A Neurotoxic Insecticide Promotes Fungal Infection in Aedes aegypti Larvae by Altering the Bacterial Community.

Symbiotic bacteria have a significant impact on the formation of defensive mechanisms against fungal pathogens and insecticides. The microbiome of the mosquito Aedes aegypti has been well studied; however, there are no data on the influence of insecticides and pathogenic fungi on its structure. The fungus Metarhizium robertsii and a neurotoxic insecticide (avermectin complex) interact synergistically, and the colonization of larvae with hyphal bodies is observed after fungal and combined (conidia + avermectins) treatments. The changes in the bacterial communities (16S rRNA) of Ae. aegypti larvae under the influence of fungal infection, avermectin toxicosis, and their combination were studied. In addition, we studied the interactions between the fungus and the predominant cultivable bacteria in vitro and in vivo after the coinfection of the larvae. Avermectins increased the total bacterial load and diversity. The fungus decreased the diversity and insignificantly increased the bacterial load. Importantly, avermectins reduced the relative abundance of Microbacterium (Actinobacteria), which exhibited a strong antagonistic effect towards the fungus in in vitro and in vivo assays. The avermectin treatment led to an increased abundance of Chryseobacterium (Flavobacteria), which exerted a neutral effect on mycosis development. In addition, avermectin treatment led to an elevation of some subdominant bacteria (Pseudomonas) that interacted synergistically with the fungus. We suggest that avermectins change the bacterial community to favor the development of fungal infection.

[Genetic characterization of clinical Klebsiella isolates circulating in Novosibirsk]. / Генетическая Ñ Ð°Ñ€Ð°ÐºÑ‚ÐµÑ€Ð¸ÑÑ‚Ð¸ÐºÐ° ÐºÐ»Ð¸Ð½Ð¸Ñ‡ÐµÑÐºÐ¸Ñ Ð¸Ð·Ð¾Ð»ÑÑ‚Ð¾Ð² клебсиелл, Ñ†Ð¸Ñ€ÐºÑƒÐ»Ð¸Ñ€ÑƒÑŽÑ‰Ð¸Ñ Ð² Новосибирске.

72 clinical strains of Klebsiella spp. isolated from samples obtained from humans in Novosibirsk, Russia, were analyzed. Species identification of strains was performed using 16S rRNA and rpoB gene sequences. It was revealed that Klebsiella pneumoniae strains were dominant in the population (57 strains), while the remaining 15 strains were K. grimontii, K. aerogenes, K. oxytoca and K. quasipneumoniae. By molecular serotyping using the wzi gene sequence, K. pneumoniae strains were assigned to twenty-one K-serotypes with a high proportion of virulent K1- and K2-serotypes. It was found that K. pneumoniae strains isolated from the hospitalized patients had a higher resistance to antibiotics compared to the other Klebsiella species. Real-time PCR revealed that the population contained genes of the blaSHV, blaTEM, blaCTX families and the blaOXA-48 gene, which are the genetic determinants of beta-lactam resistance. It has been shown that the presence of the blaCTX sequence correlated with the production of extended-spectrum beta-lactamases, and phenotypic resistance to carbapenems is due to the presence of the blaOXA-48 gene. At the same time, the carbapenemase genes vim, ndm, kpc, imp were not detected. Among the aminoglycoside resistance genes studied, the aph(6)-Id and aadA genes were found, but their presence did not always coincide with phenotypic resistance. Resistance to fluoroquinolones in the vast majority of strains was accompanied by the presence of the aac(6')-IB-cr, oqxA, oqxB, qnrB, and qnrS genes in various combinations, while the presence of the oqxA and/or oqxB genes alone did not correlate with resistance to fluoroquinolones. Thus, the detection of blaCTX and blaOXA-48 can be used to quickly predict the production of extended-spectrum beta-lactamases and to determine the resistance of Klebsiella to carbapenems. The detection of the aac(6')-Ib-cr and/or qnrB/qnrS genes can be used to quickly determine resistance to fluoroquinolones.

Taxonomic composition and biodiversity of the gut microbiome from patients with irritable bowel syndrome, ulcerative colitis, and asthma.

To date, the association of an imbalance of the intestinal microbiota with various human diseases, including both diseases of the gastrointestinal tract and disorders of the immune system, has been shown. However, despite the huge amount of accumulated data, many key questions still remain unanswered. Given limited data on the composition of the gut microbiota in patients with ulcerative colitis (UC) and irritable bowel syndrome (IBS) from different parts of Siberia, as well as the lack of data on the gut microbiota of patients with bronchial asthma (BA), the aim of the study was to assess the biodiversity of the gut microbiota of patients with IBS, UC and BA in comparison with those of healthy volunteers (HV). In this study, a comparative assessment of the biodiversity and taxonomic structure of gut microbiome was conducted based on the sequencing of 16S rRNA genes obtained from fecal samples of patients with IBS, UC, BA and volunteers. Sequences of the Firmicutes and Bacteroidetes types dominated in all samples studied. The third most common in all samples were sequences of the Proteobacteria type, which contains pathogenic and opportunistic bacteria. Sequences of the Actinobacteria type were, on average, the fourth most common. The results showed the presence of dysbiosis in the samples from patients compared to the sample from HVs. The ratio of Firmicutes/Bacteroidetes was lower in the IBS and UC samples than in HV and higher the BA samples. In the samples from patients with intestinal diseases (IBS and UC), an increase in the proportion of sequences of the Bacteroidetes type and a decrease in the proportion of sequences of the Clostridia class, as well as the Ruminococcaceae, but not Erysipelotrichaceae family, were found. The IBS, UC, and BA samples had signif icantly more Proteobacteria sequences, including Methylobacterium, Sphingomonas, Parasutterella, Halomonas, Vibrio, as well as Escherichia spp. and Shigella spp. In the gut microbiota of adults with BA, a decrease in the proportion of Roseburia, Lachnospira, Veillonella sequences was detected, but the share of Faecalibacterium and Lactobacillus sequences was the same as in healthy individuals. A signif icant increase in the proportion of Halomonas and Vibrio sequences in the gut microbiota in patients with BA has been described for the f irst time.

Bacteriophages as Therapeutic Preparations: What Restricts Their Application in Medicine.

The increasing prevalence of bacterial pathogens with multiple antibiotic resistance requires development of new approaches to control infections. Phage therapy is one of the most promising approaches. In recent years, research organizations and a number of pharmaceutical companies have intensified investigations aimed at developing bacteriophage-based therapeutics. In the United States and European countries, special centers have been established that experimentally apply phage therapy to treat patients who do not respond to antibiotic therapy. This review describes the features of bacteriophages as therapeutic tools, critically discusses the results of clinical trials of bacteriophage preparations, and assesses the prospects for using phage therapy to treat certain types of infectious diseases.

[Oncolytic enteroviruses].

Increasing information concerning molecular biology of viruses and virus-cell interactions makes it possible to use viruses as a tool in effort to treat cancer diseases. As a rule, tumor cells are highly sensitive to viruses that may be used in cancer therapy. Therewith, applications of viral oncolysis in treatment of cancer diseases assume maximum possible safety of used viruses for patient and environment. Human enteroviruses are one of the most convenient sources to generate oncolytic viruses. Many of enteroviruses are non-pathogenic for humans or cause mild disease. Progress in genetic engineering permits to develop attenuated enterovirus variants with high safety and selectivity. This review focuses on the main members of Enterovirus genus, such as Coxsackieviruses, and vaccine strains as promising source for development of oncolytic agents, applicable for cancer therapy. It reviews data concerning recently developed and tested oncolytic variants of enteroviruses and discusses perspectives of their application in cancer therapy and problems, concerning their improvement and practical use.

[Combinatorial libraries of phage antibodies: application for analysis of gene segments encoding variable domains of autoantibodies to tumor necrosis factor].

Twenty unique phage antibodies to human tumor necrosis factor alpha were selected from a naive combinatorial library of human single chain fragment variable. Analysis of gene segments encoding selected antibodies shown that repertoire of variable domains of heavy and light chains included variable domains of both naive autoantibodies and antibodies produced as a result of somatic hypermutagenesis.

Selection of naturally occurring autoantibodies to interleukin-18 from phage display library.

Four unique phage single-chain variable fragments (scFvs) to recombinant human interleukin 18 (IL-18) has been selected from a naïve combinatorial library of human scFvs. Binding of unique phage antibodies with IL-18 was tested by ELISA and Western-blotting. No cross reactivity with tumor necrosis factor α, interferons α and γ was shown for the selected antibodies. The gene segments encoding V(D)J regions of selected antibodies exhibited a high degree of homology to germline genes, therefore we suggest that the selected scFv's belong to repertoire of naïve autoantibodies.

[Influence of the cycle number in processing of cellulose from waste paper on its ability to hydrolysis by cellulases].

Hydrolytic ability of laboratory enzyme preparations from fungus of the Penicillium genus was investigated using kraft pulp from nonbleached softwood and bleached hardwood cellulose as substrates. The enzyme preparations were shown to efficiently hydrolyze both softwood and hardwood cellulose. The yields of glucose and reducing sugars were 24-36 g/l and 27-37 g/l from 100 g/l of dry substrate in 48 h, respectively, and depended on the number of substrate grinding cycles.

Isolation and properties of fungal beta-glucosidases.

Using chromatography on different matrixes, three beta-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) beta-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (beta-glucan from barley and laminarin); ii) beta-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).

Phage display on the base of filamentous bacteriophages: application for recombinant antibodies selection.

The display of peptides and proteins on the surface of filamentous bacteriophage is a powerful methodology for selection of peptides and protein domains, including antibodies. An advantage of this methodology is the direct physical link between the phenotype and the genotype, as an analyzed polypeptide and its encoding DNA fragment exist in one phage particle. Development of phage display antibody libraries provides repertoires of phage particles exposing antibody fragments of great diversity. The biopanning procedure facilitates selection of antibodies with high affinity and specificity for almost any target. This review is an introduction to phage display methodology. It presents recombinant antibodies display in more details:, construction of phage libraries of antibody fragments and different strategies for the biopanning procedure.

[Use of a preparation from fungal pectin lyase in the food industry].

A new enzyme preparation of fungal pectin lyase (EC 4.2.2.10) was shown to be useful for the production of cranberry juice and clarification of apple juice in the food industry. A comparative study showed that the preparation of pectin lyase is competitive with commercial pectinase products. The molecular weight of homogeneous pectin lyase was 38 kDa. Properties of the homogeneous enzyme were studied. This enzyme was most efficient in removing highly esterified pectin.

[Application of neutral-alkaline pectate lyases to cotton fabric boil off].

Commercial and pilot pectate lyase preparations (EC 4.2.2.2) have been compared. They differ in their effect on pectins with different esterification degrees (ED). The activity of the pilot preparation with respect to a substrate with ED = 70% is tenfold lower than with respect to unesterified polygalacturonic acid. For commercial preparations, this activity ratio ranged within 1.5-2. At equal pectate lyase activities, the commercial preparations better remove pectin from crude cotton fabric during its boil off. The laboratory preparation is more efficient for improving the capillarity (wettability) of the fabric owing to the cooperative effect of the pectate lyase, cellulase, and hemicellulase present in the preparation.

[Construction and characterization of combinatorial naive phage library of human scFvs].

A combinatorial phage display library of human single-chain antibody fragments (scFv) was constructed on the basis of variable domains of heavy (Vh) and light (VI) genes cloned from the lymphocytes of six healthy donors. The size of the library was 2? 10(8) independent clones. Single-chain antibodies against recombinant human TNF?, vaccinia virus and virus-like particles formed by core protein of hepatitis B virus were selected from the library. Unique scFv sequences were identified using the HaeIII fingerprinting. The specificity of the selected clones was proved by the Western-blot analysis.

[Human recombinant antibodies to variola virus].

Eight specific antibodies to live variola virus (VV), Ind-3a strain, and 7 antibodies to VV, Butler strain, were selected from the synthetic combinatorial phage display library on single-chain (scFv) human antibodies. Indirect solid-phase enzyme immunoassay showed the ability of these antibodies to bind the VV strains Ind-3a, Butler, Brazil-131, Kuw-5, and Congo-2. Moreover, earlier selected human scFv antibodies were also tested in the reaction of binding to the above VV strains. The experiments could reveal the antibodies that bound alastrim strains more effectively that did other VV strains. The nucleotide sequences encoding for the selected scFv antibodies were determined.

[Human mini-antibodies to orthopoxviruses]. / Mini-antitela cheloveka protiv ortopoksvirusov.

A library of human scFv antibodies displayed on the surface of bacteriophages (MRC, Cambridge, England) was panned against the Elstree strain of vaccinia virus (VACV), which resulted in the phage repertoire enriched with clones positive to the strain. Individual clones from the repertoire were screened for binding, independently, to the vaccinia and ectromelia viruses; phage antibodies to the orthopoxviruses were selected. Ten unique antibodies were identified after their Vh- and Vl-genes were sequenced. All selected antibodies were assayed by ELISA for binding to the vaccinia, cowpox and ectromelia viruses. Furthermore, all selected antibodies were assayed for binding with major alastrim strains of the live variola virus. According to the results, the above phage antibodies recognized genus-specific epitopes, some of which differed in their conformation.

[Correlation between the dimensions of the human paranasal sinuses and the skull type]. / Zavisimost' razmerov okolonosovykh pazukh cheloveka ot tipa cherepa.

To detect the dependence of the dimensions of maxillar and frontal paranasal sinuses on the age, sex, and the skull type in population inhabiting Karelya, 1566 maxillar sinuses and 1404 frontal sinuses were studied in radiographs of practically healthy subjects aged 3-85 years. The correlation between age, sex, dimensions of maxillar and frontal sinuses, and the skull type was established that is revealed in that the latitudinal dimensions of the sinuses prevail in brachiomorphous skull type, while the altitude dimensions are prevalent in dolichomorphous type regardless the sex and age. Total dimensions of maxillar sinuses are larger in dolichomorphous skull type: in males that is especially pronounced for maxillar sinuses, while in females--for frontal sinuses.

[Changes in paranasal sinuses during postnatal ontogenesis of inhabitants of Karelia ]. / Izmeneniia okolonosovykh pazukh v postnatal'nom ontogeneze u zhitele Karelii.

Roentgenographs of 1566 maxillary, 1404 frontal and 131 sphenoid sinuses (a total of 3101 sinuses) belonging to the practically healthy individuals of both sexes aged 3-85 years were studied to reveal the regularities of growth of paranasal sinuses in inhabitants of Karelya. It was established that the dimensions of paranasal sinuses depended upon person's age and gender. The growth of paranasal sinuses in women was completed 1.5-2.0 years earlier than in men. In persons of elderly and senile age there was a secondary increase in sinuses' dimensions, associated with osteoporosis phenomena. In women this secondary increase was more pronounced than in men and started on average 5 years earlier.

Phage antibodies from combinatorial library neutralize vaccinia virus.

The library of human scFv antibodies displayed on the surface of bacteriophages was panned against Vaccinia virus (VACV), strain Elstree. 75% binding with Vaccinia virus. 5 clones were characterized for their binding with VACV and their ability to neutralize VACV in plaque reduction neutralization test (PRNT). Antibodies from the clones were obtained as soluble individual molecules and their binding activities were confirmed in ELISA.