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3. Zebrafish are an organism capable of heart regeneration. Since heart disease is a leading cause of death in the United States, understanding the mechanisms of zebrafish heart regeneration could lead to new therapies for humans. In a recent study, researchers investigated the role of autophytes. Autophygi is a process where cells break down and recycle damaged components, which supports healthy cell cycle progression and tissue repair. Autophygi is linked to cell cycle regulation by providing the necessary cellular resources and removing damaged cells that could disrupt normal cell division. This process is especially important during tissue repair, like when the heart is regenerating. (a) Identify the cellular organelle that contains the hydrolytic enzymes important in autophygi: intracellular digestion, recycling of cell materials, and programmed cell death (apotosis). (I/P) To study autophygi in zebrafish hearts, scientists used a method called Westem blotting. Western blotting starts with gel electrophoresis, where proteins are separated as they move through a gel. Once the proteins are separated, they are transferred to a special membrane. Scientists then use antibodies that bind to specific proteins, which are later visualized as bands. The darker or more intense the band, the higher the amount of that protein in the sample. (b) Gel electrophoresis is a laboratory method used to separate DNA fragments, RNA, or proteins. Describe how gel electrophoresis separates molecules as they move through the gel matrix. (I/P) In this study, the researchers measured three important proteins related to autophagy: Becilin, which starts the process and helps cells divide; L-C3,1, in theactive form of LC3; II, the active form that signals autophagy. The results of the Westem bar also shown in Figure 1. GAPDL was used as a loading control, a way to make sure the protein amounts were accurately compared across all samples. "S" stands for sham-treated hearts (uninjured), and "A" stands for amputated hearts (injured). The researchers checked protein levels at different days after injury, called days post-amputation (dpa). The results of the Western blue reveal how autophagy-related proteins change over time after heart injury. This gives scientists clues about how autophagy helps zebrafish heal their hearts. Figure 1: Western blot analysis of Beclin-1, LC3-1, and LC3II levels at 3 dpa, 7 dpa, and 28 dpa. (c) (i) Identify the time point(s) (3,7 , or 28 dpa) with the largest difference in LC3-II levels between sham (S) and mputated (A) hearts? (I p) (ii) Based on the results of the Western blot, determine the timeline of autophagy activation and completion during zebrafish heart regeneration. (I pt) (d) Recent research suggests that the regulation of autophagy-related genes may be linked to histone modifications, like methylation and acetylation. Explain how histone modifications affect gene expression. (I pt)

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3. Zebrafish are an organism capable of heart regeneration. Since heart disease is a leading cause of death in the United States, understanding the mechanisms of zebrafish heart regeneration could lead to new therapies for humans. In a recent study, researchers investigated the role of autophytes. Autophygi is a process where cells break down and recycle damaged components, which supports healthy cell cycle progression and tissue repair. Autophygi is linked to cell cycle regulation by providing the necessary cellular resources and removing damaged cells that could disrupt normal cell division. This process is especially important during tissue repair, like when the heart is regenerating. (a) Identify the cellular organelle that contains the hydrolytic enzymes important in autophygi: intracellular digestion, recycling of cell materials, and programmed cell death (apotosis). (I/P) To study autophygi in zebrafish hearts, scientists used a method called Westem blotting. Western blotting starts with gel electrophoresis, where proteins are separated as they move through a gel. Once the proteins are separated, they are transferred to a special membrane. Scientists then use antibodies that bind to specific proteins, which are later visualized as bands. The darker or more intense the band, the higher the amount of that protein in the sample. (b) Gel electrophoresis is a laboratory method used to separate DNA fragments, RNA, or proteins. Describe how gel electrophoresis separates molecules as they move through the gel matrix. (I/P) In this study, the researchers measured three important proteins related to autophagy: Becilin, which starts the process and helps cells divide; L-C3,1, in theactive form of LC3; II, the active form that signals autophagy. The results of the Westem bar also shown in Figure 1. GAPDL was used as a loading control, a way to make sure the protein amounts were accurately compared across all samples. "S" stands for sham-treated hearts (uninjured), and "A" stands for amputated hearts (injured). The researchers checked protein levels at different days after injury, called days post-amputation (dpa). The results of the Western blue reveal how autophagy-related proteins change over time after heart injury. This gives scientists clues about how autophagy helps zebrafish heal their hearts. Figure 1: Western blot analysis of Beclin-1, LC3-1, and LC3II levels at 3 dpa, 7 dpa, and 28 dpa. (c) (i) Identify the time point(s) (3,7 , or 28 dpa) with the largest difference in LC3-II levels between sham (S) and mputated (A) hearts? (I p) (ii) Based on the results of the Western blot, determine the timeline of autophagy activation and completion during zebrafish heart regeneration. (I pt) (d) Recent research suggests that the regulation of autophagy-related genes may be linked to histone modifications, like methylation and acetylation. Explain how histone modifications affect gene expression. (I pt)

3. Zebrafish are an organism capable of heart regeneration. Since heart disease is a leading cause of death in the United States, understanding the mechanisms of zebrafish heart regeneration could lead to new therapies for humans. In a recent study, researchers investigated the role of autophytes. Autophygi is a process where cells break down and recycle damaged components, which supports healthy cell cycle progression and tissue repair. Autophygi is linked to cell cycle regulation by providing the necessary cellular resources and removing damaged cells that could disrupt normal cell division. This process is especially important during tissue repair, like when the heart is regenerating. (a) Identify the cellular organelle that contains the hydrolytic enzymes important in autophygi: intracellular digestion, recycling of cell materials, and programmed cell death (apotosis). (I/P) To study autophygi in zebrafish hearts, scientists used a method called Westem blotting. Western blotting starts with gel electrophoresis, where proteins are separated as they move through a gel. Once the proteins are separated, they are transferred to a special membrane. Scientists then use antibodies that bind to specific proteins, which are later visualized as bands. The darker or more intense the band, the higher the amount of that protein in the sample. (b) Gel electrophoresis is a laboratory method used to separate DNA fragments, RNA, or proteins. Describe how gel electrophoresis separates molecules as they move through the gel matrix. (I/P) In this study, the researchers measured three important proteins related to autophagy: Becilin, which starts the process and helps cells divide; L-C3,1, in theactive form of LC3; II, the active form that signals autophagy. The results of the Westem bar also shown in Figure 1. GAPDL was used as a loading control, a way to make sure the protein amounts were accurately compared across all samples. "S" stands for sham-treated hearts (uninjured), and "A" stands for amputated hearts (injured). The researchers checked protein levels at different days after injury, called days post-amputation (dpa). The results of the Western blue reveal how autophagy-related proteins change over time after heart injury. This gives scientists clues about how autophagy helps zebrafish heal their hearts. Figure 1: Western blot analysis of Beclin-1, LC3-1, and LC3II levels at 3 dpa, 7 dpa, and 28 dpa. (c) (i) Identify the time point(s) (3,7 , or 28 dpa) with the largest difference in LC3-II levels between sham (S) and mputated (A) hearts? (I p) (ii) Based on the results of the Western blot, determine the timeline of autophagy activation and completion during zebrafish heart regeneration. (I pt) (d) Recent research suggests that the regulation of autophagy-related genes may be linked to histone modifications, like methylation and acetylation. Explain how histone modifications affect gene expression. (I pt)

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(a) The cellular organelle that contains the hydrolytic enzymes important in autophagy, intracellular digestion, recycling of cell materials, and programmed cell death (apoptosis) is the lysosome. Lysosomes are responsible for breaking down and recycling cellular waste, damaged organelles, and macromolecules.<br /><br />(b) Gel electrophoresis separates molecules based on their size and charge as they move through a gel matrix. The gel acts as a sieve, allowing smaller molecules to move faster and farther than larger ones. An electric field is applied across the gel, causing charged molecules to migrate towards the opposite charge: negatively charged molecules move towards the positive electrode, and positively charged molecules move towards the negative electrode. The separation occurs because molecules with different sizes and charges will travel at different rates through the gel.<br /><br />(c)<br />(i) To identify the time point(s) with the largest difference in LC3-II levels between sham (S) and amputated (A) hearts, you would need to examine Figure 1 from the study. Typically, this involves comparing the intensity of the bands representing LC3-II at each time point (3 dpa, 7 dpa, and 28 dpa). The time point with the most significant difference in band intensity between S and A indicates the largest difference in LC3-II levels.<br /><br />(ii) Based on the results of the Western blot, the timeline of autophagy activation and completion during zebrafish heart regeneration can be determined by observing changes in protein levels over time. Generally, an increase in LC3-II levels indicates autophagy activation, while a return to baseline levels suggests completion. By analyzing the Western blot data, one can infer when autophagy is most active and when it subsides during the regeneration process.<br /><br />(d) Histone modifications, such as methylation and acetylation, affect gene expression by altering the chromatin structure and accessibility of DNA to transcription machinery. Acetylation of histones typically leads to a more open chromatin structure, facilitating access to transcription factors and promoting gene expression. In contrast, methylation can either activate or repress gene expression, depending on the specific amino acids modified and the context within the chromatin. These modifications serve as signals that regulate the transcriptional activity of genes, including those involved in autophagy.
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