SciCombinator

Discover the most talked about and latest scientific content & concepts.

Journal: Methods in molecular biology (Clifton, N.J.)

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Shoot apical meristems (SAMs) of higher plants harbor a set of stem-cells and provide cells for the development of all the above-ground biomass of plants. Most of the important pattern formation events such as maintenance of stem-cell identity, specification and differentiation of leaf/flower primordia, and temporal control of the transition from vegetative to reproductive program are determined in SAMs. Genetic analysis has revealed molecular and hormonal pathways involved in stem-cell maintenance, organ differentiation, and flowering time. However, limited information is available as to how different pathways interact with each other to function as a network in specifying different cell types and their function. Deciphering gene networks that underlie cell fate transitions requires new approaches aimed at assaying genome-scale expression patterns of genes at a single cell-type resolution. Here we provide details of experimental methods involved in protoplasting of SAM cells, generating cell type-specific gene expression profiles, and analysis platforms for identifying and inferring gene networks.

Concepts: DNA, Gene, Gene expression, Cell, Organism, Stem cell, Cellular differentiation, Meristem

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The formation of protein aggregates is linked to the onset of several human disorders of increasing prevalence, ranging from dementia to diabetes. In most of these diseases, the toxic effect is exerted by the self-assembly of initially soluble proteins into insoluble amyloid-like fibrils. Independently of the protein origin, all these macromolecular assemblies share a common supersecondary structure: the cross-β-sheet conformation, in which a core of β-strands is aligned perpendicularly to the fibril axis forming extended regular β-sheets. Due to this ubiquity, the presence of cross-β-sheet conformational signatures is usually exploited to detect, characterize, and screen for amyloid fibrils in protein samples. Here we describe in detail some of the most commonly used methods to analyze such supersecondary structure.

Concepts: Protein structure, Disease, Nutrition, Cell biology, Tertiary structure, Prion, Biochemistry, Beta sheet

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Phytoplasma collections are a vital resource for researchers and diagnosticians studying phytoplasma diseases. They provide material as a point of reference and a research tool to increase our understanding of phytoplasmas and the diseases they cause. This chapter describes the techniques required to create and maintain collections of phytoplasma-infected Catharanthus roseus (Madagascar periwinkle).

Concepts: Research, Medicinal plants, Phytoplasma, Catharanthus roseus, Apocynaceae, Sugarcane Grassy Shoot Disease, Catharanthus

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Maintenance of phytoplasma strains in tissue culture is achievable for all strains transmitted to periwinkle (Catharanthus roseus), and also for other naturally infected plant host species. Shoots of 1-3 cm length are grown in a solid medium containing Murashige and Skoog (MS) micro- and macroelements and 0.12 mg/L benzylaminopurine. The continued presence of phytoplasmas in infected shoots of periwinkle that have been maintained in micropropagation for up to 20 years can be shown by diagnostic methods such as nested PCR tests using the 16S rDNA gene (see Chapters 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,and 26 for phytoplasma diagnostic methods).

Concepts: Polymerase chain reaction, 16S ribosomal RNA, Phytoplasma, 22, 1, Catharanthus roseus, Apocynaceae, Catharanthus

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In this chapter, we describe a real-time PCR detection system for fast, reliable, specific, and sensitive detection and discrimination of ‘Candidatus Phytoplasma mali’, ‘Ca. P. prunorum’, and ‘Ca. P. pyri’ from the 16SrX (apple proliferation-AP) group. These phytoplasmas are causal agents of fruit tree diseases within the Rosaceae family, namely apple proliferation, European stone fruit yellows, and pear decline. The assays use (hydrolysis) TaqMan(®) minor groove binder probes. The panel of assays comprises the same set of primers and specific probes for species-specific amplification, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The assays described can be used in routine phytoplasma surveys and in certification programmes.

Concepts: DNA, Polymerase chain reaction, Fruit, Rosaceae, Coconut, Phytoplasma, Apple, Candidatus

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Phytoplasmas are routinely detected by nucleic acid-based techniques. These approaches rely on enriched phytoplasma DNA extracts of good quality, following labor intensive and time-consuming purification protocols. Here we describe a very rapid, specific, sensitive, and reliable method for flavescence dorée phytoplasma detection, based on real-time Taqman(®) reverse transcription-PCR of the 16S rRNA. The protocol is particularly useful for large-scale screening of vineyards and nurseries, pathogen surveys, and field epidemiological studies.

Concepts: Biology, Ribosomal RNA, 16S ribosomal RNA, 30S, English-language films, Protocol, Phytoplasma

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Double-stranded RNA (dsRNA) is involved in different biological processes. At least three different pathways can respond to dsRNA in mammals. One of these pathways is RNA interference (RNAi) where long dsRNA induces sequence-specific degradation of transcripts carrying sequences complementary to dsRNA. Long dsRNA is also a potent trigger of the interferon pathway, a sequence-independent response that leads to global suppression of translation and global RNA degradation. In addition, dsRNA can be edited by adenosine deamination, which may result in nuclear retention and degradation of dsRNA or in alteration of RNA coding potential. Here, we provide a technical review summarizing different strategies of long dsRNA usage. While the review is largely focused on long dsRNA-induced RNAi in mammalian cells, it also provides helpful information on both the in vitro production and in vivo expression of dsRNAs. We present an overview of currently available vectors for dsRNA expression and provide the latest update on oocyte-specific transgenic RNAi approaches.

Concepts: DNA, Gene, Bacteria, Molecular biology, Virus, RNA, Small interfering RNA, RNA interference

28

We describe two improved assays for in vitro and in vivo screening of inhibitors and chemicals for antimalarial activity against blood stages of the rodent malaria parasite, Plasmodium berghei. These assays are based on the determination of bioluminescence in small blood samples that is produced by reporter parasites expressing luciferase. Luciferase production increases as the parasite develops in a red blood cell and as the numbers of parasites increase during an infection. In the first assay, in vitro drug luminescence (ITDL) assay, the in vitro development of ring-stage parasites into mature schizonts in the presence and absence of candidate inhibitor(s) is quantified by measuring luciferase activity after the parasites have been allowed to mature into schizonts in culture. In the second assay, the in vivo drug luminescence (IVDL) assay, in vivo parasite growth (using a standard 4-day suppressive drug test) is quantified by measuring the luciferase activity of circulating parasites in samples of tail blood of drug-treated mice.

Concepts: Immune system, Bacteria, Malaria, Plasmodium falciparum, Plasmodium, Red blood cell, Bioluminescence, Plasmodium berghei

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Assessing the ability of human spermatozoa to acquire fertilizing potential (capacitation) by stimulating exocytosis of the contents of the acrosome (acrosome reaction) is thought to have diagnostic potential (De Jonge, Reprod Med Rev 3:159-178, 1994). Calcium-mobilizing agents, such as calcium ionophores (A23187) and progesterone, stimulate the acrosome reaction in vitro (Brucker and Lipford, Hum Reprod Update 1:51-62, 1995). Acrosomal status is easily detected using Pisum sativum Agglutinin labeled with fluorescein isothiocyanate (Cross and Meizel, Biol Reprod 41:635-641, 1989). Herein we describe a procedure for assessing capacitation and the acrosome reaction of human spermatozoa in vitro.

Concepts: Sperm, Spermatozoon, In vitro fertilisation, Germ cells, Acrosome, Acrosome reaction

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The Martini force field is a coarse-grained force field suited for molecular dynamics simulations of biomolecular systems. The force field has been parameterized in a systematic way, based on the reproduction of partitioning free energies between polar and apolar phases of a large number of chemical compounds. In this chapter the methodology underlying the force field is presented together with details of its parameterization and limitations. Then currently available topologies are described with a short overview of the key elements of their parameterization. These include the new polarizable Martini water model. A set of three selected ongoing studies using the Martini force field is presented. Finally the latest lines of development are discussed.

Concepts: Mathematics, Molecular dynamics, Water, Molecule, Chemistry, Chemical element, Chemical compound, CHARMM