scholarly journals Hierarchy and extremes in selections from pools of randomized proteins

2016 ◽  
Vol 113 (13) ◽  
pp. 3482-3487 ◽  
Author(s):  
Sébastien Boyer ◽  
Dipanwita Biswas ◽  
Ananda Kumar Soshee ◽  
Natale Scaramozzino ◽  
Clément Nizak ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Scaling Law ◽  
Natural Populations ◽  
Value Theory ◽  
Darwinian Evolution ◽  
Future Evolution ◽  
Synthetic Protein ◽  
Simple Scaling ◽  
Protein Libraries

Variation and selection are the core principles of Darwinian evolution, but quantitatively relating the diversity of a population to its capacity to respond to selection is challenging. Here, we examine this problem at a molecular level in the context of populations of partially randomized proteins selected for binding to well-defined targets. We built several minimal protein libraries, screened them in vitro by phage display, and analyzed their response to selection by high-throughput sequencing. A statistical analysis of the results reveals two main findings. First, libraries with the same sequence diversity but built around different “frameworks” typically have vastly different responses; second, the distribution of responses of the best binders in a library follows a simple scaling law. We show how an elementary probabilistic model based on extreme value theory rationalizes the latter finding. Our results have implications for designing synthetic protein libraries, estimating the density of functional biomolecules in sequence space, characterizing diversity in natural populations, and experimentally investigating evolvability (i.e., the potential for future evolution).

scholarly journals The cyanobacterial circadian clock follows midday in vivo and in vitro

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Eugene Leypunskiy ◽  
Jenny Lin ◽  
Haneul Yoo ◽  
UnJin Lee ◽  
Aaron R Dinner ◽  
...  
Keyword(s):  
Circadian Clock ◽  
Scaling Law ◽  
Clock Cycle ◽  
Circadian Clocks ◽  
Day Length ◽  
Mathematical Framework ◽  
Simple Scaling ◽  
Biochemical Oscillator

Circadian rhythms are biological oscillations that schedule daily changes in physiology. Outside the laboratory, circadian clocks do not generally free-run but are driven by daily cues whose timing varies with the seasons. The principles that determine how circadian clocks align to these external cycles are not well understood. Here, we report experimental platforms for driving the cyanobacterial circadian clock both in vivo and in vitro. We find that the phase of the circadian rhythm follows a simple scaling law in light-dark cycles, tracking midday across conditions with variable day length. The core biochemical oscillator comprised of the Kai proteins behaves similarly when driven by metabolic pulses in vitro, indicating that such dynamics are intrinsic to these proteins. We develop a general mathematical framework based on instantaneous transformation of the clock cycle by external cues, which successfully predicts clock behavior under many cycling environments.

Dwindling status of Epimedium Elatum (Morren & Decne) and its geographical distribution in Kashmir Himalaya, India

2018 ◽  
pp. 47-52
Keyword(s):  
Medicinal Plant ◽  
Biological Activities ◽  
Conservation Status ◽  
Natural Populations ◽  
Culture Techniques ◽  
Kashmir Himalayas ◽  
Near Future ◽  
First Time ◽  
Tissue Culture Techniques

Epimedium elatum (Morren & Decne) of family Berberidaceace is a rare perennial medicinal plant, endemic to high altitude forests of Northwestern Himalayas in India. Ethnobotanically, it has been used as an ingredient for treatment of bone-joint disorders, impotence and kidney disorders in Kashmir Himalayas. Phytochemically, it is rich in Epimedin ABC and Icariin; all of these have been demonstrated to possess remarkable biological activities like PDE-5 inhibition (treatment of erectile dysfunction), anticancer, antiosteoporosis antioxidant and antiviral properties. The present investigation reports its traditional usage, comprehensive distribution and conservation status from twenty ecogeographical regions in Kashmir Himalayas, India. The species was reported from Gurez valley for the first time. Numerous threats like excessive grazing, deforestration, habitat fragmentation, tourism encroachment, landslides and excessive exploitation have decreased its natural populations in most of the surveyed habitats. Consequently, its existence may become threatened in near future if timely conservation steps are not taken immediately by concerned stakeholders involved in medicinal plant research. Moreover, use of plant tissue culture techniques is recommended for development of its in vitro propagation protocols. Therefore, introduction of this medicinal plant in botanical gardens, protected sites and development of monitoring programmes are needed for its immediate conservation in Northwestern Himalayas, India.

scholarly journals Aspirin enhances the therapeutic efficacy of cisplatin in oesophageal squamous cell carcinoma by inhibition of putative cancer stem cells

2021 ◽  
Author(s):  
Zhigeng Zou ◽  
Wei Zheng ◽  
Hongjun Fan ◽  
Guodong Deng ◽  
Shih-Hsin Lu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Squamous Cell Carcinoma ◽  
Cancer Stem Cells ◽  
Cell Carcinoma ◽  
Cell Lines ◽  
Squamous Cell ◽  
High Throughput Sequencing ◽  
Combined Treatment ◽  
Oesophageal Squamous Cell Carcinoma

Abstract Background Cancer stem cells (CSCs) are related to the patient’s prognosis, recurrence and therapy resistance in oesophageal squamous cell carcinoma (ESCC). Although increasing evidence suggests that aspirin (acetylsalicylic acid, ASA) could lower the incidence and improve the prognosis of ESCC, the mechanism(s) remains to be fully understood. Methods We investigated the role of ASA in chemotherapy/chemoprevention in human ESCC cell lines and an N-nitrosomethylbenzylamine-induced rat ESCC carcinogenesis model. The effects of combined treatment with ASA/cisplatin on ESCC cell lines were examined in vitro and in vivo. Sphere-forming cells enriched with putative CSCs (pCSCs) were used to investigate the effect of ASA in CSCs. Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) was performed to determine the alterations in chromatin accessibility caused by ASA in ESCC cells. Results ASA inhibits the CSC properties and enhances cisplatin treatment in human ESCC cells. ATAC-seq indicates that ASA treatment results in remarkable epigenetic alterations on chromatin in ESCC cells, especially their pCSCs, through the modification of histone acetylation levels. The epigenetic changes activate Bim expression and promote cell death in CSCs of ESCC. Furthermore, ASA prevents the carcinogenesis of NMBzA-induced ESCC in the rat model. Conclusions ASA could be a potential chemotherapeutic adjuvant and chemopreventive drug for ESCC treatment.

scholarly journals Selection of aptamers against triple negative breast cancer cells using high throughput sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Débora Ferreira ◽  
Joaquim Barbosa ◽  
Diana A. Sousa ◽  
Cátia Silva ◽  
Luís D. R. Melo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
High Throughput ◽  
Triple Negative Breast Cancer ◽  
High Throughput Sequencing ◽  
Triple Negative ◽  
Metastatic Breast ◽  
Target Cells ◽  
Cancer Tissue ◽  
Surface Receptors

AbstractTriple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with $$K_{d}$$ K d values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis.

scholarly journals The Banana Root Endophytome: Differences between Mother Plants and Suckers and Evaluation of Selected Bacteria to Control Fusarium oxysporum f.sp. cubense

2021 ◽  
Vol 7 (3) ◽  
pp. 194
Author(s):  
Carmen Gómez-Lama Cabanás ◽  
Antonio J. Fernández-González ◽  
Martina Cardoni ◽  
Antonio Valverde-Corredor ◽  
Javier López-Cepero ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Canary Islands ◽  
High Throughput Sequencing ◽  
Fungal Communities ◽  
Phenological Stage ◽  
Mother Plants ◽  
Fusarium Wilt Of Banana ◽  
Identification And Characterization

This study aimed to disentangle the structure, composition, and co-occurrence relationships of the banana (cv. Dwarf Cavendish) root endophytome comparing two phenological plant stages: mother plants and suckers. Moreover, a collection of culturable root endophytes (>1000) was also generated from Canary Islands. In vitro antagonism assays against Fusarium oxysporum f.sp. cubense (Foc) races STR4 and TR4 enabled the identification and characterization of potential biocontrol agents (BCA). Eventually, three of them were selected and evaluated against Fusarium wilt of banana (FWB) together with the well-known BCA Pseudomonas simiae PICF7 under controlled conditions. Culturable and non-culturable (high-throughput sequencing) approaches provided concordant information and showed low microbial diversity within the banana root endosphere. Pseudomonas appeared as the dominant genus and seemed to play an important role in the banana root endophytic microbiome according to co-occurrence networks. Fungal communities were dominated by the genera Ophioceras, Cyphellophora, Plecosphaerella, and Fusarium. Overall, significant differences were found between mother plants and suckers, suggesting that the phenological stage determines the recruitment and organization of the endophytic microbiome. While selected native banana endophytes showed clear antagonism against Foc strains, their biocontrol performance against FWB did not improve the outcome observed for a non-indigenous reference BCA (strain PICF7).

scholarly journals A Review on the Methods Used for the Detection and Diagnosis of Rabbit Hemorrhagic Disease Virus (RHDV)

2021 ◽  
Vol 9 (5) ◽  
pp. 972
Author(s):  
Joana Abrantes ◽  
Ana M. Lopes
Keyword(s):  
Disease Virus ◽  
Diagnostic Tests ◽  
High Throughput Sequencing ◽  
Current Knowledge ◽  
European Rabbit ◽  
Rabbit Hemorrhagic Disease Virus ◽  
Hemorrhagic Disease ◽  
Rabbit Hemorrhagic Disease ◽  
Detection And Diagnosis

Since the early 1980s, the European rabbit (Oryctolagus cuniculus) has been threatened by the rabbit hemorrhagic disease (RHD). The disease is caused by a lagovirus of the family Caliciviridae, the rabbit hemorrhagic disease virus (RHDV). The need for detection, identification and further characterization of RHDV led to the development of several diagnostic tests. Owing to the lack of an appropriate cell culture system for in vitro propagation of the virus, much of the methods involved in these tests contributed to our current knowledge on RHD and RHDV and to the development of vaccines to contain the disease. Here, we provide a comprehensive review of the RHDV diagnostic tests used since the first RHD outbreak and that include molecular, histological and serological techniques, ranging from simpler tests initially used, such as the hemagglutination test, to the more recent and sophisticated high-throughput sequencing, along with an overview of their potential and their limitations.

scholarly journals EJE Prize 2013: Regulation of aldosterone secretion: from physiology to disease

2013 ◽  
Vol 168 (6) ◽  
pp. R85-R93 ◽  
Author(s):  
Felix Beuschlein
Keyword(s):  
High Throughput Sequencing ◽  
Secondary Hypertension ◽  
Small Sample ◽  
Cellular Systems ◽  
Common Disease ◽  
Aldosterone Secretion ◽  
Technical Developments ◽  
Starting Point

Arterial hypertension is a major cardiovascular risk factor that affects between 10 and 40% of the population in industrialized countries. Primary aldosteronism (PA) is the most common form of secondary hypertension with an estimated prevalence of around 10% in referral centers and 4% in a primary care setting. Despite its high prevalence until recently, the underlying genetic and molecular basis of this common disease had remained largely obscure. Over the past decade, a number of insights have been achieved that have relied onin vitrocellular systems, wild-type and genetically modifiedin vivomodels, as well as clinical studies in well-characterized patient populations. This progress has been made possible by a number of independent technical developments including that of specific hormone assays that allow measurement in small sample volumes as well as genetic techniques that enable high-throughput sequencing of a large number of samples. Furthermore, animal models have provided important insights into the physiology of aldosterone regulation that have served as a starting point for investigation of mechanisms involved in autonomous aldosterone secretion. Finally, national and international networks that have built up registries and biobanks have been instrumental in fostering translational research endeavors in PA. Therefore, it is to be expected that in the near future, further pathophysiological mechanisms that result in autonomous aldosterone secretion will be unraveled.

scholarly journals Evolutionary paths to macrolide resistance in a Neisseria commensal converge on ribosomal genes through short sequence duplications

PLoS ONE ◽  
2022 ◽  
Vol 17 (1) ◽  
pp. e0262370
Author(s):  
Jordan C. Raisman ◽  
Michael A. Fiore ◽  
Lucille Tomin ◽  
Joseph K. O. Adjei ◽  
Virginia X. Aswad ◽  
...  
Keyword(s):  
Tandem Duplication ◽  
Natural Populations ◽  
Growth Defect ◽  
Duplication Events ◽  
Pathogen Populations ◽  
High Level ◽  
Evolutionary Paths ◽  
Azithromycin Resistance ◽  
Short Tandem

Neisseria commensals are an indisputable source of resistance for their pathogenic relatives. However, the evolutionary paths commensal species take to reduced susceptibility in this genus have been relatively underexplored. Here, we leverage in vitro selection as a powerful screen to identify the genetic adaptations that produce azithromycin resistance (≥ 2 μg/mL) in the Neisseria commensal, N. elongata. Across multiple lineages (n = 7/16), we find mutations that reduce susceptibility to azithromycin converge on the locus encoding the 50S ribosomal L34 protein (rpmH) and the intergenic region proximal to the 30S ribosomal S3 protein (rpsC) through short tandem duplication events. Interestingly, one of the laboratory evolved mutations in rpmH is identical (7LKRTYQ12), and two nearly identical, to those recently reported to contribute to high-level azithromycin resistance in N. gonorrhoeae. Transformations into the ancestral N. elongata lineage confirmed the causality of both rpmH and rpsC mutations. Though most lineages inheriting duplications suffered in vitro fitness costs, one variant showed no growth defect, suggesting the possibility that it may be sustained in natural populations. Ultimately, studies like this will be critical for predicting commensal alleles that could rapidly disseminate into pathogen populations via allelic exchange across recombinogenic microbial genera.

scholarly journals CDK10 Regulates Gastric Cancer Metastasis By Inhibiting lncRNA-C5ORF42-5 Via Phosphorylation Level of AKT/ERK

2021 ◽  
Author(s):  
Zi-Jian Deng ◽  
Dong-Wen Chen ◽  
Xi-Jie Chen ◽  
Jia-Ming Fang ◽  
Liang Xv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
High Throughput ◽  
Cancer Metastasis ◽  
High Throughput Sequencing ◽  
Gastric Cancer Cells ◽  
Related Proteins

Abstract Background: Gastric cancer is the fourth most common malignant disease. Both CDK10 and long noncoding RNAs (lncRNAs) have been found to exert biological functions in multiple cancers. However, it is still unclear whether CDK10 represses tumor progression in gastric cancer by reducing potential targeting lncRNAs.Methods: The functions of CDK10 and lncRNA-C5ORF42-5 in proliferation, invasion and migration were assessed by MTS assays, colony formation assays, cell cycle and apoptosis assays, Transwell assays, wound healing assays and animal experiments. We used high-throughput sequencing to confirm the existence of lncRNA-C5ORF42-5 and quantitative real-time PCR was used to evaluate lncRNA expression. Then, with RNA-seq sequencing as well as GO function and KEGG enrichment analysis, we identified the signaling pathways in which lncRNA-C5ORF42-5 was involved in gastric cancer. Finally, western blotting was used to identify the genes regulated by lncRNA-C5ORF42-5.Results: Our results showed that CDK10 is expressed at relatively low levels in gastric cancer cell lines and inhibits the progression of gastric cancer cells both in vitro and in vivo. Next, based on high-throughput sequencing, we identified a novel lncRNA, lncRNA-C5ORF42-5, in the stable CDK10-overexpressing cell line compared with the CDK-knockdown cell line and their controls. Additionally, we confirmed that lncRNA-C5ORF42-5 acts as an oncogene to promote metastasis in gastric cancer in vitro and in vivo. We then ascertained that lncRNA-C5ORF42-5 is a major contributor to the function of CDK10 in gastric cancer metastasis by upregulating lncRNA-C5ORF42-5 to reverse the effects of CDK10 overexpression. Finally, we explored the mechanism by which lncRNA-C5ORF42-5 overexpression affects gastric cancer cells to elucidate whether lncRNA-C5ORF42-5 may increase the activity of the SMAD pathway of BMP signaling and promote the expression of EMT-related proteins, such as E-cadherin. Additionally, overexpression of lncRNA-C5ORF42-5 affected the phosphorylation levels of AKT and ERK.Conclusion: Our findings suggest that CDK10 overexpression represses gastric cancer tumor progression by reducing lncRNA-C5ORF42-5 and hindering activation of the related proteins in metastatic signaling pathways, which provides new insight into developing effective therapeutic strategies in the treatment of metastatic gastric cancer.

scholarly journals Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering

2017 ◽  
Author(s):  
Sean A. Higgins ◽  
Sorel Ouonkap ◽  
David F. Savage
Keyword(s):  
Structure Function ◽  
Sequence Space ◽  
Protein Function ◽  
Fluorescent Protein ◽  
One Pot ◽  
Protein Mutagenesis ◽  
Protein Mutants ◽  
Protein Libraries

ABSTRACTComprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. However, current techniques enabling comprehensive protein mutagenesis are based on PCR and require in vitro reactions involving specialized protocols and reagents. This has complicated efforts to rapidly and reliably produce desired comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and unbiased, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple one-pot protocol.

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