cnwokemodoih's blog

Biodiversity is the number of various species of organisms in a given place. Biodiversity ensures that every organism within an ecosystem has a niche in which it can thrive. Generally, the more biodiversity an area has, the more sustainable it is and the more it is able to recover from disaster. With more variation and numbers, an illness for example could infect one species but it would not severely hurt the ecosystem it is in. Lawns have become pervasive with their growing significance in human experiences. Biodiversity is also a feature that accompanies the presence of lawns because various species of flora and fauna exist in these miniature habitats. Plant diversity on lawns is impacted significantly by human disturbances like mowing and use of herbicides which are characteristic of common maintenance practices. These effects have been studied extensively in urban areas but not so much in small towns like Amherst, Massachusetts. In this study, we will investigate the role of these human disturbances here at the University of Massachusetts Amherst and around. We will start by obtaining relevant data about maintenance practices and carrying out sampling experiments in 6 selected lawns. Analysis and comparison of the obtained data will reveal if, in fact, lawn maintenance practices negatively affect plant species diversity. This will provide a framework and resources for future decision-making in terms of biodiversity conservation.

Whole mount in-situ hybridization starts off with two independent procedures that eventually converge: probe generation and preparation of embryos. The entire procedure becomes one during the hybridization step, with I will describe in greater detail.

Probe generation will allow me to synthesis the antisense RNA probes that will bind the alpha-4 gene and help visualize gene expression. The step begins with obtaining the cDNA of the alpha-4 gene. Since the cDNA has been obtained by reverse transcription of mature alpha-4 mRNA, the issue of having introns within the sequence is circumvented. The polymerase chain reaction (PCR) of the cDNA sequence will allow me to amplify the sequence and introduce the RNA polymerase promoter. After PCR, I will purify the resultant sequences before performing an in-vitro transcription to synthesize the antisense RNA probe. Once I’ve made the RNA probes, all further steps will be performed after the application of RNAse-away on all surfaces to avoid probe degradation; RNAse degrades RNA molecules. I will also use RNAse-free water and filter tips as further precautionary measures. Observing the necessary precautions, I will label the RNA probes with digoxigenin uridine-5’-triphosphate, which will enable visualization of alpha-4 gene expression alter on.

The zebrafish  (Dania rerio) model has become popular for studying epilepsy and even other neurodevelopmental disorders, as they possess certain features that make them ideal for the study of neural circuits. Zebrafish embryos and larvae develop externally and are optically transparent. This makes it easy to monitor the development of major organs as the zebrafish embryos progress from stage to stage (Monesson-Olson et al., 2018). Moreover, larval zebrafish undergo rapid development; sensory and motor systems are present and functional within five days post-fertilization (Monesson-Olson et al., 2018). The ability to generate large clutch sizes of 50-200 embryos adds to the favorability of zebrafish. Zebrafish, as model organisms, also hold great advantage because they have less complex central nervous systems compared to mammals, yet the cell types and mechanisms are conserved among vertebrates. Logistically, their small size and low cost of maintaining large colonies of adult wildtype and mutant fish make the use of zebrafish viable (reviewed by Baraban et al., 2010).

As a neurotransmitter, GABA acts across the synaptic cleft, the junction between the presynaptic and postsynaptic neurons. On being released, GABA binds to special protein receptors to trigger inhibitory postsynaptic potential (IPSP). GABA receptors are classified into two subtypes: GABAA and GABAB receptors. GABAB receptors are metabotropic G-coupled receptors that regulate the slow component of inhibitory responses (Wu and Sun, 2015). In contrast, GABAA receptors are ionotropic ligand-gated chloride channels. In mammals, GABA­A receptors are Heteropentameric; they are composed of various combinations of 19 subunits: α1-6, β1-3, γ1-3, δ, ε, π, θ and ρ1-3 (Monesson-Olson et al., 2018). Different subunit compositions are differentially localized in the brain and have varying functionality (Wu and Sun, 2015). Mutations that affect GABAA receptors have been implicated in different forms of epilepsy. For instance, mutated α1 subunit of the GABAA receptor have been discovered in patients suffering from early infantile  epileptic encephalopathy, juvenile myoclonic epilepsy and other types of seizure disorders (reviewed by Braat and Kooy, 2015). As a result, many pharmacological compounds have been developed to target GABAA ­receptors, by binding allosteric sites. Nonetheless, even with the established anticonvulsant activity of these compounds, adverse side effects still emerge (Braat and Kooy, 2015).

Much of the epilepsy research done today focuses on understanding biological and neural structures and networks. Researchers have progressively investigated the links between neurotransmitter signaling and epilepsy. These biochemical messengers, neurotransmitters, offer a gateway to understanding the mechanisms that underlie epilepsy. Notably, GABAergic signaling has become of great interest in epilepsy research. γ-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the adult brain. It performs its functions by maintaining an inhibitory tone that counterbalances neuronal excitation (Treiman et al., 2001). Perturbation of this balance between inhibition and excitation may result in seizures. GABA is a versatile neurotransmitter. Early on in development, it performs some excitatory functions that trigger corticogenesis (Wang and Kriegstein, 2009). GABAergic signaling plays a crucial role in the origin and spread, or suppression, of epilepsy. This is revealed in studies that used chemical agents to impair GABAergic signaling in animal models (Meldrum, 1989). Furthermore, GABA-mimetic molecules have been shown to be potent anticonvulsant therapies in seizure-prone mammals (Meldrum, 1989).

Epilepsy is the fourth most common disorder of the Central Nervous System. It occurs in patients of all ages but is most commonly experienced in childhood or late adulthood (Shaikh et al., 2018). It is characterized by recurrent, spontaneous episodes of excessive electrical discharge, in the brain, called seizures. (Cunliffe, 2015). The effects of epilepsy range from temporary loss of consciousness, or abnormal motor activity observed in minor involuntary movements, to whole-body convulsions (Cunliffe et al., 2012). Problems could also be psychological in nature, experienced by patients as anxiety and depression (Shaikh et al., 2018). Persistent, prolonged seizures often cause further damage to the CNS, affecting not just the quality of life for patients but also for their carers, thus revealing the far-reaching effects of the disorder. While brain abnormalities and the manifestation of brain lesions, such as brain tumors, do play roles in triggering epileptic symptoms, many forms of epilepsy are due to genetic factors (Reid et al., 2009). About 1% of the general population suffers from epilepsy but one-third of patients are unable to benefit from available treatment due to the nature of their disorder (Cunliffe et al., 2015). Some patients are unable to respond to anti-epileptic drugs (AEDs) due to some level of pharmacoresistance while others experience adverse side effects. This necessitates the exploration of new pathways through which epilepsy may occur and development of novel therapies to combat epilepsy with greater efficacy.

The first step in establishing mutant lines, in any organism, is the induction of mutations. Here, I'll narrow down on just zebrafish, one of the many model organisms used for scientific investigation. There are a number of ways to create mutations. A highly effective one is CRISPR/Cas9. It involves the injection of guide RNAs, Cas9 protein and stop oligos into embryos at the single-cell stage. CRISPR/Cas9 gives a high level of specificity to mutation induction. A wide variety of mutations are induced; some can be innocous point mutations and others can be harmful insertions/deletions of varying lengths. The injected generation of fish is referred to as G₀ and they are mosaic organisms, meaning that different populations of cells have different genotypes (i.e. carry different mutations). To clean this up and have organisms with uniform genotypes across all cell populations, the G₀ adult zebrafish have to be outcrossed with wildtype fish. Ideally, the next generation of zebrafish (F₁) laid should have heterozygotes carrying one wild-type allele and one mutant allele, along with homozygotes carrying only wildtype alleles. Owing to the unpredictability of the process, all the embryos obtained from a cross could very well be wildtype. This might be an indication that the induced mutation did not go germline. By that, I mean that the gametes never picked up the mutation. To determine the alleles being carried by the progeny, a process called genotyping must be performed. That will be the discussion for the next segment. 

The first step in establishing mutant lines is the induction of mutations in wildtype zebrafish. There are a number of ways to create mutations. A highly effective method is CRISPR/Cas9. It involves the injection of guide RNAs, Cas9 protein and stop oligos into embryos at the single-cell stage. CRISPR/Cas9 gives a high level of specificity to mutation induction. A wide variety of mutations are induced; some can be innocous point mutations and others can be insertions and deletions of varying lengths. This injected generation of fish is referred to as G₀. The injected adult fish that develop are mosaic organisms, meaning that different populations of cells have different genotypes (i.e. carry different mutations). To clean this up and have organisms with uniform genotypes, the G₀ adult zebrafish have to be outcrossed with wildtype fish. Ideally, the next generation of zebrafish (F₁) laid should have heterozygotes carrying one wild-type allele and one mutant allele, along with homozygotes carrying only wildtype alleles. Owing to the unpredictability of the process, all the embryos obtained from a cross could be wildtype. This might be an indication that the induced mutation did not go germline. To determine the alleles being carried by the progeny, a process called genotyping must be performed. That will be the discussion for the next segment. 

The fact that the replicator observed a similar interaction, different from that which was originally observed, shows that this interspecific interaction is rather common. The tree photographed in the replicated figure has a narrower trunk than that in the original figure. This difference is likely due to insufficient detail given in the initial methods, as to the exact location of the tree observed. On the lawn between the Morrill Science Center and the University Club, there are several trees, so it is easy for a replicator to choose the wrong tree. Hence, in subsequent procedures, numerically quantifiable directions may enhance accuracy. The difference in arrangements can be attributed to the lack of arrangement specifications in the initial methods description. The absence of letter boxes in the replicated figure is because the methods description followed by the replicator did not include instructions about lettering. This should be included in subsequent method descriptions to enable more compliance with the original figure. The methods description also did not mention anything about spacing the panels in the figure; as such, the replicator had room to make assumptions. To curtail this, subsequent methods description must specify details about spacing. The initial methods followed by the replicator did not include adequate description of how to depict magnification, hence the absence of it in the replicate. More detail must be provided in future methods to enable proper execution.

As a class activity for the Biology Junior Writing course at the University of Massachusetts Amherst, the methods project was assigned to help students build their procedure writing skills. This is an essential part of scientific investigation because it enables scientists to replicate experiments accurately. Replicability aids in establishing the authenticity of findings made during scientific inquiry. Here, I observed and recorded the interspecific relationship between a tree and green lichens found near the roots of the tree. In thinking of what interspecific relationship to observe, I considered the mobility of the interaction. I knew it was important to choose two species that did not move from their point of primary observation. I also ensured that the interaction was in a common area of campus, such as behind the Morrill Science Center, for convenience and accessibility purposes. During the period of observation, the weather had to be taken into account. To this effect, I chose an interspecific interaction that was not affected by sudden changes in weather but remained apparent for a prolonged period of time. As a result, the interaction was observable at the time of replication.