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NeuroBio Write Up #2

Submitted by dkotorobay on Fri, 03/24/2017 - 11:46

Title: Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina

Main Points:

The question the paper is trying to answer is whether or not using red-shifted channelrhodopsin to treat degenerative blindness will work as it did in mice.

This study is important because it could possibly be a way to treat vision loss caused by retinal degeneration.

The results were that red-shifted channelrhodopsin also drives neuronal responses in macaque retinae as well as in the central human retina, the site of high-acuity vision, demonstrating the therapeutic potential of the red-shifted channelrhodopsin molecule.

Methods:

The models used were rd1 mice, macaque retinal explants and humans.

The techniques:

For the blind mice AAV2 injections through the sclera.

For the primates they were terminally anesthetized and their eyes were removed and the retina was isolated from the vitreous humor and cut into 1 cm pieces. The retinal explants were infected with the AAV2 and AAV8 until the day of electrophysiological recordings or fixation, the AAV infections were performed within 2 hours of the retina explants being put in tissue culture.

 For the human experiments postmortem human ocular globes were acquired from the school of surgery, 6 donors, 63 – 95 years old, postmortem delays 9 – 38 hours. Similar proceedings to the primate experiment.

Shortcomings/Weaknesses:

The age range constrained to older ages in humans, the pool of human subjects was small, only 6 people. There were also no live subjects after the mice. This may be an ethical issue.

The controls were appropriate, they tested multiple models in the same manner, though the mice were alive and the primates and humans were not alive.

Figures:

Figure 1: 3 panels, showing that the channelrhodopsin can be efficiently targeted to the RGC membrane and dendritic arbor of blind mice.

Figure 2: Graphs showing the responses triggered by optogenetic stimulation of the retina.

Figure 3: Different types of graphs showing the triggered responses in the blind mice that were treated with the AAV2.

Figure 4: One of the figures shows the locomotive behavior of the blind mice before and after treatment as well as graphs that correspond with those results.

Figure 5: Graphs showing the light responses in the AAV-infected primate retinae.

Figure 6: I believe these graphs and pictures are demonstrating where and what is activated when the explant has been infected with the AAV.

Figure 7: Pictures of the retina showing part of the foveal pit.

Figure 8: AAV- mediated optogenetic activation of the human retina after it had been incubated for 12 days.

Questions:

How would such an experiment work on a live human? Would it be conducted more like the mouse experiment?

Keywords:

Treating degenerative blindness, channelrhodopsin used to treat vision loss

Mammalogy Notes Part 1

Submitted by dkotorobay on Fri, 03/24/2017 - 11:05

Ungulates: mammals with hooves. Claws, nails, and hooves: unguis- keratinized. subunguis- transition to skin. pad- skin (may be sensitive). There are two orders of ungulates: order Perissodactyla: odd-toes ungulates, order Artiodactyla: even-toed ungulates. Order Perissodactyla: mesaxonic foot, order Artiodactyla: paraxonic foot. (the astragalus is called the talus in humans). Convergent adaptations for cursoriatlity (running): the development of the astragalus as the main weight bearing bone (the calcaneus in humans), reduction/fusion of the metapodials into a cannon bone. The shape of the astragalus in perissodactyls and artiodactyls: O. Perissodactyla: top surface pulley-shaped, bottom surface is flat. Limits movement between the leg and ankle to flexion and extension. Less mobility between ankle and foot, limits movement to flexion and extension. Shared derived characteristics: enlarged astragalus with pulley-shaped upper surface only, mesaxonic feet with a reduction of digits, all are hindgut fermenters (enlarged caecum with commensal bacteria). Family: Tapiridae- tapirs (Tapirus): they are morphologically primitive: unspecialized limbs, primitive tooth number (44), simple loph pattern to teeth (like rhinos). They have: reduced nasal bones, elongated proboscis formed by nose and upper lip. They generally live near swamps, rivers or other wet areas where they eat succulent plant material and fruit, all are either endangered or vulnerable, all pops. are decreasing. Family: Rhinocerotidae: Live in a variety of habitats but need permanent water, prefer to eat leaves and grass but will eat woody vegetation and fruit. Diceros bicornis (black African and Asian rhinos): pointed, prehensile upper lip for browsing. Ceratotherium simum (white African rhino): square lip for cropping grasses (grazing). Behavior: solitary or mom/offspring groups, females breed at 5/6 years, males at 10, usually a baby every 2-4 years. Horns: mass of long hair-like fibers fused together, composed of mineralized keratin with no bony core or sheath. Family: Equidae: the most cursorial perissodactyl: calcaneum is long and posteriorly placed, astragalus is weight-bearing, foot greatly elongated, only 3rd digit is functional. All equids are grazers. Behavior: polygynous (1 male, many females), strong social hierarchy- led by single stallion., herds based on extended family groups/ “clans”, social structure regulated by complex behaviors and vocalizations. The development of grasslands in the early Miocene is thought to be the driving force behind the evolution of horses. Were highly diverse in the past, Baluchitherium (Peracetatherium), from the Oligocene of Pakistan, is the largest known land mammal. Megacerops (Brontops) was the equivalent of a forest elephant in the late Eocene of N. America. 

Lab Report Genetics

Submitted by dkotorobay on Mon, 03/06/2017 - 14:48

Abstract:

This paper focuses on the mutations in genes in the adenine that induce a red color in yeast. The mutations were induced with a UV light and then the yeast colonies were allowed to grow on a nutrient rich media. After sufficient growth time was allowed the yeast was then transferred to nutrient poor media without adenine to see if nutrient availability would affect color. In addition, the yeast was also transferred to a nutrient poor media with adenine to determine if the mutations involved adenine or if the mutations were caused by a different gene. The results showed that mutations can be induced through UV radiation and then when these mutations are crossed with other mutations and controls it is evident which mutations did or did not involve mutations in the adenine.

Introduction:

Mutations are random changes in the base code of a DNA molecule, they are the ultimate source of genetic variability. Some of the time mutations will result in a change of phenotype. Visible mutations are rare events because of DNA’s ability to replicate without mistakes is near perfect but occasionally mistakes in replication do occur. The chances of finding a specific mutation are little to none. It is possible to increase the occurrence of them by various mutagens such as chemicals, x-rays, or ultra violet radiation (Loomis, Mutation Protocol 1).

The experiment conducted had three parts. The first part was to induce mutations using UV-light, the second part is to screen for mutations, and the last part is to categorize the mutations by complementation testing.

Exposure to UV radiation was the method used to produce mutations in haploid cells, but even with exposure to radiation mutation is still a random event so looking at multiple yeast colonies to find specific mutations is necessary. The results from the two class sections were pooled to obtain the five mutations that were found.

Screening was done through visual examination. Looking for mutated yeast that should have been red or pink. Yeast strains from the opposite mating type were also exposed to UV-radiation so that the resulting mutants could be crossed, one class section induced mutations on the “a” mating type and the other class section on the “a” mating type.

When the two mutant strains are crossed the F1 generation is analyzed. If the F1generation expresses the wild type phenotype, it can be concluded that each mutation is in one of two possible genes necessary for the wild type phenotype. When it is shown that genetically two or more genes control a phenotype, the genes are said to form a complementation group. On the other hand, if the F1 generation does not express the wild type phenotype, but a mutant phenotype instead it can be concluded that both mutations occur in the same gene (www.ndsu.edu).

By observing the results of the F1 generation in the mutant yeast crosses conclusions will be drawn on whether complementation occurred or if both mutations to the DNA occurred on the same gene.

 

Methods Project Introduction

Submitted by dkotorobay on Wed, 03/01/2017 - 11:50

INTRODUCTION:

This experiment was conducted to see what, if any, differences were to arise from instructions written by one student and followed by another. Instructions were written and then assigned to another student to follow. In the instructions it was required to take three pictures. One being a close up of moss showing and labeling the sporophytes and gametophytes, another showing the general location of where the moss was found with an arrow pointing to the moss, and the last picture was to be an aerial view of the area, either on Google maps or Google Earth, with an arrow on the map pointed to where the moss can be found. The next part of the experiment was to create a composite image, the student following the instructions was unable to ask for clarification on any point and was then required to recreate the composite image. The resulting image was then compared to the original image to see what differences there were. The author of the instructions then had to speculate as to why the differences occurred and what could have been more clear in the methods or any other reasons that could have caused differences between the images.  

NeuroBio Lecture 2/27 Part 2

Submitted by dkotorobay on Tue, 02/28/2017 - 13:20

A good question is how do tatse signals get to our brain. And the answer is through the cranial nerves 7, 9, and 10, not through the spinal cord. The gustatory nucleus is in the medulla, there are additional  projection to salivation adn vomiting centers. The ipsilateral VPM thalamus is also involved. And the primary gustatory cortex is where the concious perception of taste occurs. Taste information shares bandwidth as it travels. Individual taste receptors are very specific, multiple taste receptor cells within a papillae may synapse onto a primary taste axon, it reduces the overall number of required neurons, enables the flexibility for new tastes within the system, neurons broadly identify tastes. It is not a 1:1 ratio for neuron to taste. A collectively activates population encodes specific taste, the response pattern is the same as population coding. 

Smell and odor identification adds a dimension to taste for identifying foods and it can help warn about danger. Many soecies produce chemical ordors for social communication called pheremones. Pheremones are used for reproductive behavior, marking territory, identification, and agression or submission. The olfactory epithelium cells detect odorants, they are supporting cells that produce mucous to dissolve odors. They are basal cells that continually produce new receptor cells every 4-8 weeks. Odorant receptors make up about 3-5% of the mammalian genome, in humans that about 350 receptors and more than 1000 receptors in rodents. This involves GPCRs, one in particular is important, Golf, or the olfactory specific Gprotein, which involves cAMP gated ion channels, which requires cation and chloride channels to depolarize. Receptors show adaptation after continuous stimulation. Which means that after you've been around a scent for a while you get acclimated to it you can't smell it anymore. 

Odor signal transfer works by first having the scent come in contact with receptor cell axons then moving on to the cribriform plate next going to cranial nerve 1 and lastly reaching the olfactory bulb. 

Olfactory pathways start when receptor cells respond to many odorants, odorants can activate many receptor types. Olfactory axons converge on glomeruli in the olfactory bulb, and the glomeruli recieve inputs from 1 type of receptor, called local modulation. The central olfactory pathway refers to when the olfactory tracts goes directly to the forebrain, including the olfactory tubercle and primary olfactory cortex, also known as the temporal lobe. It reaches the thalamus before the association areas. 

There are two types of population coding. Spatial and temporal. Odors show sensory maps, and different odors activate different populations. Temporal coding involves differential spike timing and within and across sensory maps. There is still a lot of information to the decode, which occurs in oscillations. 

NeuroBio Lecture 2/27

Submitted by dkotorobay on Mon, 02/27/2017 - 23:34

Chemoreception: Sensing chemicals in our environment is helpful, chemoreceptors help us gauge whether things are edible or not, or in some causes even dangerous. It is the oldest and most prevalent sensory system found across may phyla and kingdoms. It is an adaptive survival mechanism, it helps attract and find mates. 

Gustation: There are five established tastes with known receptors. They are salty, sour, sweet, bitter, and umami. Umami is Japanese and refers to the savory taste. There are possibly more receptors for fat, starch, carbonation, calcium, and water, but they are currently unknown. Similar tasting compounds have similar chemistry, acids are sour and natural poisons are often bitter. Flavors are combinations of receptor activation, smell, texture and temperature also contribute to differentiating between flavors. 

Taste is related to the papillae. There are thousands of papillae across the tongue. With three different structures, foliate which are ridges, vallate which are pimples, and fungiform which are mushroom shaped. There are 1 to 100 taste buds per papillae and on every taste bud there are 50  to 150 taste receptor cells that are specialized for taste sensitivity like sweet or sour or etc. Taste receptors are made of neuroepithelial cells, the apical region (top) of a taste receptor has microvilli that project into the taste pore, and these cells are renewed every two weeks. 

Taste receptor signals: Taste receptors change voltage upon contact, chemical and electrical synapses onto sensory afferents. Chemicals that we taste are passed through ion channels, block ion channels and bind GPCRs. 

Salt and sour receptors work in similar ways. For salt it involves the Na+ channel which is voltage insensitive, meaning that if a charge is applied it won't make a difference to the channel. It can be blocked by amiloride, which is also used to treat high blood pressure, and it releases serotonin. For sour it involves the H+ proton channel and it blocks the K+ channel and serotonin is also released in this case. The bitter, sweet, and umami receptors work in similar ways. What they have in common is there are dimerized taste reeceptor proteins, Gq bound GPCRs adn T1R and T2R gene families. For the bitter taste receptors it uses T2R's, there are more that 25 genes dedicated to this type of taste receptors. For the sweet taste receptors it utilizes T1R2 and T1R3 dimers, lack of T1R2 limits sweet sensation. And for umami it utilizies T1R1 and T1R3, and it recognizes amino acids. MSG, and savoury tastes. 

 

 

 

Manuscript Submission Guidelines

Submitted by dkotorobay on Fri, 02/24/2017 - 12:44

The Journal of Molecular Biology has an eighteen page packet for manuscript submission guidelines, which can be found here https://www.elsevier.com/wps/find/journaldescription.cws_home/622890?gen...

As breifly as I can summarize eighteen pages any manuscript that is submitted must be reviewed by at least two other editors. There are also guidelines for composite images, they are not allowed to have more than four parts, graphs should be able to be edited. Footnotes should be used sparingly as the guidelines say, enough detail should be provided in the methods and materials section that the work could be reproduced. The discussion section should explore the significance of the results of the work, and a combined results and discussion section is usually appropriate and extensive citing and discussion of published literature shoudl be avoided.

The manuscript guidelines are extensive and thorough, answering any question imaginable relating to formatting and what is acceptable when attempting to submit a manuscript. 

Update #3 on Worm-like Organism

Submitted by dkotorobay on Fri, 02/24/2017 - 12:14

The worm-like organsim has now half covered in an orange tinted, transparent shell. This confirms that the worm is in a larval state and is becoming a pupa. The half shell appeared almost overnight and I expect to be done developing by the end of the day. I am unsure of how long it was stay a pupa but if it survives this stage I expect it to be out of this stage in about a week, at which point it will then be a bettle. This is all speculation but at this point I'm pretty sure that the worm is a meal worm. 

Methods Project Discussion Section

Submitted by dkotorobay on Fri, 02/24/2017 - 12:07

In pictures A and B the differences in amount of light can be chalked up to the fact that the weather may have been different when the originals and replicates were taken, or it may have been later in the day when the originals were taken, time of day was not specified in the methods section. All three pictures were taken from further away for the replicates and that may just be because the person wanted to show more of the surrounding environment. The font used to label the sporophytes and gametophytes was smaller and not bolded, font size and bolding was not mentioned in the methods section, in the original it was bolded and larger to make it stand out on the green background of the moss. In picture C the replicated picture was that of a screenshot of Google Earth while the original is that of a screenshot of Google maps, the methods section did specify to use Google maps so the reason for this discrepancy is unknown. Differences that were consistent between both images was the labels for the images were smaller and closer to the corner for the originals while they were larger and further away for the replicates. Also the orientation of the arrows was different between the two pictures, these differences can be contributed to the fact that instructions were not specified in the methods section and the person replicating the image through the methods section made those decisions based on personal preference.

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