scholarly journals High-throughput functional variant screens via in vivo production of single-stranded DNA

2021 ◽  
Vol 118 (18) ◽  
pp. e2018181118
Author(s):  
Max G. Schubert ◽  
Daniel B. Goodman ◽  
Timothy M. Wannier ◽  
Divjot Kaur ◽  
Fahim Farzadfard ◽  
...  
Keyword(s):  
High Throughput ◽  
Quantitative Measurement ◽  
Single Stranded Dna ◽  
Relative Growth ◽  
Relative Growth Rates ◽  
Functional Variant ◽  
Causal Variants ◽  
Targeted Deep Sequencing ◽  
Functional Screens

Creating and characterizing individual genetic variants remains limited in scale, compared to the tremendous variation both existing in nature and envisioned by genome engineers. Here we introduce retron library recombineering (RLR), a methodology for high-throughput functional screens that surpasses the scale and specificity of CRISPR-Cas methods. We use the targeted reverse-transcription activity of retrons to produce single-stranded DNA (ssDNA) in vivo, incorporating edits at >90% efficiency and enabling multiplexed applications. RLR simultaneously introduces many genomic variants, producing pooled and barcoded variant libraries addressable by targeted deep sequencing. We use RLR for pooled phenotyping of synthesized antibiotic resistance alleles, demonstrating quantitative measurement of relative growth rates. We also perform RLR using the sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for causal variants, demonstrating that RLR is uniquely suited to utilize large pools of natural variation. Using ssDNA produced in vivo for pooled experiments presents avenues for exploring variation across the genome.

scholarly journals High throughput functional variant screens via in-vivo production of single-stranded DNA

2020 ◽  
Author(s):  
Max G. Schubert ◽  
Daniel B. Goodman ◽  
Timothy M. Wannier ◽  
Divjot Kaur ◽  
Fahim Farzadfard ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
High Throughput ◽  
Entire Genome ◽  
Single Stranded Dna ◽  
Functional Variant ◽  
Continuous Generation ◽  
Sources Of Variation ◽  
Targeted Deep Sequencing ◽  
High Throughput Screens

AbstractTremendous genetic variation exists in nature, but our ability to create and characterize individual genetic variants remains far more limited in scale. Likewise, engineering proteins and phenotypes requires the introduction of synthetic variants, but design of variants outpaces experimental measurement of variant effect. Here, we optimize efficient and continuous generation of precise genomic edits in Escherichia coli, via in-vivo production of single-stranded DNA by the targeted reverse-transcription activity of retrons. Greater than 90% editing efficiency can be obtained using this method, enabling multiplexed applications. We introduce Retron Library Recombineering (RLR), a system for high-throughput screens of variants, wherein the association of introduced edits with their retron elements enables a targeted deep sequencing phenotypic output. We use RLR for pooled, quantitative phenotyping of synthesized variants, characterizing antibiotic resistance alleles. We also perform RLR using sheared genomic DNA of an evolved bacterium, experimentally querying millions of sequences for antibiotic resistance variants. In doing so, we demonstrate that RLR is uniquely suited to utilize non-designed sources of variation. Pooled experiments using ssDNA produced in vivo thus present new avenues for exploring variation, both designed and not, across the entire genome.

A Measure of Economic Growth in East and in West Pakistan

1961 ◽  
Vol 1 (2) ◽  
pp. 49-54
Author(s):  
S. U. Khan
Keyword(s):  
Regional Planning ◽  
National Income ◽  
Future Planning ◽  
Correct Evaluation ◽  
Relative Growth ◽  
Relative Growth Rates ◽  
National Planning ◽  
Income Data ◽  
Regional Basis ◽  
The Difference

It is sometimes said that "national planning will simply have no meaning if it completely ignores the economic disparities between the two wings and fails to evolve a sensible pattern of regional planning"2. The lack of much essential data on a regional basis, however, renders any precise estimate of the relative growth rates almost impossible. Data either are not available or are inadequate on such important variables as production, income, consumption and trade, so that even a correct evaluation of past development efforts is not possible. The implications of such a situation for future planning are not difficult to understand. In this article an attempt is made to estimate the absorption of specified commodities in East and West Pakistan separately3. This will indicate the pattern of consumption and also give a rough idea about the growth rate of the two wings. With this purpose in view, quantity indices of absorption are prepared for each wing separately, taking data on availability of goods and prices from the Institute's monograph on Inflation. The quantity indi¬ces, however, are not of course strictly comparable with national income estimates because of the difference in coverage of the two series. National income data include government, services, trade, etc., while the quantity indices cover only specified goods available for each region.

CHANGES IN ENZYMES AND PROTEINS IN SALT-SHOCKED VS. GRADIENT INCREASES IN NACL IN `POINSETT' CUCUMBER ROOTS

HortScience ◽  
1990 ◽  
Vol 25 (9) ◽  
pp. 1091G-1091
Author(s):  
Anne K. Hurley ◽  
B. Greg Cobb
Keyword(s):  
Invertase Activity ◽  
Shock Treatment ◽  
Cucumis Sativus L ◽  
Relative Growth ◽  
Relative Growth Rates ◽  
Salt Shock ◽  
Gradient Solution ◽  
Cucumber Roots ◽  
Average Plant ◽  
Plastochron Index

Cucumis sativus, L., `Poinsett' seedlings were grown under artificial light in 40% modified Hoagland's solution until an average plant plastochron index of 4.73 was reached. Plants were then placed in solutions of (1) 0 mM NaCl, (2) 80 mM NaCl (salt-shock), or (3) placed in a dropwise gradient solution of NaCl and Hoagland's until the final concentration of 80 mM NaCl was reached at 41 hours. Leaves of the 80 mM shock treatment wilted immediately, but recovered turgor within 6 hours. Leaves of 80 mM gradient did not wilt at anytime. The control and gradient treatments had relative growth rates which were similar to each other, but RGR decreased in the shock treatment. Invertase activity was measured in the roots at 24, 41, and 48 hours after initial treatment. Invertase activity of shock treatment increased significantly over the controls at 24 hours. The 80mM gradient was not significantly different than either treatment. Four isozymes of α– galactosidase were detected. The relative intensities of the bands varied with time and treatment. One invertase band was resolved in roots on 8% native acrylamide gels. SDS gels indicated increases in proteins in the gradient treatment compared to the control and the 80 mM shock treatment.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

scholarly journals Organ-specific, multimodal, wireless optoelectronics for high-throughput phenotyping of peripheral neural pathways

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Woo Seok Kim ◽  
Sungcheol Hong ◽  
Milenka Gamero ◽  
Vivekanand Jeevakumar ◽  
Clay M. Smithhart ◽  
...  
Keyword(s):  
High Throughput ◽  
Low Cost ◽  
Optoelectronic Device ◽  
Antenna System ◽  
Neural Pathways ◽  
Organ Specific ◽  
Coil Antenna ◽  
Sensory Fibers

AbstractThe vagus nerve supports diverse autonomic functions and behaviors important for health and survival. To understand how specific components of the vagus contribute to behaviors and long-term physiological effects, it is critical to modulate their activity with anatomical specificity in awake, freely behaving conditions using reliable methods. Here, we introduce an organ-specific scalable, multimodal, wireless optoelectronic device for precise and chronic optogenetic manipulations in vivo. When combined with an advanced, coil-antenna system and a multiplexing strategy for powering 8 individual homecages using a single RF transmitter, the proposed wireless telemetry enables low cost, high-throughput, and precise functional mapping of peripheral neural circuits, including long-term behavioral and physiological measurements. Deployment of these technologies reveals an unexpected role for stomach, non-stretch vagal sensory fibers in suppressing appetite and demonstrates the durability of the miniature wireless device inside harsh gastric conditions.

scholarly journals O23: CHARACTERISING PATIENT-DERIVED COLORECTAL CANCER TISSUE-ORIGINATED ORGANOIDAL SPHEROIDS FOR HIGH-THROUGHPUT MICROFLUIDIC APPLICATIONS

2021 ◽  
Vol 108 (Supplement_1) ◽  
Author(s):  
MI Khot ◽  
M Levenstein ◽  
R Coppo ◽  
J Kondo ◽  
M Inoue ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Fluid Flow ◽  
High Throughput ◽  
Microfluidic Devices ◽  
In Vitro Models ◽  
Fluorescent Imaging ◽  
Cancer Tissue ◽  
Cell Models

Abstract Introduction Three-dimensional (3D) cell models have gained reputation as better representations of in vivo cancers as compared to monolayered cultures. Recently, patient tumour tissue-derived organoids have advanced the scope of complex in vitro models, by allowing patient-specific tumour cultures to be generated for developing new medicines and patient-tailored treatments. Integrating 3D cell and organoid culturing into microfluidics, can streamline traditional protocols and allow complex and precise high-throughput experiments to be performed with ease. Method Patient-derived colorectal cancer tissue-originated organoidal spheroids (CTOS) cultures were acquired from Kyoto University, Japan. CTOS were cultured in Matrigel and stem-cell media. CTOS were treated with 5-fluorouracil and cytotoxicity evaluated via fluorescent imaging and ATP assay. CTOS were embedded, sectioned and subjected to H&E staining and immunofluorescence for ABCG2 and Ki67 proteins. HT29 colorectal cancer spheroids were produced on microfluidic devices using cell suspensions and subjected to 5-fluorouracil treatment via fluid flow. Cytotoxicity was evaluated through fluorescent imaging and LDH assay. Result 5-fluorouracil dose-dependent reduction in cell viability was observed in CTOS cultures (p<0.01). Colorectal CTOS cultures retained the histology, tissue architecture and protein expression of the colonic epithelial structure. Uniform 3D HT29 spheroids were generated in the microfluidic devices. 5-fluorouracil treatment of spheroids and cytotoxic analysis was achieved conveniently through fluid flow. Conclusion Patient-derived CTOS are better complex models of in vivo cancers than 3D cell models and can improve the clinical translation of novel treatments. Microfluidics can streamline high-throughput screening and reduce the practical difficulties of conventional organoid and 3D cell culturing. Take-home message Organoids are the most advanced in vitro models of clinical cancers. Microfluidics can streamline and improve traditional laboratory experiments.

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