artemisinin on the blood stages of P. falciparum were analysed. The cytostatic effects of amitozyn on parasites and various cancerous and non-cancerous human cells were compared and their toxic effects on unparasitized human red blood cells were analysed. RESULTS: Obtained results demonstrate that amitozyn effectively inhibits the growth of blood-stage parasites with IC50 9.6 ± 2, 11.3 ± 2.8 and 10.8 ± 1.8 µg/mL using CS2, 3G8 and NF54 parasite lines, respectively. The median IC50 for 14 tested human cell lines was 33-152 µg/mL. Treatment of uninfected red blood cells with a high dose of amitozyn (500 µg/mL) did not change cell morphology, demonstrating its non-toxicity for erythrocytes. The synergistic impact of the amitozyn/chloroquine combination was observed at growth inhibition levels of 10-80 %, while demonstrating a nearly additive effect at a growth inhibition level of 90 %. The combination of amitozyn with pyrimethamine has a synergistic effect at growth inhibition levels of 10-70 % and a nearly additive effect at a growth inhibition level of 90 %. The synergistic anti-malarial effect of the amitozyn/artemisinin combination was observed at growth inhibition levels of 10-40 % and a nearly additive effect at growth inhibition levels of 50-90 %. CONCLUSIONS: These in vitro results suggest that the semi-synthetic drug amitozyn, typically used for the treatment of tumours, is a potential anti-malarial candidate and warrants more detailed laboratory and pre-clinical investigations.

Rates of gyrase supercoiling and transcription elongation control supercoil density in a bacterial chromosome.

Gyrase catalyzes negative supercoiling of DNA in an ATP-dependent reaction that helps condense bacterial chromosomes into a compact interwound "nucleoid." The supercoil density (σ) of prokaryotic DNA occurs in two forms. Diffusible supercoil density (σ(D)) moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σ(C)) results from binding abundant proteins that bend, loop, or unwind DNA at many sites. Diffusible and constrained supercoils contribute roughly equally to the total in vivo negative supercoil density of WT cells, so σ = σ(C)+σ(D). Unexpectedly, Escherichia coli chromosomes have a 15% higher level of σ compared to Salmonella enterica. To decipher critical mechanisms that can change diffusible supercoil density of chromosomes, we analyzed strains of Salmonella using a 9 kb "supercoil sensor" inserted at ten positions around the genome. The sensor contains a complete Lac operon flanked by directly repeated resolvase binding sites, and the sensor can monitor both supercoil density and transcription elongation rates in WT and mutant strains. RNA transcription caused (-) supercoiling to increase upstream and decrease downstream of highly expressed genes. Excess upstream supercoiling was relaxed by Topo I, and gyrase replenished downstream supercoil losses to maintain an equilibrium state. Strains with TS gyrase mutations growing at permissive temperature exhibited significant supercoil losses varying from 30% of WT levels to a total loss of σ(D) at most chromosome locations. Supercoil losses were influenced by transcription because addition of rifampicin (Rif) caused supercoil density to rebound throughout the chromosome. Gyrase mutants that caused dramatic supercoil losses also reduced the transcription elongation rates throughout the genome. The observed link between RNA polymerase elongation speed and gyrase turnover suggests that bacteria with fast growth rates may generate higher supercoil densities than slow growing species.

Identification of the UDP-N-acetylglucosamine 4-epimerase involved in exosporium protein glycosylation in Bacillus anthracis.

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a loosely fitting exosporium composed of a basal layer and an external hair-like nap. The filaments of the nap are formed by trimers of the collagen-like glycoprotein BclA. The side chains of BclA include multiple copies of two linear rhamnose-containing oligosaccharides, a trisaccharide and a pentasaccharide. The pentasaccharide terminates with the unusual deoxyamino sugar anthrose. Both oligosaccharide side chains are linked to the BclA protein backbone through an N-acetylgalactosamine (GalNAc) residue. To identify the gene encoding the epimerase required to produce GalNAc for BclA oligosaccharide biosynthesis, three annotated UDP-glucose 4-epimerase genes of B. anthracis were cloned and expressed in Escherichia coli. The candidate proteins were purified, and their enzymatic activities were assessed. Only two proteins, encoded by the BAS5114 and BAS5304 genes (B. anthracis Sterne designations), exhibited epimerase activity. Both proteins were able to convert UDP-glucose (Glc) to UDP-Gal, but only the BAS5304-encoded protein could convert UDP-GlcNAc to UDP-GalNAc, indicating that BAS5304 was the gene sought. Surprisingly, spores produced by a mutant strain lacking the BAS5304-encoded enzyme still contained normal levels of BclA-attached oligosaccharides. However, monosaccharide analysis of the oligosaccharides revealed that GlcNAc had replaced GalNAc. Thus, while GalNAc appears to be the preferred amino sugar for the linkage of oligosaccharides to the BclA protein backbone, in its absence, GlcNAc can serve as a substitute linker. Finally, we demonstrated that the expression of the BAS5304 gene occurred in a biphasic manner during both the early and late stages of sporulation.

The spore-specific alanine racemase of Bacillus anthracis and its role in suppressing germination during spore development.

Spores of Bacillus anthracis are enclosed by an exosporium composed of a basal layer and an external hair-like nap. The nap is apparently formed by a single glycoprotein, while the basal layer contains many different structural proteins and several enzymes. One of the enzymes is Alr, an alanine racemase capable of converting the spore germinant l-alanine to the germination inhibitor d-alanine. Unlike other characterized exosporium proteins, Alr is nonuniformly distributed in the exosporium and might have a second spore location. In this study, we demonstrated that expression of the alr gene, which encodes Alr, is restricted to sporulating cells and that the bulk of alr transcription and Alr synthesis occurs during the late stages of sporulation. We also mapped two alr promoters that are differentially active during sporulation and might be involved in the atypical localization of Alr. Finally, we constructed a Deltaalr mutant of B. anthracis that lacks Alr and examined the properties of the spores produced by this strain. Mature Deltaalr spores germinate more efficiently in the presence of l-alanine, presumably because of their inability to convert exogenous l-alanine to d-alanine, but they respond normally to other germinants. Surprisingly, the production of mature spores by the Deltaalr mutant is defective because approximately one-half of the nascent spores germinate and lose their resistance properties before they are released from the mother cell. This phenotype suggests that an important function of Alr is to produce D-alanine during the late stages of sporulation to suppress premature germination of the developing spore.

Anthrose biosynthetic operon of Bacillus anthracis.

The exosporium of Bacillus anthracis spores consists of a basal layer and an external hair-like nap. The nap is composed primarily of the glycoprotein BclA, which contains a collagen-like region with multiple copies of a pentasaccharide side chain. This oligosaccharide possesses an unusual terminal sugar called anthrose, followed by three rhamnose residues and a protein-bound N-acetylgalactosamine. Based on the structure of anthrose, we proposed an enzymatic pathway for its biosynthesis. Examination of the B. anthracis genome revealed six contiguous genes that could encode the predicted anthrose biosynthetic enzymes. These genes are transcribed in the same direction and appear to form two operons. We introduced mutations into the B. anthracis chromosome that either delete the promoter of the putative upstream, four-gene operon or delete selected genes in both putative operons. Spores produced by strains carrying mutations in the upstream operon completely lacked or contained much less anthrose, indicating that this operon is required for anthrose biosynthesis. In contrast, inactivation of the downstream, two-gene operon did not alter anthrose content. Additional experiments confirmed the organization of the anthrose operon and indicated that it is transcribed from a sigma(E)-specific promoter. Finally, we demonstrated that anthrose biosynthesis is not restricted to B. anthracis as previously suggested.

Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana.

Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumefaciens-mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2-mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Co-immunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Co-immunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of Bs2. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1-containing protein complex that is required for the expression of bacterial disease resistance.

A cytokinetic function of Drosophila ORC6 protein resides in a domain distinct from its replication activity.

Coordination between separate pathways may be facilitated by the requirements for common protein factors, a finding congruent with the link between proteins regulating DNA replication with other important cellular processes. We report that the smallest of Drosophila origin recognition complex subunits, Orc6, was found in embryos and cell culture localized to the cell membrane and cleavage furrow during cell division as well as in the nucleus. A two-hybrid screen revealed that Orc6 interacts with the Drosophila peanut (pnut), a member of the septin family of proteins important for cell division. This interaction, mediated by a distinct C-terminal domain of Orc6, was substantiated in Drosophila cells by coimmunoprecipitation from extracts and cytological methods. Silencing of Orc6 expression with double-stranded RNA resulted in a formation of multinucleated cells and also reduced DNA replication. Deletion of the C-terminal Orc6-peanut interaction domain and subsequent overexpression of the Orc6 mutant protein resulted in the formation of multinucleated cells that had replicated DNA. This mutant protein does not localize to the membrane or cleavage furrows. These results suggest that Orc6 has evolved a domain critical mainly for cytokinesis.