scholarly journals Development and Application of a Quantitative PCR Detection Method to Quantify Venturia oleaginea in Asymptomatic Olive (Olea europaea) Leaves

2020 ◽  
Vol 110 (3) ◽  
pp. 547-555
Author(s):  
Silvia Scibetta ◽  
Giovanni E. Agosteo ◽  
Ahmed Abdelfattah ◽  
Maria G. Li Destri Nicosia ◽  
Santa O. Cacciola ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
High Throughput Sequencing ◽  
Current Knowledge ◽  
Leaf Growth ◽  
Its Region ◽  
Qpcr Assay ◽  
Detection Methods ◽  
Effective Control ◽  
Its2 Region

Olive leaf spot (OLS), caused by Venturia oleaginea, is one of the most common and serious diseases of olive trees in the Mediterranean region. Understanding the pathogen life cycle is important for the development of effective control strategies. Current knowledge is incomplete owing to a lack of effective detection methods. It is extremely difficult to culture V. oleaginea in vitro, so primers were designed to amplify and sequence the internal transcribed spacer ITS1-5.8S-ITS2 region of the fungus directly from infected olive leaves. Sanger sequencing indicated a unique ITS region present in the European strains screened, confirming the appropriateness of the target region for developing a quantitative PCR (qPCR) assay. Furthermore, high-throughput sequencing of the same region excluded the presence of other Venturia species in the olive phyllosphere. The qPCR assay proved very specific and sensitive, enabling the detection of approximately 26 copies of target DNA. The analysis of symptomless leaves during early stages of the epidemic from the end of winter through spring revealed a similar quantity of pathogen DNA regardless of the leaf growth stage. In contrast, the pathogen titer changed significantly during the season. Data indicated that leaf infections start earlier than expected over the season and very young leaves are as susceptible as adult leaves. These findings have important practical implications and suggest the need for improved scheduling of fungicide treatments. The qPCR assay represents a valuable tool providing quantitative results and enables detection of V. oleaginea in all olive organs, including those in which OLS cannot be studied using previously available methods.

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.

Fruit Volatiles to Control Postharvest Rot of Stone Fruits and Pears

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 469a-469
Author(s):  
L.J. Skog ◽  
D.P. Murr ◽  
B.E. Digweed
Keyword(s):  
Volatile Compounds ◽  
Isoamyl Acetate ◽  
Penicillium Expansum ◽  
Effective Control ◽  
In Vitro Tests ◽  
Stone Fruits ◽  
In Situ Tests ◽  
Highly Effective ◽  
Postharvest Rot

Volatile compounds are ubiquitous in plants, giving fruits their characteristic aroma and flavor. There is increasing evidence that these compounds can protect plants from pathogenic organisms. In this trial ≈25 volatile compounds were tested for efficacy against Monilinia fructicola and Penicillium expansum. Both in vitro tests on agar plugs of actively growing pathogens and in situ tests on inoculated stone fruits and pears were conducted. The volatile compounds were grouped into three categories based upon fungicidal activity in vitro: highly effective (fungicidal concentration ≤100 M), moderately effective (fungicidal concentration between 100–200 M) and ineffective (fungicidal concentration >200 M). Highly effective compounds included: acetaldehyde, citral, 2-ethyl-1-hexanol, 2,exadienal, E-2-hexenal, 4-hexen-3-one, linalool, (E,E)2,4-nonadienal, E-2-nonenal, E-3-none-2-one, salicylaldehyde, and valeraldehyde. Moderately effective compounds included: (E,Z) 2,6-nonadienal, propionaldehyde, terpinene, butyl acetate, E-cinnamaldehde, hexanal, E-2-hexen-1-ol, Z-3-hexen-1-ol and isoamyl acetate. Ineffective compounds included: butyrolactone, ethanol, ethyl acetate, and methyl acetate. Effectiveness of the compounds varied with both strain and type of microorganism tested. Concentraions required for effective control were much higher when the compounds were tested on inoculated fruit. Phytotoxicity was a problem with some compounds.

Modulation of Matrix Metalloproteinases by Plant-Derived Products

2020 ◽  
Vol 20 ◽  
Author(s):  
Nur Najmi Mohamad Anuar ◽  
Nurul Iman Natasya Zulkafali ◽  
Azizah Ugusman
Keyword(s):  
Clinical Trials ◽  
Natural Products ◽  
Matrix Metalloproteinases ◽  
Animal Studies ◽  
Current Knowledge ◽  
In Vitro Models ◽  
In Vivo Studies ◽  
Extracellular Matrix Components

: Matrix metalloproteinases (MMPs) are a group of zinc-dependent metallo-endopeptidase that are responsible towards the degradation, repair and remodelling of extracellular matrix components. MMPs play an important role in maintaining a normal physiological function and preventing diseases such as cancer and cardiovascular diseases. Natural products derived from plants have been used as traditional medicine for centuries. Its active compounds, such as catechin, resveratrol and quercetin, are suggested to play an important role as MMPs inhibitors, thereby opening new insights into their applications in many fields, such as pharmaceutical, cosmetic and food industries. This review summarises the current knowledge on plant-derived natural products with MMP-modulating activities. Most of the reviewed plant-derived products exhibit an inhibitory activity on MMPs. Amongst MMPs, MMP-2 and MMP-9 are the most studied. The expression of MMPs is inhibited through respective signalling pathways, such as MAPK, NF-κB and PI3 kinase pathways, which contribute to the reduction in cancer cell behaviours, such as proliferation and migration. Most studies have employed in vitro models, but a limited number of animal studies and clinical trials have been conducted. Even though plant-derived products show promising results in modulating MMPs, more in vivo studies and clinical trials are needed to support their therapeutic applications in the future.

Bone Marrow Niches for Skeletal Progenitor Cells and their Inhabitants in Health and Disease

2019 ◽  
Vol 14 (4) ◽  
pp. 305-319 ◽  
Author(s):  
Marietta Herrmann ◽  
Franz Jakob
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Adult Stem Cells ◽  
Current Knowledge ◽  
Cell Populations ◽  
Surface Markers ◽  
Bone Marrow Niches

The bone marrow hosts skeletal progenitor cells which have most widely been referred to as Mesenchymal Stem or Stromal Cells (MSCs), a heterogeneous population of adult stem cells possessing the potential for self-renewal and multilineage differentiation. A consensus agreement on minimal criteria has been suggested to define MSCs in vitro, including adhesion to plastic, expression of typical surface markers and the ability to differentiate towards the adipogenic, osteogenic and chondrogenic lineages but they are critically discussed since the differentiation capability of cells could not always be confirmed by stringent assays in vivo. However, these in vitro characteristics have led to the notion that progenitor cell populations, similar to MSCs in bone marrow, reside in various tissues. MSCs are in the focus of numerous (pre)clinical studies on tissue regeneration and repair.Recent advances in terms of genetic animal models enabled a couple of studies targeting skeletal progenitor cells in vivo. Accordingly, different skeletal progenitor cell populations could be identified by the expression of surface markers including nestin and leptin receptor. While there are still issues with the identity of, and the overlap between different cell populations, these studies suggested that specific microenvironments, referred to as niches, host and maintain skeletal progenitor cells in the bone marrow. Dynamic mutual interactions through biological and physical cues between niche constituting cells and niche inhabitants control dormancy, symmetric and asymmetric cell division and lineage commitment. Niche constituting cells, inhabitant cells and their extracellular matrix are subject to influences of aging and disease e.g. via cellular modulators. Protective niches can be hijacked and abused by metastasizing tumor cells, and may even be adapted via mutual education. Here, we summarize the current knowledge on bone marrow skeletal progenitor cell niches in physiology and pathophysiology. We discuss the plasticity and dynamics of bone marrow niches as well as future perspectives of targeting niches for therapeutic strategies.

Subnuclear compartmentalization of sequence-specific transcription factors and regulation of eukaryotic gene expression

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Current Knowledge ◽  
Nuclear Architecture ◽  
Subnuclear Localization ◽  
Modular Structures

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.

scholarly journals Pan-American Lancehead Pit-Vipers: Coagulotoxic Venom Effects and Antivenom Neutralisation of Bothrops asper and B. atrox Geographical Variants

Toxins ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 78
Author(s):  
Lachlan A. Bourke ◽  
Christina N. Zdenek ◽  
Edgar Neri-Castro ◽  
Melisa Bénard-Valle ◽  
Alejandro Alagón ◽  
...  
Keyword(s):  
Evolutionary Biology ◽  
Current Knowledge ◽  
Clinical Manifestations ◽  
In Vivo Studies ◽  
Factor X ◽  
Clotting Factors ◽  
Bothrops Asper ◽  
Species Specific

The toxin composition of snake venoms and, thus, their functional activity, can vary between and within species. Intraspecific venom variation across a species’ geographic range is a major concern for antivenom treatment of envenomations, particularly for countries like French Guiana that lack a locally produced antivenom. Bothrops asper and Bothrops atrox are the most medically significant species of snakes in Latin America, both producing a variety of clinical manifestations, including systemic bleeding. These pathophysiological actions are due to the activation by the venom of the blood clotting factors Factor X and prothrombin, thereby causing severe consumptive coagulopathy. Both species are extremely wide-ranging, and previous studies have shown their venoms to exhibit regional venom variation. In this study, we investigate the differential coagulotoxic effects on human plasma of six venoms (four B. asper and two B. atrox samples) from different geographic locations, spanning from Mexico to Peru. We assessed how the venom variation of these venom samples affects neutralisation by five regionally available antivenoms: Antivipmyn, Antivipmyn-Tri, PoliVal-ICP, Bothrofav, and Soro Antibotrópico (SAB). The results revealed both inter- and intraspecific variations in the clotting activity of the venoms. These variations in turn resulted in significant variation in antivenom efficacy against the coagulotoxic effects of these venoms. Due to variations in the venoms used in the antivenom production process, antivenoms differed in their species-specific or geographical neutralisation capacity. Some antivenoms (PoliVal-ICP, Bothrofav, and SAB) showed species-specific patterns of neutralisation, while another antivenom (Antivipmyn) showed geographic-specific patterns of neutralisation. This study adds to current knowledge of Bothrops venoms and also illustrates the importance of considering evolutionary biology when developing antivenoms. Therefore, these results have tangible, real-world implications by aiding evidence-based design of antivenoms for treatment of the envenomed patient. We stress that these in vitro studies must be backed by future in vivo studies and clinical trials before therapeutic guidelines are issued regarding specific antivenom use in a clinical setting.

scholarly journals Seminal Plasma: Relevant for Fertility?

2021 ◽  
Vol 22 (9) ◽  
pp. 4368
Author(s):  
Heriberto Rodriguez-Martinez ◽  
Emilio A. Martinez ◽  
Juan J. Calvete ◽  
Fernando J. Peña Vega ◽  
Jordi Roca
Keyword(s):  
Seminal Plasma ◽  
Current Knowledge ◽  
Inorganic Ions ◽  
Vitro Fertilization ◽  
Dna And Rna ◽  
Model Animal ◽  
Sexual Glands ◽  
Species Specific

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA—the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.

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).

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