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De novo transcriptome analysis using 454 pyrosequencing of the Himalayan Mayapple,Podophyllum hexandrum
Get PDFBackground: The Himalayan or Indian Mayapple (Podophyllum hexandrum Royle) produces podophyllotoxin, which
is used in the production of semisynthetic anticancer drugs. High throughput transcriptome sequences or genomic
sequence data from the Indian Mayapple are essential for further understanding of the podophyllotoxin
biosynthetic pathway.
Results: 454 pyrosequencing of a P. hexandrum cell culture normalized cDNA library generated 2,667,207 raw reads
and 1,503,232 high quality reads, with an average read length of 138 bp. The denovo assembly was performed by
Newbler using default and optimized parameters. The optimized parameter generated 40, 380 assembled
sequences, comprising 12,940 contigs and 27,440 singlets which resulted in better assembly as compared to default
parameters. BLASTX analysis resulted in the annotation of 40,380 contigs/singlet using a cut-off value of ≤1E-03.
High similarity to Medicago truncatula using optimized parameters and to Populus trichocarpa using default
parameters was noted. The Kyoto encyclopedia of genes and genomes (KEGG) analysis using KEGG Automatic
Annotation Server (KAAS) combined with domain analysis of the assembled transcripts revealed putative members
of secondary metabolism pathways that may be involved in podophyllotoxin biosynthesis. A proposed schematic
pathway for phenylpropanoids and podophyllotoxin biosynthesis was generated. Expression profiling was carried
out based on fragments per kilobase of exon per million fragments (FPKM). 1036 simple sequence repeats were
predicted in the P. hexandrum sequences. Sixty-nine transcripts were mapped to 99 mature and precursor microRNAs
from the plant microRNA database. Around 961 transcripts containing transcription factor domains were noted. High
performance liquid chromatography analysis showed the peak accumulation of podophyllotoxin in 12-day cell
suspension cultures. A comparative qRT-PCR analysis of phenylpropanoid pathway genes identified in the present data
was performed to analyze their expression patterns in 12-day cell culture, callus and rhizome.
Conclusions: The present data will help the identification of the potential genes and transcription factors involved in
podophyllotoxin biosynthesis in P. hexandrum. The assembled transcripts could serve as potential candidates for
marker discovery and conservation, which should form the foundations for future endeavors
A transient reversal of miRNA-mediated repression controls macrophage activation
No full textIn mammalian macrophages, the expression of a number of
cytokines is regulated by miRNAs. Upon macrophage activation, proinflammatory cytokine mRNAs are translated,
although the expression of miRNAs targeting these mRNAs
remains largely unaltered. We show that there is a transient
reversal of miRNA-mediated repression during the early phase of the inflammatory response in macrophages, which leads to the protection of cytokine mRNAs from miRNA-mediated repression. This derepression occurs through Ago2 phosphorylation, which results in its impaired binding to miRNAs and to the corresponding target mRNAs. Macrophages expressing a mutant, non-phosphorylatable AGO2—which remains bound to miRNAs during macrophage activation—have a weakened inflammatory response and fail to prevent parasite invasion. These findings highlight the relevance of the transient relief of miRNA repression for macrophage functio
Noise-Aided Logic in an Electronic Analog of Synthetic Genetic Networks
Get PDFWe report the experimental verification of noise-enhanced logic behaviour in an electronic analog of a synthetic genetic network, composed of two repressors and two constitutive promoters. We observe good agreement between circuit measurements and numerical prediction, with the circuit allowing for robust logic operations in an optimal window of noise. Namely, the input-output characteristics of a logic gate is reproduced faithfully under moderate noise, which is a manifestation of the phenomenon known as Logical Stochastic Resonance. The two dynamical variables in the system yield complementary logic behaviour simultaneously. The system is easily morphed from AND/NAND to OR/NOR logi
NF-kappaB Mediated Transcriptional Repression of Acid Modifying Hormone Gastrin
Get PDFHelicobacter pylori is a major pathogen associated with the development of gastroduodenal diseases. It has been
reported that H. pylori induced pro-inflammatory cytokine IL1B is one of the various modulators of acid secretion in
the gut. Earlier we reported that IL1B-activated NFkB down-regulates gastrin, the major hormonal regulator of acid
secretion. In this study, the probable pathway by which IL1B induces NFkB and affects gastrin expression has been
elucidated. IL1B-treated AGS cells showed nine-fold activation of MyD88 followed by phosphorylation of TAK1 within
15 min of IL1B treatment. Furthermore, it was observed that activated TAK1 significantly up-regulates the NFkB
subunits p50 and p65. Ectopic expression of NFkB p65 in AGS cells resulted in about nine-fold transcriptional
repression of gastrin both in the presence and absence of IL1B. The S536A mutant of NFkB p65 is significantly less
effective in repressing gastrin. These observations show that a functional NFkB p65 is important for IL1B-mediated
repression of gastrin. ChIP assays revealed the presence of HDAC1 and NFkB p65 along with NCoR on the gastrin
promoter. Thus, the study provides mechanistic insight into the IL1B-mediated gastrin repression via NFk
Observation of Synchronization in Coupled Chaotic Oscillators
No full textThe word synchronization comes from Greek word “sunkhronos: sun-, syn- + khronos, time” meaning sharing a common time. In general, it maintains the same meaning, as correlation or agreement in time of different processes [1]. Studies of synchronization started in the 17th century when Christiaan Huygens first discovered that two pendulum clocks hanging from a wooden beam synchronized either inphase or antiphase [2]. Two other very interesting cases were reported much later, the flashing of fireflies in unison [3] and the peculiarities of adjustment of organ pipes [4].
Many more examples of synchronization those exist in nature were reported in the last two decade such as cardio-respiratory synchrony [5], the crowd synchrony [6] in the
millennium bridge [7], London and quorum sensing of bacteria [8]. In the last two decades, the interest in the studies of synchronization also included chaotic
systems. A dynamical system is called chaotic, in simple words, if its solution is sensitive to initial conditions but still bounded. Another important characteristic of chaos is its broad frequency spectrum. It was a common notion that the chaotic systems evolving in time, started from two different initial conditions, cannot synchronize. So the studies of collective behavior of chaotic systems were of great interest. Finally, it was first reported
in 1990 [9] that synchronization of two chaotic systems is possible under strong coupling. Although a similar work on chaos synchronization was published earlier in 1983[10] but
it was not well known to the nonlinear dynamics community
Dual Functionalized Graphene Oxide Serves as a Carrier for Delivering Oligohistidine- and Biotin-Tagged Biomolecules into Cells
No full textA versatile method of dual chemical functionalization of graphene oxide (GO) with Tris-[nitrilotris(acetic acid)] (Tris-NTA) and biotin for cellular delivery of oligohistidine- and biotintagged biomolecules is reported. Orthogonally functionalized GO surfaces with Tris-NTA and
biotin to obtain a dual-functionalized GO (DFGO) are prepared and characterized by various spectroscopic and microscopic techniques. Fluorescence microscopic images reveal that DFGO surfaces are capable of binding oligohistidine-tagged biomolecules/proteins and avidin/
biotin-tagged biomolecules/proteins orthogonally. The
DFGO nanoparticles are noncytotoxic in nature and can
deliver oligohistidine- and biotin-tagged biomolecules
simultaneously into the cel
Reduced Virulence of the Vibrio cholerae fadD Mutant Is Due to Induction of the Extracytoplasmic Stress Response
No full textVibrio cholerae, an important human intestinal pathogen, is responsible for the diarrheal disease cholera. The pathogenesis of V.cholerae is a highly coordinated process that involves diverse regulatory factors. It has recently been demonstrated that disruption of the V. cholerae fadD gene, encoding a long-chain fatty acyl coenzyme A (acyl-CoA) ligase, drastically reduces expression of the major virulence genes and in vivo lethality of this important human pathogen. This effect was due to reduced membrane
localization of the central virulence regulator TcpP. In this study, the reason for the impaired membrane localization of TcpP in the fadD mutant was investigated. We demonstrate that extracytoplasmic stress is induced in the V. cholerae �fadD strain. In response to the extracytoplasmic stress, the integral membrane protease RseP is activated and degrades the membrane-localized
TcpP in the fadD mutant strain. Indeed, disruption of the rseP gene in a fadD mutant background restored membrane localization of TcpP and expression of the downstream virulence genes toxT, ctxA, and tcpA. Increased expression of the �E regulon genes in ethanol-treated wild-type V. cholerae indicated that ethanol exposure could induce an extracytoplasmic stress response in V. cholerae. Ethanol treatment also led to activation of the RseP protease activity and resulted in degradation of membranelocalized
TcpP and subsequent reduction in expression of the virulence genes. Taken together, these results suggest that extracytoplasmic stress response per se reduces virulence of V. cholerae by impairing membrane localization of Tcp
Potential Contribution of SIM2 and ETS2 Functional Polymorphisms in Down Syndrome Associated Malignancies
No full textProper expression and functioning of transcription factors (TFs) are essential for regulation of different
traits and thus could be crucial for the development of complex diseases. Subjects with Down syndrome (DS) have
a higher incidence of acute lymphoblastic leukemia (ALL) while solid tumors, like breast cancer (BC) and oral cancer
(OC), show rare incidences. Triplication of the human chromosome 21 in DS is associated with altered genetic
dosage of different TFs. V-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2) and Single Minded 2 (SIM2) are
two such TFs that regulate several downstream genes involved in developmental and neurological pathways. Here
we studied functional genetic polymorphisms (fSNP) in ETS2 and SIM2 encoding genes in a group of patients and
control subjects to better understand association of these variants with DS phenotypes.We employed an in silico approach to identify potential target pathways of ETS2 and SIM2. fSNPs in genes encoding for these two TFs were identified using available databases. Selected sites were genotyped in individuals with DS, their parents, ALL, BC, OC as well as ethnically matched control individuals. We further analyzed these data by population-based statistical methods.Allelic/genotypic association analysis showed significant (P < 0.03) differences of rs2070530, rs1051476, rs11254, rs711 for DS subjects compared to control. rs711 also exhibited significantly different genotypic distribution pattern in parents of DS probands (P < 0.02) and BC patients (P < 0.02). Interaction analysis revealed independent main effect of rs711 in all the groups, while rs11254 exhibited independent main effect in DS subjects only. High entropy values were noticed for rs461155 in the solid tumor groups. Significant interactive effects of rs2070531 with rs1051475, rs1051476, rs11254 were observed in all the groups except DS.
We infer from the present investigation that the difference in frequencies of fSNPs and their independent as well as interactive effects may be the cause for altered expression of SIM2 and ETS2 in DS and malignant groups, which affects different downstream biological pathways. Thus, altered expression of SIM2 and ETS2 could be one of the reasons for variable occurrence of different malignant conditions in DS
Indirect Read-out of the Promoter DNA by RNA Polymerase in the Closed Complex
No full textTranscription is initiated when RNA polymerase
recognizes the duplex promoter DNA in the closed
complex. Due to its transient nature, the closed
complex has not been well characterized. How the
initial promoter recognition occurs may offer
important clues to regulation of transcription initiation.
In this article, we have carried out single-base
pair substitution experiments on two Escherichia coli
promoters belonging to two different classes, the
�35 and the extended �10, under conditions which
stabilize the closed complex. Single-base pair substitution
experiments indicate modest base-specific
effects on the stability of the closed complex of both
promoters. Mutations of base pairs in the �10 region
affect the closed complexes of two promoters differently,
suggesting different modes of interaction of
the RNA polymerase and the promoter in the two
closed complexes. Two residues on p70 which have
been suggested to play important role in promoter
recognition, Q437 and R436, were mutated and
found to have different effects on the closedcomplex
stability. DNA circular dichroism (CD) and
FRET suggest that the promoter DNA in the closed
complex is distorted. Modeling suggests two
different orientations of the recognition helix of the
RNA polymerase in the closed complex. We propose
that the RNA polymerase recognizes the sequence
dependent conformation of the promoter DNA in
the closed comple
Identification and Characterization of the Enzymatic and Immunoregulatory Role of Plasmodial Macrophage Migration Inhibitory Factor in Host Pathology
No full textMalaria remains one of the world’s most devastating infectious diseases. Approximately 3
billion people, one half of the world’s population, live in at-risk regions for malaria infection.
This leads to about 250 million malaria cases every year and nearly one million deaths (World
Malaria Report, WHO, 2011,
http://www.who.int/malaria/world_malaria_report_2011/en/index.html). The etiologic agent
Plasmodium falciparum (P. falciparum) is the major organism responsible for the majority of
deaths due to malaria, 90% of which occur in Africa with more than 85% in children under
the age of 5. The presence of widespread drug resistance is hampering the effectiveness of
most of the available drug arsenal (1, 2) with the notable exception of artemisinin-based
derivatives. In the battle to fight malaria, introduction of new drugs with novel mechanisms of
action is essential