Protein Energy Malnutrition Ravina Group Introduction Protein energy malnutrition (PEM) is a major problem facing the developing world. PEM results from insufficient consumption of proteins to meet the body’s nutritional needs, which is at least 0.8g/kg or 0.37g/lb according to the RDA requirement. It affects more than 500 million people, causing 10 million deaths every year worldwide. Children are the individuals most at risk; in some countries more than 25% of the children are affected by protein energy malnutrition. In these countries, PEM is responsible for the deaths of the majority of children under five years old. The two main clinical syndromes of protein energy malnutrition are kwashiorkor and marasmus, which are common in these underdeveloped countries. These two syndromes are directly related to the functional compartments of protein in the body, namely the somatic and visceral compartments. Proteins in the somatic compartment refer to protein in the skeletal muscles, while proteins in the visceral compartment refer to proteins in the deeper organs, primarily the liver. There are different methods of assessing protein energy malnutrition; when the child’s weight falls below 60% of normal that child is diagnosed with marasmus, which results in growth retardation and a loss of muscle mass and subcutaneous fats. In kwashiorkor the weight loss is masked by the extensive edema which results form this condition. Here the visceral proteins, such as albumin are loss resulting in hypoalbuminemia. In contrast to marasmus, not much skeletal muscle mass or subcutaneous fat is lost. The Role of Cassava in Diet

The diets in these underdeveloped countries are usually rich in carbohydrates such as rice, yams and other tubers which are readily available and poor in proteins, vitamins, and minerals. Among the yams and tubers which make of the bulk of the diet, is cassava, (Manihot esculenta Crantz) an all season crop grown throughout the tropics. Cassava roots are the third most important source of carbohydrate after rice and corn in these countries in Africa, Asia, and Latin America. Cassava roots provide up to 60% of their daily energy-needs.

Figure 1. Cassava (http://www.biology-blog.com/blogs/permalinks/5-2006/cassava-plants-to-fight-hunge.html) Ironically, the Cassava leaves, which are not as widely used as the roots, have high protein content (17-34% dry weight, depending on the varieties). The essentials and non-essentials amino-acids profile of the Cassava leaves, with a few exceptions, are comparable to those of milk, cheese, soybean, fish and egg. (See Table 1.) Their

protein values exceed those of the FAO reference protein. In addition, Cassava leaves are very rich in minerals (Ca, Zn, Ni, and K), in vitamins B1, B2, C, and in carotene. Analysis also shows the presence of gamma amino butyric acid (GABA) and alpha-amino butyric acid (a-ABA).

Food (100g) Protein (g)

Almonds 21

Salmon 20.2

Beef (lean) 23

Eggs (1 medium) 8.1

Chicken breast (no skin) 30.1

Turkey breast (no skin) 29.9

Cod fillet 19.4

Lentils (cooked) 7.6

Cassava Leaves Crude 36.3g/kg

Table 1. A comparison of protein content in selected foods. Amino Acid content Soybean meal Dry Cassava Leaves

Alanine 4.4 5.7

Asparagine NA NA

Cystine 1.6 1.4

Glycine 4.4 4.8

Isolucine 4.7 4.5

Lysine 6.5 5.9

Proline 5.6 NA

Serine 5.5 NA

Tryptophan NA 2.0

Valine 5.1 5.6

Arginine 7.5 5.3

Aspartic acid 11.9 9.8

Glutamine 19.0 12.3

Histidine 2.8 2.3

Leucine 7.1 8.2

Methionine 1.6 1.9

Phenylalanine 5.6 5.4

Threonine 4.2 4.4

Tyrosine 4.7 4.0

Table 2. A comparison of amino acid content of soybean and dry cassava leaves

Activities

Choose two of the following extended activities to explore with your group. Be prepared to to present your findings to the class.

1. Some amino acids are considered non-essential, which ones are these? Why are

they considered non-essential?

2. How might starvation affect the availability of these non-essential amino acids? How might a diet containing cassava leaves address this problem?

3. Cassava leaves contain linamarin, a cryogenic glucoside. How does low protein intake contribute to the development of neuropathologies (Konzo) on prolonged cassava leaves consumption?

4. The mitochondrial enzyme rhodanese, is a sulfurtransferase. What is the role of the enzyme in cyanide metabolism? How does a adequate or inadequate protein diet contribute to the action of this enzyme?

5. Rhodanese catalyzes the transfer of sulfane sulfur from thiofulfate. 3-mercaptiopyruvate sulfurtransferase catalyzes a similar reaction. Are there regions of homology between these two molecules? If these regions exist, what might they represent?

6. How is PEM diagnosed? What clinical tests would help support a diagnosis of PEM? Why?

7. Identify five countries where PEM is currently a concern. Make an argument for the efficacy of genetically improving a local crop versus providing diet supplements to these populations.

Part II. Production of Methionine-rich Cassava leaves

Cassava leaves, cheap and abundant in developing countries, represent a highly sustainable strategy to address the PEM challenges, and offer a significant alternative source of proteins to replace the costly conventional source of proteins. Cassava leaves may constitute a solution to alleviate PEM-related disease due to their high protein content (17-34% dry weight, depend on the varieties). However, the nutritional value of cassava leaves is limited because of the deficiency in methionine content in relation to the FAO recommendation for protein. Golden rice, genetically fortified in beta-carotene (pro-vitamin A) to address the occurrence of blindness, is an example of one strategy to alleviate nutrient deficiencies in developing countries An international team of scientists led by Richard Sayre, supported by a grant of $12.1 M from the Melinda & Bill Gates Foundation, works to “fortify the cassava plant, a staple root crop in many developing countries, with enough vitamins, minerals and protein to provide the poor and malnourished with a day's worth of nutrition in a single meal.” (To find out more about this effort, go to: http://esciencenews.com/articles/2006/06/30/fortified.cassava) Activity 1: Producing Methionine-rich Cassava Leaves Recombinant DNA technology and tissue culture methods will allow scientists to transfer genes into plant cells and then select plants expressing useful traits such as resistance to disease or improved nutritional qualities. The researchers above plan to use plant genetic engineering techniques to reach their goals. You need to know how the researchers introduce transgenes into a cassava plants that will encode increased methionine production in the leaves.

1. The following four enzymes are used by genetic engineers. Explain why by describing the function of each enzyme in the table below.

Enzyme Function

DNA polymerase

Restriction enzymes

Ligase

Reverse transcriptase

2. List at least three techniques used to transform plants (alter their DNA) with a brief explanation of each.

Activity 2: Inserting Genes with Agrobacterium tumefaciens

1. Agrobacterium tumefaciens is a widely used bacterium in biotechnology due to its ability to insert foreign DNA into plants. Briefly describe how it is helpful for the bacterium to be able to do this.

Figure 2. Gall produced by A. tumefaciens

2. What is the role of the Ti plasmid?

Figure 3. The Ti plasmid from a strain of Agrobacterium tumefaciens

3. According to fig.3 it is necessary to replace T-DNA, the tumor -producing gene

with the gene of interest (methionine in this case) and two other genes. Give their names and describe their roles.

4. Suppose that you were able to make a mini-plasmid recombining the three genes and transfer it back into A. tumefaciens to replace its own T-DNA. What will be the next step to modify the cassava leaves. What do you need to put in the medium to kill the bacteria cells.

Activity 3: Isolating a Suitable Methionine Gene You are assigned to finding out the sequence for the methionine gene in cassava, so you look it up in an online database or from a publication.

1. Explain how researchers determine a sequence.

2. High levels of methionine can be found in brazil nuts, fish, and several meat sources. Which of these would be most likely to contain DNA that might be useful to increase methionine production in cassava? Why?

Your research turns up a paper by Gander et al.(1991) on the success of transgenic beans with a 23% increase in methionine content. The researchers used the plasmid pEA23 containing the glucuronidase (GUS) coding region as well as the 35S CaMV promoter and the 2S-albumin gene from the Brazil nut (be2s2). This doubled the activity of the 35S CaMV promoter plus the AMV enhancer sequence resulting in more methionine. So you are ready to begin!

3. How would you introduce a plasmid containing the new DNA into the plant cells? Explain the sequence.

Your plants are ready after the plasmid experimental procedure above. They all look green and healthy.

4- How can you determine which plant is expressing the new methionine gene?

8. It is not easy to carry out the experiments in a laboratory because of time constraints

and limited chemical supplies, you will run a molecular biology simulation developed

by Mark Bergland and Karen Klyczek from UWRF to select the transgenic plant containing the methionine Elisa and Western blot using an anti-2S-BN polyclonal antibody(Aragao et al.,1999) 6. Describe two other methods to insert gene in plants

Activity 4. Are Genetically Modified Organisms (GMO) safe?

Figure 4. a. Glofish: These are first genetically modified animals to be sold as pets.

Figure 4. b. Kenyans examining insect-resistant transgenic Bt corn.

See Figure 4. (http://en.wikipedia.org/wiki/Genetically_modified_organism The genetic engineering techniques that you studied to produce the

methionine-enriched cassava (a GMO product) have expanded rapidly and its applications have widespread in many humanitarian areas such as production of golden rice to fight blindness, insulin for the diabetics, plant resistant to pest, herbicides, harsh environment to mention few. In USA alone, 89% of soya beans and 60% of corn are GMO products

However the expansion has not been without controversy, certain countries have even banned or instituted moratoria on the use or production of GMO. Some public interest group have protested against GMO products.

Group Activity: Outline the pros and cons of the following project: You may use some of

the questions below to defend your arguments

• Genetically modified organisms have potential to help prevent diseases. Can these food items be used effectively to prevent disease in at-risk populations?

• GMOs have potential to create less expensive foods that contain the appropriate amount of nutrients. Can this translate into appropriate food supplies for people with limited economic resources?

• GMOs could produce more food from the same amount or less cropland. What is the economic impact to U.S. and world agricultural economies?

• GMOs could be developed that can survive droughts or floods on lands that are currently unable to sustain crops. What are the environmental impacts of bringing this land into production?

• GMOs augment certain properties of foods through genetic manipulation. Can we understand interactions with other systems of the body, other foods, pharmaceuticals, or allergic reactions?

http://ohioline.osu.edu/hyg-Fact/5000/5058.html

Should bananas be modified to produce human vaccines against infectious diseases?

PROS CONS

PART III: Cassava’s Hidden Risk Have you ever eaten tapioca or used tapioca to bake a delicious dessert?

Tapioca is a derivative of the Cassava root and in many places of the world it is a staple food source. Despite the food security it provides, Cassava roots and leaves contain potentially toxic levels of cyanogenic glycosides [linamarin (95%) and lotaustralin (5%)] (Conn, 1979, 1994; Balagopalan et al., 1988). Depending on the variety of the cassava, the cyanogenic glucoside content of the leaves could be as much as six times that of the root.

The consumption of poorly processed cassava has not only been associated with

insufficient amino acids such as Cys and Met in human diets, but also cyanide-poisoning leading to a neurological disease called konzo. This paralytic disease can sometimes result in death ((Howlett etal.,1990) and has reached epidemic proportions in some countries. Cassava has caused other diseases such as tropical ataxic neuropathy, and hyperthyroidism as well.

Figure 5. Due to the toxicity of cyanogens in cassava, these Konzo patients are treated at Liupo Rehabilitation Centre, Mozambique. (See http://www.anu.edu.au/BoZo/CCDN/index.html)

Activity 1: Group Investigations

Develop your own question for study in the use of cassava with respect to global health.

Example 1. Extend your exploration of the problem of cyanogenic glucosides by developing a pamphlet for distribution that describes how the preparation of the roots reduces the potential for cyanide poisoning. Include images and online resources for the reader.

Example 2. Write a short article suitable for the public on why cassava poisonings increase in drought seasons. Be sure to include graphics with a brief explanation for why they are helpful.

Example 3. Put together a poster for an organic chemistry class explaining cassava cyanide production. Also add your own global health focus.

Example 4. Cassava leaves contain linamarin, a cyanogenic glucoside. How does low protein intake contribute to the development of neuropathologies (Konzo) on prolonged cassava leaves consumption?

Activity 2: Exploring Cassava Chemistry

Due to the importance of cassava as a source of dietary energy and cash crop in the tropics, the reduction of cyanogens in cassava is a critical research area. Researchers all over the world have worked to study the biosynthetic pathway of cyanide and reduce cyanogens content of cassava using different transgenic strategies.

Figure 6. Cyanogenic glycoside (linamarin) synthesis and turnover pathways in cassava. (From http://www.ncbi.nlm.nih.gov/pubmed/15630626)

1. How is cyanide produced in Cassava?

Activity 3: Bioinformatics: Exploring DNA Sequences in Cassava and Other Plants Producing Cyanogenic Glycosides

Beside cassava, other economically important crops such as sorghum, almonds, lima bean, white clover, and the rubber tree also produce cyanogenic glycosides. The availability of online molecular data such as DNA sequences will allow you to compare gene sequences from different organisms that have the same function. In Figure 6. you can see that the gene encoding Cytochromes P-450 (CYP79D1 and CPY79D2) catalyze the first steps in the biosynthesis of the cyanogenic glucoside linamarin in cassava.

Begin by comparing Sorgum cytochromes CYP79A1 and two Cassava cytochromes CYP79D1/ CYP79D2 using SWAMI, a suite of bioinformatics tools that enable you to locate, compare, and organize sequence information. Steps:

1. Log in to SWAMI: http://www.ngbw.org/web/login!input.action a. Register for a free user account (If don’t already have an account) b. Log in

2. Create a new folder a. Click Create New Folder b. Type a name for the folder. For instance “Sorghum and Cassava

Cytochromes” 3. Upload Cytochrome sequences

a. On the left side of the screen, you should see the folder you just created. b. Under the folder, click Data. c. Click Upload/Enter Data

i. Type in the label: “Sorghum CYP79A1” ii. Do a copy and paste of the Sorghum CYP79A1 sequence into the

“enter your data” area. iii. Choose entity type: “Protein” iv. Choose data type: “Sequence” v. Leave data format as: “unknown”

d. Click Return to Data e. Repeat steps i. through v. with the other two sequences.

i. Label them Cassava CYP79D1 and Cassava CYP79D2 4. Create two tasks to compare the Protein Sequences

a. Click the Toolkit tab b. Click the Protein Sequences Tools tab c. Choose Align

i. Type a description: “Sorghum CYP79A1 vs. Cassava CYP79D1”

ii. Click Set Description iii. Click Select Input Data

1. Check Sorghum sequence 2. Click Select Data tab

iv. Click Set Parameters 1. Choose Cassava CYP79D1 as the second sequence 2. Click Save Parameters

v. If Align is not on the button next to tool: Click Select Tool and Choose Align

d. Click Save Task e. Click Create New Task and repeat steps for “Sorghum CYP79A1 vs.

Cassava CYP79D2” 5. Run the tasks and save the output

a. Click Task link, if necessary, to return to task list b. Click Run the task for each task

i. Might have to click Refresh Task , if you see the View Task option but not the View Output option

c. Click View Output d. Click filename “align.txt” e. Click Save to Current Folder

i. Label as “Sorghum CYP79A1 vs. Cassava CYP79D1 output” and“Sorghum CYP79A1 vs. Cassava CYP79D2 output”

ii. Choose entity type “protein” iii. Choose data type “sequence alignment” iv. Click Save

6. Try doing an alignment of the two Cassava Cytochromes vs each other 7. Try doing the alignment by choosing a Cassava Cytochrome as the input and

then the Sorghum as the second sequence 8. Questions:

a. How similar is the Sorghum Cytochrome to each Cassava Cytochrome? (answer 54% from literature)

b. How similar are the Cassava Cytochromes to each other? (answer 85% similarities)

c. Are the results different if you choose Cassava as the input and Sorghum as the second sequence?

Activity 4: Silencing the Killer CY79D1 and CYP9D1 Genes: Production of Cyanogenic Glycoside Free Cassava

An European team led by Birger Moller produced transgenic cassava plants with a 92% reduction in cyanogenic glucoside content in tubers and acyanogenic (less than 1%) leaves. These results were obtained by RNA interference (RNAi) to block the expression of CYP79D1 and CYP79D2. (To find out more, see: http://www.plantphysiology.org.)

RNAi is a natural process of gene silencing that occurs in nearly all plant and animal cells. RNAi protects the genome not only from virus and transposable elements but also plays a role in developmental control. The term RNAi was first used by Andrew Fire and Craig Mello who received the Nobel Prize in Physiology and Medicine in 1998 for their work in silencing specific genes expression homologous to the double strand RNA delivered into C.elegans. RNAi was called “an electrifying discovery”, the third revolution in biotechnology and became a powerful research tool use with tremendous potential in biotechnology and medicine for diagnostics and therapeutics In the following animation www.hhmi.org/biointeractive/ma/mai/index.html and the figures below, you are going to explore the mechanism of RNAi

Fig 1: RNAi pathway Fig 2: Double-strand RNA cut by Dicer enzyme Figure credits: ?

Understanding the Animation

Part 1.

1. Write a brief summary of the different steps of RNAi pathway. 2. Understanding transcription and translation of the molecular dogma, justify the

use of post-transcriptional gene silencing (PTSG) term used previously for RNAi.

3. Give the names of two other methods used to modulate mRNA

Part 2.

1. What is the fate of the dsRNA for the targeted gene after its introduction into the

cells? 2. What are the nature and function of the dicer? Is it appropriate to call the dicer

as “molecular ruler? Justify your answer

3. Are there endogenous (originating in the cells) dsRNA? If yes, what are they called?

Part 3. Dicer cleaves the dsRNA into fragments of 20-23 nt or small interfering RNA (siRNA) with 2-3’ overhang on each end.. 1. What may be the benefits for having these short double-stranded RNA? 2. Can siRNA silence non-targeted gene (off-target) which have homologous

sequence to the targeted one?

3. What are the possible consequences of “off-target” or on-specific of siRNA? Part 4.

The siRNA are incorporated into the RNA-induced silencing complex (RISC). RISC contains an argonaute.

1. What is the function of the argonaute?

2. What is the fate of the sense and antisense from the dsRNA?

3. Now that you have successfully reviewed the RNAi mechanisms, describe how you might apply this technology to help solve real biological problems. Scientists could use RNAi technology to reduce the amount of cyanide in the cassava leaves, produce tomatoes rich in omega-3-omega 6 or treat diseases such as HIV or heart attack.

4. Choose one of the projects proposed above and describe the different steps you

are going to undertake.