Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.     

Fusinite of fossil coals as an information source about the anatomy of ancient plants

“Clarification” and ion and high-frequency etching of fusinite are described. These methods allow microscopic examination of fusinitized plant tissues in fine anatomical detail, revealing the outlines of cells, the structure of cell walls, the outlines of bordered pits, and, occasionally, even cellular microstructures. It is supposed that the effects of “clarification” and etching are caused by those optical differences between the composition of organic biopolymers that constituted the anatomical structures of plants that have been preserved in the fossil state.     

Efficient Cargo Delivery into Adult Brain Tissue Using Short Cell-Penetrating Peptides

Zebrafish brains can regenerate lost neurons upon neurogenic activity of the radial glial progenitor cells (RGCs) that reside at the ventricular region. Understanding the molecular events underlying this ability is of great interest for translational studies of regenerative medicine. Therefore, functional analyses of gene function in RGCs and neurons are essential. Using cerebroventricular microinjection (CVMI), RGCs can be targeted efficiently but the penetration capacity of the injected molecules reduces dramatically in deeper parts of the brain tissue, such as the parenchymal regions that contain the neurons. In this report, we tested the penetration efficiency of five known cell-penetrating peptides (CPPs) and identified two– polyR and Trans – that efficiently penetrate the brain tissue without overt toxicity in a dose-dependent manner as determined by TUNEL staining and L-Plastin immunohistochemistry. We also found that polyR peptide can help carry plasmid DNA several cell diameters into the brain tissue after a series of coupling reactions using DBCO-PEG4-maleimide-based Michael’s addition and azide-mediated copper-free click reaction. Combined with the advantages of CVMI, such as rapidness, reproducibility, and ability to be used in adult animals, CPPs improve the applicability of the CVMI technique to deeper parts of the central nervous system tissues.     

The effect of vitamin E treatment during preovulatory period on reproductive performance of goats following estrous synchronization using intravaginal sponges

The objectives of this study were to investigate whether the use of intravaginal sponge for estrous synchronization of goats causes oxidative stress, and to examine the effect of administering vitamin E during preovulatory period on reproductive performance of estrous synchronized goats. Estrus was synchronized in 36 non-lactating adult does using intravaginal sponges containing 30 mg of fluorogestane acetate (FGA) for 14 days. All females received 500 IU of eCG at the sponge withdrawal. The goats were allocated at random to two groups balanced for breed, age and body weight. Treatment group (n=18) received 200mg of vitamin E i.m. at the time of sponge removal and again at the time of second artificial insemination. The other 18 goats (control) were administered 1 ml of physiological saline instead of vitamin E on each of these two occasions. All does in estrus was intracervically inseminated at 12 and 24h after the onset of estrus. Blood samples were collected every 72h during the experimental period for evaluation of malondialdehyde (MDA) and vitamin E concentrations. Serum MDA level increased and vitamin E concentration decreased during the period of vaginal sponge application. Following the sponge removal, MDA level declined rapidly to below basal level in the treatment group but remained high in the control group. Conversely, vitamin E concentration increased in the treatment group after the sponge withdrawal and remained at a low level in the control group. No statistically significant differences (P>0.05) were observed between groups in terms of estrous response, conception rate, gestation length or kidding rate. However, the number of multiple births (70.0% versus 50.0%) and prolificacy rate (2.40+/-0.37 versus 1.63+/-0.26 kids per kidding) were significantly higher (P<0.05) for the treatment group than those of the control group. The results indicate that the use of intravaginal sponges for estrous synchronization of goats causes an increase in level of oxidative stress. However, the vitamin E treatment during preovulatory period can prevent the overproduction of reactive oxygen species (ROS), and it may improve the multiple birth rates and the number of kids born in estrous synchronized goats.     

Antimicrobial activity of the resins obtained from the roots and stems of Cedrus libani and Abies cilicica

The antimicrobial activity of the ethanol extract of resins obtained from the roots and stems of Cedrus libani and Abies cilicica was investigated using a disc diffusion method. Results revealed that crude extracts of the resins of both plants are highly effective against tested microorganisms by preventing their growth to a greater extent.     

Cerebroventricular microinjection (CVMI) into adult zebrafish brain is an efficient misexpression method for forebrain ventricular cells

The teleost fish Danio rerio (zebrafish) has a remarkable ability to generate newborn neurons in its brain at adult stages of its lifespan-a process called adult neurogenesis. This ability relies on proliferating ventricular progenitors and is in striking contrast to mammalian brains that have rather restricted capacity for adult neurogenesis. Therefore, investigating the zebrafish brain can help not only to elucidate the molecular mechanisms of widespread adult neurogenesis in a vertebrate species, but also to design therapies in humans with what we learn from this teleost. Yet, understanding the cellular behavior and molecular programs underlying different biological processes in the adult zebrafish brain requires techniques that allow manipulation of gene function. As a complementary method to the currently used misexpression techniques in zebrafish, such as transgenic approaches or electroporation-based delivery of DNA, we devised a cerebroventricular microinjection (CVMI)-assisted knockdown protocol that relies on vivo morpholino oligonucleotides, which do not require electroporation for cellular uptake. This rapid method allows uniform and efficient knockdown of genes in the ventricular cells of the zebrafish brain, which contain the neurogenic progenitors. We also provide data on the use of CVMI for growth factor administration to the brain--in our case FGF8, which modulates the proliferation rate of the ventricular cells. In this paper, we describe the CVMI method and discuss its potential uses in zebrafish.     

Molecular characterization of rhizospheric soil streptomycetes isolated from indigenous Turkish plants and their antimicrobial activity

As part of a research program whose aim is to determine the diversity of streptomycetes in order to discover new bioactive secondary metabolites, rhizosphere soils of three indigenous plants were analyzed. A total of 55 actinomycetes were isolated using three different medium from the samples. The rhizospheric soil of the plant Aethionema dumanii gave the highest number of actinomycetes, i.e., 42% versus 27% and 31% for the soils from Salvia aytachii and Achillea ketenoglui, respectively. The AIA is the most favorable medium for the isolation of the actinomycetes from different rhizospheric soils. 16S rDNA sequence analysis revealed that while some isolates belong to different cluster groups such as Streptomyces lydicus, S. rochei, S. microflavus, S. griseoflavus, S. albidoflavus and S. violaceusniger, the majority of the sequences did not considerable clustered with the member of different Streptomyces groups. The in vitro antimicrobial activities of the crude organic and aqueous extracts of isolates were screened using a disc diffusion assay against a panel of bacteria and C. albicans. A total of 22 isolates showed antimicrobial activity. The antibacterial action of the extracts is more pronounced on Gram-positive than on Gram-negative bacteria in most cases. About 18% of the actinomycetes showed also antifungal activity. Study of the influence of two different culture media on production of bioactive molecules showed that the higher antimicrobial activity was obtained in M2 when compared to TSB. The results from this study provide evidence that the streptomycetes in the rhizosphere soils could be promising sources for antimicrobial bioactive agents.     

Antimicrobial Activity of Resins Obtained from the Roots and Stems of Cedrus libani and Abies cilicia

The antimicrobial activity of the ethanol extract of resins obtained from the roots and stems of Cedrus libani and Abies cilicia has been studied, using the disc diffusion method. The results obtained indicate that crude extracts of the resins of both plants are highly efficient in preventing the growth of microorganisms.