1) The document discusses molecular identification and phylogenetic analysis of marine DNA viruses from sea water samples collected off the coast of India.
2) Metagenomic techniques including concentration, filtration, viral DNA extraction and PCR amplification were used to obtain DNA sequences from viruses.
3) Three viral sequences were analyzed - an adenovirus targeting the hexon protein gene, an iridovirus targeting the major capsid protein gene, and a phycodnavirus targeting the major capsid protein gene.
4) BLAST analysis showed the adenovirus sequence was closely related to other adenoviruses. Phylogenetic trees grouped the sequences with known viral families.
Microbiome Identification to Characterization: Pathogen Detection Webinar Ser...QIAGEN
The research community has begun correlating the makeup of individual microbiomes with disorders and diseases such as autism, atherosclerosis, obesity and cancer. To accomplish this, researchers must first identify and characterize these microbial communities. This slidedeck will begin with a general introduction of metagenomics and an overview of experimental strategies. Following this, a comprehensive microbiome assay pipeline will be introduced. We conclude with application-based examples that demonstrate how to identify and characterize microbiome profiles.
K-mers in metagenomics
K-mers play a critical role in the exploration of metagenomic data. They have been widely used to assign taxonomic attributions to the short genomic fragments characteristic of shotgun (metagenomic) sequencing. These approaches provide an assembly-free method for profiling microbial communities, and have helped elucidate the factors driving microbial community composition across biogeochemical gradients. Advances in sequencing technology are now making it cost-effective to sequence microbial communities at sufficient depths to allow for the assembly of high-quality contigs. This has made it possible to adopt k-mer based approaches to enable reliable binning of contigs originating from a single microbial population within a community. In this session, I will present both an overview of how k-mers can be used to assign taxonomic attributions to short metagenomic reads, and discuss how these approaches have advanced to a point where population genomes can be recovered en masse from even complex microbial communities.
Microbiome Identification to Characterization: Pathogen Detection Webinar Ser...QIAGEN
The research community has begun correlating the makeup of individual microbiomes with disorders and diseases such as autism, atherosclerosis, obesity and cancer. To accomplish this, researchers must first identify and characterize these microbial communities. This slidedeck will begin with a general introduction of metagenomics and an overview of experimental strategies. Following this, a comprehensive microbiome assay pipeline will be introduced. We conclude with application-based examples that demonstrate how to identify and characterize microbiome profiles.
K-mers in metagenomics
K-mers play a critical role in the exploration of metagenomic data. They have been widely used to assign taxonomic attributions to the short genomic fragments characteristic of shotgun (metagenomic) sequencing. These approaches provide an assembly-free method for profiling microbial communities, and have helped elucidate the factors driving microbial community composition across biogeochemical gradients. Advances in sequencing technology are now making it cost-effective to sequence microbial communities at sufficient depths to allow for the assembly of high-quality contigs. This has made it possible to adopt k-mer based approaches to enable reliable binning of contigs originating from a single microbial population within a community. In this session, I will present both an overview of how k-mers can be used to assign taxonomic attributions to short metagenomic reads, and discuss how these approaches have advanced to a point where population genomes can be recovered en masse from even complex microbial communities.
Speeding up sequencing: Sequencing in an hour enables sample to answer in a w...Thermo Fisher Scientific
At this time next generation sequencing (NGS) is hindered by slow and often manual workflow procedures. Decreasing overall workflow times is critical for the widespread adoption of targeted and whole genome sequencing (WGS) for many time-sensitive applications, in particular for infectious disease analysis. To this end, we describe improvements to the four main steps of the NGS workflow: i) library preparation; ii) template preparation, iii) sequencing; iv) and data analysis. Together, these advances dramatically decrease the overall turnaround times.
Ion Torrent semiconductor-based sequencing instruments utilities flow sequencing with speed largely dependent on and the number of nucleotide flows (one flow produces ~0.5 base) and the speed of the flows (Figure 2).
Environmental dissipation of dsRNA in soil, aquatic systems and plants - Pame...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
10.02.19
Invited talk
Symposium #1816, Managing the Exaflood: Enhancing the Value of Networked Data for Science and Society
Title: Advancing the Metagenomics Revolution
San Diego, CA
Validation of RNA interference by RNA-Seq: How to see the big picture - Brend...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
Variation in responsiveness to environmental RNAi in insects - University of ...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
Next Generation Sequencing for Identification and Subtyping of Foodborne Pat...nist-spin
"Next Generation Sequencing for Identification and Subtyping of Foodborne Pathogens" presentation at the Standards for Pathogen Identification via NGS (SPIN) workshop hosted by National Institute for Standards and Technology October 2014 by Rebecca Lindsey, PhD from Enteric Diseases Laboratory Branch of the CDC.
Next generation sequencing for Identification and Characterization of plant v...Malyaj R Prajapati
Plant viruses have always been a challenge to plant growth and crop production in several parts of the world. Virus can be transmitted by vegetative propagation, fungi, nematodes, aphids, leaf hoppers, plant hoppers, beetles, white flies, and so forth. Viruses symptoms vary with the infecting virus and the infected part which includes leaf spots, leaf blights, root rots, fruit rots, fruit spots, wilt, dieback and decline. It is causing economic losses by reducing crop quality, quantity and nutritional value. Thus, their reliable detection is of a crucial importance for plant protection. While the adoption of molecular techniques such as RT-PCR has increased the speed and accuracy of virus diagnostics, such techniques only allow the detection of known viruses, i.e., each test is specific to one or a small number of related viruses. Therefore, unknown virus can be missed and testing can be slow and expensive if molecular tests are unavailable. NGS technology is one of the most popular tools for virus diagnostics. It is highly efficient, rapid diagnostics tools, and low-cost high-throughput and deep RNA sequencing. Due to the capacity to target multiple unique signature loci of virus in an infected plant metagenome and also useful for discovery of new virus and new hosts. It is including virus genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. By using deep RNA-seq requires fast and robust bioinformatics methods to enable host sequence removal and virus classification. Future developments in this area, including the use of bioinformatics tools for identification and characterization of multiple plant virus and analysis of diversity of plant viruses.
The purpose of this study was to investigate species specific inhibitory effects of esDNA isolated from two conspecific organisms: Vibrio parahaemolyticus (VP) and Vibrio harveyi (VH), and to assess the functional role of esDNA to enhance the survival rate of Artemia sp. In an in vitro study, nine doses of Extracellular self-DNA of Vibrio parahaemolyticus (esDNAVP) and Vibrio harveyi (esDNAVH) were used as the target for the challenge test with the conspecific bacteria. In an in vivo study, the protective effect of esDNA was then tested in nauplii of the brine shrimp Artemia at various priming times and concentrations of esDNA under gnotobiotic conditions prior to challenge with VP and VH at the concentration of 5 × 105 CFU mL-1. The results from in vitro study showed that the use of esDNAVP at levels of 24.02 and 48.05 ng µl-1 and esDNAVH at concentrations of 13.33 and 26.67 ng µl-1 were able to inhibit the growth of the conspecific species when added to the culture medium at the concentration level of 5 × 105 CFU mL-1. The results from in vivo study showed that the use of 24.02; 48.05 and 72.07 ng µl-1 of esDNAVP as well as the use of 13.33; 26.67 and 40.00 ng µl-1 of esDNAVH inhibited the growth of VP and VH and enhanced the survival rate of Artemia sp compared to the control treatment (P<0.05). Taken together, we confirmed that esDNA obtained from the extraction and random fragmentation from esDNAVP and esDNAVH, produces a species-specific inhibitory effect on the same species and can serve as a potential alternative strategy for disease control to deliver the functionality of esDNA to the fish and shrimp.
Speeding up sequencing: Sequencing in an hour enables sample to answer in a w...Thermo Fisher Scientific
At this time next generation sequencing (NGS) is hindered by slow and often manual workflow procedures. Decreasing overall workflow times is critical for the widespread adoption of targeted and whole genome sequencing (WGS) for many time-sensitive applications, in particular for infectious disease analysis. To this end, we describe improvements to the four main steps of the NGS workflow: i) library preparation; ii) template preparation, iii) sequencing; iv) and data analysis. Together, these advances dramatically decrease the overall turnaround times.
Ion Torrent semiconductor-based sequencing instruments utilities flow sequencing with speed largely dependent on and the number of nucleotide flows (one flow produces ~0.5 base) and the speed of the flows (Figure 2).
Environmental dissipation of dsRNA in soil, aquatic systems and plants - Pame...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
10.02.19
Invited talk
Symposium #1816, Managing the Exaflood: Enhancing the Value of Networked Data for Science and Society
Title: Advancing the Metagenomics Revolution
San Diego, CA
Validation of RNA interference by RNA-Seq: How to see the big picture - Brend...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
Variation in responsiveness to environmental RNAi in insects - University of ...OECD Environment
10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.
Next Generation Sequencing for Identification and Subtyping of Foodborne Pat...nist-spin
"Next Generation Sequencing for Identification and Subtyping of Foodborne Pathogens" presentation at the Standards for Pathogen Identification via NGS (SPIN) workshop hosted by National Institute for Standards and Technology October 2014 by Rebecca Lindsey, PhD from Enteric Diseases Laboratory Branch of the CDC.
Next generation sequencing for Identification and Characterization of plant v...Malyaj R Prajapati
Plant viruses have always been a challenge to plant growth and crop production in several parts of the world. Virus can be transmitted by vegetative propagation, fungi, nematodes, aphids, leaf hoppers, plant hoppers, beetles, white flies, and so forth. Viruses symptoms vary with the infecting virus and the infected part which includes leaf spots, leaf blights, root rots, fruit rots, fruit spots, wilt, dieback and decline. It is causing economic losses by reducing crop quality, quantity and nutritional value. Thus, their reliable detection is of a crucial importance for plant protection. While the adoption of molecular techniques such as RT-PCR has increased the speed and accuracy of virus diagnostics, such techniques only allow the detection of known viruses, i.e., each test is specific to one or a small number of related viruses. Therefore, unknown virus can be missed and testing can be slow and expensive if molecular tests are unavailable. NGS technology is one of the most popular tools for virus diagnostics. It is highly efficient, rapid diagnostics tools, and low-cost high-throughput and deep RNA sequencing. Due to the capacity to target multiple unique signature loci of virus in an infected plant metagenome and also useful for discovery of new virus and new hosts. It is including virus genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. By using deep RNA-seq requires fast and robust bioinformatics methods to enable host sequence removal and virus classification. Future developments in this area, including the use of bioinformatics tools for identification and characterization of multiple plant virus and analysis of diversity of plant viruses.
The purpose of this study was to investigate species specific inhibitory effects of esDNA isolated from two conspecific organisms: Vibrio parahaemolyticus (VP) and Vibrio harveyi (VH), and to assess the functional role of esDNA to enhance the survival rate of Artemia sp. In an in vitro study, nine doses of Extracellular self-DNA of Vibrio parahaemolyticus (esDNAVP) and Vibrio harveyi (esDNAVH) were used as the target for the challenge test with the conspecific bacteria. In an in vivo study, the protective effect of esDNA was then tested in nauplii of the brine shrimp Artemia at various priming times and concentrations of esDNA under gnotobiotic conditions prior to challenge with VP and VH at the concentration of 5 × 105 CFU mL-1. The results from in vitro study showed that the use of esDNAVP at levels of 24.02 and 48.05 ng µl-1 and esDNAVH at concentrations of 13.33 and 26.67 ng µl-1 were able to inhibit the growth of the conspecific species when added to the culture medium at the concentration level of 5 × 105 CFU mL-1. The results from in vivo study showed that the use of 24.02; 48.05 and 72.07 ng µl-1 of esDNAVP as well as the use of 13.33; 26.67 and 40.00 ng µl-1 of esDNAVH inhibited the growth of VP and VH and enhanced the survival rate of Artemia sp compared to the control treatment (P<0.05). Taken together, we confirmed that esDNA obtained from the extraction and random fragmentation from esDNAVP and esDNAVH, produces a species-specific inhibitory effect on the same species and can serve as a potential alternative strategy for disease control to deliver the functionality of esDNA to the fish and shrimp.
Presentation 8: Vibrio parahaemolyticus: a versatile pathogen that can adapt ...ExternalEvents
http://www.fao.org/documents/card/en/c/28b6bd62-5433-4fad-b5a1-8ac61eb671b1/
International Technical Seminar/Workshops on Acute hepatopancreatic necrosis disease (AHPND)
DNA Barcoding of Stone Fish Uranoscopus Oligolepis: Intra Species Delineation...journal ijrtem
Abstract: The present study addresses this issue by examining the patterning of Cytochrome Oxidase I diversity in the stone fish Uranoscopus oligolepis the structurally diverse group of Family Uranoscopidae. The sequences were analyzed for their species identification using BOLD’s identification engine. The COI sequences of U. oligolepis from different geographical regions were extracted from NCBI for intra species variation analysis. All sequences were aligned using Clustal W. The sequences were trimmed using software and phylogenetic tree was constructed with bootstrap test. The results showed that the cytosine content was high (31%). The least molar concentration was observed in guanine (19.5%) and Adenine (19.6%). Thymine was the second predominant in molar concentration next to thymine which is followed by adenine. The G+C content was found to be 49.6% and A+T content was 50.4%. Leucine and Alanine content was high in the amino acid composition. From the study it is assumed that the mitochondrial gene COI can be the potential barcoding region to identify an organism up to the species level. Keywords: COI, intra species, Uranoscopus oligolepis, barcoding, phylogenetic
Identification of causative agent for fungal infection and effect of disinfec...AbdullaAlAsif1
Common carp (Cyprinus carpio) is one of the commercially important and commonly cultured fish. In the hatchery intensive incubation leads to microbial overgrowth in C. carpio eggs that hamper egg development, hatchability and larval survivability. The aim of this study is to find out causes of mass mortality in C. carpio eggs during peak- breeding season between March to May 2015 at Mafatema fish hatchery, Chanchra, Jessore sadar upazilla. In the present study three disinfectants with three different concentrations in each such as methylene blue 1, 3 and 5mg/L., malachite green 1, 3 and 5mg/L., sodium chloride 1, 2 and 3g/L were used to observe the hatching rate of fertilized eggs and survival rate of larvae. Bacterial load of culture water was examined during the induced breeding of C. carpio with mycological examination of egg samples with different disinfectants. The total bacterial count fluctuated from 3.4 x 108 CFU/ml to 32.7 x 108 CFU/ml during the period of fertilization to 4days of hatching. The fertilized eggs infected by Saprolegnia spp. were appeared as tuft hairy like balls with a white cottony envelop. Among all the treatment 1mg/L methylene blue, 3mg/L malachite green and 1g/L sodium chloride showed significantly better (P<0.05) hatching rate 95·33±2·08, 88.00±2.64 and 92.33±4.04% respectively. The same concentration of methylene blue, malachite green and sodium chloride showed significantly better (P<0.05) better survival rate 95·00±4.35, 75.00±3.00 and 87.00±6.24% respectively. Finally among all the treatment 1mg/L of methylene blue showed significantly better (P<0.05) hatching and survival rate 95·33±2·08% and 95·00±4.35 % respectively. So 1mg/L of methylene blue is the best disinfectant for C. carpio fertilized egg treatment.
screening model for Parkinson's disease.pptxAHEMANTHBABU
Parkinson's disease (PD) is a complex neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra region of the brain. This degeneration results in a wide range of motor and non-motor symptoms, including bradykinesia, rigidity, tremors, and postural instability. PD not only affects motor function but also leads to cognitive and psychiatric impairments, significantly reducing the quality of life for those afflicted.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
3. Why marine
Virus???
Higher Concentration
(~108 viruses ml–1 ) but less
is known.
DNA containing viruses
are abundant in Marine
system.
Causative agent of disease
in aquaculture.
Less in known about
diversity and evolution.
Important for
biogeochemical cycles.
Metagenomics (Jo
Handelsman, Jon Clardy et
al. 1998) study came in light
to address the challenge of
studying unculturable viral
particles (<99%).
Metagenomics is an
alternative culture-
independent and sequence-
independent approach that
does not rely on the
presence of any particular
gene in all the subject
entities.
Why Metagenomics??
4. A virus particle, called a virion, can be thought of as a delivery
system that surrounds a payload.
Virus as “a piece of bad news wrapped in a protein
coat.”
The delivery system consists of structural components used by
the virus to survive in the environment and bind to host cells
The payload contains the viral genome and often includes
enzymes required for the initial steps in virus replication
Virus looks like!!!!!!
5. Examples of the main types of viruses:
Tailed phage that infect bacteria .
Filamentous viruses that infect all domains of life, and
Enveloped viruses that infect animal and plant cells.
6. Origin of Viruses
• DNA part coding for important cellular machinery escape
from cellular control gained essential viral gene. These
genes replicated by cellular host and form virus like
particles.
Alternative
hypothesis
• Primitive cells having essential cellular machinery decreased in
size and genome get encapsulated by endosymbiont that
develop in primitive viral like particles.
Reductive
evolution
• Primitive atmosphere contain micelles (can trap nucleic acid
like particles), with the passing of time as trapped material
in micelles increased ribozyme activity evolved and micelles
become protovirus, form protein and fused to share
information, this way slowly-slowly protovirus evolved in to
modern virus.
Origin
based on
micelles
11. Marine Viruses:
Spencer 1955- The first phage isolated from the marine
environment was reported more than 50 years ago but the
abundance of viruses was recognized in the late 1980s.
Bergh et al. 1989 - Published a paper in journal Nature “High
abundance of viruses found in aquatic environments”.
This paper gives insight that viruses are abundant and
ecologically important components of the marine environment.
Marine viruses includes eukaryotic viruses, phage and
generalized transducing agents (GTAs) and infect all organisms
from bacteria to whales.
Pascal Hingamp et. al. 2013. Exploring nucleo-cytoplasmic
large DNA viruses in Tara Oceans microbial metagenomes.
Nature (2013).
12. R Danovaro et al. Nature .2008
Relationships between viruses and prokaryotes in deep-sea sediments
worldwide.
13. Importance of Viruses
Phage Therapy Atmospheric C02
Viral lysis diverts carbon
from the POC to the DOC
pool, effectively “short-
circuiting” the microbial
loop away from the
grazers.
Aquaculture as
disease controlling
weapon.
Detection and
diagnostics of disease
causing agents, as
antimicrobial agent
Viral lysis of phytoplankton
DMS- gas that influences cloud
formation. Viral lysis releases
organic Fe complexes which is
1000 times more bioavailable
and efficiently assimilated by
bacterial cells than Fe(III)
Carbon , Fe and
Nitrogen cycle
14. Eukaryotic viruses having large double
stranded DNA (dsDNA) genome ranging
from 100kb up to 1.26Mb.
Host range of these viruses is from
microscopic unicellular eukaryotes to
larger animals, including humans.
Nuclear cytoplasmic large DNA virus (NCLDV):
Virus of theses group replicate either
exclusively in the cytoplasm, or in both
cytoplasm and in nucleus of host cell
15. Virus family Host range Genome
size range,
kb
Replication site Virion
Phycodnaviridae Green algae; algal
symbionts of
paramecia and
hydras
150-400 Nucleus and
cytoplasm
isometric
Poxviridae Animals: insects,
reptiles, birds,
mammals
130-380 Cytoplasm isometric
Asfarviridae Mammals 170 Cytoplasm isometric
Ascoviridae Insects, mainly,
Noctuids
150-190 Nucleus and
cytoplasm
isometric
Iridoviridae Insects, cold-
blooded
vertebrates
100-220 Nucleus and
cytoplasm
isometric
Mimiviridae Acanthamoeba 1,180 Cytoplasm isometric
Marseillevirus Acanthamoeba 370 Nucleus and
cytoplasm
16. Metagenomics
Metagenomics is an alternative culture-independent and sequence-
independent approach that does not rely on the presence of any
particular gene in all the subject entities.
Why Metagenomics???
Metagenomics study came in light
to address the challenge of studying
unculturable prokaryotes (<99%).
17. The Global Ocean Sampling Expedition (GOS)
With the goal of assessing the genetic diversity in marine
microbial communities and to understand their role in
nature's fundamental processes in Sargasso Sea.
Started in August 2003.
The GOS datasets were submitted to
both NCBI and Community Cyber
infrastructure for Advanced Marine
Microbial Ecology Research and
Analysis (CAMERA)
1,800 microbial species were
discovered including 148 novel
phylotypes, encoding more than 1.2
million genes.
This study expanded our knowledge of
ocean photobiology, microbial diversity,
and evolution.
18. Sampling Route of The Sorcerer II
Onboard scientists take a 200 to 400 liters water sample
approximately every 200 miles, filter it through progressively
smaller filters to capture the various sized organisms, and then freeze
the filters with the captured microorganisms.
22. Overview of the present work
New sequence
and Viral
relation
Phylogeny
DNA
Virus
23. Study Site & Samples used:
50 liters of surface sea water sample was
collected in polyethylene containers from
the Cochin Barmouth region, India (at
latitude 9° 58' 0" North and longitude 76°
15' 0" East) during September 2013.
Purification and Concentration of Samples:
Using tangential flow filtration system of 0.2 µM filters and
flocculation process.
Using this system ~50 liters of pre-filtered sea water was
concentrated down to ~20 ml from which viral particles were
pelleted by ultracentrifugation at 14, 000 x g. The viral pellet was re-
suspended with ~2 ml of sterile PBS buffer.
Sampling and purification
24. Nature protocol 2009
Hose clamp
>0.2 μm bacteria,
protists
Flow
Retentate
Peristaltic
pump
Pressure gauge
Filtrate
0.2 μm(viral
particles
Whole water sample
>0.2 μm (bacteria, protists
<0.2 μm (viral particles
Tangential Flow Filtration Unit
50 liter of
sea water
Next Step
26. Due to small genomic content in virus, we followed QIAGEN MiniElute virus spin
procedure rather than rigorous manual laboratory protocol for viral DNA extraction.
Viral sample (already kept in -20oC) was lysed by pulse-vortexing with 25 µl of
QUIAGEN protease and 200 µl of buffer AL for 15 sec in 2 ml centrifuge tubes.
This sample was incubated at 56 oC for 15 minute and mixed with 250 µl of 100%
ethanol and kept 5 minute at room temperature now this lysate was carefully
transferred in QUIAGEN mini elute column.
Buffer AW1 and AW2 (provided with kit) was added on column sequentially and
centrifuged 800 rpm for 1 minute with each. Filtrate was discarded and column was
placed in fresh collecting tube.
The column was washed by adding 500 µl 100% ethanol and centrifuging at 8000 rpm,
collection tube was discarded.
QUIAGEN mini elution kit was dried by centrifuging at 14,000rpm for 3 minutes and
incubating at 56oC for 3 minutes.
For elution of DNA, 150 µl of elution buffer was applied on dried column and column
was centrifuged at 14,000 rpm.
Filtrate containing viral DNA sample was collected in autoclaved microcentrifuge tube
and stored at -20oC.
Viral DNA Isolation
27. Viral DNA quantified by spectrophotometer
Viral DNA was quantified and qualified by spectrophotometric
analysis at 260 & 280 nm in a 1-cm light-path length quartz
cuvette.
SAMPLE IN ELUTION BUFFER
O.D AT 260 nm = 0.18
CONCENTARTION OF DNA = 0.D AT 260nm X 50 X DILUTION FACTOR
(IN OUR CASE 50)
= 0.18 X 50 X 50
= 450 MICROG / 1 ML
= 450 nanog/microl
SAMPLE IN MILLIQ
O.D AT 260 nm = 0.14
= 350 nanog/microl
concentration required for PCR = 100ng DNA
28. 40 primers were designed based on conserved regions of marine
DNA viruses viz. DNA polymerase gene, major capsid proteins,
Hexon Protein gene etc. were synthesized at Sigma Inc.
Primer Length: optimal length of PCR primers is 18-22 bp.
Primer Melting Temperature: Primers with melting temperatures
in the range of 52- 65 oC.
GC Content: should be 40-60%.
Primer Designing and Synthesis
Gene Tool Software
Standardization of PCR conditions:
PCR conditions were standardized by varying annealing
temperatures for various primer sets.
31. Cloning, Ligation and Sequencing
The amplified DNA fragments
were cloned in pGEM-T Easy
vector by TA cloning.
The ligation reaction mixture
contained 3.5µl of PCR product
and .5µl of pGEM-T Easy vector,
5µl 2x rapid ligation buffer and
1µl of T4 DNA ligase in a final
volume of 10 ml.
Positive amplicons were
cloned on to pGEMT Easy
vector.
32. 1.5 ml bacterial culture was transferred to a 2 ml autoclaved eppendorf tube, and
centrifuged at 6,000 rpm for 5 min, supernatant was removed.200 μl of resuspension
solution (1M Tris-cl,pH-8.0,0.5M EDTA,1M Glucose, RNase, MilliQ) was added into
each tube, and vortex to completely resuspend cell pellet.
Plasmid was extracted from positive clones using standard laboratory procedure.
Immediately after adding 200 μl of lysis buffer (10N NaOH,25% SDS, MilliQ) tubes
were gently inverted 4-5 times and 350 μl of neutralizing solution (5M Pottassium
acetate, Glacial acetic acid, MilliQ) was added (mixed gently by inverting the tubes 4-5
times).The tubes were centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to
a new labeled 1.5 ml Eppendorf tube containing 600 μl of ice cold isopropanol (mixed by
gentle taping) and tubes were placed in ice for 10 min.
Plasmid DNA precipitate (transparency pellet) was done by centrifuging tubes at 12,000
rpm for 10 min. Supernatant was discarded and the pellet was washed with 70% ethanol
by centrifuging at 12000 rpm for 10 mint.
Tubes were air dried by keeping invert position on a piece of paper towel for 10-20 min.
20-30 μl of 10Mm Tris-cl was added in air dried DNA pellet and the tubes were kept in
4°C for overnight to completely dissolve the pellet and stored at -200C.
Presence of plasmid was also confirmed by running plasmid sample on 1% agarose gel
and visualised under UV light.
Plasmid isolation and Amplification
34. Sequencing and Analysis of Sequence
10µl of plasmid was handed over for sequencing at
SciGenom, Cochin, India.
The sequences were analysed, trimmed and assembled
using GeneTool software.
The nucleotide sequence homology and the translated
amino acid sequence comparisons were performed
using BLAST algorithm (BLASTn and BLASTp) of the
National Center for Biotechnology Information (NCBI)
(http://www.ncbi.nlm.nih.gov/blast).
Gene translation and prediction of deduced proteins
were performed with ExPASy
(http://www.au.expasy.org/).
35. Continued……
The multiple sequence alignments of nucleotide and amino
acid sequence were performed with sequences retrieved from
NCBI and multi-aligned using ClustalW and GeneDoc
computer programmes.
Phylogenetic and molecular evolutionary analyses was
conducted by the Neighbor-Joining (NJ) and Maximum-
Likelihood (ML) methods using MEGA version 5.
The nucleotide sequences described in this study were
deposited in to GenBank and were assigned accession numbers
KJ958986 and KJ958987.
37. Adenovirus: Nucleotide & Amino acid sequence of hexon protein
Results:
Results will not be shared here
Sorry , Hope soon you will se
them in Publication………..
55. Future Dimensions and Use of this
work
Open the door of metagenomic viral study in Cochin bar
mouth region since my work clearly indicate presence of
large viruses in this area.
Discovery of new viral gene.
The application of metagenomic sequence information will
facilitate the design of better culturing strategies to link
genomic analysis with pure culture studies.
Reassembly of multiple genomes will provide insight into
energy and nutrient cycling within the community, genome
structure, gene function, population genetics and
microheterogeneity, and lateral gene transfer among
members of an uncultured community.
56. Paper Communicated in Peer-viewed Journals
Marine Virus: Larger genome with bigger impact - A review
Dhirendra Kumar Singh1, Swapna P. Antony1*and I.S. Bright Singh1
1National Center for Aquatic Animal Health, Cochin University of Science
and Technology, Cochin-682016, Kerala, India
Yes, I am a virus and I can encode eukaryotic proteins.
Dhirendra Kumar Singh1 and Swapna P. Antony1*
1National Center for Aquatic Animal Health, Cochin University of Science
and Technology, Cochin-682016, Kerala, India
Submitted in: Archives in Virology - Elsevier. Impact Factor- 2.6
Review’s
57. Continued…..
Research Article for Publication:
International Conference:
Major Capsid Protein Gene: A reliable phylogenetic biomarker for marine
viruses“
Abstract Submitted for Power Point Presesntation in “International Conference
on emerging trend in Biotechnology” (ICETB-2014) going to held in JNU-
Delhi on 6-9 November 2014
Molecular characterization and phylogenetic analysis of Family
Iridoviridae from marine environment as inferred from the major capsid
protein gene
Dhirendra Kumar Singh1 and Swapna P. Antony1*
1National Center for Aquatic Animal Health, Cochin University of Science and
Technology, Cochin-682016, Kerala, India
Communicated in: Journal of Experimental Marine Biology and Ecology, Impact- 2.4
58. Molecular characterization and phylogenetic
analysis of Family Adenoviridae major capsid
protein has been done, writing of paper is
remaing.
Analysis of Some other sequences might be
analysed and cab be written as paper.
59. My special Thanks goes to….
thanks to Ramya Chechi and
all NCAAH scholars for their kind help and support.