Nei-like 1 (NEIL1) excises 5-carboxylcytosine directly and stimulates TDG-mediated 5-formyl and 5-carboxylcytosine excision

Thymine DNA glycosylase (TDG) and Nei-like 1 (NEIL1) have both been implicated in the base excision repair step of active DNA demethylation. The robust glycosylase activity of TDG on DNA substrates containing 5-formylcytosine (5fC) or 5-carboxylcytosine (5caC) is universally accepted, but the mode of action of NEIL1 is still debated. Based on genetic experiments, it has been suggested that NEIL1 acts redundantly with TDG and excises 5fC and 5caC directly. However, this result has been disputed, and it was suggested instead that NEIL1 is recruited by the monofunctional TDG for the 2′-deoxyribose excision step. Using purified human NEIL1 and its catalytically impaired P2T and E3Q variants as controls, we detect NEIL1 activity on 5caC, but not a 5fC containing dsDNA substrate. We confirm direct NEIL1 TDG binding and NEIL1 mediated 2′ deoxyribose excision downstream of TDG glycosylase activity. NEIL1 acts not only downstream of TDG, but also enhances TDG activity on 5fC or 5caC containing DNA. NEIL1 mediated enhancement of the TDG glycosylase activity is substrate specifc and does not occur for dsDNA with a T/G mismatch

Mating of 2 Laboratory Saccharomyces cerevisiae Strains Resulted in Enhanced Production of 2-Phenylethanol by Biotransformation of L-Phenylalanine

2-Phenylethanol (2-PE) is an aromatic alcohol with a rosy scent which is widely used in the food, fragrance, and cosmetic industries. Promising sources of natural 2-PE are microorganisms, especially yeasts, which can produce 2-PE by biosynthesis and biotransformation. Thus, the first challenging goal in the development of biotechnological production of 2-PE is searching for highly productive yeast strains. In the present work, 5 laboratory Saccharomyces cerevisiae strains were tested for the production of 2-PE. Thereafter, 2 of them were hybridized by a mating procedure and, as a result, a new diploid, S. cerevisiae AM1-d, was selected. Within the 72-h batch culture in a medium containing 5 g/L of L-phenylalanine, AM1-d produced 3.83 g/L of 2-PE in a shaking flask. In this way, we managed to select the diploid S. cerevisiae AM1-d strain, showing a 3- and 5-fold increase in 2-PE production in comparison to parental strains. Remarkably, the enhanced production of 2-PE by the hybrid of 2 yeast laboratory strains is demonstrated here for the first time

On the role of steric clashes in methylation control of restriction endonuclease activity

Restriction-modification systems digest non-methylated invading DNA, while protecting host DNA against the endonuclease activity by methylation. It is widely believed that the methylated DNA would not 'fit' into the binding site of the endonuclease in the productive orientation, and thus steric clashes should account for most of the protection. We test this concept statistically by grafting methyl groups in silico onto non-methylated DNA in co-crystal structures with restriction endonucleases. Clash scores are significantly higher for protective than non-protective methylation (P < 0.05% according to the Wilcoxon rank sum test). Structural data alone are sufficient to distinguish between protective and non-protective DNA methylation with 90% confidence and decision thresholds of 1.1 Å and 48 Å3 for the most severe distance-based and cumulative volume-based clash with the protein, respectively (0.1 Å was deducted from each interatomic distance to allow for coordinate errors). The most severe clashes are more pronounced for protective methyl groups attached to the nitrogen atoms (N6-methyladenines and N4-methylcytosines) than for C5-methyl groups on cytosines. Cumulative clashes are comparable for all three types of protective methylation

Development of a Continuous System for 2-Phenylethanol Bioproduction by Yeast on Whey Permeate-Based Medium

2-Phenylethanol (2-PE) is an alcohol with a rosy scent and antimicrobial activity, and therefore, it is widely used in the food and cosmetic industries as an aroma and preservative. This work was aimed to draw up a technology for 2-PE bioproduction on whey permeate, which is waste produced by the dairy industry, rich in lactase and proteins. Its composition makes it a harmful waste to dispose of; however, with a properly selected microorganism, it could be converted to a value-added product. Herein, two yeast Kluyveromyces marxianus strains and one Kluyveromyces lactis, isolated from dairy products, were tested for 2-PE production, firstly on standard media and then on whey permeate based media in batch cultures. Thereafter, the 2-PE bioproduction in a continuous system in a 4.8 L bioreactor was developed, and subsequently, the final product was recovered from culture broth. The results showed that the yield of 2-PE production increased by 60% in the continuous culture compared to batch culture. Together with a notable reduction of chemical oxygen demand for whey permeate, the present study reports a complete, effective, and environmentally friendly strategy for 2-PE bioproduction with a space-time yield of 57.5 mg L−1 h−1

Structural basis of the methylation specificity of R.DpnI

R.DpnI consists of N-terminal catalytic and C-terminal winged helix domains that are separately specific for the Gm6ATC sequences in Dam-methylated DNA. Here we present a crystal structure of R.DpnI with oligoduplexes bound to the catalytic and winged helix domains and identify the catalytic domain residues that are involved in interactions with the substrate methyl groups. We show that these methyl groups in the Gm6ATC target sequence are positioned very close to each other. We further show that the presence of the two methyl groups requires a deviation from B-DNA conformation to avoid steric conflict. The methylation compatible DNA conformation is complementary with binding sites of both R.DpnI domains. This indirect readout of methylation adds to the specificity mediated by direct favorable interactions with the methyl groups and solvation/desolvation effects. We also present hydrogen/deuterium exchange data that support ‘crosstalk’ between the two domains in the identification of methylated DNA, which should further enhance R.DpnI methylation specificity

Orbital cellulitis as a complication of ethmoiditis in a 6-year-old patient – case report

Ethmoid sinuses are one of the first formed of all the paranasal sinuses, therefore they are most frequently affected by inflammation when it comes to children. The diagnosis of acute ethmoiditis is based mainly on clinical presentation. Due to the specific anatomical conditions of the ethmoid sinuses, inflammation is spreading fast onto the orbital structures and the surrounding tissues. It is estimated that in approximately 91% of cases, orbital cellulitis is related to sinusitis. This work aims at drawing attention to the importance of early diagnosis and appropriate treatment of a common illness that is acute rhinosinusitis. The presentedcase report discusses the course of the disease in a 6-year-old boy who additionally developed orbital cellulitis – a rare complication that concerns approximately 0.5–3.9% of all cases. We can distinguish two types of orbital complications: preseptal cellulitis (anterior to the orbital septum) and posterior to the orbital septum ex. orbital cellulitis (inside the septum). The first group contains inflammatory eyelid oedema, relatively mild and most frequently observed, characterized by favourable prognosis. The complications belonging to the second group are decidedly less frequent and more serious. These are: orbital cellulitis, subperiosteal abscess, orbital abscess and cavernous sinus thrombosis. What helps to differentiate between the two groups are proptosis, eye movement disorder and decreased visual acuity. The systemic treatment consists in the parenteral administration of broad spectrum antibiotics. Despite available modern treatment options one should not forget the fact that late intervention may lead to septic complications such as meningitis, cavernous sinus thrombosis or osteomyelitis. Moreover, discontinuation of treatment may cause permanent, but very rare sequelae including loss of vision and glaucoma. Therefore, acute ethmoiditis is to be treated as a paediatric and rhinological emergency condition.Spośród wszystkich zatok obocznych nosa najwcześniej rozwijają się zatoki sitowe i to one są najczęściej objęte procesem zapalnym u dzieci. Rozpoznanie ethmoiditis acuta opiera się na obrazie klinicznym i wywiadzie. Ze względu na szczególne warunki anatomiczne komórek sitowia proces zapalny szybko szerzy się na struktury oczodołów oraz tkanki otaczające. Szacunkowo za około 91% przypadków zapaleń tkanek miękkich oczodołu odpowiada zapalenie zatok. W pracy pragniemy zwrócić uwagę na konieczność wczesnego diagnozowania i odpowiedniego leczenia ostrego zapalenia jam nosa i zatok przynosowych. Przedstawiamy opis przebiegu choroby u 6-letniego chłopca, u którego rozwinęło się rzadkie powikłanie w postaci zajęcia procesem zapalnym oczodołów, dotyczące około 0,5–3,9% przypadków. Wyróżniamy dwa typy powikłań oczodołowych: przedprzegrodowe i zaprzegrodowe. Do pierwszej grupy należy stosunkowo łagodny i najczęściej obserwowany zapalny obrzęk powiek, cechujący się pomyślnym rokowaniem. Powikłania zaklasyfikowane do drugiej grupy, czyli obejmujące przegrodę oczodołową, są zdecydowanie rzadziej spotykane i poważniejsze. Należą do nich: zapalenie tkanek miękkich, ropień podokostnowy i ropień oczodołu oraz zakrzepowe zapalenie zatoki jamistej mózgu. Wytrzeszcz, zaburzenia ruchomości gałki ocznej oraz osłabienie ostrości wzroku pozwalają na zróżnicowanie obu grup. Leczenie ogólne polega na parenteralnym podawaniu antybiotyków o szerokim spektrum działania. Nawet przy współczesnych możliwościach leczniczych nie należy zapominać, że opóźnienie wdrożenia leczenia może doprowadzić do powikłań septycznych, takich jak zapalenie opon mózgowo-rdzeniowych oraz zakrzepowe zapalenie zatoki jamistej mózgu, a także zapaleń szpiku i kości. Obserwowano również trwałe następstwa zaniechania leczenia pod postacią utraty wzroku lub jaskry. Z tego względu ostre zapalenie zatok sitowych musi być traktowane jako naglący stan pediatryczny i rynologiczny

Valorisation of Whey Permeate in Sequential Bioprocesses towards Value-Added Products–Optimisation of Biphasic and Classical Batch Cultures of <i>Kluyveromyces marxianus</i>

Whey permeate is categorised as hazardous wastewater for aquatic environments, mainly due to its high lactose content. Therefore, it must be valorised before being released into the environment. One pathway for whey permeate management is its use in biotechnological processes. Herein, we present roads for whey permeate valorisation with the K. marxianus WUT240 strain. The established technology is based on two bioprocesses. During first, 2.5 g/L 2-phenylethanol and fermented plant oils enriched with different flavourings are obtained after 48 h biphasic cultures at 30 °C. The second process leads to a maximum of 75 g ethanol/L (YP/S = 0.53 g/g) after 96 h at 30 °C. Moreover, established whey permeate valorisation pathways reduced its biochemical oxygen demand and chemical oxygen demand values by 12- to 3-fold, respectively. Together, the present study reports a complete, effective, and environmentally friendly whey permeate management strategy while simultaneously enabling the acquisition of valuable compounds with substantial application potential

Rosiglitazone Ameliorates Cardiac and Skeletal Muscle Dysfunction by Correction of Energetics in Huntington&rsquo;s Disease

Huntington&rsquo;s disease (HD) is a rare neurodegenerative disease that is accompanied by skeletal muscle atrophy and cardiomyopathy. Tissues affected by HD (central nervous system [CNS], skeletal muscle, and heart) are known to suffer from deteriorated cellular energy metabolism that manifests already at presymptomatic stages. This work aimed to test the effects of peroxisome proliferator-activated receptor (PPAR)-&gamma; agonist&mdash;rosiglitazone on grip strength and heart function in an experimental HD model&mdash;on R6/1 mice and to address the mechanisms. We noted that rosiglitazone treatment lead to improvement of R6/1 mice grip strength and cardiac mechanical function. It was accompanied by an enhancement of the total adenine nucleotides pool, increased glucose oxidation, changes in mitochondrial number (indicated as increased citric synthase activity), and reduction in mitochondrial complex I activity. These metabolic changes were supported by increased total antioxidant status in HD mice injected with rosiglitazone. Correction of energy deficits with rosiglitazone was further indicated by decreased accumulation of nucleotide catabolites in HD mice serum. Thus, rosiglitazone treatment may not only delay neurodegeneration but also may ameliorate cardio- and myopathy linked to HD by improvement of cellular energetics