Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three genomic nomenclature systems to all sequence data from the World Health Organization European Region available until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation, compare the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2

Evolutionary Engineering Of Phenylethanol-resistant Saccharomyces Cerevisiae

Thesis (M.Sc.) -- İstanbul Technical University, Institute of Science and Technology, 2013Saccharomyces cerevisiae, genetik ve moleküler biyoloji çalışmalarında çok sık kullanılan, özellikleri iyi bilinen model organizmalardan biridir. Bilimsel araştırmalardaki kullanım alanlarının yanı sıra, S. cerevisiae endüstriyel üretimde de önemli bir yere sahiptir. Özellikle etanol üretimi ve ekmek yapımında yaygın olarak kullanılmaktadır. S. cerevisiae, tek hücreli bir ökaryotik mikrorganizma olup, tomurcuklanma yolu ile hem eşeysiz, hem de mayoz bölünme gerçekleştirerek eşeyli olarak çoğalabilmektedir. S. cerevisiae’nin yüksek ökaryotların genomu ile gösterdiği yüksek homoloji de bir çok bilimsel çalışmada yarar sağlamaktadır. Özellikle insan genomu ile olan benzerliği sebebiyle, kanser, yaşlanma ve birçok hastalık mekanizmaları S. cerevisiae hücreleri kullanılarak araştırılmaktadır. Mikroorganizmalar, doğal ve endüstriyel ortamlarda sıkça stres koşullarına maruz kalmaktadır. Bunlar, yüksek yada düşük sıcaklık, ozmolarite, oksidatif stres, mekanik stres ve metal stresi gibi streslerdir. Araştırmacılar, mikrobiyel stres direnç mekanizmalarını araştırmakta ve aynı zamanda çeşitli streslere karşı direnç düzeylerini arttırmaya çalıştırmaktadırlar. Aynı zamanda, endüstriyel verimin arttırılması amacıyla, üreticiler de stres direnci yüksek mikroorganizmalar aramaktadırlar. Bu tez çalışmasında, feniletanole dirençli maya hücreleri elde edilerek feniletanole karşı geliştirilen direncin moleküler mekanizmalarının incelenmesi amaçlandı. Bunun için ilk olarak evrimsel mühendislik yöntemi ile feniletanole dirençli S. cerevisiae mutantları elde edildi. Ardından, feniletanole dirençli S. cerevisiae mutantlarında, feniletanol direncinin moleküler mekanizmasını anlamak amacıyla fenotip analizleri, fizyolojik ve transkriptomik analizler gerçekleştirildi. Çalışma başlangıcında evrimsel mühendislik yaklaşımı yaban tip S. cerevisiae hücreleri üzerinde gerçekleştirildi. Bu amaçla ilk olarak başlangıç popülasyonunda genetik çeşitliliği arttırmak için kimyasal bir mutajen olan etil metan sülfonat (EMS) yaban tip maya hücrelerine uygulandı. Elde edilen mutajenize edilmiş maya kültürü, sonrasında seçilime maruz bırakılarak, kültür içinden istenen fenotipteki bireylerin seçilmesi planlandı. Seçilim süresince, ilk başta düşük konsantrasyonlarda (1.5 mL/L) feniletanol kültüre uygulandı ve inkübasyon gerçekleştirildi. Sonraki basamakta, hayatta kalan maya hücreleri, daha yüksek bir feniletanol konsantrasyonunda tekrar inkübe edildi. Her basamakta, OD600 değerleri ölçüldü ve hayatta kalma oranları kritik bir seviyeye düşene kadar bu seçilim işlemleri devam edildi. En son 3.6 mL/L feniletanol konsantrasyonuna kadar gelindi ve 56. nesilde seçilim işlemi durduruldu. Bu elde edilen son popülasyondan rastgele 10 birey seçildi ve direnç yeteneklerine göre kıyaslandı. On mutant birey, yaban tip ve son popülasyonun feniletanol dirençleri damlatma ve en muhtemel sayı (MPN) yöntemleri ile ölçüldü ve karşılaştırıldı. Elde edilen 10 birey arasından en yüksek direnci gösteren birey seçildi ve “C9” olarak adlandırıldı. C9 bireyinde genetik kararlılık testi uygulandı. Bu test ile maya mutantının feniletanol direncinin kalıcı olup olmadığı belirlendi. Damlatma ve MPN çalışmaları, bu bireyin feniletanol direncinin değişmediğini gösterdi. İlgili mutantta feniletanol direnci genetik olarak kararlı bulundu. Feniletanole dirençli maya mutantının çapraz direnç özellikleri de incelendi. Bunun için çapraz direnç testi uygulandı. Bu testte, seçilen mutant ve yaban tip, farklı konsantrasyonlarda feniletanol (2.5 mL/L ve 3 mL/L), etanol (8%, 10% ve 12% v/v), asetat (0.004% v/v), kobalt (1 mM ve 3 mM), bor (80 mM), bakır (0.5 mM), hidrojen peroksit (0.5 mM) ve nikel’e (0.2 mM) maruz bırakıldı, hayatta kalma oranları kıyaslandı. Tüm bu stres faktörleri içinde, feniletanol dirençli mutant, etanole karşı da direnç gösterdi. Etanol ve feniletanol’ün hücresel etki mekanizmalarının muhtemel benzerliklerinden dolayı bu iki stres faktörünün çapraz dirence neden olması beklenen bir durum olarak nitelendirilebilir. Feniletanol dirençli C9 mutantı, aynı zamanda kobalt’a karşı belirgin bir hassasiyet göstermektedir. Feniletanole dirençli mutantın direnç mekanizmasının moleküler düzeyde incelenmesi için transkriptomik analiz gerçekleştirilmiştir. Bu amaçla, DNA mikroarray yaklaşımı kullanılmış ve C9 ile yaban tip arasında, kontrol koşullarındaki transkripsiyon profilleri karşılaştırılmıştır. Analiz sonucunda, C9’un genel transkripsiyon profilinde ilgi çekici sonuçlara rastlanmıştır. Bu sonuçlardan biri, çok yüksek sayıda gende transkripsiyon artışı görülmesidir. S. cerevisiae genomunda bulunun yaklaşık 6000 gen içerisinde 1000 kadar genin anlatımı artarken 800’e yakın gende de anlatımda azalış olmuştur. Tüm bu genler, maya genomunun yaklaşık %30’una denk gelmektedir. Bu yüksek transkripsiyon profili, maya hücrelerinin stres anında gösterdiği kısa süreli cevaplar ile benzerlik göstermektedir. Normalde kısa süren ve çok sayıda kendini gösteren bu reaksiyonlar çevresel stres cevabı (Environmental stress response, ‘ESR’) olarak bilinmektedir. Feniletanole dirençli mutantta ESR’den sorumlu genler önemli düzeyde aktif durumdadır. Feniletanole dirençli mutanta ait transkripsiyon profilinde ilk göze çarpan anlatımı artan 1000 kadar gen arasında, karbonhidrat metabolizması ile ilgili genlerin önemli bir yer kaplamasıdır. Anlatımı artan 166 gen ile karbonhidrat metabolizmasından sorumlu genler, C9’un anlatımı artmış tüm genlerinin yaklaşık %20’sini oluşturmaktadır. Bunu 98 gen ile oksidatif stres cevabı izlemektedir. Aynı zamanda anlatımı artmış genler arasında 63 tanesi genel stres cevabından, 35 tanesi hücre duvarı organizasyonundan, 21 gen ise otofaji ve mitokondri yıkımından sorumludur. Belirtilen %20’lik katkı karbonhidrat metabolizmasının, C9 mutantında önemli bir şekilde tetiklenmiş olduğunu göstermektedir. Benzer durum, daha önce tanımlanan ESR koşullarında da görülmüştür. Hücreler, stres altında kısa süreliğine glikoz metabolizmasını hızlandırmaktadır. Ancak, C9 mutantında bu genlerin anlatımları ortamda stres koşulları bulunmasa da aralıksız olarak gerçekleşmiştir. Benzer durum, anlatımı azalan genlerde de görülmüştür. Analiz sonuçlarına göre, C9 bireyinde özellikle nükleik asit metabolizması ve protein, ribozom sentezinde görev alan çoğu genin anlatımı ciddi oranda azalmıştır. Anlatımı azalan 821 genin %81’i rRNA ve tRNA’ların sentezi ve bağlanmasında, translasyonun başlamasında, RNA-DNA bağlanmasında, helikaz aktivitesinde görev almaktadır. C9’da protein sentezini azaltacak yönde görülen bu değişiklikler aynı zamanda genel ESR koşullarında da görülmektedir. Bu sonuçlar da feniletanol dirençli C9 bireylerinin sürekli bir ESR durumunda olduğu görüşünü desteklemektedir. C9’un aynı zamanda, özelleşmiş stres cevapları da verdiği görülmüştür. Yaban tipe kıyasla, aldehit dehidrogenaz 3 adlı genin 234 kat daha fazla anlatımı gerçekleşmiştir. Alkolün yıkılması sırasında ortaya çıkan bir toksik madde olan aldehidin yıkılmasından sorumlu bu genin yüksek şekilde anlatılması, C9’un sahip olduğu feniletanol direnci için önemli olabilir. Bu tez çalışmasında, feniletanol dirençli S. cerevisiae hücreleri evrimsel mühendislik yöntemleri ile elde edilmiş ve transkripsiyon seviyesinde karakterizasyonu gerçekleştirilmiştir. Dirençli maya mutantının yüksek seviyede gösterdiği gen anlatımı, yaban tip hücrelerin stres anında verdiği anlık tepkilerle benzerlik göstermektedir. Anlatımın yüksek ve karmaşık olması, feniletanol direncinin tek bir gen veya gen grubu ile ilişkilendirilmesini zorlaştırmaktadır. Bu sebeple, çevresel stres cevaplarının daha iyi anlaşılması ve feniletanol direncinin temel kökeninin bulunması için anlatımı önemli ölçüde artmış ya da azalmış genlerin delesyonu ya da aşırı anlatımı gibi ilave moleküler araştırmaların yapılması önerilebilir.Saccharomyces cerevisiae is one the most widely used model organisms in genetics, molecular biology and metabolic studies. In addition to its use in scientific research, it is one of the oldest microorganisms used for ages for industrial applications. S. cerevisiae is a unicellular eukaryotic organism, which can be found in haploid and diploid form, and can induce meiosis to generate new progeny of haploid from diploids (so called sporulation event) or reproduce asexually by budding. It shares high degree of homologies with higher eukaryotes like human. Due to these functional similarities, S. cerevisiae can be used in research related to cancer, aging and other human diseases. In natural environment and in industrial applications, S. cerevisiae cells are often under stress resistance that results them environmental changes. These changes can be named as osmotic, high or low temperature, dehydration, starvation, metal ion stresses etc. Researchers are interested in the microbial resistance mechanisms to these different types of stresses. Additionally, they are searching for strategies to increase stress tolerance. Producers are also interested in increasing yield and for this reason; they are searching for stress-resistant microorganisms. The aim of the present study was to obtain phenylethanol (PEA) resistant yeast strains via evolutionary engineering approach and perform transcriptomic and metabolic characterization to identify responsible pathways ans molecular factors in this resistance. In this thesis study, firstly, phenylethanol-resistant S. cerevisiae mutants were obtained by evolutionary engineering approach. Phenotypic and genetic characterization was then carried out to identify the molecular principles of phenylethanol resistance in S. cerevisiae. To apply evolutionary engineering to wild type S. cerevisiae cells, these cells were treated with a chemical mutagen EMS (Ethyl Methane Sulfonate) to increase the genetic diversity of the initial population to which selection would be applied. This mutagenized culture was cultivated at increasing phenylethanol concentrations in the culture medium along with the wild type to determine the initial stress level to be applied. Phenylethanol stress was then applied to this mutagenized culture. The phenylethanol concentration was increased gradually for each successive population. The first population was obtained upon 1.5 mL/L exposure to phenylethanol and the final 56th population was obtained upon exposure to 3.6 mL/L PEA. The final population was used for randomly selecting ten individual mutants. Those ten individual mutants, wild type and the final population were tested for phenylethanol resistance and it was observed that the evolved strain and the final population could grow at high phenylethanol concentrations at which the wild type could not show any sign of survival. One of the individual mutants which showed highest phenylethanol-resistance was chosen and genetic stability assay was applied. It was shown that phenylethanol-resistance was a genetically stable trait in the mutant tested. This evolved strain was termed C9. In this study, PEA-resistant strain C9 was analyzed according to its cross-resistance abilities against various metals and organic compounds and compared with the wild type. Different concentrations of phenylethanol (2.5 mL/L and 3 mL/L), ethanol (8%, 10% and 12% v/v), acetate (0.004% v/v), cobalt (1 mM and 3 mM), boron (80 mM), copper (0.5 mM), hydrogen peroxide (0.5 mM) and nickel (0.2 mM) were used. It was observed that, phenylethanol-resistant mutant also show had cross-resistance to ethanol. Besides, C9 had increased sensitivity to cobalt stress. To investigate the molecular mechanisms of phenylethanol resistance of the evolved strain, whole genome transcriptomic analysis was conducted for wild type and C9. Sampling for microarray analysis was carried out when the cultures were in their exponential phase of growth. The expression profile of the mutant was compared to that of the wild type. The results showed that, phenylethanol-resistant C9 strain had immense amount of upregulated and downregulated genes in its genome under control conditions without any external stress. DNA microarray analysis showed that C9 had about 1000 upregulated and 800 downregulated genes which make up about 30% of whole genome. Such large scale changes in transcription levels indicate that some global expression response was always active in C9. That genome-wide expression program resembles a highly known large-scale stress reaction called “environmental stress response” (ESR). DNA microarray analysis results indicated that there were about 1000 upregulated genes in C9 compared to wild type and majority of these genes were responsible for carbohydrate metabolism. With upregulated 166 genes, carbohydrate metabolism contributes to about 20% of all upregulated genes in C9. Following with 98 genes responsible for oxidative stress response, 63 genes for general stress response, 35 genes for cell wall reorganization and renewal, 21 genes for degradation of mitochondria and cell itself were found to be upregulated. With 20% contribution, genes in carbohydrate metabolism were shighly upregulated in phenylethanol resistant C9 strain. In addition to increased activity of genes involved in glycolysis, many other genes associated with hexose transport, alternative carbon source utilization were also over-expressed. Same cellular states were also observed under ESR conditions which may indicate that C9 strain apparently induces ESR actively and continuously. Additionally, many putative genes involved in cell wall biosynthesis, autophagy, DNA damage response were up-regulated. Same similarities were also observed in repression profile of C9 compared to wild type. Interpretation of downregulated genes showed that C9 strain selectively repressed major nucleic acid metabolism and ribosome synthesis. More than 81% of 821 downregulated genes were related to synthesis and binding of rRNA and tRNA, initiation of translation, RNA-DNA binding, and helicase activity. Additionally, similar regulations have also been observed previously during ESR in stressed-wild type strains upon initial stress exposure. C9 also showed unique stress responses against alcohol stress. In comparison with wild type, phenylethanol-resistant C9 strain showed 234-fold higher expression of ALD3 gene. This gene might be related to main resistance mechanisms against phenylethanol and ethanol. Increased ALD3 gene expression may prepare cells to overcome excess amounts of aldehyde byproducts of alcohol degradation. In this thesis study, a phenylethanol hyper-resistant S. cerevisiae mutant was obtained and characterized at transcriptomic level. Duw to the complexity and the large size of change in the transcriptomic response of the resistant mutant, it is not likely to point out one or a few genes that are crucial for phenylethanol resistance. However, it was shown that continuous induction of ESR genes may provoke specific resistance mechanisms. It could therefore be recommended to continue molecular research to enlighten the mechanism of phenylethanol resistance, for example, by overexpression/deletion of genes that were highly upregulated/downregulated according to transcriptomic analysis results.Yüksek LisansM.Sc

Stemness Related Genes Cause Resistance to SMAC mimetics in Neuroblastoma Cells.

Stemness Related Genes Cause Resistance to SMAC mimetics in NeuroblastomaCells</p

Evolutionary engineering and molecular characterization of a caffeine-resistant Saccharomyces cerevisiae strain

Caffeine is a naturally occurring alkaloid, where its major consumption occurs with beverages such as coffee, soft drinks and tea. Despite a variety of reports on the effects of caffeine on diverse organisms including yeast, the complex molecular basis of caffeine resistance and response has yet to be understood. In this study, a caffeine-hyperresistant and genetically stable Saccharomyces cerevisiae mutant was obtained for the first time by evolutionary engineering, using batch selection in the presence of gradually increased caffeine stress levels and without any mutagenesis of the initial population prior to selection. The selected mutant could resist up to 50 mM caffeine, a level, to our knowledge, that has not been reported for S. cerevisiae so far. The mutant was also resistant to the cell wall-damaging agent lyticase, and it showed cross-resistance against various compounds such as rapamycin, antimycin, coniferyl aldehyde and cycloheximide. Comparative transcriptomic analysis results revealed that the genes involved in the energy conservation and production pathways, and pleiotropic drug resistance were overexpressed. Whole genome re-sequencing identified single nucleotide polymorphisms in only three genes of the caffeine-hyperresistant mutant; PDR1, PDR5 and RIM8, which may play a potential role in caffeine-hyperresistance. Graphic abstrac

Evolutionary Engineering of an Iron-Resistant Saccharomyces cerevisiae Mutant and Its Physiological and Molecular Characterization

Iron plays an essential role in all organisms and is involved in the structure of many biomolecules. It also regulates the Fenton reaction where highly reactive hydroxyl radicals occur. Iron is also important for microbial biodiversity, health and nutrition. Excessive iron levels can cause oxidative damage in cells. Saccharomyces cerevisiae evolved mechanisms to regulate its iron levels. To study the iron stress resistance in S. cerevisiae, evolutionary engineering was employed. The evolved iron stress-resistant mutant &ldquo;M8FE&rdquo; was analysed physiologically, transcriptomically and by whole genome re-sequencing. M8FE showed cross-resistance to other transition metals: cobalt, chromium and nickel and seemed to cope with the iron stress by both avoidance and sequestration strategies. PHO84, encoding the high-affinity phosphate transporter, was the most down-regulated gene in the mutant, and may be crucial in iron-resistance. M8FE had upregulated many oxidative stress response, reserve carbohydrate metabolism and mitophagy genes, while ribosome biogenesis genes were downregulated. As a possible result of the induced oxidative stress response genes, lower intracellular oxidation levels were observed. M8FE also had high trehalose and glycerol production levels. Genome re-sequencing analyses revealed several mutations associated with diverse cellular and metabolic processes, like cell division, phosphate-mediated signalling, cell wall integrity and multidrug transporters

Isolation and characterization of severe acute respiratory syndrome coronavirus 2 in Turkey

Copyright: © 2020 Pavel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated with severe respiratory illness emerged in Wuhan, China, in late 2019. The virus has been able to spread promptly across all continents in the world. The current pandemic has posed a great threat to public health concern and safety. Currently, there are no specific treatments or licensed vaccines available for COVID-19. We isolated SARS-CoV-2 from the nasopharyngeal sample of a patient in Turkey with confirmed COVID-19. We determined that the Vero E6 and MA-104 cell lines are suitable for supporting SARS-CoV-2 that supports viral replication, development of cytopathic effect (CPE) and subsequent cell death. Phylogenetic analyses of the whole genome sequences showed that the hCoV-19/Turkey/ERAGEM-001/2020 strain clustered with the strains primarily from Australia, Canada, England, Iran and Kuwait and that the cases in the nearby clusters were reported to have travel history to Iran and to share the common unique nucleotide substitutions

Genomic, transcriptomic, and metabolic characterization of 2-Phenylethanol-resistant Saccharomyces cerevisiae obtained by evolutionary engineering

International audience2-Phenylethanol is an aromatic compound commonly used in the food, cosmetic, and pharmaceutical industries. Due to increasing demand for natural products by consumers, the production of this flavor by microbial fermentation is gaining interest, as a sustainable alternative to chemical synthesis or expensive plant extraction, both processes relying on the use of fossil resources. However, the drawback of the fermentation process is the high toxicity of 2-phenylethanol to the producing microorganism. The aim of this study was to obtain a 2-phenylethanol-resistant Saccharomyces cerevisiae strain by in vivo evolutionary engineering and characterize the adapted yeast at the genomic, transcriptomic and metabolic levels. For this purpose, the tolerance to 2-phenylethanol was developed by gradually increasing the concentration of this flavor compound through successive batch cultivations, leading to an adapted strain that could tolerate 3.4 g/L of 2-phenylethanol, which was about 3-times better than the reference strain. Genome sequencing of the adapted strain identified point mutations in several genes, notably in HOG1 that encodes the Mitogen-Activated Kinase of the high-osmolarity signaling pathway. As this mutation is localized in the phosphorylation lip of this protein, it likely resulted in a hyperactive protein kinase. Transcriptomic analysis of the adapted strain supported this suggestion by revealing a large set of upregulated stress-responsive genes that could be explained in great part by HOG1 -dependent activation of the Msn2/Msn4 transcription factor. Another relevant mutation was found in PDE2 encoding the low affinity cAMP phosphodiesterase, the missense mutation of which may lead to hyperactivation of this enzyme and thereby enhance the stressful state of the 2-phenylethanol adapted strain. In addition, the mutation in CRH1 that encodes a chitin transglycosylase implicated in cell wall remodeling could account for the increased resistance of the adapted strain to the cell wall-degrading enzyme lyticase. Finally, the potent upregulation of ALD3 and ALD4 encoding NAD + -dependent aldehyde dehydrogenase together with the observed phenylacetate resistance of the evolved strain suggest a resistance mechanism involving conversion of 2-phenylethanol into phenylacetaldehyde and phenylacetate implicating these dehydrogenases

Genomic, transcriptomic and physiological analyses of silver‐resistant Saccharomyces cerevisiae obtained by evolutionary engineering

International audienceSilver is a non-essential metal used in medical applications as an antimicrobial agent, but it is also toxic for biological systems. To investigate the molecular basis of silver resistance in yeast, we employed evolutionary engineering using successive batch cultures at gradually increased silver stress levels up to 0.25-mM AgNO(3)in 29 populations and obtained highly silver-resistant and genetically stableSaccharomyces cerevisiaestrains. Cross-resistance analysis results indicated that the silver-resistant mutants also gained resistance against copper and oxidative stress. Growth physiological analysis results revealed that the highly silver-resistant evolved strain 2E was not significantly inhibited by silver stress, unlike the reference strain. Genomic and transcriptomic analysis results revealed that there were mutations and/or significant changes in the expression levels of the genes involved in cell wall integrity, cellular respiration, oxidative metabolism, copper homeostasis, endocytosis and vesicular transport activities. Particularly the missense mutation in theRLM1gene encoding a transcription factor involved in the maintenance of cell wall integrity and with 707 potential gene targets might have a key role in the high silver resistance of 2E, along with its improved cell wall integrity, as confirmed by the lyticase sensitivity assay results. In conclusion, the comparative physiological, transcriptomic and genomic analysis results of the silver-resistantS. cerevisiaestrain revealed potential key factors that will help understand the complex molecular mechanisms of silver resistance in yeast