Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization

<p>Abstract</p> <p>Background</p> <p>RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet.</p> <p>Results</p> <p>Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of λRed-driven recombination between the plasmid and a constructed <it>in vitro </it>linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, <it>repB</it>, the plasmid loci <it>oriT</it>, <it>mobC </it>and <it>mobA </it>were substituted by the DNA fragment containing P_{<it>lac</it>UV5}→<it>lacI</it>. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after <it>lacI </it>elimination. High stability of both constructed plasmids has been demonstrated in <it>Escherichia coli </it>and <it>Pantoea ananatis</it>. Design of RSFmob allows easy substitution of P_{<it>lac</it>UV5}by any desirable promoter for construction of novel derivatives with changed copy number or host range.</p> <p>Conclusion</p> <p>Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in <it>E. coli </it>and <it>P. ananatis </it>have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production.</p

Optimization of cultivation medium for the production of Bacillus intermedius 3-19 glutamyl endopeptidase | Optimizatsiia sredy kul'tivirovaniia dlia produktsii glutamiléndopeptidazy Bacillus intermedius 3-19.

The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of glutamyl endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of glutamyl endopeptidase

The Infestation of Fleas of the Long-Tailed Souslik with Entomoparasitic Nematodes in Tuva Mountain Natural Plague Focus

Entomoparasitic nematodes are supposed to be a link between parts of Yersinia pestis population in the environment and the flea vector. The aim of the study was to assess the prevalence and intensity of infestation in the fleas of the long-tailed souslik with entomoparasitic nematodes on the territory of Mongun-Taiginsky station in the Tuva natural plague focus. Materials and methods. Fleas were collected during the scheduled epizootiological surveys in 2019–2021. In the course of taxonomic identification the presence of parasitic nematodes was registered. In order to evaluate the intensity of nematode invasion, a total of 190 fleas were dissected. The number of adult parasitizing females and presence of larvae was recorded. Statistical processing of the data was performed with the help of conventional methods using the Excel software. The criterion χ2 was applied; the influence of various factors (species, gender of fleas) on the studied parameters was assessed through single- and two-factor analysis of variance. Results and discussion. During three years of observations, entomoparasitic nematodes were found in six species of fleas: Citellophilus tesquorum, Frontopsylla elatoides, Rhadinopsylla li transbaikalica, Frontopsylla hetera, Oropsylla alaskensis, and Neopsylla mana. The differences in infestation with nematodes between the species are presented. The highest invasion rate – 25.1–25.6 % – is observed in Rh. li transbaikalica. The gender of leas does not influence their infestation. It is established that invaded fleas are more often found in the nest than in the fur of animals, they are less actively migrate to the burrow entrance compared to not invaded ones. Evaluation of infestation prevalence has revealed that fleas Rh. li transbaikalica are the hosts for nematodes of mono- or oligoxenic species, which do not occur in other fleas

Optimization of cultivation medium for the production of Bacillus intermedius 3-19 glutamyl endopeptidase

The effect of some components of cultivation medium on the growth of the streptomycin-resistant Bacillus intermedius strain 3-19 and on the production of glutamyl endopeptidase was investigated using factorial experimental design, which allowed the concentrations of peptone and inorganic phosphate to be optimized for the maximum production of the enzyme. Experiments with different peptones and casamino acids showed that the enzyme production is maximum with peptone 3 of plant origin. The addition of casamino acids or amino acids to the peptone-containing cultivation medium inhibited the production of glutamyl endopeptidase. © 2002 MAIK "Nauka/Interperiodica"

PECULIARITIES OF INTERACTION BETWEEN CITELLOPHILUS TESQUORUM ALTAICUS (IOFF, 1936) FLEA AND YERSINIA PESTIS WITH VARIOUS PLASMID COMPOSITION

Objective of the study is in vitro investigation of mutual relations between Citellophilus tesquorum altaicus and Yersinia pestis with various plasmid composition: influence of the strain on flea alimentary activity and mortality rate, frequency and dynamics of biofilm formation.Materials and methods. C. tesquorum altaicus were infected by three Yersinia pestis strains: virulent triple-plasmid I-3230 isolated in Mongolia, referential for the Tuva focus I-2638 carrying four plasmids (pYT, pYV, pYP, pTP 33) and also selected from it avirulent isogenic clone I-3480 that lost two plasmids (pYV, pYP). Peculiarities of interaction between fleas and Y. pestis strains were estimated through the lens of specimens with «conglomerates» and “blocks” for feeding, the period from infection prior to the beginning of conglomerates’ formation, alimentary activity, and mortality rate of the infected fleas.Results and conclusions. It was revealed that alimentary activity of the infected insects was higher than that of the control group, and the highest – in fleas infected with I-2638 strain. Greater numbers of dead fleas at feeding was noted in specimens inoculated with I-3230 strain. Predominant significance of I-2638 strain was established in C. tesquorum altaicus biofilm formation both as «conglomerates» and “blocks”. I-3480 strain also formed the conglomerates in fleas more actively than I-3230 lacking pTP33 plasmid. Thus, four-plasmid I-2638 strain surpassed triple-plasmid I-3230 and two-plasmid I-3480 strains in reference to all tested indicators except flea mortality rates. It may testify to co-adaptation of Y. pestis and C. tesquorum altaicus from the Tuva plague focus and to the possibility of a pTP33 functional role in enhancement of a biofilm formation in vivo

Synthesis and secretion of proteinases by Bacillus intermedius in the late stages of sporulation

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating β-galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites

Synthesis and secretion of proteinases by Bacillus intermedius in the late stages of sporulation

In the late stages of sporulation, cells of Bacillus intermedius 3-19 secreted into the medium two proteinases, glutamyl endopeptidase and subtilisin, whose maximum activities were recorded in the 40th and 44th hours of growth, respectively. By estimating β-galactosidase activity as a marker of cytoplasmic membrane integrity, it was revealed that the accumulation of these proteinases in the medium was a result of their secretion and not of lysis of the cell envelope. Concentrations of peptone and inorganic phosphate ensuring the maximum production of the enzymes were established. Ammonium ions were shown to inhibit the production of proteinases by the mechanism of repression by nitrogen metabolites

A prospective randomized controlled trial of bone metabolism in patients with charcot foot

BACKGROUND: Diabetic neuroosteoarthropathy (DNOAP, Charcot&rsquo;s foot) - is a progressive destructive inflammatory disease of the osteoarticular apparatus of the foot, untimely and inadequate treatment of which can lead to the formation of gross deformities. More often, DNOAP is unilateral, bilateral lesion is relatively rare. It is not always possible to trace the relationship between the debut of DNOAP with trauma and chronic hyperglycemia. There is data demonstrating the role of individual pro-inflammatory factors in the pathogenesis of DNOAP, however, studies combining the evaluation of various metabolic markers of Charcot&rsquo;s foot formation are currently extremely poor. AIM: To evaluate the hormonal and metabolic markers of bone formation and resorption in patients with DNOAP and without this diabetic complication. METHODS: A prospective, controlled trial included 70 patients with type 2 diabetes mellitus (37 men and 43 women) who formed 2 groups: group 1 included patients with DNOAP, group 2 was formed by patients with diabetes without foot skeleton damage. All patients underwent a study of 1,25-OH-vitamin D, sclerostin, pro-MMP-1, C-terminal propeptide type 1 collagen (PICP), type 1 collagen, osteocalcin, PTH, 25-OH-vitamin D, beta-cross-slaps. RESULTS: The results of the studies confirmed the presence of vitamin D deficiency in all patients with diabetes mellitus included in the study, revealed the absence of statistically significant differences between the groups in the values of sclerostin, pro-MMP-1; 25-OH-vitamin D, type I collagen, and osteocalcin (p &gt; 0.05). However, significant differences were found in the 1.25-OH vitamin D levels: patients with DNOAP presented the lower rates of 1,25-OH-vitamin D in comparison to control group (p &lt;0.05). Beta-cross and PICP levels were significantly higher in DNOAP patients as well (p &lt;0.05). Those findings show the more severe collagen degradation in patients with DNOAP and can be the genetically predisposed cause of DNOAP development. Though further studies are needed. CONCLUSION: In patients with DNOAP a decrease in 1,25-OH-vitamin D levels was found, as well as the alteration of the synthesis and destruction of collagen (beta-cross-slaps and PICP) compared to patients with diabetes mellitus without osteoarticular disorders

Characteristics of an antioxidant activity of Yersinia pestis with different plasmid spectrum

Experimental data concerning complex study of antioxidant activity of Y. pestis with different plasmid spectrum (wild-type Yersinia pestis subsp. pestis, Yersinia pestis subsp. altaica and their isogenic variants) are represented in the article. Superoxide dismutase activity of the tested Y. pestis strains was from 6.0 to 9.0 x 109 microbe cells. Significant differences between the Y. pestis strains with different plasmid composition were not detected by this parameter. Our results, consistent with the data of other authors, tell that superoxide dismutase activity is a thermo-inducible feature and does not depend on a plasmid spectrum. High peroxide destroying activity was also detected in all tested Y. pestis strains. The differences between the strains regarding their common peroxide destroying activity were found. This parameter of the plague microbe strains lacking pYP plasmid was at least 3 times lower than common peroxide destroying activity in strains with this plasmid in the genome. In our opinion these revealed differences were caused by characteristics of plasmid spectrum. The common peroxide destroying activity's degree of Y. pestis strains can be associated with the presence of pYP plasmid in the genome. The isogenic variants of Y. pestis strains lacking one of the plasmids had smaller pathogenic activity. This fact points to the need for further study of these strains

Diagnosis and rational treatment of painful diabetic peripheral neuropathy: an interdisciplinary expert consensus

Diabetic peripheral neuropathy is a common chronic complication of diabetes mellitus, significantly impairing well-being, quality of life and functioning of patients. The prevalence of diabetic peripheral neuropathy in the Russian Federation ranges from 0.1% to 67.2% in type 1 and from 0.1 to 42.4% in type 2 diabetes mellitus. However, based on the large-scale epidemiological studies, the true prevalence of diabetic peripheral neuropathy is much higher (50 to 70%), with its painful variant occurring in 16% to 30% of patients. Despite the fact that diabetic peripheral neuropathy remains the most common chronic complication of diabetes mellitus, its diagnosis and therapy leave much to be desired. To optimize diagnostic and treatment approaches to painful diabetic peripheral neuropathy, a group of experts representing the leading Russian professional medical associations has developed clinical guidelines for the diagnosis and rational therapy of patients with painful diabetic peripheral neuropathy. This document presents practical aspects of the clinical diagnosis of painful diabetic peripheral neuropathy and an algorithm for differential diagnosis of pain in the lower extremities in patients with diabetes mellitus. The use of symptomatic analgesics with central action, such as anticonvulsants, antidepressants and opioids, is based on the main aspects of neuropathic pain pathophysiology. The characteristics of each drug class are given, with consideration of evidence on their efficacy, tolerability, and the possibility of combination therapy. The data on the first, second, and third lines of agents is presented in accordance with several international clinical guidelines. The need for a tailored drug choice, taking into account the evidence-based data on their efficacy and safety, concomitant drug therapy, tolerability, cost and preferences of the patient, age of the patient and concomitant disorders, is emphasized