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November 7-11

On Monday in molecular biology, Dr. Peng discussed translation in bacteria and eukaryotes. The third test in that class was on Wednesday and covered site-specific recombination and translocation of DNA, transcription mechanisms, RNA splicing, and translation.

In biochemistry on Tuesday and Thursday, we learned about lipids’ roles in membranes, including the phospholipid bilayer of biological membranes and the fluid mosaic model. In bacterial physiology, we began with the structure of microbial communities, particularly biofilm formation. We continued with cell-to-cell communication systems, such as quorum sensing, which allows bacteria to sense how many of their own kind are in the population. Quorum sensing is used to coordinate varied activities such as competence, virulence, biofilm formation, sporulation, and bacteriocin production. We also discussed symbiosis such as mutualism, cooperation, commensalism, predation, and parasitism.

On Friday, the bacterial genetics lab met in the computer lab to analyze the Sanger sequences of the RFLP assay. The data did not work out as well as expected, probably because the PCR product was not incubated long enough with the restriction enzyme and the enzyme did not cut all the sequences. We have the final test for the lab next week, and then the class will be completed.

Article Review 7

A Trp574 to Leu Amino Acid Substitution in the ALS Gene of Annuai Biuegrass (Poa annua) Is Associated with Resistance to ALS-lnhibiting Herbicides

ALS-inhibiting herbicides target the ALS enzyme, which controls branched chain amino acid (Val, Leu, Ile) synthesis, causing plant death. The most common mechanism of resistance in plants is a point mutation in the target gene, and 6 mutations have been associated with herbicide resistance in several weed species. Annual bluegrass is one of the most troublesome weeds, especially in the southeastern United States, and several resistant populations have recently been reported. The purpose of this study was to determine the mechanism of resistance in an annual bluegrass population from Alabama. Resistance was confirmed in the AL population compared with a susceptible population, and the ALS gene was then sequenced. The resistant population showed a mutation at Trp574, and a smaller amplicon surrounding the mutation was cloned and sequenced to look at different forms of ALS. The sequence confirmed the Trp to Leu mutation at position 574 in the ALS gene. This mutation has been shown to confer resistance to ALS-inhibiting herbicides in other plant populations, indicating that the mutation may be the cause of resistance in the AL population.

The authors sequenced 15 plaques at each stage of amplification, so the results should be reliable. The results also agree with previously published studies. The research is definitely relevant, as annual bluegrass is resistant to herbicides with many different mechanisms of action. There has not been much research into nonchemical mechanisms of control, so there may come a point where annual bluegrass has developed resistance to most or all herbicides. Recently, resistant populations have been found in Mississippi, Tennessee, and Georgia as well as the original population in Alabama.

More research should be done into nonchemical mechanisms of annual bluegrass control, as it develops resistance rapidly. Alternatively, more research could be done to develop an herbicide with a novel mechanism of action.

McElroy, J. Scott, et al. “A Trp574 To Leu Amino Acid Substitution In The ALS Gene Of Annual Bluegrass (Poa Annua) Is Associated With Resistance To ALS-Inhibiting Herbicides.” Weed Science 61.1 (2013): 21-25. Environment Complete. Web. 7 Nov. 2016.

October 31-November 4

In molecular biology on Monday and Wednesday, Dr. Peng lectured about translation in prokaryotes and eukaryotes. He discussed the structure of tRNAs as well as tRNA charging by class I and class II aminoacyl-tRNA synthetases. We also learned about ribosome structure and translation initiation, elongation, and termination.

In biochemistry on Tuesday and Thursday, Dr. Popescu discussed lipids, including sections on the biological roles of lipids and structure and properties of storage, membrane, and signaling lipids. We learned about all 6 classes of lipids – fatty acids, triacylglycerols, wax esters, glycerophospholipids, sphingolipids, and isoprenoids. On Tuesday in bacterial physiology, we learned about bacterial two-component signaling systems such as the Arc, Nar, Che, Pho, Bvg, Agr, Spo, PhoQ/PhoP, and EnvZ/OmpF systems. We also began to discuss microbial communities and environmental stresses such as heat shock response, SOS response, and oxidative stress responses.

Bacterial genetics lab was cancelled on Friday because the Sanger sequencing results weren’t ready. We had planned to do computer analysis of the results.

October 24-28

On Monday and Wednesday in molecular biology, Dr. Peng lectured on RNA splicing mechanisms and RNA editing. He reviewed important proteins and nucleic acids in the spliceosome such as snRNPs (small nuclear RNA paired with protein). He also went over the three ways of splicing an RNA – nuclear pre-mRNA, group I introns, and group II introns. We then moved on to alternative splicing and RNA editing such as site-specific deamination and RNA-directed uridine insertion/deletion.

On Tuesday and Thursday in biochemistry, Dr. Popescu began the chapter on carbohydrates and glycobiology. We started with basic structures of monosaccharides including open chain and ring forms and moved to disaccharides formed via glycosidic bonds, before looking at structures and metabolism of common polysaccharides including glycogen, starch, cellulose, and chitin. We concluded the chapter discussing the biological function of glycoconjugates – glycolipids, glycoproteins, and proteoglycans. In bacterial physiology, we concluded the section on metabolism by learning about the tricarboxylic acid cycle (TCA cycle), the electron transport chain, oxidative phosphorylation, and the 5 types of fermentation in bacteria (lactic acid, ethanol, butyric acid, mixed acid, and propionic acid fermentation).

On Friday in bacterial genetics lab, we performed plasmid preparation to separate the plasmids from the E. coli and chromosomal DNA and proteins. Dr. Brown also lectured on Sanger sequencing in preparation for analysis of our sequences.

October 10-21

The Monday before fall break, I had a test in molecular biology on DNA mutability and repair, homologous recombination, site-specific recombination, and transposition of DNA. The Monday and Wednesday after fall break, Dr. Peng reviewed transcription mechanisms in bacteria and eukaryotes.

In biochemistry on Tuesday, we had a review for the test on Thursday. The test covered protein metabolism, enzymes, nucleotides and nucleic acids, regulation of gene expression, and DNA-based technologies. In bacterial physiology, Dr. Roberts reviewed bacterial metabolism, including the Embden–Meyerhof–Parnas (EMP) pathway, the Entner–Doudoroff (ED) pathway, and the pentose phosphate pathway (PPP), focusing on key intermediates, enzymes, and regulators.

In bacterial genetics lab on Friday, we prepared a restriction fragment length polymorphism (RFLP) assay on our PCR products.

 

Article Review 6

Global Methylation In The Placenta And Umbilical Cord Blood From Pregnancies With Maternal Gestational Diabetes, Preeclampsia, And Obesity.

Metabolic disorders such as global diabetes mellitus (GDM), preeclampsia, and obesity in pregnant women are risk factors for future diseases in the infant such as metabolic challenges and neurobehavioral impairments in childhood. While the biological mechanisms linking the metabolic problems in mothers to future diseases in the child have not yet been elucidated, epigenetic factors such as DNA methylation may play a part. This study investigates the relationships between global methylation in placenta and umbilical cord blood; maternal GDM, preeclampsia, and obesity; and newborn phenotypes such as weight, gestational age at birth, body length, and head circumference at birth. There was no correlation between global methylation in placenta and umbilical cord blood. There were no significant differences in global methylation levels in umbilical cord blood in women with metabolic disorders compared with controls. However, in women with GDM and preeclampsia, the global methylation levels in the placenta were lower than in controls, and in obese women, the methylation levels were higher than in controls. Methylation levels in umbilical cord blood were not associated with any infant phenotypes, but methylation levels in the placenta were associated with head circumference and body length. Specifically, a greater methylation level was associated with smaller head circumference and body length in the infants.

Metabolic disorders such as GDM and obesity have become more frequent as people consume more sugar, saturated fats, and processed foods. This research is, therefore, relevant to today’s world as more and more women will have metabolic disorders before and during their pregnancy. The sample size for this study was small, though it was only intended to be a preliminary study.

This study should be replicated with a larger sample of women and a longer follow up period for evaluation of development in the child. Additionally, gene-specific methylation studies should be performed on candidate genes to determine if there is correlation between methylation in either the placenta and umbilical cord blood and maternal metabolic disorders.

Nomura, Yoko, et al. “Global Methylation In The Placenta And Umbilical Cord Blood From Pregnancies With Maternal Gestational Diabetes, Preeclampsia, And Obesity.” Reproductive Sciences (Thousand Oaks, Calif.) 21.1 (2014): 131-137. MEDLINE. Web. 20 Oct. 2016.

Article Review 5

Cerebral Perfusion Pressure In Women With Preeclampsia Is Elevated Even After Treatment Of Elevated Blood Pressure

Maternal deaths from preeclampsia occur mainly due to cerebral infarction and hemorrhage, possibly due to increased cerebral perfusion pressure (CPP). High CPP has been linked to preeclamptic symptoms and eclamptic seizures. Labetalol and MgSO4, drugs used to control hypertension in preeclamptic patients, cause a decrease in CPP, while another antihypertensive drug, nimodipine, causes CPP to increase. Nimodipine is associated with more eclamptic seizures, possibly due to the increase in CPP. The purpose of the study was to evaluate the CPP in preeclamptic women with blood pressure controlled by antihypertensives. The CPP was calculated using zero-flow pressure and mean arterial blood pressure measurements. The zero flow pressure, arterial blood pressure, CPP, and CFI were all higher in preeclamptic women than control women. Additionally, a correlation was found between mean arterial blood pressure and CPP in preeclamptic patients, but not in controls.

As cerebrovascular complications are the main cause of death to preeclamptic women, and preeclampsia affects 3-8% of all pregnancies, this study is relevant. The authors used two methods to calculate the CPP, and, in both cases, the CPP was higher for preeclamptic women. Additionally, the authors report that the CPP calculated for the control patients was similar to that reported in other studies, which shows consistency between studies. The authors did, however, use non-invasive methods to measure the CPP, which have not been validated in humans. Nevertheless, they got similar results with both calculations of CPP.

Future studies could include trials with additional or alternative antihypertensive medications to bring the CPP to normal levels along with the blood pressure. The effect of lower CPP on the risk of cerebrovascular complications can then be observed. Additionally, studies could be done to validate the techniques for non-invasive measurement of CPP in humans.

Sonneveld, Milan J., et al. “Cerebral Perfusion Pressure In Women With Preeclampsia Is       Elevated Even After Treatment Of Elevated Blood Pressure.” Acta Obstetricia Et Gynecologica Scandinavica 93.5 (2014): 508-511. Academic Search Complete. Web. 20 Oct. 2016.

Article Review 4

CXCR4, CXCR7, and CXCL12 Are Associated with Trophoblastic Cells Apoptosis and Linked to Pathophysiology of Severe Preeclampsia

The purpose of this paper was to examine the expression of the chemokine CXCL12 and its receptors, CXCR4 and CXCR7, in placental tissues and primary trophoblasts from preeclamptic and normal pregnancies. CXCL12 and its receptors are involved not only in the immune system, but also in various other processes, including remodeling the spiral arteries to form the placenta. CXCL12 and CXCR4 have been shown to be involved in trophoblast proliferation and invasion and decidual stromal cell migration and motility; lack of either of these functions is associated with preeclampsia. The authors examined the association between these proteins and preeclampsia.

Placentas were obtained after a cesarean section (21 normal pregnant, 11 mild preeclamptic, and 18 severe preeclamptic). A scanning electron microscope was used to observe morphological changes of trophoblast cells in preeclamptic placentas that caused a significant tendency towards apoptosis. Immunohistochemistry showed that there were lower levels of CXCR4, CXCR7, and CXCR12 were lower in severe preeclamptic placentas than in normal placentas. RT-qPCR was used to show that the same pattern was true for mRNA expression of the proteins, and Western blot analysis confirmed for protein expression levels in trophoblasts. The authors concluded that apoptosis of the cytotrophoblast cells may be regulated by expression of CXCR4, CXCR7, and CXCL12.

All three proteins have been shown to be important for proper placenta formation, so I think the research to compare expression between normal and preeclamptic patients is necessary and relevant. I think the study was well designed, especially by using different techniques to confirm results. However, the number of samples was small, only 50 in total, probably because the placenta was required for testing.

It would be interesting to see if the results in this study were replicated in a future study with more samples. While the study observes a decrease in the three proteins studied, more studies are required to understand the how they relate to the pathophysiology of preeclampsia. Additional studies should be done to understand the mechanism of cytotrophoblast invasion and how CXCL12 and its receptors relate to this.

 

Lu, Jing, et al. “CXCR4, CXCR7, And CXCL12 Are Associated With Trophoblastic Cells Apoptosis And Linked To Pathophysiology Of Severe Preeclampsia.” Experimental And Molecular Pathology 100. (2016): 184-191. ScienceDirect. Web. 9 Oct. 2016.