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T-Blue and Ph-HCl staining

Submitted by ewinter on Thu, 04/25/2019 - 05:35

Toluidine blue (T-blue) stains polysaccharides and phloroglucinol HCL (Ph-HCL) stains lignin. In the NaN1793 mutants, both the Ph-HCL and T-blue stains appear a much lighter color at the distal cortex as opposed to the proximal cortex (Fig. 7B, 7D). In the wild - type plants, this contrast is not observed to as great of an extent (Fig. 7). The NaN1793 mutation is predicted to be a nonsense mutation, resulting in a truncated, nonfunctional protein. This phenotypic difference suggests that Bradi1g25180 may be involved in formation of cell walls of the distal cortex.

 

Confirming NaN1783 mutation

Submitted by ewinter on Wed, 04/24/2019 - 22:52

Using the Primer3 software, primers were created to amplify the region of the NaN1793 mutation. These primers were chosen with respect to the fact that Sanger sequencing can only sequence about 1000 b.p maximum, and the mutation should not be too close to either primer since the beginning and end of sequencing data is usually less accurate than the middle. The expected size of the amplicon is 874 b.p. In order to confirm that DNA was extracted from the plants and to decide which samples to take for sequencing, gel electrophoresis was run. Two gels were run. One gel contains PCR products in a ¼ dilution in T10E1 (TE) buffer while the other contains PCR products in a ¼ dilution with H2O as a control. T10E1 buffer protects nucleic acids from degradation.

 

Sleep

Submitted by ewinter on Fri, 04/19/2019 - 13:26

There are five major functions of sleep as defined by neuroscientists. These are energy conservation, reinforcement of ecological niche, body restoration, brain restoration, and memory consolidation. There are four major brain regions that are responsible for sleep. The basal forebrain causes slow wave sleep via GABAergic projections to the hypothalamus. The brainstem, specifically the reticular formation, activates forebrain to wakefulness via acetylcholine and norepinephrine. The pons induces REM sleep via GABAergic and glycinergic projections to the spinal motor neurons to suppress motor activity. The hypothalamus coordinates the circadian rhythm and switches between states of sleep via hypocretin projections to the other three brain regions aforementioned.

 

Three classes of Hormones

Submitted by ewinter on Fri, 04/19/2019 - 11:57

There are three major classes of hormones. Peptide hormones are small protein molecules. Their mechanism of action is binding to G-protein coupled receptors for fast effects. An example of an amine is acetylcholine. Amines are modified amino acids. Their mechanism of action is the same as peptides. Common amines include epinephrine and norepinephrine. Steroids are derivatives of cholesterol. Their mechanism of action includes diffusing across the cell membrane and binding to cytosolic receptors. They affect gene expression by contributing to chromatin remodeling. Cortisol is a well known steroid that regulates glucose levels and promotes learning during stressful situations.

Signalaing in Neuroscience

Submitted by ewinter on Fri, 04/19/2019 - 11:45

There are three types of signaling in neuroscience. Neurocrine signaling, also known as synaptic transmission, is when neurotransmitters are released by a presynaptic neuron into the synapse, and bind to the postsynaptic neuron. The presynaptic neuron fires an action potential for the neurotransmitters to be released, and to postsynaptic neuron depolarizes when the neurotransmitters bind. Endocrine signaling is when endocrine glands release hormones into the bloodstream. Hormones may have differing effects, and effect any cell type that expresses their receptor. This type of signaling results in slower, widespread effects. The third type of signaling is neuroendocrine signaling. This is when neurons integrate postsynaptic potentials, fire action potentials, and release hormones into the bloodstream.

 

AT1g61610 has ZCP4 Promoter cis-element

Submitted by ewinter on Thu, 04/18/2019 - 23:49

AT1g61610 is paralogous to AT4g21390, and AT4g21390 is gene B120, so it is homologous to gene B120 in Brachypodium distachyon, which is our gene of interest. AT1g61610 was found to have a Zinniacysteine protease 4 (ZCP4) promoter cis-element-like sequence 1000 b.p. upstream, and the ZCP4 promoter governs tracheary element differentiation (Pyo et al. 2007). This cis-element is known as tracheary-element-regulating-cis-element (TERE). 61 genes in Arabidopsis thaliana were found to include this cis-element 1000 b.p. upstream. These genes were Arabidopsis tracheary element differentiation related genes. Their functions include programmed cell death, cell wall biosynthesis and modification, lignification, phosphorylation, photosynthesis, and unclassified function. The presence of this cis-element-like sequence implicates AT1g61610 in the development of xylem, although nothing further is known to this end. Xylem are the vesicles in plants that transport water up from the roots to the leaves. The development of xylem includes lignification and secondary cell wall formation. This finding may help design experiments to elucidate the function of gene B120 in B. distachyon, which remains largely unknown.

Confirming Mutation Discussion

Submitted by ewinter on Thu, 04/18/2019 - 19:49

The bright bands on the gel indicate that DNA was successfully extracted. This was expected, despite some worry about self complementation due to the “any th” value of 8.16 in the primer3 output. We do not know why lane number 2 on the TE gel did not show a band. We recall loading sample into this lane. It is possible that something in the DNA extraction protocol failed, so no DNA of large band size was extracted. We preferentially chose mutants from the TE gel to send for sequencing because TE stabilizes DNA. All three of the trustworthy sequence results confirmed that the mutant plants were homozygous for the NaN1793 mutation.

Gel Extraction

Submitted by ewinter on Thu, 04/18/2019 - 19:49

By looking at the gels, we decided to extract M1 from H20 because it did not show up on TE. We then chose to take M2, M6, and M7 from the TE gel. After purifying the DNA from the gel using the gel extraction protocol. The concentration of M1 was 30.2 ng/uL. The concentration of M2 was 37.3 ng/uL. The concentration of M6 is 24.1 ng/uL. The concentration of M7 is 25.1 ng/uL. The A260/280 ratios are all near 2.0. This is usually considered pure for RNA. In this case, the fact that they are consistent across all four samples is a good indicator that we have pure DNA.

 

ZCP4 promoter

Submitted by ewinter on Wed, 04/17/2019 - 00:08

AT1g61610 was found to have a Zinniacysteine protease 4 (ZCP4) promoter cis-element-like sequence 1000 b.p. upstream, and the ZCP4 promoter governs tracheary element differentiation (Pyo et al. 2007). The presence of this cis-element-like sequence implicates AT1g61610 in the development of xylem, although nothing further is known to this end. Xylem are the vesicles in plants that transport water up from the roots to the leaves. The development of xylem includes lignification and secondary cell wall formation.

Poster Writing and Content

Submitted by ewinter on Fri, 04/12/2019 - 10:51

In my opinion, the writing is very clear. In each of the four sections outlined above, the writing is organized by bullet points with one sentence following. This length of writing seems suitable for a poster. The figures are high resolution, notably the picture of the immunohistochemical expression of PD-L1 in squamous cell carcinoma. No raw data is shown. The only data shown includes four odds ratios with confidence intervals and P-values. The data for the figures is not displayed in its own chart. The bar graphs serve to visually engage the viewer to the results. Without reading the text, the viewer can see the PD-L1 expression levels in tumor cells and lymphocytes, and how this varies across tumor type.  

 

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