International Journal of

Advanced Biotechnology

and Bioinformatics

Chief Patron Editor-in-Chief Prof. P. B. Sharma Prof. Samir K. Brahmachari

Executive Editor Dr. Yasha Hasija

Aim and Scope

INTERNATIONAL JOURNAL OF ADVANCED BIOTECHNOLOGY &

BIOINFORMATICS is an online, freely available, peer reviewed journal that endeavors

to rapidly publish original, authentic and fundamental research papers, review articles,

case studies and short communications in all the spheres of biotechnology and

bioinformatics. With biotechnology transforming every sphere of our lives by its

innovations, the journal attempts to provide a platform to both academic and industrial

laboratories to share their research and provide useful insights. The emphasis is to

capture the novel thoughts and ideas from all across the globe and make science

available to the best of minds. The journal is an effort to enlighten its audience with the

recent trends in the spheres of biotechnology and bioinformatics.

Topics of interest cover different aspects of life sciences and biomedical research

including genomics, proteomics, metabolomics, bioinformatics, bioprocess technology,

drug designing and discovery, systems biology and others. Inimitable attributes of the journal:

• The journal aims to bring together the best of research in advanced biotechnology

and bioinformatics, with greater emphasis on the usefulness and applicability of the

work. • Each research theme is collected from both biotechnology and bioinformatics sectors

and aims to be a useful resource for both the scientific and academic community.

• Apart from contemporary research, the journal also emphasizes the latest

developments in biotechnology and bioinformatics. • It also aims to become a benchmark of quality research at both the national and

international levels in the coming years.

The contents of this journal are available for free at www.ijabb.dce.edu

International Journal of

Advanced Biotechnology and

Bioinformatics

Volume 2, Issue 1, November 2013 Chief Patron:

Prof. P. B. Sharma Vice Chancellor, Delhi Technological University, Shahbad Daulatpur, Delhi, India

Editor-in-Chief

Prof. Samir K. Brahmachari Founder Director, CSIR-Institute of Genomics and Integrative Biology (IGIB) Chief Mentor, CSIR-Open Source Drug Discovery (OSDD) Director General, Council of Scientific and Industrial Research

(CSIR) New Delhi -110001, India Executive Editor

Dr. Yasha Hasija Assistant Professor & Associate Head, Department of Biotechnology, Delhi Technological University, Shahbad Daulatpur, Delhi, India

Publisher Delhi Technological University

Shahbad Daulatpur, Bawana Road, Delhi-110042, India

Copyright © 2013 by the International Journal of Advanced

Biotechnology and Bioinformatics

All rights reserved. Subject to statutory exception and the provisions of

relevant collective licensing agreements, no part of this publication may

be reproduced, stored in a retrieval system, or transmitted in any form, without the prior written permission of the publisher.

International Journal of Advanced Biotechnology and Bioinformatics

is an online, freely available, peer reviewed journal that endeavors to

rapidly publish original, authentic and fundamental research papers, review articles, case studies and short communications in all the spheres of

biotechnology and bioinformatics.

This journal is published annually. Online access to the publication is

available free of charge at www.ijabb.dce.edu. For further information:

Dr. Yasha Hasija Department of Biotechnology

Delhi Technological University

Shahbad Daulatpur, Bawana Road Delhi-110042, India

Email: yashahasija@dce.edu; yashahasija@gmail.com

Table of Contents

EDITORIAL BOARD ................................................................................................................................................................................. 3

• Review Articles

RESVERATROL AND ITS IMPACT ON PROSTATE CANCER

James A. Stokes III, Udai P. Singh, Elijah Nyairo, Manoj K. Mishra1................................................................................... 6-13

PARTICIPATORY GENOMICS IN THE ERA OF PERSONAL GENOMES

Vinod Scaria ......................................................................................................................................................................... 14-18

• Research Articles

“MIR”ACLES IN NEURO MOLECULAR MEDICINE

Harrisham Kaur, Richa Sharm1, Asmita Das....................................................................................................................... 19-26

ISOLATION AND SCREENING OF BIOSURFACTANT PRODUCING BACTERIA FROM POLLUTED LAKE

Prachi Jain, Abha Saxena, Ishwar Chandra, Bijoyata Borah, J.Jemila1............................................................................. 27-32

INTERNATIONAL JOURNAL OF ADVANCED BIOTECHNOLOGY AND BIOINFORMATICS VOL.2, ISSUE.1, NOVEMBER 2013

RESVERATROL AND ITS IMPACT ON PROSTATE CANCER

James A. Stokes III1, Udai P. Singh2, Elijah Nyairo3, Manoj K. Mishra1*

1Department of Biological Sciences,

2 Physical Sciences, Alabama State University, Montgomery, AL 36104, USA,

3Pathology, Microbiology, and Immunology, School of Medicine, University of South Carolina, Columbia, SC

29208, USA

ABSTRACT

Cancer is a serious threat to human health in populations across the globe. Chances are that at some point cancer will

have an effect on every person’s life in one form or another. Current treatments are incredibly inefficient because of

the disease’s unique adaptive capabilities, which render many treatments useless. Still other treatment methods, such

as chemotherapy, often make the patient more ill than the disease itself. Resveratrol (RES), a polyphenolic

compound naturally found in a variety of red fruits and some vegetables presents an effective non-toxic alternative,

which would allow physicians to treat the disease non-intermittently. Recently, much research has been conducted

on the cancer-fighting properties of RES, but very little is known about the mechanism(s) in which RES utilizes to

express its numerous anti-cancer properties. In this review, we are focusing on some of the mechanisms utilized by

RES to sensitize Prostate cancer (PCa) cells.

Key words: Prostate cancer, Resveratrol, Aryl Hydrocarbon Receptor

1. Introduction

Cancer is a major threat to the health of human

populations all over the world, but it is especially

prevalent in populations of the western hemisphere.

Cancer is extremely tough to prevent because there

are so many factors that contribute toits different

types including: lung, breast, cervical, and prostate.

In this review paper, we will be focusing on the

mechanisms used in the sensitization of mainly

prostate cancer cells, and how scientists are

uncovering new hope for a cure to this type of cancer,

utilizing natural remedies. Prostate cancer (PCa) is

the most common form of cancer among U.S. men 50

years of age or older. It is most commonly presents as

an adenocarcinoma (a tumor formed on the glandular

structures of epithelial tissue) of the male prostate,

which changes the morphology of the gland [1-3].

*Corresponding author: Manoj K. Mishra, Ph.D.

Department of Biological Sciences

Alabama State University

Montgomery, AL 36104, USA

Email: mmishra@alasu.edu

Tel: 1-334-229-5085

This can lead to complications of the urinary system

and if not treated early can spread to other parts of

the body [4, 5]. Studies are still being conducted as to

what are the direct causative factors for the disease.

Thus far, not much progress has been made in the

search for a solution and researchers in both the

scientific arena as well as the medical establishment

are becoming increasingly aware of the relationship

between a proper diet and good health [6-8].

Researchers and physicians are beginning to

investigate the potency of more natural compounds

against diseases such as cancer. One such compound

is resveratrol (RES). RES is a polyphenolic

compound found in the skins of red grapes (which are

used to make red wine), some berries, select

vegetables, and even peanuts. The chemical has

natural anti-oxidative, anti-inflammatory, and anti-

aging properties [9-14]. In its most pristine form the

compound protects the plant(s), which produces it,

from harmful invasive pathogenic organisms. Researchers seek to harness RES’s disease/ pathogen

fighting power in hopes of revolutionizing the way

cancer is treated. Ambitious research endeavors such

as the conjugation of RES with various carbon

nanoparticles (poly epsilon-caprolactone, PCL and

poly lactic glycolic acid, PLGA) illustrates this point.

The resulting data from such work have shown the

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Stokes III J. A. et. al. RESVERATROL AND ITS IMPACT ON PROSTATE CANCER

significant increase in the overall stability and target

efficiency of the compound. The study also

demonstrated that conjugated RES increased

cytotoxic conditions in cancer cell lines more so than

non-conjugated RES [15]. Furthermore, RES is

attractive to researchers because of its low probability

of harmful side effects. Current cancer treatment

methodologies put the patient at risk often making

the patient more ill than the cancer itself. When

someone is diagnosed with cancer the individual is

often put through a rigorous regimen of

chemotherapy. The chemotherapy treatment is toxic

to both cancerous and non-cancerous cells, which

causes severe morbidity in the patient to the point

where the treatment must be halted intermittently to

allow the patient to recover [16-18]. This period of

intermission also allows the cancer cells to recover

often with more resistant properties than the

preceding generation. This is why current cancer

treatment methodologies have had such a historically

low rate of success. In recent years, research has

shown that RES is an effective inhibitor of several

types of cancers including prostate. Not much is

understood about how RES mediates the tumor

sensitization, but it is clear that RES acts to prevent

cancer proliferation on a variety of levels including

nuclear/genetic. This review paper is an attempt to

highlight the mechanism(s) of tumor clearance by

RES.

2. RES inhibition at DNA/nuclear level

Several studies have reported that RES acts through a

variety of biochemical pathways, both intrinsic and

extrinsic to the cell to inhibit PCa development.

Perhaps amongst the most intriguing finding is the

fact that RES has the ability to illicit changes within

the target cell at the DNA/nuclear level [19-24].

There are a variety of ways in which RES achieves

this but the general idea is that the chemotherapeutic

agent acts as a ligand and binds to a number of

different cellular receptors initiating sequential

phosphorylating events inside the cell that eventually

make their way into the nucleus. Once inside the

nucleus, it activates pro-apoptotic transcription

factor(s) which help in expressing anti-cancer genes

that result in cell death (figure 1). A perfect example

would be a previous study involving a novel RES

analogue (HS-1793) which exhibited the ability to

inhibit the expression of the proteins: hypoxia-

inducible factor-1alpha (HIF-1α) and vascular

endothelial growth factor (VEGF) in human prostate

cancer cells (PC-3). These transcriptional factors

have been implicated for their role in the metastasis

and angiogenesis of tumor cells [25-27]. Some of the

molecules involved in DNA damage-dependent

apoptosis are described below.

Figure 1: Illustrates the generic ligand-receptor interaction

which leads to an intracellular cascade of phosphorylating

signaling events. In this case, RES the ligand causes a chain of

cellular reactions that eventually leads to apoptosis.

2.1. Role of PTEN/AKT Pathway

One such example is the regulation of the

phosphatase and tensin homolog deleted on

chromosome 10 (PTEN)/AKT pathway by RES [28,

29]. In this pathway, RES directly binds to the

Epidermal Growth Factor Receptor (EGFR) rapidly

inhibiting EGFR phosphorylation, which results in

decreased AKT phosphorylation and activity. It must

be understood that inside the cell there are many

pathways that are initiated simultaneously that often

intersect and produce the same biochemical results.

PTEN is a known tumor-suppressor gene and it is

concurrently up-regulated when RES binds to its

receptor. Another one of those receptors is the

Androgen Receptor (AR) [11, 30-32]. RES inhibits

the phosphorylation of this receptor which

subsequently results in PTEN transcriptional activity

and the suppression of tumor formation. Binding to

AR yields the same result as in the previously

described EGFR pathway [33-35]. Studies indicate

that an imbalance of AR activity is a major factor in

tumor formation; PTEN, through the activation of its

pathway by RES, facilitates a balance for this

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INTERNATIONAL JOURNAL OF ADVANCED BIOTECHNOLOGY AND BIOINFORMATICS VOL.2, ISSUE.1, NOVEMBER 2013

receptor’s activity. RES produces a conformational

change of AR (Hsp90/70) in the cytosol of the target

cell, which then translocates into the nucleus to then

express specific anti-cancer genes. In short, RES

induces PTEN activity by AR modulation in PCa

cells [28].

In one study, RES was used to target cells with PTEN

mutations. The inactivation of AKT by the binding of

RES and the up-regulation of PTEN induces the

activation of FOXO gene transcription factors [29,

36]. FOXO gene transcription factors are proteins

that were conserved throughout evolution. These

genes are common to mammalian cells and are

known to scientist as “housekeeping” genes. These

genes regulate fundamental cellular functions such as

DNA damage repair, cell cycle arrest, cellular stress

responses, and apoptosis. Researchers found that

FOXO genes are functionally redundant; this simply

means that several genes can regulate the same

pathway and produce similar transcription factors

[29, 37]. During this study when the FOXO gene was

knocked down murine (mouse) model cells exhibited

the first stages of tumor development. When FOXO

expression was artificially induced the model cells

did not exhibit tumorogenesis. This indicates that the

FOXO gene could be an important mediator for

cellular apoptosis and RES activity. This study shows

that RES induces apoptosis and growth arrest through

yet another cellular mechanism, this time via the

activation of FOXO transcription factors. For many

human PCa models the PTEN gene is inactivated

through some type of mutation this causes

phosphorylation/activation of pro-cancer pathways

such as P13K/AKT, which as a result are catalysts for

unregulated cell growth and tumor progression [29].

2.2. Role of P53

Another widely known gene concerning cancer and

its progression is p53. This gene is associated with

many cancers; in fact, p53 mutations are present in

over half of all human cancers [38-43]. P53 is

responsible for mediating cell cycle arrest and

apoptosis in response to specific stimuli such as

cellular and genetic stress. Prior studies have shown

that the administration of RES produced an effect on

all cell lines involved suggesting that RES has the

capacity to mediate anti-cancer effects through a

wide array of biochemical pathways [38-40, 44].

However, the effect of RES was somewhat truncated

in the cell line with the partially knocked down p53

gene. Constitutive p53 mRNA expression was

observed at much higher levels in the cell lines

without partially deleted p53 [44]. This indicates that

the dietary compound acts in concert with p53 on

some molecular level, which is not yet fully

understood.

2.3. Role of micro RNAs

Recently, RES has shown the capability to regulate

several microRNAs. MicroRNAs are small non-

coding RNAs, which regulate coding RNAs at the

post-transcriptional level [45]. MicroRNAs originate

from primary transcripts converted in the nucleus into

precursor miRNAs [46-49]. They are then exported

to the cytoplasm where they are cleaved and

transcribed. MicroRNAs are associated inmany gene

regulatory functions/networks and their malfunction

has been linked to a bevy of different cancers. The

over expression microRNAs have been implicated in

the progression of several types of leukemias,

lymphomas, and PCa [50-52]. In certain cases of

chronic inflammation, the inflammation has been

found to be a significant factor in the increased

expression of some microRNAs. By far the

mostresearchedof these mysterious bits of non-coding

RNA is miR21 [50, 53]. Because of its impactful role

in oncogenesis researchers often use miR21 as a

biomarker for cancer identification. These regulatory

RNAs induce oncogenesis by down regulating tumor-

suppressor genes such as programmed cell death 4

(PDCD-4), mapsin, and the previously mentioned

PTEN; the down regulation of these gene’s products

creates an environment perfect for the onset of cancer

proliferation. Studies have shown miR21 to be a

major catalyst in the metastasis of some forms of

colon cancer by disrupting apoptosis via the

mechanism previously stated [45-47, 53]. Apoptosis

in human gliablastoma cells was restored when the

miR21 gene was knocked down providing further

evidence of miR21’s role in oncogenesis. In this

particular study, researchers found that when model

cells were treated with RES, the tumor formation was

greatly inhibited. In this finding, RES actually down

regulatedAKT, which is an upstream regulator of the

miR21 gene. This of course decreased the formation

of miR21’s anti-apoptotic products including Bcl-2

proteins and nuclear factor (NF-kB) [54-56]. In

addition to this mechanism RES also showed the

capacity to upregulate miR663, a microRNA that acts

antagonistically to the anti-apoptotic effects of

miR21 [45, 57].

8

Stokes III J. A. et. al. RESVERATROL AND ITS IMPACT ON PROSTATE CANCER

2.4. Role of Aryl Hydrocarbon Pathway

Polycyclic aromatic hydrocarbons (PAHs) are

environmental poisons such as those produced from

byproducts of the chemical, nuclear, and textile

industries. PAHs and other environmental toxicants

(such as cigarette smoke) act as ligands for the Aryl

Hydrocarbon Receptor (AhR) [58-61]. When bound

to the cellular receptors, these toxins can cause

immunosuppression and endocrine malfunction,

which has been linked to the development of some

cancers [62-64]. Prior studies have been carried out

to examine what component of red wine was

responsible for its anti-cancer properties [4, 10, 12,

19, 20, 65, 66]. One group of researchers isolated a

variation of RES-trihydroxystilbene resveratrol (3, 5,

4’-trihydroxystilbene resveratrol RES), which is

found in red wine, and according to their study, is a

competitive antagonist of PAHs for AhR binding [22,

23, 67]. In addition, this form of RES is very potent

and vegetables it is also nontoxic which is key to its

and since it is naturally found in some edible fruits

potential treatment of human cancer. This study

demonstrated that RES binds to AhR in the cytosol

and translocates the RES-AhR complex into the cell’s

nucleus. A whole-cell binding competitive assay was

designed in vitroto compare the binding efficiencies

of RES, TCDD (tetrachlorodibenzo-p-dioxin-a type

of PAH), and indole-3-carbinol (an established

natural AhR ligand) to AhR [23]. The study revealed

that RES did not displace the other two ligands from

binding with AhR. All three compounds bound to the

receptor at a similar rate. This indicated that the

inhibitory effect of RES took place at the DNA level

between both the RES-AhR and transcriptional

complexes within the nucleus. To confirm this a GFP

fluorescent tag was placed on an AhR vector to track

the movement and destination of the ligand-receptor

complexes within the cell [67]. Thus, RES

mechanisms have been shown to work through AhR

pathway.

3. RES sensitization of cancer cells

Cancer is notoriously difficult to treat because of the

innate ability of its cells to develop multiple forms of

resistance to modern treatments. Among other

mechanisms cancer cells possess an efflux pump that

is responsible for immediately pumping out any

agents that are toxic to the cell. Cancer cells have

also been documented to possess enhanced DNA

repair mechanisms, which in effect neutralize the

main mode of effectiveness of many available

cancer-fighting drugs [68-73]. The expression of

glutathione/glutathione S-transferase (GSH/GST)

within the cancer cell presents yet another problem to

cancer researchers because this allows the cancer cell

to inactivate chemotherapeutic agents through the

cleavage of chemical structures. To put it concisely,

the medical establishment is losing an arms race with

an innate cellular intelligence that wishes to survive

and proliferate at all costs. That is why dietary

compounds such as RES present such promise

because it has shown the versatility to combat this

disease through multiple channels [72, 74-76].

Research has documented RES’s ability to modulate

apoptotic pathways, and down-regulate both proteins

responsible for molecular transportation and tumor

proliferation. RES sensitizes cancer cells to anti-

cancer agents by modulating or changing the effects

of certain proteins responsible for the survival of the

cell thereby making the cell more susceptible to the

induction of apoptosis [29, 40, 77-79]. As we have

previously discussed, RES has the ability to up-

regulate pro-apoptotic genes, such as p53, PTEN, and

PDCD-4. RES has the ability to bypass or eliminate

chemoresistance mechanisms by modulating the

actions of transmembrane and multi-drug resistance

proteins (MRPs). Additionally, RES mediated the

activation of caspase proteins, which are responsible

for the initial stages of cellular degradation and DNA

fragmentation [80].

4. RES and the mitochondrial pathway

Additional studies demonstrated that RES sensitizes

mammalian cells to apoptosis through the

mitochondrial pathway. In healthy cells the

mitochondrial membrane contains proteins from the

Bcl-2 family [56, 81, 82]. These proteins help

maintain the integrity of the mitochondrial

membrane. In response to internal cell stressors Bax

proteins make their way to the mitochondrial

membrane and deactivate the Bcl-2 proteins thereby

releasing pro-apoptotic factors such as, cytochrome c.

These apoptotic factors then bind to initiator caspases

and then later effector caspases, which subsequently

induce cell death [66, 83-85]. Unpublished

experimental data from our group demonstrated the

active involvement of mitochondrial and caspase

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INTERNATIONAL JOURNAL OF ADVANCED BIOTECHNOLOGY AND BIOINFORMATICS VOL.2, ISSUE.1, NOVEMBER 2013

pathways. RES produced cell death not only through

the mitochondrial pathway, which was more active,

but also through a caspase-dependent pathway. This

suggests a synergistic role of caspase-dependent and

caspase-independent mechanisms in RES-dependent

sensitization of tumor cells.

5. Summary/ Conclusion

RES is truly a remarkable compound that utilizes

many different biochemical pathways to inhibit a

number of cancers. Its versatility far exceeds

anything ever manufactured in man-made

laboratories. The question now becomes, how does

this amazingly effective dietary compound mediate

its incredible inhibitory effects on cancer cells? And

by what mechanism(s) does this chemotherapeutic

agent use to induce apoptosis in cancer cells

exclusively. Advancements in this area of research

have the potential to revolutionize cancer diagnosis

and treatment.

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mutant p53 prostate cancer cell line.J Urol,

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INTERNATIONAL JOURNAL OF ADVANCED BIOTECHNOLOGY AND BIOINFORMATICS VOL.2, ISSUE.1, NOVEMBER 2013

PARTICIPATORY GENOMICS IN THE ERA OF PERSONAL

GENOMES

Vinod Scaria*

GN Ramachandran Knowledge Center for Genome Informatics, CSIR Institute of Genomics and

Integrative Biology, Mathura Road, Delhi 1100025, India.

ABSTRACT

The recent advancements in the field of nucleotide sequencing has tremendously improved the scale of genome

sequencing and consequent drastic reduction in the costs, making whole genome/exome sequencing affordable.

Though most of the genomic data presently resides in well-protected silos, prohibiting cross-comparison and

efficient data mining. This significantly limits the realisation of the wealth of information encoded in the genome. In

this commentary, the author argues how a participatory approach could significantly impact healthcare and research.

Key words: Next-Generation Sequencing, Personal genomics, Exomes

1. Introduction

Tremendous developments in the scale, throughput

and accuracy of nucleotide sequencing in the recent

years have accelerated the understanding of genome

sequence, genome structure and biology. These new

technologies have been popularly dubbed as next-

generation sequencing technologies, differentiating it

from the earlier sequencing technologies which were

time-limiting, costly and many times laborious. Next-

generation sequencing technologies has today found

applications beyond human genomics in a wide

variety of areas, including research applications,

microbial identification, biosurveillance and

industrial quality control.

Apart from the advances in scale and throughput of

whole genome sequencing, a number of technologies

and datasets developed immediately after the

availability of the reference Human genome sequence

has provided immense insights into the genetic

variations in human populations and their

associations with diseases or in general human traits.

*Corresponding author: Vinod Scaria, Ph.D.

GN Ramachandran Knowledge Center for Genome

Informatics, CSIR Institute of Genomics and

Integrative Biology, Mathura Road, Delhi 1100025,

India.

Email: vinods@igib.in

This include miniaturisation of genotyping assays in

the form of genotyping microarrays clubbed with

statistical genetics methods to identify genetic

variations associated with human traits. Presently

there are over 80 diseases and traits with genome-

wide estimates of genetic risk . The recent years have

seen an increasing number of traits studied for their

genetic predisposition has and independent

revalidation of the risk in different genetically

unrelated populations, significantly adding to the

repertoire of genetic variants showing association

with human traits.

Presently technologies allow a wide variety of scales

and resolutions of understanding genomic variations.

On one extreme is typing individual variations or a

set of variations in genomes. The next higher

resolution is offered by exome capture and

sequencing which offers complete sequences of the

exons of protein-coding genes. The highest resolution

is by and large achieved through whole genome

sequencing. Each of the resolution of genome

analysis offers characteristic advantages and widely

used to address characteristic questions. While the

genotyping approach has been widely applied for

understanding ancestry and predisposition to traits,

exome and whole genome sequencing approaches

have been used for understanding rare genetic

variations, including somatic variations in cancers.

Recent high throughput sequencing expeditions

including the 1000 genome sequencing, regional

sequencing initiatives including the Pan-Asian

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Scaria V. PARTICIPATORY GENOMICS IN THE ERA OF PERSONAL GENOMES

personal genomics initiative and many country-wide

sequencing projects including the Arab genome

projects aim to improve the repertoire of the human

genetic variants which would the starting point to

understand genetic variations and their phenotypic

consequences.

The present manuscript discusses how improved

scale, accuracy and reducing costs would provide a

new opportunity towards understanding genomes at

an individual level, and how a participatory approach

could significantly impact healthcare and research.

2. Personal Genomics

The advent of sequencing technology which offer fast

and efficient sequencing of whole human genomes

and consequent drastic reduction in cost has opened

up a new opportunity towards understanding the

genome sequence, variations and the biological

consequences of the variations at an individual level.

This has many implications and applications in

healthcare and has been one of the widely discussed

topics in recent years. The availability of whole

genome sequencing technology initially saw the

sequencing of personal genomes including individual

genomes from different populations around the

world.

The availability of whole genome sequencing at

affordable costs has made possible sequencing of

whole genomes or exomes of a number of

individuals. This includes research applications

including population scale projects as well as Cancer

Genome projects around the world. Genome/exome

sequencing is also presently being done routinely for

diagnosis of rare genetic diseases. Many of the

genomes/ exomes sequenced as part of research

projects have been collected from anonymous

volunteers and apart from analysis of the variants

focussed on the research problem in question. The

vast majority of the variations generated as part of the

study, though could be of immense use to the

subject/volunteer under question, remain behind

privacy and security firewalls.

The application of genome sequencing in regular

clinical settings, though is its infancy, has shown

promises, especially in fine-tuning the diagnosis and

therapy. Nevertheless, the routine application would

require tremendous investments in handling, long-

term storage and analysis of data and possibly

complete automation of the analysis and reporting

pipeline. This would only be possible with

widespread application of genomics through a further

reduction in the cost of genome sequencing and

better, faster and more informative analysis pipelines

for interpretation of the functional genetic variants

with faster turnaround times.

3. Evidence/accuracy and interpretation

The evidence on genetic variants and their effects

have been improving significantly in the recent past,

thanks to genome-wide association studies, and an

increasing number of genetic variants identified

through exome sequencing of rare genetic diseases.

In addition, evidence based collection of genetic

variants, for example, pharmacogenetic variants have

been systematically curated in databases. As a matter

of fact, many pharmacogenetic markers are presently

advised and included in the drug labels. Availability

of tools and resources in open source and ubiquitous

computing has made it possible to analyse and

interpret whole genome or exome data sets in clinical

settings.

A number of initiatives have spearheaded the

standardisation of evidence and interpretation of

genetic variations. The foremost in this include the

GET-Evidence initiative supported by the Personal

Genomics Project which aims to provide resources

and tools for analysis of genetic variations. A number

of crowdsourcing initiatives including SNPedia ,

annotating and curating genetic evidence from

literature, OpenPGx which has been manually

curating pharmacogenetic variant information and

OpenSNP which has been curating personal

genotype datasets are initiatives worth mentioning.

4. Cost of genome sequencing

It is widely assumed that genome sequencing or

genotyping is an expensive proposition and

accessible to a very few, well-to-do and technology

savvy individuals. This elitist concept is not founded

on hard facts. In fact the cost of whole genome

sequencing has exponentially fallen over the last

decade, with good quality human exomes/genomes

presently available at near 1000-3000 US$ in

commercial settings and is expected to significantly

fall in the coming decade, with the wide acceptability

and applicability of genome sequencing for clinical

diagnostics.

Though hard evidence do not exist on the potential

cost-advantage of genome sequencing in terms of the

value it brings to healthcare improvement,

nevertheless a number of ongoing studies aim to fill

in this gap. In many markets, routine exome/genome

sequencing for genetic diseases have been covered by

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INTERNATIONAL JOURNAL OF ADVANCED BIOTECHNOLOGY AND BIOINFORMATICS VOL.2, ISSUE.1, NOVEMBER 2013

insurance, suggesting a potential economic benefit of

sequencing.

5. Disclosure, Privacy and Ethics

Privacy of genetic information forms the key in any

genomics study or analysis. Nevertheless, a

significant number of individuals have made

available the genome sequences in public domain.

There is no hard evidence to show that disclosure of

genome information in public domain could be

harmful. And in fact more revealing information

regarding an individual could be potentially gleaned

through social networks and other online sources.

The advantages of disclosure including including

potential availability of a higher level of annotation

through application of novel methodologies and tools

far outweighs the potential harm implicated by

making the genome sequences available in public

domain . The disclosure and availability of genome

sequences in public domain also attains enormous

importance as no single organisation would be able to

provide all the potential applications and annotations

possible on genomes as any point in time. The

dynamic nature of the analysis pipelines and

annotation data sets used for the analysis limits a

comprehensive analysis of genome at any point in

time. Nevertheless a continuous update and re-

analysis would be impossible to be performed every

time a new toolkit or annotation dataset is made

available. Nevertheless availability of a dataset in

public domain, including the Human reference

genome and other personal genomes has made

possible in-depth analysis of datasets on a continuous

basis as they would form a corpus for testing toolkits

and annotation data sets. The advantages and

implications of making available genome sequences

of not just individuals, but even other members of a

family has been recently discussed in much great

detail.

6. Availability of data in public domain

The availability of genome sequences in public

domain would significantly add to the available

repertoire of genomic variations and would be the

much needed starting point towards standardising and

applying tools and annotation data sets. This could

potentially help in a variety of ways not limited to

personal benefits. For example a corpus of data sets

from a particular population or group would help to

understand variations prevalent in the particular

population, say for example a pharmacogenetic

variant. This would help physicians to plan treatment

regimen for the patient group as a whole, which has

the potential to drastically improve the quality of

healthcare by avoiding adverse drug reactions, for

example and reducing the cost by avoiding individual

testing. Many ethnic groups already have such

population level predispositions known at least for

some diseases , which could be improved with high-

quality genomics datasets.

7. Future perspectives

The routine application of personal genomics in

clinical settings and participatory genomics would in

the future be dependent on five key factors which

include technological advancements and changes in

the social outlook.

1) Cost of genome sequencing: A significant

reduction in the cost of genome sequencing

commensurate with the economic benefits accrued

through genome sequencing and also tagged to the

affordability in markets where healthcare insurance

does not support genome sequencing would decide

the tipping point for widespread adoption and

practice of genome sequencing in regular clinical

settings.

2) Scale and turnaround time: One of the major

technical limitations of whole genome sequencing is

the time taken for the sequencing and analysis of

data, which needs to significantly improve to provide

clinical turnaround times for data generation and

interpretation. This would require significant

investments in the area of information technology

and availability of rich resources for evidence on

clinical actionability of genetic variations.

3) Genetic education and conducive social

outlook: Genetic education on the pitfalls and

advantages of personal genomics is absolutely

essential to abrogate mistrust and sharing of

information based on free-will.

4) Economic benefit for genome sequencing: The

long term sustenance of any technology and its

application would depend on the economic benefits

accrued by screening apparently normal individuals.

In depth studies on the personal benefits of whole

genome sequencing and benefits of sharing data

versus confidentiality of the data needs to be

estimated to estimate the cost at which genome

sequencing would be economically sustainable in

routine clinical applications. The economic benefit

would also in turn depend on the depth of annotation

possible and the accuracy of the analysis and its

clinical applicability in preventive strategies.

16

Scaria V. PARTICIPATORY GENOMICS IN THE ERA OF PERSONAL GENOMES

5) Availability of large secure and interoperable

databases and resources: The key to participatory

personal genomics is the availability of a common

and accessible repository of genomic variations

available for researchers. Initiatives in this direction

including the Personal Genome Project and

OpenSNP which collects personal datasets and

limited phenotype information in public domain are

worth mention.

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“MIR”ACLES IN NEURO MOLECULAR MEDICINE

Harrisham Kaur1, Richa Sharma1, Asmita Das1*

1Deparment of Biotechnology, Delhi Technological University, Bawana Road, New Delhi, Delhi-110042, INDIA

ABSTRACT

MicroRNAs (miRNAs) are transcriptional and post-transcriptional regulators that negatively control the expression

of a gene. These small non-coding RNAs are 20 to 26 nucleotides long and regulate gene expression by binding

specifically to the 3-prime untranslated region of a gene transcript, leading to its translational repression or

degradation. Studies demonstrate that miRNAs form an integral part of gene regulatory system of multicellular

organisms and consequently play a pivotal role in the expression of protein coding genes. Many of the miRNAs are

reportedly involved in mammalian Central Nervous System (CNS) and mediate the function and regulation of the

nervous system. The disruption of miRNA based gene regulatory system has been implicated in numerous CNS

disorders as a single miRNA can control the expression of several downstream mRNA targets. In the present study

we report a bioinformatics pipeline for identification of miRNAs from the EST dataset of medicinal plant Curcuma

longa. The putative miRNAs identified can be exploited in molecular therapeutics for CNS disorders.

Key words: miRNA, CNS, gene-silencing, Bioinformatics

1. Introduction

Translational control and mRNA transcript

degradation is an imperative part of gene regulation

machinery of a cell. Small non coding RNA

molecules are effective modulators of transcriptional

activities of a gene [1]. These RNA molecules are

reportedly involved in the cytoplasmic control of

mRNA translation and degradation, a phenomenon

known as RNA interference or RNA silencing. A cell

exhibits RNA interference with the help of either

endogenous or exogenous micro RNAs (miRNAs)

and by short interfering RNAs (siRNAs). These 20 to

26 nucleotides long, single-stranded effector

molecule alters the transcriptional rate [2]. A stable

mRNA transcript can be decapped by the interfering

RNAs or can be cleaved by an endonuclease [3].

Since their discovery in Caenorhabditis elegans,

hundreds of miRNAs are now being reported in

genomes of plants and animals [4]. Studies prove that

upto 30 % of human genes are regulated by miRNAs

[5].

*Corresponding Author

Email: asmita1710@gmail.com,

asmitadas1710@dce.ac.in

Fax: 91-11278271023

Tel: 91-1127294668

Increasing evidence suggests that miRNAs play a key

role in cell growth, tissue differentiation, embryonic

development, cell proliferation, and apoptosis [6].

The dysregulation and impaired function of miRNAs

and their targets may result in various diseases

including cancer [7], cardiovascular diseases [8],

schizophrenia and Fragile- X mental retardation [9]

and AIDS [10]. Recent studies carried out on rat

spinal cord reveals the presence of 350 miRNAs.

This suggests that many miRNAs are implied in the

regulation and development of the Central Nervous

System [11-12]. miRNAs govern diverse set of

neurobiological functions such as neuronal growth

and apoptosis. Krichevsky et al. suggests that the

expression of miR-124 and miR-9 increases during

differentiation of mouse embryonic stem (ES) cell-

derived neural progenitors [13]. Micro RNAs have

been found to be associated in CNS and progressive

neurodegenerative disorders. In Parkinson‟s disease

which is characterised by the progressive neuro-

degeneration of dopaminergic neurons in the

substantia nigra (a structure located in the mid brain)

a micro RNA miR-133b is under-expressed [14].

miR-133b regulates the function and maturation of

dopaminergic neurons by negative feedback circuit

including a paired like homeodomain transcription

factor Pitx3. Decreased levels of miR-133b in the

mid brain disrupts the negative feedback loop of Pitx

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Kaur H. et. al. “MIR”ACLES IN NEURO MOLECULAR MEDICINE

leading to degradation of dopaminergic neurons and

producing the multitude of symptoms associated with

Parkinson Disease [14]. Huntington‟s disease is

caused mainly by a mutation in the gene which codes

for the protein huntingtin. This mutation is

responsible for the occurrence of excess of Glutamine

residues in a protein thus disrupting its normal

function. The disruption in the function of huntingtin

protein causes abnormal neuronal cell death by an

unknown mechanism. Extensive research on testing

of RNAi (RNA interference) technology for reducing

the abnormal production of huntingtin protein is

underway as a plausible cure for Huntington‟s

disease. Preclinical studies on animal models in

which the mutant huntingtin was suppressed showed

considerable improvement in motor dysfunction and

neuro-pathology. A group of researchers have

collaborated with CHDI Foundation (Cure

Huntington's Disease Initiative) to develop an

implatable infusion system to deliver a siRNA

molecule which will target mutated huntingtin

directly in the brain of patients with Huntington‟s

disease. Similarly in Spinocerebellar ataxia, a

mutation in a gene called SCA1 increases the number

of CAG repeats on the DNA leading to the

production of an abnormal ataxin-1 protein. Hence,

downregulating the expression of such mutated genes

could reduce the effect of spinocerebellar ataxia.

Mouse stage III preclinical trials involving a miRNA

silencing the mutated SCA1 gene showed improved

motor co-ordination as compared to untreated mice

[15]. Similar associations of miRNAs and their

possible RNAi are reported in diseases like

schizophrenia, Alzheimer‟s and Parkinson‟s [12].

Various genes like MAPK12, NOP56 are known to

associate with CNS and neurodegenerative disorders.

Thus, present study aimed to explore different

miRNAs that govern the silencing of genes that are

responsible for CNS disorders. We found 12

miRNAs from medicinal plant Curcuma longa

(Indian Turmeric) which were able to silencing

human gene transcripts involved in CNS and other

Neuro-degenerative disorders. Curcuma longa is a

medicinal plant which is known to treat various

ailments for centuries in Ayervedic medicine, the

traditional Indian system of healing. The major

component of Curcuma longa Curcumin is

responsible for its biological activities. In-vitro data

suggests that curcumin, the active component of the

Zingiberaceae family plant has antioxidant, anti-

inflammatory and anti-amyloid activity. Commonly

known as the blood-purifier turmeric preparations are

used to cure a variety of diseases like dyspepsia,

flatulence, liver disease, urinary tract disease [16].

Due to the anti-oxidant properties of Curcumin, the

plant preparations have also been known to prevent

CNS disorders [16-17]. In addition to its anti-oxidant

properties, the homeostatic, the cholesterol lowering

properties and the anti-amyloid properties of

Curcumin make it an attractive source for treating

CNS and neurodegenerative disorders. A recent

study in animal models reports a decrease in amyloid

pathology in Alzhemier Disease by the use of

Curcumin [18]. Hence Curcuma longa can be

explored as a potential molecular therapeutic agent in

the treatment of neurodegenerative and CNS

disorders.

In the present study, we have attempted to explore

the medicinal uses of Curcuma longa by the in-silico

identification of miRNAs from the Expressed

Sequence Tags dataset of the plant. These putative

miRNAs identified by bioinformatics approaches and

in-house developed perl scripts were screened for the

potential human targets. Computational identification

of miRNAs exploits the fact that 3-prime UTR of

human mRNAs contains a 2-8 nucleotides long seed

region which shows complementarity with the 5-

prime region of their respective miRNAs. Hence,

most computational algorithms designed to predict

the miRNA targets rely upon the basic rule of base-

complementarity. The human targets which were

identified by the in-silico pipeline were then screened

for their involvement in CNS disorders and the linked

pathways. We report 12 putative miRNAs potentially

involved in neurodegenerative and CNS disorder

pathways. These RNAs can prove to be a potent tool

in neuro-molecular therapeutics.

2. Materials and Methods

2.1 Curcuma longa EST dataset

As of January 1st 2013 dbEST (database of Expressed

Sequence Tags) [19]; contained 74,186,692 ESTs in

the database. Among them, 12,593 EST‟s of

Curcuma longa were downloaded from dbEST

database of NCBI. The dbEST has the highest set of

impurities associated with it including the vectors,

linkers, adaptors and the primer sequences which has

to trimmed before further analysis [20]. Seqclean was

used to remove impurities and redundant sequences.

Seqclean [21] utilizes BLAST (Basic Local

Alignment Search Tool) [22] to screen sequences

highly similar (default parameter: 94% identity) to a

given list of vectors, linkers, adaptors and primer

sequences. Seqclean takes care of the polyA repeats

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that associted with the mRNA transcripts and uses

low complexity filtering for picking similar vector

segments in sample ESTs [22]. The cleaned and

trimmed ESTs were then processed via

RepeatMasker [23]. Sequencing process of ESTs may

yield some artifacts and many distinct ESTs may be a

part of the same mRNA of an organism. To

undermine this sequencing error, assembling of the

ESTs into contiguous contigs and scaffolds was done

by TGICL clustering tool [24].

2.2 microRNA sequence dataset

Mature and precursor plant miRNAs were

downloaded from Plant MicroRNA Database

(PMRD). PMRD is a database of validated and

published plant miRNAs [25].

2.3 Searching miRNAs homologs and their

secondary structure prediction

Contigs and Singletons obtained as an output of

TGICL were blasted against PMRD miRNA dataset

of plant miRNA to identify EST homologs in our set.

Initial blast search was done with stringent

parameters of evalue 2e-4, word size 7 and 90%

identity. EST database was also blasted with e-value

1000 and 90% identity to remove false-negatives

predicted in first blast search. Secondary structures of

predicted miRNAs were calculated and their

Minimum Folding Energy (MFE) scores were

recorded using mfold and RNAfold web servers [26].

2.4 Hybridization with human 3’UTRs using

miRanda

The non-redundant human UTRs were downloaded

from UTRdb database. The targets for putative

miRNAs were then scanned in the 3‟UTR with the

help miRanda software. Hydridization energy and

percent identity for each hit EST were calculated and

used to further screen the possible mRNA-miRNA

targets. The target genes were then scanned by

KEGG database for their involvement in CNS and

neurodegenerative disorders.

Figure 1 Bioinformatics Pipeline for in-silico identification of

miRNAs from EST datasets

3. Results and Discussions

Out of the numerous miRNAs satisfying the BLAST

search and the minimum energy folding criteria, a

total of 23 putative miRNAs were found to hybridize

with the human 3‟UTR sequences downloaded from

human UTR database. The hybridization was carried

out using miRanda software which runs a simple base

complementarity algorithm at its back end. The

targets were screened based on blast score and

hybridization energy parameters of miRanda

software. The hybridized UTRs of human genes as

predicted by miRanda were fed into Kyoto

Encyclopedia of Genes and Genomes (KEGG) to

track the metabolic pathways of the target genes. 12

miRNAs were found to be directly involved in

pathways of CNS disorders and neuro-degenerative

disorders. The result is in accordance to the known

anti-anxiety, anti-amyloid and anti-oxidant activity of

Curcumin, as about 50 % of the predicted miRNAs

were potent at silencing the expression of target

genes involved jointly or independently in CNS

disorders. Table 1 depicts the sequence, the length

and the minimum folding energy of the predicted

miRNAs.

21

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Kaur H. et. al. “MIR”ACLES IN NEURO MOLECULAR MEDICINE

Table 1: Statistics/ Details of the predicted

miRNAs. With the increase in length of the

mature miRNA, the minimum folding energy

tends to decrease. LGMD, Limb-Girdle Muscular

Dystrophy; ALS, Amyotrophic Lateral Sclerosis;

PME, Progressive Myoclonus Epilepsy; MPS3

Mucopolysaccharidosis Type III; CMT Charcot-

Marie-Tooth disease.

The AU content of pre-miRNA should range within

30% to 70% as unstable pre-miRNA structures are

essential to produce mature single stranded miRNAs

by endonuclease decay [27]. Our results are thus in

accordance with thermodynamic properties of

secondary structure and to the total AU acceptable

content. Figure 2 shows the secondary structures of

the putative miRNAs. These structures were

predicted using RNAfold and Mfold webserver. The

consensus Minimum Folding Energy (MFE) was then

chosen for further analysis of miRNAs.

Figure 2 Secondary Structures of putative miRNAs generated by

RNAfold web server.

ath-miRf10621-akr was found to be complementary

to the 2 to 8 nucleotide seed region of human PERK

gene. Recent Studies on PERK pathways indicate

that PERK can be a potential neuro molecular target

for the treatment of Alzheimer‟s disease [28].

miRNA ptc-miRf12223-akr was found to be

complementary to the ATPO gene (ATP oligomycin

sensitivity conferral protein: OSCP). OSCP is a

mitochondrial matrix protein and is a subunit of

F1F0-ATPase complex. Accumulation of

ubiquitinated OSCP in mitochondria of patients

suffering from neurodegenerative diseases is

common. A growing body of evidence suggests that

defect in the ubiquitination-proteosome pathway of

cells may lead to accumulation of matrix associated

proteins in mitochondria of cells, which consequently

may lead to cancer and neurodegenerative diseases

[29]. Figure 3 shows the involvement of ATPO gene

in neurodegenerative diseases. Specifically targeting

this gene can ameliorate the neurodegenerative

tendency of the CNS disorders.

Figure 3 ATPO and neurodegenerative disorders.(picture

adopted from KEGG)

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Figure 4 Ubiquitin Pathway playing a pivotal role in Parkinson’s

disease (picture adopted from KEGG)

Several theories prove the evolutionary conservation

of microRNA non-coding sequences among plant

families. To substantiate this fact, a phylogenetic tree

was constructed from the 12 putative miRNAs.

Figure 5 shows the phylogenetic tree obtained by

MEGA software [30].

Figure 5 Phylogenetic Tree of the predicted miRNAs constructed

using MEGA software

MicroRNAs regulating a particular set of disorders

share considerable sequence similarity. This is due to

the fact that the genes governing a particular set of

disorders are linked together at the pathway level. For

example the pathway of Alzheimer‟s disease reveals

a connection between the human ATPO gene (ATP

Synthase O subunit) and the human PERK gene. This

suggests that genes involved in CNS or

neurodegenerative disorders are linked together and

are thus regulated together by a set of closely related

miRNAs. Analysis of phylogenetic tree (Figure 5) of

predicted miRNAs showed that these miRNAs are

closely related to each other. The result of the

phylogenetic tree analysis strongly reveals that

predicted miRNAs are regulating a set of genes

associated with the CNS Disorders.

4. Conclusion

A revolution in CNS drug discovery was anticipated

with the mapping of the human genome. It was

believed that the data would account for

understanding disease pathophysiology and for

discovery of certain disease associated drugs [31].

But the neuropsychiatric conditions have dozens of

discrete, casual, and susceptibility related genetic

associations. For schizophrenia, a total of 30 gene

associations are reported, none of which have been

proved to be a potent drug target [32]. Interestingly

the currently used target for treatment of

schizophrenia, the dopamine D2 receptor, was not

present among these reported genetic associations.

The catechol-O-methyltransferase V158

polymorphism found in some patients with

schizophrenia have been associated with a variety of

disease conditions like breast cancer, myocardial

infarction, anxiety and Alzheimer‟s disease, limiting

its use as a drug target for treating any of these

conditions [32]. Even the most apparently simple

genetic disorder can be caused by a variety of

mutations. Mutations can vary in type. One mutation

can alter the maximum velocity of a protein, other,

the Michaelis constant, and lastly may produce a

functional enzyme or a receptor with different

stability or a propensity to aggregate. The chance that

a single drug target could reverse these three different

mutations is very meagre. Thus, collective tools of

translational and regulational medicine,

pharmacogenomics, neuroimaging, and molecular

therapeutics can be considered as better promise for

multi-target disorders [33].

The present study was aimed at in silico prediction of

miRNAs in Curcuma longa and their implication in

CNS and neuro-degenerative disorders. The

methodology predicted 12 putative miRNAs, the

human gene targets of which were directly involved

in pathways of neuro-degenerative and CNS

disorders.

The putative miRNAs can be effective RNAi based

drugs in the following CNS and neuro-degenrative

disorders:

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Kaur H. et. al. “MIR”ACLES IN NEURO MOLECULAR MEDICINE

1. Amyotrophic lateral sclerosis (ALS)

The predicted miRNAs „ath-miRf10590‟ and „ath-

miRf11012-akr‟ in Curcuma longa were found to

silence human MAPK12 gene which induces the

abnormal activation of SOD1 gene responsible for

causing Amyotrophic lateral sclerosis disease

(adopted from Kyoto Encyclopedia of genes and

genomes KEGG). The inhibition of MAPK12

signalling pathway in brains of ALS disease patients

would provide a breakthrough in ALS prevention and

cure.

2. Spinocerebellar ataxia (SA)

Spinocerebellar ataxia is a type of neuro-degenerative

disease which is characterized by slow progressive

incoordination of gait and is often associated with

poor coordination of hands, speech, and eye

movements. It can be caused by mutations in many

genes known to regulate body co-ordination. For

example NOP56 (nucleolar protein 56) gene mutation

in Spinocerebellar ataxia causes a large expansion of

an intronic GGCCTG hexanucleotide repeat which

leads to abnormal ribosome biogenesis and genetic

information processing in eukaryotes [34].

Spinocerebellar ataxia is also caused by a mutation in

the gene SCA7 which leads to CAG repeats in the

DNA and in turn augments the production of

abnormal ataxin-7 protein. miRanda results showed

that predicted miRNAs „ath-miRf10730-akr‟ and

„ptc-miRf12248-akr‟ bind to NOP56 and SCA7 gene

respectively and can thus be exploited in RNAi based

therapeutics for spinocerebellar ataxia.

3. Lissencephaly

Lissencephaly is a severe neuronal migration disorder

that ranges from agyria/pachygyria to subcortical

band heterotopia. Human PAFAH (platelet-activating

factor acetylhydrolase IB subunit alpha) gene is a key

mediator in lipid metabolism pathway of humans.

The mutated PAFAH gene causes dysfunctional lipid

metabolism which is responsible for Lissencephaly

and Miller-Dieker syndrome. Putative miRNA „osa-

miRf11323-akr‟ silencing mutated PAFAH gene can

provide a cure for Lissencephaly.

4. Alcoholism

Alcoholism is a term used to describe the obsessive

and uncontrollable consumption of alcohol. It‟s an

addictive or substance-dependent disorder normally

referred as the „Alcohol Dependence Syndrome‟ in

medical terms. src homology 2 domain-containing

transforming protein C (SHC3) gene is a major

intermediate in the pathway of human substance

dependence. This pathway is responsible for alcohol

addiction in humans (adopted from KEGG). The

miRNA „ath-miRf10147-akr‟ showed efficient

binding to SHC3 gene transcript and can be delivered

in brains of alcohol addicts to disrupt the addiction

pathway of Alcoholism.

5. Mucopolysaccharidosis type III

Mucopolysaccharidosis type III is an autosomal

recessive lysosomal storage disorder caused by a

defect in one of the four enzyme genes involved in

glycosaminoglycan degradation. The defect results in

the accumulation of heparan sulfate in many organs,

as well as elevated metabolite levels in urine.

Common signs and symptoms include mental

retardation, behavior and aggression problems, and

seizures. The mutated N-sulfoglucosamine

sulfohydrolase gene (SGSH) causes abnormal

Glycosaminoglycan degradation which leads to

abnormal accumulation of heparan sulphate and the

apparent symptoms associated with the disease. The

miRNA „ath-miRf10354-akr‟ based RNAi

therapeutic could help in alleviating the symptoms

and be a possible cure for the disease.

6. Progressive myoclonic epilepsy (PME)

The ceroid-lipofuscinosis neuronal protein 8 (CLN8)

gene cause fatty acid elongation in mitochondria

which is the sole cause of Progressive myoclonic

epilepsy. miRNA „ath-miRf10158-akr‟ could be used

to regulate the pathway of fatty acid synthesis in

patients suffering from epilepsy.

7. Hereditary Spastic Paraplegia

Hereditary Spastic Paraplegia is a nervous system

disorder characterized by progressive distal limb

weakness and lower extremity spasticity. AP5Z1 gene

(adaptor-related protein complex 5, zeta 1 subunit) is

an important gene associated with spastic paraplegia.

Defects in this gene are the cause of autosomal

recessive form of spastic paraplegia. Putative miRNA

„ptc-miRf12150-akr‟ was predicted to silence this

gene.

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8. Parkinson’s, Alzheimer’s and Huntington’s

disease

Human ATPO gene (ATP synthase) is associated

with the mitochondrial F1 complex. This complex

plays a key role in metabolic pathways and oxidative

phosphorylation. Defects in ATPO gene can affect

ubiquitin-proteosomal pathway of cell and can cause

accumulation of matrix associated proteins in the

mitochondria of the cells. The result can be an

increased propensity to neuro-degenerative disorders

like Alzheimer‟s Parkinson‟s and Huntington‟s

disease. The defective ATPO gene is found to be a

potential target for silencing by the putative miRNA

„ptc-miRf12223-akr‟. RNAi based therapeutics

specically targeting the damaged ATPO gene could

pacify the symptoms and slow the rate of progression

of the CNS disorders [35].

9. Charcot-Marie-Tooth disease

A recent study states that the mutations in

Ganglioside-induced differentiation associated

protein-1 (GADP1L1) causes a rare form autosomal

recessive Charcot-Marie-Tooth diseases [36].

Charcot-Marie-Tooth disease is a hereditary motor

and sensory neuropathy characterised by progressive

loss of muscle tissue and touch sensation across the

body parts [37]. Putative miRNA „ptc-miRf10122-

akr‟ targets the gene which causes the CMT disease.

The miRNA can be an effective drug-target to be

explored for human CMT disorder. miRNA based therapeutics could accelerate the

research in CNS disorder prevention and therapy.

The putative miRNAs predicted by the in-silico

method from EST dataset of Curcuma longa (a well-

known panacea for many human ailments) could be

excellent drug source for RNAi based neuro-

therapeutics. In-silico identification of miRNA in

medicinal plants could be useful not only in

predicting new drug molecules and targets for CNS

and neuro-degenerative disorders but also for other

fatal multifactorial diseases like cancer, HIV and

Tuberculosis.

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INTERNATIONAL JOURNAL OF ADVANCED BIOTECHNOLOGY AND BIOINFORMATICS VOL.2, ISSUE.1, NOVEMBER 2013

ISOLATION AND SCREENING OF BIOSURFACTANT

PRODUCING BACTERIA FROM POLLUTED LAKE

Prachi Jain1, Abha Saxena1, Ishwar Chandra2*, Bijoyata Borah1, J.Jemila1

1 Amity Institute of Biotechnology, Amity University, Noida, U.P.

2 Dept. of Biotechnology, Sir M Visvesvaraya Institute of Technology, Bangalore, Karnataka

ABSTRACT

Present work emphasizes on isolation and screening of biosurfactant producing microorganism. Four different

strains were isolated for the study AW1, PR2, BF3, PL4. Out of all the strains PL4 which was isolated from a lake

polluted with municipal waste was characterized. The isolated bacteria showed high oil displacement activity 7.5

cm, when cultured on minimal salt medium. Surface tension calculated was 21.16 dynes/cm, which was least among

all other strains and it has highest emulsifying capability of diesel oil because its emulsification index was maximum

i.e. 60%. Glucose and diesel are two important media ingredients essential for production of biosurfactant for this

strain.

Key words: Biosurfactant, Bacteria, Polluted lake

1. Introduction

The term surfactant is an abbreviation of the

expression “surface active agents” [1]. They are

fundamentally distinguished by their amphiphilic and

amphipathic characteristics and by their ability to

decrease surface and interfacial tensions of liquids.

Biosurfactants or microbial surfactants are surface

metabolites that produced by bacteria, yeast and

fungi having very different chemical structures and

properties [2]. They are amphiphilic molecules

consisting of hydrophobic and hydrophilic domains

that find application in an extremely wide variety of

industrial process involving emulsification, foaming,

detergency, wetting, dispersing or solubilization [3].

These are produced mainly by hydrocarbon

producing microorganisms which includes

Arthrobacter spp., Bacillus spp., Candida spp.,

Clostridium spp., Cornybacterium spp., Nocardia

spp., Pseudomonas spp. and more other genera have

been reviewed [5-14].

*Corresponding author: Ishwar Chandra

Dept. of Biotechnology, Sir M Visvesvaraya Institute

of Technology, Bangalore, Karnataka

Email: ishwar.bt016@gmail.com

The characteristic property of a biosurfactant is its

ability to reduce surface and interfacial tension by

accumulating at the interfaces between liquids, solids

and gases. They reduce the repulsive forces between

two dissimilar phases, which allows them to mix

more easily4-6. Major classes of Biosurfactants

include glycolipids, phospholipids, lipopeptides,

lipoproteins, fatty acids and polymeric biosurfactants

[15].

Biosurfactants have many environmental applications

such as recovery of hydrocarbons from dregs &

muds, removal of heavy metals from sediments,

enhancement of oil recovery [16-18]. Other potential

applications of biosurfactants relate to food,

cosmetics, pharmaceuticals, agriculture, mining,

transportation of crude oil [12-14]. Some

biosufactants are known to have therapeutic

applications as antibiotics, antiviral and antifungal

compounds [19].

They are potential alternatives of chemically

synthesized surfactant in a variety of application

because of their advantages such as lower toxicity,

higher biodegradability, higher foaming, better

environmental compatibility, the possibility of their

production through fermentation, lower critical

micelle concentration, ability to be synthesized from

renewable resources, higher selectivity, specific

activity at extreme temperatures, pH and salinity [20-

28].

27

Jain P. et. al. ISOLATION AND SCREENING OF BIOSURFACTANT PRODUCING BACTERIA FROM POLLUTED LAKE

Our objective is to isolate hydrocarbon utilizing or

biosurfactant producing microorganism from various

sites including polluted lake, automobile workshop,

refinery, etc and then performing various primary and

secondary screening tests. Also the best medium

selection and effect of different carbon sources on the

growth followed by the production of biosurfactant is

studied in this work.

2. Materials and Methods

2.1 Sample collection

Soil and water samples were collected from

Automobile workshop located in Punjabi bagh (west)

, New Delhi (AW1), Hindustan petroleum refinery,

Shakurbasti, New Delhi (PR2), Hydrocarbon

contaminated Soil near the ETP of Bhomia buttons

pvt.ltd, Bahadurgarh, Haryana (BF3), Polluted lake,

Noida (PL4). The samples were stored in sterile

polythene bags in refrigerator at 4°C.

2.2 Enrichment and isolation of culture

One gram of soil sample was dispensed in 250 ml

shake flask containing 100ml of the minimal salt

medium (MSM: Glucose 30 g/l, Sodium nitrate 3.6

g/l, Yeast extract 0.5 g/l, Potassium chloride 1.1 g/l,

Sodium chloride 1.1 g/l, Ferrous sulphate

heptahydrate 0.00028 g/l, Magnesium sulphate

heptahydrate 0.5 g/l, Trace elements solution 5ml/l,

Zinc sulphate heptahydrate 0.29 g/l, Calcium

chloride tetrahydrate 0.24 g/l, Copper sulphate

pentahydrate 0.25 g/l, Manganese sulphate

monohydrate 0.17 g/l), enriched with 1% diesel. The

media was incubated at 37⁰C on incubator shaker at

150rpm for four days. After four cycles of

enrichment, serially diluted (10‾⁵, 10‾⁷, 10‾⁸) cultures

were plated on MSM agar. The plates were overlayed

with 100µl of diesel oil and incubated at 37⁰C for 3

days. Similarly, the water samples were also used for

the isolation of microorganism by spread plate

technique on MSM agar plates and incubated at 37°C

for 2-3 days. The single colony from the plates were

streaked into nutrient agar plates and incubated

overnight. The culture was maintained on nutrient

agar plates and periodically subcultured.

2.3 Screening of biosurfactant producing

microorganisms

Loopfull of culture was inoculated in nutrient broth

from nutrient agar plate and incubated till the OD

reaches 0.6. 1ml of the culture was then transferred to

250 ml Erlenmeyer flask containing 50 ml of MSM

supplemented with 1ml diesel incubated along with

control at 37⁰C in incubator shaker at 150rpm for 96

hrs. The cells were harvested by centrifugation at

7,000 rpm for 15 minutes at 4⁰C, dried in oven to

determine dry biomass weigh. The culture filtrate

was used for further investigations.

Primary screening was done by Cetyl Trimethyl

Ammonium Bromide (CTAB) method developed by

Siegmund and Wagner,1991 where blue agar plates

were prepared by supplementing cetyl trimethyl

ammonium bromide (0.5mg/ml) and methylene blue

(6µl/50ml) in minimal salt (MS) agar containing 1%

diesel. The culture filtrate was loaded into the wells

bored in MS agar plate and incubated overnight at

37⁰C followed by flushing with CTAB-MB solution

and incubation at 4⁰C for 24 hours in order to

observe a dark halo around the wells.

Oil spread method (OSM) developed by Morikawa et

al, 2000, was also used where, petri plate base was

filled with 15ml distilled water followed by addition

of 500µl cedar oil to the surface of water. Further,

5µl of supernatant from culture broth was added at

different spots on cedar oil. The occurrence of clear

zone was an indication of a biosurfactant activity.

The diameter of the clear zones was visualized under

visible light and measured after 30 seconds, which

correlates to the surfactant activity, also known as oil

displacement activity.

Secondary screening was done using Emulsification

activity by Cooper and Goldenberg, 1987, was

measured at 540nm by optical density method. 2ml

samples of cell free supernatant were added to a

boiling test tubes containing 2ml distilled water and

the solution was mixed with 1ml of diesel. After

vigorous vortex for 2 minutes, the tubes were allowed

to stand for 1 hour to separate aqueous and oil phase.

Aqueous phase is carefully removed and OD is

measured and compared with uninoculated broth

used a negative control. The calculations were done

for all the cultures individually and their

emulsification activities were compared with each

other.

The emulsifying capacity was evaluated by an

emulsification index (E24). 2 ml of diesel was added

to 2 ml of the cell – free solution in test tube, vortex

mixing for 2 min and allowed to stand for 24 hr. The

E24 index is given as percentage of the height of

emulsified layer (cm) divided by the total height of

the liquid column (cm). The percentage of

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INTERNATIONAL JOURNAL OF ADVANCED BIOTECHNOLOGY AND BIOINFORMATICS VOL.2, ISSUE.1, NOVEMBER 2013

emulsification index calculated by using the

following equation:

E24 = 𝐻𝑒𝑖𝑔𝑕𝑡 𝑜𝑓 𝑒𝑚𝑢𝑙𝑠𝑖𝑜𝑛 𝑓𝑜𝑟𝑚𝑒𝑑

𝑇𝑜𝑡𝑎𝑙 𝑕𝑒𝑖𝑔𝑕𝑡 𝑜𝑓 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 x100

Surface tension was measured by Stalagmometer.

This device is essentially a pipette with a broad

flattened tip, which permits large drops of re-

producible size to form and finally drop under the

action of gravity. The surface tension can be

determined on the basis of the number of drops which

fall per volume, the density of the sample and the

surface tension of a reference liquid, e.g., water.

2.4 Growth kinetics study of isolate (PL4 strain

500µl inoculum was transferred to 50ml minimal salt

medium (MSM) and 50ml nutrient broth (NB)

separately. Biomass analysis for every one hour was

done spectrophotometrically at 595nm and plotted

versus time.

2.5 Medium Selection for biosurfactant

producing isolate

Three different media minimal salt medium g/l

(MSM: ), Bushnell Hass medium g/l (BH:

Magnesium sulphate 0.200, Calcium chloride 0.020,

Monopotassium phosphate 1.000, Dipotassium

phosphate 1.000, Ammonium nitrate 1.000, Ferric

chloride 0.050, Agar 20, at pH 7 ± 0.2 ) and

Proteose peptone-glucose-ammonium salts medium

g/l (PPGASM: Glucose 5, Ammonium chloride 1,

Peptone 10, Potassium chloride 1.5, Magnesium

sulphate 0.4 ) were investigated for biosurfactant

production.

The biosurfactant producing isolate PL4 was

inoculated in nutrient broth and incubated till OD

reached 0.6. Then, 1ml of the culture was inoculated

to the flasks containing MSM, BH and PPGASM

supplemented with 1% diesel and incubated at 37⁰C

at 150 rpm for 96 hours. CTAB, OSM,

Emulsification activity, emulsification index and

surface tension measurements were carried out and

results were analysed for optimum medium.

In order to Optimization Medium effect of

combination of glucose and diesel on biosurfactant

production was also studied in order to optimize

carbon content. Three different combinations i.e, 3%

glucose (no diesel) 1% diesel (no glucose) and 3%

glucose (1% diesel) were investigated by

supplementing them in MSM media separately. One

ml of the grown culture (OD 0.6) was inoculated into

the flasks and incubated for 96 hour and harvested.

The harvested culture filtrate was analyzed for

maximum biosurfactant production in order to obtain

the best combination.

Effect of different carbon sources (3%) incorporated

in Minimal salt medium (MSM) on biosurfactant

production was studied, where sucrose, glucose and

starch were investigated. 5% stock solution of each

carbon source was prepared and autoclaved

separately along with minimal salt broth in three

different flasks. 30 ml stock solution was added to 20

ml minimal salt medium (MSM) incorporated with

1% diesel to achieve the desired concentration of 1 %

(w/v). 1% of the isolated culture PL4 was inoculated

in the MS medium containing flasks respectively and

incubated at 37⁰C for 4 days. After four days, the

broth was centrifuged and supernatant was analyzed.

All surface activity tests were performed to find the

best carbon source.

3. RESULTS AND DISCUSSIONS

3.1 Screening of biosurfactant producers

Out of 12 isolates, 4 were able to grow on MSM

media with diesel as a carbon source. Oil spreading

for PL4 strain showed a positive result by displacing

the oil and hence increasing the diameter by 5 cm as

shown by figure 1whereas the other isolates were

unable to displace the oil thereby showing negative

result. Table 1 shows diameters of Displacement

Zone of oil on water by biosurfactant. PL4 strain

showed the maximum emulsification activity and

emulsification index, table 2 shows the surface

tension of culture filtrates of respective isolates. PL4

strain has the highest capability to emulsify the diesel

oil because its emulsification index was calculated

maximum i.e 60%.

The dark blue halo is observed due to ion pairing

complexation. PL4 strain showed a larger dark blue

halo than other isolates as shown by figure 2. Hence

the isolate is an anionic biosurfactant producer as it

was able to form an ion pair complexation with

cationic methylene blue and CTAB. Moreover

surface tension of cell free broth containing

biosurfactant was reduced to 21.16 dynes/cm.

PL4 strain showed higher oil displacement activity,

emulsification activity and reduction in the surface

tension when grown on MSM medium hence it is the

optimum medium for it to culture. Table 3 shows the

results on different media. In Optimization of culture

medium when both glucose and diesel together used

as the sole carbon source for optimum growth and

29

Jain P. et. al. ISOLATION AND SCREENING OF BIOSURFACTANT PRODUCING BACTERIA FROM POLLUTED LAKE

production of biosurfactant in MSM medium was

observed. The isolated strain was cultured on media

different media containing carbon substrates such as

glucose, starch and sucrose and on glucose maximum

production of biosurfactant was observed. Table 4

shows the effect of different carbon sources.

Table 1: Diameter of Displacement Zone of oil on

water by biosurfactant

Sample Initial

diameter

(cm)

Diameter after

displacement

(cm)

Interpretation

AW1 2.5 2.5 -

PR2 2.5 2.5 -

BF3 2.3 2.5 -

PL4 2.5 7.5 +

Table 2: Surface Tension of culture filtrates of

different isolates

Isolates No. of

drops

Surface tension

(dynes/ cm)

Interpretation

Uninoculated 32 64.33 Control

AW1 29 76.98 -

PR2 35 68.76 -

BF3 41 50.14 -

Table 3: Properties of different media

S.No. Medium OSM E24 Surface

tension

(dynes/cm)

1. BH Negative 1.6 46

2. PPGASM Negative 1.7 29.03

3. MSM Positive 3.01 24.26

Table 4: Effect of Carbon sources

S.No. Carbon

source

OSM E24 Emulsifi-

cation

activity

Surface

tension

(dynes/c

m)

1. Glucose Best 60% 0.61 31.76

2. Starch Negative 46% 0.32 39.86

3. Sucrose Better 52% 0.47 49.24

1(a): without culture

filtrate

1(b): with culture filtrate

Figure 1: Primary screening: oil spread method

(PL4)

Figure 2: Primary screening: Blue agar plate

method (PL4)

4. CONCLUSION

The potent biosurfactant producer PL4 was

successfully isolated from a lake polluted with

municipal waste, and characterized successfully. The

bacteria showed high oil displacement activity when

cultured on minimal salt medium. Surface tension

was also least with this strain which is the most

desirable character required for the perfect

biosurfactant producing microorganism.

It was also observed from the study that both glucose

(major carbon source) and diesel are essential for

production of biosurfactant. As biosurfactant is

produced in the stationary phase of the growth cycle

and glucose is required for the growth of the strain

followed by the degradation of the diesel thereby

producing the biosurfactant extracellularly in the

surrounding medium.

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