Investigaciones – Ciencias y Tecnología

Bioprospecting for Antibiotics from Marine Bacteria

Dra. Prachi Tripathi
prachitripathi@br.inter.edu

Actinomycetes are a group of Gram-positive bacteria that are the source of over 70% of the antibiotics we use. Members of the group have the potential to produce a range of secondary metabolites with bioactive properties. Although much of the earth’s surface is covered by marine habitats, actinomycetes from these environments have remained relatively underexplored.
In this research project, a number of marine habitats close to the shorelines of Puerto Rico will be investigated for the presence of antibiotic-yielding actinomycetes. The project will be carried out in four phases: (1) site selection and collection of seawater samples, (2) isolation and culture of bacteria, (3) study of antagonistic activity of cultured bacteria against selected pathogenic bacteria and (4) Atomic Force Microscopy (AFM) study of bacteria found to have such antagonistic activities. The project is also expected to result in the establishment of a culture collection of marine actinomycetes at IAUPR Barranquitas Campus.

Increasing Student Engagement and Retention in STEM Through Advancing Research Experience and Knowledge in Marine Biotechnology (NSF Award# 1928792)

Dra. Prachi Tripathi
prachitripathi@br.inter.edu

With support from the Improving Undergraduate STEM Education: Hispanic-Serving Institutions (HSI Program), this Track 1 project aims to increase undergraduate student interest, retention and success in Biological Sciences. This collaborative project at the Barranquitas and Guayama campuses of the Inter American University of Puerto Rico will carry forward the goals of the HSI Program in economically disadvantaged, rural areas of Puerto Rico. The project will target those points along the academic pipeline that represent critical transitions at which students are most likely to switch to non-STEM major or drop-out of college and will do so through innovative changes in STEM teaching and learning.

Puerto Rico being an island offers a plethora of opportunities in Marine Biotechnology from the standpoint of research as well as industrial applications. However, there has been very little progress in this direction, chiefly due to a serious lack of educational offerings in the area of marine biotechnology at institutions of higher education in Puerto Rico, which in turn translates into lack of trained personnel in this area. By introducing a minor concentration in Marine Biotechnology, along with an elective course in Blue Chemistry at the undergraduate level, this project aims to revitalize the undergraduate biology program, as well as train undergraduates to assume roles in academia, policy-making and industry.

The specific goals of this project are (1) enhancing Hispanic student opportunities attaining degrees in STEM education through critical transition utilizing new courses and curriculum revision; and 2) improving the Hispanic students’ STEM learning experience by research/problem-based learning, collaboration between two campuses and modern laboratory activities including a teaching museum. The project will generate new knowledge about how a research-focused learning experience at the undergraduate level combined with the support from tutorial services and faculty trained in modern scientific tools and techniques can contribute to higher retention and degree attainment in STEM. Further, it will generate knowledge about the impact of introducing a minor in an applied, job-oriented area of study, such as Marine Biotechnology, on the number of students choosing a STEM major. In addition, the project will assess if and how a community science center such as a marine museum can encourage high school students to enroll in STEM courses in college. The effectiveness of these interventions will be evaluated through a mixed-methods evaluation and will include a combination of the before-and-after study design and both quantitative and qualitative methods providing data for both formative and summative evaluation. The findings of this project will be disseminated widely through symposia, publications and through a dedicated website.

Fish Species Diversity at Usabón River Barranquitas, Aibonito; Puerto Rico

Prof. Antonio E. Carro Anzalotta
acarro@br.inter.edu

An inventory of fish species at the lower Usabón river ecosystem was done by capturing fish with nets and electrofishing equipment. Fish at the lower Usabón river included a total of 12 species (all of them exotic). Native fish species have not been documented during the time of the study at the Usabón river either because they have never been present or because manmade dam structures prevent native fish migration.

Fish species inventory were done at the Usabón river through a 25 year period show a diverse array of introduced fish populations with no representatives of native species.  Fish were sampled using cast nets, hook and line and electrofishing equipment.  Some information was also obtained by interviewing local fishermen.  Fish species present at Usabon river respond to various periods of fish introductions to Puerto Rico.  (1) the prehatchery phase, with limited introductions from the United States to rivers and earliest reservoirs; (2) the coldwater phase, with primary emphasis on trout species for high-altitude river introductions; (3) the early warm water phase, with generous species introductions and supplementation without significant evaluation; and (4) the current modern phase, with primary focus on largemouth bass and prey species along with significant assessment, evaluation, and research on stocking efficacy. These species have impacted mostly reservoirs but some have become established on rivers. There is another group of fishes that have been introduced for aquaculture purposes and as aquarium pets.

The largest streams of Puerto Rico have been impacted by the construction of reservoirs for domestic, irrigation and power generation uses.  The upstream portion of the dams generally loses its native fish populations.  Usabón river is an effluent to La Plata river which is one of the biggest rivers in the Island with several small and big dams.  Dams without spillway act as an impermeable barriers that eliminate all native fish and shrimp fauna from upstream reaches.  This aspect poses a great threat on the life cycle of anphidromous species and gives advantage to exotic or introduced species that can complete their lifecycles in freshwater.  Exotic species invasions is another force against  native freshwater fishes. The impact of exotic fish populations on native species has been poorly studied.

Secuenciación del genoma del cloroplasto de Arracacia xanthorrhiza Bancroft: Importancias biotecnológicas y filogenéticas

Profa. María M. Meléndez Ortega
mmmelendez@br.inter.edu

La planta Arracacia xanthorrhiza, conocida internacionalmente como Zanahoria Peruana y en Puerto Rico como apio, pertenece a la familia Apiaceae y es originaria de los Andes. La planta genera un tubérculo que tiene un alto valor económico y nutricional, siendo el segundo cultivo de mayor demanda en América del Sur y en la zona central de Puerto Rico. En los últimos años se ha observado el aumento de agentes patógenos que provocan en ciertos casos, la pudrición del cormo teniendo como efecto una marcada disminución en la siembra y producción del apio. La ausencia de prácticas de manejo preventivo y propio de los tallos aéreos, utilizados en la propagación de la planta hace que las infecciones causadas por patógenos dañen el tubérculo y otras partes de la planta. A pesar de la importancia económica y agrícola del apio, no existe ninguna estrategia biotecnológica para mejorar o proteger al cultivo. La falta de investigación enmarcada en el aspecto molecular y biotecnológico son las bases para iniciar el proyecto de secuenciación del genoma del cloroplasto de A. xanthorrhiza. La secuenciación del genoma del cloroplasto permitirá reconocer los metabolitos que se sintetizan cuando la planta: (1) interacciona con el ambiente, (2) activa los mecanismos de defensa ante la invasión de patógenos, (3) se adapta ante los efectos del cambio climático y (4) a su vez permitirá la identificación, conservación y estudio filogenéticos. Es por tanto que el objetivo principal de la investigación es establecer el genoma completo de A. xanthorrhiza, caracterizar la organización del genoma del cloroplasto y compararlo con Daucus carota y Solanum tuberosum. Para esto, utilizando el Illumina’s NexteraXT DNA Sample Prep kit y la plataforma MiSeq de Illumina del Instituto de Biotecnología Sustentable (UIPR, Barranquitas) se realizó la experimentación. Los resultados indicaron que el genoma completo del cloroplasto de A. xanthorrhiza posee 143,989bp (Genbank: NC_032364.1) con una estructura cuatripartita conservada que contiene 111 regiones codificantes, 83 regiones únicas, 12 regiones repetidas, 33 tRNAs y un contenido de 37.5% GC. Mientras que al comparar el genoma completo de A. xanthorrhiza mediante programas computadorizados indican que D. carrota presente menos regiónes intergénica que S. tuberosum al comparase con A. xanthorrhiza. En tanto que, D. carrota tienen un porcentaje de divergencias más alta que S. tuberosum. Los estudios filogenéticos indican que A. xanthorrhiza está estrechamente relacionada con las especies de Angélica, Glenhnia, Ostericum, Ledebouriella y Peucedamun y es adyacente a la especie D. carrota. Los resultados de este estudio son la base para iniciar el desarrollo de técnicas moleculares que desencadenen la obtención de mejores cultivos y productos de A. xanthorrhiza.

The role of the interleukin-12/STAT4 axis in breast cancer

Dr. Ángel Núñez Marrero
anunez@br.inter.edu

Breast cancer (BC) is one of the leading causes of cancer-related deaths among women worldwide. This cancer is highly heterogeneous and the most aggressive subtypes have reduced therapeutic alternatives. Interleukin-12 (IL-12) is a proinflammatory cytokine that links effective innate and adaptive immune responses against tumor cells. Genetic variation, immune-responses downmodulation and tumor microenvironment dynamics have been identified as key modulators of cytokine anti-tumor responses. Single Nucleotide Polymorphisms (SNPs) in IL-12 genes have been associated with cancer risk. However, limited studies have assessed the role of IL-12 in BC risk comprehensively, and these were done in European and Asian populations. In addition, IL-12 relates to an anti-tumor microenvironment. This research aims were (1) to assess whether SNPs in IL-12/STAT4 signaling axis candidate genes were associated with BC risk among the genetically unique Puerto Rican (PUR) women population, and (2) to assess if the IL-12 signaling was downregulated at the breast tumor microenvironment by doing in vitro and in vivo analyses. We were expecting an association of IL-12/STAT4 axis SNPs and BC risk in PUR women and a lower expression of the IL-12/STAT4 axis in the most aggressive BC molecular subtypes. A candidate-gene association study was performed to assess the association of the IL-12/STAT4 tag SNPs and BC risk in 463 cases and 461 controls. We also assessed the in vivo and in vitro expression of the IL-12/STAT4 axis by molecular analyses and by co-culture system. SNPs on the IL12-STAT4 axis such as IL12A, TYK2 and IL12RB1 were associated with high BC risk among PUR women. Whereas SNPs on the IL12B, IL12RB1, JAK2, STAT4 and IFNG genes were associated with a decreased risk. Key IL-12/STAT4 axis molecules such as STAT4 and IFNG expression were reduced in BC regardless of the BC molecular subtype status. IL-12 enhanced anti-tumor responses in co-culture models. The IL-12/STAT4 axis is associated with BC risk in PUR women. The BC tumor microenvironment has a reduced IL-12/STAT4 signaling. Further functional studies will clarify the specific genetic and molecular mechanisms related to the role of the IL-12/STAT4 axis in BC, which potentially may benefit IL-12 related immunotherapeutic approaches.

Isolation and Characterization of Biofertilizers from the Coastal Areas of Puerto Rico

Alok Arun, Ph. D.
alok_arun@br.inter.eduu

Seaweeds are abundant on the coastal areas of Puerto. Seaweeds have been used in the industry for several purposes for over decades across the world. Seaweeds are used as source for food (sushi), industrial raw materials and therapeutic and botanical applications. The current market values of seaweeds are estimated to be more than US$60 billion dollar.  Previous research works have shown that seaweeds contain a number of plant growth-stimulating compounds and hence have been used as amendment in crop production. Seaweed Liquid Extract (SLE), obtained from a variety of seaweeds, is used as a nutrient supplement or biofertilizer in agriculture to increase plant growth and yield. The fertilizers obtained from seaweeds are ecofriendly and has almost zero-effect on the environment. Puerto Rico tourism and coastal environment is severely affected by the abundance of seaweed, Sargassum fluitans, on its coastal areas. We hypothesized that the waste of Sargassum fluitans collected from the coastal areas of Puerto Rico can be utilized to locally produce eco-friendly biofertilizers. Therefore, we studied Sargassum fluitans to assess its role as a biofertilizers on the tomato plant. We prepared seaweed liquid extract of Sargassum fluitans and measured the effect of varying concentrations of S.fluitans SLE on growth and development of model plant Solanum Lycopersicum (tomato). Our results indicated that a concentration of 5% of SLE exponentially increased the shoot length when compared with controls indicating a potential role of SLE obtained from S. fluitans in plant growth and development. We further assessed if the bacterial microbial communities associated with the tomato plant is affected in the presence of varying concentrations of SLE. Our preliminary metagenomic data suggests that application of SLE changes the bacterial communities of the host plant treated with SLE and consequently SLE may enhance the growth of the plant by controlling the association of microbial communities. In future, the use of bio-fertilizers from seaweed would not only contribute to the blue economy but would provide an ecofriendly method of keeping the coasts of Puerto Rico clean.

De novo transcriptome sequencing and gene expression profiling of Arracacia xanthorrhiza reveals candidate genes for secondary metabolite production and starch biosynthesis.

Dr. Alok Arun y Profa. María M. Meléndez Ortega
alok_arun@br.inter.edu, mmmelendez@br.inter.edu

The Peruvian carrot (Arracacia xanthorrhiza) commonly referred, as apio is a crop that is a secondary food item for over 100 million people, mainly in South America and Puerto Rico. While the storage roots are the main product, the rootstock and leaves are used as animal feed and the aerial stems are used as propagules. The crop has low input requirements and can be grown in a variety of frost-free tropical highland environments. However, in the absence of preventive management practices and proper handling, infections caused by pathogens severely damage the storage roots. The lack of certainty about the agents and the process of infection is a major gap to understanding how to prevent infection at its very onset. Despite the economical and agricultural importance of apio, any biotechnological strategy aimed at improving the yield or protecting the crop from pest damage is non-existent, partly due to lack of any genomic tools and techniques. For non-model plants like apio that has very limited genomic/transcriptomic information available, developing transcriptome databases might improve its cultivation and storage. In our study, we characterized the global gene expression profile of A. xanthorrhiza tissues (tuber, leaf, flower and cormel) using Illumina high-throughput RNA sequencing platform. The study generated 74,533 contigs corresponding to approximately 72 million paired-end reads. Functional annotation of the dataset identified 12 of the 15 homologous enzymes involved in flavonoid biosynthesis pathway in plants and six gene families involved in starch biosynthesis. We also detected 5,525 SSR distributed on 6,247 Unigenes and predicted 1,826 transcription factor coding unigenes. This study is the first global report on transcriptome dataset in Arracacia species. The gene transcript dataset will provide a resource for further research on improving the quality and storage life of the crop.

Isolation and Characterization of Essential oils from the leaf of Arracacia xanthorriza

Michelle Cartagena Rivera, Ph. D.
mcartagena@br.inter.edu

Arracacia xanthorrhiza, commonly known as apio, is a Peruvian carrot that develops a tuber with high levels of starch. It is and understudied plant that could contain potential medicinal compounds, such as essential oils. Essential oils are chemical substances developed in plant cells, and can be used in medicine or aromatherapy. Also, essential oils are secondary metabolites founded in aromatic plants that can be extracted using different types of distillations.  This research is focused on isolating the essential oils, specifically from the leaf of apio. For the isolation of these oils, both steam distillation and Soxhlet distillation were performed. Also, solvent extraction and a simple distillation were used for reducing the solvent volume in the sample. The results showed a dark-green extract when using both techniques which could possibly contain essential oils.  These samples will be analyzed by Gas Chromatography Mass Spectrometry.

Fatty acid characterization of the leaf of Arracacia xanthorriza

Michelle Cartagena Rivera, Ph. D.
mcartagena@br.inter.edu

Plants produce the majority of the world’s lipids, and most animals, including humans, depend on these lipids major source of calories and essential fatty acids. In the other hand, fatty acids have many biological properties. Many of them have antimicrobial and anticancer properties. It has been shown that fatty acids can induce apoptosis in cancer cells. Also, it has been shown that fatty acids can be used as topical agents to treat acne. In plants there are rare unusual occurring fatty acids that have not been study in terms of its biological activity. Apio (Arracacia xanthorriza) is a common plant that grows mostly in Puerto Rico’s central area, like Barranquitas. It is composed of roots, tuber, flowers and “fruit”. Many projects have been developed with it, but nothing is known about its fatty acid profile nor if these fatty acids have potential biological activity. In order to know about the fatty acid profile and its biological activity experiments will be done.

Extraction of heavy metals from the plant Theobroma cacao

Michelle Cartagena Rivera, Ph. D.
mcartagena@br.inter.edu

Theobroma cacao plant is native to Mexico and extends into the Amazon rainforest. This plant has had a great industrial and economic impact in South America, especially in the countries of Colombia, Venezuela and Perú. In addition, several investigations have been conducted in this area and it has been found that the cocoa plant has high amounts of heavy metals, especially cadmium (Cd). Therefore, cacao is grown in Puerto Rico, but there is no research to present and stipulate information on heavy metals presented by the plant. The objectives of this research are to determine the heavy metals (Cd) in the Theobroma cacao plant through ICP instrumentation.

Cadmium toxicity in mammalian cells at the genetic and molecular level

Juan A. Negrón Berríos, Ph.D.
janegron@br.inter.edu

Cadmium is a toxic heavy metal that in humans comes from smoking and from agricultural crops in the food chain.  In spite the fact that cadmium toxic effects have been widely demonstrated, its specific mechanism remain unknown. Exposition to cadmium has been linked to tumor development, mainly in kidney and thyroid tissues, osteoporosis, emphysema, anemia, liver inflammation, and others.  At the molecular level in mammalian cells, it has been demonstrated that cadmium induces cell apoptosis, genomic instability, cytoskeletal disruption, and oxidative stress.  In this study we have used Chinese Hamsters Ovary Cells (CHOs) to investigate the genetic and molecular mechanisms of cadmium toxicity.  Cadmium cell viability assays, gene expression of metal binding proteins, non-coding RNAs analysis, and DNA methylation assays have been done to correlate cadmium mechanisms with epigenetic changes.  Exposition of CHOs to µM concentrations of cadmium, showed a correlation with cell death, overexpression of metallothioneins, specific DNA methylation profiles, and down-regulation and up-regulation of small non-coding RNAs.  These results suggest an epigenetic effect associated to cadmium toxicity in the animal cells.

Temporary immersion bioreactor techniques for the micropropagation of plantains

Molecular DNA barcoding of mosquito and bacterial species to identify alternative mechanism to control human diseases in Puerto Rico

Juan A. Negrón Berríos, Ph.D. and Alok Arun
janegron@br.inter.edu, alok_arun@br.inter.edu

There are several mosquito species in Puerto Rico, which are vectors of human pathogens, including Aedes aegypti L.  These mosquito species carry pathogens such as Zika, Dengue, or Chikungunya viruses.  The aims of this project are to 1) apply molecular genetics techniques, such as DNA barcoding as a supplement to the traditional morphological identification, 2) obtain a collection of mosquitos from different geographical areas in Puerto Rico, 3)   determine the presence of the bacteria Wolbachia sp. in mosquito bodies. Important information is needed in Puerto Rico about the taxonomy and distribution of mosquito species.  In collaboration with the Center for Disease Control and Prevention, mosquito samples were obtained from different geographical areas of Puerto Rico.  These mosquitos were identified, dissected, and preserved for analysis.   Using DNA as template from mosquitos’ tissues, the COX1 gene fragment was amplified by Polymerase Chain Reaction.  The amplified sequences of the COX1 gene are being correlated with mosquito’s identification.    Analyses are in process to tried to try to detect in mosquito, the presence of Wolbachia sp., an intracellular bacterium commonly present in arthropods.