ResearchPad - Recent Articles https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[An automated aquatic rack system for rearing marine invertebrates]]> https://www.researchpad.co/article/elastic_article_12087 One hundred years ago, marine organisms were the dominant systems for the study of developmental biology. The challenges in rearing these organisms outside of a marine setting ultimately contributed to a shift towards work on a smaller number of so-called model systems. Those animals are typically non-marine organisms with advantages afforded by short life cycles, high fecundity, and relative ease in laboratory culture. However, a full understanding of biodiversity, evolution, and anthropogenic effects on biological systems requires a broader survey of development in the animal kingdom. To this day, marine organisms remain relatively understudied, particularly the members of the Lophotrochozoa (Spiralia), which include well over one third of the metazoan phyla (such as the annelids, mollusks, flatworms) and exhibit a tremendous diversity of body plans and developmental modes. To facilitate studies of this group, we have previously described the development and culture of one lophotrochozoan representative, the slipper snail Crepidula atrasolea, which is easy to rear in recirculating marine aquaria. Lab-based culture and rearing of larger populations of animals remain a general challenge for many marine organisms, particularly for inland laboratories.ResultsHere, we describe the development of an automated marine aquatic rack system for the high-density culture of marine species, which is particularly well suited for rearing filter-feeding animals. Based on existing freshwater recirculating aquatic rack systems, our system is specific to the needs of marine organisms and incorporates robust filtration measures to eliminate wastes, reducing the need for regular water changes. In addition, this system incorporates sensors and associated equipment for automated assessment and adjustment of water quality. An automated feeding system permits precise delivery of liquid food (e.g., phytoplankton) throughout the day, mimicking real-life feeding conditions that contribute to increased growth rates and fecundity.ConclusionThis automated system makes laboratory culture of marine animals feasible for both large and small research groups, significantly reducing the time, labor, and overall costs needed to rear these organisms. ]]> <![CDATA[NADPH Oxidase RbohD and Ethylene Signaling are Involved in Modulating Seedling Growth and Survival Under Submergence Stress]]> https://www.researchpad.co/article/elastic_article_12086 In higher plants under low oxygen or hypoxic conditions, the phytohormone ethylene and hydrogen peroxide (H2O2) are involved in complex regulatory mechanisms in hypoxia signaling pathways. The respiratory burst oxidase homolog D (RbohD), an NADPH oxidase, is involved in the primary stages of hypoxia signaling, modulating the expression of downstream hypoxia-inducible genes under hypoxic stress. In this study, our data revealed that under normoxic conditions, seed germination was delayed in the rbohD/ein2-5 double mutant, whereas postgermination stage root growth was promoted. Under submergence, the rbohD/ein2-5 double mutant line had an inhibited root growth phenotype. Furthermore, chlorophyll content and leaf survival were reduced in the rbohD/ein2-5 double mutant compared with wild-type plants under submerged conditions. In quantitative RT-PCR analysis, the induction of Ethylene-responsive factor 73/hypoxia responsive 1 (AtERF73/HRE1) and alcohol dehydrogenase 1 (AtADH1) transcripts was lower in the rbohD/ein2-5 double mutant during hypoxic stress than in wild-type plants and in rbohD and ein2-5 mutant lines. Taken together, our results indicate that an interplay of ethylene and RbohD is involved in regulating seed germination and post-germination stages under normoxic conditions. Moreover, ethylene and RbohD are involved in modulating seedling root growth, leaf chlorophyll content, and hypoxia-inducible gene expression under hypoxic conditions.

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<![CDATA[Validation of a Preformulated, Field Deployable, Recombinase Polymerase Amplification Assay for <i>Phytophthora</i> Species]]> https://www.researchpad.co/article/elastic_article_12085 Recombinase polymerase amplification (RPA) assays are valuable molecular diagnostic tools that can detect and identify plant pathogens in the field without time-consuming DNA extractions. Historically, RPA assay reagents were commercially available as a lyophilized pellet in microfuge strip tubes, but have become available in liquid form more recently—both require the addition of primers and probes prior to use, which can be challenging to handle in a field setting. Lyophilization of primers and probes, along with RPA reagents, contained within a single tube limits the risk of contamination, eliminates the need for refrigeration, as the lyophilized reagents are stable at ambient temperatures, and simplifies field use of the assays. This study investigates the potential effect of preformulation on assay performance using a previously validated Phytophthora genus-specific RPA assay, lyophilized with primers and probes included with the RPA reagents. The preformulated lyophilized Phytophthora RPA assay was compared with a quantitative polymerase chain reaction (qPCR) assay and commercially available RPA kits using three qPCR platforms (BioRad CFX96, QuantStudio 6 and Applied Biosystems ViiA7) and one isothermal platform (Axxin T16-ISO RPA), with experiments run in four separate labs. The assay was tested for sensitivity (ranging from 500 to 0.33 pg of DNA) and specificity using purified oomycete DNA, as well as crude extracts of Phytophthora-infected and non-infected plants. The limit of detection (LOD) using purified DNA was 33 pg in the CFX96 and ViiA7 qPCR platforms using the preformulated kits, while the Axxin T16-ISO RPA chamber and the QuantStudio 6 platform could detect down to 3.3 pg with or without added plant extract. The LOD using a crude plant extract for the BioRad CFX96 was 330 pg, whereas the LOD for the ViiA7 system was 33 pg. These trials demonstrate the consistency and uniformity of pathogen detection with preformulated RPA kits for Phytophthora detection when conducted by different labs using different instruments for measuring results.

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<![CDATA[Correction to: Indirect choroidal neovascularization secondary to a posterior-segment intraocular foreign body – case report]]> https://www.researchpad.co/article/elastic_article_12084 <![CDATA[A Systematic Review of Studies Published between 2016 and 2019 on the Effectiveness and Efficacy of Pneumococcal Vaccination on Pneumonia and Invasive Pneumococcal Disease in an Elderly Population]]> https://www.researchpad.co/article/elastic_article_12083 Adult vaccination is high on the agenda in many countries. Two different vaccines are available for the prevention of pneumococcal disease in adults: a 23-valent polysaccharide vaccine (PPV23), and a 13-valent conjugated vaccine (PCV13). The objective of this review is to update the evidence base for vaccine efficacy and effectiveness of PPV23 and PCV13 against invasive pneumococcal disease and pneumonia among an unselected elderly population. We systematically searched for clinical trials and observational studies published between January 1 2016 and April 17 2019 in Pubmed, Embase, Cinahl, Web of Science, Epistemonikos and Cochrane databases. Risk of bias was assessed using Cochrane Risk of Bias tool for and the Newcastle–Ottawa Scale. Results were stratified by vaccine type and outcome. We identified nine studies on PCV13 and six on PPV23. No new randomized clinical trials were identified. Due to different outcomes, it was not possible to do a meta-analysis. New high-quality observational studies indicate protective vaccine effectiveness for both vaccines against vaccine type pneumonia. Our estimates for the protective vaccine efficacy and effectiveness (VE) of PPV23 on pneumonia and pneumococcal pneumonia overlap with results from previously published reviews. Some of the results indicate that the effectiveness of the PPV23 is best in younger age groups, and that it decreases over time.

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<![CDATA[Prediction model construction of mouse stem cell pluripotency using CpG and non-CpG DNA methylation markers]]> https://www.researchpad.co/article/elastic_article_12082 Genome-wide studies of DNA methylation across the epigenetic landscape provide insights into the heterogeneity of pluripotent embryonic stem cells (ESCs). Differentiating into embryonic somatic and germ cells, ESCs exhibit varying degrees of pluripotency, and epigenetic changes occurring in this process have emerged as important factors explaining stem cell pluripotency.ResultsHere, using paired scBS-seq and scRNA-seq data of mice, we constructed a machine learning model that predicts degrees of pluripotency for mouse ESCs. Since the biological activities of non-CpG markers have yet to be clarified, we tested the predictive power of CpG and non-CpG markers, as well as a combination thereof, in the model. Through rigorous performance evaluation with both internal and external validation, we discovered that a model using both CpG and non-CpG markers predicted the pluripotency of ESCs with the highest prediction performance (0.956 AUC, external test). The prediction model consisted of 16 CpG and 33 non-CpG markers. The CpG and most of the non-CpG markers targeted depletions of methylation and were indicative of cell pluripotency, whereas only a few non-CpG markers reflected accumulations of methylation. Additionally, we confirmed that there exists the differing pluripotency between individual developmental stages, such as E3.5 and E6.5, as well as between induced mouse pluripotent stem cell (iPSC) and somatic cell.ConclusionsIn this study, we investigated CpG and non-CpG methylation in relation to mouse stem cell pluripotency and developed a model thereon that successfully predicts the pluripotency of mouse ESCs. ]]> <![CDATA[Rapid Spread of Classical Swine Fever Virus among South Korean Wild Boars in Areas near the Border with North Korea]]> https://www.researchpad.co/article/elastic_article_12080 There has been a rapid increase in the number of classical swine fever (CSF) sero-positive wild boars captured near the demilitarized zone (DMZ), located the border with North Korea. In 2015–2016, few CSFV-positive antibody boars were detected; however, the number has increased steeply since 2017. Most occurred in the northern region of Gyeonggi before spreading slowly to Gangwon (west to east) in 2018–2019. Multi-distance spatial cluster analysis provided an indirect estimate of the time taken for CSFV to spread among wild boars: 46.7, 2.6, and 2.49 days/km. The average CSF serum neutralization antibody titer was 4–10 (log 2), and CSFV Ab B-ELISA PI values ranged from 65.5 to 111.5, regardless of the age and sex of wild boars. Full genome analysis revealed that 16 CSFV strains isolated from wild boars between 2017 and 2019 were identical to the YC16CS strain (sub-genotype 2.1d) isolated from an outbreak in breeding pigs near the border with North Korea in 2016. The rapid increase in CSF in wild boars may be due to a continuously circulating infection within hub area and increased population density. The distribution pattern of CSFV in Korean wild boars moves from west to southeast, affected by external factors, including small-scale hunting, geographical features and highways.

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<![CDATA[The Complex Relationship between HTLV-1 and Nonsense-Mediated mRNA Decay (NMD)]]> https://www.researchpad.co/article/elastic_article_12079 Before the establishment of an adaptive immune response, retroviruses can be targeted by several cellular host factors at different stages of the viral replication cycle. This intrinsic immunity relies on a large diversity of antiviral processes. In the case of HTLV-1 infection, these active innate host defense mechanisms are debated. Among these mechanisms, we focused on an RNA decay pathway called nonsense-mediated mRNA decay (NMD), which can target multiple viral RNAs, including HTLV-1 unspliced RNA, as has been recently demonstrated. NMD is a co-translational process that depends on the RNA helicase UPF1 and regulates the expression of multiple types of host mRNAs. RNA sensitivity to NMD depends on mRNA organization and the ribonucleoprotein (mRNP) composition. HTLV-1 has evolved several means to evade the NMD threat, leading to NMD inhibition. In the early steps of infection, NMD inhibition favours the production of HTLV-1 infectious particles, which may contribute to the survival of the fittest clones despite genome instability; however, its direct long-term impact remains to be investigated.

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<![CDATA[Prevention and Control of <i>Legionella</i> and <i>Pseudomonas</i> spp. Colonization in Dental Units]]> https://www.researchpad.co/article/elastic_article_12078 Introduction: Dental Unit Waterlines (DUWLs) have shown to be a source of Legionella infection. We report the experience of different dental healthcare settings where a risk management plan was implemented. Materials and methods: In a Hospital Odontostomatology Clinic (HOC) and three Private Dental Clinics (PDCs) housing 13 and six dental units (DUs), respectively, an assessment checklist was applied to evaluate staff compliance with guideline recommendations. DUWLs microbial parameters were investigated before and after the application of corrective actions. Results: In the HOC a poor adherence to good practices was demonstrated, whereas protocols were carefully applied in PDCs. L. pneumophila sg 2–15 was isolated in 31% (4/13) and 33% (2/6) of DUs in HOC and PDCs, respectively, mainly from handpieces (32%, 6/19) with counts >102 colony-forming units per milliliter (CFU/L), often associated with P. aeruginosa (68%, 13/19). The shock disinfection with 3% v/v hydrogen peroxide (HP) showed a limited effect, with a recolonization period of about 4 weeks. Legionella was eradicated only after 6% v/v HP shock disinfection and filters-installation, whilst P. aeruginosa after the third shock disinfection with a solution of 4% v/v HP and biodegradable surfactants. Conclusions: Our data demonstrate the presence and persistence of microbial contamination within the DUWLs, which required strict adherence to control measures and the choice of effective disinfectants.

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<![CDATA[Detecting PCOS susceptibility loci from genome-wide association studies via iterative trend correlation based feature screening]]> https://www.researchpad.co/article/elastic_article_12077 Feature screening plays a critical role in handling ultrahigh dimensional data analyses when the number of features exponentially exceeds the number of observations. It is increasingly common in biomedical research to have case-control (binary) response and an extremely large-scale categorical features. However, the approach considering such data types is limited in extant literature. In this article, we propose a new feature screening approach based on the iterative trend correlation (ITC-SIS, for short) to detect important susceptibility loci that are associated with the polycystic ovary syndrome (PCOS) affection status by screening 731,442 SNP features that were collected from the genome-wide association studies.ResultsWe prove that the trend correlation based screening approach satisfies the theoretical strong screening consistency property under a set of reasonable conditions, which provides an appealing theoretical support for its outperformance. We demonstrate that the finite sample performance of ITC-SIS is accurate and fast through various simulation designs.ConclusionITC-SIS serves as a good alternative method to detect disease susceptibility loci for clinic genomic data. ]]> <![CDATA[The Role of Long Noncoding RNAs in Human Papillomavirus-associated Pathogenesis]]> https://www.researchpad.co/article/elastic_article_12076 Infections with high-risk human papillomaviruses cause ~5% of all human cancers. E6 and E7 are the only viral genes that are consistently expressed in cancers, and they are necessary for tumor initiation, progression, and maintenance. E6 and E7 encode small proteins that lack intrinsic enzymatic activities and they function by binding to cellular regulatory molecules, thereby subverting normal cellular homeostasis. Much effort has focused on identifying protein targets of the E6 and E7 proteins, but it has been estimated that ~98% of the human transcriptome does not encode proteins. There is a growing interest in studying noncoding RNAs as biochemical targets and biological mediators of human papillomavirus (HPV) E6/E7 oncogenic activities. This review focuses on HPV E6/E7 targeting cellular long noncoding RNAs, a class of biologically versatile molecules that regulate almost every known biological process and how this may contribute to viral oncogenesis.

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<![CDATA[Novel Coronavirus: Current Understanding of Clinical Features, Diagnosis, Pathogenesis, and Treatment Options]]> https://www.researchpad.co/article/elastic_article_12075 Since December 2019, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in devastating consequences worldwide and infected more than 350,000 individuals and killed more than 16,000 people. SARS-CoV-2 is the seventh member of the coronavirus family to affect humans. Symptoms of COVID-19 include fever (88%), cough (68%), vomiting (5%) and diarrhoea (3.7%), and transmission of SARS-CoV-2 is thought to occur from human to human via respiratory secretions released by the infected individuals when coughing and sneezing. COVID-19 can be detected through computed tomography scans and confirmed through molecular diagnostics tools such as polymerase chain reaction. Currently, there are no effective treatments against SARS-CoV-2, hence antiviral drugs have been used to reduce the development of respiratory complications by reducing viral load. The purpose of this review is to provide a comprehensive update on the pathogenesis, clinical aspects, diagnosis, challenges and treatment of SARS-CoV-2 infections.

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<![CDATA[Comparative Studies of <i>Fraxinus</i> Species from Korea Using Microscopic Characterization, Phytochemical Analysis, and Anti-Lipase Enzyme Activity]]> https://www.researchpad.co/article/elastic_article_12074 Fraxinus species belongs to the Oleaceae family, commonly known as Ash tree, and has been utilized as a folk medicine with various medicinal properties, including anti-obesity activity. The goal of the present study was to establish quality control parameters using microscopic characterization, phytochemical differentiation, and anti-lipase activity evaluation of five Fraxinus plants in Korea. Microscopic evaluation of the lower surface, petiole, and midrib of leaves, and stem bark showed discriminative anatomical characteristics, such as the stomatal index of the lower leaf surface; the number of sclerenchyma cells, and the diameter of parenchyma cells in the petiole and midrib; and the cork cell size and fiber frequency in the stem bark. Phytochemical analysis using high-performance liquid chromatography revealed the significant variation in the chemical profiles of the 12 major secondary metabolites among the samples. The orthogonal projections to latent structure-discrimination analysis efficiently differentiated each group belonging to each Fraxinus plant with the anatomical and quantification data. F. rhynchophylla and ligstroside showed the most potent anti-lipase activity among the plants and the 12 major metabolites, respectively. These findings could serve as the scientific criteria for the appropriate identification and establishment of standards for the use of Fraxinus species as medicinal plants.

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<![CDATA[Hybrid Nanoparticles of Poly (Methyl Methacrylate) and Antimicrobial Quaternary Ammonium Surfactants]]> https://www.researchpad.co/article/elastic_article_12073 Quaternary ammonium surfactants (QACs) are microbicides, whereas poly (acrylates) are biocompatible polymers. Here, the physical and antimicrobial properties of two QACs, cetyl trimethyl ammonium bromide (CTAB) or dioctadecyl dimethyl ammonium bromide (DODAB) in poly (methyl methacrylate) (PMMA) nanoparticles (NPs) are compared to those of QACs alone. Methyl methacrylate (MMA) polymerization using DODAB or CTAB as emulsifiers and initiator azobisisobutyronitrile (AIBN) yielded cationic, nanometric, homodisperse, and stable NPs. NPs’ physical and antimicrobial properties were assessed from dynamic light scattering (DLS), scanning electron microscopy, and viability curves of Escherichia coli, Staphylococcus aureus, or Candida albicans determined as log(colony-forming unities counting) over a range of [QACs]. NPs were spherical and homodisperse but activity for free QACs was higher than those for QACs in NPs. Inhibition halos against bacteria and yeast were observed only for free or incorporated CTAB in NPs because PMMA/CTAB NPs controlled the CTAB release. DODAB displayed fungicidal activity against C. albicans since DODAB bilayer disks could penetrate the outer glycoproteins fungus layer. The physical properties and stability of the cationic NPs highlighted their potential to combine with other bioactive molecules for further applications in drug and vaccine delivery.

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<![CDATA[Resistance of Tick Gut Microbiome to Anti-Tick Vaccines, Pathogen Infection and Antimicrobial Peptides]]> https://www.researchpad.co/article/elastic_article_12072 Ixodes scapularis ticks harbor microbial communities including pathogenic and non-pathogenic microbes. Pathogen infection increases the expression of several tick gut proteins, which disturb the tick gut microbiota and impact bacterial biofilm formation. Anaplasma phagocytophilum induces ticks to express I. scapularis antifreeze glycoprotein (IAFGP), a protein with antimicrobial activity, while Borrelia burgdorferi induces the expression of PIXR. Here, we tested the resistance of I. scapularis microbiome to A. phagocytophilum infection, antimicrobial peptide IAFGP, and anti-tick immunity specific to PIXR. We demonstrate that A. phagocytophilum infection and IAFGP affect the taxonomic composition and taxa co-occurrence networks, but had limited impact on the functional traits of tick microbiome. In contrast, anti-tick immunity disturbed the taxonomic composition and the functional profile of tick microbiome, by increasing both the taxonomic and pathways diversity. Mechanistically, we show that anti-tick immunity increases the representation and importance of the polysaccharide biosynthesis pathways involved in biofilm formation, while these pathways are under-represented in the microbiome of ticks infected by A. phagocytophilum or exposed to IAFGP. These analyses revealed that tick microbiota is highly sensitive to anti-tick immunity, while it is less sensitive to pathogen infection and antimicrobial peptides. Results suggest that biofilm formation may be a defensive response of tick microbiome to anti-tick immunity.

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<![CDATA[ggroups: an R package for pedigree and genetic groups data]]> https://www.researchpad.co/article/elastic_article_12071 R is a multi-platform statistical software and an object oriented programming language. The package archive network for R provides CRAN repository that features over 15,000 free open source packages, at the time of writing this article (https://cran.r-project.org/web/packages, accessed in October 2019). The package ggroups is introduced in this article. The purpose of this package is providing functions for checking and processing the pedigree, calculation of the additive genetic relationship matrix and its inverse, which are used to study the population structure and predicting the genetic merit of animals. Calculation of the dominance relationship matrix and its inverse are also covered. A concept in animal breeding is genetic groups, which is about the inequality of the average genetic merits for groups of unknown parents. The package provides functions for the calculation of the matrix of genetic group contributions (Q). Calculating Q is computationally demanding, and depending on the size of the pedigree and the number of genetic groups, it might not be feasible using personal computers. Therefore, a computationally optimised function and its parallel processing alternative are provided in the package.ResultsUsing sample data, outputs from different functions of the package were presented to illustrate a real experience of working with the package.ConclusionsThe presented R package is a free and open source tool mainly for quantitative geneticists and ecologists, who deal with pedigree data. It provides numerous functions for handling pedigree data, and calculating various pedigree-based matrices. Some of the functions are computationally optimised for large-scale data. ]]> <![CDATA[Cellular and Subcellular Compartmentation of the 2<i>C</i>-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle]]> https://www.researchpad.co/article/elastic_article_12070 The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis.

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<![CDATA[Constitutive and Induced Expression of Total Phenol and Phenol Oxidases in Wheat Genotypes Ranging in Resistance/Susceptibility to the Root-Lesion Nematode <i>Pratylenchus thornei</i>]]> https://www.researchpad.co/article/elastic_article_12069 Plant-derived phenolic compounds contribute to the defense against various pathogens, including root-lesion nematodes (Pratylenchus spp.). However, there are no reports on the role of phenolic compounds in wheat (Triticum aestivum) against Pratylenchus thornei. In this study, wheat genotypes ranging from resistant to very susceptible to P. thornei were used to investigate the level of total phenols and phenol oxidases, polyphenol oxidase (PPO), and peroxidase (POD) expressed in root tissues when grown in the presence and absence of P. thornei over time (2–8 weeks). Higher constitutive levels of total phenols were found in resistant synthetic hexaploid wheats CPI133872 (576 µg gallic acid equivalent (GAE)/g root) and CPI133859 (518 µg GAE/g root) at 8 weeks after sowing, compared with moderately resistant and susceptible genotypes (192 to 390 µg GAE/g root). The activity of PPO was induced in resistant (CPI133872) and moderately resistant (GS50a and its derivate QT8343) genotypes, becoming maximal at 4 weeks after P. thornei inoculation. The activity of POD was induced in CPI133872 at 6 weeks after P. thornei inoculation. Different genetic sources of resistance to P. thornei showed diverse defense mechanisms and differences in timing responses. The combined effects of total phenols and oxidative enzymes could be important for defense against P. thornei in some resistant wheat genotypes.

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<![CDATA[In Vitro Anti-NTHi Activity of Haemophilin-Producing Strains of <i>Haemophilus haemolyticus</i>]]> https://www.researchpad.co/article/elastic_article_12068 Nontypeable Haemophilus influenzae (NTHi) is a leading causative organism of opportunistic respiratory tract infections. However, there are currently no effective vaccination strategies, and existing treatments are compromised by antibiotic resistance. We previously characterized Haemophilus haemolyticus (Hh) strains capable of producing haemophilin (HPL), a heme-binding protein that restricts NTHi growth by limiting its access to an essential growth factor, heme. Thus, these strains may have utility as a probiotic therapy against NTHi infection by limiting colonization, migration and subsequent infection in susceptible individuals. Here, we assess the preliminary feasibility of this approach by direct in vitro competition assays between NTHi and Hh strains with varying capacity to produce HPL. Subsequent changes in NTHi growth rate and fitness, in conjunction with HPL expression analysis, were employed to assess the NTHi-inhibitory capacity of Hh strains. HPL-producing strains of Hh not only outcompeted NTHi during short-term and extended co-culture, but also demonstrated a growth advantage compared with Hh strains unable to produce the protein. Additionally, HPL expression levels during competition correlated with the NTHi-inhibitory phenotype. HPL-producing strains of Hh demonstrate significant probiotic potential against NTHi colonization in the upper respiratory tract, however, further investigations are warranted to demonstrate a range of other characteristics that would support the eventual development of a probiotic.

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<![CDATA[Estimation of amyloid aggregate sizes with semi-denaturing detergent agarose gel electrophoresis and its limitations]]> https://www.researchpad.co/article/elastic_article_12067 Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan’s laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.

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