Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe

The frequency of Duchenne muscular dystrophy/Becker muscular dystrophy and Pompe disease in children with isolated transaminase elevation: results from the observational VICTORIA study

IntroductionElevated transaminases and/or creatine phosphokinase can indicate underlying muscle disease. Therefore, this study aims to determine the frequency of Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD) in male children and Pompe disease (PD) in male and female children with isolated hypertransaminasemia.MethodsThis multi-center, prospective study enrolled patients aged 3–216 months with serum alanine transaminase (ALT) and/or aspartate transaminase (AST) levels >2× the upper limit of normal (ULN) for ≥3 months. Patients with a known history of liver or muscle disease or physical examination findings suggestive of liver disease were excluded. Patients were screened for creatinine phosphokinase (CPK) levels, and molecular genetic tests for DMD/BMD in male patients and enzyme analysis for PD in male and female patients with elevated CPK levels were performed. Genetic analyses confirmed PD. Demographic, clinical, and laboratory characteristics of the patients were analyzed.ResultsOverall, 589 patients [66.8% male, mean age of 63.4 months (standard deviation: 60.5)] were included. In total, 251 patients (188 male and 63 female) had CPK levels above the ULN. Of the patients assessed, 47% (85/182) of male patients were diagnosed with DMD/BMD and 1% (3/228) of male and female patients were diagnosed with PD. The median ALT, AST, and CPK levels were statistically significantly higher, and the questioned neurological symptoms and previously unnoticed examination findings were more common in DMD/BMD patients than those without DMD/BMD or PD (p < 0.001).DiscussionQuestioning neurological symptoms, conducting a complete physical examination, and testing for CPK levels in patients with isolated hypertransaminasemia will prevent costly and time-consuming investigations for liver diseases and will lead to the diagnosis of occult neuromuscular diseases. Trial RegistrationClinicaltrials.gov NCT04120168

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

The Philosophical Analysis of Reinhart P. A. Dozy's History of Islamic Works (Example İsmail Fenni, Manastirli İsmail Hakki, Filibeli Ahmet Hilmi)

Dozy'nin "Tarih-i İslamiyet" kitabında İslamiyet ve Müslümanlar hakkındaki ifadeleri bilimsel ve felsefi olarak birçok çelişik ifadeye sahiptir. Ancak bu durum ülkemizde bazı düşünürler tarafından normal karşılanırken, bazı milliyetçi ve İslamcı düşünürler tarafından yoğun şekilde eleştirilmiş ve bu düşüncelere reddiyeler yazılma ihtiyacı hissedilmiştir. Abdullah Cevdet'in Dozy'nin kitabını tercüme etmesi ve bu kitaba karşı yapmış olduğu aşırı övgüler, İsmail Fenni Ertuğrul, Filibeli Ahmet Hilmi, Manastırlı İsmail Hakkı gibi İslamcı ve milliyetçi düşünürleri harekete geçirmiştir. Tezimizde, bu düşünürlerin eleştirilerinin amacını, bunları hangi felsefi ve bilimsel yöntemler ile yapmaya çalıştıklarını ve Dozy'e eleştirilerini incelemeye çalıştık.In Dozy's "History of Islam" his statements about Islam and Muslims have many contradictory expressions, scientifically and philosophically. However, while this situation was considered normal by some thinkers in our country, it was extensively criticized by some nationalist and Islamist thinkers and it was felt the need to write rejections to these ideas. Abdullah Cevdet's translation of Dozy's book and the excessive praise he made against this book provoked Islamist and nationalist thinkers such as İsmail Fenni Ertuğrul, Ahmet Hilmi from Filibeli and İsmail Hakkı from Manastir. In our thesis, we tried to examine the purpose of the criticism of these thinkers, what philosophical and scientific methods they try to do and their criticism to Dozy

The experimental research of combined effect of various dispersive agents and cement on undrained shear strength of high plasticity clayey soil

Bu tez çalışmasında, kohezyonlu zeminlere kimyasal ajan ve çimento eklendiğinde zeminin dayanım parametrelerini nasıl etkilediği incelenmiştir. Çalışmada malzeme olarak Gazi Üniversitesi merkez yerleşkesi içerisinde açılan hafriyat çukurundan temin edilen kil, bağlayıcı malzeme olarak puzolanik çimento ve kimyasal katkı kullanılmıştır. Çalışma kapsamında kil üzerinde ilk olarak sınıflama deneyleri yürütülmüş ve zemin sınıfının yüksek plastisiteli kil olduğu saptanmıştır. Daha sonra kil üzerinde standart Proctor deneyi yapılarak kilin optimum su muhtevası %29,81 ve maksimum kuru birim hacim ağırlığı 13.42 kN/m3 olarak belirlenmiştir. Drenajsız dayanım deneyleri için numuneler, kilin optimum su muhtevası ve maksimum kuru birim hacim ağırlık değerleri kullanılarak hazırlanmıştır. Dört farklı kimyasal ajan (Sodyum hegzametafosfat, Alüminyum sülfat, Sodyum karbonat, Sodyum silikat), üç farklı solüsyon miktarı (%5,%10 ve %20) iki faklı çimento oranında (Çimento/(Çimento+Kil) = %5 ve %10) hazırlanan kimyasal ajan-çimento-kil karışımlarının standart Proctor enerjisi ile sıkıştırıldıktan sonra 28 gün kürde bekletilmiştir. Daha sonra numuneler drenajsız üç eksenli dayanım deneylerine tabi tutulmuştur. Deney sonuçlarına v göre sadece çimento kullanılarak elde edilen çimento-kil karışımlarının Çimento/(Çimento+Kil) oranı arttırıldığında içsel sürtünme açısı yaklaşık %40 artmış ve kohezyon dayanımı değişmemiştir. Çimento/(Çimento+kil) oranı %5 olduğunda tüm karışımlar için kohezyon dayanımları, tüm kimyasal ajanlar ve tüm solüsyon oranları için azalmıştır. Bu oran %10 olduğunda kohezyon dayanımları bir numune haricinde azalmıştır. Bu numunenin kohezyon dayanımı da, sadece çimento katılarak oluşturulan karışımın kohezyon dayanımı ile kıyaslandığında yaklaşık %50 artmıştır. Kimyasal ajan türü ve kimyasal ajan solüsyon miktarının içsel sürtünme açısı üzerindeki etkisi belirli bir eğilim göstermemiştir.In this thesis study, the effect of chemical agent and cement addition to cohesive soils on the resistance parameters of the soil was examined. As study material, clay obtained from the excavation pit in the Gazi University central settlement, pozzolanic cement as binding material and chemical additive was used. Under the scope of the study, initially classification experiments were held on clay and it was understood that the soil class is high plasticized clay. Afterwards, standard Proctor experiment was applied on clay and the optimum water content of the clay was found to be 29.8 % and maximum dry unit volume weight was found to be 13.4 kN/m3. For the undrained resistance experiments, the samples were prepared by using the optimum water content and maximum dry unit volume weight of clay. The chemical agent cement clay mixtures prepared by using four different chemical agent (Sodium hexametaphosphate, aluminum sulphate, sodium carbonate, sodium silicate), three different solution amounts (5%, 10% and 20%) and two different cement ratio, (cement/(cement+clay) = 5% and 10%) were pressed by Proctor energy and kept in curing for 28 days. Afterwards the samples were exposed to undrained three axis resistance experiments. According to experiment results, when the cement/(cement+clay) ratio of the vii cement-clay mixtures obtained by using only cement are increased the inner friction angle increased approximately 40% and cohesion resistance didn't change. When the cement/(cement+clay) ratio becomes 5% the cohesion resistances for all mixtures decreased for all chemical agents and all solution ratios. When this ratio becomes 10%, cohesion resistances decreased except one sample and the cohesion resistance of that sample increased approximately 50% compared to the cohesion resistance of the mixture prepared by adding only cement. The effect of chemical agent type and chemical agent solution amount on the inner friction angle, did not show a certain tendency

Ggenotoxicity assessment of heavy metals (zn, cr, pb) on strawberry plants using rapd assay

The aim of the present study is to assess DNA damage in leaves of strawberry (Fragaria × ananassa Duch.) seedlings treated with different heavy metals (zinc, chromium, lead) using random amplified polymorphic DNA (RAPD) assay and analysis of total soluble protein content. For this purpose, strawberry seedlings were treated with 400 and 800 μM Zn, Pb and Cr for 7 days. Thirty four RAPD primers produced 218 bands at molecular weight ranging from 183 bp to 5180 bp. Compared with control, RAPD patterns of heavy metal exposed groups showed differences in band loss, gain of new bands and increase and decrease of band intensity. The highest polymorphism rate (32.11%) was observed in 800 μM Pb applied strawberry seedlings. To evaluate the alterations in RAPD profiles qualitatively, genomic template stability (GTS) was performed and the values were 75.08% and 69.59% for 400 and 800 μM Zn treatments, 74.52% and 68.60% for 400 and 800 μM Cr treatments, 70.11% and 63.78% for 400 and 800 μM Pb treatments, respectively. Total soluble protein content in heavy metal-treated groups also showed a similar correlation to GTS values. RAPD analyses are useful biomarker assays to determine the genotoxicity induced by environmental pollutants such as heavy metals in plant model system

Alleviation of copper stress with homobrassinosteroid in germinating sunflower roots

We investigated potential alleviation effects of homobrassinosteroid (HBR) hormone on sunflower (Helianthus annuus L. cv. TR003) grown under copper (Cu) stress conditions. Seeds were grown under Cu (30 µM and 40 µM) and also HBR (2 µM) treatments to analyse primary root lengths, protein contents, antioxidant system enzymes' activities (superoxide dismutase-SOD and catalaseCAT) and expressions of these enzymes at 48 h and 72 h. When compared to control, Cu applications decreased primary root lengths. On the other hand, HBR application increased primary root lengths of Cu-stressed samples at 48 h and 72 h. Depending on concentration and timing, protein contents and enzyme activities showed varying results in Cutreated and Cu+HBR-applied samples. In addition to enzyme activities, SOD and CAT expression profiles were also investigated by RT-PCR. Expected band sizes for SOD (309 bp) and CAT (248 bp) were observed in all samples. These results indicate a positive role of HBR on Custressed sunflower for agricultural applications