text:
Mimotopes selected with a neutralizing antibody against urease B from Helicobacter pylori induce enzyme inhibitory antibodies in mice upon vaccination.
ABSTRACT Urease B is an important virulence factor that is required for Helicobacter pylori to colonise the gastric mucosa. Mouse monoclonal antibodies (mAbs) that inhibit urease B enzymatic activity will be useful as vaccines for the prevention and treatment of H. pylori infection. Here, we produced murine mAbs against urease B that neutralize the enzyme's activity. We mapped their epitopes by phage display libraries and investigated the immunogenicity of the selected mimotopes in vivo.
The urease B gene was obtained (GenBank accession No. DQ141576) and the recombinant pGEX-4T-1/UreaseB protein was expressed in Escherichia coli as a 92-kDa recombinant fusion protein with glutathione-S-transferase (GST). Five mAbs U001-U005 were produced by a hybridoma-based technique with urease B-GST as an immunogen. Only U001 could inhibit urease B enzymatic activity. Immunoscreening via phage display libraries revealed two different mimotopes of urease B protein; EXXXHDM from ph.D.12-library and EXXXHSM from ph.D.C7C that matched the urease B proteins at 347-353 aa. The antiserum induced by selected phage clones clearly recognised the urease B protein and inhibited its enzymatic activity, which indicated that the phagotope-induced immune responses were antigen specific.
The present work demonstrated that phage-displayed mimotopes were accessible to the mouse immune system and triggered a humoral response. The urease B mimotope could provide a novel and promising approach for the development of a vaccine for the diagnosis and treatment of H. pylori infection.
- [Show abstract] [Hide abstract]
ABSTRACT: In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193 to 204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of Taenia solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile.Experimental Parasitology 05/2013; · 1.86 Impact Factor - SourceAvailable from: Vanete Thomaz-SoccolSilvana M Alban, Juliana Ferreira de Moura, João Carlos Minozzo, Marcelo Távora Mira, Vanete Thomaz Soccol[Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. METHODS: Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. RESULTS: Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5) and peptide 1B (1/5). In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. CONCLUSIONS: The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.BMC Infectious Diseases 01/2013; 13(1):42. · 2.56 Impact Factor - [Show abstract] [Hide abstract]
ABSTRACT: Helicobacter pylori (H. pylori) infection remains a significant global public health problem. Vaccine, especially edible vaccine, is considered to be effective in the management of H. pylori infections. By using recombinant technology, Lactococcus lactis (L. lactis) could serve as an antigen-delivering vehicle for the development of edible vaccine. The aim of this study was to produce edible UreB (urease B) vaccine derived from L. lactis against H. pylori. The UreB subunit is the most effective and common immunogen of all strains of H. pylori. The UreB was produced as a chimeric protein fused with IL-2 (human interleukin 2) as the mucosal adjuvant. Mucosal immunization of mice with recombinant L. lactis NZ9000 containing the UreB-IL-2 protein elicited more anti-UreB antibody that specifically bounded to the purified bacterial UreB protein and more cytokines such as IFN-γ, IL-4, and IL-17, and had a lower H. pylori burden and urease activity than control mice. These results suggest that the recombinant L. lactis expressing UreB-IL-2 can be potentially used as an edible vaccine for controlling H. pylori infection.Molecular and Cellular Probes 09/2013; · 1.87 Impact Factor
Page 1
RESEARCH ARTICLEOpen Access
Mimotopes selected with a neutralizing antibody
against urease B from Helicobacter pylori induce
enzyme inhibitory antibodies in mice upon
Yan Li1, Yunshan Ning1*, Yundan Wang1, Dandan Peng2, Yaodong Jiang3, Lili Zhang4, Min Long5, Jun Luo5,
Ming Li1*
Background: Urease B is an important virulence factor that is required for Helicobacter pylori to colonise the gastric
mucosa. Mouse monoclonal antibodies (mAbs) that inhibit urease B enzymatic activity will be useful as vaccines for
the prevention and treatment of H. pylori infection. Here, we produced murine mAbs against urease B that
neutralize the enzyme's activity. We mapped their epitopes by phage display libraries and investigated the
immunogenicity of the selected mimotopes in vivo.
Results: The urease B gene was obtained (GenBank accession No. DQ141576) and the recombinant pGEX-4T-1/
UreaseB protein was expressed in Escherichia coli as a 92-kDa recombinant fusion protein with glutathione-S-
transferase (GST). Five mAbs U001-U005 were produced by a hybridoma-based technique with urease B-GST as an
immunogen. Only U001 could inhibit urease B enzymatic activity. Immunoscreening via phage display libraries
revealed two different mimotopes of urease B protein; EXXXHDM from ph.D.12-library and EXXXHSM from ph.D.
C7C that matched the urease B proteins at 347-353 aa. The antiserum induced by selected phage clones clearly
recognised the urease B protein and inhibited its enzymatic activity, which indicated that the phagotope-induced
immune responses were antigen specific.
Conclusions: The present work demonstrated that phage-displayed mimotopes were accessible to the mouse
immune system and triggered a humoral response. The urease B mimotope could provide a novel and promising
approach for the development of a vaccine for the diagnosis and treatment of H. pylori infection.
Helicobacter pylori is a helical Gram-negative bacillus
that was originally discovered by Marshall and Warren
in the stomach of patients with gastritis and peptic
ulceration [1]. H. pylori has subsequently been recog-
nised as the major aetiological determinant of various
gastroduodenal diseases. Approximately half of the
world's population has been estimated to be infected by
H. pylori and harbours the bacterium in their upper
gastrointestinal tract [2]. Even though antibiotic-based
triple therapy is still the most effective treatment for
H. pylori infection, it seems that it is not feasible for
large-scale control of infection, partly because of the
high cost, poor compliance, and emergence of antibio-
tic-resistant strains. Increasing rates of therapeutic treat-
ment failure and high rates of re-infection, together with
low hygiene standards in developing countries have
made it imperative to develop vaccines to control infec-
tion [3].
Currently, most H. pylori vaccines in animal models
have utilised whole-cell preparation of native or recom-
binant proteins from the bacterium, together with
mucosal adjuvant. In general, these vaccines are
designed from a natural form of the pathogen after lysis
or inactivation that differs from natural epitopes [4]. In
response to H. pylori infection, the host triggers
* Correspondence: email@example.com; firstname.lastname@example.com
1School of Biotechnology, Southern Medical University, Guangzhou
Dadaobei No.1838, Guangzhou, China, 510515
Full list of author information is available at the end of the article
Li et al. BMC Biotechnology 2010, 10:84
© 2010 Li et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Page 2
vigorous humoral and cellular immune responses.
Although H. pylori-specific antibodies have been
detected at high titres in inflamed gastric mucosa and in
the serum, the infection can persist and/or never
resolve. This suggests that H. pylori can evade the innate
and adaptive immune responses, and the latter
responses triggered by H. pylori via this natural
approach do not elicit effective immunity [5]. Therefore,
we hypothesise that modified immunity might be
achieved via the use of mimotopes that differ from nat-
ural epitopes. This approach might be able to trigger an
effective immune response that is absent in natural
infections and natural-immunity-based approaches.
Phage display peptide libraries are usually employed to
select epitopes, which mimic the epitopes of natural
proteins recognised by the immune system. Such mimo-
topes are widely used in the development of vaccines
against many diseases [6-8], the design of molecules that
act as agonists or antagonists to many key biomolecules,
and the development of diagnostic reagents [9-12]. It
has been reported that mimotopes induce production of
protective antibodies, and consequently, become candi-
dates for the development of potential vaccines [13,14].
Mimotopes selected from random peptide libraries can
drive an active immune response towards the original
antigen and lead to effective immunity [15-17].
Urease plays a central role in the pathogenesis of
H. pylori infection and promotes colonisation of the sto-
mach and gut. Urease enzymatically hydrolyses urea to
form ammonia and bicarbonate, which neutralise gastro-
intestinal acids and protect the bacteria against the
acidic environment of the stomach. Urease is composed
of two major subunits, urease A and urease B, and the
latter is considered to be an excellent antigen for the
induction of protective immune responses [18,19].
Mucosal vaccination with Lactococcus lactis that
expresses urease B induces the production of IgG in
blood and urease-B-specific faecal IgA against H. pylori
infection [20]. Recently, by transformation of the gene
of urease B into carrot, Zhang et al. have found that
transgenic carrot plants can express the protein of
urease B and effectively induce immune responses in
mice [21]. In addition, immunisation of mice with the
trivalent fusion vaccine that was constructed by geneti-
cally linking heat shock protein A (HspA), H. pylori
adhesion (HpaA) and urease B414 (250-387 aa), has
been shown to protect mice from H. pylori infection
[22]. Thus, urease B appears to be an important target
for the design of a prophylactic vaccine for H. pylori.
Using this technique, we have already obtained the
neutralising mimotopes of Lpp20 and catalase for
H. pylori [23,24]. Whether such mimotopes of urease B
can be exploited to elicit functional antibody responses
against H. pylori has yet to be fully investigated.
In the present study, we applied the phage display
technology to identify the neutralising epitope of
H. pylori urease B with specific monoclonal antibody
(mAb) U001 that has been shown to inhibit significantly
urease activity. Two phage-displayed libraries were
immunoscreened and the selected phage clones were
analysed. The mimotopes were used to immunise
BALB/c mice and the immune responses for the phago-
topes were evaluated.
Expression and purification of recombinant protein
Genomic DNA extracted from H. pylori was used as a
template for PCR. To amplify the DNA that encoded
urease B, which included a major part of the active site,
primers were designed as described in the Methods. An
aliquot of the PCR mixture was analysed by agarose gel
electrophoresis and a DNA fragment of about 1700 bp
was detected, which corresponded to the expected posi-
tion of urease B. The DNA sequence was submitted to
GenBank (accession No. DQ141576). Expression of
recombinant protein urease B-GST was harvested from
Escherichia coli (Top10) and analysed by SDS-PAGE.
We observed a band of 92 kDa, which corresponded to
the predicted molecular size of the fusion protein
(Figure 1). Soluble urease B-GST protein was purified
by glutathione Sepharose (Figure 2) and used as the
immunogen. Western blotting confirmed that sera from
an H. pylori-infected patient specifically recognised the
recombinant urease B protein, whereas the negative sera
could not (Figure 2).
Production and characterisation of anti-urease B mAbs
Five hybridomas, U001-U005, were obtained by cell
fusion. All mAbs were IgG1 (?) and showed high speci-
ficity to urease B. The titre of culture supernatant and
ascites fluid was 1:32-1:64 and 1:32,000-1:128,000,
GST- UreaseBGST- UreaseBGST- UreaseB
Figure 1 SDS-PAGE analysis of expression of pGEX-4T-1/urease
B in E. coli. 1: pGEX-4T-1 before induction; 2: pGEX-4T-1 after
induction; 3: pGEX-4T-1/urease B before induction; 4: pGEX-4T-1/
urease B after induction; 5: protein marker.
Li et al. BMC Biotechnology 2010, 10:84
Page 2 of 12
Page 3
respectively. Triple serial concentrations of the antigen
and each mAb were selected to construct the corre-
sponding curves and to extrapolate the Kaffvalues using
the formula given in the Methods section. The apparent
Kavalues for the five mAbs varied from 1×108/M to
1×1012/M. mAb U001 displayed the highest affinity of
~1012/M (data not shown).
Inhibition and competition studies using increasing
concentrations of inhibitor or competitor mAbs revealed
that four of the five mAbs (U001, U003, U004 and
U005) competed significantly with each other, which
indicated recognition of the same or overlapping epi-
topes. However, U002 did not compete with the other
antibodies and recognised a distinct epitope.
Urease B enzymatic activity was measured and the
inhibitory effects of U001 and U002 were quantitated by
means of an inhibition assay. The inhibition rates were
calculated by comparing mAb-treated and control rates
for Urease B activity (Figure 3). U001 possessed the
strongest inhibitory effect on urease B enzymatic activ-
ity, whereas mAb U002 had a very moderate effect.
Screening of phage display peptide libraries and
characterisation of recombinant phage
To map the neutralising epitopes of urease B that were
recognised by mAb U001, the Ph.D.-12 and Ph.D.-C7C
libraries were screened with this purified mAb. After three
rounds of biopanning, the ratio of output/input increased
(Table 1) and 70 clones (35 each from the Ph.D.-12 and
Ph.D.-C7C libraries) were randomly selected. When tested
by ELISA for their immunoreactivity with mAb U001, 29
dodecapeptide clones and 28 heptapeptide clones gave
positive signals (Figures 4 and 5) with BSA cross-reactivity,
and sequences of 15 clones for each library were tested.
The deduced amino acid sequences of the corresponding
inserts were identified as six different sequences for
the Ph.D.-12 library and five for the Ph.D.-C7C library
(Table 2). The deduced amino acid sequences of selected
phage clones were aligned and analysed. The amino acid
sequences appearing in more than three different selected
clones and matched with urease B protein were grouped
as the consensus residues and +summarised with bold
letters (Table 2). Although these clones did not match the
urease B protein sequence, they shared extensive homol-
ogy with each other and were clustered and classified as
the conformational epitopes of urease B. Peptide
EXXXHDM from the Ph.D.-12 library and EXXXHSM
from the Ph.D.-C7C library showed a good match with the
urease B protein at 347-353 aa. Amino acids E, D, H, M
and S are key residues of the urease B epitope.
Competitive ELISA was performed to confirm the spe-
cificity of the positive clones. The results showed that the
recombinant phage D1 clone (with EHWSHDMFSPGD
sequence) and phage H1 clone (with EKLKHSM
sequence) all inhibited the mAb U001 binding to recom-
binant urease B protein, whereas wild-type M13 phage
had no effect (Figure 6).
Immune response induced by the selected phage clones
To evaluate the immune responses induced by the
selected mimotopes as vaccine candidates, phage clones
Figure 2 SDS-PAGE analysis of purified urease B and Western
blot analysis of urease B reactivity with patient serum.
(A) Bacteria expressing GST-urease B were lysed by sonication, and
the supernatant was applied to a Glutathione Sepharose 4B column.
Purified urease B was analysed on a 10% SDS-PAGE. 1: purified
urease B; 2: protein marker. (B and C) The gels were transferred to
nitrocellulose membranes and probed with diluted human sera
(1:50) from a normal donor or an H. pylori-infected patient.
Detection was performed with a goat anti-human antibody linked
to HRP (1:10,000), followed by staining with 3-amino-9-
ethylcarbozole. 1: GST; 2: urease B; 3: protein marker.
Figure 3 Inhibition of urease B enzymatic activity by specific
mAb U001. Urease (25 μl) was incubated with 25 μl mAb U001 or
U002 (equivalent to 0-25 μg) in 96-well microtitre plates overnight
at 4°C, with PBS as a control. On the following day, 50 μl 50 mM
phosphate buffer (pH 6.8) that contained 500 mM urea, 0.02%
phenol red, and 0.1 mM DTT was added to each well. The colour
development was monitored at 550 nm with a microplate reader.
The inhibition ratio was determined by the following equation:
[(activity without Ab - activity with Ab)/(activity without Ab)] × 100.
Li et al. BMC Biotechnology 2010, 10:84
Page 3 of 12
Page 4
D1 and H1 were chosen to immunise BALB/c mice by
intraperitoneal injection. Western blot analysis con-
firmed urease B recognition by the antisera (raised by
phage clones and the controls; Figure 7). The sera from
clone D1 (lane 1) and clone H1 (lane 2) reacted specifi-
cally with urease B protein at ~92 kDa, whereas the
pre-immune serum (lane 3), and the serum induced by
wild-type M13 phage (lane 4) or TBS (lane 5) were
negative. Triplicate ELISA measurements demonstrated
significant serum responses against urease B for each
sample and specific antibody responses from clone D1
and H1 were higher than others. The immune response
to wild-type M13 phage control was very low (Figure 8).
Alignment results from MIMOX
Computer-based analysis was performed to define the
consensus amino acid residues of the mimotopes.
Mimox wrapped ClustalW to align a set of mimotope
sequences that were analysed using an embedded ver-
sion of JalView. Figure 9 shows the resultant analysis,
which identified E, H, D and M as the consensus amino
acid residues in the aligned mimotopes.
Investigators have demonstrated previously that peptide
mimotopes obtained from phage epitope libraries or by
chemical synthesis can bind antibodies raised against
native structures. In fact, peptide mimotopes have been
found for most, if not all, anti-carbohydrate mAbs [16].
Moreover, peptide epitopes can mimic glycosphingoli-
pids and oligonucleotide structures [15,25]. In the case
of mimotope immunisation, several studies have shown
effective responses in vivo [26,27]. Furthermore, protec-
tive immune responses by mimotope immunisation have
been verified in many infectious diseases [7,28-30].
In the field of vaccine design, the phage-displayed
mimotopes have recently been shown to be possible vac-
cine components that do not necessarily represent the
structural equivalents of the original antigen, but provide
functional images that could replace the original epitopes
Table 1 Enrichment of phage displaying epitope by panning over mAb U001 from ph.D.-12 and Ph.D.-C7C
LibraryCyclecoating mAb (mg/L)Washing (TBST)Input phage (pfu)
1.5 × 1011
1.5 × 1011
1.5 × 1011
1.5 × 1011
1.5 × 1011
1.5 × 1011
Output phage (pfu)
8.4 × 103
8.0 × 104
3.0 × 105
7.6 × 103
7.2 × 104
3.5 × 105
Recovery rate (%)
5.6 × 10-6
5.3 × 10-5
2.0 × 10-4
5.4 × 10-6
4.8 × 10-5
2.3 × 10-4
????? ? ? ? ?
??????? ???????? ???????????? ?? ??
Figure 4 Binding specificity of selected phage from the Ph.D.-12 library. Wells of 96-well microtitre plates were coated with mAb U001 and
BSA (10 μg/ml, 100 μl) by incubation at 4°C overnight and blocked with 5% BSA in TBS. Affinity-selected phage were added to the wells and
allowed to bind at 37°C for 1 h. After the unbounded phage was removed, the bound phage was detected by incubation with peroxidase-
labelled murine anti-M13 antibodies. The bound peroxidase was determined by incubation with O-phenylenediamine dihydrochloride and the
reaction was determined in an ELISA reader at OD492. All the assays were carried out in triplicate and the error bars indicate standard deviation.
Li et al. BMC Biotechnology 2010, 10:84
Page 4 of 12
Page 5
for vaccine development [31-33]. Application of phage dis-
play technology to elucidate the essential chemical features
of conformational or discontinuous epitopes that are
recognised by some disease-related antibodies can provide
important information about the molecular mechanisms
of these diseases. The selected sets of peptides might also
lead to the future design of more effective reagents for
therapeutic and/or diagnostic purposes. The main advan-
tage of the phage-based approach is that it does not pro-
duce a consensus sequence of a large pool of peptides that
share a common property, but rather yields a list of pep-
tide sequences, each of which is endowed with the same
property. Alignment and comparison of the amino acid
sequences of the selected clones can then determine one
or more consensus sequences. This approach enabled us
to obtain the epitope profile of H. pylori in an efficient
manner. The profiles not only reveal the important roles
of each antigen, but also provide the epitope structures
within the antigenic protein that is involved in the induc-
tion of successful immune responses.
In this study, we first cloned and expressed urease B
of H. pylori, which was then used as an immunogen to
produce mAbs against this recombinant antigen by
hybridoma technology. We obtained two types of mAb
against different epitopes of urease B and observed that
mAb U001 strongly inhibited its enzymatic activity,
whereas mAb U002 did not induce this inhibitory activ-
ity. Therefore, mAb U001 could be beneficial for the
prevention of bacterial growth and attachment to the
gastric mucosa, thus, we identified the neutralising epi-
tope of this mAb via two phage display libraries (Ph.D.-
12 and Ph.D.-C7C). The two mimotopes of urease B
were characterised via affinity screening, binding, com-
petitive inhibition and DNA sequencing. Most clones
from the Ph.D.-12 library share the core EXXXHDM
sequence, therefore, we consider that EXXXHDM might
be a motif of urease B because clone D1 (with this
sequence) competitively inhibited U001 binding to
urease B and induced an immune response in mice.
Clone H1 (EKLKHSM sequence) from the Ph.D.-C7C
library appeared at a high frequency and had the same
function as clone D1. The mimotopes EXXXHDM and
EXXXHSM, which are highly homologous with urease B
residues 347-353 aa are also adjacent to a neutralising
epitope on urease B (321-339 aa), as reported previously
[34]. Li et al. have identified a B-cell-neutralising
??????????? ?? ???? ?? ???? ????????
Figure 5 Binding specificity of selected phage from the Ph.D.-C7C library. Wells of 96-well microtitre plates were coated with mAb U001
and BSA (10 μg/ml, 100 μl) by incubation at 4°C overnight and blocked with 5% BSA in TBS. Affinity-selected phage were added to the wells
and allowed to bind at 37°C for 1 h. After the unbounded phage was removed, the bound phage was detected by incubation with peroxidase-
labelled murine anti-M13 antibodies. The bound peroxidase was determined by incubation with O-phenylenediamine dihydrochloride and the
reaction was determined in an ELISA reader at OD492. All the assays were carried out in triplicate and the error bars indicate standard deviation.
Table 2 The sequences of ph.D.-12 and ph.D.-C7C phage-
displaying peptides from the recombinant phage clones
randomly selected through biopanning
LibraryClone no.Sequence
UreaseB protein
Li et al. BMC Biotechnology 2010, 10:84
Page 5 of 12
Page 6
epitope of urease B (211-225 aa) [35]. These observa-
tions suggest that there are predominant antigenic epi-
topes on the urease B protein that are effectively
The mimotopes selected from the above-mentioned
phage display libraries are not completely homologous
with the known amino acid sequence of urease B. Many
researchers have demonstrated similar results to ours
using antibodies as a tool to screen phage display library
mimotopes for other target protein antigens. One reason
for the disparity in mimotope urease B sequences might
be that the epitopes that are recognised by the gener-
ated mAbs could be a linear or conformational epitope
that consists of a discontinuous amino acid sequence. In
the latter case, the discontinuous amino acids could be
in close spatial proximity due to the folding of peptides.
We therefore speculate that amino acids E, D, H, M and
S are key residues for the urease B epitope. Our results
suggest that sequences that contain EXXXHDM and
EXXXHSM with potential antigenicity and immunogeni-
city should be further studied for their use in the devel-
opment of a diagnostic reagent, and vaccine that targets
H. pylori urease B. It is particularly interesting that H3
and H12 screened from the Ph.D.-C7C library (Table 2)
are exactly the same but in inverse order. Unlike the Ph.
D.-12 phage display library, the randomised sequence of
the Ph.D-C7C library is flanked by a pair of cysteine
Figure 7 Western blot analysis of antiserum induced by
phagotopes. Two phage (clones D1 and H1) were used to
immunise BALB/c mice through intraperitoneal administration. For
each immunisation, 1012pfu phage (in 50 μl) was used, with TBS
and wild-type M13 phage as controls. Three mice were immunised
with each sample and pre-immune sera were obtained. The mice
were bled once (pre-booster), and they were given a booster in the
same manner. The immune mice were bled every 7 days after
booster immunisation, and western blotting was performed to test
each serum sample. 1: serum from D1 phagotope; 2: serum from H1
phagotope; 3: pre-immune serum; 4: serum from wild M13phage; 5:
serum from TBS.
Figure 6 Phage inhibition of mAb U001 binding to urease B protein. Wells of microtitre plates were coated with urease B protein and
incubated with the mAb U001 (0.5 μg) plus increasing amounts (109-1015pfu) of the selected phage (clones D1 and H1). The binding was
monitored by OD492measurements after reaction with the peroxidase-conjugated goat anti-mouse IgG. The inhibition ratio was determined by
the following equation: [(activity without Ab - activity with Ab)/(activity without Ab)] × 100.
Li et al. BMC Biotechnology 2010, 10:84
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Page 7
residues. Under non-reducing conditions, the cysteine
residues spontaneously form a disulfide cross-link,
which results in phage display of cyclized peptides. Dis-
ulphide-constrained peptide libraries have previously
proven useful in identification of structural epitopes.
From this study, we presume that the epitope recog-
nised by mAb U001 is a structural epitope. Amio acid S
and M, which are also included in H1, are the key
amino acids that constitute this structural epitope.
Although the immune systems in mice and humans
differ, it is still informative to check the immune
responses and evaluate the potential of these experimen-
tal vaccine candidates in mice. Recombinant phage can
display epitopes and also bear significant adjuvant activ-
ity [36], therefore, we directly used phagotopes as anti-
gens to immunise mice. Our findings showed that the
phage-displayed epitope was able to mimic immunologi-
cal properties of the native epitope on H. pylori urease
B. Furthermore, using phage-displayed epitopes as anti-
gens, we were able to detect the antibody in the sera
from immunised animals. The results suggest that the
phagotopes also provide a convenient and economic
approach to prepare antigens, and could be applied in
an ELISA-based method for the rapid diagnosis of
H. pylori infection.
We demonstrated the feasibility of identifying important
mimotopes of urease B for vaccine development by
screening phage-displayed random peptide libraries with
mAb. The results also indicate the potential application
of phagotopes as alternative vaccine components. Com-
parison of mimotopes selected from a peptide library
with mAb and the original antigen sequence could lead
to a better understanding of the molecular mechanisms
that participate in the immune response and to the
design of specific peptides for diagnostic purposes and
vaccine development.
Bacterial strains and growth conditions
H. pylori strain NCTC11639 was obtained from the
Department of Microbiology, Southern Medical Uni-
versity (Guangzhou, China). This cell line was routi-
nely cultured on Columbia agar plates supplemented
with 7% saponin-lysed horse blood, 0.004% triphenyl-
tertrazolium chloride, and Dent selective supplement
(Oxoid, Basingstoke, UK) for 3 days at 37°C under a
micoaerophilic atmosphere (5% O2,15% CO2 and
80% N2). A colony was picked up and inoculated into
20 ml Brucella broth (Becton Dickinson, Cockeysville,
MD, USA) that contained 0.1% b-cyclodextrin supple-
mented with 5% (v/v) horse serum for 24 h. Two milli-
litres of culture medium was transferred to 40 ml fresh
medium and the same processes were repeated twice.
One millilitre of the incubated cell-containing medium
(mostly spiral rather than coccoid bacteria) was plated
on Brucella agar (Becton Dickinson) that contained 7%
(v/v) defibrinated horse blood, and cultured for an
additional 3 days at 37°C in a microaerophilic atmo-
sphere. Bacterial cells were harvested and washed
twice with cold PBS (25 mM sodium phosphate, pH
?????????? ????? ??? ????????????
Figure 8 Serum antibodies induced by phagotopes. Two purified phage clones (D1 and H1) were used to immunise BALB/c mice through
intraperitoneal administration. For each immunisation, phage (1012pfu/50 μl) were used, with TBS and wild-type M13 phage as controls. Three
mice were immunised with each sample and pre-immune sera were obtained. The mice were bled once (pre-booster), and they were given a
booster in the same manner. The immune mice were bled every 7 days after the booster immunisation, and triplicate ELISAs were performed to
test each serum sample with urease B as coated protein. All assays were carried out in triplicate and the error bars indicate standard deviation.
Li et al. BMC Biotechnology 2010, 10:84
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Page 8
7.2, 0.9% NaCl, pH 7.0). The cells were sedimented by
centrifugation (5,000×g for 10 min at 4°C) and stored
at -80°C.
Cloning, expression and enzymatic activity of
recombinant urease B protein
Genomic H. pylori DNA was prepared using a DNA
extraction kit (Takara, Belmont, Japan) according to the
manufacturer's instructions. Extracted genomic DNA
was then used as the template for amplification of the
urease B coding region using the Taq DNA polymerase
PCR kit (Takara). Two primer sequences that corre-
sponded to the 5' and 3' end of coding gene plus
EcoRI and SalI restriction sites were P1: 5'-TGCGAA
TTCAAAAAGATTAGCAGAAAA-3' and P2: 5'-GG
GTCGACGAAAATGCTAAAGAGTTG-3'. The PCR
products were cloned into an EcoRI- and SalI-digested
pGEX-4T-1 expression vector (Amersham Biosciences).
The plasmid was then transformed into E. coli strain
TOP10 (Invitrogen BV, Leek, The Netherlands) and
urease B expression was induced with 1 mM IPTG
when the cells were in log phase growth at 37°C. After
4 h, the cells were harvested by centrifugation at 7000
rpm/min for 10 min and washed with ice-cold PBS that
contained 5 mM EDTA and 2 mM PMSF. All subse-
quent procedures were performed at 4°C.
To purify recombinant urease B, the cells were resus-
pended in PBS that contained 5 mM EDTA and 2 mM
PMSF and lysed by sonication on ice, and centrifuged
at 10,000 rpm for 20 min to sediment cell debris. The
supernatant was applied to Glutathione Sepharose 4B
(Pharmacia, Uppsala, Sweden) to purify urease B
Figure 9 Mimotopes selected out with U001 were input and aligned with ClustalW. (A) Alignment of multiple sequences by ClustalW.
(B) The frequency of a given amino acid at each position of the alignment was calculated and displayed in a table. (C) A 3D bar figure was
created. The x axis represents the 20 amino acid types and gap, whereas the y axis is the frequency of occurrence and the z axis is the position
of the aligned sequences. (D) The alignment could be managed with JalView.
Li et al. BMC Biotechnology 2010, 10:84
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Page 9
protein according to the manufacturer's protocol.
Urease B samples were analysed by electrophoresis on
a 10% SDS-polyacrylamide gel. The gels were stained
for protein with 0.25% Coomassie Blue or transferred
to a nitrocellulose membrane (Millipore, Bedford, MA,
USA) and probed with diluted human sera (1:50 dilu-
tion with PBS) from a patient diagnosed with H. pylori
infection (positive serum) and a normal donor without
infection (negative serum). Detection was performed
with a goat anti-human antibody linked to horseradish
peroxidase (1:10,000) followed by staining with
3-amino-9-ethylcarbozole (Sigma, St Louis, MO, USA)
as a chromogen.
Production of the mAb and urease B inhibition test
BALB/c female mice, 4-6 weeks old, were purchased
from the Animal Center of Southern Medical University.
The mice were immunised by intraperitoneal injection
with 50 μg purified urease B-GST, which was emulsified
with an equal volume of Freund's complete adjuvant.
Three additional booster injections were administered
using incomplete adjuvant every 2 weeks. Three days
after the last injection, the spleen cells were harvested
and fused with the Sp 2/0 mouse myeloma cell line.
After 10-14 days, the culture supernatants were
screened for positive mAbs against recombinant urease
B-GST by ELISA. In the screening process, the mAbs
that reacted with urease B-GST but not with GST were
selected. Positive colonies were subcloned twice by lim-
iting dilution. Ascitic fluid was obtained from mice
primed with a 0.5-ml intraperitoneal injection of Pris-
tane and then injected with 106hybridoma cells. The
class and subclass of each mAb were determined with a
mouse monoclonal antibody isotyping kit (HyCult Bio-
technology, Uden, The Netherlands). The mAbs were
purified using protein G-Sepharose 4FF (Pharmacia)
according to the manufacturer's instructions, to remove
components that might have interfered with the biopan-
ning experiments.
The antibody titres were determined by ELISA. Puri-
fied urease B-GST antigen was coated on the microti-
tre plates (100 μl/well, 5 μg/ml in coating solution, 0.1
M sodium bicarbonate, pH 9.6) (Corning, Corning,
NY, USA) and incubated at 4°C overnight. The super-
natants of the cell culture as well as the ascitic fluids
were incubated with pre-coated urease B-GST (100 μl/
well) for 2 h at 37°C. Goat anti-mouse IgG HRP
(1:10,000 dilution with washing buffer) was used to
detect urease B-GST mAbs and O-phenylenediamine
dihydrochloride (Pierce Chemicals) was used as sub-
strate for HRP and the reaction was monitored at an
absorption of 492 nm using a microplate reader
(Model 550; Bio-Rad).
The affinity constant (Ka) of mAbs was determined by
using a method that was developed by Beatty [37].
Urease B-GST protein was coated on the ELISA plates
at three different concentrations ([Ag], [Ag'], and [Ag''])
followed by incubation with each mAb at different
concentrations. The Kaof each mAb was calculated
based upon the sigmoid curves that represented the
relationship between the concentration of mAb and the
OD values. The antibody concentration at half maximal
absorption for each curve was obtained by extrapolating
the curves and named ([Ab], [Ab'], and [Ab'']). The Ka
was calculated using the following equation: K = (n-1)/2
(n [Ab'] -[Ab]), where n = Ag/Ag'.
Using competitive ELISA, the topology of the epitopes
that were recognised by the mAbs was studied. Competi-
tive tests compared the ability of the various unlabeled
mAbs (competitors) to compete with the peroxidase-
conjugated mAbs for binding to urease B that was immo-
bilised on ELISA plates, in which saturating amounts of
unlabelled mAbs were added to the plates coated with
urease B protein before addition of labelled mAbs. Differ-
ent concentrations of each competitor mAb in PBS,
which contained a constant concentration of a labelled
mAb, were co-incubated with immobilised urease B for
40 min at 37°C. Following extensive washing with PBS,
the plates were developed with O-phenylenediamine
dihydrochloride (Pierce Chemicals) as mentioned above.
Percentage inhibition was finally estimated based on the
following calculation: percentage inhibition = [(OD with-
out competitor) - (OD with competitor)/OD without
competitor] ×100.
Purified H. pylori urease B was prepared. Stored cell
pellets were thawed, resuspended in 10% n-octyl b-D-
glucopyranoside (Wako Pure Chemical Industries),
vortexed five times for 10 s each, and centrifuged at
10,000 × g for 20 min at 4°C). DL-Dithiothreitol (DTT;
0.1 mM) (Sigma) was added to the supernatant, and the
mixture was applied to a PD-10 column (Sephadex G-25;
Pharmacia Biotech, Uppsala, Sweden), and the eluate was
dialysed against PBS that contained DTT. The eluate was
applied to a column (Shoei Works Co.) with Affi-Gel Hz
hydrazide gel (Bio-Rad) coupled to D12(S3) overnight in
a cold room. The column was washed with PBS that con-
tained DTT, and elution was performed with 5 M urea in
0.1 M potassium phosphate buffer (pH 6.5) that con-
tained 0.1 mM DTT. The eluate was dialysed with the
PD-10 column. The amount of protein was determined
by Lowry's method.
The H. pylori urease inhibition test was carried out
using the mAbs prepared above. Urease (25 μl) was
incubated with 25 μl anti-urease B epitope mAb U001,
U002 (equivalent to 0-25 μg) in 96-well microtitre plates
overnight at 4°C, with PBS as the control. On the
Li et al. BMC Biotechnology 2010, 10:84
Page 9 of 12
Page 10
following day, 50 μl 50 mM phosphate buffer (pH 6.8)
that contained 500 mM urea, 0.02% phenol red, and 0.1
mM DTT was added to each well. The colour develop-
ment was monitored at 550 nm with a microplate
reader (Model 550; Bio-Rad). Percentage inhibition was
determined by the following equation: [(activity without
Ab - activity with Ab)/(activity without Ab)] × 100.
Phage display library screening using specific mAb as a
target molecule
The Ph.D.-12 and Ph.D.-C7C libraries were obtained
from New England BioLabs. They were combinatorial
peptides that were fused to the minor coat protein (pIII)
of M13 phage, and consisted of about 1.5×1013and
2×1013pfu/ml electroporated sequences, respectively.
Three rounds of biopanning were undertaken according
to the manufacturer's instructions, with some modifica-
tions. A plate well was coated with 100 μg/well of anti-
urease B mAb U001 in 0.1 M NaHCO3, pH 8.6, and
incubated overnight at 4°C. The well was washed 10
times with TBST (50 mM Tris, 150 mM NaCl, pH 7.5,
and 0.1% Tween-20), filled with 400 μl blocking buffer
(0.1 M NaHCO3, pH 8.6, 5 mg/ml BSA, and 0.02%
NaN3), and incubated for 1 h at 4°C. In the first round
of biopanning, 10 μl phage (1.5×1011) from the initial
library, in 100 μl TBST, was incubated with the well-
bound mAb for 1 h at room temperature, under rocking
conditions. After repeated washes with TBST, the
bound phage was eluted from the well with 100 μl elu-
tion buffer (0.2 M glycine-HCl, pH 2.2). After neutrali-
sation with 15 μl 1 M Tris-HCl, pH 9.1, the eluted
phage were amplified by infecting 20 ml of a 1:100 dilu-
tion of an overnight culture of E. coli ER2738. The cul-
ture was incubated for 4.5 h at 37°C with vigorous
shaking. The supernatants were obtained and precipi-
tated with PEG/NaCl.
In the second and third rounds of biopanning, 20% of
the amplified phage from the preceding round was incu-
bated with mAb U001 overnight at 4°C as described
above. The procedures were identical to the first round
except that the coated mAb concentration was 10 mg/l
and 1 mg/l, and the washing steps were carried out with
0.3% and 0.5% TBST for the second and third round of
biopanning, respectively.
Binding specificity of the selected phage by ELISA and
DNA sequencing
Ninety-six-well plates were coated with mAb U001 and
BSA (10 μg/ml, 100 μl) by incubation at 4°C overnight,
and blocked with 5% BSA in TBS. Affinity-selected phage
were added to the wells and allowed to bind at 37°C for
1 h. After the unbounded phage was removed with 5%
TBST, the bound phage were detected by incubation
with peroxidase-labelled murine anti-M13 antibodies
(Promega, Madison, WI, USA). The bound peroxidase
was determined by incubation with O-phenylenediamine
dihydrochloride (Pierce Chemicals) in buffer (30 mM
citrate, 70 mM Na2HPO4, and 0.02% H2O2, pH 5.5).
After the reaction was stopped by the addition of 3 N
HCl, A492was determined with an ELISA reader (model
550; Bio Rad). All the assays were carried out in triplicate.
The phage from the third biopanning eluate were
cloned and amplified for DNA sequencing and immu-
noanalysis. The nucleotide sequence of the gene III
insert was determined as per the instruction manual.
The amino acid sequence of the insert was deduced
from the nucleotide sequence and was compared with
native urease B sequence using DNAMAN version 6.0
software. The sequences that appeared >3 times among
the selected phage clones were classified as the consen-
sus sequence. The aligned amino acid sequences shared
by three (or more) identical amino acids within the
dodecapeptides (heptapeptides) were determined as the
mimotopes of the matched protein sequences.
Competitive ELISA analysed the binding specificities
of the recombinant phage to the screened mAb U001.
The wells of microtitre plates were coated with urease B
protein and incubated with mAb U001 (0.5 μg) plus
increasing amounts of the selected phage. The binding
was monitored by OD492measurements after reaction
with the peroxidase-conjugated goat anti-mouse IgG
Animal immunisation and preparation of antisera
To evaluate the potential of the selected mimotopes as
experimental vaccine candidates, two purified phage
clones (D1 and H1) were used to immunise female
inbred specific-pathogen-free BALB/c mice through
intraperitoneal administration. For each immunisation,
phage (~1012pfu/50 μl) was used, and TBS and wild-
type M13 phage were used as the negative controls.
Three mice were immunised with each sample and pre-
immune sera (negative control) were obtained. The
mice were bled once (pre-booster), and then they were
given booster injections in the same manner. The
immune mice were bled every 7 days after the booster
immunisation, and ELISA and western blotting were
performed to test each serum sample for H. pylori
urease B-specific antibody activity.
Analyzing mimotopes with MIMOX
MIMOX (http://immunet.cn/mimox/intro.html#7) is a
web tool for bioinformatics analysis of peptide
sequences derived from phage display. The interface
ClustalW can align a set of mimotope sequences and
deduce consensus sequences based on the aligned
mimotopes through a simple statistical method. A set of
mimotopes obtained above (including 6 dodecapeptides
Li et al. BMC Biotechnology 2010, 10:84
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Page 11
and 5 heptapeptides) was input and aligned with all
parameters as defaults. The alignment can be viewed
and managed through JAIView.
This study was supported by National Natural Science Foundation of China
(31070119) and also supported by grants from State Key Development
Program of Basic Research of China (2007CB513101).
Author details
1School of Biotechnology, Southern Medical University, Guangzhou
Dadaobei No.1838, Guangzhou, China, 510515.2Department of Cardiology,
Guangzhou General Hospital of Guangzhou Military Command, Guangzhou,
China.3Department of Urology, Nanfang Hospital, Southern Medical
University, Guangzhou, China.4Cancer Center, Nanfang Hospital, Southern
Medical University, Guangzhou, China.5Department of Microbiology,
Southern Medical University, Guangzhou, China.
Authors' contributions
YL performed the majority of the bench work except for the preparation of
mouse antibodies against urease B. YW also contributed the majority of the
work except for screening the phage library. DP participated in cloning and
expressing recombinant urease B. YJ and LZ performed ELISA and western
blotting. ML and JL performed the culture of H. pylori and immunised the
animals. YN and ML designed the experiments. YL wrote the manuscript and
all authors have read and approved the final manuscript.
Received: 28 March 2010 Accepted: 30 November 2010
Published: 30 November 2010
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Cite this article as: Li et al.: Mimotopes selected with a neutralizing
antibody against urease B from Helicobacter pylori induce enzyme
inhibitory antibodies in mice upon vaccination. BMC Biotechnology 2010
Submit your next manuscript to BioMed Central
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Li et al. BMC Biotechnology 2010, 10:84
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text:
Silchoro Apartments
TripAdvisor Reviews Silchoro Apartments Albufeira
Travel Blogs from Albufeira
... to work hard at not succumbing too often. Coffee is about a euro or two depending on where you have it and there are loads of untouristy coffee shops and restaurants which is just a door in a wall, no advertising, complete with tiles on the walls and rickety chairs and a cat asleep on a table.There are loads of free roaming dogs. Some are typical medium height, long legged, brown mongrels. Others might walk past you on ...
Hello again, After I published the first blog of today I noticed my heading was wrong. Sometimes the computer changes a word to what it thinks I meant and I neglected to proof read my entry. So the title has been changed to what it should have been in the first place! So only a couple of photos taken a couple of weeks ago and I just never included them before. So again, Happy New Year, Adventure ...
Feliz Another Novo!!!
... Portugal! Not a very good history lesson.
Tomorrow I take the bus to Seville. It is about a 4 hour ride in a giant bus, the long distance buses are huge. The cost again is inexpensive...about $16.
Seville should be packed with places to explore and things to see. I have booked a room for a week, but may stay longer before I head down to Cadiz on the coast...to be seen!
Enjoy the few photos. Happy New Year, Adventure ...
Flea market in Monte Gordo
... they must have dragged the boats up, now there is a big tractor that pulls the boats out of the water onto the beach and then returns them to the sea after the catch is uploaded. A short entry today, but I still hope you enjoy it. My best, Adventure ...
Bon Natal from Portugal!
... and gave them both the little birds. They seemed so pleased they stood up and gave me a hug and a kiss on each cheek. They seemed very photo worthy and through hand jestures agreed to having their photo taken. I hope you enjoy the photo as much as I enjoyed the giving of the birds and ...

subset: commoncrawl


text:
Climate change study examines economic effect
AFBF: High prices for calves, fed cattle will continue through 2015
Lower cotton price likely to limit season's seeded acres
Network lets Indiana farms test practices toward water quality
Ive seen a great deal of misinformation on bloat prevention over the years. You would think, as common a problem as bloat is in cattle and sheep, we would all be on the same page with the information.
Years ago, we were told that research had been done to show that frost did not increase the incidence of bloat in livestock. Farmers would tell me that was just plain wrong. Universities all pretty much agree now that frost, and even dew, on legumes increase the incidence of bloat.
Bloat is caused by an accumulation of gas in the first two stomachs of a ruminant animal. Production of carbon dioxide occurs as a natural part of digestion and is usually discharged by belching. If the animal cant get rid of the gas, it builds up putting pressure on the diaphragm. This can be caused by a froth that layers on top of the rumen content preventing the gas bubbles from rising to the top and bursting. Since the animal cant belch, the gas is trapped in the bubbles and the condition gets worse.
Pasture bloat occurs when the forage is high in protein and low in fiber. It is most common in pastures that have substantial amounts of alfalfa or clovers in the sward. Immature legumes, which are low in fiber, are most likely to cause the problem. In areas where trefoil can be used as a pasture legume, it is a good choice since it does not cause bloat, by most accounts. Bloat seldom occurs in pastures that are predominantly grass.
Most experts agree that bloat occurs more often when animals go into the pasture hungry and when the animals are first turned into the pasture. Greedy feeders are most likely to get into problems. A rapid intake of lush green legume is often the cause. Frost, dew or even rain can increase the incidence.
To prevent bloat, in the spring, graze pastures, which are 50 percent or more grass. When you first turn animals into a new pasture, try to be sure that they are not hungry. You may even need to feed long stem hay. This reduces the excessive intake. Do not turn into a new pasture when dew or frost is on the legumes. Try to stick to a routine of moving animals from paddock to paddock.
One of the best practices for prevention of bloat in cattle or sheep is feeding poloxalene. It is a surfactant that helps prevent the formation of foam in the rumen.
This farm news was published in the May 24, 2006 issue of Farm World.


text:
Birmingham Baptist AssociationChurches working together...to meet needs...to change lives.The Birmingham Baptist Association is a partnership of 135 congregations in the Metro-Birmingham area.
Ministers Conference CalendarWinter Schedule 2015
All ministers are invited to our weekly Ministers Conference for a time of prayer, fellowship and encouragement.
We meet on Mondays at 11:00 am, at the BBA building. Please give us your first and second choice for the sessions you would like to attend. Attendence is limited to one session per calendar year. (Please note that the schedule is subject to change.) JanuaryJanuary 12 - Harper Shannon, Retired
January 19 - Office closed for Martin Luther King Day. January 26 - Executive Board meeting, Shades Mountain Baptist Church FebruaryFebruary 2 - Rev. Nathan Daniels, Westmont Baptist ChurchFebruary 9 - Rev. Jon Killingsworth, Pastor, Springville Baptist Church February 16 - Rev. Steven Weems, Pastor, Huffman Baptist Church February 23 - State Evangelism Conference Please visit our Community Events Info page to learn about exciting events happening in the Greater Birmingham area and beyond!Helen White - Evie EubankOffering for Associational MissionsSupport local missions in our Jerusalem


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To prevent a top-heavy turkey breast from tilting over onto its side, use kitchen shears to trim an inch or two from the bony lower portion of the rib cage—just enough to level the underside of the breast so that it rests securely in the pan or on the grill.
Southern Living DECEMBER 2010
1. Combine light brown sugar and next 5 ingredients. Rub mixture over turkey breast; cover and chill 8 hours.
2. Preheat oven to 350°. Let turkey breast stand at room temperature 30 minutes. Place turkey breast on a rack in a roasting pan, breast side up. Pour chicken broth in bottom of pan. Cover turkey loosely with aluminum foil.
3. Bake at 350° for 1 hour and 45 minutes or until a meat thermometer inserted into thickest portion registers 170°. Transfer turkey to a serving platter, and let stand 15 minutes before slicing.
Go to Full Version of
Sugar-and-Spice Cured Turkey Breast Recipe


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Arcadia Wildlife Sanctuary
127 Combs Rd., Easthampton, MA 01027
3 people saved this
Seven hundred and twenty-five acres of woods, meadow, and marsh; five miles of trails; observation tower. Look for signs of black bear, otter, beaver, and deer.
Of Note:
- Amenities: Accessible Facilities
10 Scenes of Massachusetts At Its Snowy Best
Sometimes, a blanket suits Mother Nature quite nicely. There hasn't been a whole lot...
Search All Listings Here.


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We stayed over the weekend two nights and paid £130 per night for the deluxe room. This was nice but not deluxe. It had a standard bed and mismatched decor but ok. No evening meal available due to illness but this also meant no bar or drinks. This is not clear on booking. The breakfast was good with a nice... More
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Race 1 will resume as soon as the track is clear, but only for Junior Dragster, Hot Rod and Stock Eliminator.
The remainder of Race 1 and Race 2 will be run at 9 a.m. Monday.
IHRA: 10 Most Recent Entries [ top ] Jan 27- San Antonio Nationals Postponed; Baton Rouge Added to 2015 Schedule Jan 25- IHRA Southwestern Nationals to Open 2015 Nitro Jam Drag Racing Series Jan 23- Team Aruba Comes Back for 11th Racing Season in the US Jan 23- IHRA Announces John Bisci as New Public and Media Relations Director Jan 23- IHRA Releases Crower Pro Mod Rules and Schedule Jan 23- IHRA Lowers Minimum Age for JR Race Car Junior Dragster Competitors Jan 20- Southwestern Int'l Raceway Hosts IHRA Nitro Jam Season Opener February 6-7 Jan 19- Sonny's Racing Engines to Sponsor IHRA Nitro Jam Bonus Programs Jan 10- IHRA Announces Super Rod Bonus Program Coming to Division 3 Dec 29- Hot Rod Power Tour Returning to MIR in 2015


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Things to see and do - Ventspils
Leaving for Baltic states
Where to sleep?
Ventspils Hotel tips
Raibie Logi from36 €
Book -
Hotel Vilnis from27 €
Book -
Olimpiska Centra Ventspils Hotel from
Ventspils Travel guide
Ventspils : Michelin's recommendations
A seaside resort with fine Art Nouveau buildings, a neo-Classical old town laid out around a busy square on which stands the town hall, and everywhere little pockets of well-kept greenery… Ventspils knows how to make the most of its virtues and is developing a well-earned reputation as a lively quality tourist destination.
Tourist attractions Ventspils : Things to see and do
Ventspils Castle
Jana Iela 17 LV - Ventspils
It took this castle three centuries (13C-16C) to assume its current aspect, but the rooms within offer a longer historical perspective, ranging from prehistory to the 19C and explaining the region's development through a rich collection of tools, jew..
Open-Air Maritime Museum
Rinka Iela 2 LV - Ventspils
Beyond the exhibition hall dedicated to the region's nautical life, the park has fishing boats displayed under awnings, as well as fishermen's cabins constituting a veritable village (with windmill and beehives). There are explanatory panels in Engli..
Hotels Ventspils
Hotel Vilnis
Talsu iela 5 LV - Ventspils LV-3602
The Vilnis is a pleasant 3-star hotel in the north of Ventspils, next to the business centre and 2 km away from the centre. The restaurant serves a wide range of traditional and creative dishes in a pleasant and calm atmosphere. The décor includes..
Ganību iela 26 LV - Ventspils LV-3601
Prix Prices from : 35 €
This modern 3-star hotel is located in the heart of Ventspils, just 150 metres from the Olympic Hall and 1.5 km from the beach. It features free Wi-Fi. Every room in the hotel has a TV and a bathroom with a shower. Some also feature a bath or a saun..
Raibie Logi
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Set in a restored 20th-century wood house in central Ventspils, Raibie Logi offers rooms with free Wi-Fi, and free private parking. It is a 10 minute walk from the beach. Rooms at Logi Raibie can accommodate various group sizes. They are tastefully ..


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This year, I noticed H.H. Shekha Moza bint Nasser of Qatar among the usual collection of European royals and movie stars that constitute Vanity Fair's annual celebration of wealthy people wearing clothes. (They're just like us. But they have more money. Ergo, tailors.)
Was it the turbans? They needed a token Arab among the royals? Were they bowled over by the grace with which she executed her duties as chairperson of the Qatar Foundation for Education, Science and Community Development? You would not believe what she had to go through to get that job!
The elect who compile this list apparently take their duties quite seriously but I am not privileged to be among them, so I don't know why they picked bint Nasser.
But I would offer to them the saga of Syria's first lady, Vogue and journalist Joan Juliet Buck as a cautionary tale. In this, according to Buck, Vogue assigned her to profile Asma al-Assad. The first lady was young and good-looking, and she wanted to talk about culture, antiquities, and museums, and Vogue was fine with that. Buck resisted, but not enough. The gauzy piece that resulted made her and Vogue look pretty bad when civil war broke out and the young, good-looking lady's husband got medieval on his citizens. Not that Vogue cared.
A civil war in Qatar, which is tiny – 300,000 citizens – and due to its oil and natural gas revenue has the world's highest per capita GDP, according to Forbes, is unlikely.
But Qatar is an absolute monarchy. The Al Thani family's got the power, unlike the Europeans who are parasites, sure, but harmless ones. There's also all kinds of exploitation of foreign laborers in Qatar.
It's true that Qatari women can vote and run for office and that they're allowed to go out and drive without a related male companion. So that is huge, and maybe if it were otherwise, bint Nasser wouldn't have made the list.
Still, her selection is just a bit too evocative of the Vogue editor's reasons for profiling Asma al-Assad. The same dynamic shapes glossy media's fascination with Jordan's Queen Noor, although she seems to be a pretty substantial person. It's myopic and self-indulgent to glorify these women for their looks and unearned titles in the face of their societies' inequality and their people's suffering. Even if they do look good in a turban.
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With one of the biggest brands in Hip-Hop, Snoop has never been one to shy away from the big screen and the bright lights of Hollywood. With roles in movies and television throughout his career, Snoop has caught the attention of audiences both young and old as Snoop has played the voice over for several… Read more »
Lil Wayne: "I Don't Know Where Hip Hop Is At"
Action Bronson Performs "Strictly 4 My Jeeps" At Nursing Home (VIDEO)
Texas rap duo V.I.P. drops their visual to "Old School Screw Tape," featuring up-and-coming Houston rapper Propain. After receiving widespread attention for the audio of the song "Old School Screw Tape," the Austin, Texas based group releases the visuals to their ode to legendary Houston Hip-Hop DJ and icon, DJ Screw. Look for the guys… Read more »
Treach Talks Watching Beyonce Naked, New Jersey Tolls + More (VIDEO)
Rapper Charged With Murder After Allegedly Rapping About The Crime In His Song
Mobb Deep's Prodigy Releases New Book 'H.N.I.C.' Through Infamous Books


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Virtual MIDI Piano Keyboard is a MIDI events generator and receiver. It
doesn't produce any sound by itself, but can be used to drive a MIDI
synthesizer (either hardware or software, internal or external). You can use
the computer's keyboard to play MIDI notes, and also the mouse. You can use
the Virtual MIDI Piano Keyboard to display the played MIDI notes from another
instrument or MIDI file player.Changes
* Option to represent MIDI velocity as highlight color tint
* Tool shortcuts, with a shortcuts editor dialog borrowed from Qtractor
* Language menu, allowing to select and change languages on the fly
* New "About Translation" dialog. Linux translation files moved to the program
data directory
* Fixed knobs and sliders tool-tip positions
* Allow to trigger defined but hidden keys
* Process incoming controller events: all sounds/notes off, and reset
* Translations updatedCopyright (C) 2008-2010, Pedro López-Cabanillas
License: GPL v3More info
Linux-audio-announce mailing list
LINUX® is a registered trademark of Linus Torvalds in the USA and other countries.
Linuxaudio.org logo copyright Thorsten Wilms © 2006.
Hosting provided by the Virginia Tech Department of Music and DISIS.


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Nestled in the charming town of Newark, the University of Delaware thrives on a combination of rich historical significance and cutting-edge research. More than 20,000 undergraduate and graduate students—affectionately know as Blue Hens&mda...
Jenna Cassara from Freehold, NJ has been named to the University of Delaware's Dean's List for the 2014 spring semester. To meet eligibility requirements for the Dean's List, a student must be enr... more
Spring 2014 Verified by University of DelawareJenna Cassara joined hundreds of Blue Hens in lending a helping hand to communities in need in various U.S. cities this spring break. The University of Delaware's alternative spring break program ... more
Spring 2014 Verified by University of DelawareJenna has not added any work experience yet.


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Al-Tira, Baysan
From Wikipedia, the free encyclopedia
- See Tira for other sites with similar names.
Al-Tira | |
Subdistrict | Baysan |
Palestine grid | 193/228 |
Population | 200 (1945) |
Date of depopulation | April 15, 1948[1] |
Cause(s) of depopulation | Whispering campaign |
Current localities | Gazit[2][3] |
Al-Tira was a Palestinian Arab village in the District of Baysan. It was depopulated by the Israel Defense Forces during the 1947–1948 Civil War in Mandatory Palestine on April 15, 1948 as part of Operation Gideon under the command of Yosef Weitz. It was located 17.5 km north of Baysan overlooking Wadi al-Bira. However, 'Ayn al-Bayda was the main source of drinking water for al-Tira inhabitants.
At the time of the 1931 census, Et Tira had 24 occupied houses and a population of 108 Muslims.[4]
In 1945 it had a population of 200.
The kibbutz Gazit was established on the land of village land in September 1948, 1.5 km southwest of the village site.[2][3]
- Hadawi, Sami (1970), Village Statistics of 1945: A Classification of Land and Area ownership in Palestine, Palestine Liberation Organization Research Center
- Khalidi, Walid (1992), All That Remains: The Palestinian Villages Occupied and Depopulated by Israel in 1948, Washington D.C.: Institute for Palestine Studies, ISBN 0-88728-224-5
- Morris, Benny (2004). The Birth of the Palestinian Refugee Problem Revisited. Cambridge University Press. ISBN 978-0-521-00967-6.
External links[edit]
This geography of Palestine article is a stub. You can help Wikipedia by expanding it. |


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24-Percent Hike For McMechen WaterDecember 7, 2013McMECHEN — McMechen City Council this week unanimously passed on second and final reading a 24-percent water increase.... VincentVegaCHRISTMAS ARRIVES EARLY IN MCMECHEN! Mayor jGracik distorts the truth of the damaging consequence this outrageous rate increase will have on senior citizens and those on fixed income. He stated, the increase will be $1.73 for the minimum users. Interestingly, what heh failed to disclose is that, perhaps only TEN PERCENT of the affected are MINIMUM users. Very few of the water users enjoy the DOUBLE PENSION and salary he receives! SHAME ON THE MAYOR & COUNCIL! 0 Agrees | 1 Disagrees | Report Abuse » RagnarSo McMechen residents & businesses use 24% of the water and will pay 37% of the overall increase? Sounds fair, right? 0 Agrees | 0 Disagrees | Report Abuse » Showing 2 of 2 comments
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Samsung 7.5 Cu. Ft. Gas Dryer
The Samsung 7.5 Cu. Ft. Front Load Gas Dryer, in white, connects your smart phone to your dryer, letting you diagnose and troubleshoot issues with ease. Nine drying cycles vary from bedding to air fluff and give you the customizability to handle any garment, no matter how soiled. Sensor dry technology automatically adjusts drying time based on how much moisture is remaining, so your clothes come out perfectly dry. Additional features include a drum light, child lock, lint filter indicator, and on/off signal. Plus, the door is reversible, making it easy to customize your laundry room.
Samsung 7.5 Cu. Ft. Gas Dryer
Additional Documents
Weights & Dimensions
Product Type: Gas | Finish: White |
Powder Coated Finish: Yes | Primary Material: Metal |
Number of Items Included: 1 | Scratch Resistant: Yes |
Rust Resistant: Yes | Pre-Set Dry Cycles: Yes
Adjustable Levels of Drying: Yes
| Adjustable Temperature: Yes
Timed Dry: Yes
| Venting System Included: Yes
Drawers Included: No | Door Window: Yes |
Door Safety Lock: Yes | Door Handle Design: Inner pocket |
Reversible Door: Yes | Drum Light: Yes |
Steam Setting: No | Control Type: Electronic; Dial |
Wrinkle Protection: Yes | Moisture Sensor: Yes |
End of Cycle Signal: Yes | Damp Dry Signal: Yes |
Lint Filter Indicator: Yes | Mixed Load Signal: Yes |
Energy Saver Option: Yes | Custom Program Setting: Yes |
Smart Care: Yes | Stackable: No |
Voltage: 120 Volts (V) | Frequency: 60 Hertz |
Capacity: 7.5 Cubic Feet | Compatible Washer Part Number: WF42H5000AW/A2 |
Country of Manufacture: Imported |
Assembly Required: No | Installation Required: Yes |
Product Warranty: 1 year parts and labor, 2 years control board, 3 years stainless steel drum and 10 years direct drive motor |
ADA Compliant: No | Energy Star Compliant: Yes |
ETL Certified: Yes | cETL Certified: Yes |
CSA Certification: Yes | CSA Certified: Yes |
NSF Certified: Yes |
Still have questions about this product? We're here to help
Samsung 7.5 Cu. Ft. Gas Dryer
Tell us what you think!
Tell us what you think about this product. It will help us provide you with better products in the future and help your fellow shoppers make better purchases.
Write the first reviewAbout the Manufacturer
For over 70 years, Samsung has been dedicated to making a better world through diverse businesses that today span advanced technology, semiconductors, skyscraper and plant construction, petrochemicals, fashion, medicine, finance, hotels, and more. Their flagship company Samsung Electronics, leads the global market in high-tech electronics manufacturing and digital media.Reference
7.5 Cu. Ft. Gas Dryer is also sometimes listed under the following manufacturer numbers: DV42H5000GW


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Statue Of Mahe De La Bourdonnais Former Colonial Governor Stdenis Reunion Island France Stock Photo 128519310Authority,Colonial Style,Color Image,Day,French Overseas Territory,Government,Governor,History,Leadership,Male Likeness,Monument,Naval Officer,No People,Outdoors,Photography,Politics,Reunion Island,Saint-Denis de La Reunion,Statue,The Past,Travel,Vertical,bertrand-francois mahe de la bourdonnaisPhotographer Danita DelimontCollection: Gallo Images Bertrand-Francois Mahe de La Bourdonnais (Saint-Malo, 11 February 1699 �?? Paris, 10 November 1753) was a French naval officer and administrator, in the service of the French East India Company.


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Scotland's favorite son, "The Bard" Robert (also Robbie, Rabbie) Burns is celebrated worldwide on his birthday January 25th. Born in 1759, he is regarded as Scotland's national poet and is best known [...read]
Susan Morrice dubbed "Female Warren Buffet"Denver based Susan Morrice is significantly involved with energy companies, jets, and other business ventures. In a recent interview with host Mike Robertson and co-host/producer Peggy Tuck on radio show "Straight Talk [...read] |
AOH Irish New Year's Eve Celebration at the Celtic TavernThe Ancient Order of Hibernians (AOH), Fr. Joseph Carrigan Division and The Celtic Tavern have joined forces for their 3rd Annual Family Irish New Year's Celebration. The event takes place from 2 [...read] |
The 29th Annual Winter Solabration December 20thBy Chris Kermiet – – - The Winter Solabration is a celebration of the Winter Solstice in music and dance. The dance consists of traditional American community dances Ð contras, squares, and [...read] |
Celtic Woman "Home For Christmas" with the Colorado SymphonyIrish music sensation CELTIC WOMAN celebrates the holiday season with "Home for Christmas, The Symphony Tour" with a stop in Colorado Wednesday, December 17, 7:30PM at Boettcher Concert Hall, at Denver Center [...read] |
Friends of Irish Studies December 2014(photo: Irish Studies student Ariana Walsh) Aithne ar ár Mic Léinn – Introducing our Students Ariana Walsh [Elementary Irish I] I joined the Friends of Irish Studies and am taking Irish language [...read] |
The Christmas Revels— An Irish Celebration of the Winter Solstice(Photo Caption: Every Christmas Revels show features the "Lord of the Dance," written by Sidney Carter to the tune of the familiar Shaker song "Simple Gifts." The song leader leads the audience [...read] |
CHEF ERIC McBRIDE & THE CELTIC CATERER GUEST CHEF at WILLIAM OLIVER'S PUBLICK HOUSE In FT. COLLINS This Holiday SeasonChef Eric W. McBride and the recipes of the Celtic Caterer will be featured throughout the month of December, as the Guest Chef at the newly renovated William Oliver's Public House (2608 [...read] |
December 2014 – Ceolta Notes— By Rodger Hara (photo: Acoustic Eidolon) Acoustic Eidolon presents its 14th Annual Holiday Concert at the Rialto Theater, 228 E. 4th St., Loveland, CO on the 5th and 6th and on [...read] |
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"Peace Meets the Street: On the Ground In Northern Ireland, 1993-2001". Rodger Hara Interviews Author Jim Lyons"Peace Meets the Street: On the Ground In Northern Ireland, 1993-2001". Rodger Hara Interviews Author Jim Lyons On a cold and snowy Veteran's Day in Jim Lyons' 30th floor office at the [...read] |
Irish Play "Juno and the Paycock" at Carsen Theatre(photo caption: Louis Clark as Captain Boyle, Kathy Reed as Mrs. Boyle, and Robert Mitchell as Joxer in The Upstart Crow's production of "Juno and the Paycock' by Sean O'Casey. Photo by [...read] |


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Birds and The Bees This animation tells you all about the birds and the bees. 6/18/2005 12:00:00 AM 236,175 1,331
Bird smashes into Windshield This racecar driver hits a bird at a very high speed. 9/23/2008 12:00:00 AM 74,088 25
Guy Tries To Wake Up Knocked Out Bird Mister...Mister please wake up, mister please wa - Oh shit! 6/17/2013 11:15:42 AM 58,630 67
Parrot Sings Bodies Hit Floor This parrot can sing the song Let the bodies hit the floor. 12/17/2010 12:00:00 AM 58,278 218
Cat vs Cat vs Bird It's an epic cat fight with some local birds getting in on the action. 11/29/2010 12:00:00 AM 57,461 115


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Go Huck Yourself & g.h.y. bikes Plus
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gohuckyourself.com is a website, ghybikes.com is a bike shop in Renton Wa. and the rest is an ever evolving entity. We mostly are into the aggressive side of biking but enjoy every aspect.
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Select () or exclude () categories to narrow your recipe search.
As you select categories, the number of matching recipes will update.
Find exactly what you're looking for with the web's most powerful recipe filtering tool.
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"Very good, I thought it was better than a lot of the overly sweet margaritas at restaurants. We used Corona too which was a nice touch!"
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"I took these into work and my boss said it was the best choc chip cookie she's ever had! Very easy and quick to make too!"


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He posted, she posted
Barbarella Fokos 9:30 a.m., Jan. 28
Posted August 28, 2012
Thursday 30One-time San Diego–based dirty-blues duo Little Hurricane blows back into town. The SDMA winners (Best New Artist) are out to tout their Homewrecker CD. After a few more West Coast dates, Tone and CC ...


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Perhaps the most difficult issue in discussing the idea of Zion is defining exactly what we mean. Even though D&C Gospel Doctrine lesson #46 is titled "Zion—The Pure in Heart," its first section is titled "The word Zion has several meanings" and lists no less than six. Of these, I've seen evidence in Mormon poetry for two or three definitions. First, the early Mormon poets used Zion in a millennial sense, to mean "The New Jerusalem." They also used ion to mean "The dwelling place of those who are exalted," or perhaps even simply "Those who are exalted." And,... Read more »
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- Seminary Woes
New Testament Gospel Doctrine Lesson #5
- Petra: Julie, you are brilliant and these posts are gems. I just bought your book and...
- Amanda: I love the image of the singles bar/dance. That had never occured to me before!
- Terry H: BTW Interview with Julie on Thursday Jan 29 at 9:00 Mountain Time. Its a call-in,...
Notes From All Over
If you serve in YW or know a YW, you will want to see these. (Julie) http://bycommonconsent.com/2015/01/26/young-women-values-not-princesses-not-for-the-faint-of-heart/ ... See MoreSee Less
At Clean Cut: "How can a 21st-century church ask people to not talk openly about things?" (Dave) ... See MoreSee Less
Kevin Barney on why faith should be good enough. We need to lower the testimony bar. (Dave) ... See MoreSee Less
Jana Riess on what the Church gets from John Dehlin's upcoming excommunication, besides bad publicity: basically nothing. (Dave) ... See MoreSee Less
Crowd sourcing my kid's seminary problems: a plea for your collective wisdom and experience. http://timesandseasons.org/index.php/2015/01/seminary-woes/#comment-529591 ... See MoreSee Less
RealClearReligion ran my post critiquing the dominant narrative (at least in the mainstream press) that John Dehlin's disciplinary council is primarily about his support for same-sex marriage and Ordain Women.
I explored some additional points on my own blog. http://difficultrun.nathanielgivens.com/2015/01/20/why-john-dehlin-faces-church-discipline/ (Nathaniel) ... See MoreSee Less
At JI, a Q&A with Jared Hickman, author of "The Book of Mormon as Amerindian Apocalypse." (Dave) ... See MoreSee Less
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In contrast to decades past, I suspect that atheism/agnosticism is a much greater threat to Mormonism than evangelical Christianity. ... See MoreSee Less
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- Mormons Around the World: Country Newsroom Websites | Jan 23 January 23, 2015
- Reid L. Neilson Named Assistant Church Historian and Recorder January 23, 2015
- Mormonism In the News: Getting It Right | January 22, 2015 January 22, 2015


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This is a great place, and very nice for the low price. The manager has bikes you can rent whenever you need them (although only a few are actually in decent riding condition), and the daily breakfast is pretty good. The rooms are comfortable and have a mini fridge. The walk to the beach is less than 5 minutes and... More
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Pryslezz - Sideways ft. Jason DeRulo & N.O.X.
Artist: Pryslezz
Featuring: Jason Derulo
Producer: Drop Dead Beats
Album: Death of a Man, Rebirth of a King
Avg Rating: 3.6 ( 13 votes )
There's no better way to relieve the stress built up over a long work week than popping a few bottles at your local club – that is, if you don't end up drinking so much that you're unable to walk straight when closing time rolls around. On a brand new single from Booth newcomer Pryslezz, the emcee is so tipsy he's leanin' Sideways. Fortunately, the Ohio native has a beautiful lady to hold him up as he continues to enjoy his night. Also accompanying Pryslezz in his drunken revelry are North Carolina emcee N.O.X. and R&B singer Jason DeRulo (who must be using a fake I.D., since he's only 19). The production, provided by Drop Dead Beats, isn't anything you haven't heard before, but it invests the record with enough club appeal to get some bodies out onto the dance floor. Sideways is set for inclusion on the emcee's sophomore album, Death of a Man, Rebirth of a King, due out February 24th.
Written by richard on 01/12/09
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King Ape/Lightyear/EMI
First DJ Booth Appearance:
"Find a Way ft. Slum Village" (2009)
Total DJ Booth Features:
1 Songs
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The view from my office on a damp misty and foggy morning 10 days ago. I live on the side of a depression between two slight hills and fog builds up inside there occasionally.
Photowalk on Saturday! See the link above and please comment if you're coming! We need numbers for the complementary lunch at the end
Aperture | ƒ/6.3 |
Camera | Canon EOS 40D |
Focal length | 48mm |
Shutter speed | 1/160s |
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"Art making is a form of alchemy, in a way, because you are trying…to make gold from nothing. When it works very well is when you manage to turn the ordinary into the extraordinary."
Drawing for "WEIGHING…and WANTING" (1997)
"That experience gave me a lot of confidence in the validity of working without a program, without the 'essay' being written in advance. So I made a decision I would never ever write a script, I would never write a storyboard, I would never ever write a proposal on the basis that even if I wrote them, the act of codifying like that some-how killed the project."
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Overview of content related to 'mimas'
This page provides an overview of 61 articles related to 'mimas', listing most recently updated content first. Note that filters may be applied to display a sub-set of articles in this category (see FAQs on filtering for usage tips). Select this link to remove all filters.
Mimas is a nationally designated academic data centre based at The University of Manchester in the United Kingdom. Its role is to support the advancement of knowledge, powering world-class research and teaching. Mimas hosts a significant number of the UK's research information assets - and builds applications to help people make the most of this rich resource. The organisation has a longstanding relationship with JISC, developing services that support teaching, learning and research, and strong connections with research councils, especially the Economic and Social Research Council (ESRC). (Excerpt from Wikipedia article: Mimas)
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