Blogs

In the unknown lab, we started by gram-staining our unknown organism. This is a way of testing for the presence of a thick polysaccharide wall. Gram positive organisms have this thick wall, while gram negative organisms do not. To gram stain, first 4-5 drops of crystal violet are added onto the organism. Then after 60 seconds it is washed off with sterile water, and 4-5 drops of iodine is added for 60 seconds. Then hydrogen peroxide is added and left on for 10 seconds. Then 4-5 drops of safran is added for 60 seconds. Once that is done, the organism is observed under a microscope. The gram positive organisms appear purple, and the gram negative organisms appear pink. Gram negative organisms consist of non-fermenters and enteric bacteria. Gram positive organisms consist of staphococci and strepococci. Oxidase tests are done on gram negative bacteria to distunguish between non-fermenters and enteric bacteria. Positive tests appear for non-fermenters. Catalase tests are done on gram positive tests to distinguisg between staphococci and strepococci. Positive tests appear for staphococci. 

In the respirartory system, the air travels into the oral and nasal cavities. In the nasal cavity there are conchea and meatus. Continuing from the nasal cavity to the larynx and trachea, there is the nasopharynx, oropharynx, and laryngealpharynx. The larynx is protected by thyroid cartiage and the trachea is protected by cricoid cartilage. From there the air enters the primary bronchi. The primary bronchi split off into secondary brochi, and the secondary bronchi split off into tertiary brochi. The tertiary bronchi break off into brochioles, where there are aveoli sacs. Oxygen and carbon dioxide exchange into and out of the blood occur here. 

I just read about NASA’s twin study and it really is incredible. I never really thought about the effect that space or zero gravity would have on someone. The twin that went into space didn’t suffer too much on upon entering space. In fact he responded relatively well to being in space. In fact the shortening of his chromosomal telomeres was reduced when in space. This means that his aging process was theoretically slowed. That being said when he returned to Earth his chromosomal telomeres shortened almost immediately and his body had an autoimmune response. His body thought that something was attacking him when in actuality he was not sick at all. He was just adjusting to the full force of gravity. His legs started to swell, he developed rashes, he was in a lot of pain. It took more than eight months for him to return to normal. It did take a toll on his body. The purpose of the study was to see how zero gravity affects the body long term. NASA wanted to know this because they want to know how plausible space travel really is.   

The authors first cloned melanopsin cDNA in rat cells to show that the protein sequence is nearly identical to that of mice. Then they generated specific antibodies targeting melanopsin to show the subset of cells that contained the protein. Tau-lacZ targeting shows the projections of melanopsin positive cells to the SCN and other regions of the brain. Lastly, they used a combination of immunofluorescence and Lucifer Yellow to show that intrinsically photosensitive RGC’s were melanopsin positive. Figure 3 shows that the authors targeted the tau-lacZ gene locus RGC’s and used immunofluorescence in mice. The structure of melanopsin positive cells was similar to that in rats. X-gal labeling not only showed retinal labeling of axons, cell bodies and dendrites, but it also showed B-galactosidase activity in parts of the brain such as the SCN, the olivary pretectal nucleus, the dorsal lateral geniculate, and other parts of the brain. This finding suggested that melanopsin positive cells are involved with processing information that is relayed to the brain.

The experiment showed that melanopsin is the photopigment present on specific RGC’s that is likely to be involved in photoentrainment. Figures 1 and 2 show melanopsin positive RGC’s using immunofluorescence in rats. Melanopsin positive cells were only about 1% of the total number of RGC’s, and only a few of the melanopsin positive cells are found in the inner nuclear layer while the rest are in the ganglion cell layer. Confocal, stained images as well as digital images of melanopsin positive RGC’s were generated. The data from figures 1 and 2 showed the abundance of melanopsin on the dendrites, axons, and cell bodies in addition to the shape and structure of RGC’s. 

The article, “Effects of instantaneous and growth CO2 levels and abscisic acid on stomatal and mesophyll conductance” by Yusuke Mizokami, Ko Noguchi, Mikiko Kojima, Hitoshi Sakakibara, and Ichiro Terashima (2017) was a research specific to analyze the trend of the mesophyll conductance changing with different CO2 levels and abscisic acid, abbreviated as ABA. The research was based on the results obtained in 2015 by Mizokami et al. that dealt with mesophyll conductance in drought environments. To understand further about the mesophyll conductance and the stomatal conductance in plants, the new case study was developed. To obtain a more accurate data than from the previous experiment, they included additional factors to consider; such as the relationship between stomatal conductance and the intercellular CO2 concentration (Mizokami et al., 2017). In the past, Mizokami et al. have concluded two points. First, if ABA level increases in a leaf, both mesophyll and stomatal conductance decreases. Secondly, ABA is not responsible for decreasing both conductance because the ABA-deficient plants also decreased both conductance in response to high CO2 levels (Mizokami et al., 2017). With these two points in mind, the new study was carried out to understand the function of mesophyll conductance (Mizokami et al., 2017)

Most plants  are composed of two very distint parts; xylem and phloem. These plants are known as being vasuclar, the simple small plants such as mosses and algae, do not have these parts and are known as non-vascular plants. Xylem tissue is mostly used for transporting water from the roots to the leaves. Phloem is used for transporting compounds produced in the leaves to the roots. This movement from leaves to the roots in the phloem is bidirectional, but in the xylem it is not. However, the xylem is made up of dead cells which are elongated and hollow. In contrast, the phloem is made up of cells such as sieve tubes and companion cells. Both of these parts of vascular plants are essential in transporting nutrients and keeping the plant hydrated. Photosynthesis in leaves requires a lot of water from the xylem and produces lots of sugar for the phloem. Although these parts are quite distinct they work together to allow the plant to flourish. 

This was also about a talk I went to at Harvard. This talk was very dense on the engineering aspect of microbiology which I found interesting because it was something that I don’t typically think about. The main point of the talk was discovering ways in which we can categorize and catalog bacteria. The proposed method was by looking at bacterial cell envelopes and inducing a dipole on them to determine how they behave. The other half of the talk was about about how we can use electric fields to help introduce new DNA and genes to cells. This is where the talk became incredibly engineering heavy and a prototype machine that could carry out multiple electroporation experiments at once was introduced. The way that bacterial cells were categorized by their cell envelope was with a technique called low frequency dielectrophoresis. With this technique a dipole is induced on the cell by creating a non-uniform gradient. The bacterial cells were then placed in tubes that had a constriction point, it is at this point that the now polarized cells feel the dielectrophoresis force and clump at the constriction point. The bacteria used in this experiment was a mutant strain of Streptococcus mitis. A mutant was used because S. mitis typically has a virulence factor that causes clumping, the mutant had this virulence factor removed so that any clumping near the constriction point would be fully attributed to the dielectrophoresis force and not the virulence factor. Two other bacterial strains were used to observe polarizability. The first, Geobacter sulfurreducens polarizability was studied by observing its extracellular electron transfer mechanics. The second, Shewanella oneidensis polarizability was studied by looking at its Mtr pathway. This gene was later inserted into Escherichia coli to see if it was also impact the polarizability of it. The engineering part was to make a machine that carried out multiple electroporation experiments at once. This was done by condensing the apparatus down to the size of a pipette tip.

There are multiple TAAs common in pancreatic cancer that have been or are being targeted for immunotherapy. Ideally, treatment can incorporate vaccines for a few TAAs to account for patient tumor diversity. Carcinoembryonic antigen (CEA) is a particularly attractive TAA to incorporate into a vaccine as it is overexpressed in over 90% of pancreatic cancer. A clinical trial with 1 mg of the CEA vaccine CAP1-6D elicited robust CD8+ T cell responses in 7 out of 19 tested patients (Geynisman et al. 2013). Other potential targets for vaccines include KIF20-A (part of the kinesin family), KRAS, WT-1, and VEGF (Banerjee et al. 2018). I need to research each of these more to see which ones seem best to incorporate into our treatment plan. Activators of the STING protein, which induces pro-inflammatory responses through the INF-beta and NF-kappaB pathways, seems to contribute to the regression of tumors via T cell recruitment as well as enhance the responses of anti-CTLA4 and anti-PDL1 immunotherapies (Banerjee et al. 2018). More research is needed in this topic as well, but perhaps a cancer vaccine and a STING activator can be used in combination in hopes of creating an overall more effective immunotherapy.