Resumen de las Investigaciones Presentadas en el Séptimo ... · analysis was performed, revealing...

Resumen de las Investigaciones Presentadas en el

Séptimo Simposio de Estudiantes Investigadores en el Área de las Biociencias

Universidad Interamericana de Puerto Rico

Recinto de Barranquitas

13 de marzo de 2015

2

Heavy metals and gene expression in plant stomata

Hernández Miranda, Yalexa M.; Negrón Berríos, Juan A.

Inter American University of Puerto Rico – Barranquitas Campus

Stomata are structures that regulate leaf gas exchange with the atmosphere, allowing the entrance

of CO2 to promote photosynthesis and the exit of O2 as a byproduct. Many studies have

documented the changes caused by environmental stress in the number of stomata on plant leaves

and in the opening and closing of these plant structures. Opening and closing of stomata is

regulated by environmental and physiological factors, such as hormones, however, many

molecular and genetic details have not been revealed. The purpose of this research is to

understand the genetic and molecular basis of the environmental stress on plant stomata. We are

focused on the effect of heavy metals on the expression of the following genes from Arabidopsis

thaliana: stomatal closure-related actin binding protein 1 (SCAB1), actin 2 (ACT2), myosin

(MYOATP), metallothionein 2B (MT2B), and nine-cis-epoxycarotenoid dioxygenase 3 (NCED3).

Plants were exposed to cadmium and total RNA from whole plant and plant stomata was isolate.

Different bioinformatics tools were used to assess and identify specific oligonucleotide sequences

for these genes. cDNA was obtained and gene expression was determined by real time PCR.

Preliminary results showed changes in the expression of SCAB1, ACT2, MYOATP, MT2B and

NCED3 due to exposure to cadmium. Experiments are underway to establish a relationship

between the opening and closing of stomata due exposition to cadmium.

3

New Bio-inorganic Antibiotic for ESKAPE super bugs

Stephanie González Morales; Ana Pérez Matos

Inter American University of PR – San German Campus

During the last thirty years, the resistance of bacteria to antibiotics has increased significantly.

Existing antibiotic molecules have been evaluated for the development of new antibiotics in order

to decrease the resistance of the organisms. Since 2010, only one new antibiotic has been approved

by the FDA (Food and Drug Administration) to help fight these bacteria. The American Society for

Infectious Diseases (IDSA) has identified six pathogenic bacteria associated with antibiotic

resistance. The acronym “ESKAPE” is utilized in order to emphasize that the majority of hospital

infections worldwide "escape" from the antibacterial effects. Microorganisms associated with

antibiotic resistance are ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus,

Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and

Enterobacteriaceae). This group of bacteria represents new paradigms in the pathogenesis,

transmission and resistance. These are responsible for a large percentage of nosocomial infections

in hospitals. Among the individuals who have been infected by these bacteria are hospitalized

patients, cancer patients, people in intensive care unit (ICU), people exposed to mechanical

ventilators, patients with bacteremia and patients with catheters. The purpose of this study is to

find a more powerful antibiotic that helps eliminate the resistance of these nosocomial bacteria. It

proposes the use of binders as a new antibiotic that could help decrease the resistance of these

microorganisms. Our preliminary studies showed that some ESKAPE bacteria are indeed resistant

to commercial antibiotics such as penicillin, novobiocin and streptomycin. It is expected that when

testing the binders with ESKAPE they can have a better effect than commercial antibiotics.

4

Cadmium Resistance of Bacterial Cells Transformed with Small Non-coding RNA’s from

Arabidopsis thaliana

Marrero Rodríguez, Alba L.; Negrón Berríos, Juan A.

Inter American University of PR – Barranquitas Campus

Non coding RNAs have been associated to the regulation of gene expression and metabolism in

eukaryotic organisms. In plants it has been found that non coding RNAs are expressed under abiotic

stress. In this study, we transformed Escherichia coli’s JM109 strain with small non-coding RNAs

extracted from Arabidopsis thaliana, exposed to the toxic heavy metal cadmium. We want to investigate

the effect of the plant non coding RNAs in the bacteria tolerance to cadmium. Small non-coding RNAs

isolated from Arabidopsis thaliana plants exposed and not exposed to cadmium were cloned in E. coli

using the pGEM-T Easy Vector System. After DNA isolation and sequencing of inserts, a bioinformatic

analysis was performed, revealing high similarity with miRNAs found in A. thaliana. Bacterial cells

carrying different plant’s RNA sequences were exposed to various cadmium concentrations. After the

generation of growth curves, we analyzed the bacterial cells viability in relation to cadmium toxicity.

Transformed cells with Arabidopsis thaliana’s small ncRNAs have shown resistance to high levels of

cadmium, which are toxic to untransformed cells. These biologic systems could be used as alternatives

for remediation environments contaminated with toxic heavy metals.

5

Potential early colon cancer detection through molecular analysis

Rosendo Mercado, Nicole Loumarinett; Negrón Berríos, Juan A.


Cancer is a group of complex diseases caused by the interaction of environmental and genetic

factors. Today colon cancer is the number one cause of death in the U.S. territory alone;

nevertheless it affects men and women throughout the globe. Data from P.R. during 2010 reported

more than 14,011 known cases of colon and rectal cancer, at the same time there were 5,197

known deaths. The main objective of this study is to identify a potential colon cancer detection

method at a molecular level and before the disease is at an advanced period. We will focus on the

level of methylation of promoter region of the following genes: APC, DCC and MSH2. It is known

that there are few, to none, methods of early diagnosis of this illness; yet the epigenetics and DNA

methylation analysis could play an important role in this recognition. The addition of methyl

groups on cytokinesis at the CpG sequences of the DNA can alter gene regulation and block the

promotor region that allows the expression of the genes; DNA methylation is maintained after DNA

replication and is a stable epigenetic mark. Through the calibration and analysis of PCR and qPCR

of methylated DNA and non-methylated DNA, from a human tissue extraction, we can determine a

High Melting Temperature Curve and the percent of methylation of the DNA. Furthermore, it is of

vast importance to have the documentation required for the extraction and manipulation of

human colon tissue. The IRB, is an ethical review board that has been designated to approve,

monitor, and review biomedical research involving humans. The key goal of the IRB is to protect

human subjects from physical or psychological harm, reviewing research protocols and materials.

6

Effects of Cadmium on Epigenetics of Cytoskeletal Genes in CHO Cells

Colón Rodríguez, Idalisse; Negrón Berríos, Juan A.


Epigenetic is a mechanism that involves changes in the expression of genes, which are not involved

in changes or alterations in the sequence of the DNA strands. DNA methylation is one of the most

studied chemical changes associated to epigenetics. Highly methylated DNA is usually associated

to inhibition of transcriptions. Actin cytoskeleton provides support and internal organization to

the cell. It is also involved in intracellular transport. The goal of this study is to investigate

epigenetics of β actin non-muscle myosin type II in cytoskeleton of Chinese Hamster Ovaries cells

(CHO cells) exposed to cadmium. In particular, we are interested in the methylation profile in β

actin and non-muscle myosin type II during the toxic conditions induced by the heavy metal.

Genomic DNA was isolated and treated with the bisulfite reaction, in which the un-methylated

cytosine were transformed in thymine, later using PCR reaction these thymine residues were

transformed in uracil, whereas, the methylated cytosine residues remain unchanged. High

Resolution Melting (HRM) analysis through real time PCR assay was used to obtain the percentage

of DNA methylation. This allows us to discriminate between methylated and non-methylated

cytosine. Bioinformatics was used to develop specific primers for the promoter region of actin and

myosin from CHO cells. Primers were calibrated using regular and real time PCR, to make sure of

their specificity. Preliminary results indicate differences in methylation profile at the promoter

region of actin and myosin genes in CHO cells during to exposition to, cadmium.

7

Anticancer properties of Bryophyllum pinnatum

Rodríguez Rivera, Julio E.; Estrella Matos, Luis A.; Negrón Berríos, Juan A.


Bryophyllum pinnatum is a common medicinal plant used in traditional medicine for treatment of

various infections, bowel diseases, healing wounds and other diseases. Previous work in our

laboratory indicates that Bryophyllum pinnatum may have some anticancer properties.

Bryophyllum pinnatum extracts seems to reduce the viability of the colorectal cell line HCT 116.

Fluorouracil (5 FU) is a compound used in chemotherapies to target and kills tumor cells. The

purpose of this research is to perform a viability analysis using 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT), to compare the effect on cell viability in HCT 116 cells by

using 5FU alone and a mixture of extracts of Bryophyllum pinnatum and 5FU. This work is could

contribute to further demonstrate the anticancer properties of Bryophyllum pinnatum.

8

Isolation and Identification of Vibrio sp. in Marine Waters of Salinas Bay, Cabo Rojo

Beata Toro, Victoria; Pérez Matos, Ana

Inter American University of Puerto Rico – San Germán Campus

Bacteria belonging to the Vibrio genus are well-known pathogens that thrive in warm saline water.

Although most species of these bacteria only affect fish and marine wildlife, V. cholerae, V.

parahaemolyticus, and V. vulnificus affect humans, producing skin rashes and gastroenteritis.

Testing for these microorganisms is crucial, especially since researchers have recently found that

other species of Vibrio, such as V. alginolyticus, V. metschnikovii and V. harveyi clade, previously

thought to only harm fish, have virulent strands that could be harmful to humans. Previous studies

have found Vibrio in oysters harvested from the beaches of Cabo Rojo, Puerto Rico near Salinas

Bay, indicating the presence of these bacteria in the water. Two stations were chosen for sampling

sites through the coast of Salinas Bay, one presenting a high level of anthropogenic activity, the

other, surrounded by mangroves. The species and concentration of bacteria will be compared by

seasons, “Wet Season” (running from August-December 2014) versus “Dry Season” (running from

January-May 2015). The “Wet Season” surface water samples were filtered and diluted with

Alkaline Peptone Water (APW), 3.5% NaCl. After a 24 hour period, the samples were transferred

to Thiosulfate-citrate-bile salts-sucrose agar (TCBS), a Vibrio selective agar. Twenty-four hours

later, colonies were transferred onto Tryptic Soy Agar (TSA), 3.5% NaCl for differentiating

colonies. All samples were incubated at 37˚ C. Visibly different colonies were analyzed

biochemically with API 20E® strips. The results demonstrate preliminary identification for V.

parahaemolyticus, V. vulnificus, V. cholerae, V. alginolyticus & V. fluvialis. The products of the 16S

rDNA PCR amplification have been sent for sequencing. Upon receiving the results, they will be

subjected to phylogenetic analysis. Previous studies suggest “Wet Season” will demonstrate higher

bacteria concentration than “Dry Season”.

9

Antioxidant Activity, Total Phenolic and Flavonoid Contents of Scutellaria havanensis

Zavala Ortiz, Yarelia; Negrón Berríos, Juan A.


Scutellaria is a genus of about 400 species and many species have been used in traditional Native

American medicine. Pharmacological studies have confirmed that total extracts or flavonoids of

the genus Scutellaria possess anti-lipoperoxidation, anti-platelet, anti-inflammatory, antitumor,

hepatoprotective, antioxidant, antibacterial and antiviral activities. It is proposed to research the

herb Scutellaria havanensis, only Scutellaria species reported from Puerto Rico. Therefore, the

objective of the present study was to determine the total phenolic and total flavonoid contents,

and to evaluate the antioxidant potential of leaf extract of S. havanensis.

Using the Folin Ciocalteu’s Reagent, total phenols of S. havanensis were measured at an

absorbance of 765nm. Also, the antioxidant activity of methanol and ethanol extracts was

evaluated by TROLOX equivalent antioxidant capacity (TEAC). The total flavonoid content of the

extracts was estimated by the Aluminum Chloride Colorimetric Method. In summary, the methanol

extracts showed the highest polyphenolic content and possessed the better antioxidant activity

than the other extracts. However, the total flavonoid content was highest in ethanol extract. The

results indicated a direct correlation between the antioxidant activity and the polyphenolic

content of the extracts. The present study confirmed that the methanol extract of S. havanensis

leaves is a potential source of natural antioxidants.

10

Compost Development based on grass and tree cortex

Jiménez Ortiz, Arleen; Sandoval Amaral, Aramis; Negrón Berríos, Juan A.


The compost is the result of the biodegradation of organic material. In this case, grass and wood

chips will be used. The Chinese used this technique during thousands of years. The biodegradation

process occurs by the action of microorganisms, in particular fungi and bacteria. There two ways

to create compost, aerobically, which occurs in presence of oxygen and anaerobically which occurs

without oxygen. The compost that is being developed is vegetative, that is, it only contains grass

and wood chips, and the process will be aerobic. The organic material that is being collected is

freshly cut grass and wood chips from Inter American University at Barranquitas Campus. This

organic material is free of pesticides and fertilizers. The main objective of this project is to

calibrate the vegetative compost production protocol. Organic materials have been mixed in three

different proportions. Different parameters like temperature, pH and humidity will be measured

in order to determine optimum conditions for the elaboration of the compost. The compost

contains materials with high nutritional value that can be used as a natural fertilizer in different

food crops or ornamental plants. In addition it can be used to restore environmental conditions

in saturated areas by the exploitation and the misuse during industrial and agricultural activities.

The project is just beginning, but is expected that once the protocol calibration has been

completed, the project will begin the stage with the macroeconomic view, since it is highly cost

effective and the materials will be obtained from the campus and water used will be from the rain.

The product will be marketed at island level in an environmentally friendly packaging and under

optimum quality control that guarantees the use of our compost in farms, homes and other places.

11

Antimicrobial Properties of Copper

Rios González, Celeny D.; Romero Pérez, Lizbeth

Inter American University of PR – Arecibo Campus

Copper is an important metal that is distributed throughout nature and whose discovery dates

from around 5000 BC. Pure copper is soft and malleable; a freshly exposed surface has a reddish-

orange color. It has been used in a variety of applications ranging from construction of household

utensils, clothing accessories and even as an electric conductor. History has shown a health related

use for copper in ancient civilizations. Copper is a powerful antimicrobial with proven rapid, board

spectrum efficacy against pathogens threatening public health and wire community. On this study,

we want to evaluate and compare, at two different times, the effect of solid copper on

Staphylococcus aureus and Enterobacter aerogenes. The antibacterial effect of copper will be

compared with plastic and stainless steel. Our results show that copper inhibits the three tested

bacteria at 1 and 3 hours of exposure. Copper surfaces showed complete inhibition of growth in

contrast with the effect observed in plastic and stainless steel. We also tested, using S. aureus and

Pseudomonas aeruginosa, the antibacterial effect of copper in solution. Inhibition of copper was

observed on S. aureus at a concentration of 15 ug/ml. Our results demonstrate the antibacterial

properties of copper on bacteria of medical important. Solid copper is more effective inhibiting

growth of bacteria than copper in solution. Further studies are required to test more

concentrations of copper in solution and other strains of the tested bacteria.

12

Micropropagación de Arracacia xanthorrhiza

Meléndez Rivera, Omaliz; Negrón Berríos, Juan A.


Arracacia xanthorrhiza is an edible tuber grown in many countries in South America, such as

Brazil, Venezuela and Perú. In Puerto Rico Arracacia is produced by a small group of farmers from

the center of the island for local distribution. The culture of the tuber in soil have been largely

affected by weather conditions, harvest time and pathogenic pests, like fungi, viruses, and bacteria.

Many of these problems would be avoided through the development of in vitro culture by

micropropagation technique in the laboratory. By in vitro culture we could obtain seeds and

plantlets for subsequent planting in farmland. In this work, the propagation of Arracacia was

achieved by the culture of meristematic tissue, petiole, and tuber. Two different disinfections

protocols were used to eliminate pathogens. Murashige & Skoog (MS) medium developed shoots

and calluses with a combination of different growth factors. Benzyladenine, and naphthalene

acetic acid with MS produced calli from petiole. However, benzyladenine with MS formed shoots

from the meristematic tissue and tuber. Finally, disease-free plantlets will help farmers to

commercialize a healthy tuber.

13

Micropropagation of Theobroma cacao

Emily Anette Collazo López, Luis O. Ríos Colón


The Theobroma cacao is a native South American plant, which belongs to the division

Magnoliophyta and the Malvaceae family. Cocoa is the main product that is derived from T. cacao

which contains a high market demand, especially for the production of chocolate. Commercial

production of plants requires and efficient and systematic strategy to generate plants with similar

phenotypic characteristics. Micropropagation techniques offers an alternative to generate

plantlets from different parts of the plant. Culture protocol is being standardized to produce

plantlets from somatic tissue and embryos. Once the plantlets have been developed, an effective

acclimation protocol will be established. Non-the less, multiplying calluses obtained for T. cacao

will be compared genetically with the parent plant. After obtaining these results, the goal is to

obtain seedlings with little genetic variation and high quality.

14

Antioxidant properties of Arracacia xanthorrhiza

Santos Hernández, Wigdalí; Negrón Berríos, Juan A.


Food from plant origin, especially fruits and vegetables, may have a protective effect against

oxidizing agents. This property is due to the presence of bioactive compounds with antioxidants

such as vitamin C, vitamin E, β-carotene, and a complex mixture of phenolic compounds. The

objective of this work is to study the relation between total polyphenol content and antioxidant

activity in the root vegetables Arracacia xanthorrhiza and Solanum tuberosum. Polyphenols were

determined after extraction by the Folin-Ciocalteau method. A standard curve was generated with

the reagent gallic acid, and by measuring the absorbance of 765 nm. A linear regression curve of

0.95 was obtained. The vegetables studied showed better absorbance and concentration in their

raw state. Results obtained indicate that Solanum tuberosum has a higher polyphenol content than

Arracacia xanthorrhiza. Searching for other antioxidant products, a carotenoids analysis was

carried out by thin layer chromatography. These results showed that Arracacia xanthorrhiza

contains more carotenoids than Solanum tuberosum.

15

Measurement of Methylation Pattern in HCT116 Cell Line Human Colon Carcinoma

Jiménez Berrios, Joel; Panzardi Ortega, Francisco; Negrón Berríos, Juan A.


Colorectal cancer is one of the most frequent cancers in the world; it is caused by genetic mutations

and epigenetic alteration of the epithelial cell of the colon. The gene K-ras plays a major role in the

development of this cancer, due to a mutation of this proto-oncogene that proceeds to

uncontrollable cell growth. Evidence shows that the mutation of this gene triggers a cascade of

alterations and lesions in DNA. HCT 116 cell line from human cancer carcinoma has a mutation on

codons 12 and 13 that is common in many cancers, including pancreas, colon, and lung cancer.

This cell line was used to extract genomic DNA to characterize this mutation. PCR calibration

showed optimal results at 25µM of primer concentration, and a band of the gene K-ras near 165bp,

as predicted by bioinformatics. In addition, on this band will be extracted and digested using

restriction enzymes in order to distinguish normal Kras from that mutated on codons 12 and 13.

The main goal of this study is to correlate the Kras mutation with the level of methylation of other

genes associated to colon/rectum cancer. In addition, the methylation profile of Kras will also be

studied. Findings will help to establish the potential implication of epigenetics in colon/rectum

cancer.

16

Somaclonal variation in Arracacia xanthorrhiza

Rivera-Zofakis, Ellena C.M.; Negrón Berríos, Juan A.


All plants regenerated from somatic cells should be clones, but a number of observations have

indicated that this is not the case. Somaclonal variation is a genetic variation observed among

progeny of plants regenerated from somatic cells cultured in vitro. This type of variation has been

proven useful in breeding programs of various crop plants, in conventional plant breeding and

solely being the avenue for crop improvement. Somaclonal variation provides great promise for

reducing the time required to produce new varieties of plants and breeding new lines that are

easily patentable due to their novel variation. Commercially somatoclonal variation could bring

difficulties to obtain plants with similar phenotype. In this work, the objective would be to find

out if it there is genetic variability in Arracacia xanthorrhiza cultured by micropropagation. This

variation will be identified by DNA fingerprinting, using Random Amplified Polymorphic DNA

(RAPD). Genomic DNA from clones and mother plant will be isolated and subjected to PCR analysis

using random primers. Results from RAPD analysis will provide preliminary data in terms of

genetic variability within the clones and also with the mother plant. Our long term goal is to obtain

plants from calluses, with little genetic variation, that could be used as starting material by farmers

during large scale production of Arracacia xanthorrhiza.

17

Micropropagación de Calathea allouia

Pérez González, Jocelys; Negrón Berríos, Juan A.


El lerén (nombre científico: Calathea allouia) crece en åreas tropicales del Caribe y América del

Sur. Su valor se debe a su tubérculo que se puede utilizar como alimento. La producción de lerén

en Puerto Rico se limita a cantidades mínimas cultivadas por agricultores independientes. Esto ha

resultado en un riesgo para la existencia de la planta ya que son menos los lugares donde se

consigue. Por lo tanto, la micropropagación podría ser una opción que pueda rescatar el cultivo

del lerén debido a que se obtendrían mayores cantidades de plantas en condiciones óptimas y

listas para plantarse en poco tiempo. Sin embargo, esta planta es propensa a desarrollar hongos y

se debe establecer un método determinado para reducir su contaminación.

18

Extraction and analysis of DNA isolated from harvestmen

García, José; Ayala, Jonathan; Romero, Lizbeth

Inter American University of PR – Arecibo Campus

Harvestmen are known to be humidity indicators. They belong to the order of Opiliones and can

be found in rocks and caves. In Puerto Rico, only 10 species have been identified. With the

advancement of molecular techniques, phylogenetic relationships among them can be easily

established. The objective of our study is to identify a method to isolate gDNA from harvestmen.

Four specimens were collected at El Yunque Rainforest or El Karso in Arecibo. One leg, two legs or

¼ of the body were used. The extraction was carried out using the DNEasy blood and tissue kit

(QIAGEN). Concentration and purity of the DNA was determined by spectrophotometry. PCR was

performed using primers for 28S rDNA gene on a reaction of 35 cycles. Our results show that gDNA

obtained from one leg is enough and has good quality to allow gene amplification. Further studies

are required to amplify other genes in order to identify unknown species of harvestmen found in

Puerto Rico.

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