Structural and Functional Characteristics of the Proline-Rich Antimicrobial Peptide Minibactenecin from Leukocytes of Domestic Goat Capra hircus.

We performed structural and functional studies of minibactenecin mini-ChBac7.5Nα, a natural proline-rich cathelicidin from domestic goat Capra hircus. To identify the key residues important for the biological action of the peptide, a panel of its alanine-substituted analogues was produced. The development of E. coli resistance to the natural minibactenecin, as well as to its analogues carrying substitutions for hydrophobic amino acids in the C-terminal residues was studied. The data obtained indicate the possibility of rapid development of the resistance to this class of peptides. The main factors in the formation of the antibiotic resistance are various mutations leading to inactivation of the SbmA transporter.

Analysis of Antibacterial Action of Mammalian Host-Defense Cathelicidins and Induction of Resistance to Them in MßL-Producing Pseudomonas aeruginosa.

Recombinant analogs of a number of natural host-defense mammalian cathelicidins were obtained and predominant mechanism of their antibacterial action was studied. The ability of cathelicidins to suppress the growth of Pseudomonas aeruginosa producing metallo-ß-lactamases (MßL) was studied, and the possibility of appearance of cathelicidin-resistant bacteria was evaluated. Among peptides with different structures and mechanisms of action, only the strains resistant to ChMAP-28 were not obtained, which indicated minimum risk of the development of natural resistance to this cathelicidin. High antibacterial activity, wide spectrum of action, and the absence of cross-resistance effects allow considering ChMAP-28 peptide as a candidate to be developed further as a therapeutic agent against MßL-producing bacteria.

New insights into ligand binding by plant lipid transfer proteins: A case study of the lentil Lc-LTP2.

Lipid transfer proteins (LTPs) are an important class of plant proteins containing an internal cavity and binding hydrophobic ligands. Although LTP structures and functions are well studied, mechanisms of ligand binding remain unclear. Earlier, we discovered the lentil lipid transfer protein Lc-LTP2 capable of binding and transfer various ligands. We have shown that the "bottom" entrance of the Lc-LTP2 cavity takes part in attachment to the micelle surface and in lipids uptake. Here, we studied the role of Arg45 and Tyr80, located at the "bottom" entrance, in Lc-LTP2 ligand binding. We obtained recombinant mutant analogs of Lc-LTP2 (R45A, Y80A, R45A/Y80A), investigated their ability to bind fatty acids and lysolipids, as well as performed molecular modeling of the protein-ligand complexes. We showed that replacement of one or both residues led to a change of the internal hydrophobic cavity dimensions. As a result, lipids may change their orientation into the protein cavity, and thereby binding ability of mutant analogs may be affected as well. In the present work, we revealed an important role of Arg45 and Tyr80 in stabilization of the Lc-LTP2 complexes with both fatty acids and lysolipids with different ligand orientation.

Identification of Na+-Pumping Cytochrome Oxidase in the Membranes of Extremely Alkaliphilic Thioalkalivibrio Bacteria.

For the first time, the functioning of the oxygen reductase Na+-pump (Na+-pumping cytochrome c oxidase of the cbb3-type) was demonstrated by examining the respiratory chain of the extremely alkaliphilic bacterium Thioalkalivibrio versutus [Muntyan, M. S., et al. (2015) Cytochrome cbb3 of Thioalkalivibrio is a Na+-pumping cytochrome oxidase, Proc. Natl. Acad. Sci. USA, 112, 7695-7700], a product of the ccoNOQP operon. In this study, we detected and identified this enzyme using rabbit polyclonal antibody against the predicted C-terminal amino acid sequence of its catalytic subunit. We found that this cbb3-type oxidase is synthesized in bacterial cells, where it is located in the membranes. The 48-kDa oxidase subunit (CcoN) is catalytic, while subunits CcoO and CcoP with molecular masses of 29 and 34 kDa, respectively, are cytochromes c. The theoretical pI values of the CcoN, CcoO, and CcoP subunits were determined. It was shown that parts of the CcoO and CcoP subunits exposed to the aqueous phase on the cytoplasmic membrane P-side are enriched with negatively charged amino acid residues, in contrast to the parts of the integral subunit CcoN adjacent to the aqueous phase. Thus, the Na+-pumping cytochrome c oxidase of T. versutus, both in function and in structure, demonstrates adaptation to extremely alkaline conditions.

Short-term outcomes of a multicentre randomized clinical trial comparing D2 versus D3 lymph node dissection for colonic cancer (COLD trial).

BACKGROUND: It remains unclear whether extended lymphadenectomy provides oncological advantages in colorectal cancer. This multicentre RCT aimed to address this issue. METHODS: Patients with resectable primary colonic cancer were enrolled in four hospitals registered in the COLD trial, and randomized to D2 or D3 dissection in a 1 : 1 ratio. Data were analysed to assess the safety of D3 dissection. RESULTS: The study included the first 100 patients randomized in this ongoing trial. Ninety-nine patients were included in the intention-to-treat (ITT) analysis (43 D2, 56 D3). Ninety-two patients received the allocated treatment and were included in the per-protocol (PP) analysis: 39 of 43 in the D2 group and 53 of 56 in the D3 group. There were no deaths. The 30-day postoperative morbidity rate was 47 per cent in the D2 group and 48 per cent in the D3 group, with a risk ratio of 1·04 (95 per cent c.i. 0·68 to 1·58) (P = 0·867). There were two anastomotic leaks (5 per cent) in the D2 group and none in the D3 group. Postoperative recovery, complication and readmission rates did not differ between the groups in ITT and PP analyses. Mean lymph node yield was 26·6 and 27·8 in D2 and D3 procedures respectively. Good quality of complete mesocolic excision was more frequently noted in the D3 group (P = 0·048). Three patients in the D3 group (5 per cent) had metastases in D3 lymph nodes. D3 was never the only affected level of lymph nodes. N-positive status was more common in the D3 group (46 per cent versus 26 per cent in D2), with a risk ratio of 1·81 (95 per cent c.i. 1·01 to 3·24) (P = 0·044). CONCLUSION: D3 lymph node dissection is feasible and may be associated with better N staging. Registration number: NCT03009227 ( http://www.clinicaltrials.gov).

ANTECEDENTES: El beneficio oncológico de la linfadenectomía extendida en el cáncer colorrectal es controvertido. Este ensayo clínico aleatorizado multicéntrico tuvo como objetivo abordar esta discrepancia. MÉTODOS: Se analizaron los datos de los primeros 100 pacientes aleatorizados en un ensayo en curso para evaluar la seguridad de la disección D3. Los pacientes con cáncer de colon primario resecable incluidos en 4 hospitales participantes en el ensayo COLD, se aleatorizaron para la disección D2 y D3 en una proporción 1: 1. RESULTADOS: Se incluyeron 99 pacientes en el análisis por intención de tratamiento (intention-to-treat, ITT) (43 en D2, 56 en D3). Un total de 92 pacientes recibieron el tratamiento asignado y se incluyeron en el análisis por protocolo (per-protocol, PP): 90,7% (39 de 43) en D2, 94,6% (53 de 56) en D3. No hubo mortalidad. La morbilidad postoperatoria a los 30 días fue del 46,5% en el grupo D2 y del 48,2% en el grupo D3 con un riesgo relativo (RR) de 1,04 (i.c. del 95%: 0,68 a 1,58), P = 0,86. Hubo dos casos de fuga anastomótica (4,7%) en el grupo D2 y ninguna en D3. La recuperación postoperatoria, las complicaciones y las tasas de reingreso no difirieron entre los análisis ITT y PP. El recuento medio de ganglios linfáticos fue 26,6 y 27,8 en D2 y D3, respectivamente. Se observó una resección completa del mesorrecto de buena calidad con mayor frecuencia en el grupo D3 (P = 0,048). En el grupo D3, 3 pacientes (5,4%) tenían metástasis en los ganglios linfáticos D3. D3 nunca fue el único nivel afectado de ganglios linfáticos. El estadio pN positivo fue más frecuente en el grupo D3: 46,4% versus 25,6% en D2, con un RR para revelar enfermedad N positiva de 1,81 (i.c. del 95% 1,01 a 3,2), P = 0,04. CONCLUSIÓN: La disección de ganglios linfáticos D3 es factible y puede estar asociada con una mejor estadificación N.

Protocol for a multicentre randomized clinical trial comparing oncological outcomes of D2 versus D3 lymph node dissection in colonic cancer (COLD trial).

Background: The extent of lymph node dissection in colonic cancer surgery remains arguable, and evidence from RCTs regarding extended lymph node dissection outcomes is lacking. This study aimed to compare the long-term results of D3 lymph node dissection with those of D2 dissection. Methods: This is a multicentre RCT. The aim is to enrol 768 patients with primary colonic cancer assigned randomly to D2 or D3 lymph node dissection. The trial is assessing the superiority of 5-year overall survival as the primary endpoint in patients undergoing D3 lymph node dissection versus D2 dissection. Secondary endpoints include disease-free survival, short-term outcomes (30-day morbidity and mortality), quality of complete mesocolic excision and lymph node dissection, pattern of lymph node metastasis and quality of life in patients following D2 and D3 lymph node dissection. Experience of 20 D3 and 20 D2 lymph node dissections is required for surgeons to participate in the trial. For surgical accreditation four non-edited videos of procedures will be assessed. Patients will be followed up for 5 years after last patient enrolment. Intention-to-treat analysis will be performed. Discussion: The results of this study will demonstrate whether extended lymph node dissection is superior to standard dissection in terms of oncological outcomes, and will also assess the impact of more extensive surgery on short-term outcomes and quality of life.

Pediocin-Like Antimicrobial Peptides of Bacteria.

Bacteriocins are bacterial antimicrobial peptides that, unlike classical peptide antibiotics, are products of ribosomal synthesis and usually have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like bacteriocins (PLBs) belong to the class IIa of the bacteriocins of Gram-positive bacteria. PLBs possess high activity against pathogenic bacteria from Listeria and Enterococcus genera. Molecular target for PLBs is a membrane protein complex - bacterial mannose-phosphotransferase. PLBs can be synthesized by components of symbiotic microflora and participate in the maintenance of homeostasis in various compartments of the digestive tract and on the surface of epithelial tissues contacting the external environment. PLBs could give a rise to a new group of antibiotics of narrow spectrum of activity.

Biotechnological Method of Preparation and Characterization of Recombinant Antimicrobial Peptide Avicin A from Enterococcus avium.

Avicin A is a bacteriocin from the gram-positive bacterium Enterococcus avium. It exhibits a high microbicidal activity against bacteria of the genus Listeria, a causative agent of the severe human infection listeriosis. We developed a biotechnological method for obtaining avicin A and characterized its structure and biological activity. We also proposed a possible mechanism of the antimicrobial action of avicin A.

Estimation of the thermal and photochemical stabilities of pheromones.

The correlation between the kinetic stability of molecules against temperature and variations in their geometric structure under optical excitation is investigated by the example of different organic pheromone molecules sensitive to temperature or ultraviolet radiation using the density functional theory. The kinetic stability is determined by the previously developed method based on the calculation of the probability of extension of any structural bond by a value exceeding the limit value Lмах corresponding to the breaking of the bond under temperature excitation. The kinetic stability calculation only requires the eigenfrequencies and vibrational mode vectors in the molecule ground state to be calculated, without determining the transition states. The weakest bonds in molecules determined by the kinetic stability method are compared with the bond length variations in molecules in the excited state upon absorption of light by a molecule. Good agreement between the results obtained is demonstrated and the difference between them is discussed. The universality of formulations within both approaches used to estimate the stability of different pheromone molecules containing strained cycles and conjugated, double, and single bonds allows these approaches to be applied for studying other molecules. Graphical Abstract Estimation of the thermal and photochemical stabilities of pheromones.

In Vitro Study of Antitumor Effect of Antimicrobial Peptide Tachyplesin I in Combination with Cisplatin.

We studied combined effect of ß-hairpin antimicrobial peptide tachyplesin I and cytotoxic agent cisplatin on tumor and normal human cell lines. MTT assay and flow cytometry showed that tachyplesin I selectively sensitized cancer cells to cisplatin in specified concentration ratios. In vitro experiments demonstrated that combined use of tachyplesin I and cisplatin allows decreasing the effective dose of the cytostatic thus reducing nonspecific toxicity.

The First Recombinant Viper Three-Finger Toxins: Inhibition of Muscle and Neuronal Nicotinic Acetylcholine Receptors.

Genes encoding two three-finger toxins TFT-AF and TFT-VN, nucleotide sequences of which were earlier determined by cloning cDNA from venom glands of vipers Azemiops feae and Vipera nikolskii, respectively, were expressed for the first time in E. coli cells. The biological activity of these toxins was studied by electrophysiological techniques, calcium imaging, and radioligand analysis. It was shown for the first time that viper three-finger toxins are antagonists of nicotinic acetylcholine receptors of neuronal and muscle type.

Analysis of Synergistic Effects of Antimicrobial Peptide Arenicin-1 and Conventional Antibiotics.

We studied combined effects of antimicrobial peptide arenicin-1 from lugworm Arenicola marina and some conventional antibiotics. A number of drug combinations with pronounced synergistic effects were revealed. The influence of antibacterial activity assessment conditions was determined and the methodology excluding false-positive test results was developed.

Effect of N- and C-Terminal Modifications on Cytotoxic Properties of Antimicrobial Peptide Tachyplesin I.

We analyze the effects of N-terminal acetylation and C-terminal amidation on the cytotoxic properties of ß-hairpin antimicrobial peptide tachyplesin I. MTT-assay showed that modified tachyplesin I exhibited increased cytotoxicity toward both tumor and normal human cells. Hemolytic activity of modified tachyplesin I was also higher than that of the initial molecule. In contrast to non-modified tachyplesin I, the peptide with C- and N-terminal modifications is resistant to proteolytic degradation in fresh human serum. C- and N-terminal modifications make tachyplesin I more attractive prototype of anticancer drug due to its more potent cytotoxic effect and better pharmacokinetic properties.

Minibactenecins ChBac7.Nα and ChBac7. Nß - Antimicrobial Peptides from Leukocytes of the Goat Capra hircus.

Antimicrobial peptides (AMPs) of neutrophils play an important role in the animal and human host defenses. We have isolated two AMPs (average molecular masses of 2895.5 and 2739.3 Da), with potent antimicrobial activity from neutrophils of the domestic goat (Capra hircus). A structural analysis of the obtained peptides revealed that they encompass N-terminal fragments (1-21 and 1-22) of the proline-rich peptide bactenecin 7.5. The primary structure of caprine bactenecin 7.5 had been previously deduced from the nucleotide sequence, but the corresponding protein had not been isolated from leukocytes until now. The obtained caprine AMPs were designated as mini-batenecins (mini-ChBac7.5Nα and mini-ChBac7.5Nß), analogously to the reported C-terminal fragment of the ovine bactenecin 7.5 named Bac7.5mini [Anderson, Yu, 2003]. Caprine mini-ChBac7.5Nα and mini-ChBac7.5Nß exhibit significant antimicrobial activity against Gram-negative bacteria, including drug-resistant strains of Pseudomonas aeruginosa, Klebsiella spp., Acinetobacter baumannii at a range of concentrations of 0.5-4 µM, as well as against some species of Gram-positive bacteria (Listeria monocytogenes EGD, Micrococcus luteus). The peptides demonstrate lipopolysaccharide-binding activity. Similarly to most proline-rich AMPs, caprine peptides inactivate bacteria without appreciable damage of their membranes. Mini-ChBac7.5Nα and mini-ChBac7.5Nß have no hemolytic effect on human red blood cells and are nontoxic to various cultured human cells. Therefore, they might be considered as promising templates for the development of novel antibiotic pharmaceuticals. Isolation of highly active fragments of the antimicrobial peptide from goat neutrophils supports the hypothesis that fragmentation of cathelicidin-related AMPs is an important process that results in the generation of potent effector molecules, which are in some cases more active than full-size AMPs. These truncated AMPs may play a crucial role in host defense reactions.

Lipid Transfer Proteins As Components of the Plant Innate Immune System: Structure, Functions, and Applications.

Among a variety of molecular factors of the plant innate immune system, small proteins that transfer lipids and exhibit a broad spectrum of biological activities are of particular interest. These are lipid transfer proteins (LTPs). LTPs are interesting to researchers for three main features. The first feature is the ability of plant LTPs to bind and transfer lipids, whereby these proteins got their name and were combined into one class. The second feature is that LTPs are defense proteins that are components of plant innate immunity. The third feature is that LTPs constitute one of the most clinically important classes of plant allergens. In this review, we summarize the available data on the plant LTP structure, biological properties, diversity of functions, mechanisms of action, and practical applications, emphasizing their role in plant physiology and their significance in human life.

Structural and Functional Characterization of Recombinant Isoforms of the Lentil Lipid Transfer Protein.

The recombinant isoforms Lc-LTP1 and Lc-LTP3 of the lentil lipid transfer protein were overexpressed in E. coli cells. It was confirmed that both proteins are stabilized by four disulfide bonds and characterized by a high proportion of the α-helical structure. It was found that Lc-LTP1 and Lc-LTP3 possess antimicrobial activity and can bind fatty acids. Both isoforms have the ability to bind specific IgE from sera of patients with food allergies, which recognize similar epitopes of the major peach allergen Pru p 3. Both isoforms were shown to have immunological properties similar to those of other plant allergenic LTPs, but Lc-LTP3 displayed a less pronounced immunoreactivity.

[Synthesis of Oligocaccharides Containing Internal and Terminal Fragment Galß1-3GlcNAc].

Synthesis of oligocaccharides Galß1-3GlcNAcß-sp, GlcNAcß1-3Galß1-3GlcNAcß-sp, Galß1-3GlcNAcß1-3Galß1-3GlcNAcß-sp, Galß1-3GlcNAcß1-3Galß1-4GlcNAcß-sp, Galß1-3GlcNAcß1-6Galß1-4GlcNAcß-sp (sp = O(CH2)3NH2 or O(CH2)2NH2) was carried out using glycosylation by N-Troc-protected derivatives of glycosamine or disaccharide Galß1-3GlcN.

Structure and Biological Functions of ß-Hairpin Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are evolutionarily ancient factors of the innate immune system that serve as a crucial first line of defense for humans, animals, and plants against infection. This review focuses on the structural organization, biosynthesis, and biological functions of AMPs that possess a ß-hairpin spatial structure. Representatives of this class of AMPs are among the most active antibiotic molecules of animal origin. Due to their wide spectrum of activity and resistance to internal environmental factors, natural ß-hairpin AMPbased compounds might become the most promising drug candidates.

[Interaction of Arenicin-1 with C1q Protein].

The interaction between arenicin-1, that is an antimicrobial peptide from polychaeta Arenicola marina, and human complement system protein C1q was studied using enzyme-linked receptor sorbent assay and ELISA. We revealed that arenicin-1 and C1q form complex that is stable in high ionic strength condition 0.5 M NaCl. The ability of C1q to interact with arenicin-1 is comparable with the binding activity of C1q towards another antimicrobial peptide, porcine cathelicidin protegrin-1, which has a similar spatial arrangement with arenicin-1. Namely, both arenicin-1 and protegrin-1 form cystine-stabilized antiparallel ß-hairpin structure.

[Altitude-belt zonality of wood vegetation within mountainous regions of the Sayan Mountains: a model of ecological second-order phase transitions ].

As a description of altitude-belt zonality of wood vegetation, a model of ecological second-order transitions is proposed. Objects of the study have been chosen to be forest cenoses of the northern slope of Kulumyss Ridge (the Sayan Mauntains), while the results are comprised by the altitude profiles of wood vegetation. An ecological phase transition can be considered as the transition of cenoses at different altitudes from the state of presence of certain tree species within the studied territory to the state of their absence. By analogy with the physical model of second-order, phase transitions the order parameter is introduced (i.e., the area portion occupied by a single tree species at the certain altitude) as well as the control variable (i.e., the altitude of the wood vegetation belt). As the formal relation between them, an analog of the Landau's equation for phase transitions in physical systems is obtained. It is shown that the model is in a good accordance with the empirical data. Thus, the model can be used for estimation of upper and lower boundaries of altitude belts for individual tree species (like birch, aspen, Siberian fir, Siberian pine) as well as the breadth of their ecological niches with regard to altitude. The model includes also the parameters that describe numerically the interactions between different species of wood vegetation. The approach versatility allows to simplify description and modeling of wood vegetation altitude zonality, and enables assessment of vegetation cenoses response to climatic changes.