7: Metabolism II - Biology

7: Metabolism II - Biology

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In the last chapter, we focused on metabolic pathways that played important oxidative/reductive roles relative to cellular energy. In this chapter, the pathways that we cover have lesser roles from an energy perspective, but important roles, nonetheless, in catabolism and anabolism of building blocks of proteins and nucleic acids, nitrogen balance, and sugar balance. In a sense, these might be thought of as the “kitchen sink" pathways, but it should be noted that all cellular pathways are important. In this second section of metabolism, we cover metabolic pathways that do not have a strong emphasis on oxidation/reduction.

  • 7.1: Carbohydrate Storage and Breakdown
    Carbohydrates are important cellular energy sources. They provide energy quickly through glycolysis and passing of intermediates to pathways, such as the citric acid cycle, amino acid metabolism (indirectly), and the pentose phosphate pathway. It is important, therefore, to understand how these important molecules are made.
  • 7.2: Pentose Phospate Pathway
    Portions of the PPP are similar to the Calvin Cycle of plants, also known as the dark reactions of photosynthesis. We discuss these reactions separately in the next section. The primary functions of the PPP are to produce NADPH (for use in anabolic reductions), ribose-5-phosphate (for making nucleotides), and erythrose-4-phosphate (for making aromatic amino acids). Three molecular intermediates of glycolysis can funnel into PPP (or be used as usual in glycolysis).
  • 7.3: Calvin Cycle
    The Calvin Cycle occurs exclusively in photosynthetic organisms and is the part of photosynthesis referred to as the “Dark Cycle." It is in this part of the process that carbon dioxide is taken from the atmosphere and ultimately built into glucose (or other sugars). Though reduction of carbon dioxide to glucose ultimately requires electrons from twelve molecules of NADPH (and 18 ATPs). One reduction occurs 12 times (1,3 BPG to G3P) to achieve the reduction necessary to make one glucose.
  • 7.4: C4 Plants
    The Calvin Cycle is the means by which plants assimilate carbon dioxide from the atmosphere, ultimately into glucose. Plants use two general strategies for doing so. The first is employed by plants called C3 plants (most plants) and it simply involves the pathway described above. Another class of plants, called C4 plants employ a novel strategy for concentrating the CO2 prior to assimilation.
  • 7.5: Urea Cycle
    Yet another cyclic pathway important in cells is the urea cycle (Figure 7.5.1). With reactions spanning the cytoplasm and the mitochondria, the urea cycle occurs mostly in the liver and kidney. The cycle plays an important role in nitrogen balance in cells and is found in organisms that produce urea as a way to excrete excess amines.
  • 7.6: Nitrogen Fixation
    The process of nitrogen fixation is important for life on earth, because atmospheric nitrogen is ultimately the source of amines in proteins and DNA. The enzyme playing an important role in this process is called nitrogenase and it is found in certain types of anaerobic bacteria called diazotrophs. Symbiotic relationships between some plants (legumes, for example) and the nitrogen-fixing bacteria provide the plants with access to reduced nitrogen.
  • 7.7: Amino Acid Metabolism
    The pathways for the synthesis and degradation of amino acids used in proteins are the most varied among the reactions synthesizing biological building blocks. We start with some terms. First, not all organisms can synthesize all the amino acids they need. Amino acids that an organism cannot synthesize (and therefore must have in their diets) are called essential amino acids. The remaining amino acids that the body can synthesize are called non-essential.
  • 7.8: Amino Acid Catabolism
    Breakdown of glutamine by glutaminase is a source of ammonium ion in the cell. The other product is glutamate. Glutamate, of course, can be converted by a transamination reaction to alpha-ketoglutarate, which can be oxidized in the citric acid cycle.
  • 7.9: Nucleotide Metabolism
    Synthesis of ribonucleotides by the de novo method occurs in two pathways – one for purines and one for pyrimidines. What is notable about both of these pathways is that nucleotides are built from very simple building blocks.
  • 7.10: Pyrimidine de novo Biosynthesis
    Starting materials for pyrimidine biosynthesis include bicarbonate, amine from glutamine, and phosphate from ATP to make carbamoyl-phosphate (similar to the reaction of the urea cycle). Joining of carbamoyl phosphate to aspartic acid (forming carbamoyl aspartate) is catalyzed by the most important regulatory enzyme of the cycle, aspartate transcarbamoylase (also called aspartate carbamoyltransferase or ATCase).
  • 7.11: Purine de novo Biosynthesis
    Synthesis of purine nucleotides differs fundamentally from that of pyrimidine nucleotides in that the bases are built on the ribose ring. The starting material is ribose 5-phosphate, which is phosphorylated by PRPP synthetase to PRPP using two phosphates from ATP. PRPP amidotransferase catalyzes the transfer of an amine group to PRPP, replacing the pyrophosphate on carbon 1. Thus begins the synthesis of the purine ring.
  • 7.12: Deoxyribonucleotide de novo Biosynthesis
    Synthesis of deoxyribonucleotides de novo requires an interesting enzyme called ribonucleotide reductase (RNR). RNR catalyzes the formation of deoxyribonucleotides from ribonucleotides. The most common form of RNR is the Type I enzyme, whose substrates are ribonucleoside diphosphates (ADP, GDP, CDP, or UDP) and the products are deoxyribonucleoside diphosphates (dADP, dGDP, dCDP, or dUDP). Thymidine nucleotides are synthesized from dUDP.

Thumbnail: Metabolic Metro Map. Image used with permission (CC BY-SA 4.0; Chakazul).​​​​​​

What is Metabolism, & How Does it Work in Human Biology?

Metabolism is the sum of all the chemical processes or chemical reactions in the cells of living organisms that allows them to sustain life.

Metabolism is the summation of 3 main functions: to convert food to energy, to convert food to building blocks for the body, and to eliminate metabolic waste.

Metabolism also includes the chemical reactions that make up digestion and the cellular transportation of different substances between cells.

These enzyme-catalyzed chemical reactions that we call “metabolism” allows organisms to grow, reproduce, maintain cellular structures, and react to environmental stimuli.

Notice that I used the word “enzyme-catalyzed” when describing metabolism. In the context of the sentence above, it means that enzymes speed up the cellular chemical reactions fast enough to sustain life. In other words, enzymes are a key part of our metabolism. Without enzymes, our metabolism would be too slow, and we couldn’t live.

Catabolism & Anabolism

Further, Metabolism is divided into two categories: catabolism and anabolism.

Catabolism is the break down of organic matter. For example, during cellular respiration glucose breaks down to pyruvate.

Anabolism is the building up of cell components. For example, a cell’s production of proteins and nucleic acids.

In general, the break down of substances releases energy, and the build up of substances consumes energy.

A good example is fat when fat is broken down, energy is released. But when there is an excess amount of energy in the body, the body stores it back as fat.

What is a Metabolic Pathway?

A missing enzyme is like a missing domino…

Finally, let me tell you about metabolic pathways. First you should know that chemical reactions of metabolism can be organized into what is called a “metabolic pathway”. And a metabolic pathway is a chain of chemical reactions that occur inside a cell, often aided with enzymes (biological catalysts). Metabolic pathways are setup such that often the product of one reaction acts as the substrate for the next chemical reaction.

Note that metabolism heavily relies on enzymes, such that a metabolic pathway can be off if the cell does not have the required enzyme for any one of the pathway’s chemical reactions.

Another way to think about enzymes and pathways are with a domino analogy. If you take out a domino out of the middle of a line of dominos, and then push over the first domino, then the line of dominos won’t all fall down. But instead stop right where you removed the middle domino. And enzymes are like that the metabolic chain of reactions stop if you are missing an enzyme.

Waste Product Metabolism

Another chemical process under what we call “metabolism” is to eliminate metabolic waste. Metabolic waste are substances left over from metabolic processes like cellular respiration. These leftover substances are either surplus or toxic, and cannot be used by the organism.

Examples of metabolic waste include nitrogen compounds, water, CO2, phosphates, sulfates, and more.

Animals excrete these metabolic wastes. However, plants can salvage some of those metabolic wastes, particularly nitrogen compounds (think of what fertilizer is made of). And soil bacteria can further deal with left over metabolic waste. This way, you can see that there is an ecological cycle when it comes to metabolic waste.

7: Metabolism II - Biology

1. Molecular biology explains living processes in terms of the chemical substances involved.

The most frequently occurring chemical elements in living things are:

  • A variety of other elements are needed by living organisms including:
    • phosphorus
    • sulfur
    • iron
    • calcium
    • sodium

    2. Carbon atoms can form four covalent bonds allowing a diversity of stable compounds to exist.

    3. Life is based on carbon compounds including carbohydrates, lipids, proteins and nucleic acids.

    4. Metabolism is the web of all the enzyme-catalysed reactions in a cell or organism.

    5. Anabolism is the synthesis of complex molecules from simpler molecules including the formation of macromolecules from monomers by condensation reactions.

    • condensation synthesis
      • monomers linked together (=anabolized) to form polymers
      • through the release of H2O
      • with energy supplied by a nucleotide sugar (e.g. ADP-glucose)

      monosaccharides, disaccharides and polysaccharides:

      • condensation synthesis reactions link two monosaccharide monomers
      • forming one disaccharide molecule and one H2O molecule
      • repeated additions of monosaccharides produces a polysaccharide

      fatty acids, glycerol and triglycerides

      • three separate condensation synthesis reactions
      • link three fatty acid monomers to a single glycerol monomer
      • forming one triglyceride molecule and three H2O molecules

      amino acids and polypeptides:

      • two amino acid monomers are linked to form a dipeptide
      • releasing one H2O molecule
      • repeated condensation synthesis reactions produce polypeptides (=proteins)

      6. Catabolism is the breakdown of complex molecules into simpler molecules including the hydrolysis of macromolecules into monomers.

      • hydrolysis
        • polymers broken down (= catabolized) into monomers (as in digestion)
        • with H2O used as a source of -H and a -OH group
        • catalyzed by enzymes
        • a polysaccharides can be broken down into monosaccharides
        • H2O molecules used as a sources of -H and a -OH groups
        • catalyzed by enzymes

        fatty acids, glycerol and triglycerides:

        • a triglyceride is broken down into one glycerol and three fatty acid molecules
        • with three H2O molecules used as a sources of -H and a -OH groups
        • catalyzed by enzymes

        amino acids and polypeptides:

        • a polypeptide is broken down into separate amino acid molecules
        • with H2O molecules used as a sources of -H and a -OH groups
        • catalyzed by enzymes

        Applications and skills:

        • Application: Urea as an example of a compound that is produced by living organisms but can also be artificially synthesised.

        Skill: Drawing molecular diagrams of glucose, ribose, a saturated fatty acid and a generalized amino acid.

        Skill: Identification of biochemicals such as sugars, lipids or amino acids from molecular diagrams.

        Guidance: Only the ring forms of D-ribose, alpha–D-glucose and beta-D-glucose are expected in drawings.

        Alcohol Fermentation

        Another familiar fermentation process is alcohol fermentation (Figure 2), which produces ethanol, an alcohol. The alcohol fermentation reaction is the following:

        Figure 2 The reaction resulting in alcohol fermentation is shown.

        The fermentation of pyruvic acid by yeast produces the ethanol found in alcoholic beverages (Figure 3). If the carbon dioxide produced by the reaction is not vented from the fermentation chamber, for example in beer and sparkling wines, it remains dissolved in the medium until the pressure is released. Ethanol above 12 percent is toxic to yeast, so natural levels of alcohol in wine occur at a maximum of 12 percent.

        Figure 3 Fermentation of grape juice to make wine produces CO2 as a byproduct. Fermentation tanks have valves so that pressure inside the tanks can be released.

        Again, the purpose of this process is not to produce ethanol, but rather to convert NADH back into NAD + so that glycolysis can continue.

        Respiration is a three-step process that includes glycolysis, the Krebs cycle, and a bunch of electrons being pushed around the membranes of mitochondria. Together they take that energy out of the sugar-related molecules. Glucose is combined with oxygen and releases usable energy, carbon dioxide, and water.

        Cells can use that extra energy to power their functions. The energy isn't just floating around. It's stored in an excitable compound called ATP (adenosine triphosphate). ATP is the power molecule used by all the cells of an organism to power the secondary reactions that keep us alive. You may also hear about other power molecules like NADH, NADPH, or FADH. These are equally important as ATP, but they are used less often. We exhale carbon dioxide when we breathe. That CO2 comes from the breakdown of glucose in our mitochondria. As we just told you, plants can take in that carbon dioxide and use it to make sugars. Did you know that plants also create CO2? They might not breathe it out the way we do, but plants need energy too. They break down sugars in their cells and release CO2 just like us.

        Part 2: Mitochondrial Metabolism and Cell Decisions

        00:00:08.09 Hi.
        00:00:09.22 My name is Jared Rutter,
        00:00:11.00 and I'm a Professor in the Department of Biochemistry
        00:00:13.06 and an Investigator of the Howard Hughes Medical Institute
        00:00:16.17 at the University of Utah.
        00:00:18.03 And I'm gonna tell you in this second part of my series
        00:00:20.28 about what I believe to be a very important role of mitochondria,
        00:00:25.14 and their metabolism,
        00:00:27.15 in controlling how cells make decisions.
        00:00:30.27 And in this talk, I'm going to allude to some data
        00:00:34.25 generated by Vettore Therapeutics,
        00:00:36.17 which is a company that I co-founded
        00:00:39.03 and I'm quite involved in.
        00:00:41.07 So, as I alluded to in my first part,
        00:00:45.02 the first part of this series,
        00:00:47.27 my laboratory made it a goal to understand some of the
        00:00:52.24 uncharacterized mitochondrial proteins
        00:00:55.10 that are conserved across evolution.
        00:00:57.02 And that has led us into thinking about a lot of different mitochondrial processes
        00:01:02.06 and making what we believe are some interesting discoveries
        00:01:04.27 about how mitochondria work
        00:01:08.06 and how they communicate with the rest of the cell.
        00:01:10.16 And what I'm going to tell you about today is a story
        00:01:13.22 about metabolite transport.
        00:01:16.14 So, when glucose is brought into the cell,
        00:01:19.03 it's converted through the actions of glycolysis
        00:01:21.27 to pyruvate.
        00:01:23.21 And that pyruvate, in most differentiated cells in our body,
        00:01:27.06 is taken into the mitochondria,
        00:01:29.04 where it's converted to acetyl-CoA,
        00:01:31.02 which then donates its carbons to the TCA cycle.
        00:01:35.27 And through this process,
        00:01:38.14 this enables highly efficient ATP production,
        00:01:41.01 as I alluded to in detail in the first part of my talk.
        00:01:47.12 So, some cells in our body, however,
        00:01:50.10 don't do this quite so efficiently,
        00:01:52.27 and instead convert pyruvate and other glycolytic intermediates
        00:01:56.24 into building blocks that help to fuel
        00:02:01.07 cell growth and proliferation.
        00:02:02.22 And this is most famous in the context of cancer,
        00:02:05.02 where this is known as the Warburg effect.
        00:02:07.23 And again, this is thought to enable
        00:02:10.20 building blocks to be produced from carbon that's brought into the cell,
        00:02:14.15 rather than just the production of ATP.
        00:02:18.16 So, I want to also point out that in this context
        00:02:22.01 some of that pyruvate can be converted to lactate,
        00:02:27.07 and that lactate be exported.
        00:02:28.06 And that will be very important
        00:02:30.22 toward the end of this talk.
        00:02:34.10 So, this is, arguably, the most well known,
        00:02:38.23 well studied metabolic pathway in all of biology.
        00:02:42.03 But surprisingly, one obligate component of this pathway
        00:02:46.27 was not molecularly identified until a few years ago,
        00:02:49.11 and that is the process by which
        00:02:52.21 pyruvate enters the mitochondria.
        00:02:54.25 Pyruvate is a charged molecule
        00:02:56.26 and doesn't pass through membranes efficiently on its own.
        00:02:59.01 It needs a protein to enable that to happen.
        00:03:01.15 And that protein was, again,
        00:03:03.24 not molecularly identified until a few years ago,
        00:03:06.15 when it turns out that two of the proteins
        00:03:09.16 that we had been studying
        00:03:11.23 as being highly conserved but uncharacterized mitochondrial proteins
        00:03:15.24 turned out to form a dimeric complex
        00:03:19.18 known as the mitochondrial pyruvate carrier, or MPC.
        00:03:23.15 The MPC is an obligate heterodimer.
        00:03:25.26 There's an MPC1 protein and an MPC2 protein.
        00:03:29.00 Those two proteins come together.
        00:03:31.14 Both of them are absolutely required
        00:03:34.04 for the function of this complex.
        00:03:35.28 And in the absence of one or the other,
        00:03:38.08 the other one just gets degraded.
        00:03:40.12 And I will allude to that later, when we talk about studies in mice.
        00:03:45.17 A graduate student, John Schell,
        00:03:48.19 was heavily involved in the discovery of the MPC
        00:03:51.07 and the early work at thinking about the roles of this complex,
        00:03:54.26 and he gives a great introduction to the discovery
        00:03:58.18 and the importance of this protein
        00:04:01.16 in mediating some of the metabolic effects
        00:04:04.01 that we see in cancer.
        00:04:05.25 And it would be great to watch.
        00:04:08.25 But I just want to summarize that and tell you
        00:04:11.20 that one thing that John found, not surprisingly perhaps,
        00:04:15.06 is that many of those cells that do this so-called Warburg effect,
        00:04:19.00 where pyruvate is maintained in the cytosol,
        00:04:21.20 not imported into the mitochondria and oxidized.
        00:04:26.02 many of those cells actually have
        00:04:30.02 low expression of the MPC.
        00:04:32.03 Or, in the case of some cancers,
        00:04:34.08 mutations or deletions that impair the activity of the MPC.
        00:04:38.06 And frequently, that is coupled with high expression
        00:04:41.28 of this MCT4 lactate exporter
        00:04:44.24 that removes lactate from the cytosol.
        00:04:48.22 But the question really is, does that matter?
        00:04:52.13 Does it matter that those cells
        00:04:55.23 have this MPC-low/MCT4-high situation,
        00:05:00.28 and as a result have a metabolic program
        00:05:04.27 that's characterized by aerobic glycolysis, so to sp.
        00:05:08.24 which is how it's known,
        00:05:11.14 as opposed to carbohydrate oxidation in the mitochondria?
        00:05:14.17 Does that metabolism actually matter
        00:05:17.07 for the behavior of those cells?
        00:05:19.10 And the system in which we've studied that in greatest detail
        00:05:22.28 is depicted here, and this is the intestinal epithelium shown here.
        00:05:27.11 And the key feature of this is that these intestinal stem cells.
        00:05:31.05 those intestinal stem cells sit here
        00:05:35.03 at the base of the crypt,
        00:05:37.08 in a protected compartment,
        00:05:39.06 and proliferate and then differentiate
        00:05:41.29 as they move up the crypt and into the villus,
        00:05:44.06 eventually forming all the mature cell types
        00:05:46.20 of the intestinal epithelium
        00:05:49.28 that perform the barrier function and all the other essential functions
        00:05:54.02 that this epithelium performs.
        00:05:57.04 There's also another great thing about studying the intestinal epithelium,
        00:06:01.05 and that is that it's very highly organized.
        00:06:03.13 Again, with the stem cells sitting at the base of this crypt,
        00:06:07.13 you know where they are, you know what they look like.
        00:06:10.08 And also, there are great ex-vivo systems for studying this system.
        00:06:15.09 And another great feature of the intestinal stem cell system
        00:06:19.19 is the abil. the ability we have to make
        00:06:22.26 these so-called intestinal organoids, as shown here,
        00:06:25.23 and these are two examples shown.
        00:06:27.25 So, these organoids are essentially an intestinal epithelium
        00:06:31.23 that is folded back on itself to create
        00:06:34.28 an enclosed structure that is complete with intestinal crypts,
        00:06:39.04 as shown here,
        00:06:41.01 where the stem cells, again,
        00:06:42.25 sit at the base of this crypt,
        00:06:45.07 and as they proliferate and differentiate
        00:06:47.01 cause the extrusion of this crypt
        00:06:50.01 from what would otherwise be a spherical organoid.
        00:06:53.03 So, one of the things that John wanted to do
        00:06:56.15 is ask the question of whether these stem cells,
        00:07:00.10 which normally have low expression of the MPC,
        00:07:03.14 actually require that low expression of the MPC
        00:07:05.29 to act like stem cells.
        00:07:07.27 So, what he did was rather simple:
        00:07:10.12 force those stem cells to express the MPC at a higher level
        00:07:14.27 and ask, what is the consequence of that?
        00:07:16.21 And what he found is that that essentially causes these stem cells
        00:07:20.03 to stop acting like stem cells.
        00:07:22.02 They lose the ability to make new crypts, as shown here.
        00:07:26.06 Those cells don't die,
        00:07:28.21 but they stop acting like stem cells
        00:07:30.24 and even stop expressing many of the molecular markers of stem cells.
        00:07:33.26 And interestingly, one thing that he found
        00:07:36.14 is that this phenotype of MPC overexpression
        00:07:39.22 was completely reversed
        00:07:42.14 when he treated these organoids with an inhibitor of the MPC
        00:07:45.17 that had been. had been discovered almost 50 years ago now,
        00:07:48.22 and we now know to be a quite specific
        00:07:52.23 and very useful inhibitor of the mitochondrial pyruvate carrier.
        00:07:57.16 And moreover, John did another experiment,
        00:08:00.17 which was to isolate stem cells from these wild type organoids,
        00:08:04.21 plate them again,
        00:08:07.08 and ask for their ability to make a new organoid.
        00:08:10.07 And what he found is that treatment with this MPC inhibitor
        00:08:14.07 in that experiment
        00:08:16.13 caused a rather dramatic increase
        00:08:18.29 in the ability of these stem cells to make a new organoid,
        00:08:21.18 to a similar or even greater level
        00:08:25.00 than the effects caused by very canonical, well known drugs
        00:08:29.10 that are used to promote stemness:
        00:08:31.10 valproic acid and an inhibitor of the GSK3-beta protein,
        00:08:35.06 which causes activation of the Wnt/beta-catenin system.
        00:08:40.15 And I won't show you the data for this, but loss.
        00:08:44.12 genetic loss of the MPC in intestinal stem cells,
        00:08:46.27 in vivo in mice,
        00:08:49.06 not surprisingly leads to an expanded and hyperproliferative stem cell compartment
        00:08:54.20 in vivo.
        00:08:56.15 And I'll allude later to some of the consequences of that, we think.
        00:08:59.14 So, the MPC sits here at this very critical juncture,
        00:09:04.20 between the metabolic programs operated by many stem cells and cancer cells,
        00:09:10.25 which require pyruvate metabolism in the cytosol,
        00:09:15.26 and those characterized by pyruvate oxidation in the mitochondria.
        00:09:20.08 It sits in this critical juncture.
        00:09:22.02 And we believe that this MPC activity
        00:09:24.16 -- the activity of this complex to promote mitochondrial pyruvate import --
        00:09:28.22 has an active role in promoting differentiation
        00:09:31.27 and limiting stemness.
        00:09:35.15 And I want to make one critical point.
        00:09:37.15 We've often thought about this,
        00:09:40.00 and people ask us all the time,
        00:09:41.22 well, does this mean these stem cells just don't have mitochondria?
        00:09:44.18 It turns out, as pointed to in.
        00:09:47.25 with yellow arrows here,
        00:09:50.07 these stem cells are chock full of mitochondria.
        00:09:53.08 They have more mitochondria
        00:09:56.18 than the differentiated cells around them,
        00:09:58.09 but it's just those mitochondria appear not to be focused
        00:10:02.08 on doing mitochondrial pyruvate oxidation. It's really fascinating to think what they might be doing
        00:10:08.17 and how that mitochondrial function is controlled.
        00:10:13.13 So, the question is how this relates
        00:10:17.09 to the signaling that goes on in stem cells,
        00:10:19.22 because we all know about the signaling
        00:10:22.12 that tells a stem cell to be maintained a stem cell.
        00:10:26.01 And how does this metabolic program interface with that?
        00:10:30.09 And I want to just point to a couple of experiments
        00:10:33.14 done by colleagues of mine,
        00:10:35.09 Roo Wisidagama, who was a graduate student in the lab of Carl Thummel
        00:10:38.11 in the Department of Human Genetics at the University of Utah.
        00:10:42.21 And they used the Drosophila system
        00:10:45.16 and have done really elegant work studying the impacts of the MPC there.
        00:10:52.04 And the system that they have employed is a system
        00:10:54.21 that enables the generation of clones in the Drosophila intestinal epithelium
        00:10:58.10 that, simultaneously to a genetic manipulation,
        00:11:01.15 also turn on the expression of GFP.
        00:11:05.06 So, you can see a clone, here, that. in the control animals,
        00:11:09.19 that generates a clone of a certain number of cells.
        00:11:13.02 And when the APC gene.
        00:11:15.03 two genes in Drosophila.
        00:11:17.17 are deleted, that clone becomes much larger.
        00:11:19.28 And the APC gene is a tumor suppressor,
        00:11:23.04 the most commonly mutated gene in colon cancer,
        00:11:25.29 which causes hyperproliferation through constitutive activation
        00:11:29.18 of the Wnt/beta-catenin pathway.
        00:11:31.25 The same thing happens in flies,
        00:11:34.08 and as a result, you get hyperproliferation
        00:11:36.14 of those stem cells and a large clone.
        00:11:40.13 And the experiment that they did, among many others,
        00:11:43.26 is now to force those stem cells to express the MPC,
        00:11:48.24 and ask, what is the effect of that?
        00:11:50.24 And the effect of that is that those stem cells
        00:11:54.04 essentially stop proliferating.
        00:11:56.16 And very interestingly, these stem cells don't die.
        00:11:59.14 They just stop proliferating, and this is quantified here.
        00:12:02.28 They just stop proliferating.
        00:12:05.25 So, even though the signaling is presumably
        00:12:10.06 telling these stem cells to proliferate.
        00:12:12.17 APC is mutated, the. the.
        00:12:16.02 presumably the transcriptional program is driving proliferation.
        00:12:19.08 But when the metabolism doesn't cooperate,
        00:12:22.03 these stem cells don't proliferate.
        00:12:26.00 I think that puts this effect of
        00:12:29.11 the MPC controlling stemness and differentiation
        00:12:32.10 into a very interesting light.
        00:12:35.24 So, I alluded to data in mammals and in flies.
        00:12:40.03 There's data that I won't show you in fish,
        00:12:43.00 which shows, similarly,
        00:12:45.18 a very important role of the MPC.
        00:12:47.13 Others have shown this effect in other stem cell types.
        00:12:50.13 So, does this actually have an impact on tumor formation?
        00:12:54.09 Does this effect of the MPC control oncogenesis, in vivo,
        00:13:00.03 in the intestine?
        00:13:02.01 So, Claire Bensard, a current MD-PhD student in the lab,
        00:13:05.02 did an experiment where she eliminated the MPC.
        00:13:09.02 again, specifically in intestinal stem cells,
        00:13:11.28 eliminated MPC1.
        00:13:14.01 It's interesting. This is a hetero. heterodimeric protein.
        00:13:17.27 So, we're deleting the MPC1 gene,
        00:13:20.10 and the mRNA for MPC1 is lost.
        00:13:22.13 MPC2 is not.
        00:13:24.21 But interestingly, this is an obligate heterodimer,
        00:13:27.04 and as a result of that even though MPC2
        00:13:31.10 presumably continues to be expressed,
        00:13:33.18 it's completely eliminated from the intestinal epithelium,
        00:13:36.18 presumably due to degradation because its partner, MPC1,
        00:13:40.29 is no longer being expressed.
        00:13:42.22 So, we end up in a situation where the MPC
        00:13:45.11 is absent from the intestinal epithelium.
        00:13:47.28 So, what effect does this have on tumorigenesis?
        00:13:51.03 So, Claire did a really nice experiment
        00:13:54.09 where she subjected these mice to an environmental,
        00:13:58.01 oncological stress in the intestine
        00:14:00.25 and asked for their ability, or their propensity,
        00:14:03.13 to generate tumors in the intestine.
        00:14:05.11 And what she observed is a dose-dependent increase
        00:14:08.05 in tumorigenesis from the wild type to the heterozygote
        00:14:12.00 to the genetic loss animals,
        00:14:14.15 as shown by the height of these bars --
        00:14:19.06 it's the number of lesions per animal.
        00:14:21.15 And the red colors indicate.
        00:14:23.23 indicate more aggressive tumors,
        00:14:25.28 again being generated in the. in those animals
        00:14:28.25 where in the stem cells lacked MP. the MPC.
        00:14:31.25 So, more tumors, and those tumors were more aggressive.
        00:14:35.02 And again, all that's happening here is loss of
        00:14:38.10 this mitochondrial pyruvate carrier specifically in the stem cells.
        00:14:42.12 I think that's a very important consequence
        00:14:46.16 of loss of the MPC.
        00:14:48.05 So, not only does the MPC
        00:14:50.13 appear to limit stemness, directly,
        00:14:53.05 but also oncogenesis.
        00:14:54.24 Most likely an indirect effect of affecting stemness.
        00:14:58.13 And I haven't told you about this,
        00:15:01.11 but it's becoming clear from others in the field
        00:15:04.22 that this process also plays a very important role
        00:15:07.25 in inflammation and fibrosis.
        00:15:10.27 So, based on this,
        00:15:13.00 we thought this would be a great idea for a way
        00:15:15.24 to maybe deal with some of the pathologies
        00:15:18.12 associated with these processes:
        00:15:21.18 oncogenesis, hyperinflammatory disease, fibrotic disease.
        00:15:26.18 And so, we decided to start a company
        00:15:29.04 along with my uncle, Bill Rutter,
        00:15:30.29 and decided to.
        00:15:33.18 can we find a way to activate the MPC?
        00:15:36.07 That seems to be what we need to do,
        00:15:38.03 to activate this process, prevent oncogenesis or reverse it,
        00:15:41.28 and potentially also prevent inflammation and fibrosis.
        00:15:47.17 So, it. we started a company and hired a fantastic scientist
        00:15:50.27 to lead the scientific operations,
        00:15:53.01 Mark Parnell.
        00:15:54.27 And we figured out very quickly that activating the MPC
        00:15:58.23 was not going to be an easy task.
        00:16:00.14 And to date, we've completely failed.
        00:16:02.11 But what Mark did instead
        00:16:05.19 was to come upon a way to perform a related metabolic manipulation
        00:16:10.11 that seems to have many of the same consequences.
        00:16:13.26 And that is through inhibition of this MCT4 protein.
        00:16:18.07 So, again, this is a lactate exporter
        00:16:20.25 that takes the lactate that's made from cytosolic pyruvate
        00:16:25.22 and exports it.
        00:16:28.10 And what appears to be the case.
        00:16:30.13 when MCT4 is inhibited,
        00:16:32.19 presumably cytosolic lactate accumulates,
        00:16:34.27 cytosolic pyruvate accumulates,
        00:16:36.19 and that perhaps just drives, by mass action,
        00:16:39.15 mitochondrial pyruvate uptake and metabolism.
        00:16:42.04 And the net effect is similar to as if.
        00:16:44.20 to what we've seen when we overexpress the MPC genetically.
        00:16:50.04 So, that's what we tried to do:
        00:16:53.18 inhibit the MCT4 protein.
        00:16:55.15 And Mark was able to develop
        00:16:58.22 some very potent and specific inhibitors of the M. of MCT4,
        00:17:02.22 and their statistics are shown here.
        00:17:04.18 The key features of this is that the MCT4 inhibitor that he found,
        00:17:09.01 this VB253 compound,
        00:17:11.04 is very potent for MCT4
        00:17:13.25 and selective over the related MCT1 protein,
        00:17:17.19 inhibition of which seems to cause some toxicity.
        00:17:21.00 So, this protein. this VB253 molecule is also.
        00:17:25.09 has quite good pharmacological properties
        00:17:27.24 and seems to be quite safe.
        00:17:29.21 So, I'm gonna show you some of the data
        00:17:32.09 that's been generated with this compound,
        00:17:34.11 again, with the idea that by manipulating these metabolic pathways
        00:17:37.16 we might be able to rewire metabolism,
        00:17:41.15 change cell behaviors in a way that would be beneficial therapeutically.
        00:17:47.25 One of the indications that we've been most interested
        00:17:51.22 in trying to treat with this VB253 compound
        00:17:55.28 is idiopathic pulmonary fibrosis.
        00:17:58.29 And there's still a lot to be understood
        00:18:01.22 about the disease pathogenesis of IPF,
        00:18:06.26 but a few things that we do know.
        00:18:10.06 it's clear that fibroblasts. fibroblasts become activated
        00:18:14.09 and form this so-called myofibroblast cell type.
        00:18:18.09 And myofibroblasts, like cancer cells and like stem cells
        00:18:22.11 that we talked about previously,
        00:18:24.20 exhibit this highly glycolytic phenotype,
        00:18:26.20 characterized by low MPC expression, high MCT4 expression.
        00:18:32.11 again, characteristic of that metabolic phenotype.
        00:18:36.05 And this disease process is also contributed to
        00:18:40.00 by pro-fibrotic macrophages,
        00:18:42.14 which also exhibit that same metabolic profile.
        00:18:46.16 So, this might be a scenario
        00:18:49.12 where, if we could inhibit this MCT4 protein in this context,
        00:18:53.02 this might reverse the pathogenic behaviors of these cells,
        00:18:58.11 limit the deposition of extracellular matrix
        00:19:01.07 and lung fibrosis.
        00:19:03.10 So, that's what we set out to test.
        00:19:05.18 So, just to show you some of the data behind what I just said.
        00:19:08.23 so, it turns out these pro-fibrotic myofibroblasts
        00:19:12.18 do express a large amount of this MCT4 protein,
        00:19:17.05 as shown by staining here,
        00:19:19.13 as well as these activated macrophages.
        00:19:21.17 Both of them show this high MCT4 staining.
        00:19:25.06 And again, this is the target of this VB253 molecule.
        00:19:29.03 So, if this mol. if this protein is inhibited,
        00:19:31.26 does it have an effect?
        00:19:33.18 And it turns out that it does.
        00:19:35.11 So, what you're looking at here is pathological scoring,
        00:19:38.03 on the left,
        00:19:40.21 of a mouse model of idiopathic pulmonary fibrosis,
        00:19:45.02 where mice are given bleomycin to induce lung fibrosis,
        00:19:48.09 and then the fibrosis is scored
        00:19:51.25 as a function of time.
        00:19:53.23 And interestingly, what was done here is to actually give bleomycin first,
        00:19:57.06 create injury,
        00:19:59.10 and then treat with this MCT4 inhibitor.
        00:20:02.14 And in spite of doing it in that order,
        00:20:05.05 which is a more challenging experimental paradigm,
        00:20:07.26 this VB253 molecule actually decreases the fibrosis score,
        00:20:10.29 a little bit better than what's the standard of care now in patients,
        00:20:15.12 which is a molecule called pirfenidone.
        00:20:18.11 And on the right, you see that smooth muscle actin,
        00:20:20.23 which again is a marker of fibrosis,
        00:20:22.24 which is almost normalized by VB253.
        00:20:29.11 This are just examples of smooth muscle actin staining.
        00:20:32.02 Again, from. compared to the control,
        00:20:35.04 bleomycin causes a dramatic increase
        00:20:39.00 in staining with smooth muscle actin,
        00:20:41.06 coincident with fibrosis.
        00:20:44.10 This is partially reversed by pirfenidone,
        00:20:47.06 but seems to be almost completely reversed
        00:20:50.00 by inhibition of MCT4.
        00:20:52.09 And this seems to be cell autonomous.
        00:20:55.04 And this was a very important result for us.
        00:20:57.07 What's being done here is to take fibroblasts from IPF patients,
        00:21:01.16 culture them in vitro, where they're the only cell type in the dish,
        00:21:04.25 and in that context inhibition of MCT4
        00:21:08.18 leads to a decrease in the production of smooth muscle actin.
        00:21:12.20 So, that tells us that this effect on decreased smooth muscle actin
        00:21:17.17 at least can be partially explained
        00:21:20.11 by actions directly on these fibroblasts.
        00:21:22.22 It's not something complex going through the brain or the liver
        00:21:26.21 or the skeletal muscle.
        00:21:28.18 This seems to be happening locally in the lung.
        00:21:31.21 Finally, the last data slide I want to show you
        00:21:36.06 is that this has an effect on the ability of the lung
        00:21:39.14 to contract in breathing.
        00:21:41.10 And this whole body plethysmography
        00:21:43.19 is a measure of bronchial obstruction.
        00:21:46.25 And you'll notice that when. upon bleomycin treatment,
        00:21:49.13 there is more bronchial obstruction,
        00:21:51.15 less breathing capacity.
        00:21:53.11 That is maybe decreased a little bit by these two molecules,
        00:21:56.10 which are, again, the standard of care
        00:21:59.07 approved for treatment in humans.
        00:22:00.25 But inhibition of MCT4 works a little bit better, even,
        00:22:03.11 to decrease this bronchial obstruction
        00:22:05.14 and promote healthy lung function.
        00:22:08.03 So, we're really excited about the idea that rewiring metabolism in this way,
        00:22:13.25 by inhibition of MCT4,
        00:22:16.16 might change the behavior of these cells.
        00:22:19.14 Again, we have no evidence that these fibroblasts die
        00:22:23.10 or that these macrophages die.
        00:22:26.08 They just change their behavior.
        00:22:28.09 And that altered behavior decreases the production
        00:22:32.18 of the extracellular matrix that promotes fibrosis
        00:22:35.24 and leads to a decrease in fibrosis itself.
        00:22:39.18 And we're really interested to try and understand
        00:22:43.12 not only the applications of this in human disease
        00:22:45.15 but also really fundamentally understand,
        00:22:47.21 how is it that by just altering the metabolism of these cells
        00:22:52.02 does that change their behavior?
        00:22:54.29 And again, this just reminds me to tell you that
        00:22:59.24 we think that this might be going on
        00:23:02.23 through the actions of the mitochondrial pyruvate carrier.
        00:23:04.29 Pyruvate that enters the mitochondria ends up being converted into
        00:23:08.26 very important signaling molecules,
        00:23:10.24 like acetyl-CoA and other TCA cycle intermediates,
        00:23:14.13 that are known to have important signaling roles in the cytosol and the nucleus.
        00:23:18.19 And perhaps, one of those molecules
        00:23:21.13 plays an important role in changing cell behavior.
        00:23:23.21 There are also very important redox effects.
        00:23:26.04 So, I think it's critical for us to understand,
        00:23:28.23 how do our cells sense their metabolic state?
        00:23:32.25 And it's something that I believe we're just beginning to understand.
        00:23:36.22 How do they know what metabolites they have?
        00:23:39.10 And I think if we could understand that,
        00:23:42.16 we might better understand
        00:23:46.06 how manipulations like inhibition of MCT4
        00:23:49.03 change their behavior.
        00:23:50.24 And maybe we'd be able to make even better manipulations,
        00:23:53.03 build better drugs that would treat people better.
        00:23:56.01 So, I also think it's. you know, the MPC is not unique
        00:24:00.23 in being an important metabolic control point
        00:24:02.18 there are many others.
        00:24:04.12 And if we can identify those metabolic control points and manipulate them,
        00:24:06.13 we might be able to make even better manipulations
        00:24:08.25 to better change the behavior of cells
        00:24:13.21 to improve human health.
        00:24:18.12 And I told you a little bit about IPF.
        00:24:20.20 We think there are many manifestations
        00:24:23.13 -- cancer being one that's perhaps the most obvious --
        00:24:26.12 where modulation of this metabolic program,
        00:24:29.22 the disposition of pyruvate,
        00:24:32.00 might have important consequences.
        00:24:34.05 And we're really anxious trying to understand
        00:24:37.00 the different ways that this can be used.
        00:24:40.00 So, I just want to thank the people that did the work.
        00:24:42.16 I alluded to many of them as we went through.
        00:24:44.29 They've been fantastic collaborators,
        00:24:47.07 and thanks to those that paid for this work to be conducted,
        00:24:50.19 and thanks to you for listening.


        Imaging of Glutamine Metabolism

        Increased recognition of the potential importance of glutamine as a metabolic substrate, as described above, has spurred the development of radiolabeled glutamine for imaging. Synthesis of both 18 F- and 11 C-labeled glutamine was first reported in 2011 (76,77). As chemically identical compounds, 11 C-labeled glutamine and unlabeled glutamine share an analogous and complex metabolism. As such, the 11 C radiolabel is rapidly passed to metabolites and distributed in numerous cellular compartments for biosynthesis, energy production, and excretion. l -[5- 11 C]-glutamine has been studied preclinically in a mouse glioma xenograft and transgenic mice bearing M/tomND spontaneous human mammary tumors (77). Such complexity, combined with a relatively short half-life, will likely confine 11 C-labeled glutamine to research applications.

        The addition of a fluorine moiety substantially changes the distribution and metabolism of glutamine, which has enabled translation for human applications. 18 F-(2S,4R)4-fluoroglutamine ( 18 F-Gln) shares the same transporters as native glutamine but is metabolized to a limited degree. 18 F-Gln has demonstrated uptake in rats bearing 9L tumor xenografts, as well as in genetically engineered mice with conditional myc gene expression (78). In humans, 18 F-Gln has been studied in a range of cancers, including glioma, pancreas, and breast (79,80). In 3 glioma patients imaged with clinical disease progression, tumors demonstrated increased 18 F-Gln uptake. Minimal or no 18 F-Gln uptake was seen in the 3 patients with stable disease. In contrast to 18 F-FDG, which demonstrates high background brain uptake, 18 F-Gln has only minimal uptake in normal brain. These promising early results suggest the utility of 18 F-Gln in identifying glioma patients at risk of progression (Fig. 3A (79)).

        (A) T1-weighted MRI with contrast shows minimal enhancement (arrowheads) along surgical cavity (dotted line) in glioma patient. Corresponding 18 F-FDG PET image shows uptake in tumor posteriorly but not anteriorly (arrowheads). Corresponding 18 F-glutamine (Gln) PET image shows tumor uptake both posteriorly and anteriorly. This patient had clinically progressive disease. (Adapted and reprinted with permission of (79).) (B) Schematic of glutamine metabolism and effect of glutaminase inhibitors. With glutaminase inhibition, cellular glutamine increases whereas cellular glutamate decreases. (C, top) 18 F-glutamine PET images of triple-negative breast cancer xenograft show increased 18 F-glutamine uptake after glutaminase inhibition reflecting increased glutamine pool size. (C, bottom) In contrast, receptor-positive breast cancer xenograft shows high uptake of 18 F-glutamine at baseline without increase after glutaminase inhibition reflecting inherently low glutaminase activity. (Adapted and reprinted with permission of (81).)

        As a minimally metabolized glutamine analog that shares the same transporters as native glutamine, 18 F-Gln uptake has been proposed as a measure of cellular glutamine pool size. In triple-negative breast cancer tumor extracts with inherently high glutamine use, 1 H MRS demonstrated a relatively small cellular glutamine pool size. After inhibition of glutaminase, the first enzyme in the glutaminolytic pathway, glutamine pool size increased. Conversely, a large glutamine pool size was observed in estrogen receptor–positive tumor extracts with low glutamine use, without a change in pool size after glutaminase inhibition. 18 F-Gln PET imaging of tumor xenografts underscored these findings, with tumor-to-blood ratios, an approximation of 18 F-Gln distribution volume, demonstrating concordant results (Figs. 3B and 3C (81)). Kinetic analysis of dynamic images in these same tumor models demonstrated largely reversible uptake of 18 F-Gln and confirmed 18 F-Gln distribution volume as a marker of glutamine pool size (82). This work provides a theoretical framework for image interpretation of 18 F-Gln, which differs greatly from the analysis of 18 F-FDG, which is trapped. Further studies are required to ensure appropriate image analysis with consideration of tracer pharmacokinetics. Estimation of changes in pool size with 18 F-Gln provides the ability to infer tumor glutaminolysis in vivo, suggesting its use as a biomarker to select patients for glutaminase therapy. Changes in pool size after glutaminase therapy can provide a measure of pharmacodynamic response to targeted glutaminase therapy. Given the prevalence of glutamine dysregulation in certain cancers, PET imaging with 18 F-Gln may have broad application beyond a targeted pairing with glutaminase inhibitors.

        Whereas imaging with 18 F-Gln has just reached human patients in early clinical trials, the Food and Drug Administration approved the use of synthetic amino acid anti-1-amino-3- 18 F-fluorocyclobutane-1-carboxylic acid (FACBC) for the detection of recurrent prostate cancer in 2016 (83). This synthetic amino acid with a 4-carbon ring (84) shares transporters with natural amino acids, most notably the alanine-serine-cysteine transporter 2 (ASCT2) (85). Anti-1-amino-3- 18 F-fluorocyclobutane-1-carboxylic acid enables the detection of persistent disease in men with biochemically recurrent of prostate cancer, leveraging the established dysregulation of amino acid use in these tumors (86).

        In like manner to 18 F-labeled glutamine, imaging with 18 F-labeled glutamate analogs has advanced into early clinical trials. (4S)-4-(3- 18 F-fluoropropyl)- l -glutamate demonstrated transport through the cystine/glutamate exchanger system xc − . This transporter, involved in glutathione biosynthesis and regulation of reactive oxygen species, has high levels of expression in several tumors. As such, this transporter makes an attractive target for tumor imaging (87). In humans, uptake of (4S)-4-(3- 18 F-fluoropropyl)- l -glutamate in breast and non–small lung cancer correlated with expression of the xc − transporter by immunohistochemistry (88). However, given the subcellular localization of glutamate in the cytosol for glutathione biosynthesis and glutamate in the mitochondria after formation from glutamine via glutaminase (89), (4S)-4-(3- 18 F-fluoropropyl)- l -glutamate appears limited in its ability to fully characterize glutamine or glutamate metabolism in malignancy and may be more effective as a biomarker of free radical regulation.

        Hyperpolarized MRI

        Hyperpolarization of 5- 13 C-glutamine has been performed, with clinical translation hampered by a short T1 and a limited polarization efficiency. Early work demonstrated the ability to image the conversion of hyperpolarized glutamine to glutamate in human hepatocellular carcinoma cells (90) and human glioma cells. A deuterated glutamine was hyperpolarized in the latter experiment, more than doubling the T1 (33 s vs. 15 s in the undeuterated compound) (91). More recently, dynamic nuclear polarization of 5- 13 C-glutamine has been translated for in vivo MRS imaging in rats. Metabolism of the parent substrate to its metabolite glutamate was detected in the rat hepatic tumor but not in normal liver (92). In addition, 1- 13 C-glutamate has been successfully hyperpolarized, enabling the unique potential to measure flux from glutamate to α-ketoglutarate in the TCA cycle (93). With continued technical innovation, hyperpolarization of glutamine or glutamate, as well as other metabolites, may hold the potential for human translation (71).

        CEST imaging of glutamate has been successfully translated into humans, demonstrating the capability to detect temporal lobe epilepsy in patients without a detectable lesion on conventional MRI. Glutamate CEST identified the laterality of a seizure focus in 4 of 4 patients with epilepsy (94). Increased glutamate in seizure foci marks mitochondrial and metabolic injury, which may represent the result of and cause of a seizure in a self-propagating process (95). In like manner to PET, glutamine CEST may have applications in oncologic imaging as a measure of tumoral glutaminolysis based on glutamate pool size.

        Imaging Acetate Metabolism

        Imaging opportunities with acetate parallel its metabolic fate, holding the potential to provide measures of TCA metabolism. Indeed, studies dating back into the 1980s demonstrated that radiolabeled acetate metabolism can estimate TCA cycle flux in the myocardium as a measure of myocardial energy metabolism that is proportional to oxygen consumption (96). Metabolism of acetate is measured as the clearance rate from the myocardium, which is indicative of labeled-acetate metabolism as the radiolabel passes to downstream TCA molecules and eventually to radiolabeled CO2, which is cleared rapidly from tissues (38). As opposed to cardiac metabolism, which uses acetate almost entirely for energy production, cancer cells also metabolize acetate for lipid synthesis (97), as a key component in membrane synthesis that is required for the proliferative phenotype (98). Unlike acetate energy metabolism, acetate incorporation into lipids and other molecules used for biogenesis results in trapping of the 11 C label, which can be measured as a trapping flux constant (Ki) or by static uptake measures late after injection (99). 11 C-acetate has been extensively studied in prostate cancer for primary staging, assessing regional lymph node involvement and distant metastatic disease, and in biochemical recurrence (98). A pilot study of 11 C-acetate in prostate cancer with bone metastases demonstrated a correlation between assessment of tumor response with 11 C-acetate and clinical response, suggesting the utility of this radiotracer for treatment response (Fig. 4 (100)). 11 C-acetate has been studied in other malignancies, notably bladder and renal cell carcinoma given the lack of urinary excretion, as well as hepatocellular carcinoma (101). These data underscore the potential role of acetate as a marker of cancer metabolism as 11 C-acetate holds great promise as a radiotracer indicating the balance of energy metabolism and biogenesis in the TCA cycle. The ability to measure both energy metabolism and biosynthetic flux in cancer using 11 C-acetate has been a challenge but may be possible with alternative approaches (102), or possibly with the combination of PET and dynamic nuclear polarization MRSI methods, which can track the biochemical fate of a labeled substrate through the detection of its metabolites (103).

        Comparison of bone scan, 18 F-FDG PET, and 11 C-acetate PET before (A) and after (B) androgen deprivation therapy in patient with osseous metastases from prostate cancer. 11 C-acetate demonstrates response to treatment. Bone scan does not demonstrate significant change, and 18 F-FDG PET fails to detect osseous metastases at either time point. PSA = prostate-specific antigen level. (Reprinted with permission of (100).)

        Topic 2: Molecular Biology

        This topic has 14% of occurrence in the papers 1 and 2.
        Below you can find the subtopics of Topic 2 and the percentage of how many times they appear on the exams from the past years.

        Every subtopic is important for the exam, but some are known to be seen more often than others.
        Here you will find some guidance on the content that you should focus more on.

        2.1 Molecules to metabolism: Least common subtopic
        Focus more on these understandings, applications and skills:

        • Urea as an example of a compound that is produced by living organisms but can also be artificially synthesized
        • Drawing molecular diagrams of glucose, ribose, a saturated fatty acid and a generalised amino acid
        • Identification of biochemicals such as sugars, lipids or amino acids from molecular diagrams

        Questions related to these are:

        • Usually there is identification or drawing of the structures such as fatty acid, amino acid, starch
        • Explain the process of Urea production.

        2.2 Water: Least common subtopic
        Focus more on these understandings, applications and skills:

        • Hydrogen bonds and bipolarity explain the cohesive, adhesive, thermal and solvent properties of water
        • Substances can be hydrophilic or hydrophobic
        • Comparison of the thermal properties of water with those of methane
        • Use of water as a coolant in sweat
        • Modes of transport of glucose, amino acids, cholesterol, fats, oxygen and sodium chloride in blood in relation to their solubility in water

        Questions related to these are:

        • Water properties and how it affects the environment, usually it comes as multiple choice or as a long answer question.

        2.3 Carbohydrates and lipids: Common Topic
        Focus more on these understandings, applications and skills:

        • Fatty acids can be saturated, monounsaturated or polyunsaturated
        • Triglycerides are formed by condensation from three fatty acids and one glycerol
        • Structure and function of cellulose and starch in plants and glycogen in humans
        • Lipids are more suitable for long-term energy storage in humans than carbohydrates
        • Determination of body mass index by calculation or use of a nomogram

        Questions related to these are:

        • Analyze nomogram
        • Compare energy of lipid against carbohydrates
        • Identify the difference between saturated and unsaturated

        2.4 Proteins Least, common subtopic
        Focus more on these understandings, applications and skills:

        • The amino acid sequence of polypeptides is coded for by genes
        • A protein may consist of a single polypeptide or more than one polypeptide linked together
        • The amino acid sequence determines the three-dimensional conformation of a protein
        • Denaturation of proteins by heat or by deviation of pH from the optimum

        Questions related to these are:

        • Describe primary and tertiary structures for proteins
        • Identify when proteins are denatured by heat or pH

        2.5 Enzymes, Least common subtopic
        Focus more on these understandings, applications and skills:

        • Temperature, pH and substrate concentration affect the rate of activity of enzymes
        • Enzymes can be denatured
        • Design of experiments to test the effect of temperature, pH and substrate concentration on the activity of enzymes
        • Experimental investigation of a factor affecting enzyme activity

        Questions related to these are:

        • Enzymes role in different processes
        • How enzymes are denatured dependent on the environment.
        • Explain the factors that affect enzyme activity, temperature, pH and substrate concentration

        2.6 Structure of DNA and RNA, Least common subtopic
        Focus more on these understandings, applications and skills:

        • DNA differs from RNA in the number of strands present, the base composition and the type of pentose
        • DNA is a double helix molecule made of two antiparallel strands of nucleotides linked by hydrogen bonding between complementary base pairs
        • Drawing simple diagrams of the structure of single nucleotides of DNA and RNA, using circles, pentagons and rectangles to represent phosphates, pentoses and bases

        Questions related to these are:

        • Difference between DNA and RNA structure
        • Be able to draw nucleotides and DNA structure

        2.7 DNA replication, transcription and translation: Very Common SubTopic
        Focus more on these understandings, applications and skills:

        • The replication of DNA is semi-conservative and depends on complementary base pairing
        • Helicase unwinds the double helix and separates the two strands by breaking hydrogen bonds
        • DNA polymerase links nucleotides together to form a new strand, using the pre-existing strand as a template
        • Transcription is the synthesis of mRNA copied from the DNA base sequences by RNA polymerase
        • Translation is the synthesis of polypeptides on ribosomes
        • The amino acid sequence of polypeptides is determined by mRNA according to the genetic code
        • Codons of three bases on mRNA correspond to one amino acid in a polypeptide
        • Translation depends on complementary base pairing between codons on mRNA and anticodons on tRNA
        • Use a table of the genetic code to deduce which codon(s) corresponds to which amino acid
        • Analysis of Meselson and Stahl’s results to obtain support for the theory of semi-conservative replication of DNA
        • Use a table of mRNA codons and their corresponding amino acids to deduce the sequence of amino acids coded by a short mRNA strand of known base sequence
        • Deducing the DNA base sequence for the mRNA strand

        Questions related to these are:

        • Transcription of mRNA and reading codons to amino acids
        • Explain DNA replication and translation, how ribosomes are important for it.
        • mRNA codes are given and students need to deduce the DNA base sequence
        • Explain the Meselson and Stahl’s results and how it support semi-conservative replication.

        2.8 Cell respiration, Very Common SubTopic
        Focus more on these understandings, applications and skills:

        • Cell respiration is the controlled release of energy from organic compounds to produce ATP
        • ATP from cell respiration is immediately available as a source of energy in the cell
        • Anaerobic cell respiration gives a small yield of ATP from glucose
        • Aerobic cell respiration requires oxygen and gives a large yield of ATP from glucose
        • Lactate production in humans when anaerobic respiration is used to maximise the power of muscle contractions

        Questions related to these are:

        • Identify Cell Respiration reactants and products
        • Explain different stages of cell respiration
        • Understand the difference between aerobic and anaerobic process

        Focus more on these understandings, applications and skills:

        • Photosynthesis is the production of carbon compounds in cells using light energy
        • Visible light has a range of wavelengths with violet the shortest wavelength and red the longest
        • Chlorophyll absorbs red and blue light most effectively and reflects green light more than other colours
        • Oxygen is produced in photosynthesis from the photolysis of water
        • Temperature, light intensity and carbon dioxide concentration are possible limiting factors on the rate of photosynthesis
        • Drawing an absorption spectrum for chlorophyll and an action spectrum for photosynthesis
        • Separation of photosynthetic pigments by chromatography

        Questions related to these are:

        • Be able to draw or identify the action and absorption spectrums
        • Explain the process of light dependent and dependent.
        • Identify and explain the factors that affect photosynthesis.

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        Biology - Fungi

        Fungi are the members of eukaryotic organisms, which includes microorganisms such as molds, yeasts, and mushrooms.

        Fungi do not photosynthesize rather they obtain their food by absorbing the dissolved molecules, usually by secreting digestive enzymes into their environment.

        Fungi are found in almost every part of the world, and they can grow in a wide range of habitats, ranging from extreme environments (such as deserts) to mild (such as temperate region).

        Fungi are the primary decomposers in most of the ecological systems.

        The study of fungi is known as mycology.

        Fungi have membrane-bound cytoplasmic organelles, for example mitochondria, sterol-containing membranes, and ribosomes.

        Fungi have also a cell wall and vacuoles (property of plants).

        Fungi have no chloroplast and they are heterotrophic organisms (property of animals) likewise, fungi have both the properties of plants and animals.


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        Keywords: infectious diseases, genome-scale metabolic networks, pathogen-host interactions, transcriptome, metabolome, gut microbiota, dual omics

        Citation: ౺kır T, Panagiotou G, Uddin R and Durmuş S (2020) Novel Approaches for Systems Biology of Metabolism-Oriented Pathogen-Human Interactions: A Mini-Review. Front. Cell. Infect. Microbiol. 10:52. doi: 10.3389/fcimb.2020.00052

        Received: 22 October 2019 Accepted: 27 January 2020
        Published: 13 February 2020.

        Philip R. Hardwidge, Kansas State University, United States

        Marat R. Sadykov, University of Nebraska Medical Center, United States
        Vໜtor Antonio Garc໚-Angulo, University of Chile, Chile

        Copyright © 2020 ౺kır, Panagiotou, Uddin and Durmuş. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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