Cas9 Cuts and Consequences; Detecting,Predicting, and Mitigating CRISPR/Cas9 On- and Off-Target Damage

Large deletions and genomic re-arrangements are increasingly recognized as common products of double-strand break repair at Clustered Regularly Interspaced, Short Palindromic Repeats - CRISPR associated protein 9 (CRISPR/Cas9) on-target sites. Together with well-known off-target editing products from Cas9 target misrecognition, these are important limitations, that need to be addressed. Rigorous assessment of Cas9-editing is necessary to ensure validity of observed phenotypes in Cas9-edited cell-lines and model organisms. Here the mechanisms of Cas9 specificity, and strategies to assess and mitigate unwanted effects of Cas9 editing are reviewed; covering guide-RNA design, RNA modifications, Cas9 modifications, control of Cas9 activity; computational prediction for off-targets, and experimental methods for detecting Cas9 cleavage. Although recognition of the prevalence of on- and off-target effects of Cas9 editing has increased in recent years, broader uptake across the gene editing community will be important in determining the specificity of Cas9 across diverse applications and organisms.     

Designer metalloenzymes for synthetic biology: Enzyme hybrids for catalysis

Combining organometallics and biology has generated broad interest from scientists working on applications from in situ drug release to biocatalysis. Engineered enzymes and biohybrid catalysts (also referred to as artificial enzymes) have introduced a wide range of abiotic chemistry into biocatalysis. Predominantly, this work has concentrated on using these catalysts for single step in vitro reactions. However, the promise of using these hybrid catalysts in vivo and combining them with synthetic biology and metabolic engineering is vast. This report will briefly review recent advances in artificial metalloenzyme design, followed by summarising recent studies that have looked at the use of these hybrid catalysts in vivo and in enzymatic cascades, therefore exploring their potential for synthetic biology.     

Studies on fragment-based design of allosteric inhibitors of human factor XIa

Human factor XIa (hFXIa) has emerged as an attractive target for development of new anticoagulants that promise higher level of safety. Different strategies have been adopted so far for the design of anti-hFXIa molecules including competitive and non-competitive inhibition. Of these, allosteric dysfunction of hFXIa’s active site is especially promising because of the possibility of controlled reduction in activity that may offer a route to safer anticoagulants. In this work, we assess fragment-based design approach to realize a group of novel allosteric hFXIa inhibitors. Starting with our earlier discovery that sulfated quinazolinone (QAO) bind in the heparin-binding site of hFXIa, we developed a group of two dozen dimeric sulfated QAOs with intervening linkers that displayed a progressive variation in inhibition potency. In direct opposition to the traditional wisdom, increasing linker flexibility led to higher potency, which could be explained by computational studies. Sulfated QAO 19S was identified as the most potent and selective inhibitor of hFXIa. Enzyme inhibition studies revealed that 19S utilizes a non-competitive mechanism of action, which was supported by fluorescence studies showing a classic sigmoidal binding profile. Studies with selected mutants of hFXIa indicated that sulfated QAOs bind in heparin-binding site of the catalytic domain of hFXIa. Overall, the approach of fragment-based design offers considerable promise for designing heparin-binding site-directed allosteric inhibitors of hFXIa.     

Malleability of the SARS-CoV-2 3CL Mpro Active-Site Cavity Facilitates Binding of Clinical Antivirals

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Additive diversity partitioning as a guide to regional montane reserve design in Asia: an example from Yunnan Province,China

Aim Data on spatial and temporal turnover in species composition within a region is essential to design regional protected areas. Montane systems are often recognized as biodiversity hotspots. The primary objective of this study is to identify patterns of montane bird diversity across multiple spatial and temporal scales using an additive diversity partitioning framework. Location The Ailao Mountains, central Yunnan Province, China. Methods We used point counts to sample bird communities in four elevational zones, on eastern and western slopes, during both the breeding and the non‐breeding seasons. Diversity (richness and Shannon) was partitioned across space (points, elevational zones and slopes) and time (seasons). We used permutation tests to compare observed values to values expected by random chance. A complementary cluster analysis was also used to evaluate beta diversity. Results Overall, the gamma diversity was attributed to significantly higher beta diversity (relative to that of randomization tests) among elevational zones and, to a lesser extent, between slopes. For Shannon–Wiener Index, beta diversity between seasons was significantly higher than expected and had a similar contribution to the gamma diversity as with beta diversity between slopes. Hierarchical cluster analysis supported the findings for Shannon–Wiener Index. The contribution of beta diversity among points to gamma diversity within each elevational zone generally lessened with increasing elevation. Main conclusions Our results show significantly high levels of beta diversity among elevational zones and between slopes, as well as between seasons for Shannon diversity, in a small area of the Ailao Mountain range. Thus, a regional montane reserve system should cover the entire elevational gradient and multiple slopes, rather than only the montane crest. Furthermore, higher pattern diversity in lower elevational zones suggests that larger areas should be preserved at lower elevational zones. Finally, the design of regional reserve systems require more studies conducted at multiple seasons at a regional scale.     

DStabilize: A Web Resource to Generate Mirror Images of Biomolecules

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Guidelines for measuring and reporting environmental conditions in controlled-environment studies

Guidelines for measuring and reporting environmental conditions in plant growth chambers are presented in tabular form. These guidelines are recommended by the North Central Region (NCR-101) Technical Committee on Growth Chamber Use, a committee formed under the Cooperative Research Program of the State Agricultural Experiment Stations (SAES) and the United States Department of Agriculture (USDA). Recommendations on location and frequency of measurements as well as suggested format and units of measurement are listed for each environmental parameter. The adoption of these standardized guidelines should greatly improve the uniformity of research conducted in controlled environments and facilitate comparison of experimental results obtained in studies conducted in different laboratories on a world-wide basis.     

Comparison of Amberchrom-CG161C and Dowex99 as the adsorbent of a four-zone simulated moving bed process for removal of acetic acid from biomass hydrolyzate

One of the important steps in the application of biomass to producing sugars, which can be converted into bio-ethanol and other valuable chemicals by fermentation, is to hydrolyze the biomass components by sulfuric acid. It was reported that such a hydrolysis entailed the generation of acetic acid, which has been recognized as a key impurity to be surely removed from the biomass hydrolyzate for ensuring high fermentability of the hydrolyzed sugars. Regarding such a removal task, there has been a previous application of a simulated moving bed (SMB) process based on the Dowex99 adsorbent, whose performance, however, was limited by low selectivity between acetic acid and sugars. To overcome such a limitation, another adsorbent alternative to Dowex99 was searched in this study. It was found that Amberchrom-CG161C allowed higher selectivity between acetic acid and sugars than Dowex99. To investigate the relative superiority of Amberchrom-CG161C over Dowex99 as the adsorbent of an SMB process for removing acetic acid from the biomass hydrolyzate, the two SMB processes based on Amberchrom-CG161C and Dowex99 were optimized using the SMB optimization tool based on standing wave design (SWD) method. The optimization results revealed that the Amberchrom-CG161C SMB outperformed the Dowex99 SMB by a wide margin.     

“七步法”课堂设计与实践：构建研究导向型微生物学“金课”课堂

研究导向型教学是以学生成长和发展为中心的创新性教育模式，其核心目标与“金课”的建设目标一致，强调培养学生的研究技能，使其具有创新性和高阶性。在微生物学一流课程建设过程中，将研究导向型教学理念与BOPPPS课堂[导入(bridge-in)、目标(objective)、前测(pre-assessment)、参与式学习(participatory-learning)、后测(post-assessment)和总结(summary)]组织形式相结合，并创新性引入“提升”模块，以问题为驱动，聚焦科学前沿，融入思政材料，形成导入—目标—前测—参与式学习—后测—提升—总结(七步法)的新型模块化闭环教学模式。该模式的实施能够有效提升课堂教学的逻辑性和组织性，提高教学效率，提升学生分析问题和解决问题的高阶性，为学生将来开展科研工作奠定良好的基础，并为打造以学生为中心的“金课”课堂实践提供借鉴参考。