Enzyme-Directed Mutasynthesis: A Combined Experimental and Theoretical Approach to Substrate Recognition of a Polyketide Synthase

Acyltransferase domains control the extender unit recognition in Polyketide Synthases (PKS) and thereby the side-chain diversity of the resulting natural products. The enzyme engineering strategy presented here allows the alteration of the acyltransferase substrate profile to enable an engineered biosynthesis of natural product derivatives through the incorporation of a synthetic malonic acid thioester. Experimental sequence−function correlations combined with computational modeling revealed the origins of substrate recognition in these PKS domains and enabled a targeted mutagenesis. We show how a single point mutation was able to direct the incorporation of a malonic acid building block with a non-native functional group into erythromycin. This approach, introduced here as enzyme-directed mutasynthesis, opens a new field of possibilities beyond the state of the art for the combination of organic chemistry and biosynthesis toward natural product analogues

ЕФЕКТИВНІСТЬ ЗАСТОСУВАННЯ КОРМОВОЇ ДОБАВКИ НА ОСНОВІ БАКТЕРІЙ РОДУ BACILLUS

The article highlights the results of using a feed additive based on spore-forming bacteria of the genus Bacillus when growing broiler chickens. The addition of the studied additive to the diet of chickens in the first group at a dose of 0.5 and to the second group at a dose of 1 g/kg of feed increased the intensity of the bird's growth. In particular, the average daily weight gain of broilers in the experimental groups was, respectively, 11 and 11.8% higher than in the control group. At the same time, the yield of gutted broiler carcasses in the first and second experimental groups was higher, compared to the control group, by 245 and 275 g, respectively. Under these conditions, in the first experimental group with an average body weight of 2411.1 g, feed consumption during the experimental period was 4310 g/head, while feed conversion was 1.78. In the second experimental group with an average body weight of 2426.9 g, the feed consumption was 4253.3 g/head and the conversion was 1.75, while in the control group with an average body weight of 2178.4 g, the feed consumption was 4433.3 g/head, and the feed conversion was 2.03. When identifying microorganisms of the Enterobacteriaceae family, in the first research group were found: Escheriсhia coli, Escheriсhia fergusonii, Escheriсhia hermannii, Escheriсhia vulneris, Serratia odorifera; in the second experimental group: Escheriсhia coli, Escheriсhia fergusonii, Serratia odorifera, Kluyvera spp.; and in the control group: Escheriсhia terioteria enorita, Serratia oderofera cloacea, Shigella spp. Microorganisms of the genus Salmonella, and also Campilobacter and C. perfringens on day 42 in broilers of all groups were not detected. Therefore, the use of a feed additive based on spore-forming bacteria of the genus Bacillus leads to a decrease in feed conversion, an increase in the body weight of birds, and an increase in total and average daily weight gain, compared to the control group. Also was found the positive effect of feed additives on the qualitative and quantitative composition of the intestinal microflora, while the microflora of the bird is corrected in the direction of an increase in the normal flora and a decrease in pathogenic and conditionally pathogenic microflora.У статті висвітлено результати застосування кормової добавки на основі спороутворюючих бактерій роду Bacillus, при вирощуванні курей-бройлерів. Внесення досліджуваної добавки у раціон курей першої групи у дозі 0,5 а другої – 1 г/кг корму підвищувало інтенсивність росту птиці. Зокрема, середньодобовий приріст маси тіла бройлерів дослідних груп був, відповідно, на 11 та 11,8 % вищим, ніж у контрольній групі. При цьому вихід потрошеної тушки бройлерів у першій та другій дослідній групах був вищим, порівняно з контрольною групою, відповідно, на 245 та 275 г. За цих умов у першій дослідній групі, при середній масі тіла 2411,1 г, витрати кормів за дослідний період становили 4310 г/гол, при цьому конверсія корму становила 1,78. У другій дослідній групі, при середній масі тіла 2426,9 г, витрати кормів складали 4253,3 г/гол, а конверсія корму становила 1,75, тоді як у контрольній групі, при середній масі тіла 2178,4 г, витрати кормів становили 4433,3 г/гол, при цьому конверсія корму становила 2,03. При ідентифікації мікроорганізмів родини Enterobacteriaceае виявили у першій дослідній групі: Escheriсhia colі, Escheriсhia fergusonii, Escheriсhia hermannii, Escheriсhia vulneris, Serratia odorifera; у другій дослідній групі: Escheriсhia coli, Escheriсhia fergusonii, Serratia odorifera, Kluyvera spp.; в контрольній групі: Escheriсhia terіoterіa enorita, Serratia oderofera cloacea, Shigella spp. Мікроорганізмів роду Salmonella, а також Campilobacter та C. perfringens на 42 добу у бройлерів усіх груп не виявляли. Отже, застосування кормової добавки на основі спороутворюючих бактерій роду Bacillus призводить до зниження конверсії корму, підвищення маси тіла птиці, збільшення загального та середньодобового приросту ваги, порівняно з контрольною групою. Встановлено позитивний вплив застосування кормової добавки на якісний та кількісний склад мікрофлори кишечнику, при цьому відбувається корекція мікрофлори птиці у бік збільшення нормофлори та зменшення патогенної і умовно-патогенної мікрофлори

Визначення активності фунгіцидних засобів до збудників дерматомікозів домашніх тварин

The aim of the study was to identify and identify the causative agents of dermatomycosis in dogs and cats and to determine their sensitivity to solutions of ketonazole and chlorhexidine digluconate, which are part of the drug Skinhard. This is a spray for external use, manufactured by PJSC “Halychpharm”. For laboratory diagnosis of dermatomycoses, samples from the affected parts of the skin and fur of domestic animals were used. To isolate a pure culture of fungi were sown on selective media: wort agar, agar Saburo, Chapek. The optimal cultivation regime for pathogenic fungi was 20–25 °С. Isolated fungal cultures were identified by the appearance and shape of colonies, their consistency, color, ability to grow at 37 °C, microscopic structure, in particular - the nature of mycelial branching and the presence of septa, location of conidiophores, spores and other signs. A microbiological examination of the affected areas of the skin of dogs and cats was performed to detect microscopic fungi. The following fungi were isolated from dogs affected by mycoses: Candida spp., Aspergillus niger, Epidermophyton spp., Microsporum spp., Mucor spp., Trichophyton spp., Mallasseria spp., Sporotrich spp, Candida albicans, and from cats: Microspor Candida spp., Spototrich spp., Rhizorus spp., Fusarium spp. Trichophyton spp. Isolated microscopic fungi of the genus Malassezia spp. belong to superficial mycoses (keratomycoses) and affect the superficial layers of skin and hair. Epidermatophytes: Trichophyton spp., Epidermophyton spp., Microsporum spp. affect the epidermis, skin and coat. In addition, opportunistic fungi of the genus Aspergillus spp, Mucor spp., Rhizorus spp. and Fusarium spp. Chlorhexidine digluconate was found to be highly active against dermatophytes (Trichophyton spp., Microsporum spp.), Less active against fungi of the genus Candida and less active against Aspergillus spp, Mucor spp., Rhizorus spp, Fusarium spp. Ketonazole is highly active against dermatophytes (Trichophyton spp., Epidermophyton spp., Microsporum spp.), Yeast (Malassezia spp., Candida spp.,) And opportunistic fungi (Aspergillus spp., Mucor spp., Rhizorus spp.).Метою дослідження було виділити та ідентифікувати збудники дерматомікозу собак та котів та визначити їх чутливість до розчинів кетоназолу та хлоргексидину диглюконату, які входять до складу препарату Скінгард. Це – спрей для зовнішнього застосування, виробництва ПАТ “Галичфарм”. Для лабораторної діагностики дерматомікозів використовували зразки з уражених частин шкіри та шерсті домашніх тварин. Для виділення чистої культури грибів проводили посіви на селективні середовища: сусло-агар, агар Сабуро, Чапека. Оптимальний режим культивування для патогенних грибів становив 20–25 °С. Виділені культури грибів ідентифікували за зовнішнім виглядом і формою колоній, їх консистенцією, кольором, здатністю до росту за температури 37 °С, мікроскопічній будові, зокрема – характером розгалуження міцелію і наявністю у ньому септ, розташуванню конідієносців, спор та іншими ознаками. Проведено мікробіологічне дослідження уражених ділянок шкіри собак та котів на предмет виявлення мікроскопічних грибків. Від собак, уражених мікозами виділяли наступні грибки: Candida spp., Aspergillus niger, Epidermophyton spp., Microsporum spp., Mucor spp., Trichophyton spp., Mallasseria spp., Sporotriх spp., Candida albicans, а від котів: Microsporum spp., Candida spp., Spototriх spp., Rhizoрus spp., Fusarium spp. Trichophyton spp. Виділені мікроскопічні грибки роду Malassezia spp. належать до поверхневих мікозів (кератомікозів) і уражають поверхневі шари шкіри та шерсть. Епідерматофіти: Trichophyton spp., Epidermophyton spp., Microsporum spp. уражають епідерміс, шкіру та шерстний покрив. Крім цього, у досліджуваних зразках виявлено опортуністичні гриби роду Aspergillus spp., Mucor spp., Rhizoрus spp. та Fusarium spp. Встановлено, що хлоргексидину диглюконат високо активний до дерматофітів (Trichophyton spp., Microsporum spp.), менше активний до грибків роду Candida та мало активний до Aspergillus spp., Mucor spp., Rhizoрus spp., Fusarium spp. Кетоназол високо активний щодо дерматофітів (Trichophyton spp., Epidermophyton spp, Microsporum spp.), дріжджів (Malassezia spp., Candida spp.) та до опортуністичних грибів (Aspergillus spp, Mucor spp., Rhizoрus spp, Fusarium spp.)

Mesoscopic scattering of spin s particles

Quantum effects in weakly disordered systems are governed by the properties of the elementary interaction between propagating particles and impurities. Long range mesoscopic effects due to multiple scattering are derived by iterating the single scattering vertex, which has to be appropriately diagonalized. In the present contribution, we present a systematic and detailed diagonalisation of the diffuson and cooperon vertices responsible for weak localisation effects. We obtain general expressions for eigenvalues and projectors onto eigenmodes, for any spin and arbitrary elementary interaction with impurities. This description provides a common frame for a unified theory of mesoscopic spin physics for electrons, photons, and other quantum particles. We treat in detail the case of spin-flip scattering of electrons by freely orientable magnetic impurities and briefly review the case of photon scattering from degenerate dipole transitions in cold atomic gases.Comment: published version, with a new figure and new section

Galaxy Clusters as Reservoirs of Heavy Dark Matter and High-Energy Cosmic Rays: Constraints from Neutrino Observations

Galaxy Clusters (GCs) are the largest reservoirs of both dark matter and cosmic rays (CRs). Dark matter self-annihilation can lead to a high luminosity in gamma rays and neutrinos, enhanced by a strong degree of clustering in dark matter substructures. Hadronic CR interactions can also lead to a high luminosity in gamma rays and neutrinos, enhanced by the confinement of CRs from cluster accretion/merger shocks and active galactic nuclei. We show that IceCube/KM3Net observations of high-energy neutrinos can probe the nature of GCs and the separate dark matter and CR emission processes, taking into account how the results depend on the still-substantial uncertainties. Neutrino observations are relevant at high energies, especially at >10 TeV. Our results should be useful for improving experimental searches for high-energy neutrino emission. Neutrino telescopes are sensitive to extended sources formed by dark matter substructures and CRs distributed over large scales. Recent observations by Fermi and imaging atmospheric Cherenkov telescopes have placed interesting constraints on the gamma-ray emission from GCs. We also provide calculations of the gamma-ray fluxes, taking into account electromagnetic cascades inside GCs, which can be important for injections at sufficiently high energies. This also allows us to extend previous gamma-ray constraints to very high dark matter masses and significant CR injections at very high energies. Using both neutrinos and gamma rays, which can lead to comparable constraints, will allow more complete understandings of GCs. Neutrinos are essential for some dark matter annihilation channels, and for hadronic instead of electronic CRs. Our results suggest that the multi-messenger observations of GCs will be able to give useful constraints on specific models of dark matter and CRs. [Abstract abridged.]Comment: 31 pages, 20 figures, 1 table, accepted for publication in JCAP, references and discussions adde

Rossby wave dynamics of the North Pacific extra-tropical response to El Niño: importance of the basic state in coupled GCMs

The extra-tropical response to El Nino in a "low" horizontal resolution coupled climate model, typical of the Intergovernmental Panel on Climate Change fourth assessment report simulations, is shown to have serious systematic errors. A high resolution configuration of the same model has a much improved response that is similar to observations. The errors in the low resolution model are traced to an incorrect representation of the atmospheric teleconnection mechanism that controls the extra-tropical sea surface temperatures (SSTs) during El Nino. This is due to an unrealistic atmospheric mean state, which changes the propagation characteristics of Rossby waves. These erroneous upper tropospheric circulation anomalies then induce erroneous surface circulation features over the North Pacific. The associated surface wind speed and direction errors create erroneous surface flux and upwelling anomalies which finally lead to the incorrect extra-tropical SST response to El Nino in the low resolution model. This highlights the sensitivity of the climate response to a single link in a chain of complex climatic processes. The correct representation of these processes in the high resolution model indicates the importance of horizontal resolution in resolving such processes

Electron and hole transmission through superconductor - normal metal interfaces

We have investigated the transmission of electrons and holes through interfaces between superconducting aluminum (Tc = 1.2 K) and various normal non-magnetic metals (copper, gold, palladium, platinum, and silver) using Andreev-reflection spectroscopy at T = 0.1 K. We analyzed the point contacts with the modified BTK theory that includes Dynes' lifetime as a fitting parameter G in addition to superconducting energy gap 2D and normal reflection described by Z. For contact areas from 1 nm^2 to 10000 nm^2 the BTK Z parameter was 0.5, corresponding to transmission coefficients of about 80 %, independent of the normal metal. The very small variation of Z indicates that the interfaces have a negligible dielectric tunneling barrier. Fermi surface mismatch does not account for the observed transmission coefficient.Comment: 9 pages, 4 figures, submitted to Proceedings of the 19th International Conference on Magnetism ICM2012 (Busan 2012

The influence of the feed additive “Metisevit” on the activity of the antioxi-dant defense system of piglets under conditions of nitrate-nitrite load

The work aimed to investigate the effect of the “Metisevit” feed additive on the activity of the enzymes of the antioxidant system of the blood of piglets under conditions of nitrate-nitrite load. The research was carried out on 60-day-old piglets of the large white breed. For the experiment, 10 clinically healthy piglets were selected, from which 2 groups of five animals were formed. Piglets of the control group were fed sodium nitrate at a dose of 0.3 g NO3-/kg body weight for three months. Piglets of the research group were also fed sodium nitrate at a dose of 0.3 g NO3-/kg of body weight, together with Metisevit at a dose of 0.36 g/kg of feed for three months. The “Metisevit” feed additive was developed at the Department of Pharmacology and Toxicology of the Stepan Gzhytskyi National University of Veterinary Medicine and Biotechnology Lviv, which contains vitamin E, selenium, and methiphene (methionine, phenarone, zeolite). Under the conditions of feeding piglets with sodium nitrate feed at a dose of 0.3 g of NO3ˉ/kg, a decrease in the activity of enzymes of the antioxidant defense system was established: superoxide dismutase by 14.8 %, catalase by 20%, glutathione peroxidase by 15.1 %. Metisevit in piglets increased the activity of the enzyme link of the antioxidant protection system, preventing the development of oxidative stress that occurs in nitrate-nitrite toxicosis. During our experiments, it was found that the introduction of Metisevit into the diet of piglets with excessive consumption of nitrates prevented the development of chronic nitrate-nitrite toxicosis. It was established that Metisevit increases the activity of enzymes – superoxide dismutase, catalase, and glutathione peroxidase. These enzymes reduce the high level of lipid peroxidation products in the body of piglets under conditions of nitrate-nitrite load

ЧУТЛИВІСТЬ МІКРООРГАНІЗМІВ, ВИДІЛЕНИХ З ПАТОЛОГІЧНОГО МАТЕРІАЛУ, ДО АНТИМІКРОБНИХ ПРЕПАРАТІВ

Excessive and uncontrolled use of antibiotics commonly results in the emergence of antimicrobial resistance (AMR). Therefore, the main aim of these studies was to determine the antibiotic susceptibility of microorganisms isolated from the pathological material. The article describes the susceptibility of isolated strains to the following antibiotics: ampicillin, amoxicillin, oxacillin, ofloxacin, streptomycin, cefazolin, gentamicin, amikacin, amoxiclav, erythromycin, enrofloxacin, lincomycin, tetracycline, doxycycline, vancomycin, rifampicin, ciprofloxacin, norfloxacin, tylosin, nitrofurantoin, polymyxin B. During the study of six Escherichia coli and three Salmonella enteritidis strains, it was ascertained that they demonstrated different susceptibility to the studied antibiotics, in particular, only two E. coli isolates, which is 33,3 %, were resistant to streptomycin, 50 % of the isolated strains were susceptible. When determining the susceptibility, it was established that all isolated E. coli isolates were resistant to cefazolin, vancomycin, oxacillin, and 83,3 % were resistant to amoxicillin, ampicillin, vancomycin, amoxiclav, rifampicin. 33,3 % of Salmonella enteritidis isolates were moderately susceptible to streptomycin, amoxicillin, and ampicillin, and 66,7 % were sensitive to ciprofloxacin, norfloxacin, and enrofloxacin. All isolated S. enteritidis strains were found to be resistant to cefazolin, tylosin, vancomycin, oxacillin, lincomycin, and erythromycin, 100 % susceptible to ciprofloxacin, amikacin, and ofloxacin and 68,7 % moderately susceptible to amoxicillin and gentamicin.Надмірне та безконтрольне використання антибіотиків часто приводить до виникнення антибіотикорезистентності. Тому основна мета цих досліджень полягала у визначенні чутливості до антибіотиків мікроорганізмів, виділених з патматеріалу. У статті наведене визначення чутливості виділених штамів до таких антибіотиків: ампіциліну, амоксициліну, оксациліну, офлоксацину, стрептоміцину, цефазоліну, гентаміцину, амікацину, амоксиклаву, еритроміцину, енрофлоксацину, лінкоміцину, тетрацикліну, доксицикліну, ванкоміцину, рифампіцину, ципрофлоксацину, норфлоксацину, тилозину, нітрофурантоїну, поліміксину В. При дослідженні шести щтамів ‒ Esсherichia coli та трьох Salmonella enteritidis, встановлено, що вони проявляли різну чутливість до досліджуваних антибіотиків, зокрема резистентними до стрептоміцину виявились лише два ізоляти E. coli, що складає 33,3 %, чутливими було 50 % виділених штамів. При визначенні чутливості встановлено, що всі виділені ізоляти E. coli були стійкими до цефазоліну, ванкоміцину, оксациліну, а 83,3 % стійкими до амоксациліну, ампіциліну, ванкоміцину, амоксиклаву, рифампіцину. 33,3 % виділених ізолятів S. enteritidis були помірно-чутливими до стрептоміцину, амоксациліну, ампіциліну, а 66,7 % ‒ чутливими до ципрофлоксацину, норфлоксацину та енрофлоксацину. Встановлено, що всі виділені штами S. enteritidis були стійкими до цефазоліну, тилозину, ванкоміцину, оксациліну, лінкоміцину та еритроміцину, 100 % чутливими до ципрофлоксацину, амікацину та офлоксацину та 68,7 % помірно-чутливими до амоксациліну та гентаміцину

Intercomparison of the northern hemisphere winter mid-latitude atmospheric variability of the IPCC models

We compare, for the overlapping time frame 1962-2000, the estimate of the northern hemisphere (NH) mid-latitude winter atmospheric variability within the XX century simulations of 17 global climate models (GCMs) included in the IPCC-4AR with the NCEP and ECMWF reanalyses. We compute the Hayashi spectra of the 500hPa geopotential height fields and introduce an integral measure of the variability observed in the NH on different spectral sub-domains. Only two high-resolution GCMs have a good agreement with reanalyses. Large biases, in most cases larger than 20%, are found between the wave climatologies of most GCMs and the reanalyses, with a relative span of around 50%. The travelling baroclinic waves are usually overestimated, while the planetary waves are usually underestimated, in agreement with previous studies performed on global weather forecasting models. When comparing the results of various versions of similar GCMs, it is clear that in some cases the vertical resolution of the atmosphere and, somewhat unexpectedly, of the adopted ocean model seem to be critical in determining the agreement with the reanalyses. The GCMs ensemble is biased with respect to the reanalyses but is comparable to the best 5 GCMs. This study suggests serious caveats with respect to the ability of most of the presently available GCMs in representing the statistics of the global scale atmospheric dynamics of the present climate and, a fortiori, in the perspective of modelling climate change.Comment: 39 pages, 8 figures, 2 table