scholarly journals Retrieval of the Complete Coding Sequence of the UK-Endemic Tatenale Orthohantavirus Reveals Extensive Strain Variation and Supports its Classification as a Novel Species

2019 ◽  
Author(s):  
Joseph G. Chappell ◽  
Theocharis Tsoleridis ◽  
Okechukwu Onianwa ◽  
Gabby Drake ◽  
Ian Ashpole ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Novel Species ◽  
Accurate Analysis ◽  
Coding Sequence ◽  
Evolutionary Origins ◽  
Pcr Product ◽  
Pcr Method ◽  
The Uk ◽  
Potential Pathogenicity

AbstractHantaviruses are a diverse group of single-stranded, negative-sensed RNA viruses, known to cause sporadic outbreaks of potentially fatal human disease. To date, only two Orthohantavirus species have been detected in the UK - Seoul virus and Tatenale. Whilst Seoul is known to be pathogenic in humans, only partial fragments of Tatenale have been recovered, precluding any accurate analysis of its phylogeny or potential pathogenicity. To overcome this shortfall we used a degenerate primer PCR method to identify Tatenale-infection in rodents living in two separate locations in the UK. PCR positive samples were then subjected to either unbiased high-throughput sequencing or overlapping PCR product sequencing to recover the complete coding sequence of the Tatenale virus. This analysis provided in-depth insight into the evolutionary origins of this recently identified UK Orthohantavirus and unequivocally showed that Tatenale virus meets the established criteria for classification as a novel species. Crucially, our data will facilitate in vitro investigation into the zoonotic potential of Tatenale virus.

scholarly journals Retrieval of the Complete Coding Sequence of the UK-Endemic Tatenale Orthohantavirus Reveals Extensive Strain Variation and Supports Its Classification as a Novel Species

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 454 ◽  
Author(s):  
Joseph G. Chappell ◽  
Theocharis Tsoleridis ◽  
Okechukwu Onianwa ◽  
Gabby Drake ◽  
Ian Ashpole ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Novel Species ◽  
Single Species ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Coding Sequence ◽  
The United Kingdom ◽  
Single Field ◽  
The Uk ◽  
Globally Distributed

Orthohantaviruses are globally distributed viruses, associated with rodents and other small mammals. However, data on the circulation of orthohantaviruses within the UK, particularly the UK-endemic Tatenale virus, is sparse. In this study, 531 animals from five rodent species were collected from two locations in northern and central England and screened using a degenerate, pan- orthohantavirus RT-PCR assay. Tatenale virus was detected in a single field vole (Microtus agrestis) from central England and twelve field voles from northern England. Unbiased high-throughput sequencing of the central English strain resulted in the recovery of the complete coding sequence of a novel strain of Tatenale virus, whilst PCR-primer walking of the northern English strain recovered almost complete coding sequence of a previously identified strain. These findings represented the detection of a third lineage of Tatenale virus in the United Kingdom and extended the known geographic distribution of these viruses from northern to central England. Furthermore, the recovery of the complete coding sequence revealed that Tatenale virus was sufficiently related to the recently identified Traemersee virus, to meet the accepted criteria for classification as a single species of orthohantavirus.

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 Multiple Types of Novel Enteric Bopiviruses (Picornaviridae) with the Possibility of Interspecies Transmission Identified from Cloven-Hoofed Domestic Livestock (Ovine, Caprine and Bovine) in Hungary

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 66
Author(s):  
Zoltán László ◽  
Péter Pankovics ◽  
Gábor Reuter ◽  
Attila Cságola ◽  
Ádám Bálint ◽  
...  
Keyword(s):  
Novel Species ◽  
Phylogenetic Analyses ◽  
Interspecies Transmission ◽  
Background Information ◽  
Type Ii ◽  
Rt Pcr ◽  
Faecal Samples ◽  
The Family ◽  
Domestic Livestock

Most picornaviruses of the family Picornaviridae are relatively well known, but there are certain “neglected” genera like Bopivirus, containing a single uncharacterised sequence (bopivirus A1, KM589358) with very limited background information. In this study, three novel picornaviruses provisionally called ovipi-, gopi- and bopivirus/Hun (MW298057-MW298059) from enteric samples of asymptomatic ovine, caprine and bovine respectively, were determined using RT-PCR and dye-terminator sequencing techniques. These monophyletic viruses share the same type II-like IRES, NPGP-type 2A, similar genome layout (4-3-4) and cre-localisations. Culture attempts of the study viruses, using six different cell lines, yielded no evidence of viral growth in vitro. Genomic and phylogenetic analyses show that bopivirus/Hun of bovine belongs to the species Bopivirus A, while the closely related ovine-origin ovipi- and caprine-origin gopivirus could belong to a novel species “Bopivirus B” in the genus Bopivirus. Epidemiological investigation of N = 269 faecal samples of livestock (ovine, caprine, bovine, swine and rabbit) from different farms in Hungary showed that bopiviruses were most prevalent among <12-month-old ovine, caprine and bovine, but undetectable in swine and rabbit. VP1 capsid-based phylogenetic analyses revealed the presence of multiple lineages/genotypes, including closely related ovine/caprine strains, suggesting the possibility of ovine–caprine interspecies transmission of certain bopiviruses.

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 Establishment of Highly Efficient Agrobacterium Rhizogenes-mediated Transformation for Stevia Rebaudiana Bertoni Explants

2016 ◽  
Vol 58 (1) ◽  
pp. 113-118 ◽  
Author(s):  
Żaneta Michalec-Warzecha ◽  
Laura Pistelli ◽  
Francesca D’Angiolillo ◽  
Marta Libik-Konieczny
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Roots ◽  
Molecular Level ◽  
Stevia Rebaudiana ◽  
Culture Conditions ◽  
Light Conditions ◽  
Inoculum Concentration ◽  
Stevia Rebaudiana Bertoni ◽  
Pcr Method

Abstract Leaves and internodes from Stevia rebaudiana Bertoni plants growing in different conditions were used for transformation with two strains of Agrobacterium rhizogenes: ATCC 15384 and LBA 9402. Hairy roots formation was observed and the percentage of the transformed explants depended on the type of explant, time of inoculation and inoculum concentration. Inoculation of explants from ex vitro and in vitro plants with LBA 9402 strain led to higher efficiency of transformation than inoculation with ATCC 15384 strain. Growth rate of hairy roots in liquid culture was assessed under light and dark conditions. It was found that the growth of hairy roots decreased significantly under light conditions. Transformation of hairy roots growing in different culture conditions was confirmed at the molecular level using PCR method with primers constructed against rolB and rolC genes from A. rhizogenes.

scholarly journals C-Repeat Binding Factor and Dehydrin Genes are Induced Co-Ordinately in Drought Tolerance Response of Wheat Cultivars

2017 ◽  
Vol 9 (2) ◽  
pp. 18 ◽  
Author(s):  
Csilla Deák ◽  
Katalin Jäger ◽  
Veronika Anna Nagy ◽  
Réka Oszlányi ◽  
Beáta Barnabás ◽  
...  
Keyword(s):  
Drought Tolerance ◽  
Mrna Level ◽  
Drought Treatment ◽  
Drought Tolerant ◽  
Aba Sensitivity ◽  
Defence Genes ◽  
Binding Factor ◽  
Tolerance Response ◽  
Pcr Method

Four bread wheat genotypes with contrasting drought stress tolerance were studied. Expression levels of dehydrin (Wdhn13) and C-repeat binding factor (Cbf14, Cbf15) genes were investigated in leaves of two drought tolerant (Plainsman V, Mv Emese) and two sensitive (GK Élet, Cappelle Desprez) cultivars by semi-quantitative RT-PCR during drought treatment at anthesis. Coordinate induction of Cbf14, Cbf15 and Wdhn13 genes occurred at a late stage of stress treatment in all cultivars except the most sensitive Cappelle Desprez, where no induction was evident. The most pronounced late induction of genes was observed in the tolerant Mv Emese genotype. Cbf14, Cbf15 and Wdhn13 showed largely parallel changes of expression in stressed adult plants. The mRNA level of the same set of genes was measured in leaves of non-stressed seedlings with qRT-PCR method. Expression level of Wdhn13 was high and low in seedlings of tolerant and sensitive cultivars, respectively. Cbf15 specific transcript was barely detectable in leaves of non-stressed seedlings. In order to shed light on any potential difference in hormone responsiveness, seedlings were subjected to ABA treatment in vitro. At low hormone concentrations (10 and 20 µM ABA) consistently weaker ABA induced root growth retardation of GK Élet was found in comparison with the other three cultivars. Results highlight pronounced and late induction of a set of defence genes and low ABA sensitivity as features appearing in drought tolerant and sensitive responses, respectively. Data is discussed in the light of multifactorial determination of the complex phenotype of drought tolerance in wheat.

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).

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