AKANKSHA fluorescence at 675nm with a large

AKANKSHA DAVE14IMSILS003BIOCHEMISTRY ASSSIGNMENT 2IMSC SEM 7QUESTION 1: Write 200 words summary of a recently discovered fluorescent protein with ~300 nanometer Stokes shift from a fish! ANSWER: In recent research, it is discovered that sandercyanin fluorescent protein (SFP) can be engineered for biological imaging applications. SFP is found on the dorsal side of walleye and is produced seasonally (mostly in late summer) and is possibly produced in sacciform cells in the epidermis layer of fish. It was found that the walleye (Sander vitreus),a golden yellow fish found in north America, which are very sensitive to light , have adapted to increased UV radiation by formation of sandercyanin-Biliverdin IX? complex in its skin mucus on the dorsal side exhibiting blue colour. Sandercyanin-BLA complex acts a natural sunscreen to the walleye. Increased UV radiation degrades heme to Biliverdin Ix? (BLA) which is a chromophore attached to sandercyanin. Sandercyanin is a monomeric protein. When BLA gets bound to sandercyanin, it forms a stable homotetramer complex in which each subunit is of 18.6KDa. The sandercyanin-BLA complex extracted from a walleye mucus shows a glycosylated protein with a lipocalin fold. Properties of sandercyanin-BLA complex are:Excitation wavelength at 375nm in UV regionEmission wavelength at 675nm which gives red fluorescenceBLA is noncovalently bound to sandercyaninLarger stokes shift (~300nm)BLA binds to hydrophobic part of sandercyaninSpecific pi-stacking interactions Water mediated hydrogen bonding between sandercyanin and BLADue to hydrophobicity, specific stacking interactions and hydrogen bonding, BLA is able to consume energy at 375nm and then generate far red fluorescence at 675nm with a large spectral shift of 300nm.REFERENCES:https://www.google.com/patents/US20140272964https://www.researchgate.net/publication/309412214_Blue_protein_with_red_fluorescenceQUESTION 2: Write 300 words summary hallmarks of cancer. ANSWER: The emerging hallmarks of cancer are:Deregulated uptake of glucose and amino acids: Two important nutrients and metabolites that fulfil the biosynthetic of cell proliferation and growth are GLUCOSE and GLUTAMINE. Increase in uptake of glucose by cancer cells compared to normal nonproliferating tissues is considered as a metabolic hallmark of cancer. In normal cells forced glucose transporter (GLUT4) occurs but in cancer cells it does not occur. Positron emission tomography uses fluorescent labelled glucose (F18-fluorodeoxyglucose) to determine uptake of glucose in cancer cells and diagnose cancer. Glutamine is second important substrate used as hallmark of cancer. High uptake of glutamine by cancer cells is considered as hallmark of cancer.Opportunistic nutrient acquisition: Cancer cells also face nutrient scarcity due to increased uptake of nutrients and inadequate tumour vascular supply. This force them to adapt other opportunistic ways for uptake of nutrients, which are suitable for them to survive and proliferate even in unfavourable conditions. If extracellular nutrients are unavailable, they adapt the inaccessible metabolic pathways to synthesize the required metabolites which help us to differentiate between normal cells and tumour cell.Use of glycolysis/TCA cycle intermediates for biosynthesis and NADPH production: In cancer cells, not only the amount of metabolites are increased but there is also a change in the way of use of those metabolites and nutrients. In quiescent cancer cells, glucose is used for acetyl-CoA synthesis in mitochondria which is then used in tricarboxylic (TCA) cycle for oxidation. Further through electron transport chain (ETC), ATP production is carried out. But in cancer cells, oxidative phosphorylation is more compared to glycolysis.Increased demand of nitrogen:  Nitrogen consumption in biosynthetic pathways in cancer cells increases which leads to increase in demand of nitrogen. This is considered as hall mark of cancer.Alterations in metabolite driven gene regulation: P53 is considered as a master regulator of cell cycle. Point mutation in P53 gene leads to higher cell division which leads to more flux through glycolysis and due to which there is in increase acetyl-CoA concentration. Increase in acetyl-CoA, leads to altered histone modification. This shows that in cancer cells a metabolite acts a signalling molecule for gene regulation.Metabolic interactions with microenvironment: Loss of contact inhibition, angiogenesis, etc.Metastasis: Cancer cells initiate from a particular cell and can spread to any tissue, organ or nearby lymph node.REFERENCES:http://www.cell.com/cell-metabolism/fulltext/S1550-4131%2815%2900621-Xhttps://www.cancer.gov/types/metastatic-cancerQUESTION 3: Write 100 words EACH for all the types of coherent and incoherent regulatory networks ANSWER: There are 4 types of coherent and 4 types of incoherent network motifs:C1FFL (Coherent 1 feed forward loop) = Most abundant type of FFL. Mostly found in E.coli and yeast. It shows positive sign for all regulations involved in circuit. In steady state, gene Z receives two inputs, one from X directly and one from Y indirectly from X. Thus X controls Z in direct pathway and X also controls Y in indirect pathway. In signal ON state in AND circuit, transcription factor X becomes active and expresses Z(direct pathway) and transcription factor X becomes active and activates Y which gets expressed and bind to Z (indirect path)which results into delayed expression of Z. In OR gate there is delayed degradation.C2FFL (coherent 2 feed forward loop) = In AND circuit, when X signal is ON, there is no gene expression. When X signal is OFF the transcription factor for X is not activated and thus it does not directly activate Z. But in AND circuit, through indirect path, there is delayed expression of Z.C3FFL (coherent 3 feed forward loop) = In AND circuit the system reaches steady state when there is inverted signal for gene X expression. In signal ON condition, there is no gene X expression in AND gate. When signal is OFF, in AND gate, through direct pathway the gene expression of gene Z is repressed. But in indirect pathway there is delayed expression of gene Z.C4FFL ( Coherent 4 feed forward loop) = In AND circuit, the system reaches steady state when signal for X expression is present and Z gene is expressed even if gene Y is repressed. When signal for gene X expression is OFF, there is no gene expression but when signal for X expression is ON, delayed expression of Z gene is there though gene Y is not expressed in indirect pathway. In direct pathway, Gene Z is expressed normally.I1FFL (Incoherent 1 feed forward loop) =AND / OR gates not applicable. When signal for gene X expression is present and there is inverted signal for gene Y expression, steady state is obtained. When gene X expression signal is ON, expression of protein Z is weak but when signal for gene X expression is OFF, no gene expression. There is no effect of gene Y signalling as gene Y is repressed.I2FFL (Incoherent 2 feed forward loop) = AND / OR gates not applicable. Steady state is obtained when signal for gene expression of both X and Y are inverted. When signal for X gene expression is ON , gene Z and gene Y are repressed, hence gene Z is not expressed. When signal for gene X expression is OFF, protein expression of gene Z is very weak. No effect of signalling of gene Y as Z is repressed by gene Y.I3FFL (Incoherent 3 feed forward loop) = AND / OR gates not applicable. No steady state. Gene X directly controls gene Z expression i.e when signal for gene X expression is ON it will repress gene Z. when signal for gene expression of X is OFF, there will be gene Z expression. No effect of signalling pathway of gene Y because it is repressed.I4FFL (Incoherent 4 feed forward loop) = AND / OR gates not applicable. No steady state. When signal for gene X expression is ON, gene Z is expressed. When signal for gene X expression is OFF, there is no response and hence no gene Z expression. No effect of signalling pathway of gene Y as it is repressing the expression of gene Z. REFERENCES:nptel.ac.in/courses/102106035/Module%204/Lecture%205/Lecture%205.pdfQUESTION 4: Write a 300 words detailing role of metabolites in regulation. ANSWER: There are critical metabolic pathways and critical metabolites which are crucial for gene regulation.Metabolites involved in glycolysis play a vital role in gene regulationDue to increased uptake of glucose there may be deregulation in cellular mechanisms which lead to cancer.Increased uptake of glutamine also changes a normal cell to tumour cell.During glycolysis, increase in acetyl-CoA leads to altered histone modifications which become very crucial for DNA packaging and thus mutation may occur.Increased production of lactic acid.Metabolites like P53 play a major role in signalling pathways.Increased oxidative phosphorylation.Increase in yield of kreb’s cycle and decrease in yield of glycolysis due to metabolic reprogramming.REFERENCES:http://www.nature.com/scitable/topicpage/cell-metabolism-14026182