QRB Discovery最新文献

{"title":"Ultrafast enzymatic digestion of deoxyribonucleic acid in aqueous microdroplets for sequence discrimination and identification.","authors":"Xiaoqin Zhong,&nbsp;Hao Chen,&nbsp;Richard N Zare","doi":"10.1017/qrd.2021.2","DOIUrl":"https://doi.org/10.1017/qrd.2021.2","url":null,"abstract":"<p><p>We report the use of aqueous microdroplets to accelerate deoxyribonucleic acid (DNA) fragmentation by deoxyribonuclease I (DNase I), and we present a simple, ultrafast approach named DNA fragment mass fingerprinting to discriminate different DNA sequences by comparing their fragment mass patterns. DNA fragmentation in tiny microdroplets, which was produced by electrosonically spraying (+3 kV) a room temperature aqueous solution containing 10 μM DNA and 10 μg ml^-1 DNase I from a homemade setup, takes less than 1 ms. High differentiation/identification fidelity could be obtained by applying a cosine correlation measure for similarity assessment between two fragment mass patterns, which compares both mass-to-charge ratios (<i>m/z</i>) with an error tolerance of 5 ppm and the peaks' relative intensities. A single-nucleotide mutation in the sequence of bases, as exemplified by the sickle cell anemia mutation, is differentiated by setting a cutoff value of similarity at 90%. The order change of two adjacent bases in the sequence could still be well discriminated with a similarity of only 62% between the fragment mass patterns of the two similar sequences, which have the same molecular weights and thus cannot be differentiated by gel electrophoresis or direct mass detection by mass spectrometry. Compared to traditional genotyping methods, such as quantitative real-time polymerase chain reaction, the identification process with our approach could be completed within several minutes without any other expensive and complicated reagents or experimental steps. The potential of our approach for convenient and fast microbe genetic discrimination or identification is further demonstrated by differentiating the Orf1ab gene fragments of two similar coronaviruses with a very high sequence homologous rate of 96%, SARS-CoV-2 and bat-SL-CoVZC45, with a similarity of 0% between their fragment mass patterns.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":" ","pages":"e4"},"PeriodicalIF":0.0,"publicationDate":"2021-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2021.2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39125273","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combined electrochemistry and mass spectrometry imaging to interrogate the mechanism of action of modafinil, a cognition-enhancing drug, at the cellular and sub-cellular level.","authors":"Elias Ranjbari,&nbsp;Mai H Philipsen,&nbsp;Zhaoying Wang,&nbsp;Andrew G Ewing","doi":"10.1017/qrd.2021.4","DOIUrl":"https://doi.org/10.1017/qrd.2021.4","url":null,"abstract":"<p><p>Modafinil is a mild psychostimulant-like drug enhancing wakefulness, improving attention and developing performance in various cognitive tasks, but its mechanism of action is not completely understood. This is the first combination of amperometry, electrochemical cytometry and mass spectrometry to interrogate the mechanism of action of a drug, here modafinil, at cellular and sub-cellular level. We employed single-cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC) to investigate the alterations in exocytotic release and vesicular catecholamine storage following modafinil treatment. The SCA results reveal that modafinil slows down the exocytosis process so that, the number of catecholamines released per exocytotic event is enhanced in the modafinil-treated cells. Also, IVIEC results offer an upregulation effect of modafinil on the vesicular catecholamine storage. Mass spectrometry imaging by time-of-flight secondary ion mass spectrometry (ToF-SIMS) illustrates that treatment with modafinil reduces the cylindrical-shaped phosphatidylcholine at the cellular membrane, while the high curvature lipids with conical structures such as phosphatidylethanolamine and phosphatidylinositol are elevated after modafinil treatment. Combining the results obtained by SCA, IVIEC and ToF-SIMS suggests that modafinil-treated cells release a larger portion of their vesicular content at least in part by changing the lipid composition of the cell membrane, suggesting regulation of cognition.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e6"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2021.4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10283663","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structural basis of anti-SARS-CoV-2 activity of HCQ: specific binding to N protein to disrupt its interaction with nucleic acids and LLPS.","authors":"Mei Dang,&nbsp;Jianxing Song","doi":"10.1017/qrd.2021.12","DOIUrl":"https://doi.org/10.1017/qrd.2021.12","url":null,"abstract":"<p><p>SARS-CoV-2 nucleocapsid (N) protein plays the essential roles in key steps of the viral life cycle, thus representing a top drug target. Functionality of N protein including liquid-liquid phase separation (LLPS) depends on its interaction with nucleic acids. Only the variants with N proteins functional in binding nucleic acids might survive and spread in evolution and indeed, the residues critical for binding nucleic acids are highly conserved. Hydroxychloroquine (HCQ) was shown to prevent the transmission in a large-scale clinical study in Singapore but so far, no specific SARS-CoV-2 protein was experimentally identified to be targeted by HCQ. Here by NMR, we unambiguously decode that HCQ specifically binds NTD and CTD of N protein with Kd of 112.1 and 57.1 μM, respectively to inhibit their interaction with nucleic acid, as well as to disrupt LLPS. Most importantly, HCQ-binding residues are identical in SARS-CoV-2 variants and therefore HCQ is likely effective to different variants. The results not only provide a structural basis for the anti-SARS-CoV-2 activity of HCQ, but also renders HCQ to be the first known drug capable of targeting LLPS. Furthermore, the unique structure of the HCQ-CTD complex suggests a promising strategy for design of better anti-SARS-CoV-2 drugs from HCQ.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e13"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10392676/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9917606","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Response to crowded conditions reveals compact nucleus for amyloid formation of folded protein.","authors":"Tony E R Werner,&nbsp;Istvan Horvath,&nbsp;Pernilla Wittung-Stafshede","doi":"10.1017/qrd.2020.17","DOIUrl":"https://doi.org/10.1017/qrd.2020.17","url":null,"abstract":"<p><p>Although the consequences of the crowded cell environments may affect protein folding, function and misfolding reactions, these processes are often studied in dilute solutions <i>in vitro.</i> We here used biophysical experiments to investigate the amyloid fibril formation process of the fish protein apo-β-parvalbumin in solvent conditions that mimic steric and solvation aspects of the <i>in vivo</i> milieu. Apo-β-parvalbumin is a folded protein that readily adopts an amyloid state <i>via</i> a nucleation-elongation mechanism. Aggregation experiments in the presence of macromolecular crowding agents (probing excluded volume, entropic effects) as well as small molecule osmolytes (probing solvation, enthalpic effects) revealed that both types of agents accelerate overall amyloid formation, but the elongation step was faster with macromolecular crowding agents but slower in the presence of osmolytes. The observations can be explained by the steric effects of excluded volume favoring assembled states and that amyloid nucleation does not involve monomer unfolding. In contrast, the solvation effects due to osmolyte presence promote nucleation but not elongation. Therefore, the amyloid-competent nuclei must be compact with less osmolytes excluded from the surface than either the folded monomers or amyloid fibers. We conclude that, in contrast to other amyloidogenic folded proteins, amyloid formation of apo-β-parvalbumin is accelerated by crowded cell-like conditions due to a nucleation process that does not involve large-scale protein unfolding.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e2"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2020.17","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10301864","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Which are the 'Hilbert Problems' of Biophysics?","authors":"Bengt Norden","doi":"10.1017/qrd.2020.15","DOIUrl":"https://doi.org/10.1017/qrd.2020.15","url":null,"abstract":"Whilst considering commissioning articles for QRBDiscovery as Editor-in-Chief, I would like to explain what I think could be interesting topics for new articles in addition to unexpected amazing discoveries. Recently, I submitted a somewhat philosophical paper with the title ‘The Mole and Albert Einstein’ to which I had been invited by the Swedish Physics Society. To prepare myself, I had been reading Einstein’s famous papers and PhD Thesis of 1905 (in German to avoid interpreters’ pedagogical improvements!) and was struck by his quite narrow approach in his attempt to address and generalize so many different problems. I do not want to diminish the impression of his genius, but I suddenly saw Einstein as a somewhat bewildered young (25 years) man eager to make an impact.When in 2005 the Physics world celebrated the anniversary of Einstein’smiracle year, hewas claimed to be aChemist byNature columnist Philip Ball because of his work and PhD Thesis on molecular diffusion inspired by Brownian motion and his goal to determine Avogadro’s number – but then why not call Einstein a Biophysicist? Following van’t Hoff, the first Nobel Laureate in Chemistry (1901), Einstein starts with the osmotic pressure and van’t Hoff’s formula pV* =nRT, which looks like the ideal gas law, and which he assumes to be a thermodynamic macroscopic equation of state integrating corresponding microscopic equations in which the colliding solvent molecules just balance the osmotic pressure. His analysis (including some strange detours) leads to the famous statistical error function for diffusion. His explanation of the Brownianmotion also results in the final acceptance by the science society of the atom and molecule concepts in the shape we know them today. The jumpy randommotions that Robert Brown observed pollen grains to performwhen suspended in water, Einstein explains, reflect the thermal motions of surrounding water molecules, an unequal number obviously colliding from opposite sides of the grain, telling something about the size of the numbers. The molecular dynamics and molecular-kinetic theory of heat are at the heart of Einstein’s thinking in 1905 andmany years later his endeavor to develop a universal theory for the fundamental forces can be seen as just an expansion of this early work on liquids and intermolecular forces, based on Newtonian kinetic theory of matter. Where am I heading with this? Well, I might ask: would a modern approach by molecular dynamics computation exactly agree with Einstein’s description of Brownian dynamics? Or might there be some deviations that could suggest additional, yet undiscoveredmechanisms, such as wave front-like attacks by many coherently coupled solvent molecules by which the momentum transfer between the small molecules and big pollen grains could occur? The biophysical impact of such amechanism could be earth-shattering, changing the rules for thermal activations of various processes inside the cell, adding large fluctuation f","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e1"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2020.15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9922985","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A large 'discovery' experiment: Gender Initiative for Excellence (Genie) at Chalmers University of Technology.","authors":"Maria Saline,&nbsp;Mary Sheeran,&nbsp;Pernilla Wittung-Stafshede","doi":"10.1017/qrd.2021.3","DOIUrl":"https://doi.org/10.1017/qrd.2021.3","url":null,"abstract":"<p><p>Sweden tops gender equality rankings, but Swedish academia is still lacking women in top positions. To address gender inequality in its faculty, Chalmers University of Technology has invested 300 million SEK (30 million Euros) over 10 years in Gender initiative for Excellence (Genie). Genie aims to increase the university's success and excellence via gender equality efforts. In this editorial, we want to share insights on explicit efforts during Genie's first 2.5 years with the goal to inspire and advise other universities and researchers.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e5"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2021.3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9926487","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Low-cooling-rate freezing in biomolecular cryo-electron microscopy for recovery of initial frames.","authors":"Chunling Wu,&nbsp;Huigang Shi,&nbsp;Dongjie Zhu,&nbsp;Kelong Fan,&nbsp;Xinzheng Zhang","doi":"10.1017/qrd.2021.8","DOIUrl":"https://doi.org/10.1017/qrd.2021.8","url":null,"abstract":"<p><p>When biological samples are first exposed to electrons in cryo-electron microcopy (cryo-EM), proteins exhibit a rapid 'burst' phase of beam-induced motion that cannot be corrected with software. This lowers the quality of the initial frames, which are the least damaged by the electrons. Hence, they are commonly excluded or down-weighted during data processing, reducing the undamaged signal and the resolution in the reconstruction. By decreasing the cooling rate during sample preparation, either with a cooling-rate gradient or by increasing the freezing temperature, we show that the quality of the initial frames for various protein and virus samples can be recovered. Incorporation of the initial frames in the reconstruction increases the resolution by an amount equivalent to using ~60% more data. Moreover, these frames preserve the high-quality cryo-EM densities of radiation-sensitive residues, which is often damaged or very weak in canonical three-dimensional reconstruction. The improved freezing conditions can be easily achieved using existing devices and enhance the overall quality of cryo-EM structures.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e11"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/df/9c/S2633289221000089a.PMC10392635.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9917603","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The unhappy chaperone.","authors":"Sara Linse,&nbsp;Kyrre Thalberg,&nbsp;Tuomas P J Knowles","doi":"10.1017/qrd.2021.5","DOIUrl":"https://doi.org/10.1017/qrd.2021.5","url":null,"abstract":"<p><p>Chaperones protect other proteins against misfolding and aggregation, a key requirement for maintaining biological function. Experimental observations of changes in solubility of amyloid proteins in the presence of certain chaperones are discussed here in terms of thermodynamic driving forces. We outline how chaperones can enhance amyloid solubility through the formation of heteromolecular aggregates (co-aggregates) based on the second law of thermodynamics and the flux towards equal chemical potential of each compound in all phases of the system. Higher effective solubility of an amyloid peptide in the presence of chaperone implies that the chemical potential of the peptide is higher in the aggregates formed under these conditions compared to peptide-only aggregates. This must be compensated by a larger reduction in chemical potential of the chaperone in the presence of peptide compared to chaperone alone. The driving force thus relies on the chaperone being very unhappy on its own (high chemical potential), thus gaining more free energy than the amyloid peptide loses upon forming the co-aggregate. The formation of heteromolecular aggregates also involves the kinetic suppression of the formation of homomolecular aggregates. The unhappiness of the chaperone can explain the ability of chaperones to favour an increased population of monomeric client protein even in the absence of external energy input, and with broad client specificity. This perspective opens for a new direction of chaperone research and outlines a set of outstanding questions that aim to provide additional cues for therapeutic development in this area.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e7"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1017/qrd.2021.5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10301862","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The G protein-first activation mechanism of opioid receptors by Gi protein and agonists.","authors":"Amirhossein Mafi,&nbsp;Soo-Kyung Kim,&nbsp;William A Goddard","doi":"10.1017/qrd.2021.7","DOIUrl":"https://doi.org/10.1017/qrd.2021.7","url":null,"abstract":"<p><p>We report the G protein-first mechanism for activation of G protein-coupled receptors (GPCR) for the three closest subtypes of the opioid receptors (OR), μOR, κOR and δOR. We find that they couple to the inactive Gi protein-bound guanosine diphosphate (GDP) <i>prior</i> to agonist binding. The inactive Gi protein forms anchors to the intracellular loops of the <i>inactive</i> apo-μOR, apo-κOR and apo-δOR, inducing opening of the cytoplasmic region to form a pre-activated state that holds Gi protein in place until agonist binds. Then, agonist binds to μOR, κOR and δOR already complexed with Gi protein, to trigger the Gαi to open up the tightly coupled GDP binding site, making GDP accessible for GTP exchange, an essential step for Gi signalling. We show that the agonist alone <i>cannot</i> open the intracellular region of μOR and κOR, requiring Gi protein to open the cytoplasmic region by itself. We consider that this G protein-first mechanism may apply to activation of other Class A GPCRs. However, for δOR, agonist binding can open up the intracellular region to encourage Gi protein recruitment. Thus, activation of Gi protein mediated by δOR favourably may proceed with either ligand-first or G protein-first activation mechanisms.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e9"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10392629/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9917607","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A multimodal electrochemical approach to measure the effect of zinc on vesicular content and exocytosis in a single cell model of ischemia.","authors":"Ying Wang,&nbsp;Chaoyi Gu,&nbsp;Andrew G Ewing","doi":"10.1017/qrd.2021.10","DOIUrl":"https://doi.org/10.1017/qrd.2021.10","url":null,"abstract":"<p><p>Zinc ion is essential for normal brain function that modulates synaptic activity and neuronal plasticity and it is associated with memory formation. Zinc is considered to be a contributing factor to the pathogenesis of ischemia, but the association between zinc and ischemia on vesicular exocytosis is unclear. In this study, we used a combination of chemical analysis methods and a cell model of ischemia/reperfusion to investigate exocytotic release and vesicular content, as well as the effect of zinc alteration on vesicular exocytosis. Oxygen-glucose deprivation and reperfusion (OGDR) was used as an <i>in vitro</i> model of ischemia in a model cell line. Exocytotic release and vesicular storage of catecholamine content were increased following OGDR, resulting in a higher fraction of release during exocytosis. However, zinc eliminated these increases following OGDR and the fraction of release remained unchanged. Understanding the consequences of zinc accumulation on vesicular exocytosis at the early stage of OGDR should aid in the development of therapeutic strategies to reduce ischemic brain injury. As the fraction released has been suggested to be related to presynaptic plasticity, insights are gained towards deciphering ischemia related memory impairment.</p>","PeriodicalId":34636,"journal":{"name":"QRB Discovery","volume":"2 ","pages":"e12"},"PeriodicalIF":0.0,"publicationDate":"2021-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10392633/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9923419","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}