110 research outputs found
Custom Design and Analysis of High-Density Oligonucleotide Bacterial Tiling Microarrays
Not until recently have custom made high-density oligonucleotide microarrays been available at an affordable price. The aim of this thesis was to design microarrays and analysis algorithms for DNA repair and DNA damage detection, and to apply the methods in real experiments.
Thomassen et al. have used their custom designed whole genome-tiling microarrays for detection of transcriptional changes in Escherichia coli after exposure to DNA damageing reagents. The transcriptional changes in E. coli treated with UV light or the methylating reagent MNNG were shown to be larger and to include far more genes than previously reported. To optimize the data analysis for the custom made arrays, Thomassen and coworkers designed their own normalization and analysis algorithms, and showed these more suitable than established methods that are currently applied on custom tiling arrays.
Among other findings several novel stress-induced transcripts were detected, of which one is predicted to be a UV-induced short transmembrane protein. Additionally, no upregulation of the previously described UV-inducible aidB is shown. In the MNNG study several genes are shown as downregulated in response to DNA damage although having upstream regulatory sequences similar to the established LexA box A and B. This indicates that the LexA regulon also might control gene repression and that the box A and B sequence can not alone answer for the LexA controlled gene regulation.
Thomassen et al. have also custom designed a microarray for oncogenic fusion gene detection. Cancer specific fusion genes are often used to subgroup cancers and to define the optimal treatment, but currently the laboratory detection procedure is both laborious and tedious. In a blinded study on six cancer cell lines proof of principle was shown by detection of six out of six positive controls. The design and analysis methods for this microarray are now being refined to make a diagnostic fusion gene detection tool
Competitive Equilibrium and Trading Networks: A Network Flow Approach
Under full substitutability of preferences, it has been shown that a competitive equilibrium exists in trading networks, and is equivalent (after a restriction to equilibrium trades) to (chain) stable outcomes. In this paper, we formulate the problem of finding an efficient outcome as a generalized submodular flow problem on a suitable network. Equivalence with seemingly weaker notions of stability follows directly from the optimality conditions, in particular the absence of improvement cycles in the flow problem. Our formulation yields strongly polynomial algorithms for finding competitive equilibria in trading networks, and testing (chain) stability
Selection of antigenically advanced variants of seasonal influenza viruses.
Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent sera to human H1N1 and H3N2 viruses. We also selected antigenic escape variants from human viruses treated with convalescent sera and from mice that had been previously immunized against human influenza viruses. Our pilot studies with past influenza viruses identified escape mutants that were antigenically similar to variants that emerged in nature, establishing the feasibility of our approach. Our studies with contemporary human influenza viruses identified escape mutants before they caused an epidemic in 2014-2015. This approach may aid in the prediction of potential antigenic escape variants and the selection of future vaccine candidates before they become widespread in nature.This work was supported by the Bill & Melinda Gates Foundation Global Health Grant OPPGH5383; National Institute of Allergy and Infectious Diseases (NIAID) Public Health Service research grants (USA); ERATO (Japan Science and Technology Agency); the Center for Research on Influenza Pathogenesis (CRIP) funded by the NIAID Contracts HHSN266200700010C and HHSN27 2201400008C; the Japan Initiative for Global Research Network on Infectious Diseases; Grants-in-Aid for Specially Promoted Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan; Grants-in-Aid from the Ministry of Health, Labour and Welfare, Japan; grants from the Strategic Basic Research Program of the Japan Science and Technology Agency; and by the Advanced Research & Development Programs for Medical Innovation from the Japan Agency for Medical Research and Development (AMED). C.A.R. was supported by a University Research Fellowship from the Royal Society. The authors acknowledge a Netherlands Organisation for Scientific Research (NWO) VICI grant, European Union (EU) FP7 programs EMPERIE (223498) and ANTIGONE (278976); Human Frontier Science Program (HFSP) program grant P0050/2008; Wellcome 087982AIA; and NIH Director's Pioneer Award DP1-OD000490-01. D.F.B and D.J.S. acknowledge CamGrid, the University of Cambridge distributed computer system. The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health.This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/nmicrobiol.2016.5
Selection of antigenically advanced variants of seasonal influenza viruses
Influenza viruses mutate frequently, necessitating constant updates of vaccine viruses. To establish experimental approaches that may complement the current vaccine strain selection process, we selected antigenic variants from human H1N1 and H3N2 influenza virus libraries possessing random mutations in the globular head of the haemagglutinin protein (which includes the antigenic sites) by incubating them with human and/or ferret convalescent se
Domain Organization of Long Signal Peptides of Single-Pass Integral Membrane Proteins Reveals Multiple Functional Capacity
Targeting signals direct proteins to their extra - or intracellular destination such as the plasma membrane or cellular organelles. Here we investigated the structure and function of exceptionally long signal peptides encompassing at least 40 amino acid residues. We discovered a two-domain organization (“NtraC model”) in many long signals from vertebrate precursor proteins. Accordingly, long signal peptides may contain an N-terminal domain (N-domain) and a C-terminal domain (C-domain) with different signal or targeting capabilities, separable by a presumably turn-rich transition area (tra). Individual domain functions were probed by cellular targeting experiments with fusion proteins containing parts of the long signal peptide of human membrane protein shrew-1 and secreted alkaline phosphatase as a reporter protein. As predicted, the N-domain of the fusion protein alone was shown to act as a mitochondrial targeting signal, whereas the C-domain alone functions as an export signal. Selective disruption of the transition area in the signal peptide impairs the export efficiency of the reporter protein. Altogether, the results of cellular targeting studies provide a proof-of-principle for our NtraC model and highlight the particular functional importance of the predicted transition area, which critically affects the rate of protein export. In conclusion, the NtraC approach enables the systematic detection and prediction of cryptic targeting signals present in one coherent sequence, and provides a structurally motivated basis for decoding the functional complexity of long protein targeting signals
Global circulation patterns of seasonal influenza viruses vary with antigenic drift.
Understanding the spatiotemporal patterns of emergence and circulation of new human seasonal influenza virus variants is a key scientific and public health challenge. The global circulation patterns of influenza A/H3N2 viruses are well characterized, but the patterns of A/H1N1 and B viruses have remained largely unexplored. Here we show that the global circulation patterns of A/H1N1 (up to 2009), B/Victoria, and B/Yamagata viruses differ substantially from those of A/H3N2 viruses, on the basis of analyses of 9,604 haemagglutinin sequences of human seasonal influenza viruses from 2000 to 2012. Whereas genetic variants of A/H3N2 viruses did not persist locally between epidemics and were reseeded from East and Southeast Asia, genetic variants of A/H1N1 and B viruses persisted across several seasons and exhibited complex global dynamics with East and Southeast Asia playing a limited role in disseminating new variants. The less frequent global movement of influenza A/H1N1 and B viruses coincided with slower rates of antigenic evolution, lower ages of infection, and smaller, less frequent epidemics compared to A/H3N2 viruses. Detailed epidemic models support differences in age of infection, combined with the less frequent travel of children, as probable drivers of the differences in the patterns of global circulation, suggesting a complex interaction between virus evolution, epidemiology, and human behaviour.T.B.
was
supported
by
a
Newton
International
Fellowship
from
the
Royal
Society
and
through
NIH
U54
GM111274.
S.R.
was
supported
by
MRC
(UK,
Project
MR/J008761/1),
Wellcome
Trust
(UK,
Project
093488/Z/10/Z),
Fogarty
International
Centre
(USA,
R01
TW008246‐01),
DHS
(USA,
RAPIDD
program),
NIGMS
(USA,
MIDAS
U01
GM110721‐01)
and
NIHR
(UK,
Health
Protection
Research
Unit
funding).
The
Melbourne
WHO
Collaborating
Centre
for
Reference
and
Research
on
Influenza
was
supported
by
the
Australian
Government
Department
of
Health
and
thanks
N.
Komadina
and
Y.‐M.
Deng.
The
Atlanta
WHO
Collaborating
Center
for
Surveillance,
Epidemiology
and
Control
of
Influenza
was
supported
by
the
U.S.
Department
of
13
Health
and
Human
Services.
NIV
thanks
A.C.
Mishra,
M.
Chawla‐Sarkar,
A.M.
Abraham,
D.
Biswas,
S.
Shrikhande,
AnuKumar
B,
and
A.
Jain.
Influenza
surveillance
in
India
was
expanded,
in
part,
through
US
Cooperative
Agreements
(5U50C1024407
and
U51IP000333)
and
by
the
Indian
Council
of
Medical
Research.
M.A.S.
was
supported
through
NSF
DMS
1264153
and
NIH
R01
AI
107034.
Work
of
the
WHO
Collaborating
Centre
for
Reference
and
Research
on
Influenza
at
the
MRC
National
Institute
for
Medical
Research
was
supported
by
U117512723.
P.L.,
A.R.
&
M.A.S
were
supported
by
EU
Seventh
Framework
Programme
[FP7/2007‐2013]
under
Grant
Agreement
no.
278433-‐PREDEMICS
and
ERC
Grant
agreement
no.
260864.
C.A.R.
was
supported
by
a
University
Research
Fellowship
from
the
Royal
Society.This is the author accepted manuscript. It is currently under infinite embargo pending publication of the final version
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